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.

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.

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.

High numbers (10 7 to 10 10 cells per g [dry weight]) of heterotrophic, gram-negative, rod-shaped, non-sporeforming, aerobic, thermophilic bacteria related to the genus Thermus were isolated from thermogenic composts at temperatures between 65 and 82؇C. These bacteria were present in different types of wastes (garden and kitchen wastes and sewage sludge) and in all the industrial composting systems studied (open-air windrows, boxes with automated turning and aeration, and closed bioreactors with aeration). Isolates grew fast on a rich complex medium at temperatures between 40 and 80؇C, with optimum growth between 65 and 75؇C. Nutritional characteristics, total protein profiles, DNA-DNA hybridization (except strain JT4), and restriction fragment length polymorphism profiles of the DNAs coding for the 16S rRNAs (16S rDNAs) showed that Thermus strains isolated from hot composts were closely related to Thermus thermophilus HB8. These newly isolated T. thermophilus strains have probably adapted to the conditions in the hot-compost ecosystem. Heterotrophic, ovalspore-forming, thermophilic bacilli were also isolated from hot composts, but none of the isolates was able to grow at temperatures above 70؇C. This is the first report of hot composts as habitats for a high number of thermophilic bacteria related to the genus Thermus. Our study suggests that Thermus strains play an important role in organic-matter degradation during the thermogenic phase (65 to 80؇C) of the composting process.

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