Evolution in the Genus Rhinella: A Total Evidence Phylogenetic Analysis of Neotropical True Toads (Anura: Bufonidae)

True toads of the genus Rhinella are among the most common and diverse group of Neotropical anurans. These toads are widely distributed throughout South America, inhabiting a great diversity of environments and ecoregions. Currently, however, the genus is defined solely on the basis of molecular characters, and it lacks a proper diagnosis. Although some phenetic species groups have traditionally been recognized within Rhinella, the monophyly of some of them have been rejected in previous phylogenetic analyses, and many species remain unassigned to these poorly defined groups. Additionally, the identity and taxonomy of several species are problematic and hinder the specific recognition and description of undescribed taxa. In this work, we first perform phylogenetic analyses of separate mitochondrial and nuclear datasets to test the possible occurrence of hybridiza-tion and/or genetic introgression in the genus. The comparative analysis of both datasets revealed unidirectional mitochondrial introgressions of an unknown parental species into R . horribilis (“ghost introgression”) and of R . dorbignyi into R . bernardoi; therefore, the mitochondrial and nuclear data-sets of these species were considered separately in subsequent analyses. We performed total-evidence phylogenetic analyses that included revised molecular (four mitochondrial and five nuclear genes) and phenotypic (90 characters) datasets for 83 nominal species of Rhinella, plus several undescribed and problematic species and multiple outgroups. Results demonstrate that Rhinella was nonmono-phyletic due to the position of R . ceratophrys, which was recovered as the sister taxon of Rhaebo nasicus with strong support. Among our outgroups, the strongly supported Anaxyrus + Incilius is the sister clade of all other species of Rhinella. Once R . ceratophrys is excluded, the genus Rhinellais monophyletic, well supported, and composed of two major clades. One of these is moderately supported and includes species of the former R . spinulosa Group (including R . gallardoi); the mono-phyletic R . granulosa, R . crucifer, and R . marina Groups; and a clade composed of the mitochondrial sequences of R . horribilis. The other major clade is strongly supported and composed of all the spe-cies from the non-monophyletic R . veraguensis and R . margaritifera Groups, the former R . acrolophaGroup, and R . sternosignata. Consistent with these results, we define eight species groups of Rhinella that are mostly diagnosed by phenotypic synapomorphies in addition to a combination of morpho-logical character states. Rhinella sternosignata is the only species that remains unassigned to any group. We also synonymize nine species, treat three former subspecies as full species, and suggest that 15 lineages represent putative undescribed species. Lastly, we discuss the apparently frequent occurrence of hybridization, deep mitochondrial divergence, and “ghost introgression”; the incomplete phenotypic evidence (including putative character systems that could be used for future phy-logenetic analyses); and the validity of the known fossil record of Rhinella as a source of calibration points for divergence dating analyses.Peer reviewe

Data from: 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

crystallin

alignment for the beta-crystallin fragmen

alphapolypeptide

alignment for the intron 1 of the A alpha polypeptide fragmen

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

<p>Abstract</p> <p>Background</p> <p>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 <it>Rhinella crucifer</it> 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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>Our results confirmed that current taxonomy does not represent evolutionary units in the <it>Rhinella crucifer</it> group. Correspondence between genetically distinguishable units and the currently recognized species is only possible for <it>Rhinella henseli</it> and <it>R. inopina</it>. The recognition of other species relies on the reassessment of the geographic range of <it>R. crucifer</it>, the examination of the type series of <it>R. ornata</it> for hybrids, and on the use of additional markers to verify the genetic distinctiveness of <it>R. abei.</it> We state that <it>R. pombali</it> should not remain a valid species since its description appears to be based on hybrids, and that the name <it>R. pombali</it> should be considered a synonym of both <it>R. crucifer</it> and <it>R. ornata</it>. The fifth inferred but undescribed genetic unit may represent a new species. Our results underscore the potential of the <it>R. crucifer</it> 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.</p

ABC_NxC

perl script for the ABC analysis with regions N and

tableS1

Voucher numbers, locality, coordinates, region, and genbank assession numbers for individuals used in this stud

ABCwithinC

perl script for the ABC analysis within region