A new species of nocturnal gecko ( Paroedura ) from karstic limestone in northern Madagascar

Paroedura hordiesi sp. n. is described from Montagne des Français, a karstic limestone massif in the far north of Madagascar recently established as nature reserve. The new species has the nostril in contact with the rostral scale and shares many characters with P. karstophila and especially with P. homalorhina which are also restricted to karstic habitats. Paroedura hordiesi differs from P. karstophila by a smoother skin on dorsum and legs, by original and regenerated tails being both entirely smooth, by colouration, and by larger snout-vent length. Morphologically the new species is most similar to P. homalorhina from the Ankarana reserve from which it can be distinguished by shorter limbs and a less slender habitus. Published molecular data place the new species as close relative of P. homalorhina and another undescribed species from Nosy Hara Island, while newly determined data of the cox1 gene for P. karstophila confirm the distinctness of the new species from this taxon. Integrating the information from published and novel molecular data, the new species differs from all nominal Paroedura (except P. vahiny for which no molecular data are available to date) by strong genetic divergences. P. hordiesi might be another microendemic species of the Montagne des Français region. We suggest its IUCN Red List classification as “Critically Endangered” on the basis that it has an extent of occurrence of at most 50 km2, it is known from a single location, and there is a continuing decline in the extent and quality of its habitat.


Introduction
The far north of Madagascar comprises a mosaic of heterogeneous landscapes ranging from rainforests on volcanic basement to deciduous dry forests in karstic massifs and littoral habitats on sandy ground (e. g.Lavranos et al. 2001, Vences et al. 2009, Crottini et al. 2012).The geological and climatic diversity of this area is reflected by a high species diversity and a high degree of microendemism (e. g.Andreone 2004, Wilmé et al. 2006, Ranaivoarisoa et al.Montagne des Français and adjacent littoral habitats (Ramanamanjato et al. 1999, Raselimanana et al. 2000, Glaw et al. 2001, 2005, 2012, 2013, Köhler et al. 2010a, 2010b, Miralles et al. 2011).These major habitat types are separated from each other by rather steep ecotones in northern Madagascar and thus in part constitute "habitat islands" for several species, possibly allowing allopatric speciation.Several taxa including dwarf frogs (Stumpffia), dwarf chameleons (Brookesia), burrowing skinks (Paracontias), leaf-tail geckos (Uroplatus), and the nocturnal geckos of the genus Paroedura have undergone remarkable diversification in northern Madagascar (Jackman et al. 2008, Köhler et al. 2010a, 2010b, Ratsoavina et al. 2011).
The genus Paroedura is widely distributed throughout Madagascar's biomes, including eastern rainforest, western dry forest, extremely arid thornbush savanna and high mountain habitats (Angel 1942, Guibé 1956, Dixon and Kroll 1974, Rösler and Krüger 1998, Nussbaum and Raxworthy 2000).Five of the 15 described species from Madagascar occur in the far north, and two additional species from the Comoro islands have close relationships to the northern P. lohatsara and P. stumpffi suggesting that they originated by two colonization events from the northern species (Hawlitschek and Glaw 2013).Recent surveys indicate that the karstic limestone massifs in this region still harbour further undescribed reptile species (e. g.Jackman et al. 2008, Recknagel et al. 2013).Some of them might be microendemic and threatened by substantial habitat destruction.In the following we describe a new Paroedura species from Montagne des Français to contribute to the taxonomic inventory of this massif, and to highlight the threats affecting this microendemic species and other biota in the region.
To obtain molecular comparisons of the new species with previously unstudied nominal Paroedura species, we sequenced a fragment of the mitochondrial gene for cytochrome oxidase subunit I (cox1) with primers and protocols defined by Nagy et al. (2012) for several Paroedura samples from Tsingy de Namoroka (corresponding voucher specimens to be catalogued in MNHN).These samples have become available through a recent herpetological survey by one of us (II) and include topotypical P. karstophila, previously unstudied from a molecular perspective.The resulting sequences were combined with those of Nagy et al. (2012) and Koubová et al. (2014) in MEGA version 5 (Tamura et al. 2011) to yield an alignment of 664 bp.We performed phylogenetic inference in MEGA under the Maximum Likelihood optimality criterion, with a general time-reversible substitution model with gamma-distributed rates and invariant sites, NNI branch swapping, and assessing robustness of nodes with 500 bootstrap replicates.Newly obtained sequences were submitted to GenBank (accession numbers: KM978078-KM978080).

Molecular differentiation in the genus Paroedura
The multigene phylogenies of Jackman et al. (2008) and Hawlitschek and Glaw (2013) as well as the combined cox1 sequences of Nagy et al. (2012), Koubová et al. (2014) and those newly obtained (Fig. 1) provide evidence of strong genetic divergence of P. hordiesi to all other described Paroedura species except P. vahiny for which DNA sequence data is not yet available.
The cox1 data place the new species sister to an undescribed candidate species from Nosy Hara (79% bootstrap support), and this clade forms part of a more inclusive clade with P. oviceps and the undescribed Ankarafantsika species (bootstrap support 69%; no cox1 sequences were available for P. homalorhina from Ankarana which in a previous study was the sister taxon of P. hordiesi, see Jackman et al. 2008).All studied species and candidate species of Paroedura included in the cox1 data set were differentiated by very high pairwise uncorrected distances.P. hordiesi differed from its Nosy Hara sister lineage by a p-distance of 16.3-16.5%,and from all other Paroedura including the relatively distantly related P. karstophila by >20%.Two samples sequenced from the Tsingy de Namoroka, the type locality of P. karstophila, were sister to each other but showed a substantial divergence of 15.9% cox1 p-distance, suggesting that possibly two cryptic species may be hiding under the name P. karstophila in this karstic massif.The sample from Ankarafantsika (P.sp.Ankarafantsika in Fig. 1), identified in Jackman et al. (2008) as P. karstophila, in fact belongs to yet another, undescribed species according to our subsequent comparisons and is only distantly related to P. karstophila (Fig. 1).The DNA barcoding voucher sequence for the karyotype of P. karstophila (Koubová et al. 2014) is identical to our sequences of P. hordiesi, suggesting that the karyotype description in this paper refers to our new species rather than to P. karstophila.
Paratypes.All paratypes were collected in the "tsingy" limestone massif at Montagne des Français, Antsiranana province, north Madagascar.Specimens were collected on the tsingy outcrops along the way between the Hotel "Kings Lodge" (12°18'44,8''S, 49°20'22,6''E, 10 m  Diagnosis.Paroedura hordiesi sp.n. is a medium-sized species (SVL up to 58 mm, tail length up to 53 mm), having moderately prominent dorsal tubercles disposed into moderately distinct, and generally regular longitudinal rows and an original tail with no spines.
The new species can be easily attributed to the genus Paroedura based on its nested phylogenetic position within the genus (Jackman et al. 2008) and its morphological similarity to other Paroedura species, especially concerning the ventral structure of their fingers and toes which comprise a pair of squarish terminal adhesive pads.Among Malagasy geckos this terminal toe structure is only found in Paroedura and the related genus Ebenavia (Glaw & Vences, 2007).The latter genus can be easily distinguished from Paroedura by its much narrower and strongly pointed head, its elongated body, and smaller size.
Comparisons.The new species can be distinguished from the 17 other currently recognized Paroedura species (including the three available junior synonyms in the genus) as follows: From P. androyensis, P. bastardi, P. ibityensis, P. lohatsara, P. maingoka, P. picta, and P. vahiny by having the nostril in contact with the rostral scale; from P. gracilis by absence of a raised vertebral ridge on the body and shorter forelimbs which are not extending forward beyond tip of snout; from P. masobe by much smaller size (SVL up to 58 mm versus 107 mm), much smaller eyes with a pigmented iris (versus black iris) and absence of a dorsal row of paired spines on the tail; from the two Comoroan species P. sanctijohannis and P. stellata by slightly smaller size (SVL up to 58 mm versus 68 mm and 62 mm, respectively) and absence of whorls with distinct spiny tubercles of the original tail; from the syntopically distributed P. stumpffi by smaller size (SVL up to 58 mm versus 70 mm) and absence of whorls with distinct spiny tubercles of the original tail; from P. tanjaka by much smaller size (SVL up to 58 mm versus 102 mm) and absence of whorls with distinct spiny tubercles of the original tail; from P. vazimba by larger size (SVL up to 58 mm versus 49 mm) and absence of whorls with distinct spines of the original tail; from P. oviceps from its type locality Nosy Be by smaller size (SVL up to 58 mm versus 69 mm) and rather regularly arranged tubercle rows on the back (versus rather irregular rows of tubercles); from P. karstophila by the absence of whorls with distinct spiny tubercles of the original tail (and by a smoother regenerated tail, see Nussbaum and Raxworthy 2000) and by colouration (see Fig. 2 versus Fig. 5); and from its close relative P. homalorhina (Jackman et al. 2008) by shorter limbs (finger tips reach the anterior margin of eye versus snout tip when forelimbs are adpressed along the body), slightly smaller size (SVL up to 58 mm versus 65 mm, see Table 2), distinct and generally regularly arranged tubercles rows on the back (versus less distinct and less regular rows), and a more slender habitus (see Fig. 2 versus Fig. 5).In addition, Paroedura hordiesi can be easily distinguished from most other Paroedura species (P.androyensis, P. bastardi, P. gracilis, P. ibityensis, P. lohatsara, P. maingoka, P. masobe, P. oviceps, P. picta, P. sanctijohannis, P. stellata, P. stumpffi, P. tanjaka, P. vahiny and P. vazimba) by adult colouration in life (see colour photographs in Glaw and Vences 2007, Schönecker 2008, Hawlitschek and Glaw 2013) and from P. androyensis, P. bastardi, P. gracilis, P. homalorhina, P. ibityensis, P. lohatsara, P. maingoka, P. masobe, P. picta, P. sanctijohannis, P. stellata, P. stumpffi, P. tanjaka, and P. vazimba by juvenile colouration (Glaw and Vences 2007, Schönecker 2008, Hawlitschek and Glaw 2013, FG pers. obs.; juvenile colouration of the other species still unknown).Genetically, P. hordiesi can be distinguished from all other species in the genus by its molecular differentiation in mitochondrial and nuclear genes (Jackman et al. 2008, Nagy et al. 2012, Hawlitschek and Glaw 2013, Fig. 1) except for P. vahiny for which DNA sequences are not yet available.
Description of the holotype.SVL 52.6 mm, further measurements and counts are given in Table 1.Holotype in good condition, with complete but broken original tail and everted hemipenes.Head distinctly wider than neck and wider than body.Snout angled downward to tip, slight depression between poorly developed canthal ridges.Ear opening is a vertical slit.Original tail slightly shorter than snout-vent length, nearly round in cross section in its proximal part, laterally compressed in its distal half, with sharply pointed tip; ventral pygal section with pair of postcloacal sacs.Digits moderately expanded at tips.Rostral scale rectangular, much wider than tall and wider than mental.Nostril in contact with Table 1.Morphometric and meristic variation of several type specimens of Paroedura hordiesi from the type locality Montagne des Français.Abbreviations for measurements and counts (see Materials and Methods for other abbreviations): ZSM = Zoologische Staatssammlung München; SVL = snout-vent length; TL = tail length; HL = maximum head length (from tip of snout to posterior margin of ear); HW = maximum head width, at widest point; HH = maximum head height; AGL = axilla-groin distance; ED = maximum eye diameter; EO = maximum ear opening diameter; FOL = forelimb length, from axilla to tip of longest finger; HIL = hindlimb length, from groin to tip of longest toe; SPL = number of supralabial scales; IFL = number of infralabial scales; NAS = number of nasals in direction from rostral to labial including nasorostrals, supranasals, postnasals; IN = number of internasals; IO = number of interorbitals; PM = number of postmentals; SLM4 = number of subdigital lamellae on fourth digit of manus; SLP4 = number of subdigital lamellae on fourth toe of pes; PCT = number of postcloacal tubercles; TLT = number of tubercle rows on tail.Counts are listed left-right.All measurements in Tables 1 and 2 were taken by H. Rösler with a caliper to the nearest 0.1 mm.HT = holotype, PT = paratype, F = female, M = male, SA = subadult.13-13 12-13 14-13 13-13 14-13 14-13 n.a.-12 13-12 14-14 NAS 5-5 5-5 5-5 4 5-5 5-5 5-5 5-5 5-5 5-5 rostral, first supralabial, and five further scales.First supralabial largest, labials smooth.Snout and interorbital scales juxtaposed, some raised, some scales in front of orbits tuberculate, as are some larger lateral occipital scales.Dorsolateral neck and body scales very heterogeneous with about eight partly poorly recognizable longitudinal rows at midbody of enlarged tubercles; en- : head dorsally beige with almost no recognizable pattern except a whitish dorsolateral spot above each ear opening and a well-defined blackish area above each eye which represents, however, no pigmentation of the skin, but is due to the blackish eyeball shining through the skin.Dorsum greyish with four poorly defined beige spots on the back which are the remains of dorsal crossbands: one distinct spot between the forelimbs, two poorly recognizable spots on the back between forelimbs and hindlimbs, and a fourth poorly recognizable spot between the hindlimbs.Dorsal surfaces of forelimbs and hindlimbs marbled with beige and grey.Flanks with similar colour as dorsum, but without any recognizable traces of light crossbands.Tail dorsally with whitish-grey and brown alternating transverse bands which are poorly delimited in the distal portion of the tail.Throat, chest, venter, ventral parts of forelimbs and hindlimbs and ventral side of tail whitish.Iris dark grey, pupil white.Colour of holotype in life unknown (no colour photographs available).Variation.Morphometric and meristic variation of ten specimens of the type series is summarized in Table 1, but there is also a remarkable individual variation in dorsal colouration pattern (Fig. 2).Paratype ZSM 2106/2007, the smallest known juvenile (SVL 28.1 mm, tail length 25.2 mm), shows a moderately distinct juvenile colouration both in life (Fig. 2) and in preservative consisting of four light crossbands on the back between the insertion of forelimbs and the insertion of hindlimbs.A similarly distinct banding on body and original tail is still visible in the larger juvenile paratype ZSM 2107/2007 (Fig. 2, SVL 35.2 mm, tail length 34.7 mm).Although the juvenile colouration is distinct compared to most adults, it is less colourful and less contrasting in comparison with many other Paroedura species.The adult paratypes usually have a less distinct dorsal colour pattern than the juveniles, ranging from poorly recognizable (e. g.ZSM 532/2000) to distinct and well delimited (e. g.ZSM 531/2000) after 14 years in alcohol.Additional photographs of living individuals are provided in Henkel and Schmidt (1995), Glaw and Vences (2007), and Schönecker (2008).Most of the adult type specimens have a regenerated tail (without distinct transversal banding of dark and white), indicating a high pressure by predation or intraspecific aggression.

Hemipenis.
The following description is based on the everted right organ of ZSM 343/2004 (Fig. 4).Hemipenis medium-sized (total length 6.4 mm, width at apex 4.5 mm), apex divided in two lobes.Sulcus spermaticus forms an S-shaped, narrow and deep groove, bordered by moderately distinct lips reaching the apex.On the apex, the sulcus is becoming broader and divides in two branches.A horn-like apical cone is present in the center of each lobe.Calyces on the apex are only present at the border of the lobes.The hemipenes of the holotype (ZSM 342/2004) are virtually identical in every respect.
Habitat and habits.Paroedura hordiesi was observed multiple times at night in karstic dry forest in the rainy season, mainly climbing on karstic rocks and the ruins of an old fort.It was found in close syntopy with P. lohatsara in the karstic limestone areas, whereas P. stumpffi was only encountered on the slope between the massif and the sea, in areas without karstic formations.
Etymology.The specific name is dedicated to Freddy Hordies, in recognition of his support for biodiversity research and conservation through the BIOPAT initiative.
Distribution and conservation status.Paroedura hordiesi is reliably known only from the recently established nature reserve of Montagne des Français.The species possibly also occurs at Ampombofofo, ca. 25 km north of this massif (Megson et al. 2009: ZSM 1531/2008), but the identity of this population needs further study as molecular data are not available thus far.Surveys in other limestone areas of northern Madagascar revealed a superficially similar Paroedura at Nosy Hara (Metcalf et al. 2007) which is, however, strongly differentiated by colouration (see photo in Glaw and Vences 2007) and mitochondrial DNA sequences (Fig. 1 and Nagy et al. 2012), and is therefore considered a further undescribed candi- date species.The Ankarana massif is known to harbour P. homalorhina, P. karstophila, and P. stumpffi (Nussbaum andRaxworthy 2000, Raselimanana 2008).Our own surveys in Ankarana revealed only homalorhina and P. stumpffi and we consider the occurrence of P. hordiesi unlikely in this massif.Rakotondravony (2006a) found Paroedura sp. at Binara in the Loky-Manambato region which potentially could refer to P. hordiesi, but the identity of this record remains to be studied.No unidentified Paroedura species was found at Analamera (Rakotondravony 2006b).Thus current evidence suggests that P. hordiesi is microendemic of Montagne des Français and perhaps the adjacent Ampombofofo region.
For consistency with the IUCN Red List Assessment for Paroedura lohatsara (Raxworthy et al. 2011) and other potential microendemic species of the Montagne des Français region, we suggest a classification as "Critically Endangered" on the basis that P. hordiesi has an extent of occurrence of at most 50 km², it is known from a single location, and there is a continuing decline in the extent and quality of its habitat.

Discussion
With the description of Paroedura hordiesi we add a further, probably microendemic new species to the herpetofauna of Montagne des Français.Although this population is already known for approximately 20 years, several factors have hampered the clarification of its identity, including the variability in colouration and in distinctiveness of longitudinal rows of dorsal tubercles of P. hordiesi, P. homalorhina and P. karstophila, the existence of several undescribed species with similar key characters, the rarity of individuals with an original tail, and the absence of genetic data and colour photographs reliably referable to P. karstophila.This situation has led to substantial uncertainty about the correct name for the species from Montagne des Français, as is reflected by the different preliminary names that have been used for this species in the literature (see above).DNA sequences are now available from two P. karstophila-like specimens from the type locality Namoroka (Fig. 1).These strongly differ from each other, suggesting that P. karstophila is possibly a composite taxon including two cryptic species which occur in syntopy at Namoroka.Although the identity of P. karstophila remains to be further studied, we have little doubts that one of the two Namoroka lineages will turn out to correspond to this taxon.
Due to PCR failure with universal reptile primers (Nagy et al. 2012), no cox1 sequences are available for several species of Paroedura, including P. homalorhina which was sister to P. hordiesi in the study of Jackman et al. (2008;as P. sp. n.).On the other hand, the new species from Nosy Hara and P. karstophila have not yet been sequenced for the genes used by Jackman et al. (2008).This unfortunate lack of overlap of the two molecular data sets, to be remedied by future sequencing efforts, hampers our ability to determine whether P. hordiesi is more closely related to P. homalorhina, or to the undescribed species from Nosy Hara.It does not, however, compromise our taxonomic conclusion of P. hordiesi being a valid species differentiated from both these close relatives by a substantial genetic divergence and morphological characters.
Paroedura hordiesi is a typical example of a microendemic karst specialist of an isolated habitat island that apparently has lost its ability to survive outside its special habitat and thus has lost the genetic exchange with populations from neighboured karstic massifs.Although the surrounding massifs are only separated by several kilometers of grassland or other non-karstic habitats, they are often populated by different species which might have evolved in isolation for millions of years.The cox1 data presented in Fig. 1 suggest that the undescribed Paroedura species from the island Nosy Hara is a close relative of P. hordiesi.The two species differ by a substantial genetic distance, and distinctly by their colouration (see photographs in Glaw and Vences 2007), although the distance between this island and Montagne des Français is just about 35 km.The distance between Montagne des Français and Ankarana is approximately 50 km, yet the latter massif is populated by P. homalorhina, the second close relative of P. hordiesi (see discussion in previous paragraph).Similar patterns of microendemism and comparable phylogenetic relationships have been found in dwarf chameleons of the genus Brookesia (Glaw et al. 2012), in a clade of dwarf frogs of the genus Stumpffia (Köhler et al. 2010a), and in lizards of the genus Zonosaurus (Raselimanana et al. 2009, Recknagel et al. 2013), suggesting that similar vicariant processes among karstic habitat islands might have affected these groups of organisms in the far north of Madagascar.The karstic massifs might have provided sufficient resources and protection from desiccation during periods of drier climate to allow the long-term survival of these populations, finally leading to completed speciation.The evolution and long-term survival of these microendemic species suggests rather stable conditions without catastrophic events over very long time periods.The apparent lack of gene flow among the karstic habitat islands also suggests a limited dispersal capacity of karst specialists across the non-karstic interspersed matrix.by a cooperation accord between the UADBA and the ZSM.Malagasy authorities are acknowledged for collection and export permits.The research F. Glaw was supported by the "Deutsche Forschungsgemeinschaft" (grant no.GL 314/1) and the European Association of Zoos and Aquaria (EAZA).Fieldwork of I. Ineich was made possible with the help of Ewe Madagascar for logistical support (Marc Gansuana), La Colas and the Société Aquamas for providing boats, thanks to them.He also wants to thank Lucile Allorge and Thomas Haevermans (MNHN) for scientific organisation, MNHN Labex for providing founds, and the Parc naturel du Namoroka, its guides and the inhabitants of the village of Vilanandro for their useful help.

Figure 1 .
Figure1.Maximum Likelihood tree inferred from 664 bp of the mitochondrial cytochrome oxidase subunit 1 (cox1) gene, showing the differentiation between Paroedura species.Note that this single-gene tree is not suitable to reconstruct the basal relationships of these geckos but is rather shown to document the large genetic divergences among all of them, and of P. hordiesi to its relatives.Numbers behind species names are sample numbers as given inNagy et al. (2012) andKoubová et al. (2014), as well as GenBank accession numbers of the respective sequences.
larged tubercles separated partly by small flat scales and smaller tubercles.Dorsal scales of forelimbs mostly flat.Dorsal scales of hindlimbs large and tuberculate, much smaller above knee.Ventral scales of forelimbs slightly smaller than surrounding ventral scales of the body.Dorsal pygal scales like dorsal body scales; lateroventral pygals tuberculate.Tail scales flat, tail segments without any transverse row of spiny tubercles.Mental triangular, bordered posteriorly by a pair of elongate, irregular hexagonal postmentals.Postmentals contact mental, first and second infralabial, one enlarged lateral gular, one smaller posterolateral gular, and one larger central gular.First three infralabials not significantly larger than others.Gulars small, granular.Ventrals of chest and abdomen flat.Proximal subdigitals in rows of 2-3.Pair of squarish, terminal pads.Claws curving downwards between terminal pads of digits.Colour after 10 years in alcohol (Fig 3)

Table 2 .
Morphometric and meristic variation of Paroedura homalorhina from the type locality Ankarana.See Table1for abbreviations and further details.