A new Gephyromantis (Phylacomantis) frog species from the pinnacle karst of Bemaraha, western Madagascar

Abstract We describe a new mantellid frog of the subfamily Mantellinae from the karstic Bemaraha Plateau, western Madagascar. The new species belongs to the genus Gephyromantis, subgenus Phylacomantis, which previously included Gephyromantis azzurrae, Gephyromantis corvus and Gephyromantis pseudoasper. Gephyromantis atsingy sp. n. has a snout-vent length of 35–43 mm and is a scansorial frog living among the Tsingy de Bemaraha pinnacles and inside the caves present in the area. A morphological analysis and biomolecular comparison revealed the degree of differentiation between these four species of the Phylacomantis subgenus.The new species seems to be endemic to Tsingy de Bemaraha.


Introduction
Th e intense herpetological activity carried out in Madagascar during the last decades, together with the wider use of integrative taxonomic tools has led to the description of an astonishingly high number of new amphibians species (Köhler et al. 2005 and to the identifi cation of numerous still undescribed candidate species (Vieites et al. 2009).
Although, the highest species richness of amphibians is typically found along the eastern rainforest belt (Andreone et al. 2005, Glaw and, an increasing number of peculiar species are known from the arid western part of Madagascar (Glaw et al. 1998, Glos et al. 2005, Mercurio and Andreone 2007, Bora et al. 2010. At these sites the research eff ort has been gradually increased in recent years, and systematic surveys have recently taken place (e.g. Mercurio et al. 2008, Raselimanana 2008, Bora et al. 2010. While the species already described from the arid West mostly belong to radiations of explosive breeders reproducing in ephemeral ponds, a special attention has been given to species ascribed to clades that are more typical of humid habitats and rainforest biomes. Th is was the case, for example, with the recent discovery of two new mantellines at the Isalo Massif (Mercurio and Andreone 2007), the peculiar Tsingymantis antitra at Ankarana , some Boophis and some cophyline microhylids ) in the huge karstic massif of Tsingy de Bemaraha, and four large-bodied cave-dwelling species of Stumpffi a from karstic regions in the North .
During recent herpetofaunal inventories we discovered a further new species of a rather inconspicuous Gephyromantis frog inhabiting the deciduous forest of the karstic Bemaraha Plateau.
Th irty-six described species are currently ascribed to Gephyromantis that is currently divided in fi ve subgenera, including Phylacomantis. Four species are currently ascribed to this subgenus: G. corvus Glaw & Vences, G. pseudoasper Guibé, G. azzurrae Mercurio & Andreone and the new species described in the present paper. With the exception of G. pseudoasper, that mostly inhabits the rainforests of the North, the other species are found only in xeric habitats in the south-western (G. corvus and G. azzurrae) and western Madagascar (the new species described herein) (Fig. 1).
Due to morphological and external similarities, the new frog was formerly believed to be related to G. corvus, a frog endemic of the Isalo Massif. Unfortunately, the secretive life of this new species prevented us from obtaining much biological informations and we still lack information about its acoustic repertoire, breeding behaviour and larval morphology. Notwithstanding these challenges, the ongoing collaborative eff ort generated by the ACSAM (A Conservation Strategy for the Amphibians of Madagascar,  allowed us to integrate the data and photographs obtained by three independent survey teams.
We present here the formal description of this new Gephyromantis species of the subgenus Phylacomantis, which diff ers from the other Phylacomantis species by a combination of morphological traits colouration and by a high divergence in mitochondrial DNA sequences.

Study site
Th e Tsingy de Bemaraha is a karstic plateau in the Melaky Region, fi ve to 15 km wide and about 100 km long, located in western Madagascar. Numerous long, sharp pinnacles of rock, that may reach 45 meters in height, outcrop along the plateau and form the characteristic landscape (the so-called "tsingy" or "atsingy" in Malagasy language). Dry, deciduous forest is the most common vegetation type but humid areas occur within some of the larger canyons. An extensive area of forest and rock outcrop is included within two adjacent protected areas (Parc National Tsingy de Bemaraha and Réserve Naturelle Intégrale du Tsingy de Bemaraha). Savanna grasslands surround the plateau and there are numerous marshy depressions, caves and gorges associated with the main outcrop. Th is area has been object of some herpetological surveys that led to the discovery and description of several new species of amphibians , Glos et al. 2005 and reptiles (Schimmenti and Jesu 1996, Nussbaum and Raxworthy 2000, Glaw et al. 2009a.

Sampling methods
We searched for frogs at night with the aid of hand torches and headlamps. Geographic coordinates were taken using a GPS device. Toponyms often follow the indications by local people, and must be therefore seen as largely unoffi cial names. Frogs were collected by hand and euthanised by immersion in chlorobutanol solution, fi xed in 5% formalin or in 90% ethanol and fi nally stored in 75% ethanol solution. Voucher specimens (Tab. 1) are currently housed at the Museo Regionale di Scienze Naturali di Torino (MRSN), Zoologische Staatssammlung München (ZSM), and Université d'Antananarivo, Dé-partement de Biologie Animale (UADBA). Original fi eld numbers are FN and FAZC (Franco Andreone Zoological Collection), FGZC (Frank Glaw Zoological Collection), BMR (Jasmin E. Randrianirina), and RBJ (Richard K. B. Jenkins). A few individuals do not bear any fi eld number (no fi eld number = NFN). Th e specimens of the type series were compared with the specimens of the other known species of the Phylacomantis subgenus (Tab. 1): six specimens of G. azzurrae from Isalo, fi ve specimens of G. corvus from Isalo, and three specimens of G. pseudoasper from Nosy Be (see Tab. 1). Th e comparative specimens of G. pseudoasper were most probably temporary stored in denaturating solutions, and the sequencing was therefore not successful. For this reason, the three specimens here analyzed were only compared morphologically (Tab. 1), and the required sequences were retrieved from GenBank (DQ987513, DQ987515, DQ987517, DQ987518; DQ926890; AY848422-AY848424). Morphological information on G. azzurrae specimens were taken from Mercurio and Andreone (2007) and sequences were retrieved from GenBank (EF222300-EF222305).

DNA analysis
A fi ngertip, o r part of the muscle of the tongue, was cut from each collected individual and stored in 99% ethanol. Total genomic DNA was extracted from the tissue samples using proteinase K digestion (10 mg/ml concentration) following Bandi et al. (1994) protocol. To sequence a fragment of ca. 550bp of the mitochondrial 16S rRNA gene, which has proven to be suitable in anuran species identifi cation  we used the primers 16SA-L 5'-CGCCTGTTTATCAAAAACAT-3' and 16SB-H 5'-CCG-GTCTGAACTCAGATCACGT-3', modifi ed from Kocher et al. (1989) and Palumbi et al. (1991). PCR reactions were performed using standard cycling protocols (Vences et al. 2003) and the light strands were sequenced using an ABI3730XL by Macrogen Inc. Sequences were blasted in GenBank, checked by eye, edited, aligned using the BioEdit sequence alignment editor (version 7.0.5.3;Hall 1999). Th e alignment of all the processed samples required the inclusion of gaps to account for indels in only a few cases in one hypervariable region. All newly determined sequences have been deposited in GenBank (HQ640413-HQ640426). Mean genetic distances matrix (uncorrected p-distance transformed into percent) between and within individuals belonging to the type series of G. atsingy (holotype and 7 paratypes) and of other species of the subgenus Phylacomantis (Gephyromantis corvus, G. pseudoasper and G. azzurrae) were computed. Etymology. Th e specifi c noun "atsingy" (pronounced: "a-tseen-jě") is a Malagasy word. Th e terms "atsingy" or "tsingy" are the common names used to refer to the pointed and sharp calcareous lime stone formations and pinnacles originated through rainfall erosion. Although present in several other localities in western Madagascar (e.g.: Ankarana), the outcrops of Bemaraha are typical of this area and the specifi c name is therefore associated with the locality of provenience of the types.

Gephyromantis
Remark. Th is species has been referred to as Gephyromantis sp. aff . corvus "Bemaraha" by , as Gephyromantis sp. 10 "Bemaraha" by Vieites et al. (2009), and Gephyromantis sp. aff . corvus by Bora et al. (2010).   Description of the holotype. Subadult male in mediocre state of preservation, with the belly opened for gonadal inspection and part of the ventral surface of thighs cut and opened to check the glands. SVL 34.8 mm; for other measurements see Tab. 1. Body slender; head longer than wide, in line with the body; snout slightly pointed in dorsal view, rather rounded in lateral view; nostrils directed laterally, much nearer to tip of snout than to eye; canthus rostralis well defi ned; tympanum distinct, rounded, its horizontal diameter about 50% of eye diameter; supratympanic fold well distinct, regularly curved; tongue distinctly bifi d posteriorly. Arms slender; subarticular tubercles single; outer and inner metacarpal tubercles paired; fi ngers without webbing; fi nger disks triangular distinctly enlarged; nuptial pads absent. Hind limbs slender; tibiotarsal articulation reaching the nostril when hindlimbs are adpressed along body; lateral metatarsalia partly connected; inner metatarsal tubercle distinct, outer metatarsal tubercle small but recognizable; webbing of foot 1(1), 2i(1), 2e(1), 3i(2), 3e(1), 4i(2), 4e(2), 5(1). Skin slightly granular on dorsum and belly, ventral skin smooth on throat and chest. Femoral glands cluster ("Type 2", according to Glaw et al. 2000) hardly recognizable from external view, but with an overall granular structure and with 4-6 single whitish granular glands of ca. 1 mm diameter scattered on thighs. Th e vocal sacs in the male holotype are indistinct. Th e live colouration, based upon the photograph taken by J.E. Randrianirina is light brownish with darker dots and marbling ( Fig. 2;  A). Th e fi nger and toe tips are lighter than the remnant parts of fore-and hindlegs. After about seven years of preservation in ethanol the holotype still conserves the original marbled-brownish colour patterns, although it showed a slight loss of colour ( Fig.  2; K-L). In particular, the belly became much whitish and inconspicuous. A rather characteristic and darker X-shaped marking is visible on the shoulder region, as well as a diff use marbling darker pattern on the back and head. Th e tympanum is whitish. Limbs are brownish, with dark brown cross-bands: 3 on femur, 3 on tibia, 5-6 on tarsus and foot, 4 on lower arm and hand. On the fl anks, the dorsal colour fades into the whitish ventral colour. Th e ventral side is uniformly cream-whitish on forelimbs and belly, while the throat is very lightly pigmented.
Variation. We based the current description of variability upon some specimens (paratypes and complementary individuals), part of which (ZSM 23/2006, 37/2006, 107/2006 were also photographed in nature, and thus provided more diagnostic characters. Th e female ZSM 23/2006 ( Fig. 2; C-E), shows a back with sparse larger warts. Its colouration appears light brown with greyish shadings, darker dots and transversal bands on the back and legs. Th ese are more evident in the preserved individual, where a pattern of darker spots is visible on the back, suggesting the presence of a darker X-shaped drawing. Th ese spots are visible in two other individuals, MRSN A5484 (a female) and in the holotype MRSN A5487 ( Fig. 2; A, K), although for the former specimen we do not have photographs taken in life. Th e tympanum is uniformly brownish, and the iris is yellowish with darker reticulations. Th e belly is comparatively smooth, with fewer warts on its lateral parts.
Th e throat is quite smooth. Th e central part of the belly is lighter than the fl anks and the ventral sides of thighs, whitish on breast and thorax, with sparse darker spots. Th e inguinal part appears yellowish. Th e throat is darker than the belly, with a median lighter (although not so contrasted) line. Th e lateral borders of the lower jaw bear darker spots. After preservation, the colouration appears substantially similar, although faded. Th e juvenile ZSM 37/2006 ( Fig. 2; E) presents a rather smooth back and fl anks with sparse and barely evident warts. Th e colouration is brownish shading to the grey on the fl anks and lateral parts of the back, with darker spots, extending around the fl anks. Th e central part of the back is crossed by a longitudinal light (almost beige) band which enlarges on the head to cover the upper eyelids. Th e posterior part of such a band narrows to shade almost totally at the level of the vent. A thin, almost continuous whitish longitudinal line runs from the tip of the snout until the groin. Th e juvenile ZSM 107/2006 ( Fig. 2; I) also shows a rather smooth back. Th e colouration is much darker, and the markings and spots are less visible. Th e tympanum is lighter than the surrounding areas, and the upper ridge is entoured by black pigment. Both these juveniles after about four years of preservation present a similar pattern of colouration as in life. In ZSM 107/2006 the central part of the back appears quite lighter than the surrounding areas, with a sort of arrow pattern. An interesting comparison is with the only mature available male (SVL 36.6 mm) photographed in life, the individual labelled UADBA 39099 ( Fig. 2; F-H). Th is male appears quite slim in the photographs (either in dorsal or ventral view), with rather uniform light brown shading to greenish in life, and a moderately glandular skin texture ( Fig. 2; F-G). Th e belly appears rather smooth in life, with the whole venter and thorax whitish ( Fig. 2; H). Th e throat is darker with a rather indistinct central whitish band and vocal sacs are not recognizable. Lower parts of arms and thighs are pinkish, while tibiae are more whitish pigmented. Th e plantar surfaces are also reddish-pink. In this male, the glands are well visible and yellowish, and appear similar to those observed in G. azzurrae, G. corvus and G. pseudoasper. In particular, they clearly belong to the gland "Type 2", sensu Glaw et al. (2000), with 70 granules counted from the inner side of the right gland itself (whose external measure is 7.5*3.1 mm). In MRSN A5484 (a female) we notice a dark bar between the eyes, and an X-shaped darker spot at mid-dorsum; quite large and isolated dark spots are visible in the posterior part of the back. Th e belly is uniformly whitish and smooth. Th e three juveniles MRSN A5483, MRSN A5482, and MRSN A5485, are similar in colouration (excepting for MRSN A5483 exhibiting a light mid-dorsal line), with dark back with sparse lighter spots and shading, and almost whitish bellies. Of MRSN A5483 we also dispose of a photo taken in life, where the longitudinal light line is evident ( Fig. 2; J).
Natural history. According to our observations, the species lives in habitats that retain some humidity, such as rock cavities and along the walls of the canyon-like formations. One important notation comes from the fact that several of the collectors, independently (JER, FG, JCR) found this species within the caves which are typical of the area. We suspect that the species uses caves because these sites presumably have a higher humidity than the surrounding areas. In such a sense it behaves similarly to G. corvus at Isalo, which is known to frequent narrow canyons and cave-like canyons (Mercurio et al. 2008). Apparently, the new species (both adults and juveniles) is not confi ned to the proximity of water, and it has been observed jumping among the tsingy pinnacles also far from water bodies. All the individuals were active at night on tsingy rocks or during the day in caves. No data are available about mating behaviour, advertisement calls and tadpole morphology.
Distribution. Only known from the localities of the type specimens within the Tsingy de Bemaraha National Park.
Comparison with other species. Gephyromantis atsingy sp. n. diff ers from G. pseudoasper, G. azzurrae and G. corvus by the lack of paired blackish skin folds (vocal sacs) along the lower jaws in adult males, and from G. azzurrae also by details of colouration (see below). Following our measurements, adult males of G. atsingy can also be diff erentiated among each other by the number of granules in the femoral glands: 70 granules in G. atsingy; 96 granules in G. corvus; 38-45 granules in G. azzurrae and 39-43 granules in G. pseudoasper. In addition, the new species diff ers from all three species by substantial genetic diff erentiation (see below).
All the described species of Gephyromantis, subgenus Phylacomantis, show similarities with G. atsingy (Tab. 2). Th e dorsal pattern is similar in all species, showing an assemblage of darker spots and reticulations on the lighter background, and barred legs and arms. Th e dorsal colouration in G. atsingy is usually light brown-beige, with a somehow greenish shading, while in G. corvus it is uniformly grey or dark grey with sparse darker (uniformly-sized) warts and dots. Notwithstanding, the examined specimens of G. atsingy have a much more contrasted X-shaped dark spot on the back. Th is is less evident in G. corvus, where the dark-light pattern is more confuse and irregular. We observed a longitudinal repetition of lighter elements, a longitudinal light band or a middorsal light line only in G. atsingy. Th e belly in both species is light, but in G. atsingy we detected more frequently the darker drawing with a lighter central area on the throat and chest. According to the original description and subsequent papers Andreone 2007, Mercurio et al. 2008), G. azzurrae has a quite variable dorsal colouration. Th e holotype of the species, as depicted by , has a wide lighter dorsal band upon a darker dorsal colouration, and the belly is reddish. Other examined specimens of G. azzurrae present more uniform dorsal colouration. In both species the dorsal skin is featured by the presence of similar larger warts. In comparison to G. atsingy, the G. pseudoasper specimens are smaller and have a more warty back. Th e colouration in G. pseudoasper is much darker and the belly is much more pigmented: the throat, the thorax and the anterior part of the belly are heavily spotted in dark, with a clear median light line on the throat. Th e posterior parts of the belly and parts of the ventral side of the legs in G. pseudoasper are often orange. Th e external vocal sacs are evident and well developed, while these are not visible in G. atsingy.
Mitochondrial variation and diff erentiation. Th e molecular data confi rm the attribution of G. atsingy to the subgenus Phylacomantis Vences 2006, Vieites et al. 2009). Th e analyzed specimens of G. atsingy, G. azzurrae, G. corvus and G. pseudoasper appear genetically very uniform and show an intraspecifi c uncorrected divergence of 0.5%, 0.4%, 0.1% and 0.1% respectively, in the 16S rRNA gene sequences. Th e genetic distance between G. atsingy and the three other Phylacomantis species ranges between 10.2% (comparison between G. atsingy and both G. corvus and G. pseudoasper) and 11.2% (comparison between G. atsingy and G. azzurrae). Among the genus Gephyromantis the smallest genetic distance is observed between G. corvus and G. azzurrae (9.1%) and the highest uncorrected divergence between G. azzurrae and G. pseudoasper (13.1%). Gephyromantis corvus and G. pseudoasper have a genetic distance of 12%. Th ese divergences are comparatively high among mantelline species (see Vences et al. 2005, Vieites et al. 2009), and corroborate the species status of G. atsingy. Th e phylogenetic relationships between the species of the Phylacomantis subgenus have been resolved recently ( N. Kaff enberger et al., in preparation). Th ese analyses confi rm the monophyly of the subgenus, provide evidence for the basal position of G. pseudoasper and uncover the sister relationship between Gephyromantis atsingy and the complex made of G. corvus and G. azzurrae. Conservation.
Th is species appears to be restricted to the Bemaraha Plateau, where it has been found in seven localities within the Tsingy de Bemaraha National Park. It may also occur in the Réserve Naturelle Intégrale, which forms the northerly limit of the Bemaraha Plateau, but survey data are lacking from this site. Within the national park, some areas of forest are damaged by conversion to agriculture and charcoal production, but the humid canyons where G. atsingy occur are generally well protected. We therefore recommend assigning a category of Near Th reatened because the species nearly qualifi es for listing as Vulnerable under D2: the species is confi ned to a single site, the Bemaraha Massif (1,577 km 2 ), with a plausible threat that could impact the species in the near future. If the threat became operational, the species would be eligible for listing as Endangered since its extent of occurrence is well within the 5,000km 2 threshold under the B criterion and it would occur at a single location (where the threat is habitat loss from agricultural activities and charcoal production) and there would be a continuing decline in the quality and area of habitat, qualifying the species for the criteria B1ab(iii).

Discussion
Th e Bemaraha plateau is one of the most peculiar areas of western Madagascar in terms of amphibian richness and endemicity (Raselimanana 2008, Bora et al. 2010. Th e new Gephyromantis species described here adds one more taxon to a list of endemics, which includes Heterixalus carbonei, Plethodontohyla fonetana, Rhombophryne sp., Stumpffi a sp. aff . helenae, Boophis tampoka , Bora et al. 2010, although some of these species might also be present at other sites of the West. Th e description of G. atsingy, and the previously mentioned works underline how little we still know about the amphibian fauna of this part of Madagascar and stresses the importance of further systematic surveys in these isolated areas.
One powerful tool is the application of an integrated taxonomy approach, which includes aspects of direct fi eld surveys, behavioural assessment, molecular screening and bioacustic analysis. Th is is what allowed Vieites et al. (2009) to identify a high number of candidate species boosting a descriptive process of a large number of poorly diff erentiated species in an astonishing short lapse of time (Mercurio and Andreone 2007, Cramer et al. 2008, 2010, Wollenberg et al. 2008, Andreone et al. 2010, Vallan et al. 2010, Vences et al. 2010a. As already stressed by  and Bora et al. (2010), Isalo and Bemaraha were probably in contact until relatively recently and were covered by humid vegetation, which allowed the existence of typical rainforest species at Isalo (e.g., Boophis luteus, Mantidactylus femoralis and M. lugubris), and rainforest-derived species at Bemaraha (e.g., Boophis tampoka, Plethodontohyla fonetana and Rhombophryne sp.). Th is hypothesis is also supported by the shared presence of typical rainforest elements, and by the presence of other species, like Mantella sp. aff . expectata and Blommersia sp. aff . wittei.
We expect that other forest blocks in western Madagascar may host further undescribed species of Gephyromantis and we highlight the need of conservation actions in Madagascar's dry forests due to the increasing deforestation rate and changing climatic scenarios.
Th e accelerated species discovery in Malagasy amphibians points to the importance of taxonomic surveys (see www.sahonagasy.org), and we like to consider G. atsingy as another "forceps delivered" species, according to the attractive defi nition given by Vallan et al. (2010) for Boophis calcaratus. In fact, we knew about the existence of G. atsingy for several years, but did not have enough data to describe it, since Gephyromantis atsingy and G. corvus appear to be rather similar to each other and share several life history traits. We here described this new species, recognizing that a species without a formal description and an attached name is simply an "invisible" species, hard to be protected and classifi ed within the IUCN Red List (IUCN 2010).