Linking genetic variation with traits of interest has been a longstanding challenge for evolutionary biologists. Here, we explore elements of genome evolution in the hero shrew (Soricidae: Scutisorex somereni): a small, elusive mammal with the most distinctive vertebral column in the entire tree of life. We first assembled and annotated a draft genome from short-read, long-read, and chromosome-conformation-capture sequencing for S. somereni. We combined this genome with publicly available genomes from 30 other mammal species spanning millions of years of evolutionary history to search for genes potentially related to the evolution of the S. somereni axial skeleton. We first performed an orthology clustering analysis to identify gene duplications and single copy orthologs. With orthogroups, we tested for gene family expansion and contraction. The single copy orthologs that we identified were used to test for positive selection or changes in selective constraint in S. somereni compared to the other species considered. We found significant gene family size change in immune, sensory, and metabolic gene families, and identified multiple HOX genes under positive selection that might help explain the evolution of the hero shrew's extreme phenotype. Overall, our study is a critical first step that has highlighted candidate genes and gene families for further genomic exploration, underscoring the continuing challenge of understanding the genomic underpinnings of unusual phenotypes.

The evolutionary dynamics of cryptic species remain poorly understood, and their detection relies primarily on methods that quantify divergence, assuming that gene flow is absent. Here, we examine how gene flow shapes the evolutionary trajectories and species boundaries in Bornean Fanged Frogs, a renowned example of cryptic diversity where a single species has been split into 18 genetically divergent yet morphologically indistinguishable species. We employed target-capture data from over 13,000 loci to assess lineage independence of 14 nominal species distributed across Malaysian Borneo by evaluating both divergence and cohesion using network multispecies coalescent (NMSC) and MSC + migration approaches. Under the Unified Species Concept, only 6 of the 14 nominal species unambiguously form independently evolving lineages; the remainder represent cohesive metapopulation lineages nested within those six species. While mitochondrial p-distances varied substantially (up to 10%), genome-wide net divergences (Da) were surprisingly consistent, ranging from 0.5 to 2%, placing all the hypothesized "cryptic species" within the empirical gray zone of the speciation continuum. We show that diversification in the gray zone is unpredictable and heavily impacted by gene flow, leading to two key phenomena that confound species delimitation: (1) the artifactual branch effect, where admixed lineages are inferred as long, early-diverging branches, creating an illusion of deep divergence; and (2) the species-definition anomaly zone, where intraspecific pairwise sequence distances exceed interspecific ones. We further demonstrate that divergence in the gray zone varies substantially among metrics and genomic regions, reflecting heterogeneity in evolutionary dynamics across the genome. Different genomic markers also vary considerably in phylogenetic discordance and their ability to retain signatures of gene flow. Loci from anchored hybrid enrichment and ultraconserved elements produced less phylogenetic discordance and retained signals of older introgression but failed to detect recent migration, making them suitable for phylogenetic reconstruction and inferring ancient introgression, but not ongoing gene flow. Recognizing the central role of gene flow reframes our understanding of cryptic species; rather than being considered as genetically distinct units that failed to evolve morphological differentiation, they can be manifestations of continuous diversification in the gray zone. This shift in perspective offers a new and dynamic evolutionary framework for identifying and interpreting cryptic biodiversity across the Tree of Life.

Anthropogenic climate change is one of the main threats to global biodiversity, with amphibians being among the most vulnerable vertebrate groups. In this context, the IUCN currently lists 69 species of Neotropical glassfrogs as threatened. However, our knowledge of how their taxonomic and phylogenetic diversity will be distributed in future climate change scenarios remains limited. In this study, we projected the future distribution of threatened species to estimate changes in taxonomic and phylogenetic diversity across geography under two climate scenarios (SSP2-4.5 and SSP3-7.0). We also identified priority areas for conservation based on phylogenetic diversity and the Evolutionary and Global Distinctiveness Index. Our results suggest that the Andes and Amazon Basin will experience the most drastic climatic changes, with at least six species projected to experience complete loss of climatic suitability across all assessed scenarios, consequently facing a high risk of extinction. Additionally, most species exhibit a tendency to shift towards higher elevations, accompanied by a significant reduction in their geographic range. On average, these changes could result in a loss of approximately 30% of their phylogenetic diversity. The northwest Andean montane forests of Ecuador and Colombia are identified as key refuges for future taxonomic and phylogenetic diversity of glassfrogs. However, less than 36% of their projected range overlaps with protected areas, highlighting the immediate need for conservation action.

We scientifically name and describe two new species of spiny frog (Mantellidae: Spinomantis) from moderately high elevations in Ranomafana National Park, in south-east Madagascar. This region has been surveyed extensively and has a remarkably high anuran diversity with many undocumented species still being regularly discovered. We describe a previously identified candidate species, S. sp. Ca07, as Spinomantis lavabato sp. nov. and a previously undiscovered species from this region as Spinomantis lakolosy sp. nov.. The new species are morphologically cryptic within the S. bertini complex but can be recognized by a combination of subtle differences in coloration such as a lack or weak expression of sharp dorsolateral color border in both species. Bioacoustically, the new species are quite different from other species in the complex: Spinomantis lakolosy sp. nov. has the longest note duration with a powerful-sounding call that rings like a bell, which can be heard over the sound of rushing water in nearby streams; while S. lavabato sp. nov. differs by having the shortest note duration that is quiet and sounds like a trill. Furthermore, both species are substantially diverged in mitochondrial DNA, with pairwise distances in a fragment of the 16S rRNA marker of 7–9% to all other related species. Furthermore, we identify a lineage for future study in the complex from the nearby high-elevation site Andemaka within Ranomafana National Park that has an intermediate sounding advertisement call between the newly described species; although we cannot formally describe this lineage herein due to unavailability of voucher specimens, this lineage emphasizes the unexpectedly high diversity of sympatric species of the S. bertini complex in the Ranomafana area. Additionally, we identify another lineage from Ambahavala in the Anosy Chain with strong mitochondrial divergence for future study. Our findings also highlight the need for continued inventory work in high elevation rainforests of Madagascar, even in relatively well-studied regions such as Ranomafana National Park.

We describe a new species of arboreal mantellid frog of the genus Guibemantis (classified in the subgenus Guibemantis) from Madagascar. The new species, Guibemantis sioka sp. nov., is morphologically similar to G. depressiceps but differs by a high uncorrected genetic distance greater than 6% in the mitochondrial 16S rRNA gene from all other related species, and by a conspicuous dark-light marbling of the flanks, relatively large hands, and the structure of its advertisement calls from its closest relatives. Single individuals of the new species have been found at each of three high-elevation sites (1210–1650 m a.s.l.) in the Southern Central East and South East of Madagascar (Ranomafana National Park, Ivohiboro Reserve, and Andohahela National Park). If this apparent rarity was confirmed by future studies, it would stand in stark contrast to other Guibemantis which are often observed in large breeding aggregations.

The Gephyromantis moseri complex, classified in the mantellid subgenus Duboi mantis , currently contains one species of frog, G. moseri (Glaw & Vences, 2002) from the Andasibe area in the Northern Central East of Madagascar, as well as several genetically divergent populations from the North East that have been provisionally assigned to the species. We here analyse DNA sequences of one mitochondrial (16S rRNA) and one nuclear -encoded gene (RAG -1), morphology, and advertisement calls of newly collected material of this species complex from various localities in Madagascar. Based on this integrative evidence, in particular concordant nuclear gene differentiation between seven highly divergent (> 4 %) mitochondrial lineages, as well as differences in advertisement call structure, body size and head shape between some of these lineages, we conclude that the G. moseri complex contains several additional species of which four are formally named and described in this study: G. fuscus sp. nov., a rather small -sized species sister to G. moseri , occurring in two sites (Mahasoa and the western part of the Makira Reserve), G. ma kira sp. nov., a species known from only one available voucher specimen from eastern Makira, G. bemiray sp. nov. from eastern Makira, Masoala, and Ambolokopatrika; and G. ampondo sp. nov. from Marojejy in the North East. Two further lineages for which voucher specimens were not available in the framework of this study are considered unconfirmed candidate species G. sp. Ca19 and G. sp. Ca33, pending the collection of further material. The revision of the G. moseri complex adds to the diversity of Duboimantis and once more demonstrates the existence of secretive or genuinely rare restricted -range species among the Malagasy frogs whose inventory can only be completed by further fieldwork and integrative taxonomic research.

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 known only from unprotected sites and require adequate conservation management.

Snake venoms are complex mixtures of toxic proteins that hold significant medical, pharmacological and evolutionary interest. To better understand the genetic diversity underlying snake venoms, we developed VenomCap, a novel exon-capture probe set targeting toxin-coding genes from a wide range of elapid snakes, with a particular focus on the ecologically diverse and medically important subfamily Hydrophiinae. We tested the capture success of VenomCap across 24 species, representing all major elapid lineages. We included snake phylogenomic probes in the VenomCap capture set, allowing us to compare capture performance between venom and phylogenomic loci and to infer elapid phylogenetic relationships. We demonstrated VenomCap's ability to recover exons from similar to 1500 target markers, representing a total of 24 known venom gene families, which includes the dominant gene families found in elapid venoms. We find that VenomCap's capture results are robust across all elapids sampled, and especially among hydrophiines, with respect to measures of target capture success (target loci matched, sensitivity, specificity and missing data). As a cost-effective and efficient alternative to full genome sequencing, VenomCap can dramatically accelerate the sequencing and analysis of venom gene families. Overall, our tool offers a model for genomic studies on snake venom gene diversity and evolution that can be expanded for comprehensive comparisons across the other families of venomous snakes.

It remains unclear how variation in the intensity of sperm competition shapes phenotypic and molecular evolution across clades. Mice and rats in the subfamily Murinae are a rapid radiation exhibiting incredible diversity in sperm morphology and production. We combined phenotypic and genomic data to perform phylogenetic comparisons of male reproductive traits and genes across 78 murine species. We identified several shifts towards smaller relative testes mass (RTM), presumably reflecting reduced sperm competition. Several sperm traits were associated with RTM, suggesting that mating system evolution selects for convergent suites of traits related to sperm competitive ability. We predicted that sperm competition would also drive more rapid molecular divergence in species with large testes. Contrary to this, we found that many spermatogenesis genes evolved more rapidly in species with smaller RTM due to relaxed purifying selection. While some reproductive genes evolved rapidly under recurrent positive selection, relaxed selection played a greater role in underlying rapid evolution in small testes species. Our work demonstrates that postcopulatory sexual selection can impose strong purifying selection shaping the evolution of male reproduction and that broad patterns of molecular evolution may help identify genes that contribute to male fertility.

Abstract An emerging challenge in interpreting phylogenomic data sets is that concatenation and multi-species coalescent summary species tree approaches may produce conflicting results. Concatenation is problematic because it can strongly support an incorrect topology when incomplete lineage sorting (ILS) results in elevated gene-tree discordance. Conversely, summary species tree methods account for ILS to recover the correct topology, but these methods do not account for erroneous gene trees (“EGTs”) resulting from gene tree estimation error (GTEE). Third, site-based and full-likelihood methods promise to alleviate GTEE as these methods use the sequence data from alignments. To understand the impact of GTEE on species tree estimation in Hylidae tree frogs, we use an expansive data set of ∼9,000 exons, introns, and ultra-conserved elements and initially found conflict between all three types of analytical methods. We filtered EGTs using alignment metrics that could lead to GTEE (length, parsimony-informative sites, and missing data) and found that removing shorter, less informative alignments reconciled the conflict between concatenation and summary species tree methods with increased gene concordance, with the filtered topologies matching expected results from past studies. Contrarily, site-based and full-likelihood methods were mixed where one method was consistent with past studies and the other varied markedly. Critical to other studies, these results suggest a widespread conflation of ILS and GTEE, where EGTs rather than ILS are driving discordance. Finally, we apply these recommendations to an R package named PhyloConfigR, which facilitates phylogenetic software setup, summarizes alignments, and provides tools for filtering alignments and gene trees.

This repository contains genetic sequences obtained from Hybrid-Enrichment and RAD sequencing protocols of the amphibian genera Discoglossus, Lissotriton, Rana and Triturus, as well as phylogenetic trees inferred from the RADseq data. This data was generated for the manuscript "Exploring the impact of read clustering thresholds on RADseq-based systematics: an empirical example from European amphibians.", in which we tested the influence of the clustering threshold used to assemble RADseq data on downstream phylogenetic inferences. Details on the data generation and analyses can be found in the manuscript and related supplementary materials. The repository is organised as follow: -> Hybrid-Enrichment: alignments of the Hybrid-Enrichment markers in phylip/fasta format (with one subdirectory for each of the four datasets assembled: Discoglossus, Lissotriton, Rana, Triturus) --> RADseq: Assemblies and phylogenetic trees obtained from a RADseq protocol --> Assemblies: RADseq assemblies (complete loci sequences and SNP matrices, spreadsheets with assembly metrics). Divided into "iCT" (assemblies produced with 23 different intra-sample Clustering Threshold [iCT] and a fixed between-samples Clustering Threshold [bCT]) and "bCT" (assemblies produced with a fixed iCT and 23 different bCT). Both iCT and bCT are further divided in four sub-directories corresponding to the four datasets: Discoglossus, Lissotriton, Rana, Triturus) --> Trees: Phylogenetic trees inferred from the aforementionned assemblies. Divided into "iCT" (RAxML concatenation trees inferred from the assemblies with different iCTs) and "bCT" (RAxML concatenation trees and Tetrad species trees inferred from the assemblies with different bCTs).

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.

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

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.

The data available for reconstructing molecular phylogenies have become wildly disparate. Phylogenomic studies can generate data for thousands of genetic markers for dozens of species, but for hundreds of other taxa, data may be available from only a few genes. Can these two types of data be integrated to combine the advantages of both, addressing the relationships of hundreds of species with thousands of genes? Here, we show that this is possible, using data from frogs. We generated a phylogenomic data set for 138 ingroup species and 3,784 nuclear markers (ultraconserved elements [UCEs]), including new UCE data from 70 species. We also assembled a supermatrix data set, including data from 97% of frog genera (441 total), with 1-307 genes per taxon. We then produced a combined phylogenomic-supermatrix data set (a "gigamatrix") containing 441 ingroup taxa and 4,091 markers but with 86% missing data overall. Likelihood analysis of the gigamatrix yielded a generally well-supported tree among families, largely consistent with trees from the phylogenomic data alone. All terminal taxa were placed in the expected families, even though 42.5% of these taxa each had >99.5% missing data and 70.2% had >90% missing data. Our results show that missing data need not be an impediment to successfully combining very large phylogenomic and supermatrix data sets, and they open the door to new studies that simultaneously maximize sampling of genes and taxa.