BackgroundAmphibians are rapidly vanishing. At the same time, it is most likely that the number of amphibian species is highly underestimated. Recent DNA barcoding work has attempted to define a threshold between intra- and inter-specific genetic distances to help identify candidate species. In groups with high extinction rates and poorly known species boundaries, like amphibians, such tools may provide a way to rapidly evaluate species richness.MethodologyHere we analyse published and new 16S rDNA sequences from 60 frog species of Amazonia-Guianas to obtain a minimum estimate of the number of undescribed species in this region. We combined isolation by distance, phylogenetic analyses, and comparison of molecular distances to evaluate threshold values for the identification of candidate species among these frogs.Principal FindingsIn most cases, geographically distant populations belong to genetically highly distinct lineages that could be considered as candidate new species. This was not universal among the taxa studied and thus widespread species of Neotropical frogs really do exist, contrary to previous assumptions. Moreover, the many instances of paraphyly and the wide overlap between distributions of inter- and intra-specific distances reinforce the hypothesis that many cryptic species remain to be described. In our data set, pairwise genetic distances below 0.02 are strongly correlated with geographical distances. This correlation remains statistically significant until genetic distance is 0.05, with no such relation thereafter. This suggests that for higher distances allopatric and sympatric cryptic species prevail. Based on our analyses, we propose a more inclusive pairwise genetic distance of 0.03 between taxa to target lineages that could correspond to candidate species.ConclusionsUsing this approach, we identify 129 candidate species, two-fold greater than the 60 species included in the current study. This leads to estimates of around 170 to 460 frog taxa unrecognized in Amazonia-Guianas.SignificanceAs a consequence the global amphibian decline detected especially in the Neotropics may be worse than realised.

Aim For many taxa, inaccuracy of species boundaries and distributions hampers inferences about diversity and evolution. This is particularly true in the Neotropics where prevalence of cryptic species has often been demonstrated. The frog genus Adenomera is suspected to harbour many more species than the 16 currently recognized. These small terrestrial species occur in Amazonia, Atlantic Forest (AF), and in the open formations of the Dry Diagonal (DD: Chaco, Cerrado and Caatinga). This widespread and taxonomically complex taxon provides a good opportunity to (1) test species boundaries, and (2) investigate historical connectivity between Amazonia and the AF and associated patterns of diversification.Location Tropical South America east of the Andes.Methods We used molecular data (four loci) to estimate phylogenetic relationships among 320 Adenomera samples. These results were integrated with other lines of evidence to propose a conservative species delineation. We subsequently used an extended dataset (seven loci) and investigated ancestral area distributions, dispersal-vicariance events, and the temporal pattern of diversification within Adenomera. ResultsOur conservative delineation identified 31 Confirmed Candidate Species (four remaining unconfirmed) representing a 94% increase in species richness. The biogeographical analysis suggested an Amazonian origin of Adenomera with as many as three dispersals to the DD and one to the AF during the Miocene. These dispersals were associated with habitat shifts from forest towards open habitats. Main conclusionsThe DD played a major role in the history of Adenomera in limiting dispersal and favouring diversification of open-habitat lineages. Moreover, a forest bridge during the Miocene Climatic Optimum may have permitted dispersal from Amazonia towards the AF and subsequent diversification. Uncovering species boundaries and distributions might drastically change inferences based on currently perceived distribution patterns.

Few studies to date have examined genetic variability of widespread tropical amphibian species over their distributional range using diVerent kinds of molecular markers. Here, we use genetic data in an attempt to delimit evolutionary entities within two groups of Neotropical frogs, the Scinax ruber species group and the Rhinella margaritifera species group. We combined mitochondrial and nuclear markers for a phylogenetic (a total of »2500 bp) and phylogeographic study (»1300 bp) to test the reliability of the currently accepted taxonomic assignments and to explore the geographic structure of their genetic variation, mainly based upon samples from the French Guianan region. Phylogenetic analyses demonstrated the polyphyly of Scinax ruber and Rhinella margaritifera. S. ruber consists of six lineages that may all merit species status. ConXicting signals of mitochondrial and nuclear markers indicated, among some Scinax lineages and species, the possibility of ongoing hybridization processes. R. margaritifera consisted of 11 lineages which might represent distinct species as well. Phylogeographic analyses added further information in support of the speciWc status of these lineages. Lineages of low divergence were found in sympatry and were reciprocally monophyletic for mitochondrial as well as nuclear genes, indicating the existence of young lineages that should be awarded species status. Our results highlight the utility of combining phylogenetic and phylogeographic methods, as well as the use of both mitochondrial and nuclear markers within one study. This approach helped to better understand the evolutionary history of taxonomically complex groups of species. The assessment of the geographic distribution of genetic diversity in tropical amphibian communities can lead to conclusions that diVer strongly from prior analyses based on the occurrence of currently recognized species alone. Such studies, therefore, hold the potential to contribute to a more objective assessment of amphibian conservation priorities in tropical areas.

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AimMapping Amazonian biodiversity accurately is a major challenge for integrated conservation strategies and to study its origins. However, species boundaries and their respective distribution are notoriously inaccurate in this region. Here, we generated a georeferenced database of short mtDNA sequences from Amazonian frogs, revised the species richness and the delimitation of bioregions of the Eastern Guiana Shield and estimated endemism within these bioregions. LocationAmazonia, with a focus on the Eastern Guiana Shield. Taxon studiedAmphibia: Anura. MethodsWe used an extensive DNA-based sampling of anuran amphibians of Amazonia using nextgeneration sequencing to delimit Operational Taxonomic Units (OTU) and their distribution. We analysed this database to infer bioregions using Latent Dirichlet Allocation modelling. We then compared endemism within these bioregions based on our results and the current IUCN database, and inferred environmental variables that contributed the most to the biogeographic pattern. ResultsThe recognized anuran species richness within the focal area increased from 440 species currently listed by the IUCN Red List to as much as 876 OTUs with our dataset. We recovered eight bioregions, among which three lie within the Eastern Guiana Shield. We estimated that up to 82% of the OTUs found in this area are endemic, a figure three times higher than the previous estimate (28%). Environmental features related to seasonal precipitations are identified as playing an important role in shaping Amazonian amphibian bioregions. Main conclusionsOur results have major implications for defining future conservation priorities of this vast area given that endemism in most Amazonian bioregions is vastly underestimated, and might therefore hide a large portion of threatened species. Moreover, these findings raise concern about meta-analyses based on public databases within Amazonia dealing with poorly known groups.

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

The Guiana Shield (GS) is one of the most pristine regions of Amazonia and biologically one of the richest areas on Earth. How and when this massive diversity arose remains the subject of considerable debate. The prevailing hypothesis of Quaternary glacial refugia suggests that a part of the eastern GS, among other areas in Amazonia, served as stable forested refugia during periods of aridity. However, the recently proposed disturbance-vicariance hypothesis proposes that fluctuations in temperature on orbital timescales, with some associated aridity, have driven Neotropical diversification. The expectations of the temporal and spatial organization of biodiversity differ between these two hypotheses. Here, we compare the genetic structure of 12 leaf-litter inhabiting frog species from the GS lowlands using a combination of mitochondrial and nuclear sequences in an integrative analytical approach that includes phylogenetic reconstructions, molecular dating, and Geographic Information System methods. This comparative and integrated approach overcomes the well-known limitations of phylogeographic inference based on single species and single loci. All of the focal species exhibit distinct phylogeographic patterns highlighting taxon-specific historical distributions, ecological tolerances to climatic disturbance, and dispersal abilities. Nevertheless, all but one species exhibit a history of fragmentation/isolation within the eastern GS during the Quaternary with spatial and temporal concordance among species. The signature of isolation in northern French Guiana (FG) during the early Pleistocene is particularly clear. Approximate Bayesian Computation supports the synchrony of the divergence between northern FG and other GS lineages. Substructure observed throughout the GS suggests further Quaternary fragmentation and a role for rivers. Our findings support fragmentation of moist tropical forest in the eastern GS during this period when the refuge hypothesis would have the region serving as a contiguous wet-forest refuge.

Dendrophryniscus is an early diverging clade of bufonids represented by few small-bodied species distributed in Amazonia and the Atlantic Forest. We used mitochondrial (414 bp of 12S, 575 bp of 16S genes) and nuclear DNA (785 bp of RAG-1) to investigate phylogenetic relationships and the timing of diversification within the genus. These molecular data were gathered from 23 specimens from 19 populations, including eight out of the 10 nominal species of the genus as well as Rhinella boulengeri. Analyses also included sequences of representatives of 18 other bufonid genera that were publically available. We also examined morphological characters to analyze differences within Dendrophryniscus. We found deep genetic divergence between an Amazonian and an Atlantic Forest clade, dating back to Eocene. Morphological data corroborate this distinction. We thus propose to assign the Amazonian species to a new genus, Amazonella. The species currently named R. boulengeri, which has been previously assigned to the genus Rhamphophryne, is shown to be closely related to Dendrophryniscus species. Our findings illustrate cryptic trends in bufonid morphological evolution, and point to a deep history of persistence and diversification within the Amazonian and Atlantic rainforests. We discuss our results in light of available paleoecological data and the biogeographic patterns observed in other similarly distributed groups.