Low levels of genetic diversity depicted from mitochondrial DNA sequences in a heavily exploited marine fish (Cynoscion acoupa, Sciaenidae) from the Northern coast of Brazil

The acoupa weakfish (Cynoscion acoupa - Sciaenidae) is a marine species of croaker with estuarine-dependent behavior, found in the western Atlantic from Panama to Argentina. It is one of the most exploited food fish on the northern coast of Brazil. In this study, DNA sequences were determined from the entire control region (D-loop) of the mitochondrial genome of 297 individuals collected during seven different months between December 2003 and August 2005 on the northern coast of Brazil (Amapá and Pará). Genetic variability expressed by haplotype (h = 0,892) and nucleotide (p = 0,003) diversities were low compared to other heavily exploited marine fish species from the western Atlantic and eastern Asia. AMOVA depicted a lack of genetic structuring among the samples from different years, indicating the presence of a single stock of C. acoupa within the sample area. The possible reasons for the low levels of genetic diversity are discussed. These results demonstrate a need for the monitoring of C. acoupa harvesting and the preservation of the estuaries within its geographic range, considering that this large fish depends on estuarine ecosystems during part of its life cycle.

A test of the utility of DNA barcoding in the radiation of the freshwater stingray genus Potamotrygon (Potamotrygonidae, Myliobatiformes)

DNA barcoding is a recently proposed global standard in taxonomy based on DNA sequences. The two main goals of DNA barcoding methodology are assignment of specimens to a species and discovery of new species. There are two main underlying assumptions: i) reciprocal monophyly of species, and ii) intraspecific divergence is always less than interspecific divergence. Here we present a phylogenetic analysis of the family Potamotrygonidae based on mitochondrial cytochrome c oxidase I gene, sampling 10 out of the 18 to 20 valid species including two non-described species. Potamotrygonidae systematics is still not fully resolved with several still-to-be-described species while some other species are difficult to delimit due to overlap in morphological characters and because of sharing a complex color patterns. Our results suggest that the family passed through a process of rapid speciation and that the species Potamotrygon motoro, P. scobina, and P. orbignyi share haplotypes extensively. Our results suggest that systems of identification of specimens based on DNA sequences, together with morphological and/or ecological characters, can aid taxonomic studies, but delimitation of new species based on threshold values of genetic distances are overly simplistic and misleading.

Identification and phylogenetic inferences on stocks of sharks affected by the fishing industry off the Northern coast of Brazil

The ongoing decline in abundance and diversity of shark stocks, primarily due to uncontrolled fishery exploitation, is a worldwide problem. An additional problem for the development of conservation and management programmes is the identification of species diversity within a given area, given the morphological similarities among shark species, and the typical disembarkation of processed carcasses which are almost impossible to differentiate. The main aim of the present study was to identify those shark species being exploited off northern Brazil, by using the 12S-16S molecular marker. For this, DNA sequences were obtained from 122 specimens collected on the docks and the fish market in Bragança, in the Brazilian state of Pará. We identified at least 11 species. Three-quarters of the specimens collected were either Carcharhinus porosus or Rhizoprionodon sp, while a notable absence was the daggernose shark, Isogomphodon oxyrhyncus, previously one of the most common species in local catches. The study emphasises the value of molecular techniques for the identification of cryptic shark species, and the potential of the 12S-16S marker as a tool for phylogenetic inferences in a study of elasmobranchs.

Methylation status of ANAPC1, CDKN2A and TP53 promoter genes in individuals with gastric cancer

Gastric cancer is the forth most frequent malignancy and the second most common cause of cancer death worldwide. DNA methylation is the most studied epigenetic alteration, occurring through a methyl radical addition to the cytosine base adjacent to guanine. Many tumor genes are inactivated by DNA methylation in gastric cancer. We evaluated the DNA methylation status of ANAPC1, CDKN2A and TP53 by methylation-specific PCR in 20 diffuse- and 26 intestinal-type gastric cancer samples and 20 normal gastric mucosa in individuals from Northern Brazil. All gastric cancer samples were advanced stage adenocarcinomas. Gastric samples were surgically obtained at the João de Barros Barreto University Hospital, State of Pará, and were stored at -80°C before DNA extraction. Patients had never been submitted to chemotherapy or radiotherapy, nor did they have any other diagnosed cancer. None of the gastric cancer samples presented methylated DNA sequences for ANAPC1 and TP53. CDKN2A methylation was not detected in any normal gastric mucosa; however, the CDKN2A promoter was methylated in 30.4% of gastric cancer samples, with 35% methylation in diffuse-type and 26.9% in intestinal-type cancers. CDKN2A methylation was associated with the carcinogenesis process for ~30% diffuse-type and intestinal-type compared to non-neoplastic samples. Thus, ANAPC1 and TP53 methylation was probably not implicated in gastric carcinogenesis in our samples. CDKN2A can be implicated in the carcinogenesis process of only a subset of gastric neoplasias.

Genetic variation in native and farmed populations of Tambaqui (Colossoma macropomum) in the Brazilian Amazon: regional discrepancies in farming systems

O tambaqui, Colossoma macropomum, é a espécie de peixes mais popularmente usada para a aquicultura no Brasil, mas não há nenhum estudo comparando a variação genética entre as populações nativas e de cultivo desta espécie. No presente estudo foram analisadas sequências de DNA mitocondrial para avaliar a diversidade genética entre duas populações selvagens, um plantel de produção de alevinos, e uma amostra de estoques de piscicultura, todos da região de Santarém, no oeste do estado do Pará. Níveis similares de diversidade genética foram encontrados em todas as amostras e, surpreendentemente, o plantel mostrou expressiva representação da diversidade genética registrada em populações selvagens. Estes resultados contrastam consideravelmente com os do estudo anterior de estoques cultivados nos estados do Amapá, Pará, Piauí, Rondônia, que registrou apenas dois haplótipos, indicando uma longa história de endogamia nas matrizes utilizadas para a produção de alevinos. Os resultados dos dois estudos mostram dois cenários distintos de aquicultura do tambaqui na Amazônia, que devem ser melhor avaliados, a fim de garantir o sucesso da expansão da atividade na região, e no resto do Brasil, já que o tambaqui e seus híbridos agora são cultivados em todo o país.

Karyotypic and morphological divergence between two cryptic species of Eigenmannia in the Amazon basin with a new occurrence of XX/XY sex chromosomes (Gymnotiformes: Sternopygidae)

Espécies de Eigenmannia estão amplamente distribuídas na região Neotropical, com oito espécies válidas atualmente reconhecidas. Populações de Eigenmannia de três localidades do leste da Amazônia foram investigadas usando técnicas citogenéticas e morfológicas, revelando dois táxons designados aqui comoEigenmannia sp. "A" e Eigenmannia sp. "B". As espécies diferem em três caracteres morfométricos, dois merísticos e um osteológico. Eigenmannia sp. "A" apresenta 2n = 34 (22 m/sm+12st/a) e Eigenmannia sp. "B" apresenta 2n = 38 (14 m/sm+24st/a) e cromossomos sexuais de diferenciação simples, do tipo XX/XY. Em ambas espécies a Heterocromatina Constitutiva (HC) rica em bases A-T está distribuída na região centromérica de todos os cromossomos. Eigenmannia sp. "B" também apresenta blocos de HC na região intersticial dos pares cromossômicos 8, 9 e X que coraram positivamente para CMA₃, indicando regiões ricas em G-C. A NOR está localizada no braço curto do par 17 em Eigenmannia sp. "A" e no braço curto do par 14 em Eigenmannia sp. "B". FISH com sondas de rDNA hibridizaram em regiões de tamanhos diferentes entre os homólogos, sugerindo heteromorfismo. A diferenciação do cromossomo X em Eigenmannia sp. "B" pode ser o resultado de amplificação de sequências repetitivas de DNA.

Mitochondrial DNA-like sequence in the nuclear genome of Saguinus (Callitrichinae, Primates): transfer estimation

Seqüências tipo mitocondriais têm comumente sido encontradas no genoma nuclear de diversos organismos. Quando acidentalmente incluídas em estudos de seqüências mitocondriais, diversas conclusões errôneas podem ser obtidas. No entanto, estes pseudogenes nucleares tipo mitocondriais podem ser usados para a estimativa da taxa relativa de evolução de genes mitocondriais e também como grupo externo em análises filogenéticas. No presente trabalho, seqüências mitocondriais com características do tipo de pseudogene, tais como deleções e/ou inserções e códons de parada, foram encontradas em tamarins (Saguinus spp., Callitrichinae, Primates). A análise filogenética permitiu a estimativa do tempo da migração da seqüência mitocondrial para o genoma nuclear e algumas inferências filogenéticas. A escolha de um grupo externo não adequado (Aotus infulatus) não permitiu uma reconstrução filogenética confiável da subfamília Callitrichinae. A divergência bastante antiga de Cebidae (Callitrichinae, Aotinae e Cebinae) pode ter favorecido o aparecimento de homoplasias, obscurecendo a análise.

Sciaenidae fish of the Caeté River estuary, Northern Brazil: mitochondrial DNA suggests explosive radiation for the Western Atlantic assemblage

Sciaenids are fish which are normally abundant in tropical estuaries of the western Atlantic. Studies on the Caeté river estuary in the northern Brazilian state of Pará have revealed that in this area Sciaenidae is the dominant family, comprising almost 50% of all teleosts sampled. In this paper we present the results of the first phylogenetic study on South American estuarine sciaenids, during which we obtained mitochondrial gene 16S sequences from 15 species belonging to eight genera occurring in the Caeté estuary. Intergeneric nucleotide divergences varied from 5 to 15%, Lonchurus and Menticirrhus being the most divergent lineages. Nucleotide divergences were quite variable amongst species of the same genus, ranging from 1.2% (Stellifer microps x Stellifer naso) to 8.4% (Menticirrhus americanus x Menticirrhus littoralis). Cladograms based on maximum parsimony, minimum evolution and maximum likelihood depicted an explosive diversification pattern for the western Atlantic sciaenid assemblage. Our analysis further reveals a very close relationship between Bairdiella and Stellifer, a monophyletic clade which emerged during the more recent diversification events of the Sciaenidae family. The phylogenetic reconstruction suggests the need for a revision of the taxonomy and nomenclature of the Bairdiella/Stellifer group.

Phylogenetic analysis of 16S mitochondrial DNA data in sloths and anteaters

We sequenced part of the 16S rRNA mitochondrial gene in 17 extant taxa of Pilosa (sloths and anteaters) and used these sequences along with GenBank sequences of both extant and extinct sloths to perform phylogenetic analysis based on parsimony, maximum-likelihood and Bayesian methods. By increasing the taxa density for anteaters and sloths we were able to clarify some points of the Pilosa phylogenetic tree. Our mitochondrial 16S results show Bradypodidae as a monophyletic and robustly supported clade in all the analysis. However, the Pleistocene fossil Mylodon darwinii does not group significantly to either Bradypodidae or Megalonychidae which indicates that trichotomy best represents the relationship between the families Mylodontidae, Bradypodidae and Megalonychidae. Divergence times also allowed us to discuss the taxonomic status of Cyclopes and the three species of three-toed sloths, Bradypus tridactylus, Bradypus variegatus and Bradypus torquatus. In the Bradypodidae the split between Bradypus torquatus and the proto-Bradypus tridactylus / B. variegatus was estimated as about 7.7 million years ago (MYA), while in the Myrmecophagidae the first offshoot was Cyclopes at about 31.8 MYA followed by the split between Myrmecophaga and Tamandua at 12.9 MYA. We estimate the split between sloths and anteaters to have occurred at about 37 MYA.

Aplicação de redes neurais artificiais na classificação de padrões filogeográficos com base na variabilidade genômica do DNA mitocondrial

Historicamente, o processo de formação das populações da Amazônia, assim como de todo território brasileiro, envolveu três grupos étnicos principais: o ameríndio, o europeu e o africano. Como conseqüência, estas populações possuem em geral constituição miscigenada do ponto de vista social e biológico. Desde o final do século passado, estudos do DNA mitocondrial (mtDNA) tem sido desenvolvidos com o propósito de estimar a mistura interétnica presente nestas populações. Para isto, é de fundamental importância a classificação de uma determinada linhagem de mtDNA em um dos mais de 250 haplogrupos/subclados propostos na literatura. Com o objetivo de desenvolver um sistema automatizado, preciso e acurado de classificação de seqüências (linhagens) de mtDNA, o presente trabalhou lançou mão da técnica de Redes Neurais Artificiais (RNA’s) tendo como base os estudos de filogeografia. Para esta classificação, foram desenvolvidas quatro redes neurais artificiais diretas, com múltiplas camadas e algoritmo de aprendizagem de retropropagação. As entradas de cada rede equivalem às posições nucleotídicas polimórficas da região hipervariável do DNA mitocondrial, as quais retornam como saída a classificação específica de cada linhagem. Posterior ao treinamento, todas as redes apresentaram índices de acerto de 100%, demonstrando que a técnica de Rede Neural Artificial pode ser utilizada, com êxito, na classificação de padrões filogeográficos com base no DNA mitocondrial.

Cytogenetic and DNA barcoding reveals high divergence within the trahira, Hoplias malabaricus (Characiformes: Erythrinidae) from the lower Amazon River

Dados moleculares e citogenéticos tem evidenciado especiação críptica na traíra sul-americana, Hoplias malabaricus. No presente estudo, cariótipos e sequências de DNA barcode de espécimes de sete populações, habitando a região do baixo rio Amazonas, foram analisadas a fim de caracterizar o nível de divergência genética dentro de um único cariomorfo. Todos os espécimes possuem 2n = 40 cromossomos (20m+20sm) os quais são inseridos no grupo de traíras do cariomorfo C. DNA barcode revelou seis haplogrupos, com clara divergência entre populações do Brasil e da Argentina. Os resultados apoiam a hipótese de complexo de espécies e indicam que um único cariomorfo de Hoplias malabaricus pode conter mais de uma espécie.