Identificação molecular (DNA barcoding) e variabilidade genética em leporinus piau (characiformes: anostomidae) de bacias hidrográficas do Estado do Maranhão, Brasil

Abstract

The Anostomidae family comprises 12 genera and approximately 140 valid species. In this Family the genus Leporinus includes about 90 valid species, grouping the largest number of taxa within the Characiformes and most diverse in the anostomidae family. The species Leporinus piau is widely distributed in watersheds of the Northeast and presents very similar morphological patterns hindering their correct identification. Therefore, on this study sequences of mitochondrial and nuclear genome were used to identify and estimate the genetic variability levels of Leporinus piau so try to resolve the taxonomic uncertainties. The DNA was isolated using the Phenol-chloroform protocol, the mitochondrial and nuclear region regions were amplified by PCR. The PCR products were sequenced using the didesoxiterminal method on ABI 3500. A total of 107 specimens were sequenced for the rRNA gene resulting in obtaining of a fragment of 516 pb, 14 haplotypes and value of haplotype diversity (h) of 0.726 and the nucleotide (π) of 0.012. The haplotype crossroad revealed three haplogroups two haplotypes formed by the basins of the Rivers Itapecuru, Pindaré and Mearim, and another formed by haplotypes of Parnaíba River basin. The rates of genetic distance between the haplogrupos ranged from 1,9 to 2,9%. The results of AMOVA revealed high structuring between the populations (FST = 0,648) and between haplogroups (0,951) with highly significant p value. A fragment of 622 pb of the COI gene was obtained for 103 specimens resulting in 17 haplotypes and high haplotype diversity values and nucleotide (һ = 0,819 and π = 0,025). The haplotypes crossroad generated also showed the formation of three distinct evolutionarily haplogrupos with genetic distance values ranging from 3,6% to 7,5%. The AMOVA indicated that most of the molecular variation (96,59%) occurs between haplogrupos (FST = 0,965) with highly significant p value. The Phylogenetic analysis obtained by the mitochondrial data generated similar topology strongly grouping haplotypes into two clades, the first consisting of the haplogroup Parnaíba (haplotypes from Parnaíba) and the second formed by haplotypes from Rivers Itapecuru, Pindaré and Mearim, subdivided into two haplogroups (ITA/PIN/MEA-I e ITA/PIN/MEA-II). The comparison of the data in the BOLD Systems platform showed a percentage of 98.84% similarity to L. piau and 99.16% with L. friderici for specimens derived from the Parnaíba Basin, to the copies from the other basins, the found similarity ranged from 97,17% to 100% with (Leporinus sp04, 06, 10 e 15) and of 99,17% to 99,34% with L. friderici. For TROP core region was obtained a fragment of 250 pb to 120 individuals, showing the occurrence of 05 haplotypes. The haplotype crossroad also showed the formation of three haplogrupos with genetic distance values between haplogrupos ranging from 0,8% to 2,1%. The AMOVA analysis also showed that the most of the molecular variation (96,54%) occurs between haplogrupos (FST = 0,965, P <0.00001). Our results confirm a process of genetic structure to L. piau, suggesting the existence of two strains for Rios Itapecuru, Pindaré and Mearim and a third of exclusive occurrence in the basin of Rio Parnaíba