The megadiverse Neotropical fish fauna lacks a comprehensive and reliable DNA reference database, which hampers precise species identification and DNA based biodiversity assessment in the region. Here, we developed a mitochondrial 12S ribosomal DNA reference database for 67 fish species, representing 54 genera, 25 families, and six major Neotropical orders. We aimed to develop mini-barcode markers (i.e. amplicons with less than 200 bp) suitable for DNA metabarcoding by evaluating the taxonomic resolution of full-length and mini-barcodes and to determine a threshold value for fish species delimitation using 12S. Evaluation of the target amplicons demonstrated that both full-length library (565 bp) and mini-barcodes (193 bp) contain enough taxonomic resolution to differentiate all 67 fish species. For species delimitation, interspecific genetic distance threshold values of 0.4% and 0.55% were defined using full-length and mini-barcodes, respectively. A custom reference database and specific mini-barcode markers are important assets for ecoregion scale DNA based biodiversity assessments (such as environmental DNA) that can help with the complex task of conserving the megadiverse Neotropical ichthyofauna.

Summary:The Atlantic goliath grouper is a critically endangered species that inhabits estuarine and reef environments and is threatened primarily by fishing activities and habitat destruction. Despite the urgent need for protection, its genetic conservation status remains unknown. The aim of the present study was to evaluate the gene flow among the populations of the species along the coast of Brazil based on the control region of the mitochondrial DNA. The results indicate low haplotype diversity (0.40-0.86) and very low nucleotide diversity (0.1-0.5%). They also show that the genetic diversity of the species varies considerably along the coast and that this finding may be especially important for the identification of priority areas for its conservation. The population analyses indicate a low but significant degree of genetic structuring (Φ ST =0.111), probably due to the occurrence of rare haplotypes at some locations, although the genetic differentiation between sites was not correlated with geographic distance (r=0.0501; p=0.7719), and the shared haplotypes indicate that gene flow occurs among all locations along the Brazilian coast. The results of the pairwise F ST indicate a high degree of genetic differentiation between locations. The incipient population structuring detected in the present study is not related systematically to the geological or physical features of the Brazilian coast. The complex interaction of fluctuations in sea level, marine currents, and the reproductive characteristics of the species hampers the identification of the specific role of each of these processes in the gene flow dynamics of the population units of the Atlantic goliath grouper. The low overall levels of genetic diversity, the pairwise F ST values and the significant population structuring among groups (Φ CT ) identified in the present study all reinforce the critically endangered status of the species and are inconsistent with the presence of a single, panmictic population of groupers on the Brazilian coast. The results of this study suggest that, though it may be incipient, the observed genetic structuring must be taken into account in order to prevent potential problems, such as outbreeding depression, in the management of wild stocks.Keywords: critically endangered species; gene flow; genetic diversity; marine fish; western Atlantic Ocean.Evidencias en el ADN mitocondrial reflejan una incipiente estructuración poblacional en el mero guasa del Atlántico (Epinephelus itajara, Epinephelidae) en Brasil Resumen: El mero guasa del Atlántico está críticamente en peligro, habita en ambientes estuarinos y arrecifes, los cuáles están amenazados principalmente por las actividades de pesca y la destrucción de su hábitat. A pesar de la necesidad urgente de protección, su estado de conservación genética aún es desconocido. El objetivo del presente estudio fue evaluar el flujo génico entre las poblaciones de esta especie a lo largo de la costa de Brasil analizando la variabilidad genética de la región control del ADN mitocondrial....

Hypomasticus copelandii is a Neotropical freshwater fish widely distributed across coastal drainages of southeastern Brazil, a highly impacted region of South America. The interspecific phylogenetic relationships within the genus and the taxonomic status of the species remain uncertain. Using two mitochondrial and one nuclear locus, we performed a phylogenetic, species delimitation, and time-calibrated analyses to test the hypothesis that H. copelandii is a species complex currently delimited by different Atlantic coastal systems. Results indicate that H. copelandii presents two well-delimited genetic lineages: one in the northern drainages of the Jucuruçu, Mucuri and Doce rivers, and the other in the southern region represented by the Paraíba do Sul River Basin. The time-calibrated phylogeny indicated a split between the two genetic lineages at around 2.8 million years ago (Ma), which might be related to headwater capture events during the Plio-Pleistocene. The discovery of a distinct genetic lineage for H. copelandii suggests distinct management plans for the northern and southern drainages. Such hidden diversity within the H. copelandii provides useful information for taxonomy and conservation across a severely impacted region of Brazil.

Roadways and road traffic modify landscapes, posing a threat to the conservation of species in different biomes. The aim of the present study was to analyze roadkill records of wild mammals and to compare the results to findings from 37 other studies conducted in Brazil, to evaluate the richness and diversity of threatened species in different eco-regions of the country. This study was conducted between June 2007 and June 2008, along 60 km of the inter-state highway BR-364, which connects the municipalities of Ouro Preto d’Oeste and Presidente Médici in the state of Rondônia (northern Brazil). Two hundred twenty roadkills were recorded involving 13 species of mammals. Cingualata, Pilosa and Carnivora were the most represented orders. The most represented were the generalist species Dasypus novemcinctus (56.7%), Tamandua tetradactyla (10.5%), and Cerdocyon thous (8.2%), reflecting the adaptability of these species to habitat changes and presence of humans. The number of roadkilled individuals and species indicated no significant differences between the dry and rainy seasons. The findings indicate a high index of roadkills (3.38 individuals/km/year) and moderate number of run over species (0.20 species/km/year) in the region in comparison to records from other roadways in Brazil, with 46% species having an endangered status. Roadways in the eco-regions of the Cerrado (Brazilian savanna) accounted a significantly larger proportion of endangered species (mean: 52%) in comparison to the Pampa (lowlands of southern Brazil; 24%), but the differences in comparison to roadways in Amazonia (37%) and the Atlantic Forest (31%) were non-significant. The present findings underscore the need for emergency measures to minimize the impact of roadkills on mammals. Moreover, priority should be given to more impacted roadways and more threaten eco-regions.

The Atlantic goliath grouper, Epinephelus itajara, is a critically endangered species, threatened by illegal fishing and the destruction of its habitats. A number of other closely related grouper species found in the western Atlantic are also fished intensively. While some countries apply rigorous legislation, illegal harvesting followed by the falsification of fish products, which impedes the correct identification of the species, is a common practice, allowing the catch to be marketed as a different grouper species. In this case, molecular techniques represent an important tool for the monitoring and regulation of fishery practices, and are essential for the forensic identification of a number of different species. In the present study, species-specific primers were developed for the Cytochrome Oxidase subunit I gene, which were applied in a multiplex PCR for the simultaneous identification of nine different species of Epinephelidae: Epinephelus itajara, E. quinquefasciatus, E. morio, Hyporthodus flavolimbatus, H. niveatus, Mycteroperca acutirostris, M. bonaci, M. marginata, and M. microlepis.Multiplex PCR is a rapid, reliable and cost-effective procedure for the identification of commercially-valuable endangered fish species, and may represent a valuable tool for the regulation and sustainable management of fishery resources.O mero, Epinephelus itajara, encontra-se criticamente ameaçado, resultado da pesca ilegal e destruição dos habitas. Filogeneticamente relacionadas a este táxon encontram-se garoupas que atualmente são intensamente pescadas no Atlântico Oeste. Apesar de leis mais restritivas aplicadas em alguns países, a captura ilegal com a descaracterização morfológica é uma prática comum que impossibilita a identificação correta da espécie permitindo que seja comercializada como garoupas, badejos ou chernes. A este respeito, técnicas moleculares representam ferramentas importantes para o monitoramento e fiscalização da pesca, provando ser essencial, na identificação forense de diversas espécies. Primers espécie-específicos foram desenvolvidos com base no gene Citocromo Oxidase subunidade I que aplicados em PCR-Multiplex possibilitam a identificação simultânea de nove espécies Epinephelidae: Epinephelus itajara, E. quinquefasciatus, E. morio, Hyporthodus flavolimbatus, H. niveatus, Mycteroperca acutirostris, M. bonaci, M. marginata e M. microlepis. A identificação via PCR multiplex de espécies de peixes ameaçadas e comercialmente importantes é um método rápido, prático, seguro e de baixo custo, que poderá ser útil o controle do uso e manejo sustentável de recursos pesqueiros.