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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.

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.

Restriction site-Associated DNA sequencing (RADseq) has great potential for genome-wide systematics studies of non-model organisms. However, accurately assembling RADseq reads into orthologous loci remains a major challenge in the absence of a reference genome. Traditional assembly pipelines cluster putative orthologous sequences based on a user-defined clustering threshold. Because improper clustering of orthologs is expected to affect results in downstream analyses, it is crucial to design pipelines for empirically optimizing the clustering threshold. While this issue has been largely discussed from a population genomics perspective, it remains understudied in the context of phylogenomics and coalescent species delimitation. To address this issue, we generated RADseq assemblies of representatives of the amphibian generaDiscoglossus, Rana, LissotritonandTriturususing a wide range of clustering thresholds. Particularly, we studied the effects of the intra-sample Clustering Threshold (iCT) and between-sample Clustering Threshold (bCT) separately, as both are expected to differ in multi-species data sets. The obtained assemblies were used for downstream inference of concatenation-based phylogenies, and multi-species coalescent species trees and species delimitation. The results were evaluated in the light of a reference genome-wide phylogeny calculated from newly generated Hybrid-Enrichment markers, as well as extensive background knowledge on the species’ systematics. Overall, our analyses show that the inferred topologies and their resolution are resilient to changes of the iCT and bCT, regardless of the analytical method employed. Except for some extreme clustering thresholds, all assemblies yielded identical, well-supported inter-species relationships that were mostly congruent with those inferred from the reference Hybrid-Enrichment data set. Similarly, coalescent species delimitation was consistent among similarity threshold values. However, we identified a strong effect of the bCT on the branch lengths of concatenation and species trees, with higher bCTs yielding trees with shorter branches, which might be a pitfall for downstream inferences of evolutionary rates. Our results suggest that the choice of assembly parameters for RADseq data in the context of shallow phylogenomics might be less challenging than previously thought. Finally, we propose a pipeline for empirical optimization of the iCT and bCT, implemented in optiRADCT, a series of scripts readily usable for future RADseq studies.

We describe a fantastic new species of forest frog (Mantellidae: Gephyromantis: subgenus: Laurentomantis) from moderately high elevations in the vicinity of Andasibe, Madagascar. This region has been surveyed extensively and has a remarkably high anuran diversity with many undocumented species still being discovered. Surprisingly, by exploring areas around Andasibe that lacked biodiversity surveys, we discovered a spectacular and clearly morphologically distinct species, previously unknown to science, Gephyromantis marokorokosp. nov., documented for the first time in 2015. The new species is well characterised by a very rugose and granular dorsum, dark brown skin with bright red mottling, sparse light orange to white spots on the ventre, vibrant red eyes and femoral glands present only in males that consist of eight medium-sized granules. Bioacoustically, the new species has a quiet advertisement call that differs from related species by having a moderate call duration, 2–4 strongly pulsed notes and a slow note repetition rate. Furthermore, it has substantial differentiation in mitochondrial DNA, with pairwise distances of 7–9% to all other related species in sequences of the mitochondrial 16S rRNA marker. Additional evidence is given through a combined four mitochondrial markers and four nuclear exons concatenated species tree, strongly supporting G. striatus as the sister species of the new species in both analyses. The discovery of this new species highlights the need for continued inventory work in high elevation rainforests of Madagascar, even in relatively well-studied regions.

Despite the prevalence of high-throughput sequencing in phylogenetics, many relationships remain difficult to resolve because of conflicting signal among genomic regions. Selection of different types of molecular markers from different genomic regions is required to overcome these challenges. For evolutionary studies in frogs, we introduce the publicly available FrogCap suite of genomic resources, which is a large collection of ~15,000 markers that unifies previous genetic sequencing efforts. FrogCap is designed to be modular, such that subsets of markers and SNPs can be selected based on the desired phylogenetic scale. FrogCap uses a variety of marker types that include exons and introns, ultraconserved elements, and previously sequenced Sanger markers, which span up to 10,000 bp in alignment lengths; in addition, we demonstrate potential for SNP-based analyses. We tested FrogCap using 121 samples distributed across five phylogenetic scales, comparing probes designed using a consensus- or exemplar genome-based approach. Using the consensus design is more resilient to issues with sensitivity, specificity, and missing data than picking an exemplar genome sequence. We also tested the impact of different bait kit sizes (20,020 vs. 40,040) on depth of coverage and found triple the depth for the 20,020 bait kit. We observed sequence capture success (i.e., missing data, sequenced markers/bases, marker length, and informative sites) across phylogenetic scales. The incorporation of different marker types is effective for deep phylogenetic relationships and shallow population genetics studies. Having demonstrated FrogCap's utility and modularity, we conclude that these new resources are efficacious for high-throughput sequencing projects across variable timescales.

Species are the fundamental units of life and their recognition is essential for science and society. DNA barcoding, the use of a single and often mitochondrial gene, has been increasingly employed as a universal approach for the identification of animal species. However, this approach faces several challenges. Here, we demonstrate with empirical data from a number of metazoan animal lineages that multiple nuclear-encoded markers, so called universal single-copy orthologs (USCOs) performs much better than the single barcode gene to discriminate closely related species. Overcoming the general shortcomings of mitochondrial DNA barcodes, USCOs also accurately assign samples to higher taxonomic levels. These loci thus provide a powerful and unifying framework for species delimitation which considerably improves the DNA-based inference of animal species.

Genome-scale data have greatly facilitated the resolution of recalcitrant nodes that Sanger-based datasets have been unable to resolve. However, phylogenomic studies continue to use traditional methods such as bootstrapping to estimate branch support; and high bootstrap values are still interpreted as providing strong support for the correct topology. Furthermore, relatively little attention has been given to assessing discordances between gene and species trees, and the underlying processes that produce phylogenetic conflict. We generated novel genomic datasets to characterize and determine the causes of discordance in Old World treefrogs (Family: Rhacophoridae)—a group that is fraught with conflicting and poorly supported topologies among major clades. Additionally, a suite of data filtering strategies and analytical methods were applied to assess their impact on phylogenetic inference. We showed that incomplete lineage sorting was detected at all nodes that exhibited high levels of discordance. Those nodes were also associated with extremely short internal branches. We also clearly demonstrate that bootstrap values do not reflect uncertainty or confidence for the correct topology and, hence, should not be used as a measure of branch support in phylogenomic datasets. Overall, we showed that phylogenetic discordances in Old World treefrogs resulted from incomplete lineage sorting and that species tree inference can be improved using a multi-faceted, total-evidence approach, which uses the most amount of data and considers results from different analytical methods and datasets.

In cryptic amphibian complexes, there is a growing trend to equate high levels of genetic structure with hidden cryptic species diversity. Typically, phylogenetic structure and distance-based approaches are used to demonstrate the distinctness of clades and justify the recognition of new cryptic species. However, this approach does not account for gene flow, spatial, and environmental processes that can obfuscate phylogenetic inference and bias species delimitation. As a case study, we sequenced genome-wide exons and introns to evince the processes that underlie the diversification of Philippine Puddle Frogs—a group that is widespread, phenotypically conserved, and exhibits high levels of geographically based genetic structure. We showed that widely adopted tree- and distance-based approaches inferred up to 20 species, compared to genomic analyses that inferred an optimal number of five distinct genetic groups. Using a suite of clustering, admixture, and phylogenetic network analyses, we demonstrate extensive admixture among the five groups and elucidate two specific ways in which gene flow can cause overestimations of species diversity: 1) admixed populations can be inferred as distinct lineages characterized by long branches in phylograms; and 2) admixed lineages can appear to be genetically divergent, even from their parental populations when simple measures of genetic distance are used. We demonstrate that the relationship between mitochondrial and genome-wide nuclear $p$-distances is decoupled in admixed clades, leading to erroneous estimates of genetic distances and, consequently, species diversity. Additionally, genetic distance was also biased by spatial and environmental processes. Overall, we showed that high levels of genetic diversity in Philippine Puddle Frogs predominantly comprise metapopulation lineages that arose through complex patterns of admixture, isolation-by-distance, and isolation-by-environment as opposed to species divergence. Our findings suggest that speciation may not be the major process underlying the high levels of hidden diversity observed in many taxonomic groups and that widely adopted tree- and distance-based methods overestimate species diversity in the presence of gene flow. [Cryptic species; gene flow; introgression; isolation-by-distance; isolation-by-environment; phylogenetic network; species delimitation.]

Adaptive radiation is a key topic at the intersection of ecology and evolutionary biology. Yet the definition and identification of adaptive radiation both remain contentious. Here, we introduce a new approach for identifying adaptive radiations that combines key aspects of two widely used definitions. Our approach compares evolutionary rates in morphology, performance, and diversification between the candidate radiation and other clades. We then apply this approach to a putative adaptive radiation of frogs from Madagascar (Mantellidae). We present new data on morphology and performance from mantellid frogs, then compare rates of diversification and multivariate evolution of size, shape, and performance between mantellids and other frogs. We find that mantellids potentially pass our test for accelerated rates of evolution for shape, but not for size, performance, or diversification. Our results demonstrate that clades can have accelerated phenotypic evolution without rapid diversification (dubbed "adaptive non-radiation"). We also highlight general issues in testing for adaptive radiation, including taxon sampling and the problem of including another adaptive radiation among the comparison clades. Finally, we suggest that similar tests should be conducted on other putative adaptive radiations on Madagascar, comparing their evolutionary rates to those of related clades outside Madagascar. Based on our results, we speculate that older Madagascar clades may show evolutionary patterns more similar to those on a continent than an island.

Made available in DSpace on 2020-12-12T01:22:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-17 The subgenus Mantidactylus is a group of frogs endemic to Madagascar, including the largest anuran species on the island. Although these frogs are common and widely distributed, their taxonomy remains unclear. Two species are currently recognised, M. grandidieri and M. guttulatus, with another available name, Rana pigra, considered to be a synonym of M. grandidieri. However, molecular studies have suggested the presence of several cryptic species within the group. Additionally, due to the lack of prominent morphological features, allocating the available names to evolutionary lineages has proven challenging. In the present study, we take a first step towards solving these problems by using fragments of the 16S mitochondrial gene and RAG1 nuclear gene from all over the range of the subgenus to describe its genetic diversity. We also use a newly designed target enrichment laboratory protocol to sequence three mitochondrial fragments from five name-holding museum specimens (as old as 120 years) in order to determine to which lineages the existing names should be applied. The study of the 16S mitochondrial gene revealed 7 geographically separated lineages, distinct enough to be considered candidate species. Out of the five museum specimens analysed, four successfully yielded DNA sequences and could be attributed to one of the aforementioned lineages. Therefore, the name Mantidactylus grandidieri should be applied to the populations from North-Eastern Madagascar, while M. guttulatus refers to populations from inland localities along the Eastern coast of the island. On the other hand, the holotype of Rana pigra did not yield enough genetic material to allow definitive identification. While our data were not sufficient to assess the status of the four lineages distributed along the Eastern coast, the populations from North-Western Madagascar were highly distinct on both the mitochondrial and nuclear markers. We thus describe them as a new species, M. radaka sp. nov. http://www.zoobank.org/urn:lsid:zoobank.org:pub:1EDDAF0D-FE37-490A-B09E-E136A0C5CB35. Zoological Institute Braunschweig University of Technology Sektion Herpetologie Zoologische Staatssammlung München (ZSM-SNSB) Institut de Systématique Biodiversité (ISYEB) Sorbonne Université Programa de Pós-Graduação em Biologia Animal Departamento de Biologia Animal Laboratório de Herpetologia Universidade Federal Rural do Rio de Janeiro Faculty of Mathematics and Natural Sciences Institute for Biochemistry and Biology University of Potsdam Department of Natural History University Museum of Bergen Depto de Zoologia Instituto de Biologia Universidade Estadual Paulista - UNESP Department of Life Sciences The Natural History Museum, Cromwell Road Museo Regionale di Scienze Naturali Research Centre in Biodiversity Genetics and Evolution Universidade do Porto Biodiversity Institute and Department of Ecology and Evolutionary Biology University of Kansas Mention Zoologie et Biodiversité Animale Faculté des Sciences Université d’Antananarivo Depto de Zoologia Instituto de Biologia Universidade Estadual Paulista - UNESP

Basic ecological data are missing from the majority of Madagascar’s snakes. We here report seven dietary records from two species of the genus Compsophis and one record from Alluaudina bellyi. Amon...

We investigate the molecular phylogeny of Boophis, a group of arboreal frogs from the Malagasy-Comoroan family Mantellidae. Based on newly acquired DNA sequences of five mitochondrial and five nuclear markers (7444 base pairs), we infer a phylogeny of Boophis with complete species-level taxon sampling. We reconstruct the phylogeny using Bayesian inference and maximum likelihood and estimate divergence dates for the major clades of the genus. The phylogenetic analyses together support the monophyly of the two subgenera (Sahona and Boophis), and provide strong support for most previously identified species groups, except that the B. ulftunni group is nested within the B. majori group. We also erect a new species group related to the B. mandraka group, the B. blommersae group, composed of small-sized, brown stream-breeding frogs previously included within the B. majori group. Finally, we use the resulting phylogeny to illustrate striking examples of repeated evolution of coloration and ventral transparency and address the biogeographic history and broad pattern of species diversification in the genus. Ancestral area reconstructions provide evidence that Boophis diversified within the Eastern highland forests of Madagascar, and we suggest that adaptation to these highland areas was important in their diversification.

As molecular methods continue to elucidate genetic structure at increasingly finer resolutions, delimiting species in the grey zone of the speciation continuum is becoming more relevant in biodiversity research, especially in under-studied biodiversity hotspots such as Southeast Asia where new species are being described at an unprecedented rate. Obvious species at both ends of the speciation continuum have mostly been described and attention is now turning towards the “grey zone:” an intermediate stage in which species criteria are in conflict and boundaries between populations and species are less clear. This study demonstrates that widely-used criteria (phylogenetic placement, genetic divergence, phylogeny- and distance-based species delimitation methods) can overestimate species diversity/boundaries when introgression is present. However, a comprehensive species delimitation framework that considers spatial and genetic population structure, introgression, and the use of species delimitation methods based on parameter estimation, can provide a more accurate characterization of species boundaries in this grey zone. We applied this approach to a group of Southeast Asian frogs from the Pulchrana picturata Complex that exhibits continuous morphological variation and high genetic divergences. Results showed that introgression was rampant among Bornean populations, which led to phylogenetic discordance and an overestimation of species. We suspect that our results do not form an isolated case; and that introgression among cryptic populations, occurring continuously across a wide geographic area (e.g., the topographically complex island of Borneo, and Earth’s major continents), may be more common than previously thought.

Despite the increasing use of high-throughput sequencing in phylogenetics, many phylogenetic relationships remain difficult to resolve because of conflict between gene trees and species trees. Selection of different types of markers (i.e. protein-coding exons, non-coding introns, ultra-conserved elements) is becoming important to alleviate these phylogenomic challenges. For evolutionary studies in frogs, we introduce the new publicly available FrogCap suite of genomic resources, which is a large and flexible collection of probes corresponding to ∼15,000 markers that unifies previous frog sequencing work. FrogCap is designed to be modular, such that subsets of markers can be selected based on the phylogenetic scale of the intended study. FrogCap uses a variety of molecular marker types that include newly obtained exons and introns, previously sequenced UCEs, and Sanger-sequencing markers, which span a range of alignment lengths (100–12,000 base pairs). We tested three probe sets from FrogCap using 105 samples across five phylogenetic scales, comparing probes designed using a consensus- or genome-based approach. We also tested the effects of using different bait kit sizes on depth of coverage and missing data. We found that larger bait kits did not result in lowered depth of coverage or increased missing data. We also found that sensitivity, specificity, and missing data are not related to genetic distance in the consensus-based probe design, suggesting that this approach has greater success and overcomes a major hurdle in probe design. We observed sequence capture success (in terms of missing data, quantity of sequence data, recovered marker length, and number of informative sites) and compared them at all phylogenetic scales. The incorporation of different molecular marker types allowed recovery of the variation required for resolving difficult phylogenetic relationships and for performing population genetic studies. Altogether, FrogCap is a valuable and adaptable resource for performing high-throughput sequencing projects across variable timescales.

Numerous types of genomic markers have been used to resolve recalcitrant nodes, yet their relative performance and congruence have rarely been compared directly. Using target-capture sequencing, we obtained more than 12,000 highly informative exons and introns, including ∼600 UCEs to address long-standing systematic problems in Southeast Asian Golden-backed frogs of the genus complex Hylarana. To reduce gene tree estimation errors, we filtered the data using different thresholds of taxon completeness and parsimony informative sites (PIS) in addition to using the best-fit models of DNA evolution to estimate individual single-locus gene trees. We then estimated species trees using concatenation (IQ-TREE), summary coalescent (ASTRAL), and distance-based methods (ASTRID). Topological incongruence among these methods and variation in nodal support were examined in detail using a suite of different measures including quartet frequencies, bootstrap, local posterior probabilities, gene concordance factors, and quartet scores. Results showed that high levels of incongruence were present along the backbone of the phylogeny, specifically surrounding short internodes. We also demonstrated that filtering data by PIS was more efficacious at improving congruence compared to filtering by missing data, and that exons were more sensitive to data filtering than introns and UCEs. Despite utilizing more than 6.9 million characters and 2.7 million PIS, analyses failed to converge on a single concordant topology. Instead, exons, introns, and UCEs produced genuinely strongly-supported yet conflicting phylogenetic signals, which affected our phylogeny estimates at different scales/levels—indicating a general, potentially alarming challenge for phylogenomics inference employing many of todays massive datasets. Additionally, bootstrap values were consistently high despite low levels of congruence and high proportions of gene trees that support conflicting topologies, indicating that traditional bootstraps are likely poor measures of congruence or branch support in large phylogenomic datasets, especially during instances of rapid diversification. Although low bootstrap values do ostensibly reflect low heuristic support, we recommend that high bootstrap support obtained from large genomic datasets be interpreted with caution. Additional complimentary measures such quartet frequencies, gene concordance factors, quartet scores, and posterior probabilities can be useful to provide a more robust and accurate representation of bipartition certainty and ultimately, evolutionary history of incompletely resolved or poorly-understood clades.

Tissue sample databases housed in biodiversity archives represent a vast trove of genetic resources, and these tissues are frequently destructively subsampled and provided to researchers for DNA extractions and subsequent sequencing. While obtaining a sufficient quantity of DNA for downstream applications is vital for these researchers, it is also important to preserve tissue resources for future use given that the original material is destructively and consumptively sampled with each use. It is therefore necessary to develop standardized tissue subsampling and loaning procedures to ensure that tissues are being used efficiently. In this study, we specifically focus on the efficiency of DNA extraction methods by using anuran liver and muscle tissues maintained at a biodiversity archive. We conducted a series of experiments to test whether current practices involving coarse visual assessments of tissue size are effective, how tissue mass correlates with DNA yield and concentration, and whether the amount of DNA recovered is correlated with sample age. We found that tissue samples between 2 mg and 8 mg resulted in the most efficient extractions, with tissues at the lower end of this range providing more DNA per unit mass and tissues at the higher end of this range providing more total DNA. Additionally, we found no correlation between tissue age and DNA yield. Because we find that even very small tissue subsamples tend to yield far more DNA than is required by researchers for modern sequencing applications (including whole genome shotgun sequencing), we recommend that biodiversity archives consider dramatically improving sustainable use of their archived material by providing researchers with set quantities of extracted DNA rather than with the subsampled tissues themselves.

Natural history and behavioral information for species of the genus Pristimantis is scarce. We conducted an extensive study of Pristimantis eremitus, a vulnerable species for which there is scant biological information. We monitored 17 individuals for movements and intraspecific interactions for 13 mo. We describe the species’ variation in dorsal color and pattern, which we used to uniquely identify individuals during the study. We also present results on the habitat utilization and home range of this species, providing evidence that this species shows a preference toward the bromeliad microhabitat when compared with other microhabitat types. We also describe the advertisement call and associated behaviors of the species. We update the known distribution of the species. Our data provide a baseline for future researchers to survey and identify individuals of P. eremitus, and which could be applied comparatively to other species where little of their natural history is known. We also show how an in...

Using FrogCap, a recently-developed sequence-capture protocol, we obtained >12,000 highly informative exons, introns, and ultraconserved elements (UCEs), which we used to illustrate variation in evolutionary histories of these classes of markers, and to resolve long-standing systematic problems in Southeast Asian Golden-backed frogs of the genus-complex Hylarana. We also performed a comprehensive suite of analyses to assess the relative performance of different genetic markers, data filtering strategies, tree inference methods, and different measures of branch support. To reduce gene tree estimation error, we filtered the data using different thresholds of taxon completeness (missing data) and parsimony informative sites (PIS). We then estimated species trees using concatenated datasets and Maximum Likelihood (IQ-TREE) in addition to summary (ASTRAL-III), distance-based (ASTRID), and site-based (SVDQuartets) multispecies coalescent methods. Topological congruence and branch support were examined using traditional bootstrap, local posterior probabilities, gene concordance factors, quartet frequencies, and quartet scores. Our results did not yield a single concordant topology. Instead, introns, exons, and UCEs clearly possessed different phylogenetic signals, resulting in conflicting, yet strongly-supported phylogenetic estimates. However, a combined analysis comprising the most informative introns, exons, and UCEs converged on a similar topology across all analyses, with the exception of SVDQuartets. Bootstrap values were consistently high despite high levels of incongruence and high proportions of gene trees supporting conflicting topologies. Although low bootstrap values did indicate low heuristic support, high bootstrap support did not necessarily reflect congruence or support for the correct topology. This study reiterates findings of some previous studies, which demonstrated that traditional bootstrap values can produce positively misleading measures of support in large phylogenomic datasets. We also showed a remarkably strong positive relationship between branch length and topological congruence across all datasets, implying that very short internodes remain a challenge to resolve, even with orders of magnitude more data than ever before. Overall, our results demonstrate that more data from unfiltered or combined datasets produced superior results. Although data filtering reduced gene tree incongruence, decreased amounts of data also biased phylogenetic estimation. A point of diminishing returns was evident, at which higher congruence (from more stringent filtering) at the expense of amount of data led to topological error as assessed by comparison to more complete datasets across different genomic markers. Additionally, we showed that applying a parameter-rich model to a partitioned analysis of concatenated data produces better results compared to unpartitioned, or even partitioned analysis using model selection. Despite some lingering uncertainties, a combined analysis of our genomic data and sequences supplemented from GenBank (on the basis of a few gene regions) revealed highly supported novel systematic arrangements. Based on these new findings, we transfer Amnirana nicobariensis into the genus Indosylvirana; and I. milleti and Hylarana celebensis to the genus Papurana. We also provisionally place H. attigua in the genus Papurana pending verification from positively identified (voucher substantiated) samples.

Reserva Las Gralarias is one of the best-studied localities of the Ecuadorian Andes in terms of its batrachofauna. However, as expected in any community, some species are rare and, therefore, their discovery and description are problematic. Herein, based on a single specimen, we describe Pristimantis gralarias sp nov. Even though we are aware of the problems associated to singleton species (i.e., unknown intraspecific variation, limited ecological information), our efforts to finding additional specimens have been unsuccessful. Thus, given the importance of species descriptions in threatened areas (e.g. Andes), and that the new taxon is supported by both morphological and genetic data, we consider that the description is justified. Pristimantis gralarias sp nov is easily distinguished from all other members of the P. myersi group by its long and slender fingers and toes, with discs that are not expanded laterally (or are only slightly expanded). Furthermore, the new species is characterized by having a black venter with minute white spots and a red groin. A molecular phylogeny corroborates the placement of the new species in the P. myersi group and its distinctiveness in relation to other species. Finally, we discuss on the limitations and advantages associated to species descriptions based on one or few specimens.

Two new species of frogs previously confused with Pristimantis calcarulatus are identified using molecular phylogenetics and described using an integrative taxonomic approach. The species are distributed in montane cloud forests of the northwestern Ecuadorian Andes. Pristimantis pahuma n. sp. and Pristimantis cedros n. sp. differ from their closest relatives by strongly supported reciprocal monophyly in mitochondrial genetic data (16S). Additionally, the genetic distance among P. cedros n. sp., P. pahuma n. sp. and P. calcarulatus (sensu stricto) is 7.1–9.5%. The advertisement call of P. pahuma n. sp. also differs from that of P. calcarulatus; the former emits single-note calls irregularly, whereas P. calcarulatus always calls in a series of 8–24 notes (calls for P. cedros n. sp. not recorded). Morphologically, the three species are almost undistinguishable; however, P. cedros n. sp. differs in life by having an iris with more numerous and smaller black reticulations, whereas the other two species have la...

We present a new molecular phylogeny of the stream treefrog genus Hyloscirtus, with an improved taxon sampling in the Hyloscirtus bogotensis group. The tree supports the existence of three clades within the genus (Hyloscirtus armatus group, H. bogotensis group and Hyloscirtus larinopygion group) in congruence with previous studies, and suggests the presence of at least three new species in the H. bogotensis group. Herein, we describe one of these species, Hyloscirtus mashpi n. sp. from the Pacific slope of the Ecuadorian Andes. The validity of the latter is supported by molecular, morphological and acoustic data. We also tested individuals of the new species for the chytrid fungus Batrachochytrium dendrobatidis, finding a prevalence of 17.6% (6 positives and 28 negatives). However, at sampled streams, frog densities were high, suggesting that H. mashpi n. sp. may be tolerant to the infection.

We describe a new frog, Pristimantis mutabilis sp. nov., from the Andes of Ecuador. Individuals of the new species are remarkable for their ability to change skin texture from tuberculate to almost smooth in a few minutes, being the first documented amphibian species to show such dramatic phenotypic plasticity. The new taxon is assigned to the P. myersi group. It differs from other members of its group by body size (adult males 17.2–17.4 mm; adult females 20.9–23.2 mm), arboreal habitat, and red flash coloration in females. We document three call types for the new species, which differ through their number of notes and amplitude peaks. The three types are pulsed calls that share a dominant frequency of 3186.9–3445.3 Hz. Surprisingly, we also document similar skin texture plasticity in species (P. sobetes) from a different species group, suggesting that this ability might be more common than previously thought. The discovery of these variable species poses challenges to amphibian taxonomists and field biologists, who have traditionally used skin texture and presence/absence of tubercles as important discrete traits in diagnosing and identifying species. Reciprocal monophyly and genetic distances also support the validity of the new species, as it has distances of 15.1–16.3% (12S) and 16.4–18.6% (16S) from the most similar species, Pristimantis verecundus. Additionally, each of the two known populations of Pristimantis mutabilis are reciprocally monophyletic and exhibit a high genetic distance between them (5.0–6.5%). This pattern is best explained by the presence of a dry valley (Guayllabamba River) that seems to be acting as a dispersal barrier. © 2015 The Linnean Society of London

On a recent expedition to eastern Madagascar, we discovered a distinct new species of the genusCalummathat we describe here using an integrative approach combining morphology, coloration, osteology and molecular genetics.Calummaroalokosp. n.has a dermal rostral appendage and occipital lobes, and belongs to theC.boettgericomplex, within the Madagascar-endemic pheneticC.nasutumspecies group. It is readily distinguished from other species of theC.boettgericomplex by a characteristic two-toned body coloration and small body size with a snout-vent length of 45.6 mm in an adult male. The osteology of the skull, with a prominent maxilla and broad parietal, is similar to the closest related species,C.uetzi. Analysis of uncorrected genetic distances within theC.nasutumgroup using the mitochondrial gene ND2 shows a minimum pairwise distance of 11.98% toC.uetzifrom the Sorata massif and Marojejy National Park >500 km north of the type locality ofC.roalokosp. n.. Given an apparently small range (potentially 2), located entirely outside of any nationally-protected areas, we recommend this new species be classified as Endangered under criterion B1ab(iii) of the IUCN Red List. The discovery of clearly distinct species likeC.roalokosp. n.in an area of Madagascar that is comparatively thoroughly surveyed highlights the critical role of continued field surveys for understanding the true extent of Madagascar’s spectacular biodiversity.

In many frog species, males defend a territory through direct male-to-male interactions and/or aggressive calling behaviour. We describe the site fidelity, vocalizations, aggressive interactions, and male combat behaviour of the glassfrog Nymphargus grandisonae. We show high specificity of males’ calling and mating sites. We then describe the temporal and spectral differences for six types of vocalizations. We link these vocalizations to behavioural observations, describing their aggressive and reproductive contexts. Additionally, we show that combat is highly variable and includes three previously described and two unreported variations. We describe injuries resulting from combat and we report the first observation of a multiple night fight between the same two males. Our observations on site fidelity and aggression provide evidence for territoriality among males. Furthermore, our results suggest that combat behaviour in glassfrogs is more complex than previously hypothesized and that hypotheses on the e...

The Tropical Andes are an important global biodiversity hotspot, harbouring extraordinarily high richness and endemism. Although elevational richness and speciation have been studied independently in some Andean groups, the evolutionary and ecological processes that explain elevational richness patterns in the Andes have not been analysed together. Herein, we elucidate the processes underlying Andean richness patterns using glassfrogs (Centrolenidae) as a model system. Glassfrogs show the widespread mid-elevation diversity peak for both local and regional richness. Remarkably, these patterns are explained by greater time (montane museum) rather than faster speciation at mid-elevations (montane species pump), despite the recency of the major Andean uplift. We also show for the first time that rates of climatic-niche evolution and elevational change are related, supporting the hypothesis that climatic-niche conservatism decelerates species' shifts in elevational distributions and underlies the mid-elevation richness peak. These results may be relevant to other Andean clades and montane systems globally.

Geographic barriers and elevational gradients have long been recognized as important in species diversification. Here, we illustrate an example where both mechanisms have shaped the genetic structure of the Neotropical rainfrog, Pristimantis ornatissimus, which has also resulted in speciation. This species was thought to be a single evolutionary lineage distributed throughout the Ecuadorian Choco and the adjacent foothills of the Andes. Based on recent sampling of P. ornatissimus sensu lato, we provide molecular and morphological evidence that support the validity of a new species, which we name Pristimantis ecuadorensis sp. nov. The sister species are elevational replacements of each other; the distribution of Pristimantis ornatissimus sensu stricto is limited to the Ecuadorian Choco ecoregion (< 1100 m), whereas the new species has only been found at Andean localities between 1450–1480 m. Given the results of the Multiple Matrix Regression with Randomization analysis, the genetic difference between P. ecuadorensis and P. ornatissimus is not explained by geographic distance nor environment, although environmental variables at a finer scale need to be tested. Therefore this speciation event might be the byproduct of stochastic historic extinction of connected populations or biogeographic events caused by barriers to dispersal such as rivers. Within P. ornatissimus sensu stricto, morphological patterns and genetic structure seem to be related to geographic isolation (e.g., rivers). Finally, we provide an updated phylogeny for the genus, including the new species, as well as other Ecuadorian Pristimantis.

Squamate reptiles (lizards and snakes) are one of the most diverse groups of terrestrial vertebrates. Recent molecular analyses have suggested a very different squamate phylogeny relative to morphological hypotheses, but many aspects remain uncertain from molecular data. Here, we analyse higher-level squamate phylogeny with a molecular dataset of unprecedented size, including 161 squamate species for up to 44 nuclear genes each (33 717 base pairs), using both concatenated and species-tree methods for the first time. Our results strongly resolve most squamate relationships and reveal some surprising results. In contrast to most other recent studies, we find that dibamids and gekkotans are together the sister group to all other squamates. Remarkably, we find that the distinctive scolecophidians (blind snakes) are paraphyletic with respect to other snakes, suggesting that snakes were primitively burrowers and subsequently re-invaded surface habitats. Finally, we find that some clades remain poorly supported, despite our extensive data. Our analyses show that weakly supported clades are associated with relatively short branches for which individual genes often show conflicting relationships. These latter results have important implications for all studies that attempt to resolve phylogenies with large-scale phylogenomic datasets.

We describe a new species of Boophis treefrog from Ranomafana National Park in the southern central east of Madagascar. This region has remarkably high anuran diversity, and along with neighbouring sites, hosts more than 35 Boophis species. Boophis boppa sp. nov. is part of the B. ankaratra sub-clade (herein named the B. ankaratra complex), previously identified within the monophyletic B. albipunctatus species group. It occurs sympatrically with two other species of the complex (B. ankaratra and B. schuboeae). Morphological differentiation of species within the B. ankaratra clade remains elusive, but species are well characterized by distinct advertisement calls, with B. boppa having the longest note duration and inter-note intervals when compared to closely related species. Furthermore, it has moderate differentiation in mitochondrial DNA, with pairwise distances of 1.9-3.7% to all other species in sequences of the mitochondrial 16S rRNA marker. Additional evidence is given by the lack of haplotype sharing with related species for the nuclear exon DNAH-3. All examples of syntopic occurrence in this complex involve species with strongly different advertisement calls, while allopatric species have more similar calls. Such a pattern might result from adaptive call co-evolution but could also be the result of non-adaptive processes. Thorough clarification of the systematics of the B. ankaratra sub-clade is required, and we outline future directions for both bioacoustic and genetic research.

We review the systematics of frogs of the Hyloscirtus larinopygion group. A new phylogenetic tree inferred frommitochondrial DNA (partial sequences of 12S rRNA, valine-tRNA, and 16S rRNA genes; ~2.3 kb) of eleven species ofthe H. larinopygion group is provided, based on maximum parsimony, maximum likelihood, and Bayesian analyses. Ourphylogeny confirms the close relationship of members of the H. larinopygion group with Andean relatives of the H. armatus group, which also occurs in the Andes. Hyloscirtus tapichalaca is placed as sister species to the rest of the H. larinopygion group, in which two clades (A+B) are evident. Although ingroup relationships are well supported, themonophyly of the H. larinopygion group and placement of H. tapichalaca require additional testing. Genetic divergencesamong species of the H. larinopygion group are shallow compared to those observed in many other anurans, with geneticdistance among sister species (H. princecharlesi and H. ptychodactylus) as low as 1.31%. However, this pattern isconcordant with radiations in other highland Andean lineages of anurans that show marked morphological or behavioraldifferentiation, but low divergence in mitochondrial markers. Divergence-time analyses (using BEAST) indicate that the Hyloscirtus clade is a relatively ancient lineage that appeared in the Eocene, at a minimum age of 51.2 million years ago(MYA), while the H. larinopygion group originated in the Middle-Late Eocene at a minimum age of 40.9 MYA. Ourresults might suggest a rapid radiation of Hyloscirtus starting in the Miocene into the Pliocene, from at least 14.2 MYA tothe most recent divergence between sister taxa at ~2.6 MYA. We also describe two sympatric new species of Hyloscirtus from northwestern Ecuador: H. criptico sp. nov. and H. princecharlesi sp. nov. We diagnose them by their phylogeneticposition (they are not sister to each other), genetic divergence, and a unique combination of color patterns, and othermorphological features. Additionally, we describe the suctorial tadpoles and the extreme ontogenic color changes in H. larinopygion, H. lindae, H. pantostictus, H. princecharlesi, H. psarolaimus, and H. tigrinus. Furthermore, we describe theosteology of H. criptico, H. lindae, H. pacha, H. pantostictus, H. princecharlesi, H. psarolaimus, H. ptychodactylus, and H. staufferorum. We describe vocalizations of H. lindae, H. pacha, H. pantostictus, H. pasarolaimus, H. staufferorum, and H. tapichalaca. Hyloscirtus tigrinus is recorded for the first time in Ecuador and its range is extended 62.4 km (airlinedistance), from its southernmost locality record in Departamento de Narino, Colombia. Most species of the H. larinopygion group are currently severely threatened by extinction, after surviving the catastrophic extinctions in the1980s and 1990s that led to the disappearance of many other sympatric anurans that bred in swiftly flowing water and hadlotic water tadpoles in the Andean highlands. Research and conservation actions are urgently needed for these species. Inorder to better call attention to these conservation issues, we name one of the new species in honor of Prince Charles ofWales, who is contributing significantly to the growth of awareness in the battle against tropical deforestation, climate change, and the catastrophic extinction of rainforest amphibians.

We describe a new species of Nymphargus from Reserva Las Gralarias, in the montane forests of the Pacific versant ofthe Andes of Ecuador. The new species, Nymphargus lasgralarias sp. nov., is nearly morphologically identical to sym-patric Nymphargus griffithsi (Goin 1961), with the exception of the following characters: (i) a gold colored iris with nu-merous small spots and lighter reticulation, (ii) absence of dark dorsal spotting, and (iii) a significantly larger body size.Additionally, we describe the vocalizations for N. griffithsi and N. lasgralarias. The call of N. lasgralarias sp. nov. is eas-ily distinguished from N. griffithsi through the following temporal and spectral characteristics: (i) calls are emitted in series, (ii) waveform shape is always pulsed, (iii) significantly shorter call duration, and (iv) lower dominant frequency.