Assessing model adequacy for Bayesian Skyline plots using posterior predictive simulation.

Bayesian skyline plots (BSPs) are a useful tool for making inferences about demographic history. For example, researchers typically apply BSPs to test hypotheses regarding how climate changes have influenced intraspecific genetic diversity over time. Like any method, BSP has assumptions that may be violated in some empirical systems (e.g., the absence of population genetic structure), and the naïve analysis of data collected from these systems may lead to spurious results. To address these issues, we introduce P2C2M.Skyline, an R package designed to assess model adequacy for BSPs using posterior predictive simulation. P2C2M.Skyline uses a phylogenetic tree and the log file output from Bayesian Skyline analyses to simulate posterior predictive datasets and then compares this null distribution to statistics calculated from the empirical data to check for model violations. P2C2M.Skyline was able to correctly identify model violations when simulated datasets were generated assuming genetic structure, which is a clear violation of BSP model assumptions. Conversely, P2C2M.Skyline showed low rates of false positives when models were simulated under the BSP model. We also evaluate the P2C2M.Skyline performance in empirical systems, where we detected model violations when DNA sequences from multiple populations were lumped together. P2C2M.Skyline represents a user-friendly and computationally efficient resource for researchers aiming to make inferences from BSP.

Sympatric and independently evolving lineages in the Thoropa miliaris - T. taophora species complex (Anura: Cycloramphidae).

Species delimitation can be challenging and affected by subjectivity. Sibling lineages that occur in sympatry constitute good candidates for species delimitation regardless of the adopted species concept. The Thoropa miliaris + T. taophora species complex exhibits high genetic diversity distributed in several lineages that occur sympatrically in the southeastern Atlantic Forest of Brazil. We used 414 loci obtained by anchored hybrid enrichment to characterize genetic variation in the Thoropa miliaris species group (T. saxatilis, T megatympanum, T. miliaris, and T. taophora), combining assignment analyses with traditional and coalescent phylogeny reconstruction. We also investigated evolutionary independence in co-occurring lineages by estimating gene flow, and validated lineages under the multispecies coalescent. We recovered most previously described lineages as unique populations in assignment analyses; exceptions include two lineages within T. miliaris that are further substructured, and the merging of all T. taophora lineages. We found very low probabilities of gene flow between sympatric lineages, suggesting independent evolution. Species tree inferences and species delimitation yielded resolved relationships and indicate that all lineages constitute putative species that diverged during the Pliocene and Pleistocene, later than previously estimated.

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.

Outstanding diversity and microendemism in a clade of rare Atlantic Forest montane frogs.

Distributed across topographically complex landscapes that vary from lowland to high elevation, the Atlantic Forest harbors one of the richest biotas worldwide. Atlantic Forest amphibians are particularly speciose, taxonomic accounts are rising and the group is used as model for biogeographic inference. Past climate-related habitat fragmentation is often invoked to explain diversification, with montane taxa expected to become more widespread during glacial times and restrained at interglacials. In this study we investigate diversification in Ischnocnema lactea and I. holti (Anura: Brachycephalidae), two rare frog species inhabiting Atlantic Forest montane regions in Southeastern Brazil. Previous phylogenetic accounts have suggested uncertain limits between these two sister species. We assembled a multilocus DNA dataset, delimited lineages in this clade, and used ecological niche modeling to explore past and future putative ranges. Assignment analyses and traditional and coalescent phylogenetic methods confirmed the existence of a species complex of Miocene origin comprising nine lineages, most of which show very narrow ranges. Lineages were fully supported as species based in coalescent species delimitation, but the phylogenetic relationships among lineages in higher elevation were unresolved. Models of past ranges suggest extensive suitable areas at the last glacial maximum which, along with phylogenetic uncertainty, are consistent with a hypothesis that climate driven vicariance at higher elevation areas resulted in hard polytomies. Species distribution models under future climates suggest narrower ranges of the lineages relative to now, but no species are currently considered endangered. Overall, our results argue in favor for the reassessment of the taxonomic and conservation status of the I. holti - I. lactea species complex.

Male-male competition and repeated evolution of terrestrial breeding in Atlantic Coastal Forest frogs.

Terrestrial breeding is a derived condition in frogs, with multiple transitions from an aquatic ancestor. Shifts in reproductive mode often involve changes in habitat use, and these are typically associated with diversification in body plans, with repeated transitions imposing similar selective pressures. We examine the diversification of reproductive modes, male and female body sizes, and sexual size dimorphism (SSD) in the Neotropical frog genera Cycloramphus and Zachaenus, both endemic to the Atlantic rainforest of Brazil. Species in this clade either breed in rocky streams (saxicolous) or in terrestrial environments, allowing us to investigate reproductive habitat shifts. We constructed a multilocus molecular phylogeny and inferred evolutionary histories of reproductive habitats, body sizes, and SSD. The common ancestor was small, saxicolous, and had low SSD. Terrestrial breeding evolved independently three times and we found a significant association between reproductive habitat and SSD, with shifts to terrestrial breeding evolving in correlation with decreases in male body size, but not female body size. Terrestrial breeding increases the availability of breeding sites and results in concealment of amplexus, egg-laying, and parental care, therefore reducing male-male competition at all stages of reproduction. We conclude that correlated evolution of terrestrial reproduction and small males is due to release from intense male-male competition that is typical of exposed saxicolous breeding.

Phylogeographic model selection leads to insight into the evolutionary history of four-eyed frogs.

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.

The evolution of phylogeographic data sets.

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. Single-species 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.

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.

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.