Integrative revision of the Blommersia wittei complex, with description of a new species of frog from western and north-western Madagascar.

Frogs of the Blommersia wittei complex are widespread in western and northern Madagascar, and are one of two clades of the family Mantellidae that have colonized the Comoran island of Mayotte. Based on a comprehensive set of DNA sequences of the mitochondrial 16S rRNA gene and the nuclear-encoded RAG1 and SACS genes, integrated with morphological and bioacoustic data, we here analyze the genetic differentiation of populations of this complex across Madagascar. We confirm that a candidate species named B. sp. Ca5 in previous studies represents a genetically well-defined evolutionary lineage distributed over much of western Madagascar, which we describe herein as Blommeria bara sp. nov. based on its molecular and bioacoustic differentiation. Blommersia wittei occurs across northern Madagascar but its type locality Ambanja, at the lower Sambirano river, is very close to the range of another, newly discovered microendemic lineage that was only found at two sites along the upper Sambirano river (here named as candidate species B. sp. Ca12). The B. wittei complex thus provides an example of a clade of closely related Malagasy frogs that contains species widespread over hundreds of kilometers, as well as extreme microendemics. For a full resolution of this species complex, more data need to be collected on the geographical contact among these two lineages, on the morphology and bioacoustics of B. sp. Ca12, and on the north-eastern populations of B. wittei at Sambava, which are weakly differentiated in mitochondrial genes but differ in bioacoustics and possibly in the extent of foot webbing.

Into the Chamber of Horrors: A proposal for the resolution of nomenclatural chaos in the Scaphiophryne calcarata complex (Anura: Microhylidae), with a new species-level phylogenetic hypothesis for Scaphiophryninae.

The genus Scaphiophryne (Anura: Microhylidae) contains at least nine species that, together with their probable sister genus Paradoxophyla, form the Madagascar-endemic subfamily Scaphiophryninae. Scaphiophryne are robust burrowing frogs with explosive breeding behavior and are characterized by a unique larval morphology, being intermediate between that of filter-feeding and generalized tadpole types. Based on tadpole morphology, the nominal subgenus Scaphiophryne can be distinguished from the subgenus Pseudohemisus, a clade occurring in arid biomes with a largely unsolved taxonomy and nomenclature. Here we combine a comprehensive molecular phylogeny of all scaphiophrynines based on five mitochondrial and two nuclear genes with a re-examination of the name-bearing type specimens of the seven species-level nomina assignable to the subgenus Pseudohemisus. Our molecular phylogeny supports monophyly of the two subgenera and assigns the enigmatic S. matsoko to the subgenus Scaphiophryne. In this subgenus, our analysis confirms all currently accepted species as independent evolutionary lineages based on concordant differentiation in mitochondrial and nuclear genes, and provides evidence for a combination of low mitochondrial divergence, distinct nuclear divergence, and high nuclear heterozygosity. Yet, contrary to previous hypotheses, we do not find evidence of ongoing hybridization. In the subgenus Pseudohemisus we find support for three clearly delimited species-level lineages, one of which contains two deep conspecific lineages that warrant further study. To stabilize the taxonomy in this subgenus, and considering the very poor state of preservation of several type series and the heterogeneity of some of them, we here designate lectotypes for the nomina: Hemisus obscurus Grandidier, 1872; Calophrynus calcaratus Mocquard, 1895; Pseudohemisus verrucosus Angel, 1930; Pseudohemisus longimanus Angel, 1930; and Pseudohemisus longimanus var. melanopleura Angel, 1934. Taxonomically, we accept Scaphiophryne brevis (Boulenger, 1896) as a well-defined species; restrict S. calcarata (Mocquard, 1995) to the southern lineage of the S. calcarata complex; resurrect Hemisus obscurus Grandidier, 1872 (previously considered to be a dubious name or nomen inquirendum) as Scaphiophryne obscura to refer to the western and north-western lineage of the S. calcarata complex; consider Pseudohemisus verrucosus Angel, 1930 as junior synonym of Scaphiophryne brevis; and consider Pseudohemisus longimanus Angel, 1930 and Pseudohemisus longimanus var. melanopleura Angel, 1934 as junior synonyms of Scaphiophryne obscura. Finally, to accelerate taxonomic progress, we (i) outline a set of criteria by which such old and ambiguous nomina can be applied to known lineages, (ii) suggest that the International Code of Zoological Nomenclature should slightly broaden the conditions under which neotypes are to be designated, and (iii) propose to redouble discussions on the introduction of the concept of the epitype to zoology.

Extreme miniaturization of a new amniote vertebrate and insights into the evolution of genital size in chameleons.

Evolutionary reduction of adult body size (miniaturization) has profound consequences for organismal biology and is an important subject of evolutionary research. Based on two individuals we describe a new, extremely miniaturized chameleon, which may be the world's smallest reptile species. The male holotype of Brookesia nana sp. nov. has a snout-vent length of 13.5 mm (total length 21.6 mm) and has large, apparently fully developed hemipenes, making it apparently the smallest mature male amniote ever recorded. The female paratype measures 19.2 mm snout-vent length (total length 28.9 mm) and a micro-CT scan revealed developing eggs in the body cavity, likewise indicating sexual maturity. The new chameleon is only known from a degraded montane rainforest in northern Madagascar and might be threatened by extinction. Molecular phylogenetic analyses place it as sister to B. karchei, the largest species in the clade of miniaturized Brookesia species, for which we resurrect Evoluticauda Angel, 1942 as subgenus name. The genetic divergence of B. nana sp. nov. is rather strong (9.9‒14.9% to all other Evoluticauda species in the 16S rRNA gene). A comparative study of genital length in Malagasy chameleons revealed a tendency for the smallest chameleons to have the relatively largest hemipenes, which might be a consequence of a reversed sexual size dimorphism with males substantially smaller than females in the smallest species. The miniaturized males may need larger hemipenes to enable a better mechanical fit with female genitals during copulation. Comprehensive studies of female genitalia are needed to test this hypothesis and to better understand the evolution of genitalia in reptiles.

'Barcode fishing' for archival DNA from historical type material overcomes taxonomic hurdles, enabling the description of a new frog species.

Taxonomic progress is often hindered by intrinsic factors, such as morphologically cryptic species that require a broad suite of methods to distinguish, and extrinsic factors, such as uncertainties in the allocation of scientific names to species. These uncertainties can be due to a wide variety of factors, including old and poorly preserved type specimens (which contain only heavily degraded DNA or have lost important diagnostic characters), inappropriately chosen type specimens (e.g. juveniles without diagnostic characters) or poorly documented type specimens (with unprecise, incorrect, or missing locality data). Thanks to modern sequencing technologies it is now possible to overcome many such extrinsic factors by sequencing DNA from name-bearing type specimens of uncertain assignment and assigning these to known genetic lineages. Here, we apply this approach to frogs of the Mantidactylus ambreensis complex, which was recently shown to consist of two genetic lineages supported by concordant differentiation in mitochondrial and nuclear genes. These lineages co-occur on the Montagne d'Ambre Massif in northern Madagascar but appear to have diverged in allopatry. We use a recently published bait set based on three mitochondrial markers from all known Malagasy frog lineages to capture DNA sequences from the 127-year-old holotype of Mantidactylus ambreensis Mocquard, 1895. With the obtained sequences we are able to assign the name M. ambreensis to the lowland lineage, which is rather widespread in the rainforests of northern Madagascar, leaving the microendemic high-elevation lineage on Montagne d'Ambre in north Madagascar in need of description. We describe this species as Mantidactylus ambony sp. nov., differing from M. ambreensis in call parameters and a smaller body size. Thus, using target enrichment to obtain DNA sequence data from this old specimen, we were able to resolve the extrinsic (nomenclatural) hindrances to taxonomic resolution of this complex. We discuss the broad-scale versatility of this 'barcode fishing' approach, which can draw on the enormous success of global DNA barcoding initiatives to quickly and efficiently assign type specimens to lineages.

Species complexes and the importance of Data Deficient classification in Red List assessments: The case of Hylobatrachus frogs.

Taxonomy is the cornerstone of extinction risk assessments. Currently, the IUCN Red List treats species complexes either under a single overarching species name-resulting in an unhelpfully broad circumscription and underestimated threat assessment that does not apply to any one species lineage-or omits them altogether-resulting in the omission of species that should be assessed. We argue that taxonomic uncertainty alone, as in species complexes, should be grounds for assessment as Data Deficient (DD). Yet, use of the DD category is currently discouraged, resulting in assessments based on poor data quality and dismissal of the importance of taxonomic confidence in conservation. This policy may be leading to volatile and unwarranted assessments of hundreds of species across the world, and needs to be revised. To illustrate this point, we here present a partial taxonomic revision of torrent frogs from eastern Madagascar in the Mantidactylus subgenus Hylobatrachus. Two named species, Mantidactylus (Hylobatrachus) lugubris and M. (H.) cowanii, and several undescribed candidate species are recognised, but the application of the available names has been somewhat ambiguous. In a recent re-assessment of its conservation status, M. (H.) lugubris was assessed including all complex members except M. (H.) cowanii within its distribution, giving it a status of Least Concern and distribution over most of eastern Madagascar. After describing two of the unnamed lineages as Mantidactylus (Hylobatrachus) atsimo sp. nov. (from southeastern Madagascar) and Mantidactylus (Hylobatrachus) petakorona sp. nov. (from the Marojejy Massif in northeastern Madagascar), we show that Mantidactylus (Hylobatrachus) lugubris is restricted to the central east of Madagascar, highlighting the inaccuracy of its current Red List assessment. We propose to re-assess its status under a more restrictive definition that omits well-defined candidate species, thus representing the actual species to which its assessment refers, to the best of current knowledge. We recommend that for species complexes in general, (1) nominal lineages that can be confidently restricted should be assessed under the strict definition, (2) non-nominal species-level lineages and ambiguous names should be prioritised for taxonomic research, and (3) ambiguous names should be assessed as DD to highlight the deficiency in data on their taxonomic status, which is an impediment to their conservation. This would reduce ambiguity and underestimation of threats involved in assessing species complexes, and place the appropriate emphasis on the importance of taxonomy in anchoring conservation.

Three new species of nocturnal geckos of the Paroedura oviceps clade from xeric environments of Madagascar (Squamata: Gekkonidae).

We describe three new gecko species of the Paroedura oviceps clade, diagnosed by deep divergences in mitochondrial DNA, absence of haplotype sharing in two nuclear genes (sacs and kiaa1239), and morphological differences. Paroedura spelaea sp. nov. is an extremely slender species from karst habitats in the limestone massif of the Tsingy de Bemaraha National Park in western Madagascar, morphologically reminiscent of P. homalorhina but distinguished by the absence of distinct dorsal rows of spiny tubercles. Paroedura fasciata sp. nov. from the small karstic island Nosy Hara in northern Madagascar is phylogenetically placed sister to P. spelaea despite radical morphological differences. It is morphologically most similar to P. hordiesi from the geographically close Montagne des Français massif, but is smaller, has slightly more spiny dorsal scales, and a distinctive colour pattern with light grey dorsal crossbands. The third species, Paroedura kloki sp. nov., is known from Ankarafantsika National Park and the western slopes of Makira, two sites of dry to transitional forest in western Madagascar. It is at least partly arboreal and morphologically very similar to P. oviceps, from which it differs by spiny scales extending over its entire tail. Although incomplete, the available phylogenetic evidence suggests that the karst specialists in the P. oviceps clade (P. fasciata, P. homalorhina, P. hordiesi, P. spelaea) form a monophyletic group which might have diversified by vicariance after becoming isolated, respectively, in their limestone habitats in Tsingy de Bemaraha, Ankarana, Nosy Hara and Montagne des Français.

The use of bioacoustics in anuran taxonomy: theory, terminology, methods and recommendations for best practice.

Vocalizations of anuran amphibians have received much attention in studies of behavioral ecology and physiology, but also provide informative characters for identifying and delimiting species. We here review the terminology and variation of frog calls from a perspective of integrative taxonomy, and provide hands-on protocols for recording, analyzing, comparing, interpreting and describing these sounds. Our focus is on advertisement calls, which serve as premating isolation mechanisms and, therefore, convey important taxonomic information. We provide recommendations for terminology of frog vocalizations, with call, note and pulse being the fundamental subunits to be used in descriptions and comparisons. However, due to the complexity and diversity of these signals, an unequivocal application of the terms call and note can be challenging. We therefore provide two coherent concepts that either follow a note-centered approach (defining uninterrupted units of sound as notes, and their entirety as call) or a call-centered approach (defining uninterrupted units as call whenever they are separated by long silent intervals) in terminology. Based on surveys of literature, we show that numerous call traits can be highly variable within and between individuals of one species. Despite idiosyncrasies of species and higher taxa, the duration of calls or notes, pulse rate within notes, and number of pulses per note appear to be more static within individuals and somewhat less affected by temperature. Therefore, these variables might often be preferable as taxonomic characters over call rate or note rate, which are heavily influenced by various factors. Dominant frequency is also comparatively static and only weakly affected by temperature, but depends strongly on body size. As with other taxonomic characters, strong call divergence is typically indicative of species-level differences, whereas call similarities of two populations are no evidence for them being conspecific. Taxonomic conclusions can especially be drawn when the general advertisement call structure of two candidate species is radically different and qualitative call differences are thus observed. On the other hand, quantitative differences in call traits might substantially vary within and among conspecific populations, and require careful evaluation and analysis. We provide guidelines for the taxonomic interpretation of advertisement call differences in sympatric and allopatric situations, and emphasize the need for an integrative use of multiple datasets (bio-acoustics, morphology, genetics), particularly for allopatric scenarios. We show that small-sized frogs often emit calls with frequency components in the ultrasound spectrum, although it is unlikely that these high frequencies are of biological relevance for the majority of them, and we illustrate that detection of upper harmonics depends also on recording distance because higher frequencies are attenuated more strongly. Bioacoustics remains a prime approach in integrative taxonomy of anurans if uncertainty due to possible intraspecific variation and technical artifacts is adequately considered and acknowledged.

A new morphologically cryptic species of Phyllomedusa (Anura: Phyllomedusidae) from Amazonian forests of northern Peru revealed by DNA sequences.

We describe and name Phyllomedusa chaparroi sp. nov., a medium-sized species (snout-vent length in adult males 67.9-77.5 mm) of monkey frog from Amazonian rainforests of northern Peru. Although morphologically most similar to P. boliviana and P. camba (indistinguishable from the latter in external qualitative and quantitative traits), phylogenetic analysis of combined mitochondrial and nuclear markers place the new species sister to a clade containing P. neildi, P. tarsius, and P. trinitatis. Phyllomedusa chaparroi can be readily differentiated from these species by having a dark reddish-brown iris with indistinct tiny orange spots versus an orange iris with marked dark reticulation found in P. neildi, P. tarsius, and P. trinitatis. Furthermore, genetic distances for a 532 bp sequence of the 16S gene between the new species and its sister species are 2.8-4.1 %, whereas distances are 4.5-5.5 % to the morphologically cryptic P. camba. We briefly discuss the importance of DNA sequences in revealing morphologically cryptic species and modify the content of the P. tarsius species group based on phylogenetic analyses and observations on iris coloration.

Off the scale: a new species of fish-scale gecko (Squamata: Gekkonidae: Geckolepis) with exceptionally large scales.

The gecko genus Geckolepis, endemic to Madagascar and the Comoro archipelago, is taxonomically challenging. One reason is its members ability to autotomize a large portion of their scales when grasped or touched, most likely to escape predation. Based on an integrative taxonomic approach including external morphology, morphometrics, genetics, pholidosis, and osteology, we here describe the first new species from this genus in 75 years: Geckolepis megalepissp. nov. from the limestone karst of Ankarana in northern Madagascar. The new species has the largest known body scales of any gecko (both relatively and absolutely), which come off with exceptional ease. We provide a detailed description of the skeleton of the genus Geckolepis based on micro-Computed Tomography (micro-CT) analysis of the new species, the holotype of G. maculata, the recently resurrected G. humbloti, and a specimen belonging to an operational taxonomic unit (OTU) recently suggested to represent G. maculata. Geckolepis is characterized by highly mineralized, imbricated scales, paired frontals, and unfused subolfactory processes of the frontals, among other features. We identify diagnostic characters in the osteology of these geckos that help define our new species and show that the OTU assigned to G. maculata is probably not conspecific with it, leaving the taxonomic identity of this species unclear. We discuss possible reasons for the extremely enlarged scales of G. megalepis in the context of an anti-predator defence mechanism, and the future of Geckolepis taxonomy.

Reconciling molecular phylogeny, morphological divergence and classification of Madagascan narrow-mouthed frogs (Amphibia: Microhylidae).

A recent study clarified several aspects of microhylid phylogeny by combining DNA sequences from Sanger sequencing and anchored phylogenomics, although numerous aspects of tree topology proved highly susceptible to data partition and chosen model. Although the phylogenetic results of the study were in conflict with previous publications, the authors made several changes to the taxonomy of Madagascar's cophyline microhylids. We re-analyzed part of their data together with our own molecular and morphological data. Based on a supermatrix of 11 loci, we propose a new phylogeny of the Cophylinae, and discuss it in the context of a newly generated osteological dataset. We found several sample misidentifications, partially explaining their deviant results, and propose to resurrect the genera Platypelis and Stumpffia from the synonymy of Cophyla and Rhombophryne, respectively. We provide support for the previous genus-level taxonomy of this subfamily, and erect a new genus, Anilany gen. nov., in order to eliminate paraphyly of Stumpffia and to account for the osteological differences observed among these groups. Deep nodes in our phylogeny remain poorly supported, and future works will certainly refine our classification, but we are confident that these will not produce large-scale rearrangements.

Integrative taxonomic revision of mantellid frogs of the genus Aglyptodactylus (Anura: Mantellidae).

Molecular genetic studies have in recent years revealed the presence of hidden species diversity in Madagascar's amphibians, and numerous genera contain candidate species awaiting description. We here revise the taxonomy of one such genus within the endemic Malagasy-Comorian family Mantellidae. The target taxon, Aglyptodactylus, is classified in the subfamily Laliostominae and to date contained three recognized nominal species. We focus in particular on clarifying the status of three candidate species proposed for the genus, and for this purpose integrate evidence from molecular genetics, bioacoustics and morphology. We furthermore summarize the information available for all species in the genus concerning their taxonomic identities, distributions and advertisement calls. Our analyses of advertisement calls revealed significant differences coinciding with major clades revealed by phylogenetic analysis of the mitochondrial 16S rRNA gene. Together with the new molecular dataset this provides clear evidence for evolutionary lineage divergence and supports the three candidate species previously characterized by mtDNA divergence of single specimens only. We also observed morphological differences among some of the lineages but these were in most cases not unambiguously diagnostic. As a taxonomic consequence we describe two new species: Aglyptodactylus australis sp. nov. from Andohahela rainforest in southeastern Madagascar, which so far is the largest species known in the genus with females reaching 82.5 mm snout-vent length; and Aglyptodactylus chorus sp. nov. from northeastern lowland rainforests in the Maroantsetra region. For the third proposed and confirmed candidate species we resurrect the name Rana inguinalis Günther, 1877 as Aglyptodactylus inguinalis (Günther, 1877) from the synonymy of A. madagascariensis. This name is applied to populations distributed in lowlands along most of the east coast of Madagascar, from Marojejy in the north southward to Tolagnaro. Genetic data indicate strong differentiation of A. inguinalis with three sub-lineages receiving support, and we cannot exclude that A. inguinalis as defined herein is a complex of species warranting further partition in the future. As a result of the revised taxonomy, A. madagascariensis remains restricted to rainforests at higher elevations (650‒1500 m a.s.l.) ranging from northern to south-central Madagascar, and showing some further differentiation with respect to a northern and a southern sub-lineage. Difficulties in the assignment of historical type specimens are discussed, as well as the biogeography and conservation status of the recognized species.

A new arboreal frog of the genus Guibemantis from the southeast of Madagascar (Anura: Mantellidae).

We describe a new species of arboreal frog of the genus Guibemantis, subgenus Guibemantis, from low altitude rainforest in Manombo Special Reserve, south-eastern Madagascar. Previously published phylogenetic analyses of mitochondrial DNA sequences have placed Guibemantis diphonus sp. nov. sister to G. timidus. The new species is distinguished from G. timidus and all other species in the subgenus by a substantial genetic differentiation (≥ 4.4% uncorrected p-distance in the mitochondrial 16S rRNA gene), strongly divergent advertisement call, and some limited morphological differences. It is the smallest known species in the subgenus, with 34-36 mm snout-vent length in adult males. Its advertisement call is unique among other species in the subgenus in being composed of two distinctly different note types (only one note type in the other species).

High levels of diversity uncovered in a widespread nominal taxon: continental phylogeography of the neotropical tree frog Dendropsophus minutus.

Species distributed across vast continental areas and across major biomes provide unique model systems for studies of biotic diversification, yet also constitute daunting financial, logistic and political challenges for data collection across such regions. The tree frog Dendropsophus minutus (Anura: Hylidae) is a nominal species, continentally distributed in South America, that may represent a complex of multiple species, each with a more limited distribution. To understand the spatial pattern of molecular diversity throughout the range of this species complex, we obtained DNA sequence data from two mitochondrial genes, cytochrome oxidase I (COI) and the 16S rhibosomal gene (16S) for 407 samples of D. minutus and closely related species distributed across eleven countries, effectively comprising the entire range of the group. We performed phylogenetic and spatially explicit phylogeographic analyses to assess the genetic structure of lineages and infer ancestral areas. We found 43 statistically supported, deep mitochondrial lineages, several of which may represent currently unrecognized distinct species. One major clade, containing 25 divergent lineages, includes samples from the type locality of D. minutus. We defined that clade as the D. minutus complex. The remaining lineages together with the D. minutus complex constitute the D. minutus species group. Historical analyses support an Amazonian origin for the D. minutus species group with a subsequent dispersal to eastern Brazil where the D. minutus complex originated. According to our dataset, a total of eight mtDNA lineages have ranges >100,000 km2. One of them occupies an area of almost one million km2 encompassing multiple biomes. Our results, at a spatial scale and resolution unprecedented for a Neotropical vertebrate, confirm that widespread amphibian species occur in lowland South America, yet at the same time a large proportion of cryptic diversity still remains to be discovered.

Resolving an enigma by integrative taxonomy: Madagascarophis fuchsi (Serpentes: Lamprophiidae), a new opisthoglyphous and microendemic snake from northern Madagascar.

Herpetological surveys in the dry forests of the limestone massif Montagne des Français in the far north of Madagascar have recently yielded a number of undescribed reptile species. Here we describe an additional new and potentially microendemic species of the snake genus Madagascarophis (Squamata: Serpentes: Pseudoxyrhophiinae) which lives in this massif syntopically with M. colubrinus septentrionalis and differs distinctly from M. colubrinus and M. meridionalis in its mitochondrial and nuclear DNA sequences. Morphologically Madagascarophis fuchsi sp. nov. is characterized by a broad contact between the posterior inframaxillaries (genials), 25 dorsal scale rows at midbody, and a low number of ventrals (171-172). We redescribe the holotype of M. ocellatus and present new data on the morphological variation of the northern subspecies M. c. septentrionalis and M. c. citrinus. Although Montagne des Français has recently been included into the network of nature reserves in Madagascar, continuous deforestation is strongly threatening this important center of reptile endemism. In line with the assessment of other microendemic reptiles of this massif we suggest to consider the new species as Critically Endangered according to the IUCN criteria and encourage new efforts to protect this area more efficiently.

Molecular, morphological and osteological differentiation of a new species of microhylid frog of the genus Stumpffia from northwestern Madagascar.

We describe a new small-sized microhylid frog of the genus Stumpffia and provide a preliminary survey of Stumpffia diversity in northern and northwestern Madagascar based on molecular and morphological data. Analysis of 68 previously published and 142 newly generated DNA sequences of a fragment of the mitochondrial 16S rRNA gene revealed a large genetic diversity within Stumpffia, with numerous strongly differentiated lineages occurring across Madagascar. The anal. ysis confirmed a clade formed exclusively by Stumpffia species occurring in north/northwestern Madagascar and contain. ing almost all species from this area. Based on DNA sequences of the RAG1 gene, we further assess that most of the deep mitochondrial lineages do not share haplotypes in this nuclear marker, even in the few cases of sympatry, which we interpret as confirmation of their status as independent evolutionary lineages. Micro-computed tomography revealed subtle differences in phalangeal formulae among some of these candidate species, providing additional taxonomic characters for future revisions of the genus. The newly described species Stumpffia analamaina sp. nov. occurs 27 km north-west of Antsohihy and is at present the southernmost confirmed Stumpffia of the north/northwestern clade. It is mainly characterized by a snout-vent length of 10-12 mm, advertisement calls consisting of a series of short melodious chirps, manus with four fingers and pes with five toes, with only slight length reduction of first finger and toe. It furthermore does not share haplotypes with any other Stumpffia species in 16S or RAG1, and has relatively longer hands and feet compared with other small-sized Stumpffia of the north/northwestern clade such as S. madagascariensis and S. pygmaea. The new species lives in an area of dry forest where it was found in leaf litter along a small stream bordered by degraded gallery vegetation. Due to the limited distributions of almost all species in the north/northwestern Stumpffia clade, the rareness of appropriate microhabitat around the type locality of S. analamaina sp. nov. and the rampant habitat destruction in this part of Madagascar, we propose a threat status of Critically Endangered for this new species.

Rivaling the world's smallest reptiles: discovery of miniaturized and microendemic new species of leaf chameleons (Brookesia) from northern Madagascar.

BACKGROUND: One clade of Malagasy leaf chameleons, the Brookesia minima group, is known to contain species that rank among the smallest amniotes in the world. We report on a previously unrecognized radiation of these miniaturized lizards comprising four new species described herein. METHODOLOGY/PRINCIPAL FINDINGS: The newly discovered species appear to be restricted to single, mostly karstic, localities in extreme northern Madagascar: Brookesia confidens sp. n. from Ankarana, B. desperata sp. n. from Forêt d'Ambre, B. micra sp. n. from the islet Nosy Hara, and B. tristis sp. n. from Montagne des Français. Molecular phylogenetic analyses based on one mitochondrial and two nuclear genes of all nominal species in the B. minima group congruently support that the four new species, together with B. tuberculata from Montagne d'Ambre in northern Madagascar, form a strongly supported clade. This suggests that these species have diversified in geographical proximity in this small area. All species of the B. minima group, including the four newly described ones, are characterized by very deep genetic divergences of 18-32% in the ND2 gene and >6% in the 16S rRNA gene. Despite superficial similarities among all species of this group, their status as separate evolutionary lineages is also supported by moderate to strong differences in external morphology, and by clear differences in hemipenis structure. CONCLUSION/SIGNIFICANCE: The newly discovered dwarf chameleon species represent striking cases of miniaturization and microendemism and suggest the possibility of a range size-body size relationship in Malagasy reptiles. The newly described Brookesia micra reaches a maximum snout-vent length in males of 16 mm, and its total length in both sexes is less than 30 mm, ranking it among the smallest amniote vertebrates in the world. With a distribution limited to a very small islet, this species may represent an extreme case of island dwarfism.

Molecular phylogeny and biogeography of Malagasy frogs of the genus Gephyromantis.

The genus Gephyromantis is a clade within the Malagasy-Comoroan family Mantellidae composed of rainforest frogs that live and breed to varying degrees independently from water. Based on DNA sequences of five mitochondrial and five nuclear genes we inferred the phylogeny of these frogs with full taxon coverage at the species level. Our preferred consensus tree from a partitioned Bayesian analysis of 5843 base pairs of 51 nominal and candidate species supports various major clades within the genus although the basal relationships among these remain unresolved. The data provide strong evidence for the monophyly of the subgenera Gephyromantis (after exclusion of Gephyromantis klemmeri), Laurentomantis, Vatomantis, and Phylacomantis. Species assigned to the subgenus Duboimantis belong to two strongly supported clades of uncertain relationships. G. klemmeri, previously in the subgenus Gephyromantis, was placed with high support sister to the Laurentomantis clade, and the Laurentomantis+G. klemmeri clade was sister to Vatomantis. A reconstruction of ancestral distribution areas indicates a diversification of several subgenera in the northern biogeographic regions of Madagascar and the dispersal out of northern Madagascar for several clades.

How to uncoil your partner--"mating songs" in giant pill-millipedes (Diplopoda: Sphaerotheriida).

The stridulation of the giant pill-millipede genus Sphaerotherium from South Africa, one of only three groups of millipedes that produce sounds, was studied. One hundred one stridulation series of a total of nine different species (Sphaerotherium dorsaloide, Sphaerotherium hanstroemi, Sphaerotherium mahaium, Sphaerotherium similare, Sphaerotherium punctulatum, Sphaerotherium convexitarsum, Sphaerotherium dorsale, Sphaerotherium rotundatum, and Sphaerotherium perbrincki) were analyzed. Stridulation sounds are produced only with a special field of ribs on the posterior surface of the posterior telopod, which is actively moved over a field of sclerotized nubs on the inner margin of the anal shield. The Sphaerotherium male usually stridulates only when in contact with a female to initiate mating. This seems to prevent the female from volvating into a ball or stimulate the female to uncoil when already rolled in. The sound analyzes revealed a broad frequency spectrum in all stridulation sounds produced, without obvious differences in frequency distribution among species. However, the temporal pattern of the stridulation varies greatly between species and seems to be species-specific, arguing for a species recognition function of the stridulation during courtship behavior. A single species (S. punctulatum) was found to stridulate during mating while three species also show postcopulatory stridulation. Apparently, pill-millipedes are not capable of acoustic perception, as no hearing organs are known, indicating that the communication is mainly based on perception of temporal vibration patterns, and not of the acoustic signal itself. The need to overcome the rolling-in reflex of the female is developed as a hypothesis why stridulation exists only in millipedes able to coil into a ball, and apparently evolved four times independently in the superorder Oniscomorpha.

A multilocus phylogeny of Malagasy scincid lizards elucidates the relationships of the fossorial genera Androngo and Cryptoscincus.

A phylogeny for 29 species of scincine lizards from Madagascar, based on 3693 bp of six mitochondrial and five nuclear genes, revealed multiple parallel evolution of adaptations for a burrowing life, and unexpected relationships of the monotypic genera Androngo and Cryptoscincus. Androngo trivittatus was sister to Pygomeles braconnieri, and Cryptoscincus minimus was deeply nested within the genus Paracontias, all of these being fossorial taxa of elongated bodies and partly or fully reduced limbs. To account for these results, we place Cryptoscincus as a junior synonym of Paracontias, and discuss possible taxonomic consequences that may affect the status of Androngo, once additional data become available.

Vast underestimation of Madagascar's biodiversity evidenced by an integrative amphibian inventory.

Amphibians are in decline worldwide. However, their patterns of diversity, especially in the tropics, are not well understood, mainly because of incomplete information on taxonomy and distribution. We assess morphological, bioacoustic, and genetic variation of Madagascar's amphibians, one of the first near-complete taxon samplings from a biodiversity hotspot. Based on DNA sequences of 2,850 specimens sampled from over 170 localities, our analyses reveal an extreme proportion of amphibian diversity, projecting an almost 2-fold increase in species numbers from the currently described 244 species to a minimum of 373 and up to 465. This diversity is widespread geographically and across most major phylogenetic lineages except in a few previously well-studied genera, and is not restricted to morphologically cryptic clades. We classify the genealogical lineages in confirmed and unconfirmed candidate species or deeply divergent conspecific lineages based on concordance of genetic divergences with other characters. This integrative approach may be widely applicable to improve estimates of organismal diversity. Our results suggest that in Madagascar the spatial pattern of amphibian richness and endemism must be revisited, and current habitat destruction may be affecting more species than previously thought, in amphibians as well as in other animal groups. This case study suggests that worldwide tropical amphibian diversity is probably underestimated at an unprecedented level and stresses the need for integrated taxonomic surveys as a basis for prioritizing conservation efforts within biodiversity hotspots.