Diversity and Molecular Evolution of Nonvisual Opsin Genes across Environmental, Developmental, and Morphological Adaptations in Frogs.

Nonvisual opsins are transmembrane proteins expressed in the eyes and other tissues of many animals. When paired with a light-sensitive chromophore, nonvisual opsins form photopigments involved in various nonvisual, light-detection functions including circadian rhythm regulation, light-seeking behaviors, and seasonal responses. Here, we investigate the molecular evolution of nonvisual opsin genes in anuran amphibians (frogs and toads). We test several evolutionary hypotheses including the predicted loss of nonvisual opsins due to nocturnal ancestry and potential functional differences in nonvisual opsins resulting from environmental light variation across diverse anuran ecologies. Using whole-eye transcriptomes of 81 species, combined with genomes, multitissue transcriptomes, and independently annotated genes from an additional 21 species, we identify which nonvisual opsins are present in anuran genomes and those that are also expressed in the eyes, compare selective constraint among genes, and test for potential adaptive evolution by comparing selection between discrete ecological classes. At the genomic level, we recovered all 18 ancestral vertebrate nonvisual opsins, indicating that anurans demonstrate the lowest documented amount of opsin gene loss among ancestrally nocturnal tetrapods. We consistently found expression of 14 nonvisual opsins in anuran eyes and detected positive selection in a subset of these genes. We also found shifts in selective constraint acting on nonvisual opsins in frogs with differing activity periods, habitats, distributions, life histories, and pupil shapes, which may reflect functional adaptation. Although many nonvisual opsins remain poorly understood, these findings provide insight into the diversity and evolution of these genes across anurans, filling an important gap in our understanding of vertebrate opsins and setting the stage for future research on their functional evolution across taxa.

Immunolocalization of Aquaporin 1, 2, and 9 in Anuran Testis of the Neotropical Pointedbelly Frog Leptodactylus podicipinus.

Many anuran survival strategies involve hydric regulation, and reproduction is not different. The aquaporin (AQP) family plays an important role in water transport and regulation in many tissues, including the male gonad. The testes undergo various stages of change during the reproductive cycle, and water balance is an important factor for ensuring reproductive success. Considering the relevance of water control in testicular development in anurans and the lack of research regarding the tissue localization of AQP in the male gonad, the present study investigated the expression of three AQPs (1, 2, and 9) in the testis of the neotropical anuran species Leptodactylus podicipinus during two different periods of the reproductive cycle (reproductive and non-reproductive). AQP1 and 2 immunoreactions were found in early germ cells, spermatozoa, Leydig cells, and Sertoli cells, which were more frequently expressed within the reproductive period. AQP1 was also found in the testicular blood vessels. AQP9 was identified predominantly in the epithelium of the intratesticular ducts of reproductive-period individuals. This study presents, for the first time, the localization of AQP1, AQP2, and AQP9 in the testes of an anuran species and the differences in their location during two distinct periods of the reproductive cycle.

Insights into the evolution of photoreceptor oil droplets in frogs and toads.

Photoreceptor oil droplets (ODs) are spherical organelles placed most commonly within the inner segment of the cone photoreceptors. Comprising neutral lipids, ODs can be either non-pigmented or pigmented and have been considered optically functional in various studies. Among living amphibians, ODs were only reported to occur in frogs and toads (Anura), while they are absent in salamanders and caecilians. Nonetheless, the limited understanding of their taxonomic distribution in anurans impedes a comprehensive assessment of their evolution and relationship with visual ecology. We studied the retinae of 134 anuran species, extending the knowledge of the distribution of ODs to 46 of the 58 currently recognized families, and providing a new perspective on this group that complements the available information from other vertebrates. The occurrence of ODs in anurans shows a strong phylogenetic signal, and our findings revealed that ODs evolved at least six times during the evolutionary history of the group, independently from other vertebrates. Although no evident correlation was found between OD occurrence, adult habits and diel activity, it is inferred that each independent origin involves distinct scenarios in the evolution of ODs concerning photic habits. Furthermore, our results revealed significant differences in the size of the ODs between nocturnal and arrhythmic anurans relative to the length of the cones' outer segment.

A cretaceous frog with eggs from northwestern China provides fossil evidence for sexual maturity preceding skeletal maturity in anurans.

Mesozoic fossils of frogs are rare in the palaeontological record, particularly those exhibiting soft tissues that offer limited insights into early life-history characteristics. Here we report on a skeletally immature frog from the Lower Cretaceous of northwest China, with egg masses in the body and eggs in the oviduct, indicative of a gravid female. CT reconstruction of the specimen allows referral to Gansubatrachus qilianensis and we assign it as a paratype complementing the diagnosis of the type species. The new fossil, which might represent a younger individual than the holotype of Gansubatrachus, shows that sexual maturation occurred before full adulthood in this frog and provides evidence of death linked to mating behaviour. We also discuss other potential sources of variation and life-history traits of Gansubatrachus. The new finding represents the oldest Early Cretaceous frog preserving in situ eggs and provides a glimpse into ancient anuran development during Mesozoic times.

Expression patterns of melanin-related genes are linked to crypsis and conspicuousness in a pumpkin toadlet.

Colour signals play pivotal roles in different communication systems, and the evolution of these characters has been associated with behavioural ecology, integumentary production processes and perceptual mechanisms of the species involved. Here, we present the first insight into the molecular and histological basis of skin colour polymorphism within a miniaturized species of pumpkin toadlet, potentially representing the lowest size threshold for colour polytypism in tetrapods. Brachycephalus actaeus exhibits a coloration ranging from cryptic green to conspicuous orange skin, and our findings suggest that colour morphs differ in their capability to be detected by potential predators. We also found that the distribution and abundance of chromatophores are variable in the different colour morphs. The expression pattern of coloration related genes was predominantly associated with melanin synthesis (including dct, edn1, mlana, oca2, pmel, slc24a5, tyrp1 and wnt9a). Up-regulation of melanin genes in grey, green and brown skin was associated with higher melanophore abundance than in orange skin, where xanthophores predominate. Our findings provide a significant foundation for comparing and understanding the diverse pathways that contribute to the evolution of pigment production in the skin of amphibians.

Countrywide screening supports model-based predictions of the distribution of Batrachochytrium dendrobatidis in Ukraine.

The chytrid Batrachochytrium dendrobatidis (Bd) is a widespread fungus causing amphibian declines across the globe. Although data on Bd occurrence in Eastern Europe are scarce, a recent species distribution model (SDM) for Bd reported that western and north-western parts of Ukraine are highly suitable to the pathogen. We verified the SDM-predicted range of Bd in Ukraine by sampling amphibians across the country and screening for Bd using qPCR. A total of 446 amphibian samples (tissue and skin swabs) from 11 species were collected from 36 localities. We obtained qPCR-positive results for 33 samples including waterfrogs (Pelophylax esculentus complex) and fire- and yellow-bellied toads (Bombina spp.) from 8 localities. We found that Bd-positive localities had significantly higher predicted Bd habitat suitability than sites that were pathogen-free. Amplification and sequencing of the internal transcribed spacer (ITS) region of samples with the highest Bd load revealed matches with ITS haplotypes of the globally distributed BdGPL strain, and a single case of the BdASIA-2/BdBRAZIL haplotype. We found that Bd was non-randomly distributed across Ukraine, with infections present in the western and north-central forested peripheries of the country with a relatively cool, moist climate. On the other hand, our results suggest that Bd is absent or present in low abundance in the more continental central, southern and eastern regions of Ukraine, corroborating the model-predicted distribution of chytrid fungus. These areas could potentially serve as climatic refugia for Bd-susceptible amphibian hosts.

A bird-like genome from a frog: Mechanisms of genome size reduction in the ornate burrowing frog, Platyplectrum ornatum.

The diversity of genome sizes across the tree of life is of key interest in evolutionary biology. Various correlates of variation in genome size, such as accumulation of transposable elements (TEs) or rate of DNA gain and loss, are well known, but the underlying molecular mechanisms driving or constraining genome size are poorly understood. Here, we study one of the smallest genomes among frogs characterized thus far, that of the ornate burrowing frog (Platyplectrum ornatum) from Australia, and compare it to other published frog and vertebrate genomes to examine the forces driving reduction in genome size. At ∼1.06 gigabases (Gb), the P. ornatum genome is like that of birds, revealing four major mechanisms underlying TE dynamics: reduced abundance of all major classes of TEs; increased net deletion bias in TEs; drastic reduction in intron lengths; and expansion via gene duplication of the repertoire of TE-suppressing Piwi genes, accompanied by increased expression of Piwi-interacting RNA (piRNA)-based TE-silencing pathway genes in germline cells. Transcriptomes from multiple tissues in both sexes corroborate these results and provide insight into sex-differentiation pathways in Platyplectrum Genome skimming of two closely related frog species (Lechriodus fletcheri and Limnodynastes fletcheri) confirms a reduction in TEs as a major driver of genome reduction in Platyplectrum and supports a macroevolutionary scenario of small genome size in frogs driven by convergence in life history, especially rapid tadpole development and tadpole diet. The P. ornatum genome offers a model for future comparative studies on mechanisms of genome size reduction in amphibians and vertebrates generally.

An integrative taxonomy study reveals a rare new species of the genus Creptotrema (Trematoda: Allocreadiidae) in an endangered frog in South America.

During an ecological study with a near-endangered anuran in Brazil, the Schmidt's Spinythumb frog, Crossodactylus schmidti Gallardo, 1961, we were given a chance to analyze the gastrointestinal tract of a few individuals for parasites. In this paper, we describe a new species of an allocreadiid trematode of the genus Creptotrema Travassos, Artigas & Pereira, 1928, which possesses a unique trait among allocreadiids (i.e., a bivalve shell-like muscular structure at the opening of the ventral sucker); the new species represents the fourth species of allocreadiid trematode parasitizing amphibians. Besides, the new species is distinguished from other congeners by the combination of characters such as the body size, ventral sucker size, cirrus-sac size, and by having small eggs. DNA sequences through the 28S rDNA and COI mtDNA further corroborated the distinction of the new species. Phylogenetic analyses placed the newly generated sequences in a monophyletic clade together with all other sequenced species of Creptotrema. Genetic divergences between the new species and other Creptotrema spp. varied from 2.0 to 4.2% for 28S rDNA, and 15.1 to 16.8% for COI mtDNA, providing robust validation for the recognition of the new species. Even though allocreadiids are mainly parasites of freshwater fishes, our results confirm anurans as hosts of trematodes of this family. Additionally, we propose the reallocation of Auriculostoma ocloya Liquin, Gilardoni, Cremonte, Saravia, Cristóbal & Davies, 2022 to the genus Creptotrema. This study increases the known diversity of allocreadiids and contributes to our understanding of their evolutionary relationships, host-parasite relationships, and biogeographic history.

Larger Common River Frogs (Amietia delalandii) have Fewer and Shorter Tissue Microplastic Fibres than Smaller Frogs.

Little is known about microplastics (MPs) in adult frogs. We investigated MPs in adult Common River Frogs (Amietia delalandii) from Potchefstroom, South Africa. Five kinds of samples were analysed: natural water, water used to rinse the skin, skin, intestine, and the remainder of the body (corpus). Tissues were digested. Microplastics occurred in all frogs and sample types (1128 MPs counted). Fibres were the most prevalent MP. Fibre lengths were between 28 and 4300 µm, either polyester or polyvinyl alcohol. MPs in skin were likely derived from the ambient, and MPs in the corpus from translocation via the skin. Fibres in tissues were significantly shorter in larger frogs, a phenomenon we provisionally assign to in situ biodegradation. Microplastics in frogs can potentially be transferred through the food web to higher trophic levels. This study provides the first evidence of MPs in adult frog tissues and avenues for further investigations.

Genomic Analyses Reveal Association of ASIP with a Recurrently evolving Adaptive Color Pattern in Frogs.

Traits shared among distantly related lineages are indicators of common evolutionary constraints, at the ecological, physiological, or molecular level. Here, we show that the vertebral stripe, a cryptic color pattern, has evolved hundreds of times in the evolutionary history of anurans (frogs and toads) and is favored in terrestrial habitats. Using a genome-wide association study, we demonstrate that variation near the Agouti signaling protein gene (ASIP) is responsible for the different vertebral stripe phenotypes in the African grass frog Ptychadena robeensis. RNAseq and real-time quantitative PCR revealed that differential expression of the gene and an adjacent long non-coding RNA is linked to patterning in this species. Surprisingly, and although the stripe phenotypes are shared with closely related species, we found that the P. robeensis alleles are private to the species and unlikely to evolve under long-term balancing selection, thus indicating that the vertebral stripe phenotypes result from parallel evolution within the group. Our findings demonstrate that this cryptic color pattern evolved rapidly and recurrently in terrestrial anurans, and therefore constitutes an ideal system to study repeated evolution.

Land-use preferences of the European green toad (Bufotes viridis) in the city of Vienna (Austria): the importance of open land in urban environments.

Urban areas are increasing worldwide, which poses threats to animal wildlife. However, in certain cases cities can provide refuges for endangered animals. The European green toad (Bufotes viridis) is one of such examples, which is known from cities throughout their distribution. In contrast, considerable areas of their former (primary) habitats have been degraded. The primary habitats of this species include steppes and wild river floodplains, both characterized by dynamic changes and the presence of open areas. We used available green toad observation data (2007-2020) to model the effects of land-use types on occurrence probability in the city of Vienna. Forest and densely populated areas were highly significantly negatively associated with green toad presence, while transformation/construction site areas showed a strong positive effect. Such occurrence pattern might be characteristic for early succession species, which depend on stochastic environmental disturbances (e.g., droughts and floods) in their primary habitats. We argue that urban landscape planning should appreciate the potential ecological value of open land in cities which is either in a transition phase or a permanent 'wasteland'. Ecological managing of such landscape could vastly increase urban biodiversity.

A Variety of Fungal Species on the Green Frogs' Skin (Pelophylax esculentus complex) in South Banat.

In the last several decades, amphibian populations have been declining worldwide. Many factors have been linked to global amphibian decline, including habitat destruction, pollution, introduced species, global environmental changes, and emerging infectious diseases. Recent studies of amphibian skin infections were mainly focused on the presence of chytridiomycosis, neglecting other members of the frogs' skin communities. The diversity pattern of fungal dwellers on the skin of green frogs (Pelophylax esculentus complex) was investigated. A total of 100 adults were sampled from three localities in South Banat (northern Serbia) over three consecutive years and detected fungal dwellers were identified using light microscopy and ITS and BenA gene sequencing. Structures belonging to fungi and fungus-like organisms including a variety of spores and different mycelia types were documented in the biofilm formed on amphibian skin, and are classified into 10 groups. In total, 42 fungal isolates were identified to species, section, or genus level. The difference in mycobiota composition between sampling points (localities and green frog taxa) was documented. The highest number of fungal structures and isolates was recorded on the hybrid taxon P. esculentus and locality Stevanove ravnice. Parental species showed a markedly lower diversity than the hybrid taxon and were more similar in diversity patterns and were placed in the same homogenous group. The locality Stevanove ravnice exhibited more pronounced differences in diversity pattern than the other two localities and was placed in a distinct and separate homogenous group. Among the fungal isolates, the highest isolation frequency was documented for Alternaria alternata, Aspergillus sp. sect. Nigri, Epicoccum nigrum, Fusarium proliferatum, and Trichoderma atroviride. Among the documented species, dematiaceous fungi, causative agents of chromomycosis in amphibians, were also recorded in this research with high isolation frequency. Also, some rare fungal species such as Quambalaria cyanescens and Pseudoteniolina globosa are documented for the first time in this research as microbial inhabitants of amphibian skin.

Evolution of hyperossification expands skull diversity in frogs.

Frogs (Anura) are one of the most diverse vertebrate orders, comprising more than 7,000 species with a worldwide distribution and extensive ecological diversity. In contrast to other tetrapods, frogs have a highly derived body plan and simplified skull. In many lineages of anurans, increased mineralization has led to hyperossified skulls, but the function of this trait and its relationship with other aspects of head morphology are largely unexplored. Using three-dimensional morphological data from 158 species representing all frog families, we assessed wide-scale patterns of shape variation across all major lineages, reconstructed the evolutionary history of cranial hyperossification across the anuran phylogeny, and tested for relationships between ecology, skull shape, and hyperossification. Although many frogs share a conserved skull shape, several extreme forms have repeatedly evolved that commonly are associated with hyperossification, which has evolved independently more than 25 times. Variation in cranial shape is not explained by phylogenetic relatedness but is correlated with shifts in body size and ecology. The species with highly divergent, hyperossified skulls often have a specialized diet or a unique predator defense mechanism. Thus, the evolution of hyperossification has repeatedly facilitated the expansion of the head into multiple new shapes and functions.

Toxicity of POEA-containing glyphosate-based herbicides to amphibians is mainly due to the surfactant, not to the active ingredient.

Current international legislation regarding agrochemicals requires thorough toxicological testing mainly of the active ingredients. In a 96-h acute toxicity test we exposed Rana dalmatina and Bufo bufo tadpoles to either one of three concentrations of glyphosate, three concentrations of the surfactant (POEA), three concentrations of the two components together, or to non-contaminated water (control), and subsequently assessed mortality and body mass. To investigate whether simultaneous exposure to another stress factor influences effects of the contaminants, we performed tests both in the presence or absence of predator chemical cues. We found that the surfactant had significant harmful effects on tadpoles; survival was lowered by the highest concentration of the surfactant in case of R. dalmatina, while in B. bufo tadpoles it reduced survival already at medium concentrations. Body mass was significantly influenced by medium and high surfactant concentrations in both species. The presence of glyphosate did not have a significant effect by itself, but it slightly increased mortality in tadpoles exposed to medium concentrations of the surfactant in both species. The presence of chemical cues did not have an effect on the examined variables. Our study confirms that the toxicity of glyphosate-based herbicides is mainly due to the examined surfactant. Nonetheless, we found that glyphosate can enhance the harmful effect of the surfactant. These results stress that during the authorization process of new pesticide formulations, not only the active ingredients would need to be examined but the excipients should also be taken into account in an obligatory and systematic manner.

Consequences of polyploidy and divergence as revealed by cytogenetic mapping of tandem repeats in African clawed frogs (Xenopus, Pipidae).

Repetitive elements have been identified in several amphibian genomes using whole genome sequencing, but few studies have used cytogenetic mapping to visualize these elements in this vertebrate group. Here, we used fluorescence in situ hybridization and genomic data to map the U1 and U2 small nuclear RNAs and histone H3 in six species of African clawed frog (genus Xenopus), including, from subgenus Silurana, the diploid Xenopus tropicalis and its close allotetraploid relative X. calcaratus and, from subgenus Xenopus, the allotetraploid species X. pygmaeus, X. allofraseri, X. laevis, and X. muelleri. Results allowed us to qualitatively evaluate the relative roles of polyploidization and divergence in the evolution of repetitive elements because our focal species include allotetraploid species derived from two independent polyploidization events - one that is relatively young that gave rise to X. calcaratus and another that is older that gave rise to the other (older) allotetraploids. Our results demonstrated conserved loci number and position of signals in the species from subgenus Silurana; allotetraploid X. calcaratus has twice as many signals as diploid X. tropicalis. However, the content of repeats varied among the other allotetraploid species. We detected almost same number of signals in X. muelleri as in X. calcaratus and same number of signals in X. pygmaeus, X. allofraseri, X. laevis as in the diploid X. tropicalis. Overall, these results are consistent with the proposal that allopolyploidization duplicated these tandem repeats and that variation in their copy number was accumulated over time through reduction and expansion in a subset of the older allopolyploids.

Identification and determination of the absolute configuration of amorph-4-en-10ß-ol, a cadinol-type sesquiterpene from the scent glands of the African reed frog Hyperolius cinnamomeoventris.

Hyperolid reed frogs are one of the few families of Anurans known to possess glands that emit volatile compounds used in chemical communication. Hyperolius cinnamomeoventris, a model species, possesses a gular gland on its vocal sac that emits chemicals, and sends visual and auditory signals during calling. Previous investigations have shown that the glandular compounds are typically macrocyclic lactones. However, in this work, we show that another major constituent of the male specific gland is (10R,1S,6R,7R,10R)-amorph-4-ene-10ß-ol [(1R,4R,4aR,8aS)-4-isopropyl-1,6-dimethyl-1,2,3,4,4a,7,8,8a-octahydronaphthalen-1-ol]. This compound was synthesized for the first time and has the opposite configuration to amorph-4-ene-10ß-ol known from plants. A short synthesis using an organocatalytic approach through a tandem Mannich/intramolecular Diels-Alder reaction led to a mixture of cadinols, which was used for the assignment of the natural cadinol structures and their stereoisomers.

A frog's eye view: Foundational revelations and future promises.

From the mid-19th century until the 1980's, frogs and toads provided important research models for many fundamental questions in visual neuroscience. In the present century, they have been largely neglected. Yet they are animals with highly developed vision, a complex retina built on the basic vertebrate plan, an accessible brain, and an experimentally useful behavioural repertoire. They also offer a rich diversity of species and life histories on a reasonably restricted physiological and evolutionary background. We suggest that important insights may be gained from revisiting classical questions in anurans with state-of-the-art methods. At the input to the system, this especially concerns the molecular evolution of visual pigments and photoreceptors, at the output, the relation between retinal signals, brain processing and behavioural decision-making.

A genomic survey of LINE elements in Pipidae aquatic frogs shed light on Rex-elements evolution in these genomes.

The transposable elements (TE) represent a large portion of anuran genomes that act as components of genetic diversification. The LINE order of retrotransposons is among the most representative and diverse TEs and is poorly investigated in anurans. Here we explored the LINE diversity with an emphasis on the elements generically called Rex in Pipidae species, more specifically, in the genomes ofXenopus tropicalis, used as a model genome in the study of anurans,the allotetraploid sister species Xenopus laevis and theAmerican species Pipa carvalhoi. We were able to identify a great diversity of LINEs from five clades, Rex1, L2, CR1, L1 and Tx1, in these three species, and the RTE clade was lost in X. tropicalis. It is clear that elements classified as Rex are distributed in distinct clades. The evolutionary pattern of Rex1 elements denote a complex evolution with independent losses of families and some horizontal transfer events between fishes and amphibians which were supported not only by the phylogenetic inconsistencies but also by the very low Ks values found for the TE sequences. The data obtained here update the knowledge of the LINEs diversity in X. laevis and represent the first study of TEs in P. carvalhoi.

Resolving the deep phylogeny: Implications for early adaptive radiation, cryptic, and present-day ecological diversity of Papuan microhylid frogs.

The microhylid frogs of the New Guinea region are the largest and most ecologically diverse subfamily (Asterophryinae) of one of the largest anuran families in the world and can live in communities of up to 20 species. While there has been recent progress in resolving the phylogenetic relationships of Asterophryinae, significant uncertainties remain, impeding further progress in understanding the evolution of microhabitat use, parental care, and life history variation in this group. In particular, the early divergences at the base of the tree remain unclear; as does the monophyly of some genera; and recent studies have discovered that species with wide geographic distribution are instead cryptic species complexes. In this study, we fortified geographic sampling of the largest previous phylogenetic effort by sequencing an additional 62 taxa and increased data quality and quantity by adding new layers of data vetting and by filling in previously incomplete loci to the five gene dataset (2 mitochondrial, 3 nuclear protein-coding genes) to obtain a dataset that is now 99% complete in over 2400 characters for 233 samples (205 taxa) of Asterophryinae and 3 outgroup taxa, and analyzed microhabitat use data for these taxa from field data and data collected from the literature. Importantly, our sampling includes complete community complements at 19 sites as well as representatives at over 80 sites across New Guinea and its offshore islands. We present a highly resolved molecular phylogeny which, for the first time, has over 95% of nodes supported (84% highly supported) whether using Maximum Likelihood or Bayesian Inference, allowing clarification of all genera (whether monophyletic or clearly not), their sister genera relationships, as well as an age estimate for the Asterophryinae at approximately 20MYA. Early generic diversification occurring between 17 and 12 MYA gave rise to a surprising diversity of about 18 genera as well as the 5 putative microhabitat types. Our tree reveals extensive cryptic diversity calling any widespread taxa into doubt, and clearly demonstrates that complex multispecies communities of Asterophryinae are ecologically diverse, are numerous, and of ancient origin across New Guinea. We discuss the implications of our phylogeny for explaining the explosive diversification of Asterophryinae as the result of adaptive radiation, niche conservatism, and non-adaptive radiation.

Ecological divergence and synchronous Pleistocene diversification in the widespread South American butter frog complex.

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.