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

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

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.

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.

Gene and species trees of a Neotropical group of treefrogs: genetic diversification in the Brazilian Atlantic Forest and the origin of a polyploid species.

The Neotropical Phyllomedusa burmeisteri treefrog group includes four diploid (P. bahiana, P. burmeisteri, P. distincta and P. iheringii) and one tetraploid (P. tetraploidea) forms. Here we use mitochondrial and nuclear sequence variation from across its range to verify if recognized morphospecies correspond to phylogenetic clades, examine the origin of the polyploid P. tetraploidea, and compare range wide patterns of diversification to those of other BAF organisms. We compared single gene trees with one Bayesian multi-gene tree, and one Bayesian species tree inferred under a coalescent framework. Our mtDNA phylogenetic analyses showed that P. bahiana, P. burmeisteri and P. iheringii correspond to monophyletic clades, while P. distincta and P. tetraploidea were paraphyletic. The nuclear gene trees were concordant in revealing two moderately supported groups including (i) P. bahiana and P. burmeisteri (northern species) and (ii) P. distincta, P.tetraploidea and P. iheringii (southern species). The multi-gene tree and the species tree retrieved similar topologies, giving high support to the northern and southern clades, and to the sister-taxa relationship between P. tetraploidea and P. distincta. Estimates of (t)MRCA suggest a major split within the P. burmeisteri group at ≈ 5 Myr (between northern and southern groups), while the main clades were originated between ≈ 0.4 and 2.5 Myr, spanning the late Pliocene and Pleistocene. Patterns of geographic and temporal diversification within the group were congruent with those uncovered for other co-distributed organisms. Independent paleoecological and geological data suggest that vicariance associated with climatic oscillations and neotectonic activity may have driven lineage divergence within the P. burmeisteri group. P. tetraploidea probably originated from polyploidization of P. distincta or from a common ancestor.

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.

Strong selection against hybrids at a hybrid zone in the Ensatina ring species complex and its evolutionary implications.

The analysis of interactions between lineages at varying levels of genetic divergence can provide insights into the process of speciation through the accumulation of incompatible mutations. Ring species, and especially the Ensatina eschscholtzii system exemplify this approach. The plethodontid salamanders E. eschscholtzii xanthoptica and E. eschscholtzii platensis hybridize in the central Sierran foothills of California. We compared the genetic structure across two transects (southern and northern Calaveras Co.), one of which was resampled over 20 years, and examined diagnostic molecular markers (eight allozyme loci and mitochondrial DNA) and a diagnostic quantitative trait (color pattern). Key results across all studies were: (1) cline centers for all markers were coincident and the zones were narrow, with width estimates of 730 m to 2000 m; (2) cline centers at the northern Calaveras transect were coincident between 1981 and 2001, demonstrating repeatability over five generations; (3) there were very few if any putative F1s, but a relatively high number of backcrossed individuals in the central portion of transects; and (4) we found substantial linkage disequilibrium in all three studies and strong heterozygote deficit both in northern Calaveras, in 2001, and southern Calaveras. Both linkage disequilibrium and heterozygote deficit showed maximum values near the center of the zones. Using estimates of cline width and dispersal, we infer strong selection against hybrids. This is sufficient to promote accumulation of differences at loci that are neutral or under divergent selection, but would still allow for introgression of adaptive alleles. The evidence for strong but incomplete isolation across this centrally located contact is consistent with theory suggesting a gradual increase in postzygotic incompatibility between allopatric populations subject to divergent selection and reinforces the value of Ensatina as a system for the study of divergence and speciation at multiple stages.

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.

Isolation and characterization of 15 polymorphic microsatellites in the Plethodontid salamander Ensatina eschscholtzii.

We developed 15 new polymorphic microsatellites for the plethodontid salamander Ensatina eschscholtzii. Loci were isolated from a genomic library from Ensatina eschscholtzii xanthoptica enriched for (AAAG)(n) repetitive elements. The number of alleles per locus ranged from 4 to 20 (mean 9) in the sampled population. Observed heterozygosity ranged from 0.37 to 1. None of the loci deviated from Hardy-Weinberg equilibrium or showed significant linkage disequilibrium after a Bonferroni correction for multiple comparisons. All loci amplified in the six other subspecies of the Ensatina eschscholtzii complex. These new markers will prove useful in measuring gene flow and population structure as well as patterns of mating and sperm use in Ensatina.

Anfíbios do Estado de São Paulo, Brasil: conhecimento atual e perspectivas / Amphibians of São Paulo State, Brazil: state-of-art and perspectives

A última lista de espécies do Estado de São Paulo foi atualizada, totalizando 236 espécies de anfíbios, das quais 230 são anuros e seis são Gymnophiona. Foram removidos da lista Bokermannohyla gouveai e Sphaenorhynchus surdus por não ocorrerem no Estado de São Paulo. O número de espécies de anuros registrado representa 27% da riqueza de espécies do país e um aumento de 31% em relação ao número de espécies registradas para o Estado em 1998, demonstrando que, a despeito do Estado de São Paulo ser a região brasileira onde os anuros foram mais estudados, o número de espécies conhecidas deve aumentar nos próximos anos. Foram evidenciadas duas lacunas geográficas importantes: a região sudoeste do Estado, principalmente na bacia hidrográfica do rio Paranapanema e a região nordeste, principalmente na divisa entre os Estados de Minas Gerais e São Paulo. Apesar de ambas terem sido amostradas recentemente, ainda há carência de informações. O estado atual do conhecimento e perspectivas nas áreas de taxonomia, sistemática, ecologia e conservação são avaliados.

The last list of species of the state of São Paulo State was updated and totaled 236 species of amphibians, 230 of which are anurans and six are caecilians. Bokermannohyla gouveai and Sphaenorhynchus surdus were removed from this list, because they did not occur in the State of São Paulo. The number of anuran species recorded comprise 27% of the species richness of the country and an increase by 31% in the number of species recorded for the state since 1998. Thus, despite the State of São Paulo be the Brazilian region where the anurans have been most studied, these data show that the number of known species tends to increase in the next years. We have identified two major geographical gaps of inventory: the southwest of the state, especially in the Paranapanema river basin and the northeast region, mainly at the border between the States of Minas Gerais and São Paulo. Although both have been sampled recently, information is still lacking. The current state of knowledge and perspectives in the areas such as taxonomy, systematics, ecology and conservation are evaluated.