Complex Patterns of Diversification in the Gray Zone of Speciation: Model-Based Approaches Applied to Patagonian Liolaemid Lizards (Squamata: Liolaemus kingii clade).

In this study we detangled the evolutionary history of the Patagonian lizard clade Liolaemus kingii, coupling dense geographic sampling and novel computational analytical approaches. We analyzed nuclear and mitochondrial data (restriction site-associated DNA sequencing and cytochrome b) to hypothesize and evaluate species limits, phylogenetic relationships, and demographic histories. We complemented these analyses with posterior predictive simulations to assess the fit of the genomic data to the multispecies coalescent model. We also employed a novel approach to time-calibrate a phylogenetic network. Our results show several instances of mito-nuclear discordance and consistent support for a reticulated history, supporting the view that the complex evolutionary history of the kingii clade is characterized by extensive gene flow and rapid diversification events. We discuss our findings in the contexts of the "gray zone" of speciation, phylogeographic patterns in the Patagonian region, and taxonomic outcomes. [Model adequacy; multispecies coalescent; multispecies network coalescent; phylogenomics; species delimitation.].

Environmental correlates of phenotypic evolution in ecologically diverse Liolaemus lizards.

Evolutionary correlations between phenotypic and environmental traits characterize adaptive radiations. However, the lizard genus Liolaemus, one of the most ecologically diverse terrestrial vertebrate radiations on earth, has so far shown limited or mixed evidence of adaptive diversification in phenotype. Restricted use of comprehensive environmental data, incomplete taxonomic representation and not considering phylogenetic uncertainty may have led to contradictory evidence. We compiled a 26-taxon dataset for the Liolaemus gracilis species group, representing much of the ecological diversity represented within Liolaemus and used environmental data to characterize how environments occupied by species' relate to phenotypic evolution. Our analyses, explicitly accounting for phylogenetic uncertainty, suggest diversification in phenotypic traits toward the present, with body shape evolution rapidly evolving in this group. Body shape evolution correlates with the occupation of different structural habitats indicated by vegetation axes suggesting species have adapted for maximal locomotory performance in these habitats. Our results also imply that the effects of phylogenetic uncertainty and model misspecification may be more extensive on univariate, relative to multivariate analyses of evolutionary correlations, which is an important consideration in analyzing data from rapidly radiating adaptive radiations.

ExplorATE: a new pipeline to explore active transposable elements from RNA-seq data.

MOTIVATION: Transposable elements (TEs) are ubiquitous in genomes and many remain active. TEs comprise an important fraction of the transcriptomes with potential effects on the host genome, either by generating deleterious mutations or promoting evolutionary novelties. However, their functional study is limited by the difficulty in their identification and quantification, particularly in non-model organisms. RESULTS: We developed a new pipeline [explore active transposable elements (ExplorATE)] implemented in R and bash that allows the quantification of active TEs in both model and non-model organisms. ExplorATE creates TE-specific indexes and uses the Selective Alignment (SA) to filter out co-transcribed transposons within genes based on alignment scores. Moreover, our software incorporates a Wicker-like criteria to refine a set of target TEs and avoid spurious mapping. Based on simulated and real data, we show that the SA strategy adopted by ExplorATE achieved better estimates of non-co-transcribed elements than other available alignment-based or mapping-based software. ExplorATE results showed high congruence with alignment-based tools with and without a reference genome, yet ExplorATE required less execution time. Likewise, ExplorATE expands and complements most previous TE analyses by incorporating the co-transcription and multi-mapping effects during quantification, and provides a seamless integration with other downstream tools within the R environment. AVAILABILITY AND IMPLEMENTATION: Source code is available at https://github.com/FemeniasM/ExplorATEproject and https://github.com/FemeniasM/ExplorATE_shell_script. Data available on request. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Rapid Radiation and Rampant Reticulation: Phylogenomics of South American Liolaemus Lizards.

Understanding the factors that cause heterogeneity among gene trees can increase the accuracy of species trees. Discordant signals across the genome are commonly produced by incomplete lineage sorting (ILS) and introgression, which in turn can result in reticulate evolution. Species tree inference using the multispecies coalescent is designed to deal with ILS and is robust to low levels of introgression, but extensive introgression violates the fundamental assumption that relationships are strictly bifurcating. In this study, we explore the phylogenomics of the iconic Liolaemus subgenus of South American lizards, a group of over 100 species mostly distributed in and around the Andes mountains. Using mitochondrial DNA (mtDNA) and genome-wide restriction site-associated DNA sequencing (RADseq; nDNA hereafter), we inferred a time-calibrated mtDNA gene tree, nDNA species trees, and phylogenetic networks. We found high levels of discordance between mtDNA and nDNA, which we attribute in part to extensive ILS resulting from rapid diversification. These data also reveal extensive and deep introgression, which combined with rapid diversification, explain the high level of phylogenetic discordance. We discuss these findings in the context of Andean orogeny and glacial cycles that fragmented, expanded, and contracted species distributions. Finally, we use the new phylogeny to resolve long-standing taxonomic issues in one of the most studied lizard groups in the New World.[Andes; ddRADSeq; introgression; lizards; mtDNA; reptiles; SNPs.].

A new species of Liolaemus (Reptilia: Squamata) of the Liolaemus capillitas clade (Squamata, Liolaemini, Liolaemus elongatus-kriegi group) from Sierra de Velasco, La Rioja Province, Argentina.

A new species of the Liolaemus capillitas clade is described. Liolaemus galactostictos sp. nov. differs from other members of its group by a combination of morphological and molecular traits, in particular its black dorsal coloration pattern not found in any other Liolaemus species. Liolaemus galactostictos sp. nov. is only known from its type locality. This new species is found in rocky fields surrounded by grasslands on the top of the Velasco Mountains, a ¨sky island environment¨, in northwestern Argentina. As well as other members of its clade this species seems to be strictly saxicolous, viviparous and feeds on insects.

An integrative approach to address species limits in the southernmost members of the Liolaemus kingii group (Squamata: Liolaemini).

Recent conceptual and methodological advances have enabled an increasing number of studies to address the problem of species delimitation in a comprehensive manner. This is of particular interest in cases of species whose divergence times are recent and/or effective population sizes are large, where the conclusions obtained from a single source of evidence may lead to erroneous estimations of true species numbers or incorrect assignment of individuals to species. Iguanian lizards of the Liolaemus kingii group (13 species) comprise an important component of the endemic fauna of Patagonia. The southernmost species of this group (namely L. baguali, L. escarchadosi, L. sarmientoi, and L. tari) show widely overlapping distributions across southern Patagonia, also, their phylogenetic relationships are ambiguous and species boundaries have not been explicitly tested. Here we use a comprehensive approach to assess species limits through the use of molecular and morphological information (mitochondrial cytb, nuclear sequences collected by ddRADseq, and linear, meristic and landmark-based morphometrics). We found support for the current taxonomy given that the different analyses recognized the nominal species (4 entities), also a candidate species was supported by mitochondrial and morphological data. In addition, we detected signs of admixture between some of the species. Our results indicate that the L. kingii group can serve as a model system in studies of diversification accompanied by hybridization in nature, which in turn might have been promoted by past climatic oscillations and generalist morphologies. We emphasize the importance of using multiple lines of evidence in order to solve evolutionary stories, and minimizing potential erroneous results that may arise when relying on a single source of information.

Phylogeographic analysis and species distribution modelling of the wood frog Batrachyla leptopus (Batrachylidae) reveal interglacial diversification in south western Patagonia.

BACKGROUND: The evolutionary history of southern South American organisms has been strongly influenced by Pleistocene climate oscillations. Amphibians are good models to evaluate hypotheses about the influence of these climate cycles on population structure and diversification of the biota, because they are sensitive to environmental changes and have restricted dispersal capabilities. We test hypotheses regarding putative forest refugia and expansion events associated with past climatic changes in the wood frog Batrachyla leptopus distributed along ∼1,000 km of length including glaciated and non-glaciated areas in southwestern Patagonia. METHODS: Using three mitochondrial regions (D-loop, cyt b, and coI) and two nuclear loci (pomc and crybA1), we conducted multilocus phylogeographic analyses and species distribution modelling to gain insights of the evolutionary history of this species. Intraspecific genealogy was explored with maximum likelihood, Bayesian, and phylogenetic network approaches. Diversification time was assessed using molecular clock models in a Bayesian framework, and demographic scenarios were evaluated using approximate Bayesian computation (ABC) and extended Bayesian skyline plot (EBSP). Species distribution models (SDM) were reconstructed using climatic and geographic data. RESULTS: Population structure and genealogical analyses support the existence of four lineages distributed north to south, with moderate to high phylogenetic support (Bootstrap > 70%; BPP > 0.92). The diversification time of B. leptopus' populations began at ∼0.107 mya. The divergence between A and B lineages would have occurred by the late Pleistocene, approximately 0.068 mya, and divergence between C and D lineages was approximately 0.065 mya. The ABC simulations indicate that lineages coalesced at two different time periods, suggesting the presence of at least two glacial refugia and a postglacial colonization route that may have generated two southern lineages (p = 0.93, type I error: <0.094, type II error: 0.134). EBSP, mismatch distribution and neutrality indexes suggest sudden population expansion at ∼0.02 mya for all lineages. SDM infers fragmented distributions of B. leptopus associated with Pleistocene glaciations. Although the present populations of B. leptopus are found in zones affected by the last glacial maximum (∼0.023 mya), our analyses recover an older history of interglacial diversification (0.107-0.019 mya). In addition, we hypothesize two glacial refugia and three interglacial colonization routes, one of which gave rise to two expanding lineages in the south.

Rivers of Indochina as potential drivers of lineage diversification in the spotted flying lizard (Draco maculatus) species complex.

Southeast Asia hosts a rich concentration of biodiversity within multiple biodiversity hotspots. Indochina, a region with remarkably high levels of in situ diversification, possesses five major rivers (Ayeyarwady, Chiang Mai, Mekong, Red, and Salween), several of which coincide with phylogenetic breaks of terrestrial taxa. Draco maculatus possesses a range that stretches across Indochina, which widespread geographic distribution along with potential discrete variation within subspecies alludes to the possibility of this taxon constituting multiple divergent lineages. Using sequence data from three mitochondrial (12S, 16S, and ND2) and three nuclear (BDNF, CMOS, and PNN) genes, we provide the first estimated phylogeny of this hypothesized species complex and examine its phylogeographic architecture with maximum likelihood and Bayes factor delimitation (BFD) approaches. Our results support multiple divergent lineages with phylogenetic breaks coincident with rivers, indicating that river barriers may be contributing to the elevated levels of in situ diversification of Indochina.

Pleistocene expansion and connectivity of mesic forests inside the South American Dry Diagonal supported by the phylogeography of a small lizard.

Traditionally focused on Amazonian and Atlantic rainforests, studies on the origins of high Neotropical biodiversity have recently shifted to also investigate biodiversity processes in the South American dry diagonal, encompassing Chaco, Cerrado savannas, and Caatinga seasonally dry tropical forests. The plateau/depression hypothesis states that riparian forests in the Brazilian Shield in central Brazil are inhabited by Pleistocene lineages, with shallow divergences and signatures of population expansion. Moreover, riparian forests may have acted as a vegetation network in the Pleistocene, allowing gene/species flow across the South American dry diagonal. We tested these hypotheses using Colobosaura modesta, a small gymnophthalmid lizard from forested habitats in the Cerrado savannas and montane/submontane forests in the Caatinga. We conducted phylogeographic analyses using a multi-locus dataset, tested alternative demographic scenarios with Approximate Bayesian Computation, and also employed species delimitation tests. We recovered a history of recent colonization and expansion along riparian forests, associated with Pleistocene climate shifts, and the existence of a new species of Colobosaura restricted to the Serra do Cachimbo region. We also present evidence that riparian forests have provided an interconnected network for forest organisms within the South American dry diagonal and that Pleistocene events played an important role in their evolutionary history.

Phylogenomic data resolve higher-level relationships within South American Liolaemus lizards.

Phylogenomic approaches now generate hundreds of loci representative of the whole genome that can be used for phylogenetic analyses. The South American lizard genus Liolaemus is the most species-rich vertebrate radiation from temperate zones (more than 265 described species), yet most higher-level phylogenetic relationships within Liolaemus remain poorly resolved. In this study, we used 584 nuclear loci collected using targeted sequenced capture to estimate the phylogenetic relationships among 26 species representing the two subgenera within Liolaemus (Eulaemus + Liolaemus), and all major groups within Eulaemus. Previous molecular and morphological-based phylogenetic analyses of Eulaemus based on a limited number of characters resolved few higher-level relationships, although one point of agreement is that the early divergence within Eulaemus corresponds to the lineomaculatus section, followed by the diversification of eight main clades that are strongly supported and recognized. Liolaemus probably experienced relatively rapid divergences during parts of its evolutionary history, and a phylogenomic approach was used to resolve the relationships among the major groups. The new analyses presented here support the division of Liolaemus into two subgenera, and resolve relationships among many of the major clades of Eulaemus with strong support. A Bayesian divergence dating analysis using 44 protein-coding genes provides an estimation of the split of the two Liolaemus subgenera of approximately 19,7 ma (95% HPD = 16,94-23,04), while diversification within Eulaemus started at 15,05 ma (95% HPD = 12,94 - 17,59) among the L. lineomaculatus and the L. montanus series by Mid Miocene. A novel phylogenetic network analyses for SNP data identified two hybridizing edges among different groups of Eulaemus at different points in time. Having a solid phylogenetic hypothesis of the main Eulaemus clades opens new opportunities to test a variety of macroevolutionary questions for this unique radiation.

The enigmatic Leiosaurae clade: Phylogeography, species delimitation, phylogeny and historical biogeography of its southernmost species.

The clade Leiosaurae is composed of poorly-known species endemic to the southern region of South America. The difficulties of finding these lizards in the field, and their highly conserved morphology, have limited our taxonomic knowledge and understanding of their evolutionary histories. Here, we use data collected over 9 years to study the phylogenetic history, genetic diversity, and biogeographic history of almost all the southernmost species of Leiosaurae (except P. nigroigulus), including: Leiosaurus bellii, Diplolaemus darwinii, D. bibronii, D. sexcinctus and D. leopardinus. We use a fragment of the mitochondrial cytochrome-b gene to resolve general phylogeographic patterns, and add another mitochondrial gene and eight nuclear genes to perform species delimitation and phylogenetic analyses associated with divergence times. We found evidence for three putative new species-level taxa within L. bellii and five within Diplolaemus species, indicating high levels of geographic structure. We use a time-calibrated phylogeny to estimate ranges of ancestral distributions and to generate new hypotheses about their historical biogeography.

Patterns, Mechanisms and Genetics of Speciation in Reptiles and Amphibians.

In this contribution, the aspects of reptile and amphibian speciation that emerged from research performed over the past decade are reviewed. First, this study assesses how patterns and processes of speciation depend on knowing the taxonomy of the group in question, and discuss how integrative taxonomy has contributed to speciation research in these groups. This study then reviews the research on different aspects of speciation in reptiles and amphibians, including biogeography and climatic niches, ecological speciation, the relationship between speciation rates and phenotypic traits, and genetics and genomics. Further, several case studies of speciation in reptiles and amphibians that exemplify many of these themes are discussed. These include studies of integrative taxonomy and biogeography in South American lizards, ecological speciation in European salamanders, speciation and phenotypic evolution in frogs and lizards. The final case study combines genomics and biogeography in tortoises. The field of amphibian and reptile speciation research has steadily moved forward from the assessment of geographic and ecological aspects, to incorporating other dimensions of speciation, such as genetic mechanisms and evolutionary forces. A higher degree of integration among all these dimensions emerges as a goal for future research.

Coalescent-based species delimitation in the sand lizards of the Liolaemus wiegmannii complex (Squamata: Liolaemidae).

Coalescent-based algorithms coupled with the access to genome-wide data have become powerful tools for assessing questions on recent or rapid diversification, as well as delineating species boundaries in the absence of reciprocal monophyly. In southern South America, the diversification of Liolaemus lizards during the Pleistocene is well documented and has been attributed to the climatic changes that characterized this recent period of time. Past climatic changes had harsh effects at extreme latitudes, including Patagonia, but habitat changes at intermediate latitudes of South America have also been recorded, including expansion of sand fields over northern Patagonia and Pampas). In this work, we apply a coalescent-based approach to study the diversification of the Liolaemus wiegmannii species complex, a morphologically conservative clade that inhabits sandy soils across northwest and south-central Argentina, and the south shores of Uruguay. Using four standard sequence markers (mitochondrial DNA and three nuclear loci) along with ddRADseq data we inferred species limits and a time-calibrated species tree for the L. wiegmannii complex in order to evaluate the influence of Quaternary sand expansion/retraction cycles on diversification. We also evaluated the evolutionary independence of the recently described L. gardeli and inferred its phylogenetic position relative to L. wiegmannii. We find strong evidence for six allopatric candidate species within L. wiegmannii, which diversified during the Pleistocene. The Great Patagonian Glaciation (∼1 million years before present) likely split the species complex into two main groups: one composed of lineages associated with sub-Andean sedimentary formations, and the other mostly related to sand fields in the Pampas and northern Patagonia. We hypothesize that early speciation within L. wiegmannii was influenced by the expansion of sand dunes throughout central Argentina and Pampas. Finally, L. gardeli is supported as a distinct lineage nested within the L. wiegmannii complex.

Evidence of body size and shape stasis driven by selection in Patagonian lizards of the Phymaturus patagonicus clade (Squamata: Liolaemini).

During the speciation process sibling lineages accumulate differences in time (e.g. genetic, morphological, and/or ecological). Phenotypic traits such as size or shape, however, could experience rapid changes or show stasis depending on their role in survival and reproduction. The clade Phymaturus patagonicus includes 26 species characterized by a conservative morphology, and all inhabit rock crevice microhabitats in arid environments. In this study we quantify levels of morphological divergence (size and shape) among the multiple species relative to interspecific molecular divergence, and show that most species have not diverged significantly in size and/or shape to permit unambiguous species diagnosis with morphological data alone. The influence of stabilizing selection for an adaptive optimum in body size and head shape was detected for 13 of the 16 variables analyzed in an Ornstein-Uhlenbeck model. The strict dependence of these species to rock-crevice microenvironments likely explains the observed morphological stasis across the many species of the Phymaturus patagonicus group.

The evolutionary history of Lygodactylus lizards in the South American open diagonal.

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).

Phylogenomic evidence for a recent and rapid radiation of lizards in the Patagonian Liolaemus fitzingerii species group.

Rapid evolutionary radiations are difficult to resolve because divergence events are nearly synchronous and gene flow among nascent species can be high, resulting in a phylogenetic "bush". Large datasets composed of sequence loci from across the genome can potentially help resolve some of these difficult phylogenetic problems. A suitable test case is the Liolaemus fitzingerii species group of lizards, which includes twelve species that are broadly distributed in Argentinean Patagonia. The species in the group have had a complex evolutionary history that has led to high morphological variation and unstable taxonomy. We generated a sequence capture dataset for 28 ingroup individuals of 580 nuclear loci, alongside a mitogenomic dataset, to infer phylogenetic relationships among species in this group. Relationships among species were generally weakly supported with the nuclear data, and along with an inferred age of ∼2.6 million years old, indicate either rapid evolution, hybridization, incomplete lineage sorting, non-informative data, or a combination thereof. We inferred a signal of mito-nuclear discordance, indicating potential hybridization between L. melanops and L. martorii, and phylogenetic network analyses provided support for 5 reticulation events among species. Phasing the nuclear loci did not provide additional insight into relationships or suspected patterns of hybridization. Only one clade, composed of L. camarones, L. fitzingerii, and L. xanthoviridis was recovered across all analyses. Genomic datasets provide molecular systematists with new opportunities to resolve difficult phylogenetic problems, yet the lack of phylogenetic resolution in Patagonian Liolaemus is biologically meaningful and indicative of a recent and rapid evolutionary radiation. The phylogenetic relationships of the Liolaemus fitzingerii group may be best modeled as a reticulated network instead of a bifurcating phylogeny.

Hybridization could be a common phenomenon within the highly diverse lizard genus Liolaemus.

Hybridization is likely to occur more often between closely related taxa that have had insufficient time to diverge to the point of reproductive incompatibility; hybridization between deeply divergent lineages is rare. In squamate reptiles, hybridization has been proposed as a possible explanation for the extensive paraphyly observed in mitochondrial gene trees in several species complexes of the South American lizard genus Liolaemus. One of the best-documented cases is within the L. boulengeri and L. rothi complexes, which diverged ~5.5 million years ago. Here, we describe a comprehensive study for approaching the hybridization hypothesis between these lizard species complexes. We explored the level of gene tree discordance using the novel 'extra lineage contribution' statistics (XLC, presented in this study) that quantifies the level of gene tree discordance contribution per individual within a species. We included molecular data (12 nuclear and two mitochondrial genes) from 127 individuals, and results of a coalescent model-based analysis show that the most likely explanation for the gene tree-species tree discordance is interspecific hybridization. Our best-supported hypothesis suggests current and past hybridization between L. rothi (rothi complex) and L. tehuelche (boulengeri complex), and independently between L. rothi and L. boulengeri and L. telsen (boulengeri complex). The hybrid descendants are characterized by intermediate phenotypes between the parental species, but are more similar to L. rothi in body size. We discuss the possible role of hybridization in Liolaemus evolution.

The shadow of the past: Convergence of young and old South American desert lizards as measured by head shape traits.

Convergence is a pervasive phenomenon in the Tree of Life, and evolution of similar phenotypes sharing the same environmental conditions is expected in phylogenetically closely related species. In contrast, contingent factors are probably more influential in shaping phenotypic diversity for distantly related taxa. Here, we test putative convergent evolution of lizard head morphologies among relatively closely related desert dwelling Liolaemus species, and the very distantly related Ctenoblepharys adspersa. We estimated a multilocus time-calibrated phylogeny of 57 species of South American liolaemus lizards, based on seven molecular markers. We collected head shape data for 468 specimens, and used three phylogenetic comparative methods (SURFACE, CONVEVOL, and WHEATSHEAF index) to test for and estimate the strength of convergence. We found strong evidence for convergence among Pacific desert lizard C. adspersa, Liolaemus audivetulatus, Liolaemus insolitus, Liolaemus poconchilensis, Liolaemus stolzmanni, and a candidate species (Liolaemus "Moquegua"). Our results suggest that, despite the long divergence and phylogenetic distance of C. adspersa with respect to convergent Liolaemus species, natural selection was probably more important than historical contingency in shaping phenotypic evolution in these desert lizards.

New species of Liolaemus (Reptilia, Squamata, Liolaemini) of the Liolaemus donosobarrosi clade from northwestern Patagonia, Neuquén province, Argentina.

Two new species of the Liolaemus donosobarrosi clade are described. Liolaemus tirantii sp. nov. and Liolaemus calliston sp. nov. differ from other members of their clade by a combination of coloration characters, morphometric and molecular traits. Liolaemus tirantii sp. nov. is known from three localities separated only a few kilometers from each other and Liolaemus calliston sp. nov. is known only from the type locality. Both species inhabit a region strongly impacted by oil and gas extraction but their conservation status is unknown.