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      Using Different Methods to Access the Difficult Task of Delimiting Species in a Complex Neotropical Hyperdiverse Group

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          Abstract

          The genus Rineloricaria is a Neotropical freshwater fish group with a long and problematic taxonomic history, attributed to the large number of species and the pronounced similarity among them. In the present work, taxonomic information and different molecular approaches were used to identify species boundaries and characterize independent evolutionary units. We analyzed 228 samples assembled in 53 distinct morphospecies. A general mixed yule-coalescent (GMYC) analysis indicated the existence of 70 entities, while BOLD system analyses showed the existence of 56 distinct BINs. When we used a new proposed integrative taxonomy approach, mixing the results obtained by each analysis, we identified 73 OTUs. We suggest that Rineloricaria probably has some complexity in the known species and several species not formally described yet. Our data suggested that other hyperdiverse fish groups with wide distributions can be further split into many new evolutionary taxonomic units.

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          An index of substitution saturation and its application.

          We introduce a new index to measure substitution saturation in a set of aligned nucleotide sequences. The index is based on the notion of entropy in information theory. We derive the critical values of the index based on computer simulation with different sequence lengths, different number of OTUs and different topologies. The critical value enables researchers to quickly judge whether a set of aligned sequences is useful in phylogenetics. We illustrate the index by applying it to an analysis of the aligned sequences of the elongation factor-1alpha gene originally used to resolve the deep phylogeny of major arthropod groups. The method has been implemented in DAMBE.
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            Sequence-based species delimitation for the DNA taxonomy of undescribed insects.

            Cataloging the very large number of undescribed species of insects could be greatly accelerated by automated DNA based approaches, but procedures for large-scale species discovery from sequence data are currently lacking. Here, we use mitochondrial DNA variation to delimit species in a poorly known beetle radiation in the genus Rivacindela from arid Australia. Among 468 individuals sampled from 65 sites and multiple morphologically distinguishable types, sequence variation in three mtDNA genes (cytochrome oxidase subunit 1, cytochrome b, 16S ribosomal RNA) was strongly partitioned between 46 or 47 putative species identified with quantitative methods of species recognition based on fixed unique ("diagnostic") characters. The boundaries between groups were also recognizable from a striking increase in branching rate in clock-constrained calibrated trees. Models of stochastic lineage growth (Yule models) were combined with coalescence theory to develop a new likelihood method that determines the point of transition from species-level (speciation and extinction) to population-level (coalescence) evolutionary processes. Fitting the location of the switches from speciation to coalescent nodes on the ultrametric tree of Rivacindela produced a transition in branching rate occurring at 0.43 Mya, leading to an estimate of 48 putative species (confidence interval for the threshold ranging from 47 to 51 clusters within 2 logL units). Entities delimited in this way exhibited biological properties of traditionally defined species, showing coherence of geographic ranges, broad congruence with morphologically recognized species, and levels of sequence divergence typical for closely related species of insects. The finding of discontinuous evolutionary groupings that are readily apparent in patterns of sequence variation permits largely automated species delineation from DNA surveys of local communities as a scaffold for taxonomy in this poorly known insect group.
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              DNA barcode-based delineation of putative species: efficient start for taxonomic workflows

              The analysis of DNA barcode sequences with varying techniques for cluster recognition provides an efficient approach for recognizing putative species (operational taxonomic units, OTUs). This approach accelerates and improves taxonomic workflows by exposing cryptic species and decreasing the risk of synonymy. This study tested the congruence of OTUs resulting from the application of three analytical methods (ABGD, BIN, GMYC) to sequence data for Australian hypertrophine moths. OTUs supported by all three approaches were viewed as robust, but 20% of the OTUs were only recognized by one or two of the methods. These OTUs were examined for three criteria to clarify their status. Monophyly and diagnostic nucleotides were both uninformative, but information on ranges was useful as sympatric sister OTUs were viewed as distinct, while allopatric OTUs were merged. This approach revealed 124 OTUs of Hypertrophinae, a more than twofold increase from the currently recognized 51 species. Because this analytical protocol is both fast and repeatable, it provides a valuable tool for establishing a basic understanding of species boundaries that can be validated with subsequent studies.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                2 September 2015
                2015
                : 10
                : 9
                : e0135075
                Affiliations
                [1 ]Universidade Estadual Paulista, Departamento de Morfologia, Laboratório de Biologia e Genética de Peixes, Botucatu, São Paulo State, Brazil
                [2 ]Universidade Federal de Viçosa, Campus de Rio Paranaíba, Rio Paranaíba, Minas Gerais State, Brazil
                University of Innsbruck, AUSTRIA
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: GJCS FFR MSR CO FF. Performed the experiments: GJCS MSR FFR. Analyzed the data: GJCS MSR CO. Contributed reagents/materials/analysis tools: MSR FF CO. Wrote the paper: GJCS MSR FFR FF CO.

                Article
                PONE-D-15-06870
                10.1371/journal.pone.0135075
                4557985
                26332320
                bc6b27ee-9424-489d-8848-603c6a9017c8
                Copyright @ 2015

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                : 25 February 2015
                : 16 July 2015
                Page count
                Figures: 2, Tables: 0, Pages: 12
                Funding
                This research was supported by the Brazilian agencies FAPESP (Fundação de Amparo à pesquisa do Estado de São Paulo, proc. 2014/05051-5 to FFR, proc. 2007/04071-2 to GJCS) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, proc. 441347/2014-2 to FFR).
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