Disentangling the contemporary and historical effects of landscape on the population genomic variation of two bird species restricted to the highland forest enclaves of northeastern Brazil.

Investigating the impact of landscape features on patterns of genetic variation is crucial to understand spatially dependent evolutionary processes. Here, we assess the population genomic variation of two bird species (Conopophaga cearae and Sclerurus cearensis) through the Caatinga moist forest enclaves in northeastern Brazil. To infer the evolutionary dynamics of bird populations through the Late Quaternary, we used genome-wide polymorphism data obtained from double-digestion restriction-site-associated DNA sequencing (ddRADseq), and integrated population structure analyses, historical demography models, paleodistribution modeling, and landscape genetics analyses. We found the population differentiation among enclaves to be significantly related to the geographic distance and historical resistance across the rugged landscape. The climate changes at the end of the Pleistocene to the Holocene likely triggered synchronic population decline in all enclaves for both species. Our findings revealed that both geographic distance and historical connectivity through highlands are important factors that can explain the current patterns of genetic variation. Our results further suggest that levels of population differentiation and connectivity cannot be explained purely on the basis of contemporary environmental conditions. By combining historical demographic analyses and niche modeling predictions in a historical framework, we provide strong evidence that climate fluctuations of the Quaternary promoted population differentiation and a high degree of temporal synchrony among population size changes in both species.

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.

Genetic traces of hybrid zone movement across a fragmented habitat.

Theoretical and empirical studies suggest that the structure and position of hybrid zones can change over time. Evidence for moving hybrid zones has been directly inferred by repeated sampling over time, or indirectly through the detection of genetic footprints left by the receding species and the resulting asymmetric patterns of introgression across markers. We here investigate a hybrid zone formed by two subspecies of the Iberian golden-striped salamander, Chioglossa lusitanica, using a panel of 35 nuclear loci (31 SNPs and 4 allozymes) and one mitochondrial locus in a transect in central Portugal. We found concordant and coincident clines for most of the nuclear loci (n = 22, 63%), defining a narrow hybrid zone of ca. 6 km wide, with the centre positioned ca. 15 km south of the Mondego River. Asymmetric introgression was observed at another 14 loci. Their clines are displaced towards the north, with positions located either close to the Mondego River (n = 6) or further northwards (n = 8). We interpret these profiles as genetic traces of the southward displacement of C. lusitanica lusitanica by C. l. longipes over the wider Mondego River valley. We noted the absence of significant linkage disequilibrium, and we inferred low levels of effective selection per locus against hybrids, suggesting that introgression in the area of species replacement occurred under a neutral diffusion process. A species distribution model suggests that the C. lusitanica hybrid zone coincides with a narrow corridor of fragmented habitat. From the position of the displaced clines, we infer that patches of locally suitable habitat trapped some genetic variants that became disassociated from the southward moving hybrid zone. This study highlights the influence of habitat availability on hybrid zone movement.

Evaluating surrogates of genetic diversity for conservation planning.

Protected-area systems should conserve intraspecific genetic diversity. Because genetic data require resources to obtain, several approaches have been proposed for generating plans for protected-area systems (prioritizations) when genetic data are not available. Yet such surrogate-based approaches remain poorly tested. We evaluated the effectiveness of potential surrogate-based approaches based on microsatellite genetic data collected across the Iberian Peninsula for 7 amphibian and 3 reptilian species. Long-term environmental suitability did not effectively represent sites containing high genetic diversity (allelic richness). Prioritizations based on long-term environmental suitability had similar performance to random prioritizations. Geographic distances and resistance distances based on contemporary environmental suitability were not always effective surrogates for identification of combinations of sites that contain individuals with different genetic compositions. Our results demonstrate that population genetic data based on commonly used neutral markers can inform prioritizations, and we could not find an adequate substitute. Conservation planners need to weigh the potential benefits of genetic data against their acquisition costs.

Evaluación de los Sustitutos de la Diversidad Genética para la Planeación de la Conservación Resumen Los sistemas de áreas protegidas deberían conservar la diversidad genética intraespecífica. Ya que para obtener datos genéticos se requieren recursos, se han propuesto distintas estrategias para generar los planes para los sistemas de áreas protegidas (priorizaciones) cuando los datos genéticos no están disponibles. A pesar de lo anterior, dichas estrategias basadas en sustitutos han sido poco evaluadas. Evaluamos la efectividad del potencial de las estrategias basadas en sustitutos cuya base son los datos genéticos de microsatélites obtenidos en toda la Península Ibérica y correspondientes a siete especies de anfibios y a tres de reptiles. La idoneidad ambiental a largo plazo no representó efectivamente los sitios que contienen una diversidad genética alta (riqueza de alelos). Las priorizaciones basadas en la idoneidad ambiental a largo plazo tuvieron un desempeño similar a las priorizaciones aleatorias. Las distancias geográficas y las distancias de resistencia basadas en la idoneidad ambiental contemporánea no siempre fueron sustitutos efectivos para la identificación de las combinaciones de sitios que contienen individuos con composiciones genéticas diferentes. Nuestros resultados demuestran que los datos genéticos de una población basados en marcadores neutrales de uso común pueden informar a las priorizaciones y que no pudimos encontrar un sustituto adecuado. Los planificadores de la conservación necesitan sopesar los beneficios potenciales de los datos genéticos contra sus costos de adquisición.

The roles of vicariance and dispersal in the differentiation of two species of the Rhinella marina species complex.

The high levels of Neotropical biodiversity are commonly associated with the intense Neogene-Quaternary geological events and climate dynamics. Here, we investigate the evolutionary history of two species of Neotropical closely related amphibians (R. horribilis and R. marina). We combine published data with new mitochondrial DNA sequences and multiple nuclear markers, including 12 microsatellites. The phylogenetic analyses showed support for grouping the samples in two main clades; R. horribilis (Central America and Mexico) and R. marina (South America east of the Andes). However, the phylogenetic inferences also show an evident mito-nuclear discordance. We use Approximate Bayesian Computation (ABC) to test the role of different events in the diversification between the two groups recovered. We found that both species were affected primarily by a recent Pleistocene divergence, which was similar to the divergence estimate revealed by the Isolation-with-Migration model, under persistent bidirectional gene flow through time. We provide the first evidence that R. horribilis is differentiated from the South American R. marina at the nuclear level supporting the taxonomic status of R. horribilis, which has been controversial for more than a century.

Discordant patterns of introgression across a narrow hybrid zone between two cryptic lineages of an Iberian endemic newt.

The study of natural hybrid zones can illuminate aspects of lineage divergence and speciation in morphologically cryptic taxa. We studied a hybrid zone between two highly divergent but morphologically similar lineages (south-western and south-eastern) of the Iberian endemic Bosca's newt (Lissotriton boscai) in SW Iberia with a multilocus dataset (microsatellites, nuclear and mitochondrial genes). STRUCTURE and NEWHYBRIDS analyses retrieved few admixed individuals, which classified as backcrosses involving parental individuals of the south-western lineage. Our results show asymmetric introgression of mtDNA beyond the contact from this lineage into the south-eastern lineage. Analysis of nongeographic introgression patterns revealed asymmetries in the direction of introgression, but except for mtDNA, we did not find evidence for nonconcordant introgression patterns across nuclear loci. Analysis of a 150-km transect across the hybrid zone showed broadly coincident cline widths (ca. 3.2-27.9 km), and concordant cline centres across all markers, except for mtDNA that is displaced ca. 60 km northward. Results from ecological niche modelling show that the hybrid zone is in a climatically homogenous area with suitable habitat for the species, suggesting that contact between the two lineages is unlikely to occur further south as their distributions are currently separated by an extensive area of unfavourable habitat. Taken together, our findings suggest the genetic structure of this hybrid zone results from the interplay of historical (biogeographic) and population-level processes. The narrowness and coincidence of genetic clines can be explained by weak selection against hybrids and reflect a degree of reproductive isolation that is consistent with cryptic speciation.

A dynamic continental moisture gradient drove Amazonian bird diversification.

The Amazon is the primary source of Neotropical diversity and a nexus for discussions on processes that drive biotic diversification. Biogeographers have focused on the roles of rivers and Pleistocene climate change in explaining high rates of speciation. We combine phylogeographic and niche-based paleodistributional projections for 23 upland terra firme forest bird lineages from across the Amazon to derive a new model of regional biological diversification. We found that climate-driven refugial dynamics interact with dynamic riverine barriers to produce a dominant pattern: Older lineages in the wetter western and northern parts of the Amazon gave rise to lineages in the drier southern and eastern parts. This climate/drainage basin evolution interaction links landscape dynamics with biotic diversification and explains the east-west diversity gradients across the Amazon.

Draft genome assembly of the invasive cane toad, Rhinella marina.

Background: The cane toad (Rhinella marina formerly Bufo marinus) is a species native to Central and South America that has spread across many regions of the globe. Cane toads are known for their rapid adaptation and deleterious impacts on native fauna in invaded regions. However, despite an iconic status, there are major gaps in our understanding of cane toad genetics. The availability of a genome would help to close these gaps and accelerate cane toad research. Findings: We report a draft genome assembly for R. marina, the first of its kind for the Bufonidae family. We used a combination of long-read Pacific Biosciences RS II and short-read Illumina HiSeq X sequencing to generate 359.5 Gb of raw sequence data. The final hybrid assembly of 31,392 scaffolds was 2.55 Gb in length with a scaffold N50 of 168 kb. BUSCO analysis revealed that the assembly included full length or partial fragments of 90.6% of tetrapod universal single-copy orthologs (n = 3950), illustrating that the gene-containing regions have been well assembled. Annotation predicted 25,846 protein coding genes with similarity to known proteins in Swiss-Prot. Repeat sequences were estimated to account for 63.9% of the assembly. Conclusions: The R. marina draft genome assembly will be an invaluable resource that can be used to further probe the biology of this invasive species. Future analysis of the genome will provide insights into cane toad evolution and enrich our understanding of their interplay with the ecosystem at large.

Improving amphibian genomic resources: a multitissue reference transcriptome of an iconic invader.

Background: Cane toads (Rhinella marina) are an iconic invasive species introduced to 4 continents and well utilized for studies of rapid evolution in introduced environments. Despite the long introduction history of this species, its profound ecological impacts, and its utility for demonstrating evolutionary principles, genetic information is sparse. Here we produce a de novo transcriptome spanning multiple tissues and life stages to enable investigation of the genetic basis of previously identified rapid phenotypic change over the introduced range. Findings: Using approximately 1.9 billion reads from developing tadpoles and 6 adult tissue-specific cDNA libraries, as well as a transcriptome assembly pipeline encompassing 100 separate de novo assemblies, we constructed 62 202 transcripts, of which we functionally annotated ∼50%. Our transcriptome assembly exhibits 90% full-length completeness of the Benchmarking Universal Single-Copy Orthologs data set. Robust assembly metrics and comparisons with several available anuran transcriptomes and genomes indicate that our cane toad assembly is one of the most complete anuran genomic resources available. Conclusions: This comprehensive anuran transcriptome will provide a valuable resource for investigation of genes under selection during invasion in cane toads, but will also greatly expand our general knowledge of anuran genomes, which are underrepresented in the literature. The data set is publically available in NCBI and GigaDB to serve as a resource for other researchers.

Integration of molecular, bioacoustical and morphological data reveals two new cryptic species of Pelodytes (Anura, Pelodytidae) from the Iberian Peninsula.

Parsley frogs (Pelodytes) comprise the only genus in the family Pelodytidae, an ancient anuran lineage that split from their closest relatives over 140 million years ago. Pelodytes is a Palearctic group restricted to Western Eurasia including three extant species: the eastern species P. caucasicus, endemic to the Caucasus area, and two closely related species inhabiting Western Europe: the Iberian endemic P. ibericus and the more widespread P. punctatus. Previous studies based on mitochondrial and nuclear DNA markers have revealed the existence of two additional lineages of Parsley frogs in the Iberian Peninsula, which have been flagged as candidate species. Here, we integrate novel molecular, morphological and bioacoustical data to assess the differentiation of the four western Parsley frog lineages. Species trees and Bayesian population assignment analyses based on nuclear markers confirm previous studies and concordantly delineate four parapatric lineages with narrow hybrid zones. Mitochondrial divergence is low (< 2% pairwise distances in the 16S rRNA gene), in line with previously reported low mitochondrial substitution rates in non-neobatrachian frogs. Based on concordance between mitochondrial and nuclear markers, we conclude that four species of Parsley frogs occur in Western Europe: Pelodytes punctatus, distributed from northern Italy to northeastern Spain; Pelodytes ibericus, inhabiting southern Spain and southern Portugal; Pelodytes atlanticus sp. nov., from the Portuguese Atlantic coast; and Pelodytes hespericus sp. nov., occurring in central and eastern Spain. However, bioacoustical and morphological differentiation of these species is low, with no obvious and qualitative diagnostic characters allowing full species discrimination. Differences in the relative size of metacarpal tubercles exist but this character is variable. Pelodytes ibericus and Pelodytes atlanticus are smaller than the other two species, and P. ibericus has shorter limbs and various distinctive osteological characters. Bioacoustically, the pattern by which two different note types are combined in advertisement calls separates P. hespericus from the remaining species. Despite these differences, we emphasize that the taxonomic status of all four western Parsley frogs requires additional investigation, especially the patterns of genetic admixture across contact zones. While a status of separate species best conforms to the currently available data, alternative hypotheses are also discussed.

De novo transcriptome assembly and polymorphism detection in two highly divergent evolutionary units of Bosca's newt (Lissotriton boscai) endemic to the Iberian Peninsula.

This article reports the de novo transcriptome assemblies of two highly divergent evolutionary units of the Iberian endemic Bosca's newt, Lissotriton boscai. These two units are distributed mostly allopatrically but overlap in the central-southwestern coastal region of Portugal. The resources we provide include the raw sequence reads, the assembled transcripts, the annotation and SNPs called for both lineages.

Concerted evolution in the mitochondrial control region of the Amazon small-bodied frog Pseudopaludicola canga (Anura, Leiuperidae).

This study presents evidence of concerted evolution in the mitochondrial control region of the frog Pseudopaludicola canga. Four repeat units of 88 bp (as well as a fifth, incomplete unit) were observed in the 5' domain, with the duplicated segments of the same specimen being more related to one another than to the equivalent regions in other specimens, as a result of concerted evolution. We highlight that drawing conclusions from phylogeographical analysis using the control region containing VNTRs must be interpreted with caution, because it violated a basic assumption of phylogeny, since the regions cannot be treated as independent characters.

Patterns of Genetic Variability in Island Populations of the Cane Toad (Rhinella marina) from the Mouth of the Amazon.

The Amazonian coast has several unique geological characteristics resulting from the interaction between drainage pattern of the Amazon River and the Atlantic Ocean. It is one of the most extensive and sedimentologically dynamic regions of the world, with a large number of continental islands mostly formed less than 10,000 years ago. The natural distribution of the cane toad (Rhinella marina), one of the world's most successful invasive species, in this complex Amazonian system provides an intriguing model for the investigation of the effects of isolation or the combined effects of isolation and habitat dynamic changes on patterns of genetic variability and population differentiation. We used nine fast-evolving microsatellite loci to contrast patterns of genetic variability in six coastal (three mainlands and three islands) populations of the cane toad near the mouth of the Amazon River. Results from Bayesian multilocus clustering approach and Discriminant Analyses of Principal Component were congruent in showing that each island population was genetically differentiated from the mainland populations. All FST values obtained from all pairwise comparisons were significant, ranging from 0.048 to 0.186. Estimates of both recent and historical gene flow were not significantly different from zero across all population pairs, except the two mainland populations inhabiting continuous habitats. Patterns of population differentiation, with a high level of population substructure and absence/restricted gene flow, suggested that island populations of R. marina are likely isolated since the Holocene sea-level rise. However, considering the similar levels of genetic variability found in both island and mainland populations, it is reliable to assume that they were also isolated for longer periods. Given the genetic uniqueness of each cane toad population, together with the high natural vulnerability of the coastal regions and intense human pressures, we suggest that these populations should be treated as discrete units for conservation management purposes.

Molecular phylogeny and diversification of a widespread Neotropical rainforest bird group: The Buff-throated Woodcreeper complex, Xiphorhynchus guttatus/susurrans (Aves: Dendrocolaptidae).

The genus Xiphorhynchus is a species rich avian group widely distributed in Neotropical forests of Central and South America. Although recent molecular studies have improved our understanding of the spatial patterns of genetic diversity in some species of this genus, most are still poorly known, including their taxonomy. Here, we address the historical diversification and phylogenetic relationships of the X. guttatus/susurrans complex, using data from two mitochondrial (cyt b and ND2) and one nuclear (ß-fibint7) genes. Phylogenetic relationships were inferred with both gene trees and a Bayesian-based species tree under a coalescent framework (∗BEAST). With exception of the nuclear ß-fibint7 gene that produced an unresolved tree, both mtDNA and the species tree showed a similar topology and were congruent in recovering five main clades with high statistical support. These clades, however, are not fully concordant with traditional delimitation of some X. guttatus subspecies, since X. g. polystictus, X. g. guttatus, and X. g. connectens are not supported as distinct clades. Interestingly, these three taxa are more closely related to the mostly trans-Andean X. susurrans than the other southern and western Amazonian subspecies of X. guttatus, which constitutes a paraphyletic species. Timing estimates based on the species tree indicated that diversification in X. guttatus occurred between the end of the Pliocene and early Pleistocene, likely associated with the formation of the modern Amazon River and its main southern tributaries (Xingu, Tocantins, and Madeira), in addition to climate-induced changes in the distribution of rainforest biomes. Our study supports with an enlarged dataset a previous proposal for recognizing at least three species level taxa in the X. guttatus/susurrans complex: X. susurrans, X. guttatus, and X. guttatoides.

Genomic resources notes accepted 1 August 2014-30 September 2014.

This article documents the public availability of (i) transcriptome sequence data, assembly and annotation, and single nucleotide polymorphisms (SNPs) for the cone snail Conus miliaris; (ii) a set of SNP markers for two biotypes from the Culex pipiens mosquito complex; (iii) transcriptome sequence data, assembly and annotation for the mountain fly Drosophila nigrosparsa; (iv) transcriptome sequence data, assembly and annotation and SNPs for the Neotropical toads Rhinella marina and R. schneideri; and (v) partial genomic sequence assembly and annotation for 35 spiny lizard species (Genus Sceloporus).

Molecular evidence for cryptic candidate species in Iberian Pelodytes (Anura, Pelodytidae).

Species delineation is a central topic in evolutionary biology, with current efforts focused on developing efficient analytical tools to extract the most information from molecular data and provide objective and repeatable results. In this paper we use a multilocus dataset (mtDNA and two nuclear markers) in a geographically comprehensive population sample across Iberia and Western Europe to delineate candidate species in a morphologically cryptic species group, Parsley frogs (genus Pelodytes). Pelodytes is the sole extant representative of an ancient, historically widely distributed anuran clade that currently includes three species: P. caucasicus in the Caucasus; P. punctatus in Western Europe, from Portugal to North-Western Italy; and P. ibericus in Southern Iberia. Phylogenetic analyses recovered four major well-supported haplotype clades in Western Europe, corresponding to well demarcated geographical subdivisions and exhibiting contrasting demographic histories. Splitting times date back to the Plio-Pleistocene and are very close in time. Species-tree analyses recovered one of these species lineages, corresponding to P. ibericus (lineage B), as the sister taxon to the other three major species lineages, distributed respectively in: western Iberian Peninsula, along the Atlantic coast and part of central Portugal (lineage A); Central and Eastern Spain (lineage C); and North-eastern Spain, France and North-western Italy (lineage D). The latter is in turn subdivided into two sub-clades, one in SE France and NW Italy and the other one from NE Spain to NW France, suggesting the existence of a Mediterranean-Atlantic corridor along the Garonne river. An information theory-based validation approach implemented in SpedeSTEM supports an arrangement of four candidate species, suggesting the need for a taxonomic revision of Western European Pelodytes.

Realized niche shift during a global biological invasion.

Accurate forecasts of biological invasions are crucial for managing invasion risk but are hampered by niche shifts resulting from evolved environmental tolerances (fundamental niche shifts) or the presence of novel biotic and abiotic conditions in the invaded range (realized niche shifts). Distinguishing between these kinds of niche shifts is impossible with traditional, correlative approaches to invasion forecasts, which exclusively consider the realized niche. Here we overcome this challenge by combining a physiologically mechanistic model of the fundamental niche with correlative models based on the realized niche to study the global invasion of the cane toad Rhinella marina. We find strong evidence that the success of R. marina in Australia reflects a shift in the species' realized niche, as opposed to evolutionary shifts in range-limiting traits. Our results demonstrate that R. marina does not fill its fundamental niche in its native South American range and that areas of niche unfilling coincide with the presence of a closely related species with which R. marina hybridizes. Conversely, in Australia, where coevolved taxa are absent, R. marina largely fills its fundamental niche in areas behind the invasion front. The general approach taken here of contrasting fundamental and realized niche models provides key insights into the role of biotic interactions in shaping range limits and can inform effective management strategies not only for invasive species but also for assisted colonization under climate change.

Cryptic genetic diversity is paramount in small-bodied amphibians of the genus Euparkerella (Anura: Craugastoridae) endemic to the Brazilian Atlantic forest.

Morphological similarity associated to restricted distributions and low dispersal abilities make the direct developing "Terrarana" frogs of the genus Euparkerella a good model for examining diversification processes. We here infer phylogenetic relationships within the genus Euparkerella, using DNA sequence data from one mitochondrial and four nuclear genes coupled with traditional Bayesian phylogenetic reconstruction approaches and more recent coalescent methods of species tree inference. We also used Bayesian clustering analysis and a recent Bayesian coalescent-based approach specifically to infer species delimitation. The analysis of 39 individuals from the four known Euparkerella species uncovered high levels of genetic diversity, especially within the two previously morphologically-defined E. cochranae and E. brasiliensis. Within these species, the gene trees at five independent loci and trees from combined data (concatenated dataset and the species tree) uncovered six deeply diverged and geographically coherent evolutionary units, which may have diverged between the Miocene and the Pleistocene. These six units were also uncovered in the Bayesian clustering analysis, and supported by the Bayesian coalescent-based species delimitation (BPP), and Genealogical Sorting Index (GSI), providing thus strong evidence for underestimation of the current levels of diversity within Euparkerella. The cryptic diversity now uncovered opens new opportunities to examine the origins and maintenance of microendemism in the context of spatial heterogeneity and/or human induced fragmentation of the highly threatened Brazilian Atlantic forest hotspot.

Cryptic patterns of diversification of a widespread Amazonian woodcreeper species complex (Aves: Dendrocolaptidae) inferred from multilocus phylogenetic analysis: implications for historical biogeography and taxonomy.

Inferring evolutionary relationships between recently diverged taxa is still challenging, especially taking into account the likely occurrence of incomplete lineage sorting and/or introgression. The Xiphorhynchus pardalotus/ocellatus species complex includes between two to three polytypic species and eight to nine subspecies distributed throughout most of lowland Amazonia and the foothills of the eastern Andes. To understand its historical diversification and address the main unsettled issues of phylogenetic relationships and taxonomy, we apply several approaches using data from two mitochondrial (Cyt b and ND2) and three nuclear genes (ß-fibint7, CPZint3 and CRYAAint1) for all described species and most subspecies of this complex. We compared single gene trees with a multilocus concatenated tree and Bayesian species tree inferred under a coalescent framework ((*)BEAST). Our results showed a general pattern of incongruence among gene trees and multilocus trees. Despite of this, the coalescent-based species tree analysis supports the sister-taxa relationship of X. ocellatus and X. chunchotambo, while X. pardalotus comes out as the basal taxon. With exception of the last, our results revealed within both X. ocellatus and X. chunchotambo high levels of genetic differentiation (p-distances 0.5-5.5%) with well-supported lineages. Our phylogenetic analyses showed several incongruences with current subspecies taxonomy, revealing that X. o. ocellatus is paraphyletic relative to X. o. perplexus, and the currently recognized subspecies X. c. napensis corresponds to two distinct evolutionary lineages, which are not supported as sister-lineages. In addition, the deep level of genetic divergence between X. o. beauperthuysii and the extant subspecies of X. ocellatus is more consistent with species-level differences found in this complex. Divergence time estimates were consistent with a historical scenario of intense population subdivision and speciation during the Early-mid Pleistocene. The spatial pattern and timing of diversification overlap broadly with that reported for other Amazonian vertebrate lineages.

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.