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.

Supplementary data are available at Bioinformatics online.

BackgroundThe rapid evolution of 454 GS-FLX sequencing technology has not been accompanied by a reassessment of the quality and accuracy of the sequences obtained. Current strategies for decision-making and error-correction are based on an initial analysis by Huse et al. in 2007, for the older GS20 system based on experimental sequences. We analyze here the quality of 454 sequencing data and identify factors playing a role in sequencing error, through the use of an extensive dataset for Roche control DNA fragments.ResultsWe obtained a mean error rate for 454 sequences of 1.07%. More importantly, the error rate is not randomly distributed; it occasionally rose to more than 50% in certain positions, and its distribution was linked to several experimental variables. The main factors related to error are the presence of homopolymers, position in the sequence, size of the sequence and spatial localization in PT plates for insertion and deletion errors. These factors can be described by considering seven variables. No single variable can account for the error rate distribution, but most of the variation is explained by the combination of all seven variables.ConclusionsThe pattern identified here calls for the use of internal controls and error-correcting base callers, to correct for errors, when available (e.g. when sequencing amplicons). For shotgun libraries, the use of both sequencing primers and deep coverage, combined with the use of random sequencing primer sites should partly compensate for even high error rates, although it may prove more difficult than previous thought to distinguish between low-frequency alleles and errors.

Microsatellites (or SSRs: simple sequence repeats) are among the most frequently used DNA markers in many areas of research. The use of microsatellite markers is limited by the difficulties involved in their de novo isolation from species for which no genomic resources are available. We describe here a high-throughput method for isolating microsatellite markers based on coupling multiplex microsatellite enrichment and next-generation sequencing on 454 GS-FLX Titanium platforms. The procedure was calibrated on a model species (Apis mellifera) and validated on 13 other species from various taxonomic groups (animals, plants and fungi), including taxa for which severe difficulties were previously encountered using traditional methods. We obtained from 11,497 to 34,483 sequences depending on the species and the number of detected microsatellite loci ranged from 199 to 5791. We thus demonstrated that this procedure can be readily and successfully applied to a large variety of taxonomic groups, at much lower cost than would have been possible with traditional protocols. This method is expected to speed up the acquisition of high-quality genetic markers for nonmodel organisms.

It has been 30 years since it was first proposed that the vertebrate genome evolved through several rounds of genome-wide duplications (polyploidizations) 1 . Despite rapid advances in genetics, including sequencing of the complete genomes of several divergent species, this hypothesis has not been tested rigorously and is still a matter of debate 2 . If polyploidizations occurred during chordate evolution, there should be a network of paralogous regions in the present-day jawed vertebrate (Gnathostomata) genomes 3 . Here we present an investigation of the major histocompatibility complex (MHC) paralogous regions, which we accomplished by characterizing the corresponding region in amphioxus by identifying nine anchor genes and sequencing both the anchor genes and the regions that flank them (a total of 400 kb). Phylogenetic analysis of 31 genes (including the anchor genes) in these regions shows that duplications occurred after the divergence of cephalochordates and vertebrates but before the Gnathostomata radiation. The distribution of human and amphioxus orthologs in their respective genomes and the relationship between these distributions support the en bloc duplication events. Our analysis represents the first step towards demonstrating that the human ancestral genome has undergone polyploidization. Moreover, reconstruction of the pre-duplicated region indicates that one of the duplicated regions retains the ancestral organization.The genomic region in amphioxus that is equivalent to the human MHC paralogous regions has been defined through several steps, including: (i) choosing anchor genes, (ii) cloning their amphioxus equivalents, (iii) isolating the corresponding amphioxus genomic regions and analyzing neighboring genes (especially their phylogenetic relationships to the human genes) and (iv) studying the distribution of the human genes that are orthologous to the amphioxus genes.We hypothesized that the MHC paralogous regions are the result of en bloc duplications that occurred before the Gnathostomata radiation. After these large-scale duplications, some of the duplicated genes probably returned to single-copy status or translocated to other chromosomal regions. We therefore used two approaches to select the anchor genes. The first approach is based on the analysis of the MHC paralogous regions. To identify paralogous genes that could result from these events, we selected Fig. 1 Definition of the anchor genes. Two main approaches were used: the analysis of the human MHC paralogous regions and establishing the preserved syntenies between human and bony fishes (Actinopterygii). a, If the paralogous regions are the result of en bloc duplications, the observed conserved syntenies were present in the ancestral genomic region before the en bloc duplication. In the case of the MHC paralogous regions, this event probably occurred at least 420 Myr ago; thus, these syntenies are also at least this old. We expanded upon previous work 13 by searching the paralogous regions to find new paralogous genes in the data...

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.

Microsatellite marker development has been greatly simplified by the use of high-throughput sequencing followed by in silico microsatellite detection and primer design. However, the selection of markers designed by the existing pipelines depends either on arbitrary criteria, or older studies on PCR success. Based on wet laboratory experiments, we have identified the following factors that are most likely to influence genotyping success rate: alignment score between the primers and the amplicon; the distance between primers and microsatellites; the length of the PCR product; target region complexity and the number of reads underlying the sequence. The QDD pipeline has been modified to include these most pertinent factors in the output to help the selection of markers. Furthermore, new features are also included in the present version: (i) not only raw sequencing reads are accepted as input, but also contigs, allowing the analysis of assembled high-coverage data; (ii) input data can be both in fasta and fastq format to facilitate the use of Illumina and IonTorrent reads; (iii) A comparison to known transposable elements allows their detection; (iv) A contamination check can be carried out by BLASTing potential markers against the nucleotide (nt) database of NCBI; (v) QDD3 is now also available imbedded into a virtual machine making installation easier and operating system independent. It can be used both on command-line version as well as integrated into a Galaxy server, providing a user-friendly interface, as well as the possibility to utilize a large variety of NGS tools.

The vairone Leuciscus souffia is a cyprinid fish that inhabits river systems in and around the Alps. The complete mitochondrial DNA control region (