Draft genome sequences of 21 Pedobacter strains isolated from amphibian specimens.

The genomes of 21 Pedobacter strains isolated from the European salamander Salamandra salamandra and different Madagascan frog species were sequenced using Illumina sequencing. Here, we report their draft genome sequences (~4.7-7.2 Mbp in size) to allow comparative genomics and taxonomic assignment of these strains.

Unexpected diversity and co-occurrence of phytotelmic frogs (Guibemantis) around Andasibe, one of the most intensively surveyed amphibian hotspots of Madagascar, and descriptions of three new species.

The area around the Malagasy village of Andasibe, which includes Analamazaotra-Mantadia National Park as well as other protected areas, is characterized by very species-rich and well-studied communities of animals and plants, but new species are still regularly discovered. Three species of phytotelmic frogs of the subgenus Pandanusicola in the genus Guibemantis are known from this area, G. flavobrunneus, G. liber, and G. pulcher. Further Pandanusicola frogs from this area have been provisionally assigned to G. bicalcaratus or G. albolineatus, pending detailed taxonomic review. During preliminary exploration of the ecology of these specialized frogs that live and reproduce in the leaf axils of Pandanus screw pines, we noticed the syntopic presence of two differently colored and differently sized Pandanusicola in Andasibe that could not be unambiguously assigned to any known species. A genetic screening revealed that these correspond to yet two further species in the area. Based on our data, seven species of Pandanusicola occur in Andasibe and nearby forests: (1) G. liber, the only non-phytotelmic species of the subgenus in the region; (2) G. flavobrunneus which is the largest species and characterized by a diagnostic yellowish brown dorsal pattern; (3) G. pulcher, characterized by translucent-green color with purplish brown spotting not observed in any other species in the area; (4) G. methueni, a brownish species usually lacking contrasted dorsolateral bands that differs from the other species in the area by emitting a characteristic trill-like advertisement call series (rather than clicks or chirps) and according to our data is widespread along Madagascars east coast; as well as three new species: (5) G. ambakoana sp. nov., a brownish species, typically with contrasted incomplete light dorsolateral bands and with single click-like advertisement calls; (6) G. vakoa sp. nov., a species that is equally brownish but lacks contrasted light dorsolateral bands and that has single click-like advertisement calls of very short duration; and (7) G. rianasoa sp. nov., a species that is smaller sized and has less distinct femoral glands than all the others, and emits a short series of soft chirp-like advertisement calls. All these species are genetically highly distinct, with >5% uncorrected pairwise distances in the mitochondrial 16S rRNA gene, and lack of haplotype sharing in two nuclear-encoded genes. The co-occurrence of seven Pandanusicola frogs in a relatively small geographic area is unprecedented in Madagascar and calls for in-depth studies of a possible differentiation in habitat use and life history.

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.

A new species of Pandanus-dwelling frog from northern Madagascar related to Guibemantis pulcher.

Populations of phytotelmic frogs from northern Madagascar assigned to Guibemantis (Pandanusicola) pulcher are known to differ genetically from populations further south in the eastern rainforest belt of the island, but to date, their status has not been analyzed in depth. We combined molecular genetic data with an examination of color pattern to clarify the taxonomy of these frogs. DNA sequences of both mitochondrial and nuclear-encoded genes were consistently differentiated between the northern populations and those occurring further south. Uncorrected pairwise distance in the 16S rRNA gene was 3.7‒4.3% and thus at a level usually characterizing distinct frog species in Madagascar. Furthermore, the northern specimens were characterized by more and smaller purplish-brown spots on their green dorsal surface, and a less distinct brown patch on the flanks. Although fully conclusive evidence for the species status of the northern lineage from bioacoustic differences, sympatric occurrence or narrow hybrid zone is currently lacking, such species-level distinctness is currently the most likely hypothesis. We therefore name the northern populations as Guibemantis (Pandanusicola) pulcherrimus sp. nov. The new species is known from Makira (type locality) and Bemanevika, and specimens morphologically assignable to this taxon have also been recorded from Masoala, Marojejy and Anjanaharibe-Sud.

Repeated divergence of amphibians and reptiles across an elevational gradient in northern Madagascar.

How environmental factors shape patterns of biotic diversity in tropical ecosystems is an active field of research, but studies examining the possibility of ecological speciation in terrestrial tropical ecosystems are scarce. We use the isolated rainforest herpetofauna on the Montagne d'Ambre (Amber Mountain) massif in northern Madagascar as a model to explore elevational divergence at the level of populations and communities. Based on intensive sampling and DNA barcoding of amphibians and reptiles along a transect ranging from ca. 470-1470 m above sea level (a.s.l.), we assessed a main peak in species richness at an elevation of ca. 1000 m a.s.l. with 41 species. The proportion of local endemics was highest (about 1/3) at elevations >1100 m a.s.l. Two species of chameleons (Brookesia tuberculata, Calumma linotum) and two species of frogs (Mantidactylus bellyi, M. ambony) studied in depth by newly developed microsatellite markers showed genetic divergence up the slope of the mountain, some quite strong, others very weak, but in each case with genetic breaks between 1100 and 1270 m a.s.l. Genetic clusters were found in transect sections significantly differing in bioclimate and herpetological community composition. A decrease in body size was detected in several species with increasing elevation. The studied rainforest amphibians and reptiles show concordant population genetic differentiation across elevation along with morphological and niche differentiation. Whether this parapatric or microallopatric differentiation will suffice for the completion of speciation is, however, unclear, and available phylogeographic evidence rather suggests that a complex interplay between ecological and allopatric divergence processes is involved in generating the extraordinary species diversity of Madagascar's biota. Our study reveals concordant patterns of diversification among main elevational bands, but suggests that these adaptational processes are only part of the complex of processes leading to species formation, among which geographical isolation is probably also important.

Gray versus yellow ventral coloration: Identity, distribution, color polymorphism and molecular relationships of the microhylid frog Platypelis mavomavo Andreone, Fenolio & Walvoord, 2003.

The Malagasy frog Platypelis mavomavo from Ambolokopatrika in the North East of Madagascar was originally diagnosed based on its bright yellow venter, but only limited information on this species has become available after its initial description in 2003. Several Platypelis specimens with yellow ventral color have been erroneously assigned to this species due to a lack of DNA sequences from the P. mavomavo type series. On the other hand, the candidate species Platypelis sp. Ca10 from Andranomapanga in the Northern Central East of Madagascar with gray ventral color has been defined based on its genetic differentiation from other nominal Platypelis species. Here we study the genetic variation of P. mavomavo and P. sp. Ca10 based on mitochondrial (16S rRNA) and nuclear-encoded (RAG-1) genes, including a newly determined sequence from the P. mavomavo holotype, which was studied using a museomics approach. We find only limited genetic variation among the samples studied, and this variation is unlinked to ventral coloration but instead reflects geographic distribution. We, therefore, conclude that P. sp. Ca10 is a gray-colored variant of P. mavomavo, and that P. mavomavo is rather widespread in the North East and Northern Central East of Madagascar, with populations in areas bordering the North West (Ambohitantely) and Sambirano (Ampotsidy) geographic regions, and the yellow-bellied morph restricted to the North East (Makira, Ambolokopatrika). Due to the range extension of P. mavomavo, the conservation status of the species requires re-assessment.

Integrative revision of the Lygodactylus madagascariensis group reveals an unexpected diversity of little brown geckos in Madagascars rainforest.

The Lygodactylus madagascariensis species group, constituting the subgenus Domerguella, currently contains five valid species of inconspicuous dwarf geckos from Madagascars humid forests, but at least 18 deep genetic lineages have been revealed by recent molecular studies. Given the high morphological similarity of these lineages, taxonomic resolution of this astonishing diversity requires efforts to correctly delimit species, as well as assigning the available nomina to the species-level lineages identified. We here combine DNA sequences of one mitochondrial and two nuclear-encoded gene fragments with morphometric measurements and scale counts, and report evidence for a species status of most of the previously identified lineages. In particular, we rely on sympatric and often even syntopic occurrence of several of these lineages without evidence for genetic admixture, and consistent with subtle morphological differences. Furthermore, the very high divergences of 7.423.8% pairwise distances in the relatively conserved mitochondrial 16S rRNA gene, combined with a lack of haplotype sharing in the nuclear-encoded genes and differences in scale counts convinced us that most of the other, allopatrically distributed lineages also represent distinct species. We elevate L. madagascariensis petteri to species level and formally name eight new species: L. salvi sp. nov., a species from the Sambirano region in northern Madagascar, previously called L. sp. 8; L. tantsaha sp. nov. (L. sp. 10), a species occurring sympatrically with L. madagascariensis and L. petteri on Montagne dAmbre in far northern Madagascar; L. roellae sp. nov. (L. sp. 17), a species characterized by a striped coloration in all known specimens, from northern Madagascar; L. winki sp. nov. (L. sp. 18), an unstriped species from northern Madagascar but belonging to a subclade mostly distributed in the eastern rainforests of the island; L. ulli sp. nov. (L. sp. 21), a species from the same subclade as L. winki but known only from the Marojejy Massif in the North East; L. fritzi sp. nov. (L. sp. 11), a further species of this subclade from coastal lowlands in the Northern Central East; L. hodikazo sp. nov. (L. sp. 23) known from a single specimen collected at the Tsingy de Bemaraha and therefore the only Domerguella species known from the West region of Madagascar; and L. hapei sp. nov. (L. sp. 26), an enigmatic species from the Sambirano region characterized by a striped pattern on the throat that is otherwise unknown in the subgenus. Three additional deep mitochondrial lineages of Domerguella were identified in our analysis, but could not be further analyzed due to the lack or scarcity of voucher specimens. More field work and collection of voucher specimens is needed to understand their status. Furthermore, the taxonomy of the Domerguella subclade occurring in eastern Madagascar, with three described species (L. guibei, L. miops, L. fritzi), two synonyms (L. septemtuberculatus, Microscalabotes spinulifer) and at least two further deep genetic lineages co-occurring in a relatively small area, requires further revisionary work, possibly aided by target-enrichment sequencing of the respective name-bearing types.

Geography-Dependent Horizontal Gene Transfer from Vertebrate Predators to Their Prey.

Horizontal transfer (HT) of genes between multicellular animals, once thought to be extremely rare, is being more commonly detected, but its global geographic trend and transfer mechanism have not been investigated. We discovered a unique HT pattern of Bovine-B (BovB) LINE retrotransposons in vertebrates, with a bizarre transfer direction from predators (snakes) to their prey (frogs). At least 54 instances of BovB HT were detected, which we estimate to have occurred across time between 85 and 1.3 Ma. Using comprehensive transcontinental sampling, our study demonstrates that BovB HT is highly prevalent in one geographical region, Madagascar, suggesting important regional differences in the occurrence of HTs. We discovered parasite vectors that may plausibly transmit BovB and found that the proportion of BovB-positive parasites is also high in Madagascar where BovB thus might be physically transported by parasites to diverse vertebrates, potentially including humans. Remarkably, in two frog lineages, BovB HT occurred after migration from a non-HT area (Africa) to the HT hotspot (Madagascar). These results provide a novel perspective on how the prevalence of parasites influences the occurrence of HT in a region, similar to pathogens and their vectors in some endemic diseases.

Population diversification in the frog Mantidactylus bellyi on an isolated massif in northern Madagascar based on genetic, morphological, bioacoustic and ecological evidence.

In the processes that give rise to new species, changes first occur at the population level. But with the continuous nature of the divergence process, change in biological properties delimiting the shift from "individuals of divergent populations" towards "individuals of distinct species", as well as abiotic factors driving the change, remain largely ambivalent. Here we study diversification processes at the population level in a semi-aquatic frog, Mantidactylus (Brygoomantis) bellyi, across the diverse vegetation types of Montagne d'Ambre National Park (MANP), Madagascar. Genetic diversity was assessed with seven newly developed microsatellite markers as well as mitochondrial DNA sequences and concordance with patterns of ecological, morphological, and bioacoustic divergence evaluated. We found M. bellyi lacking mitochondrial differentiation within MANP, while microsatellite datasets partitioned them into three highly differentiated, geographically separated subpopulations (with indications for up to five subpopulations). The molecular grouping-primarily clustering individuals by geographic proximity-was coincident with differences in mean depth and width of waters, suggesting a possible role of fluvial characteristics in genetic exchange in this stream-breeding species. Genetic clustering not consistent with differences in call properties, except for dominant call frequencies under the two-subpopulations model. Morphological divergence was mostly consistent with the genetic clustering; subpopulations strongly differed by their snout-vent length, with individuals from high-elevation subpopulations smaller than those from populations below 1000 m above sea level. These results exemplify how mountains and environmental conditions might primarily shape genetic and morphological divergence in frog populations, without strongly affecting their calls.

An unexpected new red-bellied Stumpffia (Microhylidae) from forest fragments in central Madagascar highlights remaining cryptic diversity.

The Madagascan endemic subfamily Cophylinae in the family Microhylidae, is an example of a taxonomic group for which much is still to be discovered. Indeed, the cophyline frogs present a large portion of Madagascar's cryptic and microendemic amphibian diversity, yet they remain understudied. A new red-bellied species of the microhylid frog genus Stumpffia is described from the central plateau of Madagascar. Visual encounter surveys in Ambohitantely and Anjozorobe in 2019 and 2020 identified this previously unknown Stumpffia species, which closely resembles Stumpffiakibomena known from Andasibe in the east. Stumpffialynnae sp. nov. adds another species to the red-bellied species complex, differing from S.kibomena by genetic differentiation in the mitochondrial 16S rRNA gene (3.6-3.9%) and distinct nuclear RAG1 haplotypes, as well as strongly by its advertisement call. The new species is known from across Ambohitantely Special Reserve and Anjozorobe Angavo protected area, but is known only from one complete specimen and eight individual tissue samples. Based on the rarity of the species, the small number of locations in which it has been found, and its disappearing forest habitat, its IUCN Red List classification is suggested as "Endangered". This species is the first Stumpffia described from Madagascar's central plateau, highlighting the importance of conserving the remnant forest fragments in this area and the ongoing need to survey and protect this threatened habitat type.

Review of threatened Malagasy freshwater fishes in zoos and aquaria: The necessity of an ex situ conservation network-A call for action.

Madagascar's biota is characterized by an extraordinary species richness, with a high degree of endemism. The island's freshwater habitats harbor numerous micro-endemic species, restricted to particular regions and thus particularly at risk of extinction, due to deforestation, overfishing, and introduction of exotic species. The present study investigates for which threatened Malagasy freshwater fish species ex situ populations have already been established, as a baseline to prioritize actions to develop an effective ex situ conservation breeding network. Populations in zoos and aquaria were primarily determined using the Zoological Information System. Of 173 fish species recorded from Malagasy freshwater habitats, 123 exclusively inhabit freshwater; 79 of these are endemic to Madagascar, and 50 are classified as threatened. Our survey found 21 Malagasy freshwater fish species kept in zoos worldwide, of which 19 are endemic and threatened (22 if counting species kept by private breeders). Nine of the 19 Malagasy freshwater fish species kept in zoos have successfully reproduced within the 12 months preceding our survey. The ex situ conservation activities for threatened Malagasy freshwater fishes thus have not improved significantly since the strong start in the early 2000s. More than half of the 50 threatened endemic Malagasy freshwater fish species (viz. 31 species) are not kept ex situ, including 11 species ranked as Critically Endangered. Based on these findings we call for a better distribution of offspring among institutions, including private breeders in the framework of citizen conservation initiatives; a closer connection of ichthyological field research in Madagascar with conservation breeding efforts to set up ex situ populations-both in Madagascar and abroad-of species not yet kept in captivity; and the development of effective, integrated in situ and ex situ conservation strategies.

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.

Towards completion of the species inventory of small-sized leaf-tailed geckos: two new species of Uroplatus from northern Madagascar.

The northern part of Madagascar is well known for its high species diversity and endemism. Exceptional species richness is related to the existence of large forest blocks and mountain complexes. These areas shelter a diverse variety of habitats occupied by a wide diversity of species, including leaf-tailed geckos of the genus Uroplatus. Based on morphological and molecular evidence, we here formally name two evolutionary lineages as new species that previously had been considered as candidate species (Uroplatus spp. Ca3 and Ca4), both small-sized species of the Uroplatus ebenaui group. Genetically, both new species are related to U. finiavana with a genetic divergence (uncorrected pairwise distance) of 10.3-12.8% in a fragment of the mitochondrial 16S rRNA gene, and separated from each other by 10.3-10.7%. Uroplatus fangorn sp. nov. is described from the Marojejy massif and is also known from Andrevorevo, Lohanandroranga and Sorata; it is similar to U. ebenaui and U. fetsy but is distinguishable by its rather short tail and only partially black pigmented oral mucosa. Uroplatus fivehy sp. nov. is described from the Sorata Massif and is wider-ranging, occurring in an area from Marotandrano and Makira to Sorata, comprising Marojejy, Anjanaharibe-Sud, Ankitsika and Betaolana; it is morphologically similar to U. finiavana but distinguishable by tail size and shape. Both newly described species are found in rainforest from mid to high elevation and range respectively from 840-1417 m for U. fivehy sp nov. and 1300-1800 m for U. fangorn sp. nov. According to their respective geographical distributions, we propose to classify the two new species as Vulnerable under the IUCN Red List criteria, due to their occurrence in some forests outside of the protected area network plus the continuing decline of forest patches in the north of Madagascar.

Sympatric lineages in the Mantidactylus ambreensis complex of Malagasy frogs originated allopatrically rather than by in-situ speciation.

Madagascar's biota is characterized by a high degree of microendemism at different taxonomic levels, but how colonization and in-situ speciation contribute to the assembly of local species communities has rarely been studied on this island. Here we analyze the phylogenetic relationships of riparian frogs of the Mantidactylus ambreensis species complex, which is distributed in the north of Madagascar and was originally described from Montagne d'Ambre, an isolated mountain of volcanic origin, currently protected within Montagne d'Ambre National Park (MANP). Data from mitochondrial DNA, and phylogenomic data from FrogCap, a sequence capture method, independently confirm that this species complex is monophyletic within the subgenus Ochthomantis, and identify two main clades within it. These two clades are separated by 5.6-6.8% pairwise distance in the mitochondrial 16S rRNA gene and co-occur in MANP, with one distributed at high elevations (940-1375 m a.s.l.) and the other at lower elevations (535-1010 m a.s.l.), but show almost no haplotype sharing in the nuclear RAG1 gene. This occurrence in syntopy without admixture confirms them as independent evolutionary lineages that merit recognition as separate species, and we here refer to them as high-elevation (HE) and low-elevation (LE) lineage; they will warrant taxonomic assessment to confidently assign the name ambreensis to one or the other. Populations of the M. ambreensis complex from elsewhere in northern Madagascar all belong to the LE lineage, although they do occur over a larger elevational range than in Montagne d'Ambre (285-1040 m a.s.l.). Within LE there are several phylogroups (LE1-LE4) of moderately deep divergence (1.5-2.8% in 16S), but phylogroup LE4 that occurs in MANP has a deeply nested phylogenetic position, as recovered separately by mitochondrial and sequence capture datasets. This suggests that HE and LE did not diverge by a local fission of lower and upper populations, but instead arose through a more complex biogeographic scenario. The branching pattern of phylogroups LE1-LE4 shows a clear south-to-north phylogeographic pattern. We derive from these results a testable hypothesis of vicariant speciation that restricted the HE lineage to MANP and the LE candidate species to a climatic refugium further south, with subsequent northwards range expansion and secondary colonization of MANP by LE. These results provide an example for complex assembly of local microendemic amphibian faunas on Madagascar.

A new species of the Spinomantis bertini species complex (Anura: Mantellidae) from Pic d'Ivohibe Special Reserve (Madagascar).

We describe a new frog species of the Spinomantis bertini species complex based on congruent genetic evidence from mitochondrial and nuclear DNA sequences as well as minor morphological differences. A molecular phylogeny derived from the mitochondrial 16S rRNA gene revealed that Spinomantis mirus sp. nov. is sister to a clade containing the other two described species of the species complex, S. bertini and S. beckei, but strongly differentiated from these two species by 8.1-9.8% pairwise distances in this gene. DNA sequences from two nuclear genes revealed that S. mirus sp. nov. shares no alleles with these two species. Phenotypically, S. mirus sp. nov. differs from the other species of the complex by its larger size and some aspects of its coloration, including clear mottling on the flanks, tri-color banding on the legs, and distinct brown dots on the dorsum. The new species is so far only known from Pic d'Ivohibe Special Reserve. Its distribution thus appears to not overlap with those of the other two species, which as far as known are restricted to the Andohahela Massif in the extreme South-East of Madagascar, at a distance of about 250 km from Ivohibe.

Description of the lucky Cophyla (Microhylidae, Cophylinae), a new arboreal frog from Marojejy National Park in north-eastern Madagascar.

We describe a new species of arboreal microhylid frog from northern Madagascar. The new species is assigned to the genus Cophyla based on molecular phylogeny and morphological similarities to other species of this genus. Molecular phylogenetic analysis resolved Cophyla fortuna sp. nov. as closely related to C. noromalalae. However, the two species were reciprocally monophyletic both in a tree reconstructed from a combination of mitochondrial genes, and in a tree based on the nuclear RAG-1 gene. The new species, previously identified as candidate species Cophyla sp. Ca4, occurs in lowland bamboo forests around the Marojejy Massif in northeastern Madagascar. It differs from the allopatric C. noromalalae, so far only known from the Montagne d'Ambre Massif in northern Madagascar, by slightly smaller body size and shorter duration and higher spectral frequency of advertisement calls. Several additional genetically divergent mitochondrial lineages of Cophyla, related to the C. fortuna/noromalalae complex and to C. phyllodactyla, occur in intervening areas between Montagne d'Ambre and Marojejy, and their status requires further study.

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.

Identification and Synthesis of Luteolide, a Highly Branched Macrolide Semiochemical from the Mantellid Frog Gephyromantis luteus.

Luteolide is a 10-membered aliphatic macrolactone, (4 R,8 S,9 S)-4,8-dimethylundecan-9-olide ((-) -17), released by the femoral gland of males of the mantellid frog Gephyromantis luteus. Its structure was established using NMR, MS, and chiral GC and confirmed by stereoselective synthesis of different stereoisomers. Among the approximately 20 current macrolides known from the Mantellidae, luteolide is the first example of a volatile macrolide furnishing three stereogenic centers and an ethyl side chain.