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Empirical phylogeographic studies have progressively sampled greater numbers of loci over time, in part motivated by theoretical papers showing that estimates of key demographic parameters improve as the number of loci increases. Recently, next-generation sequencing has been applied to questions about organismal history, with the promise of revolutionizing the field. However, no systematic assessment of how phylogeographic data sets have changed over time with respect to overall size and information content has been performed. Here, we quantify the changing nature of these genetic data sets over the past 20 years, focusing on papers published in Molecular Ecology. We found that the number of independent loci, the total number of alleles sampled and the total number of single nucleotide polymorphisms (SNPs) per data set has improved over time, with particularly dramatic increases within the past 5 years. Interestingly, uniparentally inherited organellar markers (e.g. animal mitochondrial and plant chloroplast DNA) continue to represent an important component of phylogeographic data. Singlespecies studies (cf. comparative studies) that focus on vertebrates (particularly fish and to some extent, birds) represent the gold standard of phylogeographic data collection. Based on the current trajectory seen in our survey data, forecast modelling indicates that the median number of SNPs per data set for studies published by the end of the year 2016 may approach~20 000. This survey provides baseline information for understanding the evolution of phylogeographic data sets and underscores the fact that development of analytical methods for handling very large genetic data sets will be critical for facilitating growth of the field.Keywords: DNA sequences, information content, phylogeography, sampling, single nucleotide polymorphisms, temporal trends IntroductionPhylogeographers have been working to collect multilocus data ever since a series of theoretical papers pertinent to the discipline demonstrated that estimates of key demographic parameters improve as the number of loci increases (e.g. Edwards & Beerli 2000;Hey & Nielsen 2004;Felsenstein 2006;Carling & Brumfield 2007). Recent improvements in DNA sequencing technology have led to platforms with greater speed, resolution and/or output (e.g. Margulies et al. 2005;Bentley et al. 2008;Rothberg et al. 2011) when compared to the traditional Sanger method. These technological advances, together with the development of general-purpose protocols for discovering and screening many DNA sequence polymorphisms arrayed across a species' genome (e.g. Baird et al. 2008;Kerstens et al. 2009;Faircloth et al. 2012;Peterson et al. 2012), are transforming the field of phylogeography to one that is no longer data limited. Investigations concerned with reconstructing long-term population history generally require large numbers of sampled alleles (i.e. many individuals and populations), across multiple loci, to adequately characterize levels of diversity and spatial genetic structuring (McCor...

The Rhinella granulosa group consists of 13 species of toads distributed throughout open areas of South America and Panama. In this paper we perform a phylogenetic analysis considering all but one species of the group, employing five nuclear and four mitochondrial genes, for up to 7910 bp per specimen. Separate phylogenetic analyses under direct optimization (DO) of nuclear and mitochondrial sequences recovered the R. granulosa group as monophyletic and revealed topological incongruence that can be explained mainly by multiple events of hybridization and introgression, both mitochondrial and nuclear. The DO combined analysis, after the exclusion of putatively introgressed or heterozygous genomes, resulted in a phylogenetic hypothesis for the R. granulosa group in which most of the species are recovered as monophyletic, but with interspecific relationships poorly supported. The optimization of morphological (adult and larval), chromosomal, and behavioural characters resulted in 12 putative phenotypic synapomorphies for this species group and some other synapomorphies for internal clades. Our results indicate the need for additional population genetic studies on R. dorbignyi and R. fernandezae to corroborate the taxonomic status of both taxa. Finally, we discuss biological and genetic characteristics of Bufonidae, as possible explanations for the common occurrence of hybridization and introgression observed in some lineages of this family.

Phylogeographic research investigates biodiversity at the interface between populations and species, in a temporal and geographic context. Phylogeography has benefited from analytical approaches that allow empiricists to estimate parameters of interest from the genetic data (e.g., θ = 4Neμ, population divergence, gene flow), and the widespread availability of genomic data allow such parameters to be estimated with greater precision. However, the actual inferences made by phylogeographers remain dependent on qualitative interpretations derived from these parameters' values and as such may be subject to overinterpretation and confirmation bias. Here we argue in favor of using an objective approach to phylogeographic inference that proceeds by calculating the probability of multiple demographic models given the data and the subsequent ranking of these models using information theory. We illustrate this approach by investigating the diversification of two sister species of four-eyed frogs of northeastern Brazil using single nucleotide polymorphisms obtained via restriction-associated digest sequencing. We estimate the composite likelihood of the observed data given nine demographic models and then rank these models using Akaike information criterion. We demonstrate that estimating parameters under a model that is a poor fit to the data is likely to produce values that lead to spurious phylogeographic inferences. Our results strongly imply that identifying which parameters to estimate from a given system is a key step in the process of phylogeographic inference and is at least as important as being able to generate precise estimates of these parameters. They also illustrate that the incorporation of model uncertainty should be a component of phylogeographic hypothesis tests. information theory | model selection | Pleurodema | site frequency spectrum | Caatinga I n biological populations with interbreeding individuals, allele frequencies will inevitably change with time, both in stochastic and systematic manners, through neutral and adaptive processes. These processes-genetic drift, gene flow, mutation, recombination, and natural selection -constitute observable phenomena that lead directly to population structure, population divergence, and eventually speciation. Phylogeography is ideally situated to investigate systems where the microevolutionary processes that act within gene pools begin to form macroevolutionary patterns and has been described as the bridge between population genetics and phylogenetics (1). The power of the discipline comes from the consideration of geographic origin of individuals and populations along the continuum between populations and species (2, 3).Phylogeographic research has progressed through several stages since Avise et al.(1) introduced the term. Initial studies were based on information that can be gathered from the genetic data under few assumptions, for example by calculating summary statistics or estimating gene trees. Inferences were then derived from qualitative interpretations ...

Aim The Caatinga is a widespread but poorly known biome in South America. Its historical stability is controversial and different types of evidence provide support to contrasting hypotheses. We investigate how past biome dynamics may have caused diversification in the endemic four-eyed frog genus Pleurodema.Location Caatinga biome.Methods We sampled 353 individuals of Pleurodema alium and Pleurodema diplolister from 60 localities and genotyped them at 12 (P. alium) or 20 (P. diplolister) microsatellite loci. We sequenced a mitochondrial fragment for a subset (199) of the samples. After exploratory analyses to infer genetic structure between and within species, we designed seven biogeographical scenarios based on the literature on species distributions, palaeomodels and geological palaeoindicators. We tested these hypotheses by calculating the posterior probability of models using multilocus approximate Bayesian computation (ABC).Results Both markers recovered well-defined interspecific limits with restricted introgression, but population structure within P. diplolister is subtle and affected by isolation by distance. Model selection using ABC supported two scenarios of diversification that included recent demographic growth. Genetic breaks at intra-and interspecific levels were geographically coincident, and correlated with past forest invasions reported in the literature.Main conclusions Our data support the idea that past expansions of the Amazon and Atlantic forests over the current Caatinga distribution shaped the genetic structure in endemic four-eyed frogs at more than one level by promoting intermittent vicariance. Additional support comes from the distribution patterns of forest and Caatinga taxa. Variation among groups suggests differential organismal responses to past habitat shifts, probably mediated by specific natural-history traits. This paper provides the first direct evidence that taxa endemic to the Caatinga were affected by past forest interactions.

BackgroundDelimiting 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.ResultsThese 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.ConclusionsOur 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.

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.

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 hybridization 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 datasets 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 nonmonophyletic 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 Rhinella is 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 monophyletic 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 species from the non-monophyletic R . veraguensis and R . margaritifera Groups, the former R . acrolopha Group, 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 morphological 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 phylogenetic...