Cambio Climático en La Alta Montaña Mediterránea. Ecología Reproductiva, Potencial Adaptativo y Viabilidad Poblacional de Silene Ciliata.

Tesis Doctoral
Cambio climtico en la alta montaa mediterrnea.

Ecologa reproductiva, potencial adaptativo y viabilidad
poblacional de Silene ciliata.

Autor:
Luis Gimnez Benavides
Marzo de 2006

Directores:
Dr. Adrin Escudero Alcntara
Dr. Jos Mara Iriondo Alegra

Departamento de Matemticas y Fsica Aplicadas y
Ciencias de la Naturaleza. rea de Biodiversidad y Conservacin
Universidad Rey Juan Carlos. Mstoles, Madrid.

D. Adrin Escudero Alcntara, Dr. en Ciencias Biolgicas, Profesor Titular del
Departamento de Matemticas y Fsica Aplicadas y Ciencias de la Naturaleza de
la Universidad Rey Juan Carlos, y D. Jos Mara Iriondo Alegra, Dr. Ingeniero
Agrnomo, Catedrtico de Escuela Universitaria del Departamento de Biologa
Vegetal de la E.U.I.T. Agrcola de la Universidad Politcnica de Madrid,

CERTIFICAN

Que la Memoria Doctoral titulada Cambio climtico en la alta montaa
mediterrnea. Ecologa reproductiva, potencial adaptativo y viabilidad
poblacional de Silene ciliata, que presenta el licenciado en Ciencias Biolgicas
Luis Gimnez Benavides, ha sido realizada en la Universidad Rey Juan Carlos
bajo su direccin. Consideramos que se encuentra concluida y, por tanto,
autorizamos su presentacin.

En Mstoles, a 10 de abril de 2006.

Adrin Escudero Alcntara

Jos Mara Iriondo Alegra

Indice

Resumen Antecedentes. Objetivos de esta tesis doctoral. Metodologa.
Conclusiones generales 1

Captulo 1. Patch dynamics and islands of fertility in a high mountain
Mediterranean community 25

Captulo 2. Seed germination of high mountain Mediterranean species:
altitudinal, interpopulation and interannual variability 55

Captulo 3. Coping with climate warming: reproductive plasticity in a high
mountain Mediterranean plant under a severe heatwave 83

Captulo 4. Limited importance of a nursery moth pollinator for the reproductive
success of its host plant. Towards an unspecialized pollination
strategy? 119

Captulo 5. Local adaptation in the rearing edge of a high mountain
Mediterranean plant 153

Captulo 6. Population dynamics at the altitudinal rear edge may cause range
contraction of a high mountain Mediterranean plant 185

Resumen

RESUMEN

1
ANTECEDENTES

A pesar de que el clima es un fenmeno en constante evolucin que ha
experimentado repetidos cambios radicales a lo largo de la historia de la Tierra, ya no
hay duda de que el proceso de calentamiento actual se encuentra acelerado de manera
anormal y no se debe exclusivamente a causas naturales. El incremento global de las
temperaturas durante el siglo XX se ha estimado en unos 0.6 0.2 C, coincidiendo
temporalmente con el progresivo aumento de las emisiones de dixido de carbono y
otros gases causantes del efecto invernadero (IPCC, 2001). En el caso de las
precipitaciones, si bien se han detectado ciertas tendencias a la baja en algunas
regiones, stas no constituyen patrones claramente definidos. Las predicciones de
cambio para el ao 2100, elaboradas por el Grupo Intergubernamental de Expertos sobre
el Cambio Climtico (IPCC, 2001), se estiman en un aumento de las temperaturas de 1.4
a 1.8 C. A nivel mundial, aumentarn el vapor de agua y las precipitaciones medias,
pero a nivel regional se prevn importantes variaciones. Tambin cabe esperar cambios
en la frecuencia e intensidad de sucesos climticos extremos. La probabilidad de un
aumento de las temperaturas mxima y mnima durante el da, junto con una mayor
frecuencia de das calientes y olas de calor ser ms alta, mientras que disminuir el
nmero de olas de fro y das de helada (Easterling y col., 2000). As mismo, la
frecuencia de las sequas estivales aumentar en muchos lugares del interior de los
continentes, y es probable que se intensifiquen las sequas, y tambin las crecidas, a raz
de sucesos relacionados con El Nio.
En la Pennsula Ibrica, los modelos ms avanzados cifran el futuro
calentamiento en 1.2 C y 2 C cada 30 aos en invierno y en verano respectivamente, lo
que constituye uno de los incrementos ms acusados previstos para el contexto europeo.
Las precipitaciones tendern a disminuir en toda la regin Mediterrnea, existiendo
regiones ms y menos desfavorecidas e incluso se espera que el rgimen de
precipitaciones aumente en ciertas zonas (IPCC, 2001; de Castro y col., 2004).
Los efectos del calentamiento global sobre los ecosistemas terrestres y acuticos
ya se han comenzado a notar, y en los ltimos aos se han acumulado innumerables
evidencias cientficas de su impacto a mltiples escalas, desde los individuos y sus
poblaciones hasta las comunidades y paisajes que los albergan (Sala, 2000). No
obstante, an se desconoce en gran medida la magnitud del impacto, as como la
capacidad de respuesta de los diferentes organismos a estos cambios.

RESUMEN

2
Respuesta de los ecosistemas de alta montaa al cambio climtico. Qu
sabemos?

Los ecosistemas rticos y de alta montaa son, junto con otros ambientes como
los sistemas ridos y semiridos, elementos especialmente vulnerables a los cambios
climticos, ya que estn controlados principalmente por las condiciones ambientales y
muchas de las especies que los colonizan crecen muy prximas a sus lmites fisiolgicos
de tolerancia. Sin embargo, por estas mismas razones los ecosistemas rticos y alpinos
se consideran especialmente interesantes para el estudio de los impactos del cambio
climtico, ya que sus efectos pueden ser detectados en cortos espacios de tiempo y a
escalas geogrficamente muy reducidas (Grabherr y col., 1994; Walther y col., 2005).
Ms que el aumento de las temperaturas en s, el efecto ms relevante del
cambio climtico sobre los ecosistemas de alta montaa viene producido por el cambio
en el rgimen de acumulacin de nieve y de su posterior deshielo. El grado de innivacin
y el momento del deshielo, ambos directamente correlacionados con la precipitacin y la
temperatura ambiental (Groisman y col., 1994; Dye, 2002), son los principales factores
que determinan los ciclos biogeoqumicos, la humedad edfica, el inicio y la duracin de
la temporada de crecimiento de las plantas, e indirectamente, la produccin anual de
biomasa (Walker y Webber, 1994; Kudo, 2003). Estos dos factores controlan tambin en
gran medida la fenologa de la floracin y fructificacin, as como el xito reproductivo de
muchas especies (Galen y Stanton, 1995; Stinson, 2004; Molau y col., 2005). As, varios
autores han puesto de manifiesto cmo las reducciones en el espesor de nieve
acumulada y el adelanto del momento del deshielo, estn provocando importantes
cambios en el xito reproductivo de las plantas de alta montaa (Inouye y McGuire,
1991; Price y Waser, 1998; Inouye y col., 2002; Kudo e Hirao, 2006).
En general, los estudios observacionales y experimentales realizados hasta la
fecha en sistemas rticos y alpinos muestran una cierta tendencia a la reduccin de las
limitaciones impuestas sobre el crecimiento y la reproduccin de las plantas (las
temporadas de crecimiento se alargan y las temperaturas resultan menos restrictivas).
Una de las consecuencias ms evidentes del aumento de las temperaturas es, por
ejemplo, la ascensin del lmite altitudinal del bosque, la cual se ha observado en
sistemas montaosos de todo el mundo (Seppa y col., 2002; Dullinger y col., 2004). Del
mismo modo, muchas especies no arbreas han ido migrando lentamente hacia las
cumbres, observndose en algunas regiones un aumento significativo en la riqueza de
especies a cotas altas (Grabherr, y col., 1994; Kullmann, 2002; Klanderud y Birks, 2003;
Walther y col., 2005). A nivel experimental, los estudios de calentamiento realizados por
el consorcio ITEX (Internacional Tundra Experiment) y por otras iniciativas
RESUMEN

3
internacionales, han revelado que en general las plantas rtico-alpinas aumentan su
crecimiento y mejoran su xito reproductivo a consecuencia del aumento de temperatura
de 2-3 C impuesto mediante el uso de OTCs (Open Top Chambers) (Arft y col., 1999).
Sin embargo, bajo esta aparente mejora de las condiciones de vida en las montaas se
esconden muchos problemas e incertidumbres. Por ejemplo, la ascensin de especies
termfilas puede traer consigo la desaparicin o reduccin por exclusin competitiva de
poblaciones de especies tpicamente orfilas, asentadas en las altas cumbres (Grabherr
y col., 1994; Pauli y col., 2003). Adems, ya que no todas las especies respondern del
mismo modo ante los cambios ambientales, las interacciones planta-planta entre
especies (facilitacin, mutualismo y competencia) pueden variar drsticamente
provocando efectos no esperados a nivel de comunidad (Heegard y Vandvik 2004;
Klanderud, 2005; Klanderud y Totland, 2006). Del mismo modo, las interacciones planta-
animal, como por ejemplo las establecidas con los polinizadores, dispersadores o
depredadores, pueden verse alteradas ocasionando importantes desacoplamientos de
consecuencias difcilmente predecibles (Totland 2001; Galen 1990, Freeman y col.
2003).

Potencial adaptativo y viabilidad poblacional

Bajo las nuevas condiciones ambientales impuestas por el calentamiento global
cabe esperar varias respuestas: desaparicin, migracin o adaptacin. Hasta la fecha,
por lo que nosotros sabemos, no ha sido descrita la extincin total de ninguna especie
por causas estrictamente derivadas del cambio climtico. Como se ha comentado
anteriormente, s existen numerosas evidencias de desplazamientos recientes en la
distribucin de especies originados por factores climticos, muchos de ellos con
reducciones importantes en la superficie de ocupacin (McDonald y Brown 1992; Pauli y
col. 2003; Lesica y McCune 2004; Breshears y col., 2005; Wilson y col., 2005). Sin
embargo, los esfuerzos de la comunidad cientfica en los ltimos aos han ido ms
encaminados a detectar la capacidad adaptativa de las especies (y las comunidades)
para enfrentarse a estos rpidos cambios ambientales. En este sentido hay que destacar
dos niveles claramente diferenciados. El primero es la capacidad fisiolgica de los
individuos para adaptarse a las condiciones cambiantes, lo que llamamos la plasticidad
fenotpica. As, cabe esperar que las especies respondan al adelantamiento del deshielo
iniciando antes su ciclo de crecimiento y reproduccin (Totland y Alatalo, 2002; Stinson,
2004), o que un descenso de la humedad edfica induzca una mayor eficiencia en el uso
del agua (Brock y Galen, 2005). El segundo nivel es el determinado por la capacidad
evolutiva de las especies, o potencial gentico para adaptarse a un cambio drstico
RESUMEN

4
cuando ste va ms all de los lmites medios de tolerancia de los individuos presentes
en una poblacin (Stanton y col., 2000). Algunos autores han argumentado que ante un
calentamiento tan extremadamente acelerado como el actual los organismos ssiles,
entre los que se encuentran las plantas, no tendrn capacidad de migrar lo
suficientemente rpido, por lo que su persistencia depender principalmente de su
plasticidad fenotpica y su potencial evolutivo (Davis y Shaw, 2001; Etterson, 2004; Davis
y col., 2005; Jump y Peuelas, 2005).
Por tanto, para obtener una evaluacin completa del impacto del cambio del
climtico sobre las plantas de alta montaa es necesario el estudio exhaustivo de los
factores que condicionan el crecimiento, el xito reproductivo, las interacciones entre
especies, o la capacidad de respuesta de los individuos y las poblaciones.
Una de las aproximaciones quiz ms integradoras y reveladoras de los procesos
que ocurren a nivel poblacional es el anlisis demogrfico. Mediante el uso de modelos
de anlisis matricial es posible conocer de una manera relativamente sencilla las
tendencias poblacionales (crecimiento, persistencia o declive), as como detectar los
estados y tasas vitales (reclutamiento, crecimiento, fecundidad y supervivencia) ms
sensibles a los cambios ambientales, o los que ms contribuyen a la tasa de crecimiento
poblacional, (Boyce, 1992; Caswell, 2001; Pic, 2002).

El caso Mediterrneo

Como ya he descrito, Espaa y por extensin toda la cuenca Mediterrnea
constituye una de las regiones de Europa ms sensibles al cambio climtico, ya que las
limitaciones ambientales vienen determinadas principalmente por el agua y no por la
temperatura como sucede en otras regiones como la Atlntica y Centro-Europea (Garca
y col., 1999, Castro y col., 2004; Thuiller y col. 2005). Mientras cabe esperar que el
calentamiento provoque un aumento en la productividad de los ecosistemas situados
ms al norte, probablemente genere una disminucin por escasez de agua en nuestra
regin (Valladares y col., 2004). Por su situacin geogrfica y su compleja orografa, la
Pennsula Ibrica alberga una extraordinaria riqueza de especies y un elevado grado de
endemicidad (Gimnez y col. 2004). Adems, muchas especies de ptimo euro-siberiano
alcanzan la pennsula en forma de poblaciones relcticas, acantonadas en refugios
climticamente favorables (Petit y col., 2003; Hewitt, 2004; Petit y col., 2005). Tal es el
caso de numerosos elementos rtico-alpinos, los cuales aparecen relegados a las
cumbres de nuestros principales sistemas montaosos constituyendo las poblaciones
ms meridionales de estas especies (Pauli y col. 2003). Los ecosistemas emplazados en
RESUMEN

5
su lmite ecolgico y/o geogrfico son los que probablemente presenten una mayor
vulnerabilidad al cambio climtico (Valladares y col., 2004), entre los que detacan las
comunidades de alta montaa mediterrnea objeto de la presente tesis doctoral. Sin
embargo, pese a los evidentes riesgos vaticinados, an son pocos los estudios que se
han desarrollado al respecto en nuestra regin (ver revisin en ECCE, 2004).

OBJETIVOS DE ESTA TESIS DOCTORAL

La presente tesis doctoral tiene como objetivos generales profundizar en el
conocimiento de las comunidades de la alta montaa mediterrnea del centro de la
pennsula Ibrica, y en especial en el impacto que el cambio climtico actual puede
generar sobre los procesos de reproducin, crecimiento, potencial adaptativo y viabilidad
poblacional. Para ello realizamos una aproximacin a dos niveles, comunidad y especie.
En los captulos 1 y 2 se presentan algunos resultados fruto de una aproximacin
preliminar a nivel comunitario. En el captulo 1 se estudia la estructura y dinmica de la
comunidad de pastos psicroxerfilos oro- y crioromediterrneos dominante en la zona de
estudio (Sierra de Guadarrama, Madrid). En el captulo 2 se analiza la ecologa de
germinacin de un buen nmero de especies de sta y otras comunidades adyacentes.
Los captulos 3 a 6 se centran en una especie caracterstica y dominante en estos
ecosistemas que fue seleccionada como modelo de estudio, Silene ciliata Pourret
(Caryophyllaceae).

METODOLOGA

rea de estudio.
El rea de estudio de esta tesis est localizada en el Parque Natural de la
Cumbre, Circo y Lagunas de Pealara (Sierra de Guadarrama, Madrid, 40
o
47 N, 3
o
57
W). No obstante, para los captulos 1 y 2 fueron seleccionadas algunas localidades fuera
de sus lmites, dentro del cercano Parque Regional de la Cuenca Alta del Manzanares.
Los datos climticos (procedentes de la estacin del Puerto de Navacerrada, situada a
1890 m, 8 km al suroeste de la zona) arrojan temperaturas medias anuales de 2 a 4 C, y
medias mximas y mnimas del mes ms fro de -3 a 1 C y -9 a -6 C respectivamente.
La precipitacin anual es de unos 1400 mm (figura 1).
Como en muchas otras zonas, el cambio climtico ha empezado ya a
manifestarse en esta regin. Aunque ciertamente escasas, algunas evidencias cientficas
RESUMEN

6
han demostrado en los ltimos aos el acusado incremento de las temperaturas, y han
descrito algunos de los efectos que a da de hoy comienzan a percibirse. En el marco
regional del Sistema Central espaol, Granados y Toro (2000) y Agust-Panareda y col.
(2002) estimaron el acusado aumento de las temperaturas que durante el ltimo siglo se
ha producido, mediante reconstrucciones climticas a partir de fsiles de quironmidos
(Diptera: Insecta) acumulados en diversas lagunas de montaa (figura 2). La primera
evidencia del efecto de este aumento de temperatura sobre los ecosistemas de la Sierra
de Guadarrama la ofreci Sanz-Elorza y col. (2003), quienes demostraron el progresivo
reemplazamiento de los pastizales de montaa cacuminales por el enebral-piornal
oromediterrneo al migrar este en altura. Posteriormente, Wilson y col. (2005)
describieron la importante contraccin de los lmites altitudinales inferiores de 16
especies de mariposas desde la dcada de los 70 hasta la actualidad.

Figura 1. Diagrama ombrotrmico del puerto
de Navacerrada (1890 m), situada a 8 km al
suroeste de la zona de estudio (fuente:
Rivas-Martnez y col., 1990).

Figura 2. Reconstruccin de la
temperatura media del aire en verano,
estimada a partir de la abundancia de
fsiles de quironmidos en lagunas de
alta montaa del Sistema Central
(modificado de Granados y Toro, 2000).

RESUMEN

7
El pastizal psicroxerfilo de alta montaa mediterrnea

Los pastizales o joragales psicroxerfilos de alta montaa mediterrnea
constituyen la vegetacin potencial de las cumbres de la Sierra de Guadarrama por
encima de los 2100-2200 m. Est compuesto principalmente por gramneas duras entre
las que destaca Festuca curvifolia, y un importante nmero de nanocamfitos
almohadillados o pulviniformes, como Silene ciliata, Armeria caespitosa, Minuartia
recurva o Jasione crispa subsp. centralis, muchos de ellos endmicos de las montaas
ibricas. Una serie de reliquias alpino-pirenaicas, como Phyteuma hemisphaericum o
Agrostis rupestres, enriquecen la composicin de especies de estos pastos dotndolos
de un valor florstico y biogeogrfico destacado. Se diferencian de las comunidades de
pastos genuinamente alpinos por presentar un periodo de sequa estival intensa, lo que,
unido a la fuerte insolacin recibida, confiere a las especies que los habitan caracteres
propios de la vegetacin xeroftica: cutculas gruesas, colores glaucos, abundancia de
especies leosas, etc. (Rivas-Martnez y col., 2000). Como consecuencia de su
singularidad florstica y su reducida superficie, estos ecosistemas estn incluidos dentro
de la Directiva Habitats de la Unin Europea (Directiva 92/43/CEE).
Desde el punto de vista del aprovechamiento, su inters pastoral es bastante alto
a pesar de su escasa productividad y su baja palatabilidad y valor nutricional. La razn
es que constituyen la nica fuente de alimento fresco para el ganado durante el duro
verano mediterrneo. Es por ello que se ha venido pastoreando desde hace siglos,
tradicionalmente con ovino pero que est siendo sustituido por bovino de razas
autctonas como la negra avilea (San Miguel, 2001).

Silene ciliata Pourret (Caryophyllaceae)

Es un elemento orfito circunmediterrneo de porte almohadillado, distribuido por
las principales regiones montaosas de la Pennsula Balcnica, los Apeninos, el Massif
Central francs y la parte norte de la Pennsula Ibrica (Tutin y col., 1995; figura 3). En
esta ltima, aparece acantonada en las principales reas montaosas por encima de los
1100 m de altitud (Pirineos, Cordillera Cantbrica, Sistema Ibrico y Sistema Central). Su
carcter fincola, su rea de ocupacin relativamente pequea y el aislamiento de sus
poblaciones han motivado su inclusin en los catlogos autonmicos del Pas Vasco y
Castilla-La Mancha como especie rara y de inters especial, respectivamente
(B.O.P.V., 1998; D.O.C.M., 1998). Fuera de Espaa, est recogida en el catlogo de
especies protegidas de la regin francesa de Auvergne (J.O.R.F., 1990), y catalogada
RESUMEN

8
como muy rara en Portugal (Dray, 1985). El Sistema Central constituye el lmite
meridional de distribucin de la especie, donde ocupa un amplio rango altitudinal, desde
los piornales y enebrales rastreros por encima de los 1900 m, justo coincidiendo con el
lmite altitudinal del bosque, hasta los pastos psicroxerfilos oro- y crioromediterrneos
hasta los 2500 m. La ecologa de esta especie nunca haba sido descrita en detalle hasta
la presente tesis doctoral.

Figura 3. Distribucin mundial de Silene ciliata (fuente: Atlas Florae Europae. Tutin y col., 1995).

La especie fue seleccionada como modelo de estudio por ser un elemento
dominante y representativo de las comunidades de pastos psicroxerfilos de montaa.
Los elementos dominantes de una comunidad a menudo influyen fuertemente sobre
otras especies presentes y pueden determinar en gran medida la funcionabilidad del
ecosistema en su conjunto (Grime, 1998; Klanderund, 2005; Eriksson, 2000). El gnero
Silene cuenta adems con una abundante literatura cientfica sobre diversos aspectos de
su biologa reproductiva y dinmica de poblaciones que permite contrastar nuestros
resultados.
Para los captulos 1, 2, 4 y 5 fueron seleccionadas tres poblaciones de S. ciliata
dentro del Parque de Pealara. La primera de ellas se situ en los depsitos morrnicos
frontales del circo glaciar de Pealara (1966 m), en claros del enebral-piornal dominante
a esta altura (Avenello iberica-Juniperetum nanae y Senecioni carpetani-Cytisetum
oromediterranei). A esta cota, S. ciliata se halla en el lmite inferior de su distribucin
local y comienza a ser muy escasa, presentando poblaciones pequeas y fragmentadas.
Las otras dos poblaciones se situaron en el pico Dos Hermanas (2282 m) y pico
Pealara (2425 m), en zonas de pastizal psicroxerfilo (Hieracio myriadeni-Festucetum
RESUMEN

9
curvifoliae), donde S. ciliata es especie frecuente y caracterstica. Las localidades se
seleccionaron a lo largo de este gradiente altitudinal (470m aprox.), tratando de abarcar
en la medida de lo posible la variabilidad de microambientes en los que aparece la
especie en esta sierra, pero evitando introducir grandes diferencias estructurales del
terreno y zonas de hidromorfa evidente. Para el captulo 3 se utiliz otra poblacin
cercana de la especie, situada a una altitud de 2100 m.

Hadena consparcatoides (Schawerda, 1928) (Lepidoptera: Noctuidae)

Es una polilla nocturna univoltina cuya distribucin y ecologa es prcticamente
desconocida. Ha sido citada en unas pocas localidades montaosas del centro
peninsular, la Cordillera Cantbrica y los Pirineos Franceses (Calle, 1982; Yela, 1991;
Lasata y col., 2001; Moya, 2001). Algunos miembros de la familia Hadenidae (Hadena y
Perizoma especialmente) son conocidos por establecer una estrecha interaccin con
plantas de la familia de las Cariofilceas, especialmente con representantes de los
gneros Silene, Dianthus, Viscaria, Lychnis y Saponaria. Los individuos femeninos liban
el nctar de las flores de estas plantas al tiempo que depositan sus huevos en el interior
del cliz, favoreciendo tambin su polinizacin con el polen que portan (figura 4). Una
vez que eclosionan los huevos, las larvas penetran en las cpsulas y depredan las
semillas en formacin (Kephart et al, 2006). La relacin establecida entre estas especies
se encuentra, por lo tanto, a caballo entre la facilitacin y el parasitismo (la planta es
polinizada mientras las larvas de la polilla se nutren a expensas de sus semillas). El
desplazamiento hacia uno u otro tipo de relacin depende en gran medida de la
presencia o ausencia de otros polinizadores que puedan ofrecer su servicio sin imponer
un coste adicional a la planta (Thompson y Pellmyr, 1992). Hasta la fecha, se desconoca
la planta nutricia de H. consparcatoides y su estrecha relacin con S. ciliata. El
descubrimiento de este polinizador-depredador interactuando con la planta objeto de
nuestro estudio fue un estmulo para iniciar un anlisis preliminar, que finalmente fragu
en unos de los captulos de esta tesis.

RESUMEN

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Figura 4. Un ejemplar hembra de Hadena consparcatoides libando nectar de
una flor de Silene ciliata (Pealara, agosto 2005).

RESUMEN

11
Estructura de esta memoria

Todos los captulos de la presente memoria han sido escritos originalmente en
ingls para su publicacin en revistas cientficas de mbito internacional. Por ello, se
presentan los manuscritos originales de estos artculos. El nombre de los coautores y su
estado de publicacin se detallan a continuacin, junto con un breve resumen de cada
unos de ellos.

Captulo 1. Escudero, A.; Gimnez-Benavides, L.; Iriondo, J.M. y Rubio, A. 2004. Patch
dynamics y islands of fertility in a high mountain Mediterranean community.
Arctic, Antarctic, y Alpine Research, 36: 488497.
En este captulo se presentan los resultados de una aproximacin a nivel comunitario,
donde se estudia la estructura parcheada de los pastos psicroxerfilos dominantes en la
Sierra de Guadarrama por encima de los 2200 metros de altitud. La estructura parcheada
(manchas de vegetacin localizadas entre zonas de suelo desnudo) ha sido
tradicionalmente interpretada como el resultado de fenmenos de interaccin entre
especies, los cuales generan la agregacin de stas en respuesta a condiciones
ambientales severas. Los mecanismos que dan lugar a esta peculiar estructuracin han
sido estudiados en ecosistemas ridos y semiridos, as como en ambientes alpinos, pero
nunca antes haban sido estudiados en zonas de alta montaa mediterrnea. Nuestra
hiptesis es que esta estructura es consecuencia de las mltiples interacciones de
facilitacin y competencia entre especies. As, el establecimiento de las especies pioneras
generar, entre otras cosas, un aumento de la fertilidad del suelo donde se asienten,
favoreciendo la entrada de especies secundarias. Mediante una aproximacin
observacional, caracterizamos la composicin y abundancia de especies en las manchas de
vegetacin y analizamos muestras de suelo bajo stas y en zonas de suelo desnudo. Con
estos datos, se propone un modelo dinmico que explica la formacin, desarrollo y sucesin
de especies en los parches de vegetacin caractersticos de esta comunidad.

Captulo 2. Gimnez-Benavides L, Escudero A, Prez-Garca F. 2005. Seed
germination of high mountain Mediterranean species: altitudinal,
interpopulation y interannual variability.
Ecological Research, 20: 433-444.
En este captulo se estudia la ecologa de germinacin de un buen nmero de especies
colonizadoras de estos ambientes. Los objetivos fueron, por un lado, determinar las
RESUMEN

12
condiciones que favorecen la germinacin de cada una de las especies (de cara a
proyectos de restauracin de la cubierta vegetal o reintroduccin de especies), y por otro,
comprobar si existe una estrategia comn de germinacin para las especies de alta
montaa mediterrnea, y si sta difiere de la correspondiente a otras especies de
ambientes rticos y alpinos. En concreto se estudia la respuesta germinativa de 20
especies orfilas cuya distribucin se encuentra por encima del lmite altitudinal del
bosque (de 1900 a 2400 m), muchas de ellas endmicas o raras en la Pennsula Ibrica.
Se examin el efecto de diferentes temperaturas (10 C, 15 C, 20 C, 25 C y 15/25 C)
as como el efecto de un pretratamiento de estratificacin hmeda en fro (4 C) sobre la
capacidad de germinacin. Adems, se estudi la variabilidad altitudinal, interpoblacional
e interanual de la germinacin para algunas de las especies.

Captulo 3. Gimnez-Benavides, L., Escudero, A. y Iriondo, J.M. Coping with climate
warming: reproductive plasticity in a high mountain Mediterranean plant
under a severe heatwave.
New Phytologist, en revisin.
En este captulo se estudia la fenologa de la floracin y los factores que determinan el
xito reproductivo de Silene ciliata en tres poblaciones a lo largo de un gradiente
altitudinal. Los registros climticos de la zona de los ltimos 45 aos establecen que se
ha producido un progresivo calentamiento (1.8 C ms de media anual) y un sustancial
adelantamiento del momento de deshielo primaveral (19.7 das antes). Se establece la
hiptesis de partida de que las plantas de alta montaa mediterrnea no podrn
beneficiarse del alargamiento de la temporada de crecimiento. Al contrario que en los
ambientes rtico-alpinos, la desaparicin temprana de la nieve, fuente principal de la
humedad edfica durante el verano, acentuar el estrs hdrico sufrido por las plantas.
Es de esperar tambin que los efectos del estrs hdrico sean ms acusados en las
poblaciones situadas a menos altitud, donde las temperaturas son sensiblemente ms
altas. El objetivo principal es por lo tanto determinar hasta qu punto la plasticidad
fenolgica de la especie puede contrarrestar los efectos que el cambio climtico pudiera
ocasionar sobre ella. Mediante el uso de modelos de ecuaciones estructurales (SEM)
construimos una serie de relaciones causales entre variables reproductivas y
microambientales, las cuales fueron evaluadas durante dos aos consecutivos, uno de
condiciones ambientales medias (2002) y otro de temperaturas extremadamente altas y
precipitaciones estivales prcticamente nulas (2003).

RESUMEN

13

Captulo 4. Gimnez-Benavides, L., Dtterl, S., Jrgens, A., Escudero, A. y Iriondo, J.M.
Limited importance of a nursery moth pollinator for the reproductive success
of its host plant. Towards an unspecialized pollination strategy?
Manuscrito indito.
En este captulo se estudia la ecologa de la polinizacin de Silene ciliata, y se describe
por primera vez la relacin existente entre Hadena consparcatoides, un noctuido de
distribucin reducida, y esta planta. Al igual que se ha observado en otros pares de
especies afines, la relacin entre este polinizador-depredador y su planta husped revela
un cierto grado de especializacin. S. ciliata presenta un marcado sndrome de floracin
nocturna. Sus flores permanecen abiertas nicamente desde el atardecer al amanecer,
emitiendo un olor intenso, y sus anteras se abren para liberar el polen durante el
crepsculo. H. consparcatoides, por su parte, deposita sus huevos nicamente en esta
planta, y sus larvas se alimentan de sus semillas. El objetivo principal del estudio fue
evaluar el grado de dependencia de S. ciliata por la polinizacin nocturna, y en especial
por la brindada por H. consparcatoides. Se estableci la hiptesis de que en presencia
de otros polinizadores no depredadores la polilla pasa a jugar un papel de parsito
potencial del que S. ciliata tender evolutivamente a escapar. Mediante la observacin
de los polinizadores naturales de S. ciliata (tanto diurnos como nocturnos), y con ayuda
de un sencillo experimento de exclusin de unos u otros, determinamos el xito
reproductivo de la planta que puede asignarse a cada grupo de polinizadores. El estudio
se complement con la caracterizacin qumica del aroma desprendido por las flores, y
su comparacin con los aromas de especies con sndromes de polinizacin similares.
Adems, se realizaron ensayos para determinar la capacidad de la polilla para reconocer
el aroma floral completo, y algunos de sus principales componentes por separado.

Captulo 5. Gimnez-Benavides, L., Escudero, A. y Iriondo, J.M. Local adaptation in the
rearing edge of a high mountain Mediterranean plant.
Evolutionary Ecology, enviado.
En este captulo se evalan las limitaciones ambientales sobre el proceso de
germinacin y supervivencia de plntulas de Silene ciliata. Como factores crticos del
proceso de regeneracin natural de las especies, es de esperar que la germinacin y el
establecimiento de las plntulas estn sometidos a procesos de seleccin natural y
evolucin adaptativa. En la alta montaa mediterrnea el factor ms limitante lo
constituye la sequa estival, y sta se acenta a medida que desciende la altitud y
RESUMEN

14
aumentan las temperaturas. Nuestras hiptesis de partida fueron: (1) las condiciones
ambientales para la germinacin y supervivencia (en especial la humedad edfica) se
vuelven ms restrictivas en las poblaciones situadas a menor altitud, y (2) a pesar de
ello, puede existir un cierto grado de adaptacin local de las semillas a las condiciones
de su poblacin de origen, de manera que el efecto negativo del estrs hdrico se vea
reducido. Mediante un experimento de siembras recprocas entre poblaciones de S.
ciliata situadas a lo largo de su rango altitudinal, determinamos dnde se generan las
condiciones ms restrictivas para la regeneracin por semillas, as como el grado de
adaptacin local de cada una de las poblaciones.

Captulo 6. Population dynamics at the altitudinal rear edge may cause range
contraction of a high mountain Mediterranean plant.
Manuscrito indito.
En este ltimo captulo se estudi la dinmica poblacional de Silene ciliata en dos
poblaciones de la especie en la Sierra de Guadarrama, una situada en su lmite inferior
de distribucin (1970 m) y otra localizada a media altura (2250 m). Los objetivos
principales fueron estudiar la dinmica demogrfica de estas poblaciones, as como
determinar qu fases de su ciclo vital resultan ms crticas para su supervivencia a largo
plazo, y si stas son las mismas para las poblaciones en su lmite de distribucin
(sometidas a condiciones ms limitantes) que para las situadas en su centro. Nuestra
hiptesis inicial fue que ante las condiciones de calentamiento actuales cabe esperar un
progresivo desplazamiento de la especie hacia cotas ms altas, el cual debera verse
reflejado en los parmetros demogrficos de las poblaciones estudiadas. Por otro lado,
algunos estudios con especies afines muestran que una alta longevidad de los individuos
puede favorecer una estrategia de persistencia de las poblaciones a largo plazo, en lugar
de un desplazamiento altitudinal. Estas hiptesis fueron evaluadas mediante la
construccin de modelos matriciales demogrficos y anlisis log-lineares a partir de
datos tomados durante un periodo de cuatro aos.

RESUMEN

15
CONCLUSIONES GENERALES

A continuacin se enumeran, por orden de aparicin en los captulos de esta
tesis, los resultados y conclusiones ms relevantes.

1.- La estructura parcheada de la vegetacin en los pastizales crioromediterrneos de la
Sierra de Guadarrama determina claramente la fertilidad del suelo. Todos los niveles de
nutrientes, as como el pH, conductividad, balance C:N, cantidad de materia orgnica y
humedad, demuestran que las condiciones edficas son mejores bajo manchas de
vegetacin que en zonas desnudas. La fertilidad del suelo viene en parte determinada por el
tamao de los parches a travs de la acumulacin de biomasa, en especial la procedente
de las gramneas presentes en la comunidad. Sin embargo, slo se hall una relacin
significativa entre la composicin de especies y el pH. En este sentido, cabe destacar la
presencia de Silene ciliata en los parches con un pH ms cido que refleja suelos menos
desarrollados. Esto sugiere que S. ciliata puede actuar como especie pionera al tener la
capacidad de establecerse en zonas de suelo muy pobres. Adems, el anlisis de
asociaciones entre pares de especies revela la existencia de fenmenos de facilitacin y
repulsin entre algunas especies. As, se sugiere un modelo por el cual ciertas especies
pioneras promueven la entrada de otras en los primeros estadios de formacin de un
parche. Sin embargo, al alcanzar un desarrollo determinado, las especies comienzan a
competir por los recursos.

2.- Para ms de la mitad de las especies cuya capacidad germinativa fue estudiada no
fue necesario ningn tipo de tratamiento pregerminativo. Al contrario de lo que sucede en
las especies mediterrneas de zonas de baja altitud, las especies de alta montaa
alcanzan mayores porcentajes de germinacin a temperaturas relativamente altas, si
bien puede hacerse un claro agrupamiento de especies con requerimientos de
temperatura bajos, altos e indiferentes. Las semillas parecen estar fisiolgicamente
preparadas para germinar inmediatamente despus de su dispersin, sin que exista un
periodo extenso de maduracin postdispersiva aparente como sucede en muchas
especies rtico-alpinas. Para la mayora las especies que presentaron una clara
dormicin, el tratamiento de estratificacin en fro aument sustancialmente la
germinacin (6 de 9 especies), al igual que en otras especies de climas polares y
alpinos. Finalmente, la germinacin result muy variable entre poblaciones, altitudes y
aos, pero no se detect un patrn consistente entre especies.

RESUMEN

16
3.- El perodo de floracin de Silene ciliata es muy tardo comparndolo con el resto de
especies de su comunidad. Este se inicia a finales de Junio, prolongndose hasta
principios de septiembre. Al contrario que para muchas especies de medios cubiertos por
la nieve durante gran parte del ao,, el deshielo y el aumento de temperaturas no parece
controlar el inicio de la floracin, por lo que suponemos que sta viene controlada por el
fotoperiodo. Esta estrategia limita fuertemente el aprovechamiento del agua del deshielo
para una fase tan demandante como la reproductiva. No obstante, los modelos SEM
construidos mostraron que el xito reproductivo individual viene determinado en gran
medida por el intervalo de tiempo comprendido entre el momento del deshielo y el inicio
de la floracin. Esto sugiere la existencia de una fuerte presin selectiva hacia una
floracin ms temprana que evite el periodo de mayor sequa estival. Adems, las
plantas de las poblaciones situadas a mayor altitud se reprodujeron significativamente
mejor que las situadas en el lmite inferior, siendo estas diferencias ms acentuadas
durante el ao de extrema sequa y altas temperaturas. En resumen, una fuerte sequa
estival puede ocasionar un alto fracaso reproductivo en S. ciliata especialmente en las
poblaciones ms bajas. A pesar de la existencia de una seleccin fenotpica hacia una
floracin temprana, el periodo reproductivo de la especie parece estar controlado
genticamente por factores que no permiten una plasticidad lo suficientemente amplia
como para aprovechar los escasos recursos hdricos. Bajo el escenario de altas
temperaturas, escasas precipitaciones estivales y deshielo progresivamente ms
temprano, S. ciliata puede ver seriamente comprometido su xito reproductivo.

4.- Silene ciliata muestra un sndrome de floracin claramente nocturno que se ajusta
muy bien al mostrado por muchas especies afines. La composicin qumica de su
esencia floral refleja fielmente la de una especie de floracin nocturna con capacidad
para atraer especialmente a polillas que acten como polinizadores. No obstante, la
ausencia en su fragancia de ciertos compuestos qumicos, atrayentes muy especficos
de especies del gnero Hadena y afines, resulta bastante sorprendente. La observacin
de polinizadores revel que pese al marcado sndrome nocturno, S. ciliata es visitada
tanto por insectos diurnos como nocturnos, entre los cuales se encuentra Hadena
consparcatoides. Los ensayos de exclusin de polinizadores revelaron que los
polinizadores diurnos producen ms frutos por flor que los nocturnos, sin embargo estos
ltimos generaran ms semillas por fruto que los primeros. Adems, las semillas
procedentes de polinizaciones diurnas fueron ms pesadas y germinaron mejor que las
generadas por visitas nocturnas, lo que confiere una ventaja a la descendencia
producida durante el da. Estos resultados, junto con la alta prdida de frutos ocasionada
por depredacin de las larvas de H. consparcatoides, demuestran que esta polilla no slo
RESUMEN

17
no es imprescindible para el xito reproductivo de S. ciliata, si no que su relacin refleja
actualmente un parasitismo en lugar de un mutualismo. En este contexto, la ausencia en
la fragancia floral de elementos de reconocimiento especficos podra ser interpretada
como una estrategia para evitar en la medida de lo posible la depredacin de sus
semillas.

5.- La germinacin en campo de las semillas de Silene ciliata se concentra
inmediatamente despus del deshielo, lo que favorece el aprovechamiento de la escasa
humedad edfica. El estrs hdrico estival constituye la principal causa de muerte de
plntulas de S. ciliata, el cual va acentundose y provocando mayor mortalidad a medida
que avanza el verano. Los resultados de nuestros experimentos de siembras recprocas
entre poblaciones demostraron que la especie germina y sobrevive mejor en el centro de
su distribucin que en los lmites inferior y superior. Sin embargo, a pesar de la
existencia de ambientes en general ms favorables para la regeneracin, detectamos
ciertos niveles de adaptacin local que afectaron a la supervivencia y crecimiento de las
plntulas emergidas. Es decir, las plntulas sobrevivieron mejor y crecieron ms cuando
se sembraron en sus lugares de origen que cuando se transplantaron a otras
poblaciones. La existencia de procesos de adaptacin evolutiva como stos pueden
ayudar a contrarrestar los efectos del cambio climtico si se hacen efectivos al menos
con la misma rapidez con la que cambian las condiciones ambientales. En este caso, un
escenario de persistencia en lugar de desplazamiento altitudinal podra ser factible.

6.- El seguimiento demogrfico mostr claras diferencias entre las plantas del lmite
inferior y las situadas en el centro del rango altitudinal. El tamao medio de las plantas
fue considerablemente mayor en la poblacin inferior, reflejando una poblacin ms
envejecida que la situada en cotas superiores, donde se observ un mayor reclutamiento
de plntulas y una predominancia de plantas de pequeo tamao. La supervivencia de
los individuos fue tambin menor en la poblacin inferior, aunque en ambas poblaciones
se observ una mayor mortalidad de individuos en las clases de tamao ms grandes.
Este proceso lleva a unos valores de longevidad individual muy inferiores a los
observados en otras especies del gnero Silene de ambientes alpinos, siendo
especialmente llamativo en el caso de la poblacin inferior. La menor longevidad y el
bajo reclutamiento de plntulas indican que las poblaciones deben estar sufriendo una
serie de condiciones ambientales que limitan enormemente su persistencia a largo plazo.
Los valores de la tasa finita de crecimiento () confirman la tendencia decreciente,
especialmente en la poblacin inferior, siendo la supervivencia de las plantas el factor
que ms repercute sobre esta tasa de crecimiento poblacional. Globalmente los
RESUMEN

18
resultados sugieren una escasa capacidad de las plantas situadas en cotas inferiores
para hacer frente a las condiciones ambientales, que las obliga a desplazarse hacia
cotas ms elevadas. Dado que actualmente las poblaciones ya se encuentran habitando
las cotas ms elevadas de la Sierra de Guadarrama, estos procesos pueden llevar a una
seria reduccin del rango altitudinal de distribucin de la especie, con la consiguiente
reduccin del rea de ocupacin.

RESUMEN

19
BIBLIOGRAFA

BOPV. 1998. ORDEN de 10 de julio de 1998, del Consejero de Industria, Agricultura y
Pesca por la que se incluyen en el Catlogo Vasco de Especies Amenazadas de la
Fauna y Flora, Silvestre y Marina, 130 taxones y 6 poblaciones de la flora vascular
del Pas Vasco.
Brock MT and Galen C. 2005. Drought tolerance in the alpine dandelion, Taraxacum
ceratophorum (Asteraceae), its exotic congener T. officinale, and interspecific
hybrids under natural and experimental conditions. American Journal of Botany 92:
1311-1321.
Castro J, Zamora R, Hdar JA, Gmez JM. 2004. Seedling establishment of a boreal tree
species (Pinus sylvestris) at its southernmost distribution limit, consequences of
being in a marginal, Mediterranean habitat. Journal of Ecology 92: 266-277.
Caswell H. 2001. Matrix population models. Sinauer Associates, Inc., Sunderland
Massachussets.
DOCM. 1998. Decreto 33/1998, de 5 de mayo, por el que crea el Catlogo Regional de
Especies Amenazadas de Castilla-La Mancha.
Dray AM 1985. Plantas a proteger em Portugal continental. Servio Nacional de Parques,
Reservas e Conservaao da Natureza, Lisboa. 56 pp.
Dullinger S, Dirnbck T and Grabherr G. 2004. Modelling climate change-driven treeline
shifts: relative effects of temperature increase, dispersal and invasibility. Journal of
Ecology. 92: 241-252.
Dye DG. 2002. Variability and trends in the annual snow cover cycle in Northern
Hemisphere land areas, 1972-2000. Hydrological Processes 16: 3065-3077.
Easterling DR, Meehl GA, Parmesan C, Changnon SA, Karl TR, Mearns LO. 2000.
Climate Extremes: Observations, Modeling, and Impacts. Science 289: 2068.
ECCE. 2004. Evaluacin de los impactos del cambio climtico en Espaa. Ministerio de
Medio Ambiente.
Etterson JR. 2004. Evolutionary potential of Chamaecrista fasciculata in relation to
climate change. I. Clinal patterns of selection along an environmental gradient in the
great plains. Evolution 58: 1446-1458.
Garca D, Zamora R, Hdar JA, Gmez JM. 1999. Age structure of Juniperus communis
L. in the Iberian peninsula: Conservation of remnant populations in Mediterranean
mountains. Biological Conservation 87: 215-220.
Grabherr G, Gottfried M, Pauli H. 1994. Climate effects on mountain plants. Nature 369:
448.
RESUMEN

20
Groisman PY, Karl TR, Knight RW, Stenchikov GL. 1994. Changes of snow cover,
temperature, and radiative heat balance over the Northern Hemisphere. Journal of
Climate 7: 1633-1656.
Heegaard E and Vandvik V. 2004. Climate change affects the outcome of competitive
interactions: an application of principal response curves. Oecologia 139: 459-466.
IPCC. 2001. Climate Change 2001: The Scientific Basis. Contribution of Working Group I
to the Third Assessment Report of the Intergovernmental Panel on Climate Change.
Cambridge,UK: Cambridge University Press.
Klanderud K. 2005. Climate change effects on species interactions in an alpine plant
community. Journal of Ecology, Blackwell Publishing Limited. 93: 127-137.
Moreno JM and Oechel WC (eds.) 1995 Global change and Mediterranean-type
ecosystems. Springer-Verlag, New York.
McDonald KA and Brown JH. 1992. Using montane mammals to model extinctions due to
global change. Conservation Biology 6: 409-415.
Pic, X. 2002. Desarrollo, anlisis e interpretacin de los modelos demogrficos
matriciales para la biologa de conservacin. Ecosistemas 3
http//www.aeet.org/ecosistemas/023/ investigacin2.htm
Rivas-Martnez S, Fernndez-Gonzlez F Snchez-Mata D and Pizarro JM. 1990.
Vegetacin de la Sierra de Guadarrama. Itinera Geobotanica 4: 3-132.
Sala OE, Chapin III FS, Armesto JJ, Berlow E, Bloomfield J, Dirzo R, Huber-Sanwald E,
Huenneke LF, Jackson RB, Kinzig A, Leemans R, Lodge DM, Mooney HA,
Oesterheld M, Poff NL, Sykes MT, Walker BH, Walker M and Wall DH. 2000. Global
Biodiversity Scenarios for the Year 2100. Science 287: 1770.
Stanton ML, Roy BA and Thiede DA. 2000. Evolution in Stressful Environments. I.
Phenotypic Variability, Phenotypic Selection, and Response to Selection in Five
Distinct Environmental Stresses. Evolution 54: 93-111.
Stinson KA. 2004. Natural selection favors rapid reproductive phenology in Potentilla
pulcherrima (Rosaceae) at opposite ends of a subalpine snowmelt gradient.
American Journal of Botany 91: 531-539.
Totland O and Alatalo JM. 2002. Effects of temperature and date of snowmelt on growth,
reproduction, and flowering phenology in the arctic/alpine herb, Ranunculus
glacialis. Oecologia 133: 168-175.
Petit RJ, Hampe A and Cheddadi R. 2005. Climate changes and tree phylogeography in
the Mediterranean. Flora 54: 877885.
Petit RJ, Aguinagalde I, de Beaulieu J-L, Bittkau C, Brewer S, Cheddadi R, Ennos R,
Fineschi S, Grivet D, Lascoux M, Mohanty A, Mller-Starck G, Demesure-Musch B,
RESUMEN

21
Palm A, Martn JP, Rendell S and Vendramin GG. 2003. Glacial refugia: hotspots
but not melting pots of genetic diversity. Science 300: 1563.
Hewitt GM. 2004. Genetic consequences of climatic oscillations in the Quaternary.
Philosophical Transactions: Biological Sciences 359: 183-195.
Thuiller W, Lavorel S, Arajo MB, Sykes MT, Prentice C. 2005. Climate change threats to
plant diversity in Europe. Proceedings of the National Academy of Sciences of the
United States of America 102: 82458250.
Kudo G. 2003. Warming effects on growth, production, and vegetation structure of alpine
shrubs, a five-year experiment in northern Japan. Oecologia 135: 280-287.
Walker MD, Webber PJ. 1994. Effects of interannual climate variation on aboveground
phytomass in alpine vegetation. Ecology 75: 393-408.
Molau U, Nordenhall U, Eriksen B. 2005. Onset of flowering and climate variability in an
alpine landscape: A 10-year study from Swedish Lapland. American Journal of
Botany 92: 422-431.
Galen C, Stanton ML. 1995. Responses of snowbed plant species to changes in growing
season length. Ecology 76: 1546-1557.
Inouye DW, McGuire AD. 1991. Effects of snowpack on the timing and abundance of
flowering in Delphinium nelsonii, implications for climate change. American Journal
of Botany 78: 997-1001.
Inouye DW, Morales MA, Dodge GJ. 2002. Variation in timing and abundance of
flowering by Delphinium barbeyi Huth (Ranunculaceae), the roles of snowpack,
frost, and La Nia, in the context of climate change. Oecologia 130: 543-550.
Price MV, Waser NM. 1998. Effects of experimental warming on plant reproductive
phenology an a subalpine meadow. Ecology 79: 1261-1271.
Dullinger S, Dirnbock T, Grabherr G. 2003. Patterns of shrub invasion into high mountain
grasslands of the Northern Calcareous Alps, Austria. Arctic, Antarctic and Alpine
Research 35: 434-441.
Klanderud, K. and Birks, H. J. B. 2003. Recent increases in species richness and shifts in
altitudinal distributions of Norwegian mountain plants. The Holocene 13: 1-6.
Kullmann L. 2002. Rapid recent range-margin rise of tree and shrub species in the
Swedish Scandes. Journal of Ecology 90: 68-77.
Walther GR, Beibner S and Burga CA. 2005. Trends in the upward shifts of alpine plants.
Journal of Vegetation Science 16: 541-548.
RESUMEN

22
Arft AM, Walker MD, Gurevitch J, Alatalo JM, Bret-Harte MS, Dale M, Diemer M, Gugerli
F, Henry GHR, Jones MH, Hollister R, Jnsdttir IS, Laine K, Lvesque E, Marion
GM, Molau U, Mlgaard P, Nordenhll U, Raszhivin V, Robinson CH, Starr G,
Stenstrm A, Stenstrm M, Totland , Turner L, Walker L, Webber P, Welker JM
and Wookey PA. 1999. Response patterns of tundra plant species to experimental
warming, a meta-analysis of the International Tundra Experiment. Ecological
Monographs 69: 491511.
Pauli H, Gottfried M, Dirnbck T, Dullinger S, Grabherr G. 2003. Assesing the long-term
dynamics of endemic plants at summit habitats. In: Nagy L, Grabherr G, Krner C,
Thompson DBA, eds. Alpine Biodiversity in Europe. Berlin, Germany: Springer-
Verlag, 95-207.
Heegaard E and Vandvik V. 2004. Climate change affects the outcome of competitive
interactions: an application of principal response curves. Oecologia 139: 459-466.
Klanderud K and Totland . 2006. Simulated climate change alter dominance hierarchies
and diversity of an alpine biodiversity hotspot. Ecology, in press.
Galen C. 1990. Limits to the distribution of alpine tundra plants, herbivores and the
skypilot, Polemonium viscosum. Oikos 59: 355-358.
Freeman RS, Brody AK, Neefus CD. 2003. Flowering phenology and compensation for
herbivory in Ipomopsis aggregata. Oecologia 136: 394-401.
Lesica P and McCune B. 2004. Decline of arctic-alpine plants at the southern margin of
their range following a decade of climatic warming. Journal of Vegetation Science
15: 679-690.
Breshears DD, Cobb NS, Rich PM, Price KP, Allen CD, Balice RG, Romme WH, Kastens
JH, Floyd ML, Belnap J, Anderson JJ, Myers OB and Meyer CW. 2005. Regional
vegetation die-off in response to global-change-type drought. PNAS 102: 15144-
15148.
Wilson RJ, Gutirrez D, Gutirrez J, Martnez D, Agudo R, Montserrat V. 2005. Changes
to the elevational limits and extent of species ranges associated with climate
change. Ecology Letters 8: 1138-1146.
Totland O and Alatalo JM. 2002. Effects of temperature and date of snowmelt on growth,
reproduction, and flowering phenology in the arctic/alpine herb, Ranunculus
glacialis. Oecologia 133: 168-175.
Stanton ML, Roy BA and Thiede DA. 2000. Evolution in Stressful Environments. I.
Phenotypic Variability, Phenotypic Selection, and Response to Selection in Five
Distinct Environmental Stresses. Evolution 54: 93-111.
Davis MB, Shaw RG. 2001. Range shifts and adaptive responses to Quaternary climate
change. Science 292, 673-679.
RESUMEN

23
Davis MB, Shaw RG and Etterson JR. 2005. Evolutionary responses to changing climate.
Ecology 86, 17041714.
J.O.R.F. 1990. Relatif la liste des espces vgtales protges en rgion Auvergne
compltant la liste nationale. Journal Officiel de la Rpublique Franaise, 10 mai
1990.
Boyce M.S. 1992. Population viability analysis. Annual Review of Ecology and
Systematics 23: 481-506.
Thompson JN and Pellmyr O. 1992. Mutualism in pollinating seed parasites amid co-
pollinators: constraints on specialization. Ecology 73: 1780-1791.
San Miguel A. 2001. Pastos naturales espaoles. Caracterizacin, aprovechamiento y
posibilidades de mejora. Fundacin Conde del Valle Salazar. Ediciones Mundi-
Prensa, Madrid. 320 pp.
Totland O. 2001. Environment-dependent pollen limitation and selection on floral traits in
an alpine species. Ecology 82: 2233-2244.
Ttland O. 2001. Environment-dependent pollen limitation and selection on floral traits in
Seppa H, Nyman M, Korhola A and Weckstrom J. 2002. Changes of treelines and alpine
vegetation in relation to post-glacial climate dynamics in northern Fennoscandia
based on pollen and chironomid records. Journal of Quaternary Science 17: 287-
301.
Gimnez E, Melendo M, Valle F, Gmez-Mercado F, Cano E. 2004. Endemic flora
biodiversity in the south of the Iberian Peninsula, altitudinal distribution, life forms
and dispersal modes. Biodiversity and Conservation 13: 2641-2660.
Valladares F, Peuelas J and Calabuig E. 2004. Impactos sobre los ecosistemas
terrestres. En: ECCE. Impactos de cambio climtico en Espaa, Ministerio de
Medio Ambiente, pp. 65-112.
de Castro M, Martn-Vide J and Alonso S. 2004. El clima de Espaa: Pasado, presente y
escenarios de clima para el siglo XXI. En: ECCE. Impactos del Cambio Climtico en
Espaa. Madrid, Ministerio de Medio Ambiente, pp. 1-64.
Jump AS and Peuelas J. 2005. Running to stand still: adaptation and the response of
plants to rapid climate change. Ecology Letters 8, 1010-1020.
Kudo G, Hirao A. 2006. Habitat-specific responses in the flowering phenology and seed
set of alpine plants to climate variation: implications for global-change impacts.
Population Ecology 48: 49-58.

RESUMEN

24
CAPITULO 1

25

Captulo 1.

Patch dynamics and islands of fertility in a high
mountain Mediterranean community.

Escudero, A.; Gimnez-Benavides, L.; Iriondo, J.M. and Rubio, A.
Arctic, Antarctic, and Alpine Research, 36: 488497.
PATCHY STRUCTURE IN A HIGH MOUNTAIN MEDITERRANEAN FELLFIELD

26
CAPITULO 1

27
SUMMARY

Vegetation in high Mediterranean mountains usually consists of patchy
communities. Patch structures have been interpreted as a result of the prevalence of
facilitation phenomena in highly stressful environments. Several mechanisms have been
proposed in order to explain the factors that control the existence of these clumped
structures. However, they have not been evaluated in these mountains. Our hypothesis is
that patchy structure in high mountain Mediterranean vegetation is a consequence of
facilitative and competitive interactions in a very harsh environment which ultimately
involve strong localized effects on soil properties.
Our results show that levels of soil nutrients were higher under vegetation patches
than in bare ground areas, confirming the hypothesis of an amelioration of soil resources
under canopies. Pairwise associations and repulsions suggest the existence of two
contrasting composition stages. Contrasting models relating patch species composition
(cover and biomass) and soil resources indicated a weak relationship between species
features and soil nutrient levels. Finally, structural modeling showed that patch size has a
relevant but indirect effect on soil resource levels through grass and total biomass.
We conclude that patch structure and dynamics in high Mediterranean mountain
communities may be partly controlled by an endogenous process involving facilitation and
competition for soil key resources. These interactions may operate through some
community traits related to patch size but not to composition.


28
INTRODUCTION

Vegetation cover frequently occurs as relatively small patches embedded in a
bare ground matrix in some alpine Iberian communities -cryoromediterranean belt sensu
Rivas-Martnez (1987)- as well as in other mountain regions of the world (Komrkov
1979, Armesto et al. 1980, Kikvidze 1993, Nez et al. 1999). This spatial pattern has
also been described in highly stressful habitats such as arid and semi-arid environments
(Sala and Aguiar 1996, Schlesinger et al. 1990), for which contrasting patch shaping
factors have been extensively studied (see Aguiar and Sala 1999). Composition, shape
and size of patches could be determined by the strength and direction of biotic
interactions (Aguiar and Sala 1999), which change over time and with environmental
conditions (Callaway 1997, Holzapfel and Mahall 1999). In the case of nutrient-limited
environments, facilitation will occur if benefits from an increase of nutrient levels within
patches overcome other problems such as deterioration of light conditions within the
vegetated patch (Holmgren et al. 1997). This positive biotic interaction may shift to
competition as plants grow (Franco and Nobel 1989, Escudero et al. 1999, 2000). In
addition, patch structure and dynamics in stressful environments have been explained on
the basis of a trade-off between facilitative and competitive interactions among plants
(Fowler 1986, Wu and Levin 1994, Ludwig et al. 1994, Couteron and Lejeune 2001).
Some authors have shown the existence of rather similar stressful environments
and demonstrated positive biotic interactions in arctic (Carlsson and Callaghan 1991) and
alpine vegetation (Holtmaier and Broll 1992, Kikvidze 1993, Nez et al. 1999, Callaway
et al. 2002). However, there is a considerable lack of evidence on spatial structuring
under the climate conditions of Mediterranean high mountains (but see Gaviln et al.
2002) because the large body of research available has mostly focused on
phytosociological and gradient vegetation analyses (Rivas-Martnez 1963, Escudero et al.
1994). The extrapolation of arctic and alpine shaping factors to Mediterranean
environments should be conducted with caution because high mountain Mediterranean
plants face specific constraints such as the development of an intense water deficit during
the short period in which temperatures are high enough to enable growth (e.g. Sierra
Nevada site in Southern Spain, Callaway et al. 2002).
Our hypothesis is that patch structure in high mountain Mediterranean vegetation
is a consequence of facilitative and competitive interactions in a very harsh environment
which ultimately comprise strong localized effects on soil properties. The importance of
plant-created soil heterogeneity has been demonstrated in many types of ecosystems
(Valiente-Banuet and Ezcurra 1991, Facelli and Brock 2000). Biomass tends to
accumulate in soils beneath vegetation patches and modifies biogeochemical processes
CAPITULO 1

29
(Schlesinger et al. 1990), generating so-called islands of fertility (Jackson and Caldwell
1993, Schlesinger et al. 1996, Kelly et al. 1996). Several plant species have been
implicated in this process (Chen and Stark 2000, Vinton and Burke 1995), including some
occurring in alpine systems (Onipchenko et al. 2000). However, to test this hypothesis
under Mediterranean conditions, a specific study under the appropriate field conditions
was needed.
Some papers have examined patch structures by analyzing species associations
in several ways (Silvertown and Wilson 1994, Gotelli 2000, Haase et al. 1996), including
in alpine systems (Kikvidze 1993, Kikvidze and Nakhutsrishvili 1998, Nez et al. 1999,
Gaviln et al. 2002). These studies have detected association and repulsion and
therefore suggested the existence of biotic interactions, such as facilitation or competition
(Freeman and Elmen 1995, Vil and Sardans 1999, Callaway and Puignaire 1999). In
contrast, others have mostly focused on the role of soil nutrients on patch formation and
death (Tongway and Ludwig 1994, Halvorson et al. 1995, Schlesinger et al. 1996). Here,
we have conducted a study which includes both complementary approaches.
Specifically, we addressed the following questions: 1) Are nutrient levels different
between skeletal bare ground surfaces and under vegetated alpine patches?, 2) We
hypothesized a monotonic improvement in soil conditions as the patch increases in size;
hence, is there a positive relationship between soil nutrients and patch size?, 3) We
evaluated the relationship between species composition (through the species-wise
biomass pool and the species-specific cover within a patch) and nutrient soil levels; so,
could both traits control the resource level of each island of fertility? 4) Finally, several
authors have shown the important role of grasses in patch dynamics (Sala et al. 1989,
Aguiar and Sala 1999) so, what is the relative importance of grasses (biomass and cover)
on soil resource levels? This question was approached by developing a causal aprioristic
model which was assessed using structural equation modeling.

METHODS

The study system
The cryoromediterranean bioclimatic belt occurs in the Sierra de Guadarrama, a
mountain range in the north of the province of Madrid, as a reduced set of conspicuous
vegetation islands in the higher summits above 2200-2300 m. The Sierra de Guadarrama
reaches its highest point at 2448 m at the peak of mount Pealara. The dominant
vegetation in snow-free zones, such as windblown slopes and crests is an extremely
short pasture (rarely exceeding three centimeters height) with several creeping

30
chamaephytes, caespitosous grasses and lichens but not mosses. Plants are organized
in small-ellipsoideal-shaped patches that usually do not surpass 50 cm in diameter (see
Gaviln et al. 2002). From a phytosociological point of view the community has been
denominated Hieracio myriadeni-Festucetum curvifoliae (Rivas-Martnez 1963) and is
endemic to the highest alpine islands of the Guadarrama sector of the Sistema Central in
Spain (Rivas-Martnez et al. 1990). This community is particularly rich in endemics, such
as Festuca curvifolia, Hieracium vahlii subsp. myriadenum, Minuartia recurva subsp.
bigerrensis and Armeria caespitosa, as well as in arctic and alpine relicts, such as
Agrostis rupestris and Phyteuma hemisphaericum. The timberline is located between
1900-2100 m, and is dominated by stunted pines (Pinus sylvestris) which appear
interspersed in a shrub matrix characterized by Cytisus oromediterraneus and Juniperus
communis subsp. alpina.
The study site is a well-conserved remnant of this community on Cerro de
Valdemartn (Madrid province, 40
o
47 34 N, 3
o
57 10 W), where no evidence of intense
anthropogenic disturbance due to human trampling or cattle grazing was found. It covers
approximately 2.5 ha. on gentle slopes (<10 %) around the summit. Altitude ranges from
2240 m to 2279 m at the summit. About 60 % of the ground is covered by pebbles or
exposed gravel. There is a sharp ecotone with a Nardus-dominated community where
snowpack is always higher. In the Navacerrada Pass (1890 m), located two kilometers to
the west, annual average air temperature is 6.3
o
C, whereas the mean monthly
temperature ranges from 1
o
C in January to 16
o
C in July. Mean annual precipitation is
1330 mm with a very marked drought from May to October (less than 10 % of the total
annual rainfall) (Rivas-Martnez et al. 1990). At our study site, which is located more than
300 meters higher, the climatic conditions should be more restrictive for plant growth.

Observational study
Transect lines running parallel to the maximum slope at 10 m intervals were
established on the stand. Every patch of vegetation in contact with transect lines was
marked and sixty of them were randomly selected in late July (2001), during the period of
maximum community vegetative activity. All patches were photographed with a digital
camera at one meter height and perpendicular to the ground surface. Digital images were
analyzed with Olympus Micro Image version 4.0. Patch size, perimeter and cover of each
species including lichens were measured. Immediately after taking the digital photos, we
collected all above-ground vegetation and necromass per patch and separated it species-
wise in the laboratory. Biomass was measured by drying samples to 70
o
C for 48 h in a
controlled oven and weighing them on a balance. Aboveground biomass is an accurate
CAPITULO 1

31
estimator of plant performance even for plants growing under stressful conditions. We
also collected lichens because in other fellfield communities, they are relevant elements
of the community (Walker et al. 2001).
A six centimeters deep soil core (6 cm in diameter) beneath the patches was
collected immediately after plant sampling. It is worth noting here that smaller patches are
present in the study area although they were not examined because soil sampling was
not feasible. To compare the characteristics of soil under patches with that outside them,
we collected twenty soil samples in bare surfaces interspersed among patches. These
sampling points were located at least 15 cm away from any patch or plant. Soil samples
were air-dried at laboratory temperature for approximately one month and then sieved.
The mass of fine soil (< 2 mm), fine-gravel (2-20 mm) and gravel (> 20 mm) fractions of
the soil samples was quantified and their relative contribution calculated. Analyses of
conductivity and pH were determined in the supernatant of a 1:2.5 soil-deionised water
(v/v) suspension with glass electrodes. An estimate of moisture percentage was
calculated gravimetrically after drying a subsample of the fine soil fraction at 105 C.
Nitrogen was determined by semi-micro Kjeldahl procedure (Bremner 1965). An acid
(concentrated H
2
SO
4
) digestion was followed by a steam distillation and NH
4
+
was
evaluated by titration with 0.1 M H
2
SO
4
. The organic carbon was determined according to
a modification of the Walkley and Black method (Walkley, 1946) as follows: 1 N K
2
Cr
2
O
7

and concentrated H
2
SO
4
were added

to a soil sample (< 2mm) and the dichromate excess
was titrated after the addition of 10 ml of concentrated H
3
PO
4
with 0.5 N
Fe(NH
4
)
2
(SO
4
)
2
.6H
2
O. Free iron was extracted with Na
2
S
2
O
4
at 3.5 pH and evaluated by
titration with 0.05 N K
2
Cr
2
O
7
(Deb, 1950). We also determined carbon:nitrogen ratio,
available phosphorous (extracted with acetic acid, sulphuric acid and carbonates, pH 3.3
(Burriel and Hernando, 1950), and determined colorimetrically (DINKO, model D 105))
and available potassium (extracted with 1M NH
4
Ac, pH 7) by flame photometry (Flame
photometer JENWAY, model PFP7) (U.S.Salinity Laboratory Staff, USDA, 1954).
To conduct pairwise comparisons between species, we randomly selected and
measured 200 additional multi-species patches. They were subjected to the condition of
being within the size range of the sixty patches used for biomass estimation (37-160 cm
of perimeter).
Numerical analyses
Previous to statistical analyses, variables were checked for normality and
transformations were performed when necessary. Differences in soil parameters between
skeletal soils and beneath-patch soils were tested by means of one-way ANOVAs or
Mann-Whitney non-parametric tests.

32
We tested the associations between species using contingency table analyses
with Fishers exact test and Phi coefficient on presence/absence data from all patches
(N=260).
An aprioristic structural modeling approach (SEM) was conducted to test the
causal hypotheses for the nutrient level reached under each patch (see Mitchell 1993,
Iriondo et al. 2003). Although our model involves a very reduced set of explaining
variables, we have preferred SEM over ordinary multiple regression because the SEM
models, among other advantages, allow the estimation of indirect effects which are
usually eclipsed even in relatively simple models (Magnusson 2002). Our causal model
included the following relationships that were derived from our hypotheses and existing
evidences: 1) the size of the patch positively affects nutrient levels, 2) the size of the
patch positively affects grass biomass, 3) the size of the patch positively affects total
biomass, 4) total grass biomass positively determines nutrient levels and 5) total biomass
affects nutrient levels (Fig. 1). We considered separately the total biomass effect from the
grass component because there is evidence that both components have a primary role on
high mountain vegetation processes (Grabherr 1989). The null hypothesis of the model is
that the observed and predicted covariances are equal. Standardized path coefficients
were estimated by the maximum likelihood method. The degree of fit between the
observed and predicted covariance structures was first examined by a
2
goodness-of-fit
test. As this test may show inadequate statistical power (Mitchell 1993), we used other fit
indices such as the Normed Fit Index (NFI) and the Goodness-of-Fit Index (GFI). Both
NFI and GFI range from 0 to 1, with values above 0.95 indicating a good fit (Tanaka
1987). The significance of each individual path coefficient was assessed through
multivariate Wald tests. Previously, we calculated the variance inflation factor for each
variable to detect multicollinearity problems. Computation of the structural equation was
performed using LISREL (version 8.51. Jreskog and Srbon 2001).
We used constrained ordinations to examine the multivariate relationships
between species biomass and cover, and soil nutrients (Legendre and Anderson 1999,
Rubio and Escudero 2000). Our null hypothesis was that the influence of environmental
variables (soil data set) on this multivariate data set (species biomass data matrix) was
not significantly different from random. With this in mind a Detrended Correspondence
Analysis (DCA; Hill and Gauch 1980) with the biomass data set was conducted by
detrending by segments and non-linear rescaling of the axes, which has the property that
the extracted axes are scaled in units of average standard deviation (Gauch 1982). As
the extracted gradients of the biomass data base were relatively short (standard deviation
units 2), we conducted a Redundancy Analysis (hereafter RDA) which is a constraining
ordination technique that assumes linear responses in relation to environmental variables.
CAPITULO 1

33
Using the soil variables as constraining matrix, the total variation explained (TVE) was
calculated as the sum of all extracted canonical axes (Borcard et al. 1992). A Monte Carlo
permutation test (1000 randomizations) was performed to determine the accuracy of the
relationship between the two data sets, using the sum of all canonical eigenvalues or
trace to build the F-ratio statistic (ter Braak 1990; Verdonschot and ter Braak 1994;
Legendre and Anderson 1999). If the RDA model was significant, a forward stepwise
procedure was carried out to select a reduced model including only significant variables.
We incorporated explanatory variables one at a time and step by step in the order of their
decreasing eigenvalues after partialling out the variation accounted by the already
included variables. The process stopped when the new variable was not significant (p
>0.01). Improvement of the reduced model with each new selected variable was
determined by a Monte Carlo permutation test with 1000 randomizations. A similar
approach was conducted using as the main matrix the specific plant cover per plant data
set. Multivariate analyses were conducted with CANOCO for Windows v. 4.0 (ter Braak
and Smilauer 1997).

RESULTS

Patch size varied from 95 cm
2
to 912 cm
2
. The total number of species in the sixty
patches for biomass and soil analyses were 13 for vascular plants and 3 for lichens,
whereas the average numbers of species per patch were 4.6 (range 2 to 8) and 1.3
(range 0 to 3) respectively (Table 1). The most important species in percentage of patch
cover were Festuca curvifolia (100%), Sedum brevifolium (67 %), Jasione crispa subsp.
centralis (57%) and Silene ciliata subsp. elegans (45 %). Among the lichens the most
dominant species were Coelocaulon aculeatum (79 %) and Cetraria islandica (38 %).
Using aboveground biomass, a rather different pattern emerges, Silene ciliata (5.1 g per
patch on average), Festuca curvifolia (3.7 g), and Minuartia bigerrensis (3.1 g) being the
main vascular plants, and C. aculeatum (0.5 g) the most important lichen species (Table
2). As expected, a highly significantly positive correlation was found between total
biomass and patch area (R= 0.472, P <0.002). However, no correlation was found
between total biomass and species richness (i.e. total number of species per patch
including lichens; R= 0.213, P >0.176).
As shown in Table 3, all soil variables apart from fine gravel and phosphorous
content were significantly different between soil beneath patches and soil in skeletal
zones. All nutrient levels were always higher under patches, the acidity was lower and the
fine-earth component contribution was greater.

34
We detected a reduced set of significant pairwise associations between plant
species using the Fishers exact test (P< 0.01). Only species occurring in more than 10 %
of samples were included (eight vascular plants and two lichens). From this subset we
found eight positive and two negative significant interactions.
Our causal model had a significant fit with the seven soil parameters tested (Fig.
1).
2
goodness of fit showed P values above 0.1, indicating that predicted and observed
covariances did not differ significantly. NFI and GFI were in all cases above 0.95 which is
the standard threshold for not rejecting the models. The power of our model mainly rests
on the important positive relationship between patch size and total biomass (standardized
path coefficient of 0.60), and patch size and grass biomass (standardized path coefficient
of 0.60). Surprisingly, there was no significant direct effect between patch size and any of
the tested soil resources, but there was a considerable indirect effect through grass and
total biomass. The direction and intensity of those effects were specific to each soil
resource. Total biomass had a significant negative effect on nitrogen content (path
coefficient -0.34), iron content (-0.37) and phosphorous (-0.36), but grass biomass had no
effect. In contrast, potassium level and pH were positively affected by total biomass (0.34
and 0.29 respectively) but negatively by grass biomass (-0.33 and -0.30). Organic matter
pools and C/N ratio showed no significant relationships with any of the biomass
components.
When the species biomass matrix was constrained by the soil data set (nine
variables), a significant model was obtained (Table 4) and TVE reached 33 %. Following
a forward stepwise procedure, only pH was selected from the soil matrix (Table 4). The
RDA model with just this variable was highly significant, the drop in TVE with respect to
the model with all soil variables being ca. 14 %. The biplot of the reduced model
including pH clearly shows that vectors for the biomass per patch of some species are
arranged following acidity levels. Silene ciliata increases in biomass in those patches with
higher levels of pH, whereas Festuca curvifolia, Coelocaulon aculeatum and Jasione
crispa perform better in patches with very acid soils (Fig. 2). The RDA model with the
species cover matrix had no significant relationships with soil variables (Table 4).

DISCUSSION

Differences between soils beneath canopies and in bare ground
Soils beneath patches in our study site are richer in nutrients than soils in
surrounding open spaces. Our results seem to support the hypothesis that, under
stressful conditions, biological activity results in the creation of high-nutrient patches in a
CAPITULO 1

35
low-nutrient matrix (Callaway et al. 1991, Belsky and Canham 1994, Facelli and Brock
2000, Hirobe et al. 2001). Enrichment of soil nutrients under perennials has been
reported in many contrasting systems, particularly in semi-arid regions (Schmida and
Whittaker 1981, Callaway et al. 1991, Puignaire et al. 1996, Moro et al. 1997) but also in
alpine and arctic environments (Chapin et al. 1994) and even under caulescent rosettes
in tropical mountain Andean ecosystems (see Prez 1992 and 1995). The potential
mechanisms involved in this pattern may be related to the so-called nutrient pumping
effect (Callaway and Puignaire 1999, Titlyanova et al. 1999). Roots take up nutrients from
deep stocks and deposit them on the soil surface. On the other hand, plants in patches
may trap streamflow nutrient particles (Whitford et al. 1997, Garner and Steinberger
1989), capture windblown materials (Coppinger et al. 1991) or even at a larger scale,
capture particles through freeze-thaw cryopedogenic cycles (Johnson and Billings 1962),
which is the main disturbance factor in relatively snow-free alpine zones (Sakai and
Larcher 1987, Krner 1999). Nitrogen atmospheric deposition may also be an important
factor in mountain regions, especially in the vicinity of large urban areas (Sievering 2001)
as in our case (Madrid, a city with a population around 4-5 million people, is just 50 km
away from our study site). This fact might be especially relevant because nitrogen is
recognized as a limiting factor in cold environments (Bowman et al. 1993, Atkin 1996), so
levels of this nutrient may be at least partly responsible for patch dynamics (Bowman et
al. 1996, Choler et al. 2001). Finally, patch formation and growth may also be shaped by
intense summer droughts suffered in this Mediterranean mountain range, as shown in
other water-limited systems (Aguiar et al. 1992, Aguiar and Sala 1994). Recently, Nez
et al. (1999) highlighted the relevance of such a type of summer droughts in the highest
Andean communities of central Argentina. Under this constraint, patches would provide a
suitable microenvironment to protect seedlings from desiccation, favoring recruitment
within patches over bare areas in alpine and arctic communities (Bliss 1971, Urbanska
and Schtz 1986). There is no doubt that the presence of plants drives amelioration
processes in these high mountain environments (Krner 1999), but, as stated by Chen
and Stark (2000), could plants detect soil heterogeneity or existing fertility sources before
patch construction? Our observational study was not able to give more information about
this point, but the fact that soil resources in the twenty bare soil samples had nutrient
levels lower than the soil beneath patches and that they showed very homogeneous
levels for almost all soil parameters, suggests that the origin of these islands of fertility is
exclusively plant-driven (Hirobe et al. 2001). In this sense, Grieve (2000) suggest that
well-vegetated ground in alpine environments promote and maintain soil nutrient
enrichment minimizing disturbance phenomena such as human trampling and
cryoturbation processes.

36
Soil amelioration process and species composition
Usually, enrichment in soil resources is associated with changes in species
composition interpreted as the result of biotic interactions (Callaway 1995, Callaway and
Walker 1997, Homgrem et al., 1997). In this sense, there are numerous studies of spatial
plant associations in arctic and alpine communities showing a large number of positive
associations which have been interpreted as evidences of facilitation (Sohlberg and Bliss
1984, Holtmaier and Broll 1992, Kikvidze 1993, Aksenova et al. 1998, Kikvidze and
Nakhutsrishvili 1998, Nez et al. 1999). On the other hand, repulsions have been
interpreted as a result of competitive exclusion and senility (Valiente-Banuet et al. 1991,
Aguiar and Sala 1994). Using contingency table analyses on presence/absence data we
detect two contrasting stages (Fig. 3). One was comprised of pioneer plants such as
Jurinea humilis and Sedum brevifolium, together with the two most abundant species
(Festuca curvifolia and Silene ciliata), and the other was dominated by creeping
chamaephytes such as Thymus penyalarensis. Similarly, Kikvidze (1993) explained the
pattern of co-occurrences in the subnival-alpine limit of the Central Caucasus as
comprising four dynamical steps based on the existence of pair-wise positive and
negative interactions. In contrast, Nez et al. (1999) only found positive associations
and noted an absence of replacement or species exclusion in a high-Andean community.
The results presented here are partly in disagreement with those from our previous
studies in Central Spain (Gaviln et al. 2002) which showed different associations and
repulsions. Those differences are probably related to the larger spatial scale approach
followed in Gaviln et al. (2002), which included several types of Mediterranean high
mountain communities within a wider geographical region.
Traditionally, patch dynamics have been considered a quasi-deterministic process
in which benefactor species modify the environment through a wide variety of direct and
indirect pathways to facilitate the recruitment, growth and survival of the so-called
beneficiary species (Callaway 1995, Callaway and Puignaire 1999, Tewksbury and Lloyd
2001). However, we have detected an evident enrichment at soil resource levels not
associated with the performance of any specific plant species, as shown by our RDA
models. In addition, the way in which cover is distributed among species in a patch
showed relatively short gradients not related to nutrient levels of soil beneath patches
(P>0.10). We expected plant species composition and structure to control local soil
properties (Chen and Stark 2000, Hirobe et al. 2001), but we only found a weak trend
between biomass distribution among species and acidity (Fig. 2). Taking into account
RDA modeling and pairwise comparison in combination, we suggest that the patch
dynamic sequence (with at least two contrasting stages) is not coupled with soil
CAPITULO 1

37
amelioration but may be a response to other limiting factors. Our contingency table
results suggest that graminoid Festuca curvifolia exerts a pioneer behavoiour probably
related to its clonal growth. Positive interactions with lichens and with outstanding pioneer
crassulacean such as Sedum brevifolium reinforce such interactions. On the other hand,
negative associations frequently appear between plants with similar requirements or
same functional groups, such as Silene ciliata and Minuartia bigerrensis, both cushion
plants with roots that capture nutrients from relatively deep layers.
Although it has been demonstrated that individual alpine plant species can modify
soil conditions under monoculture (Onipchenko et al. 2000), it is reasonable that under
natural conditions plant-specific effects may be overshadowed by those of the complete
set of species, as shown in some semi-arid systems (Padien and Lajtha 1992, Vinton and
Burke 1995).

Patch and soil dynamics in a high mountain Mediterranean fellfield community
We hypothesized a linear enrichment in soil nutrients due to a direct effect of
patch size (Kikvidze 1993) and an indirect effect of size through two contrasting
community traits: grass biomass and total biomass. However, our results do not support
this expected direct effect for any of the modeled variables. Furthermore, the strength of
the model is based on an indirect effect of size through total biomass and grass biomass.
These findings agree with Chiarucci et al. (1999), who suggested that the most
meaningful measure of abundance when exploring community structures is biomass.
Our findings show the possible existence of senility processes coupled to a key
soil resource such as nitrogen (Bowman et al. 1993), phosphorous or iron content (Fig.
1). It is worth noting that this decay is mediated only by total biomass because grass
biomass had no significant effects (Wald test), despite the existence of positive path
coefficients. Facelli and Brock (2000) provided the first characterization of the
development and decline of fertility islands in arid lands and demonstrated that it is an
endogenous process. The main difference with our results is that there the process was
controlled by a keystone species, Acacia papyrocarpa, whereas in our case it is not
controlled by any particular species but by plant growth forms - grasses versus total
vegetation- (see Aguiar and Sala 1999). Thus it can be concluded that although soil
nutrient amelioration occurs after patch establishment, there is an endogenous process
that determines the decomposition of the patch probably due to competition among plants
when patches reach a limiting size. This process leads the bigger patches to an observed
loss of integrity and consequent fragmentation in small ones.

38
At least for these key soil resources, our initial hypothesis needs to be
reconsidered because patch size does not positively affect nutrient levels but instead
determines a conspicuous decline through indirect effects mediated by total biomass (see
Rehder 1976). We propose a model for patch and soil dynamics (Fig. 4). This suggests
an amelioration process through facilitation during the first steps of patch formation and a
subsequent competitive decline by means of an unimodal relationship between patch size
indirectly through total biomass or other non-controlled community traits- and nutrient
level. It has been previously shown that nutrient availability is higher in mid-successional
stages at least in forests (Walker 1989, van Cleve et al. 1991, Chapin et al. 1994).
Probably we were able to fit significant linear models to our data because the smaller
patches were not surveyed. They mainly reflect the right half side of the distribution of the
redrawn model (Fig. 4). Nevertheless, we built models including quadratic transformations
of variables but this approach did not significantly improve the fit relative to the linear
approach.
It is also worth noting that the effect of grass biomass significantly balances the
positive effect of total biomass in the case of potassium content and acidity. This is in line
with the complementary role of contrasting functional types in patch dynamics (Grabherr
1989, Aguiar and Sala 1999). Thus, facilitative recruitment in the so-called pioneer stage
and the subsequent competitive exclusion to the secondary stage is not coupled to soil
resources but the total biomass and the balance with grass biomass. Finally, we did not
detect any significant effect of grass biomass in organic matter and C/N ratio even though
total soil N is usually a function of soil organic concentration (Rehder 1976, Krner 1999).
Although some authors have highlighted the shift from competition to facilitation with
abiotic stress (see Callaway et al. 2002), a temporal perspective at the scale of the patch
suggests that competition may also play a key role in very stressful sites (Tielborger and
Kadmon 2000).
To conclude, our study shows that patch dynamics in high Mediterranean
mountain communities may be partly controlled by an endogenous process involving soil
key resources through facilitation and competition. These interactions may operate
through some community traits related to patch size but not species composition. This
process determines the existence of conspicuous islands of fertility interspersed in a bare
ground matrix.

CAPITULO 1

39
ACKNOWLEDGEMENTS

We thank Dr. Isabel Martnez for the determination of lichen specimens and Dr.
David Gutirrez for his careful and fruitful revision of a preliminary draft. Esteban Surez
from Cornell University and an anonymous referee provided valuable comments on the
manuscript. Dr. Wilson carefully revises the English. The study was partially financed by
project REN 2000-0254-P4-03 project of the Spanish Ministry of Science and
Technology.

40
REFERENCES

Aguiar, M. R. and Sala, O. E., 1999: Patch structure, dynamics and implications for the
functioning of arid ecosystems. Tree, 14: 273-277.
Aguiar, M. R. and Sala, O. E., 1994: Competition, facilitation, seed distribution, and the
origin of patches at the Patagonian steppe. Oikos, 70: 26-34.
Aguiar, M. R., Soriano, A. and Sala, O. E., 1992: Competition and facilitation in the
recruitment of grass seedlings in Patagonia. Functional Ecology, 6: 66-70.
Aksenova, A. A., Onipchenko, V. G. And Blinnikov, M. S., 1998: Plant interactions in
alpine tundra: 13 years of experimental removal of dominant species. Ecoscience,
5: 258-270.
Armesto, J. J., Arroyo, M. K. and Villagrn, C., 1980: Altitudinal distribution, cover and
size structure of umbelliferous cushion plants in the High Andes of Central Chile.
Acta Oecologica, 1: 327-332.
Atkin, O. K., 1996: Reassessing the nitrogen relations of Arctic plants. A mini-review.
Plant Cell and Environment, 19: 695-704.
Belsky, A. J. and Canham, C. D., 1994: Forest gaps and isolated savanna trees.
BioScience, 44: 77-84.
Bliss, L. C., 1971: Arctic and alpine plant life cycles. Annual Review of Ecology
Systematics, 2: 405-438.
Borcard, D., Legendre, P. and Drapeau, P., 1992: Partialling out the spatial component of
ecological variation. Ecology, 73: 1045-1055.
Bowman, W. D., Schardt, J. C. and Schmidt, S. K., 1996: Symbiotic N2-Fixation in Alpine
Tundra: Ecosystem Input and Variation in Fixation Rates Among Communities.
Oecologia, 108: 345-350.
Bowman, W. D., Theodose, T. A., Schardt, J. C., Conant, R. T., 1993: Constraints of
nutrient availability on primary production in two alpine tundra communities.
Ecology, 74: 2085-2097.
Bremner, J. M., 1965: Inorganic forms of nitrogen. In Black, C. A. et al. (eds.) Methods of
soil analysis. Part 2. Agron. Monogr. 9. ASA, 11791237.
Burriel, F. and Hernando, V., 1950: El fsforo en los suelos espaoles; V. Un nuevo
mtodo para determinar el fsforo asimilable en los suelos. Anales Edafologa, 9:
611-622.
Callaway, R. M., 1997: Positive interactions in plant communities and the individualistic-
continnum concept. Oecologia, 112: 143-149.
CAPITULO 1

41
Callaway, R. M. and Pugnaire, F. I., 1999: Facilitation in plant communities. In Pugnaire,
F. I. and Valladares, F. (eds), Handbook of functional plant ecology. Marcel Dekker,
623648.
Callaway, R. M., Brooker, R. W., Choler, P., Kikvidze, Z., Lortie, C. J., Michalet, R.,
Paolini, L., Pugnaire, F. I., Newingham, B., Aschehoug, E. T., Armas, C., Kikodze,
D. and Cook, B. J., 2002: Positive interactions among alpine plants increase with
stress. Nature, 417: 844-848.
Callaway, R. M., 1995: Positive interactions among plants. The Botanical Review, 61:306-
349.
Callaway, R. M. and Walker, L. R., 1997: Competition and facilitation: a synthetic
approach to interactions in plant communities. Ecology, 1958-1965.
Callaway, R. M., Nadkarni, N. M. and Mahall, B. E., 1991: Facilitation and interference of
Quercus douglasii on understory productivity in central California. Ecology, 72:
1484-1499.
Carlsson, B. A. and Callaghan, T. V., 1991: Positive plant interactions in tundra
vegetation and the importance of shelter. Journal of Ecology, 79: 973-983.
Chapin, F. S., Walker, L. R., Fastie, C. R. and Sharman, L. C., 1994: Mechanisms of
primary sucession following deglaciation at Glacier Bay, Alaska. Ecological
Monographs, 64: 149-175.
Chen, J. and Stark, J. M., 2000: Plant species effect and carbon and nitrogen cycling in a
sagebrush-crested wheatgrass soil. Soil Biology and Biochemistry, 32: 47-57.
Chiarucci, A., Wilson, J. B., Anderson, B. J. and De Dominicis, V., 1999: Cover versus
biomass as an estimate to species abundance: does it make a difference to the
conclusions? Journal of Vegetation Science, 10: 35-42.
Choler, P., Michalet, R. and Callaway, R. M., 2001: Facilitation and competition on
gradients in alpine plant communities. Ecology, 82: 3295-3308.
Coppinger, K. D., Reiners, W. A., Burke, I. C. and Olson, R. K., 1991: Net erosion on a
sagebrush steppe landscape as determined by cesium-137 distribution. Soil
Science Society American Journal, 55: 254258.
Couteron, P. and Lejeune, O., 2001: Periodic spotted patterns in semi-arid vegetation
explained by a propagation-inhibition model. Journal of Ecology, 89: 616-628.
Deb, B. C., 1950: Estimation of free iron oxides in soils and clays and their removal.
Journal of Soil Science, 1(2): 212-220.
Escudero, A., Iriondo, J. M., Olano, J. M., Rubio, A. and Somolinos, R., 2000: Factors
affecting the establishment of a gypsophyte: The case of Lepidium subulatum.
American Journal of Botany, 87(6): 861-871.

42
Escudero, A., Pajarn, S. and Gaviln, R., 1994: Saxicolous communities in the Sierra
del Moncayo (Spain): a classification study. Coenoses, 9(1): 15-24.
Escudero, A. , Somolinos, R. C. and Rubio, A., 1999: Factors controlling the
establishment of Helianthemum squamatum (L.) Dum., an endemic gypsophile of
semi-arid Spain. Journal of Ecology, 87 (2): 290-302.
Facelli, J. M. and Brock, D. J., 2000: Patch dynamics in arid lands: localized effects of
Acacia papyrocarpa on soils and vegetation of open woodlands of south Australia.
Ecography, 23: 479-491.
Fowler, N., 1986: The role of competition in plant communities in arid and semiarid
regions. Annual Review of Ecology and Systematics, 17: 9-110.
Franco, A. C. and Nobel, P. S., 1989: Effect of nurse plants on the microhabitat and
growth of cacti. Journal of Ecology, 77: 870-886.
Freeman, D. C. and Emlen, J. M., 1995: Assessment of interspecific interactions in plant
communities: an illustration from the cold desert saltbush grasslands of North
America. Journal of Arid Environments, 31, 179-198.
Garner, W., and Steinberger, Y., 1989: A proposed mechanism for the formation of
Fertile Islands in the desert ecosystem. Journal of Arid Environments, 16: 257-
262.
Gauch, H. G., 1982: Multivariate analysis in community ecology. London: Cambridge
University Press.
Gaviln, R. G., Snchez-Mata, D., Escudero, A. and Rubio, A., 2002: Spatial structure
and interactions in Mediterranean high mountain vegetation (Sistema Central,
Spain). Israel Journal of Plant Sciences 50: 217-228.
Gotelli, N. J., 2000: Null model analysis of species co-occurrence patterns. Ecology, 81:
2606-2621.
Grabherr, G., 1989: On community structure in high alpine grasslands. Vegetatio, 83:
223-227.
Grieve, I. C., 2000: Effects of human disturbance and cryoturbation on soil iron and
organic matter distributions and on carbon storage at high elevations in the
Cairngorn Mountains, Scotland. Geoderma, 95: 1-14.
Haase, P., Pugnaire, F. I., Clark, S. C. and Incoll, L. D., 1996: Spatial pattern in a two-
tiered semi-arid shrubland in southeastern Spain. Journal of Vegetation Science, 7:
527534.
Halvorson, J. J., Smith, J. L., Bolton, H. and Rossi, R. E., 1995: Evaluating shrub-
associated spatial patterns of soil properties in a shrub-steppe ecosystem using
multiple-variable geostatistics. Soil Science Society American Journal, 59: 1476-
1487.
CAPITULO 1

43
Hill, M. O. and Gauch, H. G., 1980: Detrended Correspondence Analysis: an improved
ordination technique. Vegetatio, 42: 47-58.
Hirobe, M., Ohte, N., Karasawa, N., Zhang, G., Wang, L. and Yoshikawa, K., 2001: Plant
species effect on the spatial patterns of soil properties in the Mu-us desert
ecosystem, Inner Mongolia, China. Plant and Soil, 234: 195-205.
Holmgren, M., Scheffer, M. and Huston, M. A., 1997: The interplay of facilitation and
competition in plant communities. Ecology, 78: 1966-1975.
Holtmaier, F. K. and Broll, G., 1992: The influence of tree islands and microtopography on
pedological conditions in the forest alpine tundra ecotone on Niwot Ridge, Colorado
Front Range, USA. Arctic and Alpine Research, 24: 216-228.
Holzapfel, C. and Mahall, B. E., 1999: Bidirectional facilitation and interference between
shrubs and annuals in the Mohave Desert. Ecology, 80: 1747-1761.
Iriondo, J. M., Albert, M. J. and Escudero, A., 2003: Structural Equation Modelling: an
alternative for assessing causal relationships in threatened plant populations.
Biological Conservation, 113(3): 367-377.
Jackson, R. B. and Caldwell, M. M., 1993: Geostatistical patterns of soil heterogeneity
around individual perennial plants. Journal of Ecology, 81:683-692.
Johnson, P. L. and Billings, W. D., 1962: The alpine vegetation of the Beartooth Plateau
in relation to cryopedogenic processes and patterns. Ecological Monographs, 32:
105-135.
Jreskog, K. G. and Srbon, D., 1996: LISREL
8 Users Reference Guide. Chicago:

Scientific Sotfware International.
Kelly, R. H., Burke, I. C. and Lauenroth, W. K., 1996: Soil organic matter and nutrient
availability responses to reduced plant inputs in shortgrass steppe. Ecology, 77:
2516-2527.
Kikvidze, Z., 1993: Plant species associations in alpine-subnival vegetation patches in the
Central Caucasus. Journal of Vegetation Science, 4: 297-302.
Kikvidze, Z. and Nakhutsrishvili, G., 1998: Facilitation in subnival vegetation patches.
Journal of Vegetation Science, 9: 261-264.
Komrkov, V., 1979: Alpine Vegetation of the Indian Peaks Area, Front Range, Colorado
Rocky Mountains. Flora et Vegetatio Mundi 7. Vaduz, Cramer.
Krner, C., 1999: Alpine Plant Life. Berlin: Springer-Verlag.
Legendre, P. and Anderson, M. J., 1999: Distance-based redundancy analysis: testing
multispecies responses in multifactorial ecological experiments. Ecological
Monographs, 69: 1-24.

44
Ludwig, J. A., Tongway, D. J. and Marsden, S. A. G., 1994: A flow-filter model for
simulating the conservation of limited resources in spatially heterogeneous, semi-
arid landscapes. Pacific Conservation Biology, 1: 209-213.
Magnusson, W. E., 2002: Categorical ANOVA: strong inference for weak data. Bulletin of
the Ecological Society of America, 83: 81-86.
Mitchel, R. J., 1993: Path analysis: pollination. In Scheiner, S. M. and Gurevitch, J. (eds)
Design and analysis of ecological experiments. New York: Chapman and Hall, 211-
231.
Moro, M. J., Pugnaire, F. I., Haase, P. and Puigdefbregas, J., 1997: Effect of the canopy
of Retama sphaerocarpa on its understorey in a semiarid environment. Functional
Ecology, 11: 175-184.
Nez, C., Aizen, M. A. and Ezcurra, C., 1999: Species associations and nurse plant
effects in patches of high-Andean vegetation. Journal of Vegetation Science, 10:
357-364.
Onipchenko, V. G., Makarov, M. I. and van der Maarel, E., 2000: Change soil properties
by alpine plant species: a monoculture experiment. Proceedings of the IAVS
Symposium. Uppsala: Opulus Press, 18-20.
Padien, D. J., and Lajtha, K., 1992: Plant spatial pattern and nutrient distribution in
pinyon-juniper woodlands along an elevational gradient in northern New Mexico.
International Journal of Plant Science, 153: 425-433.
Prez, F. L., 1992: The influence of organic matter addition by caulescent Andean
rossetes on surfacial soil properties. Geoderma, 54: 151-171.
Prez, F. L., 1995: Plant-induced spatial patterns of surface soil properties near
caulescent Andean rossetes. Geoderma, 68: 101-121.
Pugnaire, F. I., Haase, P. and Puigdefbregas, J., 1996: Facilitation between higher plant
species in a semiarid environment. Ecology, 7: 1420-1426.
Rehder, H., 1976: Nutrient turnover studies in alpine ecosystems. I. Phytomass and
nutrient relations in four mat communities of the northern calcareous Alps.
Oecologia, 22: 411-423.
Rivas-Martnez, S., 1963: Estudio de la vegetacin y flora de las Sierras de Guadarrama
y Gredos. Anales del Instituto Botnico Cavanilles, 21(1): 5-330.
Rivas-Martnez, S., 1987: Mapa de las series de vegetacin de Espaa (escala 1:
400.000). Madrid: ICONA.
Rivas-Martnez, S., Fernndez-Gonzlez, F. Snchez-Mata, D. and Pizarro, J. M., 1990:
Vegetacin de la Sierra de Guadarrama. Itinera Geobotanica, 4: 3-132.
CAPITULO 1

45
Rubio, A. and Escudero, A., 2000: Small-scale spatial soil-plant relationship in semi-arid
gypsum environments. Plant and Soil, 220: 139-150.
Sakai, A. and Larcher, W., 1987: Frost survival of plants. Berlin: Springer-Verlag.
Sala, O. E. and Aguiar, M. R., 1996: Origin, maintenance, and ecosystem effect of
vegetation patches in arid lands. In West, N. (ed). Rangelands in a Sustainable
Biosphere. Proceedings of the Fifth International Rangeland Congress vol. 2.
Denver: Society for Range Management, 29-32.
Sala, O. E., Golluscio, R. A., Lauenroth, W. K. and Soriano, A., 1989: Resource
partitioning between shrubs and grasses in the Patagonian steppe. Oecologia, 81:
501-505.
Schlesinger, W. H., Raikes, J. A., Hartley, A. E. and Cross, A. F., 1996: On the spatial
pattern of soil nutrients in desert ecosystems. Ecology, 7(2): 364-374.
Schlesinger, W. H., Reynolds, J. F., Cunningham, G. L., Huenneke, L. F., Jarrell, W. M.,
Virginia, R. A. and Whitford, W. G., 1990: Biological feedbacks in global
desertification. Science, 247: 1043-1048.
Schmida, A., Whittaker, R. H., 1981: Pattern and biological microsite effects in two shrub
communities, southern California. Ecology, 62: 234-251.
Sievering, H., 2001: Atmospheric chemistry and deposition. In Bowman, W. D. and
Seastedt, T. R. (eds) Structure and function of an alpine ecosystem. New York:
Oxford University Press.
Silverton, J. and Wilson, J. B., 1994: Community stucture in a desert perennial
community. Ecology, 72: 409-417.
Sohlberg, E. H. and Bliss, L. C., 1984: Microscale pattern of vascular plant distribution in
two high arctic plant communities. Canadian Journal of Botany, 62: 14-20.
Tanaka, J. S., 1987: How big is big enough?: Sample size and goodness of fit in
structural equation models with latent variables. Child Development, 58:134-146.
ter Braak, C. J. F., 1990: Update notes: CANOCO version 3.1. - Ithaca, New-York:
Microcomputer Power.
ter Braak, C. J. F. and Smilauer, P., 1997: Canoco for Windows version 4.0. Wagenigen,
The Netherlands: Centre for Biometry.
Tewksbury, J. J. and Lloyd, J. D., 2001: Positive interactions under nurse-plants: spatial
scale, stress gradients and benefactor size. Oecologia, 127: 425-434.
Tielborger, K. and Kadmon, R., 2000: Temporal environmental variation tips the balance
between facilitation and interference in desert plants. Ecology, 81: 1544-1553.
Titlyanova, A. A., Romanova, I. P., Kosykh, N. P. and Mironycheva-Tokareva, N. P.,
1999: Pattern and process in above-ground and bellow-ground components of
grassland ecosystems. Journal of Vegetation Science, 10: 307-320.

46
Tongway, D. J. and Ludwig, J. A., 1994: Small-scale resource heterogeneity in semi-arid
landscapes. Pacific Conservation Biology, 1: 201-208.
U.S. Salinity Laboratory Staff, 1954: Diagnosis and improvement of saline and alkali soils.
Agricultural Hanbook 60, USDA. Washington, D. C.: U.S. Gov. Print. Off.
Urbanska, K. M. and Schtz, M., 1986: Reproduction by seed in alpine plants and
revegetation research above timberline. Botanica Helvetica, 96: 43-60.
Valiente-Banuet, A. and Ezcurra, E., 1991: Shade as a cause of of the association
between the cactus Neobuxbaumia tetetzo and the nurse-plant Mimosa luisana in
the Tehuacan Valley, Mexico. Journal of Ecology, 79: 961-971.
Valiente-Banuet, A., Vite, F. and Zavala-Hurtado, A., 1991: Interaction between the
cactus Neobuxbaumia tetetzo and the nurse shrub Mimosa luisana. Journal of
Vegetation Science, 2: 11-14.
van Cleve, K., Chapin, F. S., Dyrness, C. T. And Vierek, L. A., 1991: Element cycling in
taiga forests: state-factor control. BioScience, 41: 78-88.
Verdonschot, P. F. M. and ter Braak, C. J. F., 1994: An experimental manipulation of
oligochaete communities in mesocosms treated with chlorpyrifos or nutrient
additions: multivariate analyses with Monte Carlo permutation tests. Hydrobiologia,
278: 251-266.
Vil, M. and Sardans, J., 1999: Plant competition in mediterranean-type vegetation.
Journal of Vegetation Science, 10: 281-294.
Vinton, M. A. and Burke, I. C., 1995: Interactions between individual plant species and
soil nutrient status in short grass steppe. Ecology, 76: 1116-1133.
Walker, L. R., 1989: Soil nitrogen changes during primary sucession on a foodplain in
Alaska, U.S.A. Arctic and Alpine Research, 21: 341-349.
Walker, M. D., Walker, D. A., Theodose, T. A. and Weber, P. J., 2001: The vegetation:
hierarchical species-environment relationships. - In Bowman, W. D. and Seastedt,
T. R. (eds) Structure and Function of an Alpine Ecosystem. Niwot Ridge, Colorado.
New York: Oxford University Press, 99-127.
Walkley, A., 1946: A critical examination of a rapid method for determining organic carbon
in soils: Effect of variations in digestion conditions and of inorganic constituents.
Soil Science, 63: 251-263.
Whitford, W.G., Anderson, J. and Rice, P.M., 1997: Stemflow contribution to the 'fertile
island' effect in creosotebush, Larrea tridentata. Journal of Arid Environents, 35:
451-457.
Wu, J. and Levin, S. A., 1994: A spatial patch dynamic modeling approach to patternand
process in an annual grassland. Ecological Monographs, 64(4): 447-464.

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Table 1. Descriptive statistics of the vegetation patches (N= 60).

Variables Min Max Mean S. D.
Patch size (cm
2
) 95 912 275 160
Patch perimeter (cm) 37 160 74 23
Total vegetation cover (%) 60 105 89 9
Total plant biomass (g)
Grass biomass (g)
3.5
0.7
21.8
17.2
9.7
3.8
3.9
3.0
Total number of species per patch 2 9 5.9 1.4
Vascular plant species per patch 2 8 4.6 1.3
Lichen species per patch 0 3 1.3 0.8


48
Table 2. Species (vascular plants and lichens) recorded in the patches sampled for
biomass assessment. Additional vascular plants were found in the 200 patches surveyed
for pairwise comparisons and are not included in the table: Armeria caespitosa, Biscutella
gredensis, Festuca iberica, Koeleria caudata subsp crassipes and Senecio boissieri.
Mean aboveground biomass per patch and frequency of occurrence in patches are
shown. Life form abbreviations are: Hm: hemicryptophyte, HM cae.: caespitosous
hemicryptophyte Cs: cushion chamaephyte, L: lichen species.

Species
Life
form
Mean
Biomass (g)
S.D.
Frequency
(%)
Festuca curvifolia Hm cae. 3.7 3.0 100
Jasione crispa subsp. centralis Cs 2.0 1.1 57
Jurinea humilis Hm 0.5 0.6 43
Thymus praecox subsp. penyalarensis Ch 0.7 1.4 43
Phyteuma hemisphaericum Hm 0.4 0.0 2
Hieracium vahlii subsp. myriadenum Hm 0.2 0.1 24
Agrostis rupestris Hm cae. 1.3 0.0 2
Poa cenisia Hm 0.3 0.0 2
Deschampsia flexulosa subsp. iberica Hm cae. 1.0 0.1 5
Minuartia recurva subsp. bigerrensis Cs 3.4 2.0 38
Sedum brevifolium DC. Hm 0.2 0.3 67
Silene ciliata subsp. elegans Cs 5.1 3.8 45
Luzula hispanica Hm 0.3 0.3 14
Coleocaulon aculeatum L 0.5 0.6 79
Cetraria islandica L 0.1 0.1 38
Cladonia ecmocyna L 0.5 1.0 14

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Table 3. Mean and standard deviation of the edaphic variables measured below canopies
(N= 60) and in bare soils (N= 20). Significant differences between samples were tested
using one-way analysis of variance or Mann-Whitney U test (noted as U).

Edaphic variables
Soil beneath
canopies Bare soil
Statistic
Sign.
Mean
S.D.
Mean S.D.

Gravel (%) 31.23 13.65 41.07 12.81 7.300 0.009

Fine gravel (%) 13.78 6.17 17.26 8.08 3.493 0.067
Fine-earth (%) 54.97 14.09 41.66 12.28 2.273 0.001

Moisture (%) 2.35 0.70 1.88 0.39 7.730 0.007

pH 5.13 0.20 4.94 0.18 13.331 0.001

Conductivity (S m
-1
) 0.039 0.023 0.029 0.006 253.0 (U) 0.007

Organic matter (%) 7.30 2.02 4.78 1.15 26.510 0.000

Nitrogen (%) 0.39 0.077 0.30 0.076 19.159 0.000

C/N ratio 10.52 1.53 8.50 2.31 16.595 0.000

Fe oxides (%)

1.10 0.33 0.93 0.40 235.5 (U) 0.005

Potassium (ppm) 142.86 38.58 92.90 67.25 13.810 0.000

Phosphorous (ppm)

219.92 154.47 169.60 82.72 402.5 (U) 0.792


50
Table 4. Results of RDA models between aboveground species biomass distribution and
species cover matrices, with soil nutrients as constraining environmental matrix.
1
,
2
,
3

represent the eigenvalues of the corresponding extracted axes. Total Variation Explained
(TVE) is the sum of all constrained axis eigenvalues. For the Monte Carlo test, F-ratio
statistic was computed using the sum or trace of all canonical eigenvalues, and p is the
significance of the model after 1000 randomizations. The forward stepwise procedure for
the significant RDA model (biomass x soil) is shown below. Drop TVE represents the
difference between the TVE of the model including all the variables and the TVE of the
reduced model.

RDA models
2

3
TVE (%)
Monte Carlo test
F-ratio p
Biomass distribution x soil nutrients 0.066 0.017 33 2.395 0.001
Species cover x soil nutrients 0.050 0.043 22.7 1.429 n.s.

Reduced model (biomass x soil)
Explanatory variables
F-ratio p TVE (%)
Drop
TVE (%)
p
Step 1 pH 5.53 0.003 18.3 14.7 0.001

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Figure 1. Path models for the effects of patch size, total biomass and gramineous biomass on
mineral nutrient levels (N, P, K and Fe), organic matter, pH and C/N ratio. Width of each arrow is
proportional to the standardized path coefficient, solid lines denote positive paths and dashed lines
denote negative paths. Asterisks indicate values significantly different from zero according to the
Wald test (p<0.05). GFI: Goodness-of-Fit Index; NFI: Normed Fit Index. NFI and GFI values above
0.95 along with a nonsignificant
2
indicate a good model fit.
0.60
*

0.60
*
n. s. 0.60
*

-0.36
*
0.60
*
n. s.
n. s.
n. s.
n. s.

0.60
*

0.60
*
-0.37
*
n. s. 0.60
*

0.60
*
0.29
*
n. s.
n. s. n. s.
n. s.
-0.33
*

0.34
*
0.60
*

0.60
*
n. s.

n. s. 0.60
*

0.60
*
n. s. 0.60
*

0.60
*
2
= 0.78
P = 0.37
GFI = 0.99
NFI = 0.98
Total
biomass
Gramineous
biomass
Organic
matter
Size
Total
biomass
Gramineous
biomass
N
-0.34
*
2
= 0.813
P = 0.36
GFI = 0.99
NFI = 0.98
Size
2
= 0.78
P = 0.37
GFI = 0.99
NFI = 0.98
Total
biomass
Gramineous
biomass
pH
Size
2
= 0.78
P = 0.37
GFI = 0.99
NFI = 0.98
Total
biomass
Gramineous
biomass
K
-0.30
*

Size
2
= 0.78
P = 0.37
GFI = 0.99
NFI = 0.98
Total
biomass
Gramineous
biomass
Fe Ox
Size
2
= 0.78
P = 0.37
GFI = 0.99
NFI = 0.98
Total
biomass
Gramineous
biomass
C/N
Size
2
= 0.78
P = 0.37
GFI = 0.99
NFI = 0.98
Total
biomass
Gramineous
biomass
P
Size

52

Figure 2. Biplot (axes 1 and 2) of the reduced RDA model including only the pH variable.
Vectors for the biomass of each species are included. Values of biomass per patch are
represented as proportional circles for the four most abundant species. Absences are not
shown.

CAPITULO 1

53

Figure 3. Significant pairwise associations between plant species (P<0.01). Solid lines
indicate positive interactions and dotted lines negative associations. Shadow indicates
lichen species.


54

K
e
y

s
o
i
l

r
e
s
o
u
r
c
e

l
e
v
e
l

(
N

a
n
d

P
)
Patch size (indirect effect through biomass)
Surveyed
interval
SEM Response
Competition F
a
c
i
l
i
t
a
t
i
o
n
Senescency limit

Figure 4. Hypothetical model indicating the effect of patch size upon key soil resource
levels. In an initial stage consisting of small patches and isolated plants, resource level
would increase with patch size mainly due to facilitation. Above a particular threshold for
patch size, competition interactions would become increasingly more important and
promote a decrease in soil resource levels as patch size increases. The negative effects
of patch size on resource levels detected by our SEM models would lie on this
competitive phase of the hypothetical model.
CAPITULO 2

55

Captulo 2.

Seed germination of high mountain Mediterranean
species: altitudinal, interpopulation and interannual
variability.

Gimnez-Benavides, L.; Escudero, A. and Prez-Garca, F.
Ecological Research (2005) 20: 433-444.
GERMINATION OF HIGH MOUNTAIN MEDITERRANEAN PLANTS

56
CAPITULO 2

57
SUMMARY

Germination response of 20 species from high altitudes of the Mediterranean-type
climate, most of them rare endemics, was studied. Our main goal was to model the
germination response of a complete set of Iberian high mountain species. The effect of
temperature and others parameters such as spatial and temporal short gradients on
germination were also evaluated. Some seed features (mass and size) were also related
to the germination response. Finally, we tested the effect of cold-wet stratification
pretreatment when germination reached small percentages under natural conditions.
Seeds were collected at four locations from 1900 to 2400 m.a.s.l. in Sierra de
Guadarrama (Spanish Central Range) along two consecutive growth seasons (2001-
2002) and submitted to different temperatures and a constant photoperiod of 16 h light/8
h darkness. Most plants readily germinate without treatment, reaching an optimum at
relatively high temperatures contrarily to genuine lowland Mediterranean species. Seeds
seem to be physiologically prepared to rapid germination even though these plants
usually face to very intense summer droughts after ripening and dispersal. Germination
was also highly variable among altitudes, populations and years, but results were
inconsistent among species. Such flexibility could be interpreted as an efficient survival
strategy for species growing under unpredictable environments (Mediterranean climate)
and will reduce the risk of seedlings to be subjected to poor growing due to the
establishment of intense competition hierarchies. Finally cold-wet stratification increased
germination capacity in 6 of 9 dormant species, as widely reported for many arctic, boreal
and alpine species. In conclusion, high mountain Mediterranean species do not differ
from alpine species but the relatively high number of species which are ready to
germinate without any treatment.

58
INTRODUCTION

High levels of endemicity are probably the most relevant feature of Mediterranean-
type climate high mountains (Vre et al. 2003) reaching values above 30 % in some
cases such as in Sierra Nevada, southeastern Spain, and frequently with very small and
isolated populations. Global warming evidences in the Mountains of central Spain
(Gaviln et al. 2001, Sanz-Elorza et al. 2003) suggest that orophilous species are being
posed at serious risk due to the advance of lowland plants as widely predicted (Grabherr
et al. 1994, Peuelas & Filella 2001). Surprisingly, there is almost no basic information for
conservation of these plants despite Mediterranean mountains are considered one of the
most threatened systems in Spain and the European Union (Gmez-Campo 1987,
European Community 1992).
Seed storage is considered the best way to store and maintain large pools of
genetic diversity in plants (Thompson et al. 1981, Bonner 1990). However appropriate
germination protocols must be developed for successful propagation and conservation
approaches. For instance reinforcement of wild populations using stored seed material
may be a valuable tool for conserving endemic and threatened species (Schemske et al.
1994, Prez-Garca et al. 1995, Cerabolini et al. 2004). On the other hand, revegetation
actions in alpine environments have been also carried out during last decades in Europe
and North America (Brown & Johnston 1979, Urbanska & Schtz 1986). Detailed
information about alpine native plants as potential species for restoring ski runs, mine
sites and other human impacts has gradually acquired great importance (Urbanska 1986,
Chambers 1997). Specific germination requirements of alpine species constitute very
valuable information for restoration managers for seeding practices, so large amounts of
data have been published (McDonough 1969, Acharya 1989, Chambers 1989, Chambers
et al. 1987). Despite an ambitious project to remove and restore an old ski resort in
central Spain has been conducted (Snchez-Herrera 2000) there is a complete lack of
knowledge about reproductive features of key species.
Previous studies on alpine flora around the world suggest the absence of a
specific alpine germination strategy (Krner 1999). Even more, germination behaviour
may largely vary within a single species from one population to another, from year to year
and also among individuals (Urbanska & Schtz 1986). There is a tendency to better
germinate under high temperatures (Mooney & Billings 1961, Billings & Mooney 1968),
but also at alternating temperatures (Amen 1966, Walter & McDonough 1969 and 1970,
Bliss 1971). Winter dormancy seems to be a common requirement for some alpine
species (Krner 1999). Both innate and enforced seed dormancy have been successfully
broken by subjecting seeds to a wide variety of treatments: gibberellins imbibition
CAPITULO 2

59
(McDonought, 1969), scarification (Pelton 1956), stratification in multiple forms (dry-hot,
dry-cold, wet-cold) (Pelton 1956, Cavieres & Arroyo 2000), or combined. Among them,
wet-cold stratification seems to produce the best results in alpine plants (Baskin & Baskin
1998). The extrapolation of these germination requirements of alpine plants to
Mediterranean environments should be conducted with caution because high mountain
Mediterranean plants face specific constraints such as the development of an intense
water deficit during the short period in which temperatures are high enough to enable
growth (e.g. Sierra Nevada site in Southern Spain, Callaway et al. 2002).
The main objectives of this work were 1) to model the germination response of
Iberian high mountain species, and compare with lowland Mediterranean species and
with plants from arctic and alpine environments; 2) to analyze effects of altitude,
population variability and year of collection upon seed germination and, additionally, 3) to
evaluate the effectiveness of the cold-wet stratification treatment for the induction of
germination in species with high levels of dormancy.

MATERIAL AND METHODS

Plant material collection and description of the field localities
We have selected 20 species, most of them endemics of the Iberian Peninsula
and with small and isolated populations above timberline. Species cover a wide variety of
functional types from perennial grasses to creeping chamaephytes and hemicriptophytes,
and also communities from rock to short pasture habitats (see Table 1). Seeds were
collected directly from mother plants during the fruiting season (from July to September)
along two consecutive years (2001 and 2002). We collected from small areas, where all
the available seeds were harvested. When possible we collected material from different
populations and years (2001 and 2002).
Five sets of 10 or 20 seeds per species and per population (enough to reach the
accuracy of the precision balance, COBOS AX120, d=0.1mg) were weighted and seed
mass calculated. Seed maximum and minimum diameter were measured over digital
images (n=25) taken from the weighted seeds and were analyzed using Olympus Micro
Image ver. 4.0.
Collection sites were located in the highest portion of Sierra de Guadarrama, a
SW-NE running mountain range located in the north of the Madrid province (40
o
N, 3
o
W)
and belonging to the Spanish Central Range. Rainfall data for specific locations are
unavailable, but annual rainfall in the closest weather station (Navacerrada Pass, 1890
m) is about 1400 mm, with a very pronounced summer drought when less that 10% of

60
annual precipitation occurs. A complete synopsis of the cryoromediterranean belt
vegetation is given in Rivas-Martnez et al. (1999).
Locality 1 was located in the vicinity of Pico Pealara (2428 m), the highest peak
of Sierra de Guadarrama. The dominant vegetation in the summit flat areas and crests,
where snow cover only remains 120-140 days/year (Palacios et al. 2003), is a
discontinuous cryophilic pasture dominated by Festuca curvifolia (association Hieracio
myriadeni-Festucetum curvifoliae Rivas-Martnez 1963). This community is particularly
rich in endemics, such as Hieracium vahlii subsp. myriadenum, Minuartia recurva subsp.
bigerrensis and Armeria caespitosa, as well as in arctic and alpine relicts, such as
Agrostis rupestris and Phyteuma hemisphaericum (see Gaviln et al. 2002 and Escudero
et al. 2004). Rockwall steps and blockfields are colonized by scattered communities
dominated by Allium schoenoprassum, Saxifraga willkommiana, Veronica fruticans
subsp. cantabrica, Silene boryi (Allietum latirifolii and Saxifragetum willkommianae
associations). Annual average air temperature is 3.8
o
C, whereas the mean monthly
temperature ranges from 5.2
o
C in January to 13.6
o
C in July.
Locality 2 was located in the summit of Dos Hermanas (2269 m), which is located
3 km far apart. Due to its lower altitude, grass-dominated communities are interspersed
with an open shrub formation dominated by Cytisus oromediterraneus and Juniperus
communis subsp. alpina (Senecioni carpetani-Cytisetum oromediterranei). Average
snowcover duration is slightly shorter (100-120 days/year). Rockfall and blockfield
habitats are also present. Annual average air temperature is 4.9
o
C, whereas the mean
monthly temperature ranges from 3.7
o
o
C in July.
Locality 3 was in Cerro de Valdemartn (2279 m), a mount located in an adjacent
mountain range running parallel 5 km away, and separated by the headwaters of the
Lozoya river valley (see Fig. 1). Vegetation, snow cover duration and temperature regime
are similar to locality 2. For further analyses populations of localities 2 and 3 were
assigned to a similar altitude.
Finally, the Locality 4 was in Hoya de Pealara, a late Pleistocene glacial cirque
situated at 1950 m, in the timberline zone. Seeds were collected from a left moraine,
covered by dispersed stunted pines (Pinus sylvestris) in a Cytisus-Juniperus shrub
matrix. Despite its lower altitude snow cover duration lasts 120-140 days/year due to its
leeward orientation that protect from prevailing westerly winds (Palacios et al. 2003).
Annual average air temperature is 6.9
o
C, whereas the mean monthly temperature ranges
from 1.9
o
o
C in July.

CAPITULO 2

61
Germination test
Ripe fruits were collected directly from mother plants in relatively small areas (50-
100 m
2
). Seeds were cleaned and stored in plastic vials at room temperature and
darkness for several months (4-5) until trials were carried out. Germination responses
were obtained under a 16-h light/8h-dark photoperiod at three constant temperature
regimes (10
o
C, 15
o
C and 20
o
C). Seeds of Silene ciliata, Silene boryi subsp.
penyalarensis, Minuartia recurva subsp. bigarrensis, Jasione crispa subsp. centralis and
Hieracium vahlii subsp. myriadenum collected in 2001 were also submitted to 25
o
C and
alternating 25/15
o
C temperatures because number of seeds was highly enough. Four
replicates of 25 seeds per treatment were placed on two layers of filter paper into 5 or 8
cm in diameter Petri dishes, depending on seed size. Filter papers were kept soaked
along the whole experimental period (35 days) and each three or four days seeds
showing radicle emergence were counted and thereafter removed from the Petri dishes.
Dishes location in the chamber was regularly changed. Tests were carried out
simultaneously in germination chambers (Selecta Hotcold GL, Barcelona, Spain)
equipped with 6 cool-white fluorescent light tubes (Philips 18 W TLD standard type,
wavelength 400 to 650 nm) providing a photon flux density of approximately 19 mol m
-2

s
-1
. Chambers temperatures were also monitored with a portable thermometer (Oregon
Scientific MTR102) in order to avoid bias due to failures in the chamber control.

Stratification pretreatment
A cold-wet stratification treatment was carried out with those species in which final
germination percentage was lower than 60% (Allium schoenoprassum, Biscutella
laevigata subsp. gredensis, Jurinea humilis, Luzula hispanica, Ranunculus ollissiponensis
subsp. alpinus, Saxifraga pentadactylis subsp. wilkommiana, Sempervivum vicentei
subsp. paui, Senecio pyrenaicus subsp. carpetanus, Silene ciliata subsp. elegans,
Solidago virgaurea subsp. fallit-tirones and Veronica fruticans subsp. cantabrica). In
order to ensure humid and dark conditions, batches from one population per taxon were
placed between two double layers of filter paper in 8 cm Petri dishes and wetted with
distilled water. Dishes were wrapped in aluminium foil and stored in a refrigerator at 4
o
C
for 3 months. Germination tests were conducted as above described.
Seed viability was performed with tetrazolium test (Hendry & Grime 1993).
Moisten seeds, sliced in two sections with a scalpel, were incubated in 10 ml of 0.1%
tetrazolium chloride for 24 h at laboratory temperature.


62
Data analysis
The effect of population of origin (locality), altitude and year upon final germination
percentage was modelled by fitting Generalised Linear Models (GLMs) (McCullagh &
Nelder 1989). Germination data follows a binomial distribution (probability ranging from 0
to 1), so the best way to achieve linearity is the use of a generalized linear model with
logit link function and binomial error distribution, setting the variance to "mean(1-mean)"
(Venables & Ripley 1998, Schtz & Rave 1999). Experimental parameters (temperature,
population, altitude and year) were included as explanatory variables (fixed factors).
Because data are overdispersed in some cases we used the quasilikelihood approach to
overcome possible difficulties (Guisan et al. 2002). Significant terms were identified using
a stepwise addition of terms to the null model, based on the magnitude of the Cp statistic
at each step (Spector 1994), until no additional terms improved the model. Regression
coefficients were tested for significance by t tests.
2
tests were also conducted to
evaluate whether or not selected predictors explain a significant fraction of the deviance
(Guisan et al. 2002). Statistical analysis was performed using the S-Plus 2000 statistical
package (MathSoft, Inc. 1999).
Seeds of different localities and years were not available for all species, so
species-specific models were performed depending on the available material. For
example, models for species with only one population were performed including only
temperature as fixed factor (e.g., Allium schoenoprassum, Sempervivum vicentei subsp.
paui, Senecio pyrenaicus subsp. carpetanus, Solidago virgaurea subsp. fallit-tirones and
Murbeckiella boryi). Species with populations located at different altitudes were analysed
setting temperature and altitude as factors (twelve species). When more than one
population per altitude were collected (populations 2 and 3, both at 2200 m), population
factor could also be added into the model (e. g., Armeria caespitosa, Festuca curvifolia,
Jurinea humilis, Silene boryi subsp. penyalarensis and Thymus praecox subsp.
penyalarensis). Finally, for those species which at least one population was collected
both years, variable year was also fitted into the model (the case of Jasione crispa subsp.
centralis and Minuartia recurva subsp. bigerrensis). Detailed information about population
of origin, altitude, and year of collection of seeds is showed in Table 1.
The Kaplan-Meier method was performed to model the germination curves. This
method allows the use of right censored data (i.e., the experiment ends before all seeds
germinate). Pairwise shape comparisons for modelled germination curves were tested by
non parametric Log-Rank tests (Pyke & Thompson 1986).

CAPITULO 2

63
RESULTS

Germination without pretreatment
Since the number of species is high, we have grouped the species accordingly to
their ability to germinate (both final germination percentage and germination rate). Mean
germination percentage is showed in Fig. 1. On average, barely a third (7 species)
presented more than 75% germination, whilst 4 species (20%) failed to achieve 25% of
seeds germinated. We also found 7 species with germinability around 60% (range 54% to
68%). Finally, only 2 species (10 %) were unable to germinate without pre-treatment. In
relation to germination rate most plants were ready to germinate rapidly (Fig. 2).
Germination curves showed a minimum period to initiate germination of 3-6 days, except
Biscutella laevigata subsp. gredensis and Ranunculus ollissiponensis subsp. alpinus that
required 9 days. No significant relationships were found between seed features
(independently size and weight) and final germination (Kendall's tau = -0.09 and 0.05
respectively). Seeds were relatively small in most cases (see Table 1).

Response to temperature
Differences between species can be summarised into three groups (see Fig. 1
and Table 2 for species-specific responses). Group 1 clusters the majority of the species,
which are able to germinate equally at the three temperatures tested (temperature factor
was not selected in the corresponding model). Neither significant difference was found on
germination curves among temperatures, except in Solidago virgaurea subsp. fallit-
tirones and Thymus praecox subsp. penyalarensis (Fig. 2). Group 2 consists of species
that better germinate at warmer temperatures. At lower temperatures, germination rates
were significantly slower. Group 3, which is represented only by two species (Jurinea
humilis and Silene ciliata) is characterised by optimum germination at relatively low (15
o
C) temperatures. Nevertheless, the latter species can reach substantially higher values
at alternating 25/15
o
C.

Altitudinal, interpopulation and interannual variability
Results of the generalized linear models show the influence of both altitude and
population variability on the germination behaviour of several species (Table 2). Seeds
collected at higher altitudes reached higher germination percentage and higher
germination rate than those from lower elevations in three of twelve species (Jasione
crispa subsp. centralis, Minuartia recurva subsp. bigerrensis and Thymus praecox subsp.
penyalarensis). On the contrary, four species better responded at lower elevations.

64
Population also explained a slight but significant fraction of variation in three of five
species (Festuca curvifolia, Thymus praecox subsp. penyalarensis and Jurinea humilis).
Seeds from a given locality in both years were only available for two species,
Minuartia recurva subsp. bigarrensis from locality 3 and Jasione crispa subsp. centralis
from locality 4 (Fig. 3). Minuartia recurva subsp. bigarrensis showed higher germination
values in 2002. Variable year was included in the final model (Table 2).

Germination after cold-wet stratification
It should be noted that germination tests could not be properly carried out for
several species below 60% germination. We lost seed batches of Jurinea humilis and
Solidago virgaurea subsp. fallit-tirones due to fungal contamination during stratification.
On the other hand, Sempervivum vicentei subsp. paui and Ranunculus ollissiponensis
subsp. alpinus surprisingly achieved high germination values during the stratification
period, leaving insufficient seeds to conduct temperature tests. Nevertheless, a positive
effect of treatment in the case of Sempervivum vicentei subsp. paui was evident
(germination reached 78% in opposition to 2% on average before treatment). On the
contrary, similar germination behaviour was obtained for Ranunculus ollissiponensis
subsp. alpinus (76% before and 75% during the treatment).
We found a significantly increase in final germination after cold-wet stratification in
6 of 9 species. Higher effects were found in Veronica fruticans subsp. cantabrica and
Luzula hispanica, in which absolute dormancy of fresh seeds (total absence of
germination) was broken after treatment (Fig. 4). Veronica fruticans subsp. cantabrica
showed the highest germination value (96%) at the low temperature (10
o
C), decreasing
progressively at warmer temperatures. On the other hand, Luzula hispanica germinated
only at 10
o
C, reaching 50%. Optimal results were also obtained for A. schoenoprassum
and Silene ciliata subsp. elegans. First one exhibited maximum germination at warmer
temperatures (98% both at 15
o
C and 20
o
C, opposite to 43% at 10
o
C) while Silene ciliata
subsp. elegans reached 96% at 15
o
C, decreasing at extreme temperatures. Germination
curves always followed these tendencies (see Fig. 5). While seed stratification in
Saxifraga pentadactylis subsp. wilkommiana caused little increase on final germination at
10
o
C and 15
o
C (but decreasing radically at 20
o
C), germination curves showed a high
germination rate at unmodified conditions. That is, Saxifraga pentadactylis subsp.
wilkommiana slightly better germinate, but slower, after stratification. Finally, Biscutella
laevigata subsp. gredensis and Senecio pyrenaicus subsp. carpetanus were unable to
germinate after stratification. A seed viability test assessed after the experiment revealed
that seeds of both species were unviable.

CAPITULO 2

65
DISCUSSION

Germination without pretreatment
Surprisingly most species germinated without pre-treatment despite germination
assays were carried out 4-5 months after seed collection (July-August). Previous results
suggest that alpine plants are unable to germinate during current season (autumn time),
so they get into a dormancy process to avoid harshness of winter time and germinate
after snowmelt in early summer (Urbanska & Schtz 1986, Krner 1999). This fact has
been interpreted by different authors as a maturation period to complete development of
embryos (Urbanska & Schtz 1986), a physical restriction of seed coats that needs
scarification by freeze-thaw cycles on soil (Urbanska et al. 1979, Zuur-Isler 1982), or
merely an environmental constrain rather than physiological requirement (Billings &
Mooney 1968). Our results show that in most high mountain Mediterranean plants
storage periods of 4-5 months may be sufficient to induce germination, even under room
temperature conditions. Since immediately post-harvest germination tests were not
carried out with these species, assumptions about pre-winter germination capability
should be taken with care. Any case, seeds seem to be physiologically prepared to
germinate after very short periods even though these plants usually suffer a very intense
summer drought along or immediately after ripening and dispersal (Gaviln et al. 2001)

Response to temperature
As a general rule alpine plants does not differ in their temperature requirements
for germination from lowland plants (Krner 1999), so most of them prefer relatively high
temperatures (Mooney & Billings 1961, Billings & Mooney 1968). This seems also the
case for Mediterranean orophyllous plants but no so sharply. Most species show
temperature indifference (Group 1, 50% of cases) or warm-preference (Group 2, 30%) for
germination, but almost no cold-adaptation. Surprisingly, this behaviour differs from the
typical of Mediterranean plants, for which germination at low temperatures is a widely
extended trait (Bell et al. 1993, Escudero et al. 1997, Thanos et al. 1995). Logically, this
must be a consequence of harsh mixture of conditions of high Mediterranean habitats.
Alternating conditions are also reported to offer very good results for many alpine species
(Amen 1966, Bliss 1970), and we have also found such a type of response in the two
tested Silene species (although only five species were tested at this temperature regime).
Seeds readily germinate in almost all cases (Fig.s 2 and 5) as similarly reported
for many alpine species around the world (Baskin & Baskin 1998). Since alpine
ecosystems are typically subjected to short growing seasons, this strategy could be
claimed to ensure enough time for plant establishment (Chambers et al. 1987, Angosto &

66
Matilla 1994, Krner 1999). Under Mediterranean harsh climate conditions, where topsoil
remains moistened only few weeks after snowmelt, number of days required for
germination may acquires special importance.

Altitudinal, interpopulation and interannual variability
Alpine plants show a very great variability in the germination behaviour which
some times has been attributed to environmental conditions but not always associated to
habitat features (Bliss, 1971, Urbanska & Schtz 1986, Chambers 1989). Our results
clearly agree with these generalities. Variation in germination of different species had
been observed in relation to altitude by several authors (Holm 1994, Vera 1997).
However, we have not found a consistent pattern in germination related to altitude
variation. Among tested, some species showed better results at higher (25%), whereas
others at lower altitudes (33%). Population variation contributes to explain germination
behaviour in three of five species. Year variable accounts for the greatest proportion of
variability in one of two species tested. The variability of germination characteristics could
be interpreted as one of the most important survival strategies for species growing under
unpredictable environmental conditions (Gutterman 1994, Kigel 1995) and will reduce the
risk of seedlings to be subjected to poor growing due to the establishment of intense
competition hierarchies.

Germination after cold-wet stratification
We found stratification enhancement in seven Mediterranean mountain species
(including S. vicentei subsp. paui, which emerged massively during treatment), whereas 2
species presented similar germination percentages and 2 else lost viability. Cold
stratification is known to improve germination percentage in many alpine and non-alpine
species of Eastern Europe and North America (Bell & Bliss 1980, Marchand & Roach
1980, Bewley & Black 1982, Baskin & Baskin 1998). Cavieres and Arroyo (2000) also
proved effective induction of Phacelia secunda seed germination by means of cold-wet
stratification in the Mediterranean type-climate Andes of Chile, with longer treatment
periods required for populations from higher elevations. Additionally, cold stratification
may level out interpopulation differences in germination patterns (Schtz & Milberg 1997,
Milberg & Andersson 1998). Unfortunately, we tested just one locality per taxon, so we
can not establish altitudinal and/or population comparisons.
On the other hand, seed coat structure has previously pointed out as a possible
cause of seed dormancy in alpine plants (Urbanska et al. 1979, Zuur-Isler 1982). Luzula
hispanica seeds are covered by a hard waxy layer that turns gelatinous when wetted.
CAPITULO 2

67
Amen (1965) reported same kind of structure for L. spicata, and proved its role on the
total dormancy of the species. Following his recommendation we tried a manual
scarification on the radicle area, obtaining similar germination percentage that reached by
cold-wet stratification.
With respect to Allium schoenoprassum, we have found contradictory results with
obtained by Specht and Keller (1997). They observed relatively high germination of two
months-stored seeds, reaching optimal results at 16
o
C and 26
o
C in absence of
stratification treatment. However, the use there of accessions grown on garden makes
these results uncomparable.
It is important to note that all large-seeded species were unable to germinate after
stratification. Seeds of Jurinea humilis and Solidago virgaurea subsp. fallit-tirones directly
died during stratification due to severe fungal attack, whereas Biscutella laevigata subsp.
gredensis and Senecio pyrenaicus subsp. carpetanus seeds lost their viability along
treatment. Contrarily, all small-seeded species improve their final germination after
stratification, except Saxifraga pentadactylis subsp. wilkommiana. Similar results were
found by Schwienbacher and Erschbamer (2001), who showed that most species of the
Central Alps tested had high germination rates after short cold treatment, with Saxifraga
oppositifolia among the exceptions. They argued that a brief cold storage might be
insufficient to break the deep dormancy of this species. These results are partially agree
with the general assumption that large heavy-weight seeds are mainly short-lived,
whereas small low-weight seeds are adapted to maintain persistent seed banks
(Thompson et al. 1993). However, results must be taken with caution because
contradictory results between laboratory and field experiments have been reported in this
sense (see Schwienbacher & Erschbamer, 2001).

CONCLUSIONS

It has been suggest the absence of a genuine alpine germination strategy (Krner
1989). High mountain Mediterranean plants do not seem to differ in germination
characteristics from other alpine plants but the relatively high number of species which
are ready to germinate without any treatment. Temperature optima differ from those of
typical Mediterranean plants. Germination response can vary between altitudes, sites and
years, but without clear trends among species. There is a considerable proportion of
species with different levels of dormancy that can be successfully broken by cold-wet
stratification. Type and duration of dormancy, as well as genotypic vs. phenotypic causes
of variation, and its implications on recruitment from seed banks need to be clarified.


68
ACKNOWLEDGEMENTS

We would like to thank undergraduate students Mara Gmez and Silvia Gonzlez
for assistance in the laboratory. Arantzazu Lpez de Luzuriaga reviewed an earlier draft
of the manuscript. This research was financed by the Spanish Ministry of Science and
Technology (project REN 2003-03366).
CAPITULO 2

69
REFERENCES

Acharya S. N. (1989) Germination response of two alpine grasses from the Rocky
Mountains of Alberta. Canadian Journal of Plant Science 69: 1165-1177.
Amen R. D. (1966) The extent and role of seed dormancy in alpine plants. Quarterly
Review of Biology 41: 271-281.
Angosto T. and Matilla A. J. (1994) Modifications in seeds of Festuca indigesta from two
different altitudinal habitats. Seed Science and Technology 22: 319-328.
Baskin C. C. and Baskin J. M. (1998) Seeds. Ecology, biogeography, and evolution of
dormancy and germination. Academic Press, San Diego.
Bell K. L. and Bliss L. C. (1980) Plant reproduction in high arctic environment. Arctic and
Alpine Research 12 (1): 1-10.
Bell D. T., Plummer J. A. and Taylor S. K. (1993) Seed germination ecology in
southwestern Australia. Botanical Review 59: 24-73.
Bewley J. D. and Black M. (1982) Physiology and Biochemistry of Seeds, 2. Viability,
Dormancy, and Environmental Control. Springer-Verlag, Berlin.
Billings W. D. and Mooney H. A. (1968) The ecology of arctic and alpine plants. Biological
Reviews 43: 481-529.
Bliss L. C. (1971) Arctic and alpine plant life cycles. Annual Review of Ecology
Systematics 2: 405-438.
Bonner F. T. (1990) Storage of seeds, Potential and limitations for germplasm
conservation. Forest Ecology and Management 35 (1-2): 35-43.
Brown R. A. and Johnston R. S. (1979) Revegetation of disturbed alpine rangelands. In:
Special management needs of alpine ecosystem. Range Science Series No. 5 (ed
Johnson D. A.) pp. 78-94. Society for Range Management, Denver, Colorado.
Cavieres L. A. and Arroyo M. T. K. (2000) Seed germination response to cold
stratification period and thermal regime in Phacelia secunda (Hydrophyllaceae).
Plant Ecology 149: 1-8.
Cerabolini B., De Andreis R., Ceriani R. M., Pierce S. and Raimondi, B. (2004) Seed
germination and conservation of endangered species from the Italian Alps,
Physoplexis comosa and Primula glaulescens. Biological Conservation 117: 351-
356.
Chambers J. C. (1989) Seed viability of alpine species, variability within and among
years. Journal of Range Management 42(4): 304-308.
Chambers J. C. (1997) Restoring alpine ecosystem in the Western United States,
environmental constrains, disturbance characteristics, and restoration success. In:

70
Restoration ecology and sustainable development (eds Urbanska K. M. et al.) pp.
161-187. University Press, Cambridge.
Chambers J. C., McMahon J. A. and Brown R. W. (1987) Germination characteristics of
alpine grasses and forbs, a comparison of early and late serial dominants with
reclamation potential. Reclamation and Revegetation Research 6: 235-249.
Escudero A., Carnes L. F. and Prez-Garca F. (1997) Seed germination of gypsophytes
and gypsovags in semi-arid central Spain. Journal of Arid Environments 36: 487-
497.
Escudero A., Gimnez-Benavides L., Iriondo J. M. and Rubio A. (2004) Patch dynamics
and islands of fertility in a high mountain Mediterranean community. Arctic, Antarctic
and Alpine Research36: 488497.
European Community (1992) Council Directive 92/43/EEC on the conservation of natural
habitats and of wild fauna and flora.
Gaviln R. G., Fernndez-Gonzlez F. and Rivas-Martnez S. (2001) Variaciones
bioclimticas en Madrid: un estudio sobre cambio climtico local. In: Vegetacin y
Cambos Climticos (eds Gmez F. and Mota J. F.) pp. 243-256. Servicio de
publicaciones de la Universidad de Almera, Almera.
Gaviln R. G., Snchez-Mata D., Escudero A. and Rubio A. (2002) Spatial structure and
interactions in Mediterranean high mountain vegetation (Sistema Central, Spain).
Israel Journal of Plant Sciences 50: 217-228.
Gmez-Campo C. (1987) Libro rojo de las especies vegetales amenazadas de Espaa
peninsular e Islas Baleares. ICONA, Madrid.
Grabherr G., Gottfried M. and Pauli H. (1994) Climate effects on mountain plants. Nature
369: 448.
Guisan A., Edwards T.C. Jr. and Hastie T. (2002) Generalized linear and generalized
additive models in studies of species distributions: setting the scene. Ecological
Modelling 157: 89-100.
Guisan A. and Zimmermann N. E. (2000) Predictive habitat distribution models in
ecology. Ecological Modelling 135: 147-186.
Gutterman Y. (1994) Strategies of seed dispersal and germination in plants inhabiting
deserts. Botanical Review 60: 373-425.
Hendry G. A. and Grime J. P. (1993) Methods in Comparative Plant Ecology. London,
Chapman and Hall.
Holm S.O. (1994) Reproductive patterns fo Betula pendula and B. pubescens coll. along
a regional altitudinal gradient in northern Sweden. Ecography 17: 60-72.
Kigel J. (1995) Seed germination in arid and semiarid regions. In: Seed Development and
Germination (eds Kigel J. and Galili G.) pp. 645-699. Marcel Dekker, New York.
CAPITULO 2

71
Krner C. (1999) Alpine Plant Life. Berlin, Springer-Verlag.
Marchand P. J. and Roach D. A. (1980) Reproductive strategies of pioneering alpine
species, seed production, dispersal, and germination. Arctic and Alpine Research
12 (2): 137-146.
MathSoft, Inc. (1999) S-Plus 2000. Guide to Statistics, Volume I. Seattle, Washington.
McCullagh P. and Nelder J.A. (1989) Generalized Linear Models. Chapman and Hall,
New York.
McDonough W. T. (1969) Effective treatments for the induction of germination in
mountain rangeland species. Northwest Science 43(1): 18-22.
McDonough W. T. (1970) Germination of 21 species collected from a high-elevation
rangeland in Utah. The American Midland Naturalist 84(2): 551-554.
Milberg P. and Andersson L. (1998) Does cold stratification level out differences in seed
germinability between populations. Plant Ecology 134: 225-234.
Mooney H. A. and Billings W. D. (1961) Comparative physiological ecology of arctic and
alpine populations of Oxyria digyna. Ecological Monographs 31: 1-29.
Palacios D., de Andrs N. and Luengo E. (2003) Distribution and effectiveness of nivation
in Mediterranean mountain, Pealara (Spain). Geomorphology 54: 157-178.
Pelton J. (1956) A study of seed dormancy in eighteen species of high altitude Colorado
plants. Butler University Botanical Studies 18(1): 74-84.
Peuelas J., Filella I. and Comas P. (2002) Changed plant and animal life cycles from
1952 to 2000 in the Mediterranean region. Global Change Biology 8: 531-544.
Prez-Garca F., Iriondo J. M., Gonzlez-Benito M. E., Carnes L. F., Tapia J, Prieto C.,
Plaza R. and Prez C. (1995) Germination studies in endemic plant species of the
Iberian Peninsula. Israel Journal of Plant Sciences 43: 239-247.
Pyke D. A. and Thompson J. N. (1986) Statistical analysis of survival and removal rate
experiments. Ecology 67(1): 240-245.
Rivas-Martnez S. (1963) Estudio de la vegetacin y flora de las Sierras de Guadarrama
y Gredos. Anales del Instituto Botnico Cavanilles 21(1): 5-330.
Rivas-Martnez S., Cant P., Fernndez-Gonzlez F., Molina J.A., Pizarro J. M. and
Puente E. (1999) Sinopsis of the Sierra de Guadarrama vegetation. Itinera
Geobotnica 13: 189-260.
Snchez-Herrera F. (2000) La restauracin ambiental de la Antigua estacin de esqu de
Valcotos y el programa de conservacin del Parque Natural de Pealara. In:
Segundas Jornadas Cientficas del Parque Natural de Pealara y del Valle del
Paular, pp. 19-25. Direccin General del Medio Natural, Madrid.

72
Sanz-Elorza M., Dana E. D., Gonzlez A. and Sobrino E. (2003) Changes in the high-
mountain vegetation of the Central Iberian Peninsula as a probable sign of global
warming. Annals of Botany 92: 273-280.
Schemske D. W., Husband B.C., Ruckelshaus C. G., Goodwillie C., Parker I. M. and
Bishop J.G. (1994) Evaluating approaches to the conservation of rare and
endangered plants. Ecology 75: 584-606.
Schtz W. and Milberg P. (1997) Seed dormancy in Carex canescens, regional
differences and ecological consequences. Oikos 78: 420-428.
Schtz W. and Rave G. (1999) The effect of cold stratification and light on the seed
germination of temperate sedges (Carex) from various habitats and implications for
regenerative strategies. Plant Ecology 144: 215-230.
Specht C. E. and Keller E. R. J. (1997) Temperature requirements for seed germination in
species of the genus Allium L. Genetic Resources and Crop Evolution 44: 509-517.
Spector P. (1994) An introduction to S and S-PLUS. Duxbury Press, Belmont.
Thanos C. A., Kadis C. C., and Skarou F. (1995) Ecophysiology of germination in the
aromatic plants thyme, savory and oregano (Labiatae). Seed Science Research 5:
161-170.
Thompson K., Band S. R. and Hodgson J. G. (1993) Seed size and shape predict
persistence in soil. Functional Ecology 7: 236-241.
Thompson P. A., Smith R. D., Dickie J. B., Sanderson R. H. and Probert R. J. (1981)
Collection and regeneration of populations of wild plants from seed. Biological
Conservation 20(3): 229-245.
Urbanska K. M. and Schtz M. (1986) Reproduction by seed in alpine plants and
revegetation research above timberline. Botanica Helvetica 96: 43-60.
Urbanska K. M. (1986) Behaviour of alpine plants and high altitude revegetation research.
In: Proceedings of the High Altitude Revegetation Workshop N
o
7 (eds. Schuster M.
A. and Zuck R. H.) pp. 215-226. Forth Collins, Colorado.
Urbanska K. M., Schwank O. and Fossati A. (1979) Variation within Lotus corniculatus L.
s. l. from Switzerland, II. Reproductive behaviour of L. alpinus (DC) Schleicher.
Berlin Geobotanical Institute ETH 46: 62-85.
Vre H., Lampinen C., Humphries C. and Williams P. (2003) Taxonomic diversity of
vascular plants in the European alpine areas. In: Alpine Biodiversity in Europe.
Ecological Studies, vol. 167 (eds Nagy L. et al.) pp. 133-148. Springer-Verlag,
Berlin.
Venables W. N. and Ripley B. D. (1998) Modern Applied Statistics with S-PLUS.
Springer-Verlag, Berlin.
CAPITULO 2

73
Vera M.L. (1997) Effect of altitude and seed size on germination and seedling survival of
heathland plants in north Spain. Plant Ecology 133: 101-106.
Zuur-Isler D. (1982) Germinating behaviour and early life phase of some species from
alpine serpentine soils. Berlin Geobotanical Institute ETH 49: 76-107.

74

Table 1. Plant life form, altitudinal range, and mean seed mass and size for 20 alpine species collected at Guadarrama mountain range.
Localities and years of collected plant material are marked for each species.

Loc.1
(2428m)
Loc. 2
(2269m)
Loc. 3
(2279m)
Loc. 4
(1950m)
Species
Life
form
Habitat
type
Altitudinal
range
Mean seed
mass mg-
(S.D.)
Mean
maximum
seed
diameter
-mm- (S.D.)
Mean
minimum
seed
diameter
-mm- (S.D.)
2001 2002 2001 2002 2001 2002 2001 2002
Allium
schoenoprassum*
G Rockwall OM-COM
1,558
(0,133)
2,755 (0,279) 1,784 (0,244) . x . . . . .
Armeria caespitosa* Cs
Dry
grassland
SM-COM
1,184
(0,067)
4,700 (0,734) 2,020 (0,341) . x . x . x . .
Biscutella laevigata
subsp. gredensis*
Hm
Blockf./ Dry
grassland
OM-COM
1,120
(0,061)
2,555 (0,199) 1,883 (0,144) x . . . x . . .
Festuca curvifolia*
Hm
Cae
Dry
grassland
SM-COM
1,032
(0,150)
4,558 (0,359) 1,244 (0,150) . x . x . x . .
Hieracium vahlii
subsp. myriadenum*
Hm
Dry
grassland
OM-COM
0,203
(0,017)
1,908 (0,164) 0,678 (0,108) x . . x x . . .
Jasione crispa subsp.
centralis
+

Cs
Dry
grassland
OM-COM
0,057
(0,011)
0,951 (0,094) 0,443 (0,083) . x . x . . x x
Jurinea humilis Hm
Dry
grassland
SM-COM
4,708
(0,276)
5,422 (0,580) 3,064 (0,462) . x . x . x . .
Luzula hispanica + Hm
Dry
grassland
COM
0,292
(0,019)
1,248 (0,103) 0,802 (0,126) . . . . x . . .
Minuartia recurva
subsp. bigerrensis *
Cs
Dry
grassland
COM
0,252
(0,016)
1,179 (0,120) 0,876 (0,094) . x . . x x . .
Murbeckiella boryi
+
A Rockwall SM-COM
0,106
(0,007)
1,235 (0,162) 0,625 (0,094) . . . . x . . .
CAPITULO 2

75
Loc.1
(2428m)
Loc. 2
(2269m)
Loc. 3
(2279m)
Loc. 4
(1950m)
Species
Life
form
Habitat
type
Altitudinal
range
Mean seed
mass mg-
(S.D.)
Mean
maximum
seed
diameter
-mm- (S.D.)
Mean
minimum
seed
diameter
-mm- (S.D.)
2001 2002 2001 2002 2001 2002 2001 2002
Ranunculus
ollissiponensis subsp.
alpinus
+

Hm
Dry
grassland
SM-COM
0,744
(0,067)
2,860 (0,270) 2,254 (0,226) . . . . x . x .
Saxifraga pentadactylis
subsp. wilkommiana
+

Cs Rockwall M-COM
0,052
(0,006)
0,811 (0,092) 0,491 (0,094) . . . . x . . .
Silene boryi subsp.
penyalarensis *
Hm
Blockf./
Rockw.
OM-COM
0,986
(0,058)
1,678 (0,147) 1,283 (0,136) . x . x . . x .
Silene ciliata subsp.
elegans
Cs
Dry
grassland
OM-COM
0,594
(0,061)
1,531 (0,496) 1,083 (0,362) . . . . x . x .
Sempervivum vicentei
subsp. paui
+

Hm
Blockf./ Dry
grass.
OM-COM
0,053
(0,010)
0,954 (0,126) 0,501 (0,088) . . . . x . . .
Senecio boissieri
+
Hm
Dry
grassland
COM
1,372
(0,148)
2,454 (1,687) 0,605 (0,455) . . . . . x . .
Senecio pyrenaicus
subsp. carpetanus *
Hm
Dry
grassland
OM-COM
2,396
(0,122)
3,917 (1,688) 1,096 (0,554) . x . . x . . .
Solidago virgaurea
subsp. fallit-tirones
+

Hm Blockfield SM-COM
0,814
(0,011)
3,503 (1,447) 0,948 (0,392) . x . . . . . .
Thymus praecox
subsp. penyalarensis *
Ch
Dry
grassland
COM
0,122
(0,016)
1,138 (0,119) 0,837 (0,102) . x . x . x . .
Veronica fruticans
subsp. cantabrica
+

Hm Blockfield COM
0,096
(0,040)
0,853 (0,107) 0,618 (0,081) . x . . . . . .

Hm: hemicryptophyte, Cs: Cushion chamaephyte, Hm Cae: Caespitous hemicryptophyte, A: annual, G: geophyte. SM: Supra-, OM: Oro- ,
COM: Cryoromediterranean belt. * Indicate endemics of Central mountain range,
+
indicate endemics of the Iberian Peninsula.


76

Table 2. Results of the reduced Generalized Linear Models for final germination
percentage of fresh seeds. Selection of variables temperature, altitude, population
(locality) and year was carried out by means of a forward stepwise procedure. Species
are grouped accordingly to temperature requirement for optimal germination. Coef. (S.E.):
coefficient value (Standard Error); t: t test for significance of the coefficients; Prob. (t): p
value of t test; Res. D. F.: Residual Degrees of Freedom; Res. Dev.: Residual Deviance;
Adj. D
2
: adjusted D
2
following Guisan and Zimmermann (2000), F:
2
tests used to
evaluate if selected predictors explain a significant fraction of the deviance; Prob. (F): p
value of
2
tests.

Species Variable Coef. (SE) t
Prob
(t)
Res.
D.F.
Res.
Dev.
Adjus.D
2
F
Prob
(F)
Group 1. No Temperature requirement
Mur Null 11 0,3411
Temp. - - - 10 0,3190 0,9681 0,5684 0,4683
Sax Null 11 0,4734
Temp. - - - 10 0,4503 0,0488 0,5055 0,4933
Semp Null 11 0,6114
Temp. - - - 10 0,5147 0,1581 1,8059 0,2087
Senb Null 11 0,1846
Temp. - - - 10 0,1052 0,4303 2,5361 0,1553
Sol Null 11 0,8298
Temp. - - - 10 0,6917 0,9308 2,0797 0,1798
Arm Null 35 3,1354
Alt.
-1,056
(0,247)
-4,2656 0,0001 34 2,1074 0,3279 18,2298 0,0001
Bis Null 20 3,1957
Alt.
-0,847
(0,439)
-1,9311 0,0677 19 2,6607 0,1674 4,0113 0,0597
Ran Null 23 1,8630
Alt.
-0,6568
(0,2363)
-2,7798 0,0104 22 1,3971 0,2501 7,9172 0,0101
Fesc Null 35 8,7073
Pobl.
1,0685
(0,2431)
4,3943 0,0001 34 5,3761 0,3826 22,9723 0,0000
Thy Null 35 6,1207
Alt.
2,4313
(0,4459)
5,4520 0,0000 34 3,9351 0,3382 25,3992 0,0000
Pobl.
0,8255
(0,2494)
3,3101 0,0021 33 2,9699 0,5005 11,2168 0,0020
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77
Species Variable Coef. (SE) t
Prob
(t)
Res.
D.F.
Res.
Dev.
Adjus.D
2
F
Prob
(F)
Group 2. High Temperature requirement (20
o
C)
Alli Null 11 1,7839
Temp.
1,5189
(0,2501)
6,0731 0,0001 10 0,3427 0,8079 53,0164 0,0000
Senp Null 11 2,7624
Temp.
1,1500
(0,1873)
6,1399 0,0001 10 0,5392 0,1664 2,0797 0,1798
Hie Null 43 6,9432
Temp. 0,3594 2,2795 0,0275 42 6,1359 0,1163 5,6993 0,0215
(0,1576)
Sbor Null 35 26,3755
Temp.
2,4412
(0,3886)
6,2808 0,0000 34 7,1796 0,7278 71,0145 0,0000
Jas Null 47 28,8022
Temp.
2,2152
(0,2023)
10,9472 0,0000 46 6,2726 0,7775 196,4671 0,0000
Alt.
0,5369
(0,1621)
3,3122 0,0002 45 4,9575 0,8241 11,4680 0,0015
Min Null 35 6,5573
Year
1,0425
(0,3301)
3,1578 0,0004 34 4,6897 0,2415 29,0067 0,0000
Temp.
1,0546
(0,2156)
4,8909 0,0000 33 2,8502 0,5390 28,5711 0,0000
Alt.
1,9933
(0,6910)
2,8844 0,0066 32 2,0757 0,6643 12,0287 0,0015
Group 3. Medium Temperature requirement (15
o
C)
Silc Null 19 7,7464
Temp.
0,6694
(0,1939)
3,4511 0,0025 18 4,5027 0,4187 14,3020 0,0014
Jur Null 34 10,5831
Alt.
-2,4180
(0,6211)
-3,8929 0,0004 33 7,6814 0,2288 19,7693 0,0001
Pobl.
0,7377
(0,3477)
2,1213 0,0411 31 5,0465 0,4934 4,5900 0,0401
Temp.
0,6445
(0,1791)
3,5972 0,0010 32 5,7202 0,4257 13,3613 0,0009


78

Figure 1. Final germination percentages for the 20 species at different temperature
regimes.
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79

Figure 2. Modelled germination curves by the Kaplan-Meier method for fresh seeds.
These four cases exemplify different germination behaviours obtained for the 20 species.


80

Figure 3. Interannual variability (2001-2002) in final germination percentage for Jasione
crispa subsp. centralis and Minuartia recurva subsp. bigerrensis.

Figure 4. Final germination percentage before and after submitting seeds to cold-wet
stratification treatment.

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81

Figure 5. Modelled germination curves by the Kaplan-Meier method before and after
submitting seeds to cold-wet stratification.

82

CAPITULO 3

83

Captulo 3.

Coping with climate warming: reproductive plasticity in a
high mountain Mediterranean plant under a severe
heatwave.

Gimnez-Benavides, L.; Escudero, A. and Iriondo, J. M.
New Phytologist, under review.
REPRODUCTIVE PLASTICITY OF SILENE CILIATA UNDER CLIMATE CHANGE

84
CAPITULO 3

85
SUMMARY

Mountain plant species are particularly sensitive to climate warming and the
increasing frequency of extreme events because snowmelt timing, which is directly
correlated to changes in temperature and precipitation, exerts a direct control on their
reproduction. Current warming trends are leading to earlier snowmelt dates, and the
subsequent lengthening of the snow-free period. Our hypothesis is that high mountain
Mediterranean plants are not able to take advantage of a lengthened growing season
because it leads to longer drought periods, which may critically reduce reproductive
output. However, phenological plasticity may somewhat mitigate these negative effects
through temporal shifts. In this study we assessed the effects of an extreme climatic
event on the flowering phenology and reproductive success of Silene ciliata, a
Mediterranean high mountain plant, compared to a climatically average year. The species
showed a late-flowering pattern that hampered the use of snowmelt water. The proportion
of flowering plants substantially decreased at the lowest-altitude population, especially in
the harsh year. Plants better performed at the highest population. Plant fitness was
largely explained by prefloration interval (i.e. time from snowmelt date to the onset of
flowering), suggesting a selective pressure towards early flowering due to soil moisture
depletion. Our results indicate that water deficit in hot and dry years could threaten the
viability of lowland populations, while high altitude environments are more stable over
time.

86
INTRODUCTION

In alpine and arctic regions, snowpack and snowmelt timing, which are directly
correlated to snowfall regime and ambient temperature (Groisman et al., 1994; Dye,
2002), are the two major factors determining soil moisture, and, indirectly, the onset of the
growing season, plant growth and annual rates of primary production (Walker & Webber,
1994; Kudo, 2003). In reproductive terms, they have been widely reported as drivers of
flowering phenology and reproductive output (Billings & Mooney, 1968; Galen & Stanton,
1995; Stinson, 2004; Molau et al., 2005). Current global warming is affecting snowmelt
timing and, subsequently, the reproductive output of high mountain plants (Inouye &
McGuire, 1991; Price & Waser, 1998; Inouye et al., 2002; Kudo & Hirao, 2006).
Latitudinal poleward shifts in distribution range of plants are not always feasible because
of orographic isolation, and upward shifts to higher summits are irremediably associated
with a decrease in habitat area (Pauli et al., 2003). Moreover, lowland species are
expanding their upper distribution limits mainly due to water availability (Pigott & Pigott,
1993) and higher temperatures (Conolly & Dahl, 1970) and thus, may push cryophilic
plant populations to the verge of extinction (Grabherr et al., 1994; Pauli et al., 2003).
As conditions for the reproduction and survival of plants are more suitable in the
centre of their distribution area than in the periphery (Lawton, 1993), viability
assessments of peripheral plant populations in global warming scenarios have become a
conservation concern. Several reproduction and regeneration studies have been carried
out on these populations (McCarty, 2001), however, most of them have focused on
northern plant limits (Arft et al., 1999) while they have rarely been conducted on southern
boundaries (Garca et al., 1999; Peuelas & Boada, 2003; Hampe, 2005). When
latitudinal gradients run parallel to climatic gradients, special adverse conditions for
regeneration often occur at the species' southernmost distribution margin (in the northern
hemisphere) (Lesica & McCune, 2004; Hampe & Petit, 2005). A complete identification of
the limitations on reproduction of high-mountain plants in the southern margin of their
distributions may provide us with valuable information on plant response plasticity and
potential to counteract rapid climate change.
The reproductive assessment of high mountain plants should consider
environmental variability at several scales: altitudinal gradient, which controls the local
climate, small-scale heterogeneity, which may mitigate adverse conditions or exacerbate
restrictions, and finally, temporal heterogeneity which may be especially important in
stochastic environments such as the Mediterranean. This environmental variability may
cause changes in flowering phenology (above cited references), pollinator frequency
(Ttland, 2001 and references therein), predator frequency (Galen, 1990; Freeman et al.,
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87
2003) and intensity of plant-plant interactions (Klanderund, 2004), affecting the overall
performance of plants.
Little is known about the potential threats of climate warming to Mediterranean
habitats (Peuelas et al., 2002, 2004; Peuelas & Boada, 2003), and no studies have
focused on Mediterranean high mountains in above-timberline habitats. Extrapolation of
arctic and alpine reproductive mechanisms and causal factors to Mediterranean high-
mountain environments should be conducted with caution because specific constraints,
such as severe water deficit during the growing season, must be taken into account.
Mediterranean-type mountains hold a great number of endemic species, many of
which are restricted to the higher summits (Gimnez et al., 2004). Many arctic and alpine
species also find their southernmost limit in these mountains, hence representing a global
conservation priority (e.g. Sierra Nevada, Pauli et al., 2003). Although these ecosystems
are among those predicted to be the most vulnerable to global warming in Europe
(Thuiller et al., 2005), our knowledge about the regeneration and reproduction of these
plants is almost null.
In this paper, we examined the reproductive performance and plasticity of Silene
ciliata, a high mountain Mediterranean plant, in contrasting environments along an
altitudinal gradient in its southern distribution limit. Our aim was to identify the main
limitations on plant fitness. Specifically, we quantified the plasticity of the flowering event
in order to assess to what extent phenological shifts can cope with a rapidly changing
environment. For this purpose, we modelled the overall reproductive response of the
species using a large set of phenological, biotic and abiotic variables at contrasting
spatial scales.
In addition to mean warming trends, one of the major concerns of current climate
change is the increasing frequency and intensity of extreme climate events (Easterling et
al. 2000). Although the global fingerprint of rising temperatures during the last century
has been observed in biological features from phenology (Parmesan & Yohe, 2003) to
range shifts (Wilson et al., 2005), there are, to our knowledge, few works dealing with the
plant response to extreme events (Guschick & BassiriRad, 2003; Reusch et al., 2005).
During the course of our phenological study in summer 2003, the greatest
heatwave registered in the last 150 years took place in Europe (Schr & Jendritzky,
2004). In central and southwestern parts of the continent, heat and drought conditions
caused severe damage to cultivated and wild plant species. This allowed us to evaluate
the species reproductive capacities under an extreme event, and gave us an opportunity
to better infer the potential threats of global warming through the rising intensity and
frequency of extreme events.

88
We hypothesize that a lengthening of the snow-free period as a result of
increasing temperatures may become a critical problem for plants occurring in
Mediterranean mountains due to harsh summer drought. We also expect warming
conditions to be especially adverse for the lowest margin populations and more intense in
years with low precipitation and high temperatures (extreme events). Survival under such
conditions is only feasible if reproduction is plastic enough to cope with short-term
environmental changes and/or selection has time to act (Jump & Peuelas, 2005).

MATERIALS AND METHODS

Species and study area
Silene ciliata Poiret (Caryophyllaceae) is a perennial cushion plant, inhabiting
main mountain ranges in the northern half of the Iberian Peninsula, the Massif Central in
France, the Appenines and the Balkan Peninsula (Tutin et al., 1995). In Central Spain,
the plant reaches its southern latitudinal limit. The species typically forms cushion
rosettes of up to 2 cm in height and 15 cm in diameter. Flowering stems reach 15 cm in
height and bear 1 to 5 flowers. Hand-crossing experiments indicate that S. ciliata is a self-
compatible species. Both fruit set and seed set are high under xenogamy and
geitonogamy treatments. However, passive autogamy is totally restricted by a
pronounced protandry. Fruit capsules open at the top when ripe and up to 100 seeds per
fruit are dispersed by stalk vibration. Seeds are relatively small (1.53 0.49 mm in
diameter, 0.59 0.06 mg in weight). Although a small fraction is able to germinate
immediately after dispersal (<25 % at 20 C), nearly 100 % of dispersed seeds germinate
after a cold-stratification period or contrasting high temperatures (Gimnez-Benavides et
al., 2005).
Three altitudes covering the local range of S. ciliata were chosen in Sierra de
Guadarrama (Circo de Pealara Natural Park), a SW-NE running mountain range located
50 km north of Madrid (40
o
N, 3
o
W). The lowest altitude (1976 m, hereafter "L") was in a
moraine deposit at Hoya de Pealara, a Pleistocene glacial cirque situated in the
timberline zone. Vegetation is dominated by stunted pines (Pinus sylvestris) interspersed
in a shrub matrix of Cytisus oromediterraneus and Juniperus communis subsp. alpina in a
Festuca curvifolia pasture. This site is the lower altitudinal limit of the species. The
intermediate altitude (2256 m, "I" hereafter) was located on the Dos Hermanas summit
about 3 km from altitude L. At this site, a patchy xerophytic pasture dominated by F.
curvifolia co-occurs with the high altitudinal limit of the Cytisus-Juniperus shrub formation.
The highest altitude (2428 m, H hereafter) was located at Pico Pealara, the highest
peak of Sierra de Guadarrama about 3 km from altitude I. The summit flat areas and
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89
crests are covered by F. curvifolia fellfields, occasionally displaced by a moister Nardus
stricta-dominated community where snowcover remains longer. This fellfield community,
which supports extreme winds and is characterized by a high diversity of cushion plants,
constitutes the main habitat of S. ciliata (see Gaviln et al. 2002 and Escudero et al. 2005
for further details).
Sierra de Guadarrama is characterized by a Mediterranean-type climate. Snowfall
generally begins in October, and snowmelt concludes in May-June. Mean annual
precipitation at the Navacerrada Pass weather station (40 46N, 4 19W; 1860 m),
located 8 km southwest of Pico Pealara, is 1350 mm and is concentrated from early
October to late May. A pronounced drought season (less than 10 % of the total annual
rainfall) occurs from June to September (Palacios et al., 2003). For annual trends, we
used data from the above-mentioned station, as it had the only long climatic series
available for the area (1960-2004, Instituto Nacional de Meteorologa). In 2002-2003,
climatic data at each study site were provided by a network of portable weather stations
of the Pealara Natural Park.

Plant monitoring
In 2002, three plots were randomly established at each altitudinal level. Plot
location was selected to minimize differences in exposure (NE), slope (0-10) and human
management (almost null) among sites. Plot size varied between 10 and 15 m
2
. At each
plot, 55-60 individuals were randomly selected and tagged for a total sample size of 166
plants at L, 165 plants at I and 171 plants at H. Plant cushions located at a minimum
distance of 2 cm from each other were considered different individuals. As the species
does not propagate vegetatively, each labelled plant was considered a genet. A detailed
measure of plant size, calculated as the horizontally projected area of the cushion in cm
2
,
was obtained from digital images taken of each plant using Image Tool 3.0 (University of
Texas). To estimate microsite conditions, a 25 x 25-cm quadrat was centred on each
plant. Cover percentage of bare soil, rocks, other S. ciliata individuals, and any other
plant species was estimated with the digital image analyzer. Each year, snowmelt date
was estimated for each altitude from digital images taken at weekly intervals and was
assigned to the date of the first picture displaying ~80% bare ground.
Flowering and fruiting phenology were monitored at weekly intervals for a total of
13 and 14 census days in 2002 and 2003 respectively. At each census, we recorded the
number of open flowers and mature fruits on each plant, as well as the number of flowers
and fruits with external signs of predation. To estimate seed production, mature and
predated fruits were collected before opening to avoid loss of seeds by dispersal.

90
Flowering phenology was characterized for each plant by measuring six variables:
a) Prefloration interval - number of days from snowmelt to first open flower; b) First
flowering date - number of days from 1
st
January to first open flower; c) Moment - number
of days from the first open flower in the population to maximum flower count on each
plant; d) Duration - number of days the plant remained in bloom; e) Flowering synchrony
- number of days that the flowering of one individual overlaps with the flowering of every
other plant in the population. We applied the following function modified from Augspurger
(1983):
=

=
1
1

1
1

n
f if
if
i
b
a
n
S

where n is the number of plants, a
ij
is the number of days individuals i and j are
simultaneously in bloom, and b
ij
is the number of days at least one of them is in bloom. S
i

ranges between 1, when flowering completely overlaps, and 0, when there is no
synchrony.
Female reproductive success was assessed for each plant by means of five
variables: flower number, fruit number and seed number (expressed as total number per
plant), fruit set (proportion of flowers setting fruit) and brood size (average proportion of
ovules setting seed in each ripe fruit). Additionally, a measure of total yield per unit of
plant area was calculated for each plot (n=9) by determining the ratio between total seed
number and total plant area (in cm
2
). This allowed us to assess differences in seed
production among populations irrespective of differences in plant size.

Statistical analysis
Table 1 summarizes the different statistical techniques used to analyze the
processes that sequentially affect reproductive success.

Flowering probability
To determine the dependence of flowering ability on plant size (Lacey, 1986) and
the effect of altitude, we modelled flowering probability by fitting Generalised Linear Mixed
Models (GLMMs). Models were built for each year with plant size and altitude as fixed
variables and plot as a random variable nested in altitude. Since the response variable
was a probability ranging from 0 to 1 (i.e. not flowering or flowering), we used a binomial
estimator, using a logit link function and setting the variance to mean (1-mean). Effects of
random factor were tested using the Wald Z-statistic test, and fixed factors were tested
with F-tests. The degrees of freedom in each level of variation (595 plants and 3 plots per
altitude) were estimated by Satterthwaites method (Littell et al., 1996).
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We also estimated flowering probability curves by fitting a new GLMM for each
altitude and year separately, maintaining plant size as a fixed variable and plot as a
random variable. Since the plot random variable was always non-significant, we plotted
the curves using the estimated parameter for plant size and the formula:

( )
x
e
P
+
+
=
1
1

where is the intercept with the X-axis, which is related to the threshold size for
reproduction, and is the slope of the curve, which is related to the percentage of
reproductive plants (Mndez & Karlsson, 2004). Flowering probability curves were
compared between years by including year as a fixed factor.

Structural equation modelling: female reproductive success and reproductive plasticity
The relationships among plant size, microsite variability (i.e. intra- and
interspecific competition and bare soil cover), phenological variables and reproductive
success were studied with a non-standard structural equation model (SEM) with a latent
variable. This technique allows the analysis of complex multivariate causal schemes
represented in a path diagram. We formulated our path diagram based on our a priori
hypothesis of causal links between variables (Fig. 1). The SEM tests whether our data
are supported by the underlying mechanisms that our specific model describes (Shipley,
1999). Our working model proposes that the absolute fitness of each plant (expressed as
fruit number) ultimately depends on plant size, flowering phenology components and
microsite conditions (Fig. 1). We hypothesized that flowering onset would depend on
reaching a resource threshold. Thus, larger plants would have an earlier flowering start,
i.e. shorter prefloration interval. Plant size might also determine flower number and
flowering period, because larger plants may invest more resources in reproduction. We
also expected prefloration interval to affect flower number both directly and/or through
duration, since early-flowering plants can have longer flowering periods than late-
flowering plants. We considered prefloration interval and duration important phenological
traits affecting synchrony among individuals, which ultimately control fruit set enabling
cross-pollination. Fruit set could also be determined by flower number (positively in terms
of attractiveness to pollinators, or negatively because of resource limitation). Finally, plant
microsite characteristics were added to the model as a latent variable to test their effect
on reproductive output. Latent variables are not measured directly but can be expressed
in terms of one or more directly measurable variables called indicators (Loehlin, 1992).
We constructed the latent variable as an estimator of microsite suitability, which was

92
comprised of abundance of surrounding plants (i.e. both intra- and interspecific plant
cover variables) as an estimator of potential plant-plant interactions, and available space
for plant development (i.e. soil cover variable). Microsite suitability was expected to
influence flower number, fruit set and fruit number mainly through resource limitation.
There is also an evident relationship between plant size and microsite characteristics, but
since the direction of causality is difficult to interpret, we modelled this relationship as an
unanalyzed correlation (Mitchell, 1993).
The model was evaluated separately for each altitude and year. For each altitude
data from the three plots were pooled together and only plants that flowered were
included in the analyses (Table 2). Sample size, ranging from 63 to 146, was at least five
times the number of variables to be estimated as recommended by Loehlin (1992). Since
the estimation method in SEM depends on the sample covariance matrix, which is a poor
estimator for distributions with high kurtosis (Bollen, 1989), we removed outliers when
necessary. We also calculated the variance inflation factor (VIFs) for each variable to
check for multicollinearity. Since all VIF values were below 10 (Petraitis et al., 1996), we
incorporated all variables in the model. The null hypothesis of the model is that the
observed and predicted covariances are equal. Standardized path coefficients (equivalent
to standardized partial regression coefficients) were estimated using the Maximum
Likelihood method. This method is considered the best option when deviations from
multivariate normality are severe and the sample size is not very large (Finch et al.,
1997). Microsite suitability is an unobserved variable with no unit of measurement. To
solve this scale indeterminacy problem, we made it equal to the unit of measurement of
the indicator variable that best represented the latent construct (that with the largest
standardized coefficient in a preliminary exploratory analysis) by fixing its path coefficient
to 1 (Hatcher, 1994).
The degree of fit between the observed and predicted covariance structures was
first assessed by a
2
goodness-of-fit test. A significant
2
indicates that the model does
not properly fit the data. However, this test may show inadequate statistical power when
data depart from multivariate normality and sample sizes are small (Loehlin, 1992). A
2
/df ratio < 2.0 is usually considered an acceptable fit to data (Hatcher, 1994). We used
two alternative fit indices that provide an accurate fit regardless of sample size: the
Comparative Fit Index (CFI) (Bentler, 1989) and the Non-normed Fit Index (NNFI)
(Bentler & Bonet, 1980). Values > 0.90 and 0.81, respectively, indicate a good fit
compared with a null model that assumes independence among all variables (Hatcher,
1994). The significance of each individual path coefficient was subsequently assessed by
a multivariate Wald test (P<0.05). This test locates the path coefficients that can be
eliminated without significantly increasing the chi-square value of the model. The effect
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93
of unexplained causes on each variable is measured as (1-R
2
)
1/2
, with R
2
being the
coefficient of determination, that is, the proportion of observed variance explained by
each equation. Model comparisons between altitudes and years were systematically
conducted by overimposing path diagrams. Computation of the structural equations was
performed using the CALIS estimation procedure with the LINEQS statement (SAS
Statistical Package, SAS Institute, 1990). For a complete description of structural
equation models in ecology see Mitchell, 1993; Shipley, 1999 and Iriondo et al., 2003.

Models for total seed production and brood size
Brood size and seed number per plant were not included in the SEM model
because sample size (number of plants bearing seeds) was below the recommended
threshold (Loehlin, 1992). We fitted GLMMs for the total seed production of each year,
using the Gamma estimation since Poisson originated a high dispersion. A log link
function was used, setting the variance to mean (Venables & Ripley, 1998). Altitude,
fruit number, brood size and the indicator variables of microsite suitability (intra- and
interspecific competition and soil cover) were included as fixed variables, and plot as a
random variable nested in altitude. For brood size we fitted the mixed models using the
binomial estimation (probability from 0 to 1), a logit link function, and setting the
variance to 1-mean. We included the following fixed variables: altitude, plant size, fruit
number, synchrony and the indicator variables of microsite suitability. Plot was again
included as a random variable nested in altitude. Effects of random and fixed factors were
tested as described above.

RESULTS

Annual trends and microclimate at study sites
In the last 45 years mean annual temperature increased ~1.8 C in the study
region (r
2
=0.41, p<0.001). This warming was especially pronounced in the last 35 years
as stated for the whole planet (IPCC, 2001). Temperature increased 2 C in the winter
months (October-March) and 1.5 C in the growing season (April-September). Seasonal
rainfall revealed no significant downward decline (Fig. 2a-b). Snow cover duration,
expressed as the accumulated number of days that snow covered the ground during the
thaw season (from April to June), decreased 19.7 days in the last 45 years (Fig. 2c), but
dropped 31.3 days when considering only the last 35 years (r
2
=0.32, p<0.001).
Temperature, precipitation and snowmelt date varied greatly between the two
years of this study. The year 2002 had average temperatures and summer rainfall,
whereas 2003 was relatively cold and rainy in winter but very hot and dry in the growing

94
season due to the most severe known European heatwave (Schr & Jendritzky, 2004).
Maximum temperatures of the complete climate series were reached in summer. No rainy
days were recorded in July, and only one in August (see Fig. 2b). As a consequence,
plants emerged from snow ~22 days earlier in 2003. Along the altitudinal gradient, the
lower site was an average 1.8 C warmer than the intermediate site which was 1.1 C
warmer than the higher site. Both years, snow disappeared from low altitude plots ~9
days earlier than from intermediate plots which emerged ~9 days earlier than high altitude
plots (Table 2).

In 2002 minimum size at reproduction coincided with the minimum monitored size
(plant size ranged from 0.8 to 216 cm
2
) at all altitudes. Hence, we considered every
monitored plant a potential reproductive individual, and, in consequence, the percentage
of flowering plants was calculated with respect to all individuals. S. ciliata showed
important altitudinal and interannual differences in the percentage of flowering plants.
While in I and H altitudes S. ciliata had a high rate of flowering plants both years, the
proportion of flowering plants at L altitude was very low, especially during the second year
(Table 2, Figure 5a).
Results of GLMMs showed a lower probability of flowering in altitude L than in H
and I (Table 3). Furthermore, flowering probability was size-dependent both years (Table
3). Altitudinal and interannual differences can be easily explored in the associated
flowering probability curves (Fig. 3). It is interesting to note the significant drop in
flowering probability experienced at altitude L between years (F-value = 24.07, P <
0.0001), while I and H remained similar (F-value = 0.58, P = 0.448 and F-value = 0.14, P
= 0.708 respectively). On the other hand, an important shift took place in the flowering
probability of smaller individuals from one year to the next (see differences in the
intercept point of curves). In 2002, all populations had a flowering probability of
approximately 0.6 for the smallest monitored plant, while in 2003 flowering probability
dropped to almost 0.25 in altitude L, remained nearly the same in I, and increased to 0.9
in H.

Flowering phenology
Populations showed marked altitudinal and interannual differences for all of the
phenological variables. The flowering season started at the end of June and extended
until the end of September in 2002, but substantially shortened to early September in
2003. In 2002, populations at altitudes I and H showed a considerable delay in the first
flowering day (8-10 days) compared to L, but this difference disappeared in the extremely
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95
dry and hot 2003 (Fig. 4). Flowering duration increased progressively with altitude in
2002, but shortened at all sites in 2003. Prefloration interval was about 2.5 months and
did not differ among altitudes within years, but was slightly longer in the second year.
Moment took place approximately in the middle of mean flowering period at each altitude
in 2002, but occurred either earlier or at the same time in 2003 (Fig. 4). Flowering
synchrony was relatively low at all altitudes. The plants of altitudes H had the highest
flowering synchrony each year (Table 2).

Female reproductive success
In general, plants performed better at the upper distributional limit. Flower number,
fruit number and seed number reached maximum values at altitude H both years (Table
2, Figure 5b). Although all populations had a similar phenological pattern, plants at
altitude I had comparatively lower values than at altitude H for all reproductive traits.
Significant interannual differences were also found. While plants at altitude L had the
highest fruit set in 2002, more fruits per flower were produced at altitude I and H in 2003
(the heatwave year) (Fig. 5c). Results of the mixed model for seed number reinforced the
altitudinal differences observed (Table 4). Fruit number and brood size also explained a
fraction of variability in seed number both years (Table 4). However, the effect of
interspecific competition was only significant in the second (heatwave) year. On the other
hand, brood size only showed significantly higher values at altitude L in 2002, and was
significantly affected by plant size, fruit number and synchrony both years (Table 5, Fig.
5d). Total yield (expressed as mean number of seeds produced per cm
2
of plant) always
reached the highest values at altitude H, followed by altitude L in 2002 and altitude I in
2003 (Figure 5e).

Path analyses
The path models provided a good overall fit for all altitudes and both years studied
(Fig. 1). Five of the six path models had an excellent fit with observed data as indicated
by their non-significant
2
(P > 0.05),
2
/df ratio < 2 (range 0.9 to 1.3), and by goodness of
fit indices (NNFIs and CFIs) values that were > 0.90. However, the model for population
H in 2003 was rejected by both the
2
test (
2
= 44.24, df = 26, P = 0.014,
2
/df = 1.7) and
by the NNFI and CFI (0.79 and 0.88 respectively) even though the
2
/df ratio was < 2
(Hatcher 1994). The strength of all six models mainly rested on the sequence of causal
relationships from plant size and prefloration interval to duration, from duration to flower
number, and from flower number to fruit number. Prefloration interval had a negative
effect on duration, which largely determined flower number (coefficients greater than 0.43
in all cases, P < 0.001) and synchrony. Although direct effects of prefloration interval on

96
flower number were non-significant in all models but one, indirect effects through duration
were negative and relatively high in some models (-0.35, -0.11, -0.18 in 2002 and -0.41, -
0.31, -0.20 in 2003, at altitudes L, I and H, respectively). On the other hand, plant size
also influenced duration in all cases, but surprisingly did not directly affect flower number
in some models. In 2002, plant size negatively affected prefloration interval in altitudes I
and H.
Fruit number was significantly affected by flower number and fruit set in all
models. However, neither synchrony nor microsite suitability influenced fruit set, except in
the second year at altitude H, where variables accounted for equal but opposite effects.
Finally, no causal relationships were found between microsite suitability and other
reproductive traits. Nevertheless, the indicator variables of the latent construct gave us
some valuable information about micro-environmental differences at each site. The effect
of soil cover was highly positive at altitude L but negative at I and H, and the contrary took
place with interspecific competition. As noted in Table 2, mean values of both variables
were comparatively higher at L.

DISCUSSION

Recent climate trends in this mountainous region suggest a very rapid warming
(Fig. 2), as reported worldwide (IPCC, 2001). As shown by Agust-Panareda et al. (2002)
and Granados & Toro (2000) by estimates from chironomid fossil records, sharp climate
change during the last century has been the norm in this part of the Iberian Peninsula.
However, the recent increase in temperature rates had been unknown of in these
mountains and the ecological consequences are extremely difficult to forecast. The first
field evidence of this recent warming in this region was reported by Sanz-Elorza et al.
(2003), who pointed out the ongoing replacement of the typical high mountain grassland
Festuca curvifolia by Cytissus oromediterraneus-Juniperus alpina, a scrubland from lower
altitudes. Wilson et al. (2005) found a marked contraction of lower elevational limits for 16
butterfly species in the same area. Similar altitudinal shifts in species distributions linked
to climate warming have been reported in other Mediterranean mountains at a lower
altitude (Peuelas & Boada, 2003), and in mountains around the globe (Grabherr et al.,
1994; Dullinger et al., 2003; Parmesan et al., 1999).
Frequency of extreme climatic events, like the heatwave that occurred in 2003, is
predicted to increase in the coming decades (Easterling et al., 2000; Schar et al., 2004).
Nevertheless, global change research has mainly focused on the long-term effects of
rising mean temperatures, undervaluating the effects of extreme events and their impact
on natural systems (Gaines, 1993; Gutschick & BassiriRad, 2003; Reusch et al., 2005).
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97
Comparison between average climate years and years with extreme heat and drought is
an efficient field approach to determine the capacity of plants to counteract rapid
warming.
One of the most significant effects of climate warming in high mountains is related
to snow cover duration. The onset of the growing season in mountains mainly depends
on snowmelt date, which has become substantially earlier in Central Spain (Fig. 2c). This
seems to be mainly related to an increase in spring temperatures, since mean seasonal
precipitation has not dropped significantly (Fig. 2b). An increase in the duration of the
growing season should benefit high mountain specialists (Krner, 1999). However, some
studies have emphasized the importance of drought stress in high Mediterranean
mountains during summer (Castro et al., 2002; Castro et al., 2004). Thus, a temperature-
wise lengthening of the growing season may be coupled with a parallel intensification of
evapotranspiration rates and topsoil desiccation (Walker et al., 1995; Starr et al., 2000),
which may lead to more intense drought stress and reduced nutrient uptakes and growth
in Mediterranean climates (Royce & Barbour, 2001a,b; Peuelas et al., 2004). In a
parallel study, we measured the volumetric soil moisture at 0-6 cm depth at the same
study sites in 2004, which was an average climate year. The results confirmed the
existence of severe topsoil water limitation during summer (<5 % in water soil content),
and an altitudinal gradient in water availability (Gimnez-Benavides, unpublished data).

Flowering phenological pattern
Silene ciliata starts flowering over two months after snowmelt, once the annual
leaf crop has been produced (Fig. 4). According to the classification of life-history
strategies made by Molau (1993) for arctic and alpine plants, S. ciliata would be a typical
late-flowering species. The prefloration interval varied according to the snowmelt date
each year. Thus, S. ciliata started flowering on similar dates in both monitored years in
spite of a much earlier snowmelt date in 2003 (Fig. 4). This indicates that in S. ciliata, as
in many late-flowering alpine plants, the onset of blooming is controlled by photoperiodic
triggers and not by snow conditions or soil moisture content (Krner, 1999; Keller &
Krner, 2003). This late-flowering pattern is unusual in Mediterranean-type climates
where flowering is bimodally concentrated in spring/early summer and in autumn, a
strategy apparently developed to avoid the difficulties of severe summer drought
(Petanidou et al., 1995; Thompson, 2005).
Early-flowering species suffer high abortion rates and the risk of unsuccessful
reproduction due to late cold episodes in early summer and/or limited availability of
pollinators. In late-flowering species reproductive output may be limited by early snow
events in late summer/early autumn, shortening the time available for seed ripening.

98
Thus, it has been hypothesised that late-flowering species in temperate mountains may
enhance their reproductive success under a climate warming scenario due to a more
extended growing season, whereas early-flowering species might not respond to the
same extent or in the same direction (Molau, 1993; Alatalo & Ttland, 1997; Molau et al.,
2005). However, as Starr et al. (2000) pointed out, the potential advantage of a longer
growing season may be seriously limited by low water availability. We found a markedly
worse reproductive performance in 2003 (characterized by an extended snow-free
season) especially at altitude L (the most adverse site). Our results suggest that summer
drought in Mediterranean high mountains imposes important reproduction limitations on
late-flowering plants (Peuelas et al., 2004). So, the assumption that late-flowering arctic
and alpine plants benefit under warming conditions should be reconsidered for
Mediterranean mountains.

Effect of size on reproductive success
Silene ciliata showed notable size-dependence at several stages of the
reproductive process. First, and surprisingly for a long-lived species, plant size played a
major role in flowering probability (Fig. 3). Secondly, although plant size did not directly
affect flower and fruit production, it did so indirectly through the onset and duration of the
flowering period (Fig. 1).
Size-dependence of flowering probability has been cited by many authors as an
important factor controlling plant fitness (Mndez & Karlsson, 2004). However,
intraspecific variation among sites and years of such size-dependence has been scarcely
studied, especially in relation to environmental factors (Mndez & Karlsson, 2004 and
references therein). Our results support the idea that flowering probability in S. ciliata
substantially varied on a spatiotemporal scale that was influenced by environmental
conditions. A marked decrease in flowering probability occurred at its lower altitudinal limit
especially in 2003, a year with an extremely dry summer. Differences in the physiological
status of plants probably induced by drought stress may explain why the fraction of
flowering plants varies among sites and between years. Likewise, if flowering a given
year is determined by reaching a resource threshold, then larger plants will have a higher
chance of flowering. Moreover, smaller plants can increase reproductive success in future
years by remaining vegetative for one season.
Surprisingly, we did not find a direct causal relationship between plant size and
total flower number in 2003 at any altitude or at population L in 2002. Such a relationship
constitutes a general pattern in most plant species and habitats (Ollerton & Lack, 1998;
Mitchell, 1994) including the Mediterranean (Albert et al., 2001; Herrera, 1991). However,
plant size exerted a constant positive effect on duration which, in turn, had a significant
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99
positive effect on flower number and flowering synchrony. Thus, despite the lack of a
direct effect, as shown in our SEM diagrams, plant size indirectly controls flower
production: larger plants have a longer flowering period due to higher resource
investment and this corresponds to higher flower production.

Importance of the prefloration interval
Although flowering start in high mountains is mainly predicted by snowmelt date
(Inouye & McGuire, 1991; Inouye et al., 2002; Stinson, 2004; Kudo, 1991), variation in
flowering start has also been reported as a heritable trait (Zimmerman, 1988). However,
this variation can also be affected by other factors such as individual plant size (Ollerton
& Lack, 1998; Stinson, 2004). Our results show that plant size partially determines the
prefloration interval (i.e. flowering start) in two of the six scenarios presented (Fig. 1).
Nevertheless, the absence of a direct causal relationship between plant size and
prefloration interval in some models could simply be a consequence of the size-
dependence of flowering probability. Plant size probably loses its predictive power on
prefloration interval in those scenarios where smaller plants simply did not flower or had a
low flowering probability.
The strength of all six SEM models mainly rested on a sequence of causal
relationships linking prefloration interval, duration, flower number and fruit number. A
close link between prefloration interval and flower and fruit production has been found in
other alpine (Stinson, 2004) and Mediterranean-type species (Volis et al., 2004). This
suggests that a short prefloration interval could be an important phenotypic trait under
selection because some fitness components markedly increase in early flowering plants.
In a greenhouse experiment, Stanton et al. (2000) concluded that phenotypic selection
may follow a stress-avoidance strategy through earlier flowering in different stressful
environments. Ttland (1999) found no evidence of selection acting on flowering time in
the alpine Ranunculus acris subjected to experimental warming, but previous studies in
the same study area found higher plant fitness in early flowering plants, suggesting that
selection could differ between seasons depending on temperature conditions (Ttland,
1994, 1997).
In S. ciliata, a shorter prefloration interval also led to higher flowering synchrony,
but contrary to our expectations, synchrony did not contribute to improving fruit set but
produced a weak negative effect in population H in 2003 (Figure 1) and in brood size
(Table 5d). The low predictive power of synchrony over fitness components has been
pointed out by many authors (see a review in Gmez, 1993), who suggest that
phenotypic selection does not operate over flowering synchrony. Our results also indicate
that fruit set was negatively influenced by flower number in the two best scenarios

100
(populations M and H in 2002, Fig. 1). Resource limitation, rather than low pollinator
availability, may explain this finding. When a plant produces more flowers than it can
support, flower abortion takes place even under optimal conditions (Volis et al., 2004).
Nevertheless, in spite of occasional higher fruit set and brood size values at population L,
total yield at the population level was significantly higher for population H because more
individuals flowered (Fig 5e). This fact also has important genetic consequences since
seed production of higher populations comes from more individuals, reinforcing the
genetic variability of offspring.

CONCLUSIONS

Our results showed that Silene ciliata presents high reproductive plasticity at its
southern marginal limit, varying its success and fitness cost along altitude and between
years. At the lowest altitudinal limit, the species showed a comparatively marked
reproductive failure especially in the extremely hot and dry year, whereas the
intermediate and high altitudinal limits tended to provide more stable reproduction over
time. Such differences are mainly mediated by the percentage of plants that flower at
each population each year.
As shown by Peuelas et al. (2005), experimental warming in water-stressed sites
does not increase flowering and plant productivity probably because such warming
increases water loss. Mediterranean summer conditions also decrease photosynthetic
rates and nutrient availability (Llorens et al., 2004). Thus, the lower S. ciliata population
suffered the most restrictive conditions during the extreme event, despite a lengthening of
the growing season.
It has been shown that late-flowering alpine and subarctic plants may enhance
their reproductive performance in warming scenarios (Molau, 1993; Alatalo & Ttland,
1997, Molau et al., 2005). However, our results suggest that in high Mediterranean
mountains summer droughts imposed by warming trends condition plant reproductive
success, especially in lower altitudinal limits. As Stinson (2004) pointed out, the extent to
which alpine plants can endure climate change may depend on their potential for adaptive
plasticity in flowering phenology under new environmental conditions. Although flowering
earlier improves plant fitness, the flowering start in S. ciliata seems to be
photoperiodically constrained toward the end of the season, preventing efficient use of
the limited water supply. Flexibility to exploit selective pressure towards early flowering in
evolutive terms is limited, especially when faced with rapid warming scenarios and
frequent extreme heat events. Thus, time scale may not be long enough to allow for local
adaptation and/or actual range shifts (Etterson & Shaw, 2001; Jump & Peuelas, 2005).
CAPITULO 3

101
Our findings highlight the existence of different response patterns to climate
warming around the globe (Peuelas et al., 2004), and the value of studies on extreme
events along altitudinal gradients to determine plant performance plasticity and capacity
to face global warming conditions (Guschick & BassiriRad, 2003; Dunne et al. 2004).

ACKNOWLEDGEMENTS

The authors thank the staff of Parque Natural de las Cumbres, Circo y Lagunas de
Pealara, for permission to work in the area; M. Gmez-Espasandn and S. Gonzlez, for
help with seed counting; and R. Garca, C. Fernndez, Dr. A.L. Luzuriaga and Dr. M.J.
Albert for help with the field work, often under less

than ideal conditions. They also thank
Dr. D. Palacios for providing the digital images for snowmelt date estimation; Dr. M.J.
Albert and two anonymous referees for helpful comments on earlier drafts of the

manuscript; and L. De Hond for her linguistic assistance. This work was supported by the
Spanish Government CICYT project REN2003-03366.

102
REFERENCES

Agust-Panareda A, Thompson R. 2002. Reconstructing air temperature at eleven remote
alpine and arctic lakes in Europe from 1781 to 1997 AD. Journal of Paleolimnology
28: 7-23.
Alatalo JM, Ttland O. 1997. Response to simulated climatic change in an alpine and
subarctic pollen-risk strategist, Silene acaulis. Global Change Biology 3: 74-79.
Albert MJ, Escudero A, Iriondo JM. 2001. Female reproductive success of narrow
endemic Erodium paularense in contrasting microhabitats. Ecology 82: 1734-1747.
Arft AM, Walker MD, Gurevitch J et al. 1999. Response patterns of tundra plant species
to experimental warming, a meta-analysis of the International Tundra Experiment.
Ecological Monographs 69: 491511.
Augspurger CK. 1981. Reproductive synchrony of a tropical shrub, experimental studies
on effects of pollinators and seed predators on Hybantus prunifolius (Violaceae).
Ecology 63: 775-788.
Bentler PM. 1989. EQS structural equations program manual. California, USA: BMDP
Statistical Software.
Bentler PM, Bonet DG. 1980. Significance tests and goodness of fit in the analysis of
covariance structures. Psychometrika 45: 289-308.
Billings WD, Mooney HA. 1968. The ecology of arctic and alpine plants. Biological
Reviews 43: 481-529.
Bollen KA. 1989. Structural equations with latent variables. New York, USA: Wiley.
Castro J, Zamora R, Hdar JA. 2002. Mechanisms blocking Pinus sylvestris colonization
of Mediterranean mountain meadows. Journal of Vegetation Science 13: 725-731.
Castro J, Zamora R, Hdar JA, Gmez JM. 2004. Seedling establishment of a boreal tree
species (Pinus sylvestris) at its southernmost distribution limit, consequences of
being in a marginal, Mediterranean habitat. Journal of Ecology 92: 266-277.
Conolly AP, Dahl E. 1970. Maximum summer temperature in relation to the modern and
quaternary distributions of certain arctic-montane species in the British Isles. In:
Walker D, West RG, eds. Studies in the Vegetational History of the British Isles.
Cambridge, UK: Cambridge University Press, 159223.
Dullinger S, Dirnbock T, Grabherr G. 2003. Patterns of shrub invasion into high mountain
grasslands of the Northern Calcareous Alps, Austria. Arctic, Antarctic and Alpine
Research 35: 434-441.
Dunne JA, Saleska SR, Fischer ML, Harte J. 2004. Integrating experimental and gradient
methods in ecological climate change research. Ecology 85: 904-916.
CAPITULO 3

103
Dye DG. 2002. Variability and trends in the annual snow cover cycle in Northern
Hemisphere land areas, 1972-2000. Hydrological Processes 16: 3065-3077.
Easterling DR, Meehl GA, Parmesan C, Changnon SA, Karl TR, Mearns LO. 2000.
Climate Extremes: Observations, Modeling, and Impacts. Science 289: 2068.
Escudero A, Gimnez-Benavides L, Iriondo JM, Rubio A. 2005. Patch dynamics and
islands of fertility in a high mountain Mediterranean community. Arctic, Antarctic and
Alpine Research 36: 518-527.
Etterson JR, Shaw RG. 2001. Constraint to adaptive evolution in response to global
warming. Science 294: 151-154.
Finch JF, West SG, MacKinnon DP. 1997. Effects of sample size and nonnormality on the
estimation of mediated effects in latent variable models. Structural Equation
Modeling 4: 87107.
Freeman RS, Brody AK, Neefus CD. 2003. Flowering phenology and compensation for
herbivory in Ipomopsis aggregata. Oecologia 136: 394-401.
Gaines SD, Denny MW. 1993. The Largest, Smallest, Highest, Lowest, Longest, and
Shortest: Extremes in Ecology. Ecology 74: 1677-1692.
Galen C, Stanton ML. 1995. Responses of snowbed plant species to changes in growing
season length. Ecology 76: 1546-1557.
Galen C. 1990. Limits to the distribution of alpine tundra plants, herbivores and the
skypilot, Polemonium viscosum. Oikos 59: 355-358.
Garca D, Zamora R, Hdar JA, Gmez JM. 1999. Age structure of Juniperus communis
L. in the Iberian peninsula: Conservation of remnant populations in Mediterranean
mountains. Biological Conservation 87: 215-220.
Gaviln RG, Snchez-Mata D, Escudero A, Rubio A. 2002. Spatial structure and
interactions in Mediterranean high mountain vegetation (Sistema Central, Spain).
Israel Journal of Plant Sciences 50: 217-228.
Gimnez E, Melendo M, Valle F, Gmez-Mercado F, Cano E. 2004. Endemic flora
biodiversity in the south of the Iberian Peninsula, altitudinal distribution, life forms
and dispersal modes. Biodiversity and Conservation 13: 2641-2660.
Gimnez-Benavides L, Escudero A, Prez-Garca F. 2005. Seed germination of high
mountain Mediterranean species, altitudinal, interpopulation and interannual
variability. Ecological Research 20: 433-444.
Gmez JM. 1993. Phenotypic selection on flowering synchrony in a high mountain plant,
Hormathophylla spinosa (Cruciferae). Journal of Ecology 81: 605-613.
Grabherr G, Gottfried M, Pauli H. 1994. Climate effects on mountain plants. Nature 369:
448.

104
Granados I, Toro M. 2000. Recent warming in a high mountain lake (Laguna Cimera,
Central Spain) inferred by means of fossil chironomids. Journal of Limnology 59:
109-119.
Groisman PY, Karl TR, Knight RW, Stenchikov GL. 1994. Changes of snow cover,
temperature, and radiative heat balance over the Northern Hemisphere. Journal of
Climate 7: 1633-1656.
Gutschick VP, BassiriRad H. 2003. Extreme events as shaping physiology, ecology, and
evolution of plants: toward a unified definition and evaluation of their consequences.
New Phytologist 160: 21-42.
Hampe A. 2005. Fecundity limits in Frangula alnus (Rhamnaceae) relict populations at
the species' southern range margin. Oecologia 143: 377-386.
Hampe A, Petit RJ. 2005. Conserving biodiversity under climate change, the rear edge
matters. Ecology Letters 8: 461-467.
Hatcher L. 1994. A step-by-step approach to using SAS for factor analysis and structural
equation modelling. North Carolina, USA: SAS Institute Inc.
Herrera CM. 1991. Dissecting factors responsible for individual variation in plant
fecundity. Ecology 72: 1436-1448.
Inouye DW, McGuire AD. 1991. Effects of snowpack on the timing and abundance of
flowering in Delphinium nelsonii, implications for climate change. American Journal
of Botany 78: 997-1001.
Inouye DW, Morales MA, Dodge GJ. 2002. Variation in timing and abundance of
flowering by Delphinium barbeyi Huth (Ranunculaceae), the roles of snowpack,
frost, and La Nia, in the context of climate change. Oecologia 130: 543-550.
IPCC 2001. Climate Change 2001: The Scientific Basis. Contribution of Working Group I
to the Third Assessment Report of the Intergovernmental Panel on Climate Change.
Cambridge,UK: Cambridge University Press.
Iriondo JM, Albert MJ, Escudero A. 2003. Structural equation modelling: an alternative for
assessing causal relationships in threatened plant populations. Biological
Conservation 113: 367-377.
Jump AS, Peuelas J. 2005. Running to stand still: adaptation and the response of plants
to rapid climate change. Ecology Letters 8: 1010-1020.
Keller F, Krner C. 2003. The role of photoperiodism in alpine plant development. Arctic,
Antarctic and Alpine Research 35: 361-368.
Klanderund K. 2004. Climate change effects on species interactions in an alpine plant
community. Journal of Ecology 93: 127-137.
Krner C. 1999. Alpine Plant Life. Berlin, Germany: Springer-Verlag.
CAPITULO 3

105
Kudo G. 1991. Effects of snow-free period on the phenology of alpine plants inhabiting
snow patches. Arctic and Alpine Research 23: 436-443.
Kudo G. 2003. Warming effects on growth, production, and vegetation structure of alpine
shrubs, a five-year experiment in northern Japan. Oecologia 135: 280-287.
Kudo G, Hirao A. 2006. Habitat-specific responses in the flowering phenology and seed
set of alpine plants to climate variation: implications for global-change impacts.
Population Ecology 48: 49-58.
Lacey EP. 1986. Onset of reproduction in plants. Size versus age-dependency. Trends in
Ecology and Evolution 1: 7276.
Lawton JH. 1993. Range, population abundance and conservation. Trends in Ecology
and Evolution 8: 409-413.
Lesica P, McCune B. 2004. Decline of arctic-alpine plants at the southern margin of their
range following a decade of climatic warming. Journal of Vegetation Science 15:
679-690.
Littell RC, Milliken GA, Stroup WW, Wolfinger RD. 1996. SAS System for Mixed Models.
North Carolina, USA: SAS Institute Inc.
Llorens L, Peuelas J, Beier C, Emmett B, Estiarte M, Tietema A. 2004. Effects of an
experimental increase of temperature and drought on the photosynthetic
performance of two ericaceous shrub species along a north-south European
gradient. Ecosystems 7: 613-624.
Loehlin JC. 1992. Latent Variable Models. An Introduction to Factor, Path, and Structural
Analysis. New Jersey, USA: Lawrence Erlbaum.
McCarthy JP. 2001. Ecological consequences of recent climate change. Conservation
Biology 15: 320-331.
Mndez M, Karlsson PS. 2004. Between-population variation in size-dependent
reproduction and reproductive allocation in Pinguicula vulgaris (Lentibulariaceae)
and its environmental correlates. Oikos 104: 5970.
Mitchell RJ. 1993. Path analysis: pollination. In: Scheiner SM, Gurevitch J, eds. Design
and analysis of ecological experiments. New York, USA: Chapman and Hall, 211
231.
Mitchell RJ. 1994. Effects of floral traits, pollinator visitation and plant size on Ipomopsis
aggregata fruit production. American Naturalist 143: 870889.
Molau U. 1993. Relationships between flowering phenology and life-history strategies in
tundra plants. Arctic and Alpine Research 25, 391-402.
Molau U, Nordenhall U, Eriksen B. 2005. Onset of flowering and climate variability in an
alpine landscape: A 10-year study from Swedish Lapland. American Journal of
Botany 92: 422-431.

106
Ollerton J, Lack AJ. 1998. Relationships between flowering phenology, plant size and
reproductive success in Lotus corniculatus (Fabaceae). Plant Ecology 139: 35-47.
Palacios D, de Andrs N, Luengo E. 2003. Distribution and effectiveness of nivation in
Mediterranean mountain, Pealara (Spain). Geomorphology 54: 157-178.
Parmesan C, Ryrholm N, Stefanescu C, Hill JK, Thomas CD, Descimon H, Huntley B,
Kaila L, Kullberg J, Tammaru T, Tennent WJ, Thomas JA, Warren M. 1999.
Poleward shifts in geographical ranges of butterfly species associated with regional
warming. Nature 399: 579.
Pauli H, Gottfried M, Dirnbck T, Dullinger S, Grabherr G. 2003. Assesing the long-term
dynamics of endemic plants at summit habitats. In: Nagy L, Grabherr G, Krner C,
Thompson DBA, eds. Alpine Biodiversity in Europe. Berlin, Germany: Springer-
Verlag, 95-207.
Peuelas J, Boada M. 2003. A global change-induced biome shift in the Montseny
mountains (NE Spain). Global Change Biology 9: 131-140.
Peuelas J, Filella I, Zhang X, Llorens L, Ogaya R, Lloret F, Comas P, Estiarte M,
Terradas J. 2004. Complex spatiotemporal phenological shifts as a response to
rainfall changes. New Phytologist 161: 837-846.
Peuelas J, Filella Y, Comas P. 2002. Change plant and animal life cycles from 1952 to
2000 in the Mediteranean Region. Global Change Biology 8: 531-544.
Peuelas J, Gordon C, Llorens L, Nielsen T, Tietema A, Beier C, Bruna P, Emmett B,
Estiarte M, Gorissen A. 2004. Nonintrusive field experiments show different plant
responses to warming and drought among sites, seasons, and species in a North-
South european gradient. Ecosystems 7: 598-612.
Petanidou T, Ellis WN, Margaris NS, Vokou D. 1995. Constraints of flowering phenology
in a phryganic (East Mediterranean shrub) community. American Journal of Botany
82: 607-620
Petraitis PS, Dunham AE, Niewiarowski PH. 1996. Inferring multiple causality: the
limitations of path analysis. Functional Ecology 10: 421-431.
Pigott CD, Pigott S. 1993. Water as a determinant of the distribution of trees at the
boundary of the Mediterranean zone. Journal of Ecology 81, 557566.
Price MV, Waser NM. 1998. Effects of experimental warming on plant reproductive
phenology an a subalpine meadow. Ecology 79: 1261-1271.
Reusch TBH, Ehlers A, Hammerli A, Worm B. 2005. Ecosystem recovery after climatic
extremes enhanced by genotypic diversity. Proceecings of the National Academy of
Science 102: 2826-2831.
Royce EB, Barbour MG. 2001. Mediterranean climate effects I. Conifer water use across
a Sierra Nevada ecotone. American Journal of Botany 88: 911-918.
CAPITULO 3

107
Royce EB, Barbour MG. 2001b. Mediterranean climate effects II. Conifer growth
phenology across a Sierra Nevada ecotone. American Journal of Botany 88: 919-
932.
Sanz-Elorza M, Dana ED, Gonzlez A, Sobrino E. 2003. Changes in the high-mountain
vegetation of the Central Iberian Peninsula as a probable sign of climate warming.
Annals of Botany 92: 273-280.
Schr C, Vidale PL, Luthi D, Frei C, Haberli C, Liniger MA, Appenzeller C. 2004. The role
of increasing temperature variability in European summer heatwaves. Nature 427:
332-336.
Shipley B. 1999. Cause and Correlation in Biology, A Users Guide to Path Analysis,
Structural Equations and Causal Inference. Cambridge, UK: Cambrigde University
Press.
Starr GRE, Oberbauer SF, Pop W. 2000. Effects of lengthened growing season and soil
warming on the phenology and physiology of Polygonum bistorta. Global Change
Biology 6: 357-369.
Thompson JD. 1995. Plant Evolution in the Mediterranean. Oxford, UK: Oxford University
Press.
Thuiller W, Lavorel S, Arajo MB, Sykes MT, Prentice C. 2005. Climate change threats to
plant diversity in Europe. Proceedings of the National Academy of Sciences of the
United States of America 102: 82458250.
Ttland O. 1994. Intraseasonal variation in pollination intensity and seed set in an alpine
population of Ranunculus acris in southwestern Norway. Ecography 17: 159-165.
Ttland O. 1997. Limitations on reproduction in alpine Ranunculus acris. Canadian
Journal of Botany 75: 137-144.
Ttland O. 1999. Effects of temperature on performance and phenotypic selection on
plant traits in alpine Ranunculus acris. Oecologia 120: 242-252.
Ttland O. 2001. Environment-dependent pollen limitation and selection on floral traits in
Tutin TG, Heywood VH, Burges NA, Valentine DH, Walters SM, Webb DA. 1995. Flora
Europaea Vol 1-5. Electronic dataset supplied by Pankhurst RJ, Royal Botanic
Garden of Edinburgh.
Venables WN, Ripley BD. 1998. Modern applied statistics with S-Plus. New York, USA:
Springer-Verlag.

108
Volis S, Verhoeven KJF, Mendlinger S, Ward D. 2004. Phenotypic selection and
regulation of reproduction in different environments in wild barley. Journal of
Evolutionary Biology 17: 11211131.
Walker MD, Webber PJ. 1994. Effects of interannual climate variation on aboveground
phytomass in alpine vegetation. Ecology 75: 393-408.
Wilson RJ, Gutirrez D, Gutirrez J, Martnez D, Agudo R, Montserrat V. 2005. Changes
to the elevational limits and extent of species ranges associated with climate
change. Ecology Letters 8: 1138-1146.
Zimmerman M. 1988. Nectar production, flowering phenology, and strategies for
pollination. In: Lovett Doust JL, Lovett Doust L, eds. Plant Reproductive Ecology.
Patterns and Strategies. Oxford, UK: Oxford University Press, pp 157178.
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Table 1. Summary of statistical techniques used in the study to cover the sequence of
reproductive events.

Reproductive
process
Flowering probability Fruit production Seed production
Statistical tool
GLMMs (binomial,
logit)
SEM (Maximum
Likelihood)
GLMMs (gamma,
log)
Predictors Plant size
Altitude
Plot (nested in
altitude)

Plant size
Duration
Prefloration interval
Synchrony
Flower number
Fruit set
Fruit number
Microsite suitability*
Soil cover
Intraspecific
competition
Interspecific
competition
Plant size
Altitude
Fruit number
Brood size
Soil cover
Intraspecific
competition
Interspecific
competition
Plot (nested in
altitude)

GLMMs, Generalized Linear Mixed Modelling; SEM, Structural Equation Modelling; *
denote a latent variable in SEM. Parentheses after the GLMMs indicate the error
distribution function of the response variable and its corresponding link function
respectively. Parentheses after SEM indicate the estimation method to calculate
standardized path coefficients (for a detailed description see text).

110

Table 2. Reproductive output, phenological traits and microsite conditions of Silene ciliata
in Sierra de Guadarrama (Madrid).

2002 2003
Variable
Low
(n=166)
Intermediate
(n=165)
High
(n=171)

Low
(n=166)
Intermediate
(n=165)
High
(n=171)
Flowering plants
108
(65.0%)
126 (75.7%) 146 (85.8%) 63 (37.9%) 120 (72.7%) 148 (86.5%)
Plant size (cm
2
)
38.4
30.4
33.0 32.3 28.0 23.0 38.4 30.4 33.0 32.3 28.0 23.0
Flowering plant
size (cm
2
)
42.5
31.1
37.2 35.2 30.2 24.0 47.0 33.9 37.6 35.3 29.9 23.9
Flower number
4.22
6.14
4.25 4.87 11.72 12.93 1.82 3.65 4.08 4.99 8.65 9.67
Fruit number
2.56
3.84
1.02 1.70 3.55 4.55 1.19 2.38 1.87 2.39 3.24 3.52
Seed number
127.54
131.97
56.16 62.94
138.35
143.00

90.87
106.89
87.27 72.91
119.46
93.42
Fruit set
0.37
0.35
0.24 0.32 0.30 0.31 0.24 0.34 0.36 0.36 0.40 0.35
Brood size
0.32
0.22
0.22 0.21 0.19 0.17 0.27 0.20 0.25 0.21 0.23 0.17
First flowering
date (day)
207.83
12.47
215.73 12.41 218.09 11.39
191.14
8.99
195.53 10.83
196.32
11.40
Prefloration
interval (day)
75.8
12.5
75. 3 12.4 71.1 11.4 86.5 9.7 84.4 9.1 76.7 8.1
Moment (day)
34.53
10.76
41.56 14.45 48.44 13.70
14.30
9.07
16.98 12.90 16.34 14.89
Duration (day)
10.40
10.45
13.37 13.61 19.02 15.00 7.42 9.11 9.31 9.73 11.18 9.70
Synchrony
0.26
0.10
0.25 0.11 0.29 0.10 0.21 0.10 0.29 0.11 0.32 0.10
Intraspecific
competition (%)*
3.8 4.1 2.2 2.9 1.6 2.6 3.5 4.2 2.2 3.0 1.5 2.5
Interspecific
competition (%)*
57.1
18.2
46.6 21.1 41.8 17.0
55.8
17.3
46.9 20.5 41.7 16.9
Bare soil cover
(%)*
20.4
18.1
12.4 10.2 13.6 9.9
21.5
18.2
12.1 10.0 14.0 9.9
Snowmelt date 13-May 21-May 28-May 19-Apr 28-Apr 9-May

Values are expressed as mean SD except flowering plants, expressed in number
(percentage of total) and snowmelt date, expressed in date. *Indicate variables used to
construct the latent variable microsite suitability for SEM models.
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Table 3. Generalized Linear Mixed Model for flowering probability of Silene ciliata in 2002
and 2003.

Solution for Fixed Effects Deviance change
Effect Coefficient S.D. D.f. t value P-value F-Value P-value
2002
alt L -1.4431 0.2995 6.61 -4.82 0.0023
alt I -0.6841 0.3041 7.19 -2.25 0.0583
alt H . . . . .
altitude 11.88 0.0099
plant size 0.0274 0.0062 349 4.41 <.0001 19.45 <.0001
2003
alt L -3.3861 0.9092 4.92 -3.72 0.0141
alt I -1.6005 0.9056 4.84 -1.77 0.1393
alt H . . . . .
altitude 6.99 0.0412
plant size 0.0260 0.0051 496 5.04 <.0001 25.39 <.0001

Altitude was added as a categorical variable (2 d.f.). The random variable plot,
nested in altitude, was non-significant for both models (0.0087 0.0726, Z-value
= 0.12, Prob. Z = 0.4519 in 2002; 1.0272 0.7769, Z-value = 1.32, Prob. Z =
0.0931 in 2003).

112

Table 4. Generalized Linear Mixed Model for seed number per plant of Silene ciliata in
2002 and 2003.

2002
alt L -0.3418 0.1241 9.23 -2.75 0.0218
alt I -0.4193 0.1080 5.64 -3.88 0.0092
alt H . . . .
fruit number 0.1828 0.0100 195 18.20 <.0001 331.29 <.0001
brood size 2.6509 0.2106 182 12.59 <.0001 158.50 <.0001
soil cover -0.0010 0.0039 154 0.27 0.7906 0.07 0.7906
intrasp. compet. -0.0171 0.0139 184 -1.22 0.2222 1.50 0.2222
intersp. compet. 0.0034 0.0027 68.6 1.23 0.2232 1.51 0.2232
2003
alt L -0.4354 0.1478 12.3 -2.95 0.0120
alt I -0.1243 0.1138 4.61 -1.09 0.3281
alt H . . . .
fruit number 0.2090 0.0102 189 20.46 <.0001 418.53 <.0001
brood size 2.3301 0.1785 191 13.06 <.0001 170.45 <.0001
soil cover 0.0029 0.0035 178 0.84 0.4021 0.71 0.4021
intrasp. compet. 0.0156 0.0117 190 1.34 0.1822 1.79 0.1822
intersp. compet. 0.0055 0.0023 114 2.41 0.0176 5.80 0.0176

Altitude was added as a categorical variable (2 d.f.). The random variable plot, nested in
altitude, was non-significant for both models (0.0004 0.0103, Z-value = 0.04, Prob. Z =
0.4835 in 2002; 0.0119 0.0127, Z-value = 0.94, Prob. Z = 0.1740 in 2003).
CAPITULO 3

113

Table 5. Generalized Linear Mixed Model for brood size of S. ciliata in 2002 and 2003.

2002
alt L 0.6767 0.2530 11 2.67 0.0216
alt I 0.0991 0.2355 8.41 0.42 0.6845
alt H 0 . . . .
plant size 0.0055 0.0026 192 2.07 0.0401 4.27 0.0401
fruit number 0.05301 0.0183 199 2.90 0.0042 8.40 0.0042
synchrony -5.0626 0.8533 192 -5.93 <0.0001 35.20 <0.0001
soil cover -0.0027 0.0071 196 -0.38 0.7041 0.14 0.7041
intrasp. compet. -5.3116 10.4291 189 -0.51 0.6111 0.26 0.6111
intersp. compet. -0.0073 0.0051 149 -1.41 0.1600 1.99 0.1600
2003
alt L 0.2199 0.2786 7.28 1.35 0.2172
alt I 0.2250 0.1644 2.94 0.98 0.3991
alt H 0 . . .
plant size 0.0094 0.00267 172 3.52 0.0006 12.38 0.0006
fruit number 0.0469 0.0227 190 2.07 0.0402 4.27 0.0402
synchrony -3.0557 0.6535 190 -4.68 <0.0001 21.86 <0.0001
soil cover -0.0146 0.0083 185 -1.75 0.0812 3.07 0.0812
intrasp. compet. -16.5975 9.8127 134 -1.69 0.0931 2.86 0.0931
intersp. compet. 0.00478 0.00501 157 0.94 0.3493 0.89 0.3493

Altitude was added as a categorical variable (2 d.f.). The random variable plot, nested in
altitude, was non-significant for both models (0.0239 0.0399, Z-value = 0.60, Prob. Z =
0.2744 in 2002; 0.0049 0.0331, Z-value = 0.15, Prob. Z = 0.4414 in 2003).

114
High 2003
NNFI: 0.79
CFI: 0.88
P
2
: 0.014
N: 143
Size Fruits
Duration
Synchrony
Prefloration
interval
Fruitset
Flowers
0.36***
-0.49***
-0.45***
-0.20*
0.45***
0.62***
0.57***
0.80
0.38
0.61
0.95
0.85
0.80
0.20*
-0.94*
0.99
Intraspecific
competition
Interspecific
competition
Soil cover
1.00
Microsite
suitability
0.99
High 2003
NNFI: 0.79
CFI: 0.88
P
2
: 0.014
N: 143
Size Fruits
Duration
Synchrony
Prefloration
interval
Fruitset
Flowers
0.36***
-0.49***
-0.45***
-0.20*
0.45***
0.62***
0.57***
0.80
0.38
0.61
0.95
0.85
0.80
0.20*
-0.94*
0.99
Intraspecific
competition
Interspecific
competition
Soil cover
1.00
Microsite
suitability
0.99
High 2002
NNFI: 0.92
CFI: 0.96
P
2
: 0.144
N: 146
Size Fruits
Duration
Synchrony
Prefloration
interval
Fruitset
Flowers
0.40*
-0.31***
0.67*** -0.30**
0.61***
0.78***
0.41***
0.63
0.21***
0.79
0.54
0.98
0.55
0.84
-0.26**
-0.62*
0.99
Intraspecific
competition
Interspecific
competition
Soil cover
1.00
Microsite
suitability
-0.17*
0.98
High 2002
NNFI: 0.92
CFI: 0.96
P
2
: 0.144
N: 146
Size Fruits
Duration
Synchrony
Prefloration
interval
Fruitset
Flowers
0.40*
-0.31***
0.67*** -0.30**
0.61***
0.78***
0.41***
0.63
0.21***
0.79
0.54
0.98
0.55
0.84
-0.26**
-0.62*
0.99
Intraspecific
competition
Interspecific
competition
Soil cover
1.00
Microsite
suitability
-0.17*
0.98
Intermediate 2002
NNFI: 0.96
CFI: 0.98
P
2
: 0.305
N: 126
Size Fruits
Duration
Synchrony
Prefloration
interval
Fruitset
Flowers
Soil cover
0.21*
-0.78***
-0.26**
0.43***
0.67***
0.59***
0.73
-0.37***
0.28***
0.88
0.60
1.00
0.97
0.64
0.93
-0.23**
-0.19**
-0.46*
0.99
Intraspecific
competition
Interspecific
competition
Microsite
suitability
-0.21*
0.97
Intermediate 2002
NNFI: 0.96
CFI: 0.98
P
2
: 0.305
N: 126
Size Fruits
Duration
Synchrony
Prefloration
interval
Fruitset
Flowers
Soil cover
0.21*
-0.78***
-0.26**
0.43***
0.67***
0.59***
0.73
-0.37***
0.28***
0.88
0.60
1.00
0.97
0.64
0.93
-0.23**
-0.19**
-0.46*
0.99
Intraspecific
competition
Interspecific
competition
Microsite
suitability
-0.21*
0.97
Intermediate 2003
NNFI: 1.04
CFI: 1.00
P
2
: 0.607
N: 115
Size Fruits
Duration
Synchrony
Prefloration
interval
Fruitset
Flowers
-0.47**
0.66***
0.60***
0.70***
0.74
-0.11***
0.99
0.50
0.99
0.91
0.86
-0.19***
0.99
Intraspecific
competition
Interspecific
competition
Soil cover
1.00
0.16*
Microsite
suitability
0.99
0.45***
Intermediate 2003
NNFI: 1.04
CFI: 1.00
P
2
: 0.607
N: 115
Size Fruits
Duration
Synchrony
Prefloration
interval
Fruitset
Flowers
-0.47**
0.66***
0.60***
0.70***
0.74
-0.11***
0.99
0.50
0.99
0.91
0.86
-0.19***
0.99
Intraspecific
competition
Interspecific
competition
Soil cover
1.00
0.16*
Microsite
suitability
0.99
0.45***
Low 2002
NNFI: 0.91
CFI: 0.95
P
2
: 0.147
N: 108
Size Fruits
Duration
Synchrony
Prefloration
interval
Fruitset
Flowers
Microsite
suitability
Intraspecific
competition
Interspecific
competition
0.26**
0.26*
0.56*** -0.58***
0.61***
0.82***
0.44***
0.72
-0.12***
0.40
0.96
-0.27***
0.99
0.89
0.77
1.00
0.99
Soil cover
0.99
Low 2002
NNFI: 0.91
CFI: 0.95
P
2
: 0.147
N: 108
Size Fruits
Duration
Synchrony
Prefloration
interval
Fruitset
Flowers
Microsite
suitability
Intraspecific
competition
Interspecific
competition
0.26**
0.26*
0.56*** -0.58***
0.61***
0.82***
0.44***
0.72
-0.12***
0.40
0.96
-0.27***
0.99
0.89
0.77
1.00
0.99
Soil cover
0.99
Low 2003
NNFI: 0.90
CFI: 0.94
P
2
: 0.278
N: 63
Size Fruits
Duration
Synchrony
Prefloration
interval
Fruitset
Flowers
0.36**
-0.59***
0.70***
0.61***
0.53***
0.63
-0.26***
0.53
0.92
-0.39***
0.99
0.92
0.73
0.99
Intraspecific
competition
Interspecific
competition
Soil cover
1.00
Microsite
suitability
0.99
0.46**
Low 2003
NNFI: 0.90
CFI: 0.94
P
2
: 0.278
N: 63
Size Fruits
Duration
Synchrony
Prefloration
interval
Fruitset
Flowers
0.36**
-0.59***
0.70***
0.61***
0.53***
0.63
-0.26***
0.53
0.92
-0.39***
0.99
0.92
0.73
0.99
Intraspecific
competition
Interspecific
competition
Soil cover
1.00
Microsite
suitability
0.99
0.46**
Full model
Size Fruits
Duration
Synchrony
Prefloration
interval
Fruitset
Flowers
Microsite
suitability
Intraspec.
competition
Interspec.
competition
Soil cover
Full model
Size Fruits
Duration
Synchrony
Prefloration
interval
Fruitset
Flowers
Microsite
suitability
Intraspec.
competition
Interspec.
competition
Soil cover
Figure 1. Path diagram representing hypothesized causal relationships
among phenotypic traits, microsite characteristics and plant fitness at
contrasting altitudes for two years. Positive effects are indicated by solid
lines and negative effects by broken lines. The effects of unexplained
causes are expressed by external arrows to each dependent variable.
Arrow widths are proportional to adjacent standardized path coefficients.
Path coefficients non-significantly different from zero are omitted. Fit
statistics (NNFI, CFI and P
2
) and sample size (N) are given on the
upper-left corner of each path. Microsite suitability is a latent variable.

CAPITULO 3

115

Figure 2. Linear trends in climatic variables over the period 1960-1994 at Puerto de
Navacerrada weather station (1860 m). Mean total precipitation and mean temperature
over (a) winter season (October-March) and (b) growing season (April-September). (c)
Mean days of snow cover during snowmelt (April-June). Last two values on each plot
(open circles) correspond to the observed years (2002 and 2003).

116

Figure 3. Size-dependent flowering probability in S. ciliata. Curves were drawn according
to the intercept and slope parameters estimated by Generalized Linear Mixed Models as
described in the text.
CAPITULO 3

117

Figure 4. Altitudinal variation in flowering phenology among populations of Silene ciliata in
2002 and 2003. Horizontal black bars refer to the start, duration and finish dates of
flowering period per altitude and year. Dotted lines represent absolute values, solid lines
mean values; diamonds inside them mark the mean flowering moment. Black arrows
indicate snowmelt date. Grey bars represent mean flower number per plant.


118

Figure 5. Altitudinal differences in reproductive traits of S. ciliata in
2002 and 2003. All variables are mean values per plant except % of
flowering plants and seeds/cm
2
, which are expressed as mean
values per plot (n=3 per altitude). Error bars represent standard
deviations.
40
60
80
100
%

F
l
o
w
e
r
i
n
g

p
l
a
n
t
s
2002 2003
F
l
o
w
e
r

n
u
m
b
e
r
/
p
l
a
n
t

2,5
5,0
7,5
10,0
12,5
F
r
u
i
t

s
e
t
/
p
l
a
n
t
0,20
0,30
0,40
Low
Low Intermediate Intermediate High High
Altitude Altitude
S
e
e
d
s
/
c
m

Low Intermediate High
0,0
1,0
2,0
3,0
Low Intermediate High

2

Altitude Altitude
B
r
o
o
d

s
i
z
e
/
p
l
a
n
t
0,20
0,25
0,30
0,35
2002 2003
a)
b)
c)
d)
e)
CAPITULO 4

119

Captulo 4.

Limited importance of a nursery moth pollinator for the
reproductive success of its host plant. Towards an
unspecialized pollination strategy?

Gimnez-Benavides, L.; Dtterl, S.; Jrgens, A.;

Escudero, A. and Iriondo, J. M.
Inedit manuscript.

DAY VS. NIGHT POLLINATION IN S. CILIATA

120
CAPITULO 4

121
SUMMARY

The pollinating seed predation in Caryophyllaceae species by noctuid moths
(Hadena and Perizoma) have been extensively described in last decades. Some
evidences across multiple pairs of species support the idea that such systems constitute
relatively specialized interactions, shifting between parasitism and mutualism depending
on the presence of effective co-pollinators.
In this work, we describe one more example of Silene - Hadena specific
interaction, the Silene ciliata - Hadena consparcatoides system. We determined
qualitatively and quantitatively the flower scent composition of S. ciliata, comparing it with
other Caryophyllaceae species that show a similar nocturnal pollination syndrome.
Laboratory bioassays were performed to identify electrophysiologically active compounds
to the moth that were present in the flower scents of S. ciliata and S. latifolia. We
experimentally induced two contrasting pollination regimes at non-overlapping day-night
periods in a S. ciliata natural population. The subsequent effects of pollination regimes on
the plant reproductive success were assessed from flower to seedling stages.
Flower scent composition and bioassays showed that S. ciliata bore a marked
nocturnal scent, although important compounds found in other nocturnal Silene species
were absent. Bioassays indicated that some dominant compounds of both Silene species
elicited strong signals in the antennae of the moth. Diurnal flower visitors produced more
fruits per flower than nocturnal visitors, but the latter produced more seeds per fruit
resulting in a similar overall fecundity. However, seeds generated from daylight pollination
were heavier and germinated better. Our results showed that S. ciliata had a typical
nocturnal pollination syndrome, although similar reproductive success and higher
offspring vigor were reached under diurnal pollination regime. Therefore, absence of
pollination benefit by H. consparcatoides would not compromise the plant reproductive
success, while seed predation exerted by the nursery pollinator shifts the interaction
towards parasitism.


122
INTRODUCTION

The evolution and maintenance of specialized pollination systems constitute a
major paradigm in plant ecology. Assuming the fact that a plant species could receive
visits by a large number of animal species, Stebbins (1970) proposed the principle that a
plant species should evolve to maximize the visits of its most effective pollinator. Since
then, in the attempt to understand the selective forces that pollinators exert on plants to
develop specialized pollination syndromes, numerous studies have evaluated the effects
of different pollinator assemblages (e.g. diurnal vs. nocturnal) on plant reproductive
success (Morse and Fritz, 1983; Jennersten, 1988; Thompson and Pellmyr, 1992;
Groman and Pellmyr, 1999; Herrera, 2000; Young, 2002; Wolff et al., 2003). Results
derived from these surveys are contradictory, showing that specialized systems are not
always adapted to the most effective pollinator, and that selection may favour flower traits
that incorporate new pollinators without excluding the previous ones (Fenster et al.,
2004).
Among specialized plant-animal interactions, the nursery pollination systems (i.e.
insects that lay eggs and rear their larvae in some of the fruits they produce when
pollinating) constitute one of the most accepted examples of coevolution (Thompson and
Pellmyr, 1992). The interactions comprised by Silene plant species and Hadena moths
(and related genera) have recently been promoted as excellent systems to study the
evolution of nursery pollination (Kephart et al, 2006). Contrarily to other evolved nursery
pollination systems (e.g. Yucca-Tegeticula moths and Ficus-Agaonid wasps), the
examples provided by Silene-Hadena pairs of species reflect an apparent primitive
stage of the potential mutualism, eventually shifting to parasitism depending on the
presence of effective co-pollinators (Dufay and Anstett, 2003). For instance, Westerbergh
(2004) found that pollination vs. seed predation trade-off exerted by Perizoma affinitatum
only resulted positive to Silene dioica if co-pollinators acted on 60 % of flowers.
Numerous studies have been carried out on this concern in last years, improving our
knowledge of the conditions that favours selection for nursery moth pollination in
Caryophyllaceae (Jennertsen, 1988; Jennertsen and Morse, 1991; Shykoff and Bucheli,
1995; Young, 2002; Barthelmess et al., 2006). However, nearly all studies have been
developed in a few northern European and American Silene species, while Mediterranean
and Middle East species remain unexplored despite those areas represent the centre of
maximum diversity. Moreover, no survey to date have specifically explored the effects of
those pollination regimes on fitness components beyond parental fertility, despite that the
effective selection by a functional group of pollinators is only possible if it leads to
differential progeny viability (see Herrera, 2000).
CAPITULO 4

123
Among the flower adaptations, the composition and time of emission of flower
scents have been found to be closely related to pollination syndromes (Knudsen and
Tollsen, 2003 and 2004). In specialized interactions, flower odours are commonly used as
communication pathways between plants and animals (Dtterl and Jrgens, 2005; Dtterl
et al., 2006). Preferences of pollinators by specific floral odours can influence the plant
reproductive success, and therefore flower scent composition is susceptible to
evolutionary adaptation (Galen, 1985).
Here we report the results of several experiments and observational data of a new
emerging example of Silene-Hadena interaction located in the Iberian Peninsula: S. ciliata
Poiret and H. consparcatoides (Schawerda, 1928). Our purpose was to test whether S.
ciliata really obtains comparatively higher reproductive and progeny fitness under
nocturnal than under diurnal pollination as we may expect based on its flowering
syndrome. We specially focus on the nature of the interaction with H. consparcatoides, in
an attempt to evaluate whether current selective pressures imposed by the actual
ecological context reinforce or contrarily swamp the potential mutualistic interaction.
Thus, we analize (1) the flower scent composition of S. ciliata compared to other
nocturnal Caryophyllaceae species, (2) the electrophysiologically active compounds of
the S. ciliata and S. latifolia flower scents to the moth, (3) the effects of diurnal vs.
nocturnal pollinator species on components of female fertility and progeny fitness,; and
(4) the consumption of S. ciliata capsules by H. consparcatoides.

METHODS

Species and study area
Silene ciliata Poiret (Caryophyllaceae) is a long-lived perennial plant, inhabiting
main mountain ranges of northern Mediterranean basin countries (Tutin et al. 1995). The
species grows in cushion rosettes of up to 2 cm in height and 15 cm in diameter.
Flowering stems have 15 cm in height and bear 1 to 5 flowers. The species can be
considered ginomonoecious. Most of individuals produce perfect flowers, but a little
proportion produces pistillate flowers (containing a functional gynoecium but an atrophied
androecium) in addition to perfect ones. Hand-crossing experiments indicate that S.
ciliata is a self-compatible species, but passive autogamy is totally restricted by a
pronounced protandry (Gimnez-Benavides, unpublished).
Hadena consparcatoides (Schawerda, 1928) (Lepidoptera: Noctuidae) is a
univoltine moth species with a practically unknown distribution and ecology. It has been
cited in a few localities in mountain ranges of Central Spain, Cantabric Range and in the
French Pyrenees (Calle, 1982; Yela, 1991; Lasata et al., 2001; Moya, 2001) but no other

124
host plant has been previously cited for the species. The scarce phenological data
available reveal that the flight period of H. consparcatoides occurs from June to October
(Yela, 1991; this study). Female moths drink nectar and lay their eggs into the flowers
(Gimnez-Benavides, pers. obs.). Once the eggs hatch, larvae penetrate into the
capsules and eat the seeds. Each larva needs more than a single fruit to complete its
development, so when it eats all the seeds in one fruit it moves to another fruit. Larvae
hibernate as pupae in the ground.

Volatile collection
Floral scent was collected using dynamic head-space methods. A single
inflorescence of 2-3 newly opened flowers was enclosed within a polyethylene oven bag
(Toppits
) during 5 minutes and the emitted volatiles were trapped during 2-5 minutes in
an adsorbent tube through the use of a membrane pump (ASF Thomas, Inc.). The flow
rate was adjusted to about 200 ml/min using a 9V battery. ChromatoProbe quartz
microvials of Varian Inc. (length: 15 mm; inner diameter: 2 mm) were used after cutting
the closed end, and filling them with a mixture (1:1) of 3 mg Tenax-TA
(mesh 60-80) and

Carbotrap
(mesh 20-40), and used them as adsorbent tubes. The adsorbents were fixed
in the tubes using glass wool. In 2004, samples were collected during 5 minutes at night
(from 22.00 to 24.00 h, 10 samples) to assess composition. In 2005, samples were
collected during 2 minutes at night (from 22.00 to 24.00 h, 5 samples) and day (from
10.00 to 16.00 h, 4 samples) to assess the rythmicity of odour emission. Additionally,
three scent samples for GC-EADs bioassays were collected in a similar way, but 30-40
flowers per sample (10-15 inflorescences) were enclosed in a polyethylene oven bag and
volatiles were trapped in large vials (length: xx mm; inner diameter: xx mm) during 4-5
hours at night (from 22.00 to 5.00 h).

Chemical analysis
The samples were analysed on a Varian Saturn 2000 System using a 1079
injector that had been fitted with the ChromatoProbe kit. This kit allows the thermal
desorption of small amounts of solids or liquids contained in quartz microvials (Micro-
SPE, Amirav and Dagan, 1997), or in our case the thermal desorption of the trapped
volatiles. The adsorbent tube was loaded into the probe, which was then inserted into the
modified GC injector. The injector split vent was opened (1/20) and the injector heated to
40C to flush any air from the system. The split vent was closed after 2 minutes and the
injector was heated at 200C/min, then held at 200C for 4,2 min, after which the split
vent was opened (1/10) and the injector cooled down.
CAPITULO 4

125
A ZB-5 column (5% phenyl polysiloxane) was used for the analyses (60 m long,
inner diameter 0.25 mm, film thickness 0.25 m, Phenomenex). Electronic flow control
(EFC) was used to maintain a constant helium carrier gas flow of 1.8 ml min
-1
. The GC
oven temperature was held for 7 min at 40C, then increased at 6C per min until 250C
and then held for 1 min. The MS interface was 260C and the ion trap worked at 175C.
The mass spectra were taken at 70 eV (in EI mode) with a scanning speed of 1 scan
-1
from m/z 30 to 350. The GC-MS data were processed using the Saturn Software package
5.2.1. Component identification was carried out using the NIST 02 mass spectral data
base (NIST algorithm), or MassFinder 2.3, and confirmed by comparison of retention
times with published data (Adams, 1995; Davies, 1990). Identification of individual
components was confirmed by comparison of both mass spectrum and GC retention data
with those of authentic standards.

Electrophysiological analysis
Experiments were performed by means of a GCEAD/FID system and a GC
EAD/MS system with floral scent extracts of S. ciliata and S. latifolia, and with additional
standard compounds. Standard compounds were chosen from the scent of the species.
Antennae from three females and three males (one antenna per specimen) of H.
consparcatoides were tested. In addition, electroantennographic (EAG) experiments were
performed with dilution series of standard compounds (Schtz et al., 1999) to compare
the sensitivity of H. consparcatoides to different compounds, and to obtain dose
response curves for the compounds tested. Antennae from two to four moths were used
in this experiment for each compound to obtain mean response values.

GCEAD/FID system
The GCEAD/FID system consisted of a gas chromatograph (Vega 6300-01,
Carlo Erba, Rodano, Italy), an EAD interface (Syntech, Hilversum, the Netherlands) and
an EAG-amplifier (Professor U.T. Koch, University of Kaiserslautern). The GC was
equipped with a capillary column (length 30 m, inner diameter 0.32 mm, DB-1), an FID
and a split-splitless injector. The injector was operated in the splitless mode. Nitrogen
was used as carrier gas at a pressure of 100 kPa (gas flow 2.5 ml min
1
, gas vector 50
cm s
1
at 50C). The following temperature programme was employed: start at 70C,
ramp 10C min
1
, end temperature 200C. A split connection at the end of the capillary
column allowed division of the GC effluent into two capillaries leading to the FID (35 cm,
0.2 mm ID) and the EAD (45 cm, 0.32 mm ID), respectively. This provided a split ratio of
1:5 (FID/EAD). The EAD interface (Weibecker et al., 1997) was under thermostat control
and guided the capillary column through the wall of the GC oven, thus preventing

126
condensation of the sample in the cooler segments of the column. The end of the column
projected into a small chamber where the effluent was mixed with cold, humidified air
(400 ml min
1
). A polytetrafluoroethylene (PTFE)-clad flow tube led the airflow to the
detector cell, where the antenna of the moth was fixed in a perspex holder modified
according to Frbert et al. (1997); Schtz et al. (1999). Both ends of the antenna were
contacted to Ag/AgCl electrodes via hemolymph Ringer solution (Kaissling & Thorson,
1980). EAG potentials of the antenna were amplified by a factor of 100 and recorded with
a Chromstar data acquisition system (Bruker, Bremen, Germany).

GCEAD/MS system
The GCEAD/MS system (Weibecker et al., 2004) consisted of a 6890 N gas
chromatograph and a 5973 N quadrupole mass spectrometer (both Agilent, Palo Alto,
CA, USA). The GC was equipped with a type 7163 autosampler, a split-splitless injector,
and a J&W Scientific HP-5MS column (Agilent; length 30 m, inner diameter 0.25 mm, film
thickness 0.25 m). The injector was operated in the pulsed splitless mode. Helium
(purity 99.999%) was used as carrier gas at a constant flow of 1 ml min
1
, gas vector 24
cm s
1
. The following temperature programme was employed: start: 50C, hold for 1.5
min, ramp 6C min
1
to 200C, hold for 5 min. A GRAPHPACK 3D/2 flow splitter (Gerstel,
Mlheim, Germany) was used to split the effluent from the column into two pieces of
deactivated capillary leading to the mass spectrometer (length 1 m, ID 0.1 mm) and to the
EAD setup (length 1 m, ID 0.2 mm). The restriction of these capillaries resulted in an
equal split of the gas flow into the two setups. The mass spectrometer used electron
ionization at 70 eV, and is operated in the scan mode with a mass range from 35 to 300
mass units at a scan speed of 2.78 scans s
1
. The EAD setup used a modified version of
an olfactory detector port (Gerstel, Mlheim, Germany). This incorporated a flexible
heating sleeve (230C) which guided the capillary out of the GC oven where the effluent
of the capillary was mixed with humidified air. The airflow was directed to the insect
antenna, which was housed in a detector cell made of PTFE. This setup is referred to
hereafter as the EAD interface. For recording EAG potentials, the same setup as
described above was used. In addition, the amplified signal passed through a high-pass
filter with a cut-off frequency of 0.01 Hz to suppress the slow drift often observed in the
EAD signal. Afterwards, a constant voltage of 0.5 V was added to the signal. These steps
were necessary to match the amplifier output to the input signal range (0-1 V) of a
35900E A/Dconverter (Agilent). The signal was recorded using the Agilent
CHEMSTATION software.

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127
Calibration of EAD and EAG analyses
Both setups described above allowed calibration of the employed antenna with
standard odour dilutions using a calibration port in the flow tube of the EAD interface
(Weibecker et al., 2004). Odour standards were produced by drenching small pieces of
filter paper (2 cm
2
) with dilutions of odour compounds in paraffin oil (Uvasol-quality,
Merck/VWR, Darmstadt, Germany). The filter paper was put into a 10 ml glass syringe,
where the odour accumulated in a concentration proportional to that of the substance in
the solution and its vapour pressure, according to Henrys law (Schtz et al., 1997). A
reproducible stimulus could be supplied by injecting a fixed volume (5 ml) of odour-loaded
air onto the antenna. To compensate for the interindividual variation in absolute
responses (mV), the signals were normalized (corrected) by use of a standard stimulus of
cis-3-hexen-1-ol at a 10
3
dilution in paraffin oil. The responses to cis-3-hexen-1-ol were
set as 100%.

Pollination exclusion experiment
The S. ciliata population used in the exclusion experiment was in the Pealara
Natural Park, Sierra de Guadarrama, a mountain range in the north of Madrid province,
Spain (40
o
N, 3
o
W). It was located at 2100 m, colonizing an old ski run which was closed
in 1998 and is actually under restoration. The area is covered by perennial grasses such
as Festuca curvifolia, Agrostis truncatula and Nardus stricta. Surrounding vegetation is
composed by a shrub community dominated by Cytisus oromediterraneus and Juniperus
communis subsp. alpina. The Sierra de Guadarrama has a Mediterranean-type climate,
characterized by a pronounced dry season from June to September (Gimnez-Benavides
et al. 2006). Mean annual precipitation is around 1350 mm. However, the presence of a
nearby late-melting snowpatch upslope the population provide it with relatively high soil
moisture during early-summer.
In July 2004, a 20x20 m area was fenced in the population to avoid livestock
grazing and vandalism. In August 2004, we randomly selected 104 adult plants within this
area. We measured plant size (multiplying maximum by perpendicular cushion diameter),
number of open flowers at the beginning of the experiment, and distance to the nearest
conspecific neighbour. Plants were randomly assigned to one of the three experimental
treatments: a) nocturnal pollination (35 plants), b) diurnal pollination (35 plants), and c)
open pollination (33 plants). Pollination exclusions were performed by setting up small
tents covering individually each of the target plants. Tents were made of fine white mesh
(55 holes per cm
2
, 1 mm in diameter), wooden sticks and nails to fix the structure on the
ground. At the beginning of the experiment (4 August), newly opened flowers and mature
flower buds (those expected to open in subsequent days) were counted and tagged in

128
each plant. Every day at 21,30h (dusk) exclusion tents were changed from plants
belonging to the night pollination treatment to plants that belonged to the day pollination
treatment The opposite operation was performed at 6,30h (dawn). Observations of flower
visitors were carried out throughout the exclusion experiment. However, censuses were
neither systematic nor comparable between day and night. Daylight observations were
performed at 5-minutes intervals centred on single plants. However, nocturnal
observations were made by active search for pollinators moving across the population
with a head lamp, since single-plant observations in darkness did not work properly. On
18 August the exclusion treatments were concluded. Mature fruits were collected ~25
days after the start of the experiment before they opened to prevent seed loss. At lab,
fruits were dissected with a scalpel. Total mature seeds were counted under a 10x
magnifying binocular and capsules showing seed predation were counted. Additionally,
pollen:ovule ratio was determined in 10 mature buds before flower opening, following
Jrgens et al., 2002b.
Pollination success was assessed at plant level by means of three variables: fruit
set (proportion of flowers setting fruit), brood size (proportion of ovules setting seed in
each ripe fruit) and seed set (proportion of total ovules setting seed). Offspring fitness
was estimated by means of two surrogates: seed mass and seed germination. Mature
seeds of each plant were pooled and five sets of 10 seeds (enough to surpass the level of
accuracy of the precision balance, COBOS AX120, d=0.1mg) were weighted after being
stored for 36h in a silica-gel drier at room temperature. Seeds were stored in paper bags
at 6 C in darkness until the germination tests were carried out. In germination tests four
replicates of 25 seeds per plant were placed on two layers of filter paper in 8-cm diameter
Petri dishes. Filter papers were kept moistened throughout the germination test (80 days)
and every 3-4 days seeds showing radicle emergence were counted and removed. At
each census the location of the dishes in the chamber was regularly changed.
Germination conditions were 15/25
o
C alternating temperature and 16-h light/8h-dark
photoperiod. These conditions are very effective for breaking seed dormancy in S. ciliata
(Gimnez-Benavides et al., 2005). Tests were carried out in a germination chamber
(Selecta Hotcold GL, Barcelona, Spain) equipped with 6 cool-white fluorescent light tubes
(Philips 18 W TLD standard type, wavelength 400 to 650 nm) providing a photon flux
density of approximately 19 mol m
-2
s
-1
.

Estimation of nursery seed predation
It is important to highlight that real seed loss caused by the nursery pollinator is
underestimated at this experiment. Fruit predation must be higher than recorded because
the caterpillars move to other host fruits once they have eaten all the seeds of the egg-
CAPITULO 4

129
layed fruit (Brantjes, 1976; Bopp and Gottsberger, 2004). By collecting fruits before
maturation to prevent seed dispersal we remove the young larvae from the plant, thus
avoiding subsequent fruit predation. For that reason, we estimate nursery predation in the
experiment by means of presence-absence in each plant. In 2002-2003, to provide a
better assessment of the importance of nursery seed predation in S. ciliata, and as part of
a parallel study, we carried out a survey in the same mountain range, in which predation
on a fruit by fruit basis by Hadena was carefully recorded along the local altitudinal range
of S. ciliata, from 1976 to 2428 m (further details on this survey can be found in Gimnez-
Benavides et al., 2006).

Statistical analysis
To compare the doseresponse curve of different compounds, the antennal
response was logarithmized to linearize the curves. ANCOVA was subsequently used in
STATISTICA to test for parallelism of regression lines.
We fitted type III generalised linear models (GLMs) for the estimators of pollination
success (fruit set, brood size and seed set), estimators of offspring fitness (seed mass
and seed germination) and plant infection by Hadena larvae. Pollination treatment (day,
night or open), plant size, number of open flowers and distance to the nearest neighbour
were included as fixed variables in the fruit set, brood size and seed set models, whereas
number of open flowers was removed in the seed mass and seed germination models.
We used the binomial error distribution and the logit link function for fruit set, brood size
and seed germination models (probability from 0 to 1), setting the variance to 1-mean.
Seed mass followed a normal distribution. Plant infection by larvae was treated as a
binomial response variable using the quasi-likelihood estimation (due to the
overdispersion of data), the logit link function and setting the variance to mean (1-
mean) (Venables and Ripley 1998). Fruit predation by larvae in the phenological study
followed a binomial distribution. Regression coefficients were tested for significance by t
tests.
2
tests were conducted to evaluate whether the selected predictors explained a
significant fraction of the deviance (Guisan et al., 2002). Plants showing less than five
sampled flowers were removed from the analysis. Statistical analyses were performed
using the SAS (SAS Institute, 1990) and the S-Plus 2000 (MathSoft, Inc. 1999) statistical
packages.


130
RESULTS

Flower scent
The flower scent composition reflected the pattern of a night pollinated plant
species (table 1) (Knudsen and Tollsen, 1993; Jrgens and Gottsberger, 2002). However,
when compared to the chemical composition of known nocturnally pollinated Silene
species, we found important differences. Floral fragrance was especially dominated by
benzaldehide (mean concentration = 71.5 %) and secondary by benzyl acetate (9.5 %).
Noteworthy is the absence of lilac compounds, typically found in many nocturnal Silene
species (Dotterl et al., 2006). Regarding the emission rythmicity, most of the scent was
released during night hours (figure 1).

Electroantennographic study
In the electroantennographic study, flowers of S. ciliata and S. latifolia were tested
on three female and three male moths of H. consparcatoides. The strongest signals in the
antennae when S. ciliata scent was tested were elicited by benzaldehyde, benzyl acetate,
benzyl alcohol and nonanal (figure 2). When tested with S. latifolia flowers, the anntenae
revealed strong reactions to phenylacetaldehyde, lilac aldehydes, benzyl benzoate and
methyl salycilate. In general, the EAG responses increased with increasing dose of tested
compounds (dose-response curve). In the special case of lilac aldehydes, antennal
response increased almost log-linearly by inreasing dose, indicating that antennae
detected differences in concentration in low as well as in high amounts of these
compounds.

Pollination exclusion experiment
Fruit set originated from diurnal pollination was significantly higher than that from
nocturnal pollination (figure 3a, table2). Plants pollinated exclusively by diurnal visitors
reached similar fruit set than those pollinated by both diurnal and nocturnal visitors.
Contrarily, brood size was significantly higher in plants pollinated during night time than in
those pollinated exclusively during day time or all day (figure 3b, table2). Seed set did not
differ among treatments (figure 3c, table2). Seeds resulting from diurnal visitors were
heavier and germinated better than seeds resulting from nocturnal visitors, but did not
differ from seeds resulting from all day visitors (figure 4, table3). In all models, neither
plant traits (plant size and number of open flowers) nor proximity to conspecific plants
explained any change in deviance.
Altogether, 11 insect species were recoded visiting S. ciliata flowers in the course
of the experiment (table 5). Diurnal assemblage was dominated by hoverflyes
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131
(Syrphidae), accounting for 80.7 % of flower visits. Among them, Sphaerophoria scripta
was the major visitor (37.2 % of visits). The hawk moth Macroglossum stellatarum
(Sphingidae) was also a common visitor (17.5 %), whereas a single visit of the butterfly
Aglais urticae (Lepidopterae) was recorded. Nocturnal assemblage was dominated by the
noctuid moths (Noctuidae), especially by H. consparcatoides (60.1 % of visits) and
Mythimna vitellina (14.3 %). Two species of geometrid moths (Geometridae) also
occurred during night (18.8 % of visits). As expected, no species appeared in both
assemblages.

Nursery seed predation
All cases of capsule predation in the exclusion experiment could be clearly
assigned to H. consparcatoides larvae. Plants excluded from nocturnal visitation
obviously had lower probability of infection by Hadena larvae than non-excluded and
diurnally-excluded plants (figure 5a). We only detected a single Hadena caterpillar
feeding in a night-excluded visitation plant (3 % of plant infection), thus proving the
effectiveness of the exclusion system. As mentioned above, real seed loss was not
quantified in our pollinator exclusion experiment, so we could not calculate pollination vs.
seed predation tradeoffs. However, rearing assays in the laboratory indicate that each H.
consparcatoides larva needs 5 to 12 whole fruits to complete development (Gimnez-
Benavides, pers. obs.). This means that each plant could have lost 5 to 24 fruits due to
larval infection (no more than two larvae were found feeding in a single plant), which
represents 11 to 100 % of fruits produced per infected plant during the experiment,
discarding possible reductions due to larval mortality. On the other hand, percentage of
fruits predated by Hadena larvae during the phenological study (and therefore Hadena
abundance) was relatively high, differing among populations but not between years (table
4, figure 5b).

DISCUSSION
S. ciliata shows a typical night-pollination syndrome, widely extended in other
congeneric species. Flowers open at sunset, remaining closed during daylight hours
(once the white to pale-pink petals roll towards the stamens). Anther dehiscence and
stigma receptivity also start at dusk, together with the initiation of scent emission. Thus,
flower phenology and rhythmicity of scent emission seem to have evolved to maximize
nocturnal pollination by moths (Young, 2002; Jrgens et al. 2002a, 2002b; Kephart et al,
2006). However, pollination syndromes should be the result of past selective forces that
may not reflect the actual ecological context of the species properly.

132
Miyake and Yahara (1999) interpreted nocturnal flower opening and anthesis as a
strategy evolved to avoid pollen removal by wasteful diurnal bees, which collect similar or
even higher amounts of pollen than nocturnal visitors without improvement of pollination
success. Jrgens et al. (2002b) found no differences in pollen grain numbers and
pollen:ovule ratios between diurnal and nocturnal Caryophilloideae to support contrasting
effectiveness in transferring pollen. However, pollen:ovule ratio in S. ciliata is 200 38
(pollen grain number = 12.870 1.224, ovule number = 66 14), very low values
compared to both groups. Thus, the diurnal-avoidance strategy proposed by Miyake and
Yahara should not be discarded as an adaptation towards pollen economy in past
situations. Actually, Fenster et al. (2004) stated that several nocturnal Silene species can
attract diurnal pollinators by remaining their flowers open through the whole day without
any cost. However, we have seen that daylight flower closure in S. ciliata does not seem
to be a barrier against diurnal visitors, since hoverflies have space enough to land and
eat the pollen from the exserted anthers and hawkmoths can easily access to the nectar
(Gimnez-Benavides, pers. obs).
The scarcity of scent emission at daytime did not represent a limitation for diurnal
visitation either. Quantitative and qualitative differences in the flower scent between night
and day revealed that most part of the scent was emitted during night (figure 1). Flower
scent composition of S. ciliata contains some compounds typically found in nocturnal
pollinated species. Some compounds commonly found in both diurnal and nocturnal
species (benzaldehydes and benzyl alcohol) were detected at especially high
concentrations (Knudsen and Tollsen, 1993; Jrgens et al. 2002a; Dtterl et al., 2006).
However, floral odour is missing some specific compounds (lilac aldehydes and
phenylacetaldehyde) found to be important olfactory guides to flower recognition and
moth attraction in Silene latifolia and other nocturnal related species (Knudsen and
Tollsen, 1993; Dtterl and Jrgens, 2005; Dtterl et al., 2006), and non-related species
(Plepys et al., 2002; Cunningham et al., 2004). Despite its absence in S. ciliata flowers,
our electrophysiological assays proved that H. consparcatoides can smell these
compounds, though behavioural tests are needed to confirm whether real attractiveness
exists. This lack of important specific compounds for the Silene-Hadena interaction,
coupled to the prevalence of more generalist molecules are striking features. Moya and
Ackerman (1993) suggested that variation in floral scent composition could be an
avoidance strategy to inhibit pollinator recognition. Since we have demonstrated how the
nursery pollinator Hadena cosparcatoides constitute a parasite in presence of co-
pollinators, a change in flower fragrance pattern could help S. ciliata to escape from this
pollinator seed-eater (Kephart et al., 2006).
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In our exclusion experiment fruit set and brood size, both surrogates of pollination
success, showed opposite effects when night and day pollination was experimentally
induced. The lower fruit set in plants excluded from diurnal visitors could be a direct
consequence of shortage of nocturnal pollinators. However, seed production per fruit
(brood size) was higher in those plants. As a result seed set, the overall estimator of
pollination success, did not differ among treatments (figure 3c, table1). One possible
explanation of these results, the resource-based hypothesis, is that plants setting more
fruits per flower (those subjected to diurnal pollination) had to develop fewer seeds per
fruit to allocate a limited pool of resources among a large number of capsules. Thus,
seeds could reach higher mass and better germination capacity. An alternative
explanation, the pollinator effectiveness hypothesis, is that diurnal visitors could be
making more visits to S. ciliata flowers than nocturnal ones but less effective in terms of
pollen deposition, reaching higher fruit set but lower brood size. Diurnal pollinators have
usually been found to be more abundant than nocturnal ones, resulting in higher visitation
rates that lead to greater yields (Jennertsen, 1988; Jennertsen and Morse, 1991; Altizer
et al. 1998; Miyake and Yahara, 1998). Pollinator abundance in our exclusion experiment
is not statistically comparable between night and day hours, but we firmly believe that
diurnal visitors were more abundant. On the contrary, several authors have found higher
pollination effectiveness (i.e. pollen transferred to stigmas) of nocturnal pollinators
compared to diurnal ones (partially compiled in Young, 2002). For instance Shykoff and
Bucheli (1995) and Young (2002) reported higher and farther pollen transfer induced by
nocturnal visitors in Silene alba, estimated by movement of a pollen-analogue fluorescent
dye powder. Barthelmess et al. (2006) also found higher outcrossing rates in nocturnally
pollinated plants, estimated by means of genetic markers in the same species. In
consequence, Young (2002) and Barthelmess et al. (2006) found that pollination success
(expressed as seed set and fruit set respectively) was higher in plants only exposed to
nocturnal pollinators. Unfortunately we can not provide pollination effectiveness data of
the different S. ciliata visitors at the moment, but if we assume that moth visitors transfer
more pollen and farther than diurnal ones as previously reported for other nocturnal
Silene species (see above-cited references), then larger amounts of outcross pollen could
lead to more seeds per ovule under the nocturnal pollination regime (Richardson and
Stephenson, 1992; Brown and Kephart, 1999; Colling et al. 2004).
Additionally, the stigma receptivity has also been discussed in other studies as
potential cause of temporal differences in female fertility. Young and Gravitz (2002)
determined the effect of stigma age on pollination success of S. alba and found that the
higher seed set reached in nocturnally pollinated plants (reported in Young, 2002) was
not likely due to a decrease in stigma receptivity. Similarly, Kwak and Jennersten (1986)

134
did not found any relation between time of the day when pollen was applied to stigmas
and pollination success in Viscaria vulgaris and Rhinanthus angustifolus. These results
suggest that time of pollination is unlikely to explain fecundity differences observed in S.
ciliata, although experimental evidences are needed to corroborate it.

CONCLUSIONS

Results reported suggest that in spite of the evident nocturnal pollination
syndrome showed, S. ciliata has presently good options for reproductive success through
unspecialized diurnal pollination. Hoverflies (Syrphidae) and hawkmoths (Sphingidae),
the dominant groups in the diurnal assemblage of S. ciliata, are common visitors to many
Caryophyllaceae (Kephart, et al. 2006), and some case studies have pointed out their
role as effective pollinators of many plant species (Jennertsen, 1988; Erhardt, 1990 and
1991; Kearns and Inouye, 1994; Gmez and Zamora, 1999; Bosch et al., 2002). Day-
night differences in pollinator abundance, effectiveness and foraging behaviour are likely
responsible for observed differences in components of female fertility (fruit set and brood
size), but anyway the overall reproductive success (i.e. seed set) showed a functional
equivalence between day and night pollinators. Since abundance of nocturnal flower
visitors have been reported to vary greatly in space and time (Petterson, 1991), generalist
diurnal co-pollinators may provide S. ciliata with reproductive assurance in absence or
shortage of moths. Moreover, seed weight and ability to germinate, both components of
the offspring fitness, were enhanced under the diurnal pollination regime. To our
knowledge, no attempts to date had explicitly explored the role of day vs. night pollination
on components of progeny viability in Silene-Hadena systems. Those findings, together
with the important seed loss caused by the larvae of the specialized nursery pollinator,
suggest potential selective forces that may promote a generalized pollination strategy.
Such transition could be driven, among other traits, by a more generalist flower scent
composition, avoiding specific olfactory cues for the pollinating seed predator. However,
the relative contribution of different pollinators to plant fitness may fluctuate in space and
time depending on species abundance (Morse and Fritz, 1983; Schemske and Horvitz,
1984; Gmez and Zamora, 1999; Herrera, 2005). Thus, additional and more detailed
surveys at broader spatio-temporal scales are needed to document the selective
pressures acting on this possible shift from a specialist to a generalist pollination system.

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ACKNOWLEDGEMENTS

Many thanks to J.L. Yela and J. Cifuentes for their careful identification of moths;
and to C. Iriarte, Y. Valiani and B. Paredes for help in the lab. This work was supported
by the Spanish Government CICYT research project REN2003-03366, and the facilities
provided by the staff of Parque Natural de las Cumbres, Circo y Lagunas de Pealara
(Madrid).


136
REFERENCES

Adams, R.P. 1995. Identification of essential oil components by gas
chromatography/mass spectrometry. Allured Publishing Corporation, Carol Stream,
Illinois.
Altizer, S., Thrall, P.H. and Antonovics, J. (1998). Pollinator behavior and disease
transmission of the anther-smut disease of Silene alba. American Midland Naturalist
139: 147-163.
Amirav, A., Dagan, S. 1997. A direct sample introduction device for mass spectrometry
studies and gas chromatography mass spectrometry analyses. Eur. Mass
Spectrom. 3, 105-111.
Barthelmess, E.L., Richards, C.M. and McCauley, D.E. (2006). Relative effects of
nocturnal vs diurnal pollinators and distance on gene flow in small Silene alba
populations. New Phytologist 169: 689-698.
Bopp, S. and Gottsberger, G. (2004). Importance of Silene latifolia ssp. alba and S. dioica
(Caryophyllaceae) as host plants of the parasitic pollinator Hadena bicruris
(Lepidoptera, Noctuidae). Oikos 105: 221-228.
Bosch, M., Simon, J., Rovira, A.M., Molero, J. and Blanch, C. (2002) Pollination ecology
of the pre-Pyrenean endemic Petrocoptis montsicciana (Caryophyllaceae): effects
of population size. Biological Journal of the Linnean Society, 76: 79-90.
Brantjes, N.B.M. (1976). Prevention of superparasitation of Melandrium flowers
(Caryophyllaceae) by Hadena (Lepidoptera). Oecologia 24: 1-6.
Brown, E. and Kephart, S. (1999). Variability in pollen load: implications for reproduction
and seedling vigor in a rare plant, Silene douglasii var. oraria. International Journal
of Plant Sciences 160: 1145-1152.
Calle, J.A. 1982. Noctuidos Espaoles. Ministerio de Agricultura, Pesca y Alimentacin.
Boletn del Servicio contra Plagas e Inspeccin Fitopatolgica. Madrid, Neografis,
S.L.
Colling, G., Reckinger, C. and Matthies, D. (2004). Effects of pollen quantity and quality
on reproduction and offspring vigor in the rare plant Scorzonera humilis
(Asteraceae). American Journal of Botany 91: 1774-1782.
Cunningham JP, Moore CJ, Zalucki MP, West SW. 2004. Learning, odour preference and
flower foraging in moths. Journal of Experimental Biology 207: 8794.
Davies, N.W. 1990. Gas-chromatographic retention indexes of monoterpenes and
sesquiterpenes on methyl silicone and Carbowax 20M phases. Journal of
Chromatography 503: 1-24.
CAPITULO 4

137
Dtterl S, Jrgens A, Seifert K, Laube T, Weibecker B, Schtz S. 2006. Nursery
pollination by a moth in Silene latifolia: the role of odours in eliciting antennal and
behavioural responses. New Phytologist. 169: 707-718.
Dtterl S, Wolfe L, Jrgens A. 2005. Qualitative and quantitative analyses of flower scent
in Silene latifolia. Phytochemistry 66: 203213.
Dtterl, S. and Jrgens, A. (2005). Spatial fragrance patterns in flowers of Silene latifolia:
Lilac compounds as olfactory nectar guides? Plant Systematics and Evolution 255:
99-109.
Dufay, M. and Anstett, M. 2003. Conflicts between plants and pollinators that reproduce
within inflorescences. Oikos 100: 3-14.
Erhardt, A. (1990) Pollination of Dianthus gratianopolitanus (Caryophyllaceae). Plant
Systematics and Evolution 170: 125-132.
Erhardt, A. (1991) Pollination of Dianthus superbus L. Flora 185: 99-106.
Frbert P, Koch UT, Frbert A, Staten RT, Carde RT. 1997. Pheromone concentration
measured with electroantennogram in cotton fields treated for mating disruption of
Pectinophora gossypiella (Lepidoptera: Gelechiidae). Environmental Entomology
26: 11051116.
Fenster C, Armbruster W, Wilson P, Dudash M, Thomson J. 2004. Pollination syndromes
and floral specialization. Annual Review of Ecology and Systematics 35: 375403.
Galen, C. (1985). Regulation of seed-set in Polemonium viscosum: floral scents,
pollination, and resources. Ecology 66: 792-797.
Gimnez-Benavides L, Escudero A, Prez-Garca, F (2005) Seed germination of high
variability. Ecological Research 20: 433-444.
Gimnez-Benavides, L., Escudero, A. and Iriondo, J. M.

(1996) Coping with extreme
climatic events. Reproductive plasticity of a high mountain Mediterranean plant
under a severe heatwave. Unpublished.
Gmez, J. M. and R. Zamora (1999) Generalization vs. specialization in the pollination
system of Hormathophylla spinosa (Cruciferae). Ecology 80: 796-805.
Groman, J. D. and Pellmyr, O. (1999). The pollination biology of Manfreda virginica
(Agavaceae): relative contribution of diurnal and nocturnal visitors. Oikos 87: 373-
381.
Groman, J. D. and Pellmyr, O. (1999). The pollination biology of Manfreda virginica
(Agavaceae): relative contribution of diurnal and nocturnal visitors. Oikos 87: 373-
381.
Herrera, C. M. (2005). Plant generalization on pollinators: species property or local
phenomenon? American Journal of Botany 92: 13-20.

138
Herrera, C.M. 2000. Flower-to-seedling consequences of different pollination regimes in
an insect-pollinated shrub. Ecology 81: 15-29.
Jennersten O. 1988. Pollination of Viscaria vulgaris (Caryophyllaceae): the contributions
of diurnal and nocturnal insects to seed set and seed predation. Oikos 52: 310327.
Jennersten, O. and D.H. Morse. 1991. The quality of pollination by diurnal and nocturnal
insects visiting common milkweed, Asclepias syriaca. American Midland Naturalist
125: 18-28.
Jrgens A, Witt T, Gottsberger G. 2002a. Flower scent composition in night-flowering
Silene species (Caryophyllaceae). Biochemical Systematics and Ecology 30: 383
397.
Jrgens A, Witt T, Gottsberger G. 2002b. Pollen grain numbers, ovule numbers and
pollen- ovule ratios in Caryophylloideae: correlation with breeding system,
pollination, life form, style number, and sexual system. Sexual Plant Reproduction
14: 279289.
Kaissling KE, Thorson J. 1980. Insect olfactory sensilla: structural, chemical and electrical
aspects of the functional organisation. In: Satelle DB, Hall LM, Hildebrand JG, eds.
Receptors for Neurotransmitters, Hormones and Pheromones in Insects.
Amsterdam, the Netherlands: Elsevier/North- Holland Biomedical Press, 261282.
Kearns, C.A. and Inouye, D.W. (1994) Fly Pollination of Linum lewisii (Linaceae).
American Journal of Botany, 81: 1091-1095.
Kephart, S., Reynolds, R. J., Rutter, M. T., Fenster, C. B. and Dudash, M. R. (2006).
Pollination and seed predation by moths on Silene and allied Caryophyllaceae:
evaluating a model system to study the evolution of mutualisms. New Phytologist
169: 667-680.
Knudsen, J.T. and Tollsten, L. (1993). Trends in Floral Scent Chemistry in Pollination
Syndromes - Floral Scent Composition in Moth-Pollinated Taxa. Botanical Journal of
the Linnean Society 113: 263-284.
Knudsen, J.T., Tollsten, L. and Bergstrm, G. (1993). Floral scents - a checklist of volatile
compounds isolated by head-space techniques. Phytochemistry 33: 253-280.
Knudsen, J.T., Tollsten, L., Groth, I., Bergstrm, G., Raguso, R.A. (2004). Trends in floral
scent chemistry in pollination syndromes: floral scent composition in hummingbird-
pollinated taxa. Botanical Journal of the Linnean Society 146: 191-199.
Kwak, M.M. and Jennersten, O. (1986). The significance of pollination time and frequency
and of purity of pollen loads for seed set in Rhinanthus angustifolius
(Scrophulariaceae) and Viscaria vulgaris (Caryophyllaceae). Oecologia 70: 502-
507.
CAPITULO 4

139
Latasa, T., Prez, I. and Garzn, A. 2001. Trabajo de campo de Lepidpteros y
Colepteros del Parque Natural Sierra de Cebollera (La Rioja). Gobierno de La
Rioja. Consejera de Turismo y Medio Ambiente.
MathSoft, Inc. (1999) S-Plus 2000. Guide to Statistics, Volume I. Seattle, Washington.
Miyake, T. and Yahara, T. (1999). Theoretical evaluation of pollen transfer by nocturnal
and diurnal pollinators: when should a flower open? Oikos 86: 233-240.
Morse, D. and Fritz, R. (1983). Contributions of diurnal and nocturnal insects to the
pollination of common milkweed (Asclepias syriaca L.) in a pollen limited system.
Oecologia 60: 190197.
Moya, M. (2001) Listado de subfamilias y especies del Concejo de Lena Fam. Noctuidae
Latreille, 1809. (Website: http://www.entomologia.net/noctus.htm).
Moya, S. and Ackerman, J.D. (1993). Variation in the floral fragance of Epidendrum ciliare
(Orchidaceae). Nordic Journal of Botany 13: 41-47.
Pettersson M. 1991. Pollination by a guild of fluctuation moth populations option for
unspecialization in Silene vulgaris. Journal of Ecology 79: 591604.
Plepys, D., Ibarra, F., Francke, W. and Lofsted, C. (2002). Odour-mediated nectar
foraging in the silver Y moth, Autographa gamma (Lepidoptera: Noctuidae):
behavioural and electrophysiological responses to floral volatiles. Oikos, 99: 75-82.
Richardson, T.E. and Stephenson, A.G. 1992. Effects of parentage and size of the pollen
load on progeny performance in Campanula americana. Evolution 46: 1731-1739.
SAS Institute, Inc. (1996) SAS/STAT Software: Changes and Enhancements through
Release 6.11. Cary, N.C.
Schemske, D.W. and Horvitz, C.C. (1984) Variation among floral visitors in pollination
ability: a precondition for mutualism specialization. Science 225: 519-521.
Schtz S, Weibecker B, Koch UT, Hummel HE. 1999. Detection of volatiles released by
diseased potato tubers using a biosensor on the basis of intact insect antennae.
Biosensors and Bioelectronics 14: 221228.
Shykoff, J. and Bucheli, E. 1995. Pollinator visitation patterns, floral rewards and the
probability of transmission of Microbotryum violaceum, a venereal disease of plants.
Journal of Ecology 83: 189198.
Stebbins, GL. 1970. Adaptive radiation of reproductive characteristics in angiosperms. I:
Pollination mechanisms. Annual Review of Ecology and Systematics 1: 30726.
Thompson, J.N. and Pellmyr, O. (1992). Mutualism in pollinating seed parasites amid co-
pollinators: constraints on specialization. Ecology 73: 1780-1791.
Tutin TG, Heywood VH, Burges NA, Valentine DH., Walters SM, Webb DA (eds.) 1995.
Flora Europaea Vol 1-5. Electronic dataset supplied by Pankhurst RJ, Royal Botanic
Garden of Edinburgh.

140
Venables WN, Ripley BD (1998) Modern applied statistics with S-Plus. Springer-Verlag,
New York.
Weibecker B, Holighaus G, Schtz S. 2004. Gas chromatography with parallel mass
spectrometric and electroantennographic detection - analysis of wood odour by
direct coupling of insect olfaction and mass spectrometry. Journal of
Chromatography A 1056: 209216.
Weibecker B, Schtz S, Klein A, Hummel HE. 1997. Analysis of volatiles emitted by
potato plants by means of a Colorado beetle electroantennographic detector.
Talanta 44: 22172224.
Westerbergh A, Westerbergh J. 2001. Interactions between seed predators/ pollinators
and their host plants: a first step towards mutualism? Oikos 95: 324334.
Westerbergh A. 2004. An interaction between a specialized seed predator moth and its
dioecious host plant shifting from parasitism to mutualism. Oikos 105: 564574.
Wolff, D., Braun, M. and Liede, S. (2003). Nocturnal versus diurnal pollination success in
Isertia laevis (Rubiaceae): A sphingophilous plant visited by hummingbirds. Plant
Biology 5: 71-78.
Yela, J. L. 1992. Los Noctuidos (Lepidoptera) de la Alcarria (Espaa Central) y su
relacin con las principales formaciones vegetales de porte arbreo. Ministerio de
Agricultura, Pesca y Alimentacin. Madrid.
Young, H. J. (2002). Diurnal and nocturnal pollination of Silene alba (Caryophyllaceae).
Young, H. J. and Gravitz, L. (2002). The effects of stigma age on receptivity in Silene alba
(Caryophyllaceae). American Journal of Botany 89: 1237-1241.

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Table 1. Average relative amounts (in %) of floral odour compounds in 10 samples of
Silene ciliata. The compounds are ordered in classes, which to some degree reflect their
biosynthetic origin (see Knudsen and Tollsten 1993). In each class, compounds are listed
according to relative retention time order (RR
t
). tr = trace amounts (< 0.1 %). Unknowns
were included when present with more than 0.1% in any sample.

Sample Nr. RRI 001 002 003 005 006 007 008 009 010 011 Mean
Total number of
compounds

FATTY ACID
DERIVATIVES

hexanal 911 0.1 0.1 0.1 tr tr 0.6 tr 0.1
cis-3-hexenol 1026 0.2 0.6 0.4 0.2 0.2 tr 0.3 0.1 0.2
1,4,-hexandiene 1028 tr 0.5 0.1
2-butanone 1028 0.3 tr
heptanal 1107 0.3 0.5 0.8 0.2 0.2 0.2 0.2
octanal 1292 0.7 0.9 1.0 1.8 0.7 0.2 0.6 0.1 0.6
nonanal 1462 0.7 2.2 1.8 2.3 4.0 1.5 0.7 0.5 3.7 0.2 1.8
decanal 1619 0.8 3.1 7.5 8.2 4.9 2.3 1.0 0.7 4.9 0.2 3.4
cis-jasmone 1907 0.1 tr tr

BENZENOIDS
ethyl benzene
a
1035 0.3 0.1 tr
p-xylene
a
1052 1.5 0.2 0.1 5.0 0.7
benzenoid compound
a
m/z: 106, 91, 77
1102 1.1 0.1 3.0 0.4
benzaldehyde 1230 67.2 72.1 68.6 65.7 57.3 78.6 91.1 82.8 51.7 79.7 71.5
benzylalkohol 1354 1.4 1.6 0.8 3.2 6.0 7.1 1.1 5.2 1.3 2.1 3.0
1,4-diethylbenzene
a
1395 0.1 0.1 tr
acetophenone
a
1413 0.1 0.2 0.1 0.4 0.3 0.2 0.1 0.1 0.2 0.2
benzyl formate 1426 0.2 0.2 0.3 0.2 0.1 0.5 0.2 0.2
2-phenyl ethyl alcohol 1494 0.3 0.2 tr 0.1 0.9 0.2 0.2
benzyl acetate 1561 4.2 15.3 12.5 17.4 18.5 2.1 0.7 3.8 9.1 11.6 9.5
benzenoid compound
m/z: 122*, 105, 77, 51
1576 tr 0.4 tr tr tr 0.9 0.4 1.1 tr 0.1 0.3
methyl salicylate 1621 tr tr tr tr tr 0.6 1.0 tr 0.2
benzenepropanol 1670 0.1 tr
benzenoid compound
m/z: 105, 77, 51
1803 2.5 2.3 5.3 3.3 2.2 2.4 3.1 1.4 2.6 2.5


142
Sample Nr. RRI 001 002 003 005 006 007 008 009 010 011 Mean
Total number of
compounds

PHENYL
PROPANOIDS

(Z)-cinnam aldehyde 1733 0.2 0.1 0.2 0.3 0.4 0.7 0.6 0.3 0.3
(Z)-cinnamyl alcohol 1777 0.1 tr 0.2 0.9 0.1 0.1

MONOTERPENOIDS
-pinene 1177 0.7 tr tr tr 0.3 0.1
5-hepten-2-one, 6-
methyl 1264 0.1

tr
p-cymene 1338 0.5 0.2 1.6 0.2
(E)-ocimene 1371 17.6 13.3 1.9 3.3

SESQUITERPENOIDS
ST m/z: 204*, 91, 105,
79,119, 161, 133, 189
1890 0.1 0.1 0.1 0.1 0.3 tr 0.1
longifolene 1914 0.1 0.2 0.3 0.4 0.2 0.3 0.2 0.5 tr 0.2
ST m/z: 204*, 105, 91,
161, 79, 121, 67
1932 0.1 0.1 0.1 0.2 0.1 0.1 0.1 0.1 0.3 tr 0.1
-gurjunene 1938 0.1 0.2 0.1 0.3 0.2 0.1 0.1 0.1 0.5 tr 0.2
(E)-caryophyllene 1950 tr tr tr

MISCELLANEOUS
naphthalene 1614 0.1 tr tr tr tr 0.1 0.1 0.1 0.3 tr

UNKNOWNS
unidentified m/z: 41,
57, 79, 67, 105, 96
1405 0.2 0.3 0.2 0.1 0.1
unidentified m/z: 162*,
91, 119, 43, 65, 105
1631 0.4 0.2 0.3 0.3 0.1 0.1
unidentified m/z: 107*,
79, 51
1847 0.1 0.4 0.2 0.1 0.2 tr 0.1

a
Compound might be of anthropogenic origin.
b
Mass fragments for unknowns are listed with the molecular ion first (if known or
inferred) marked by an asterisk, followed by the base peak and other fragments in order
of decreasing abundance.

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Table 2. Results of GLM models for pollination success estimators in the pollinator
exclusion experiment (Guadarrama, Madrid). N, D and O denote nocturnal, diurnal and
open pollination treatments.

Fruit set
Variable d.f. Estimate SE
2
P
Intercept 1 1.830 0.310 34.86 < 0.0001
Pollination regime (N) 1 -0.960 0.294 10.65 0.0011
Pollination regime (D) 1 0.281 0.369 0.58 0.4475
Pollination regime (O) . . . .
Plant size 1 -0.001 0.001 0.74 0.3890
Open flowers 1 0.001 0.003 0.14 0.7120
Nearest neighbour distance 1 0.009 0.011 0.65 0.4193
Brood size
Intercept 1 -0.831 0.136 37.28 < 0.0001
Pollination regime (N) 1 0.319 0.140 5.17 0.0230
Pollination regime (D) 1 -0.046 0.142 0.10 0.7478
Plant size 1 -0.000 0.001 0.12 0.7284
Open flowers 1 -0.001 0.002 0.36 0.5499
Seed set
Intercept 1 -1.085 0.163 44.36 < 0.0001
Pollination regime (N) 1 0.016 0.165 0.01 0.9217
Plant size 1 -0.001 0.001 0.69 0.4050
Open flowers 1 -0.001 0.002 0.00 0.9585


144

Table 3. Results of GLM models for offspring fitness estimators in the pollinator exclusion
experiment (Guadarrama, Madrid). N, D and O denote nocturnal, diurnal and open
pollination treatmets.

Seed mass
2
P
Intercept 1 0.017 0.001 444.89 < 0.0001
Pollination regime (N) 1 -0.005 0.001 31.13 < 0.0001
Plant size 1 0.000 0.000 0.96 0.3279
Nearest neighbour distance 1 -0.003 0.000 2.09 0.1485
Seed germination
Intercept 1 2.244 0.252 79.04 < 0.0001
Pollination regime (N) 1 -0.801 0.252 10.08 0.0015
Pollination regime (D) 1 0.069 0.281 0.06 0.8014
Plant size 1 0.000 0.001 0.03 0.8646

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Table 4. Effects of year and altitude on the percentage of fruits predated during the
course of a phenological study involving three populations and two years. N, D and O
denote nocturnal, diurnal and open pollination treatments.

2
P
Intercept 1 -3.596 0.255 199.59 < 0.0001
Year (2002) 1 0.328 0.269 1.49 0.2229
Year (2003) . . . .
Altitude (1970m) 1 1.619 0.292 30.78 < 0.0001
Altitude (2250m) 1 0.942 0.329 8.20 0.0042
Altitude (2420m) . . . .

146

Table 5. Flower visitors of S. ciliata observed in the area used in the pollination exclusion
experiment.

Flower visitors Eating habit
Frequency
(% of visits)*
Nocturnal
Noctuidae
Hadena consparcatoides (Schawerda, 1928) Nursery pollinator 60.1
Mythimna vitellina (Hbner, 1808) Nectarivore 14.3
Standfussiana lucernea (Linnaeus, 1758) Nectarivore 6.8
Geometridae
Gnophos obscurata ([Denis & Schiffermller], 1775) Nectarivore 15.0
Perconia baeticaria (Staudinger, 1871) Nectarivor 3.8

Diurnal
Syrphidae
Sphaerophoria scripta (Linnaeus, 1758) Pollinivore 37.2
Eupeodes corollae (Fabricius, 1794) Pollinivore 18.4
Scaeva pyrastri (Linnaeus, 1758) Pollinivore 16.1
Eristalis tenax (Linnaeus, 1758) Pollinivore 9.0
Sphingidae
Macroglossum stellatarum (Linnaeus, 1758) Nectarivore 17.5
Macrolepidopterae
Aglais urticae (Linnaeus, 1758) Nectarivore 1.8

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Figure 1. Total amount of emitted volatiles at daytime (samples 1-17) and at nightime
(samples 18-21). Samples 1 to 10 are expressed as ng/5 minutes, while samples 13-21
are ng/2 minutes.


148

Figure 2. Electroanntenogram of Hadena consparcatoides males and females subjected
to floral scents of Silene ciliata (above) and Silene latifolia (below). Arrows indicate
electrophysiological active compounds. Name of the chemical compounds that elicited a
measurable signal are noted.
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Figure 3. Components of pollination success in the pollination exclusion experiment. a)
fruit set, b) brood size, and c) seed set. Mean standard deviation.

Open Diurnal Nocturnal
Pollination regime
0,95
0,90
0,85
0,80
0,75
0,70
0,65
M
e
a
n

f
r
u
i
t

s
e
t
a)
Control Diurnal Nocturnal
Pollination regime
0,36
0,34
0,32
0,30
0,28
0,26
0,24
M
e
a
n

b
r
o
o
d

s
i
z
e
b)
Pollination regime
0,30
0,28
0,26
0,24
0,22
0,20
0,18
M
e
a
n

s
e
e
d

s
e
t
c)

150

Figure 4. Components of offspring fitness in the pollination exclusion experiment. a) seed
mass, and b) seed germination. Mean standard deviation.

Pollination regime
1,00
0,95
0,90
0,85
0,80
M
e
a
n

s
e
e
d

g
e
r
m
i
n
a
t
i
o
n

(
%
)
b)
Pollination regime
0,018
0,017
0,016
0,015
0,014
0,013
0,012
M
e
a
n

s
e
e
d

m
a
s
s

(
m
g
/
1
0

s
e
e
d
s
)
a)
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Figure 5. Estimation of fruit predation by Hadena consparcatoides larvae, a) in the
pollination exclusion experiment, and b) during the course of a phenological study
involving three populations and two years.

Pollination regime
25
20
15
10
5
0
%

P
l
a
n
t
s

i
n
f
e
c
t
e
d

b
y

H
a
d
e
n
a

l
a
r
v
a
e
a)
b)
2420 2250 1970
Altitude (m)
20
15
10
5
0
%

F
r
u
i
t
s

i
n
f
e
c
t
e
d

b
y

H
a
d
e
n
a

l
a
r
v
a
e
2003
2002
year
276
127
518
79
237
486

152
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153

Captulo 5.

Local adaptation in the rearing edge of a high mountain
Mediterranean plant.

Gimnez-Benavides, L.; Escudero, A. and Iriondo, J. M.
Evolutionary Ecology, submitted.
LOCAL ADAPTATION IN A HIGH MOUNTAIN MEDITERRANEAN PLANT

154
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SUMMARY

In the regeneration process of plant populations, germination and seedling
establishment are critical steps that may be subjected to natural selection and adaptive
evolution. To test for evidence of local adaptation of these traits, we carried out reciprocal
sowing experiments among three populations of Silene ciliata, a high mountain
Mediterranean plant. Populations were located in the southernmost margin of the species
range, along the local altitude gradient that leads to a drought stress gradient. The aims
of this work were to assess the main limitations on S. ciliata offspring performance, and to
test whether local adaptation has a significant effect on establishment success. Drought
stress in summer was the main cause of seedling mortality even though germination was
concentrated immediately after snowmelt to make best use of soil moisture. Our results
support the hypothesis that species perform better at the centre of their altitudinal range
than at the boundaries. Despite the existence of more favourable environments for
regeneration, we found evidence of local adaptation in seedling survival and growth along
the whole gradient, thus favouring the persistence of remnant populations on the rearing
edge of the species. These findings suggest that for S. ciliata not only altitudinal shifts but
also a certain degree of local adaptation may help to counteract the effects of current
global warming.

156
INTRODUCTION

Seed germination and seedling survival are probably the most critical stages in the
life-history of plant populations (Kitajima and Fenner, 2000). Species distribution,
colonization capability and range shifts are thus partially determined by limitations on
progeny viability (Weltzin

and McPherson, 1999, 2000). The centre-periphery hypothesis
proposes that conditions for the regeneration of plant populations are less suitable on the
boundaries than in the centre of the distribution area (Lawton, 1993; Vucetich and Waite,
2003; Angert and Schemske, 2005). Similarly, in species with a wide altitudinal
distribution, a suitability gradient is also found within each mountain range (Krner, 1999).
Thus, high mountain plants occurring at the species southernmost margin and lowest
altitudinal limits, should face especially harsh constraints on seed emergence and
seedling survival (Lesica and McCune, 2004; Hampe and Petit, 2005) mainly due to water
shortage (Pigott and Pigott, 1993; Garca et al., 1999, Castro et al., 2004) and high
temperatures (Conolly and Dahl, 1970; Peuelas and Boada, 2003).
Special interest has recently arisen on how plants respond to climate warming.
Recruitment in marginal populations may inform us whether poleward and/or upward
shifts are taking place, or whether local adaptation under unfavourable conditions is
playing a mayor role in their long-term persistence (Davis and Shaw, 2001; Davis et al.,
2005; Jump and Peuelas, 2005). However, regeneration studies have rarely been
conducted at southern range limits (Garca et al., 1999; Castro et al., 2004; Peuelas and
Boada, 2003; Hampe, 2005) and hardly ever at their lowest altitudinal limit. Alpine
habitats are especially useful for evaluating range shifts and local adaptive responses
because they provide sharp environmental gradients (Grabherr et al., 1994; Callaway et
al., 2002; Walther et al., 2005). Mediterranean-type mountains offer additional interest
because water supply is limited, unlike other alpine environments (Lavorel et al., 1998;
Michalet, 2006).
Silene ciliata Poiret (Caryophyllaceae) is a perennial long-lived plant, inhabiting
main mountain ranges in the northern half of the Mediterranean Basin. Its latitudinal
range covers a relatively narrow band from 40 N to 46 N (Tutin et al., 1995).
Populations in northern Spain (Pyrennes and Cantabric Range), France (Massif Central)
and Italy (Appenines) usually occur from 1200 m to 3000 m, whereas in Central Spain,
where the species reaches its southernmost margin, it grows from ~1900 m to the highest
peaks (up to 2590m in Gredos Range). This suggests that lower latitudinal boundaries of
the species are highly limited by environmental conditions and that persistence in the
southernmost margin is restricted to isolated refuges at high altitude.
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The aim of this paper was to assess the main limitations on seed emergence and
seedling performance along an altitudinal gradient in the southern boundary of the
species, and to test whether local adaptation favours the persistence of remnant
populations under warming conditions. For that purpose, we monitored seed emergence,
seedling survival and cause of death in field sowing experiments in Sierra de
Guadarrama (Madrid, 40
o
N, 3
o
W) where the species reaches its southernmost
geographical limit. We used the reciprocal transplant approach, the most commonly
applied method for testing local adaptation (Primack and Kang, 1989). A previous study in
this area had shown that female reproductive success was very restricted at low-altitude
marginal populations probably due to water shortage (Gimnez-Benavides et al.,
unpublished). Likewise, we expect summer drought to be the main factor constraining
progeny viability in populations located at the lowest altitudinal limits.
Specifically, the questions addressed here were: (1) Does altitude represent a
stress gradient for progeny viability of S. ciliata as previously reported for reproductive
success? (2) Do S. ciliata populations present distinct ecotypes that exhibit home site
advantage? (3) What are the main causes of mortality and how do they differ along such
gradient?

MATERIAL AND METHODS

The species
S. ciliata grows in cushion rosettes of up to 2 cm in height and 15 cm in diameter.
A previous study suggested that the onset of flowering is mediated by photoperiodic
triggers (Gimnez-Benavides et al., unpublished). The flowering period begins in late
June and lasts about 8 weeks. Flowering stems reach 15 cm in height and bear 1 to 5
protandrous flowers. Hand-crossing experiments indicate that S. ciliata is a self-
compatible species (Gimnez-Benavides, unpublished data). Mean flower number per
plant ranges from 4.5 to 13.7. Fruit capsules contain up to 100 viable seeds (1.53 0.49
mm in diameter, 0.59 0.06 mg in weight). These capsules open at the top when ripe
and seeds are shortly dispersed by stalk vibration. No evidence of vegetative propagation
has been observed in this species (Gimnez-Benavides, personal observation).

Seed collection and description of the sowing sites
Three populations were selected along the altitudinal gradient. In each population,
seeds were collected directly from 20-25 mother plants from relatively small areas (300-
400 m
2
) during the fruiting season (July-August). The aim of the study was to compare
progeny responses at population level rather than to isolate maternal effects. Therefore,

158
seeds from each population were pooled and thoroughly mixed before sowing to obtain
three seed families. The lowest population was in the vicinity of Laguna de Pealara, a
lake situated in a late Pleistocene glacial cirque at 1950 m, in the timberline zone. Here,
S. ciliata colonizes pasture patches interspersed in a Cytisus oromediterraneus-Juniperus
communis subsp. alpina shrub matrix with dispersed stunted pines (Pinus sylvestris). The
intermediate population was on the summit of Dos Hermanas (2270 m), 3 km from the
low population. This site is covered by grass-dominated communities and C.
oromediterraneus-J. communis subsp. alpina shrub formations. The highest population
was on the summit of Pealara (2420 m), located 2 km from the intermediate population.
Summit flat areas and crests are covered by a discontinuous cryophilic pasture
dominated by Festuca curvifolia (Escudero et al., 2005). These three localities form the
widest altitudinal gradient found in the region (~470 m). Average snowcover duration is
100-140 days/year, usually from November to March (Palacios et al., 2003).

Measurement of environmental variables
Several environmental parameters were monitored during the experiment.
Snowmelt date at each site was determined from early survey inspections and digital
images of the areas taken at weekly intervals (provided by Dr. D. Palacios, Universidad
Complutense de Madrid). Air temperature was recorded by portable stations in the vicinity
of the planting sites. Precipitation was recorded only at the intermediate site (40 50' 13''
N, 3 54' 15'' W, 2236 m). Volumetric soil water content was measured next to one corner
of each plot on every census day during the first growing season (0-6 cm depth, Delta-T
HH2 moisture meter, Cambridge, UK). To assess edaphic properties, three soil samples
were randomly collected from bare-ground areas (top 10-cm soil) at each of the three
planting sites. Texture (percent of fine soil, fine-gravel and gravel), pH, conductivity,
organic matter, nitrogen, phosphorous and potassium content were determined using
standard soil analytical procedures.

Reciprocal sowing experiments
Seeds were weighted prior to any experiment. Five sets of 10 seeds per
population (enough to surpass the level of accuracy of the precision balance, COBOS
AX120, d=0.1mg) were weighed after being stored for 36h in a silica-gel drier at room
temperature.
In 2002 we conducted a preliminary sowing experiment to assess the most
appropriate sowing season for S. ciliata. Seed batches, collected the previous growing
season (July-September 2001) and stored at 6 C, were sown at their corresponding
home site on 25 May 2002, after the snowmelt. Seedling emergence (defined as the first
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159
census date at which cotyledons were seen above the soil surface) and seedling survival
were recorded at 10-15 day intervals throughout the growing season.
In 2003 seeds were collected on July-August and stored at room temperature until
sowing. On 11 September seeds were sown in their population of origin and in the
nearest population in altitude, following the reciprocal experiments presented in figure 1.
Seed emergence and seedling survival were recorded for two years. In 2004 censuses
were conducted at 10-day intervals throughout the growing season, from 10 May to 10
September. In 2005 censuses were carried out at the beginning and the end of the
growing season (29 June and 28 September 2005, respectively). In these two censuses
seedling size was determined by the number of leaves. The most likely cause of death
(summer drought, winter frost or herbivore/pathogen attack) was assigned to each dead
seedling.
Experimental plots consisted of 16x16 cm quadrats randomly distributed in small
bare areas at each site. To establish the quadrats, a 5x5 grid of 1-cm holes, 3-cm apart,
was made on a transparent plastic sheet. This plastic sheet was used for accurate seed
placement and subsequent monitoring. Two seeds were sown in each hole to ensure an
adequate sample size of seedlings. Seeds were individually sown ~3 mm beneath the
surface and slightly covered with sieved local topsoil. At each site four plots were
randomly assigned to each seed source. Thus, a total of 600 seeds were sown in 2002
and 1400 in 2003. All S. ciliata flowers within a 1-m radius around each plot were
removed periodically to avoid contamination by natural seed dispersal. In each survey, all
germinations were recorded but only the first emerged seedling per hole was allowed to
avoid sibling competition.

Laboratory germination test
In 2003 we simultaneously conducted a germination test under controlled
conditions with a subset of the seeds sown in the in situ reciprocal experiment. Seeds
were subjected to a cold-wet stratification treatment and incubated at alternating 15/25
o
C
temperature with a 16-h light/8h-dark photoperiod. These conditions have shown to be
very effective for breaking seed dormancy in S. ciliata (Gimnez-Benavides et al., 2005).
Seeds from each population were placed between two double layers of filter paper in 8-
cm Petri dishes and irrigated with distilled water. Dishes were wrapped in aluminium foil
and stored at 4
o
C. The length of the stratification period simulated natural conditions.
Thus, seeds were placed at 4C when the first snowfall occurred at the study area (5
November 2003), and the germination test was initiated at the start of snowmelt (12 May
2004). Another subset of the seed batches was stored in plastic vials at 6 C in darkness
until the germination tests were carried out (after-ripening treatment).

160
In germination tests four replicates of 25 seeds per population and treatment were
placed on two layers of filter paper in 8-cm diameter Petri dishes. Filter papers were kept
moistened throughout the germination test (75 days) and every 3-4 days seeds showing
radicle emergence were counted and removed. At each census the location of the dishes
in the chamber was regularly changed. Tests were carried out in a germination chamber
(Selecta Hotcold GL, Barcelona, Spain) equipped with 6 cool-white fluorescent light tubes
(Philips 18 W TLD standard type, wavelength 400 to 650 nm) providing a photon flux
density of approximately 19 mol m
-2
s
-1
.

Data analysis
Differences in soil properties among sowing sites were assessed by means of
one-way ANOVAs. Soil water content was analysed using a repeated-measures ANOVA
setting sowing site as between-subject factor and date as within-subject factor. The F-
statistics for within-subject factor and interaction term were corrected for violation of
sphericity assumption (i.e. data collected on adjacent sampling dates are more highly
correlated than data from separated sampling dates) (von Ende, 2001).
Differences in seed mass among the seeds of the three sites were analysed using
one-way ANOVAs. The effects of population of origin (seed source) and population of
sowing (sowing site) upon seed emergence, progeny viability, cause of death and
seedling size were modelled by fitting Generalised Linear Models (GLMs) (McCullagh and
Nelder, 1989). When the distribution of the response variable was a probability ranging
from 0 to 1 (seed emergence, progeny viability, summer drought mortality), we used the
quasi-likelihood estimation instead of the binomial distribution because our data were
under-dispersed (Venables and Ripley, 1998; Guisan et al., 2002). Poisson estimation,
using a "log" link function and setting the variance to "mean", was used when the
distribution of the response variable was Poisson-like (seedling size). Seed source,
sowing site and the interaction term were included as explanatory variables (fixed
factors). Regression coefficients were tested for significance by t tests.
2
tests were also
conducted to evaluate whether the selected predictors explained a significant fraction of
the deviance (Guisan et al., 2002). Progeny viability (number of established seedlings per
seeds sown, sensu Herrera, 2000) was calculated at three intervals: at the end of the first
growing season (2004), after the first winter season (2005) and after the second growing
season (2005). Statistical analyses were performed using the S-Plus 2000 statistical
package (MathSoft, Inc., 1999).
Emergence rate and survival rate were analysed using an accelerated failure-time
model (Fox, 1993). This method allows the use of right-censored data (i.e., experiments
that end before all seeds germinate or all seedlings die) to estimate parametric regression
CAPITULO 5

161
models using a maximum likelihood approach. We chose the best failure-time
distributions for our data sets based on the comparison of possible distributions with a
likelihood ratio test (Fox, 1993). Thus, we used a log-logistic distribution for the seed
emergence data set and a Weibull distribution for the seedling survival data set.
Computations were performed using the SAS LIFEREG procedure (SAS Institute, 1996).

RESULTS

Environmental differences among sowing sites
From 2003 to 2005, the low site was on average 1.8 C warmer than the
intermediate site, and 2.9 C warmer than the high site. Snowmelt at the low site started 7
days earlier than at the intermediate site, and 18 days earlier than at the high site. In
2004, mean temperatures during the growing season were 0.2 to 3.8 C lower than in
2003 and the summer was comparatively rainy (figure 2).
Soil water content significantly differed between sowing sites (F
2,25
= 27.32, P <
0.001) and between sampling dates (F
7,175
= 814.67, P < 0.001 after Greenhouse-Geisser
and Huynh-Feldt corrections for departure from sphericity). The interaction term date x
sowing site was also significant (F
14,175
= 3.78, P < 0.006 after correction), indicating that
soil water content varied at different rates at each site. Soil water content dropped sharply
in June, reaching minimum values (< 5 %) in late-July even at the highest locality.
However, the copious rainfall event in early-August partially mitigated the intense topsoil
desiccation (figure 2).
Soil analyses revealed some differences among sowing sites. Texture significantly
differed among altitudes (% gravel: F=8.56, p=0.017; % fine-gravel: F=8.79; p=0.016; %
fine-earth: F=7.68, p=0.022). The high site had higher gravel content and lower fine-earth
content than the other sites, while the intermediate site had the highest fine-gravel
content. A weak acidity gradient was observed from low to high sites (F=5.15, p=0.05).
No significant differences were found in conductivity and potassium content among sites
(F=2.79, p=0.139; F=4.71, p=0.089, respectively). Organic matter, nitrogen and
phosphorous content at the low and high sites were higher than at the intermediate site
(F=57.50, p<0.001; F=36.03, p<0.003; F=8.79, p<0.034, respectively).

Seed mass and seed emergence
Mean seed mass across sites and years was 0.39 mg. Significant differences
among sites were only found for seeds collected and planted in 2002 at the intermediate
site (F = 5.27, P = 0.023).

162
In the laboratory germination tests only 28 % of the seeds germinated after simple
storage at 6 C, but nearly 70 % germinated after the cold-wet stratification treatment,
when data from the three seed provenances was pooled together. Both seed emergence
(table 1) and emergence rate (table 2) significantly improved after cold-wet stratification in
all populations. The source of seeds explained differences in germination rate but not in
germination percentage.
In the preliminary sowing experiment conducted in 2002, barely 4 % of sown
seeds emerged across the three sowing sites. However, in the 2003 reciprocal
experiments overall seed emergence averaged 47.7 % 19.6 across all planting sites
and seed sources. In this successful sowing the germination period began immediately
after snowmelt and lasted until mid-June (figure 3). Significant differences in seed
emergence were found between sowing sites in both experiments and between seed
sources in experiment 1 (table 1). Seed source had a significant but marginal effect on
emergence rate in both experiments (table 2). However, sowing site accounted for an
important fraction of the variation found in experiment 2 (seeds germinated at a slower
rate when sown at a higher altitude) (table 2). As shown in figure 3, all seed families
germinate better in Dos Hermanas (the intermediate site).

Progeny viability
Only 10 % of germinated seedlings survived until the end of the experiment. On
average, the highest mortality occurred during the first growing season (77 %). During the
following winter, seedling mortality dropped to 9 %, and then to 4% during the second
growing season.
The effect of sowing site on progeny viability was significant and consistent across
time in experiment 1 (table 3). Seedling survival was higher when sown in the
intermediate site (figure 4). Seed source was non-significant after the first growing
season, but this effect could not be analyzed thereafter as all seedlings from Dos
Hermanas died the following winter when outplanted. Nevertheless, the significant site x
source interaction found in experiment 1 implies that differences in progeny viability
between sources of seeds did not remain consistent across sowing sites (table 3). None
of the variables in experiment 2 were significant (data not shown). Seedling survival rate
was explained by sowing site and seed source in experiment 1 but only by seed source in
experiment 2 (table 4). Survival curves in figure 4 show that mortality slows down after
mid-summer precipitations and the subsequent increase in soil moisture (figure 2).
Analyses of seedling size at the end of the experiment revealed that sowing site had a
marginal effect in experiment 1, seedlings sown in the high site being larger than those
sown in the other sites (Table 5). The effect of the interaction term could not be
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163
computed. In experiment 2, both sowing site and the interaction term were significant.
Local seedlings developed more leaves than foreign seedlings in both sowing sites (figure
5).
Summer drought was the most important cause of seedling mortality across sites
(86.5 %), followed by winter frost (10.5 %) and herbivore/pathogen attack (3 %). GLMs
were conducted to know whether sowing site and seed source could explain the
susceptibility to each cause of death. No significant differences were found among
sowing sites and seed sources in mortality due to winter frost and herbivore/pathogen
attack (data not shown), despite slight trends along the altitude gradient observed (figure
6). The results of the summer drought mortality model indicated that sowing site (and
weakly seed source) partially explained the deaths due to drought in experiment 1 (table
6). Neither site nor seed source significantly differed in experiment 2.

DISCUSSION

Germination in high mountain areas is mainly restricted to short periods in early-
summer, when temperatures are high enough and soil water from snowmelt is still
available (Bliss, 1971). Consequently, seed dormancy, which prevents immediate post-
dispersal germination, is a common trait in alpine plant species (Pelton, 1956; Amen,
1966; Bliss, 1971). We found that most S. ciliata seeds follow this pattern: not only do
they require a post-dispersal maturation period to germinate, but also a cold-wet
stratification for several months (i.e. winter season) (tables 1 and 2, figure 3), as reported
for many other alpine species (Bell and Bliss, 1980; Marchand and Roach, 1980;
Gimnez-Benavides et al., 2005; Shimono and Kudo, 2005). After the cold-wet period
and snowmelt, germination occurs at a high rate to ensure enough soil moisture (figure
3). This is especially critical in Mediterranean mountains where soil desiccation is
common during the growth season (figure 2).
Results of the reciprocal sowing experiments clearly highlight some local
adaptation processes operating in favour of offspring success. Although progeny viability
was low in both the low and intermediate seed families when sown at the low site, a
significant interaction between seed source and altitude was detected (table 3). This
suggests a certain level of local adaptation as seedling performance was higher for home
seeds than for foreign seeds at both sites (figure 4). Several authors have argued that
selection for local adaptation is theoretically favoured in rear edge populations, where
reduced gene flow due to isolation promotes the existence of distinct ecotypes
(Dynessius and Jansson, 2000; Hampe and Petit, 2005; Jakobsson and Dinnetz, 2005).
The low S. ciliata population is located on the rear edge of the suspected upward

164
elevation shifting, restricted to small open interspaces within a matrix of unsuitable
habitat. Nonetheless, home seed superiority in progeny viability was only expressed in a
first stage, because after two years the number of established seedlings was similar and
dramatically low irrespective of seed provenance (figure 4). Another evidence of local
adaptation in progeny viability was found in the high seed family (i.e. in the leading edge
of local distribution), which had in its home site a higher seedling survival than the
intermediate seed family (figure 4). Despite such a type of home vs. foreign effects has
been interpreted as a consequence of local adaptation (Kawecki and Ebert, 2004) results
should be taken with caution because they could merely reflect differences in home
habitat quality. Nevertheless, seedling size, a surrogate of plant performance, also
revealed a similar local adaptation effect when intermediate and high seed families were
reciprocally sown (table 5, figure 5).
In spite of the local adaptation effects detected, certain seed families perform
better in all situations, and certain sites are clearly more suitable for species regeneration.
We found a significant seed source effect in experiment 1. Seed emergence and
emergence rate reached higher values in the intermediate seed family than in the low
seed family regardless of the sowing site (tables 1 and 2), suggesting a seed quality
effect. On the other hand, seed emergence and progeny viability decreased significantly
when seeds from both low and intermediate populations were sown on the low site,
suggesting that the low site is the most stressful (figures 3 and 4). Conversely, the
intermediate and to a lesser extent the high site were favourable environments for S.
ciliata regeneration. Soil analyses showed that the low site holds the largest amount of
fine-earth fraction and higher values of organic matter, nitrogen and phosphorous content
than the intermediate site, all surrogates of soil development. This suggests that site
suitability for germination and seedling survival is not determined by those edaphic
properties. Contrarily, lower soil water content and higher temperature are likely the main
limitations for seedling establishment in the low site. In this sense, we recently found that
reproductive success of the low population was extremely low especially in dry years,
when many mature plants were simply unable to flower. Contrarily, the intermediate and
high populations provided better and more stable seed production over time (Gimnez-
Benavides et al., unpublished). All this supports the hypothesis that species perform
better at their range centre than at the periphery, and that severe abiotic limitations
constrain the lowland boundary (Hampe and Petit, 2005).
It is noteworthy that the great impact of summer drought on progeny viability even
though summer 2004 was a relatively rainy season. Among sites, the low site had a
significantly higher mortality due to water shortage than the other sites (table 6, figure 6),
supporting our initial assumption and previous findings on the S. ciliata reproductive
CAPITULO 5

165
output (Gimnez-Benavides et al., unpublished). Since our sowing experiments were
carried out in bare soil patches, the major cause of seedling mortality during growing
season can be reliably assigned to drought stress rather than to biotic causes (i.e.
competition). High levels of drought-induced mortality are a common feature, from
lowland to mountainous habitats, in Mediterranean-type ecosystems (Dunne and Parker,
1999; Escudero et al., 1999, 2000; Castro et al., 2004, 2005). However, it has rarely been
experimentally documented in high mountain Mediterranean-type environments (see
Cavieres et al., 2005 in Mediterranean-type mountains in Central Chile). Our results
support the recently suggested hypothesis that water shortage is a key factor controlling
plant performance in high Mediterranean mountains (Cavieres et al., 2006; Michalet,
2006). Mountain habitats have been considered extremely stressful for plant life but
normally not limited by water availability (Korner, 1999). However, Mediterranean
mountain specialists cope with both a very short growth season, typical of all mountain
environments, and severe water shortage in summer. An increasing number of studies
are currently reporting the prevalence of facilitative interactions that partially counteract
the effects of stressful conditions in Mediterranean mountains (Pugnaire et al., 1996;
Castro et al., 2004; Cavieres et al., 2005, 2006; Gmez-Aparicio et al., 2005a, 2005b),
but none of them has implicitly explored the potential role of species local adaptation in
offspring establishment success. S. ciliata populations in Central Spain are a clear
example of remnant populations within the plant distribution area, a factor commonly
associated with regression dynamics due to climatic stress (Eriksson, 1996; Garca et al.,
1999; Hampe and Petit, 2005). Recent evidences have shown plant and animal range
contractions coupled with altitudinal shifts at the warmest margins in these Mediterranean
mountains (Sanz-Elorza et al., 2003; Wilson et al., 2005) but our findings are, to our
knowledge, the first evidence of persistence under unfavourable conditions by local
adaptation (see Davis and Shaw, 2001; Davis et al., 2005).
On the other hand, studies on local adaptation in populations located on an
elevation gradient are very scarce (Clausen et al., 1940; Galen et al., 1991; Etterson,
2004; Angert and Schemske, 2005). Surprisingly, these studies have commonly used
juvenile or adult plants as starting point in reciprocal transplant experiments instead of
seeds. Such approaches do not properly explore the role of natural selection in critical
earlier life-history stages (Primack and Kang, 1989; Angert and Schemske, 2005).
Although seedling establishment has been predicted to be rare in alpine habitats because
of dominant life-history trade-offs, recent evidence suggests that limitations on early life-
history stages in mountainous habitats must not be undervalued (Forbis, 2003;
Chambers, 1995; Castro et al., 2004). The lack of evidence of local adaptation in some

166
previous works along altitudinal gradients may have resulted from the use of reciprocal
transplants instead of sowing experiments (Clausen et al., 1940; Galen et al., 1991).
In conclusion, local adaptation acting upon seed emergence and seedling survival
and growth may help to partially counteract environmental limitations in S. ciliata, allowing
the local persistence of peripheral populations. Understanding the limitations on
recruitment in plant populations at the extreme margins of their species distributions may
provide valuable information on plant response plasticity and evolutionary potential to
cope with rapid climate warming (Jump and Peuelas, 2005). Nevertheless, population
dynamics and range shift processes cannot be properly estimated through simple
recruitment rates, at least for long-lived species such as S. ciliata (Garca et al., 1999;
Garca and Zamora, 2003; Hampe and Petit, 2005). Long-term demographic monitoring,
in conjunction with the assessment of genetic patterns and ecosystem processes
operating at various spatiotemporal scales are needed to integrate current knowledge of
climate change impacts across the species ranges.

ACKNOWLEDGEMENTS

The authors thank the staff of Parque Natural de las Cumbres, Circo y Lagunas de
Pealara for permission to work in the area, and C. Iriarte, B. Martnez, Y. Valiani and B.
Paredes for the analyses of soil samples. They also thank D. Palacios for providing the
digital images for snowmelt date estimation, M.J. Albert for helpful comments on earlier
drafts of the

manuscript, and L. De Hond for her linguistc assistance. This work was
supported by the Spanish Government CICYT project REN2003-03366.

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REFERENCES

Amen, R. D. (1966) The extent and role of seed dormancy in alpine plants. Quarterly
Review of Biology 41, 271-281.
Angert, A. L., Schemske, D. W. and Geber, M. A. (2005) The evolution of species'
distributions: reciprocal transplants across the elevation ranges of Mimulus
cardinalis and M. lewisii. Evolution 59, 1671-1684.
Baskin C. C. and Baskin J. M. (1998) Seeds. Ecology, biogeography, and evolution of
dormancy and germination. Academic Press, San Diego, USA.
Bell K. L. and Bliss L. C. (1980) Plant reproduction in high arctic environment. Arctic and
Alpine Research 12, 1-10.
Bennington, C. C. and McGraw, J. B. (1995) Natural Selection and Ecotypic
Differentiation in Impatiens pallida. Ecological Monographs 65, 303-324.
Bliss L. C. (1971) Arctic and alpine plant life cycles. Annual Review of Ecology
Systematics 2, 405-438.
Callaway, R. M., Brooker, R. W., Choler, P., Kikvidze, Z., Lortie, C. J., Michalet, R.,
Paolini, L., Pugnaire, F. I., Newingham, B., Aschehoug, E. T., Armas, C., Kikodze,
D. and Cook, B. J. (2002) Positive interactions among alpine plants increase with
stress. Nature 417, 844-848.
Castro, J., Zamora, R. and Hdar, J. A. (2002) Mechanisms blocking Pinus sylvestris
colonization of Mediterranean mountain meadows. Journal of Vegetation Science
13, 725-731.
Castro, J., Zamora, R., Hdar, J. A. and Gmez, J. M. (2004) Seedling establishment of a
boreal tree species (Pinus sylvestris) at its southernmost distribution limit:
consequences of being in a marginal, Mediterranean habitat. Journal of Ecology 92,
266-277.
Castro, J., Zamora, R., Hdar, J. A. and Gmez, J. M. (2005) Alleviation of summer
drought boots establishment success of Pinus sylvestris in a Mediterranean
mountain: an experimental approach. Plant Ecology 181, 191-202.
Cavieres, L. A., Badano, E. I., Sierra-Almeida, A., Gmez-Gonzlez, S. and Molina-
Montenegro, M. A. (2006) Positive interactions between alpine plant species and
the nurse cushion plant Laretia acaulis do not increase with elevation in the Andes
of central Chile. New Phytologist 169, 59-69.
Cavieres, L. A., Quiroz, C. L., Molina-Montenegro, M. A., Muoz, A. A. and Pauchard, A.
(2005) Nurse effect of the native cushion plant Azorella monantha on the invasive
non-native Taraxacum officinale in the high-Andes of central Chile. Perspectives in
Plant Ecology, Evolution and Systematics 7, 217-226.

168
Chambers, J. C. (1995) Disturbance, life history strategies, and seed fates in alpine
herbfield communities. American Journal of Botany 82, 421-433.
Clausen, J. D., Keck, D. And Hiesey, W. M. (1940) Experimental studies on the nature of
species. I. The effect of varied environments on western North American plants.
Publications of the Carnegie Institution 520.
Conolly A. P, Dahl E. (1970) Maximum summer temperature in relation to the modern and
quaternary distributions of certain arctic-montane species in the British Isles. In
Studies in the Vegetational History of the British Isles. (D. Walker and R. G. West,
eds.), pp. 159223. Cambridge University Press, Cambridge.
Davis, M. B., Shaw, R. G. (2001) Range shifts and adaptive responses to Quaternary
climate change. Science 292, 673-679.
Davis, M. B., Shaw, R. G. and Etterson, J. R. (2005) Evolutionary responses to changing
climate. Ecology 86, 17041714.
Dunne, J. A. and Parker, V. T. (1999) Species-mediated soil moisture availability and
patchy establishment of Pseudotsuga menziesii in chaparral. Oecologia, 119: 36-45.
Dynesius, M. and Jansson, R. (2000) Evolutionary consequences of changes in species'
geographical distributions driven by Milankovitch climate oscillations. Proc. Nat.
Acad. Sci. U.S.A. 97, 9115-9120.
Eriksson, O. (1996) Regional dynamics of plants: a review of evidence for remnant,
source-sink and metapopulations. Oikos 77, 248-258.
Escudero, A., Gimnez-Benavides, L., Iriondo, J. M., Rubio, A. (2005) Patch dynamics
and islands of fertility in a high mountain Mediterranean community. Arctic, Antarctic
and Alpine Research 36, 518-527.
Escudero, A., Iriondo, J. M., Olano, J. M., Rubio, A. and Somolinos, R. C. (2000) Factors
affecting establishment of a gypsophyte: the case of Lepidium subulatum
(Brassicaceae). American Journal of Botany 87, 861-871.
Escudero, A., Somolinos, R. C., Olano, J. M. and Rubio, A. (1999) Factors controlling the
establishment of Helianthemum squamatum (L.) Dum., an endemic gypsophile of
semi-arid Spain. Journal of Ecology 87, 290-302.
Etterson, J. R. (2004) Evolutionary potential of Chamaecrista fasciculata in relation to
climate change. I. Clinal patterns of selection along an environmental gradient in the
great plains. Evolution 58, 1446-1458.
Forbis, T. A. (2003) Seedling demography in an alpine ecosystem. American Journal of
Botany 90, 1197-1206.
Fox, G. A. (2001) Failure-time analysis: emergence, flowering, survivorship, and other
waiting times. In Design and Analysis of Ecological Experiments, 2
nd
edition (S. M.
Scheiner and J. Gurevitch, eds.), pp. 235266. Oxford University Press, New York.
CAPITULO 5

169
Garca, D. and Zamora, R. (2003) Persistence, multiple demographic strategies and
conservation in long-lived Mediterranean plants. Journal of Vegetation Science 14,
921-926.
Garca, D., Zamora, R., Hdar, J. A. and Gmez, J. M. (1999) Age structure of Juniperus
communis L. in the Iberian peninsula: Conservation of remnant populations in
Mediterranean mountains. Biological Conservation 87, 215-220.
Gimnez-Benavides, L., Escudero, A. and Iriondo, J. M.

(1996) Coping with extreme
climatic events. Reproductive plasticity of a high mountain Mediterranean plant
under a severe heatwave. Unpublished.
Gimnez-Benavides, L., Escudero, A., Prez-Garca, F. (2005) Seed germination of high
variability. Ecological Research 20, 433-444.
Gmez-Aparicio, L., Gmez, J. M. and Zamora, R. (2005) Microhabitats shift rank in
suitability for seedling establishment depending on habitat type and climate. Journal
of Ecology 93, 1194-1202.
Gmez-Aparicio, L., Valladares, F. Zamora, R. and Quero, J. L. (2005) Response of tree
seedlings to the abiotic herogeneity generated by nurse shrubs: an experimental
approach at different scales. Ecography 28, 1-12.
Grabherr, G., Gottfried, M. and Pauli, H. (1994) Climate Effects on Mountain Plants.
Nature 369, 448-448.
Guisan, A., Edwards, T. C. and Hastie, T. (2002) Generalized linear and generalized
additive models in studies of species distributions: setting the scene. Ecological
Modelling 157, 89-100.
Hampe, A. and Petit, R. J. (2005) Conserving biodiversity under climate change: the rear
edge matters. Ecology Letters 8, 461-467.
Herrera, C. M. (2000) Individual differences in progeny viability in Lavandula latifolia: a
long-term field study. Ecology 81, 3036-3047.
Jakobsson, A. and Dinnetz, P. (2005) Local adaptation and the effects of isolation and
population size - the semelparous Perennial Carlina vulgaris as a study case.
Evolutionary Ecology 19, 449-466.
Jump, A. S. and Peuelas, J. (2005) Running to stand still: adaptation and the response
of plants to rapid climate change. Ecology Letters 8, 1010-1020.
Kitajima, K. and Fenner, M. (2000) Ecology of seedling regeneration. In Seeds: the
Ecology of Regeneration in Plant Communities, 2nd ed. (M. Fenner, ed.), pp. 331
359. CAB International, Wallingford.

170
Lavorel, S., Canadell, J., Rambal, S. and Terradas, J. (1998) Mediterranean terrestrial
ecosystems: research priorities on global change effects. Global Ecology and
Biogeography 7, 157-166.
Lawton, J. H. (1993) Range, population abundance and conservation. Trends in Ecology
and Evolution 8, 409-413.
Marchand P. J. and Roach D. A. (1980) Reproductive strategies of pioneering alpine
species, seed production, dispersal, and germination. Arctic and Alpine Research
12, 137-146.
MathSoft, Inc. (1999) S-Plus 2000. Guide to Statistics, Volume I. Seattle, Washington,
USA.
McCullagh, P. and Nelder, J. A. (1989) Generalized Linear Models. Chapman and Hall,
New York, USA.
Michalet, R. (2006) Is facilitation in arid environments the result of direct or complex
interactions? New Phytologist 169, 3-6.
Palacios, D., de Andrs, N. and Luengo, E. (2003) Distribution and effectiveness of
nivation in Mediterranean mountain, Pealara (Spain). Geomorphology 54, 157-178.
Pelton, J. (1956) A study of seed dormancy in eighteen species of high altitude Colorado
plants. Butler University Botanical Studies 18, 74-84.
Peuelas, J. and Boada, M. (2003) A global change-induced biome shift in the Montseny
mountains (NE Spain). Global Change Biology 9, 131-140.
Pigott C. D, Pigott, S. (1993) Water as a determinant of the distribution of trees at the
boundary of the Mediterranean zone. Journal of Ecology 81, 557566.
Primack, R. B. and Kang, H. (1989) Measuring fitness and natural-selection in wild plant-
populations. Annual Review of Ecology and Systematics 20, 367-396.
SAS Institute, Inc. (1996) SAS/STAT Software: Changes and Enhancements through
Release 6.11. Cary, N. C.
Shimono, Y. and Kudo, G. (2005) Comparisons of germination traits of alpine plants
between fellfield and snowbed habitats. Ecological Research 20, 189-197.
Venables, W. N. and Ripley, B. D. (1998) Modern Applied Statistics with S-PLUS.
Springer-Verlag, Berlin, Germany.
Von Ende, C. N. (2001) Repeated-measures analysis. Growth and other time-dependent
measures. In Design and Analysis of Ecological Experiments, 2
nd
ed. (S. M.
Scheiner and J. Gurevitch, eds.), pp. 134-157. Oxford University Press, New York.
Vucetich, J. A. and Waite, T. A. (2003) Spatial patterns of demography and genetic
processes across the species range: null hypothesis for landscape conservation
genetics. Conservation Genetics 4, 639-645.
CAPITULO 5

171
Walther, G. R., Beiner, S. and Burga, C. A. (2005) Trends in the upward shifts of alpine
plants. Journal of Vegetation Science 16, 541-548.
Weltzin, J. F. and McPherson, G. R. (1999) Facilitation of conspecific seedling
recruitment and shifts in temperate savanna ecotones. Ecological Monographs. 69,
513534.
Weltzin, J. F. and McPherson, G. R. (2000) Implications of precipitation redistribution for
shifts in temperate savanna ecotones. Ecology 81, 1902-1913.


172

Table 1. Results of GLMs for seed emergence in reciprocal sowing experiments and in
laboratory germination tests.

Experiment 1
Variable d.f. Res.
Dev.
F Prob (F) Estimate SE t-value Prob (t)
Intercept 2.871 -0.346 0.130 -2.653 0.0173
Sowing site 1 1.924 16.631 0.0015 0.523 0.130 4.013 0.0010
Seed source 1 0.850 18.872 0.0009 0.540 0.130 4.137 0.0007
Sowing site x Seed source 1 0.714 2.392 0.1478 0.202 0.130 1.547 0.1411
Experiment 2
Intercept 1.792 0.159 0.145 1.099 0.2879
Sowing site 1 1.223 7.192 0.0199 -0.390 0.145 -2.690 0.0160
Seed source 1 1.095 1.616 0.2276 -0.189 0.145 -1.308 0.2092
Sowing site x Seed source 1 0.973 1.542 0.2380 0.179 0.145 1.238 0.2334
Laboratory tests
Intercept 6.042 4.239 1.133 3.741 0.0010
Treatment 1 2.187 44.084 <0.0000 -3.050 0.736 -4.142 0.0003
Seed source 1 2.153 0.392 0.5381 -0.889 0.509 -1.744 0.0939
Treatment x Seed source 1 1.782 4.241 0.0527 0.670 0.328 2.043 0.0521

Resid. Dev.: Residual Deviance; F: test used to evaluate if selected predictors explain a
significant fraction of the deviance; Prob. (F): p value of F test; Estimate SE: coefficient
value standard error; t-value: t test for significance of the coefficients; Prob (t): p value
of t test.
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Table 2. Results of accelerated failure-time analyses (log-logistic distribution) for S. ciliata
emergence rate in reciprocal sowing experiments and in laboratory germination tests.

Experiment 1
Variable d.f. Chi-square P Estimate SE
Intercept 6833.44 <.0001 0.708 0.008
Sowing site 1 3.98 0.0460 -0.023 0.011
Seed source 1 4.11 0.0425 0.024 0.011
Scale 1 0.057 0.003
Log likelihood = 156.218
Experiment 2
Intercept 6174.17 <.0001 1.122 0.014
Sowing site 1 631.89 <.0001 -0.379 0.015
Seed source 1 4.10 0.0428 -0.030 0.015
Scale 1 0.078 0.004
Log likelihood = 111.120
Laboratory tests
Intercept 534.89 <.0001 2.657 0.114
Treatment 1 27.40 <.0001 -0.599 0.114
Seed source 2 7.94 0.0188 -0.321 0.120
Scale 1 0.480 0.024
Log likelihood = -352.855


174

Table 3. Results of GLMs for progeny viability after the first growing season, after the first
winter season and after the second growing season. Models for experiment 2 (not
showed) resulted non-significant in all cases. Seed source and interaction term could not
been computed in last two models because all seedlings from Dos Hermanas died when
planted in Laguna study site.

After first growing season
Variable d.f. Res. Dev. F Prob (F) Estimate SE t-value Prob (t)
Intercept 1.702 -2.912 0.361 -8.066 <0.0001
Sowing site 1 0.692 28.819 0.0001 1.261 0.361 3.494 0.0029
Seed source 1 0.672 0.563 0.4671 -0.411 0.361 -1.140 0.2706
Sowing site x Seed
source
1 0.448 6.402 0.0264
0.707 0.361
1.958
0.0577
After first winter season
Intercept 1.581 -3.088 0.415 -7.431 <0.0000
Sowing site 1 0.790 10.994 0.0055 1.096 0.415 2.637 0.0179
After second growing season
Intercept 1.247 -4.121 0.585 -7.043 <0.0000
Sowing site 1 0.315 34.999 <0.0000 1.867 0.585 3.191 0.0031

of t test.
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Table 4. Results of accelerated failure-time analyses (Weibull distribution) for S. ciliata
seedling survival in reciprocal sowing experiments.

Experiment 1
Variable d.f. Chi-square P Estimate SE
Intercept 1978.03 <.0001 2.138 0.048
Sowing site 1 55.39 <.0001 -0.480 0.064
Seed source 1 4.84 0.0279 0.143 0.065
Scale 1 0.450 0.026
Experiment 2
Intercept 1318.15 <.0001 2.040 0.056
Sowing site 1 0.70 0.4038 -0.052 0.063
Seed source 1 6.06 0.0138 0.152 0.062
Scale 1 0.467 0.025


176
Table 5. Results of GLM for seedling size at the end of the experiments.

Experiment 1
Variable d.f. Res. Dev. Prob (
2
) Estimate SE t-value Prob (t)
Intercept 162.428 2.426 0.068 35.615 < 0.0001
Sowing site 1 155.579 0.0088 0.146 0.072 2.014 0.0466
Seed source 1 154.858 0.3959 0.028 0.033 0.845 0.3996
Experiment 2
Intercept 183.958 2.305 0.050 45.936 < 0.0001
Sowing site 1 177.555 0.0113 0.154 0.050 3.082 0.0031
Seed source 1 171.938 0.0177 0.056 0.050 1.121 0.2665
Sowing site x Seed
source
1 151.951 < 0.0001
0.215 0.050
4.284 < 0.0001

of t test.
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Table 6. Results of GLM for differences in percentage of mortality due to summer
drought at the end of the experiment.

Experiment 1
Variable d.f. Res. Dev. F Prob (F) Estimate SE t-value Prob (t)
Intercept 1.551 1.509 0.127 11.801 <.0001
Sowing site 1 0.662 26.744 0.0002 -0.623 0.127 -4.875 <.0001
Seed source 1 0.488 5.245 0.0409 0.271 0.127 2.120 0.0429
Sowing site x Seed
source
1 0.488 0.001 0.9729
-0.004 0.127
-0.034 0.9726
Experiment 2
Intercept 1.519 1.134 0.194 5.823 <.0001
Sowing site 1 1.431 0.796 0.3897 -0.175 0.194 -0.899 0.3761
Seed source 1 1.430 0.007 0.9316 0.028 0.194 0.146 0.8848
Sowing site x Seed
source
1
1.382 0.434 0.5221
-0.128 0.194
-0.658 0.5153

of t test.

178

Figure 1. Scheme of the reciprocal sowing experiments conducted in 2003 in Sierra de
Guadarrama (Madrid, Spain). Experiment 1: reciprocal sowing between the low and
intermediate sites; Experiment 2: reciprocal sowing between the intermediate and high
sites.

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Figure 2. Evolution of volumetric soil water content during the first growing season in the
three sowing sites under study (n = 8 sampling plots in Laguna and Pealara, n = 12 in
Dos Hermanas). Grey bars denote summer precipitation at Dos Hermanas (2236 m).


180

Figure 3. Emergence rate of S. ciliata seeds. a) experiment 1: low-intermediate reciprocal
sowing; b) experiment 2: intermediate-high reciprocal sowing. Black head arrows
represent the snowmelt date on each site.
a)
b)

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Figure 4. S. ciliata seedling survival. a) experiment 1: low-intermediate reciprocal sowing;
b) experiment 2: intermediate-high reciprocal sowing. Black head arrows represent a
profuse rainfall event in mid-summer.

a)
b)

182

Figure 5. Size of surviving seedlings of the experiment 2 after second growing season
(expressed as the total number of leaves).

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Figure 6. Causes of mortality in the three sowing sites after two years.


184

CAPITULO 6

185

Captulo 6.

Population dynamics at the altitudinal rear edge may
cause range contraction of a high mountain
Mediterranean plant

Gimnez-Benavides, L.; Albert, M. J.; Escudero, A. and Iriondo, J. M.
Inedit manuscript
POPULATION DYMAMICS ALONG AN ALTITUDINAL REAR EDGE

186
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187

SUMMARY

We analyzed demographic data of a long-lived high mountain Mediterranean plant,
Silene ciliata Poirret, over a 4-year period. Selected populations were located at
contrasting altitudes within the southernmost margin of the species, representing a local
altitudinal range within the rear edge of its overall distribution. Previous studies suggest
that differences in the reproduction and performance of individuals at upper and lower
populations may have implications for population dynamics. We used matrix analysis to
asses its demographyc behaviour. Loglinear analyses were used to identify the effects of
size classes, populations and years on the performance of individuals. Variance
decomposition analyses were used to identify the life history stages more relevant to the
observed variance in population growth rate between populations. Transition matrices
revealed great spatio-temporal differences in demographic traits. Seedling recruitment
was very low all years and populations. Plant size strongly affected survival, growth rate
and inflorescence production. Smaller plants at the peripheral, low population grew faster
than smaller plants at the central one, and the contrary was observed for larger plants. S.
ciliata individuals suffered a serious reduction in their life span comparetd to other long-
lived alpine species, mainly as a consequence of an extremely high adult mortality.
Population growth rate () showed a declining trend at the lowest altitude and a
population dynamic close to stability at Dos Hermanas. Our results suggest that rear
edge populations of S. ciliata at Sierra de Guadarrama are suffering demographic
processes that may force them to an altitudinal range displacement.


188
INTRODUCTION

It has been recently well-established the relevance of species peripheral
populations as important reservoirs of intraspecific genetic diversity and evolutionary
potential (Lesica & Allendorf, 1995; Hampe & Petit, 2005; Jump & Peuelas, 2005), as
well as functionally drivers of ecosystem stability (Eriksson, 2000). Current climate
warming and other anthropogenic impacts such as habitat fragmentation and land use
change are currently threatening peripheral populations, causing the species range
contraction (Lesica and McCune, 2004). However, while demographic and evolutionary
traits underlying the expansion of species at their leading edge have been extensively
studied in last years (Petit et al., 2004), population dynamics responsible for range
contractions at the rear margins have received insufficient attention (Hampe and Petit,
2005).
Under current climatic conditions, populations at rear edge of the species ranges
(southernmost populations in the northern hemisphere) are usually constrained by water
availability (Pigott and Pigott, 1993; Eriksson, 1996; Garca et al., 1999, Castro et al.,
2004, 2005; Breshears et al., 2005), and are characterized by small population sizes and
a high degree of isolation, leading to reduced within-population genetic diversity but
enhanced among-population genetic differentiation (Hampe and Petit, 2005). It is also
expected that rear edge populations in long-lived species should be affected by
population aging, and their long-term persistence may be only feasible by a prolonged
longevity of established individuals rather than by seeding regeneration (Garca, 2003;
Garca and Zamora, 2003; Hampe and Petit, 2005). Therefore, besides assessing
reproductive status and recruitment rates, long-term demographic monitoring is needed to
ascertain the viability of these populations.
In mountain plants, conditions for regeneration and survival are hierarchically
arranged within their distribution range; firstly, they are more suitable in the centre of
distribution area than in the periphery (Lawton, 1993; Vucetich and Waite, 2003) but,
secondly, they also appear structured within each mountain island. As occur with the
latitudinal range displacements, recent upward shifts in the abundance and distribution of
species inhabiting mountain environments have been documented during the last decade
(Grabherr et al., 1994; Kullmann, 2002; Klanderud and Birks, 2003; Sanz-Elorza et al.,
2003; Dullinger et al. 2003; Wilson et al., 2005) but once more, few studies have
empirically documented the population-level dynamics driving such displacements
(Diemer, 2002; Doak and Morris, 1999).
The Mediterranean Basin is known as an extraordinary hotspot of relict
populations of temperate taxa, harbouring large amounts of the species genetic diversity
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189
(Petit et al., 2003; 2005). Among other factors, the presence of the Mediterranean sea
together with southern mountain ranges of the continent has historically shaped the
current distribution of large number of species, from animals to plants (Hewitt, 2004). For
instance, many arctic and alpine species find their southernmost limits in isolated refuges
at the summits of Mediterranean mountains (e.g. Sierra Nevada, Pauli et al., 2003).
In the present work, we analyzed demographic data of a long-lived high mountain
Mediterranean plant, Silene ciliata Poirret, over a 4-year period. Selected populations
were located at contrasting altitudes within the southernmost margin of the species, thus
representing a local altitudinal range within the rear edge of its overall distribution.
Several features of the reproductive cycle along this altitudinal gradient suggest that
differences in the reproductive behaviour and performance of individuals at upper and
lower populations may have implications for population dynamics (Gimnez-Benavides et
al., 2006). Specifically, questions addressed here were: (1) What are the demographic
processes driving population dynamics at the altitudinal rear edge of S. ciliata in Sierra de
Guadarrama? (2) Are upper and lower populations within an altitudinal gradient suffering
the same demographic processes? (3) What is the temporal variability in factors
determining population growth rate at different altitudes?

METHODS

Plant species and study site
Silene ciliata Poirret (Caryophyllaceae) is a long-lived perennial plant that grows in
main mountain ranges of the Balkan Peninsula, the Appenines, the Massif Central in
France and the northern half of the Iberian Peninsula, covering a latitudinal range from
40 N to 46 N (Tutin et al., 1995). In Central Spain, where the species reaches its
southernmost margin, it grows from ~1900 m up to the highest summits (ca. 2600 m).
The species typically grows in cushion, compact rosettes. Its flowering period extends
from late June to early-middle September. Flowering stems (from 1 to 33 per reproductive
plant) have ~15 cm in height and bear 1 to 5 flowers. Fruit capsules have up to 100 seeds
that are dispersed by stalk vibration. Seeds are relatively small (1.53 0.49 mm in
diameter, 0.59 0.06 mg in weight) and nearly all require a cold-stratification period or
contrasting high temperatures to germinate (Gimnez-Benavides et al., 2005). No
evidence of vegetative propagation has been observed in this species (Gimnez-
Benavides, pers. obs.).
The area of study was in the Sierra de Guadarrama (Circo de Pealara Natural
Park), a SW-NE running mountain range located in Central Spain, 50 km north of Madrid
city (40
o
N, 3
o
W). Two S. ciliata populations were selected for the study. The first

190
population was located in the vicinity of Laguna Chica (hereafter Laguna), a small glacial
lake situated in a Pleistocene moraine deposit in the timberline zone (1970 m).
Vegetation is dominated by a shrub matrix of Cytisus oromediterraneus and Juniperus
communis subsp. alpina intermingled with Pinus sylvestris. The second population was
located in the Dos Hermanas peak (hereafter Dos Hermanas), a summit flat area situated
at 2250 m. Here, the Cytisus-Juniperus shrub formation is replaced by a patchy
xerophytic fellfield dominated by Festuca curvifolia. This fellfield community supports
extreme winds and a relatively short snowcover period, and is characterized by a high
diversity of cushion plants (Escudero et al. 2004). Mean annual precipitation at
Navacerrada Pass weather station (1800 m, 8 km southwest of study site) is 1350 mm,
and is concentrated from late autumn to early winter. A marked drought season (less than
10% of the total annual rainfall) occurs from late May to October. Snowfall generally
begins in October, and snow-free season begins in May-June (Palacios et al. 2003).
Laguna population conforms the lowest altitudinal margin of S. ciliata in Sierra de
Guadarrama, and Dos Hermanas can be considered a central-altitudinal population within
this mountain. The uppest S. ciliata population is found in the summit of the highest peak
of the mountain range, at 2.440 m.
One permanent plot was established in each population. At Laguna, we initially
monitored all plants available within a subpopulation (128 individuals), while at Dos
Hermanas we took a sample size of 266 individuals within a larger, continuous
population. All plants found within each plot were drawn in detailed maps to allow
subsequent location. Plants were monitored every year at the end of the reproductive
season (September-October). Plant size was estimated as maximum diameter of the
cushion. Total number of inflorescences per plant was counted as an estimate of
reproductive output. Seedlings of S. ciliata emerge at the beginning of the growing
season, suffering an extremely high mortality during summer (Gimnez-Benavides et al.,
2006). Thus, all seedlings found within the plots at the end of the growing season were
registered as a basis to estimate recruitment rates. Data were collected from 2002-2005
at Dos Hermanas and from 2003-2005 at Laguna but number of inflorescences could only
be estimated in 2003 and 2005, due to adverse climatic conditions.

Matrix construction
We constructed Lefkovitch matrices with four stage classes, one vegetative class
and three reproductive classes. The first corresponded to the seedling class, i.e., plants
of about 1 cm in diameter, germinating the previous year and growing into a different
class during the next period. Reproductive classes were obtained by classification of all
individuals in the population, except seedlings, using k-means clusters. Number of
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191
inflorescences and plant size were entered as variables of interest. Optimum number of
reproductive clusters obtained was three: small (plants of 1.5-5.5 cm in diameter),
medium (6-11 cm) and large (>11 cm). Although it would be preferable to use a larger
number of classes to detect more real changes in survival with size (Morris and Doak
1998), our size-stage partition adjusted well to the size structure of the populations.
Transition matrix elements describing survival (stasis) and growth are not independent,
and thus the number of stages used influences their relative contribution to the population
growth rate (Enright et al. 1995). Anyway, as our aim was to study the demographic
behaviour of Laguna and Dos Hermanas plants especially in comparative terms, we
rested importance of possible increases in the apparent relevance of stasis.
Fecundity values were estimated as follows. Mean number of inflorescences per
class was used to calculate the proportional contribution of each adult class to total
reproductive effort, measured as the sum of the mean number of inflorescences per
class. Reproductive effort was calculated for 2003 and 2005, and mean values were used
to estimate reproductive effort in 2004 (and in 2002 for Dos Hermanas).
Fecundity values for each reproductive class and time interval was estimated
following the equation:
=
+
+
=
3
1
, ,
, 1
1 , ,
) * (
* sdl

i
t i t i
t i t
t t i
n R
R
F ,

where F
i,t,t+1
is the fecundity value of class i (small, medium or large) for the period
t to t+1, sdl
t+1
is the total number of seedlings censused in the population in time t+1, R
i,t

is the reproductive effort of class i, and n
i,t
is the total number of individuals in class i and
time t. Thus, seedlings counted the following year were allocated among the three
reproductive classes according to their proportional reproductive effort in the previous
year.
These estimations assumed that seedlings in time t+1 germinated from seeds
(inflorescences) produced in time t, as occurs in absence of a permanent soil seed bank.
Field and lab assays showed that germination in S. ciliata averages more than 70%
during one year period (Gimnez-Benavides et al., 2005 and unpublished data). Thus, we
assumed that soil seed bank does not play an important role in the dynamic of S. ciliata
populations.
Transition matrix models project population size according to the equation:

x(t+1) = Ax(t),


192
where x is a vector of the number of individuals in different plant stages (stage
distribution at time t) and A is a matrix of probabilities and fecundities that defines the
survival and reproduction of individuals in each stage between time t and time t+1 (i.e.,
matrix elements). The transition matrix A is derived from a life cycle graph that shows the
possible transitions between stages (Caswell 2001). The life cycle graph for our model
system is shown in Figure 1.

Basic demographic traits: survival, growth and fecundity
Growth rate of individual plants was calculated as size (t, t+1) / size (t). Plant
longevity was estimated following Forbis and Doak (2004), using a starting vector of one
seedling and zero adults and multiplying this vector by each matrix with all fecundities set
to zero (Caswell 2001). This iterative process was done until the summed probability of
survival for all stage classes reached 0.01. Longevity was estimated for each population
separately using the mean matrix for all transition intervals. Mean time spent in each
class can be calculated from mean annual size increases in each stage class.

To assess the dependence of flowering on plant size, and whether it varies
between populations and years, we modelled flowering probability by fitting Generalised
Linear Models. First, a complete model was built setting plant size, population and year
as explanatory variables. Second, to estimate size-dependent flowering probability curves
a single model was built for each population and year separately. We plotted the
flowering probability curves using the estimated parameter for plant size and the formula:

( )
x
e
P
+
+
=
1
1

where is the intercept with the X-axis, which is related to the threshold size for
reproduction, and is the slope of the curve, which is related to the percentage of
reproductive plants (Mndez & Karlsson 2004). Since the response variable was a
probability ranging from 0 to 1 (i.e. not flowering or flowering), we used a binomial error
distribution and a logit link function, setting the variance to mean (1-mean) (Venables
and Ripley, 1998). Effects of fixed factors were tested with F-tests (Gimnez-Benavides
et al. 2006). Models were carried out using S-Plus 2000 (MathSoft, Inc., 1999) and curves
were plotted using Sigma Plot 8.0.

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193
Analyses of plant fates
Loglinear analyses were used to identify stage, population and time effects on the
performance of individuals (Caswell 2001). Individual transitions were estimated from
counts of observed transitions. These data compound a four-way table of counts, were
n
ijkl
is the number of individuals arriving at fate i from initial stage j at time k in population l.
Impossible transitions were excluded from the analyses by assigning them structural
zeros. Loglinear models were written by including or excluding the effects of the
explanantory categorical variables [stage S (stage
t
, four stage classes), time T (time
intervals), population L (two population), and their interactions] on the response variable
fate F (stage
t+1
, four stage classes + dead). In another set of analyses, we examined the
effects of time and population on fate for each of the four stages individually. Time
intervals included in the analysis were 2003-2004 and 2004-2005. Following Caswell
(2001), we considered only hierarchical models, in which the presence of a given
interaction implied the presence of all lower-level interactions involving those factors.
Hypotheses were tested with likelihood ratio tests (G
2
). Models were ranked according to
the Akaike information criterion (AIC) in order to choose the model that fitted the data
best. The best loglinear model is the one that minimizes AIC. The AIC was calculated as
a relative value, and criteria for accepting or rejecting models followed Caswell (2001).
Analyses were performed with SPSS 13.0.

Analyses of historical effects
In perennial plants, past events such as morphological development, resource
allocation patterns, or past evironmental conditions can influence the performance of
individuals and their current rates of growth, fecundity and survivorship (Hughes and
Jackson 1985, Peterson and Black 1988, Karlsson et al. 1990, Briske and Anderson
1992, Geber et al. 1997, Rose et al. 1998, Ehrln 2000). Thus, several evidences show
that individual history is not irrelevant (Tanner et al. 1996) and transition probabilities may
significatively change as a function of individuals prior status or demographic history.
However, few studies have included the history of individuals to examine effects at the
population level (Bullock et al. 1995, Crone 1997, Rose at al. 1998), and little is known
about how historical effects influence population dynamics in most perennial plants
(Ehrln 2000, Menges and Quintana-Ascencio 2004).
We aimed to include historical effects in order to understand long-term population
dynamics of S. ciliata. Thus, population dynamics with and without considering one years
history were compared. In this case, loglinear models were written by including or
excluding effects of the explanatory variables stage S, population L and history H, on the
response variable fate F. History was a factor of the stage of individuals in time t-1

194
(stage
t-1
, four stage classes). Seedling stage was excluded for the analyses because it
has no possible history. Analyses were afterwards performed for each reproductive stage
separately.

Population dynamics
The dominant eigenvalue for each transition matrix was used to calculate the finite
rate of increase, . Bootstrapped matrices were generated by randomly sampling
individuals with replacement within stage classes. Two-thousand bootstrapped transition
matrices were used to obtain 95% confidence intervals for .
Population parameters calculated from projection matrices also include matrix
element sensitivities (s
ij
) and elasticities (e
ij
). We used elasticities to explore the relative
importance of different stages of the life cycle on the population dynamic. Elasticities (e
ij
=s
ij
*a
ij
/) measure the effects of proportional changes in matrix element a
ij
on (de Kroon
et al. 1986, Caswell 2001). We also conducted perturbation analysis of the elasticity
patterns observed, in order to know how elasticities themselves responded to changes in
matrix elements (Caswell 1996). This analysis allows to solve the problem of situational
elasticity sensu Stearn (1992), and requires calculation of the sensitivities of the
elasticities, i.e., of the second derivatives of the rate of increase with respect to the matrix
elements (Caswell 1996). Analyses were performed on the mean elasticity matrix for
each population.

Variance decomposition analysis
Variance decomposition analyses were used to identify the life history stages more
relevant to the observed variance in population growth rate between populations.
Contrary to prospective analyses (sensitivity and elasticity), this type of retrospective
methods looks back at an observed pattern of variation in vital rates and asks how that
pattern has affected the variation in (Caswell 2000).
We focused in a variance decomposition approach in a random design, with the
aim of decomposing the variance in into contributions from the variances in (and
covariances among) the matrix entries (Brault and Caswell 1993, Caswell 2001). The
variability in generated by the two different sets of matrix elements (from Laguna and
Dos Hermanas) was characterized by the variance:

V()

+
kl if
kl if kl if
if
if
if
s s a a Cov s a J ) , ( ) (
2

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195
where V (a
ij
) is the variance in the ijth matrix entry, Cov (a
ij
, a
kl
) denotes the
covariance between the ijth and klth matrix entries, and s
ij
and s
kl
are the sensitivities
evaluated at the mean matrix. All matrix analyses were performed with MATLAB 6.1
(MatWorks 1997).

RESULTS

Demographic traits
Throughout the four-year study period, the fates of 1033 plants in two populations
were followed. S. ciliata individuals were significantly larger at the lowest altitude, all
years studied (Table 1). Size structure differences can be observed in Figure 2. The great
majority of plants composed the small stage class at Dos Hermanas, while plants at
Laguna population integrated the small and medium classes in a similar way, with a
notable proportion of individuals in the large class.
Transition matrices revealed great spatio-temporal differences in demographic
traits (see Appendix A). Adult survival was higher at Dos Hermanas for all stage classes
and all time intervals. Mean survival per class decreased at Dos Hermanas from small to
large plants, while survival was higher for medium plants at Laguna. Seedling recruitment
and seedling survival showed high variability between years and populations (Appendix
A).
Estimated plant longevity inferred from transition matrices was 115 years at Dos
Hermanas and only 23 years at Laguna. In addition to these stricking differences in plant
life span between populations, mean time spent in each class showed that plants at Dos
Hermanas stayed at the smaller class aprox. three times more than in Laguna (Figure 3).
Growth rate of small plants at Laguna doubled that of plants at Dos Hermanas, while the
negative mean growth rate of large plants was much lower at Dos Hermanas (Figure 3).
Thus, plants at Laguna grew faster and so quickly changed to another class when small,
while plants at Dos Hermanas spent longer time being small, growing faster than Laguna
plants when large.
Number of seedlings censused was quite low for all years and populations
(Laguna 2004:3, 2005:2; Dos Hermanas 2003:17, 2004:4, 2005:2). Reproductive values
increased with stage class at both populations, but globally they were higher at Dos
Hermanas due to higher seedling recruitment. However, mean inflorescence production
(number of inflorescences/maximum plant diameter) was significantly higher at the lower
population in 2005 (Table 1).


196
Minimum size at reproduction was 2 cm in diameter in Laguna and Dos Hermanas
every year. Results of the complete model of flowering probability showed that plant size
accounted for the highest change in deviance (adjusted D
2
: 0.142, P(
2
)< 0.001). There
was also a significant effect of year (adjusted D
2
: 0.062, P(
2
)< 0.001) and population x
year interaction (adjusted D
2
: 0.026, P(
2
)< 0.001), while population did not explain any
change in deviance (adjusted D
2
: -0.001, P
2
) = 0.155). Curves showed a marked
reduction in flowering probability of smaller individuals in 2003 compared to 2005, being
the drop more significantly pronounced in Laguna (Figure 4).

Loglinear models
Loglinear analyses showed significant differences among transition matrices
(Figure 5A). Differences were explained both by the effect of population and time interval.
Seedlings were always affected by population and time interval, while large plants were
never affected by these factors. The fate of small and medium plants was only affected by
time (Table 2).
In order to achieve a better understanding of the effect of these factors, we carried
out loglinear analyses for each population separately, i.e., considering only the effect of
stage and time interval on fate. Results showed a significant effect of time interval at Dos
Hermanas but not at Laguna (Figure 5B). Seedling class was the only stage affected by
time interval (Table 3).
Following AIC and AIC values, models having substantial support were FSL, STL
and FST, FSL, STL for the complete analysis (Figure 5A), suggesting that the inclusion of
the population effect is the best choice to explain the fate of S. ciliata individuals, both as
a single population effect (marginal test) or as interaction with time interval (conditional
test). Thus, there were notable differences in the performance of individuals between
populations, being such differences explained by the temporal variability of the seedling
fate at Dos Hermanas, an effect that was not observed at Laguna (Figure 5B; Table 3).

Historical effects
Loglinear models including historical effects (transition from t-1 to t) showed the
relevance of considering one years history to explain the performance of individuals
(Figure 6A). The fate of S. ciliata plants was also affected by population. However, only
small plants contributed to the total G
2
for the significant models (Table 4).
Decomposed analysis for each population showed again significant effects of
history at Dos Hermanas but not at Laguna (Figure 6B). In Laguna, small plants and, in a
lesser extent, medium plants were the only stages affected by history (Table 5). AIC
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197
values confirmed that the saturated model FSH (i.e., the effect of stage and history
included) was a good model to explain the fate of plants at Dos Hermanas (Figure 6B).
Thus, variability found in the stage of plants the previous year affected the fate of small
and medium plants but only at the highest population.

Population dynamics
Population growth rates () showed a declining trend at the lowest altitude and a
population dynamic close to stability at Dos Hermanas (Table 6). Particularly stricking
was the variability in values observed, with higher values at Laguna for the 2004-2005
transition, while the highest growth rates were obtained for the 2003-2004 transition at
Dos Hermanas.
Elasticity analyses indicated that joint matrix element elasticities representing
stasis gathered up to 80% of total elasticity (see Appendix B); growth globally accounted
for about 10%, and fecundity for less than 2%. This pattern was highly consistent among
populations and years. Stasis of small and medium plants gained the highest elasticity
values, but their relative relevance varied among years and populations.
Sensitivity analyses of the highest elasticity value for mean matrices showed that
stasis of both small and medium plants complemented each other (Table 7). Globally, the
biggest effect came from the stasis of small and medium plants themselves, although
they were positive or negative effects depending on the stage class considered. Thus, the
elasticity of stasis in the small class is large when the probability of remaining in this class
is high, while growth to another class and survival of medium plants are low. Recruitment
of small plants also have positive effects. The same pattern could be applied to medium
plants. Results did not notably differ between populations (Table 7).

Variance decomposition analysis
Total contributions to variance in explained 46% and 40% of the observed
variance for 2003-2004 and 2004-2005, respectively. Total contributions followed quite
the same pattern for the two transition intervals, although globally they were higher for the
2003-2004 period (Figure 7). Most of V () was explained by the variance in matrix entries
(sum of contributions = 0.0146 and 0.0057 for 2003-2004 and 2004-2005, respectively).
The major contribution was made by transition entries representing stasis (Figure 7A),
that covaried negatively with other matrix entries (data not shown). Contributions of
summed transitions for growth also had a positive effect. Matrix entries representing
regression contributed with a decreasing variance in . Fecundity made lower contribution
to V (), although with different sign for the two transition intervals.

198
Stage class that most contributed to V () was that of small plants (Figure 7B).
Contributions of medium and large stage classes had negative effects, and variance from
seedlings had negligible effects.

DISCUSSION

Plant survival and longevity
Our results clearly revealed contrasting demographic behaviour of S. ciliata
populations, reflecting differences in demographic traits at the individual level. However, it
must be firstly remarked that some demographic traits observed globally at both
populations denoted quite distinct demographic behaviour of S. ciliata plants relative to
other high mountain closed species. Plant size strongly affected survival, growth rate and
inflorescence production. Size-dependence of demographic fates defined as transition
probabilities has been commonly observed in a great variety of plants and environments
(Hortvitz & Schemske 1995). Surprisingly, plant size effects on demographic variables of
S. ciliata were contrary to that expected. The decreasing in plant survival as plant size
increases (especially at the central population where large plants survived, on average,
20% less than small plants; see Appendix A) is not an expected value for a long-lived
perennial. Greater adult survival has been observed in other cushion, alpine
Caryophyllaceae such as Silene acaulis (Morris and Doak 1998), Minuartia obtusiloba
and Paronychia pulvinata (Forbis and Doak 2004).
Smaller stages are expected to be more vulnerable to losses of above-ground
tissues during adverse environmental conditions (p.e. seasonal drought), and
consequently are expected to show higher rates of mortality. However, higher S. ciliata
survival for the smaller classes suggests that these individuals may have accumulated
enough reserves to survive, although those resource gains were not translated into a
greater above-ground increase. Thus, resource provisioning might have allowed them to
survive in spite of their smaller size.
Great differences in plant growth rates between populations were observed.
Smaller plants at the peripheral, low population grew faster than smaller plants at the
central one, and the contrary was observed for larger plants (Figure 3). Environmental
conditions at the lowest altitude, with a longer snow-free period (Palacios, 2003), allowed
plants to grow faster, taking advantage of favorable conditions by rapidly increasing their
size. On the other hand, longer snow-cover period at upper populations provided plants
with less favorable conditions for growth, and it seems they invested more in resource
provisioning than in vegetative growth (Krner, 1999). These different strategies may
have long term consequences, as adult plant survival was quite lower at the rear edge.
CAPITULO 6

199
Rapid growth of these plants when small might imply their weakness at maturity, while
slower increase in size and greater resource supplies for plants at upper altitude provided
them with higher survival probabilities when large. Patterns of resource allocation in S.
ciliata and its relationships with plant survival must be studied in detail.
Total longevity of S. ciliata individuals inferred from transition matrices seemed
also extremely low as compared with other Silene cushion plant species, S. acaulis
(Morris and Doak 1998). Higher values of adult mortality in S. ciliata may be partly
responsible for this fact. In that Silene species, individuals may reach more than 300
years of life, and in demographic studies carried out with them no mortality among large
plants was detected (Morris and Doak 1998). Thus, S. ciliata individuals suffered a
serious reduction in their life span, mainly as a consequence of an extremely high adult
mortality, unusual among other long-lived alpine species.

Reproduction and recruitment
The significant population x year interaction in the model of flowering probability
revealed that differences in flowering ability between populations did not remain
consistent between years. In other words, interannual differences observed at the lowest
population were higher than those at the upper one. These results agree with those
obtained by Gimnez-Benavides et al. (2006), who pointed out the manifest reduction of
reproductive individuals in extremely dry years, especially at low altitude. However, mean
inflorescence production at the lower altitude was higher in 2005 (Table 1); this was a
surprising result that we are not able to explain under current lack of knowledge
concerning resource allocation for growth and reproduction.
Seedling recruitment observed was highly variable and mean values for all years
studied were very low. This is a common feature in long-lived, high mountain species
(Forbis, 2003; Forbis and Doak, 2004). Furthermore, seedling recruitment in
Mediterranean mountains have found to be seriously limited by high seedling mortality
during summer drought (Castro et al., 2004, 2005; Cavieres et al., 2005; Gimnez-
Benavides et al., 2006). As observed from our loglinear models, temporal variability in the
seedling stage was the most important feature explaining the performance of individuals
(Table 3), and pointed out its relevance at the central population but not at the peripheral
one, where number of seedlings was much lower or absent. Using a reciprocal sowing
experiment, Gimnez-Benavides et al. (2006) detected some evidences of local
adaptation favouring S. ciliata seedling establishment along the same altitudinal gradient
used in this study. However, our matrix analyses proved that annual recruitment in S.
ciliata had a negligible effect on the population growth rate. Variance decomposition
analyses also showed that observed variance in between populations could not be

200
explained by the variability in the seedling stage nor in the fecundity vital rate (Figure 7).
As predicted by life history theory (Charnov and Schaffer 1973), perenniality or high
longevity is expected to allow persistence in an environment with high interannual climate
variation, where conditions may be often unfavorable for sexual reproduction (Morris and
Doak 1998). Furthermore, it has been argumented that demographic trends of rear edge
populations cannot simply be inferred from their current recruitment rates but better by
adult mortalities (Hampe and Petit 2005). Thus, longevity should be the main strategy to
assure long-term persistence of remnant populations while waiting for eventual
recruitment episodes (Watson et al., 1997; Wiegand et al. 2004). Globally, the situation
observed at S. ciliata populations does not agree with the general fecundity/survival
trade-off pattern (Charnov 1991, Forbis and Doak 2004), as neither recruitment nor adult
survival seems to be actually favouring population persistence.

The fate of plants: including historical effects
Loglinear models emphasized the relevance of past events on the performance of
individuals, although the effects were only manifested at the central population and not at
the rear edge. Small plants were the main stage affected by history as expected from
their lower values of plant growth rate. Hence, plants that grew into a new stage were
more likely to return to the original stage the next season than individuals with a history of
no change (data from second-order matrices not shown in this paper). The lack of effect
at the lower population may be reflecting a heterogeneous demographic behaviour within
stage classes, while the effect of history at the central population denotes common
patterns of growth rate.
These findings are in agreement with those found in Lathyrus vernus (Ehrln
2000) and reflect the capacity of individuals to buffer environmental changes.
Furthermore, changes in size during one time interval were partly reversed during the
next interval, as we found a negative Spearman rank correlation between size
t+1
and size
t

(Dos Hermanas: r=-0.379, P>0.001, n=246 for growth in 2003-2004 vs growth in 2002-
2003) that was not supported when changes two intervals later were considered (r=-
0.078, P=0.225, n=243 for growth in 2004-2005 vs growth in 2002-2003). Overall, results
outlined the relevance of considering past events for a better understanding of long-lived
plant population dynamics. It would be also interesting to include historical effects in
transition matrix models to explore their implications on finite growth rate in detail.

Population dynamics: towards an altitudinal range contraction
Elasticity analyses showed that, under the current conditions, changes in the
survival of small and medium plants would have the greatest effects on finite growth of
CAPITULO 6

201
increase, . The reliability of this vital rate was further confirmed by the perturbation
analyses of elasticities, and thus we could assert that it is not only a situational elasticiticy
(Caswell 1996). The relevance of plant survival and the negligible effect of reproduction
on have been often found in other long-lived perennials (Morris and Doak, 1998).
Since survival is the major responsible for future changes in at both populations,
it is not surprising that current variance in between populations is mainly caused by
variance in stasis and growth. Although results come from different type of analyses
(prospective and retrospective), together they highlight the relevance of individual survival
and longevity in two principal perspectives. One is their obvious role for population
persistence; the other comes from the differential population behaviour at both altitudes,
which allows detecting the processes that are driving so distinct population dynamics.
Differences in finite growth rates between populations, together with all
demographic parameters described, were large enough to suggest that the studied low
peripheral population of S. ciliata is not able to withstand actual conditions, leading it to
an inevitable local extinction. Although rate of increase at the central population was
closed to stability, processes ocurring at the individual level -especially those concerning
plant survival and total individual longevity- denoted that the same mechanisms are
ongoing, and a similar population dynamic is expected in a medium term. Following this
demographic trend as upwards, a better situation at the highest population (summit of
Pealara peak) is expected. In fact, data from a vandalized plot that could not be
monitored any longer (Gimnez-Benavides, unpublished data), reflected extremely higher
values of adult survival and a finite rate of increase = 1.03 for the 2002-2003 period.
This findings contrast with those reported by Diemer (2002), who noted that populations
of long-lived alpine species will not likely be altered substantially in response to climate
warming.
Thus, our results suggest that rear edge populations of S. ciliata at Sierra de
Guadarrama are suffering demographic processes that may force them to an altitudinal
range displacement. These populations inhabiting the trailing edge (sensu Hampe and
Petit, 2005) could become completely extirpated if processes driving current population
dynamics prevail. As upward shift is unviable, since the species actually colonizes the
higher summits of the major mountain ranges in its southern margin, range contraction
would be therefore irremediably associated with a decrease in habitat area (Pauli et al.
2003, McDonald and Brown 1992). Moreover, biotic causes apart from direct climatic
causes may be also involved in the expected habitat contraction. For instance, a current
altitudinal replacement of the high mountain xerophytic pastures (the main niche of S.
ciliata) by montane shrub species have been detected in the area of study (Sanz-Elorza
et al. 2003). As a result, those remnant populations in the shrubland-grassland ecotone

202
should be excluded by competition. Urgent demographic studies considering the upper
populations of S. ciliata are needed. Knowledge of long-term dynamics of the species in
its southernmost limit, compared to that observed at central and northern populations,
could advice us whether the species is also facing a latitudinal range contraction.

ACKNOWLEDGEMENTS

The authors specially thank Pedro Quintana-Ascencio and Xavier Pic, who kindly
provided MATLAB routines for matrix analyses. Nuria Ortega, Vera Ortega and Ral
Garca-Camacho helped with the field work, and the staff of Parque Natural de las
Cumbres, Circo y Lagunas de Pealara gave us permission to work in the area. They
also thank for helpful comments on earlier drafts of the

manuscript; and Lori De Hond
for her linguistic assistance. This work was supported by the Spanish Government CICYT
project REN2003-03366.

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REFERENCES

Brault, S. and Caswell, H. (1993). Pod-specific demography of killer whales (Orcinus
orca). Ecology 74: 1444-1454.
Breshears, D. D., Cobb, N. S., Rich, P. M., Price, K. P., Allen, C. D., Balice, R. G.,
Romme, W. H., Kastens, J. H., Floyd, M. L., Belnap, J., Anderson, J. J., Myers, O.
B. and Meyer, C. W. (2005). Regional vegetation die-off in response to global-
change-type drought. PNAS 102(42): 15144-15148.
Briske, D. D. and Anderson, V. J. (1992). Competitive ability of the bunchgrass
Schizachyrium scoparium as affected by grazing history and defoliation. Vegetatio
103: 41-49.
Bullock, J. M., Mortimer, A. M. and Begon, M. (1995). Carry-over effects on interclonal
competition in the clonal grass Holcus lanatus: a response to surface analysis.
Oikos 72: 411-421.
Castro, J., Zamora, R., Hdar, J. A. and Gmez, J. M. (2004). Seedling establishment of
a boreal tree species (Pinus sylvestris) at its southernmost distribution limit:
consequences of being in a marginal, Mediterranean habitat. Journal of Ecology 92:
266-277.
Castro, J., Zamora, R., Hdar, J. A. and Gmez, J. M. (2005). Alleviation of summer
drought boots establishment success of Pinus sylvestris in a Mediterranean
mountain: an experimental approach. Plant Ecology 181: 191-202.
Caswell, H. (1996). Second derivatives of population growth rate: calculation and
applications. Ecology 77: 870-879.
Caswell, H. and Hole, W. (2000). Matrix Population Models: Construction, Analysis, and
Interpretation., Sinauer Associates, Inc.
Cavieres, L. A., Quiroz, C. L., Molina-Montenegro, M. A., Muoz, A. A. and Pauchard, A.
(2005). Nurse effect of the native cushion plant Azorella monantha on the invasive
non-native Taraxacum officinale in the high-Andes of central Chile. Perspectives in
Plant Ecology, Evolution and Systematics 7: 217-226.
Charnov, E. L. (1991). Evolution of life history variation among female mammals.
Proceedings of the National Academy of Science, USA 88: 1134-1137.
Charnov, E. L. and Schaffer, W. M. (1973). Life history consequences of natural
selection: Cole's result revisited. The American Naturalist 107: 791-793.
Crone, E. E. (1997). Parental environmental effects and cyclical dynamics in plant
populations. The American Naturalist 150: 708-729.

204
De Kroon, H., Plaisier, A., Groenendael, J. V. and Caswell, H. (1986). Elasticity: the
relative contribution of demographic parameters to population growth rate. Ecology
67: 1427-1431.
Diemer, M. (2002). Population stasis in a high-elevation herbaceous plant under
moderate climate warming. Basic and Applied ecology 3: 77-84.
Doak, D.F. and Morris, W.F. (1998). Detecting population-level consequences of ongoing
environmental change without long-term monitoring. Ecology 80: 1537-1551.
Dullinger, S., Dirnbock, T. and Grabherr, G. (2003). Patterns of shrub invasion into high
mountain grasslands of the Northern Calcareous Alps, Austria. Arctic Antarctic and
Alpine Research 35(4): 434-441.
Ehrln, J. (2000). The dynamics of plant populations: does the history of individuals
matter? Ecology 81: 1675-1684.
Enright, N. J., Franco, M. and Silvertown, J. (1995). Comparing plant life histories using
elasticity analysis: the importance of life span and the number of life-cycle stages.
Oecologia 104: 79-84.
Eriksson, O. (2000). Functional roles of remnant plant populations in communities and
ecosystems. Global Ecology and Biogeography. 00009: 443-450.
Escudero, A., Gimenez-Benavides, L., Iriondo, J. M. and Rubio, A. (2004). Patch
Dynamics and Islands of Fertility in a High Mountain Mediterranean Community.
Arctic, Antarctic, and Alpine Research 36(4): 518-527.
Forbis, T. A. (2003). Seedling demography in an alpine ecosystem. American Journal of
Botany 90(8): 1197-1206.
Forbis, T. A. and Doak, D. F. (2004). Seedling establishment and life history trade-offs in
alpine plants. Am. J. Bot. 91(7): 1147-1153.
Garca, D. and Zamora, R. (2003). Persistence and multiple demographic strategies in
long-lived mediterranean plants. Journal of Vegetation Science 14: 921-926.
Garcia, D., Zamora, R., Hodar, J. A. and Gomez, J. M. (1999). Age structure of Juniperus
communis L. in the Iberian peninsula: Conservation of remnant populations in
Mediterranean mountains. Biological Conservation 87: 215-220.
Garca, M. B. (2003). Demographic Viability of a Relict Population of the Critically
Endangered Plant Borderea chouardii. Conservation Biology 17: 1672-1680.
Geber, M. A., de Kroon, H. and Watson, M. A. (1997). Organ preformation in mayapple
as a mechanism for historical effects on demography. Journal of Ecology 85: 211-
223.
Gimnez-Benavides, L., Escudero, A. and Prez-Garca, F. (2005). Seed germination of
high mountain Mediterranean species: altitudinal, interpopulation and interannual
variability. Ecological Research 20(4): 433-444.
CAPITULO 6

205
Grabherr, G., Gottfried, M. and Pauli, H. (1994). Climate Effects on Mountain Plants.
Nature 369(6480): 448-448.
Hampe, A. and Petit, R. J. (2005). Conserving biodiversity under climate change: the rear
edge matters. Ecol Letters 8(5): 461-467.
Hewitt, G. M. (2004). Genetic consequences of climatic oscillations in the Quaternary.
Philosophical Transactions: Biological Sciences 359(1442): 183-195.
Horvitz, C. C. and Schemske, D. W. (1995). Spatiotemporal variation in demographic
transitions of a tropical understory herb: projection matrix analysis. Ecological
Monographs 65: 155-192.
Hughes, T. P. and Jackson, J. B. C. (1985). Population dynamics and life histories of
foliaceous corals. Ecological Monographs 55: 141-166.
Jump, A. S. and Peuelas, J. (2005). Running to stand still: adaptation and the response
of plants to rapid climate change. Ecology Letters 8: 1010-1020.
Karlsson, P. S., Svensson, B. M., Carlsson, B. A. and Nordell, K. O. (1990). Resource
investment in reproduction and its consequences in three Pinguicola species. Oikos
59: 393-398.
Klanderud, K. and Birks, H.J.B. 2003. Recent increases in species richness and shifts in
altitudinal distributions of Norwegian mountain plants. The Holocene 13: 1-6.
Kullmann, L. 2002. Rapid recent range-margin rise of tree and shrub species in the
Swedish Scandes. Journal of Ecology 90: 68-77.
Lawton, J. H. (1993) Range, population abundance and conservation. Trends in Ecology
and Evolution 8, 409-413.
Lesica, P. and Allendorf, F. W. (1995). When are peripheral populations valuable for
conservation? Conervation biology 9(4): 753-760.
Lesica, P. and McCune, B. (2004). Decline of arctic-alpine plants at the southern margin
of their range following a decade of climatic warming. Journal of Vegetation Science.
15: 679-690.
McDonald, K. A. and Brown, J. H. (1992). Using montane mammals to model extinctions
due to global change. Conservation Biology 6: 409-415.
Mndez, M. and Karlsson, P. S. (2004). Between-population variation in size-dependent
reproduction and reproductive allocation in Pinguicula vulgaris (Lentibulariaceae)
and its environmental correlates. Oikos 104: 5970.
Menges, E. S. and Quintana-Ascencio, P. F. (2004). Population viability with fire in
Eryngium cuneifolium: deciphering a decade of demographic data. Ecological
Monographs 74: 79-99.

206
Morris, W. F. and Doak, D. F. (1998). Life history of the long-lived gynodioecious cushion
plant Silene acaulis (Caryophyllaceae), inferred from size-based population
projection matrices. American Journal of Botany 85(6): 784-793.
Palacios, D., de Andrs, N. and Luengo, E. (2003). Distribution and effectiveness of
nivation in Mediterranean mountain, Pealara (Spain). Geomorphology 54: 157-178.
Pauli, H., Gottfried, M., Dirnbck, T., Dullinger, S. and Grabherr, G. (2003). Assesing the
long-term dynamics of endemic plants at summit habitats. Alpine Biodiversity in
Europe. L. Nagy, G. Grabherr, C. Krner and D. Thompson. Berlin, Springer-Verlag:
95-207.
Peterson, C. H. and Black, R. (1988). Density-dependent mortality caused by physical
stress interacting with biotic history. The American Naturalist 131: 257-270.
Petit, R. J., Bialozyt, R., Garnier-Gr, P. and Hampe, A. (2004). Ecology and genetics of
tree invasions: from recent introductions to Quaternary migrations. Forest Ecology
and Management 197: 117137.
Petit, R. J., Hampe, A. and Cheddadi, R. (2005). Climate changes and tree
phylogeography in the Mediterranean. Flora 54(4): 877885.
Petit, R. M. J., Aguinagalde, I., de Beaulieu, J.-L., Bittkau, C., Brewer, S., Cheddadi, R.,
Ennos, R., Fineschi, S., Grivet, D., Lascoux, M., Mohanty, A., Mller-Starck, G.,
Demesure-Musch, B., Palm, A., Martn, J. P., Rendell, S. and Vendramin, G. G.
(2003). Glacial Refugia: Hotspots But Not Melting Pots of Genetic Diversity.
Science, 300: 1563.
Pigott, C. D. and Pigott, S. (1993). Water as a determinant of the distribution of trees at
the boundary of the Mediterranean zone. Journal of Ecology 81: 557-566.
Rose, R. J., Clarke, T. and Chapman, S. B. (1998). Individual variation and the effects of
weather, age and flowering history on survival and flowering of the long-lived
perennial Gentiana pneumonanthe. Ecography 21: 317-326.
Sanz-Elorza, M., Dana, E. D., Gonzlez, A. and Sobrino, E. (2003). Changes in the high-
mountain vegetation of the Central Iberian Peninsula as a probable sign of climate
warming. Annals of Botany 92: 273-280.
Stearns, S. C. (1992). The evolution of life histories. Oxford, England, Oxford University
Press.
Tanner, J. E. and Hughes, T. P. (1996). The role of history in community dynamics: a
modelling approach. Ecology 77: 108-117.
Tutin, T., Heywood, V., Burges, N., Valentine, D., Walters, S. and Webb, D. (1995). Flora
Europaea Vol 1-5., Electronic dataset supplied by Pankhurst, R.J.
Venables, W. N. and Ripley, B. D. (1998). Modern applied statistics with S-Plus. New
York, USA, Springer-Verlag.
CAPITULO 6

207
Vucetich, J. A. and Waite, T. A. (2003) Spatial patterns of demography and genetic
processes across the species range: null hypothesis for landscape conservation
genetics. Conservation Genetics 4, 639-645.
Wilson, R. J., Gutirrez, D., Gutirrez, J., Martnez, D., Agudo, R. and Montserrat, V.
(2005). Changes to the elevational limits and extent of species ranges associated
with climate change. Ecology Letters 8: 1138-1146.


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Table 1. Mean s.d. values for plant size (maximum diameter in cm) and inflorescence
production (a corrected value to avoid plant size bias: number of inflorescences/maximum
diameter) of Silene ciliata, each studied year. Sample sizes between brackets. Mann-
Whitney test for comparison of means between populations (Laguna=1970 m, Dos
Hermanas=2250 m).

Population
Variable, year Laguna Dos Hermanas Mann-Whitney U

Plant size, 2002 - 4.61 2.85 (256) -

Plant size, 2003 7.52 4.25 (125) 4.72 2.95 (236) U=9631.5 ***

Plant size, 2004 7.00 4.21 (106) 4.34 2.78 (258) U=8000.5 ***

Plant size, 2005 7.16 3.33 (91) 4.61 2.85 (253) U=5982.5***

Infloresc., 2003 0.10 0.22 (123) 0.15 0.26 (241) U=11942.0 ns

Infloresc., 2005 0.39 0.39 (91) 0.22 0.35 (242) U=7681.0 ***

*** P<0.00025, ns=not significant. The Bonferroni method was conducted to
adjust the significance level for multiple comparisons.

Table 2. Loglinear analysis for the effect of stage (S), population (L) and time interval (T)
on fate (transition from t to t+1) (F), for matrices decomposed by stage.

Stage
Seedling Small Medium Large
Model d.f. G
2
d.f. G
2
d.f. G
2
d.f. G
2

FS, STL 6 26.194*** 12 45.058*** 12 33.067** 12 13.003 ns
FSL, STL 4 17.620** 8 7.403 ns 8 13.199 ns 8 7.772 ns
FST, STL 4 15.617** 8 39.450*** 8 22.408** 8 11.310 ns
FST, FSL, STL 2 10.148** 4 0.955 ns 4 2.536 ns 4 6.008 ns

*** P<0.001, ** P<0.01, ns not significant.
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Table 3. Loglinear analysis for the effect of stage (S) and time interval (T) on fate
(transition from t to t+1) (F) for each population, for matrices decomposed by stage.

Stage
Model d.f. G
2
d.f. G
2
d.f. G
2
d.f. G
2

FS, ST
Laguna 4 6.820 ns 3 2.055 ns 4 4.849 ns 4 0.939 ns
Dos Hermanas 3 12.448** 3 5.348 ns 4 8.350 ns 3 6.833 ns

** P<0.01, ns not significant.

Table 4. Loglinear analysis for the effect of stage (S), history (transition from t-1 to t) (H)
and population (L) on fate (transition from t to t+1) (F), for matrices decomposed by
stage.

Stage
Small Medium Large
Model d.f. G
2
d.f. G
2
d.f. G
2

S, SHL 21 72.310*** 15 23.500 ns 9 9.920 ns
FSL, SHL 18 48.350*** 12 20.156 ns 6 4.749 ns
FSH, SHL 12 33.243** 9 4.908 ns 6 6.385 ns
FSH, FSL, SHL 9 12.400 ns 6 1.552 ns 3 0.000 ns

*** P<0.001, ** P<0.01, ns not significant.


210

Table 5. Loglinear analysis for the effect of stage (S) and history (transition from t-1 to t)
(H) on fate (transition from t to t+1) (F) for each population, for matrices decomposed by
stage.

Stage

Small Medium Large
Model d.f. G
2
d.f. G
2
d.f. G
2

FS, SH
Laguna 6 7.814 ns 6 5.835 ns 3 4.749 ns
Dos Hermanas 6 40.536*** 6 14.321 * - -

*** P<0.001, * P>0.05, ns not significant.
Analyses could not be performed for large stage in Dos Hermanas

due to lack of data.

Table 6. Confidence intervals (95%) for population growth rates () of S. ciliata, each
studied year. Intervals were generated by 2000 bootstrapped transition matrices.

Matrix transition
Population 2002-2003 2003-2004 2004-2005
Laguna - 0.8329-0.8363 0.8814-0.8844
Dos Hermanas 0.9872-0.9878 0.9820-0.9915 0.9767-0.9780

CAPITULO 6

211
Table 7. Second derivatives of the higher elasticity values of transition matrices at both
populations, calculated from the mean matrices of all time intervals.

Laguna
Stage in year t
Stasis of small plants (e
2,2
) Stasis of medium plants (e
3,3
)
Stage in year t+1 Seedling Small Medium Large Seedling Small Medium Large
Seedling -0.034 0.306 -0.589 -0.161 0.000 -0.446 0.373 -0.021
Small -0.003 1.644 0.252 -0.001 -0.025 -1.281 -0.039 -0.154
Medium -0.071 0.317 -1.281 -0.338 0.030 -0.071 1.355 0.107
Large -0.069 0.003 -1.294 -0.328 -0.008 -0.884 0.346 -0.075

Dos Hermanas
Stage in year t
Stasis of small plants (e
2,2
) Stasis of medium plants (e
3,3
)
Stage in year t+1 Seedling Small Medium Large Seedling Small Medium Large
Seedling -0.031 0.330 -0.291 -0.036 -0.002 -0.706 0.163 0.006
Small -0.032 1.078 -0.285 -0.046 0.004 -0.954 0.321 0.014
Medium -0.100 -0.971 -0.954 -0.092 0.067 0.910 0.929 0.056
Large -0.105 -1.294 -0.996 -0.092 0.025 -0.205 0.494 0.027


212

S
2,3
S
1,3
S
1,2
S
2,4
S
3,4
S
4,3
S
3,2
S
2,2
S
3,3
Seedling
Small Medium Large
F
4,1
F
3,1
F
2,1
S
4,2
S
4,4
S
2,3
S
1,3
S
1,2
S
2,4
S
3,4
S
4,3
S
3,2
S
2,2
S
3,3
Seedling
Small Medium Large
F
4,1
F
3,1
F
2,1
S
4,2
S
4,4

Figure 1. Life cycle graph of Silene ciliata. Each arrow represent a one-year transition. S
denotes survival (stasis in the same class, and growth or regression to a different class)
and F fecundity.

CAPITULO 6

213

0
10
20
30
40
50
60
70
80
90
100
I
n
d
i
v
i
d
u
a
l
s

(
%
)
Stage classes
Laguna
2003
2004
2005

0
10
20
30
40
50
60
70
80
90
100
I
n
d
i
v
i
d
u
a
l
s

(
%
)
Stage classes
Dos Hermanas
2002
2003
2004
2005

Figure 2. Size structure of S. ciliata populations for 2002 to 2005, according to stage
classes established in transition matrices. N = Laguna 2003: 128, 2004: 108, 2005: 106;
Dos Hermanas 2002: 266, 2003: 267, 2004: 264, 2005: 255.


214

Figure 3. Mean growth rate (size (t, t+1) / size (t)) of plants in each stage class, for
Laguna and Dos Hermanas populations. Mean values for the two time intervals (2003-
2004 and 2004-2005). Bars represent estandar deviations.
-0,4
-0,3
-0,2
-0,1
0
0,1
0,2
0,3
0,4
Small Medium Large
Laguna
Dos Hermanas
M
e
a
n

g
r
o
w
t
h

r
a
t
e

Stage class
CAPITULO 6

215

Plant size (max. diameter in cm)
0 2 4 6 8 10 12 14 16 18 20
F
l
o
w
e
r
i
n
g

p
r
o
b
a
b
i
l
i
t
y
0,0
0,2
0,4
0,6
0,8
1,0
Laguna 2003
Laguna 2005
Dos Hermanas 2003
Dos Hermanas 2005

Figure 4. Flowering probability curves for S. ciliata individuals at Laguna and Dos
Hermanas, in 2003 and 2005 (n = 729). Curves in 2003 were significantly different
between populations. Difference between years was also significant.


216

Figure 5. Standard loglinear analysis. a) Effect of stage (S), population (L) and time
interval (T) on fate (transition from t to t+1) (F). b) Effect of stage (S) and time interval (T)
on fate (F) for each population. ***P<0.001, **P<0.01, ns not significant. AIC=G
2
-2(df).
AIC=relative value to the best model.

FST, STL
G
2
88.786***
df28
AIC32.786
AAIC42.792
FS, STL
G
2
117.321***
df42
AIC33.321
AAIC43.327
FSL, STL
G
2
45.994**
df28
AIC-10.006
AAIC0
FST, FSL, STL
G
2
19.647 ns
df14
AIC-8.353
AAIC1.653
FSTL
G
2
0
df0
AIC0
AAIC10.006
FL, FSL
AG
2
71.33
Adf14
P0
FTL, FSTL
AG
2
19.65
Adf14
P0.1415
FL, FSL
AG
2
69.14
Adf14
P-0
FT, FST
AG
2
26.35
Adf14
P0.023
FT, FST
AG
2
28.53
Adf14
P0.012
FS, ST
G
2
14.664 ns
df16
AIC-17.336
AAIC0
FST
G
2
0
df0
AIC0
AAIC-0.979
FS, ST
G
2
32.979**
df16
AIC0.979
AAIC0
FST
G
2
0
df0
AIC0
AAIC17.336
FT
AG
2
32.979
Adf16
P0.007
FT
AG
2
14.664
Adf16
P0.5493
Laguna Dos Hermanas
b
a
CAPITULO 6

217

Figure 6. Historical effects loglinear analysis. a) Effect of stage (S), population (L) and
history (transition from t-1 to t) (H) on fate (transition from t to t+1) (F). b) Effect of stage
(S) and history (transition from t-1 to t) (H) on fate (F) for each population. **P<0.01, ns
not significant. AIC=G
2
-2(df). AIC=relative value to the best model.

FSH, SHL
G
2
44.536 ns
df36
AIC-27.464
AAIC18.215
FS, SHL
G
2
106.795**
df69
AIC-31.205
AAIC14.474
FSL, SHL
G
2
74.321 ns
df60
AIC-45.679
AAIC0
FSH, FSL,
SHL
G
2
13.952 ns
df27
AIC-40.048
AAIC5.631
FSHL
G
2
0
df0
AIC0
AAIC45.679
FL, FSL
AG
2
32.47
Adf9
P0.0001
FHL, FSHL
AG
2
13.95
Adf27
P0.9817
FL, FSL
AG
2
30.58
Adf9
P0.0003
FH, FSH
AG
2
60.37
Adf33
P0.0025
FH, FSH
AG
2
62.26
Adf33
P0.0015
FS, SH
G
2
18.398 ns
df18
AIC-17.602
AAIC0
FSH
G
2
0
df0
AIC0
AAIC-0.857
FS, SH
G
2
54.857**
df27
AIC0.857
AAIC0
FSH
G
2
0
df0
AIC0
AAIC17.602
FH
AG
2
54.86
Adf27
P0.0011
FH
AG
2
18.40
Adf18
P0.4297
Laguna Dos Hermanas
b a

218

Figure 7. Total contributions (variance in and covariance among matrix entries) to the
variance in , V (), from transitions in each time interval. a) Contributions to V () are
grouped per vital rate, i.e., joint matrix entries representing fecundity, growth, regression
and stasis. b) Contributions to V () are grouped per stage class.
Vital rate
a
-0,004
-0,002
0
0,002
0,004
0,006
0,008
0,01
0,012
Fecundity Growth Regression Stasis
C
o
n
t
r
i
b
u
t
i
o
n

t
o

)

2003-2004
2004-2005
Stage class
b
-0,004
-0,002
0
0,002
0,004
0,006
0,008
0,01
0,012
Seedling Small
Medium Large
C
o
n
t
r
i
b
u
t
i
o
n

t
o

)

2003-2004
2004-2005
CAPITULO 6

219
APPENDIX

Appendix A. Transition probabilities of Silene ciliata individuals
from year t to year t+1. Nonexisting matrix entries are indicated by
dashes.

Laguna 2003-2004
Stage in year t
Stage in year t+1 Seedling Small Medium Large
Seedling - 0,000 0,017 0,091
Small 0,000 0,711 0,220 0,045
Medium 0,000 0,133 0,542 0,182
Large - 0,000 0,085 0,455

Laguna 2004-2005
Stage in year t
Seedling - 0,000 0,023 0,063
Small 0,333 0,630 0,116 0,063
Medium 0,667 0,130 0,744 0,250
Large - 0,022 0,047 0,500

Dos Hermanas 2002-2003
Stage in year t
Seedling - 0,026 0,118 0,333
Small 1,000 0,840 0,153 0,000
Medium 0,000 0,115 0,671 0,333
Large - 0,000 0,059 0,333

Stage in year t
Seedling - 0,006 0,025 0,111
Small 0,824 0,919 0,375 0,111
Medium 0,000 0,056 0,575 0,444
Large - 0,000 0,025 0,333

Stage in year t
Seedling - 0,000 0,017 0,167
Small 0,000 0,866 0,183 0,000
Medium 0,000 0,113 0,767 0,000
Large - 0,000 0,017 0,833

220
Appendix B. Elasticity values for the transition matrices of S. ciliata.
Nonexisting matrix entries are indicated by dashes.

Laguna 2003-2004
Stage in year t
Seedling - 0.000 0.000 0.000
Small 0.000 0.549 0.090 0.004
Medium 0.000 0.094 0.204 0.015
Large - 0.000 0.020 0.024

Laguna 2004-2005
Stage in year t
Seedling - 0.000 0.017 0.007
Small 0.006 0.158 0.052 0.004
Medium 0.019 0.054 0.559 0.029
Large - 0.008 0.033 0.054

Stage in year t
Seedling - 0.017 0.031 0.008
Small 0.057 0.549 0.040 0.000
Medium 0.000 0.079 0.186 0.008
Large - 0.000 0.016 0.008

Stage in year t
Seedling - 0.004 0.003 0.001
Small 0.008 0.805 0.047 0.001
Medium 0.000 0.051 0.075 0.002
Large - 0.000 0.003 0.002

Stage in year t
Seedling - 0.000 0.000 0.000
Small 0.000 0.593 0.070 0.000
Medium 0.000 0.070 0.267 0.000
Large - 0.000 0.000 0.000

Cambio Climático en La Alta Montaña Mediterránea. Ecología Reproductiva, Potencial Adaptativo y Viabilidad Poblacional de Silene Ciliata.

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Cambio Climático en La Alta Montaña Mediterránea. Ecología Reproductiva, Potencial Adaptativo y Viabilidad Poblacional de Silene Ciliata.

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Tesis Doctoral

Cambio climtico en la alta montaa mediterrnea.

8 Users Reference Guide. Chicago:

(mesh 60-80) and

Analyses could not be performed for large stage in Dos Hermanas

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