Karyotypic variation of two populations of the small freshwater stingray Potamotrygon wallacei Carvalho, Rosa & Araújo 2016: A classical and molecular approach

PLOS ONE RESEARCH ARTICLE Karyotypic variation of two populations of the small freshwater stingray Potamotrygon wallacei Carvalho, Rosa & Araújo 2016: A classical and molecular approach Alex M. V. Ferreira ID1*, Patrik F. Viana2, Leandro Marajó ID1, Eliana Feldberg2 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Programa de Pós-Graduação em Genética Conservação e Biologia Evolutiva – PPG GCBEv, Instituto Nacional de Pesquisas da Amazônia – INPA, Manaus, Amazonas, Brazil, 2 Laboratório de Genética Animal, Coordenação de Biodiversidade, Instituto Nacional de Pesquisas da Amazônia – INPA, Manaus, Amazonas, Brazil * Abstract OPEN ACCESS Citation: Ferreira AMV, Viana PF, Marajó L, Feldberg E (2023) Karyotypic variation of two populations of the small freshwater stingray Potamotrygon wallacei Carvalho, Rosa & Araújo 2016: A classical and molecular approach. PLoS ONE 18(1): e0278828. https://doi.org/10.1371/ journal.pone.0278828 Editor: Dengcai Liu, Sichuan Agricultural University at Chengdu, CHINA Received: August 29, 2022 Accepted: November 24, 2022 Published: January 20, 2023 Copyright: © 2023 Ferreira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Potamotrygoninae comprises a group of Neotropical fishes with an ancient relationship with marine environments. In the last few years, 11 new Potamotrygon species were described, including Potamotrygon wallacei Carvalho, Araújo e Rosa 2016. Cytogenetic data about this species are limited to classical markers (Giemsa, C-Banding and Ag-NOR techniques), these studies highlighted a rare sexual chromosome system XX/X0 with males presenting 67 chromosomes and females 68 chromosomes. The classical analyses performed here reveled populational variation in the karyotype formula, as well as, in the heterochromatin regions. Besides the classical markers, our molecular experiments showed multiple sites for 18S rDNA sequence (including in the X chromosomes) and single sites for 5S rDNA sequence, we did not find interstitial telomeric sequences. In addition, (AC)15, (AG)15, and (CAC)15 microsatellites showed association with the several autosome pair, and the (GT)15 clutters were found in only one population. On the other hand, (GATA)4 sequence showed association with the sexual chromosomes X in all males and females analyzed. Our results showed that pericentric inversions, in addition to fusions, shaped the karyotype of P. wallacei once we found two populations with distinct karyotype formula and this could be a result of the past events recovered by our modeling experiments. Besides, here we described the association of 18S and (GATA)4 motifs with sexual chromosomes, which indicated that these sequences had a novel in the differentiation of sexual chromosomes in P. wallacei. Data Availability Statement: All relevant data are within the paper and its Supporting information files. Funding: This study was financed by the Centro de Estudos de Adaptação às Mudanças Ambientais na Amazonia (INCT ADAPTA II, FAPEAM/CNPq 573976/2008-2), FAPEAM/SEPLANCTI/Governo do Estado do Amazonas – POSGRAD Res. No. 003/ 2019, Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), PAPAC-FAPEAM Introduction The subfamily Potamotrygoninae is a group of South American stingray lineages of marine origin that comprises over 40 species restricted to freshwater environments [1–7]. Currently, four genera, Heliotrygon, Plesiotrygon, Paratrygon, and Potamotrygon are recognized for this subfamily [8, 9], occurring in different South America river basins that flow into the Caribbean PLOS ONE | https://doi.org/10.1371/journal.pone.0278828 January 20, 2023 1 / 16 PLOS ONE (Edital PAPAC 005/2019), Universal/FAPEAM (EDITAL N. 002/2018), and Fundação de Amparo à Pesquisa do Estado do Amazonas for the scholarship awarded to the student (Edital 003/ 2019). EF was the recipient of a fellowship from CNPq (Grant #301886/2019-9). Genetics, Conservation and Evolutionary Biology (INPA/ GCBEv), given institutional support for this study. The funding agencies that financed this study had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Karyotypic variation of two populations of Potamotrygon wallacei and Atlantic Sea [2, 10]. In the Amazon basin, these stingrays are found in different types of water, such as black water, clear water and white water [2]. Additionally, some physiological and ecological traits, such as their low fecundity, late maturation and small home range size increase the vulnerability to indiscriminate fishing [11–14]. In the past years, some studies have revealed a hitherto unknown diversity into the Potamotrygoninae subfamily, with the description of a new genus and several new species [7, 8, 15]. The genus Potamotrygon, for instance, is the most speciose among all potamotrygonins, with 11 new described species only in the past decade [8, 16–20], including Potamotrygon wallacei Carvalho, Araújo e Rosa 2016 [21], one of the most iconic freshwater stingray species in the Amazon. Potamotrygon wallacei is the smallest stingray of the Potamotrygon genus, and although it has been known since the Alfred Russel Wallace’s journey into the Amazon, the species was only named (in honor of Wallace) in 2016 [13, 21]. P. wallacei is an endemic species, with distribution apparently restricted to the Rio Negro basin [21], however, the real extent of its distribution as well as many aspects of its natural history remains unknown. Due to its small size, Potamotrygon wallacei is a very appreciated species in the aquarist market, and although it is not even listed in the IUCN, this species has been legally and illegally exported to supply the international market even before its formal description [11, 13, 21]. Cytogenetic data about this species revealed the presence of an intriguing and rare XX/X0 sex chromosome system, and diploid number of 68 for females and 67 for males [22], and although there is evidence of karyotypic variations between populations, the chromosomal data are limited to the description of the diploid number and pattern of constitutive heterochromatin [22, 23]. Molecular cytogenetics approaches using repetitive sequences, such as 18S and 5S rDNAs and simple short repeats (SSRs or microsatellites), surely provides important information about chromosome evolution and sex chromosome origin [24–27]. Unfortunately, most cytogenetic studies in freshwater stingrays, including Potamotrygon wallacei, are scant (only 11 species were karyotyped—see Table 1 of Cruz et al. [28]) and limited to classical techniques (Giemsa staining, C-banding, and Ag-NOR) [22, 23, 29, 30] and only recentl (...truncated)