Diversification of the terrestrial frog genus Anomaloglossus (Anura, Aromobatidae) in the Guiana Shield proceeded from highlands to lowlands, with successive loss and reacquisition of endotrophy.

Two main landscapes emerge from the Guiana Shield: the highlands to the west called the Pantepui region and the Amazonian lowlands to the east, both harbouring numerous endemic species. With 32 currently recognized species, the genus Anomaloglossus stands out among Neotropical frogs as one that diversified only within the Guiana Shield both in the highlands and the lowlands. We present a time-calibrated phylogeny obtained by using combined mitogenomic and nuclear DNA, which suggests that the genus originates from Pantepui where extant lineages started diversifying around 21 Ma, and subsequently (ca. 17 Ma) dispersed during the Miocene Climatic Optimum to the lowlands of the eastern Guiana Shield where the ability to produce endotrophic tadpoles evolved. Further diversification within the lowlands in the A. stepheni group notably led to an evolutionary reversal toward exotrophy in one species group during the late Miocene, followed by reacquisition of endotrophy during the Pleistocene. These successive shifts of reproductive mode seem to have accompanied climatic oscillations. Long dry periods might have triggered evolution of exotrophy, whereas wetter climates favoured endotrophic forms, enabling colonization of terrestrial habitats distant from water. Acquisition, loss, and reacquisition of endotrophy makes Anomaloglossus unique among frogs and may largely explain the current species diversity. The micro evolutionary processes involved in these rapid shifts of reproductive mode remain to be revealed.

Does heat tolerance actually predict animals' geographic thermal limits?

The "climate extremes hypothesis" is a major assumption of geographic studies of heat tolerance and climatic vulnerability. However, this assumption remains vastly untested across taxa, and multiple factors may contribute to uncoupling heat tolerance estimates and geographic limits. Our dataset includes 1000 entries of heat tolerance data and maximum temperatures for each species' known geographic limits (hereafter, Tmax). We gathered this information across major animal taxa, including marine fish, terrestrial arthropods, amphibians, non-avian reptiles, birds, and mammals. We first tested if heat tolerance constrains the Tmax of sites where species could be observed. Secondly, we tested if the strength of such restrictions depends on how high Tmax is relative to heat tolerance. Thirdly, we correlated the different estimates of Tmax among them and across species. Restrictions are strong for amphibians, arthropods, and birds but often weak or inconsistent for reptiles and mammals. Marine fish describe a non-linear relationship that contrasts with terrestrial groups. Traditional heat tolerance measures in thermal vulnerability studies, like panting temperatures and the upper set point of preferred temperatures, do not predict Tmax or are inversely correlated to it, respectively. Heat tolerance restricts the geographic warm edges more strongly for species that reach sites with higher Tmax for their heat tolerance. These emerging patterns underline the importance of reliable species' heat tolerance indexes to identify their thermal vulnerability at their warm range edges. Besides, the tight correlations of Tmax estimates across on-land microhabitats support a view of multiple types of thermal challenges simultaneously shaping ranges' warm edges for on-land species. The heterogeneous correlation of Tmax estimates in the ocean supports the view that fish thermoregulation is generally limited, too. We propose new hypotheses to understand thermal restrictions on animal distribution.

Relicts in the mist: Two new frog families, genera and species highlight the role of Pantepui as a biodiversity museum throughout the Cenozoic.

The iconic mountains of the Pantepui biogeographical region host many early-diverging endemic animal and plant lineages, concurring with Conan Doyle's novel about an ancient "Lost World". While this is the case of several frog lineages, others appear to have more recent origins, adding to the controversy around the diversification processes in this region. Due to its remoteness, Pantepui is challenging for biological surveys, and only a glimpse of its biodiversity has been described, which hampers comprehensive evolutionary studies in many groups. During a recent expedition to the Neblina massif on the Brazil-Venezuela border, we sampled two new frog species that could not be assigned to any known genus. Here, we perform phylogenetic analyses of mitogenomic and nuclear loci to infer the evolutionary relationships of the new taxa and support their description. We find that both species represent single lineages deeply nested within Brachycephaloidea, a major Neotropical clade of direct-developing frogs. Both species diverged >45 Ma from their closest relatives: the first is sister to all other Brachycephaloidea except for Ceuthomantis, another Pantepui endemic, and the second is sister to Brachycephalidae, endemic to the Brazilian Atlantic Forest. In addition to these considerable phylogenetic and biogeographic divergences, external morphology and osteological features support the proposition of two new family and genus-level taxa to accommodate these new branches of the amphibian tree of life. These findings add to other recently described ancient vertebrate lineages from the Neblina massif, providing a bewildering reminder that our perception of the Pantepui's biodiversity remains vastly incomplete. It also provides insights into how these mountains acted as "museums" during the diversification of Brachycephaloidea and of Neotropical biotas more broadly, in line with the influential "Plateau theory". Finally, these discoveries point at the yet unknown branches of the tree of life that may go extinct, due to global climate change and zoonotic diseases, before we even learn about their existence, amphibians living at higher elevations being particularly at risk.

Dehydration alters behavioral thermoregulation and the geography of climatic vulnerability in two Amazonian lizards.

High temperatures and low water availability often strike organisms concomitantly. Observing how organisms behaviorally thermohydroregulate may help us to better understand their climatic vulnerability. This is especially important for tropical forest lizards, species that are purportedly under greater climatic risk. Here, we observed the influence of hydration level on the Voluntary Thermal Maximum (VTmax) in two small Amazonian lizard species: Loxopholis ferreirai (semiaquatic and scansorial) and Loxopholis percarinatum (leaf litter parthenogenetic dweller), accounting for several potential confounding factors (handling, body mass, starting temperature and heating rate). Next, we used two modeling approaches (simple mapping of thermal margins and NicheMapR) to compare the effects of dehydration, decrease in precipitation, ability to burrow, and tree cover availability, on geographic models of climatic vulnerability. We found that VTmax decreased with dehydration, starting temperature, and heating rates in both species. The two modeling approaches showed that dehydration may alter the expected intensity, extent, and duration of perceived thermal risk across the Amazon basin for these forest lizards. Based on our results and previous studies, we identify new evidence needed to better understand thermohydroregulation and to model the geography of climatic risk using the VTmax.

Testing assertions of widespread introgressive hybridization in a clade of neotropical toads with low mate selectivity (Rhinella granulosa species group).

Discordance between different genomic regions, often identified through multilocus sequencing of selected markers, presents particular difficulties in identifying historical processes which drive species diversity and boundaries. Mechanisms causing discordance, such as incomplete lineage sorting or introgression due to interspecific hybridization, are better identified based on population-level genomic datasets. In the toads of the Rhinella granulosa species group, patterns of mito-nuclear discordance and potential hybridization have been reported by several studies. However, these patterns were proposed based on few loci, such that alternative mechanisms behind gene-tree heterogeneity cannot be ruled out. Using genome-wide ddRADseq loci from a subset of species within this clade, we found only partial concordance between currently recognized species-level taxon boundaries and patterns of genetic structure. While most taxa within the R. granulosa group correspond to clades, genetic clustering analyses sometimes grouped distinct taxonomic units into a single cluster. Moreover, levels of admixture between inferred clusters were limited and restricted to a single taxon pair which is best explained by incomplete lineage sorting as opposed to introgressive hybridization, according to D-statistics results. These findings contradict previous assertions of widespread cryptic diversity and gene flow within the R. granulosa clade. Lastly, our analyses suggest that diversification events within the Rhinella granulosa group mostly dated back to the early Pliocene, being generally younger than species divergences in other closely related clades that present high levels of cross-species gene flow. This finding uniquely contradicts common assertions that this young clade of toads exhibits interspecific hybridization.

Morphological variation and genealogical discordance in Caatinga sand lizards Calyptommatus Rodrigues 1991 (Squamata: Gymnophthalmidae) with the description of a new species.

In the last decades, a remarkable fauna of psammophilous and fossorial squamates was discovered in sandy habitats of the semiarid Caatinga of northeast Brazil. Despite the increasing accumulation of genetic data from this unique fauna, an incomplete knowledge of its diversity still hampers a better understanding of its origins and diversification. The fossorial lizard genus Calyptommatus (Gymnophthalmidae) is endemic to sandy habitats of the Caatinga, being currently represented by four allopatric species. In this study, we used morphological and molecular data to assess population-level variation in Calyptommatus. We found a new morphotype of Calyptommatus from the state of Bahia, Brazil, readily distinguished from congeners by the presence of a frontal scale. Morphological, nuDNA and geographic data support the recognition of a new species herein described as Calyptommatus frontalis sp. nov. Nevertheless, genetic data revealed mito-nuclear+morphology discordance, with populations with frontal scales distributed in three distantly related mtDNA clades, suggesting either potential historical and/or current introgressions or incomplete lineage sorting. Further data are needed to clarify the status of the two other mtDNA clades displaying a frontal scale.

Convergent and lineage-specific genomic differences in limb regulatory elements in limbless reptile lineages.

Loss of limbs evolved many times in squamate reptiles. Here we investigated the genomic basis of convergent limb loss in reptiles. We sequenced the genomes of a closely related pair of limbless-limbed gymnophthalmid lizards and performed a comparative genomic analysis including five snakes and the limbless glass lizard. Our analysis of these three independent limbless lineages revealed that signatures of shared sequence or transcription factor binding site divergence in individual limb regulatory elements are generally rare. Instead, shared divergence occurs more often at the level of signaling pathways, involving different regulatory elements associated with the same limb genes (such as Hand2 or Hox) and/or patterning mechanisms (such as Shh signaling). Interestingly, although snakes are known to have mutations in the Shh ZRS limb enhancer, this enhancer lacks relevant mutations in limbless lizards. Thus, different mechanisms could contribute to limb loss, and there are likely multiple evolutionary paths to limblessness in reptiles.

New species of flea-toad, genus Brachycephalus (Anura: Brachycephalidae) from the Atlantic Forest of Esprito Santo, Brazil.

We describe a new species of dull-colored flea-toad, genus Brachycephalus, from the Atlantic Forest of Capara mountains in southeastern Brazil. The new species is characterized by its diminutive size, leptodactyliform body, brownish color with an inverted V-shaped dark mark on dorsum, skin smooth, hyperossification and dorsal shield absent, linea masculinea absent, Fingers I and IV vestigial, Toe I externally absent, Toe II reduced but functional, Toes III and IV with pointed tips, Toe V vestigial, and ventral color uniformly brown. It is a leaf litter dweller, known only from type locality in the humid forests on the eastern slopes of Parque Nacional do Capara mountains, a protected area in the states of Esprito Santo and Minas Gerais, southeastern Brazil. It is the third flea-toad occurring in the state of Esprito Santo recovered as sister to all other Brachycephalus distributed from the state of So Paulo northward in the Atlantic Forest.

The first mitochondrial genome of a South America parthenogenetic lizard (Squamata: Gymnophthalmidae).

The mitogenome of the South American parthenogenetic lizard Loxopholis percarinatum Müller, 1923 (Squamata: Gymnophthalmidae), a uni-bisexual species complex, was recovered for three individuals from Rio Negro region, Amazonas, Brazil. The content and order of genes are typical for vertebrate mitochondrial genomes, and we recovered 13 protein-coding genes, 22 tRNA, and two rRNA (12S and 16S), in addition to partial fragments of the Control Region. A maximum likelihood phylogenetic analysis with mitogenomes of selected lizard families recovered L. percarinatum with Iphisa elegans Gray, 1851, the only other Gymnophthalmidae species available in GenBank.

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.

Long known, brand new, and possibly threatened: a new species of watersnake of the genus Helicops Wagler, 1828 (Serpentes; Xenodontinae) from the Tocantins-Araguaia River Basin, Brazil.

We describe a new species of watersnake of the genus Helicops from the Tocantins-Araguaia River Basin based on characters of coloration and hemipenial morphology, also corroborated by a molecular phylogenetic analysis including 18 terminals of 10 additional congeners. The new species superficially resembles the spotted-patterned He. leopardinus, but differs from it in exhibiting dorsal spots fused into irregular bands, and a unique hemipenial structure with two pairs of distinctly enlarged basal spines. In addition, despite the superficial resemblance of color pattern, our genetic evidence indicates that the new species does not belong to the He. leopardinus radiation, representing the sister group of a component including terminals of this species, He. modestus, and He. infrataeniatus. Finally, we discuss the apparent restriction of the new species to the Tocantins-Araguaia Basin with focus on vicariant processes that may have promoted its differentiation, as well as on the conservation challenges in an area severely affected by anthropic impacts associated with the rapid expansion of hydroelectric and agricultural sectors in the Brazilian Cerrado.

A new large canopy-dwelling species of Phyllodytes Wagler, 1930 (Anura, Hylidae) from the Atlantic Forest of the state of Bahia, Northeastern Brazil.

The known diversity of treefrogs of the genus Phyllodytes has rapidly increased in recent years, currently comprising 14 species. Recent field work in the Atlantic Rainforest of the state of Bahia lead to the discovery of a new large species of Phyllodytes which is herein described based on multiple evidence including morphological, acoustical and genetic data. Phyllodytes sp. nov. is one of the largest species within the genus and presents immaculate yellowish dorsum and limbs. The advertisement call of the species is composed of 7-31 notes (half pulsed/pulsatile-half harmonic) with frequency-modulated harmonics. Phyllodytes sp. nov. has a karyotype of 2n = 22 chromosomes, as also found in other species of the genus. Genetic distance values of the 16S mitochondrial rRNA among Phyllodytes sp. nov. and its congeners range between 6.4 to 10.2%. The description of another new species for this state reinforces the need for further taxonomic work with Phyllodytes in this region that has been revealed as a priority area for research and conservation of this genus.

The complete mitochondrial genome of Iphisa elegans (Reptilia: Squamata: Gymnophthalmidae).

The complete mitogenome of the lizard Iphisa elegans Gray, 1851 was sequenced using a shotgun approach on an Illumina HiSeq 3000 platform, providing the first mitogenome for Gymnophthalmidae. The genome was 18,622 bp long, with 13 protein-coding genes, two rRNA (12S and 16S), and 22 tRNA, as well as the control region. A maximum likelihood phylogenetic analysis including I. elegans and all other available mitogenomes of Squamata provided a tree in accordance with previous phylogenetic relationships inferred for Squamata.

Effects of climate and geography on spatial patterns of genetic structure in tropical skinks.

Knowledge of how contemporary and historical factors drive patterns of genetic structure across geographic space can shed light on the processes underlying diversification. This approach is especially fruitful in studies of widespread species or species clades that occur across multiple environmental conditions and biomes. In the Neotropics, specifically, molecular data from widespread vertebrate species have revealed high levels of lineage diversity and spatial genetic structure - yet studies that explore the possible correlates of local structure patterns are lacking. We investigate the distribution of lineage diversity within two widespread South American skink species complexes of the genus Mabuya. We characterize genetic structure and diversity in these widely ranged lizards, and identify potential geographic and environmental correlates, to shed light on the processes that promote lineage diversification across the heterogeneous landscapes which they occupy. In both groups, we found mitochondrial lineages to be spatially structured along the coastal forests and the savannas of Brazil. These mtDNA patterns are, however, not shared with those inferred from nuclear DNA markers. The geographic location of major mitochondrial genetic breaks is consistent with those of other taxa, suggesting common responses to former landscape change in eastern South America, particularly along geological faults. Genetic differentiation is correlated with environmental turnover and geographic separation in one, but not in the other, group of skinks. Compared to other studies of similarly widely distributed organisms, the link between spatial environmental gradients and genetic differentiation is not as strong or consistent, suggesting a more complex history underlying current phylogeographic patterns. Our genetic data indicate the existence of yet undescribed diversity in wide-ranging lizards, and the value of phylogenetic and phylogeographic studies of similarly understudied species.

Phylogeography of the endangered sand dune whiptail lizard Glaucomastix abaetensis (Dias, Rocha amp; Vrcibradic, 2002) with the description of a new species.

Several species of Glaucomastix lizards have been described over the past decade, most of these inhabit inland Brazil. Glaucomastix abaetensis is a threatened whiptail endemic to Brazilian coastal "restinga" (sandy habitats) from Bahia State to Sergipe, with a distribution limited by riverine barriers. In order to investigate the differentiation and relationships in G. abaetensis, we integrated phylogeographic analysis, Bayesian species delimitation and morphological data to detect geographical patterns and historical events responsible for its present distribution. We recovered two highly divergent clades along its range, one of them unnamed. Glaucomastix itabaianensis sp. nov. has a a yellowish green tail, 13-16 scales in the lateral flank, 22-33 scales around tail, 28-35 femoral pores and usually four supraocular scales with the smaller one disposed posteriorly. Our results retrieved the monophyly of Glaucomastix, with G. venetacauda and G. cyanurus being sister species to a clade formed by G. littoralis and G. abaetensis. Divergence between Glaucomastix abaetensis and the new species occurred roughly 2.39 Myr ago; posterior shallow genetic divergences occurred mainly in Pleistocene. Finally, we present data on the conservation of this clade of whiptail lizards.

Chance, luck and a fortunate finding: a new species of watersnake of the genus Helicops Wagler, 1828 (Serpentes: Xenodontinae), from the Brazilian Pantanal wetlands.

We describe a new watersnake of the genus Helicops based on a single specimen found in the northern limit of the Brazilian Pantanal. Immediately after collection, the unique features of color pattern and head proportions prevented us to attribute this specimen to any other congener. Further comparisons revealed that the combination of entire nasal scales, a distinctively acuminate snout, high dorsal and supralabial counts, as well as a dorsal pattern with chain-like spot rows and a venter with vivid and peculiar orange markings confirmed that the specimen represented a new species. Molecular data supported our morphological conclusion recovering the new species deeply nested within Helicops terminals, sister to a clade composed by H. carinicaudus and H. nentur. Our discovery represents a rare instance of a snake species restricted to the Brazilian Pantanal, but we refrain from considering it a Pantanal endemic until further records allow more considerations on distributional patterns.

A dune with a view: the eyes of a neotropical fossorial lizard.

BACKGROUND: Lizards are excellent models to study the adaptations of the visual system to different scenarios, and surface-dwelling representatives have been relatively well studied. In contrast, very little is known about the functional anatomy of the eyes of fossorial lineages, and properties such as the light transmission by the ocular media have never been characterised in any fossorial species. Some lizards in the family Gymnophthalmidae endemic to the sand dunes of North Eastern Brazil have evolved sand-burrowing habits and nocturnal activity. Lizards in the sister group to Gymnophthalmidae, the family Teiidae, have decidedly diurnal and epigeal lifestyles, yet they are equally poorly known in terms of visual systems. We focussed on the eye anatomy, photoreceptor morphology and light transmittance properties of the ocular media and oil droplets in the gymnophthalmid Calyptommatus nicterus and the teiid Ameivula ocellifera. RESULTS: The general organisation of the eyes of the fossorial nocturnal C. nicterus and the epigeal diurnal A. ocellifera is remarkably similar. The lenses are highly transmissive to light well into the ultraviolet part of the spectrum. The photoreceptors have the typical cone morphology, with narrow short outer segments and oil droplets. The main difference between the two species is that C. nicterus has only colourless oil droplets, whereas A. ocellifera has colourless as well as green-yellow and pale-orange droplets. CONCLUSIONS: Our results challenge the assumption that fossorial lizards undergo loss of visual function, a claim that is usually guided by the reduced size and external morphology of their eyes. In the case of C. nicterus, the visual system is well suited for vision in bright light and shows specialisations that improve sensitivity in dim light, suggesting that they might perform some visually-guided behaviour above the surface at the beginning or the end of their daily activity period, when light levels are relatively high in their open dunes habitat. This work highlights how studies on the functional anatomy of sensory systems can provide insights into the habits of secretive species.

A new four-pored Amphisbaena (Squamata: Amphisbaenidae) from northeastern Brazil.

A new species of Amphisbaena from the Espinhaço Mountain Range in Bahia State is described based on morphological and molecular data. Amphisbaena caetitensis sp nov. is a small four-pored amphisbaenian with 186-194 body annuli, 10-12 tail annuli, 16 dorsal and 19-22 ventral segments on a midbody annulus and a strikingly distinctive tail tip. The most similar species is A. uroxena, but they can be distinguished by some morphological features besides the genetic divergence of 7.65% on the mitochondrial 16S rRNA.

Phenotype loss is associated with widespread divergence of the gene regulatory landscape in evolution.

Detecting the genomic changes underlying phenotypic changes between species is a main goal of evolutionary biology and genomics. Evolutionary theory predicts that changes in cis-regulatory elements are important for morphological changes. We combined genome sequencing, functional genomics and genome-wide comparative analyses to investigate regulatory elements in lineages that lost morphological traits. We first show that limb loss in snakes is associated with widespread divergence of limb regulatory elements. We next show that eye degeneration in subterranean mammals is associated with widespread divergence of eye regulatory elements. In both cases, sequence divergence results in an extensive loss of transcription factor binding sites. Importantly, diverged regulatory elements are associated with genes required for normal limb patterning or normal eye development and function, suggesting that regulatory divergence contributed to the loss of these phenotypes. Together, our results show that genome-wide decay of the phenotype-specific cis-regulatory landscape is a hallmark of lost morphological traits.

Geographic variation and taxonomy of red-tailed Gymnophthalmus (Squamata: Gymnophthalmidae) from Amazonian Savannas.

Gymnophthalmus represents a challenging group for systematics of microteiid lizards. Due to scarcity of exemplars and molecular data, the taxonomy of the genus has been unstable, and six of its eight species are considered part of a poorly-delimited complex of unisexual and bisexual species. Unnamed populations of red-tailed Gymnophthalmus from savanna enclaves in Amazon, similar to G. vanzoi but with differences in color pattern, have been noted in the literature. Here, we used molecular and morphological data to test the taxonomic status of the Central Amazonian red-tailed Gymnophthalmus (G. sp.). Our molecular analysis recovered a close relationship between G. vanzoi and G. sp. from Central Amazon, with minimal divergence. Samples were similar in scale counts but presented significant variation in color pattern and morphometry. Despite the geographic isolation, individuals of both populations cannot be fully differentiated based on morphology. Thus, our results suggest that G. vanzoi has a wider distribution, including the Central Amazonian red-tailed Gymnophthalmus and likely morphologically similar populations from other enclaves of Amazonian savannas, which may have been isolated recently. Based on our comparative analysis, we highlight some characters proved useful for differentiating Brazilian Gymnophthalmus and discuss some prospects for the taxonomy of the genus.