Interspecific cytogenomic comparison reveals a potential chromosomal centromeric marker in Proceratophrys frog species.

Among the repetitive elements, satellite DNA (SatDNA) emerges as extensive arrays of highly similar tandemly repeated units, spanning megabases in length. Given that the satDNA PboSat01-176, previously characterized in P. boiei, prompted our interest for having a high abundance in P. boiei and potential for centromeric satellite, here, we employed various approaches, including low coverage genome sequencing, followed by computational analysis and chromosomal localization techniques in four Proceratophrys species and, investigating the genomic presence and sharing, as well as its potential for chromosomal centromere marker in Proceratophrys frog species. Our findings demonstrate that PboSat01-176 exhibits high abundance across all four Proceratophrys species, displaying distinct characteristics that establish it as the predominant repetitive DNA element in these species. The satellite DNA is prominently clustered in the peri/centromeric region of the chromosomes, particularly in the heterochromatic regions. The widespread presence of PboSat01-176 in closely related Proceratophrys species reinforces the validity of the library hypothesis for repetitive sequences. Thus, this study highlighted the utility of the satDNA family PboSat01-176 as a reliable centromeric marker in Proceratophrys species, with potential to be applied in other species of anuran amphibians. The observed sharing and maintenance of this sequence within the genus suggest possibilities for future research, particularly through expanded sampling to elucidate parameters that underlie the library hypothesis and the evolutionary dynamics of satDNA sequences.

Diversity and Molecular Evolution of Nonvisual Opsin Genes across Environmental, Developmental, and Morphological Adaptations in Frogs.

Nonvisual opsins are transmembrane proteins expressed in the eyes and other tissues of many animals. When paired with a light-sensitive chromophore, nonvisual opsins form photopigments involved in various nonvisual, light-detection functions including circadian rhythm regulation, light-seeking behaviors, and seasonal responses. Here, we investigate the molecular evolution of nonvisual opsin genes in anuran amphibians (frogs and toads). We test several evolutionary hypotheses including the predicted loss of nonvisual opsins due to nocturnal ancestry and potential functional differences in nonvisual opsins resulting from environmental light variation across diverse anuran ecologies. Using whole-eye transcriptomes of 81 species, combined with genomes, multitissue transcriptomes, and independently annotated genes from an additional 21 species, we identify which nonvisual opsins are present in anuran genomes and those that are also expressed in the eyes, compare selective constraint among genes, and test for potential adaptive evolution by comparing selection between discrete ecological classes. At the genomic level, we recovered all 18 ancestral vertebrate nonvisual opsins, indicating that anurans demonstrate the lowest documented amount of opsin gene loss among ancestrally nocturnal tetrapods. We consistently found expression of 14 nonvisual opsins in anuran eyes and detected positive selection in a subset of these genes. We also found shifts in selective constraint acting on nonvisual opsins in frogs with differing activity periods, habitats, distributions, life histories, and pupil shapes, which may reflect functional adaptation. Although many nonvisual opsins remain poorly understood, these findings provide insight into the diversity and evolution of these genes across anurans, filling an important gap in our understanding of vertebrate opsins and setting the stage for future research on their functional evolution across taxa.

Diversity and Evolution of Frog Visual Opsins: Spectral Tuning and Adaptation to Distinct Light Environments.

Visual systems adapt to different light environments through several avenues including optical changes to the eye and neurological changes in how light signals are processed and interpreted. Spectral sensitivity can evolve via changes to visual pigments housed in the retinal photoreceptors through gene duplication and loss, differential and coexpression, and sequence evolution. Frogs provide an excellent, yet understudied, system for visual evolution research due to their diversity of ecologies (including biphasic aquatic-terrestrial life cycles) that we hypothesize imposed different selective pressures leading to adaptive evolution of the visual system, notably the opsins that encode the protein component of the visual pigments responsible for the first step in visual perception. Here, we analyze the diversity and evolution of visual opsin genes from 93 new eye transcriptomes plus published data for a combined dataset spanning 122 frog species and 34 families. We find that most species express the four visual opsins previously identified in frogs but show evidence for gene loss in two lineages. Further, we present evidence of positive selection in three opsins and shifts in selective pressures associated with differences in habitat and life history, but not activity pattern. We identify substantial novel variation in the visual opsins and, using microspectrophotometry, find highly variable spectral sensitivities, expanding known ranges for all frog visual pigments. Mutations at spectral-tuning sites only partially account for this variation, suggesting that frogs have used tuning pathways that are unique among vertebrates. These results support the hypothesis of adaptive evolution in photoreceptor physiology across the frog tree of life in response to varying environmental and ecological factors and further our growing understanding of vertebrate visual evolution.

Caecilian Genomes Reveal the Molecular Basis of Adaptation and Convergent Evolution of Limblessness in Snakes and Caecilians.

We present genome sequences for the caecilians Geotrypetes seraphini (3.8 Gb) and Microcaecilia unicolor (4.7 Gb), representatives of a limbless, mostly soil-dwelling amphibian clade with reduced eyes, and unique putatively chemosensory tentacles. More than 69% of both genomes are composed of repeats, with retrotransposons being the most abundant. We identify 1,150 orthogroups that are unique to caecilians and enriched for functions in olfaction and detection of chemical signals. There are 379 orthogroups with signatures of positive selection on caecilian lineages with roles in organ development and morphogenesis, sensory perception, and immunity amongst others. We discover that caecilian genomes are missing the zone of polarizing activity regulatorysequence (ZRS) enhancer of Sonic Hedgehog which is also mutated in snakes. In vivo deletions have shown ZRS is required for limb development in mice, thus, revealing a shared molecular target implicated in the independent evolution of limblessness in snakes and caecilians.

A staging table of Balkan crested newt embryonic development to serve as a baseline in evolutionary developmental studies.

There is an increased interest in the evolution and development of newts from the genus Triturus because: (1) morphological differentiation among the nine constituent species largely corresponds to different ecological preferences, (2) hybridization between different species pairs has various evolutionary outcomes in terms of life history traits and morphology, and (3) the genus expresses a balanced lethal system that causes arrested growth and death of half of the embryos. These features provide natural experimental settings for molecular, morphological, and life-history studies. Therefore, we produce a staging table for the Balkan crested newt (T. ivanbureschi). We provide detailed descriptions of 34 embryonic stages based on easily observable and interpretable external morphological characters, to ensure reproducibility. Compared with previous staging tables for Triturus, we include a vastly increased sample size and provide high-resolution photographs in lateral, ventral, and dorsal view, complemented by videos of specific developmental periods, and accompanied by detailed explanations on how to delineate the specific stages. Our staging table will serve as a baseline in comparative studies on Triturus newts: an emerging model system in evolutionary and developmental studies.

Diversification of the terrestrial frog genus Anomaloglossus (Anura, Aromobatidae) in the Guiana Shield proceeded from highlands to lowlands, with successive loss and reacquisition of endotrophy.

Two main landscapes emerge from the Guiana Shield: the highlands to the west called the Pantepui region and the Amazonian lowlands to the east, both harbouring numerous endemic species. With 32 currently recognized species, the genus Anomaloglossus stands out among Neotropical frogs as one that diversified only within the Guiana Shield both in the highlands and the lowlands. We present a time-calibrated phylogeny obtained by using combined mitogenomic and nuclear DNA, which suggests that the genus originates from Pantepui where extant lineages started diversifying around 21 Ma, and subsequently (ca. 17 Ma) dispersed during the Miocene Climatic Optimum to the lowlands of the eastern Guiana Shield where the ability to produce endotrophic tadpoles evolved. Further diversification within the lowlands in the A. stepheni group notably led to an evolutionary reversal toward exotrophy in one species group during the late Miocene, followed by reacquisition of endotrophy during the Pleistocene. These successive shifts of reproductive mode seem to have accompanied climatic oscillations. Long dry periods might have triggered evolution of exotrophy, whereas wetter climates favoured endotrophic forms, enabling colonization of terrestrial habitats distant from water. Acquisition, loss, and reacquisition of endotrophy makes Anomaloglossus unique among frogs and may largely explain the current species diversity. The micro evolutionary processes involved in these rapid shifts of reproductive mode remain to be revealed.

The cardiorespiratory system of miniature frogs.

Anurans of the genus Brachycephalus are among the smallest vertebrates in the world, due to an extreme process of miniaturization. As an example of this process, Brachycephalus species show loss of fingers, loss of the eardrum and middle ear, bone fusions, and the presence of paravertebral plates and parotic plaque. However, no studies addressing the consequences of miniaturization on internal organs, such as the lungs and heart, are currently available. Thus, this study aimed to investigate if overall small body size has affected the cardiorespiratory system. We investigated, via dissections, individuals of four Brachycephaloidea species: Brachycephalus rotenbergae, B. pitanga, Eleutherodactylus johnstonei, and Ischnocnema parva. We observed that B. rotenbergae and B. pitanga present a reduction of the atrial septum and absence of the carotid body. On the other hand, despite being a member of the sister genus to Brachycephalus (both genera belong to the Brachycephalidae), individuals of Ischnocnema present a heart with a complete septum and carotid body; this is also observed in E. johnstonei (Eleutherodactylidae). We observed that B. rotenbergae and B. pitanga have thin skin with a one to two cell thick germ layer, and their lungs likely exhibit lower blood supply when compared to individuals of the E. johnstonei and I. parva species. Based on the observed structures, we suggest that in species of Brachycephalus, respiration is performed mainly through the skin, and their lungs may have a reduced respiratory function.

Chytridiomycosis causes high amphibian mortality prior to the completion of metamorphosis.

Amphibian populations are undergoing extensive declines globally. The fungal disease chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), is a primary contributor to these declines. The amphibian metamorphic stages (Gosner stages 42-46) are particularly vulnerable to a range of stressors, including Bd. Despite this, studies that explicitly examine host response to chytridiomycosis throughout the metamorphic stages are lacking. We aimed to determine how Bd exposure during the larval stages impacts metamorphic development and infection progression in the endangered Fleay's barred frog (Mixophyes fleayi). We exposed M. fleayi to Bd during pro-metamorphosis (Gosner stages 35-38) and monitored infection dynamics throughout metamorphosis. We took weekly morphological measurements (weight, total body length, snout-vent-length and Gosner stage) and quantified Bd load using qPCR. While we observed minimal impact of Bd infection on animal growth and development, Bd load varied throughout ontogeny, with an infection load plateau during the tadpole stages (Gosner stages 35-41) and temporary infection clearance at Gosner stage 42. Bd load increased exponentially between Gosner stages 42 and 45, with most exposed animals becoming moribund at Gosner stage 45, prior to the completion of metamorphosis. There was variability in infection outcome of exposed individuals, with a subgroup of animals (n = 5/29) apparently clearing their infection while the majority (n = 21/29) became moribund with high infection burdens. This study demonstrates the role that metamorphic restructuring plays in shaping Bd infection dynamics and raises the concern that substantial Bd-associated mortality could be overlooked in the field due to the often cryptic nature of these latter metamorphic stages. We recommend future studies that directly examine the host immune response to Bd infection throughout metamorphosis, incorporating histological and molecular methods to elucidate the mechanisms responsible for the observed trends.

Subcutaneous occurrence of encysted Acanthocephala in an anuran amphibian from Brazil.

Acanthocephalans, in their adult stage, are obligatory parasites of many types of vertebrates, including anuran amphibians. Their complex life cycle always involves an arthropod intermediate host but may include non-obligatory strategies that could improve transmission success, such as paratenic infections. In paratenic hosts, these parasites are normally found loose in the body cavity or encysted in internal organs. Here, we present the first report of acanthocephalans found encysted under the skin of an amphibian (i.e., external to its body cavity). The specimen, a clay robber frog [Haddadus binotatus (Spix, 1824)], had been collected in an Atlantic Forest area in southeastern Brazil. Upon examination of the frog, we recovered two specimens of acanthocephalan (Order Echinorhynchida) encysted under the skin of its venter. Considering the host's relatively small size and its thin ventral musculature, we believe that the acanthocephalans may have accidentally trespassed the muscular tissue while attempting to encyst in the frog's internal body wall.

Land-use preferences of the European green toad (Bufotes viridis) in the city of Vienna (Austria): the importance of open land in urban environments.

Urban areas are increasing worldwide, which poses threats to animal wildlife. However, in certain cases cities can provide refuges for endangered animals. The European green toad (Bufotes viridis) is one of such examples, which is known from cities throughout their distribution. In contrast, considerable areas of their former (primary) habitats have been degraded. The primary habitats of this species include steppes and wild river floodplains, both characterized by dynamic changes and the presence of open areas. We used available green toad observation data (2007-2020) to model the effects of land-use types on occurrence probability in the city of Vienna. Forest and densely populated areas were highly significantly negatively associated with green toad presence, while transformation/construction site areas showed a strong positive effect. Such occurrence pattern might be characteristic for early succession species, which depend on stochastic environmental disturbances (e.g., droughts and floods) in their primary habitats. We argue that urban landscape planning should appreciate the potential ecological value of open land in cities which is either in a transition phase or a permanent 'wasteland'. Ecological managing of such landscape could vastly increase urban biodiversity.

Neuroprotective effects on microglia and insights into the structure-activity relationship of an antioxidant peptide isolated from Pelophylax perezi.

Tryptophyllins constitute a heterogeneous group of peptides that are one of the first classes of peptides identified from amphibian's skin secretions. Here, we report the structural characterization and antioxidant properties of a novel tryptophyllin-like peptide, named PpT-2, isolated from the Iberian green frog Pelophylax perezi. The skin secretion of P. perezi was obtained by electrical stimulation and fractionated using RP-HPLC. De novo peptide sequencing was conducted using MALDI MS/MS. The primary structure of PpT-2 (FPWLLS-NH2 ) was confirmed by Edman degradation and subsequently investigated using in silico tools. PpT-2 shared physicochemical properties with other well-known antioxidants. To test PpT-2 for antioxidant activity in vitro, the peptide was synthesized by solid phase and assessed in the chemical-based ABTS and DPPH scavenging assays. Then, a flow cytometry experiment was conducted to assess PpT-2 antioxidant activity in oxidatively challenged murine microglial cells. As predicted by the in silico analyses, PpT-2 scavenged free radicals in vitro and suppressed the generation of reactive species in PMA-stimulated BV-2 microglia cells. We further explored possible bioactivities of PpT-2 against prostate cancer cells and bacteria, against which the peptide exerted a moderate antiproliferative effect and negligible antimicrobial activity. The biocompatibility of PpT-2 was evaluated in cytotoxicity assays and in vivo toxicity with Galleria mellonella. No toxicity was detected in cells treated with up to 512 µg/ml and in G. mellonella treated with up to 40 mg/kg PpT-2. This novel peptide, PpT-2, stands as a promising peptide with potential therapeutic and biotechnological applications, mainly for the treatment/prevention of neurodegenerative disorders.

Savanna vegetation increase triggers freshwater community shifts.

Tropical savannas are globally extensive and ecologically invaluable ecosystems. As most ecosystems however, they are subject to serious anthropogenic stress. Defaunation, and especially the loss of large mammals, is pervasive in tropical savannas and known to trigger wide-ranging ecological effects, from vegetation changes to the loss of ecosystem function. Despite what is currently known about the terrestrial consequences of defaunation, and the potential cross-ecosystem influence of large mammals, virtually no research has investigated associated effects on small adjacent water bodies. This research gap persists because (1) tropical savannas have been historically neglected, (2) the ecological value of small water bodies (e.g. ponds) is only recently being recognized, and (3) empirical baseline data are often lacking. In this paper, we compared a rare pre-change dataset with newly collected data on 213 freshwater assemblages, to investigate community structure and composition before and after a major defaunation event. Our research focused on a diverse species assemblage of amphibian larvae (i.e. tadpoles) in temporary savanna ponds. We found that pond vegetation cover increased from 16.0% to 45.6% post-defaunation, that is, a near three-fold increase. Such habitat changes seemed to have benefitted those species that use vegetation during reproduction (e.g. the leaf-folding Afrixalus spp.), while others have declined. Interestingly, we found a strong correlation between tadpole community shifts and other freshwater organisms, which indicates that habitat changes have affected a wide variety of aquatic organisms. Given that organisms inhabiting temporary aquatic habitats often have complex life histories with terrestrial adult life stages, we propose that the terrestrial effects of defaunation have indirectly led to distinct aquatic communities, in addition to direct habitat effects. These results shed new light on the potential role of large-bodied mammals in shaping adjacent ecosystems, and raise important questions concerning the functioning of temporary aquatic systems in the Anthropocene.

Les savanes tropicales sont des écosystèmes étendus à l'échelle mondiale et d'une valeur écologique inestimable, mais qui sont soumis à une pression anthropique croissante. La défaunation, en particulier la perte de grands mammifères, est omniprésente dans les savanes tropicales et pouvant déclencher des effets écologiques de grande envergure allant des changements de végétation à la perte des fonctions écosystémiques. Malgré ce qui est connu des conséquences terrestres de la défaunation, presque aucune recherche n'a étudié les effets de la défaunation sur les plans d'eau temporaires adjacents qui sont utilisés par les grands mammifères. Cette lacune persiste parce que (1) les savanes tropicales ont été historiquement négligées, (2) la valeur écologique des plans d'eau temporaires a souvent été sous-estimée et (3) les données empiriques de référence sont souvent absentes. Dans l'étude présente, nous avons utilisé des données pré/post-défaunation sur 213 assemblages aquatiques de savane, dans le but d'étudier la structure et la composition de ces communités avant et après qu'un événement majeur de défaunation ait eu lieu. Notre recherche se focalise sur des plans d'eau temporaire comptant un nombre important d'espèces de larves d'amphibiens (têtards). Nous avons détecté une multiplication moyenne par près de trois de la couverture végétale des plans d'eau après la défaunation (16,0% à 45,6%). Ces changements d'habitat semblent avoir profité aux espèces qui utilisent la végétation pour leur reproduction (par exemple, Afrixalus spp.), tandis que d'autres espèces avec d'autres préférences d'habitat ont décliné. Nous avons calculé une forte corrélation entre la composition des têtards et celle de leurs prédateurs, ce qui indique que les changements d'habitat ont affecté la plupart des membres de ces communautés aquatiques. Étant donné que les organismes d'habitats aquatiques temporaires ont pour la plupart un cycle biologique complexe figurant à la fois un stade larvaire aquatique et un stade adulte terrestre, nous proposons qu'en plus des effets directs sur l'habitat aquatique (augmentation de la végétation), les effets terrestres de la défaunation ont indirectement altéré les communautés. Ces résultats suggèrent un rôle important des grands mammifères par leur influence sur les écosystèmes aquatique adjacents et soulèvent des questions urgentes concernant la fonctionnalité des systèmes aquatiques temporaires dans l'Anthropocène.

Symbiotic skin bacteria as a source for sex-specific scents in frogs.

Amphibians are known to possess a wide variety of compounds stored in their skin glands. While significant progress has been made in understanding the chemical diversity and biological relevance of alkaloids, amines, steroids, and peptides, most aspects of the odorous secretions are completely unknown. In this study, we examined sexual variations in the volatile profile from the skin of the tree frog Boana prasina and combined culture and culture-independent methods to investigate if microorganisms might be a source of these compounds. We found that sesquiterpenes, thioethers, and methoxypyrazines are major contributors to the observed sex differences. We also observed that each sex has a distinct profile of methoxypyrazines, and that the chemical origin of these compounds can be traced to a Pseudomonas sp. strain isolated from the frog's skin. This symbiotic bacterium was present in almost all individuals examined from different sites and was maintained in captive conditions, supporting its significance as the source of methoxypyrazines in these frogs. Our results highlight the potential relevance of bacteria as a source of chemical signals in amphibians and contribute to increasing our understanding of the role that symbiotic associations have in animals.

Codon usage pattern and evolutionary forces of mitochondrial ND genes among orders of class Amphibia.

In this study, we used a bioinformatics approach to analyze the nucleotide composition and pattern of synonymous codon usage in mitochondrial ND genes in three amphibian groups, that is, orders Anura, Caudata, and Gymnophiona to identify the commonality and the differences of codon usage as no research work was reported yet. The high value of the effective number of codons revealed that the codon usage bias (CUB) was low in mitochondrial ND genes among the orders. Nucleotide composition analysis suggested that for each gene, the compositional features differed among Anura, Caudata, and Gymnophiona and the GC content was lower than AT content. Furthermore, a highly significant difference (p < .05) for GC content was found in each gene among the orders. The heat map showed contrasting patterns of codon usage among different ND genes. The regression of GC12 on GC3 suggested a narrow range of GC3 distribution and some points were located in the diagonal, indicating both mutation pressure and natural selection might influence the CUB. Moreover, the slope of the regression line was less than 0.5 in all ND genes among orders, indicating natural selection might have played the dominant role whereas mutation pressure had played a minor role in shaping CUB of ND genes across orders.

A closer look at pupil diversity and evolution in frogs and toads.

The eyes of frogs and toads (Anura) are among their most fascinating features. Although several pupil shapes have been described, the diversity, evolution, and functional role of the pupil in anurans have received little attention. Studying photographs of more than 3200 species, we surveyed pupil diversity, described their morphological variation, tested correlation with adult habits and diel activity, and discuss major evolutionary patterns considering iris anatomy and visual ecology. Our results indicate that the pupil in anurans is a highly plastic structure, with seven main pupil shapes that evolved at least 116 times during the history of the group. We found no significant correlation between pupil shape, adult habits, and diel activity, with the exception of the circular pupil and aquatic habits. The vertical pupil arose at least in the most-recent common ancestor of Anura + Caudata, and this morphology is present in most early-diverging anuran clades. Subsequently, a horizontal pupil, a very uncommon shape in vertebrates, evolved in most neobatrachian frogs. This shape evolved into most other known pupil shapes, but it persisted in a large number of species with diverse life histories, habits, and diel activity patterns, demonstrating a remarkable functional and ecological versatility.

The peptide secreted at the water to land transition in a model amphibian has antioxidant effects.

In addition to the morphophysiological changes experienced by amphibians during metamorphosis, they must also deal with a different set of environmental constraints when they shift from the water to the land. We found that Pithecopus azureus secretes a single peptide ([M + H]+ = 658.38 Da) at the developmental stage that precedes the onset of terrestrial behaviour. De novo peptide and cDNA sequencing revealed that the peptide, named PaT-2, is expressed in tandem and is a member of the tryptophyllins family. In silico studies allowed us to identify the position of reactive sites and infer possible antioxidant mechanisms of the compounds. Cell-based assays confirmed the predicted antioxidant activity in mammalian microglia and neuroblast cells. The potential neuroprotective effect of PaT-2 was further corroborated in FRET-based live cell imaging assays, where the peptide prevented lipopolysaccharide-induced ROS production and glutamate release in human microglia. In summary, PaT-2 is the first peptide expressed during the ontogeny of P. azureus, right before the metamorphosing froglet leaves the aquatic environment to occupy terrestrial habitats. The antioxidant activity of PaT-2, predicted by in silico analyses and confirmed by cell-based assays, might be relevant for the protection of the skin of P. azureus adults against increased O2 levels and UV exposure on land compared with aquatic environments.

Phylogenomic inference of species and subspecies diversity in the Palearctic salamander genus Salamandra.

The salamander genus Salamandra is widespread across Europe, North Africa, and the Near East and is renowned for its conspicuous and polymorphic colouration and diversity of reproductive modes. The phylogenetic relationships within the genus, and especially in the highly polymorphic species S. salamandra, have been very challenging to elucidate, leaving its real evolutionary history and classification at species and subspecies levels a topic of debate and contention. However, the distribution of diversity and species delimitation within the genus are critically important for identifying evolutionarily significant units for conservation and management, especially in light of threats posed by the pathogenic chytrid fungus Batrachochytrium salamandrivorans that is causing massive declines of S. salamandra populations in central Europe. Here, we conducted a phylogenomic analysis from across the taxonomic and geographic breadth of the genus Salamandra in its entire range. Bayesian, maximum likelihood and network-based phylogenetic analyses of up to 4905 ddRADseq-loci (294,300 nucleotides of sequence) supported the distinctiveness of all currently recognised species (Salamandra algira, S. atra, S. corsica, S. infraimmaculata, S. lanzai, and S. salamandra), and all five species for which we have multiple exemplars were confirmed as monophyletic. Within S. salamandra, two main clades can be distinguished: one clade with the Apenninic subspecies S. s. gigliolii nested within the Iberian S. s. bernardezi/fastuosa; and a second clade comprising all other Iberian, Central and East European subspecies. Our analyses revealed that some of the currently recognized subspecies of S. salamandra are paraphyletic and may require taxonomic revision, with the Central- and Eastern-European subspecies all being poorly differentiated at the analysed genomic markers. Salamandra s. longirostris - sometimes considered a separate species - was nested within S. salamandra, consistent with its subspecies status. The relationships identified within and between Salamandra species provide valuable context for future systematic and biogeographic studies, and help elucidate critical evolutionary units for conservation and taxonomy.

Phylotranscriptomic evidence for pervasive ancient hybridization among Old World salamanders.

Hybridization can leave genealogical signatures in an organism's genome, originating from the parental lineages and persisting over time. This potentially confounds phylogenetic inference methods that aim to represent evolution as a strictly bifurcating tree. We apply a phylotranscriptomic approach to study the evolutionary history of, and test for inter-lineage introgression in the Salamandridae, a Holarctic salamanders group of interest in studies of toxicity and aposematism, courtship behavior, and molecular evolution. Although the relationships between the 21 currently recognized salamandrid genera have been the subject of numerous molecular phylogenetic studies, some branches have remained controversial and sometimes affected by discordances between mitochondrial vs. nuclear trees. To resolve the phylogeny of this family, and understand the source of mito-nuclear discordance, we generated new transcriptomic (RNAseq) data for 20 salamandrids and used these along with published data, including 28 mitochondrial genomes, to obtain a comprehensive nuclear and mitochondrial perspective on salamandrid evolution. Our final phylotranscriptomic data set included 5455 gene alignments for 40 species representing 17 of the 21 salamandrid genera. Using concatenation and species-tree phylogenetic methods, we find (1) Salamandrina sister to the clade of the "True Salamanders" (consisting of Chioglossa, Mertensiella, Lyciasalamandra, and Salamandra), (2) Ichthyosaura sister to the Near Eastern genera Neurergus and Ommatotriton, (3) Triturus sister to Lissotriton, and (4) Cynops paraphyletic with respect to Paramesotriton and Pachytriton. Combining introgression tests and phylogenetic networks, we find evidence for introgression among taxa within the clades of "Modern Asian Newts" and "Modern European Newts". However, we could not unambiguously identify the number, position, and direction of introgressive events. Combining evidence from nuclear gene analysis with the observed mito-nuclear phylogenetic discordances, we hypothesize a scenario with hybridization and mitochondrial capture among ancestral lineages of (1) Lissotriton into Ichthyosaura and (2) Triturus into Calotriton, plus introgression of nuclear genes from Triturus into Lissotriton. Furthermore, both mitochondrial capture and nuclear introgression may have occurred among lineages assigned to Cynops. More comprehensive genomic data will, in the future, allow testing this against alternative scenarios involving hybridization with other, extinct lineages of newts.

Phylogenomics and evolutionary history of Oreobates (Anura: Craugastoridae) Neotropical frogs along elevational gradients.

Mountain ranges offer opportunities for understanding how species evolved and diversified across different environmental conditions. Neotropical frogs of the genus Oreobates (Anura: Craugastoridae) are adapted to highland and lowland habitats along the Andes, but many aspects of their evolution remain unknown. We studied their evolutionary history using ~18,000 exons enriched by targeted sequence-capture. Since capture success was very variable across samples, we evaluated to what degree differing data filtering produced robust inferences. The inferred evolutionary framework evidenced phylogenetic discordances among lowland species that can be explained by taxonomic misidentification or admixture of ancestral lineages. Highland species showed smaller effective populations than lowland frogs, probably due to greater habitat fragmentation in montane environments. Stronger genetic drift likely decreased the power of purifying selection and led to an increased proportion of nonsynonymous mutations in highland populations that could play an important role in their adaptation. Overall, our work sheds light on the evolutionary history and diversification of this group of Neotropical frogs along elevational gradients in the Andes as well as on their patterns of intraspecific diversity.

Parallel diversification of the African tree toad genus Nectophryne (Bufonidae).

African amphibian diversity remains underestimated with many cryptic lineages awaiting formal description. An important hotspot of amphibian diversification is the Guineo-Congolian rainforest in Central Africa, its richness attributable to present day and ancestral range fragmentation through geological barriers, habitat expansion and contraction, and the presence of steep ecological gradients. The charismatic Nectophryne tree toads present an interesting case study for diversification in this region. The two formally described species comprising this genus show nearly identical geographic distributions extending across most of the Guineo-Congolian rainforest, but show little morphological disparity. Both species harbour extensive genetic diversity warranting taxonomic revisions, and interestingly, when comparing the subclades within each, the two species show remarkably parallel diversification histories, both in terms of timing of phylogenetic splits and their geographic distributions. This indicates that common processes may have shaped the evolutionary history of these lineages.