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P o l l i n a t i o n b i o l o g y o f t h re e B r a z i l i a n s p e c i e s o f M i c r a n t h o c e r e u s B a c k e b . ( C e re e a e , C a c t o i d e a e ) e n d e m i c t o t h e c a m p o s r u p e s t re s
Lidyanne Yuriko Saleme Aona1, Marlon Machado2, Emerson Ricardo Pansarin1, Cibele Cardoso de Castro3, Daniela Zappi4 and Maria do Carmo E. do Amaral1
Dept. de Botnica, IB, Universidade Estadual de Campinas, Caixa Postal 6109, Cidade Universitria s.n., 13084-970, Campinas-SP, Brazil (email: lidyanne.aona@gmail.com). 2 University of Zurich, Institute for Systematic Botany, Zollikerstrasse 107, CH-8008, Zurich, Switzerland. 3 Departamento de Biologia, Universidade Federal Rural de Pernambuco R. Dom Manoel de Medeiros, s/n - Dois Irmos, 52171-900, Recife-Pernambuco, Brazil. 4 Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AE, UK.
1

Summary: The genus Micranthocereus includes nine endemic Brazilian species distributed in the states of Bahia, Minas Gerais, Gois and Tocantins. In the present study the floral biology of M. flaviflorus, M. steckeri and M. purpureus from campo rupestre vegetation of the Chapada Diamantina, Bahia, was investigated. Data on floral morphology and nectar production (concentration and volume) were collected throughout anthesis. Flowers of all species are tubular and present coloured perianth-segments. Nectar is accumulated within a nectar-chamber, located inside the floral tube. Flowers of M. flaviflorus and M. streckeri are predominantly nocturnal, opening in the night (at about 7 p.m.). Each flower lasts c.36 hours, with a concentration of sugar in the nectar between approximately 1824%. Both species present floral features typical for ornithophily, and were pollinated by the hummingbird Clorostilbon aureoventris, whose visits occurred in regular intervals and were characterized by territorialist behaviour. Species of bees and butterflies were also observed visiting flowers of M. flaviflorus and M. streckeri. M. streckeri was additionally visited by the hummingbird Phaethornis squalidus, mainly in the afternoon. Flowers of M. purpureus are nocturnal, and each flower lasts c.17 hours and present longer corollas with wider diameter than those of the other two species studied; sugar concentration in the nectar was about 2122%. Three

sphingid and one bat species were observed visiting flowers of M. purpureus, with a greater frequency of visits of the sphingids. Pollen viability was high (8090%) and similar between the three species of Micranthocereus studied.

Zusammenfassung: Die Gattung Micranthocereus umfasst neun in Brasilien endemische Arten, und ist in den Bundesstaaten Bahia, Minas Gerais, Gois und Tocantins verbreitet. In der vorliegenden Arbeit wurde die Bltenbiologie der Arten M. flaviflorus, M. purpureus und M. streckeri aus den campos rupestres (Felssavannen) der Chapada Diamantina (Bahia) untersucht. Whrend der gesamten Blhphase wurden Daten ber Bltenmorphologie und Nektarproduktion (Konzentration und Volumen) gesammelt. Die Blten aller Arten sind rhrenfrmig and haben ein farbiges Perianth. Der Nektar sammelt sich in der Nektarkammer, die sich im Inneren der Bltenrhre befindet. Die Blten von M. flaviflorus and M. streckeri sind hauptschlich nachts geffnet. Die Anthese beginnt abends gegen ungefhr 19.00. Eine Blte dauert etwa 36 Stunden und hat eine Zuckerkonzentration im Nektar zwischen 1824%. Beide Arten weisen Bltenmerkmale auf, die typisch fr Vogelblumen sind. Sie wurden von der Kolibriart Clorostilbon aureoventris bestubt, dessen Besuche in regelmssigen Intervallen erfolgten und durch

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territoriales Verhalten gekennzeichnet waren. Ausserdem wurden verschiedene Arten von Bienen und Schmetterlingen bei Bltenbesuchen von M. flaviflorus und M. streckeri beobachtet. Besonders am Nachmittag wurde M. streckeri zustzlich von der Kolibriart Phaethornis squalidus besucht. M. purpureus ist nachtblhend, und jede Blte bleibt etwa 17 Stunden geffnet. Ihre Bltenrhren sind lnger und weisen auch einen grsseren Durchmesser auf als diejenigen der beiden anderen Arten. Die Zuckerkonzentration des Nektars betrug 2122%. Drei Arten von Schwrmern und eine Fledermausart wurden bei Bltenbesuchen beobachtet, wobei die Schwrmer hufigere Besuche zeigten als die Fledermuse. Die Lebensfhigkeit des Pollens aller drei untersuchten Arten war hoch und betrug ca. 8090%.

diurnas e a concentrao de acares varia entre 21 e 22%. Foram observadas visitas de trs espcies de esfingdeos e de uma espcie de morcego. Foi observada uma maior freqncia de visitas dos esfingdeos em relao ao morcego. O teste de viabilidade polnica foi semelhante entre as espcies estudadas (8090%). Introduction The family Cactaceae forms a monophyletic group (Wallace & Cota, 1996) with approximately 100 genera and 1300 species distributed nearly exclusively in the New World (Barthlott & Hunt, 1993). Eastern Brazil presents the third main geographical centre of diversity of the family (Zappi, 1994; Taylor & Zappi, 2004). The core geographical area defined within Eastern Brazil has been recognized primarily for its remarkable endemism of Cactaceae genera and species, especially those of the caatingas of the North-eastern states and associated highland known as campos rupestres (Taylor & Zappi, 2004). Cactaceae present a wide range of floral features regarding shape, colour, size, odour, time of anthesis and position of the reproductive parts, which indicate pollination by several groups of biotic agents (Porsch, 1939; Hunt & Taylor, 1990; Vogel, 1990), like medium-sized (McFarland et al., 1989) and large bees (Viana et al., 2001), sphingids and other moths (Suzan et al., 1994; Holland & Fleming, 1999), hummingbirds (Scobell & Scott, 2002), bats (Ruiz et al., 1997; Tschapka et al., 1999), or even nocturnal and diurnal vectors in the same species, such as by hummingbirds and bats (Fleming et al., 1996; Sahley, 2001). According to Vogel (1990), around 9% of the species of Cactaceae are pollinated by hummingbirds. Other studies on Cactaceae also reported several reproductive strategies like selfcompatibility (Bianchi et al., 2000) and selfincompatibility (Boyle, 1996; Metz et al., 2000), as well as the presence of sexual and asexual reproduction in the same species (Mandujano et al., 1998; Negron-Ortiz, 1998). The Brazilian genus Micranthocereus Backeb. (Cactoideae, Cereeae) includes nine species distributed in the states of Minas Gerais, Bahia, Gois and Tocantins (Taylor & Zappi, 2004). Studies concerning the reproductive biology of Brazilian Cactaceae recorded melitophily in Opuntia Mill. (Schlindwein & Wittmann, 1995, 1997), sphingophily in Cereus Mill. (Silva & Sazima, 1995; Locatelli & Machado, 1999a) and Selenicereus (Berg.) Britton & Rose (Barthlott et

Resumo: O gnero Micranthocereus inclui nove espcies endmicas do Brasil e restritas aos Estados da Bahia, Minas Gerais, Gois e Tocantins. Foi estudada a biologia floral de M. flaviflorus, M. steckeri e M. purpureus em campos rupestres na Chapada Diamantina, BA. Anlises da morfologia floral e medies do volume e concentrao do nctar desde a antese at o pr-fenescimento das flores foram realizadas. As flores das trs espcies estudadas so tubulares, apresentando segmentos do perianto diversamente coloridos. O nctar armazenado na cmara nectarfera, localizada no interior do tubo floral. As flores de M. flaviflorus e M. streckeri apresentam antese noturna, abrindo no incio da noite (aproximadamente 7 horas da noite). Cada flor dura cerca de 36 horas e apresenta concentrao de acares entre 18 e 24%. Ambas as espcies apresentam caractersticas tpicas de flores polinizadas por aves e so visitadas e polinizadas pela mesma espcie de beija-flor, Clorostilbon aureoventris. As visitas ocorreram em intervalos regulares e o beija-flor apresentou comportamento territorialista, quando dois indivduos visitavam simultaneamente as flores. Visitas de abelhas e borboletas tambm foram observadas nestas espcies. M. streckeri tambm foi visitado por outro beija-flor, Phaethornis squalidus, que efetuou um maior nmero de visitas no perodo vespertino. As flores de M. purpureus apresentam antese noturna, e cada flor dura aproximadamente 17 horas. O comprimento e dimetro floral so superiores aos das espcies

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al., 1997), ornithophily in Melocactus Link & Otto and Opuntia (Locatelli & Machado, 1999b; Raw, 1996), and chiropterophily in Pilosocereus Byles & Rowley (Locatelli et al., 1997). The reproductive biology of Micranthocereus species is unknown, but the genus presents great diversity in flower morphology. The main goal of this study was to investigate and compare the pollination biology of three Brazilian Micranthocereus species: M. flaviflorus Buining & Brederoo, M. streckeri van Heek & van Criekinge and M. purpureus (Grke) Ritter endemic to northeast Brazil.
Material and Methods The floral biology of the species studied was investigated in the Chapada Diamantina (Bahia State), a mountain complex that presents a large diversity of Cactaceae species (Machado, 1999; Taylor & Zappi, 2004) and which is located in the Cadeia do Espinhao, a mountain range which extends over c.1000 km from NE to SE Brazil. The study was conducted in August 2001 for M. flaviflorus and M. streckeri and in October 2003 for M. purpureus. The studied population of M. flaviflorus consisted of c. 20 individuals located in the municipal district of Morro do Chapu (1050S1200S 1 and 4040W4130W) . The plants grow in quartzitic sand in an area occupied by transitional vegetation including cerrado (savanna), campo rupestre (upland or highland savanna) and caatinga (deciduous low thorn forest). The population of M. streckeri consisted of 40 individuals and was located upon rocks in an area of campo rupestre vegetation surrounded by forest areas, being endemic to the study area, in the Municipality of Seabra (122552S and 415936W). M. purpureus presents a much larger distribution range, occurring from Rio de Contas to Jacobina (Aona, 2003). The population studied consisted of 40 individuals and is located in the Municipality of Morro do Chapu (113603S and 410952W), occurring in an area of campo rupestre vegetation. The growth habit, phenology and floral features, such as morphology, colour and scent, were recorded through field observation. Floral lifespan and anthesis events were recorded for a population of each species through the observation of
1

tagged flowers from pre-anthesis until flower senescence. Fresh flowers of each species were randomly collected in a minimum of 1530 individuals/species, fixed in FAA 50, stored in alcohol 70% and used for illustrations and for floral measurements (flower-tube length and diameter, 1732 flowers/species). Floral measurements were obtained with a digital calliper (error 0.01 mm) and compared between species with an ANOVA analysis coupled with a t-Test LSD (Sokal & Rohlf, 1969). Nectar concentration and volume were estimated in flowers bagged from the pre-anthesis stage, using a pocket refractometer and microliter syringes, respectively. Data were collected c. 24 hours after the beginning of the anthesis for M. flaviflorus (n=12 flowers) and M. streckeri (n=10 flowers, five individuals for both), and 3 hours (n=10, eight individuals) and 9 hours (n=8, six individuals) after anthesis for M. purpureus. Pollen viability was estimated using the acetatecarmine technique (Radford et al., 1974, 5-7 flowers/species, 200 pollen grains/flower). Floral visitors were recorded through direct observations in the field, in a total of 10h for M. flaviflorus, 40h for M. streckeri and 40h for M. purpureus. Time and duration of visiting bouts were recorded, as well as the visitors behaviour on the flowers, such as contact with the anthers and the stigma, type of floral resource collected and interactions with other visitors. Buds/mature flowers were tagged in the afternoon and examined early in the morning for detecting possible nocturnal pollination in M. flaviflorus and M. streckeri. The hummingbirds were identified in the field and using photographs taken during their visits, then compared with ilustrations in Ruschi (1986) and Grantsau (1989). Others floral visitors of M. purpureus were identified using photos taken during their visits to the flowers; these photos were also used to check the sites of pollen deposition on their bodies. Insect visitors of all species were collected and identified as far as possible through comparison with specimens deposited in the Museum of the Universidade Estadual of Campinas (ZUEC). Pollinators were characterized based on their frequency of visits and visiting behaviour. Frequency of visits of the main floral visitors of

This population belongs to the taxon recently recognized as M. polyanthus (Werderm.) Backeb. subsp. alvinii M. Machado & Hofacker. These plants are to some degree intermediate between M. flaviflorus and M. polyanthus (Werderm.) Backeb. which are similar species (Taylor & Zappi, 2004: 355).

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3mm 3mm

Figure 1. Longitudinal section of flower of M. flaviflorus. Note the nectar chamber (arrow). Illustration by R. Lupo. each species was compared using a Students t test (Sokal & Rohlf, 1969). Voucher specimens were deposited in the Herbarium of the Universidade Estadual de Campinas (M. flaviflorus: UEC 120736, M. streckeri: UEC 139459 and M. purpureus UEC 132282). Results Morphology and floral biology The plants of all studied species are columnar, branching at the base, with decumbent or erect stems and covered with flexible, golden to pale golden spines. Flowers are born in a specialized fertile zone (cephalium), more or less perpendicular and lateral to the stem axis. The flowers present a nectariferous chamber with a nectary inside the base of the flower-tube, where the nectar is produced and accumulated (Figures 13). All the species presented high pollen viability

Figure 2. Longitudinal section of flower of M. streckeri. Note the nectar chamber (arrow). Illustration by R. Lupo. (7790%) which is similar between species (F=3.35, P=0.06). In M. flaviflorus and M. streckeri the fertile zone consists of a lateral, superficial to sunken cephalium with flower-bearing areoles, which have white to yellowish hairs with long, pale to brownish golden bristles in M. flaviflorus, and compact, white to brownish hairs and long, pale golden to reddish brown bristles in M. streckeri. In M. purpureus, the fertile portion of the stem is a lateral cephalium sunken into the stem and composed of congested flower-bearing areoles which bear pale brown, pinkish or grey, abundant, loose or compact hair and few long golden or brownish bristles. M. flaviflorus flowered from May to September and produced fruits from July to November, whereas M. streckeri produced flowers from May to September and fruits from August to December. Both species present an annual

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5mm

Figure 3. Longitudinal section of flower of M. purpureus. Note the nectar chamber (arrow). Illustration by R. Lupo. flowering pattern (sensu Newstron et al., 1994). Flowers of M. flaviflorus have outer perianthsegments spreading to erect, reddish pink; the inner perianth-segments opened very slightly and are yellow to pale cream (Figures 4 & 5); those of M. streckeri have outer perianth-segments spreading and deep pink, the inner perianth-segments are erect and purplish pink (Figures 69). The flowers of both species are tubular, the stamens are positioned inside the floral tube; the stigma is 4- to 6-lobed, and the stigma-lobes are also inserted (Figures 13). Flower length of M. flaviflorus (12.3 0.1mm, mean SE) was smaller than that of M. streckeri (13.1 1.2mm, mean SE, t=-2.618, P=0.01, n=32 for both), whereas the diameter was similar (1.8 0.2mm for M. flaviflorus, 1.8 0.9mm for M. streckeri, mean SE, t=-1.593, P>0.01, n=32 for both, 1). Flowers of M. flaviflorus and M. streckeri started to open at about 7 p.m. (510 flowers each individual daily), and lasted c.36 hours, wilting in the morning of the third day. In recently opened flowers the stigma-lobes were closed and the pollen was released; between the end of the first day and the beginning of the second day of the anthesis, the stigma-lobes opened and

became perceptibly moist. The flowers of both species were scentless and produced similar nectar volumes, whereas the concentration of sugar was higher in M. flaviflorus (Table 1). The flowering period of M. purpureus occurred from May to November, and the plants developed fruits between July and December, indicating an annual flowering pattern (sensu Newstron et al., 1994). The floral tube of M. purpureus is deep pink-magenta, the outer perianthsegments are recurved and deep pink to reddish; the inner perianth-segments are spreading or recurved, white. The stamens are numerous (c.400), being disposed as a dense ring inside the tube. The style is long, tube-like and exceeds the anthers by about 10mm; the exerted stigma is 10lobed. Flowers are larger than those of the other two species (F=793.64 for corolla length and F=239.81 for corolla diameter, P=0 for both, Table 1). Individuals of Micranthocereus purpureus opened 2-3 flowers each in the afternoon (5 p.m.), which reached full anthesis 2 hour later, remained open during the night, and were completely closed by 1011 a.m. the next morning. When the flower was completely open, the stigmas were moist (indicating receptivity), laterally or adaxially displaced; after c. 2 hours it began to move slowly towards the center of the flower, where it remained until the end of anthesis. The flowers of M. purpureus presented a slightly sweet and sour scent. Sugar concentration in the nectar decreased during the period between 8 p.m. and 2 a.m. Nectar volume was higher in M. purpureus than in the other two species (F=142.16, P=0), which presented similar volume values (P=0.21); on the other hand, the highest nectar concentration was observed in M. flaviflorus, being followed by M. purpureus and M. streckeri (F=18.29, P<0.05 for all comparisons, Table 1). Pollination process The main floral visitors of Micranthocereus flaviflorus were males of the hummingbird Chlorostilbon aureoventris Boucier & Mulsant (Figure 4), which were observed from early morning until 2 p.m. This hummingbird visited the flowers at regular intervals of approximately 1030 minutes. The females visited the flowers only sporadically, being frequently chased away by the males during the visits. Chlorostilbon aureoventris has a straight bill (19.09 2.1mm length, mean SE, n = 5), that contacted the

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Figure 4. Female of Chlorostilbon aureoventris visiting flower of M. flaviflorus. Figure 5. The butterfly Phoebis philea on flowers of Micranthocereus flaviflorus. Figure 6. Males of Chlorostilbon aureoventris visiting flower of M. streckeri. Figure 7. Males of Chlorostilbon aureoventris on flower of M. streckeri. Note the presence of pollen grains on the bill of the hummingbird. Photos by E.R. Pansarin.

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10

11

Figure 8. The hummingbird Phaethornis squalidus visiting flower of M. steckeri. Figure 9. The hesperid Vettius sp. (arrow) on flowers of M. streckeri. Figure 10. The hawkmoth Agrius cingulatus visiting a flower of M. purpureus. Note the extend proboscis being introduced in the perianth. Figure 11. The hawkmoth Erynnyis alope visiting a flower of M. purpureus. Note the extend proboscis being introduced in the perianth. Photos 8 & 9: E.R. Pansarin; 10 & 11: L.Y.S. Aona.

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Table 1 . Floral morphometrics and nectar features (mean sd for both) of three species of Micranthocereus in northeast Brazil. Different letters in the same column indicate statistically different data. Vol.: volume, Conc.: concentration. Corolla Species Length (mm) Diameter (mm) 1.76 0.25b 1.85 0.18
b

Nectar Vol (l) 5.17 2.25b 7.3 3.8

b

Conc (%) 23.33 1.28b 19.3 1.83c 21.18 1.54a

M. flaviflorus M. streckeri M. purpureus

12.35 0.99b 13.09 1.23

c

31.88 1.87a

16.35 1.49a

143.81 49.32a

anthers and the stigma when collecting floral nectar. The visitor prevented access by other individuals of the same species to the population of plants which it defended. The butterfly Phoebis philea Johansson (Figure 5) was occasionally observed visiting M. flaviflorus flowers during the hottest hours of the day (12 to 3 p.m.), when it collected nectar and contacted the sexual parts of the flower. The bee Trigona spinipes Fabricius was observed visiting flowers of M. flaviflorus mainly from 10.30 to 11 a.m. and from 2 to 5 p.m., when it collected pollen directly from the anthers, often damaging both anthers, perianth-segments and floral tube, but without contacting the stigma. Flowers of M. streckeri were also pollinated by Chlorostilbon aureoventris (Figures 6 & 7). In this species the females were more frequent as pollinators than males. The visits occurred between 6 a.m. and 4 p.m., being more frequent in the morning and occuring in intervals of approximately 30 minutes, rarely up to 60 minutes. In the afternoon the visits were rare and took place at intervals of 1hour 10 minutes, approximately. Females of C. aureoventris presented the same territorialist behaviour observed in males in M. flaviflorus, mostly during the morning, when they defended the population from males. The hummingbird Phaethornis squalidus Temminck was sporadically observed pollinating M. streckeri flowers in late afternoon (Figure 8), in a similar frequency to that of Chlorostilbon aureoventris (t=0.896, P=0.394, Figure 12); birds of this species were also chased away by females of C. aureoventris. All hummingbird species had the pollen grains deposited on the bill. In M. flaviflorus and M. streckeri, the visits by both species of hummingbird to each flower were brief (c. 2 seconds). In most visits both hummingbirds visit-

ed more than one flower of each plant. Some insects, like the skipper Vettius sp. (Lepidoptera: Hesperiidae: Hesperiinae) species (Figure 5), occasionally visited M. streckeri flowers along the day, collected nectar and sometimes touched the sexual parts of the flower with their proboscis, being considered occasional pollinators. Honey bees, Apis melifera, were observed on flowers of M. streckeri mainly in the morning, when they visited more than one flower of each individual, or even flowers of the same cephalium. They landed at the apex of the floral-tube and collected large amounts of pollen directly from the anthers, sometimes causing anther damage, and rarely contacted the stigma, thus acting mainly as pollen robbers. Trigona spinipes was also observed visiting flowers of M. streckeri at the same period of the day and presenting similar behaviour to that observed with M. flaviflorus. Other occasional visitors were some nonidentified ants, beetles and caterpillar species, which were observed cutting (ants) and eating (beetles and caterpillars) floral parts. Flowers of M. purpureus were visited by the bat Lonchophylla mordax Thomas (Phyllostomidae, Glossophaginae), which was observed after dusk until 5 a.m., in intervals of 3060 minutes, often approaching the plant from the same direction of the cephalium, hovering in front of the flowers and visiting different individuals at each bout. During its visits, this bat introduced its head inside the floral-tube, touching the stigma and receiving the pollen grains on the head, neck and shoulders. Lonchophylla mordax also visited flowers of Encholirium brachypodum L. B. Sm. & Read (Bromeliaceae) in a higher frequency (intervals of 530 minutes) than that observed for M. purpureus, but displaying a similar behaviour. The sphingids Agrius cingulata Fabricius

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(Figure10), Erynnyis alope Drury (Figure11), Erynnyis ello L. (Sphingidae) were observed visiting the flowers of M. purpureus, between 9 p.m. and 4.30 a.m., with a peak between 9 p.m. and 12 a.m. (Figure 13). The hawkmoths approached the flower and introduced the proboscis into the nectar chamber, hovering at variable distances in front of the flower. Photographs of moths foraging at flowers reveal thick deposits of pollen along their proboscis. The hawkmoths sometimes touched the flowers with their forelegs, mainly E. ello, with visits lasting up to three seconds. All hawkmoths displayed a similar visitation pattern, visiting the flowers more or less in the same sequence and at intervals varying from 530 minutes, rarely up to 60 minutes. Agrius cingulata was the most commonly observed visitor, with visits lasting up to 1520 seconds while making circular movements with the proboscis. Frequency of visits of sphingids was greater than that of the bats (t = -2.24, P=0.02, Figure 13). During anthesis a large number of small beetles were found inside the flowers of M. purpureus, remaining there even after flower senescence. They were never seen flying from flower to flower or touching the stigma. It was not possible to observe what kind of floral resource these insects explored. The flowers of M. purpureus were also visited by the hummingbirds Chlorostilbon aureoventris in the late afternoon of the first day and Augastes lumachellus Lesson in the early morning of the following day. However, the hummingbird visits were rare and it was not possible to observe whether contact of anthers or stigmas occurred. Apis melifera and Trigona spinipes were observed between 58 a.m. and 4 and 5 p.m. (Figure 13), respectively, displaying the same behaviour as was observed with M. flaviflorus and M. streckeri (Figure 12). Discussion Most floral features of M. streckeri and M. flaviflorus are similar to those displayed by species pollinated by hummingbirds in tropical communities, such as the easily accessible and unscented flowers, with tubular and conspicuously coloured perianth, clear separation between floral resource (nectar) and sexual parts and nectar with a relatively low concentration of sugars (Faegri & van der Pijl, 1980; Arzimendi & Ornelas, 1990; Araujo, 1996, Sazima et al., 1996; Locatelli & Machado, 1999b; Buzatto et al., 2000; Castro & Oliveira, 2001, 2002; Castro & Araujo, 2004), including Cactaceae species (Scogin, 1985).

The short-tubed flowers of M. flaviflorus and M. streckeri allow the visits of both short- and long-billed visitors, like Chlorostilbon aureoventris and Phaethornis squalidus, respectively (Grantsau, 1989) however only M. streckeri was visited by both species. The absence of P. squalidus on M. flaviflorus flowers may be related to the lack of forest habitats near the studied population, since this species is a typical pollinator in Atlantic forests in Brazil (Buzato et al., 2000). This hummingbird seemed to prefer individuals of M. streckeri located in areas at lower elevations, which are close to a wooded area. The period in which P. squalidus and Trigona spinipes visited M. streckeri flowers (after 2 p.m.), and that in which T. spinipes visited M. flaviflorus flowers coincided with the decrease in the number of C. aureoventris visits, a strategy that probably minimizes the chances of being attacked by this hummingbird. It was observed in the populations of Micranthocereus streckeri that Phaethornis squalidus invades the territory of Chlorostilbon aureoventris after 11 a.m. (Figure 12). According to Buzato et al. (2000), hermit hummingbirds such as P. squalidus are the major pollinators of hummingbird-pollinated of floras in Brazilian forests. Phaethornis squalidus visited the plants that occur near to the valley, nearby the forested area. This behaviour might be related with the visits decrease of C. aureoventris after 3 p.m. and the visits increase of P. squalidus. The decrease in the frequency of visits of Chlorostilbon aureoventris to the flowers of M. flaviflorus after 2 p.m. could be explained by the presence of a sympatric Cactaceae species, Melocactus paucispinus Heimen. & R. J. Paul, whose flowers opened approximately at 2 p.m. and were also visited by C. aureoventris. However, it is necessary to test if this hummingbird really prefers M. paucispinus flowers in comparison to those of M. flaviflorus. The visiting behaviour of Chlorostilbon aureoventris to the flowers of M. flaviflorus and M. streckeri revealed two types of feeding strategies: territoriality (sensu Feinsinger, 1978; Stiles, 1981) and traplining. The traplining strategy favours cross-pollination because it maximizes pollen dispersion among different individuals (Janzen, 1971), whereas the territorialist behaviour may reduce pollen flow in a population (Linhart, 1973). The occurrence of these two types of foraging behaviour (territorialist and traplining) was also recorded for C. aureoventris

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Number of Visits

0 8-9 9-10 10-11 11-12 12-13 13-14 14-15 15-16 16-17

Time Interval

Figure 12. Frequency of visiting bouts of M. flaviflorus and M. streckeri pollinators in NE Brazil. Males of Chlorostilbon aureoventris on flowers of M. flaviflorus (hatched bars); Phaethornis squalidus (grey bars) and male of C. aureoventris (black bars) on M. streckeri flowers.

12 12

Number of Visits

8 6 4 2 0 17-18 18-19 19-20 20-21 21-22 22-23 23-24 24-01 01-01 02-03 03-04 04-05 05-06

Time Interval

Figure 13. Frequency of visiting bouts of M. purpureus pollinators in NE Brazil. Sphingids (hatched bars), Loncophylla mordax (white bars), Chlorostilbon aureoventris (black bars) and Augastes lumachellus (grey bars).

in the flowers of Melocactus zehntneri (Britton & Rose) Luetzelb. and Opuntia palmadora Britton & Rose by Locatelli & Machado (1999b). The protandry observed in M. flaviflorus and M. streckeri is considered a reproductive strategy that minimizes the interference between male and female functions as well as the chances of self-pollination (Webb & Lloyd, 1986). Protandry was not found in M. purpureus, where the position of the stigma (adaxial or lateral) in relation to the stamens changed from adaxial or lateral to central in the flowers lifespan. Flowers of M. purpureus present some typical

chiropterophilous features such as pale colour, a short floral tube with wide aperture, nocturnal anthesis and large amounts of nectar (Vogel, 1969; Faegri & van der Pijl, 1980). There is evidence that the presence of a lateral sunken cephalium, composed by congested, hairy flowerbearing areoles, forming a cushion-like structure is related with the protection of bats wings during pollination (Zappi, 1994). The exerted position of the stigma of M. purpureus seems to favour cross-pollination because it is the first reproductive part to be touched by the floral visitors (Sazima & Sazima, 1975). However, a

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relatively low frequency of visits by bats was recorded. The horizontally disposed position of the flowers, as well as its presence at only on one side of the plants of M. purpureus plants are features expected in chiropterophilous species (Vogel, 1969, Sazima et al., 1995; Machado et al., 1998), because it seems that the one-side presentation of the flowers facilitates their access by bats, which are less able to maneuver in flight than bees or hummingbirds. The low number of flowers and sweet scent presented during the anthesis by M. purpureus was found in bat-pollinated species in general (Sazima & Sazima, 1978; Machado et al., 1998; Sazima et al., 1999) as well as in other bat-pollinated Cactaceae species (Locatelli et al., 1997). The regular paths coupled with the intervals between visiting bouts observed for Lonchophylla mordax visits to M. purpureus indicate a trapline behaviour, as has been recorded for Glossophagine species when feeding on flowers (Baker, 1973; Fleming, 1982; Sazima et al., 1999). This foraging pattern was already recorded in several studies on bat-pollinated flowers in the Brazilian Atlantic rainforest (Sazima et al., 1994, Machado et al., 1998; Sazima et al., 1999), restinga (Locatelli et al., 1997) and for L. bokermanii in campo rupestre (Sazima et al., 1989). The flowers of bromeliad Encholirium brachypodum are in the great number (c.30 flowers/inflorescence daily) than M. purpureus and have disagreeable odour, more typical of bat-pollinated species. These characters observed in E. brachypodium at the study site may partially explain the preference of the L. mordax for the bromeliad and the irregular intervals in feeding bouts on M. purpureus. Despite the hawkmoths being generally considered less efficient and less important pollinators than bats in chiropterophilous species (sensu Baker et al., 1971; Sazima & Sazima, 1978; Sazima et al., 1994), these insects were more frequently observed visiting M. purpureus flowers than bats. These results indicate that M. purpureus may have, at the sudy site, a mixed bathawkmoth pollination system, similarly to the one described for Lafoensia densiflora Pohl (Silberbauer-Gottsberger & Gottsberger, 1975) and for Cereus peruvianus auctt. [=C. hildmannianus K. Schum.] (Silva & Sazima, 1995). In fact, M. purpureus seems to present floral features such as the flower odour, which is weak and sweet, rather than strong and disagreeable that are somewhat ambiguous between bat and hawk-

moth syndromes, suggesting an adaptation to attract both pollinator types. Moreover, the traplining strategy presented by many hawkmoth species (Janzen, 1971), that was also observed in this study, coupled with their quick movements and long flight distances (Linhart & Mendenhall, 1977), favour cross-pollination. Despite the frequent presence of beetles within the flowers of Micranthocereus purpureus, similarly observed in other species of Cactaceae (Silva & Sazima, 1995; Locatelli et al., 1997), some studies suggested that these insects are not important pollen vectors for most cacti species (Grant & Hurt, 1979), but seem to act only as robbers (Locatelli et al., 1997). The hummingbirds and bees that were observed contacting anthers and stigma during their visits to M. purpureus flowers should not be considered pollinators because no stigma receptivity was observed in these periods of anthesis (Figure 13). Moreover, the flowers had nearly empty anthers when visited by Augastes lumachellus Lesson, which was observed only once; this Trochilidae species was observed using M. purpureus hairs for building its nest (Machado et al., 2003). The potential for using both nocturnal and diurnal pollinators is known in Cactaceae (McGregor et al., 1962) and in other chiropterophilous angiosperms (Buzato et al., 1994; Sazima et al., 1994). The range of nectar concentration of M. purpureus was slightly higher than the values recorded for some Neotropical bat-pollinated species (5%29%, often 15%17%, von Helversen, 1993; Sazima et al., 1999). Scogin (1985) compared nectar analyses in some Cactaceae species as 2230%, 1534% and 1726%, for those respectively pollinated by hummingbirds, moths and bats. The value found for M. purpureus was similar to that found in other Brazilian bat-pollinated Cactaceae (Locatelli et al., 1997) and in species of other families in Brazil (Sazima et al., 2003). The present study found overlapping values of sugar concentration between Micranthocereus, with the two hummingbird pollinated species (M. flaviflorus and M. streckeri) at each range of the spectrum, while the bat pollinated M. purpureus had intermediate values between the two (Table 1). It was also found that the range of variation in sugar concentrations in Micranthocereus may favour relationships with less specific pollinators than was initially expected. Tribe Cereeae (Taylor & Zappi, 2004) comprises several closely

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related genera, each one with more than one pollinator and sometimes displaying gradients between pollination syndromes. For example, Stephanocereus and Coleocephalocereus are likely to be pollinated by bats and hummingbirds, and species of Pilosocereus are pollinated by bats and hawkmoths was studied by Locatelli et al. (1997), Locatelli & Machado (1999a) and E. A. Rocha, pers. com.). Our results show a remarkable variability in the flower biology within the genus Micranthocereus. Acknowledgements We thank Dr. Nigel Taylor and Dr. Volker Bittrich for kindly read the manuscript, Dr. Keith J. Brown for identification of the species sphingids, Dr. Ivan Sazima and Dr. Marlies Sazima for identification of the bat species, Ludmila Mickeliunas and Renato P. Oliveira for their assistance in the field studies and Rogrio Lupo for the illustrations. This study is part of a Master thesis by the first author for the postgraduation course in Plant Biology at the Universidade Estadual de Campinas (So Paulo, Brazil). This work was supported by the Research Support Foundation of So Paulo State - FAPESP (process no. 00/00369-7). References AONA, L. Y. S. (2003). Caracterizao e delimitao do gnero Micranthocereus Backeb. (Cactaceae) baseados em caracteres morfolgicos e moleculares. M. S. Thesis, Universidade Estadual de Campinas, SP. ARAJO, A. C. (1996). Beija-flores e seus recursos florais numa rea de plancie costeira do litoral norte de So Paulo. M. S. Thesis, Universidade Estadual de Campinas, SP. ARZIMEDI, M. C. & ORNELAS, J. F. (1990). Hummingbirds and their flower resources in a tropical dry forest in Mexico. Biotropica 22: 172180. BAKER, H. G., CRUDEN, R. W. & BAKER, I. (1971). Minor parasitism in pollination biology and its community function: the case of Ceiba acuminata. BioScience 21: 11271129. BAKER, H. G. (1973). Evolutionary relationships between flowering plants and animals in American and Africa tropical forests. In Meggers, B. J., Ayensu, E. S., & Duckworth, W. D. (Eds.), Tropical forest ecosystems in Africa and South America: A comparative review. Smithsonian Institute Press, Washington, DC., pp. 145159.

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