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Traditional methods of phylogenetic reconstruction and species delimitation may be impeded by frequent hybridization among lineages. In this study, we conducted phylogenetic and clustering analyses of ddRAD genomic data on the entire genus Salamandra, which includes six species and over 25 subspecies of terrestrial salamanders. We expanded previous datasets to include missing subspecies and incorporated new samples, with an emphasis on secondary contact zones. Results obtained from a full dataset of 392 individuals (356,874 bp; 24,192 SNPs) were compared with those obtained after excluding substantially admixed individuals (n = 95; 835,467 bp; 51,557 SNPs) to explore the consequences of introgression on phylogenetic inference and taxonomic arrangement of subspecies. We found conflicting phylogenetic placements for taxa represented by many admixed individuals (identified by clustering ancestries). In contrast, a time-calibrated tree constructed without hybrids largely agrees with previous phylogenetic hypotheses. Within S. atra, we found paraphyly of S. atra atra, suggesting an additional candidate subspecies. Within S. infraimmaculata, two lineages are assignable to known subspecies and we additionally identified a third, deeply diverged lineage sampled near the Turkish/Syrian border. In S. algira, we found limited admixture between the subspecies S. a. tingitana and S. a. splendens despite their geographic proximity. Finally, within S. salamandra, we detected significant levels of hybridization between subspecies, which blurred their phylogenetic relationships, although the removal of admixed samples in subset analyses clarified the situation in most cases. Monophyly was recovered for subspecies that were previously found paraphyletic, including S. s. salamandra, S. s. gallaica, and S. s. fastuosa. Salamandra s. "alfredschmidti" was confirmed to be a junior synonym of S. s. bernardezi. Previously disputed subspecies, like S. s. "molleri" and S. s. "hispanica", correspond to separated lineages but are affected by admixture with other lineages. Further newly identified candidate subspecies in S. salamandra included a southern lineage within S. s. wer-neri and a western lineage within S. s. bernardezi. Finally, we re-evaluate the status of recognized subspecies in Salamandra, based on evidence from multiple delimitation criteria. Given that the evolutionary history could not be resolved for all subspecies, we highlight taxa within Salamandra that warrant further molecular examination and taxonomic revision, notably within the S. s. gallaica/"molleri"/bejarae complex. This study illustrates the impact of hybridization in phylogenetic analyses and its downstream effects in the identification of conservation units and their naming in the Linnean classification. [ABSTRACT FROM AUTHOR]

We re-evaluate the taxonomic status of specimens of Rhinella collected from rainforests on the eastern Andean slopes and foothills of central Bolivia. The respective populations have been allocated to different nominal taxa, namely Rhinella fissipes and R. leptoscelis, in the past. By detailed morphological comparisons, including the type specimens of crucial taxa, as well as a phylogenetic analysis based on the mitochondrial 16S rRNA gene, we here demonstrate that the focal populations represent a divergent lineage within the Rhinella veraguensis species group, being apparently closely related to R. rumbolli from northern Argentina, but only distantly related to R. leptoscelis, with which they share several morphological character states. Also, the specimens of the focal lineage differ by a unique combination of morphological character states from all known nominal species currently assigned to the R. veraguensis group. Consequently, we describe this lineage as a new species. We discuss the history of its changing taxonomic allocations and demonstrate that former conclusions were barely justified and wrong. We also provide a description of the osteology of this species using computer tomography (micro-CT). Furthermore, we discuss the status of the nominal taxon Bufo pleuropterus Schmidt, 1857, currently considered to represent a junior synonym of Rhinella margaritifera (Laurenti, 1768), and provide arguments for the specific distinctness of these two taxa. Consequently, we regard Rhinella pleuroptera (Schmidt, 1857) a valid species. Based on its external morphology, which is rather similar to that of R. inca, we speculate that R. pleuroptera is possibly related to species in the R. veraguensis group, although this remains in need of further research. [ABSTRACT FROM AUTHOR]

We describe a large new species of leaf-tailed gecko endemic to northern Madagascar. Uroplatus garamaso sp. n. is the sister species of U. henkeli but differs by a genetic divergence > 8% in the mitochondrial 16S rRNA gene, by the absence of haplotype sharing in four nuclear-encoded genes analyzed, a smaller body size, slightly narrower tail, a more pronounced yellowish/reddish iris colour in most individuals, and lack of black pigmentation on the tip of the tongue. The new species also appears to consistently differ from U. henkeli in genital morphology, with the apex of the hemipenis ending in two terminal elements consisting of 4-5 rotulae and longitudinal rows of calyces (versus an apex with irregularly distributed calyces and ending in two symmetrical structures consisting of two broader serrated fleshy elements in U. henkeli). We genetically confirm the occurrence of U. henkeli over a rather wide area, ranging from Tsingy de Bemaraha in the West to Nosy Be in the Sambirano region of northern Madagascar. Both, U. henkeli and the new species contain several deep mitochondrial lineages that are considered as conspecific due to extensive haplotype sharing and lack of obvious morphological differences among them. [ABSTRACT FROM AUTHOR]

Based on sequences of three mitochondrial and two nuclear-encoded genes, we examine genetic variation in the leaf tailed geckos Uroplatus sameiti and U. sikorae, and morphological and chromatic characteristics of the genetic clusters identified. The mitochondrial phylogeny reveals a puzzling diversity of 16 deep lineages (4 in U. sameiti and 12 in U. sikorae) differing by 2.9–9.9% uncorrected pairwise distance in a fragment of the 16S rRNA gene. Populations from Analalava in the North and Zahamena in the Northern Central East form two mitochondrial lineages clustering with U. sameiti but being deeply divergent (>8% 16S distance); however, no information on their morphology is available. In U. sikorae, the mitochondrial lineages identified form several major geographic clades, two of which (from the northernmost and southernmost populations, respectively) received substantial support in the phylogenetic analysis. No instance of sympatry of two or more mitochondrial lineages was observed, precluding an unambiguous assessment of species status under the biological species criterion without experimental approaches or detailed hybrid zone analyses. In the fragment of the nuclear encoded gene SACS we observed haplotype sharing between species and mitochondrial lineages, while in the fragment of KIAA1239 no haplotype sharing was detected although neither species nor mitochondrial lineages formed coherent phylogroups in the respective network. A screening of colour patterns from live photos, partly of the genotyped individuals, confirmed a large variation within species and populations, with a possible sexual dichromatism where a longitudinally striped phenotype is restricted to males. All individuals from populations of the U. sikorae clade from the Southern Central East and South East had an unpigmented oral mucosa just like U. sameiti, while all other U. sikorae populations are characterized by a black oral mucosa pigmentation. The extremely strong phylogeographic structure in the U. sikorae complex without obvious species-level divergences is unprecedented for large-sized squamates in Madagascar and calls for further taxonomic scrutiny using phylogenomic approaches; and it exemplifies how the loss of any major block of the remaining rainforests in Madagascar will inevitably lead to a substantial loss of genetic diversity – even if often intraspecific – in rainforest-specialized species. [ABSTRACT FROM AUTHOR]

The Malagasy genus Gephyromantis contains 51 species of primarily terrestrial or scansorial frogs. Although many species are morphologically weakly divergent from each other, the combination of molecular and bioacoustic evidence has led to a continuous flow of species discoveries in the last years. Previous works have notably shown the existence of numerous additional deep mitochondrial lineages of uncertain status in the nominal subgenus Gephyromantis, some of these considered as confirmed or unconfirmed candidate species, some as deep conspecific lineages. Here we use DNA sequences of one mitochondrial and one nuclear marker, as well as morphological and bioacoustic data, to conduct an integrative revision of the subgenus Gephyromantis. The analyses reveal at least 12 distinct and independent evolutionary lineages belonging to the G. blanci and G. boulengeri species complexes. Evidence for the species status of these lineages included multiple cases of syntopic occurrence without genetic admixture, as well as differences in advertisement calls or morphological differentiation without intermediate forms, suggesting reproductive isolation. We discuss the relevance of these different lines of evidence and describe six new species of Gephyromantis. [ABSTRACT FROM AUTHOR]

The composition of cutaneous (skin-associated) bacterial communities of amphibians has been intensively studied in light of the potential of some of these commensal bacterial taxa to mitigate infection with the chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal). However, surprisingly, the absolute densities in which these bacteria occur on the skin are only poorly known. We here combine quantification of bacterial 16SrDNA copies from skin swabs by quantitative PCR with counts from scanning electron microscopy (SEM) images to assess and compare bacterial abundances on the skin of various central European amphibians. We focus on the fire salamander (Salamandra salamandra) which is severely threatened by the spread of Bsal. Based on counts from SEM pictures of selected skin parts, local densities of ~43,000 bacteria per mm² were ascertained in frogs, although the data are insufficient to understand whether these values apply to the whole body surface. Bacterial densities are doubtless much lower in fire salamanders in which we observed almost no bacteria in SEM examination. From qPCR data, we find: (i) statistically relevant differences in bacterial abundances among species, with the lowest abundances in terrestrial salamanders and the highest abundances in toads; (ii) higher bacterial loads in captive compared to wild fire salamanders, and much higher loads in Bsal-infected captive specimens, in agreement with the hypothesis that Bsalinfection leads to blooming of opportunistic bacteria that may cause secondary infection; (iii) an only weak decrease of bacterial load after repeated swabbing, in agreement with the hypothesis that skin swabs capture only a part of the bacteria of the swabbed skin surface. We discuss the multiple sources of uncertainty in absolute estimates of abundances of cutaneous bacteria and suggest further research to clarify and reduce these uncertainties. [ABSTRACT FROM AUTHOR]

We describe a new subspecies of day gecko, Phelsuma dorsivittata paradoxa ssp. n. (Squamata: Gekkonidae) from Nosy Be Island and the Sambirano region in northwestern Madagascar. Due to its colouration and small size, Phelsuma d. paradoxa shares at first glance more similarities with P. parva or P. quadriocellata than with P. d. dorsivittata. Yet, phylogenetic analysis of mitochondrial and nuclear-encoded DNA sequences unambiguously shows that P. d. paradoxa is related to the allopatrically distributed P. d. dorsivittata. Due to the substantial and constant morphological divergence and the geographic separation between the two taxa we concluded that assigning them an initial status of subspecies is adequate, although they are not reciprocally monophyletic in mitochondrial DNA. Subsequently we discuss possible scenarios for eyespot convergence and mitochondrial paraphyly within P. dorsivittata. Furthermore, the molecular data presented here confirm the separate position of P. lineata punctulata within the P. dorsivittata complex. In order to resolve the paraphyly of the Phelsuma lineata complex, we here elevate this taxon to species rank, as Phelsuma punctulata Mertens, 1970. [ABSTRACT FROM AUTHOR]

New genetic, bioacoustic and morphological data on green-coloured Boophis treefrogs from eastern Madagascar reveal an additional level of cryptic diversity in these frogs. Two candidate species, Boophis sp. Ca36 and Ca37, are closely related to each other and to B. sandrae, with uncorrected pairwise distances in the mitochondrial 16S rRNA gene as low as 2.2% between some individuals. However, the three lineages show full concordance between differentiation in the 16S and the nuclear-encoded SACS gene, despite confirmed syntopy of B. sandrae and B. sp. Ca37 in the Ranomafana region, and probable syntopy of B. sp. Ca36 and B. sp. Ca37 in the Andasibe region. Most likely, these lineages are also divergent in advertisement calls, but the available recordings cannot be reliably assigned to either of them. Based on new material collected from various new sites, we here formally name B. sp. Ca36 as new species B. asquithi sp. n., and suggest targeted fieldwork on calls and larval stages to allow for a complete and fully conclusive taxonomic revision of this species complex. The example of these frogs illustrates how continued underestimation of cryptic diversity in anurans can lead to incorrect assignment of specimens, and leads us to emphasize the importance of designating as name-bearing types (holotypes) of anurans only individuals whose identity is unambiguous by genetic data or, at least, call recordings reliably assignable to the type specimen. [ABSTRACT FROM AUTHOR]

We studied the status of toads of the genus Rhinella collected in the southern Cordillera Azul, central Peru. Molecular analysis of the mitochondrial 16S rRNA gene revealed them to be members of the recently proposed Rhinella festae species group, and sister to R. lilyrodriguezae, a species known from northern areas of the Cordillera Azul. The new specimens are differentiated from R. lilyrodriguezae and other species of Rhinella by substantial genetic divergence in the studied gene fragment (> 5% uncorrected pairwise distance) and several qualitative morphological characters, providing combined evidence for a divergent evolutionary lineage. We consequently describe the specimens from the southern part of the Cordillera Azul in Departamento Huánuco as a new species, Rhinella chullachaki sp. n. We briefly discuss the definition and content of species groups in Rhinella as well as the difficulties hampering taxonomic resolution within this species-rich genus. [ABSTRACT FROM AUTHOR]

Experimentally assessing the preferred body temperature (Tpref) of ectothermic animals is important to understand thermal adaptation. In lizards, this variable is usually estimated by measuring body temperature in thermal gradients. To quantify the extent to which different experimental setups influence the inferred Tpref values we submitted 65 individual lizards of three species to randomized tests using six different experimental setups at Oukaimeden, Morocco, including setup variants similar to those that have been most often used in lacertids. Among-treatment differences were substantial. Using an infrared bulb as heat source in combination with artificial cold lighting yielded about 5°C lower Tpref estimates than photothermal treatments with an incandescent bulb as heat and light source, possibly because lizards thermoregulated differently without a visual cue related to the heat source, or due to the absence of a natural photoperiod. Photothermal assays in which Tpref was assessed by hourly cloacal measurements over a 10h period yielded 2.2°C lower Tpref estimates than 2h treatments where body temperature was measured every minute with a thermocouple attached to the belly. This probably reflects that the 2h treatments targeted lizards in the initial warming-up phase, whereas the 10h treatment attempts to capture the preferred temperature of a lizard over its entire daily cycle including phases of inactivity. Lastly, we observed large differences among treatments with contact thermometers versus infrared laser thermometer measurements, calling for caution when the latter are used with artificial heat sources. Our data do not provide thorough tests of the physical, behavioural or physiological causes underlying the observed differences between treatments, but illustrate that for meta-analyses where detailed comparisons are needed, a rigorous consideration of the optimal experimental setup and its consistent use will remain necessary. [ABSTRACT FROM AUTHOR]

Sample type and preservation methods are likely to influence the results of microbiome analyses. Here, we sampled tadpoles of Nanorana parkeri non-invasively for feces, and subsequently for hindgut through dissection. Gut and fecal samples were stored in ethanol, and additionally, part of the gut samples were frozen, but temporarily thawed during transport as it often happens under difficult field conditions. Our results showed that both substrate (gut vs. feces) as well as preservation method can influence the analysis of intestinal microbiomes. Frozen gut samples strongly differed from ethanol-stored samples, and most of the frozen samples were dominated (in relative abundance) by a set of Proteobacteria OTUs that were completely absent from the ethanol-stored samples. This blooming of contaminant bacteria occurred after less than 12 h of thawing, thus caution should be taken when constancy of cold temperatures cannot be maintained in the field for sample preservation purposes. Among ethanol-stored samples, whereas the recovered bacterial richness and phylogenetic diversity did not differ, the community structure varied significantly. Thus, for meaningful gut microbiome meta-analyses it is important to acknowledge the effect of the sampling substrate for the microbial community composition. [ABSTRACT FROM AUTHOR]

The chameleon Furcifer voeltzkowi (Boettger, 1893) from northwestern Madagascar was considered to be a synonym of Furcifer rhinoceratus for many decades and was resurrected only recently based on studies of the morphology and osteology of a few male specimens, which were collected more than 100 years ago. However, basic data on this species remain unavailable, including its conservation status, life history, colouration in life, morphology of the female, genital morphology of the male, phylogenetic affinities, and genetic differentiation from F. labordi and other congeners. During a targeted expedition, we rediscovered F. voeltzkowi in its natural habitat close to its type locality, allowing us to fill some gaps of knowledge. Furcifer voeltzkowi is a sexually dimorphic species. The life colouration of males is largely green, whereas that of females is highly variable and can be extremely colourful. Both, morphology and life colouration of males and females show close similarities to F. labordi from west and southwest Madagascar, but also a number of distinct differences (e.g. a larger size of F. voeltzkowi and a smaller rostral appendage in both sexes), enabling a clear distinction of the two species in both sexes. DNA sequences of the nuclear CMOS gene and two mitochondrial markers (16S rRNA and ND4) also confirm that F. voeltzkowi is a distinct species and sister to F. labordi (pairwise differences in the 16S gene 3.5-3.6%). We estimate the distribution range (extent of occurrence) of F. voeltzkowi to comprise ca. 1.000 km2 and suggest that it qualifies as Endangered B1ab(iii) under the IUCN Red List Criteria as its populations are expected to be severely fragmented, and there is continuing decline in the extent and quality of its habitat. Similar to F. labordi, F. voeltzkowi might be an extremely short-living chameleon with a post hatching life span of only several months in the rainy season. The assumed short life might also partly explain why this splendid species got "lost" for many decades, since most roads in its habitat are not accessible in the wet season. [ABSTRACT FROM AUTHOR]

Chytridiomycosis, a disease induced by the chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), has strongly contributed to the ongoing worldwide amphibian conservation crisis. While Bd infection has caused amphibian declines for decades on several continents, Bsal is a novel threat to Central European salamanders and newts, being responsible for the collapse of Fire Salamander populations in the Netherlands, Belgium, and Germany. However, numerous other parasites causing harm to amphibians exist, yet have received much less attention than the chytrid fungi. The goal of the present study was to contribute to the understanding of declines of the Green Toad, Bufotes viridis, at its northwestern distribution border, in the area of Cologne, Germany. We combined the data from four years of Bd monitoring with a metabarcoding approach to detect other, mainly unicellular parasites, from amphibian feces and environmental samples, and also report results from Bsal testing in 2019. Skin swabs of approximately 280 amphibians were tested for Bd and 66 for Bsal, and 150 cloacal swabs and environmental samples from five sites were tested for other pathogens and parasites.We found Bd in all sampled locations with high prevalences and partly high individual infection loads but without clinical signs of chytridiomycosis. None of the samples tested for Bsal was positive for this pathogen. We further detected eight additional potential amphibian pathogens from fecal samples: three metamonads (Tritrichomonas augusta, Trichomitus batrachorum and Hexamita inflata), three ciliates (Balantidium duodeni, Nyctotherus cordiformis and N. hubeiensis), one stramenopile (Blastocystis sp.) and one metazoan (the nematode Rhabdias ranae). In the environmental samples, we detected OTUs of nine organisms potentially harmful for amphibians: Blastocystis sp., Hexamita inflata, Tritrichomonas augusta, Trichomitus batrachorum, two oomycetes (Leptolegnia sp., Saprolegnia sp.), two ichthyosporeans (Amphibiocystidium ranae, Anurofeca sp.) and the myxozoan Myxobolus sp. [ABSTRACT FROM AUTHOR]

The chytrid fungus Batrachochytrium salamandrivorans (Bsal), recently introduced from Asia to Europe, causes mortality in numerous species of salamanders and newts and has led to catastrophic declines and local extinctions of the European fire salamander (Salamandra salamandra) in the Netherlands, Belgium, and Germany. Due to the continuous spread of the pathogen, Germany can be considered as the current 'hotspot' of Bsal-driven salamander declines. The pathogen was detected in 2015 in the Eifel Mountains where it probably has been present at least since 2004. Moreover, Bsal was found in 2017 in the Ruhr District where it also might occur since 2004. The Ruhr District is a heavily urbanized and industrialized region in western Germany, which offers an unprecedented opportunity to monitor range expansion and infection dynamics of Bsal in an area affected by intense human activities. We here review the current knowledge on Bsal in the Ruhr District where the pathogen by now has been recorded based on qPCR data from 18 sites distributed over eight cities. Transect counts (adult salamanders) and larval removal-sampling at two sites where Bsal was recorded in 2017 and 2018, confirm that fire salamander populations at the affected sites have declined dramatically. However, single negative-tested individuals were still observed and reproduction could be ascertained. Moreover, we successfully detected Bsal by analysing environmental DNA (eDNA) from samples obtained from a standing water body as well as a stream. Detailed monitoring of a site in Essen (Kruppwald) from January to May 2019 provided data on infection and disease dynamics during an acute Bsal-outbreak in a population of European fire salamanders. After initial observation of single dead infected salamanders in January and February 2019, the maximum Bsal loads in the population ranged from 7.90E+03 ITS copies in early March to 2.29E+09 ITS copies at the end of March. Prevalence of infection ranged from 4% to 50% and significantly increased over time; prevalence of externally visible disease symptoms peaked on May 2 and May 8. Single dead salamanders were encountered throughout the monitoring period. Recaptures of two infected salamanders indicated an increase of Bsal load by about one order of magnitude within one week. Infected salamanders showed small-sized regular round ulcerations usually of 0.25-1 mm but sometimes up to 2.5 mm in diameter, which gave the impression of outward growth from the centre of each ulceration. Among salamander individuals monitored in the Kruppwald, such ulcerations were only found in infected salamanders, but we found no significant correlation between the intensity of the ulcerations and Bsal load. Heat treatment proved effective to cure even deep ulcerations when salamanders were kept for 10 days at 25-27°C or 14 days at 25°C, but infection persisted and ulcerations reappeared six weeks after the end of the treatment; only heat treatment at 25°C for 21 days proved effective to reliably clear the infection in three tested salamanders. [ABSTRACT FROM AUTHOR]

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The Green Toad (Bufotes viridis) in Germany is mostly confined to secondary habitats and is experiencing severe population declines especially at its northwestern range boundary in the city of Cologne. As a basis for conservation management of this species, viz. to investigate the status of the population structure, we used a library of over 15,000 short tandem repeats to establish a new set of 12 microsatellite markers for this species. We applied these markers to B. viridis individuals from six sampling sites in Cologne, and included outgroup samples from sites in northern and central Germany. The results suggest the sampled Cologne toads form four distinct populations, without an obvious distinction between sites east and west of the river Rhine. Only the green toads of three neighbouring sampling sites clustered as one population, supporting the initial hypothesis that individuals from proximate sampling sites are genetically more similar than those from more isolated sampling sites. However, mitochondrial DNA sequences revealed a haplotype east of the Rhine that was absent west of the river. Toads from the northernmost sampled population in Cologne "Ginsterpfad" were not genetically depauperate, indicating that the severe declines at this site did not lead to a loss of genetic variation and were not related to inbreeding. Some Ginsterpfad individuals were genetically highly distinct from others, which may be explained by human introduction. Despite the encountered population structure, the low number of private alleles in the populations west of the Rhine, and the uniformity in mitochondrial haplotypes with the exception of one deviant allele east of the Rhine, lead us to recommend a pragmatic approach for conservation management. We suggest to consider all Cologne Green toads as belonging to a single management unit, but propose that, wherever possible, populations east and west of the Rhine should not be mixed in reintroduction measures. [ABSTRACT FROM AUTHOR]

Phelsuma guttata and Paroedura gracilis are two species of geckos endemic to lowland and mid-elevation rainforests of the northern half of Madagascar's rainforest band. To test for concordant phylogeographic patterns in the two species and to assess intraspecific phylogeny and genetic diversity, we sequenced DNA from two mitochondrial genes (16S and ND4) and three nuclear genes (CMOS, PDC, RAG1). The mtDNA trees of both species suggest a strong phylogeographic split between phylogroups from the North East and Northern Central East biogeographic regions of Madagascar. However, this pattern is not mirrored in the nuclear gene dataset, where common alleles can be found across the two biogeographic regions. Private alleles characterize some of the northernmost and southernmost populations, especially the Montagne d'Ambre population of P. gracilis. In this species the two main mitochondrial clades agree with two distinct dorsal colour pattern phenotypes - striped versus cross-banded - separated at the junction of North East and Northern Central East. As no obvious geographic barrier to gene flow is apparent, the observed concordant phylogeographic pattern indicates that the border between these two regions might represent an area of secondary contact after divergence in refugia. Both nuclear and mitochondrial DNA reveal a higher genetic divergence among lineages of P. gracilis as compared to Ph. guttata, calling for a more detailed taxonomic assessment of the former species. [ABSTRACT FROM AUTHOR]

We describe the new gecko species Paroedura neglecta sp. n. from the Tsingy de Bemaraha National Park in western Madagascar, belonging to the P. bastardi/tanjaka clade. The species in this clade are characterized by three light dorsal crossbands on the dorsum of juveniles and subadults whereas all other Paroedura species have four such bands. The new species differs from all species in the P. bastardi complex in having the nostril in contact with the rostral scale. It is most similar to the sympatric P. tanjaka, from which it differs by the presence of prominent dorsal tubercles arranged in regular longitudinal rows (versus rather irregular rows of dorsal tubercles), smaller size, details of the dorsal colour pattern and strong genetic divergence. [ABSTRACT FROM AUTHOR]

Stump-toed frogs (genus Stumpffia Boettger, 1881) are a diverse group of small-bodied frogs endemic to Madagascar. Seven species of this genus occur on Marojejy, a steep massif in northeastern Madagascar. Here we examine the elevational distribution, phylogenetic position, biogeographic origin, and genetic differentiation of this Stumpffia assemblage. We show that none of these species is another's closest relative, but rather they are all independent lineages that probably colonised the Marojejy Massif through repeated immigration events. All of the lineages on Marojejy are most closely related to species south and southwest of the massif, except one lineage, formerly known as Stumpffia sp. Ca07, but here assigned to S. sorata as a deep conspecific lineage (and referred to as S. cf. sorata), which occurs also in Sorata, 90 km north of Marojejy. The species on Marojejy are typically restricted to narrow elevational ranges, but at least two species, S. cf. sorata and S. tridactyla, occur over elevations spanning 1000 metres. We assessed the genetic variability of these populations, and found considerable haplotype separation in fragments of the mitochondrial 16S rRNA and nuclear Rag-1 genes, suggesting some disruption of gene flow associated with elevation. We discuss the biogeographic implications of our findings and, based on previously published data, the evolution of non-overlapping bioacoustic parameters among the diverse assemblage of Stumpffia species on the Marojejy massif. [ABSTRACT FROM AUTHOR]

We examine the taxonomic status of two Malagasy leaf chameleon taxa, Brookesia antakarana Raxworthy & Nussbaum, 1995 and B. ambreensis Raxworthy & Nussbaum, 1995, integrating morphological and genetic evidence. Specimens assigned to these species occur in syntopy in Montagne d'Ambre, northern Madagascar, and were originally described based on differences in the shape of their pelvic shields. We found that the shape of these shields falls on a continuous spectrum, and detected only weak differences between the two taxa in a few other morphological features, all of which were correlated with shield length. Members of the two taxa (as assigned based on pelvic shield morphology) also showed extensive haplotype sharing in one nuclear and one mitochondrial marker. We conclude that at present there is no convincing evidence that these species are distinct, and act as first revisers in the sense of the International Code of Zoological Nomenclature to place B. ambreensis into the synonymy of B. antakarana [ABSTRACT FROM AUTHOR]

This study summarizes the data of multiple surveys carried out at the Isalo Massif (southwestern Madagascar), both within and outside the National Park limits between 2004 and 2014, providing an updated list of the amphibians and non-avian reptiles present within the area. We used an integrative taxonomic approach, combining morphological and molecular data sets to identify collected samples as either described species, or as undescribed candidate species. We here report on one new amphibian candidate species (Mantidactylus sp. aff. multiplicatus Ca65 "Isalo"), and two new amphibian distribution records (Mantella sp. aff. expectata "South", Scaphiophryne sp. aff. calcarata Ca02) compared to the former inventories of the area. Additionally, we provide molecular reference sequences for 23 of the 24 amphibian taxa and for 40 of the 47 reptile taxa known to occur in the area, combining all available sources. With this study, we validate Isalo as an important centre of biodiversity and micro-endemism within Madagascar, with four described endemic amphibian (Gephyromantis azzurrae, Mantella expectata, Mantidactylus noralottae, and Scaphiophryne gottlebei) and one reptile species (Trachylepis nancycoutuae). Of the taxa here listed for Isalo, seven amphibians and six reptiles are new candidate species, and among them at least one amphibian (Mantidactylus sp. aff. multiplicatus Ca65 "Isalo") and three reptiles (Lygodactylus sp. aff. tuberosus Ca02 "Isalo", Paroedura sp. aff. bastardi Ca02 "Isalo" and P. sp. aff. bastardi Ca03 "Ilakaka") are currently known only from Isalo. The unique biodiversity of the Isalo Massif was likely favoured by the confluence of different biomes in the area and/or its role as a refugium during palaeoclimatic oscillations. Our study highlights the crucial importance of applying molecular taxonomic identification in updating the species lists even of previously well surveyed areas, and of maintaining and increasing museum voucher collections as a permanent and accessible testimony of the biodiversity inventory. [ABSTRACT FROM AUTHOR]

We describe a large and distinctive new treefrog species with blue webbing from the west coast of the Masoala peninsula in northeastern Madagascar. Boophis masoala sp. n. is morphologically similar to the other species of the Boophis albilabris group but can be distinguished from them easily by several chromatic characters of the eyes. Despite its similar morphology, it is genetically highly differentiated (10.5-13.3% pairwise p-distance in a segment of the 16S rRNA gene) from all other species in the B. albilabris group including the morphologically most similar Boophis praedictus. Both species share the blue webbing between toes and are distributed on the Masoala peninsula, but so far were not found in close sympatry. Although we recorded the new species only from the unprotected areas near the coast, we are confident that it also occurs within the adjacent Masoala National Park. We discuss the importance of eye colouration as a predictor of specific distinctness in the genus Boophis and that of webbing colouration as taxonomic characters of large treefrogs. Based on a micro-CT scan we provide a comprehensive description of the osteology of the new species, which is the first for any Boophis species, and furthermore describe its distress call which consists of three distinct sections corresponding to (1) the starting phase with closed mouth, (2) the opening of the mouth and (3) the final section with an open mouth. [ABSTRACT FROM AUTHOR]

Systematics and classification of Asian frogs of the genus Fejervarya and related genera (family Dicroglossidae; hereafter referred to as fejervaryan frogs) have been the subject of intensive debates in the past few years. We complement previous phylogenetic studies with analyses of concatenated sequences from 14 nuclear loci and mitochondrial gene fragments, totaling 12,752 nucleotides for 46 species representing all major lineages and relevant outgroups. We find strong support for two major clades within Fejervarya: a South Asian clade and a Southeast Asian clade. Previously, South Asian species have been hypothesized to constitute a separate genus, Zakerana (currently considered a junior synonym of Fejervarya), and also include species previously described as members of the genus Minervarya. Although parsimony and species tree analyses found support for the monophyly of Fejervarya as currently understood, partitioned Bayesian inference and unpartitioned Maximum Likelihood analyses of concatenated sequences recovered Southeast Asian species as a clade sister to the genus Sphaerotheca, albeit with low nodal support. We discuss the advantages and disadvantages of alternative classification schemes in light of previously proposed criteria for naming supraspecific taxa. The current single-genus taxonomy would impart desirable economy of nomenclatural change, and morphological diagnosability. However, other taxon naming criteria such as support for monophyly, temporal framework for diversification, and biogeographic regionalism would support a contrasting two-genus alternative. Because new species of Fejervarya are increasingly being discovered and described, and because a single-genus classification (Fejervarya) will remain controversial, given ambiguous support for its inferred monophyly, we propose recognizing two genera: Southeast Asian Fejervarya, and South Asian Minervarya. This classification results in two genera whose monophyly is strongly supported, respectively, and unlikely to be challenged by future analyses. Accordingly, we transfer all species of the South Asian clade to the genus Minervarya. [ABSTRACT FROM AUTHOR]

Frogs assigned to the monotypic genus Madecassophryne (Anura, Microhylidae, Cophylinae), and possibly belonging to Madecassophryne truebae, were found in December 2016 in two low-altitude localities, Ambahavala and Kapilavato, in the Anosy Mountain in southeastern Madagascar. This poorly known genus was described in 1974 based on osteology, and neither verifiably identified photos of living specimens nor molecular information were available until now. We here update the available information on these enigmatic frogs and provide new data on morphology, osteology, bioacoustics and observations on their habitat, together with a preliminary molecular phylogenetic study, suggesting that Madecassophryne is highly divergent from other members of the cophyline clade. [ABSTRACT FROM AUTHOR]

We integrate molecular, bioacoustic and morphological data to provide a systematic revision of the frogs classified in the Gephyromantis asper clade (Anura: Mantellidae), endemic to Madagascar. Based on concordant differentiation in a mitochondrial and a nuclear gene (16S rRNA and Rag1) we distinguish six different species in this clade: G. ambohitra, G. asper, G. tahotra, G. spinifer, G. ceratophrys bona species, and an undescribed species. Gephyromantis ceratophrys (Ahl, 1929) is resurrected from the synonymy of G. asper and refers to the southernmost populations previously assigned to that species (i.e., from Ranomafana National Park in the southern central east of Madagascar). We provide several new geographical records verified by molecular sequence identity: G. asper is confirmed from the localities Anjozorobe, Mandraka, and Tsinjoarivo; G. spinifer is confirmed from Pic Ivohibe, a record previously considered questionable and representing the northernmost locality known for this species; G. ambohitra is recorded from the Masoala Peninsula, extending its known distribution range eastward; and G. tahotra (previously only known from its type locality Marojejy) is confirmed for the Tsaratanana and Sorata Massifs. An undescribed species of the group occurs at sites south and southwest of the Tsaratanana massif. It is differentiated in mitochondrial and nuclear DNA, but no bioacoustic and insufficient morphological data are thus far available for its formal taxonomic description. Because species in the G. asper clade, within Gephyromantis, are phylogenetically distant from other species of the subgenus Duboimantis, we propose a new subgenus Asperomantis for this clade of Malagasy rainforest frogs. Most individuals belonging to this subgenus are easily distinguished from other species in the genus Gephyromantis and most other mantellids by the presence of a light spot in or near the centre of the tympanum. [ABSTRACT FROM AUTHOR]

We provide an update of the geographical distribution and phylogenetic relationships of mitochondrial haplotype groups of the European common frog, Rana temporaria, one of the most widespread amphibians on Earth. Our data set of 378 newly determined DNA sequences of the cytochrome b gene is combined with 640 previously published sequences, mainly filling a sampling gap in the Spanish Basque country. A haplotype network based on 331 bp provides evidence for a previously unknown, deeply divergent haplogroup in this region, which in the eastern part of its range occurs sympatrically with a widespread group of haplotypes from the Pyrenean mountains, and to the West contacts a haplogroup typically for the central Cantabrian mountains. In contrast, samples from the isolated Montseny massif in the Spanish region of Catalonia are not differentiated from Pyrenean populations. Samples from Scotland cluster in a widespread haplogroup with samples from northern France, western Germany, and Ireland. Our data furthermore suggest that previous records of haplotype sharing of British R. temporaria populations with those from northwestern Spain are probably erroneous. A phylogenetic analysis based on 4,413 bp of mitochondrial DNA confirms the strong divergence of the newly discovered Basque haplogroup and indicates that it might be the sister group of a clade containing all other R. temporaria haplogroups. [ABSTRACT FROM AUTHOR]

We describe two new microhylid frog species of the genus Rhombophryne from the humid forests of northeastern Madagascar: Rhombophryne botabota sp. n. and R. savaka sp. n. The former is a medium-sized species, characterised by darkened lateral sides of the head (present in only one other congener, R. laevipes) and a unique combination of morphological, osteological, and molecular characters. The latter is a rather small species, characterised by medially undivided vomerine teeth with two large lateral diastemata, and presence of inguinal spots. Rhombophryne savaka sp. n. is the first species of the genus known from Makira Natural Park, and is reported also from Marojejy National Park and Ambolokopatrika (Betaolana Forest). Although its distribution range is relatively large compared to those of congeners, its known extent of occurrence is less than 2,000 km². As deforestation and habitat degradation persist as threats despite formal legal protection, we suggest an IUCN Red List status of Vulnerable for this species. Rhombophryne savaka sp. n. is possibly endemic to the Marojejy National Park, like several other Rhombophryne species, but is unusual in being found at a relatively low altitude. As such, it is likely to be at high risk of habitat loss and decreasing range, and we propose a status of Endangered for it. We discuss the affinities of these new species and the variability of calls in this genus. [ABSTRACT FROM AUTHOR]

The strawberry poison frog (Oophagapumilio), occurring from Panama to northern Nicaragua, is characterized by an extraordinary variation of aposematic colouration and pattern in some Panamanian populations and has become a model system for studying evolutionary and ecological processes. Phylogeographic analyses of this species so far included populations from Panama and Costa Rica only. Based on newly collected samples from representative sites across Nicara-gua, we here extend an existing data set of mitochondrial DNA sequences of the cytochrome b gene to analyse the phylo-geographic history of O. pumilio. The 62 sequenced samples from seven localities are more closely related to the northern clade of O. pumilio, four haplotypes are shared amongst populations from Costa Rica and southern Nicaragua, and one haplotype is shared amongst Costa Rica and northern Nicaragua. An analysis of colour patterns reveals slight differences within and amongst Nicaraguan populations, but all of them conform to the general pattern found also in Costa Rica, with a reddish dorsum and blue coloured limbs. Phylogeographic analysis suggests that the Nicaraguan populations originated by a Late Pleistocene range expansion northwards, and reconstructs the origin of the species in southeastern Costa Rica [ABSTRACT FROM AUTHOR]

The article focuses on a study conducted on an embryonic development and morphology of Mantidactylus betsileanus, also known as Malagasy frog. Topics discussed include documentation of stages of larval development of the species, the absence of external gills in the embryos, description of stage 25 of larval development from captive-bred individuals and stage 36 from wild individuals of the species.

The article discusses Madagascar's leaf tailed geckos of genus Uroplatus along with the information on geographical distribution, species boundries and high intraspecific genetic variability. It focuses on high intraspecific genetic variability to offer information on future taxonomic revisions, spatial prioritisation of conservation efforts. It mentions 14 species which has been allocated in five species group which include U. ebenaui group, U. alluaudi group and U. lineatus group.

The article discusses a study on mantellid frogs found in Madagascar. As per the study, the pattern of eye color of the frogs is significantly correlated with its ecology and habitats. In the study, it was found that significant number tree frogs of the genus Boophis have a bright colored iris, often with annular elements and a blue-colored iris periphery (sclera) diurnal, terrestrial and aposematic Mantella frogs have a uniformly black iris, and terrestrial leaf-litter dwellers have an iris horizontally divided into an upper light and lower dark part. The study presents a correlation between the presence of a frenal streak and a dark tympanic patch in the frogs. Further as per the study frenal streak and tympanic patch were associated with the horizontally divided iris color.

The article reports on the study of frogs of the Eleutherodactylus limbatus group from Cuba concerning molecular diversity, geographic distribution and colour variation. It cites several species that contain Cuban Eleutherodactylus limbatus including E. orientalis, E. limbatus and E. iberia. It notes that several of the said species have contrasting colour patterns with white stripes and bright yellow on a dark dorsum. It mentions that the evolution of contrasting dorsal colour pattern is characterized by homoplasy.

The article presents a study on the expansion of molecular systematics and known distribution of Eleutherodactylus adelus. It mentions that the phylogeny reconstruction was done through the Bayesian inference (BI) and maximum likelihood (ML). It states that the Antarctic Phosphatase (AP) and Exonuclease I and Shrimp Alkaline Phosphatase (SAP) was used to purified the amplified products. The result of the study indicates that E. adelus was not a close relative of E. varleyi and a member of the E. planirostris series.

The article presents a study which describes two new bright-eyed treefrog species from the Boophis (B.) guodoti group based on molecular genetics and morphology. It mentions that the specimens were immersed in a solution of MS222 or robutanol, and preserved in 70% ethanol. It notes that the Boophis fayi specie (sp.) exhibits a green outer iris and male lacks supratymnpanic fold while the Boophis popi sp. is characterized with red iris and elevated reticulations on the dorsums. It adds that the species are smaller than those small-to-medium members of B. goudoti group.

The article offers information on the study concerning the breeding habitats and tadpole morphology of relict frog Tsingymantis antitra in the Ankarana area in Madagascar. The tadpoles were identified using a DNA barcoding approach based on a fragment of the mitochondrial 16S rRNA gene and were stained with methylene blue to determine developmental stages and assess characters of the oral disc. It mentions that Tsingymantis has a strongly enlarged and keratinised jaw sheaths with strong serrations in the lower and upper jaw, which are typical for oophagous tadpoles. Moreover, it adds that the combination of larval habitats and oral morphology could be an indication for oophagy or a predatory feeding modes in Tsingymantis tadpoles.

The article discusses a study concerning the ecology and morphology of the sand-eating tadpoles of the treefrog Boophis picturatus in Ranomafana National Park (RNP) in the southern central east of Madagascar. It mentions that the tadpoles were euthanised by immersing into chlorobutanol solution and a principal components analysis (PCA) was performed to analyse the breeding site choice of B. picturatus frogs. It describes the buccal anatomy and external morphology of the tadpole wherein the B. picturatus has an extremely derived oral disc without keratodonts and jaw sheaths. It shows that B. picturatus tadpoles were mainly found in streams with low incline forest areas.

The article presents the results of a survey of reptiles and amphibians at several lowland habitats along the east coast of Madagascar in 2010. The survey yielded 45 reptiles and 61 amphibians, which totals 106 species. Structural differences between littoral forests and lowland forests appear to cause a difference in the composition of species. The survey shows that the most remarkable records were those of Blaesodactylus antongilensis, Uroplatus lineatus and Mantidactylus majori in the Sahafina forest which comprise important range extensions for the species.

The article presents a study on the morphologically distinct Calumma tarzan (C. tarzan), a chameleon species of the Calumma furcifer, which is located at the rainforest fragments of the Anosibe An'Ala region in central eastern Madagascar. The study involves the collection of the specimens, which are detected in the vegetation using torches and headlamps at night during the rainy seasons. It states that C. tarzan was named from the fictional forest man Tarzan to promote awareness and conservation activities for the species. Furthermore, the study shows that C. tarzan is closely related to a barely studied population of chameleons from Marolambo in Madagascar.

The article focuses on the study of dwarf species of day geckos of the genus Phelsuma from the Ankarana Massif in Madagascar. It states that Phelsuma species, a conspicuous lizard was originated in northern Madagascar and probably a sister species of P. laticauda. Snout vent length of 32.6 mm, tail length of 36.4, head width of 6.4 mm, and with a total legth of 62-72 mm. It says that Phelsuma species differ from subspecies P. lineate bombetokensis from northwestern of Madagascar by unkeeld ventrals and subcaudals, the absence of dark lateral spots. P. laticuada group was characterized by being "non-gluers" with subcaudal scales and smooth ventral, three nasals and without contact nostril-rostract. It adds that the distributions of P. laticuad reliably known from their type of locality.

The article discusses a study on the snake species called Liophidium pattoni sp. n., particularly on its phylogenetic relationships. It says that the snake species is regarded to have a unique colour pattern as compared with other snakes found in Madagascar. The study also included snake samples from the Malagasy pseudoxyrhophiine genera aside from the ones representing the genus Liophidium. It states that the scientific methods used include polymerase chain reactions (PCRs), parsimony analysis, and Bayesian phylogeny inference. Results reveal that L. pattoni has several meristic characteristics comparable to L. torquatum inducing scale counts, neck band, and transversal spot lines on the venter.

The article discusses the wasp predation on egg clutches of Guibemantis, a genus of mantellid frogs in Madagascar. It says that Malagasy frogs display various behavioural patterns such as diverse reproductive behavior, wherein they can exhibit 11 reproductive modes. It cites that predation on frog egg clutches deposited outside of water has been recorded for a few snakes and never by wasps. Meanwhile, it states that a Ropalidia wasp predation was recorded near Andasibe, Madagascar on February 2, 2007. It concludes that wasp predation of arboreal eggs left on vegetation over-hanging swamps or ponds has been found only in the Neotropics.