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Abstract The Malagasy stream-breeding treefrog species Boophis marojezensis contains bioacoustically and genetically highly divergent populations. Some of these populations have been defined as candidate species and emit somewhat bizarre advertisement calls consisting of multiple whistle-notes. We here enable a long-overdue taxonomic revision of this species complex by applying a museomics approach to sequence DNA from the holotype of B. marojezensis. Based on an integrative approach that combines divergence levels in mitochondrial DNA and in three nuclear-encoded genes, morphological data, and bioacoustic comparisons, we conclude that eight different species exist in this complex, seven of which are formally described herein as new. Although morphological differences between species are small and mainly separate small-sized from larger-sized species, conclusive evidence for the new species comes from their sympatric and sometimes syntopic occurrence without haplotype sharing in three nuclear genes and under maintenance of bioacoustic differences. Uncorrected genetic divergences in the mitochondrial 16S rRNA gene are >3% in almost all cases, and in some cases up to 8%. In reference to the otherworldly sounds by which these frogs fill Malagasy rainforests, some of them reminiscent of sounds of technical equipment in the fictional “Star Trek” universe, we here name and describe the seven new species in honor of fictional captains of starships, namely B. kirki sp. nov., B. picardi sp. nov., B. siskoi sp. nov., B. janewayae sp. nov., B. archeri sp. nov., B. pikei sp. nov., and B. burnhamae sp. nov. The majority of these species occur in northern Madagascar, where up to three species can occur in immediate geographical proximity, e.g., B. marojezensis, B. burnhamae sp. nov. and B. pikei sp. nov. at different elevations in the Marojejy Massif. South of 16°S latitude, only B. janewayae sp. nov., B. picardi sp. nov., and B. kirki sp. nov. are found, with the latter extending southwards to Ranomafana National Park. Our study confirms the existence of numerous morphologically cryptic and microendemic species among Madagascar’s amphibians, some of which are known only from unprotected sites and require adequate conservation management.

We describe a new frog species that is the sibling of Pristimantis reichlei. These two sister species inhabit the Amazonian lowlands and adjacent foothills of the Andes, from central Bolivia to central Peru. Pristimantis reichlei occurs from central Bolivia to southern Peru (Alto Purús National Park), while the new species occurs from northern Bolivia (Departamento Pando) to Panguana in central Peru (Departamento Huánuco), at elevations between 220 and 470 m a.s.l. In spite of their morphological crypsis, these siblings occur in syntopy without evidence of interbreeding (in the Alto Purús area) and are recovered as reciprocally monophyletic. Their uncorrected pairwise genetic distances in the 16S rRNA gene range from 9.5–13.5%, and their advertisement calls differ in both qualitative and quantitative traits. Moreover, our study found uncorrected pairwise distances within the new species of up to 5.0% and up to 9.3% within P. reichlei. We therefore cannot rule out the possible existence of hybrids or additional species-level lineages hidden in this complex. Furthermore, we found another potential pair of sibling species composed of P. danae and an unnamed lineage, with divergences of 9.4% in the 16S gene, whose in-depth analysis and taxonomic treatment are pending future revision. With the new nominal species, the Pristimantis danae species group now includes 20 species, distributed across the upper Amazon basin and in the eastern Andes, from western Brazil to Bolivia and Peru. Our study, together with an increasing number of other studies, indicates that sibling species are far from being rare among Amazonian amphibians and that species resolution remains low even for groups that have received considerable attention in recent years.

We contribute transcriptomic data for two species of Ostracoda, an early-diverged group of small-sized pancrustaceans. Data include new reference transcriptomes for two asexual non-marine species (Dolerocypris sinensis and Heterocypris aff. salina), as well as single-specimen transcriptomic data that served to analyse gene expression across four developmental stages in D. sinensis. Data are evaluated by computing gene expression profiles of the different developmental stages which consistently placed eggs and small larvae (at the stage of instar A-8) similar to each other, and apart from adults which were distinct from all other developmental stages but closest to large larvae (instar A-4). We further evaluated the transcriptomic data with two newly sequenced low-coverage genomes of the target species. The new data thus document the feasibility of obtaining reliable transcriptomic data from single specimens – even eggs – of these small metazoans.

Abstract Tropical ecosystems are challenging for pinnipeds due to fluctuating food availability. According to previous research, the Galapagos sea lion (GSL, Zalophus wollebaeki) adopts trophic flexibility to face such conditions. However, this hypothesis comes from studies using traditional methods (hard‐parts analysis of scat and isotopic analysis from tissue). We studied the diet of five rookeries in the southeastern Galapagos bioregion (which harbors the highest GSL density), via DNA‐metabarcoding of scat samples. The DNA‐metabarcoding approach may identify consumed prey with a higher taxonomic resolution than isotopic analysis, while not depending on hard‐parts remaining through digestion. Our study included five different rookeries to look for evidence of trophic flexibility at the bioregional level. We detected 98 prey OTUs (124 scats), mostly assigned to bony‐fish taxa; we identified novel prey items, including a shark, rays, and several deep‐sea fish. Our data supported the trophic flexibility of GSL throughout the studied bioregion since different individuals from the same rookery consumed prey coming from different habitats and trophic levels. Significant diet differentiations were found among rookeries, particularly between Punta Pitt and Santa Fe. Punta Pitt rookery, with a more pronounced bathymetry and lower productivity, was distinguished by a high trophic level and consumption of a high proportion of deep‐sea prey; meanwhile, Santa Fe, located in more productive, shallow waters over the shelf, consumed a high proportion of epipelagic planktivorous fish. Geographic location and heterogeneous bathymetry of El Malecon, Española, and Floreana rookeries would allow the animals therein to access both, epipelagic prey over the shelf, and deep‐sea prey out of the shelf; this would lead to a higher prey richness and diet variability there. These findings provide evidence of GSL adopting a trophic flexibility to tune their diets to different ecological contexts. This strategy would be crucial for this endangered species to overcome the challenges faced in a habitat with fluctuating foraging conditions.

Abstract We studied the taxonomic status of a population of Pristimantis from the southern Cordillera Azul, Departamento Huánuco, central Peru. A phylogenetic analysis based on the mitochondrial 16S rRNA gene revealed that it represents a lineage within the Pristimantis lacrimosus species group, being the closest relative of a species of uncertain taxonomic status from a lowland rainforest in central Peru (Panguana), and P. pulchridormientes from the Tingo Maria National Park. However, the focal lineage is divergent from all nominal species in the P. lacrimosus group for which respective data are available by >7.9% uncorrected pairwise distance in the 16S rRNA gene fragment. An integrative taxonomic approach, including morphological and bioacoustic analyses, provided multiple lines of evidence for the focal specimens belonging to an unnamed evolutionary lineage at the species level that we describe and name herein. The systematics of Peruvian populations associated with the P. lacrimosus group are discussed, particularly highlighting problematic taxa with uncertain taxonomic status and unknown relationships. We point to scientific challenges and actions needed to achieve a better taxonomic resolution of this species-rich clade of frogs.

We studied the taxonomic status of glassfrogs collected in Departamento Huánuco, central Peru, which in the field were tentatively allocated to Chimerella, one of the twelve genera currently recognized in the family Centrolenidae. Detailed analyses of their morphology, bioacoustics, and molecular genetics supported their generic allocation and provided evidence for them representing a divergent and unnamed evolutionary lineage within Chimerella. We herein describe this lineage as a new species, being mainly distinguished from the two other known congeners, C. corleone and C. mariaelenae, by details of colouration in life and preservative, substantial differences in advertisement call, and differentiation in mitochondrial markers (12S rRNA, 16S rRNA, cytochrome b) and a nuclear-encoded marker (Rag-1). The new species is the southernmost distributed species in the genus and was found in a swampy habitat at the bank of the Río Patay Rondos, a tributary of the Río Monzon, in rainforest at the Andean-Amazon foothills at 798 m above sea level. Aspects of species delimitation within Chimerella and related future research are briefly addressed and discussed.

The small arboreal frog Guibemantis liber (Anura: Mantellidae) has served as an example for the existence of deep conspecific lineages that differ by a substantial amount in mitochondrial DNA but are similar in morphology and bioacoustics and thus are assigned to the same nominal species. During fieldwork in northern Madagascar, we identified additional such lineages and surprisingly, observed close syntopy of two of these at various sites. In-depth study based on DNA sequences of the mitochondrial cytochrome b gene from 338 specimens of G. liber sensu lato from across its range, sequences of four nuclear-encoded markers for 154‒257 of these specimens, a phylogenomic dataset obtained by the FrogCap target capture approach, and additional mitochondrial genes for representatives of most mitochondrial lineages, as well as bioacoustic and morphological comparisons, revealed concordant differentiation among several lineages of the G. liber complex. We identify nine lineages differing by 5.3‒15.5% in cytochrome b and 2.4‒10.1% in the 16S rRNA gene, and find that several of these lack or have only limited allele sharing in the nuclear-encoded genes. Based on sympatric or parapatric occurrence without genetic admixture, combined with differences in bioacoustic and morphological characters, we scientifically name three lineages from northern Madagascar as new species: G. razoky sp. nov., G. razandry sp. nov., and G. fotsitenda sp. nov. Of these new species, G. razoky sp. nov. and G. razandry sp. nov. show widespread syntopy across northern Madagascar and differ in body size and advertisement calls. Guibemantis fotsitenda sp. nov. is sister to G. razandry sp. nov., but appears to occur at lower elevations, including in close geographic proximity on the Marojejy Massif. We also detected subtle differences in advertisement calls among various other mitochondrial lineages distributed in the Northern Central East and Southern Central East of Madagascar, but the status and nomenclatural identity of these lineages require further morphological and bioacoustic study of reliably genotyped individuals, and assignment of the three available names in the complex: Rhacophorus liber Peracca, 1893, Gephyromantis albogularis Guibé, 1947, and Gephyromantis variabilis Millot and Guibé, 1951. We discuss the identity and type material of these three nomina, designate a lectotype for Gephyromantis variabilis from Itremo, and flag the collection of new material from their type localities, Andrangoloaka and Itremo, as paramount for a comprehensive revision of the G. liber complex.

Background The Galapagos sea lion, Zalophus wollebaeki, is an endemic and endangered otariid, which is considered as a sentinel species of ecosystem dynamics in the Galapagos archipelago. Mitochondrial DNA is an important tool in phylogenetic and population genetic inference. In this work we use Illumina sequencing to complement the mitogenomic resources for Zalophus genus—the other two species employed Sanger sequencing—by a complete mitochondrial genome and a molecular clock of this species, which is not present in any case. Materials and Methods We used DNA obtained from a fresh scat sample of a Galapagos sea lion and shotgun-sequenced it on the Illumina NextSeq platform. The obtained raw reads were processed using the GetOrganelle software to filter the mitochondrial Zalophus DNA reads (∼16% survive the filtration), assemble them, and set up a molecular clock. Results From the obtained 3,511,116 raw reads, we were able to assemble a full mitogenome of a length of 16,676 bp, consisting of 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNA), and two ribosomal RNAs (rRNA). A time-calibrated phylogeny confirmed the phylogenetic position of Z. wollebaeki in a clade with Z. californianus, and Z. japonicus, and sister to Z. californianus; as well as establishing the divergence time for Z. wollebaeki 0.65 million years ago. Our study illustrates the possibility of seamlessly sequencing full mitochondrial genomes from fresh scat samples of marine mammals.

Abstract Species are the fundamental units of life and evolution. Their recognition is essential for science and society. Molecular methods have been increasingly used for the identification of animal species, despite several challenges. Here, we explore with genomic data from nine animal lineages a set of nuclear markers, namely metazoan‐level universal single‐copy orthologs (metazoan USCOs), for their use in species delimitation. Our data sets include arthropods and vertebrates. We use various data assembly strategies and use coalescent‐based species inference as well as population admixture analyses and phenetic methods. We demonstrate that metazoan USCOs distinguish well closely related morphospecies and consistently outperform classical mitochondrial DNA barcoding in discriminating closely related species in different animal taxa, as judged by comparison with morphospecies delimitations. USCOs overcome the general shortcomings of mitochondrial DNA barcodes, and due to standardization across Metazoa, also those of other approaches. They accurately assign samples not only to lower but also to higher taxonomic levels. Metazoan USCOs provide a powerful and unifying framework for DNA‐based species delimitation and taxonomy in animals and their employment could result in a more efficient use of research data and resources.

Abstract Microbial diversity positively influences community resilience of the host microbiome. However, extinction risk factors such as habitat specialization, narrow environmental tolerances, and exposure to anthropogenic disturbance may homogenize host-associated microbial communities critical for stress responses including disease defense. In a dataset containing 43 threatened and 90 non-threatened amphibian species across two biodiversity hotspots (Brazil’s Atlantic Forest and Madagascar), we found that threatened host species carried lower skin bacterial diversity, after accounting for key environmental and host factors. The consistency of our findings across continents suggests the broad scale at which low bacteriome diversity may compromise pathogen defenses in species already burdened with the threat of extinction.

The taxonomic status of the strabomantid frog species Pristimantis nebulosus (Henle, 1992), originating from the southern Cordillera Azul in central Peru, is investigated based on examination of the holotype and its comparison with freshly collected topotypic material. Following current standards, we provide a redescription of the holotype. It is in a rather poor state and exhibits certain damages and preservation artifacts, conditions that have hampered an allocation of this nominal taxon to any known living population of Pristimantis in the past. Our detailed specimen-to-specimen comparison provided morphological evidence for the conspecifity of the holotype and freshly collected topotypes. Molecular phylogenetic analysis, based on the mitochondrial 16S gene fragment places P. nebulosus in the P. conspicillatus species group, being closely related to P. bipunctatus and an undescribed candidate species from the Cordillera de Carpish. From both, P. nebulosus differs by considerable divergence in the 16S gene (p-distance 4.1–6.2%). Based on the specimens available, we provide an updated diagnosis for P. nebulosus, compare it to other species in the P. conspicillatus group and describe its advertisement call. In addition, we describe and name the closely related candidate species from the Cordillera de Carpish. It is sister to P. bipunctatus and P. nebulosus, but differs from both mainly by its tuberculate dorsal skin (versus shagreen) and divergence in the 16S gene (3.3–4.1%). We briefly discuss cryptic species diversity in the P. conspicillatus and P. danae species groups and provide justification for the description of a singleton species.

Abstract 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.

The subgenus Laurentomantis in the genus Gephyromantis contains some of the least known amphibian species of Madagascar. The six currently valid nominal species are rainforest frogs known from few individuals, hampering a full understanding of the species diversity of the clade. We assembled data on specimens collected during field surveys over the past 30 years and integrated analysis of mitochondrial and nuclear-encoded genes of 88 individuals, a comprehensive bioacoustic analysis, and morphological comparisons to delimit a minimum of nine species-level lineages in the subgenus. To clarify the identity of the species Gephyromantis malagasius, we applied a target-enrichment approach to a sample of the 110 year-old holotype of Microphryne malagasia Methuen and Hewitt, 1913 to assign this specimen to a lineage based on a mitochondrial DNA barcode. The holotype clustered unambiguously with specimens previously named G. ventrimaculatus. Consequently we propose to consider Trachymantis malagasia ventrimaculatus Angel, 1935 as a junior synonym of Gephyromantis malagasius. Due to this redefinition of G. malagasius, no scientific name is available for any of the four deep lineages of frogs previously subsumed under this name, all characterized by red color ventrally on the hindlimbs. These are here formally named as Gephyromantis fiharimpe sp. nov., G. matsilo sp. nov., G. oelkrugi sp. nov., and G. portonae sp. nov. The new species are distinguishable from each other by genetic divergences of >4% uncorrected pairwise distance in a fragment of the 16S rRNA marker and a combination of morphological and bioacoustic characters. Gephyromantis fiharimpe and G. matsilo occur, respectively, at mid-elevations and lower elevations along a wide stretch of Madagascar’s eastern rainforest band, while G. oelkrugi and G. portonae appear to be more range-restricted in parts of Madagascar’s North East and Northern Central East regions. Open taxonomic questions surround G. horridus, to which we here assign specimens from Montagne d’Ambre and the type locality Nosy Be; and G. ranjomavo, which contains genetically divergent populations from Marojejy, Tsaratanana, and Ampotsidy.

The stump-toed frogs of the Madagascar-endemic genus Stumpffia are mostly diminutive in size, but there is one group of comparatively large frogs within the genus, which we herein refer to as the Stumpffia hara species group. Each of the four known members of this species group is endemic to a single location of deciduous dry forest with exposed karstic limestone rock. Here, we report on the discovery of members of this species group on Montagne d’Ambre, a rainforest-covered extinct volcano in the North of Madagascar that has a rich Stumpffia fauna but has been thought to lack members of the S. hara species group until now. We found two members of the species group, one at the peak, and one in transitional and dry deciduous forest on the west and northern slopes of the mountain. The high-elevation species is new to science, and we here describe it as Stumpffia bishopi sp. nov. It occupies a highly distinct position in the phylogeny of these frogs, characterized by ≥ 9.8% uncorrected pairwise distance from all other nominal Stumpffia in a fragment of the mitochondrial 16S rRNA gene. It is also the smallest of the members of the S. hara species group. Our genetic results show that the low-elevation species is Stumpffia megsoni, constituting a range expansion of that species and considerably expanding our understanding of its morphology and ecology. We report its advertisement call for the first time. Our results highlight the importance of continued surveys of even well-sampled localities, with special attention on the high elevation sites of northern massifs and collection of voucher specimens, and how much there still remains to understand about even the largest of Madagascar’s small frogs.

Abstract Diverse communities of symbiotic microbes inhabit the digestive systems of vertebrates and play a crucial role in animal health, and host diet plays a major role in shaping the composition and diversity of these communities. Here, we characterized diet and gut microbiome of fire salamander populations from three Belgian forests. We carried out DNA metabarcoding on fecal samples, targeting eukaryotic 18S rRNA of potential dietary prey items, and bacterial 16S rRNA of the concomitant gut microbiome. Our results demonstrated an abundance of soft-bodied prey in the diet of fire salamanders, and a significant difference in the diet composition between males and females. This sex-dependent effect on diet was also reflected in the gut microbiome diversity, which is higher in males than female animals. Proximity to human activities was associated with increased intestinal pathogen loads. Collectively, the data supports a relationship between diet, environment and intestinal microbiome in fire salamanders, with potential health implications.

Abstract Dwelling in a variety of aquatic habitats, one of the most abundant groups of microcrustaceans, ostracodes, are widely used indicator organisms in paleolimnological research. Typically, they are identified via traditional methods using morphological features but this may be excessively time‐consuming and prone to inter‐investigator variation. DNA barcoding and metabarcoding have become important tools for specimen identification, with a great impact in the field of taxonomy, (paleo‐)ecology and evolution. Despite its potential, metabarcoding has been rarely used to analyze the community structure of ostracodes. Here, we evaluate the performance of a metabarcoding approach for ostracode identification in surface sediment samples collected from Lake Nam Co on the Tibetan Plateau. We tested six different primer pairs amplifying fragments of three different genes, and compared their success in inferring ostracode communities, coupled with morphological identification of ostracodes from the same sediment samples. In total, depending on the primer pair used, seven to nineteen ostracode amplicon sequence variants (ASVs) were identified. Via microscopy, eight morphospecies were identified. We found considerable differences between primer pairs in yielding ostracode sequences via metabarcoding. In general, the highest proportions of ostracode reads and ASVs were found with primers amplifying fragments of the 18S rRNA gene, whereas primers for COI gene had the highest in silico amplification success and highest sequencing depth per sample but only contained

Abstract Climate has a large impact on diversity and evolution of the world’s biota. The Eocene–Oligocene transition from tropical climate to cooler, drier environments was accompanied by global species turnover. A large number of Old World lacertid lizard lineages have diversified after the Eocene–Oligocene boundary. One of the most speciose reptile genera in the arid Palearctic, Acanthodactylus, contains two sub-Saharan species with unresolved phylogenetic relationship and unknown climatic preferences. We here aim to understand how and when adaptation to arid conditions occurred in Acanthodactylus and when tropical habitats where entered. Using whole mitogenomes from fresh and archival DNA and published sequences we recovered a well-supported Acanthodactylus phylogeny and underpinned the timing of diversification with environmental niche analyses of the sub-Saharan species A. guineensis and A. boueti in comparison to all arid Acanthodactylus. We found that A. guineensis represents an old lineage that splits from a basal node in the Western clade, and A. boueti is a derived lineage and probably not its sister. Their long branches characterize them—and especially A. guineensis—as lineages that may have persisted for a long time without further diversification or have undergone multiple extinctions. Environmental niche models verified the occurrence of A. guineensis and A. boueti in hot humid environments different from the other 42 arid Acanthodactylus species. While A. guineensis probably remained in tropical habitat from periods prior to the Eocene–Oligocene boundary, A. boueti entered tropical environments independently at a later period. Our results provide an important baseline for studying adaptation and the transition from humid to arid environments in Lacertidae.

The Paroedura bastardi clade, a subgroup of the Madagascan gecko genus Paroedura, currently comprises four nominal species: P. bastardi, supposedly widely distributed in southern and western Madagascar, P. ibityensis, a montane endemic, and P. tanjaka and P. neglecta, both restricted to the central west region of the island. Previous work has shown that Paroedura bastardi is a species complex with several strongly divergent mitochondrial lineages. Based on one mitochondrial and two nuclear markers, plus detailed morphological data, we undertake an integrative revision of this species complex. Using a representative sampling for seven nuclear and five mitochondrial genes we furthermore propose a phylogenetic hypothesis of relationships among the species in this clade. Our analyses reveal at least three distinct and independent evolutionary lineages currently referred to P. bastardi. Conclusive evidence for the species status of these lineages comes from multiple cases of syntopic occurrence without genetic admixture or morphological intermediates, suggesting reproductive isolation. We discuss the relevance of this line of evidence and the conditions under which concordant differentiation in unlinked loci under sympatry provides a powerful approach to species delimitation, and taxonomically implement our findings by (1) designating a lectotype for Paroedura bastardi, now restricted to the extreme South-East of Madagascar, (2) resurrecting of the binomen Paroedura guibeae Dixon & Kroll, 1974, which is applied to the species predominantly distributed in the South-West, and (3) describing a third species, Paroedura rennerae sp. nov., which has the northernmost distribution within the species complex.

Abstract 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.

Abstract In Brazil’s Atlantic Forest (AF) biodiversity conservation is of key importance since the fungal pathogen Batrachochytrium dendrobatidis (Bd) has led to the rapid loss of amphibian populations here and worldwide. The impact of Bd on amphibians is determined by the host's immune system, of which the skin microbiome is a critical component. The richness and diversity of such cutaneous bacterial communities are known to be shaped by abiotic factors which thus may indirectly modulate host susceptibility to Bd. This study aimed to contribute to understanding the environment-host–pathogen interaction determining skin bacterial communities in 819 treefrogs (Anura: Hylidae and Phyllomedusidae) from 71 species sampled across the AF. We investigated whether abiotic factors influence the bacterial community richness and structure on the amphibian skin. We further tested for an association between skin bacterial community structure and Bd co-occurrence. Our data revealed that temperature, precipitation, and elevation consistently correlate with richness and diversity of the skin microbiome and also predict Bd infection status. Surprisingly, our data suggest a weak but significant positive correlation of Bd infection intensity and bacterial richness. We highlight the prospect of future experimental studies on the impact of changing environmental conditions associated with global change on environment-host–pathogen interactions in the AF.

Lake sediments represent valuable and widely used archives for tracking environmental and biotic changes over time. Past aquatic communities are traditionally studied via morphological identification of the remains of organisms. However, molecular identification tools, such as DNA metabarcoding, have revolutionized the field of biomonitoring by enabling high-throughput and fast identification of organisms from environmental samples (e.g., sediments and soil). Sedimentary ancient DNA (sedaDNA) metabarcoding, an approach to track the biodiversity of target organisms from sediment cores, spanning thousands of years, has been successfully applied in many studies. However, researchers seldom explore how well the signals from sedaDNA data correlate with the fossil records of target organisms. This information is essential to infer past environmental conditions and community changes of bioindicators when the increasingly popular molecular identification method, metabarcoding, is desired instead of a morphological identification approach. In this study, we explore the correlations of diatom valve records across the last ∼940 years with the diatom sedaDNA metabarcoding data from the same sediment core from lake Nam Co (Tibetan Plateau). Overall, the results from valve vs. sedaDNA data revealed concordant diatom richness as well as community patterns. However, several mismatches in the diatom taxonomic composition existed between the data sets. In general, sedaDNA data harbored much higher diatom diversity, but due to the lack of reference sequences in public databases, many molecular units (amplicon sequence variants) remained unclassified to lower taxonomic levels. As our study lake, Nam Co, is characterized by brackish water and alkaline pH, some likely cases for the observed taxonomic composition mismatches may be due to a valve dissolution issue. Nevertheless, significant drivers for the diatom richness and community structure largely corresponded between data sets. Both valve and sedaDNA data demonstrated similar breakpoints for historical diatom community shifts. A particularly strong shift in the diatom community structure occurred after ∼1950 CE, which may be associated with abrupt environmental changes on the Tibetan Plateau. Altogether, our study indicates that environmentally driven signals reflected by the diatom communities are successfully recovered via microfossil as well as molecular identification methods.

Zoologists have widely acknowledged the utility of classification systems for characterising variation in anuran egg and clutch types, tadpole morphotypes, embryonic and tadpole development, amplexus types and reproductive modes. These classification systems have facilitated unambiguous communication between researchers, often working in completely different fields (e.g. taxonomy, ecology, behaviour), as well as comparisons among studies. A syntactic system, classifying anuran call guilds, is so far lacking. Based on examination of the calls of 1253 anuran species we present a simple, easy to use dichotomous key and guild system for classifying anuran advertisement calls – the call type most frequently emitted by anurans and studied by researchers. The use of only three call elements, namely clearly-defined calls, notes, and pulses, plus presence or absence of frequency modulation, allows assigning all currently known anuran advertisement calls to one of eight distinct call guilds defined here. This novel toolkit will facilitate comparative studies across the many thousand anuran species, and may help to unravel drivers of anuran call evolution, and to identify ecological patterns at the level of acoustic communities.

Abstract The Andaman day gecko (Phelsuma andamanensis) is endemic to the Andaman Archipelago, located ~ 6000 km away from Madagascar where the genus Phelsuma likely evolved. We complemented existing phylogenetic data with additional markers to show that this species consistently branches off early in the evolution of the genus Phelsuma, and this early origin led us to hypothesize that island populations within the Andaman Archipelago could have further diversified. We sampled the Andaman day gecko from all major islands in the Andamans, developed new microsatellite markers and amplified mitochondrial markers to study population diversification. We detected high allelic diversity in microsatellites, but surprisingly poor geographical structuring. This study demonstrates that the Andaman day gecko has a panmictic population (K = 1), but with weak signals for two clusters that we name ‘North’ (North Andaman, Middle Andaman, Interview, Baratang, Neil, and Long Islands) and ‘South’ (Havelock, South Andaman, Little Andaman Islands). The mitochondrial COI gene uncovered wide haplotype sharing across islands with the presence of several private haplotypes (except for the Little Andaman Island, which only had an exclusive private haplotype) signalling ongoing admixture. This species differs from two other Andaman endemic geckos for which we provide comparative mitochondrial data, where haplotypes show a distinct phylogeographic structure. Testing population history scenarios for the Andaman day gecko using Approximate Bayesian Computation (ABC) supports two possible scenarios but fails to tease apart whether admixture or divergence produced the two weak clusters. Both scenarios agree that admixture and/or divergence prior to the onset of the last glacial maximum shaped the genetic diversity and structure detected in this study. ABC supports population expansion, possibly explained by anthropogenic food subsidies via plantations of cash crops, potentially coupled with human mediated dispersal resulting in the observed panmictic population. The Andaman day gecko may thus be a rare example of an island endemic reptile benefiting from habitat modification and increased movement in its native range.

We describe a new species of arboreal microhylid frog, genus Platypelis, from northeastern Madagascar and report the expansion of distribution ranges of two other species. Platypelis laetus sp. nov. is small to medium-sized (24.3–25.6 mm snout-vent length) compared to other Platypelis, exhibits a greenish colored throat and was found in bamboo forest of the Sorata Massif. Its advertisement call consists of a single short tonal note repeated at regular intervals in long call series. Based on DNA sequences of a fragment of the mitochondrial 16S rRNA gene, the new species was placed in a clade with Platypelis olgae from the Tsaratanana Massif, and with two other, unconfirmed candidate species from the Sorata Massif and from Andravory, herein named Platypelis sp. Ca12 and Ca13. Molecular divergences among these lineages were substantial, amounting to 7.6‒8.1% uncorrected 16S p-distance to the closest nominal species, P. olgae, from which the new species is also distinguished by a lack of allele sharing in the nuclear RAG-1 gene. We also provide new records of Platypelis alticola and P. tsaratananaensis from the Sorata Massif, supported by molecular analysis. This confirms a wider distribution of these two species that previously were considered to be endemic to the Tsaratanana Massif. However, their populations in Sorata were characterized by a certain degree of genetic differentiation from Tsaratanana populations suggesting they require more detailed taxonomic assessment.

Abstract Background Host-associated microbiomes, the microorganisms occurring inside and on host surfaces, influence evolutionary, immunological, and ecological processes. Interactions between host and microbiome affect metabolism and contribute to host adaptation to changing environments. Meta-analyses of host-associated bacterial communities have the potential to elucidate global-scale patterns of microbial community structure and function. It is possible that host surface-associated (external) microbiomes respond more strongly to variations in environmental factors, whereas internal microbiomes are more tightly linked to host factors. Results Here, we use the dataset from the Earth Microbiome Project and accumulate data from 50 additional studies totaling 654 host species and over 15,000 samples to examine global-scale patterns of bacterial diversity and function. We analyze microbiomes from non-captive hosts sampled from natural habitats and find patterns with bioclimate and geophysical factors, as well as land use, host phylogeny, and trophic level/diet. Specifically, external microbiomes are best explained by variations in mean daily temperature range and precipitation seasonality. In contrast, internal microbiomes are best explained by host factors such as phylogeny/immune complexity and trophic level/diet, plus climate. Conclusions Internal microbiomes are predominantly associated with top-down effects, while climatic factors are stronger determinants of microbiomes on host external surfaces. Host immunity may act on microbiome diversity through top-down regulation analogous to predators in non-microbial ecosystems. Noting gaps in geographic and host sampling, this combined dataset represents a global baseline available for interrogation by future microbial ecology studies.

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.

In order to improve the molecular resources available for conservation management of Madagascar's threatened ichthyofauna, we elaborated a curated database of 2860 mitochondrial sequences of the mitochondrial COI, 16S and ND2 genes of Malagasy fishes, of which 1141 sequences of freshwater fishes were newly sequenced for this data set. The data set is mostly composed of COI (2015 sequences) while 16S and ND2 sequences from partly the same samples were used to match the COI sequences to reliably identified reference sequences of these genes. We observed COI uncorrected pairwise genetic distances of 5.2‒31.0% (mean 20.6%) among species belonging to different genera, and 0.0‒22.4% (mean 6.4%) for species belonging to the same genus. Deeply divergent mitochondrial lineages of uncertain attribution were found among Malagasy freshwater eleotrids and gobiids, confirming these groups are in need of taxonomic revision. DNA barcodes assigned to introduced cichlids (tilapias) included Coptodon rendallii, C. zillii, Oreochromis aureus (apparently a new country record), O. cf. mossambicus, O. niloticus, and one undetermined species of Oreochromis, with sequences of up to three species found per location. In aplocheiloid killifishes of the genus Pachypanchax, most species from northern Madagascar had only low mitochondrial divergences, three of these species (P. omalonotus, P. patriciae, and P. varatraza) were not reciprocally monophyletic, and one genetically deviant lineage was discovered in a northern locality, suggesting a need for partial taxonomic revision of this genus. While the lack of voucher specimens for most of the samples sequenced herein precludes final conclusions, our first step towards a DNA barcoding reference library of Madagascar's fishes already demonstrates the value of such a data set for improved taxonomic inventory and conservation management. We strongly suggest further exploration of Madagascar's aquatic environments, which should include detailed photographic documentation and tissue sampling of large numbers of specimens, and collection of preserved voucher specimens as well as of living fish for the buildup of ex situ assurance populations of threatened species complying with the One Plan Approach proposed by the IUCN SSC Conservation Breeding Specialist Group (CBSG).

Biological invasions are on the rise, with each invader carrying a plethora of associated microbes. These microbes play important, yet poorly understood, ecological roles that can include assisting the hosts in colonization and adaptation processes or as possible pathogens. Understanding how these communities differ in an invasion scenario may help to understand the host’s resilience and adaptability. The Asian common toad, Duttaphrynus melanostictus is an invasive amphibian, which has recently established in Madagascar and is expected to pose numerous threats to the native ecosystems. We characterized the skin and gut bacterial communities of D. melanostictus in Toamasina (Eastern Madagascar), and compared them to those of a co-occurring native frog species, Ptychadena mascareniensis, at three sites where the toad arrived in different years. Microbial composition did not vary among sites, showing that D. melanostictus keeps a stable community across its expansion but significant differences were observed between these two amphibians. Moreover, D. melanostictus had richer and more diverse communities and also harboured a high percentage of total unique taxa (skin: 80%; gut: 52%). These differences may reflect the combination of multiple host-associated factors including microhabitat selection, skin features and dietary preferences.

Predicted responses to climate change may be informed by evolutionary history. Here, the authors reconstruct the phylogeny of lacertid lizards and investigate how the evolution of this clade has varied with paleoclimates and how closely adapted extant species are to modern climates.

We describe a new species of arboreal narrow-mouthed frog, genus Platypelis, from Ambodivoangy near Maroantsetra in northeastern Madagascar. The new species, Platypelis ando sp. nov., is characterised by small body size (under 19 mm), a generally rather slender body, yellowish finger and toe tips, and a dark brown dorsal chevron. Its advertisement call is a single, moderately long, high-pitched whistle repeated at regular intervals. It is the sister species of P. ravus from Marojejy National Park, but differs from that species by considerable pairwise genetic distances (7.9%) in a fragment of the mitochondrial 16S rRNA gene, and also in bioacoustic and morphological features, especially the absence of yellow on the posterior abdomen. It is also surprisingly similar in external appearance to Cophyla occultans and C. maharipeo, to which it is not, however, closely related; these species are most easily discerned based on their calls. Platypelis ando sp. nov. joins the ranks of several species recently described from Ambodivoangy with close affiliations to species in the nearby Marojejy National Park, that are still divergent at species level. The species qualifies as Critically Endangered according to the IUCN Red List criteria, in line with other species recently assessed from this area, but we urge that more research be conducted in the nearby forests to extend the range of this and other species known only from Ambodivoangy.

We describe a new species of dwarf chameleon from the Brookesia minima species group. Brookesia tedi sp. n. occurs above 1300 m above sea level on the Marojejy massif in northeastern Madagascar. It is genetically sister to B. peyrierasi, a species occurring in lowlands around the Baie de Antongil, but is genetically strongly divergent from that species in both nuclear (c-mos) and mitochondrial (16S, ND2) genes, and morphologically in its smaller size and distinctly different hemipenis. It is the second species of the B. minima species group from Marojejy National Park, but is not known to occur in syntopy with the other species, B. karchei, due to elevational segregation.

Diatoms (Bacillariophyceae) are characterized by silicified cell walls that favor their long-term preservation in sediments, therefore widely used as bioindicators of present and past water conditions. Alongside with traditional morphological analyses, metabarcoding has become a valuable tool to study the community structures of various organisms, including diatoms. Here, we test whether the quantity of sediment sample used for DNA extraction affects the results obtained from high-throughput sequencing (metabarcoding) of the diatom rbcL region by isolating DNA from 10 g and 0.5 g (wet weight) of lake surface sediment samples. Because bioinformatics processing of metabarcoding data may affect the outcome, we also tested the consistency of the results from three different pipelines: 1) ESVs (exact sequence variants) pipeline; 2) clustering sequences at 95% sequence identity to form OTUs (operational taxonomic units; 95% OTUs); and 3) 97% OTUs pipeline. Additionally, the agreement between metabarcoding data and morphological inventories of corresponding samples were compared. Our results demonstrate highly uniform patterns between the diatom rbcL amplicons from 10 g and 0.5 g of sedimentary DNA (sedDNA) extracts (HTS 10 and HTS 0.5, respectively). Furthermore, after the careful curation of the sequencing data, metabarcoding results were highly consistent among the data sets produced by different bioinformatics pipelines. Comparing results from metabarcoding and microscopy, we identified some taxonomic mismatches: morphological analyses identified 59 diatom genera, whereas metabarcoding 49 to 54 genera. These mismatches are related to incompleteness of the sequence databases, but also to inconsistencies in diatom taxonomy in general and potential dissolution effects of diatom valves caused by high alkalinity of the investigated lake waters. Nevertheless, multivariate community analysis revealed consistent results between data sets identified by microscopy and metabarcoding – water depth and conductivity as the most significant variables in driving diatom communities in Lake Nam Co – further confirming that metabarcoding is a viable method for identifying diatom-environment relationships.

We describe a new species of the genus Gephyromantis, subgenus GephyromantisVatomantis (Mantellidae, Mantellinae), from moderately high elevation (1164–1394 m a.s.l.) on the Marojejy, Sorata, and Andravory Massifs in northern Madagascar. The new species, Gephyromantis (Vatomantis) lomorina sp. n. is highly distinct from all other species, and was immediately recognisable as an undescribed taxon upon its discovery. It is characterised by a granular, mottled black and green skin, reddish eyes, paired subgular vocal sacs of partly white colour, bulbous femoral glands present only in males and consisting of three large granules, white ventral spotting, and a unique, amplitude-modulated advertisement call consisting of a series of 24–29 rapid, quiet notes at a dominant frequency of 5124–5512 Hz. Genetically the species is also strongly distinct from its congeners, with uncorrected pairwise distances ≥10 % in a fragment of the mitochondrial 16S rRNA gene to all other nominal Gephyromantis species. A molecular phylogeny based on 16S sequences places it in a clade with species of the subgenera Laurentomantis and Vatomantis, and we assign it to the latter subgenus based on its morphological resemblance to members of Vatomantis. We discuss the biogeography of reptiles and amphibians across the massifs of northern Madagascar, the evidence for a strong link between Marojejy and Sorata, and the role of elevation in determining community sharing across this landscape.

The genus Brachycephalus is a fascinating group of miniaturized anurans from the Brazilian Atlantic Forest, comprising the conspicuous, brightly colored pumpkin-toadlets and the cryptic flea-toads. Pumpkin-toadlets are known to contain tetrodotoxins and therefore, their bright colors may perform an aposematic function. Previous studies based on a limited number of mitochondrial and nuclear-encoded markers supported the existence of two clades containing species of pumpkin-toadlet phenotype, but deep nodes remained largely unresolved or conflicting between data sets. We use new RNAseq data of 17 individuals from nine Brachycephalus species to infer their evolutionary relationships from a phylogenomic perspective. Analyses of almost 5300 nuclear-encoded ortholog protein-coding genes and full mitochondrial genomes confirmed the existence of two separate pumpkin-toadlet clades, suggesting the convergent evolution (or multiple reversals) of the bufoniform morphology, conspicuous coloration, and probably toxicity. In addition, the study of the mitochondrial gene order revealed that three species (B. hermogenesi, B. pitanga, and B. rotenbergae) display translocations of different tRNAs (NCY and CYA) from the WANCY tRNA cluster to a position between the genes ATP6 and COIII, showing a new mitochondrial gene order arrangement for vertebrates. The newly clarified phylogeny suggests that Brachycephalus has the potential to become a promising model taxon to understand the evolution of coloration, body plan and toxicity. Given that toxicity information is available for only few species of Brachycephalus, without data for any flea-toad species, we also emphasize the need for a wider screening of toxicity across species, together with more in-depth functional and ecological study of their phenotypes.

In animals with complex life cycles, selection on one life phase may constrain adaptation in another phase. Here the authors find that, during the adaptive radiation of mantellid frogs, the evolution of tadpole and adult morphologies has been uncoupled through phase-specific gene expression.

The contents of the gular glands of the male African reed frog Hyperolius cinnamomeoventris consist of a mixture of aliphatic macrolides and sesquiterpenes. While the known macrolide gephyromantolide A was readily identified, the structure of another major component was suggested to be a tetradecen-13-olide. The synthesis of the two candidate compounds (Z)-5- and (Z)-9-tetradecen-13-olide revealed the former to be the naturally occurring compound. The synthesis used ring-closing metathesis as key step. While the Hoveyda–Grubbs catalyst furnished a broad range of isomeric products, the (Z)-selective Grubbs catalyst lead to pure (Z)-products. Analysis by chiral GC revealed the natural frog compound to be (5Z,13S)-5-tetradecen-13-olide (1). This compound is also present in the secretion of other hyperoliid frogs as well as in femoral glands of male mantellid frogs such as Spinomantis aglavei. The mass spectra of the synthesized macrolides as well as their rearranged isomers obtained during ring-closing metathesis showed that it is possible to assign the location of the double bond in an unsaturated macrolide on the basis of its EI mass spectrum. The occurrence of characteristic ions can be explained by the fragmentation pathway proposed in the article. In contrast, the localization of a double bond in many aliphatic open-chain compounds like alkenes, alcohols or acetates, important structural classes of pheromones, is usually not possible from an EI mass spectrum. In the article, we present the synthesis and for the first time elucidate the structure of macrolides from the frog family Hyperoliidae.

Host-associated microbial communities can shift in structure or function when hosts change locations. Bletzet al. reciprocally transfer salamander larvae between pond and stream habitats to show that gut microbiomes shift in function, but not necessarily taxonomic identities, when hosts encounter a new environment.

In a previous study, Miralles and Vences (2013) compared seven different methods of species delimitation applied to the genus Madascincus. While focusing on methodological aspects their study involved an extensive data set of multilocus DNA sequences and of comparative morphology. On this basis they emphasized the need of revising the taxonomy of Madascincus, and revealed the existence of at least two well-supported candidate species. The present paper provides formal descriptions of these two taxa: (1) M. miafina sp. n., a species from dry areas of northern Madagascar, morphologically very similar to M. polleni (although both species are not retrieved as sister taxa), and (2) M. pyrurus sp. n., a montane species occurring >1500 m above sea level, endemic to the central highlands of Madagascar (Ibity and Itremo Massifs). Phylogenetically, M. pyrurus is the sister species of M. igneocaudatus, a taxon restricted to the dry littoral regions of the south and south-west of Madagascar in lowlands

We report the mitogenomes for five species of the Ischnocnema guentheri series, being the first described for this genus of brachycephalid frogs. We assembled mitogenomes from anchored hybrid enrichment data and recovered the 13 protein-coding genes, 22 tRNA genes, and two rRNA genes for all species. The general structure agrees with most previously sequenced neobatrachians, with two exceptions: the origin of replication of L-strand (OL) was found between tRNA-A and tRNA-N, and the position of tRNA-L and tRNA-T, which are dispersed in the control region. We provide a phylogenetic tree with outgroups, which is consistent with previous phylogenetic hypotheses.

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.

Immediate and reliable pathogen detection in large numbers of samples is essential in wildlife disease monitoring and is often realized by DNA-based techniques. Pooling samples increases processing efficiency and reduces processing costs, and has been suggested as a viable technique for quantitative PCR detection of fungal amphibian pathogens of the genus Batrachochytrium. For these fungi, this diagnostic method has been validated by in vitro set ups that provided controlled test conditions but did not take into account potential effects from amphibian skin compounds (e.g. skin secretions and Microbiota) on the approach. Some of these skin compounds are known to cause PCR inhibition in single sample applications and could lead to false negative reactions and thereby hamper pathogen detection. In this study we examined the effect of skin compounds on the pooled extraction method by swabbing individuals of seven amphibian species (one Anura and six Caudata) prior to the inoculation of the swabs with chytrid zoospores. For each species, swabs were extracted in pools of different sizes (from one to four swabs) with only one swab per pool being inoculated with zoospores. There were no significant differences regarding the ability to detect zoospores when comparing pool sizes for any species, with a tendency for more false negatives when the inoculated swab had been inoculated with a single zoospore. This study provides further in vivo evidence for the viability of the pooled extraction method for DNA-based detection of pathogens.

Miniaturised frogs form a fascinating but poorly understood amphibian ecomorph and have been exceptionally prone to taxonomic underestimation. The subfamily Cophylinae (family Microhylidae), endemic to Madagascar, has a particularly large diversity of miniaturised species which have historically been attributed to the single genus Stumpffia largely based on their small size. Recent phylogenetic work has revealed that several independent lineages of cophyline microhylids evolved towards highly miniaturised body sizes, achieving adult snout-vent lengths under 16 mm. Here, we describe five new species belonging to three clades that independently miniaturised and that are all genetically highly divergent from their relatives: (i) a new genus (Mini gen. nov.) with three new species from southern Madagascar, (ii) one species of Rhombophryne, and (iii) one species of Anodonthyla. Mini mum sp. nov. from Manombo in eastern Madagascar is one of the smallest frogs in the world, reaching an adult body size of 9.7 mm in males and 11.3 mm in females. Mini scule sp. nov. from Sainte Luce in southeastern Madagascar is slightly larger and has maxillary teeth. Mini ature sp. nov. from Andohahela in southeast Madagascar is larger than its congeners but is similar in build. Rhombophryne proportionalis sp. nov. from Tsaratanana in northern Madagascar is unique among Madagascar's miniaturised frogs in being a proportional dwarf, exhibiting far less advanced signs of paedomorphism than other species of similar size. Anodonthyla eximia sp. nov. from Ranomafana in eastern Madagascar is distinctly smaller than any of its congeners and is secondarily terrestrial, providing evidence that miniaturisation and terrestriality may be evolutionarily linked. The evolution of body size in Madagascar's microhylids has been more dynamic than previously understood, and future studies will hopefully shed light on the interplay between ecology and evolution of these remarkably diverse frogs.

The bacterial communities colonizing amphibian skin have been intensively studied due to their interactions with pathogenic chytrid fungi that are causing drastic amphibian population declines. Bacteria of the family Alcaligenaceae, and more specifically of the genus Pigmentiphaga, have been found to be associated specifically to arboreal frogs. Here we analyze their occurrence in a previously assembled global skin microbiome dataset from 205 amphibian species. Pigmentiphaga made up about 5% of the total number of reads in this global dataset. They were mostly found in unrelated arboreal frogs from Madagascar (Mantellidae and Hyperoliidae), but also occurred at low abundances on Neotropical frogs. Based on their 16S sequences, most of the sequences belong to a clade within Pigmentiphaga not assignable to any type strains of the five described species of the genus. One isolate from Madagascar clustered with Pigmentiphaga aceris (>99% sequence similarity on 16S rRNA gene level). Here, we report the full genome sequence of this bacterium which, based on 16S sequences of >97% similarity, has previously been found on human skin, floral nectar, tree sap, stream sediment and soil. Its genome consists of a single circular chromosome with 6,165,255 bp, 5,300 predicted coding sequences, 57 tRNA genes, and three rRNA operons. In comparison with other known Pigmentiphaga genomes it encodes a higher number of genes associated with environmental information processing and cellular processes. Furthermore, it has a biosynthetic gene cluster for a nonribosomal peptide syntethase, and bacteriocin biosynthetic genes can be found, but clusters for β-lactones present in other comparative Pigmentiphaga genomes are lacking.

We reconstructed nearly complete mitogenomes for three species of poison frogs, Dendrobates auratus, D. leucomelas, and D. tinctorius, from RNAseq data. We recovered the 13 protein-coding genes, 22 tRNA genes (except tRNA-Val for D. leucomelas), and two rRNA genes for all three species, plus partial sequences of the control region. The order of genes agrees with that known from a previously sequenced D. auratus, being the most commonly found for neobatrachian frogs. Based on full-sibling comparisons we estimate the probable error rate of Illumina-RNAseq reconstructed mitogenomes of up to 0.15%.

The nearly complete mitogenomes of the two species of North African Atlas geckos, Quedenfeldtia moerens and Q. trachyblepharus were assembled from anchored hybrid enrichment data and RNAseq data. Congruent assemblies were obtained for four samples included in both datasets. We recovered the 13 protein-coding genes, 22 tRNA genes, and two rRNA genes for both species, including partial control region. The order of genes agrees with that of other geckos.

Following publication of the original paper [1], it was reported that an error in the processing of Fig. 8 occurred. In the online HTML version of the article, Fig. 8 was presented as a duplication of Fig. 7. The original article [1] has been corrected.

We describe two new frog species of the genus Guibemantis Dubois, 1992 (Mantellidae) from northern Madagascar. Both species are placed in the subgenus Pandanusicola Glaw & Vences, 1994 and, like most of their relatives, appear to only inhabit the leaf axils of Pandanus plants. Guibemantis albomaculatus sp. nov. is distinguished from other closely related species by light colored dorsolateral stripes and abundant small white spots found on its flanks and limbs. Guibemantis woosteri sp. nov. also has light-colored dorsolateral stripes and small white spots, but differs by having strongly banded forelimbs and a reddish coloration around the eye orbits. Mitochondrial DNA sequences corroborate the identity of these two new species, which appear to be regional endemics in northern and north-eastern Madagascar, respectively. These descriptions bring the species count in Pandanusicola to thirteen.

Poison frogs acquire chemical defenses from the environment for protection against potential predators. These defensive chemicals are lipophilic alkaloids that are sequestered by poison frogs from dietary arthropods and stored in skin glands. Despite decades of research focusing on identifying poison frog alkaloids, we know relatively little about how environmental variation and subsequent arthropod availability impacts alkaloid loads in poison frogs. We investigated how seasonal environmental variation influences poison frog chemical profiles through changes in the diet of the Climbing Mantella (Mantella laevigata). We collected M. laevigata females on the Nosy Mangabe island reserve in Madagascar during the wet and dry seasons and tested the hypothesis that seasonal differences in rainfall is associated with changes in diet composition and skin alkaloid profiles of M. laevigata. The arthropod diet of each frog was characterized into five groups (i.e. ants, termites, mites, insect larvae, or 'other') using visual identification and cytochrome oxidase 1 DNA barcoding. We found that frog diet differed between the wet and dry seasons, where frogs had a more diverse diet in the wet season and consumed a higher percentage of ants in the dry season. To determine if seasonality was associated with variation in frog defensive chemical composition, we used gas chromatography / mass spectrometry to quantify alkaloids from individual skin samples. Although the assortment of identified alkaloids was similar across seasons, we detected significant differences in the abundance of certain alkaloids, which we hypothesize reflects seasonal variation in the diet of M. laevigata. We suggest that these variations could originate from seasonal changes in either arthropod leaf litter composition or changes in frog behavioral patterns. Although additional studies are needed to understand the consequences of long-term environmental shifts, this work suggests that alkaloid profiles are relatively robust against short-term environmental perturbations.

The number of species of frogs in the South American genus Adelophryne has increased in recent years, and it has become apparent that this group contains a substantial amount of undescribed diversity. Currently the genus contains nine described species and five candidate species. Here we describe the tenth species of the genus Adelophryne from the municipality of Igrapiúna, southern Bahia state, Brazil. The new species is characterized by its small body size, indistinct tympanum, and two phalanges in the finger IV. The species of the genus are distributed in three groups, Northern Amazonia Clade, Northern Atlantic Forest Clade and Southern Atlantic Forest Clade. The new species is phylogenetically related to species of the Northern Atlantic Forest Clade of Adelophryne and restricted to forested habitat, as typical for other Adelophryne. The species is restricted to the pristine forests in the type locality, and we consider its conservation status as Near Threatened. New morphological and molecular data of other Adelophryne species are presented, extending the distribution of Adelophryne sp. 2, Adelophryne sp. 4, Adelophryne mucronata and Adelophryne glandulata. However, a more comprehensive revision of the diversity and phylogenetic position of most Adelophryne species is needed, and the evolutionary relationships of A. meridionalis and A. pachydactyla remain unknown.

The Madagascar-endemic microhylid genus Rhombophryne consists of a range of partly or completely fossorial frog species. They lead a poorly known, secretive lifestyle, and may be more diverse than previously thought. We describe a new species from the high altitude forests of the Sorata massif in north Madagascar with unusual characteristics for this genus; R. longicrus sp. n. has long, slender legs, unlike most of its fossorial or semi-fossorial congeners. The new species is closely related to R. minuta, a much smaller frog from the Marojejy massif to the southeast of Sorata with similarly long legs. We discuss the morphology of these species relative to the rest of the genus, and argue that it suggests adaptation away from burrowing and toward a more saltatorial locomotion and an accordingly more terrestrial lifestyle. If this is the case, then these frogs represent yet more ecological diversity within the already diverse Cophylinae. We recommend an IUCN Red List status of Endangered B1ab(iii) for R. longicrus sp. n., because it is known only from a single site in a forested area of roughly 250 km2, which is not yet incorporated into any protected area.