Genetic Variants Underlying Plasticity in Natural Populations of Spadefoot Toads: Environmental Assessment versus Phenotypic Response.

Many organisms facultatively produce different phenotypes depending on their environment, yet relatively little is known about the genetic bases of such plasticity in natural populations. In this study, we describe the genetic variation underlying an extreme form of plasticity--resource polyphenism--in Mexican spadefoot toad tadpoles, Spea multiplicata. Depending on their environment, these tadpoles develop into one of two drastically different forms: a carnivore morph or an omnivore morph. We collected both morphs from two ponds that differed in which morph had an adaptive advantage and performed genome-wide association studies of phenotype (carnivore vs. omnivore) and adaptive plasticity (adaptive vs. maladaptive environmental assessment). We identified four quantitative trait loci associated with phenotype and nine with adaptive plasticity, two of which exhibited signatures of minor allele dominance and two of which (one phenotype locus and one adaptive plasticity locus) did not occur as minor allele homozygotes. Investigations into the genetics of plastic traits in natural populations promise to provide novel insights into how such complex, adaptive traits arise and evolve.

Contrasting nidification behaviors facilitate diversification and colonization of the Music frogs under a changing paleoclimate.

In order to cope with the complexity and variability of the terrestrial environment, amphibians have developed a wide range of reproductive and parental behaviors. Nest building occurs in some anuran species as parental care. Species of the Music frog genus Nidirana are known for their unique courtship behavior and mud nesting in several congeners. However, the evolution of these frogs and their nidification behavior has yet to be studied. With phylogenomic and phylogeographic analyses based on a wide sampling of the genus, we find that Nidirana originated from central-southwestern China and the nidification behavior initially evolved at ca 19.3 Ma but subsequently lost in several descendants. Further population genomic analyses suggest that the nidification species have an older diversification and colonization history, while N. adenopleura complex congeners that do not exhibit nidification behavior have experienced a recent rapid radiation. The presence and loss of the nidification behavior in the Music frogs may be associated with paleoclimatic factors such as temperature and precipitation. This study highlights the nidification behavior as a key evolutionary innovation that has contributed to the diversification of an amphibian group under past climate changes.

Development of molecular identification methods for Dryophytes suweonensis and D. japonicus, and their hybrids.

Background: As hybridization can reduce biodiversity or cause extinction, it is important to identify both purebred parental species and their hybrids prior to conserving them. The Suwon tree frog, Dryophytes suweonensis, is an endangered wildlife species in Korea that shares its habitat and often hybridizes with the Japanese tree frog, D. japonicus. In particular, D. suweonensis, D. japonicus, and their hybrids often have abnormal ovaries and gonads, which are known causes that could threaten their existence. Methods: We collected 57 individuals from six localities where D. suweonensis is known to be present. High-resolution melting curve (HRM) analysis of the mitochondrial 12S ribosomal RNA gene was performed to determine the maternal species. Thereafter, the DNA sequences of five nuclear genes (SIAH, TYR, POMC, RAG1, and C-MYC) were analyzed to determine their parental species and hybrid status. Results: The HRM analysis showed that the melting temperature of D. suweonensis was in the range of 79.0-79.3 °C, and that of D. japonicus was 77.7-78.0 °C, which clearly distinguished the two tree frog species. DNA sequencing of the five nuclear genes revealed 37 single-nucleotide polymorphism (SNP) sites, and STRUCTURE analysis showed a two-group structure as the most likely grouping solution. No heterozygous position in the purebred parental sequences with Q values ≥ 0.995 were found, which clearly distinguished the two treefrog species from their hybrids; 11 individuals were found to be D. suweonensis, eight were found to be D. japonicus, and the remaining 38 individuals were found to be hybrids. Conclusion: Thus, it was possible to unambiguously identify the parental species and their hybrids using HRM analysis and DNA sequencing methods. This study provided fundamental information for D. suweonensis conservation and restoration research.

Karyotypes of water frogs from the Pelophylax esculentus complex: results of cross-species chromosomal painting.

Amphibian species have the largest genome size enriched with repetitive sequences and relatively similar karyotypes. Moreover, many amphibian species frequently hybridize causing nuclear and mitochondrial genome introgressions. In addition, hybridization in some amphibian species may lead to clonality and polyploidization. All such events were found in water frogs from the genus Pelophylax. Among the species within the genus Pelophylax, P. esculentus complex is the most widely distributed and well-studied. This complex includes two parental species, P. ridibundus and P. lessonae, and their hybrids, P. esculentus, reproducing hemiclonally. Parental species and their hybrids have similar but slightly polymorphic karyotypes, so their precise identification is still required. Here, we have developed a complete set of 13 chromosome painting probes for two parental species allowing the precise identification of all chromosomes. Applying chromosomal painting, we identified homologous chromosomes in both parental species and orthologous chromosomes in their diploid hemiclonal hybrids. Comparative painting did not reveal interchromosomal exchanges between the studied water frog species and their hybrids. Using cross-specific chromosome painting, we detected unequal distribution of the signals along chromosomes suggesting the presence of species-specific tandem repeats. Application of chromosomal paints to the karyotypes of hybrids revealed differences in the intensity of staining for P. ridibundus and P. lessonae chromosomes. Thus, both parental genomes have a divergence in unique sequences. Obtained chromosome probes may serve as a powerful tool to unravel chromosomal evolution in phylogenetically related species, identify individual chromosomes in different cell types, and investigate the elimination of chromosomes in hybrid water frogs.

Biogeography and the evolution of acoustic communication in the polyploid North American grey treefrog complex.

After polyploid species are formed, interactions between diploid and polyploid lineages may generate additional diversity in novel cytotypes and phenotypes. In anurans, mate choice by acoustic communication is the primary method by which individuals identify their own species and assess suitable mates. As such, the evolution of acoustic signals is an important mechanism for contributing to reproductive isolation and diversification in this group. Here, we estimate the biogeographical history of the North American grey treefrog complex, consisting of the diploid Hyla chrysoscelis and the tetraploid Hyla versicolor, focusing specifically on the geographical origin of whole genome duplication and the expansion of lineages out of glacial refugia. We then test for lineage-specific differences in mating signals by applying comparative methods to a large acoustic data set collected over 52 years that includes >1500 individual frogs. Along with describing the overall biogeographical history and call diversity, we found evidence that the geographical origin of H. versicolor and the formation of the midwestern polyploid lineage are both associated with glacial limits, and that the southwestern polyploid lineage is associated with a shift in acoustic phenotype relative to the diploid lineage with which they share a mitochondrial lineage. In H. chrysoscelis, we see that acoustic signals are largely split by Eastern and Western lineages, but that northward expansion along either side of the Appalachian Mountains is associated with further acoustic diversification. Overall, results of this study provide substantial clarity on the evolution of grey treefrogs as it relates to their biogeography and acoustic communication.

Unravelling the limb regeneration mechanisms of Polypedates maculatus, a sub-tropical frog, by transcriptomics.

BACKGROUND: Regeneration studies help to understand the strategies that replace a lost or damaged organ and provide insights into approaches followed in regenerative medicine and engineering. Amphibians regenerate their limbs effortlessly and are indispensable models to study limb regeneration. Xenopus and axolotl are the key models for studying limb regeneration but recent studies on non-model amphibians have revealed species specific differences in regeneration mechanisms. RESULTS: The present study describes the de novo transcriptome of intact limbs and three-day post-amputation blastemas of tadpoles and froglets of the Asian tree frog Polypedates maculatus, a non-model amphibian species commonly found in India. Differential gene expression analysis between early tadpole and froglet limb blastemas discovered species-specific novel regulators of limb regeneration. The present study reports upregulation of proteoglycans, such as epiphycan, chondroadherin, hyaluronan and proteoglycan link protein 1, collagens 2,5,6, 9 and 11, several tumour suppressors and methyltransferases in the P. maculatus tadpole blastemas. Differential gene expression analysis between tadpole and froglet limbs revealed that in addition to the expression of larval-specific haemoglobin and glycoproteins, an upregulation of cysteine and serine protease inhibitors and downregulation of serine proteases, antioxidants, collagenases and inflammatory genes in the tadpole limbs were essential for creating an environment that would support regeneration. Dermal myeloid cells were GAG+, EPYC+, INMT+, LEF1+ and SALL4+ and seemed to migrate from the unamputated regions of the tadpole limb to the blastema. On the other hand, the myeloid cells of the froglet limb blastemas were few and probably contributed to sustained inflammation resulting in healing. CONCLUSIONS: Studies on non-model amphibians give insights into alternate tactics for limb regeneration which can help devise a plethora of methods in regenerative medicine and engineering.

Genomic differentiation with isolation by distance along a latitudinal gradient in the spotted-leg treefrog Polypedates megacephalus.

The patterns of isolation by distance (IBD) entailing increased genetic differentiation among populations have aroused extensive concerns for evolutionary biologists. Although the IBD may act on spatial processes contributing to the genetic differentiation among populations in anuran species, the factors shaping the IBD of frogs among populations in natural systems are largely unknown. Here, we studied the genetic differentiation among six populations with 24 individuals of the spotted-leg treefrog along a latitudinal gradient (1860.31 km) based on 1020 single nucleotide polymorphisms from restriction site-associated DNA sequencing. The results showed that the genetic diversity differed significantly among populations and that the insular populations had higher genetic diversity than the mainland populations. Furthermore, we also found a significant genetic differentiation among populations (FST = 0.277) and no sign of inbreeding (FUNI = -0.145). The IBD was detected for all populations, and a higher degree of the IBD was indicated when controlling for the effects of the isolation between Hainan and mainland populations caused by the Qiongzhou Strait. Our findings suggest that the form of the Qiongzhou Strait plays a key role in shaping the genetic diversity and population differentiation in treefrogs.

The highest-elevation frog provides insights into mechanisms and evolution of defenses against high UV radiation.

Defense against ultraviolet (UV) radiation exposure is essential for survival, especially in high-elevation species. Although some specific genes involved in UV response have been reported, the full view of UV defense mechanisms remains largely unexplored. Herein, we used integrated approaches to analyze UV responses in the highest-elevation frog, Nanorana parkeri. We show less damage and more efficient antioxidant activity in skin of this frog than those of its lower-elevation relatives after UV exposure. We also reveal genes related to UV defense and a corresponding temporal expression pattern in N. parkeri. Genomic and metabolomic analysis along with large-scale transcriptomic profiling revealed a time-dependent coordinated defense mechanism in N. parkeri. We also identified several microRNAs that play important regulatory roles, especially in decreasing the expression levels of cell cycle genes. Moreover, multiple defense genes (i.e., TYR for melanogenesis) exhibit positive selection with function-enhancing substitutions. Thus, both expression shifts and gene mutations contribute to UV adaptation in N. parkeri. Our work demonstrates a genetic framework for evolution of UV defense in a natural environment.

Chromosomes in the African frog genus Tomopterna (Pyxicephalidae) and probing the origin of tetraploid Tomopterna tandyi.

Speciation by polyploidization has been documented to have independently occurred in 12 families of anuran amphibians. Tomopterna tandyi was described as a South African allotetraploid species of sand frogs in the family Pyxicephalidae. Recent taxonomic revisions and new species descriptions in the genus present problems with respect to the evolution of this tetraploid species. Chromosomes, mitochondrial and nuclear gene sequences, isozymes, and male mating calls were examined for T. tandyi and for diploid species of Tomopterna. Mitochondrial sequences confirmed the diploid species, T. adiastola, to be the maternal ancestor that gave rise to the tetraploid about 5 mya. Nuclear sequences and isozymes reveal a complex reticulation of paternal ancestry that may be explained by occasional hybridization of T. tandyi with diploid species of Tompoterna at various times in sympatric populations. Interspecific diploid to tetraploid gene introgression is suspected to have also occurred in Australian and North American tetraploid species of frogs. Diploid to tetraploid introgression is facilitated through triploid hybrids that are more viable than diploid hybrids and produce unreduced triploid eggs.

Resolving the deep phylogeny: Implications for early adaptive radiation, cryptic, and present-day ecological diversity of Papuan microhylid frogs.

The microhylid frogs of the New Guinea region are the largest and most ecologically diverse subfamily (Asterophryinae) of one of the largest anuran families in the world and can live in communities of up to 20 species. While there has been recent progress in resolving the phylogenetic relationships of Asterophryinae, significant uncertainties remain, impeding further progress in understanding the evolution of microhabitat use, parental care, and life history variation in this group. In particular, the early divergences at the base of the tree remain unclear; as does the monophyly of some genera; and recent studies have discovered that species with wide geographic distribution are instead cryptic species complexes. In this study, we fortified geographic sampling of the largest previous phylogenetic effort by sequencing an additional 62 taxa and increased data quality and quantity by adding new layers of data vetting and by filling in previously incomplete loci to the five gene dataset (2 mitochondrial, 3 nuclear protein-coding genes) to obtain a dataset that is now 99% complete in over 2400 characters for 233 samples (205 taxa) of Asterophryinae and 3 outgroup taxa, and analyzed microhabitat use data for these taxa from field data and data collected from the literature. Importantly, our sampling includes complete community complements at 19 sites as well as representatives at over 80 sites across New Guinea and its offshore islands. We present a highly resolved molecular phylogeny which, for the first time, has over 95% of nodes supported (84% highly supported) whether using Maximum Likelihood or Bayesian Inference, allowing clarification of all genera (whether monophyletic or clearly not), their sister genera relationships, as well as an age estimate for the Asterophryinae at approximately 20MYA. Early generic diversification occurring between 17 and 12 MYA gave rise to a surprising diversity of about 18 genera as well as the 5 putative microhabitat types. Our tree reveals extensive cryptic diversity calling any widespread taxa into doubt, and clearly demonstrates that complex multispecies communities of Asterophryinae are ecologically diverse, are numerous, and of ancient origin across New Guinea. We discuss the implications of our phylogeny for explaining the explosive diversification of Asterophryinae as the result of adaptive radiation, niche conservatism, and non-adaptive radiation.

Fragments of bombinakinin M exist in lipopolysaccharide-stimulated skin secretions of Bombina maxima and show lipopolysaccharide-neutralizing activity.

Lipopolysaccharide (LPS) is a major pathogen-associated pattern molecule that can initiate lethal sepsis. Bioactive peptides in amphibian skin secretions, especially antimicrobial peptides, are essential components of the host immune system and help fight the microbial invasion. In this study, two peptides: peptide 1 (KINRKGPRPPG) and peptide 2 (INRKGPRPPG) were isolated, from skin secretions of the Chinese red belly frog (Bombina maxima). After stimulation with LPS, peptide 1 showed direct LPS-binding activity, low cytotoxicity, immunoregulatory functions in vitro, and neutralizing LPS effects in animal models. Thus, natural peptide 1 exhibits potential as an ideal candidate against LPS.

Early exposure to UV radiation causes telomere shortening and poorer condition later in life.

Determining the contribution of elevated ultraviolet-B radiation (UVBR; 280-315 nm) to amphibian population declines is being hindered by a lack of knowledge about how different acute UVBR exposure regimes during early life-history stages might affect post-metamorphic stages via long-term carryover effects. We acutely exposed tadpoles of the Australian green tree frog (Litoria caerulea) to a combination of different UVBR irradiances and doses in a multi-factorial laboratory experiment, and then reared them to metamorphosis in the absence of UVBR to assess carryover effects in subsequent juvenile frogs. Dose and irradiance of acute UVBR exposure influenced carryover effects into metamorphosis in somewhat opposing manners. Higher doses of UVBR exposure in larvae yielded improved rates of metamorphosis. However, exposure at a high irradiance resulted in frogs metamorphosing smaller in size and in poorer condition than frogs exposed to low and medium irradiance UVBR as larvae. We also demonstrate some of the first empirical evidence of UVBR-induced telomere shortening in vivo, which is one possible mechanism for life-history trade-offs impacting condition post-metamorphosis. These findings contribute to our understanding of how acute UVBR exposure regimes in early life affect later life-history stages, which has implications for how this stressor may shape population dynamics.

Phylogeny of Telmatobius marmoratus complex (Anura, Telmatobiidae) reveals high cryptic diversity in the Andean Altiplano.

Telmatobius is the most diverse group of anurans in the Andean Altiplano (highlands) Morphologically, these amphibians have a generally conserved morphology but in turn present large intraspecific variation, which has led to a complex taxonomy and systematics. T. marmoratus has the widest distribution of the genus and forms a complex composed of at least two Telmatobius species. Partial systematic studies based on molecular evidence reveal the existence of three lineages with a complex spatial distribution. However, these studies did not include the entire distribution of T. marmoratus. Our study aims to reassess the current systematic scenario including the complete distribution of the complex. For this, we used a multilocus approach based on mitochondrial (16S, Cytb) and nuclear (RAG1-1, BFIB) DNA sequences to build a phylogenetic hypothesis based on Bayesian inference, maximum likelihood and maximum parsimony. Subsequently, we performed single-locus (ABGD and PTP) and multilocus (STACEY) species delimitation analyses to verify the diversity of nominal species within the complex. The analyses suggest seven non-sibling lineages and 6-10 candidate species within the marmoratus complex. Only one of the two lineages restricted to the central northern plateau correspond to T. marmoratus sensu stricto. South-central marbled water frogs belong to completely new lineages closer to T. gigas and T. culeus, evidencing the polyphyletic condition of the marmoratus complex. The findings of several sympatric lineages in some localities reveal a complex history of ancient water connections in south-central Altiplano.

Molecular and epigenetic regulation of seasonal reproduction in Terai tree frog (Polypedates teraiensis).

Seasonal breeders predominantly use photoperiod as the predictable environmental cue to time their reproduction. Terai tree frogs are long-day seasonal breeders, but the molecular mechanism is unknown. We tested the role of different photoperiodic conditions on expression levels of candidate genes involved in seasonal reproduction and epigenetic regulation. Four experiments were performed. In experiment 1, frogs were exposed to long (LD: 16L:8D) or short photoperiod (SD: 8L:16D). In experiment 2, animals were procured at four different phases of breeding, i.e., during April (emergence just after hibernation), June (breeding phase), August (post-breeding), and October (just before hibernation). In experiments 3 and 4, frogs were exposed to equinox photoperiod but different (10, 100, or 500 lx) light intensities (exp. 3) or wavelength (red: 640 nm, green: 540 nm, blue: 450 nm or white; exp. 4). After 2 weeks, animals were euthanized, and their brain was harvested. mRNA levels of transcripts involved in photoperiodic transduction (Eya3 and Opn5), reproduction (Tshß, GnRH, Dio2, and Dio3), and epigenetics regulation (Dnmt1, Dnmt3a, Hdac1, Hdac3, and Tet2) were measured. Results show that LD promotes the upregulation of Eya3, Opn5, Tshß, GnRH, and Dio2. Differential expression of Opn5 during LD and SD suggests its involvement in light perception. Dio3 levels were upregulated in SD (exp.1) and during the post-breeding phase (exp. 2). These results employ the limited role of light intensity and spectrum in reproduction. This is the first study showing molecular machinery involved in the amphibian system's seasonal reproduction and epigenetic regulation.

Impact of the Miocene orogenesis on Kaloula spp. radiation and implication of local refugia on genetic diversification.

The phylogeography of the Kaloula genus in East Asia is still poorly understood. One of the difficulties is the absence of fossils to corroborate molecular dating estimates. Here, we examined the mitochondrial structure of Kaloula spp. in East Asia and focused on the impact of glaciations on the northernmost species: Kaloula borealis. We determined the phylogenetic relationships, molecular dating, and genetic connectivity assessments within the genus from 1211 bp of concatenated mitochondrial 12S and 16S. The relaxed clock analyses reveal the emergence of Kaloula spp. common ancestor in East and Southeast Asia between the Eocene and Oligocene, c. 38.47 Ma (24.69-53.65). The genetic diversification of lineages then increased on the East Asian Mainland during the Lower Miocene, c. 20.10 (8.73-30.65), most likely originating from the vicariance and radiation triggered by the orogeny of the Qinghai-Tibetan Plateau. Later, the dispersal towards the North East Asian Mainland during the Upper Miocene drove the population diversification of K. borealis c. 9.01 Ma (3.66-15.29). Finally, the central mainland population became isolated following orogenesis events and diverged into K. rugifera during the Pliocene, c. 3.06 Ma (0.02-10.90). The combination of population genetic and barrier analyses revealed a significant genetic isolation between populations of Kaloula spp. matching with the massive Qinling-Daba Mountain chain located in south-central China. Finally, we highlight a young divergence within the Eastern Mainland population of K. borealis, possibly attributed to refugia in south eastern China from which populations later expanded.

The Complex History of Genome Duplication and Hybridization in North American Gray Treefrogs.

Polyploid speciation has played an important role in evolutionary history across the tree of life, yet there remain large gaps in our understanding of how polyploid species form and persist. Although systematic studies have been conducted in numerous polyploid complexes, recent advances in sequencing technology have demonstrated that conclusions from data-limited studies may be spurious and misleading. The North American gray treefrog complex, consisting of the diploid Hyla chrysoscelis and the tetraploid H. versicolor, has long been used as a model system in a variety of biological fields, yet all taxonomic studies to date were conducted with only a few loci from nuclear and mitochondrial genomes. Here, we utilized anchored hybrid enrichment and high-throughput sequencing to capture hundreds of loci along with whole mitochondrial genomes to investigate the evolutionary history of this complex. We used several phylogenetic and population genetic methods, including coalescent simulations and testing of polyploid speciation models with approximate Bayesian computation, to determine that H. versicolor was most likely formed via autopolyploidization from a now extinct lineage of H. chrysoscelis. We also uncovered evidence of significant hybridization between diploids and tetraploids where they co-occur, and show that historical hybridization between these groups led to the re-formation of distinct polyploid lineages following the initial whole-genome duplication event. Our study indicates that a wide variety of methods and explicit model testing of polyploid histories can greatly facilitate efforts to uncover the evolutionary history of polyploid complexes.

Identification of the taxonomic status of Scinax nebulosus and Scinax constrictus (Scinaxinae, Anura) based on molecular markers / Identificação do status taxonômico de Scinax nebulosus e Scinax constrictus (Scinaxinae, Anura), baseado em marcadores moleculares

Abstract The validation of many anuran species is based on a strictly descriptive, morphological analysis of a small number of specimens with a limited geographic distribution. The Scinax Wagler, 1830 genus is a controversial group with many doubtful taxa and taxonomic uncertainties, due a high number of cryptic species. One example is the pair of species Scinax constrictus and Scinax nebulosus, which share a similar morphology. Scinax constrictus is restricted to the Brazilian Cerrado savanna, while S. nebulosus is widely distributed throughout northern South America. Despite the validation of many anuran species, discriminations based only on morphological traits is quite difficult due to the high conservative morphology of some groups. In this context, the present study uses mitochondrial and nuclear genes to provide a more consistent diagnosis and test the validity of S. constrictus as a distinct species from S. nebulosus, as well as evaluate the position of these taxa within the Scinax genus. The topologies obtained herein uphold the monophyletic status of Scinax based on all molecular markers assessed in this study, in all analytical approaches, with high levels of statistical support.

Resumo A validação de muitas espécies de anuros é baseada em uma análise morfológica e descritiva de um pequeno número de espécimes com uma distribuição geográfica limitada. O gênero Scinax Wagler, 1830 é um grupo controverso com muitos táxons duvidosos e incertezas taxonômicas devido ao grande número de espécies crípticas. Um exemplo são as espécies, Scinax constrictus e Scinax nebulosus, que compartilham uma morfologia similar. Scinax constrictus é restrito à savana do Cerrado brasileiro, enquanto S. nebulosus é amplamente distribuído pelo norte da América do Sul. Apesar da validação de muitas espécies de anuros, a discriminação baseada apenas em características morfológicas é bastante difícil, devido à alta morfologia conservadora de alguns grupos. Neste contexto, o presente estudo utiliza genes mitocondriais e nucleares para fornecer um diagnóstico mais consistente e para testar a validade de S. constrictus como uma espécie distinta de S. nebulosus, bem como avaliar a posição destes táxons dentro do gênero Scinax. As topologias obtidas confirmaram o status monofilético de Scinax com base em todos os marcadores moleculares, em todas as abordagens analíticas, com altos níveis de suporte estatístico.

Anuran Vocal Communication: Effects of Genome Size, Cell Number and Cell Size.

Significant variation in genome size occurs among anuran amphibians and can affect cell size and number. In the gray treefrog complex in North America, increases in cell size in autotriploids of the diploid (Hyla chrysoscelis) altered the temporal structure of mate-attracting vocalizations and auditory selectivity for these properties. Here, we show that the tetraploid species (Hyla versicolor) also has significantly fewer brain neurons than H. chrysoscelis. With regard to cell size in tissues involved in vocal communication, spinal motor neurons were larger in tetraploids than in diploids and comparable to differences in erythrocyte size; smaller increases were found in one of the three auditory centers in the torus semicircularis. Future studies should address questions about how environmental conditions during development affect cell numbers and size and the causal relationships between these cellular changes and the vocal communication system.

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.

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.