Morphological, cytochemical and ultrastructural aspects of blood cells in freshwater stingray species in the middle Rio Negro basin of Amazonian Brazil.

In the present work, we examined the morphology, dimensions, cytochemical staining reactions and ultrastructure of blood cells from three freshwater stingray species, Potamotrygon wallacei, Potamotrygon motoro and Paratrygon aiereba, living in the waters of the middle Rio Negro basin (Barcelos, Amazonas, Brazil). We identified erythrocytes, erythroblasts, thrombocytes and four types of leukocytes (basophils, heterophils, lymphocytes and monocytes) in the blood of these stingray species. In all the freshwater stingray species studied, the shapes and dimensions of these cells were similar to those of marine elasmobranchs. Positive PAS staining occurred in heterophils and thrombocytes, and weak staining occurred in lymphocytes and monocytes, while metachromasia only occurred in basophils. Positive Sudan Black B staining was observed in thrombocytes and lymphocytes, and weak staining occurred in heterophils. Basophils and heterophils were the only cells with positive bromophenol blue staining, while no peroxidase staining was observed in any of the four leukocyte types. This is the first study to establish the dimensions and cytochemical staining profiles of blood cells in Amazonian stingray species. Because these elasmobranch species are exported as ornamental fish to countries worldwide, this study can contribute to establishing standards for blood constituents that may be helpful in assessing the health and welfare of these fish in artificial systems.

Sex steroid hormones and the associated morphological changes in the reproductive tract of free-living males of the cururu stingray Potamotrygon wallacei.

This study analyzed sex steroid hormones and morphology of the reproductive tract in free-living adult males of the cururu stingray (Potamotrygon wallacei). These stingrays were captured during the transition between drought and rising waters in the middle Rio Negro basin, which coincides with the reproductive period (spermatogenesis and copulation) of this endemic species. Levels of the plasma steroid hormones testosterone (T), 17 ß-estradiol (E2), and progesterone (P4), the gonadosomatic and hepatosomatic indices, as well as histological characteristics of the testis and epididymis were evaluated. Three reproductive maturational stages were observed: regression males, capable to reproduce males, and active males. Plasma T levels were increasing and parallel with the rising water period in the middle Rio Negro basin and were associated with the progress of spermatogenesis. This was not observed for E2, which did not vary significantly among the different reproductive stages. The high P4 concentrations in males in regression and males capable of reproducing appeared to regulate the progression of spermatogenesis. Increases in this hormone in the plasma of active males was associated with spermiogenesis and spermiation events. These findings have not been previously observed in Amazonian freshwater stingrays and demonstrate that the reproductive activity of P. wallacei males is concomitant to the period of rising waters in the middle Rio Negro basin, and is regulated by the action of gonadal steroids, mainly testosterone.

Phylogeography and conservation genetics of the Amazonian freshwater stingray Paratrygon aiereba Müller & Henle, 1841 (Chondrichthyes: Potamotrygonidae)

The family Potamotrygonidae is monophyletic comprising three genera: Paratrygon Duméril, Potamotrygon Garman and Plesiotrygon Rosa, Castello & Thorson. The distribution of most species in this family is restricted to a single basin or fluvial system. Only Potamotrygon motoro, Potamotrygon orbignyi and Paratrygon aiereba are found in more than one river basin. In this study we investigate genetic structuring of Paratrygon aiereba, from five rivers of the Amazon region: Negro, Solimões-Amazon-Estuary system, Tapajós, Xingu and Araguaia. Sixty-three individuals were sequenced for ATPase 6, and a representative subsample of 27 individuals was sequenced for COI. The COI dataset analysis indicated that Paratrygon is sister to all other potamotrygonid genera and species. Population parameters inferred from the analysis of ATPase 6 sequences revealed that the populations of this species are structured within each river, with no or nearly non-existent gene flow occurring between rivers and a positive correlation between geographic and genetic distances. Paratrygon aiereba is comprised of three geographically restricted clades with K2P interclade distances of at least 2%. Intraspecific divergence within P. aiereba is similar to the interspecific divergence observed in Potamotrygon spp. sampled throughout the same geographic area. Using the premises of COI barcoding and the allopatric distribution of the three P. aiereba clades, the taxon P. aiereba most likely comprises three distinct biological species. Since freshwater stingrays of the family Potamotrygonidae are highly exploited for the aquarium trade, management and conservation strategies need to be implemented at the level of each river basin, rather than at the level of the Amazon basin.

A família Potamotrygonidae forma um clado monofilético com três gêneros: Paratrygon Duméril, Potamotrygon Garman e Plesiotrygon Rosa, Castello & Thorson. A maioria das espécies dessa família possui distribuição restrita a uma única bacia ou sistema fluvial, e somente as espécies Potamotrygon motoro, Potamotrygon orbignyi e Paratrygon aiereba estão presentes em mais de uma bacia hidrográfica. O presente estudo teve como objetivo investigar a estrutura genética de Paratrygon aiereba em alguns rios da região Amazônica: Negro, sistema Solimões-Amazonas, Tapajós, Xingu, e Araguaia. Para tal foram utilizados como marcador molecular os genes de ATPase subunidade 6, e COI. As análises com o fragmento de COI indicaram que o gênero Paratrygon é grupo irmão dos outros gêneros da família potamotrygonidae. Os resultados para o fragmento de ATPase mostraram que essas populações estão estruturadas dentro dos rios, com fluxo gênico restrito, ou mesmo sem fluxo gênico, apresentando uma correlação positiva entre distância genética e distância geográfica. Paratrygon aiereba é composta por três clados com distância genética de pelo menos 2%. A divergência encontrada dentro desse grupo é semelhante à observada entre Potamotrygon spp. Segundo as premissas para barcoding COI e a distribuição alopátrica de três clados em P. aiereba indicam que esse grupo pode ser um complexo de espécies. O rio Negro é conhecido por sua pesca ornamental, e na calha Solimões-Amazonas, esses animais são utilizados como fonte de proteína e sofrem com a pesca comercial. Em vista disso medidas de conservação para esta espécie devem ser tomadas em nível local, considerando cada rio separadamente, ao invés de empregar escalas regionais maiores.

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.