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Chromosomal characterization of Amazonian freshwater stingrays with evidence for new karyomorphs and XX/XY sex chromosomes

Abstract

Cytogenetic studies in the subfamily Potamotrygoninae have provided valuable insights into the understanding of the evolution and diversification of its species. In the present study, the chromosomal features of seven nominal potamotrygonin species are provided: Plesiotrygon iwamae (2n=74, FN=120), Potamotrygon amazona (2n=66, FN=107), P. constellata (2n=66, FN=110), P. leopoldi (2n=64, FN=102), P. motoro (2n=66, FN=106) from four different localities, and P. orbignyi (2n=66, FN=106), P. scobina (2n=66, FN=104), from Central Amazon. Additionally, we found a new karyomorph in P. wallacei. We considered the localization of Nucleolus Organizer Regions (NORs), as well as the pattern of constitutive heterochromatin, as species-specific characters. We found an XX/XY sex chromosome system in P. orbignyi, and we suggest that P. scobina and P. amazona also possess the same sex chromosome system. Overall, the chromosomal evolution in this group appears to have progressed towards a reduction in diploid number, with a concomitant increase in the number of bi-armed and nucleolar chromosomes.

Keywords:
Elasmobranchs; cytogenetics; chromosomal rearrangements; sex chromosomes; karyotypic diversity

Introduction

The monophyly of the order Myliobatiformes, which includes the freshwater stingrays of the subfamily Potamotrygoninae, is well established (Carvalho et al., 2016bCarvalho MR, Loboda TS and Silva JPCB (2016b) A new subfamily, Styracurinae, and new genus, Styracura, for Himantura schmardae (Werner, 1904) and Himantura pacifica (Beebe and Tee-Van 1941) (Chondrichthyes: Myliobatiformes). Zootaxa 4175:201–221.; Nelson et al., 2016Nelson JS, Grande TC and Wilson MVH (2016) Fishes of the World. John Wiley & Sons, Hoboken, 752 pp.). The subfamily Styracurinae, represented by the marine genus Styracura (S. schmardae and S. pacifica), is more closely related to the Neotropical freshwater stingrays, as confirmed by morphological (Lovejoy, 1996Lovejoy NR (1996) Systematics of myliobatoid elasmobranchs: With emphasis on the phylogeny and historical biogeography of Neotropical freshwater stingrays (Potamotrygonidae: Rajiformes). Zool J Linn Soc 117:207-257.; Carvalho et al., 2004Carvalho MR, Maisey JG and Grande L (2004) Freshwater stingrays of the Green River Formation of Wyoming (Early Eocene), with the description of a new genus and species and an analysis of its phylogenetic relationships (Chondrichthyes: Myliobatiformes). Bull Am Mus Nat Hist 284:1-36.; Aschliman et al., 2012Aschliman NC, Nishida M, Miya M, Inoue JG, Rosana KM and Naylor GJP (2012) Body plan convergence in the evolution of skates and rays (Chondrichthyes: Batoidea). Mol Phylogenet Evol 63:28–42.) and molecular phylogenies (Naylor et al., 2012aNaylor GJP, Caira JN, Jensen K, Rosana KAM, Straube N and Lakner C (2012a) Elasmobranch phylogeny: A mitochondrial estimate based on 595 species. In: Carrier JC, Musick JA and Heithaus MR (eds) Biology of sharks and their relatives. 2nd edn. CRC Press, Boca Raton, pp 31–56.; bNaylor GJP, Caira JN, Jensen K, Rosana KAM, White WT and Last PR (2012b) A DNA sequence-based approach to the identification of shark and rays species and its implication of global elasmobranch diversity and parasitology. Bull Am Mus Nat Hist 367:1–262.; Bertozzi et al., 2016Bertozzi T, Lee MSY and Donnellan SC (2016) Stingray diversification across the end-Cretaceous extinctions. Memoirs of Museum Victoria 74:379–390.; Last et al., 2016Last PR, Naylor GJP and Manjaji-Matsumoto BM (2016) A revised classification of the family Dasyatidae (Chondrichthyes: Myliobatiformes) based on new morphological and molecular insights. Zootaxa 413:345–368.). However, Marramà et al. (2018)Marramà G, Klug S, De Vos J and Kriwet J (2018) Anatomy, relationships and palaeobiogeographic implications of the first Neogene holomorphic stingray (Myliobatiformes: Dasyatidae) from the early Miocene of Sulawesi, Indonesia, SE Asia. Zool J Linnean Soc 20:1–27. do not recognize Styracurinae as a genuine member of Potamotrygonidae.

The subfamily Potamotrygoninae (35 species in Paratrygon, Potamotrygon, Plesiotrygon, and Heliotrygon) is considered monophyletic and represents today the unique group of elasmobranchs that evolved in freshwater habitats. Potamotrygonins are adapted entirely to freshwater environments, and are restricted to the continental waters of South America, where they inhabit the hydrographic basins that drain into the Atlantic Ocean and the Caribbean Sea with the exception of the São Francisco River basin and coastal drainage to the west and south of the Parnaíba River (Thorson et al., 1983Thorson TB, Brooks DR and Mayes MA (1983) The evolution of freshwater adaptation in stingrays. Nat Geogr Soc Res Rep 15:663-694.; Carvalho et al., 2003Carvalho MR, Lovejoy NR and Rosa RS (2003) Family Potamotrygonidae. In: Reis RE, Ferraris Jr CJ and Kullander SO (eds) Checklist of the Freshwater Fishes of South and Central America. Editora da Pontifícia Universidade Católica, Porto Alegre, pp 22-29.; Rosa et al., 2010Rosa RS, Charvet-Almeida P and Quijada CCD (2010) Biology of the South American potamotrygonid stingrays. In: Carrier JC, Musick JA and Heithaus MR (eds) Sharks and Their Relativies II: Biodiversity, Adaptative Physiology, and Conservation. CRC Press, Boca Raton, 241-281 pp.). The lack of an adequate management plan, combined with the increasing harvesting and trade of these fish, not only as food but also for the ornamental fish market, as well as their biological characteristics, such as their reduced fecundity, slow growth, and late sexual maturation, make them extremely vulnerable to extinction (Araújo et al., 2004Araújo MLG, Charvet-Almeida P, Almeida MP and Pereira H (2004) Freshwater stingrays (Potamotygonidae): Status, conservation and management challenges. Information document AC 20:1-6.; Charvet-Almeida et al., 2005Charvet-Almeida P, Araújo MLG and Almeida MP (2005) Reproductive aspects of freshwater stingrays (Chondrichthyes: Potamotrygonidae) in the Brazilian Amazon Basin. J Northwest Atl Fish Sci 35:165-171.; Duncan et al., 2010Duncan WP, Inomata SO and Fernandes NM (2010) Comércio de Arraias de água doce na Região do médio rio Negro, estado do Amazonas, Brasil. Rev Bras Eng Pesca 5:13-22.).

The increasing interest in the evolution of Potamotrygoninae has emphasized the need for a proper taxonomic classification and a well-supported phylogeny to better address the issues related to the evolutionary history of the group and the diversification of the stingrays in freshwater environments (Carvalho et al., 2003Carvalho MR, Lovejoy NR and Rosa RS (2003) Family Potamotrygonidae. In: Reis RE, Ferraris Jr CJ and Kullander SO (eds) Checklist of the Freshwater Fishes of South and Central America. Editora da Pontifícia Universidade Católica, Porto Alegre, pp 22-29.; Lovejoy et al., 2006Lovejoy NR, Albert JS and Crampton WGR (2006) Miocene marine incursions and marine/freshwater transitions: Evidence from Neotropical fishes. J South Am Earth Sci 21:5-13.; Toffoli et al., 2008Toffoli D, Hrbek T, Araújo MLG, Almeida MP, Charvet-Almeida P and Farias IP (2008) A test of the utility of DNA barcoding in the radiation of the freshwater stingray genus Potamotrygon (Potamotrygonidae, Myliobatiformes). Genet Mol Biol 31:324-336.; Rosa et al., 2010Rosa RS, Charvet-Almeida P and Quijada CCD (2010) Biology of the South American potamotrygonid stingrays. In: Carrier JC, Musick JA and Heithaus MR (eds) Sharks and Their Relativies II: Biodiversity, Adaptative Physiology, and Conservation. CRC Press, Boca Raton, 241-281 pp.; Carvalho and Lovejoy, 2011Carvalho MR, Perez MHS and Lovejoy NR (2011) Potamotrygon tigrina, a new species of freshwater stingray from the upper Amazon basin, closely related to Potamotrygon schroederi Fernandez-Yépez, 1958 (Chondrichthyes: Potamotrygonidae). Zootaxa 2827:1-30.; Cruz et al., 2012Cruz VP, Oliveira C and Foresti F (2012) The genetic dynamics and recent colonization of Southeastern Brazil by stingrays (Chondrichthyes: Potamotrygonidae). ScienceMED 3:79-185.; Fontenelle and Carvalho, 2016Fontenelle JP and Carvalho MR (2016) Systematic implications of brain morphology in Potamotrygonidae (Chondrichthyes: Myliobatiformes). J Morph 277:252-263.; Carvalho et al., 2016bCarvalho MR, Loboda TS and Silva JPCB (2016b) A new subfamily, Styracurinae, and new genus, Styracura, for Himantura schmardae (Werner, 1904) and Himantura pacifica (Beebe and Tee-Van 1941) (Chondrichthyes: Myliobatiformes). Zootaxa 4175:201–221.).

Indeed, taxonomic problems still exist, as many species present considerable variation in color patterns, and the presence of several mid patterns of dorsal pigmentation yields various errors and misidentifications. Even though, the number of potamotrygonins recently described is remarkable (Carvalho and Lovejoy, 2011Carvalho MR and Lovejoy NR (2011) Morphology and phylogenetic relationships of a remarkable new genus and two new species of Neotropical freshwater stingrays from the Amazon basin (Chondrichthyes: Potamotrygonidae). Zootaxa 2776:13-48.; Carvalho et al., 2011Carvalho MR and Lovejoy NR (2011) Morphology and phylogenetic relationships of a remarkable new genus and two new species of Neotropical freshwater stingrays from the Amazon basin (Chondrichthyes: Potamotrygonidae). Zootaxa 2776:13-48.; Carvalho and Ragno, 2011Carvalho MR and Ragno MP (2011) An unusual, dwarf new species of Neotropical freshwater stingray, Plesiotrygon nana sp. nov., from the upper and mid Amazon basin: the second species of Plesiotrygon (Chondrichthyes: Potamotrygonidae). Pap Avulsos Zool 51:101-138.; Loboda and Carvalho, 2013Loboda TS and Carvalho MR (2013) Systematic revision of the Potamotrygon motoro (Müller and Henle 1841) species complex in the Paraná-Paraguay basin, with description of two new ocellated species (Chondrichthyes: Myliobatiformes: Potamotrygonidae). Neotrop Ichthyol 11:693-737.; Fontenelle et al., 2014Fontenelle JP, da Silva JPCB and Carvalho MR (2014) Potamotrygon limai, sp. nov., a new species of freshwater stingray from the upper Madeira River system, Amazon basin (Chondrichthyes: Potamotrygonidae). Zootaxa 3765:249–268.; Carvalho et al., 2016aCarvalho MR, Rosa RS and Araújo MLG (2016a) A new species of Neotropical freshwater stingray (Chondrichthyes: Potamotrygonidae) from the Rio Negro, Amazonas, Brazil: The smallest species of Potamotrygon. Zootaxa 4107:566-586.; bCarvalho MR, Loboda TS and Silva JPCB (2016b) A new subfamily, Styracurinae, and new genus, Styracura, for Himantura schmardae (Werner, 1904) and Himantura pacifica (Beebe and Tee-Van 1941) (Chondrichthyes: Myliobatiformes). Zootaxa 4175:201–221.; Fontenelle and Carvalho, 2017Fontenelle JP and Carvalho MR (2017) Systematic revision of the Potamotrygon scobina Garman, 1913 species-complex (Chondrichthyes: Myliobatiformes: Potamotrygonidae), with the description of three new freshwater stingray species from Brazil and comments on their distribution and biogeography. Zootaxa 431:1-63.).

The use of cytogenetic tools for studies concerning the number, structure, function, and origin of chromosomal variation, and the evolution of rays has been proposed since the 1960’s (reviewed in Valentim et al., 2014Valentim FCS, Gross MC and Feldberg E (2014) Estatus cario-evolutivo de las rayas con énfasis en las especies de agua dulce (Potamotrygonidae). In: Lasso CA, Rosa RS, Sánchez-Duarte P, Morales-Betancourt MA and Agudelo-Córdoba E (eds) IX. Rayas de agua dulce (Potamotrygonidae) de Suramérica Parte I. Colombia, Venezuela, Ecuador, Perú, Brasil, Guyana, Surinam y Guyana Francesa: diversidad, bioecologia, uso y conservación. Serie editorial Recursos Hidrobiológicos y Pesqueros Continentales de Colombia. Instituto de Investigación de los Recursos Biológicos Alexander von Humboldt, Bogotá, 297-309 pp.). However, rays are one of the fish groups for which the fewest chromosomal data are available (Rocco et al., 2005Rocco L, Costagliola D, Fiorillo M, Tinti F and Stingo V (2005) Molecular and chromosomal analysis of ribosomal cistrons in two cartilaginous fish, Taeniura lymma and Raja montagui (Chondrichthyes, Batoidea). Genetica 123:245-253.). Yet, it is already clear that its karyotypes vary considerably, with no modal chromosome number or any foremost karyotype formula (Stingo and Rocco, 2001Stingo V and Rocco L (2001) Selachian cytogenetics: A review. Genetica 111:329-347.; Rocco et al., 2005Rocco L, Costagliola D, Fiorillo M, Tinti F and Stingo V (2005) Molecular and chromosomal analysis of ribosomal cistrons in two cartilaginous fish, Taeniura lymma and Raja montagui (Chondrichthyes, Batoidea). Genetica 123:245-253.).

To date, only six out of 35 species of Neotropical freshwater stingrays have been cytogenetically studied. However, the level of karyotypic variation detected showed distinct diploid numbers, karyotype formulas, and sex chromosomal systems, which represents an essential source of variation that can be used as markers for the characterization of evolutionary processes within different potamotrygonins (Valentim et al., 2006Valentim FCS, Falcão JN, Porto JIR and Feldberg E (2006) Chromosomes of three freshwater stingrays (Rajiformes, Potamotrygonidae) from the Rio Negro Basin, Amazon, Brazil. Genetica 128:33-39.; 2013Valentim FCS, Porto JIR, Bertollo LAC, Gross MC and Feldberg E (2013) XX/XO, a rare sex chromosome system in Potamotrygon freshwater stingray from the Amazon Basin, Brazil. Genetica 141:381-387.; 2014Valentim FCS, Gross MC and Feldberg E (2014) Estatus cario-evolutivo de las rayas con énfasis en las especies de agua dulce (Potamotrygonidae). In: Lasso CA, Rosa RS, Sánchez-Duarte P, Morales-Betancourt MA and Agudelo-Córdoba E (eds) IX. Rayas de agua dulce (Potamotrygonidae) de Suramérica Parte I. Colombia, Venezuela, Ecuador, Perú, Brasil, Guyana, Surinam y Guyana Francesa: diversidad, bioecologia, uso y conservación. Serie editorial Recursos Hidrobiológicos y Pesqueros Continentales de Colombia. Instituto de Investigación de los Recursos Biológicos Alexander von Humboldt, Bogotá, 297-309 pp.; Cruz et al., 2011Cruz VP, Shimabukuro-Dias CK, Oliveira C and Foresti F (2011) Karyotype description and evidence of multiple sex chromosome system X1X1X2X2/X1X2Y in Potamotrygon aff. motoro and P. falkneri (Chondrichthyes: Potamotrygonidae) in upper Paraná River basin, Brazil. Neotrop Ichthyol 9:201-208.; 2015Cruz VP, Oliveira C and Foresti F (2015) An intriguing model for 5S rDNA sequences dispersion in the genome of freshwater stingray Potamotrygon motoro (Chondrichthyes: Potamotrygonidae). Mol Biol 49:466-469.; Aichino et al., 2013Aichino DR, Pastori MC, Roncati HA, Ledesma MA, Swarça AC and Fenocchio AS (2013) Characterization and description of a multiple sex chromosome system in Potamotrygon motoro (Chondrichtyes, Myliobatiformes) from the Paraná River Argentina. Genet Mol Biol 2:2368-2375.).

The present study aimed to increment cytogenetic data on the Potamotrygoninae and improve our understanding of the chromosomal evolution and taxonomy of this critical and poorly studied Neotropical fish group.

Material and Methods

We conducted cytogenetic analyses on Plesiotrygon iwamae, and on seven Potamotrygon species: P. amazona, P. constellata, P. leopoldi, P. motoro, P. orbignyi, P. scobina, and P. aff. wallacei collected in the Amazon region (Table 1, Figure 1). The collection of these individuals was authorized by the Information System of the Chico Mendes Institute for Biodiversity Conservation (ICMBio), an organ of the Brazilian federal government, through license number 28.095-1. The individuals were euthanized after clove oil anesthesia and deposited in the ichthyological collection of the National Institute of Amazonian Research (INPA) in Manaus (Table 1).

Figure 1
Stingray species analyzed in the present study.
Table 1
List of the species and the number of individuals analyzed in the present study by sampling locations.

Mitosis was induced with biological yeast, according to Oliveira et al. (1988)Oliveira C, Almeida-Toledo LF, Foresti F and Toledo-Filho SA (1988) Supernumerary chromosomes, Robertsonian rearrangement and multiple NORs in Corydoras aeneus (Pisces, Siluriformes, Callichthyidae). Caryologia 41:227-236.. Mitotic chromosomes were obtained using the air drying approach described by Bertollo et al. (1978)Bertollo LAC, Takahashi CS and Moreira-Filho O (1978) Cytotaxonomic considerations on Hoplias lacerdae (Pisces, Erytrinidae). Braz J Genet 7:103-120. and adapted for stingrays by Valentim et al. (2006)Valentim FCS, Falcão JN, Porto JIR and Feldberg E (2006) Chromosomes of three freshwater stingrays (Rajiformes, Potamotrygonidae) from the Rio Negro Basin, Amazon, Brazil. Genetica 128:33-39.. The Nucleolus Organizer Region (NOR) was detected using silver nitrate (Ag-NOR), following Howell and Black (1980)Howell WM and Black DA (1980) Controlled silver staining of nucleolus organizer regions with a protective colloidal developer: a 1-step method. Experientia 36:1014-1015., while C banding for the detection of constitutive heterochromatin was conducted using barium hydroxide, as recommended by Sumner(1972)Sumner AT (1972) A simple technique for demonstrating centromeric heterochromatin. Exp Cell Res 75:304-306.. We arranged the karyotypes according to the size of chromosomes and sorted into metacentric (m), submetacentric (sm), subtelocentric (st), and acrocentric (a) types, based on the criteria proposed by Levan et al. (1964)Levan A, Fregda K and Sandberg HA (1964) Nomenclature for centromeric position on chromosomes. Hereditas 52:201-220.. To determine the fundamental number (FN) or the number of chromosome arms, we considered the metacentric, submetacentric, and subtelocentric chromosomes as having two arms, and the acrocentric chromosomes, a single arm.

Results

Plesiotrygon iwamae (Rosa, Castello and Thorson 1987) has a diploid number of 74 chromosomes in both males and females, with a karyotype formula of 26m+8sm+12st+28a and FN = 120 (Figure 2A). Multiple NORs were detected in five sites over the terminal region of the long arms of pairs 1 and 6, and a single homolog of pair 29 (Figure 2B). Blocks of constitutive heterochromatin were detected in the centromeric region of most chromosomes, although some were relatively pale. In pairs 1 and 6, heterochromatic blocks were also observed in the terminal region of the long arms, coinciding with the NORs (Figure 2C).

Figure 2
Karyotype of Plesiotrygon iwamae: (A) conventional Giemsa staining; (B) sequential AgNO3; (C) sequential C-banding.

For Potamotrygon amazona (Fontenelle and Carvalho 2017Fontenelle JP and Carvalho MR (2017) Systematic revision of the Potamotrygon scobina Garman, 1913 species-complex (Chondrichthyes: Myliobatiformes: Potamotrygonidae), with the description of three new freshwater stingray species from Brazil and comments on their distribution and biogeography. Zootaxa 431:1-63.) only a single individual (male) was analyzed, and the diploid number was 66 chromosomes, with a karyotype formula of 21m+8sm+12st+25a and FN = 107 (Figure 3A). We detected the presence of two chromosomes with no homology, a large metacentric (pair 1) and a small acrocentric (pair 29). A priori, this variation could represent an XX/XY system of sex chromosomes, although this cannot be confirmed because there is no female karyotype confirmed yet for comparison. The individual presented multiple NORs located in the terminal region of the long arms of two chromosome pairs, corresponding to pairs 5 and 24, and one homologous of pair 8 (Figure 3B). Blocks of constitutive heterochromatin were detected in the centromeric region of most chromosomes, although some were relatively pale (Figure 3C).

Figure 3
Karyotype of Potamotrygon amazona (male): (A) conventional Giemsa staining; (B) sequential AgNO3; (C) sequential C-banding.

For Potamotrygon constellata (Vaillant 1880) only females were analyzed, and the diploid number was 66 chromosomes, with a karyotype formula of 22m+8sm+14st+22a and FN = 110 (Figure 4A). The NOR is multiple, with up to six signals located in the terminal region of the long arms of pairs 2, 5, and 32, and the terminal region of the short arms of pair 22 (Figure 4B). Heterochromatin was found in the centromeric region of all the chromosomes (Figure 4C).

Figure 4
Karyotype of Potamotrygon constellata (female): (A) conventional Giemsa staining; (B) AgNO3; (C) C-banding.

Potamotrygon leopoldi (Castex and Castello 1970) has a diploid number of 64 chromosomes, in both males and females, with a karyotype formula of 24m+4sm+10st+26a, and a FN = 102 (Figure 5A). The NOR is multiple, with as many as six signals in the long arms of pairs 3 and 7, and in one homologous of pairs 23 and 32 (Figure 5B). We observed the constitutive heterochromatin in the centromeric region of all the chromosomes (Figure 5C).

Figure 5
Karyotype of Potamotrygon leopoldi: (A) conventional Giemsa staining; (B) sequential AgNO3; (C) sequential C-banding.

Potamotrygon motoro (Müller and Henle 1841Loboda TS and Carvalho MR (2013) Systematic revision of the Potamotrygon motoro (Müller and Henle 1841) species complex in the Paraná-Paraguay basin, with description of two new ocellated species (Chondrichthyes: Myliobatiformes: Potamotrygonidae). Neotrop Ichthyol 11:693-737.) is the most widely distributed species of freshwater stingray. In the present study, individuals from four different localities in the Amazon basin were analyzed, including three from the middle Negro River and one from the Solimões River (upper Amazon) near the city of Manaus (Table 1). All the individuals showed a diploid number of 66 chromosomes, with a karyotype formula of 18m+12sm+10st+26a and FN = 106, with no differentiation of males and females (Valentim et al., 2006Valentim FCS, Falcão JN, Porto JIR and Feldberg E (2006) Chromosomes of three freshwater stingrays (Rajiformes, Potamotrygonidae) from the Rio Negro Basin, Amazon, Brazil. Genetica 128:33-39.). Constitutive heterochromatin was observed in the centromeric region of all the chromosomes, with no variation among localities (Figure 6), except for the sample from the Jauaperi River (a tributary of Negro River), which presented additional heterochromatic blocks in the terminal region of the long arms of pair 11 (Figure 6B).

Figure 6
Karyotypes of Potamotrygon motoro: C-banding of individuals from different localities: (A) middle Negro River, Barcelos region; (B) Jauaperi River, middle Negro basin; (C) Catalão Lake, lower Negro basin; (D) Janauacá Lake, Solimões basin.

In Potamotrygon orbignyi (Castelnau 1855) all males and females individuals showed a diploid number of 66 chromosomes with a karyotype formula of 22m+10sm+8st+26a, and FN = 106. However, an XX/XY system of sex chromosomes is present (Figure 7A, B). The NOR is multiple, with as many as eight signals in the long arms of pair 2 and one homologous of pairs 3, 8, 12, and 30 (Figure 7, inbox). Constitutive heterochromatin was observed in the centromeric region of all the chromosomes (Figure 7 C, D).

Figure 7
Karyotypes of Potamotrygon orbignyi: (A) Giemsa staining (male); (B) Giemsa staining (female); (C) C banding (male); (D) C-banding (female); AgNO3 inbox.

For Potamotrygon scobina (Garman 1913) only males were analyzed, and they had a diploid number of 66 chromosomes with a karyotype formula of 21m+7sm+12st+28a and FN = 108 (Figure 8A). As in the case of P. amazona we detected the presence of two chromosomes with no homology, a large metacentric (pair 1) and a small submetacentric (pair 14). A priori, this variation could represent an XX/XY system of sex chromosomes, although this cannot be confirmed because there are no female karyotypes available yet for comparison. All the individuals presented multiple NORs located in the terminal region of the long arms of two chromosome pairs, corresponding to pairs 2 and 6, and one homologous of pairs 26 and 33 (Figure 8B). Blocks of constitutive heterochromatin were detected in the centromeric region of most chromosomes, although some were relatively pale. The chromosome pair 1, which lacks a homologous, had a larger block of heterochromatin in the region of the centromere, which extends to the proximal regions in both arms (Figure 8C).

Figure 8
Karyotype of Potamotrygon scobina: (A) conventional Giemsa staining; (B) AgNO3; (C) sequential C-banding.

For Potamotrygon aff. wallacei (Carvalho, Rosa, Araújo 2016), one individual (female) that was morphologically similar to Potamotrygon wallacei, presented a diploid number of 68 chromosomes, but with a different karyotype formula (22m+10sm+8st+28a and FN = 108) of those previously reported by Valentim et al. (2013)Valentim FCS, Porto JIR, Bertollo LAC, Gross MC and Feldberg E (2013) XX/XO, a rare sex chromosome system in Potamotrygon freshwater stingray from the Amazon Basin, Brazil. Genetica 141:381-387. (Figure 9A). The NOR is multiple, with up to six signals located in the terminal region of the long arms of pairs 2 and 8, and one of the homologous of pair 30, as well as the short arm of one of the homologs of pair 18 (Figure 9B). Constitutive heterochromatin is in the centromeric region of all the chromosomes (Figure 9C).

Figure 9
Karyotype of Potamotrygon aff. wallacei: (A) conventional Giemsa staining; (B) AgNO3; (C) C-banding.

DISCUSSION

Only few karyological studies have been performed so far on potamotrygonins. Our study now increases this number from six to 15 cytogenetically studied species/morphospecies, as shown in Table 2. Except for the genus Heliotrygon, we have thus considerably advanced the knowledge on freshwater stingrays karyotypes, noting that they have different diploid numbers (varying from 2n=66-90) and very different karyotype formulas (from FN=104-120).

Table 2
Compilation of the karyotype data available for the freshwater stingrays and the data obtained in the present study (2n = diploid number; FN: fundamental number; NOR: Nucleolus Organizer Region; T: terminal; p: short arm; q: long arm; m: metacentric; sm: submetacentric; st: subtelocentric; a: acrocentric).

Morphological and molecular phylogenies recover two clades in the Potamotrygoninae: Paratrygon + Heliotrygon and Potamotrygon + Plesiotrygon (Carvalho and Lovejoy, 2011Carvalho MR and Lovejoy NR (2011) Morphology and phylogenetic relationships of a remarkable new genus and two new species of Neotropical freshwater stingrays from the Amazon basin (Chondrichthyes: Potamotrygonidae). Zootaxa 2776:13-48.). If we superpose the chromosomal data over the phylogenies, it is possible to observe that the reduction in diploid number correlates with a decrease in the number of acrocentric chromosomes. For instance, Paratrygon aiereba (2n = 90; 4m+2sm+10st+74a) possesses the highest amount of chromosomes, mostly acrocentric, among potamotrygonins (Valentim et al., 2006Valentim FCS, Falcão JN, Porto JIR and Feldberg E (2006) Chromosomes of three freshwater stingrays (Rajiformes, Potamotrygonidae) from the Rio Negro Basin, Amazon, Brazil. Genetica 128:33-39.). Since we still do not know the karyotypes of Heliotrygon species, comparisons in this clade are not possible. In the other clade, Plesiotrygon iwamae (2n = 74; 26m+8sm+12st+28a) is the only cytogenetically analyzed species of the genus (P. nana remains to be karyotyped). It possesses a higher diploid number than the genus Potamotrygon, its sister group. Apparently, the potamotrygonins have undergone a reduction in diploid number (2n = 90 => 2n = 74 => 2n = 64–68) along their evolutionary history.

The fundamental chromosome numbers of these different taxa are highly consistent with Robertsonian chromosomal rearrangements that could have occurred during evolution. In the clade formed by Plesiotrygon iwamae (FN = 120) and Potamotrygon species (FN = 101–122), the differentiation of the karyotypes is based on centric fusions, in addition to possible pericentric inversions. The comparison of the two clades (Paratrygon and Potamotrygon + Plesiotrygon) reveals that, while there is a significant reduction in chromosome number, the number of arms varies, but not as drastically.

Overall, the available chromosomal data for the genus Potamotrygon indicate that, based on their diploid numbers, the species are organized in three groups, i.e., 2n = 64, 66, and 68 chromosomes. There are internal deviations, however, associated with heteromorphism of the sex chromosomes (Cruz et al., 2011Cruz VP, Shimabukuro-Dias CK, Oliveira C and Foresti F (2011) Karyotype description and evidence of multiple sex chromosome system X1X1X2X2/X1X2Y in Potamotrygon aff. motoro and P. falkneri (Chondrichthyes: Potamotrygonidae) in upper Paraná River basin, Brazil. Neotrop Ichthyol 9:201-208.; Valentim et al., 2013Valentim FCS, Porto JIR, Bertollo LAC, Gross MC and Feldberg E (2013) XX/XO, a rare sex chromosome system in Potamotrygon freshwater stingray from the Amazon Basin, Brazil. Genetica 141:381-387.), although the analysis of this arrangement is still preliminary, given that only 15 of the 30 valid Potamotrygon species have been karyotyped to date. Individuals allocated to the same species have been distinguished by the analysis of karyotypes, as in the cases of Potamotrygon wallacei and Potamotrygon aff. wallacei, and P. motoro and P. aff. motoro (Table 2).

A remarkable feature in the cytotaxonomic study of fish is the presence of heteromorphic sex chromosomes in males and females, in contrast to the normal homomorphic chromosomes present in the vast majority of species (Kitano and Peichel, 2012Kitano J and Peichel CL (2012) Turnover of sex chromosomes and speciation in fishes. Environ Biol Fish 94:549-558.). Heteromorphic sex chromosomes have been described in few marine and freshwater stingrays (for reference see Valentim et al., 2014Valentim FCS, Gross MC and Feldberg E (2014) Estatus cario-evolutivo de las rayas con énfasis en las especies de agua dulce (Potamotrygonidae). In: Lasso CA, Rosa RS, Sánchez-Duarte P, Morales-Betancourt MA and Agudelo-Córdoba E (eds) IX. Rayas de agua dulce (Potamotrygonidae) de Suramérica Parte I. Colombia, Venezuela, Ecuador, Perú, Brasil, Guyana, Surinam y Guyana Francesa: diversidad, bioecologia, uso y conservación. Serie editorial Recursos Hidrobiológicos y Pesqueros Continentales de Colombia. Instituto de Investigación de los Recursos Biológicos Alexander von Humboldt, Bogotá, 297-309 pp.). In Potamotrygoninae, the sex chromosome is an apomorphic trait detected only in Potamotrygon, in which three different sex-determination systems have been observed: XX/XY, XX/X0, and X1X1X2X2/X1X2Y (Valentin et al., 2014). The first was found in P. orbignyi and possibly also P. scobina and P. amazona (present study). The second, in Potamotrygon wallacei (Valentim et al., 2013Valentim FCS, Porto JIR, Bertollo LAC, Gross MC and Feldberg E (2013) XX/XO, a rare sex chromosome system in Potamotrygon freshwater stingray from the Amazon Basin, Brazil. Genetica 141:381-387.). The third, in P. aff. motoro and P. falkneri (Cruz et al., 2011Cruz VP, Shimabukuro-Dias CK, Oliveira C and Foresti F (2011) Karyotype description and evidence of multiple sex chromosome system X1X1X2X2/X1X2Y in Potamotrygon aff. motoro and P. falkneri (Chondrichthyes: Potamotrygonidae) in upper Paraná River basin, Brazil. Neotrop Ichthyol 9:201-208.) and in P. motoro (Aichino et al., 2013Aichino DR, Pastori MC, Roncati HA, Ledesma MA, Swarça AC and Fenocchio AS (2013) Characterization and description of a multiple sex chromosome system in Potamotrygon motoro (Chondrichtyes, Myliobatiformes) from the Paraná River Argentina. Genet Mol Biol 2:2368-2375.). Presently, the simple sex-determination systems were found in rays from the Amazon basin (Valentim et al., 2006Valentim FCS, Falcão JN, Porto JIR and Feldberg E (2006) Chromosomes of three freshwater stingrays (Rajiformes, Potamotrygonidae) from the Rio Negro Basin, Amazon, Brazil. Genetica 128:33-39.; 2013Valentim FCS, Porto JIR, Bertollo LAC, Gross MC and Feldberg E (2013) XX/XO, a rare sex chromosome system in Potamotrygon freshwater stingray from the Amazon Basin, Brazil. Genetica 141:381-387.; present study), whereas the multiple sex-determination system was found in rays from the Paraná basin (Cruz et al., 2011Cruz VP, Shimabukuro-Dias CK, Oliveira C and Foresti F (2011) Karyotype description and evidence of multiple sex chromosome system X1X1X2X2/X1X2Y in Potamotrygon aff. motoro and P. falkneri (Chondrichthyes: Potamotrygonidae) in upper Paraná River basin, Brazil. Neotrop Ichthyol 9:201-208.; Aichino et al., 2013Aichino DR, Pastori MC, Roncati HA, Ledesma MA, Swarça AC and Fenocchio AS (2013) Characterization and description of a multiple sex chromosome system in Potamotrygon motoro (Chondrichtyes, Myliobatiformes) from the Paraná River Argentina. Genet Mol Biol 2:2368-2375.).

Despite the advances in the cytogenetics of freshwater stingrays and comparisons with data available for the Rajiformes and Myliobatiformes (Valentim et al., 2014Valentim FCS, Gross MC and Feldberg E (2014) Estatus cario-evolutivo de las rayas con énfasis en las especies de agua dulce (Potamotrygonidae). In: Lasso CA, Rosa RS, Sánchez-Duarte P, Morales-Betancourt MA and Agudelo-Córdoba E (eds) IX. Rayas de agua dulce (Potamotrygonidae) de Suramérica Parte I. Colombia, Venezuela, Ecuador, Perú, Brasil, Guyana, Surinam y Guyana Francesa: diversidad, bioecologia, uso y conservación. Serie editorial Recursos Hidrobiológicos y Pesqueros Continentales de Colombia. Instituto de Investigación de los Recursos Biológicos Alexander von Humboldt, Bogotá, 297-309 pp.), the direction of chromosomal evolution in Potamotrygonidae still remains unclear, given the lack of chromosome data from the Styracura species. Available chromosomal data indicates a progressive reduction in the number of chromosomes, given that most representatives of other myliobatiform families have low diploid numbers.

In both freshwater and marine Batoidea, the constitutive heterochromatin was invariably located in the centromeric region of all the chromosomes, and the NORs were multiple (Stingo et al., 1995Stingo V, Rocco L, Odierna G and Bellitti M (1995) NOR and heterochromatin analysis in two cartilaginous fishes by C-, Ag- and RE (restriction endonuclease)-banding. Cytogenet Cell Genet 71:228-234.; Rocco et al., 2002Rocco L, Costagliola D, Marsilio A and Stingo V (2002) Localizzazione cromosomica dei cistroni ribosomali in due Batoidea. Atti 63 Convegno UZI:120.; 2005Rocco L, Costagliola D, Fiorillo M, Tinti F and Stingo V (2005) Molecular and chromosomal analysis of ribosomal cistrons in two cartilaginous fish, Taeniura lymma and Raja montagui (Chondrichthyes, Batoidea). Genetica 123:245-253.; 2007Rocco L, Liguori I, Costagliola D, Morescalchi MA, Tinti F and Stingo V (2007) Molecular and karyological aspects of Batoidea (Chondrichthyes, Elasmobranchi) phylogeny. Gene 389:80-86.; Valentim et al., 2006Valentim FCS, Falcão JN, Porto JIR and Feldberg E (2006) Chromosomes of three freshwater stingrays (Rajiformes, Potamotrygonidae) from the Rio Negro Basin, Amazon, Brazil. Genetica 128:33-39.; 2013Valentim FCS, Porto JIR, Bertollo LAC, Gross MC and Feldberg E (2013) XX/XO, a rare sex chromosome system in Potamotrygon freshwater stingray from the Amazon Basin, Brazil. Genetica 141:381-387.; Cruz et al., 2011Cruz VP, Shimabukuro-Dias CK, Oliveira C and Foresti F (2011) Karyotype description and evidence of multiple sex chromosome system X1X1X2X2/X1X2Y in Potamotrygon aff. motoro and P. falkneri (Chondrichthyes: Potamotrygonidae) in upper Paraná River basin, Brazil. Neotrop Ichthyol 9:201-208.; Aichino et al., 2013Aichino DR, Pastori MC, Roncati HA, Ledesma MA, Swarça AC and Fenocchio AS (2013) Characterization and description of a multiple sex chromosome system in Potamotrygon motoro (Chondrichtyes, Myliobatiformes) from the Paraná River Argentina. Genet Mol Biol 2:2368-2375.). This pattern was also found in the present study, although individuals of P. motoro from one locality (Jauaperi River) presented terminal heterochromatic blocks on the long arms of a single chromosome pair.

It is interesting to note that differentiated heterochromatin segments were not found in the probable sex chromosomes of P. amazona, P. orbignyi, and P. scobina, even though such segments are found in many other species of Neotropical fish (Born and Bertollo, 2000Born GG and Bertollo LAC (2000) An XX/XY sex chromosome system in a fish species, Hoplias malabaricus, with a polymorphic nor-bearing X chromosome. Chromosome Res 8:111-118.; Almeida-Toledo and Foresti, 2001Almeida-Toledo LF and Foresti F (2001) Morphologically differentiated sex chromosomes in Neotropical freshwater fish. Genetica 111:91-100.; Terencio et al., 2013Terencio ML, Schneider CH, Gross MC, Vicari MR, Farias IP, Passos KB and Feldberg E (2013) Evolutionary dynamics of repetitive DNA in Semaprochilodus (Characiformes, Prochilodontidae): A fish model for sex chromosome differentiation. Sexual Dev 7:325-333.). Cruz et al. (2011)Cruz VP, Shimabukuro-Dias CK, Oliveira C and Foresti F (2011) Karyotype description and evidence of multiple sex chromosome system X1X1X2X2/X1X2Y in Potamotrygon aff. motoro and P. falkneri (Chondrichthyes: Potamotrygonidae) in upper Paraná River basin, Brazil. Neotrop Ichthyol 9:201-208. and Aichino et al. (2013)Aichino DR, Pastori MC, Roncati HA, Ledesma MA, Swarça AC and Fenocchio AS (2013) Characterization and description of a multiple sex chromosome system in Potamotrygon motoro (Chondrichtyes, Myliobatiformes) from the Paraná River Argentina. Genet Mol Biol 2:2368-2375. recorded a similar lack of differentiation in the multiple sex chromosomes of the Potamotrygon species from the Paraná basin. Overall, a small amount of heterochromatin with an essentially centromeric distribution appears to be a plesiomorphic trait in the stingrays.

All the freshwater stingray species analyzed up to now present multiple NORs located in the terminal region of the long arms. However, Potamotrygon constellata and Potamotrygon aff. wallacei presented one chromosome pair, in which the NOR is located on the short arms. The range of active NORs (Ag-NORs) varied, from four sites in Paratrygon aiereba (Valentim et al., 2006Valentim FCS, Falcão JN, Porto JIR and Feldberg E (2006) Chromosomes of three freshwater stingrays (Rajiformes, Potamotrygonidae) from the Rio Negro Basin, Amazon, Brazil. Genetica 128:33-39.), six sites in Plesiotrygon iwamae, and up to eight sites in the Potamotrygon species.

The karyotype diversity found in the Potamotrygoninae indicates that the occurrence of chromosomal rearrangements, both Robertsonian and non-Robertsonian, is strictly associated with the karyotypic evolution of the group. These rearrangements would also account for the variability in the NORs and the presence of sex determination mechanisms, given the sex chromosomes were not differentiated through heterochromatinization, but rather by rearrangements. The simple and multiple sex chromosome systems are derived traits in the chromosomal evolution of this group, and were only found in Potamotrygon. Therefore, all these features qualify the freshwater stingrays, especially Potamotrygon, as a promising model to investigate the evolution of sex chromosomes and chromosomal evolution in general.

Acknowledgments

We are grateful to Dr. Jansen Zuanon for identifying the species. The following Brazilian entities supported this study: the National Institute for Amazonian Research/Graduate program of Genetics, Conservation and Evolutionary Biology (INPA/PPG-GCBEv), FAPEAM (PRONEX-FAPEAM/CNPq 003/2009), CNPq/475919/2013-0, CAPES/Programa Pro-Amazônia: Biodiversidade e Sustentabilidade, Edital No. 047/2012. This manuscript was edited for proper English language by Stephen Ferrari.

Conflict of Interest

The authors declare that there is no conflict of interest in this paper.

Author contributions

FCSV, JIRP and EF conceived and designed the study. FCSV and EF conducted experiments. FCSV, JIRP and EF analyzed data and wrote the manuscript. All authors read and approved the manuscript.

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  • Associate Editor: Yatiyo Yonenaga-Yassuda

Publication Dates

  • Publication in this collection
    14 Nov 2019
  • Date of issue
    Jul-Sep 2019

History

  • Received
    22 Aug 2018
  • Accepted
    07 Feb 2019
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