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Cryptic species complexes, widespread species and conservation: lessons from Amazonian frogs of the Leptodactylus marmoratus group (Anura: Leptodactylidae)
Ariadne Angulo & Javier Icochea
a a b

Departamento de Herpetologa, Museo de Historia Natural de San Marcos, Apartado, 140434, Lima 14, Peru
b

Calle Arica 371 dpto U-2, Lima 18, Peru

Version of record first published: 15 Sep 2010.

To cite this article: Ariadne Angulo & Javier Icochea (2010): Cryptic species complexes, widespread species and conservation: lessons from Amazonian frogs of the Leptodactylus marmoratus group (Anura: Leptodactylidae), Systematics and Biodiversity, 8:3, 357-370 To link to this article: http://dx.doi.org/10.1080/14772000.2010.507264

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Systematics and Biodiversity (2010), 8(3): 357370

Research Article Cryptic species complexes, widespread species and conservation: lessons from Amazonian frogs of the Leptodactylus marmoratus group (Anura: Leptodactylidae)

ARIADNE ANGULO1 & JAVIER ICOCHEA2

1 2

Departamento de Herpetologa, Museo de Historia Natural de San Marcos, Apartado 140434, Lima 14, Peru Calle Arica 371 dpto U-2, Lima 18, Peru

(Received 17 September 2009; revised 10 May 2010; accepted 30 June 2010)

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Morphologically cryptic species act as a wild card when it comes to biodiversity assessments and conservation, with the capacity to dramatically alter our understanding of the biological landscape at the taxonomic, ecological, biogeographic, evolutionary, and conservation levels. We discuss the potential effects that cryptic species may have on biodiversity assessments and conservation, as well as some of the current issues involving the treatment of cryptic species both at taxonomic and conservation levels. In addition, using a combination of advertisement call and morphological data, we describe a new species of the Leptodactylus marmoratus group from the upper Amazon basin, and we assess how cryptic species can affect conservation assessments of species in the Leptodactylus marmoratus group by examining how recent ndings affect our understanding of the distribution of what is assumed to be a widespread Amazonian species, Leptodactylus andreae. Key words: Adenomera, advertisement call, anurans, conservation, cryptic species complexes, IUCN Red List, Leptodactylus marmoratus group, taxonomy, widespread species

Introduction
With nearly a third of all nominal amphibian species considered to be globally threatened (Stuart et al., 2004), amphibian declines and extinctions are now a recognized phenomenon at the forefront of the biodiversity crisis. A critical aspect of engaging in effective conservation action, however, depends on our ability to correctly identify species as such, given that different characteristics and life histories may lead to differing conservation needs among species. Morphologically cryptic species (two or more distinct species that are classied as a single species) act as a wild card when it comes to biodiversity assessments and conservation (Bickford et al., 2007), with the capacity to dramatically alter our perception of the biological landscape at the taxonomic, ecological, biogeographic, evolutionary and conservation levels. Cryptic species are a familiar part of the amphibian landscape as any amphibian eld researcher will attest, but they are by no means exclusive to this class. There are numerous
Correspondence to: Ariadne Angulo. E-mail: ariadne.angulo@ utoronto.ca

examples of cryptic species reported for a very broad range of living beings [e.g. birds (Cuervo et al., 2005), shes (Colborn et al., 2001), fungi (Pringle et al., 2005), insects (Copren et al., 2005; Paredes-Esquivel et al., 2009), molluscs (Calvo et al., 2009) and reptiles (Torres-P rez et al., e 2009), and see also Pfenninger & Schwenk (2007) and Trontelj & Fier (2009) for meta-analyses of cryptic species s reports across major metazoan taxa], which suggests not only that this is a widespread phenomenon across the taxonomic board, but also that we may be vastly underestimating the planets species richness and current biodiversity loss. Candidates for cryptic species complexes are often concealed within widespread species. Therefore, what appears to be a single, nominal widespread species may in fact be comprised of morphologically similar but geographically restricted species (Funk et al., 2008). Widespread species do, in fact, occur; even in organisms traditionally considered to be of relatively low vagility, such as amphibians (see e.g. Gower et al., 2007); however, it is not known what proportion of cases agged as widespread species are truly genuine instances of widespread taxa, and what proportion comprises species complexes.

ISSN 1477-2000 print / 1478-0933 online C 2010 The Natural History Museum DOI: 10.1080/14772000.2010.507264

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A. Angulo and J. Icochea regards to amphibians, as numerous cases of such instances have been reported throughout this taxonomic class (e.g. Heyer et al., 1996; Ron et al., 2006, and references within; Stuart et al., 2006; Elmer et al., 2007; Fouquet et al. 2007; Sumida et al., 2007; Fu & Zeng, 2008; Inger et al., 2009; Padial & De la Riva, 2009; Vieites et al., 2009). The impacts and implications of this biodiversity in hiding can be considerable, affecting everything from estimates of total number of species, biogeography, evolution, etc. to economic prospecting of this biodiversity, conservation, and, of course, extinction rates. Two dramatic examples of the magnitude of this phenomenon are illustrated by Fouquet et al. (2007), who begin their study with 60 nominal species but estimate the existence of 129 species upon concluding the study, and by Vieites et al. (2009), who begin their study with 244 described species but upon conclusion of the study estimate that the number of species should be somewhere between 373 and 465. In both these studies, the estimated total number of species comprises roughly a twofold increase from the original sampled number of nominal species. Morphologically cryptic species complexes lurking under the guise of a presumed widespread species may have the potential to signicantly change our understanding of biodiversity as a whole. If the numbers of known or suspected cryptic amphibian species are anything to go by, the answers to questions such as why and how these forms evolved, how did they get to be where they are, how they differ among themselves, and whether or not they require conservation efforts may be a long way coming in view of the current global economic climate, limited amount of resources and available expertise that can be dedicated to address these questions at a global level. This is of particular concern in view of the current amphibian decline and extinction crisis. In their study of distribution patterns of amphibians from the Kakamega Forest in Kenya, Schick et al. (2005) assess how our understanding of biogeographic patterns can be affected by cryptic species concealed under nominal widespread species, where particular distribution patterns may be more common than initially thought, and their unveiling may better support particular biogeographic hypotheses. From a conservation perspective, Angulo (2002) provides some hypothetical examples in which cryptic species hiding under the guise of widespread nominal species can affect biodiversity assessments. Expanding on this argument, we provide a modied version of the illustrated example in Angulo (2002) and an additional example based on habitat use (Figs 1 and 2). The rst example (Fig. 1) depicts a scenario where multiple populations of a presumed widespread species (species X) are assessed over a given geographical area. If only a very limited number of these populations show some evidence of decline, the species as a whole may still be assessed as being not threatened because most of its populations may be considered as being stable. However, if the populations that are in

A growing use of alternative (non-morphological) and complementary suites of features, such as genetic and/or call data, are useful in the identication of cryptic species in amphibians (e.g. Angulo et al., 2003; Camargo et al., 2006; Ron et al., 2006; Fouquet et al., 2007; Padial et al., 2008). Many members of the Leptodactylus marmoratus group (formerly assigned to the genus Adenomera) constitute good candidates for harbouring morphologically cryptic species complexes. This is largely because (1) there is considerable morphological variation within and among populations (De la Riva, 1996; Kwet & Angulo, 2002; Kwet, 2007; Ponssa & Heyer, 2007; Kwet et al., 2009), (2) there is an overall similarity among several species, and (3) many of the nominal species are considered to occur over vast tracts of land (e.g. the Amazon basin, Atlantic rainforests of Brazil and cerrado/chaco environments). In the specic case of the Amazon basin, for example, two widespread species of the L. marmoratus group have been traditionally considered to occur in sympatry: Leptodactylus andreae M ller, 1923, and Leptodactylus hylaedactylus u (Cope, 1868), with one species thought to occur in forests [L. andreae] and the other one occurring in open environments [L. hylaedactylus] (Heyer, 1984). Recent studies on the L. marmoratus group that made use of different suites of features (acoustic signals, life history traits) have, in fact, unearthed cryptic species for geographical regions that were considered to harbour one or two widespread species (Kwet & Angulo, 2002; Angulo & Icochea, 2003; Angulo et al., 2003; Kokubum & Giaretta, 2005; Almeida & Angulo, 2006; Kwet, 2007; Angulo & Reichle, 2008). In the course of several biological inventory eld expeditions conducted in a natural gas prospecting area near Rio Camisea and Rio Urubamba, region of Cusco, upper Amazon basin of Peru, a previously unknown population of the Leptodactylus marmoratus group was observed, recorded and collected. Morphological differences with other members of the L. marmoratus group suggested that this population merited further attention (Icochea et al., 2001). Examination of advertisement call features, voucher specimens and comparisons with other species, in addition to information in the primary literature, reveal that this population is a new taxon in the Leptodactylus marmoratus group. We describe the new species in the present paper. In addition, we assess how recent ndings affect our understanding of the distribution of what is assumed to be a widespread Amazonian species, Leptodactylus andreae, and how cryptic species can affect conservation assessments of species in the Leptodactylus marmoratus group based on the IUCN Red List Categories and Criteria (IUCN, 2001).

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Cryptic species complexes and their biodiversity impacts

The phenomenon of undetected cryptic species richness is likely to be more of a norm rather than an exception with

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Fig. 1. One possible scenario of the way in which cryptic species complexes can affect conservation assessments: Where several populations of a presumed widespread species (species X) are assessed and declines are detected in only a limited number of populations, which pertain to cryptic species Y (modied from Angulo, 2002).

threats and/or declines may only be occurring in part of the nominal species range, and not affecting all populations equally. The nominal threatened species may therefore consist of both threatened and non-threatened cryptic species. While it is clear that cryptic species complexes are a common phenomenon and that they have the potential to affect many aspects of our interpretations of the natural world, one major issue is that until they are formally recognized as full species it will be difcult to allocate resources for their conservation, should they need it.

Cryptic species, taxonomy and conservation

With regards to amphibian taxonomy, the IUCN Red List of Threatened Species generally follows Amphibian Species of the World (Frost, 2009) as a taxonomic standard. The IUCN Red List encourages that, where there are instances of known species complexes, details of these undescribed cryptic species, especially pertaining to distribution, habitat and ecology, threats and conservation status, as well as details of the person providing the information, be provided under the account of the nominal species to which the cryptic species have been associated. This is a compromise, but allows the nominal species to be split quickly in the Red List upon publication of the cryptic species, with all of the supporting data in place already. However, the conservation assessment is ultimately made on the nominal species. The IUCN Red List does not normally assess undescribed species separately (it only does so under exceptional circumstances and a number of strict conditions must be met for this to happen) (IUCN Standards and Petitions Working Group, 2008), and there are valid reasons for this, such as the nomenclatural and practical issues that would arise from such a practice (e.g. one researchers Leptodactylus sp. 1 may be another researchers Leptodactylus sp. Z and there would be no published source to go to so as to ensure that they are the same species; the ever-increasing number of undescribed species; the proliferation of temporary designations and their management within the Red Lists database; and the further merging and splitting of undescribed taxa), and the chaos that would ensue from implementing this approach across the taxonomic board within the Red List. In light of this scenario, one alternative is to encourage resolution of these species complexes and publication of their component cryptic species. However, in many instances this is no simple matter, either. Often times taxonomists do not have easy or direct access to type material to compare against their own samples, the taxonomic history of such cryptic groups can be quite complex and the provenance of some nominal species can be obscure at best, and researchers do not have the time and/or are not rewarded for publishing species descriptions, as these are not as valued (and therefore not encouraged) as

decline pertain to a cryptic species with a more restricted geographic range (species Y), the cryptic species conservation needs may go unnoticed. The second example (Fig. 2) depicts an apparently single population of a widespread species (species X) occupying a variety of different habitats. However, it is possible that cryptic species Y may be restricted to a specic habitat type within this range of habitats, affecting our understanding of habitat associations and needs for both the widespread and the cryptic species. There are other potential scenarios involving the IUCN Red List Categories and Criteria. One such scenario involves a nominal species already assessed as threatened, but which could comprise more than one morphologically cryptic species that, if recognized as distinct, would each qualify for a higher threat category than that assigned to the original nominal species. An example of this would be an instance where a nominal species may be assessed as Endangered (EN) or Vulnerable (VU) but is later found to comprise three separate cryptic species, all of which meet the Criteria for Critically Endangered (CR). Alternatively, a nominal species may be assessed as threatened, but upon review it might be that not all of the composite species meet the IUCN Red List Criteria, i.e.

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Fig. 2. A second scenario of how cryptic species complexes may affect assessments: where a single population of a widespread species (species X) appears to occupy several different habitats, but cryptic species Y is restricted to a specic habitat type within its range.

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publications in more trendy and modern approaches to biodiversity issues. Unfortunately, an additional, recent issue stemming from a combination of some of these factors is that studies using phylogenetic approaches with molecular and/or other characters focusing on addressing a specic question are often built around that question, with secondary (taxonomic) ndings being relegated to a second plane. In many instances these studies identify populations that pertain to cryptic species, but because their descriptions are not within the scope of the study and/or the numbers of species involved can be potentially high, these taxa often go undescribed (and the senior author will be the rst one to admit to having done this in the past, e.g. Angulo et al., 2003; Angulo, 2004). Published results from some recent molecular studies have often led to splitting of the original nominal taxa, better circumscribing the name carriers, but leaving all other cryptic species in taxonomic and therefore conservation limbos. The combination of the issues outlined above makes morphologically cryptic species the castaway species of the taxonomic and conservation realms. In many instances describing new cryptic species identied in the course of a targeted study may not be practical or even possible; however, given the current crisis facing amphibians in particular and biodiversity as a whole, we urge researchers to consider describing and validating cryptic species whenever possible within the scope of their study and within reason.

Fig. 3. Holotype of Leptodactylus simonstuarti sp. nov.

Materials and methods

Field work was conducted in the localities of Segakiato (11 48 23 S, 72 52 51 W, c. 340 m asl; Valencia & Alonso, 1998), Konkariari (11 48 S, 72 52 W, 300 m asl; Valencia & Alonso, 1998), Cashiriari II (11 51 51 S, 72 46 45 W, 469 m asl; Comiskey et al., 2001) and Cashiriari III (11 52 56.693 S, 72 39 2.132 W, c. 694 m asl), in or near Machiguenga territory, near Ro Camisea and Ro Urubamba, District of Echarate, Province of La Convenci n, Region of Cusco, Peru, and in San Sebasti n o a (11 24 S, 69 01 W, c. 280 m asl; Alverson et al., 2000), close to Ro Tahuamanu, Department of Pando, Bolivia. Specimens were xed in 10% formalin and preserved in 70% ethanol; these are deposited at the Museo de Historia Natural de la Universidad Nacional Mayor de San Marcos (MHNSM/MUSM), Lima, Peru and Museo Noel Kempff Mercado (NKA), Santa Cruz, Bolivia. Referred specimens are deposited at MHNSM, NKA and Colecci n o Boliviana de Fauna (CBF) in La Paz, Bolivia. Other specimens used for comparative purposes are deposited at: CBF; NKA; the Academy of Natural Sciences of Philadelphia (ANSP), United States; Mus um National dHistoire Nae turelle (MNHN), Paris, France; Royal Ontario Museum

(ROM), Toronto, Canada; and Zoologische Staatssammlung M nchen (ZSM), M nchen, Germany. u u Where possible, specimens were sexed by examining for presence of vocal slits and callus on snout. With the exception of snoutvent length (SVL), tibia length (TL) and foot length (FL), which were measured with digital callipers, all other measurements were taken with an ocular micrometer tted to a dissecting microscope, as follows: head length (HL), head width (HW), eye diameter (ED), tympanic diameter (TYD), eyenostril distance (END), interorbital distance (IOD), internarial distance (IND), forearm length (FAL), hand length (HDL), thigh length (THL), tibia length (TL), tarsus length (TSL) and foot length (FL). Measurements were made to the nearest 0.1 mm and followed Heyer et al. (1990). Vocalizations were recorded using a Sony Walkman professional WM D6C tape recorder and an AKG D 1200E microphone and were later digitized at a sampling rate of 44.1 kHz and 16-bit precision. Call analysis was performed using the Macintosh-based software Signalyze (Keller, 1994), and calls were illustrated using a combination of DADiSP (DSP Development Corporation, 1998) and Corel Draw. Call parameters used to describe the call are: call length, call rate, call rise time, fundamental frequency, dominant frequency, change in dominant frequency and other frequencies. Our use of terminology follows mostly Cocroft & Ryan (1995); we follow Ewing (1989) for fundamental frequency and Almeida & Angulo (2006) for other frequencies.

Results
Leptodactylus simonstuarti sp. nov.

HOLOTYPE. Adult male, MHNSM 18218, collected at Campamento Segakiato, c. 340 m asl, Ro Camisea, District of Echarate, Province of La Convenci n, Region o

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Fig. 4. Dorsal view of Leptodactylus simonstuarti sp. nov.

Fig. 5. Ventral view of Leptodactylus simonstuarti sp. nov.

of Cusco, Peru, on 11 October 1997 at 18:20 h by Javier Icochea. PARATYPES. MHNSM 18221 collected at Campamento Konkariari, Ro Urubamba, on 21 October 1997 at 13:30 h by Javier Icochea; MHNSM 18220, collected at Cashiriari II on 11 October 1998 at 12:20 h by Alfredo Portilla; and MHNSM 18229 collected at Cashiriari III on 1 September 1998 by Eliana Quispitupac. REFERRED MATERIAL. NKA 3597 collected in San Sebasti n, Department of Pando, Bolivia, on 11 March 2001 a at 23:53 h by Ariadne Angulo, and CBF 4294, collected in 1999 by Steffen Reichle. DIAGNOSIS. Leptodactylus simonstuarti sp. nov. can be distinguished from other congeners by the following combination of features: (1) a distinct advertisement call (see under Comparison with other advertisement calls), (2) a relatively large SVL (maximum SVL in males 26.2 mm; in females 25.2 mm), (3) very dark dorsal colouration, and (4) very dark, nearly solid stripes on undersides of arms, running from wrist to arm insertion. Within Amazon-associated species, it can be distinguished from Leptodactylus heyeri (Boistel, Massary (de) & Angulo, 2006) in lacking two distinct pairs of dorsolateral folds and males lacking a yellow throat and belly (Boistel et al., 2006), and from Leptodactylus lutzi (Heyer, 1975) by having a smaller body size (SVL in L. lutzi males 30 mm) and absence of distinct spotting on the posterior face of the thigh (Heyer, 1975). Leptodactylus simonstuarti differs from Leptodactylus coca Angulo & Reichle, 2008 in having a relatively more developed inner metacarpal tubercle, dark dorsum, and a dark, continuous stripe running the length of the underside of the arm, and it differs from Leptodactylus andreae, Leptodactylus diptyx Boettger, 1885 and Leptodactylus hylaedactylus by having a larger SVL (L. andreae 21 mm, L. diptyx 22 mm and L. hylaedactylus 24.6 mm; see Angulo & Reichle, 2008 for details), dark dorsum, and very dark stripe running along the underside of the arm from the wrist to arm insertion. DESCRIPTION OF HOLOTYPE. Body small but robust; limbs relatively short. Head marginally wider than long; dorsal outline of snout rounded; in prole snout nearly acuminate; nostrils oriented dorsolaterally, approximately equidistant between tip of snout and anterior corner of eye; internarial distance >25% of head width. Tympanum distinct, nearly 60% of eye diameter; supratympanic fold well developed, extending from back of eye to arm insertion; dark, contouring outline above fold following the extent of the fold; oval cream-coloured gland below angle of jaw and supratympanic fold present and distinct; canthus rostralis indistinct. Single, internal vocal sac; paired elongate vocal slits present. Vomerine teeth posterior to choanae, arranged in transverse series parallel to choanae. Inner edge of series slightly convergent to each other. Arms short, robust; ngers slender, lacking fringes, nger lengths III

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Fig. 7. Ventral view of sole of left foot of holotype of Leptodactylus simonstuarti sp. nov. Scale bar equals 1 mm.

Fig. 6. Ventral view of palm of left hand of holotype of Leptodactylus simonstuarti sp. nov. Scale bar equals 1 mm. Note dark stripe running along arms back (white arrow).

> I = II > IV; nger tips rounded and without expansions; palm of hand with two large ovoid-shaped creamcoloured metacarpal tubercles; inner metacarpal tubercle more elliptical in shape and slightly larger at greatest diameter than outer metacarpal tubercle; conspicuous, distinct cream-coloured rounded subarticular tubercles on underside of ngers, nuptial asperities absent. Hindlimbs robust, tibia longer than thigh; toe lengths IV > III > V > II > I; toe tips slightly attened or not, with visible expansions (character states CD of Heyer, 1973); toes without fringes; underside of toes with dark lines. Metatarsal tubercles distinct; inner tarsal fold lined with scant, small white tubercles; which are also present on tarsus and sole of foot. Skin on dorsal surface smooth and glandular/tuberculate with very small tubercles sparsely distributed from about the shoulder down to the vent; thighs mostly smooth, shanks with very small, white-tipped tubercles. Venter surface smooth.

COLOUR IN LIFE. Figure 3 shows the male holotype in life. Dorsum a greyish dark brown, with irregular dark markings on the lower back running from mid-dorsum to groin. A tan-orange discontinuous dorsolateral glandular line on each side of the body, running from behind the eye to just anterior to the groin. Dorsal surfaces of hind limbs with irregular dark crossbars, white-tipped tubercles noticeable on shanks. Dorsal surfaces of arms and heels an orangereddish colour; arms with two dark, incomplete crossbars. Tympanum dark brown, supratympanic fold lined with a dark stripe running from behind the tympanum to the arm insertion and continuing irregularly from behind the arm to the groin, running roughly parallel to the discontinuous dorsolateral glandular line above. A second, marginally lighter stripe runs roughly parallel and ventral to the rst stripe, lining the boundary with the ventral surface. A small orange gland is located under each supratympanic fold and anterior to the arm, between the tympanum and arm. Two dark bars below each eye, each of them roughly diagonal to anterior and posterior angles of eye. Some white spotting on edges of upper and lower lips. A dark, nearly continuous stripe along the length of the underside of the arm, from the wrist to arm insertion, with a slight break or narrowing at the elbow level. A yellowish, mid-dorsal hairline stripe from below the shoulders to the vent. Two dark glands ank the cloaca on each side. Venter greyish-white. COLOUR IN PRESERVATIVE. Dorsum dark brown, with dark melanophore marks as described for live specimen.

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Table 1. Morphometric measurements for Leptodactylus simonstuarti sp. nov. Numbers on rst row are means standard deviation, numbers in parentheses are ranges. All measurements expressed in mm. Males (N = 2) Snoutvent length (SVL) Head length (HL) Head width (HW) Eye diameter (ED) Tympanum diameter (TYD) Eyenostril distance (END) Interorbital distance (IOD) Internarial distance (IND) Forearm length (FAL) Hand length (HDL) Thigh length (THL) Tibia length (TL) Tarsus length (TSL) Foot length (FL) 26.03 0.25 (25.926.2) 9.52 0.45 (9.209.84) 9.95 0.76 (9.4210.49) 2.78 0.30 (2.573.00) 1.71 0.0 (1.711.71) 2.03 0.15 (1.932.14) 2.57 0.30 (2.352.78) 2.46 0.15 (2.352.57) 5.03 0.45 (4.715.35) 5.35 0.0 (5.355.35) 10.17 0.76 (9.6310.70) 11.16 0.56 (10.7611.56) 6.42 0.30 (6.216.63) 11.69 0.80 (11.1312.26) Females (N = 4) 24.07 1.095 (23.225.2) 8.61 0.27 (8.358.99) 9.42 0.17 (9.209.63) 2.51 0.11 (2.352.57) 1.71 0.17 (1.501.93) 2.19 0.20 (1.932.35) 2.46 0.12 (2.352.57) 2.27 0.10 (2.142.35) 4.92 0.17 (4.715.14) 5.24 0.12 (5.145.35) 10.71 0.41 (10.2711.18) 11.32 0.38 (10.9311.69) 5.78 0.0 (5.785.78) 11.48 0.54 (10.8612.17)

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Venter white, immaculate; throat white, some mottling along the edges of the lower lip. Fingers IIIII with short, dark lines on palm of hand. SECONDARY SEXUAL CHARACTERS. Male holotype and male paratype MHNSM 18220 have a projecting ridge on snout, above upper lip, similar to L. lutzi (Kok et al., 2007 and Fig. 8, this paper). We agree with Kok et al. that this callus-like ridge, present in adult males, is probably used to excavate nesting chambers. A similar ridge used for nest construction is found in the L. fuscus group (Reading & Jofr , 2003). It is not known if these ridges are a permanent e feature or develop only for the breeding season. MEASUREMENTS OF HOLOTYPE (IN MM). SVL 25.9; HL 9.2; HW 9.4; ED 3.0; TYD 1.7; END 1.9; IOD 2.8; IND

Fig. 8. Lateral view of head of holotype of Leptodactylus simonstuarti sp. nov. Note hardened ridge above upper lip (white arrow).

2.6; FAL 4.7; HDL 5.4; THL 10.7; TL 11.6; TSL 6.2; FL 11.1 MORPHOLOGICAL VARIATION. Table 1 shows morphometric variation for Leptodactylus simonstuarti sp. nov. As for other variations, paratypes from Peru as for holotype with the following exceptions: MHNSM 18229 with a slightly lighter dorsum; MHNSM 1822018221 with short, dark spots or lines on palm of hand (ngers IIIII or IV), MHNSM 18229 without these. MHNSM 18221 with well developed supratympanic fold above the tympanum but weakly marked from tympanum to arm insertion; in the other two paratypes fold well developed from back of the eye to the arm insertion. Male paratype with small, whitetipped tubercles on the dorsal surface from shoulder level to the vent, other paratypes with either isolated or nearly absent indistinct dorsal tubercles and a mostly smooth dorsal appearance. The Bolivian specimens, both females, as for holotype, with the following variations: Specimen CBF 4294 without well marked diagonal dark bars below the eye; thin middorsal light hairline stripe running from the lumbar region down to the vent. Neither specimen with dark spots or lines on palm of hand, and both with well developed supratympanic fold from the back of the eye to arm insertion. Both with dark mottling lining the edges of the lower lip, although less than in specimens from Peru. In life, specimen NKA 3597 with very dark iris, with small and sparse yellow freckles. ADVERTISEMENT CALL. A calling male, holotype MHNSM 18218, was recorded at 18:20 h on 11 October 1997 at an ambient air temperature of 27 C, beneath the leaf litter next to a rotting log, in an old abandoned cacao plantation with some secondary growth, in the vicinity of a gas exploration campsite. The advertisement call of L. simonstuarti (Fig. 9 and Table 2 for call parameter measurements) was rst described under Adenomera Camisea I by Angulo (2004).

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Fig. 9. Advertisement call of Leptodactylus simonstuarti sp. nov. (a) Sequence of three calls, (b) one of these calls at higher resolution, (c), section of call in (b) showing detail of waveform, (d) power spectrum and (e) spectrogram of the calls in (a). Upper harmonic frequencies not depicted in (d) as they fall below the cutoff point of 30 dB.

Compared with other species of the L. marmoratus group, L. simonstuarti has a shorter mean call duration (65 ms) repeated at a high call rate (c. 231 calls/min). There are three to four pulse-like, strong amplitude modulations in the

call. The dominant frequency is the fundamental frequency (18132035 Hz), and the call may have at least two additional harmonically related frequencies at 37074049.5 Hz and 49566024 Hz, at a lower intensity than the

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Table 2. Measurement of advertisement call parameters for the holotype of Leptodactylus simonstuarti sp. nov. Numbers in rst line are means standard deviation while numbers in parentheses are ranges. Recording Number of calls Temperature ( C) Call length (ms) Call rise time (ms) Call rate (calls/min) Fundamental frequency (Hz)-Dominant frequency (Hz) Additional frequency 1 Additional frequency 2 Change in dominant frequency (Hz) Total number of harmonics detected MHNSM 18218 (male) 62 27 65.3 3.7 (57.171.4) 13.6 6.8 (4.844.8) 231 (216246) 1962.7 43.7 (1813.22034.8) 3892.8 86.9 (37074049.5) 5773.4 213.4 (49566023.8) 298 63.7 (170.9430) 3

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ancestral-descendant sequence of populations) evolving separately from others and with its own unitary evolutionary role and tendencies] as a primary concept (for a discussion on the use of species concepts in species descriptions in the L. marmoratus group see Angulo & Reichle, 2008).

Conservation status
While there is limited information on the species population status and geographic distribution, there are some inferences that can be made based on the habitats and localities which it occupies. It has been found in intervened forest macrohabitats (i.e. an old abandoned cacao plantation with some secondary growth), which suggests that it can tolerate some degree of habitat disturbance in the context of a forest environment. It has also been found in ve localities, four that are geographically close and the more distant locality in Bolivia. If there is sufcient suitable habitat between the Peruvian and Bolivian localities, the new species could also occur in between known localities. All four Peruvian localities are within the boundaries of the Camisea Natural Gas Projects Block 88 [see g. 4 in Finer et al. (2008); Block 88 corresponds to the yellow square pointed as Camisea], which started production in 2004 and has had a reopening of another two wells over the last two years, diverting gas via pipelines to the main plant in Malvinas, on the Rio Urubamba. Already there have been several pipeline ruptures, affecting both the immediate environment and local communities, posing a real, tangible threat to local ora and fauna and indigenous communities. Finer et al. (2008) identify pending oil and gas projects as currently being the primary threat to Perus Camisea region, among other western Amazonian areas. Neighbouring blocks 56, 57 and 58 are all already under exploration. In addition, there are other designated hydrocarbon blocks in between the Peruvian and Bolivian localities (blocks 113 and 111 in Peru). The Peruvian localities are also relatively close to Manu National Park, so L. simonstuarti could also occur there, although there are currently no known records for this Park. Given our limited data at this time, we suggest that L. simonstuarti be preliminarily considered as Data Decient (DD) following IUCNs Red List Categories and Criteria (IUCN, 2001). If future surveys fail to record the species in between the known sites it could meet the IUCN Criteria for a threat category; alternatively, it could be assessed as Least Concern if the species is found to occur more widely and most of the populations are found to be under no signicant threat, or possibly Near Threatened (NT) if intense hydrocarbon explorations expand to other blocks.

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fundamental frequency. Call frequency increases slightly during the call (171430 Hz). NATURAL HISTORY. The species inhabits lowland Amazonian primary and secondary terra rme forests, including regenerating cacao plantations. Individuals can be found on the forest and cacao plantation oors within the leaf litter, and calling activity is initiated at dusk. GEOGRAPHIC DISTRIBUTION. Leptodactylus simonstuarti is known from ve localities in the Amazonian lowlands east of the Andes: four geographically close localities in southern Peru (300694 m) and one in northern Bolivia (280 m) (Fig. 10). The holotype locality is separated from the one in Bolivia by c. 407 km. The species may possibly occur elsewhere in this region given that much of the area in between these sites is still thought to contain suitable habitat. ETYMOLOGY. The new species is named after Dr Simon N. Stuart, in recognition of his considerable efforts to highlight the global amphibian crisis and to encourage amphibian research and conservation efforts across the world.

Species diagnosis and species concepts

Leptodactylus simonstuarti was initially thought to be a new undescribed species on the basis of its advertisement call and morphological differences. Subsequent comparisons of these features among the L. marmoratus group support this inference. Given that advertisement calls were the primary line of evidence to suggest the biological identity of L. simonstuarti, we refer to Patersons Recognition Species Concept (RSC) (that most inclusive population of individual biparental organisms which share a common fertilization system; Paterson & McEvey, 1993: 147) as an operational species concept, while using Simpsons (1961) Evolutionary Species Concept (ESC) [a lineage (an

Discussion
There have been seven additions to the L. marmoratus group over the last 5 years: Leptodactylus nanus (as Adenomera

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Fig. 10. Map depicting the known localities (black circles) for Leptodactylus simonstuarti sp. nov. Localities Segakiato and Konkariari are very close, so they are depicted as a single circle.

nana, Kwet, 2007), Leptodactylus heyeri (as Adenomera heyeri, Boistel et al., 2006), Leptodactylus thomei Almeida & Angulo, 2006, Leptodactylus ajurauna Berneck, Costa & Garcia, 2008 (Berneck et al., 2008), Leptodactylus coca (Angulo & Reichle, 2008) Leptodactylus engelsi (as Adenomera engelsi Kwet, Steiner & Zillikens, 2009) and now L. simonstuarti. There are currently 15 nominal species recognized for the group, including L. simonstuarti as the latest, but by no means last, addition. Other species await formal description/resurrection (e.g. Angulo & Icochea, 2003; An-

gulo et al., 2003; Kokubum & Giaretta, 2005; Kwet, 2007), and while not yet ofcially tallied, it is clear that the Leptodactylus marmoratus group comprises many more species than previously thought. Prior to assigning a new name to Leptodactylus simonstuarti we attempted to determine whether we could associate an existing name to the new species [several names are available under synonymy within Leptodactylus hylaedactylus and Leptodactylus diptyx (Frost, 2009)]. Comparisons of type images kindly provided by W.R. Heyer

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Fig. 11. Map depicting current distribution for Leptodactylus andreae (IUCN, 2009) and individual localities for recently recognized species masquerading under this name. Localities are as follows: = type locality for Leptodactylus andreae; = report for L. andreae (M rquez et al., 1995) now known to pertain to L. diptyx (I. De la Riva, pers. comm.); = Tambopata Reserved Zone, Madre de Dios, a Peru, where three recently recognized forest-associated cryptic species are found (Angulo et al., 2003); = localities for L. simonstuarti sp. nov. (this study).

suggested that those of the lectotype of Leptodactylus glandulosus appeared most similar to L. simonstuarti. We were able to access seven of the 14 specimens comprising the type series of L. glandulosus, so it was possible to compare specimens directly. Upon examination, Leptodactylus glandulosus specimens were found to be much smaller

than any of those available for L. simonstuarti [SVL = 14.421.8 mm (n = 7)], lacked a nearly solid dark stripe on the underside of arms, and had unexpanded, unattened toe tips, leading us to conclude that L. simonstuarti is a different species from L. glandulosus. Heyer (1973) suggested that all of the L. glandulosus type specimens are juveniles. We

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A. Angulo and J. Icochea and French Guiana) and covering a vast geographic area of c. 5 976 957 km2. Accordingly, it is currently considered to be a Least Concern (LC) species, given its wide distribution, tolerance of a range of habitats, presumed large population, and because it is unlikely to be declining fast enough to qualify for listing in a more threatened category (IUCN, 2009). However, the geographic distribution of L. andreae may require a reassessment in light of the recent ndings involving forest-associated members of the L. marmoratus group. Figure 11 also plots the individual localities for those species that are now known to be different from the nominal L. andreae but whose populations had, in the past, been considered to be (or allied to) L. andreae by eld researchers. Acoustic data suggest that L. andreae does not occur in these specic localities and that it may have a considerably more restricted distribution than the currently accepted range. If it does not occur in Bolivia or southeastern Peru, its geographic range would be reduced by several thousand square kilometres. While it may have a lesser, yet still relatively widespread distribution, its current IUCN status may change (or remain the same); further eld work is needed to determine its current distribution. The main issue is that unrecognized cryptic species may remain undetected, and the risk is that they may even become extinct, because vital data on distributions, population status, life histories, habitat associations and threats are unknown.

concur with this assessment for the subsample that we had access to.

Comparison of advertisement calls

Angulo & Reichle (2008) provided data (their table 4) summarizing some key acoustic parameters for members of the Leptodactylus marmoratus group with an Amazonian distribution, we refer to this table for more detailed information on other species. The most notable difference between the call of Leptodactylus simonstuarti and other Amazonian calls for the group, with the exception of Adenomera cf. andreae [Angulo & Icochea, 2003, or Adenomera Camisea II sensu Angulo (2004)], is the dominant frequency [fundamental frequency in L. simonstuarti; second harmonic in other Amazonian species (Angulo & Reichle, 2008: 70)]. The call of Adenomera cf. andreae differs from that of L. simonstuarti in having a shorter call length (2138 vs. 5771 ms, respectively), lower call rate (3078 vs. 216246 calls/min, respectively) and lacking observable amplitude modulation. A second key difference between the call of L. simonstuarti and those of other Amazonian species of the L. marmoratus group comprises the call rate: the new species has an extremely high call rate (216246 calls/min), comparable only to that of Leptodactylus hylaedactylus from Tambopata, Peru (162252 calls/min).

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Geographic patterns and their implications for conservation

Based on acoustic information currently available, the total species richness for the L. marmoratus group in southeastern Peru would be at least six species, three times as many species as the currently accepted widespread Amazonian nominal species Leptodactylus andreae and Leptodactylus hylaedactylus. Most of these cryptic species are likely to have been assigned to the forest-dwelling nominal Leptodactylus andreae given that this is also the broad general habitat that these previously undetected taxa occupy. Assuming that recorded acoustic signals from the type locality of L. andreae (Peixe-boi, Par , Brazil) may pertain to this nominal a species (Kokubum, Angulo & Kok, unpubl data), this would permit resolution of the taxonomic status of most other forest-dwelling Amazonian populations for whom acoustic data are available. We use the widely distributed L. andreae as a model species as a basis for comparison with other morphologically cryptic species complexes in the genus Leptodactylus. The IUCN Red List of Threatened Species (IUCN, 2009) provides an estimate of the geographic range for Leptodactylus andreae (Fig. 11). This nominal species is currently considered to occur over most of the Amazon Basin, encompassing nine countries (Bolivia, Brazil, Peru, Ecuador, Colombia, Venezuela, Guyana, Suriname

Acknowledgements
We thank the Museo de Historia Natural de San Marcos, Museo Noel Kempff Mercado and Coleccon boliviana de fauna for the loan of type specimens. C sar Aguilar, Jes s e u C rdova, Frank Glaw, Ned Gilmore, Ross MacCulloch, o Rossy Monta o and Annemarie Ohler facilitated loan of n specimens under their care. IUCN kindly facilitated use of their shapeles and allowed use of GIS software for elaboration of maps. The Man and Biosphere Program (Smithsonian Institution) and the Shell Oil Company provided logistic support, while the Instituto Nacional de Recursos Naturales (INRENA) granted appropriate research permits. Parts of the call description and call gures were rst developed in AAs doctoral thesis; Leptodactylus simonstuarti is referred to as Adenomera Camisea I in this work. AA is grateful to Steffen Reichle for rst pointing out where to nd L. simonstuarti in Bolivia, for support while in this country, and for facilitating specimen loans, and to Gonzalo Calder n and Jhonny for help in the eld. We are grateful o to Axel Kwet and Neil Cox for providing feedback on an earlier version of this manuscript and to Vineet Katariya for help with GIS software and maps. AA is grateful to Mike Hoffmann, Neil Cox and Marinus Hoogmoed for insightful discussions on morphologically cryptic species in the IUCN Red List. We thank two anonymous reviewers and the Associate Editor, Dr Barry Clarke, for their helpful revision of our manuscript.

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