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The South American Dry Diagonal, also called the Diagonal of Open Formations, is a large region of seasonally dry vegetation extending from northeastern Brazil to northern Argentina, comprising the Caatinga, Cerrado, and Chaco subregions. A growing body of phylogeography literature has determined that a complex history of climatic changes coupled with more ancient geological events has produced a diverse and endemic-rich Dry Diagonal biota. However, the exact drivers are still under investigation, and their relative strengths and effects are controversial. Pleistocene climatic fluctuations structured lineages via vegetation shifts, refugium formation, and corridors between the Amazon and Atlantic forests. In some taxa, older geological events, such as the reconfiguration of the São Francisco River, uplift of the Central Brazilian Plateau, or the Miocene inundation of the Chaco by marine incursions, were more important. Here, we review the Dry Diagonal phylogeography literature, discussing each hypothesized driver of diversification and assessing degree of support. Few studies statistically test these hypotheses, with most support drawn from associating encountered phylogeographic patterns such as population structure with the timing of ancient geoclimatic events. Across statistical studies, most hypotheses are well supported, with the exception of the Pleistocene Arc Hypothesis. However, taxonomic and regional biases persist, such as a proportional overabundance of herpetofauna studies, and the under-representation of Chaco studies. Overall, both Pleistocene climate change and Neogene geological events shaped the evolution of the Dry Diagonal biota, though the precise effects are regionally and taxonomically varied. We encourage further use of model-based analyses to test evolutionary scenarios, as well as interdisciplinary collaborations to progress the field beyond its current focus on the traditional set of geoclimatic hypotheses., (© 2024 The Author(s). Molecular Ecology published by John Wiley & Sons Ltd.)

Divergence dating analyses in systematics provide a framework to develop and test biogeographic hypotheses regarding speciation. However, as molecular datasets grow from multilocus to genomic, sample sizes decrease due to computational burdens, and the testing of fine-scale biogeographic hypotheses becomes difficult. In this study, we use coalescent demographic models to investigate the diversification of poorly known rice paddy snakes from Southeast Asia (Homalopsidae: Hypsiscopus), which have conflicting dates of origin based on previous studies. We use coalescent modeling to test the hypothesis that Hypsiscopus diversified 2.5 mya during the Khorat Plateau uplift in Thailand. Additionally, we use ecological niche analyses to identify potential differences in the niche space of the two most widely distributed species in the past and present. Our results suggest Hypsiscopus diversified ~ 2.4 mya, supporting that the Khorat Plateau may have initiated the diversification of rice paddy snakes. We also find significant niche differentiation and shifts between species of Hypsiscopus, indicating that environmental differences may have sustained differentiation of this genus after the Khorat Plateau uplift. Our study expands on the diversification history of snakes in Southeast Asia, and highlights how results from smaller multilocus datasets can be useful in developing and testing biogeographic hypotheses alongside genomic datasets., (© 2024. The Author(s).)

Geographic barriers are frequently invoked to explain genetic structuring across the landscape. However, inferences on the spatial and temporal origins of population variation have been largely limited to evolutionary neutral models, ignoring the potential role of natural selection and intrinsic genomic processes known as genomic architecture in producing heterogeneity in differentiation across the genome. To test how variation in genomic characteristics (e.g. recombination rate) impacts our ability to reconstruct general patterns of differentiation between species that cooccur across geographic barriers, we sequenced the whole genomes of multiple bird populations that are distributed across rivers in southeastern Amazonia. We found that phylogenetic relationships within species and demographic parameters varied across the genome in predictable ways. Genetic diversity was positively associated with recombination rate and negatively associated with species tree support. Gene flow was less pervasive in genomic regions of low recombination, making these windows more likely to retain patterns of population structuring that matched the species tree. We further found that approximately a third of the genome showed evidence of selective sweeps and linked selection, skewing genome-wide estimates of effective population sizes and gene flow between populations toward lower values. In sum, we showed that the effects of intrinsic genomic characteristics and selection can be disentangled from neutral processes to elucidate spatial patterns of population differentiation., Competing Interests: Conflict of Interest None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Many phylogeographic studies on species with large ranges have found genetic-geographic structure associated with changes in habitat and physical barriers preventing or reducing gene flow. These interactions with geographic space, contemporary and historical climate, and biogeographic barriers have complex effects on contemporary population genetic structure and processes of speciation. While allopatric speciation at biogeographic barriers is considered the primary mechanism for generating species, more recently it has been shown that parapatric modes of divergence may be equally or even more common. With genomic data and better modeling capabilities, we can more clearly define causes of speciation in relation to biogeography and migration between lineages, the location of hybrid zones with respect to the ecology of parental lineages, and differential introgression of genes between taxa. Here, we examine the origins of three Nearctic milksnakes (Lampropeltis elapsoides, Lampropeltis triangulum and Lampropeltis gentilis) using genome-scale data to better understand species diversification. Results from artificial neural networks show that a mix of a strong biogeographic barrier, environmental changes, and physical space has affected genetic structure in these taxa. These results underscore conspicuous environmental changes that occur as the sister taxa L. triangulum and L. gentilis diverged near the Great Plains into the forested regions of the Eastern Nearctic. This area has been recognized as a region for turnover for many vertebrate species, but as we show here the contemporary boundary does not isolate these sister species. These two species likely formed in the mid-Pleistocene and have remained partially reproductively isolated over much of this time, showing differential introgression of loci. We also demonstrate that when L. triangulum and L. gentilis are each in contact with the much older L. elapsoides, some limited gene flow has occurred. Given the strong agreement between nuclear and mtDNA genomes, along with estimates of ecological niche, we suggest that all three lineages should continue to be recognized as unique species. Furthermore, this work emphasizes the importance of considering complex modes of divergence and differential allelic introgression over a complex landscape when testing mechanisms of speciation. [Cline; delimitation; Eastern Nearctic; Great Plains; hybrids; introgression; speciation.]., (© The Author(s) 2021. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Phylogeographic studies primarily focus on the major role of landscape topography in driving lineage diversification. However, populational phylogeographic breaks may also occur as a result of either niche conservatism or divergence, in the absence of geographic barriers to gene flow. Furthermore, these two factors are not mutually exclusive and can act in concert, making it challenging to evaluate their relative importances on explaining genetic variation in nature. Herein, we use sequences of two mitochondrial and four nuclear genes to investigate the timing and diversification patterns of species pertaining to the Leptodactylus latrans complex, which harbors four morphologically cryptic species with broad distributions across environmental gradients in eastern South America. The origin of this species complex dates back to the late Miocene (ca. 5.5 Mya), but most diversification events occurred synchronically during the late Pleistocene likely as the result of ecological divergence driven by Quaternary climatic oscillations. Further, significant patterns of environmental niche divergences among species in the L. latrans complex imply that ecological isolation is the primary mode of genetic diversification, mostly because phylogenetic breaks are associated with environmental transitions rather than topographic barriers at both species and populational scales. We provided new insights about diversification patterns and processes within a species complex of broadly and continuously distributed group of frogs along South America., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Phylogeography investigates historical drivers of the geographical distribution of intraspecific lineages. Particular attention has been given to ecological, climatic and geological processes in the diversification of the Neotropical biota. Several species sampled across the South American diagonal of open formations (DOF), comprising the Caatinga, Cerrado and Chaco biomes, experienced range shifts coincident with Quaternary climatic changes. However, comparative studies across different spatial, temporal and biological scales on DOF species are still meagre. Here, we combine phylogeographical model selection and machine learning predictive frameworks to investigate the influence of Pleistocene climatic changes on several plant and animal species from the DOF. We assembled mitochondrial/chloroplastic DNA sequences in public repositories and inferred the demographic responses of 44 species, comprising 70 intraspecific lineages of plants, lizards, frogs, spiders and insects. We then built a random forest model using biotic and abiotic information to identify the best predictors of demographic responses in the Pleistocene. Finally, we assessed the temporal synchrony of species demographic responses with hierarchical approximate Bayesian computation. Biotic variables related to population connectivity, gene flow and habitat preferences largely predicted how species responded to Pleistocene climatic changes, and demographic changes were synchronous primarily during the Middle Pleistocene. Although 22 (~31%) lineages underwent demographic expansion, presumably associated with the spread of aridity during the glacial Pleistocene periods, our findings suggest that nine lineages (~13%) exhibited the opposite response due to taxon-specific attributes., (© 2021 John Wiley & Sons Ltd.)

Tropical mountains hold more biodiversity than their temperate counterparts, and this disparity is often associated with the latitudinal climatic gradient. However, distinguishing the impact of latitude versus the background effects of species history and traits is challenging due to the evolutionary distance between tropical and temperate assemblages. Here, we test whether microevolutionary processes are linked to environmental variation across a sharp latitudinal transition in 21 montane birds of the southern Atlantic Forest in Brazil. We find that effective dispersal within populations in the tropical mountains is lower and genomic differentiation is better predicted by the current environmental complexity of the region than within the subtropical populations. The concordant response of multiple co-occurring populations is consistent with spatial climatic variability as a major process driving population differentiation. Our results provide evidence for how a narrow latitudinal gradient can shape microevolutionary processes and contribute to broader scale biodiversity patterns., (© 2021. The Author(s).)

We evaluated the role of Quaternary climatic fluctuations on the demographic history and population structure of amphibian species endemic to the 'campo rupestre' in the Neotropics, evaluating their distributional shifts, demographic changes, and lineage formation from the end of Pleistocene to present. We chose two anurans endemic to the high-elevation 'campo rupestre' in the Espinhaço Range (ER) in northeastern and southeastern Brazil (Bokermannohyla alvarengai and Bokermannohyla oxente), as models to test the role of Quaternary climatic fluctuations over their distribution range in this region. We collected tissue samples throughout their distribution range and used statistical phylogeography to examine processes of divergence and population demography. We generated spatial-temporal reconstructions using Bayesian inference in a coalescent framework in combination with hind-cast projections of species distribution models (SDMs). We also used the results and literature information to test alternative diversification scenarios via approximate Bayesian computation (ABC). Our results show that Quaternary climatic fluctuations influenced the geographic ranges of both species showing population expansion during the last glacial maximum (LGM) and range contraction during interglacial periods, as inferred from selected ABC models and from past projections of SDMs. We recovered Pleistocene diversification for both species occuring in distinctly unique periods for each taxon. An older and range-restricted lineage was recovered in a geographically isolated geological massif, deserving conservation and further taxonomic study. The diversification and distribution of these amphibian species endemic to the Neotropical 'campo rupestre' were influenced by Quaternary climatic fluctuations. The expansion of cold adapted species restricted to higher elevations during glacial periods and their concomitant retraction during interglacial periods may have been crucial for producing patterns of species richness and endemism along elevation gradients in tropical and subtropical domains. Such processes may influence the evolution of the biota distributed in heterogeneous landscapes with varied topography., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Species are being lost at an unprecedented rate during the Anthropocene. Progress has been made in clarifying how species traits influence their propensity to go extinct, but the role historical demography plays in species loss or persistence is unclear. In eastern North America, five charismatic landbirds went extinct last century, and the causes of their extinctions have been heavily debated. Although these extinctions are most often attributed to post-colonial human activity, other factors such as declining ancestral populations prior to European colonization could have made these species particularly susceptible. We used population genomic data from these extinct birds and compared them with those from four codistributed extant species. We found extinct species harboured lower genetic diversity and effective population sizes than extant species, but both extinct and non-extinct birds had similar demographic histories of population expansion. These demographic patterns are consistent with population size changes associated with glacial-interglacial cycles. The lack of support for overall population declines during the Pleistocene corroborates the view that, although species that went extinct may have been vulnerable due to low diversity or small population size, their disappearance was driven by human activities in the Anthropocene.

Inferring the history of divergence between species in a framework that permits the presence of gene flow has been crucial for characterizing the "gray zone" of speciation, which is the period of time where lineages have diverged but have not yet achieved strict reproductive isolation. However, estimates of both divergence times and rates of gene flow often ignore spatial information, for example when considering the location and width of hybrid zones with respect to changes in the environment between lineages. Using population genomic data from the North American ratsnake complex (Pantherophis obsoletus), we connected phylogeographic estimates of lineage structure, migration, historical demography, and timing of divergence with hybrid zone dynamics. We examined the spatial context of diversification by linking migration and timing of divergence to the location and widths of hybrid zones. Artificial neural network approaches were applied to understand how landscape features and past climate have influenced population genetic structure among these lineages. We found that rates of migration between lineages were associated with the overall width of hybrid zones. Timing of divergence was not related to migration rate or hybrid zone width across species pairs but may be related to the number of alleles weakly introgressing through hybrid zones. This research underscores how incomplete reproductive isolation can be better understood by considering differential allelic introgression and the effects of historical and contemporary landscape features on the formation of lineages as well as overall genomic estimates of migration rates through time., (© 2020 The Authors. Evolution © 2020 The Society for the Study of Evolution.)

Montane organisms responded to Quaternary climate change by tracking suitable habitat along elevational gradients. However, it is unclear whether these past climatic dynamics generated predictable patterns of genetic diversity in co-occurring montane taxa. To test if the genetic variation is associated with historical changes in the elevational distribution of montane habitats, we integrated paleoclimatic data and a model selection approach for testing the demographic history of five co-distributed bird species occurring in the southern Atlantic Forest sky islands. We found that changes in historical population sizes and current genetic diversity are attributable to habitat dynamics among time periods and the current elevational distribution of populations. Taxa with populations restricted to the more climatically dynamic southern mountain block (SMB) had, on average, a six-fold demographic expansion, whereas the populations from the northern mountain block (NMB) remained constant. In the current configuration of the southern Atlantic Forest montane habitats, populations in the SMB have more widespread elevational distributions, occur at lower elevations, and harbor higher levels of genetic diversity than NMB populations. Despite the apparent coupling of demographic and climatic oscillations, our data rejected simultaneous population structuring due to historical habitat fragmentation. Demographic modeling indicated that the species had different modes of differentiation, and varied in the timing of divergence and the degree of gene flow across mountain blocks. Our results suggest that the heterogeneous distribution of genetic variation in birds of the Atlantic Forest sky islands is associated with the interplay between topography and climate of distinct mountains, leading to predictable patterns of genetic diversity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Genetic structure can be influenced by local adaptation to environmental heterogeneity and biogeographic barriers, resulting in discrete population clusters. Geographic distance among populations, however, can result in continuous clines of genetic divergence that appear as structured populations. Here, we evaluate the relevant importance of these three factors over a landscape characterized by environmental heterogeneity and the presence of a hypothesized biogeographic barrier in producing population genetic structure within 13 codistributed snake species using a genomic data set. We demonstrate that geographic distance and environmental heterogeneity across western North America contribute to population genomic divergence. Surprisingly, landscape features long thought to contribute to biogeographic barriers play little role in divergence community wide. Our results suggest that isolation by environment is the most important contributor to genomic divergence. Furthermore, we show that models of population clustering that incorporate spatial information consistently outperform nonspatial models, demonstrating the importance of considering geographic distances in population clustering. We argue that environmental and geographic distances as drivers of community-wide divergence should be explored before assuming the role of biogeographic barriers., (© 2019 John Wiley & Sons Ltd.)

For many species, climate oscillations drove cycles of population contraction during cool glacial periods followed by expansion during interglacials. Some groups, however, show evidence of uniform and synchronous expansion, while others display differences in the timing and extent of demographic change. We compared demographic histories inferred from genetic data across marine turtle species to identify responses to postglacial warming shared across taxa and to examine drivers of past demographic change at the global scale. Using coalescent simulations and approximate Bayesian computation (ABC), we estimated demographic parameters, including the likelihood of past population expansion, from a mitochondrial data set encompassing 23 previously identified lineages from all seven marine turtle species. For lineages with a high posterior probability of expansion, we conducted a hierarchical ABC analysis to estimate the proportion of lineages expanding synchronously and the timing of synchronous expansion. We used Bayesian model averaging to identify variables associated with expansion and genetic diversity. Approximately 60% of extant marine turtle lineages showed evidence of expansion, with the rest mainly exhibiting patterns of genetic diversity most consistent with population stability. For lineages showing expansion, there was a strong signal of synchronous expansion after the Last Glacial Maximum. Expansion and genetic diversity were best explained by ocean basin and the degree of endemism for a given lineage. Geographic differences in sensitivity to climate change have implications for prioritizing conservation actions in marine turtles as well as for identifying areas of past demographic stability and potential resilience to future climate change for broadly distributed taxa., (© 2019 John Wiley & Sons Ltd.)

We reconstruct the molecular phylogeny of Near Eastern mountain brook newts of the genus Neurergus (family Salamandridae) based on newly determined RADseq data, and compare the outcomes of concatenation-based phylogenetic reconstruction with species-tree inference. Furthermore, we test the current taxonomy of Neurergus (with four species: Neurergus strauchii, N. crocatus, N. kaiseri, and N. derjugini) against coalescent-based species-delimitation approaches of our genome-wide genetic data set. While the position of N. strauchii as sister species to all other Neurergus species was consistent in all of our analyses, the phylogenetic relationships between the three remaining species changed depending on the applied method. The concatenation approach, as well as quartet-based species-tree inference, supported a topology with N. kaiseri as the closest relative to N. derjugini, while full-coalescent species-tree inference approaches supported N. crocatus as sister species of N. derjugini. Investigating the individual signal of gene trees highlighted an extensive variation among gene histories, most likely resulting from incomplete lineage sorting. Coalescent-based species-delimitation models suggest that the current taxonomy might underestimate the species richness within Neurergus and supports seven species. Based on the current sampling, our analysis suggests that N. strauchii, N. derjugini and N. kaiseri might each be subdivided into further species. However, as amphibian species are known to be composed of deep conspecific lineages that do not always warrant species status, these results need to be cautiously interpreted in an integrative taxonomic framework. We hypothesize that the rather shallow divergences detected within N. kaiseri and N. derjugini likely reflect an ongoing speciation process and thus require further investigation. On the contrary, the much deeper genetic divergence found between the two morphologically and geographically differentiated subspecies of N. strauchii leads us to propose that N. s. barani should be considered a distinct species, Neurergus barani Öz, 1994., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The Pleistocenic Arc Hypothesis (PAH) posits that South American Seasonally Dry Tropical Forests (SDTF) were interconnected during Pleistocene glacial periods, enabling the expansion of species ranges that were subsequently fragmented in interglacial periods, promoting speciation. The lizard genus Lygodactylus occurs in Africa, Madagascar, and South America. Compared to the high diversity of African Lygodactylus, only two species are known to occur in South America, L. klugei and L. wetzeli, distributed in SDTFs and the Chaco, respectively. We use a phylogenetic approach based on mitochondrial (ND2) and nuclear (RAG-1) markers covering the known range of South American Lygodactylus to investigate (i) if they are monophyletic relative to their African congeners, (ii) if their divergence is congruent with the fragmentation of the PAH, and (iii) if cryptic diversity exists within currently recognized species. Maximum likelihood and Bayesian phylogenetic analyses recovered a well-supported monophyletic South American Lygodactylus, presumably resulting from a single trans-Atlantic dispersal event 29 Mya. Species delimitation analyses supported the existence of five putative species, three of them undescribed. Divergence times among L. klugei and the three putative undescribed species, all endemic to the SDTFs, are not congruent with the fragmentation of the PAH. However, fragmentation of the once broader and continuous SDTFs likely influenced the divergence of L. wetzeli in the Chaco and Lygodactylus sp. 3 (in a SDTF enclave in the Cerrado)., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Most phylogenies are typically represented as purely bifurcating. However, as genomic data have become more common in phylogenetic studies, it is not unusual to find reticulation among terminal lineages or among internal nodes (deep time reticulation; DTR). In these situations, gene flow must have happened in the same or adjacent geographic areas for these DTRs to have occurred and therefore biogeographic reconstruction should provide similar area estimates for parental nodes, provided extinction or dispersal has not eroded these patterns. We examine the phylogeny of the widely distributed New World kingsnakes (Lampropeltis), determine if DTR is present in this group, and estimate the ancestral area for reticulation. Importantly, we develop a new method that uses coalescent simulations in a machine learning framework to show conclusively that this phylogeny is best represented as reticulating at deeper time. Using joint probabilities of ancestral area reconstructions on the bifurcating parental lineages from the reticulating node, we show that this reticulation likely occurred in northwestern Mexico/southwestern US, and subsequently, led to the diversification of the Mexican kingsnakes. This region has been previously identified as an area important for understanding speciation and secondary contact with gene flow in snakes and other squamates. This research shows that phylogenetic reticulation is common, even in well-studied groups, and that the geographic scope of ancient hybridization is recoverable.

Spatial patterns of genetic variation can help understand how environmental factors either permit or restrict gene flow and create opportunities for regional adaptations. Organisms from harsh environments such as the Brazilian semiarid Caatinga biome may reveal how severe climate conditions may affect patterns of genetic variation. Herein we combine information from mitochondrial DNA with physical and environmental features to study the association between different aspects of the Caatinga landscape and spatial genetic variation in the whiptail lizard Ameivula ocellifera. We investigated which of the climatic, environmental, geographical and/or historical components best predict: (1) the spatial distribution of genetic diversity, and (2) the genetic differentiation among populations. We found that genetic variation in A. ocellifera has been influenced mainly by temperature variability, which modulates connectivity among populations. Past climate conditions were important for shaping current genetic diversity, suggesting a time lag in genetic responses. Population structure in A. ocellifera was best explained by both isolation by distance and isolation by resistance (main rivers). Our findings indicate that both physical and climatic features are important for explaining the observed patterns of genetic variation across the xeric Caatinga biome.

Many studies propose that Quaternary climatic cycles contracted and/or expanded the ranges of species and biomes. Strong expansion-contraction dynamics of biomes presume concerted demographic changes of associated fauna. The analysis of temporal concordance of demographic changes can be used to test the influence of Quaternary climate on diversification processes. Hierarchical approximate Bayesian computation (hABC) is a powerful and flexible approach that models genetic data from multiple species, and can be used to estimate the temporal concordance of demographic processes. Using available single-locus data, we can now perform large-scale analyses, both in terms of number of species and geographic scope. Here, we first compared the power of four alternative hABC models for a collection of single-locus data. We found that the model incorporating an a priori hypothesis about the timing of simultaneous demographic change had the best performance. Second, we applied the hABC models to a data set of seven squamate and four amphibian species occurring in the Seasonally Dry Tropical Forests (Caatinga) in northeastern Brazil, which, according to paleoclimatic evidence, experienced an increase in aridity during the Pleistocene. If this increase was important for the diversification of associated xeric-adapted species, simultaneous population expansions should be evident at the community level. We found a strong signal of synchronous population expansion in the Late Pleistocene, supporting the increase of the Caatinga during this time. This expansion likely enhanced the formation of communities adapted to high aridity and seasonality and caused regional extirpation of taxa adapted to wet forest., (© 2017 John Wiley & Sons Ltd.)

The Atlantic Forest (AF) of Brazil has long been recognized as a biodiversity conservation hotspot. Despite decades of studies the species inventory of this biome continues to increase with the discovery of cryptic diversity and the description of new species. Different diversification mechanisms have been proposed to explain the diversity in the region, including models of forest dynamics, barriers to gene flow and dispersal. Also, sea level change is thought to have influenced coastal diversification and isolated populations on continental islands. However, the timing and mode of diversification of insular populations in the AF region were rarely investigated. Here, we analyze the phylogeography and species diversity of the small-sized direct-developing frog Ischnocnema parva. These frogs are independent from water bodies but dependent on forest cover and high humidity, and provide good models to understand forest dynamics and insular diversification. Our analysis was based on DNA sequences for one mitochondrial and four nuclear genes of 71 samples from 18 localities including two islands, São Sebastião, municipality of Ilhabela, and Mar Virado, municipality of Ubatuba, both in the state of São Paulo. We use molecular taxonomic methods to show that I. parva is composed of six independently evolving lineages, with the nominal I. parva likely endemic to the type locality. The time-calibrated species tree shows that these lineages have diverged in the Pliocene and Pleistocene, suggesting the persistence of micro-refuges of forest in the AF. For the two insular populations we used approximate Bayesian computation to test different diversification hypotheses. Our findings support isolation with migration for São Sebastião population, with ∼1Mya divergence time, and isolation without migration for Mar Virado population, with ∼13Kya divergence time, suggesting a combination of different processes for diversification on AF islands., (Copyright © 2017. Published by Elsevier Inc.)

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.

We aim to evaluate the genetic structure of an Atlantic Forest amphibian species, Scinax eurydice, testing the congruence among patterns identified and proposed by the literature for Pleistocene refugia, microrefugia, and geographic barriers to gene flow such as major rivers. Furthermore, we aim to evaluate predictions of such barriers and refugia on the genetic structure of the species, such as presence/absence of dispersal, timing since separation, and population expansions/contractions. We sequenced mitochondrial and nuclear genetic markers on 94 tissue samples from 41 localities. We inferred a gene tree and estimated genetic distances using mtDNA sequences. We then ran population clustering and assignment methods, AMOVA, and estimated migration rates among populations identified through mtDNA and nDNA analyses. We used a dated species tree, skyline plots, and summary statistics to evaluate concordance between population's distributions and geographic barriers and Pleistocene refugia. Scinax eurydice showed high mtDNA divergences and four clearly distinct mtDNA lineages. Species tree and population assignment tests supported the existence of two major clades corresponding to northeastern and southeastern Atlantic Forest in Brazil, each one composed of two other clades. Lineage splitting events occurred from late Pliocene to Pleistocene. We identified demographic expansions in two clades, and inexistent to low levels of migrations among different populations. Genetic patterns and demographic data support the existence of two northern Refuge and corroborate microrefugia south of the Doce/Jequitinhonha Rivers biogeographic divide. The results agree with a scenario of recent demographic expansion of lowland taxa. Scinax eurydice comprises a species complex, harboring undescribed taxa consistent with Pleistocene refugia. Two rivers lie at the boundaries among populations and endorse their role as secondary barriers to gene flow.

Two main hypotheses have been proposed to explain the diversification of the Caatinga biota. The riverine barrier hypothesis (RBH) claims that the São Francisco River (SFR) is a major biogeographic barrier to gene flow. The Pleistocene climatic fluctuation hypothesis (PCH) states that gene flow, geographic genetic structure and demographic signatures on endemic Caatinga taxa were influenced by Quaternary climate fluctuation cycles. Herein, we analyse genetic diversity and structure, phylogeographic history, and diversification of a widespread Caatinga lizard (Cnemidophorus ocellifer) based on large geographical sampling for multiple loci to test the predictions derived from the RBH and PCH. We inferred two well-delimited lineages (Northeast and Southwest) that have diverged along the Cerrado-Caatinga border during the Mid-Late Miocene (6-14 Ma) despite the presence of gene flow. We reject both major hypotheses proposed to explain diversification in the Caatinga. Surprisingly, our results revealed a striking complex diversification pattern where the Northeast lineage originated as a founder effect from a few individuals located along the edge of the Southwest lineage that eventually expanded throughout the Caatinga. The Southwest lineage is more diverse, older and associated with the Cerrado-Caatinga boundaries. Finally, we suggest that C. ocellifer from the Caatinga is composed of two distinct species. Our data support speciation in the presence of gene flow and highlight the role of environmental gradients in the diversification process., (© 2015 John Wiley & Sons Ltd.)

Scincine lizards in Madagascar form an endemic clade of about 60 species exhibiting a variety of ecomorphological adaptations. Several subclades have adapted to burrowing and convergently regressed their limbs and eyes, resulting in a variety of partial and completely limbless morphologies among extant taxa. However, patterns of limb regression in these taxa have not been studied in detail. Here we fill this gap in knowledge by providing a phylogenetic analysis of DNA sequences of three mitochondrial and four nuclear gene fragments in an extended sampling of Malagasy skinks, and microtomographic analyses of osteology of various burrowing taxa adapted to sand substrate. Based on our data we propose to (i) consider Sirenoscincus Sakata & Hikida, 2003, as junior synonym of Voeltzkowia Boettger, 1893; (ii) resurrect the genus name Grandidierina Mocquard, 1894, for four species previously included in Voeltzkowia; and (iii) consider Androngo Brygoo, 1982, as junior synonym of Pygomeles Grandidier, 1867. By supporting the clade consisting of the limbless Voeltzkowia mira and the forelimb-only taxa V. mobydick and V. yamagishii, our data indicate that full regression of limbs and eyes occurred in parallel twice in the genus Voeltzkowia (as hitherto defined) that we consider as a sand-swimming ecomorph: in the Voeltzkowia clade sensu stricto the regression first affected the hindlimbs and subsequently the forelimbs, whereas the Grandidierina clade first regressed the forelimbs and subsequently the hindlimbs following the pattern prevalent in squamates. Timetree reconstructions for the Malagasy Scincidae contain a substantial amount of uncertainty due to the absence of suitable primary fossil calibrations. However, our preliminary reconstructions suggest rapid limb regression in Malagasy scincids with an estimated maximal duration of 6 MYr for a complete regression in Paracontias, and 4 and 8 MYr respectively for complete regression of forelimbs in Grandidierina and hindlimbs in Voeltzkowia.

Species distributed across vast continental areas and across major biomes provide unique model systems for studies of biotic diversification, yet also constitute daunting financial, logistic and political challenges for data collection across such regions. The tree frog Dendropsophus minutus (Anura: Hylidae) is a nominal species, continentally distributed in South America, that may represent a complex of multiple species, each with a more limited distribution. To understand the spatial pattern of molecular diversity throughout the range of this species complex, we obtained DNA sequence data from two mitochondrial genes, cytochrome oxidase I (COI) and the 16S rhibosomal gene (16S) for 407 samples of D. minutus and closely related species distributed across eleven countries, effectively comprising the entire range of the group. We performed phylogenetic and spatially explicit phylogeographic analyses to assess the genetic structure of lineages and infer ancestral areas. We found 43 statistically supported, deep mitochondrial lineages, several of which may represent currently unrecognized distinct species. One major clade, containing 25 divergent lineages, includes samples from the type locality of D. minutus. We defined that clade as the D. minutus complex. The remaining lineages together with the D. minutus complex constitute the D. minutus species group. Historical analyses support an Amazonian origin for the D. minutus species group with a subsequent dispersal to eastern Brazil where the D. minutus complex originated. According to our dataset, a total of eight mtDNA lineages have ranges >100,000 km2. One of them occupies an area of almost one million km2 encompassing multiple biomes. Our results, at a spatial scale and resolution unprecedented for a Neotropical vertebrate, confirm that widespread amphibian species occur in lowland South America, yet at the same time a large proportion of cryptic diversity still remains to be discovered.

The genus Salamandra represents a clade of six species of Palearctic salamanders of either contrasted black-yellow, or uniformly black coloration, known to contain steroidal alkaloid toxins in high concentrations in their skin secretions. This study reconstructs the phylogeny of the genus Salamandra based on DNA sequences of segments of 10 mitochondrial and 13 nuclear genes from 31 individual samples representing all Salamandra species and most of the commonly recognized subspecies. The concatenated analysis of the complete dataset produced a fully resolved tree with most nodes strongly supported, suggesting that a clade composed of the Alpine salamander (S. atra) and the Corsican fire salamander (S. corsica) is the sister taxon to a clade containing the remaining species, among which S. algira and S. salamandra are sister species. Separate analyses of mitochondrial and nuclear data partitions disagreed regarding basal nodes and in the position of the root but concordantly recovered the S. atra/S. corsica as well as the S. salamandra/S. algira relationship. A species-tree analysis suggested almost simultaneous temporal splits between these pairs of species, which we hypothesize was caused by vicariance events after the Messinian salinity crisis (from late Miocene to early Pliocene). A survey of toxins with combined gas chromatography/mass spectroscopy confirmed the presence of samandarine and/or samandarone steroidal alkaloids in all species of Salamandra as well as in representatives of their sister group, Lyciasalamandra. Samandarone was also detected in lower concentrations in other salamandrids including Calotriton, Euproctus, Lissotriton, and Triturus, suggesting that the presence and possible biosynthesis of this alkaloid is plesiomorphic within the Salamandridae., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The Apennine Peninsula is one of Europe's main glacial refugial areas and harbors a large number of lineages and species. Here, a pattern of higher genetic diversity in the south compared to that of the north is characteristic of most vertebrates; however, most studies that have produced these results have relied only on inferences based on mitochondrial DNA. The spectacled salamanders (genus Salamandrina) are endemic to the Apennine Peninsula and have diverged into two sibling species: S. terdigitata (in the south) and S. perspicillata (in the north), presumably in the late Miocene or early Pliocene. By sequencing one mitochondrial (cytb) and two nuclear genes (RAG1 and POMC) and genotyping 10 microsatellite loci, we traced the evolution of these sibling species from their divergence to their contemporary population structure at a fine scale. Using a multilocus coalescent-based approach, we estimated the temporal divergence of both species at approximately 2.25 mya (million years ago), which, hence, is much younger than previous estimates. The classical pattern of high genetic diversity in the south and lower diversity in the north was confirmed only for some markers, and the demographic histories of the two species differed substantially. Whereas S. perspicillata (north) expanded from a single major refugium in the center of the Apennine Peninsula, populations of S. terdigitata (south) persisted through cooler periods in multiple refugia. Further, the fine-scale population genetic structure of 16 S. perspicillata populations revealed significant genetic differentiation, even across short geographic distances. The results of our study stress that for a better understanding of phylogeographic patterns and past demographic processes, both mitochondrial and multiple nuclear loci should be analyzed to avoid gene-specific, and possibly biased results., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The miniaturized frogs of the Eleutherodactylus limbatus group, endemic to Cuba, have recently been shown to contain alkaloids in their skin, representing the fifth independent evolutionary origin of alkaloid sequestering in anurans. Based on a comprehensive sampling of all known species of the group we here assess their range-wide phylogeography using DNA sequences of the mitochondrial cytochrome b and the nuclear Rag-1 gene. We found E. etheridgei, E. cubanus, and E. orientalis to be genetically highly distinct, with uncorrected pair-wise distances >12% in the cytochrome b gene and >5% in the 16S rRNA gene, and without haplotype sharing in the Rag-1 gene. On the contrary, E. iberia, E. jaumei, E. limbatus, and one unnamed lineage from Gran Piedra mountains had lower mitochondrial divergences to each other (<6% in cytochrome b; <3% in 16S) and extensive Rag-1 haplotype sharing, suggesting that their species status requires careful revision. All species and lineages of the group occur in the mountain areas of eastern Cuba and only one of them, E. limbatus, colonized central and western Cuba. Populations of this latter lineage are only weakly genetically differentiated, and its range expansion to western Cuba is probably Pleistocenic, demonstrating that also miniaturized frog species are able of considerable range expansions (over hundreds of kilometers) in short time spans. A phylogeny based on about 5 kb of mitochondrial and nuclear genes places, with maximum support, E. etheridgei basalmost in the group, followed by E. cubanus, E. orientalis, and the E. iberia-jaumei-limbatus clade. The screening of skin alkaloids revealed the presence of alkaloids in all lineages of the group, but alkaloids known to be sequestered by other anurans were not found in the basalmost E. etheridgei. Furthermore, individuals of the derived lineages of the E. iberia-jaumei-limbatus clade had a much larger variety of different alkaloid compounds (3-18) than the other three species (0-4). Traces of alkaloids were found in other species only remotely related to the E. limbatus group, i.e., E. caspari, E. goini, E. ricordii, and E. tetajulia. According to these findings, it can be hypothesized that the capacity to sequester dietary alkaloids into the skin evolved first in an ancestral, litter-dwelling and small-sized Cuban Eleutherodactylus, followed by evolution of miniaturization and diurnality in the early representatives of the E. limbatus group, and eventually by increased brightness of color with probable aposematic function and a highly efficient mechanism of alkaloid sequestering., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We reconstruct range-wide phylogeographies of two widespread and largely co-occurring Western Palearctic frogs, Rana temporaria and R. dalmatina. Based on tissue or saliva samples of over 1000 individuals, we compare a variety of genetic marker systems, including mitochondrial DNA, single-copy protein-coding nuclear genes, microsatellite loci, and single nucleotide polymorphisms (SNPs) of transcriptomes of both species. The two focal species differ radically in their phylogeographic structure, with R. temporaria being strongly variable among and within populations, and R. dalmatina homogeneous across Europe with a single strongly differentiated population in southern Italy. These differences were observed across the various markers studied, including microsatellites and SNP density, but especially in protein-coding nuclear genes where R. dalmatina had extremely low heterozygosity values across its range, including potential refugial areas. On the contrary, R. temporaria had comparably high range-wide values, including many areas of probable postglacial colonization. A phylogeny of R. temporaria based on various concatenated mtDNA genes revealed that two haplotype clades endemic to Iberia form a paraphyletic group at the base of the cladogram, and all other haplotypes form a monophyletic group, in agreement with an Iberian origin of the species. Demographic analysis suggests that R. temporaria and R. dalmatina have genealogies of roughly the same time to coalescence (TMRCA ~3.5 mya for both species), but R. temporaria might have been characterized by larger ancestral and current effective population sizes than R. dalmatina. The high genetic variation in R. temporaria can therefore be explained by its early range expansion out of Iberia, with subsequent cycles of differentiation in cryptic glacial refugial areas followed by admixture, while the range expansion of R. dalmatina into central Europe is a probably more recent event., (Copyright © 2013 Elsevier Inc. All rights reserved.)