An Acad Bras Cienc (2021) 93(3): e20191325 DOI 10.1590/0001-3765202120191325 Anais da Academia Brasileira de Ciências | Annals of the Brazilian Academy of Sciences Printed ISSN 0001-3765 I Online ISSN 1678-2690 www.scielo.br/aabc | www.fb.com/aabcjournal CELLULAR AND MOLECULAR BIOLOGY Mitochondrial DNA suggests Hybridization Running title: HYBRIDIZATION in Freshwater Stingrays Potamotrygon AND SPECIATION IN STINGRAYS OF XINGU (POTAMOTRYGONIDAE: MYLIOBATIFORMES) from the Xingu river, Amazonia and reveals speciation in Paratrygon aireba Academy Section: CELLULAR AND MOLECULAR BIOLOGY DAMIRES SANCHES, THAIS MARTINS, ÍTALO LUTZ, IVANA VENEZA, RAIMUNDO DA SILVA, FELIPE ARAÚJO, JANICE MURIEL-CUNHA, IRACILDA SAMPAIO, MAGALI e20191325 GARCIA, LEANDRO SOUSA & GRAZIELLE EVANGELISTA-GOMES Abstract: In the Xingu river basin, Brazil, occurs two genera of Potamotrygonidae 93 family: Potamotrygon and Paratrygon. In this region, the taxa have signifi cant economic (3) importance for the ornamental fi shing industry, being intensively captured, especially 93(3) the species Potamotrygon leopoldi, which is endemic to this basin. In the attempt to propose a species-specifi c DNA marker for the species from Xingu, as well as ensuring DOI a robust and reliable molecular identifi cation, the present work analysed mitochondrial 10.1590/0001-3765202120191325 gene portions Cytochrome Oxidase C – subunit I (COI) and Cytochrome B (Cytb) of fi ve species: P. leopoldi; Potamotrygon orbignyi; Potamotrygon motoro; Potamotrygon scobina and Paratrygon aiereba. We found haplotype sharing, with a total absence of reciprocal monophyly in the majority of taxa. Individuals morphologically identifi ed as a species showed mitochondrial DNA from another, suggesting the fi rst record of hybridization amongst freshwater stingrays of Xingu. Also, we detected a deep divergence among Paratrygon aiereba haplotypes, indicative of speciation, suggesting the possibility of a new species for the Xingu river. Therefore, although the is still confusing and controversial taxonomy of freshwater stingrays, and evidencing hybridization processes that may have shaped the evolutionary history of this Family, the genes COI and Cytb can successfully help in the their species identifi cation. Key words: Potamotrygonidae, Potamotrygon, Paratrygon aiereba, Xingu river. INTRODUCTION inclusion of a new genus (Styracura) and subfamily (Styracurinae) in Potamotrygonidae, The Potamotrygonidae family currently these taxa were initially placed in the marine includes 32 species, organized into four genera: stingray family Dasyatidae. Paratrygon Duméril (1865), a monospecific The freshwater stingrays have a restricted genus; Potamotrygon Garman (1877), composed distribution to the South American rivers, which of 27 species, also the genera Pleisiotrygon fl ow into the Caribbean and the Western Atlantic Rosa, Castello & Thorson (1987) and Heliotrygon Ocean. Generally, they do not occur in the São Carvalho & Lovejoy (2011) with two described Francisco river basin and coastal drainages from species each (Fontenelle & Carvalho 2017). the east and south of Parnaíba river (Rosa et Recently Carvalho et al. (2016) proposed the al. 2010). Some Potamotrygonidae species are An Acad Bras Cienc (2021) 93(3) DAMIRES SANCHES et al. HYBRIDIZATION AND SPECIATION IN STINGRAYS OF XINGU endemic to a single river basin, such as the marker (Chen et al. 2014, Palacios-Barreto et al. White-blotched river stingray (Potamotrygon 2017). Moreover, its accentuated polymorphism leopoldi), which is restricted to the Xingu river, can provide information about population in the Brazilian Amazon (Lasso et al. 2016, Rosa structuring (Da Silva et al. 2015, 2016, 2018, Silva 1985, not published in indexed journals). et al. 2018). Additionally, some of the species have In the case of the neotropical freshwater economic importance as ornamental fish in ichthyofauna, many studies have confirmed both national and international scenarios the utility of COI as a Barcode method (Pereira (Duncan et al. 2010). Ornamental fishing occurs et al. 2013). However in Potamotrygonidae, extensively in the Xingu river basin. Thus, the especially to the species from Potamotrygon intense capture of some taxa could compromise genus, this marker did not show the ability to the sustainability of entire species populations, discriminate the taxa, particularly the species P. likewise impacting on the maintenance of this motoro, P. scobina, P. orbiginyi which constitutes commercial activity in the region. the rosette-spot clade (Toffoli et al. 2008). The Furthermore, Potamotrygonidae is a group rosette-spot clade is a group of species that do in which the taxonomy is still poorly understood. not have reciprocal monophyly, which can be the Recently it was subjected to many taxonomic result of recent phylogenetic radiation (Schluter revisions (Almeida et al. 2008, Loboda & 2000). Considering these pointed issues, Carvalho 2013, Lasso et al. 2016, Fontenelle & followed by the dubiousness around the family Carvalho 2017). The most extreme taxonomic taxonomy may collaborate as to the incapability revision was proposed by Carvalho et al. (2016), of Barcode to separate the species (Toffoli et al. where it was proposed that a subdivision of 2008). In addition to this, hybridization cases Potamotrygonidae family be made into two and/or incomplete lineages shared among subfamilies with the inclusion of marine species, Potamotrygonidae can also cover up the DNA previously placed in Dasyatidae family. Barcode efficiency (Pereira et al. 2013, Cruz et al. On the face of it, it is essential to use robust 2015). molecular tools capable of providing species- There is not any genetic characterization specific DNA marker which can be applied to found in the literature for the Potamotrygonidae the identification of freshwater stingrays. It species from the Xingu river basin. Even though can be the basis for conservation mitigation the freshwater stingrays have a wide distribution initiatives, contribute to export control, the over this basin, there is a record of an endemic sustainable maintenance of stocks, and effective species to the basin (P. leopoldi), as well as, acknowledgement of group diversity. the intense and deregulated exploitation of The fragment from the mitochondrial fish species for the ornamental fishing market. gene Cytochrome C oxidase subunit I (COI), an Consequently, many species are captured intraspecific conserved sequence that has the and commercialized without a secure record capability to show interspecific variations, was of what species are being taken, also, there chosen to serve as standard DNA Barcode pattern, is the possibility of hybrids individuals be (Hebert et al. 2003, Ward et al. 2005, Pereira et compounding this commerce. This raises many al. 2013). Besides COI, Cytochrome b (Cytb) has concerns about the impacts of ornamental been successfully applied in the identification fishing and its sustainability, the impact on the of fish, showing as well an efficiency barcode maintenance of the overexploited stocks and An Acad Bras Cienc (2021) 93(3) e20191325 2 | 18 DAMIRES SANCHES et al. HYBRIDIZATION AND SPECIATION IN STINGRAYS OF XINGU the threats to cryptic ichthyofauna yet to be Potamotrygon orbignyi (26); Potamotrygon described. motoro (11); Potamotrygon scobina (10); In this study, we employ molecular markers Paratrygon aiereba (4) and Potamotrygon sp1 from the mitochondrial genome (Barcode (4); Potamotrygon sp2 (4). The sample collection fragments from COI and Cytb genes) for delimiting was distributed along 20 localities over the main species of Potamotrygon and Paratrygon channel of the Xingu river basin, Brazil, as well as genera. We also, discuss the effectiveness of in their tributary’s river, Bacajá river in the east these mitogenomic regions as Barcode tool, and margin and Iriri river in the west side (Figure 1). the possibility of speciation and hybridization The individuals were measured, in the Potamotrygonidae population along the photographed and identified checking the Xingu river. identification key species available for the Potamotrygonidae family (Loboda & Carvalho 2013, Fontenelle & Carvalho 2017). Tissue MATERIALS AND METHODS samples were collected from the pelvic fin, also, Sampling we verified if the individuals had already been One hundred and sisty one samples were sampled according to the presence of scars in obtained for this study, which included the pelvic fins. All individuals were released the species: Potamotrygon leopoldi (102); back into their respective sampling locations. The tissue samples were preserved in cryogenic Figure 1. The study area map showing the collection sampling localities and where the respective species were found (Potamotrygon leopoldi=yellow; Potamotrygon orbignyi=blue; Potamotrygon motoro=green; Potamotrygon scobina=pink; Paratrygon aiereba=red). An Acad Bras Cienc (2021) 93(3) e20191325 3 | 18 DAMIRES SANCHES et al. HYBRIDIZATION AND SPECIATION IN STINGRAYS OF XINGU tubes with ethyl alcohol, commercial (95%) and sequenced using the dideoxy method (Sanger stored at -20 for posterior molecular procedures. et al. 1977), with the Big Dye kit (ABI PrismTM Dye Terminator Cycle Sequencing Reading Reaction Laboratory procedures – PE Thermo Fisher). The precipitated product Whole genome was isolated using the Wizard was submitted to capillary electrophoresis in ABI Genomic kit (Promega, Fitchburg, WI, USA), 3500XL automatic sequencer (Thermo Fisher). according to the manufacturer’s instructions.