Molecular evolution of male reproduction across species with highly divergent sperm morphology in diverse murine rodents.

Sperm competition can drive rapid evolution of male reproductive traits, but it remains unclear how variation in sperm competition intensity shapes phenotypic and molecular diversity across clades. Old World mice and rats (subfamily Murinae) comprise a rapid radiation and exhibit incredible diversity in sperm morphology and production. We combined phenotype and sequence data to model the evolution of reproductive traits and genes across 78 murine species. We identified several shifts towards smaller relative testes mass, a trait reflective of reduced sperm competition. Several sperm traits were associated with relative testes mass, suggesting that mating system evolution likely selects for convergent traits related to sperm competitive ability. Molecular evolutionary rates of spermatogenesis proteins also correlated with relative testes mass, but in an unexpected direction. We predicted that sperm competition would result in rapid divergence among species with large relative testes mass, but instead found that many spermatogenesis genes evolve more rapidly in species with smaller relative testes mass due to relaxed purifying selection. While some reproductive genes evolved under positive selection, relaxed selection played a greater role underlying rapid evolution in small testes species. Our work demonstrates that sexual selection can impose strong purifying selection shaping the evolution of male reproduction.

A new species of Pandanus-dwelling frog from northern Madagascar related to Guibemantis pulcher.

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.

Filtration of Gene Trees From 9,000 Exons, Introns, and UCEs Disentangles Conflicting Phylogenomic Relationships in Tree Frogs (Hylidae).

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.

Redefining Possible: Combining Phylogenomic and Supersparse Data in Frogs.

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.

FrogCap: A modular sequence capture probe-set for phylogenomics and population genetics for all frogs, assessed across multiple phylogenetic scales.

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.

Testing for adaptive radiation: A new approach applied to Madagascar frogs.

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.

Gene Flow Increases Phylogenetic Structure and Inflates Cryptic Species Estimations: A Case Study on Widespread Philippine Puddle Frogs (Occidozyga laevis).

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.].

Target-capture phylogenomics provide insights on gene and species tree discordances in Old World treefrogs (Anura: Rhacophoridae).

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.

Gene flow creates a mirage of cryptic species in a Southeast Asian spotted stream frog complex.

Most new cryptic species are described using conventional tree- and distance-based species delimitation methods (SDMs), which rely on phylogenetic arrangements and measures of genetic divergence. However, although numerous factors such as population structure and gene flow are known to confound phylogenetic inference and species delimitation, the influence of these processes is not frequently evaluated. Using large numbers of exons, introns, and ultraconserved elements obtained using the FrogCap sequence-capture protocol, we compared conventional SDMs with more robust genomic analyses that assess population structure and gene flow to characterize species boundaries in a Southeast Asian frog complex (Pulchrana picturata). Our results showed that gene flow and introgression can produce phylogenetic patterns and levels of divergence that resemble distinct species (up to 10% divergence in mitochondrial DNA). Hybrid populations were inferred as independent (singleton) clades that were highly divergent from adjacent populations (7%-10%) and unusually similar (<3%) to allopatric populations. Such anomalous patterns are not uncommon in Southeast Asian amphibians, which brings into question whether the high levels of cryptic diversity observed in other amphibian groups reflect distinct cryptic species-or, instead, highly admixed and structured metapopulation lineages. Our results also provide an alternative explanation to the conundrum of divergent (sometimes nonsister) sympatric lineages-a pattern that has been celebrated as indicative of true cryptic speciation. Based on these findings, we recommend that species delimitation of continuously distributed "cryptic" groups should not rely solely on conventional SDMs, but should necessarily examine population structure and gene flow to avoid taxonomic inflation.

Larger, unfiltered datasets are more effective at resolving phylogenetic conflict: Introns, exons, and UCEs resolve ambiguities in Golden-backed frogs (Anura: Ranidae; genus Hylarana).

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.

Improving sustainable use of genetic resources in biodiversity archives.

Tissue sample databases housed in biodiversity archives represent a vast trove of genetic resources, and these tissues are often 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 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.

Sympatric lineages in the Mantidactylus ambreensis complex of Malagasy frogs originated allopatrically rather than by in-situ speciation.

Madagascar's biota is characterized by a high degree of microendemism at different taxonomic levels, but how colonization and in-situ speciation contribute to the assembly of local species communities has rarely been studied on this island. Here we analyze the phylogenetic relationships of riparian frogs of the Mantidactylus ambreensis species complex, which is distributed in the north of Madagascar and was originally described from Montagne d'Ambre, an isolated mountain of volcanic origin, currently protected within Montagne d'Ambre National Park (MANP). Data from mitochondrial DNA, and phylogenomic data from FrogCap, a sequence capture method, independently confirm that this species complex is monophyletic within the subgenus Ochthomantis, and identify two main clades within it. These two clades are separated by 5.6-6.8% pairwise distance in the mitochondrial 16S rRNA gene and co-occur in MANP, with one distributed at high elevations (940-1375 m a.s.l.) and the other at lower elevations (535-1010 m a.s.l.), but show almost no haplotype sharing in the nuclear RAG1 gene. This occurrence in syntopy without admixture confirms them as independent evolutionary lineages that merit recognition as separate species, and we here refer to them as high-elevation (HE) and low-elevation (LE) lineage; they will warrant taxonomic assessment to confidently assign the name ambreensis to one or the other. Populations of the M. ambreensis complex from elsewhere in northern Madagascar all belong to the LE lineage, although they do occur over a larger elevational range than in Montagne d'Ambre (285-1040 m a.s.l.). Within LE there are several phylogroups (LE1-LE4) of moderately deep divergence (1.5-2.8% in 16S), but phylogroup LE4 that occurs in MANP has a deeply nested phylogenetic position, as recovered separately by mitochondrial and sequence capture datasets. This suggests that HE and LE did not diverge by a local fission of lower and upper populations, but instead arose through a more complex biogeographic scenario. The branching pattern of phylogroups LE1-LE4 shows a clear south-to-north phylogeographic pattern. We derive from these results a testable hypothesis of vicariant speciation that restricted the HE lineage to MANP and the LE candidate species to a climatic refugium further south, with subsequent northwards range expansion and secondary colonization of MANP by LE. These results provide an example for complex assembly of local microendemic amphibian faunas on Madagascar.

Species complexes and the importance of Data Deficient classification in Red List assessments: The case of Hylobatrachus frogs.

Taxonomy is the cornerstone of extinction risk assessments. Currently, the IUCN Red List treats species complexes either under a single overarching species name-resulting in an unhelpfully broad circumscription and underestimated threat assessment that does not apply to any one species lineage-or omits them altogether-resulting in the omission of species that should be assessed. We argue that taxonomic uncertainty alone, as in species complexes, should be grounds for assessment as Data Deficient (DD). Yet, use of the DD category is currently discouraged, resulting in assessments based on poor data quality and dismissal of the importance of taxonomic confidence in conservation. This policy may be leading to volatile and unwarranted assessments of hundreds of species across the world, and needs to be revised. To illustrate this point, we here present a partial taxonomic revision of torrent frogs from eastern Madagascar in the Mantidactylus subgenus Hylobatrachus. Two named species, Mantidactylus (Hylobatrachus) lugubris and M. (H.) cowanii, and several undescribed candidate species are recognised, but the application of the available names has been somewhat ambiguous. In a recent re-assessment of its conservation status, M. (H.) lugubris was assessed including all complex members except M. (H.) cowanii within its distribution, giving it a status of Least Concern and distribution over most of eastern Madagascar. After describing two of the unnamed lineages as Mantidactylus (Hylobatrachus) atsimo sp. nov. (from southeastern Madagascar) and Mantidactylus (Hylobatrachus) petakorona sp. nov. (from the Marojejy Massif in northeastern Madagascar), we show that Mantidactylus (Hylobatrachus) lugubris is restricted to the central east of Madagascar, highlighting the inaccuracy of its current Red List assessment. We propose to re-assess its status under a more restrictive definition that omits well-defined candidate species, thus representing the actual species to which its assessment refers, to the best of current knowledge. We recommend that for species complexes in general, (1) nominal lineages that can be confidently restricted should be assessed under the strict definition, (2) non-nominal species-level lineages and ambiguous names should be prioritised for taxonomic research, and (3) ambiguous names should be assessed as DD to highlight the deficiency in data on their taxonomic status, which is an impediment to their conservation. This would reduce ambiguity and underestimation of threats involved in assessing species complexes, and place the appropriate emphasis on the importance of taxonomy in anchoring conservation.

Morphological and ecological convergence at the lower size limit for vertebrates highlighted by five new miniaturised microhylid frog species from three different Madagascan genera.

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

Molecular phylogeny and diversification of Malagasy bright-eyed tree frogs (Mantellidae: Boophis).

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.

A new (singleton) rainfrog of the Pristimantis myersi Group (Amphibia: Craugastoridae) from the northern Andes of Ecuador.

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.

Rapid Diversification and Time Explain Amphibian Richness at Different Scales in the Tropical Andes, Earth's Most Biodiverse Hotspot.

The Tropical Andes make up Earth's most species-rich biodiversity hotspot for both animals and plants. Nevertheless, the ecological and evolutionary processes underlying this extraordinary richness remain uncertain. Here, we examine the processes that generate high richness in the Tropical Andes relative to other regions in South America and across different elevations within the Andes, using frogs as a model system. We combine distributional data, a newly generated time-calibrated phylogeny for 2,318 frog species, and phylogenetic comparative methods to test the relative importance of diversification rates and colonization times for explaining Andean diversity at different scales. At larger scales (among regions and families), we find that faster diversification rates in Andean clades most likely explain high Andean richness. In contrast, at smaller temporal and spatial scales (within family-level clades within the Andes), diversification rates rarely explain richness patterns. Instead, we show that colonization times are important for shaping elevational richness patterns within the Andes, with more species found in habitats colonized earlier. We suggest that these scale-dependent patterns might apply to many other richness gradients. Recognition of this scale dependence may help to reconcile conflicting results among studies of richness patterns across habitats, regions, and organisms.

Diversification of the rainfrog Pristimantis ornatissimus in the lowlands and Andean foothills of Ecuador.

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 Chocó 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 Chocó 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.

A new species of bright-eyed treefrog (Mantellidae) from Madagascar, with comments on call evolution and patterns of syntopy in the Boophis ankaratra complex.

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.

Explaining Andean megadiversity: the evolutionary and ecological causes of glassfrog elevational richness patterns.

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.