Comparative molecular and conventional cytogenetic analyses of three species of Rhinella (Anura; Bufonidae).

The genus Rhinella corresponds to a group of anurans characterized by numerous taxonomic and systemic challenges, leading to their organization into species complexes. Cytogenetic data for this genus thus far are limited to the diploid number and chromosome morphology, which remain highly conserved among the species. In this study, we analyse the karyotypes of three species of the genus Rhinella (Rhinella granulosa, Rhinella margaritifera, and Rhinella marina) using both classical (conventional staining and C-banding) and molecular (FISH-fluorescence in situ hybridization with 18S rDNA, telomeric sequences, and microsatellite probes) cytogenetic approaches. The aim of this study is to provide data that can reveal variations in the distribution of repetitive sequences that can contribute to understanding karyotypic diversification in these species. The results revealed a conserved karyotype across the species, with 2n = 22 and FN = 44, with metacentric and submetacentric chromosomes. C-banding revealed heterochromatic blocks in the pericentromeric region for all species, with a proximal block on the long arms of pairs 3 and 6 in R. marina and on the short arms of pairs 4 and 6 in R. margaritifera. Additionally, 18S rDNA probes hybridized to pair 5 in R. granulosa, to pair 7 in R. marina, and to pair 10 in R. margaritifera. Telomeric sequence probes displayed signals exclusively in the distal region of the chromosomes, while microsatellite DNA probes showed species-specific patterns. These findings indicate that despite a conserved karyotypical macrostructure, chromosomal differences exist among the species due to the accumulation of repetitive sequences. This variation may be attributed to chromosome rearrangements or differential accumulation of these sequences, highlighting the dynamic role of repetitive sequences in the chromosomal evolution of Rhinella species. Ultimately, this study emphasizes the importance of the role of repetitive DNAs in chromosomal rearrangements to elucidate the evolutionary mechanisms leading to independent diversification in the distinct phylogenetic groups of Rhinella.

Field-based molecular detection of Batrachochytrium dendrobatidis in critically endangered Atelopus toads and aquatic habitats in Ecuador.

Batrachochytrium dendrobatidis (Bd) is a lethal fungal species that parasitizes vertebrates and is associated with the worldwide decline of amphibian populations. The development of sensitive, rapid detection methods, particularly DNA-based techniques, is critical for effective management strategies. This study evaluates the efficacy of DNA extraction and a portable PCR device in a mountable field laboratory setup for detecting Bd near the habitats of three critically endangered Atelopus toad species in Ecuador. We collected skin swabs from Atelopus balios, A. nanay, and A. bomolochos, and environmental DNA (eDNA) samples from streams in Andean and coastal regions of Ecuador. For eDNA, a comparison was made with duplicates of the samples that were processed in the field and in a standard university laboratory. Our findings revealed Bd detection in eDNA and swabs from 6 of 12 water samples and 10 of 12 amphibian swab samples. The eDNA results obtained in the field laboratory were concordant with those obtained under campus laboratory conditions. These findings highlight the potential of field DNA-based monitoring techniques for detecting Bd in amphibian populations and their aquatic habitats, particularly in remote areas. Furthermore, this research aligns with the National Action Plan for the Conservation of Ecuadorian Amphibians and contributes to the global effort to control this invasive and deadly fungus.

Phylogenomics, introgression, and demographic history of South American true toads (Rhinella).

The effects of genetic introgression on species boundaries and how they affect species' integrity and persistence over evolutionary time have received increased attention. The increasing availability of genomic data has revealed contrasting patterns of gene flow across genomic regions, which impose challenges to inferences of evolutionary relationships and of patterns of genetic admixture across lineages. By characterizing patterns of variation across thousands of genomic loci in a widespread complex of true toads (Rhinella), we assess the true extent of genetic introgression across species thought to hybridize to extreme degrees based on natural history observations and multilocus analyses. Comprehensive geographic sampling of five large-ranged Neotropical taxa revealed multiple distinct evolutionary lineages that span large geographic areas and, at times, distinct biomes. The inferred major clades and genetic clusters largely correspond to currently recognized taxa; however, we also found evidence of cryptic diversity within taxa. While previous phylogenetic studies revealed extensive mitonuclear discordance, our genetic clustering analyses uncovered several admixed individuals within major genetic groups. Accordingly, historical demographic analyses supported that the evolutionary history of these toads involved cross-taxon gene flow both at ancient and recent times. Lastly, ABBA-BABA tests revealed widespread allele sharing across species boundaries, a pattern that can be confidently attributed to genetic introgression as opposed to incomplete lineage sorting. These results confirm previous assertions that the evolutionary history of Rhinella was characterized by various levels of hybridization even across environmentally heterogeneous regions, posing exciting questions about what factors prevent complete fusion of diverging yet highly interdependent evolutionary lineages.

Uma atenção crescente tem sido dada aos efeitos da introgressão genética nos limites das espécies e como eles afetam a integridade e a persistência das espécies ao longo do tempo evolutivo. A crescente disponibilidade de dados genômicos revelou padrões contrastantes de fluxo gênico entre regiões do genoma, o que impõe desafios às inferências de relações evolutivas e de padrões de mistura genética entre linhagens. Com base em padrões de variação em milhares de marcadores genômicos em um complexo amplamente distribuído de sapos (Rhinella), avaliamos a extensão de introgressão genética entre espécies que, acredita-se, hibridizam amplamente com base em observações de história natural e análises multi-locus. Nossa amostragem geográfica abrangente de cinco táxons neotropicais revelou várias linhagens evolutivas distintas que abrangem grandes áreas geográficas e, por vezes, biomas distintos. Os principais clados e grupos genéticos inferidos correspondem em grande parte aos táxons atualmente reconhecidos; no entanto, também encontramos evidência de diversidade críptica. De acordo com estudos filogenéticos anteriores que revelaram extensa discordância mitonuclear, nossas análises de agrupamento genético revelaram vários indivíduos geneticamente misturados. Adicionalmente, análises demográficas históricas sugerem que a história evolutiva desses sapos envolveu fluxo gênico entre táxons tanto em épocas antigas quanto recentes. Por fim, testes ABBA-BABA revelaram amplo compartilhamento de alelos entre espécies, um padrão que pode ser atribuído à introgressão genética ao invés de sorteamento incompleto de alelos entre linhagens. Esses resultados confirmam sugestões anteriores de que a história evolutiva de Rhinella foi caracterizada por vários níveis de hibridização, mesmo entre ambientes distintos, levantando questões sobre quais fatores impedem a fusão completa de linhagens evolutivas divergentes porém altamente interdependentes.

A new species of Atelopus (Amphibia: Bufonidae) from eastern Panama.

A new species of the genus Atelopus, Atelopus fronterizo sp. nov., from eastern Panama is described herein based on molecular, morphological, and bioacoustic evidence. The new species can be distinguished from its congeners occurring in the region by a combination of the following characters: (1) phalangeal reduction in thumb; (2) SVL (females only) (35.1-50.1; n=13), HW/SVL (0.23-0.34; n=59), EYND/HW (0.27-0.39; n=60), TIBL/SVL (0.41-0.56; n=58), and HAL/SVL (0.22-0.28; n=49); (3) dorsal color pattern with green or yellow background and extensive dark olive blotches forming transversal bands or mottling; (4) advertisement call duration 176-235 ms with 19-34 pulses, average pulse rate 131.69 pulses/s, and dominant frequency 2 422.50-2 606.50 Hz. The new species is nested within the Central American clade of Atelopus. The minimum Kimura-2-parameter (K2P) genetic divergence between Atelopus fronterizo sp. nov. and its most phylogenetically similar congeners ( A. certus and A. glyphus) is >2.6% for 16S and >4.9% for COI (Table 1). The phylogenetic relationship is strongly supported by ultrafast bootstrap values for the maximum-likelihood trees of both genetic markers (16S, 96; COI, 100, Figure 1A). Bayesian analysis of the concatenated sequences resulted in a tree with similar topology and high posterior probability support (0.99; Supplementary Figure S1). In addition, haplotype networks inferred from COI and 16S (Supplementary Figure S2) showed a well-separated clade containing the new species (two for COI, four for 16S). The number of mutational steps between haplotypes for the new species samples is very low (1-4 in 16S; one in COI), and the minimum number of mutational steps from the nearest species is nine for 16S (distance to A. certus) and 28 for COI (distance to A. glyphus).

Finding complexity in complexes: Assessing the causes of mitonuclear discordance in a problematic species complex of Mesoamerican toads.

Mitonuclear discordance is a frequently encountered pattern in phylogeographic studies and occurs when mitochondrial and nuclear DNA display conflicting signals. Discordance among these genetic markers can be caused by several factors including confounded taxonomies, gene flow, and incomplete lineage sorting. In this study, we present a strong case of mitonuclear discordance in a species complex of toads (Bufonidae: Incilius coccifer complex) found in the Chortís Block of Central America. To determine the cause of mitonuclear discordance in this complex, we used spatially explicit genetic data to test species limits and relationships, characterize demographic history, and quantify gene flow. We found extensive mitonuclear discordance among the three recognized species within this group, especially in populations within the Chortís Highlands of Honduras. Our data reveal nuclear introgression within the Chortís Highlands populations that was most probably driven by cyclical range expansions due to climatic fluctuations. Though we determined introgression occurred within the nuclear genome, our data suggest that it is not the key factor in driving mitonuclear discordance in the entire species complex. Rather, due to a lack of discernible geographic pattern between mitochondrial and nuclear DNA, as well as a relatively recent divergence time of this complex, we concluded that mitonuclear discordance has been caused by incomplete lineage sorting. Our study provides a framework to test sources of mitonuclear discordance and highlights the importance of using multiple marker types to test species boundaries in cryptic species.

The Rhinella arenarum transcriptome: de novo assembly, annotation and gene prediction.

The common toad Rhinella arenarum is widely distributed in Argentina, where it is utilised as an autochthonous model in ecotoxicological research and environmental toxicology. However, the lack of a reference genome makes molecular assays and gene expression studies difficult to carry out on this non-model species. To address this issue, we performed a genome-wide transcriptome analysis on R. arenarum larvae through massive RNA sequencing, followed by de novo assembly, annotation, and gene prediction. We obtained 57,407 well-annotated transcripts representing 99.4% of transcriptome completeness (available at http://rhinella.uncoma.edu.ar). We also defined a set of 52,800 high-confidence lncRNA transcripts and demonstrated the reliability of the transcriptome data to perform phylogenetic analysis. Our comprehensive transcriptome analysis of R. arenarum represents a valuable resource to perform functional genomic studies and to identify potential molecular biomarkers in ecotoxicological research.

Species delimitation, phylogeny and evolutionary demography of co-distributed, montane frogs in the southern Brazilian Atlantic Forest.

The Brazilian Atlantic Forest (BAF) is recognized as one of the world's biodiversity hotspots, with even more species per unit of area than the Amazon, however the mechanisms that led to such astonishing diversity are yet to be fully understood. In this study, we investigate the diversification of two co-distributed frog genera associated with montane areas of southern BAF: Melanophryniscus (Bufonidae) and Brachycephalus (Brachycephalidae). Species delimitation methods using mitochondrial and nuclear loci supported the existence of a remarkable number of highly endemic species in each genus, most of which occupy only one or a few adjacent mountaintops. Their timing of diversification was highly congruent, supporting recent speciation events within the past 600 thousand years. Extended Bayesian skyline plots indicate that most populations have remained relatively stable in size across the evolutionary past, with recent growth after 0.15My, suggesting that the drastic changes found in previous studies on lowland frog species were not shared by these montane taxa. These results are consistent with the existence of a montane refugium in southern BAF, allowing species persistence through the climatic shifts experienced along the BAF during the Quaternary.

Postcranial osteogenesis of the helmeted water toad Calyptocephalella gayi (Neobatrachia: Calyptocephalellidae) with comments on the osteology of australobatrachians.

Calyptocephalella gayi is one of over 6,000 neobatrachians arranged into two main groups, Hyloides and Ranoides. Phylogenetically, C. gayi is placed in Australobatrachia, a Gondwanan clade that is either the most basal clade of Hyloides or the sister group of Hyloidea, depending on the cladistic hypothesis; as such, this species is a key taxon in the study of the early evolution of Neobatrachia. The ontogeny of the postcranial skeleton of C. gayi is described in this article. The description is based on pattern of chondrification and ossification of skeletal elements in a growth series of tadpoles, on juveniles and adult individuals. Particular attention was devoted to some developmental aspects and morphological traits of the adult skeleton. The body of Presacral Vertebra VIII is formed from three centers of ossification, in contrast to the usual two dorsolateral centers observed in the remaining vertebrae of C. gayi, as well as in most anuran taxa for which the development of the axial skeleton is known. Each half of the pelvic girdle arises from a single cartilaginous element. The early development of the autopodia of both the forelimb and hindlimb includes the presence of an additional chondral element, which occurs during the formation of Distal Carpal 5 and the transient formation of Distal Tarsal 4 before the latter is incorporated in the cartilaginous distal end of the fibular. Some osteological aspects of other australobatrachian anurans also are reviewed (e.g., presence of intervertebral discs) based on reports in the literature, as well as first hand observations. In the course of this study, it became evident that further osteological studies are needed to formulate a clear picture of the evolution of skeletal characters not only within Australobatrachia, but also within Neobatrachia.

Two new species of Amazophrynella (Amphibia: Anura: Bufonidae) from Loreto, Peru.

Amazophrynella is a taxonomically poorly known bufonid genus with a pan-Amazonian distribution. A large part of this ambiguity comes from taxonomic uncertainties regarding the type species A. minuta. In this study we compare morphological and molecular data of topotypic specimens of A. minuta with all other nomical congeneric species. Based on these comparisons, we describe two new species. The first species, A. amazonicola sp. nov., differs from other recognized congeners by having a tip of snout with a small triangular protrusion (in dorsal and lateral view), spiculated body and basal webbing on fingers I and II. The second species, A. matses sp. nov., differs from congeners by the smallest snout to vent length of the genus, edges of nasal protrusion dilated and elliptical shape palmar tubercles. The two species are allopatric, where the first species is known to be associated with white sand forests (=campinaranas), while the second inhabits upland (=terra firme) forests. Both species are diagnosable by a series of substitutions in the 16S rDNA, and both species are highly divergent from their sister taxa (p-distances range from 7-14%).

Barriers, rather than refugia, underlie the origin of diversity in toads endemic to the Brazilian Atlantic Forest.

In this study, we investigated the relative contribution of geographic barriers and Pleistocene refuges in the diversification of the Rhinella crucifer species complex, a group of endemic toads with a widespread distribution in the Brazilian Atlantic Forest (AF). We used intensive sampling and multilocus DNA sequence data to compare nucleotide diversity between refuge and nonrefuge areas, investigate regional demographic patterns, estimate demographic parameters related to genetic breaks and test refuge versus barrier scenarios of diversification using approximate Bayesian computation. We did not find higher levels of genetic diversity in putative refuge areas, either at regional or biome scale. Rather, the demographic history of the species complex supports regional differences with moderate population growth in the north and central regions and stability in southern AF. Genetic breaks were dated to the Plio-Pleistocene; however, our analyses rejected the role of refuges in creating a northern and central divergence, supporting a recent colonization scenario at a smaller scale within the central AF. Overall, our data rule out massive climatically driven fragmentation and large-scale recolonization events for populations across the biome. We confirmed the importance of geographic barriers in creating main divergences and underscored the importance of searching for cryptic discontinuities in the landscape. Comparison of our results with those of other AF taxa indicates organismal specific responses to moderate shifts in habitat and that multiple refuges may constitute a more realistic model for diversification of Atlantic Forest biota.

A new species of Amazophrynella (Anura:Bufonidae) from the southwestern part of the Brazilian Guiana Shield.

Amazophrynella is a genus of the family Bufonidae, currently represented by three species. The type species of the genus, Amazophrynella minuta, however, is a complex of species occurring throughout the Amazonian biome. This group remains problematic taxonomically; the difficulty lays principally in the lack of diagnostic characters in the original description of A. minuta, the lack of molecular data and refined taxonomic comparison of individuals through its wide distribution. We describe a new species of the genus Amazophrynella, distributed in the southwestern part of the Guiana Shield of Brazil based on a series of morphological and molecular characters. The new species differs from others of the genus by presenting a slightly truncated triangular snout, ventral texture covered by many fine granules, white belly covered with black spots and by fifteen molecular autapomorphies in the 16S rDNA fragment. Uncorrected p-distances of a fragment of the 16S mitochondrial rDNA gene revealed high divergence among other Amazophrynella species (9%-14%). Additionally we provide a new diagnosis of topotypic material of Amazophrynella minuta including molecular data. Our results show the existence of a subestimated diversity in the genus Amazophrynella. 

Development of 22 polymorphic microsatellite loci for the critically endangered Morato's Digger Toad, Proceratophrys moratoi.

The Morato's digger toad (Proceratophrys moratoi) inhabits Brazilian moist savannas and is critically endangered due to its very limited geographic distribution, reduced number of isolated populations, and evidence of population decline and local extinctions. With the objective of providing tools for the genetic study of the species, 22 polymorphic microsatellite loci were isolated and screened using DNA extracted from samples of oral mucosa cells obtained from 113 individuals representing five remnant P. moratoi populations in the Brazilian state of São Paulo. These markers presented 2-18 alleles per locus, polymorphism information content (PIC) of 0.02-0.87, observed heterozygosity of 0.02-0.96 and expected heterozygosity of 0.02-0.87. Three of the loci deviated significantly from Hardy-Weinberg equilibrium in one of the populations, possibly due to the presence of null alleles. Significant linkage disequilibrium was also detected between three pairs of loci. The molecular markers developed in this study were able to discriminate each of the individuals sampled (identity analysis). This means that they will be extremely useful for future genetic studies applied to the conservation of P. moratoi, providing a baseline for estimating the levels of genetic diversity, pedigrees, inbreeding, and population structure, which will be essential for the development of effective genetic management programs.

Delimiting genetic units in Neotropical toads under incomplete lineage sorting and hybridization.

BACKGROUND: Delimiting genetic units is useful to enhance taxonomic discovery and is often the first step toward understanding evolutionary mechanisms generating diversification. The six species within the Rhinella crucifer group of toads were defined under morphological criteria alone. Previous data suggest limited correspondence of these species to mitochondrial lineages, and morphological intergradation at transitions between forms suggests hybridization. Here we extensively sampled populations throughout the geographic distribution of the group and analyzed mitochondrial and nuclear sequence data to delimit genetic units using tree-based and allele frequency-based approaches. RESULTS: These approaches yielded complementary results, with allele frequency-based methods performing unexpectedly well given the limited number of loci examined. Both mitochondrial and nuclear markers supported a genetic structure of five units within the group, with three of the inferred units distributed within its main range, while two other units occur in separate isolates. The inferred units are mostly discordant with currently described forms: unequivocal association exists for only two of the six species in the group. Genetic evidence for hybridization exists for two pairs of units, with clear cyto-nuclear allele mixing observed in one case. CONCLUSIONS: Our results confirmed that current taxonomy does not represent evolutionary units in the Rhinella crucifer group. Correspondence between genetically distinguishable units and the currently recognized species is only possible for Rhinella henseli and R. inopina. The recognition of other species relies on the reassessment of the geographic range of R. crucifer, the examination of the type series of R. ornata for hybrids, and on the use of additional markers to verify the genetic distinctiveness of R. abei. We state that R. pombali should not remain a valid species since its description appears to be based on hybrids, and that the name R. pombali should be considered a synonym of both R. crucifer and R. ornata. The fifth inferred but undescribed genetic unit may represent a new species. Our results underscore the potential of the R. crucifer species group to contribute to a better understanding of diversification processes and hybridization patterns in the Neotropics, and provide the basis for future evolutionary and taxonomic studies.

Species limits in the Andean toad genus Osornophryne (Bufonidae).

As Darwin observed, the differentiation among varieties, subspecies, and species seems, often times, arbitrary. Nowadays, however, novel tools provide the possibility of testing hypotheses of species. Using the Andean toad genus Osornophryne, we address the following questions: (1) How many species are within the genus? (2) Are morphological and molecular traits congruent when delimiting species? (3) Which morphological traits are the most divergent among species? We use recently developed methods for testing species boundaries and relationships using a multilocus data set consisting of two mitochondrial genes (12S, 16S; 1647bp aligned matrix), one exon (RAG-1; 923 aligned matrix), and one intron (RPL3Int5; 1410bp aligned matrix). As another line of evidence for species delimitation, we integrated analyses of 12 morphometric variables and 10 discrete traits commonly used in amphibian systematics. The molecular and morphological approaches support the validity of most of the described species in Osornophryne. We find, however, contradictory lines of evidence regarding the status of O. angel. Within O. guacamayo, we found a genetically divergent population that, we argue, represents a new species. We consider that O. bufoniformis represents a species complex that deserves further study. We highlight the importance of incorporating morphological data when delimiting species, especially for lineages that have a recent origin and have not achieved reciprocal monophyly in molecular phylogenies. Finally, the most divergent morphological traits among Osornophryne species are associated with locomotion (finger, toes and limbs) and feeding (head), suggesting an association between morphology and the ecological habits of the species.

A multilocus perspective on the speciation history of a North American aridland toad (Anaxyrus punctatus).

Interpretations of phylogeographic patterns can change when analyses shift from single gene-tree to multilocus coalescent analyses. Using multilocus coalescent approaches, a species tree and divergence times can be estimated from a set of gene trees while accounting for gene-tree stochasticity. We utilized the conceptual strengths of a multilocus coalescent approach coupled with complete range-wide sampling to examine the speciation history of a broadly distributed, North American warm-desert toad, Anaxyrus punctatus. Phylogenetic analyses provided strong support for three major lineages within A. punctatus. Each lineage broadly corresponded to one of three desert regions. Early speciation in A. punctatus appeared linked to late Miocene-Pliocene development of the Baja California peninsula. This event was likely followed by a Pleistocene divergence associated with the separation of the Chihuahuan and Sonoran Deserts. Our multilocus coalescent-based reconstruction provides an informative contrast to previous single gene-tree estimates of the evolutionary history of A. punctatus.

Molecular phylogeny and morphometric analyses reveal deep divergence between Amazonia and Atlantic Forest species of Dendrophryniscus.

Dendrophryniscus is an early diverging clade of bufonids represented by few small-bodied species distributed in Amazonia and the Atlantic Forest. We used mitochondrial (414 bp of 12S, 575 bp of 16S genes) and nuclear DNA (785 bp of RAG-1) to investigate phylogenetic relationships and the timing of diversification within the genus. These molecular data were gathered from 23 specimens from 19 populations, including eight out of the 10 nominal species of the genus as well as Rhinella boulengeri. Analyses also included sequences of representatives of 18 other bufonid genera that were publically available. We also examined morphological characters to analyze differences within Dendrophryniscus. We found deep genetic divergence between an Amazonian and an Atlantic Forest clade, dating back to Eocene. Morphological data corroborate this distinction. We thus propose to assign the Amazonian species to a new genus, Amazonella. The species currently named R. boulengeri, which has been previously assigned to the genus Rhamphophryne, is shown to be closely related to Dendrophryniscus species. Our findings illustrate cryptic trends in bufonid morphological evolution, and point to a deep history of persistence and diversification within the Amazonian and Atlantic rainforests. We discuss our results in light of available paleoecological data and the biogeographic patterns observed in other similarly distributed groups.

Hybridization and massive mtDNA unidirectional introgression between the closely related Neotropical toads Rhinella marina and R. schneideri inferred from mtDNA and nuclear markers.

BACKGROUND: The classical perspective that interspecific hybridization in animals is rare has been changing due to a growing list of empirical examples showing the occurrence of gene flow between closely related species. Using sequence data from cyt b mitochondrial gene and three intron nuclear genes (RPL9, c-myc, and RPL3) we investigated patterns of nucleotide polymorphism and divergence between two closely related toad species R. marina and R. schneideri. By comparing levels of differentiation at nuclear and mtDNA levels we were able to describe patterns of introgression and infer the history of hybridization between these species. RESULTS: All nuclear loci are essentially concordant in revealing two well differentiated groups of haplotypes, corresponding to the morphologically-defined species R. marina and R. schneideri. Mitochondrial DNA analysis also revealed two well-differentiated groups of haplotypes but, in stark contrast with the nuclear genealogies, all R. schneideri sequences are clustered with sequences of R. marina from the right Amazon bank (RAB), while R. marina sequences from the left Amazon bank (LAB) are monophyletic. An Isolation-with-Migration (IM) analysis using nuclear data showed that R. marina and R. schneideri diverged at ≈ 1.69 Myr (early Pleistocene), while R. marina populations from LAB and RAB diverged at ≈ 0.33 Myr (middle Pleistocene). This time of divergence is not consistent with the split between LAB and RAB populations obtained with mtDNA data (≈ 1.59 Myr), which is notably similar to the estimate obtained with nuclear genes between R. marina and R. schneideri. Coalescent simulations of mtDNA phylogeny under the speciation history inferred from nuclear genes rejected the hypothesis of incomplete lineage sorting to explain the conflicting signal between mtDNA and nuclear-based phylogenies. CONCLUSIONS: The cytonuclear discordance seems to reflect the occurrence of interspecific hybridization between these two closely related toad species. Overall, our results suggest a phenomenon of extensive mtDNA unidirectional introgression from the previously occurring R. schneideri into the invading R. marina. We hypothesize that climatic-induced range shifts during the Pleistocene/Holocene may have played an important role in the observed patterns of introgression.

Phylogeography of endemic toads and post-Pliocene persistence of the Brazilian Atlantic Forest.

The Plio-Pleistocene refugia hypothesis recently gained support in explaining Brazilian Atlantic Forest megadiversity from combined analyses of species paleodistributions and genetic diversity. Here we examine genetic differentiation and historical distributions in the Rhinella crucifer group of toads, endemic to and widely distributed within this biome. We analyzed sequences of mitochondrial (control region, ND1, and ND2) and nuclear (beta-crystallin and rhodopsin) DNA markers from 65 individuals representing five species. We found deep structure across the range at mitochondrial markers; genetic diversity is geographically structured in four main haplotype clades with the oldest divergence, dated to the Pliocene, between the southernmost populations and other regions of the species' range. Remaining populations are distributed in haplotype clades that may have diverged throughout the Pleistocene. Our paleoecological distribution models support a scenario of habitat fragmentation associated with glacial cycling, but we found limited congruence of phylogeographic patterns with the refugia. We found that some genetic breaks geographically coincide with putative barriers associated to neotectonic activity, but finer-scale sampling will be necessary to test the relative importance of distinct isolation mechanisms. Overall, the data refute the recently proposed hypothesis of a southern Holocene colonization of the Atlantic Forest from northern refugia, suggesting instead persistence of forested habitats in the south. Our unexpected results underscore the need to consider distinct organismal histories in planning biome-level conservation. We discuss species correspondence to clades recovered in our phylogenetic analyses.

Gradual adaptation toward a range-expansion phenotype initiated the global radiation of toads.

Recent studies have identified range expansion as a potential driver of speciation. Yet it remains poorly understood how, under identical extrinsic settings, differential tendencies for geographic movement of taxa originate and subsequently affect diversification. We identified multiple traits that predict large distributional ranges in extant species of toads (Bufonidae) and used statistical methods to define and phylogenetically reconstruct an optimal range-expansion phenotype. Our results indicate that lineage-specific range-shifting abilities increased through an accumulation of adaptive traits that culminated in such a phenotype. This initiated the episode of global colonization and triggered the major radiation of toads. Evolution toward a range-expansion phenotype might be crucial to understanding both ancient widespread radiations and the evolutionary background of contemporary invasive species such as the cane toad.

Chromosomal studies in four species of genus Chaunus (Bufonidae, Anura): localization of telomeric and ribosomal sequences after fluorescence in situ hybridization (FISH).

Fluorescence in situ hybridization (FISH) using telomeric and ribosomal sequences was performed in four species of toad genus Chaunus: C. ictericus, C. jimi, C. rubescens and C. schneideri. Analyses based on conventional, C-banding and Ag-NOR staining were also carried out. The four species present a 2n = 22 karyotype, composed by metacentric and submetacentric chromosomes, which were indistinguishable either after conventional staining or banding techniques. Constitutive heterochromatin was predominantly located at pericentromeric regions, and telomeric sequences (TTAGGG)(n) were restricted to the end of all chromosomes. Silver staining revealed Ag-NORs located at the short arm of pair 7, and heteromorphism in size of NOR signals was also observed. By contrast, FISH with ribosomal probes clearly demonstrated absence of any heteromorphism in size of rDNA sequences, suggesting that the difference observed after Ag-staining should be attributed to differences in chromosomal condensation and/or gene activity rather than to the number of ribosomal cistrons.