Abstract

Stingrays genus Hypanus currently encompasses nine valid species from the Atlantic and Pacific oceans, though the phylogenetic relationships amongst some of them were based on a single mitochondrial gene and did not involve all putative Hypanus species. To address the monophyly of the genus and its relationship to other Dasyatinae genera, we sequenced the whole mitochondrial genomes of all species that supposedly belong to this genus and representatives of Dasyatinae, Neotrygoninae, and, as an outgroup, Fontitrygon (Urogymninae). Based on phylogenetic analyses, Hypanus is the sister-genus to all other Dasyatinae, and this subfamily is closely-related to Neotrygoninae within the family Dasyatidae. The species F. geijskesi is closely related to H. guttatus rather than to its congeners and should be allocated to Hypanus as H. geijskesi for the genus monophyly. After lineage delimitation analyses, we identified three species complexes composed of H. americanus, H. guttatus, and H. say, with two distinct evolutionary lineages within each, leaving the genus with 13 evolutionary units, of which six are currently under threat and only H. sabinus is of least concern. The urgency in identifying these new lineages lies in the fact they might already be under threat before being formally described.

Keywords:
Atlantic Ocean; Conservation; Cryptic species; Diversification; Elasmobranchs

Resumo

As raias com ferrão do gênero Hypanus atualmente compreendem nove espécies válidas nos oceanos Atlântico e Pacífico, embora as relações filogenéticas entre algumas delas tenha sido baseada em apenas um gene mitocondrial e não envolvia todas as possíveis espécies de Hypanus. Para avaliar o monofiletismo do gênero e sua relação com outros Dasyatinae, sequenciamos os genomas mitocondriais de todas as espécies que supostamente compõem o gênero e representantes de Dasyatinae, Neotrygoninae e, como grupo externo, Fontitrygon (Urogymninae). Baseados em análises filogenéticas, Hypanus é o gênero-irmão de todos os outros Dasyatinae e essa subfamília é proximamente relacionada à Neotrygoninae dentro da família Dasyatidae. A espécie F. geijskesi é mais relacionada a H. guttatus que a outras congêneres e deve ser alocada em Hypanus como H. geijskesi para que o gênero seja monofilético. Após análises de delimitações de linhagens, identificamos três complexos de espécies formados por H. americanus, H. guttatus e H. say, com duas linhagens evolutivas distintas em cada, deixando o gênero com 13 unidades evolutivas, das quais seis estão atualmente sob risco de extinção e somente o estado de H. sabinus é pouco preocupante. A urgência na identificação dessas linhagens reside no fato que podem já estar ameaçadas antes de serem formalmente descritas.

Palavras-chave:
Conservação; Diversificação; Elasmobrânquios; Espécies crípticas; Oceano Atlântico

INTRODUCTION

Speciation in marine environments is usually a complex process involving geographic isolation and ecological adaptation (Bowen et al., 2013Bowen BW, Rocha LA, Toonen RJ, Karl SA. The origins of tropical marine biodiversity. Trends Ecol Evol. 2013; 28(6):359–66. https://doi.org/10.1016/j.tree.2013.01.018
https://doi.org/10.1016/j.tree.2013.01.0... ) mediated by an organism’s life history characteristics and biology (Craig et al., 2006Craig MT, Hastings PA, Pondella DJ, Robertson DR, Rosales-Casián JA. Phylogeography of the flag cabrilla Epinephelus labriformis (Serranidae): Implications for the biogeography of the Tropical Eastern Pacific and the early stages of speciation in a marine shore fish. J Biogeogr. 2006; 33(6):969–79. https://doi.org/10.1111/j.1365-2699.2006.01467.x
https://doi.org/10.1111/j.1365-2699.2006... ). Some para- or sympatric lineages with incipient genetic differentiation might be considered as different species, not only populations (Avise, 2000Avise JC. Phylogeography: the history and formation of species. Cambridge, MA: Harvard University Press; 2000. Available from: https://doi.org/10.2307/j.ctv1nzfgj7
https://doi.org/10.2307/j.ctv1nzfgj7... ; Potkamp, Fransen, 2019Potkamp G, Fransen CHJM. Speciation with gene flow in marine systems. Contrib to Zool. 2019; 88:133–72. https://doi.org/10.1163/18759866-20191344
https://doi.org/10.1163/18759866-2019134... ). This scenario has already been documented in some sharks (Corrigan, Beheregaray, 2009Corrigan S, Beheregaray LB. A recent shark radiation: Molecular phylogeny, biogeography and speciation of wobbegong sharks (family: Orectolobidae). Mol Phylogenet Evol. 2009; 52(1):205–16. https://doi.org/10.1016/j.ympev.2009.03.007
https://doi.org/10.1016/j.ympev.2009.03.... ) and rays (Kashiwagi et al., 2012Kashiwagi T, Marshall AD, Bennett MB, Ovenden J.R. The genetic signature of recent speciation in manta rays (Manta alfredi and M. birostris). Mol Phylogenet Evol. 2012; 64(1):212–18. https://doi.org/10.1016/j.ympev.2012.03.020
https://doi.org/10.1016/j.ympev.2012.03.... ) that move extensively but are genetically restrained by environmental forces (Bowen et al., 2013Bowen BW, Rocha LA, Toonen RJ, Karl SA. The origins of tropical marine biodiversity. Trends Ecol Evol. 2013; 28(6):359–66. https://doi.org/10.1016/j.tree.2013.01.018
https://doi.org/10.1016/j.tree.2013.01.0... ). During the process of divergence, lineages incrementally acquire genotypic and phenotypic characteristics that make them distinct from each other, creating a grey zone where the definition of a species is ambiguous (De Queiroz, 2007De Queiroz K. Species concepts and species delimitation. Syst Biol. 2007; 56(6):879–86. https://doi.org/10.1080/10635150701701083
https://doi.org/10.1080/1063515070170108... ). In such cases, a limited number of genetic loci are often insufficient to resolve taxonomic issues and semi-isolated lineages prevail until genomic studies are accomplished, making the delineation of species a challenging task. So, conservation should be a priority and not be constrained by a lack of clarity in species boundaries (Roux et al., 2016Roux C, Fraïsse C, Romiguier J, Anciaux Y, Galtier N, Bierne N. Shedding light on the grey zone of speciation along a continuum of genomic divergence. PLoS Biol. 2016; 14(12):e2000234. https://doi.org/10.1371/journal.pbio.2000234
https://doi.org/10.1371/journal.pbio.200... ).

Dasyatid stingrays are globally distributed batoids that vary in size (from 23 cm to 2.2 m disc width), weigh up to 600 kg, and vary in disc shape from circular to rhombic. They primarily occur in coastal marine environments (down to 400 m), but can also be found in freshwater (Last et al., 2016bLast PR, Naylor GJP, Séret B, White WT, de Carvalho MR, Stehmann MFW. Rays of the World. Clayton South VIC: CSIRO Publishing; 2016b.). Until recently, the genus Dasyatis Rafinesque, 1810 was indicated as a paraphyletic group of stingrays based on morphological data (Rosenberger, 2001Rosenberger LJ. Phylogenetic relationships within the stingray genus Dasyatis (Chondrichthyes: Dasyatidae). Copeia. 2001; 2001(3):615–27. https://doi.org/10.1643/0045-8511(2001)001[0615:PRWTSG]2.0.CO;2
https://doi.org/10.1643/0045-8511(2001)0... ), and subsequent studies based on the mitochondrial gene NADH dehydrogenase 2 (mt-nd2) corroborated this hypothesis (Naylor et al., 2012Naylor GJP, Caira JN, Jensen K, Rosana KAM, White WT, Last PR. A DNA sequence-based approach to the identification of shark and ray species and its implications for global elasmobranch diversity and parasitology. Bull Am Museum Nat Hist. 2012; 2012(367):1–262. https://doi.org/10.1206/754.1
https://doi.org/10.1206/754.1... ). Last et al. (2016aLast PR, Naylor GJP, Manjaji-Matsumoto BM. A revised classification of the family Dasyatidae (Chondrichthyes: Myliobatiformes) based on new morphological and molecular insights. Zootaxa. 2016a; 4139(3):345–68. https://doi.org/10.11646/zootaxa.4139.3.2
https://doi.org/10.11646/zootaxa.4139.3.... ) and Lim et al. (2015Lim KC, Lim PE, Chong VC, Loh KH. Molecular and morphological analyses reveal phylogenetic relationships of stingrays focusing on the family Dasyatidae (Myliobatiformes). PLoS ONE. 2015; 10(4):1–21. https://doi.org/10.1371/journal.pone.0120518
https://doi.org/10.1371/journal.pone.012... ) revised Dasyatidae based on morphological and molecular data and divided it into four subfamilies (Dasyatinae, Hypolophinae, Neotrygoninae, and Urogymninae). Moreover, what was previously known as Dasyatis was separated into eight genera (Dasyatis, Pteroplatytrygon Fowler, 1910Fowler HW. Notes on batoid fishes. Proc Acad Nat Sci Philadelphia. 1910; 62:468–75. , TaeniuropsGarman, 1913Garman S. The Plagiostomia. (Sharks, Skates, and Rays). Cambridge, USA: Memoirs of the Museum of Comparative Zoology at Harvard College XXXVI; 1913. , Bathytoshia Whitley, 1933, Hemitrygon Müller & Henle, 1838, Hypanus Rafinesque, 1818, Telatrygon Last, Naylor & Manjaji-Matsumoto, 2016, and Megatrygon Last, Naylor & Manjaji-Matsumoto, 2016) in the subfamily Dasyatinae, grouped by morphological similarities and molecular clusters.

Despite the resurrection of a monophyletic Hypanus, the most species-rich Dasyatinae genus around the American continent, relationships among its species and theirphylogenetic position within Dasyatidae were based on a single mitochondrial marker (mt-nd2), with few representatives per independent evolutionary lineage, and some missing ones due to lack of sampling (Last et al., 2016aLast PR, Naylor GJP, Manjaji-Matsumoto BM. A revised classification of the family Dasyatidae (Chondrichthyes: Myliobatiformes) based on new morphological and molecular insights. Zootaxa. 2016a; 4139(3):345–68. https://doi.org/10.11646/zootaxa.4139.3.2
https://doi.org/10.11646/zootaxa.4139.3.... ).

Currently, Hypanus encompasses nine recognized species: H. americanus (Hildebrand & Schroeder, 1928Hildebrand SF, Schroeder WC. Fishes of Chesapeake Bay. US Government Printing Office; 1928. ), H. berthalutzae Petean, Naylor & Lima, 2020, H. dipterurus (Jordan & Gilbert, 1880), H. guttatus Bloch & Schneider (1801), H. longus (Garman, 1880Garman S. New species of selachians in the museum collection. Bull Mus Comp Zool. 1880; 6(11):167–72. ), H. marianae (Gomes, Rosa & Gadig, 2000Gomes UL, Rosa RS, Gadig OBF.. Dasyatis marianae sp. n.: A new species of stingray (Chondrichthyes: Dasyatidae) from the Southwestern Atlantic. Copeia. 2000; 2000(2):510–15. https://doi.org/10.1643/0045-8511(2000)000[0510:DMSNAN]2.0.CO;2
https://doi.org/10.1643/0045-8511(2000)0... ), H. rudis (Günther, 1870Günther A. Catalogue of the fishes in the British Museum. Catalogue of the Physostomi, containing the families Gymnotidae, Symbranchidae, Muraenidae, Pegasidae, and of the Lophobranchii, Plectognathi, Dipnoi, ...[thru] ... Leptocardii, in the British Museum. V. 8. 1870. ), H. sabinus (Lesueur, 1824), and H. say (Lesueur, 1817). Except for H. rudis from Guinea Gulf, on the western coast of the African continent, and H. dipterurus and H. longus from the Pacific Ocean, all other six species occur on the Atlantic coast of America. Even though six of these species were sampled and included in the analysis by Last et al. (2016aLast PR, Naylor GJP, Manjaji-Matsumoto BM. A revised classification of the family Dasyatidae (Chondrichthyes: Myliobatiformes) based on new morphological and molecular insights. Zootaxa. 2016a; 4139(3):345–68. https://doi.org/10.11646/zootaxa.4139.3.2
https://doi.org/10.11646/zootaxa.4139.3.... ), the placement of H. marianae was not tested, and it was considered a Hypanus species based on morphological data, as well as H. rudis, which was recently corroborated as a Hypanus species by Petean et al. (2020Petean FF, Naylor GJP, Lima SMQ. Integrative taxonomy identifies a new stingray species of the genus Hypanus Rafinesque, 1818 (Dasyatidae, Myliobatiformes) from the Tropical Southwestern Atlantic. J Fish Biol. 2020; 97(4):1120–42. https://doi.org/10.1111/jfb.14483
https://doi.org/10.1111/jfb.14483... ), who also described a new one (H. berthalutzae).

Precise delimitation and identification of these stingrays are crucial for their conservation since they are frequently the targets of fisheries where they are harvested for food and clothing (Costa et al., 2015Costa TLA, Thayer JA, Mendes LF. Population characteristics, habitat and diet of a recently discovered stingray Dasyatis marianae: Implications for conservation. J Fish Biol. 2015; 86(2):527–43. https://doi.org/10.1111/jfb.12572
https://doi.org/10.1111/jfb.12572... ; Last et al., 2016bLast PR, Naylor GJP, Séret B, White WT, de Carvalho MR, Stehmann MFW. Rays of the World. Clayton South VIC: CSIRO Publishing; 2016b.; Ceretta et al., 2020Ceretta BF, Fogliarini CO, Giglio VJ, Maxwell MF, Waechter LS, Bender MG. Testing the accuracy of biological attributes in predicting extinction risk. Perspect Ecol Conserv. 2020; 18(1):12–18. https://doi.org/10.1016/j.pecon.2020.01.003
https://doi.org/10.1016/j.pecon.2020.01.... ; Oliveira et al., 2021Oliveira CDL, Oliveira CYB, Camilo JPG, Batista VS. Demographic analysis reveals a population decline of the longnose stingray Hypanus guttatus in Northeastern Brazil. Reg Stud Mar Sci. 2021; 41:101554. https://doi.org/10.1016/j.rsma.2020.101554
https://doi.org/10.1016/j.rsma.2020.1015... ). More than half of Hypanus species are evaluated as threatened in the Red List of Threatened Species by IUCN: three are Vulnerable (H. berthalutzae, H. dipterurus, and H. longus (Charvet et al., 2020Charvet P, Derrick D, Faria V, Motta F, Dulvy NK.. Hypanus berthalutzae. IUCN Red List Threat Species 2020. 2020. https://doi.org/10.2305/IUCN.UK.2020-3.RLTS.T181244306A181246271.en
https://doi.org/10.2305/IUCN.UK.2020-3.R... ; Pollom et al., 2020aPollom R, Avalos C, Bizzarro J, Burgos-Vázquez MI, Cevallos A, Espinoza M et al. Hypanus longus. IUCN Red List Threat Species 2020. 2020a. https://doi.org/10.2305/IUCN.UK.2020-3.RLTS.T60157A124445324.en
https://doi.org/10.2305/IUCN.UK.2020-3.R... ,cPollom R, Bizzarro J, Burgos-Vázquez MI, Cevallos A, Velez-Zuazo X, Avalos C et al. Hypanus dipterurus. IUCN Red List Threat Species 2020. 2020c. https://doi.org/10.2305/IUCN.UK.2020-3.RLTS.T60152A80677563.en
https://doi.org/10.2305/IUCN.UK.2020-3.R... )), one is Endangered (H. marianae (Pollom et al., 2020bPollom R, Barreto R, Charvet P, Faria V, Herman K, Marcante F et al. Hypanus marianae. IUCN Red List Threat Species 2020. 2020b:e.T45925A104130004. https://doi.org/10.2305/IUCN.UK.2020-3.RLTS.T45925A104130004.en
https://doi.org/10.2305/IUCN.UK.2020-3.R... )), and one is Critically Endangered (H. rudis (Jabado et al., 2021cJabado RW, De Bruyne G, Derrick D, Doherty P, Diop M, Leurs GHL et al. Hypanus rudis. IUCN Red List Threat Species 2021. 2021c:e.T161620A124516434. https://dx.doi.org/10.2305/IUCN.UK.2021-2.RLTS.T161620A124516434.en
https://dx.doi.org/10.2305/IUCN.UK.2021-... )); three are classified as Near Threatened (H. americanus, H. guttatus, and H. say (Carlson et al., 2020aCarlson J, Charvet P, Blanco-Parra M, Briones Bell-lloch A, Cardenosa D, Derrick D et al.. Hypanus americanus. IUCN Red List Threat Species 2020. 2020a. https://doi.org/10.2305/IUCN.UK.2020-3.RLTS.T181244884A104123787.en
https://doi.org/10.2305/IUCN.UK.2020-3.R... ,bCarlson J, Charvet P, Blanco-Parra M, Briones Bell-lloch A, Cardenosa D, Derrick D et al.. Hypanus guttatus. IUCN Red List Threat Species 2020. 2020b. https://doi.org/10.2305/IUCN.UK.2020-3.RLTS.T44592A104125629.en
https://doi.org/10.2305/IUCN.UK.2020-3.R... ,cCarlson J, Charvet P, Blanco-Parra M, Briones Bell-lloch A, Cardenosa D, Derrick D et al.. Hypanus say. IUCN Red List Threat Species 2020. 2020c. https://doi.org/10.2305/IUCN.UK.2020-3.RLTS.T60159A3090316.en
https://doi.org/10.2305/IUCN.UK.2020-3.R... )); and only one is clearly under no risk of extinction: H. sabinus (Least Concern, Carlson et al., 2020dCarlson J, Charvet P, Blanco-Parra M, Briones Bell-lloch A, Cardenosa D, Derrick D et al. Hypanus sabinus. IUCN Red List Threat Species 2020. 2020d. https://doi.org/10.2305/IUCN.UK.2020-3.RLTS.T60158A124445557.en
https://doi.org/10.2305/IUCN.UK.2020-3.R... )).

Another dasyatid genus, in the subfamily Urogymninae, with a similar pattern of species diversification in the Atlantic Ocean and facing risks of extinction is Fontitrygon Last, Naylor & Manjaji-Matsumoto, 2016, which currently contains six species (Last et al., 2016aLast PR, Naylor GJP, Manjaji-Matsumoto BM. A revised classification of the family Dasyatidae (Chondrichthyes: Myliobatiformes) based on new morphological and molecular insights. Zootaxa. 2016a; 4139(3):345–68. https://doi.org/10.11646/zootaxa.4139.3.2
https://doi.org/10.11646/zootaxa.4139.3.... ). Four occur in western Africa: Fontitrygon margarita (Günther, 1870Günther A. Catalogue of the fishes in the British Museum. Catalogue of the Physostomi, containing the families Gymnotidae, Symbranchidae, Muraenidae, Pegasidae, and of the Lophobranchii, Plectognathi, Dipnoi, ...[thru] ... Leptocardii, in the British Museum. V. 8. 1870. ) (Vulnerable, Jabado et al., 2021aJabado RW, Badji L, Chartrain E, De Bruyne G, Derrick D, Dia M et al.. Fontitrygon margarita. IUCN Red List Threat Species 2021. 2021a:e.T161495A104172843. https://dx.doi.org/10.2305/IUCN.UK.2021-2.RLTS.T161495A104172843.en
https://dx.doi.org/10.2305/IUCN.UK.2021-... ), F. margaritella (Compagno & Roberts, 1984Compagno LJV, Roberts TR. Marine and freshwater stingrays (Dasyatidae) of West Africa, with description of a new species. Proc Calif Acad Sci. 1984; 43(18):283–300. ) (Near Threatened, Jabado et al., 2021bJabado RW, Badji L, Chartrain E, De Bruyne G, Derrick D, Dia M et al. Fontitrygon margaritella. IUCN Red List Threat Species 2021. 2021b:e.T161520A124498844. https://dx.doi.org/10.2305/IUCN.UK.2021-2.RLTS.T161520A124498844.en
https://dx.doi.org/10.2305/IUCN.UK.2021-... ), F. ukpam (Smith, 1863) (Critically Endangered, Jabado et al., 2021dJabado RW, Chartrain E, De Bruyne G, Derrick D, Diop M, Doherty P et al. Fontitrygon ukpam. IUCN Red List Threat Species 2021. 2021d:e.T39414A104174049. https://dx.doi.org/10.2305/IUCN.UK.2021-2.RLTS.T39414A104174049.en
https://dx.doi.org/10.2305/IUCN.UK.2021-... ), and F. garouaensis (Stauch & Blanc, 1963) (Critically Endangered, Jabado et al., 2021eJabado RW, Keith Diagne L, Sayer C, Tamo A, Williams AB. Fontitrygon garouaensis. IUCN Red List Threat Species 2021. 2021e:e.T39406A104171509. https://dx.doi.org/10.2305/IUCN.UK.2021-2.RLTS.T39406A104171509.en
https://dx.doi.org/10.2305/IUCN.UK.2021-... ) while two occur along the Northern coast of South America: F. colarensis (Santos, Gomes & Charvet-Almeida, 2004Santos HRS, Gomes UL, Charvet-Almeida P. A new species of whiptail stingray of the genus Dasyatis Rafinesque, 1810 from the Southwestern Atlantic Ocean (Chondrichthyes: Myliobatiformes: Dasyatidae). Zootaxa. 2004; 492(1):1–12. https://doi.org/10.11646/zootaxa.492.1.1
https://doi.org/10.11646/zootaxa.492.1.1... ) (Santos et al., 2004Santos HRS, Gomes UL, Charvet-Almeida P. A new species of whiptail stingray of the genus Dasyatis Rafinesque, 1810 from the Southwestern Atlantic Ocean (Chondrichthyes: Myliobatiformes: Dasyatidae). Zootaxa. 2004; 492(1):1–12. https://doi.org/10.11646/zootaxa.492.1.1
https://doi.org/10.11646/zootaxa.492.1.1... ) (Critically Endangered, Pollom et al., 2020dPollom R, Charvet P, Barreto R, Faria V, Herman K, Marcante F et al. Fontitrygon colarensis. IUCN Red List Threat Species 2020. 2020d. https://doi.org/10.2305/IUCN.UK.2020-3.RLTS.T60151A104170822.en
https://doi.org/10.2305/IUCN.UK.2020-3.R... ) and F. geijskesi (Boeseman, 1948Boeseman M. Some preliminary notes on Surinam sting rays, including the description of a new species. Zool Meded. 1948; 30(2):31–47. https://repository.naturalis.nl/pub/318932
https://repository.naturalis.nl/pub/3189... ) (Critically Endangered, Pollom et al., 2020ePollom R, Charvet P, Faria V, Herman K, Lasso-Alcalá O, Marcante F et al. Fontitrygon geijskesi. IUCN Red List Threat Species 2020. 2020e. https://doi.org/10.2305/IUCN.UK.2020-3.RLTS.T60153A104172152.en
https://doi.org/10.2305/IUCN.UK.2020-3.R... ). Nevertheless, only three of these species were included in Last et al. (2016aLast PR, Naylor GJP, Manjaji-Matsumoto BM. A revised classification of the family Dasyatidae (Chondrichthyes: Myliobatiformes) based on new morphological and molecular insights. Zootaxa. 2016a; 4139(3):345–68. https://doi.org/10.11646/zootaxa.4139.3.2
https://doi.org/10.11646/zootaxa.4139.3.... ) Dasyatidae revision due to a lack of samples, and both American species were provisionally positioned in Fontitrygon. Despite the incomplete taxon sampling represented, the phylogenetic relationships provided by those authors indicated that some members of Fontitrygon might be misclassified, leaving it as a possible paraphyletic genus.

A useful genetic marker for investigating phylogenetic relationships and species identities is the mitochondrial DNA (mtDNA) due to its high evolutionary rate, maternal inheritance, intraspecific polymorphisms, and genes arrangement (Avise et al., 1987Avise JC, Arnold J, Ball RM, Bermingham E, Lamb T, Neigel JE et al. Intraspecific phylogeography: The mitochondrial DNA bridge between population genetics and systematics. Annu Rev Ecol Syst. 1987; 18(1):489–522. https://doi.org/10.1146/annurev.es.18.110187.002421
https://doi.org/10.1146/annurev.es.18.11... ; Harrison, 1989Harrison RG. Animal mitochondrial DNA as a genetic marker in population and evolutionary biology. Trends Ecol Evol. 1989; 4(1):6–11. https://doi.org/10.1016/0169-5347(89)90006-2
https://doi.org/10.1016/0169-5347(89)900... ; Boore, Brown, 1998Boore JL, Brown WM. Big trees from little genomes: mitochondrial gene order as a phylogenetic tool. Curr Opin Genet Dev. 1998; 8(6):668–74. https://doi.org/10.1016/s0959-437x(98)80035-x
https://doi.org/10.1016/s0959-437x(98)80... ; Satoh et al., 2016Satoh TP, Miya M, Mabuchi K, Nishida M. Structure and variation of the mitochondrial genome of fishes. BMC Genomics. 2016; 17:719. https://doi.org/10.1186/s12864-016-3054-y
https://doi.org/10.1186/s12864-016-3054-... ). Even though a phylogeny based on mtDNA is a story of modifications on a small portion of DNA of maternal transmission, it has not been puzzled by recombination (Avise et al., 1987Avise JC, Arnold J, Ball RM, Bermingham E, Lamb T, Neigel JE et al. Intraspecific phylogeography: The mitochondrial DNA bridge between population genetics and systematics. Annu Rev Ecol Syst. 1987; 18(1):489–522. https://doi.org/10.1146/annurev.es.18.110187.002421
https://doi.org/10.1146/annurev.es.18.11... ). Species in which females are more stationary than males, mtDNA can provide distinct information than nuclear markers due to biased dispersal by sexes (Moritz et al., 1987Moritz C, Dowling TE, Brown WM. Evolution of animal mitochondrial DNA: Relevance for population biology and systematics. Annu Rev Ecol Syst. 1987; 18(1):269–92. https://doi.org/10.1146/annurev.es.18.110187.001413
https://doi.org/10.1146/annurev.es.18.11... ). However, studies on H. americanus from Central America have shown little to no philopatric behavior, with both males and females contributing to gene flow (Corcoran et al., 2013Corcoran MJ, Wetherbee BM, Shivji MS, Potenski MD, Chapman DD, Harvey GM. Supplemental feeding for ecotourism reverses diel activity and alters movement patterns and spatial distribution of the southern stingray, Dasyatis americana. PLoS ONE. 2013; 8(3):e59235. https://doi.org/10.1371/journal.pone.0059235
https://doi.org/10.1371/journal.pone.005... ; Flowers et al., 2016Flowers KI, Ajemian MJ, Bassos-Hull K, Feldheim KA, Hueter RE, Papastamatiou YP et al. A review of batoid philopatry, with implications for future research and population management. Mar Ecol Prog Ser. 2016; 562:251–61. https://doi.org/10.3354/meps11963
https://doi.org/10.3354/meps11963... ; Schwanck et al., 2020Schwanck TN, Schweinsberg M, Lampert KP, Guttridge TL, Tollrian R, O’Shea O. Linking local movement and molecular analysis to explore philopatry and population connectivity of the southern stingray. J Fish Biol. 2020; 96(6):1475–88. https://doi.org/10.1111/jfb.14325
https://doi.org/10.1111/jfb.14325... ). So, evolutionary studies on Hypanus stingrays based on mtDNA might tell a similar story to nuclear markers, to be further assessed. Recently, mitogenomes have been widely used for phylogenetic inferences in distinct metazoan clades: Diptera (da Silva et al., 2020da Silva FS, Cruz ACR, Almeida Medeiros DB, Silva SP, Nunes MRT, Martins LC et al. Mitochondrial genome sequencing and phylogeny of Haemagogus albomaculatus, Haemagogus leucocelaenus, Haemagogus spegazzinii, and Haemagogus tropicalis (Diptera: Culicidae). Sci Rep. 2020; 10:16948. https://doi.org/10.1038/s41598-020-73790-x
https://doi.org/10.1038/s41598-020-73790... ), Rodentia (Abramson et al., 2021Abramson NI, Bodrov SY, Bondareva OV, Genelt-Yanovskiy EA, Petrova TV. A mitochondrial genome phylogeny of voles and lemmings (Rodentia: Arvicolinae): Evolutionary and taxonomic implications. PLoS ONE. 2021; 16(11):e0248198. https://doi.org/10.1371/journal.pone.0248198
https://doi.org/10.1371/journal.pone.024... ), Coleoptera (Nie et al., 2021Nie R, Vogler AP, Yang XK, Lin M. Higher-level phylogeny of longhorn beetles (Coleoptera: Chrysomeloidea) inferred from mitochondrial genomes. Syst Entomol. 2021; 46(1):56–70. https://doi.org/10.1111/syen.12447
https://doi.org/10.1111/syen.12447... ), and Elasmobranchii (Palacios-Barreto et al., 2023Palacios-Barreto P, Mar-Silva AF, Bayona-Vasquez NJ, Adams DH, Díaz-Jaimes P. Characterization of the complete mitochondrial genome of the Brazilian cownose ray Rhinoptera brasiliensis (Myliobatiformes, Rhinopteridae) in the western Atlantic and its phylogenetic implications. Mol Biol Rep. 2023; 50(5):4083–95. https://doi.org/10.1007/s11033-023-08272-0
https://doi.org/10.1007/s11033-023-08272... ).

Since the massive use of molecular methods, such as DNA barcoding (Hebert, Gregory, 2005Hebert PDN, Gregory TR. The promise of DNA barcoding for taxonomy. Syst Biol. 2005; 54(5):852–59. https://doi.org/10.1080/10635150500354886
https://doi.org/10.1080/1063515050035488... ), to identify and classify species, organisms with comparable phenotypes that could have been considered as unique species are recognized as genetically diverse, a concept known as “cryptic species”. Sáez, Lozano (2005Sáez AG, Lozano E. Body doubles. Nature. 2005; 433:111. https://doi.org/10.1038/433111a
https://doi.org/10.1038/433111a... ) described these as “groups of organisms that are morphologically indistinguishable from each other, yet found to belong to different evolutionary lineages”. Sphyrna gilbertiQuattro, Driggers, Grady, Ulrich & Roberts, 2013Quattro JM, Driggers WBI, Grady JM, Ulrich GF, Roberts MA.. Sphyrna gilberti sp. nov., a new hammerhead shark (Carcharhiniformes, Sphyrnidae) from the western Atlantic Ocean. Zootaxa. 2013; 3702(2):159–78. https://doi.org/10.11646/zootaxa.3702.2.5
https://doi.org/10.11646/zootaxa.3702.2.... and Squalus suckleyi (Girard, 1855) are examples of shark species with circumtropical distributions in which morphological analyses subsequently to identifications of genetic lineages corroborated the existence of more than one entity (Ebert et al., 2010Ebert DA, White WT, Goldman KJ, Compagno LJV, Daly-Engel TS, Ward RD. Resurrection and redescription of Squalus suckleyi (Girard, 1854) from the North Pacific, with comments on the Squalus acanthias subgroup (Squaliformes: Squalidae). Zootaxa. 2010; 2612(1):22–40. https://doi.org/10.11646/zootaxa.2612.1.2
https://doi.org/10.11646/zootaxa.2612.1.... ; Quattro et al., 2013Quattro JM, Driggers WBI, Grady JM, Ulrich GF, Roberts MA.. Sphyrna gilberti sp. nov., a new hammerhead shark (Carcharhiniformes, Sphyrnidae) from the western Atlantic Ocean. Zootaxa. 2013; 3702(2):159–78. https://doi.org/10.11646/zootaxa.3702.2.5
https://doi.org/10.11646/zootaxa.3702.2.... ; Gaither et al., 2016Gaither MR, Bowen BW, Rocha LA, Briggs JC. Fishes that rule the world: circumtropical distributions revisited. Fish Fish. 2016; 17(3):664–79. https://doi.org/10.1111/faf.12136
https://doi.org/10.1111/faf.12136... ).

A concept to be explored is that of taxonomic gap, in which there is a space between the extant biodiversity and what is actually known about it (Dubois, 2010Dubois A. Zoological nomenclature in the century of extinctions: priority vs.‘usage’. Org Divers Evol. 2010; 10:259–74. https://doi.org/10.1007/s13127-010-0021-3
https://doi.org/10.1007/s13127-010-0021-... ; Raposo et al., 2020Raposo MA, Kirwan GM, Lourenco ACC, Sobral G, Bockmann FA, Stopiglia R. On the notions of taxonomic ‘impediment’, ‘gap’, ‘inflation’ and ‘anarchy’, and their effects on the field of conservation. Syst Biodivers. 2020; 19(3):296–311. https://doi.org/10.1080/14772000.2020.1829157
https://doi.org/10.1080/14772000.2020.18... ). This gap regards both the universe of unknown species and those susceptible to changes due to more studies. As many authors have said, “taxonomic stability is ignorance” (Dominguez, Wheeler, 1997Dominguez E, Wheeler QD. Taxonomic stability is ignorance. Cladistics. 1997; 13(4):367–72. https://doi.org/10.1111/j.1096-0031.1997.tb00325.x
https://doi.org/10.1111/j.1096-0031.1997... ; Benton, 2000Benton MJ. Stems, nodes, crown clades, and rank-free lists: is Linnaeus dead? Biol Rev Camb Philos Soc. 2000; 75(4):633–48. https://doi.org/10.1111/j.1469-185x.2000.tb00055.x
https://doi.org/10.1111/j.1469-185x.2000... ; Dubois, 2010Dubois A. Zoological nomenclature in the century of extinctions: priority vs.‘usage’. Org Divers Evol. 2010; 10:259–74. https://doi.org/10.1007/s13127-010-0021-3
https://doi.org/10.1007/s13127-010-0021-... ;) since with more data and analyses the gap might increase or decrease in a continuous progress of Science. It is indisputable that the lack of specimens that could serve as vouchers for each molecular sample could have consequences for taxonomy (Amorim et al., 2016Amorim DS, Santos CMD, Krell F-T, Dubois A, Nihei SS, Oliveira OMP et al. Timeless standards for species delimitation. Zootaxa. 2016; 4137(1):121–28. https://doi.org/10.11646/zootaxa.4137.1.9
https://doi.org/10.11646/zootaxa.4137.1.... ) due to the impossibility of checking the morphology of all individuals. However, given the urgency in closing this taxonomic gap to recognize the world’s biodiversity before more extinctions take place, even tissue samples could corroborate the once-existing variety of species (Engel et al., 2021Engel MS, Ceríaco LMP, Daniel GM, Dellapé PM, Löbl I, Marinov M et al. The taxonomic impediment: a shortage of taxonomists, not the lack of technical approaches. Zool J Linn Soc. 2021; 193(2):381–87. https://doi.org/10.1093/zoolinnean/zlab072
https://doi.org/10.1093/zoolinnean/zlab0... ).

Due to the taxonomic uncertainties in Dasyatinae (Lim et al., 2015Lim KC, Lim PE, Chong VC, Loh KH. Molecular and morphological analyses reveal phylogenetic relationships of stingrays focusing on the family Dasyatidae (Myliobatiformes). PLoS ONE. 2015; 10(4):1–21. https://doi.org/10.1371/journal.pone.0120518
https://doi.org/10.1371/journal.pone.012... ; Last et al., 2016aLast PR, Naylor GJP, Manjaji-Matsumoto BM. A revised classification of the family Dasyatidae (Chondrichthyes: Myliobatiformes) based on new morphological and molecular insights. Zootaxa. 2016a; 4139(3):345–68. https://doi.org/10.11646/zootaxa.4139.3.2
https://doi.org/10.11646/zootaxa.4139.3.... ; Pavan-Kumar et al., 2022Pavan-Kumar A, Singh S, Mishra A, Suman S, Gireesh-Babu P, Chaudhari A et al. Characterization of mitochondrial genome of Indian Ocean blue-spotted maskray, Neotrygon indica and its phylogenetic relationship within Dasyatidae Family. Int J Biol Macromol. 2022; 223:458–67. https://doi.org/10.1016/j.ijbiomac.2022.10.277
https://doi.org/10.1016/j.ijbiomac.2022.... ), the absence of a complete sampling of all Hypanus species in other published works, and the risks of extinction these stingrays are facing, our goal is to use mitogenomes to define the relationships among Hypanus species, identifying possible cryptic ones, and their relationships to other Dasyatinae genera. Afterward, species can be properly identified and (re)evaluated for adequate conservation measures.

MATERIAL AND METHODS

Sampling, DNA isolation, and sequencing. To test the monophyly of the genus Hypanus and the subfamily Dasyatinae, we sampled 124 specimens from all nine valid species belonging to Hypanus, six representatives of almost all Dasyatinae genera (Hemitrygon akajei (Bürger, 1841), Telatrygon acutirostra (Nishida & Nakaya, 1988), Pteroplatytrygon violacea (Bonaparte, 1832), Batytoshia lata (Garman, 1880Garman S. New species of selachians in the museum collection. Bull Mus Comp Zool. 1880; 6(11):167–72. ), Taeniurops grabatus (Geoffroy St. Hilaire, 1817), Dasyatis hypostigmaSantos & Carvalho, 2004Santos HRS, Gomes UL, Charvet-Almeida P. A new species of whiptail stingray of the genus Dasyatis Rafinesque, 1810 from the Southwestern Atlantic Ocean (Chondrichthyes: Myliobatiformes: Dasyatidae). Zootaxa. 2004; 492(1):1–12. https://doi.org/10.11646/zootaxa.492.1.1
https://doi.org/10.11646/zootaxa.492.1.1... ; except Megatrygon), and both Neotrygoninae genera (Taeniura lymma (Fabricius, 1775) and Neotrygon kuhlii (Müller & Henle, 1841)). As outgroup, we included representatives of each Fontitrygon species, subfamily Urogymninae (one sample of F. margarita, F. margaritella, F. garouaensis,and six of F. geijskesi; except F. colarensis and F. ukpam). Species distributions and sampling localities are provided in Tab. 1 (details in Tab. S1) and sample locations of Hypanus and Fontitrygon in Fig. 1. Valid names and distributions were obtained from Last et al. (2016aLast PR, Naylor GJP, Manjaji-Matsumoto BM. A revised classification of the family Dasyatidae (Chondrichthyes: Myliobatiformes) based on new morphological and molecular insights. Zootaxa. 2016a; 4139(3):345–68. https://doi.org/10.11646/zootaxa.4139.3.2
https://doi.org/10.11646/zootaxa.4139.3.... ) and Eschmeyer’s Catalog of Fishes (Fricke et al., 2023Fricke R, Eschmeyer WN, Van der Laan R. Eschmeyer’s catalog of fishes: genera, species, references [Internet]. San Francisco: California Academy of Science; 2023. Available from: http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatmain.asp
http://researcharchive.calacademy.org/re... ). Nearly all tissues were collected in fish markets, making it unfeasible to preserve most of the specimens; however, we performed barcode analyses (described below) to compare clades to examined specimens deposited in collections. Lineages for which we could provide vouchers are H. americanus, H. berthalutzae, H. geijskesi, H. guttatus, H. sabinus,and H. say;even though there is no voucher for H. marianae, tissues came from the specimens identified and collected by Costa et al. (2022Costa TLA, Petean FF, Berbel-Filho WM, Solé-Cava AM, Mendes LF, Lima SMQ. Molecular testing of the São Francisco River as an ecological filter for the Brazilian large-eyed stingray Hypanus marianae (Dasyatidae, Myliobatiformes). Hydrobiologia. 2022; 849:2435–48. https://doi.org/10.1007/s10750-022-04828-6
https://doi.org/10.1007/s10750-022-04828... ). Before mitochondrial gene capture, samples were genetically identified based on Sanger sequencing of the mitochondrial marker mt-nd2, as described by Petean et al. (2020Petean FF, Naylor GJP, Lima SMQ. Integrative taxonomy identifies a new stingray species of the genus Hypanus Rafinesque, 1818 (Dasyatidae, Myliobatiformes) from the Tropical Southwestern Atlantic. J Fish Biol. 2020; 97(4):1120–42. https://doi.org/10.1111/jfb.14483
https://doi.org/10.1111/jfb.14483... ), and compared to the database from Naylor et al. (2012Naylor GJP, Caira JN, Jensen K, Rosana KAM, White WT, Last PR. A DNA sequence-based approach to the identification of shark and ray species and its implications for global elasmobranch diversity and parasitology. Bull Am Museum Nat Hist. 2012; 2012(367):1–262. https://doi.org/10.1206/754.1
https://doi.org/10.1206/754.1... ). After capture, we performed barcode analyses comparing to data from GenBank (detailed as it follows) as another approach to verifying species’ identities. When we directly removed tissues from specimens through diving or trawling, we morphologically identified them. Data collection was under SISBIO permit 54254-3 and supported by Atlantis Divers in Fernando de Noronha, Brazil.

FIGURE 1 |
Sampling locations of all Hypanus and Fontitrygon lineages (new name combinations are used in the figure, as discussed in the text). A.Hypanus americanus, H. aff. americanus, H. berthalutzae, H. longus, and H. rudis; B. H. guttatus, H. aff. guttatus,and H. geijskesi;C. H. marianae, Fontitrygon garouaensis, F. margarita,and F. margaritella;D. H. say, H. aff. say, H. dipterurus,and H. sabinus.

From genomic DNA extraction to the alignment of protein-coding gene sequences of mitochondrial genomes, all protocols and procedures followed Li et al. (2013Li C, Hofreiter M, Straube N, Corrigan S, Naylor GJP. Capturing protein-coding genes across highly divergent species. Biotechniques. 2013; 54(6):321–26. https://doi.org/10.2144/000114039
https://doi.org/10.2144/000114039... , 2015Li C, Corrigan S, Yang L, Straube N, Harris M, Hofreiter M et al. DNA capture reveals transoceanic gene flow in endangered river sharks. Proc Natl Acad Sci. 2015; 112(43):13302–07. https://doi.org/10.1073/pnas.1508735112
https://doi.org/10.1073/pnas.1508735112... ) and Petean et al. (2020Petean FF, Naylor GJP, Lima SMQ. Integrative taxonomy identifies a new stingray species of the genus Hypanus Rafinesque, 1818 (Dasyatidae, Myliobatiformes) from the Tropical Southwestern Atlantic. J Fish Biol. 2020; 97(4):1120–42. https://doi.org/10.1111/jfb.14483
https://doi.org/10.1111/jfb.14483... ). Extracted DNA was sheared to 500 bp in an M220 Focused-ultrsonicator (Covaris, Inc., Wobuern, Massachusetts, USA) as the first step for library preparation, followed by the selection of > 200 bp fragments with solid-phase reversible immobilization beads (Li et al., 2015Li C, Corrigan S, Yang L, Straube N, Harris M, Hofreiter M et al. DNA capture reveals transoceanic gene flow in endangered river sharks. Proc Natl Acad Sci. 2015; 112(43):13302–07. https://doi.org/10.1073/pnas.1508735112
https://doi.org/10.1073/pnas.1508735112... ). We performed a series of reactions in each sample for mitochondrial gene capture using biotinylated RNA baits (Mycroarray, Ann Arbor, Michigan) (Li et al., 2013Li C, Hofreiter M, Straube N, Corrigan S, Naylor GJP. Capturing protein-coding genes across highly divergent species. Biotechniques. 2013; 54(6):321–26. https://doi.org/10.2144/000114039
https://doi.org/10.2144/000114039... ). For sequencing, we deployed an Illumina MiSeq Next Generation Sequencer and, from each read, removed low-quality reads (with Phred quality scores lower than 30; Illumina 2011, https://www.illumina.com/documents/products/technotes/technote_Q-Scores.pdf) and adaptors using Trim Galore 0.6.4 (Krueger, 2020Krueger F. Trim Galore. 2020. Available from: https://github.com/FelixKrueger/TrimGalore
https://github.com/FelixKrueger/TrimGalo... ) then mapped to the mitochondrial genome of a closely-related species, Hemitrygon akajei (NC_021132), from the GenBank using Geneious 7.9.1 (http://www.geneious.com). Finally, we used a pipeline (MitoAnnotator, (Iwasaki et al., 2013Iwasaki W, Fukunaga T, Isagozawa R, Yamada K, Maeda Y, Satoh TP et al. MitoFish and mitoannotator: A mitochondrial genome database of fish with an accurate and automatic annotation pipeline. Mol Biol Evol. 2013; 30(11):2531–40. https://doi.org/10.1093/molbev/mst141
https://doi.org/10.1093/molbev/mst141... )) to annotate sequences, which are available on GenBank under accession numbers provided in Tab. S1.

Phylogenetic reconstructions. To study the relationships within the genus Hypanus, its relationships within the subfamily Dasyatinae, and to Neotrygoninae, the whole mitogenome sequences of all 135 specimens were aligned in GENEIOUS 7.9.1 using the MUSCLE algorithm (Edgar, 2004Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004; 32(5):1792–97. https://doi.org/10.1093/nar/gkh340
https://doi.org/10.1093/nar/gkh340... ). After annotation we identified and excluded from all sequences the mitochondrial control region (CR), tRNA, and rRNA. Control region was deleted because it is highly variable among individuals and its coverage after mitochondrial capture was too low; RNAs regions were eliminated because the indels present in these regions make alignment difficult. The final alignment had 11,471 base pairs in 13 protein-coding genes.

To select the best-fitting model of molecular evolution we used PartitionFinder2 (Lanfear et al., 2017Lanfear R, Frandsen PB, Wright AM, Senfeld T, Calcott B. Partitionfinder 2: New methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Mol Biol Evol. 2017; 34(3):772–73. https://doi.org/10.1093/molbev/msw260
https://doi.org/10.1093/molbev/msw260... ) and selected the best scheme for each protein-coding gene under Bayesian Inference Criteria: GTR+gamma+invariant sites for mt-nd1 and mt-nd5, HKY+gamma for mt-nd2, mt-atp8, mt-atp6, mt-coiii, mt-nd6, and mt-cytb, HKY+gamma+invariant sites for mt-nd3, mt-nd4l, and mt-nd4, and TN93+gamma for mt-coi and mt-coii. Maximum Likelihood analyses were conducted using RAxML version 8. (Stamatakis, 2014Stamatakis A. RAxML Version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014; 30(9):1312–13. https://doi.org/10.1093/bioinformatics/btu033
https://doi.org/10.1093/bioinformatics/b... ) in CIPRES Science Gateway (Miller et al., 2010Miller MA, Pfeiffer W, Schwartz T. Creating the CIPRES Science gateway for inference of large phylogenetic trees. Proc Gatew Comput Environ Work. New Orleans, LA: 2010. p.1–08. ), with bootstrap and consensus calculations based on a 1000-generation search of tree space.

Bayesian Inferences were carried out in BEAST 2.5 (Bouckaert et al., 2019Bouckaert R, Vaughan TG, Barido-Sottani J, Duchêne S, Fourment M, Gavryushkina A et al. BEAST 2.5: An advanced software platform for Bayesian evolutionary analysis. PLoS Comput Biol. 2019; 15(4):e1006650. https://doi.org/10.1371/journal.pcbi.1006650
https://doi.org/10.1371/journal.pcbi.100... ) using Yule model as the prior tree as we are not considering known extinctions (μ = 0) and there is a reasonable sampling for analyses (ρ = 1) (Drummond, Bouckaert, 2015Drummond AJ, Bouckaert RR. Bayesian evolutionary analysis with BEAST. Bayesian Evol Anal with BEAST. 2015:1–249. https://doi.org/10.1017/CBO9781139095112
https://doi.org/10.1017/CBO9781139095112... ) in 1,000,000,000 generations with 5 chains resampled every 10,000. The software MEGA X (Kumar et al., 2018Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018; 35:1547–49. https://doi.org/10.1093/molbev/msy096
https://doi.org/10.1093/molbev/msy096... ) was used to calculate uncorrected genetic p-distances and analyze intra- and interspecific genetic differences between Hypanus lineages.

Lineage delimitation methods. By analyzing the relationships among species, we noticed some valid species could be either paraphyletic or have long branches within them, suggesting possible distinct lineages. Therefore, we decided to do five species delimitation analyses within the clades of H. guttatus and H. say independently: multiple- and single-threshold Generalized Mixed Yule Coalescent (m-GMYC and s-GMYC, Fujisawa, Barraclough, 2013Fujisawa T, Barraclough TG. Delimiting species using single-locus data and the generalized mixed yule coalescent approach: A revised method and evaluation on simulated data sets. Syst Biol. 2013; 62(5):707–24. https://doi.org/10.1093/sysbio/syt033
https://doi.org/10.1093/sysbio/syt033... ), multi-rate Poisson Tree Process (mPTP, Kapli et al., 2017Kapli P, Lutteropp S, Zhang J, Kobert K, Pavlidis P, Stamatakis A et al. Multi-rate Poisson tree processes for single-locus species delimitation under maximum likelihood and Markov chain Monte Carlo. Bioinformatics. 2017; 33(11):1630–38. https://doi.org/10.1093/bioinformatics/btx025
https://doi.org/10.1093/bioinformatics/b... ), Bayesian Poisson Tree Process (bPTP, Zhang et al., 2013Zhang J, Kapli P, Pavlidis P, Stamatakis A. A general species delimitation method with applications to phylogenetic placements. Bioinformatics. 2013; 29(22):2869–76. https://doi.org/10.1093/bioinformatics/btt499
https://doi.org/10.1093/bioinformatics/b... ), and Assemble Species by Automatic Partitioning (ASAP,(Puillandre et al., 2021Puillandre N, Brouillet S, Achaz G. ASAP: assemble species by automatic partitioning. Mol Ecol Resour. 2021; 21(2):609–20. https://doi.org/10.1111/1755-0998.13281
https://doi.org/10.1111/1755-0998.13281... ). For each clade’s data (H. guttatus and H. say) we selected an outgroup based on the results of our phylogenetic analysis and Last et al. (2016aLast PR, Naylor GJP, Manjaji-Matsumoto BM. A revised classification of the family Dasyatidae (Chondrichthyes: Myliobatiformes) based on new morphological and molecular insights. Zootaxa. 2016a; 4139(3):345–68. https://doi.org/10.11646/zootaxa.4139.3.2
https://doi.org/10.11646/zootaxa.4139.3.... ): Hypanus marianae for H. guttatus and H. sabinus for H. say analyses. Most of these analyses (except ASAP) are performed on a tree topology: both GMYC methods rely on an ultrametric tree, which was built under a Bayesian Inference analysis in BEAST 2.5, and both PTP on a tree with nucleotides’ substitutions, built with a Maximum Likelihood analysis in RAxML version 8. For such phylogenetic analyses before delimitation ones, we used jModelTest2 (Darriba et al., 2012Darriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012; 9(8):772. https://doi.org/10.1038/nmeth.2109
https://doi.org/10.1038/nmeth.2109... ) to select the best molecular evolution model for each clade. The ASAP method depends on an alignment matrix instead of a tree. For more details on delimitation methods, refer to Petean et al. (2020Petean FF, Naylor GJP, Lima SMQ. Integrative taxonomy identifies a new stingray species of the genus Hypanus Rafinesque, 1818 (Dasyatidae, Myliobatiformes) from the Tropical Southwestern Atlantic. J Fish Biol. 2020; 97(4):1120–42. https://doi.org/10.1111/jfb.14483
https://doi.org/10.1111/jfb.14483... ).

DNA Barcode analyses. The mitochondrial protein-coding gene region cytochrome c oxidase subunit I (mt-co1) was identified and extracted from some sequences for comparisons to those available at GenBank (Tab. S2). The goal was to use the molecular clusters as support for the verification of taxonomic status when vouchers were available for at least one sequence in a clade. Sequences of Fontitrygon geijskesi sampled by this study were aligned with a sample from Guyana (GN17902) and four samples from Rodrigues-Filho et al. (2020Rodrigues Filho LFS, Feitosa LM, Nunes JLS, Palmeira ARO, Martins APB, Giarrizzo T et al. Molecular identification of ray species traded along the Brazilian Amazon coast. Fish Res. 2020; 223:105407. https://doi.org/10.1016/j.fishres.2019.105407
https://doi.org/10.1016/j.fishres.2019.1... ) (GenBank numbers MN105749, MN105812, MN105813, MN105819), who provided a voucher for the species. The alignment was performed in MEGA X using the MUSCLE algorithm, which had 587 base pairs, 12 F. geijskesi samples, and one outgroup. The genetic p-distance was also performed in MEGA X and, to estimate species identities based on sequences’ similarities, a Neighbor-Joining (Saitou, Nei, 1987Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987; 4(4):406–25. https://doi.org/10.1093/oxfordjournals.molbev.a040454
https://doi.org/10.1093/oxfordjournals.m... ) tree was built in GENEIOUS 7.9.1 with 1,000 bootstrap replicas and edited in FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/).

The same barcode analyses were performed for the three clades containing species-complexes (H. guttatus, H. say, and H. americanus) identified by abovementioned analyses and Petean et al. (2020Petean FF, Naylor GJP, Lima SMQ. Integrative taxonomy identifies a new stingray species of the genus Hypanus Rafinesque, 1818 (Dasyatidae, Myliobatiformes) from the Tropical Southwestern Atlantic. J Fish Biol. 2020; 97(4):1120–42. https://doi.org/10.1111/jfb.14483
https://doi.org/10.1111/jfb.14483... ). Hypanus berthalutzae (GN18496) was used as an outgroup for all independent analyses, except for H. americanus, for which it was also the ingroup and H. guttatus (GN18434) was then used as an outgroup.

For H. guttatus, besides the 33 samples from this study, we included 52 mt-co1 sequences from GenBank, totaling 85 samples (and one outgroup) in 524 base pairs. To analyze the clade containing H. say, we extracted the mt-co1 region from mitogenomes of this species, H. dipterurus, and H. sabinus and added 13 mt-co1 sequences from H. say, two H. dipterurus, and eight H. sabinus from GenBank. The alignment had 40 samples (and one outgroup) in 547 base pairs. Finally, to investigate H. americanus, we not only added 38 mt-co1 sequences from GenBank, but we also included 23 H. berthalutzae, four H. longus, and four H. rudis, in a total of 77 samples as the ingroup in 599 base pairs.

RESULTS

Phylogenetic inferences. The genus HypanussensuLast et al. (2016aLast PR, Naylor GJP, Manjaji-Matsumoto BM. A revised classification of the family Dasyatidae (Chondrichthyes: Myliobatiformes) based on new morphological and molecular insights. Zootaxa. 2016a; 4139(3):345–68. https://doi.org/10.11646/zootaxa.4139.3.2
https://doi.org/10.11646/zootaxa.4139.3.... ) was recovered as monophyletic and sister to all other genera (except Megatryon, not sampled for this study) within the subfamily Dasyatinae (Fig. 2), which is a sister-group to Neotrygoninae. These results were already suggested by Last et al. (2016aLast PR, Naylor GJP, Manjaji-Matsumoto BM. A revised classification of the family Dasyatidae (Chondrichthyes: Myliobatiformes) based on new morphological and molecular insights. Zootaxa. 2016a; 4139(3):345–68. https://doi.org/10.11646/zootaxa.4139.3.2
https://doi.org/10.11646/zootaxa.4139.3.... ) and are now corroborated by mitogenomes through the same resulting topologies by Maximum Likelihood and Bayesian Inference phylogenetic analyses with all nodes’ values higher than 88% bootstrap and 0.995 of posterior probability. Maximum Likelihood and Bayesian Inference trees topologies with all taxa and nodes values are available in Figs. S3 and S4, respectively.

FIGURE 2 |
Maximum Likelihood tree topology of mtDNA with representatives of Hypanus species, dasyatine genera, neotrygonine genera, and urogymnine genus Fontitrygon as an outgroup. New name combinations are used in the figure in red, as discussed in the text. For each node, the maximum likelihood bootstrap value is given first, followed by the Bayesian inference posterior probability. Clade A (H. americanus complex) taken from Petean et al. (2020Petean FF, Naylor GJP, Lima SMQ. Integrative taxonomy identifies a new stingray species of the genus Hypanus Rafinesque, 1818 (Dasyatidae, Myliobatiformes) from the Tropical Southwestern Atlantic. J Fish Biol. 2020; 97(4):1120–42. https://doi.org/10.1111/jfb.14483
https://doi.org/10.1111/jfb.14483... ).

There are clear unique lineages that correspond to valid species names: Hypanus berthalutzae, H. dipterurus, H. longus, H. marianae, H. rudis, and H. sabinus.Hypanus americanus, the Southern stingray, nonetheless, is not a single evolutionary lineage (Clade A in Fig. 2), as suggested by previous mitogenomes delimitation analyses and haplotype network based on mt-nd2 (Petean et al., 2020Petean FF, Naylor GJP, Lima SMQ. Integrative taxonomy identifies a new stingray species of the genus Hypanus Rafinesque, 1818 (Dasyatidae, Myliobatiformes) from the Tropical Southwestern Atlantic. J Fish Biol. 2020; 97(4):1120–42. https://doi.org/10.1111/jfb.14483
https://doi.org/10.1111/jfb.14483... ; Figs, 2–3, respectively), which showed 8 unsampled haplotypes and mutational steps between both lineages, while there are four between H. berthalutzae and H. rudis; and a phylogeographic study based on the mitochondrial control region by Richards et al. (2019Richards VP, DeBiasse MB, Shivji M. Deep mitochondrial lineage divergence among populations of the southern stingray (Hypanus americanus (Hildebrand & Schroeder, 1928)) throughout the Southeastern United States and Caribbean. Mar Biodivers. 2019; 49:1627–34. https://doi.org/10.1007/s12526-018-0930-5
https://doi.org/10.1007/s12526-018-0930-... ) that found three populations of this species in the USA’s coast and Caribbean. The species H. marianae, which was not included in the previous molecular study (Last et al., 2016aLast PR, Naylor GJP, Manjaji-Matsumoto BM. A revised classification of the family Dasyatidae (Chondrichthyes: Myliobatiformes) based on new morphological and molecular insights. Zootaxa. 2016a; 4139(3):345–68. https://doi.org/10.11646/zootaxa.4139.3.2
https://doi.org/10.11646/zootaxa.4139.3.... ), is a monophyletic lineage and sister to the clade containing H. americanussensu lato, H. longus, H. berthalutzae, and H. rudis. This clade is, then, closely-related to a group containing H. guttatus, which now is suggested to harbor two lineages: one distributed from Central America to the south of Brazil and another of specimens from Belize (Clade B in Fig. 2).

FIGURE 3 |
Candidate species of the clade Hypanus guttatus species complex (Clade B), according to five lineage delimitation analyses using the mtDNA. Possible species found in each analysis are portrayed as colored boxes in columns. In blue, H. guttatus; red, H. aff. guttatus. The same colors are used to represent sampled specimens in the map to the right: H. guttatus, blue circles in the Brazilian coast; H. aff. guttatus, red circles in Central America. Blue star is the holotype location of the valid species, which was not sampled, in southeastern Brazilian coast.

Two species of Hypanus occur along the Pacific coast, H. dipterurus and H. longus, both with similar evolutionary histories since they are independent sister-groups to Atlantic clades: H. say and H. berthalutzae + H. rudis, respectively. Moreover, within the clade H. say, there is a clear divergence of two lineages separated by the Peninsula of Florida (Clade C in Fig. 2).

Six representatives of Fontitrygon geijskesi, subfamily Urogymninae, which was not included in the Dasyatidae revision by Last et al. (2016aLast PR, Naylor GJP, Manjaji-Matsumoto BM. A revised classification of the family Dasyatidae (Chondrichthyes: Myliobatiformes) based on new morphological and molecular insights. Zootaxa. 2016a; 4139(3):345–68. https://doi.org/10.11646/zootaxa.4139.3.2
https://doi.org/10.11646/zootaxa.4139.3.... ), formed a sister-clade to H. guttatus, within the genus Hypanus, but not Fontitrygon. So, for Hypanus to be monophyletic, this species should be reclassified as Hypanus geijskesi. The subfamily Urogymninae is then represented by three Fontitrygon species occurring in Africa, which formed a cluster: F. margarita, F. margaritella,and F. garouaensis.

Delimitation of lineages. Candidate species of both species complexes, H. guttatus and H. say, were analyzed by combining all five delimitation methods (Figs. 3–4); analyses of H. americanus complex were performed by Petean et al. (2020Petean FF, Naylor GJP, Lima SMQ. Integrative taxonomy identifies a new stingray species of the genus Hypanus Rafinesque, 1818 (Dasyatidae, Myliobatiformes) from the Tropical Southwestern Atlantic. J Fish Biol. 2020; 97(4):1120–42. https://doi.org/10.1111/jfb.14483
https://doi.org/10.1111/jfb.14483... ). The observed new lineages, sister to known species, are named as affinis to those they are closely related to. We kept the valid name according to the type-locality of each species: type of H. guttatus from Brazil, so H. aff. guttatus from Central America; and type of H. say from Egg Harbor (USA), H. aff. say from the Gulf of Mexico.

FIGURE 4 |
Candidate species of the clade Hypanus say species complex (Clade C), according to five lineage delimitation analyses using the mtDNA. Possible species found in each analysis are portrayed as colored boxes in columns. In blue, H. say; red, H. aff. say. The same colors are used to represent sampled specimens in the map to the right: H. say, blue circles in USA’s Eastern coast; H. aff. say, red circles in Gulf of Mexico. Blue star is the holotype location of the valid species, which was not sampled, in USA’s Northeastern coast.

We selected those results which were more consistent among methods, with similar branches’ division, and those that provided the least number of lineages within a species complex to avoid over-splitting taxa due to mere genetic structure. mPTP and ASAP were the most conservative analyses suggesting only two lineages within each species complex; however, while bPTP agreed with mPTP in delimiting H. say two lineages, it was a less stringent method when analyzing H. guttatus data, pointing to 25 entities (almost one per individual). Both GMYC methods, single and multiple thresholds, resulted in similar groupings within each complex and proposed only one or two lineages more than we accepted.

Intraspecific average pairwise distances vary from 0.036% in H. longus to 0.26% in H. aff. guttatus (Tab. 2), while in interspecific average pairwise, the smallest distances are 0.82% between H. rudis and H. berthalutzae, 0.83% between H. americanus and H. aff. americanus, and 0.95% between H. say and H. aff. say (Tab. 3). Interestingly, the distance between two geographically distant species as H. longus, from the Pacific, and H. rudis, from Africa is only 2.4% (Petean et al., 2020Petean FF, Naylor GJP, Lima SMQ. Integrative taxonomy identifies a new stingray species of the genus Hypanus Rafinesque, 1818 (Dasyatidae, Myliobatiformes) from the Tropical Southwestern Atlantic. J Fish Biol. 2020; 97(4):1120–42. https://doi.org/10.1111/jfb.14483
https://doi.org/10.1111/jfb.14483... ), while H. sabinus has the highest distances to all other Hypanus lineages (11.74% from H. dipterurus is the smallest).

DNA Barcoding. By analyzing the protein-coding region mt-co1 of 11 samples of “Fontitrygon geijskesi”, we obtained a monophyletic group by a Neighbor-Joining analysis with 100% of bootstrap value (Fig. 5), a result similar to that using mitogenomes. Besides, the genetic distances among all sequences varied from 0 to 0.17%, with an average of 0.03% (Tabs. 4, S5). Given the genetic similarity of these sequences and since Rodrigues Filho et al. (2020Rodrigues Filho LFS, Feitosa LM, Nunes JLS, Palmeira ARO, Martins APB, Giarrizzo T et al. Molecular identification of ray species traded along the Brazilian Amazon coast. Fish Res. 2020; 223:105407. https://doi.org/10.1016/j.fishres.2019.105407
https://doi.org/10.1016/j.fishres.2019.1... ), from which came four of those samples, could provide a voucher specimen for one of them, we have enough support to suggest that it is indeed a valid species. However, it should be considered as Hypanus geijskesi due to its close relationship to H. guttatus, as suggested by the abovementioned phylogenetic analyses.

FIGURE 5 |
Neighbor-Joining tree based on mt-co1 from samples identified as Hypanus geijskesi, with H. berthalutzae as an outgroup. Nodes’ numbers correspond to bootstrap values in percentage; only those higher than 85% are shown.

Regarding the species H. guttatus, the scenario is convoluted: the lineage H. aff. guttatus identified by mitogenomic delimitation analyses was supported by the inclusion of more samples, as shown by the Neighbor-Joining tree with 89.2% of bootstrap value (Fig. 6). The genetic distance between these two samples (GN13939, GN13946) was 0.38%, which was the same distance between GN13946 and ten other samples (GN19470, MN105788, MN105792, MN105794, MN105808, MN105817, MN105869–71, MN105875), while the distance between GN13939 and the same ten samples was 0.76%. However, the distances between these two (GN13939, GN13946) and the other 73 were higher than 1.15%, distances between nine of those abovementioned (except GN19470) and the others varied from 0.76% to 0.95%, and distances between those 73 samples varied from 0 to 0.19% (Tabs. 5, S6).

FIGURE 6 |
Neighbor-Joining tree based on mt-co1 from samples identified as Hypanus guttatus, with H. berthalutzae as an outgroup. Nodes’ numbers correspond to bootstrap values in percentage; only those higher than 85% are shown. Examined vouchers with an asterisk.

Therefore, based on these results, we suggest that, besides the lineage H. aff. guttatus, there could also be some hybridization or incomplete lineage sorting driving the evolution of H. guttatus, which could be undergoing diversifications into distinct ecological niches (Nosil, Harmon, 2009Nosil P, Harmon L. Niche dimensionality and ecological speciation. In: Butlin R, Bridle J, Schluter D, editors. Speciation and Patterns of Diversity. Cambridge: Cambridge University Press; 2009. p.127–54. Available from: https://doi.org/10.1017/CBO9780511815683.009
https://doi.org/10.1017/CBO9780511815683... ); such processes could only be understood through more sampling and markers. Given the data we have, we can corroborate the lineage H. guttatus by examination of three vouchers by one of us (FFP) (GN18451, GN18458–9; deposited at the fish collection at Universidade Federal do Rio Grande do Norte under respective codes CIUFRN 4442, 4449–50).

Through the extraction of the mt-co1 region from samples of the species-complex H. say, the result agrees with our previous outcome: groups separated by the Peninsula of Florida (Fig. 7), with high bootstrap values for each clade, H. say and H. aff. say, of 97% and 97.5%, respectively. The genetic p-distances between both lineages varied from 0.93% to 1.09%, while within lineage values ranged from 0 to 0.31% (Tabs. 6, S7). For the lineage occurring on USA’s East coast, which should bear the name H. say, one of the samples (MH378605) came from a specimen deposited at the Smithsonian Fish Collection (USNM433289), from a place close to type’s location. This specimen was examined by FFP, who identified it as H. say, thus serving as a voucher for the lineage.

FIGURE 7 |
Neighbor-Joining tree based on mt-co1 from samples identified as Hypanus say, H. dipterurus, and H. sabinus, with H. berthalutzae as an outgroup. Nodes’ numbers correspond to bootstrap values in percentage; only those higher than 85% are shown. Examined vouchers with an asterisk.

Sequences of the species H. dipterurus and H. sabinus were analyzed together with H. say complex, and analyses suggested the southernmost sample of H. dipterurus(MH 194454, from the Peruvian coast, Pacific Ocean) could be another lineage since it has an average of 3.84% of genetic distance to the other five samples of H. dipterurus from Baja California and California coast. Moreover, the inclusion of eight sequences of H. sabinus still leaves it monophyletic, with samples from both the East coast of the USA and the Gulf of Mexico. One of these samples (MT 455431) was extracted from a specimen deposited at the Smithsonian Fish Collection (USNM 426256), which was examined by FFP, hence could serve as a voucher for the clade.

Within H. americanus species-complex there seem to be two sympatric clades: one that should bear the species name, H. americanus (82.9% of bootstrap value) (Fig. 8), since mt-co1 sequences of some analyzed specimens by FFP at the Smithsonian Fish Collection (USNM 433102, USNM 433338–9) fall within it (KT 075327, MH 378683–4) and they were collected close to the species type-locality. The other clade is composed of three samples (two previously identified as H. aff. americanus by Petean et al. (2020Petean FF, Naylor GJP, Lima SMQ. Integrative taxonomy identifies a new stingray species of the genus Hypanus Rafinesque, 1818 (Dasyatidae, Myliobatiformes) from the Tropical Southwestern Atlantic. J Fish Biol. 2020; 97(4):1120–42. https://doi.org/10.1111/jfb.14483
https://doi.org/10.1111/jfb.14483... ), and one sample deposited at GenBank, MG837920). Genetic distance among these three H. aff. americanus samples is 0, and their distance to other H. americanus vary from 0.33% to 0.67%; while distances within H. americanus vary from 0 to 0.17%. Regardless of which H. americanus clade, sequences belonging to this species-complex have more than 1.5% distance to any other sequence belonging to H. berthalutzae, H. longus, and H. rudis (Tabs. 7, S8). Besides, some sequences previously identified and submitted to GenBank as H. americanus should be reallocated to H. berthalutzae (MK085594, MK085604, MK085629, MK085636, MK085638, MK085641, MK085657, MK085659, MK085662, MK085669, MK085672, MK085684, MK085742, MN105805, MN105821, MN105822, MN105823, MN105824, MN105839, MN105842, MN105845, MN105846, MN105847). Some of these sequences were used by Rodrigues Filho et al. (2020Rodrigues Filho LFS, Feitosa LM, Nunes JLS, Palmeira ARO, Martins APB, Giarrizzo T et al. Molecular identification of ray species traded along the Brazilian Amazon coast. Fish Res. 2020; 223:105407. https://doi.org/10.1016/j.fishres.2019.105407
https://doi.org/10.1016/j.fishres.2019.1... ) to suggest the existence of two lineages of H. americanus in Northern Brazil; and one of them was described as H. berthalutzae (H. americanus 1). The clade they called H. americanus 2 is what we identified as H. americanus. The lineage identified by Petean et al. (2020Petean FF, Naylor GJP, Lima SMQ. Integrative taxonomy identifies a new stingray species of the genus Hypanus Rafinesque, 1818 (Dasyatidae, Myliobatiformes) from the Tropical Southwestern Atlantic. J Fish Biol. 2020; 97(4):1120–42. https://doi.org/10.1111/jfb.14483
https://doi.org/10.1111/jfb.14483... ) and hereby sustained as H. aff. americanus was not sampled by those authors.

FIGURE 8 |
Neighbor-Joining tree based on mt-co1 from samples identified as Hypanus americanus, H. berthalutzae, H. rudis and H. longus, with H. guttatus as an outgroup. Nodes’ numbers correspond to bootstrap values in percentage; only those higher than 85% are shown. Examined vouchers with an asterisk.

DISCUSSION

Phylogenetic considerations. Hypanus was recovered as monophyletic by using mitochondrial genome sequences of all species previously attributed to it in addition to another species that was provisionally allocated in Fontitrygon by Last et al. (2016aLast PR, Naylor GJP, Manjaji-Matsumoto BM. A revised classification of the family Dasyatidae (Chondrichthyes: Myliobatiformes) based on new morphological and molecular insights. Zootaxa. 2016a; 4139(3):345–68. https://doi.org/10.11646/zootaxa.4139.3.2
https://doi.org/10.11646/zootaxa.4139.3.... ) due to a lack of sampling and morphological similarities. These authors revised the family Dasyatidae and used the mt-nd2 gene to identify chondrichthyans’ lineages, as suggested by Naylor et al. (2012Naylor GJP, Caira JN, Jensen K, Rosana KAM, White WT, Last PR. A DNA sequence-based approach to the identification of shark and ray species and its implications for global elasmobranch diversity and parasitology. Bull Am Museum Nat Hist. 2012; 2012(367):1–262. https://doi.org/10.1206/754.1
https://doi.org/10.1206/754.1... ). Our results indicate the use of this marker is reliable for identifying Chondrichthyes species, especially when resources are unavailable for genome sequencing. Some lineages suggested to belong to Hypanus, but unsampled by Last et al. (2016aLast PR, Naylor GJP, Manjaji-Matsumoto BM. A revised classification of the family Dasyatidae (Chondrichthyes: Myliobatiformes) based on new morphological and molecular insights. Zootaxa. 2016a; 4139(3):345–68. https://doi.org/10.11646/zootaxa.4139.3.2
https://doi.org/10.11646/zootaxa.4139.3.... ), were hereby included. Hypanus marianae was identified as a Hypanus species, as implied by morphological similarities by Last et al. (2016aLast PR, Naylor GJP, Manjaji-Matsumoto BM. A revised classification of the family Dasyatidae (Chondrichthyes: Myliobatiformes) based on new morphological and molecular insights. Zootaxa. 2016a; 4139(3):345–68. https://doi.org/10.11646/zootaxa.4139.3.2
https://doi.org/10.11646/zootaxa.4139.3.... ), andit is a sister-species to the clade containing H. americanus species complex, H. longus, H. rudis,and H. berthalutzae, a recently described species (Petean et al., 2020Petean FF, Naylor GJP, Lima SMQ. Integrative taxonomy identifies a new stingray species of the genus Hypanus Rafinesque, 1818 (Dasyatidae, Myliobatiformes) from the Tropical Southwestern Atlantic. J Fish Biol. 2020; 97(4):1120–42. https://doi.org/10.1111/jfb.14483
https://doi.org/10.1111/jfb.14483... ) whose phylogenetic position was also corroborated by the inclusion of representatives of the whole genus.

For the monophyly of the genus Hypanus, the species “Fontitrygon geijskesi” should be considered a member of Hypanus, as it is found to be sister to H. guttatus. Due to morphological similarities, this species was expected to be related to its African congeners F. margarita, F. margaritella, and F. garouaensis (Last et al., 2016aLast PR, Naylor GJP, Manjaji-Matsumoto BM. A revised classification of the family Dasyatidae (Chondrichthyes: Myliobatiformes) based on new morphological and molecular insights. Zootaxa. 2016a; 4139(3):345–68. https://doi.org/10.11646/zootaxa.4139.3.2
https://doi.org/10.11646/zootaxa.4139.3.... ). However, we suggest the reallocation of F. geijskesi to the genus Hypanus with a new name combination as Hypanus geijskesi (Boeseman, 1948Boeseman M. Some preliminary notes on Surinam sting rays, including the description of a new species. Zool Meded. 1948; 30(2):31–47. https://repository.naturalis.nl/pub/318932
https://repository.naturalis.nl/pub/3189... ). This result had already been observed by Rodrigues Filho et al. (2020Rodrigues Filho LFS, Feitosa LM, Nunes JLS, Palmeira ARO, Martins APB, Giarrizzo T et al. Molecular identification of ray species traded along the Brazilian Amazon coast. Fish Res. 2020; 223:105407. https://doi.org/10.1016/j.fishres.2019.105407
https://doi.org/10.1016/j.fishres.2019.1... ) on a Neighbor-Joining analysis using the mitochondrial marker COI (mt-co1), in which “F. geijskesi” specimens resulted as a sister group to H. guttatus within the genus Hypanus; however, their analysis lacked a phylogenetic inference of its relationships to other Hypanus lineages. Through a combination of mt-co1 sequences by Rodrigues Filho et al. (2020Rodrigues Filho LFS, Feitosa LM, Nunes JLS, Palmeira ARO, Martins APB, Giarrizzo T et al. Molecular identification of ray species traded along the Brazilian Amazon coast. Fish Res. 2020; 223:105407. https://doi.org/10.1016/j.fishres.2019.105407
https://doi.org/10.1016/j.fishres.2019.1... ) to those hereby provided, we noticed H. geijskesi intraspecific distances ranging from 0 to 0.17% and, as they have provided a voucher for one of their samples, we have support for the species reallocation. This change leaves the genus Fontitrygon with five species, of which four occur in the African continent and one in South America (F. colarensis, not sampled by this study).

As already suggested by Lim et al. (2015Lim KC, Lim PE, Chong VC, Loh KH. Molecular and morphological analyses reveal phylogenetic relationships of stingrays focusing on the family Dasyatidae (Myliobatiformes). PLoS ONE. 2015; 10(4):1–21. https://doi.org/10.1371/journal.pone.0120518
https://doi.org/10.1371/journal.pone.012... ), Last et al. (2016aLast PR, Naylor GJP, Manjaji-Matsumoto BM. A revised classification of the family Dasyatidae (Chondrichthyes: Myliobatiformes) based on new morphological and molecular insights. Zootaxa. 2016a; 4139(3):345–68. https://doi.org/10.11646/zootaxa.4139.3.2
https://doi.org/10.11646/zootaxa.4139.3.... ), and Pavan-Kumar et al. (2022Pavan-Kumar A, Singh S, Mishra A, Suman S, Gireesh-Babu P, Chaudhari A et al. Characterization of mitochondrial genome of Indian Ocean blue-spotted maskray, Neotrygon indica and its phylogenetic relationship within Dasyatidae Family. Int J Biol Macromol. 2022; 223:458–67. https://doi.org/10.1016/j.ijbiomac.2022.10.277
https://doi.org/10.1016/j.ijbiomac.2022.... ), but including more representatives of the subfamily Dasyatinae, we also recognized its monophyly and close relationship to Neotrygoninae, with Hypanus as the sister-group to all other genera within the first subfamily; and the subfamily Urogymninae, represented by the genus Fontitrygon, supporting the subfamilies’ rooting.

Two Hypanus species that used to have the largest geographic distributions, H. americanussensu lato (Petean et al., 2020Petean FF, Naylor GJP, Lima SMQ. Integrative taxonomy identifies a new stingray species of the genus Hypanus Rafinesque, 1818 (Dasyatidae, Myliobatiformes) from the Tropical Southwestern Atlantic. J Fish Biol. 2020; 97(4):1120–42. https://doi.org/10.1111/jfb.14483
https://doi.org/10.1111/jfb.14483... ) from Massachusetts (USA) to São Paulo (Brazil) and H. guttatus from Mexico to Southeastern Brazil, are now recognized to encompass more than one lineage each. The lineage of H. americanussensu (Petean et al., 2020Petean FF, Naylor GJP, Lima SMQ. Integrative taxonomy identifies a new stingray species of the genus Hypanus Rafinesque, 1818 (Dasyatidae, Myliobatiformes) from the Tropical Southwestern Atlantic. J Fish Biol. 2020; 97(4):1120–42. https://doi.org/10.1111/jfb.14483
https://doi.org/10.1111/jfb.14483... ) occurring at the Brazilian coast was recently described as H. berthalutzae, a sister-species to H. rudis in Eastern Atlantic. What was left of H. americanus in Central and North America also represents more lineages, as suggested by our phylogenetic analyses (Clade A, Fig. 2), delimitations done by Petean et al. (2020Petean FF, Naylor GJP, Lima SMQ. Integrative taxonomy identifies a new stingray species of the genus Hypanus Rafinesque, 1818 (Dasyatidae, Myliobatiformes) from the Tropical Southwestern Atlantic. J Fish Biol. 2020; 97(4):1120–42. https://doi.org/10.1111/jfb.14483
https://doi.org/10.1111/jfb.14483... ) (Figs. 2–3), and phylogeographic studies by Richards et al. (2019Richards VP, DeBiasse MB, Shivji M. Deep mitochondrial lineage divergence among populations of the southern stingray (Hypanus americanus (Hildebrand & Schroeder, 1928)) throughout the Southeastern United States and Caribbean. Mar Biodivers. 2019; 49:1627–34. https://doi.org/10.1007/s12526-018-0930-5
https://doi.org/10.1007/s12526-018-0930-... ) (Figs. 1–2). Richards et al. (2019Richards VP, DeBiasse MB, Shivji M. Deep mitochondrial lineage divergence among populations of the southern stingray (Hypanus americanus (Hildebrand & Schroeder, 1928)) throughout the Southeastern United States and Caribbean. Mar Biodivers. 2019; 49:1627–34. https://doi.org/10.1007/s12526-018-0930-5
https://doi.org/10.1007/s12526-018-0930-... ) found three lineages of H. americanus occurring in the USA and Caribbean; however, based on our smaller sampling and distinct markers (that did not involve the mitochondrial control region used by those authors), we noticed two sympatric clades (Fig. 1A) where they named “Clade 3” (Richards et al., 2019Richards VP, DeBiasse MB, Shivji M. Deep mitochondrial lineage divergence among populations of the southern stingray (Hypanus americanus (Hildebrand & Schroeder, 1928)) throughout the Southeastern United States and Caribbean. Mar Biodivers. 2019; 49:1627–34. https://doi.org/10.1007/s12526-018-0930-5
https://doi.org/10.1007/s12526-018-0930-... ) (Figs. 1–2) and we could not recover their “Clades 1 and 2”.

The second species with a wide distribution, H. guttatus, has also been shown to contain two lineages, as presented here and by Rodrigues Filho et al. (2020Rodrigues Filho LFS, Feitosa LM, Nunes JLS, Palmeira ARO, Martins APB, Giarrizzo T et al. Molecular identification of ray species traded along the Brazilian Amazon coast. Fish Res. 2020; 223:105407. https://doi.org/10.1016/j.fishres.2019.105407
https://doi.org/10.1016/j.fishres.2019.1... ). Therefore, two wide-ranging marine coastal species were recently shown to occupy smaller areas than previously described, with currently valid species probably harboring more than one lineage and increasing the known diversity within the genus Hypanus.

Lineage delimitations. Based on five distinct lineage delimitation methods (sGMYC, mGMYC, mPTP, bPTP, and ASAP), we analyzed two clades within Hypanus that showed deeper divergences in phylogenetic analysis than what is expected for intraspecific evolution, since branches are longer between these lineages than within each one of them (Schwartz, Mueller, 2010Schwartz RS, Mueller RL. Branch length estimation and divergence dating: estimates of error in Bayesian and maximum likelihood frameworks. BMC Evol Biol. 2010; 10:5. https://doi.org/10.1186/1471-2148-10-5
https://doi.org/10.1186/1471-2148-10-5... ).

Within the clade composed of H. guttatus, a species that supposedly occurs from Mexico to Southeastern Brazil, all analyses suggested a deeper divergence separating Central America’s samples from Brazilian ones than those within each area of occurrence. This scenario was also observed by Rodrigues Filho et al. (2020Rodrigues Filho LFS, Feitosa LM, Nunes JLS, Palmeira ARO, Martins APB, Giarrizzo T et al. Molecular identification of ray species traded along the Brazilian Amazon coast. Fish Res. 2020; 223:105407. https://doi.org/10.1016/j.fishres.2019.105407
https://doi.org/10.1016/j.fishres.2019.1... ), besides a high genetic similarity among stingrays southern of the Amazon river mouth (0.19% of mt-co1 intraspecific distance, Tab. 5, . 3), which would be left under the valid name H. guttatus due to the species’ type-locality: “Brazil”. We infer that, even though H. guttatus is a marine and estuarine species that tolerates low salinities environments, the great freshwater and nutrients influx of the Amazon system may be a barrier for these stingrays, which resulted in isolated northern and southern lineages ( Rocha, 2003Rocha LA. Patterns of distribution and processes of speciation in Brazilian reef fishes. J Biogeogr. 2003; 30(8):1161–71. https://doi.org/10.1046/j.1365-2699.2003.00900.x
https://doi.org/10.1046/j.1365-2699.2003... ; Hoorn et al., 2010Hoorn C, Wesselingh FP, Ter Steege H, Bermudez MA, Mora A, Sevink J et al. Amazonia through time: Andean uplift, climate change, landscape evolution, and biodiversity. Science. 2010; 330(6006):927–31. https://doi.org/10.1126/science.1194585
https://doi.org/10.1126/science.1194585... ). It was also shown by Tosetto et al. (2022Tosetto EG, Bertrand A, Neumann-Leitão S, Nogueira Júnior M. The Amazon River plume, a barrier to animal dispersal in the Western Tropical Atlantic. Sci Rep. 2022; 12(1):1–12. https://doi.org/10.1038/s41598-021-04165-z
https://doi.org/10.1038/s41598-021-04165... ) that the small number of species in common between the Caribbean Sea and the Brazilian coast demonstrates their high isolation by the Amazon River Plume. This differentiation suggested by genetic analysis might be supported by morphological dissimilarities as well, such as morphometric differences in nostrils, spiracles, and caudal structures (FFP, pers. obs.; review in progress).

With regards to H. say, the lineage from the eastern coast of the USA has a different evolutionary history than that from the Gulf of Mexico, which is supported by all genetic analyses conducted here. Morphological differences could also justify this finding, such as differences in snout and caudal morphometries (FFP, pers. obs.; review in progress), leaving the lineage from eastern USA under the valid species name H. say according to its type-locality (New Jersey, USA), and that occurring in the Gulf of Mexico as H. aff. say until further analyses can be performed, and its taxonomic status evaluated. These results agree with the biogeographic proposals of Spalding et al. (2007Spalding MD, Fox HE, Allen GR, Davidson N, Ferdaña ZA, Finlayson M et al. Marine ecoregions of the world: A bioregionalization of coastal and shelf areas. Bioscience. 2007; 57(7):573–83. https://doi.org/10.1641/B570707
https://doi.org/10.1641/B570707... ) as both populations occur in the Warm Temperate North Atlantic province but in distinct ecoregions and separated by the Floridian one. The southernmost portion of the Florida Peninsula is known to have a detached ecosystem from the adjacent USA coast (Bowen, Avise, 1990Bowen BW, Avise JC. Genetic structure of Atlantic and Gulf of Mexico populations of sea bass, menhaden, and sturgeon: Influence of zoogeographic factors and life-history patterns. Mar Biol. 1990; 107:371–81. https://doi.org/10.1007/BF01313418
https://doi.org/10.1007/BF01313418... ). This can also be seen in H. americanus in which samples from the Bahamas (~50 Km from the US coast) are more closely related to those from the US Virgin Islands than those from Florida (Richards et al., 2019Richards VP, DeBiasse MB, Shivji M. Deep mitochondrial lineage divergence among populations of the southern stingray (Hypanus americanus (Hildebrand & Schroeder, 1928)) throughout the Southeastern United States and Caribbean. Mar Biodivers. 2019; 49:1627–34. https://doi.org/10.1007/s12526-018-0930-5
https://doi.org/10.1007/s12526-018-0930-... ). Despite these results, gene transfer among lineages cannot be ruled out, which could result in hybridization and species not achieving reproductive isolation. As a consequence, there would be a disagreement between gene trees and species trees, a situation that might be underlying the evolution of freshwater stingrays Potamotrygoninae, as well as incomplete lineage sorting and diversification times (Fontenelle et al., 2021Fontenelle JP, Lovejoy NR, Kolmann MA, Marques FPL. Molecular phylogeny for the Neotropical freshwater stingrays (Myliobatiformes: Potamotrygoninae) reveals limitations of traditional taxonomy. Biol J Linn Soc. 2021; 134(2):381–401. https://doi.org/10.1093/biolinnean/blab090
https://doi.org/10.1093/biolinnean/blab0... ). These hypotheses should be further tested with nuclear genetic data (Petean and collaborators, working in progress).

The phylogenetic analysis of manta rays based on mitogenomes and nuclear exons (White et al., 2018White WT, Corrigan S, Yang L, Henderson AC, Bazinet AL, Swofford DL, Naylor GJP. Phylogeny of the manta and devilrays (Chondrichthyes: Mobulidae), with an updated taxonomic arrangement for the family. Zool J Linn Soc. 2018; 182(1):50–75. http://dx.doi.org/10.1093/zoolinnean/zlx018
http://dx.doi.org/10.1093/zoolinnean/zlx... ) found pairwise distances between Mobula birostris (Walbaum, 1792) and M. alfredi (Krefft, 1868) as 0.4%; even though this distance might seem small for two species, their taxonomic identities as distinct species had already been suggested by morphometric, meristic, two mitochondrial, and one nuclear gene (Marshall et al., 2009Marshall AD, Compagno LJV, Bennett MB. Redescription of the genus Manta with resurrection of Manta alfredi (Krefft, 1868) (Chondrichthyes; Myliobatoidei; Mobulidae). Zootaxa. 2009; 28(2301):1–28. https://doi.org/10.3161/000345409X484856
https://doi.org/10.3161/000345409X484856... ; Kashiwagi et al., 2012Kashiwagi T, Marshall AD, Bennett MB, Ovenden J.R. The genetic signature of recent speciation in manta rays (Manta alfredi and M. birostris). Mol Phylogenet Evol. 2012; 64(1):212–18. https://doi.org/10.1016/j.ympev.2012.03.020
https://doi.org/10.1016/j.ympev.2012.03.... ). Likewise, the pairwise distances between the three cryptic lineages of Hypanus and the valid species to which they currently belong are small: between H. guttatus and H. aff. guttatus, 1.37%, H. say and H. aff. say,0.95%, and H. americanus and H. aff. americanus, 0.83%. Simultaneously, the largest distance between two Hypanus species is 13.55% in H. sabinus and H. marianae. Interspecific genetic distances within the genus vary from 0.82% (H. berthalutzae and H. rudis, Petean etal., 2020Petean FF, Naylor GJP, Lima SMQ. Integrative taxonomy identifies a new stingray species of the genus Hypanus Rafinesque, 1818 (Dasyatidae, Myliobatiformes) from the Tropical Southwestern Atlantic. J Fish Biol. 2020; 97(4):1120–42. https://doi.org/10.1111/jfb.14483
https://doi.org/10.1111/jfb.14483... ) to 13.55%, which is a high variation. However, intraspecific variations in Hypanus range from 0.047% in H. aff. say to 0.26% in H. aff. guttatus, which are values at least four times smaller than the interspecific distances.

There are many definitions of what “cryptic species” are and, even though there is still no consensus on their meaning (Struck et al., 2018Struck TH, Feder JL, Bendiksby M, Birkeland S, Cerca J, Gusarov VI et al. Finding evolutionary processes hidden in cryptic species. Trends Ecol Evol. 2018; 33(3):153–63. https://doi.org/10.1016/j.tree.2017.11.007
https://doi.org/10.1016/j.tree.2017.11.0... ), they could be “erroneously classified (and hidden) under one species name” (Bickford et al., 2007Bickford D, Lohman DJ, Sodhi NS, Ng PKL, Meier R, Winker K et al. Cryptic species as a window on diversity and conservation. Trends Ecol Evol. 2007; 22(3):148–55. https://doi.org/10.1016/j.tree.2006.11.004
https://doi.org/10.1016/j.tree.2006.11.0... ). Both sibling lineages to currently valid species H. guttatus and H. say are yet undescribed due to a lack of taxonomic studies and poor sampling. Subtle differences between species, with some of them being separated only by morphometrics, suggest a conservative morphology, making it more difficult to identify the species complex despite the allopatric pattern. Therefore, molecular markers such as mtDNA can be used to confirm species identification. Scenarios of cryptic speciation, with DNA sequences showing deep genetic divergences and morphological data revealing subtle diversity, have been observed in many non-elasmobranch fish clades: bonefish Albula Scopoli, 1777(Colborn et al., 2001Colborn J, Crabtree RE, Shaklee JB, E, Pfeiler E, Bowen BW. The evolutionary enigma of bonefishes (Albula spp.): cryptic species and ancient separations in a globally distributed shorefish. Evolution. 2001; 55(4):807–20. https://doi.org/10.1554/0014-3820(2001)055[0807:teeoba]2.0.co;2
https://doi.org/10.1554/0014-3820(2001)0... ),catfish Noturus Rafinesque, 1818(Egge, Simons, 2006Egge JJD, Simons AM. The challenge of truly cryptic diversity: diagnosis and description of a new madtom catfish (Ictaluridae: Noturus). Zool Scr. 2006; 35(6):581–95. https://doi.org/10.1111/j.1463-6409.2006.00247.x
https://doi.org/10.1111/j.1463-6409.2006... ), tubenose goby Proterorhinus Smitt, 1900 (Neilson, Stepien, 2009Neilson ME, Stepien CA. Evolution and phylogeography of the tubenose goby genus Proterorhinus (Gobiidae: Teleostei): evidence for new cryptic species. Biol J Linn Soc. 2009; 96(3):664–84. https://doi.org/10.1111/j.1095-8312.2008.01135.x
https://doi.org/10.1111/j.1095-8312.2008... ).

Genetic data have also been showing a higher species diversity in Elasmobranchs than formerly known, indicating the necessity of taxonomic work (Richards et al., 2009Richards VP, Henning M, Witzell W, Shivji MS. Species delineation and evolutionary history of the globally distributed spotted eagle ray (Aetobatus narinari). J Hered. 2009; 100(3):273–83. https://doi.org/10.1093/jhered/esp005
https://doi.org/10.1093/jhered/esp005... , 2019Richards VP, Henning M, Witzell W, Shivji MS. Species delineation and evolutionary history of the globally distributed spotted eagle ray (Aetobatus narinari). J Hered. 2009; 100(3):273–83. https://doi.org/10.1093/jhered/esp005
https://doi.org/10.1093/jhered/esp005... ; Dudgeon et al., 2012Dudgeon CL, Blower DC, Broderick D, Giles JL, Holmes BJ, Kashiwagi T et al. A review of the application of molecular genetics for fisheries management and conservation of sharks and rays. J Fish Biol. 2012; 80(5):1789–843. https://doi.org/10.1111/j.1095-8649.2012.03265.x
https://doi.org/10.1111/j.1095-8649.2012... ; Borsa et al., 2016Borsa P, Shen K-N, Arlyza IS, Hoareau TB. Multiple cryptic species in the blue-spotted maskray (Myliobatoidei: Dasyatidae: Neotrygon spp.): An update. Comptes Rendus - Biol. 2016; 339(9–10):417–26. https://doi.org/10.1016/j.crvi.2016.07.004
https://doi.org/10.1016/j.crvi.2016.07.0... ; Henderson et al., 2016Henderson AC, Reeve AJ, Jabado RW, Naylor GJP. Taxonomic assessment of sharks, rays and guitarfishes (Chondrichthyes: Elasmobranchii) from south-eastern Arabia, using the NADH dehydrogenase subunit 2 (NADH2) gene. Zool J Linn Soc. 2016; 176(2):399–442. https://doi.org/10.1111/zoj.12309
https://doi.org/10.1111/zoj.12309... ; Sales et al., 2019Sales JBL, Oliveira CN, Santos WCR, Rotundo MM, Ferreira Y, Ready J et al. Phylogeography of eagle rays of the genus Aetobatus: Aetobatus narinari is restricted to the continental western Atlantic Ocean. Hydrobiologia. 2019; 836:169–83. https://doi.org/10.1007/s10750-019-3949-0
https://doi.org/10.1007/s10750-019-3949-... ; Fahmi et al., 2021Fahmi, Tibbetts IR, Bennett MB, Dudgeon CL. Delimiting cryptic species within the brown-banded bamboo shark, Chiloscyllium punctatum in the Indo-Australian region with mitochondrial DNA and genome-wide SNP approaches. BMC Ecol Evol. 2021; 21:121. https://doi.org/10.1186/s12862-021-01852-3
https://doi.org/10.1186/s12862-021-01852... ; Gonzalez et al., 2021Gonzalez C, Postaire B, Domingues RR, Feldheim KA, Caballero S, Chapman D. Phylogeography and population genetics of the cryptic bonnethead shark Sphyrna aff. tiburo in Brazil and the Caribbean inferred from mtDNA markers. J Fish Biol. 2021; 99(6):1899–911. https://doi.org/10.1111/jfb.14896
https://doi.org/10.1111/jfb.14896... ; Vilasboa et al., 2022Vilasboa A, Lamarca F, Solé-Cava AM, Vianna M. Genetic evidence for cryptic species in the vulnerable spiny butterfly ray Gymnura altavela (Rajiformes: Gymnuridae). J Mar Biol Assoc United Kingdom. 2022; 102(5):345–49. https://doi.org/10.1017/S002531542200056X
https://doi.org/10.1017/S002531542200056... ; Kottillil et al., 2023Kottillil S, Rao C, Bowen BW, Shanker K. Phylogeography of sharks and rays: a global review based on life history traits and biogeographic partitions. PeerJ. 2023; 11:e15396. https://doi.org/10.7717/peerj.15396
https://doi.org/10.7717/peerj.15396... ). Through the combination of distinct tools, species’ hypotheses have been corroborated by independent studies, such as Gymnura van Hasselt, 1823 (Yokota, Carvalho, 2017Yokota L, Carvalho MR. Taxonomic and morphological revision of butterfly rays of the Gymnura micrura (Bloch & Schneider, 1801) species complex, with the description of two new species (Myliobatiformes: Gymnuridae). Zootaxa. 2017; 4332(1):1–74. https://doi.org/10.11646/zootaxa.4332.1.1
https://doi.org/10.11646/zootaxa.4332.1.... , morphology; Rodrigues Filho et al., 2020Rodrigues Filho LFS, Feitosa LM, Nunes JLS, Palmeira ARO, Martins APB, Giarrizzo T et al. Molecular identification of ray species traded along the Brazilian Amazon coast. Fish Res. 2020; 223:105407. https://doi.org/10.1016/j.fishres.2019.105407
https://doi.org/10.1016/j.fishres.2019.1... , genetics; Vilasboa et al., 2022Vilasboa A, Lamarca F, Solé-Cava AM, Vianna M. Genetic evidence for cryptic species in the vulnerable spiny butterfly ray Gymnura altavela (Rajiformes: Gymnuridae). J Mar Biol Assoc United Kingdom. 2022; 102(5):345–49. https://doi.org/10.1017/S002531542200056X
https://doi.org/10.1017/S002531542200056... , genetics), Aetobatus Blainville, 1816(Richards et al., 2009Richards VP, Henning M, Witzell W, Shivji MS. Species delineation and evolutionary history of the globally distributed spotted eagle ray (Aetobatus narinari). J Hered. 2009; 100(3):273–83. https://doi.org/10.1093/jhered/esp005
https://doi.org/10.1093/jhered/esp005... , genetics; White et al., 2010White WT, Last PR, Naylor G, Jensen K, Caira J. Clarification of Aetobatus ocellatus (Kuhl, 1823) as a valid species, and a comparison with Aetobatus narinari (Euphrasen, 1790) (Rajiformes: Myliobatidae). In: Last PR, White WT, Pogonoski JJ, editors. Sharks and rays of Borneo. CSIRO Marine and Atmospheric Research; 2010. p.141–65. , 2013White WT, Furumitsu K, Yamaguchi A. A new species of eagle ray Aetobatus narutobiei from the northwest Pacific: an example of the critical role taxonomy plays in fisheries and ecological sciences. PLoS ONE. 2013; 8(12):e83785. https://doi.org/10.1371/journal.pone.0083785
https://doi.org/10.1371/journal.pone.008... , morphology and genetics; Sales et al., 2019Sales JBL, Oliveira CN, Santos WCR, Rotundo MM, Ferreira Y, Ready J et al. Phylogeography of eagle rays of the genus Aetobatus: Aetobatus narinari is restricted to the continental western Atlantic Ocean. Hydrobiologia. 2019; 836:169–83. https://doi.org/10.1007/s10750-019-3949-0
https://doi.org/10.1007/s10750-019-3949-... , genetics), Rhizoprionodon Whitley, 1929 (Springer, 1964Springer VG. A revision of the carcharhinid shark genera Scoliodon, Loxodon, and Rhizoprionodon. Proc United States Natl Mus. 1964; 115(3493):559–632. , morphology; Mendonça et al., 2011Mendonça FF, Oliveira C, Burgess G, Coelho R, Piercy A, Gadig OBF et al. Species delimitation in sharpnose sharks (genus Rhizoprionodon) in the western Atlantic Ocean using mitochondrial DNA. Conserv Genet. 2011; 12:193–200. https://doi.org/10.1007/s10592-010-0132-6
https://doi.org/10.1007/s10592-010-0132-... , genetics, Pseudobatos Last, Séret & Naylor, 2016 (Rutledge, 2019Rutledge KM. A new guitarfish of the genus Pseudobatos (Batoidea: Rhinobatidae) with key to the guitarfishes of the Gulf of California. Copeia. 2019; 107(3):451–63. https://doi.org/10.1643/CI-18-166
https://doi.org/10.1643/CI-18-166... , morphology; Sandoval-Castillo, Beheregaray, 2020Sandoval-Castillo J, Beheregaray LB. Oceanographic heterogeneity influences an ecological radiation in elasmobranchs. J Biogeogr. 2020; 47(7):1599–611. https://doi.org/10.1111/jbi.13865
https://doi.org/10.1111/jbi.13865... , genetics).

Our findings regarding these independent evolutionary units in Hypanus are only hypotheses of possible species as morphological and ecological data are recommended to be included since the use of exclusively molecular tools might lead to over or under-estimations of species (Carstens et al., 2013Carstens BC, Pelletier TA, Reid NM, Satler JD. How to fail at species delimitation. Mol Ecol. 2013; 22(17):4369–83. https://doi.org/10.1111/mec.12413
https://doi.org/10.1111/mec.12413... ). This is due to species delimitation methods being unable to distinguish deep structure as a result of population-level processes or species boundaries (Sukumaran, Knowles, 2017Sukumaran J, Knowles LL. Multispecies coalescent delimits structure, not species. Proc Natl Acad Sci. 2017; 2016:201607921. https://doi.org/10.1073/PNAS.1607921114
https://doi.org/10.1073/PNAS.1607921114... ). Therefore, to avoid failure, we are not describing any species until more data can be combined (Carstens et al., 2013Carstens BC, Pelletier TA, Reid NM, Satler JD. How to fail at species delimitation. Mol Ecol. 2013; 22(17):4369–83. https://doi.org/10.1111/mec.12413
https://doi.org/10.1111/mec.12413... ).

Conservation. There is no threshold of genetic distances between lineages that should be regarded as populations and those that should receive a species status, since this is a faint boundary (De Queiroz, 2007De Queiroz K. Species concepts and species delimitation. Syst Biol. 2007; 56(6):879–86. https://doi.org/10.1080/10635150701701083
https://doi.org/10.1080/1063515070170108... ; Roux et al., 2016Roux C, Fraïsse C, Romiguier J, Anciaux Y, Galtier N, Bierne N. Shedding light on the grey zone of speciation along a continuum of genomic divergence. PLoS Biol. 2016; 14(12):e2000234. https://doi.org/10.1371/journal.pbio.2000234
https://doi.org/10.1371/journal.pbio.200... ). As mitochondrial evolution rates are slower in elasmobranchs than in other vertebrates (Martin et al., 1992Martin AP, Naylor GJP, Palumbi SR. Rates of mitochondrial DNA evolution in sharks are slow compared with mammals. Nature. 1992; 357(6374):153–55. https://doi.org/10.1038/357153a0
https://doi.org/10.1038/357153a0... ), those mtDNA differences found here may represent distinct species. It is undoubtful that more studies are needed for a resolution of their taxonomic status; however, despite being categorized as species or populations, these lineages should be considered for conservation purposes (Henderson et al., 2016Henderson AC, Reeve AJ, Jabado RW, Naylor GJP. Taxonomic assessment of sharks, rays and guitarfishes (Chondrichthyes: Elasmobranchii) from south-eastern Arabia, using the NADH dehydrogenase subunit 2 (NADH2) gene. Zool J Linn Soc. 2016; 176(2):399–442. https://doi.org/10.1111/zoj.12309
https://doi.org/10.1111/zoj.12309... ).

The urgency in identifying these lineages is because each entity in a threatened species complex might be even more endangered than the nominal species as a whole, and may need distinct conservation measures (Bickford et al., 2007Bickford D, Lohman DJ, Sodhi NS, Ng PKL, Meier R, Winker K et al. Cryptic species as a window on diversity and conservation. Trends Ecol Evol. 2007; 22(3):148–55. https://doi.org/10.1016/j.tree.2006.11.004
https://doi.org/10.1016/j.tree.2006.11.0... ). All current valid Hypanus species have been recently evaluated by elasmobranch specialists at IUCN (2020International Union for Conservation of Nature (IUCN). The IUCN Red List of Threatened Species. Version 2020. 2020. Available from: https://www.iucnredlist.org/
https://www.iucnredlist.org/... ). Of the 13 evolutionary units identified in this study, only one is clearly under low risk, H. sabinus (Least Concern), while six are under some risk of extinction, with criteria used to evaluate each species threatened category in parenthesis: Hypanus berthalutzae (A2d), H. dipterurus (A2d), and H. longus (A2d) are Vulnerable, H. marianae (A2cd) is Endangered, and H. rudis (A2d) and H. geijskesi (A2d)are Critically Endangered. A concerning situation regards the three species-complexes (H. americanus (A2bd), H. guttatus (A2d), and H. say (A2bd)) since they had their threatened status recently evaluated and were considered as Near Threatened (Carlson et al., 2020aCarlson J, Charvet P, Blanco-Parra M, Briones Bell-lloch A, Cardenosa D, Derrick D et al.. Hypanus americanus. IUCN Red List Threat Species 2020. 2020a. https://doi.org/10.2305/IUCN.UK.2020-3.RLTS.T181244884A104123787.en
https://doi.org/10.2305/IUCN.UK.2020-3.R... ,bCarlson J, Charvet P, Blanco-Parra M, Briones Bell-lloch A, Cardenosa D, Derrick D et al.. Hypanus guttatus. IUCN Red List Threat Species 2020. 2020b. https://doi.org/10.2305/IUCN.UK.2020-3.RLTS.T44592A104125629.en
https://doi.org/10.2305/IUCN.UK.2020-3.R... ,cCarlson J, Charvet P, Blanco-Parra M, Briones Bell-lloch A, Cardenosa D, Derrick D et al.. Hypanus say. IUCN Red List Threat Species 2020. 2020c. https://doi.org/10.2305/IUCN.UK.2020-3.RLTS.T60159A3090316.en
https://doi.org/10.2305/IUCN.UK.2020-3.R... ). However, after this and previous studies (Petean et al., 2020Petean FF, Naylor GJP, Lima SMQ. Integrative taxonomy identifies a new stingray species of the genus Hypanus Rafinesque, 1818 (Dasyatidae, Myliobatiformes) from the Tropical Southwestern Atlantic. J Fish Biol. 2020; 97(4):1120–42. https://doi.org/10.1111/jfb.14483
https://doi.org/10.1111/jfb.14483... ; Richards et al., 2019Richards VP, DeBiasse MB, Shivji M. Deep mitochondrial lineage divergence among populations of the southern stingray (Hypanus americanus (Hildebrand & Schroeder, 1928)) throughout the Southeastern United States and Caribbean. Mar Biodivers. 2019; 49:1627–34. https://doi.org/10.1007/s12526-018-0930-5
https://doi.org/10.1007/s12526-018-0930-... ; Rodrigues Filho et al., 2020Rodrigues Filho LFS, Feitosa LM, Nunes JLS, Palmeira ARO, Martins APB, Giarrizzo T et al. Molecular identification of ray species traded along the Brazilian Amazon coast. Fish Res. 2020; 223:105407. https://doi.org/10.1016/j.fishres.2019.105407
https://doi.org/10.1016/j.fishres.2019.1... ), we recognized each of them might be, at least, two evolutionary lineages. Therefore, the possible restriction of each clade’s geographic range could have an impact on their threatened categories, with consequences on management proposals. The recently described H. berthalutzae is the most recent example of this scenario since it was considered H. americanus and encompassed the largest species distribution within the genus. Soon after its description, the species was evaluated and already classified as Vulnerable (Charvet et al., 2020Charvet P, Derrick D, Faria V, Motta F, Dulvy NK.. Hypanus berthalutzae. IUCN Red List Threat Species 2020. 2020. https://doi.org/10.2305/IUCN.UK.2020-3.RLTS.T181244306A181246271.en
https://doi.org/10.2305/IUCN.UK.2020-3.R... ), thus demonstrating that lineages currently unknown can already be under threat before their formal description and evaluation as a species.

Throughout evolution, some lineages might be described as species due to population isolation, while others present high genetic variability and each may be named Evolutionarily Significant Unit (ESU) (Coates et al., 2018Coates JD, Byrne M, Moritz C. Genetic diversity and conservation units: dealing with the species-population continuum in the age of genomics. Front Ecol Evol. 2018; 6(165):1–13. https://doi.org/https://doi.org/10.3389/fevo.2018.00165
https://doi.org/https://doi.org/10.3389/... ) in an attempt to identify independent entities for conservation and perpetuation of their evolutionary history (Diniz-Filho et al., 2013Diniz-Filho JAF, Loyola RD, Raia P, Mooers AO, Bini LM. Darwinian shortfalls in biodiversity conservation. Trends Ecol Evol. 2013; 28(12):689–95. https://doi.org/10.1016/j.tree.2013.09.003
https://doi.org/10.1016/j.tree.2013.09.0... ; Hoezel, 2023Hoezel AR. Where to now with the evolutionarily significant unit? Trends Ecol Evol. 2023; 38(12):1134–42. https://doi.org/10.1016/j.tree.2023.07.005
https://doi.org/10.1016/j.tree.2023.07.0... ). These ESUs should be the focus of management efforts (Ryder, 1986Ryder OA. Species conservation and systematics – the dilemma of subspecies. Trends Ecol Evol. 1986; 1(1):9–10. https://doi.org/10.1016/0169-5347(86)90059-5
https://doi.org/10.1016/0169-5347(86)900... ; Waples, 1991Waples RS. Pacific salmon, Oncorhynchus spp., and the definition of “species” under the Endangered Species Act. Mar Fish Rev. 1991; 53:11–22. , 1995Waples RS. Evolutionarily significant units and the conservation of biological diversity under the Endangered Species Act. In: Nielsen J, editor. Evolution and the aquatic ecosystem: defining unique units in population conservation. Bethesda, Maryland: American Fisheries Society; 1995. p.8–27. ; Moritz, 1994Moritz C. Defining “Evolutionary Significant Units” for conservation. Trends Ecol Evol. 1994; 9(10):373–75. https://doi.org/10.1016/0169-5347(94)90057-4
https://doi.org/10.1016/0169-5347(94)900... ).

Currently, conservation aims mostly on valid species, ignoring genetic diversity. Due to the existence of several species concepts and species’ delimitation methods, there are many conflicts within taxonomy; besides, scientists do not comply on how to deal with ESUs leading to difficulties in actually applying measurements (Coates et al., 2018Coates JD, Byrne M, Moritz C. Genetic diversity and conservation units: dealing with the species-population continuum in the age of genomics. Front Ecol Evol. 2018; 6(165):1–13. https://doi.org/https://doi.org/10.3389/fevo.2018.00165
https://doi.org/https://doi.org/10.3389/... ). Therefore, we suggest the evolutionary lineages hereby identified, even if not formally described as species, to be treated as ESUs and thus be the target of threat evaluation.

To conclude, based on 13 protein-coding mitochondrial genes, there is enough support for the monophyly of the resurrected genus Hypanus by Last et al. (2016aLast PR, Naylor GJP, Manjaji-Matsumoto BM. A revised classification of the family Dasyatidae (Chondrichthyes: Myliobatiformes) based on new morphological and molecular insights. Zootaxa. 2016a; 4139(3):345–68. https://doi.org/10.11646/zootaxa.4139.3.2
https://doi.org/10.11646/zootaxa.4139.3.... ) after the description of a new species (H. berthalutzae) and the transference of Fontitrygon geijskesi to Hypanus, becoming Hypanus geijskesi due to its close relationship to H. guttatus. Besides the recognition of a cryptic species within H. americanus by Petean et al. (2020Petean FF, Naylor GJP, Lima SMQ. Integrative taxonomy identifies a new stingray species of the genus Hypanus Rafinesque, 1818 (Dasyatidae, Myliobatiformes) from the Tropical Southwestern Atlantic. J Fish Biol. 2020; 97(4):1120–42. https://doi.org/10.1111/jfb.14483
https://doi.org/10.1111/jfb.14483... ), we have also identified evolutionary lineages that represent currently known species, as well as suggested two putatively new ones not detected until now, which are sister-lineages to H. guttatus and H. say,thus reducing their geographic distribution, with possible impacts on their conservation status.

These results leave the genus Hypanus with 13 independent evolutionary units, of which 10 are valid species and three “affinis” to their siblings (H. aff. americanus, H. aff. guttatus, and H. aff. say). Further formal descriptions of these new lineages will have consequences on their conservation status since current areas of distribution of valid species will decrease with their division into more than one entity, leading to an urgency in evaluating their threatened status and proposing conservation measures, actions that could already begin with ESUs before descriptions. Even though we have delimited some evolutionary lineages within the genus, maybe more could be found with wider sampling. Finally, to rigorously evaluate these species complexes, morphological studies, the examination of type series, and ecological niche modeling should be performed to better define these stingray species and their geographic distributions.

ACKNOWLEDGEMENTS

We thank our many collaborators who provided us with tissue samples so we could carry out our analyses: Tiego Costa, Carolina Puppin, Marcelo Carvalho, Mônica Oliveira (UFRN), Kirsten Jensen (Univ. of Kansas), Janine Caira (Univ. of Connecticut), João B. L. Sales (UFPA), Vicente Faria (UFC), Patrícia Charvet (UFPR), Paulo Mello Affonso (UESB), Otto B. F. Gadig (UNESP), Matthew Kolmann (Univ. of Louisville), Dean Grubbs (FSU), Ross Robertson, Cristina Castillo, Ruth Gibbons, Natalia Agudelo (Smithsonian Inst.), Vibha Thakur (U. Auckland), Samuel Iglésias (MNHN), Arinze Uche (U. Port Harcourt), Arinze Uche (U. Port Harcourt), Laura Jordan (U. California). We also thank Luiz A. Rocha and Hudson Pinheiro (CAS), Karla Soares (UFRJ), Vicente Faria (UFC), Françoise Lima (UFRN), Jürgen Kriwet (University of Vienna), Matthew Kolmann (Univ. of Louisville), and anonymous reviewers for insightful comments to improve this manuscript. This study is part of FFP Ph.D. thesis at the Graduate Program of Systematics and Evolution (UFRN) and was supported by the Fulbright Commission (to FFP); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (grant number 001 to FFP); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (grant number 312066/2021–0 to SQML); and the University of Florida (to GJPN).

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