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

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

Multiple contact zones and karyotypic evolution in a neotropical frog species complex.

Previous studies of DNA sequence and karyotypic data have revealed high genetic diversity in the Physalaemus cuvieri - Physalaemus ephippifer species complex-a group of small leptodactylid frogs in South America. To date, seven major genetic lineages have been recognized in this group, with species delimitation tests supporting four to seven of them as valid species. Among these, only P. ephippifer shows heteromorphic sex chromosomes, but the implications of cytogenetic divergence for the evolution of this group are unknown. We analyzed karyotypic, mitochondrial DNA, and 3RAD genomic data to characterize a putative contact zone between P. ephippifer and P. cuvieri Lineage 1, finding evidence for admixture and karyotypic evolution. We also describe preliminary evidence for admixture between two other members of this species complex-Lineage 1 and Lineage 3 of P. cuvieri. Our study sheds new light on evolutionary relationships in the P. cuvieri - P. ephippifer species complex, suggesting an important role of karyotypic divergence in its evolutionary history and underscoring the importance of hybridization as a mechanism of sex chromosome evolution in amphibians.

Convergence of gut microbiota in myrmecophagous amphibians.

The gut microbiome composition of terrestrial vertebrates is known to converge in response to common specialized dietary strategies, like leaf-eating (folivory) or ant- and termite-eating (myrmecophagy). To date, such convergence has been studied in mammals and birds, but has been neglected in amphibians. Here, we analysed 15 anuran species (frogs and toads) representing five Neotropical families and demonstrated the compositional convergence of the gut microbiomes of distantly related myrmecophagous species. Specifically, we found that the gut microbial communities of bufonids and microhylids, which have independently evolved myrmecophagy, were significantly more similar than expected based on their hosts' evolutionary divergence. Conversely, we found that gut microbiome composition was significantly associated with host evolutionary history in some cases. For instance, the microbiome composition of Xenohyla truncata, one of the few known amphibians that eat fruits, was not different from those of closely related tree frogs with an arthropod generalist diet. Bacterial taxa overrepresented in myrmecophagous species relative to other host families include Paludibacter, Treponema, and Rikenellaceae, suggesting diet-mediated selection and prey-to-predator transmission likely driving the observed compositional convergence. This study provides a basis for examining the roles of the gut microbiome in host tolerance and sequestration of toxic alkaloids from ants and termites.

Host macrocyclic acylcarnitines mediate symbiotic interactions between frogs and their skin microbiome.

The host-microbiome associations occurring on the skin of vertebrates significantly influence hosts' health. However, the factors mediating their interactions remain largely unknown. Herein, we used integrated technical and ecological frameworks to investigate the skin metabolites sustaining a beneficial symbiosis between tree frogs and bacteria. We characterize macrocyclic acylcarnitines as the major metabolites secreted by the frogs' skin and trace their origin to an enzymatic unbalance of carnitine palmitoyltransferases. We found that these compounds colocalize with bacteria on the skin surface and are mostly represented by members of the Pseudomonas community. We showed that Pseudomonas sp. MPFS isolated from frogs' skin can exploit acylcarnitines as its sole carbon and nitrogen source, and this metabolic capability is widespread in Pseudomonas. We summarize frogs' multiple mechanisms to filter environmental bacteria and highlight that acylcarnitines likely evolved for another function but were co-opted to provide nutritional benefits to the symbionts.

Evolution of territoriality in Hylinae treefrogs: Ecological and morphological correlates and lineage diversification.

Given the diverse nature of traits involved in territorial defence, they may respond to different selective pressures and then exhibit distinct patterns of evolution. These selective pressures also may cause territorial behaviour to be associated with environmental and morphological variables. Such associations, however, have mostly been studied at the intraspecific level, being phylogenetic analyses of territoriality in a broad taxonomic framework rare in the literature. We used the anuran subfamily Hylinae to test (1) whether two territorial-behaviour traits with different levels of aggression-territorial call and physical combat-are evolutionarily more labile than a morphological trait used in physical combat-the spine-shaped prepollex; (2) whether reproduction in lentic waters and phytotelmata, as well as resource scarcity, might favour the occurrence of territoriality; (3) if physical combat is more important than territorial call for the evolution of body size and sexual size dimorphism and (4) the relationships between territorial-behaviour traits and lineage diversification. We mainly used the literature to build two datasets with different levels of certainty. Territorial-behaviour traits exhibited intermediate levels of phylogenetic signal in Hylinae, whereas the phylogenetic signal for the presence of the spine-shaped prepollex was strong. We found support for the hypothesis that reproduction in lentic water favours the occurrence of territorial behaviour, because the expression of territorial-behaviour traits was more associated with reproduction in lentic than in lotic waters. Territorial-behaviour traits were not correlated with annual precipitation nor with habitat complexity. Body size and sexual size dimorphism were not correlated with the presence of territorial call nor with physical combat. We identified negative correlations between diversification rates and physical combat. Relationships of territorial call and physical combat with diversification rates suggest that these territorial behaviours influence evolutionary processes in different ways.

Host immune responses to enzootic and invasive pathogen lineages vary in magnitude, timing, and efficacy.

Infectious diseases of wildlife continue to pose a threat to biodiversity worldwide, yet pathogens are far from uniform in virulence or host disease outcome. Within the same pathogen species, virulence can vary considerably depending on strain or lineage, in turn eliciting variable host responses. One pathogen that has caused extensive biodiversity loss is the amphibian-killing fungus, Batrachochytrium dendrobatidis (Bd), which is comprised of a globally widespread hypervirulent lineage (Bd-GPL), and multiple geographically restricted, enzootic lineages. Whereas host immunogenomic responses to Bd-GPL have been characterized in a number of amphibian species, immunogenomic responses to geographically restricted, enzootic Bd lineages are less clear. To examine lineage-specific host immune responses to Bd, we exposed a species of pumpkin toadlet, Brachycephalus pitanga, which is endemic to Brazil's Southern Atlantic Forest, to either the Bd-GPL or the enzootic Bd-Asia-2/Brazil (hereafter Bd-Brazil) lineage. Using temporal samples from early, mid, and late infection stages, we quantified functional immunogenomic responses over the course of infection using differential gene expression tests and coexpression network analyses. Host immune responses varied significantly with Bd lineage. Relative to controls, toadlet responses to Bd-Brazil were weak at early infection (25 genes significantly differentially expressed), peaked by mid-stage infection (414 genes), and were nearly fully resolved by late-stage infection (nine genes). In contrast, responses to Bd-GPL were magnified and delayed; toadlets significantly differentially expressed 111 genes early, 87 genes at mid-stage infection, and 726 genes by late-stage infection relative to controls. Given that infection intensity did not vary between mid- and late-stage disease in either Bd-Brazil or Bd-GPL treatments, this suggests that pumpkin toadlets may be at least partially tolerant to the enzootic Bd-Brazil lineage. In contrast, late-stage immune activation against Bd-GPL was consistent with immune dysregulation previously observed in other species. Our results demonstrate that both the timing of immune response and the particular immune pathways activated are specific to Bd lineage. Within regions where multiple Bd lineages co-occur, and given continued global Bd movement, these differential host responses may influence not only individual disease outcome, but transmission dynamics at the population and community levels.

Habitat split as a driver of disease in amphibians.

Anthropogenic habitat disturbance is fundamentally altering patterns of disease transmission and immunity across the vertebrate tree of life. Most studies linking anthropogenic habitat change and disease focus on habitat loss and fragmentation, but these processes often lead to a third process that is equally important: habitat split. Defined as spatial separation between the multiple classes of natural habitat that many vertebrate species require to complete their life cycles, habitat split has been linked to population declines in vertebrates, e.g. amphibians breeding in lowland aquatic habitats and overwintering in fragments of upland terrestrial vegetation. Here, we link habitat split to enhanced disease risk in amphibians (i) by reviewing the biotic and abiotic forces shaping elements of immunity and (ii) through a spatially oriented field study focused on tropical frogs. We propose a framework to investigate mechanisms by which habitat split influences disease risk in amphibians, focusing on three broad host factors linked to immunity: (i) composition of symbiotic microbial communities, (ii) immunogenetic variation, and (iii) stress hormone levels. Our review highlights the potential for habitat split to contribute to host-associated microbiome dysbiosis, reductions in immunogenetic repertoire, and chronic stress, that often facilitate pathogenic infections and disease in amphibians and other classes of vertebrates. We highlight that targeted habitat-restoration strategies aiming to connect multiple classes of natural habitats (e.g. terrestrial-freshwater, terrestrial-marine, marine-freshwater) could enhance priming of the vertebrate immune system through repeated low-load exposure to enzootic pathogens and reduced stress-induced immunosuppression.

Low microbiome diversity in threatened amphibians from two biodiversity hotspots.

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

Draft genome and multi-tissue transcriptome assemblies of the Neotropical leaf-frog Phyllomedusa bahiana.

Amphibians are increasingly threatened worldwide, but the availability of genomic resources that could be crucial for implementing informed conservation practices lags well behind that for other vertebrate groups. Here, we describe draft de novo genome, mitogenome, and transcriptome assemblies for the Neotropical leaf-frog Phyllomedusa bahiana native to the Brazilian Atlantic Forest and Caatinga. We used a combination of PacBio long reads and Illumina sequencing to produce a 4.74-Gbp contig-level genome assembly, which has a contiguity comparable to other recent nonchromosome level assemblies. The assembled mitogenome comprises 16,239 bp and the gene content and arrangement are similar to other Neobratrachia. RNA-sequencing from 8 tissues resulted in a highly complete (86.3%) reference transcriptome. We further use whole-genome resequencing data from P. bahiana and from its sister species Phyllomedusa burmeisteri, to demonstrate how our assembly can be used as a backbone for population genomics studies within the P. burmeisteri species group. Our assemblies thus represent important additions to the catalog of genomic resources available from amphibians.

Temporal and spatial diversification along the Amazonia-Cerrado transition in Neotropical treefrogs of the Boana albopunctata species group.

Despite extensive research on biodiversity in Neotropical forests, biodiversity in seasonally dry, open biomes in South America has been underestimated until recently. We leverage a widespread group, Boana albopunctata, to uncover cryptic lineages and investigate the timing of diversification in Neotropical anurans with a focus on dry diagonal biomes (Cerrado, Caatinga and Chaco) and the ecotone between Amazonia and the Cerrado. We inferred a multilocus phylogeny of the B. albopunctata species group that includes 15 of 18 described species, recovered two cryptic species, and reconstructed the timing of diversification among species distributed across multiple South American biomes. One new potential species (B. aff. steinbachi), sampled in the Amazonian state of Acre, clustered within the B. calcara-fasciata species complex and is close to B. steinbachi. A second putative new species (B. aff. multifasciata), sampled in the Amazonia-Cerrado ecotone, is closely related to B. multifasciata. Lastly, we place a recently identified Cerrado lineage (B. aff. albopuncata) into the B. albopunctata species group phylogeny for the first time. Our ancestral range reconstruction showed that species in the B. albopuctata group likely dispersed from Amazonia-Cerrado into the dry-diagonal and Atlantic Forest. Intraspecies demography showed, for both B. raniceps and B. albopunctata, signs of rapid expansion across the dry diagonal. Similarly, for one clade of B. multifasciata, our analyses support an invasion of the Cerrado from Amazonia, followed by a rapid expansion across the open diagonal biomes. Thus, our study recovers several recent divergences along the Amazonia-Cerrado ecotone in northern Brazil. Tectonic uplift and erosion in the late Miocene and climate oscillations in the Pleistocene corresponded with estimated divergence times in the dry diagonal and Amazonia-Cerrado ecotone. Our study highlights the importance of these threatened open formations in the generation of biodiversity in the Neotropics.

Signatures of functional bacteriome structure in a tropical direct-developing amphibian species.

BACKGROUND: Host microbiomes may differ under the same environmental conditions and these differences may influence susceptibility to infection. Amphibians are ideal for comparing microbiomes in the context of disease defense because hundreds of species face infection with the skin-invading microbe Batrachochytrium dendrobatidis (Bd), and species richness of host communities, including their skin bacteria (bacteriome), may be exceptionally high. We conducted a landscape-scale Bd survey of six co-occurring amphibian species in Brazil's Atlantic Forest. To test the bacteriome as a driver of differential Bd prevalence, we compared bacteriome composition and co-occurrence network structure among the six focal host species. RESULTS: Intensive sampling yielded divergent Bd prevalence in two ecologically similar terrestrial-breeding species, a group with historically low Bd resistance. Specifically, we detected the highest Bd prevalence in Ischnocnema henselii but no Bd detections in Haddadus binotatus. Haddadus binotatus carried the highest bacteriome alpha and common core diversity, and a modular network partitioned by negative co-occurrences, characteristics associated with community stability and competitive interactions that could inhibit Bd colonization. CONCLUSIONS: Our findings suggest that community structure of the bacteriome might drive Bd resistance in H. binotatus, which could guide microbiome manipulation as a conservation strategy to protect diverse radiations of direct-developing species from Bd-induced population collapses.

Ecological divergence and synchronous Pleistocene diversification in the widespread South American butter frog complex.

Phylogeographic studies primarily focus on the major role of landscape topography in driving lineage diversification. However, populational phylogeographic breaks may also occur as a result of either niche conservatism or divergence, in the absence of geographic barriers to gene flow. Furthermore, these two factors are not mutually exclusive and can act in concert, making it challenging to evaluate their relative importances on explaining genetic variation in nature. Herein, we use sequences of two mitochondrial and four nuclear genes to investigate the timing and diversification patterns of species pertaining to the Leptodactylus latrans complex, which harbors four morphologically cryptic species with broad distributions across environmental gradients in eastern South America. The origin of this species complex dates back to the late Miocene (ca. 5.5 Mya), but most diversification events occurred synchronically during the late Pleistocene likely as the result of ecological divergence driven by Quaternary climatic oscillations. Further, significant patterns of environmental niche divergences among species in the L. latrans complex imply that ecological isolation is the primary mode of genetic diversification, mostly because phylogenetic breaks are associated with environmental transitions rather than topographic barriers at both species and populational scales. We provided new insights about diversification patterns and processes within a species complex of broadly and continuously distributed group of frogs along South America.

Sympatric and independently evolving lineages in the Thoropa miliaris - T. taophora species complex (Anura: Cycloramphidae).

Species delimitation can be challenging and affected by subjectivity. Sibling lineages that occur in sympatry constitute good candidates for species delimitation regardless of the adopted species concept. The Thoropa miliaris + T. taophora species complex exhibits high genetic diversity distributed in several lineages that occur sympatrically in the southeastern Atlantic Forest of Brazil. We used 414 loci obtained by anchored hybrid enrichment to characterize genetic variation in the Thoropa miliaris species group (T. saxatilis, T megatympanum, T. miliaris, and T. taophora), combining assignment analyses with traditional and coalescent phylogeny reconstruction. We also investigated evolutionary independence in co-occurring lineages by estimating gene flow, and validated lineages under the multispecies coalescent. We recovered most previously described lineages as unique populations in assignment analyses; exceptions include two lineages within T. miliaris that are further substructured, and the merging of all T. taophora lineages. We found very low probabilities of gene flow between sympatric lineages, suggesting independent evolution. Species tree inferences and species delimitation yielded resolved relationships and indicate that all lineages constitute putative species that diverged during the Pliocene and Pleistocene, later than previously estimated.

Molecular basis of a bacterial-amphibian symbiosis revealed by comparative genomics, modeling, and functional testing.

The molecular bases for the symbiosis of the amphibian skin microbiome with its host are poorly understood. Here, we used the odor-producer Pseudomonas sp. MPFS and the treefrog Boana prasina as a model to explore bacterial genome determinants and the resulting mechanisms facilitating symbiosis. Pseudomonas sp. MPFS and its closest relatives, within a new clade of the P. fluoresens Group, have large genomes and were isolated from fishes and plants, suggesting environmental plasticity. We annotated 16 biosynthetic gene clusters from the complete genome sequence of this strain, including those encoding the synthesis of compounds with known antifungal activity and of odorous methoxypyrazines that likely mediate sexual interactions in Boana prasina. Comparative genomics of Pseudomonas also revealed that Pseudomonas sp. MPFS and its closest relatives have acquired specific resistance mechanisms against host antimicrobial peptides (AMPs), specifically two extra copies of a multidrug efflux pump and the same two-component regulatory systems known to trigger adaptive resistance to AMPs in P. aeruginosa. Subsequent molecular modeling indicated that these regulatory systems interact with an AMP identified in Boana prasina through the highly acidic surfaces of the proteins comprising their sensory domains. In agreement with a symbiotic relationship and a highly selective antibacterial function, this AMP did not inhibit the growth of Pseudomonas sp. MPFS but inhibited the growth of another Pseudomonas species and Escherichia coli in laboratory tests. This study provides deeper insights into the molecular interaction of the bacteria-amphibian symbiosis and highlights the role of specific adaptive resistance toward AMPs of the hosts.

From genes to ecosystems: a synthesis of amphibian biodiversity research in Brazil / De genes a ecossistemas: uma síntese da pesquisa em biodiversidade de anfíbios no Brasil

Abstract Here, we summarize examples of significant advances in amphibian research supported by the São Paulo Research Foundation (FAPESP), focusing on recent discoveries in the fields of community ecology, habitat change, infection diseases, and multipurpose DNA sequencing. We demonstrated that FAPESP has been fundamental not only by directly funding research projects and scholarships, but also through its science training policy, fostering international collaborations with world-class research institutions, improving and consolidating new lines of research that often depended on a synergetic combination of different knowledge and complex tools. We emphasized that future studies will continue to focus on basic questions, such as description of new species, as well as taxonomic and systematic corrections. Furthermore, we also expect that there will be a strong integration among different disciplines using novel bioinformatics tools and modeling approaches, such as machine learning. These new approaches will be critical to further develop our understanding of foundational questions of amphibian life-history trait variation, disease transmission, community assembly, biogeography, and population forecasts under different global change scenarios such as agricultural expansion, agrochemical use, habitat loss, and climate change.

Resumo No presente estudo apresentamos exemplos de avanços significativos nas pesquisas com anfíbios financiadas pela Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), focando em descobertas recentes nos campos de ecologia de comunidades, modificação do habitat, doenças infecciosas e o sequenciamento de DNA com múltiplos propósitos. Demonstramos que a FAPESP tem sido fundamental não somente pelo financiamento direto de projetos de pesquisa e bolsas de estudo, mas também através de sua política de formação científica, fomentando colaborações internacionais com instituições de pesquisa de excelência mundial, melhorando e consolidando novas linhas de pesquisa que frequentemente dependem da combinação sinérgica entre diferentes linhas de conhecimento e ferramentas complexas. Enfatizamos que futuros estudos continuem com foco em questões básicas, como a descrição de novas espécies, bem como correções taxonômicas e sistemáticas. Além disso, esperamos uma forte integração entre diferentes disciplinas usando novas ferramentas de bioinformática e abordagens de modelagem, como o aprendizado de máquina. Essas novas abordagens serão críticas para desenvolver ainda mais nossa compreensão a respeito de questões fundamentais sobre as características da história de vida dos anfíbios, transmissão de doenças, estrutura de comunidades, biogeografia e previsões populacionais em diferentes cenários de mudanças globais, como a expansão da agricultura, uso de agrotóxicos, perda de habitat e mudanças climáticas.

Initial Phylotranscriptomic Confirmation of Homoplastic Evolution of the Conspicuous Coloration and Bufoniform Morphology of Pumpkin-Toadlets in the Genus Brachycephalus.

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

Microbiome associated with the tetrodotoxin-bearing anuran Brachycephalus pitanga.

The genus Brachycephalus includes small species of aposematic anurans known as microendemic, occurring in the mountains of the Atlantic Forest. Brachycephalus ephippium, B. nodoterga and B. pernix have been reported to contain the neurotoxin tetrodotoxin in skin and viscera. The biological conservation of several Brachycephalus species is currently threatened by climate change, deforestation, and the pandemic caused by the fungus Batrachochytrium dendrobatidis (Bd). Despite the well-known importance of amphibians' associated bacteria in the defensive role against pathogens, there is still a poor understanding of amphibian microbiome composition. The present study investigated the composition of B. pitanga microbial community and the presence of TTX in the host and in cultures of bacterial isolates, using a combination of metagenomics, bacterial culture isolation, mass spectrometry and metabolomic analyses. Results of culture-dependent and -independent analyses characterized the microbial communities associated with the skin and viscera of B. pitanga. Mass spectrometry analysis indicated the presence of TTX in host tissues, while bacterial production of TTX was not observed under the experimental conditions used in this investigation. This is the first report confirming the occurrence of TTX in B. pitanga.

The phylogeny of Dendropsophini (Anura: Hylidae: Hylinae).

The relationships of the hyline tribe Dendropsophini remain poorly studied, with most published analyses dealing with few of the species groups of Dendropsophus. In order to test the monophyly of Dendropsophini, its genera, and the species groups currently recognized in Dendropsophus, we performed a total evidence phylogenetic analysis. The molecular dataset included sequences of three mitochondrial and five nuclear genes from 210 terminals, including 12 outgroup species, the two species of Xenohyla, and 93 of the 108 recognized species of Dendropsophus. The phenomic dataset includes 46 terminals, one per species (34 Dendropsophus, one Xenohyla, and 11 outgroup species). Our results corroborate the monophyly of Dendropsophini and the reciprocal monophyly of Dendropsophus and Xenohyla. Some species groups of Dendropsophus are paraphyletic (the D. microcephalus, D. minimus, and D. parviceps groups, and the D. rubicundulus clade). On the basis of our results, we recognize nine species groups; for three of them (D. leucophyllatus, D. microcephalus, and D. parviceps groups) we recognize some nominal clades to highlight specific morphology or relationships and facilitate species taxonomy. We further discuss the evolution of oviposition site selection, where our results show multiple instances of independent evolution of terrestrial egg clutches during the evolutionary history of Dendropsophus.

The phylogeny of the Casque-headed Treefrogs (Hylidae: Hylinae: Lophyohylini).

The South American and West Indian Casque-headed Treefrogs (Hylidae: Hylinae: Lophyohylini) include 85 species. These are notably diverse in morphology (e.g. disparate levels of cranial hyperossification) and life history (e.g. different reproductive modes, chemical defences), have a wide distribution, and occupy habitats from the tropical rainforests to semiarid scrubland. In this paper, we present a phylogenetic analysis of this hylid tribe based on sequence fragments of up to five mitochondrial (12S, 16S, ND1, COI, Cytb) and six nuclear genes (POMC, RAG-1, RHOD, SIAH, TNS3, TYR). We included most of its species (> 96%), in addition to a number of new species. Our results indicate: (i) the paraphyly of Trachycephalus with respect to Aparasphenodon venezolanus; (ii) the nonmonophyly of Aparasphenodon, with Argenteohyla siemersi, Corythomantis galeata and Nyctimantis rugiceps nested within it, and Ap. venezolanus nested within Trachycephalus; (iii) the polyphyly of Corythomantis; (iv) the nonmonophyly of the recognized species groups of Phyllodytes; and (v) a pervasive low support for the deep relationships among the major clades of Lophyohylini, including C. greeningi and the monotypic genera Itapotihyla and Phytotriades. To remedy the nonmonophyly of Aparasphenodon, Corythomantis, and Trachycephalus, we redefined Nyctimantis to include Aparasphenodon (with the exception of Ap. venezolanus, which we transferred to Trachycephalus), Argenteohyla, and C. galeata. Additionally, our results indicate the need for taxonomic work in the following clades: (i) Trachycephalus dibernardoi and Tr. imitatrix; (ii) Tr. atlas, Tr. mambaiensis and Tr. nigromaculatus; and (iii) Phyllodytes. On the basis of our phylogenetic results, we analyzed the evolution of skull hyperossification and reproductive biology, with emphasis on the multiple independent origins of phytotelm breeding, in the context of Anura. We also analyzed the inter-related aspects of chemical defences, venom delivery, phragmotic behaviour, co-ossification, and prevention of evaporative water loss.

A role of asynchrony of seasons in explaining genetic differentiation in a Neotropical toad.

The process of diversification can be studied at the phylogeographic level by attempting to identify the environmental features that promote and maintain population divergence. Here we investigate diversification in Rhinella granulosa, a Neotropical toad from northeastern Brazil, by testing a range of hypotheses that encompass different putative mechanisms reducing gene flow among populations. We sequenced single nucleotide polymorphisms and examined individual predictions related to the role of geographic barriers (rivers), ecological gradients, historical habitat stability, and spatial variation in climate seasonality, also known as the asynchrony of seasons hypothesis. This hypothesis postulates that temporal asynchrony of wet and dry seasons over short distances causes parapatric populations to become isolated by time. After determining genetic structure, inferring past distributions, ranking demographic models, and estimating the power of monthly climatic variables, our results identified two populations that are not associated with geographic barriers, biome gradients, or historical refugia. Instead, they are predicted by spatial variation in monthly rainfall and minimum temperature, consistent with the asynchrony of seasons hypothesis, supported also by our comparative framework using multiple matrix regression and linear mixed effects modeling. Due to the toad's life history, climate likely mediates gene flow directly, with genetic differentiation being provoked by neutral mechanisms related to climate driven population isolation, and/or by natural selection against migrants from populations with different breeding times. The asynchrony of seasons hypothesis is seldom considered in phylogeographic studies, but our results indicate that it should be tested in systems where breeding is tightly coupled with climate.