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      Satellite DNA Mapping in Pseudis fusca (Hylidae, Pseudinae) Provides New Insights into Sex Chromosome Evolution in Paradoxical Frogs


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          In the frog genus Pseudis, previous works found a sex-linked heteromorphism of the PcP190 satellite DNA in the nucleolus organizer region (NOR)-bearing chromosome pairs of Pseudis bolbodactyla and Pseudis tocantins, which possess a ZZ/ZW sex determination system. A pericentromeric inversion was inferred to have occurred during W chromosome evolution, moving a chromosomal cluster enriched by the PcP190 from the short arm (as observed in P. bolbodactyla) to the NOR-bearing long arm (as observed in P. tocantins). However, whether such an inversion happened in P. tocantins or in the common ancestor of Pseudis fusca and P. tocantins remained unclear. To assess this question, we mapped PcP190 in the karyotype of P. fusca from three distinct localities. Southern blotting was used to compare males and females. The mitochondrial H1 fragment (which contains the 12S ribosomal RNA (rRNA), tRNAval, and 16S rRNA genes) and cytochrome b gene were partially sequenced, and a species tree was inferred to guide our analysis. Pseudis fusca specimens were placed together as the sister group of P. tocantins, but based on genetic distance, one of the analyzed populations is probably an undescribed species. A cluster of PcP190, located in the long arm of chromosome 7, is sex linked in this putative new species but not in the remaining P. fusca. We could infer that the pericentromeric inversion that moved the PcP190 site to the NOR-bearing chromosome arm (long arm) occurred in the common ancestor of P. fusca, the putative undescribed species, and P. tocantins.

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            Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers.

            With a standard set of primers directed toward conserved regions, we have used the polymerase chain reaction to amplify homologous segments of mtDNA from more than 100 animal species, including mammals, birds, amphibians, fishes, and some invertebrates. Amplification and direct sequencing were possible using unpurified mtDNA from nanogram samples of fresh specimens and microgram amounts of tissues preserved for months in alcohol or decades in the dry state. The bird and fish sequences evolve with the same strong bias toward transitions that holds for mammals. However, because the light strand of birds is deficient in thymine, thymine to cytosine transitions are less common than in other taxa. Amino acid replacement in a segment of the cytochrome b gene is faster in mammals and birds than in fishes and the pattern of replacements fits the structural hypothesis for cytochrome b. The unexpectedly wide taxonomic utility of these primers offers opportunities for phylogenetic and population research.
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              Underestimation of Species Richness in Neotropical Frogs Revealed by mtDNA Analyses

              Background Amphibians 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. Methodology Here 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 Findings In 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. Conclusions Using 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. Significance As a consequence the global amphibian decline detected especially in the Neotropics may be worse than realised.

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                Genes (Basel)
                Genes (Basel)
                19 February 2019
                February 2019
                : 10
                : 2
                : 160
                [1 ]Laboratory of Chromosome Studies, Department of Structural and Functional Biology, Institute of Biology, University of Campinas, Campinas 13083-863, SP, Brazil; karinsbio@ 123456yahoo.com.br
                [2 ]Department of Zoology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte 31270-901, MG, Brazil; pcagarcia@ 123456gmail.com
                Author notes
                [* ]Correspondence: kaleb.gatto@ 123456gmail.com (K.P.G.); bolsoni@ 123456unicamp.br (L.B.L.); Tel.: +55-19-35216108 (L.B.L.)
                Author information
                © 2019 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                : 24 December 2018
                : 08 February 2019

                chromosome evolution,chromosome rearrangements,phylogeny,satellite dna,sex chromosomes


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