Articulo

Neotropical Ichthyology Original article https://doi.org/10.
1590/1982-0224-2023-0046
How many lineages are there

of the stingrays genus Hypanus
(Myliobatiformes: Dasyatidae) and why
does it matter?
Correspondence: Flávia F. Petean1,2,3, Lei Yang4, Shannon Corrigan4,
Flávia F. Petean
ffpetean@gmail.com Sergio M. Q. Lima3 and Gavin J. P. Naylor4
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
Submitted May 3, 2023 within each, leaving the genus with 13 evolutionary units, of which six are
Accepted November 22, 2023 currently under threat and only H. sabinus is of least concern. The urgency in
by Toby Daly-Engel identifying these new lineages lies in the fact they might already be under threat
Epub March 11, 2024 before being formally described.
Keywords: Atlantic Ocean, Conservation, Cryptic species, Diversification,

Elasmobranchs.
1 Instituto Tecnológico de Chascomús, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de San
Martín, Av. Intendente Marino km 8.2, 7130 Chascomús, Buenos Aires, Argentina. (FFP) ffpetean@gmail.com (corresponding
Online version ISSN 1982-0224 author).
Print version ISSN 1679-6225 2 Escuela de Bio y Nanotecnologías, Universidad Nacional de San Martín.
3 Laboratório de Ictiologia Sistemática e Evolutiva, Departamento de Botânica e Zoologia, Centro de Biociências, Universidade
Federal do Rio Grande do Norte, Campus Universitário, BR-101 s/n, 59078-900 Lagoa Nova, Natal, RN, Brazil. (SMQL) sergio.
lima@ufrn.br.
Neotrop. Ichthyol.
4 Florida Program for Shark Research, Florida Museum of Natural History, University of Florida, 1659 Museum Road, 32611
vol. 22, no. 1, Maringá 2024 Gainesville, FL, USA. (LY) lyang@floridamuseum.ufl.edu, (SC) shancorrigan1@gmail.com, (GJPN) gnaylor@flmnh.ufl.edu.
Neotropical Ichthyology, 22(1):e230046, 2024 1/31

Diversification of Hypanus stingrays
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., 2013) mediated by an organism’s
life history characteristics and biology (Craig et al., 2006). Some para- or sympatric
lineages with incipient genetic differentiation might be considered as different species,
not only populations (Avise, 2000; Potkamp, Fransen, 2019). This scenario has already
been documented in some sharks (Corrigan, Beheregaray, 2009) and rays (Kashiwagi et
al., 2012) that move extensively but are genetically restrained by environmental forces
(Bowen et al., 2013). 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, 2007).
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., 2016).
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., 2016b). Until recently, the genus Dasyatis Rafinesque,
1810 was indicated as a paraphyletic group of stingrays based on morphological data
(Rosenberger, 2001), and subsequent studies based on the mitochondrial gene NADH
2/31 Neotropical Ichthyology, 22(1):e230046, 2024 ni.bio.br | scielo.br/ni

Flávia F. Petean, Lei Yang, Shannon Corrigan, Sergio M. Q. Lima and Gavin J. P. Naylor
dehydrogenase 2 (mt-nd2) corroborated this hypothesis (Naylor et al., 2012). Last et al.
(2016a) and Lim et al. (2015) 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, 1910, Taeniurops Garman, 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
their phylogenetic 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., 2016a).
Currently, Hypanus encompasses nine recognized species: H. americanus (Hildebrand
& Schroeder, 1928), H. berthalutzae Petean, Naylor & Lima, 2020, H. dipterurus (Jordan
& Gilbert, 1880), H. guttatus Bloch & Schneider (1801), H. longus (Garman, 1880), H.
marianae (Gomes, Rosa & Gadig, 2000), H. rudis (Günther, 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. (2016a), 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. (2020), 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., 2015; Last et al., 2016b; Ceretta et al., 2020; Oliveira
et al., 2021). 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., 2020; Pollom et al., 2020a,c)), one is Endangered (H.
marianae (Pollom et al., 2020b)), and one is Critically Endangered (H. rudis (Jabado et
al., 2021c)); three are classified as Near Threatened (H. americanus, H. guttatus, and H. say
(Carlson et al., 2020a,b,c)); and only one is clearly under no risk of extinction: H. sabinus
(Least Concern, Carlson et al., 2020d)).
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., 2016a). Four occur in western Africa: Fontitrygon margarita (Günther, 1870)
(Vulnerable, Jabado et al., 2021a), F. margaritella (Compagno & Roberts, 1984) (Near
Threatened, Jabado et al., 2021b), F. ukpam (Smith, 1863) (Critically Endangered, Jabado
et al., 2021d), and F. garouaensis (Stauch & Blanc, 1963) (Critically Endangered, Jabado
et al., 2021e) while two occur along the Northern coast of South America: F. colarensis
(Santos, Gomes & Charvet-Almeida, 2004) (Santos et al., 2004) (Critically Endangered,
Pollom et al., 2020d) and F. geijskesi (Boeseman, 1948) (Critically Endangered, Pollom et
al., 2020e). Nevertheless, only three of these species were included in Last et al. (2016a)
ni.bio.br | scielo.br/ni Neotropical Ichthyology, 22(1):e230046, 2024 3/31

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., 1987;
Harrison, 1989; Boore, Brown, 1998; Satoh et al., 2016). 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., 1987). 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., 1987). 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.,
2013; Flowers et al., 2016; Schwanck et al., 2020). 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., 2020), Rodentia (Abramson et al.,
2021), Coleoptera (Nie et al., 2021), and Elasmobranchii (Palacios-Barreto et al., 2023).
Since the massive use of molecular methods, such as DNA barcoding (Hebert,
Gregory, 2005), 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 (2005) described these as “groups
of organisms that are morphologically indistinguishable from each other, yet found to
belong to different evolutionary lineages”. Sphyrna gilberti Quattro, Driggers, Grady,
Ulrich & Roberts, 2013 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., 2010; Quattro et al., 2013; Gaither et al., 2016).
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, 2010; Raposo et
al., 2020). 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, 1997; Benton, 2000; Dubois, 2010;) 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., 2016)
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., 2021).
Due to the taxonomic uncertainties in Dasyatinae (Lim et al., 2015; Last et al., 2016a;
Pavan-Kumar et al., 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, 1880), Taeniurops
grabatus (Geoffroy St. Hilaire, 1817), Dasyatis hypostigma Santos & Carvalho, 2004;
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. (2016a) and Eschmeyer’s Catalog of Fishes (Fricke et al.,
2023). 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. (2022). Before mitochondrial
gene capture, samples were genetically identified based on Sanger sequencing of the
mitochondrial marker mt-nd2, as described by Petean et al. (2020), and compared to
the database from Naylor et al. (2012). 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.
From genomic DNA extraction to the alignment of protein-coding gene sequences
of mitochondrial genomes, all protocols and procedures followed Li et al. (2013, 2015)
and Petean et al. (2020). 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., 2015). We performed a series of reactions in each sample
for mitochondrial gene capture using biotinylated RNA baits (Mycroarray, Ann Arbor,
Michigan) (Li et al., 2013). 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, 2020) 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., 2013)) to
annotate sequences, which are available on GenBank under accession numbers provided
in Tab. S1.

TABLE 1 | Sampled species of the genera Hypanus, Telatrygon, Hemitrygon, Taeniurops, Pteroplatytrygon, Bathytoshia, Dasyatis, Neotrygon,
Taeniura, and Fontitrygon, their location and geographic distributions. *non-sampled species by Last et al. (2016). EA: Eastern Atlantic, NWA:
Northwestern Atlantic, SWA: Southwestern Atlantic, EP: Eastern Pacific, WA: Western Atlantic, WP: Western Pacific.
Species N Sampled locality Distribution
Hypanus americanus (Hildebrand & Schroeder, 1828) 8 Virginia (USA) to Nicaragua NWA: Massachusetts (USA) North of South America
*Hypanus berthalutzae Petean, Naylor & Lima, 2020 23 From Pará to Bahia (Brazil) SWA: Pará to São Paulo (Brazil)
EP: Hawaii (USA), California (USA) to northern Chile,

Hypanus dipterurus (Jordan & Gilbert, 1880) 4 Baja California (Mexico)
including the Galápagos Islands
*Hypanus geijskesi (Boeseman, 1948) 6 North of Brazil WA: Venezuela and Suriname to Northern Brazil
Hypanus guttatus (Bloch & Schneider, 1801) 34 From Belize to Bahia (Brazil) EA: Gulf of Mexico to Paraná (Brazil)
EP: Baja California (Mexico) to Ecuador, including the

Hypanus longus (Garman, 1880) 4 Baja California (Mexico)
Galápagos Islands
*Hypanus marianae (Gomes, Rosa & Gadig, 2000) 29 From Ceará to Bahia (Brazil) SWA: Northeastern Brazil
Hypanus rudis (Günther, 1870) 4 Senegal and Ghana EA: Gulf of Guinea
South Carolina and
Hypanus sabinus (Lesueur, 1824) 4 NWA: Delaware (USA) to Gulf of Mexico
Mississippi (USA)
South Carolina and
Hypanus say (Lesueur, 1817) 8 WA: Massachusetts (USA) to Brazil
Mississippi (USA)
Telatrygon acutirostra (Nishida & Nakaya, 1988) 1 Ariake Bay (Japan) WP: China and southern Japan
Hemitrygon akajei (Müller & Henle, 1841) 1 Ariake Bay (Japan) WP: China and Japan to Malasia
EA: Mediterranean Sea, Madeira, and Canary Islands to

Taeniurops grabata (Geoffroy St. Hilaire, 1817) 1 Senegal
Angola
Pteroplatytrygon violacea (Bonaparte, 1832) 1 California (USA) Cosmopolitan in tropical and warm temperate seas
Indo-West Pacific, Hawaii (USA), Eastern Atlantic, and

Bathytoshia lata (Garman, 1880) 1 Hawaii (USA)
Mediterranean Sea
Dasyatis hypostigma Santos & Carvalho, 2004 1 Uruguay SWA: South of Brazil
WP: Solomon Islands, Red Sea, Indo-West Pacific: East

Neotrygon kuhlii (Müller & Henle, 1841) 1 Malasia Africa, east to the Philippines and Mariana Islands,
north to Japan, Australia, and New Caledonia
Red Sea, Indo-West Pacific: East and South Africa, east

Taeniura lymma (Forsskål, 1775) 1 Indonesia to the Philippines and Papua New Guinea, north to the
Philippines, south to northern Australia
*Fontitrygon garouaensis (Stauch & Blanc, 1962) 1 Nigeria EA: Nigeria and Cameroon
Fontitrygon margarita (Günther, 1870) 1 Senegal EA: Senegal to Congo
Fontitrygon margaritella (Compagno & Roberts, 1984) 1 Senegal EA: Mauritania to Angola
Total 135

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.
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, 2004). 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., 2017) 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, 2014) in CIPRES Science Gateway (Miller et al., 2010), 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., 2019) 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, 2015) in

1,000,000,000 generations with 5 chains resampled every 10,000. The software MEGA
X (Kumar et al., 2018) 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, 2013), multi-rate Poisson Tree Process (mPTP, Kapli et al.,
2017), Bayesian Poisson Tree Process (bPTP, Zhang et al., 2013), and Assemble Species
by Automatic Partitioning (ASAP, (Puillandre et al., 2021). 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. (2016a): 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., 2012) 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. (2020).
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. (2020) (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, 1987) 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. (2020). 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 Hypanus sensu Last et al. (2016a) 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. (2016a) 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. (2020).

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., 2020; 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. (2019) 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., 2016a), is a monophyletic lineage
and sister to the clade containing H. americanus sensu 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).
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. (2016a), 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. (2020). 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.
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

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.

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.

between two geographically distant species as H. longus, from the Pacific, and H. rudis,
from Africa is only 2.4% (Petean et al., 2020), while H. sabinus has the highest distances
to all other Hypanus lineages (11.74% from H. dipterurus is the smallest).
TABLE 2 | Pairwise average distances of mitogenome within Hypanus species in %.
Species % of average divergence within each species
H. americanus 0.092
H. aff. americanus 0.070
H. longus 0.036
H. rudis 0.098
H. berthalutzae 0.121
H. marianae 0.068
H. guttatus 0.200
H. aff. guttatus 0.262
H. geijskesi 0.167
H. dipterurus 0.163
H. say 0.061
H. aff. say 0.047
H. sabinus 0.134
TABLE 3 | Pairwise distances of mitogenome between pairs of species in %. Hamer, Hypanus americanus; Haffamer, H. aff. americanus; Hlon,
H. longus; Hrud, H. rudis; Hbert, H. berthalutzae; Hmari, H. marianae; Hgut, H. guttatus; Haffgut, H. aff. guttatus; Hgeij, H. geijskesi; Hdipt, H.
dipterurus; Hsay, H. say; Haffsay, H. aff. say; Hsab, H. sabinus; Dhypo, Dasyatis hypostigma.
% Hamer Haffamer Hlon Hrud Hbert Hmari Hgut Haffgut Hgeij Hdipt Hsay Haffsay Hsab
Haffamer 0.83
Hlon 2.69 2.69
Hrud 3.11 3.12 2.40
Hbert 3.08 3.14 2.43 0.82
Hmari 4.91 4.94 4.61 4.96 4.95
Hgut 6.84 6.86 6.72 7.01 6.94 7.19
Haffgut 7.02 7.04 6.88 7.21 7.11 7.24 1.37
Hgeij 6.49 6.44 6.40 6.67 6.56 6.97 5.35 5.53
Hdipt 11.24 11.20 11.02 11.16 11.15 11.14 11.57 11.75 11.71
Hsay 10.89 10.89 10.60 10.80 10.85 10.66 11.13 11.28 11.08 4.57
Haffsay 10.97 11.01 10.80 11.00 11.06 10.83 11.35 11.53 11.39 4.76 0.95
Hsab 13.05 13.03 12.80 12.93 12.96 13.55 13.21 13.38 13.01 11.74 11.81 11.89
Dhypo 14.06 14.09 13.85 14.09 14.07 13.91 14.20 14.32 14.02 13.50 13.06 13.19 14.77

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. (2020), 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.
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
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.
TABLE 4 | Average pairwise distances of the mitochondrial marker mt-co1 between eleven samples of
Hypanus geijskesi, with H. berthalutzae as an outgroup for comparison. Values in %: interspecific in first
cell; intraspecific in second bold cell (with standard error estimate in parenthesis).
% H. berthalutzae H. geijskesi
H. geijskesi 4.6 0.03 (±0.03)

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.

TABLE 5 | Average pairwise distances of the mitochondrial marker mt-co1 between 85 samples of
Hypanus guttatus and H. aff. guttatus, with H. berthalutzae as an outgroup for comparison. Values in %:
interspecific below diagonal; intraspecific bold diagonal (with standard error estimate in parenthesis).
% H. berthalutzae H. aff. guttatus H. guttatus
H. aff. guttatus 5.73 0.38 (±0.26)
H. guttatus 5.92 1.27 0.19 (±0.07)
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, 2009); 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.
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. (2020), 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
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.
TABLE 6 | Average pairwise distances of the mitochondrial marker mt-co1 between six samples of
Hypanus dipterurus, 13 H. sabinus, 18 H. say and three H. aff. say, with H. berthalutzae as an outgroup for
comparison. Values in %: interspecific below diagonal; intraspecific bold diagonal (with standard error
estimate in parenthesis).
% H. berthalutzae H. dipterurus H. sabinus H. say H. aff. say
H. dipterurus 10.76 1.43 (±0.29)
H. sabinus 15.61 13.47 0.19 (±0.11)
H. say 11.06 5.63 13.92 0.15 (±0.1)
H. aff. say 10.60 5.12 14.05 1.21 0

these sequences were used by Rodrigues Filho et al. (2020) 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. (2020) 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.

TABLE 7 | Average pairwise distances of the mitochondrial marker mt-co1 between 46 samples of
Hypanus berthalutzae, 20 H. americanus, three H. aff. americanus, four H. rudis, and four H. longus, with
H. guttatus as an outgroup for comparison. Values in %: interspecific below diagonal; intraspecific bold
diagonal (with standard error estimate in parenthesis).
H. H. H. aff.
% H. guttatus H. rudis H. longus
berthalutzae americanus americanus
H. berthalutzae 6.09 0.17 (±0.08)
H. americanus 7.18 1.83 0.04 (±0.02)
H. aff. americanus 6.68 1.69 0.50 0.00 (±0.00)
H. rudis 6.47 0.59 1.96 1.79 0.17 (± 0.11)
H. longus 5.51 1.48 2.34 2.17 1.29 0 (± 0.00)
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. (2016a)
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. (2012). 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. (2016a), were hereby included. Hypanus marianae was identified as a Hypanus
species, as implied by morphological similarities by Last et al. (2016a), and it 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., 2020) 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., 2016a). However, we suggest
the reallocation of F. geijskesi to the genus Hypanus with a new name combination as
Hypanus geijskesi (Boeseman, 1948). This result had already been observed by Rodrigues
Filho et al. (2020) 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. (2020) 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. (2015), Last et al. (2016a), and Pavan-Kumar et
al. (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.
americanus sensu lato (Petean et al., 2020) 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. americanus sensu (Petean et al., 2020)
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. (2020) (Figs. 2–3), and phylogeographic
studies by Richards et al. (2019) (Figs. 1–2). Richards et al. (2019) 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., 2019) (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. (2020). 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, 2010).
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. (2020), besides a
high genetic similarity among stingrays southern of the Amazon river mouth (0.19%
of mt-co1 intraspecific distance, Tab. 5, Fig. 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, 2003;
Hoorn et al., 2010). It was also shown by Tosetto et al. (2022) 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. (2007)
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, 1990). 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., 2019). 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., 2021). 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., 2018) 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., 2009; Kashiwagi et al., 2012). 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
et al., 2020) 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., 2018), they could be “erroneously
classified (and hidden) under one species name” (Bickford et al., 2007). 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., 2001), catfish Noturus Rafinesque, 1818 (Egge, Simons, 2006), tubenose
goby Proterorhinus Smitt, 1900 (Neilson, Stepien, 2009).

Genetic data have also been showing a higher species diversity in Elasmobranchs
than formerly known, indicating the necessity of taxonomic work (Richards et al.,
2009, 2019; Dudgeon et al., 2012; Borsa et al., 2016; Henderson et al., 2016; Sales et al.,
2019; Fahmi et al., 2021; Gonzalez et al., 2021; Vilasboa et al., 2022; Kottillil et al., 2023).
Through the combination of distinct tools, species’ hypotheses have been corroborated
by independent studies, such as Gymnura van Hasselt, 1823 (Yokota, Carvalho, 2017,
morphology; Rodrigues Filho et al., 2020, genetics; Vilasboa et al., 2022, genetics),
Aetobatus Blainville, 1816 (Richards et al., 2009, genetics; White et al., 2010, 2013,
morphology and genetics; Sales et al., 2019, genetics), Rhizoprionodon Whitley, 1929
(Springer, 1964, morphology; Mendonça et al., 2011, genetics, Pseudobatos Last, Séret
& Naylor, 2016 (Rutledge, 2019, morphology; Sandoval-Castillo, Beheregaray, 2020,
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., 2013). 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, 2017). Therefore, to avoid failure, we are not
describing any species until more data can be combined (Carstens et al., 2013).
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, 2007; Roux et al., 2016). As mitochondrial evolution
rates are slower in elasmobranchs than in other vertebrates (Martin et al., 1992), 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., 2016).
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., 2007). All current valid Hypanus
species have been recently evaluated by elasmobranch specialists at IUCN (2020). 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., 2020a,b,c). However,
after this and previous studies (Petean et al., 2020; Richards et al., 2019; Rodrigues Filho
et al., 2020), 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., 2020), 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., 2018) in an attempt to
identify independent entities for conservation and perpetuation of their evolutionary
history (Diniz-Filho et al., 2013; Hoezel, 2023). These ESUs should be the focus of
management efforts (Ryder, 1986; Waples, 1991, 1995; Moritz, 1994).
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., 2018).
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. (2016a) 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. (2020), 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.
ACKNOWLEDGMENTS
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).
REFERENCES
• Abramson NI, Bodrov SY, Bondareva • Borsa P, Shen K-N, Arlyza IS, Hoareau
OV, Genelt-Yanovskiy EA, Petrova TV. A TB. Multiple cryptic species in the
mitochondrial genome phylogeny of voles blue-spotted maskray (Myliobatoidei:
and lemmings (Rodentia: Arvicolinae): Dasyatidae: Neotrygon spp.): An update.
Evolutionary and taxonomic implications. Comptes Rendus - Biol. 2016; 339(9–
PLoS ONE. 2021; 16(11):e0248198. https:// 10):417–26. https://doi.org/10.1016/j.
doi.org/10.1371/journal.pone.0248198 crvi.2016.07.004
• Amorim DS, Santos CMD, Krell • Bouckaert R, Vaughan TG, Barido-
F-T, Dubois A, Nihei SS, Oliveira Sottani J, Duchêne S, Fourment M,
OMP et al. Timeless standards for Gavryushkina A et al. BEAST 2.5: An
species delimitation. Zootaxa. 2016; advanced software platform for Bayesian
4137(1):121–28. https://doi.org/10.11646/ evolutionary analysis. PLoS Comput
zootaxa.4137.1.9 Biol. 2019; 15(4):e1006650. https://doi.
org/10.1371/journal.pcbi.1006650
• Avise JC. Phylogeography: the history
and formation of species. Cambridge, MA: • Bowen BW, Avise JC. Genetic structure of
Harvard University Press; 2000. https://doi. Atlantic and Gulf of Mexico populations
org/10.2307/j.ctv1nzfgj7 of sea bass, menhaden, and sturgeon:
Influence of zoogeographic factors and life-
• Avise JC, Arnold J, Ball RM, Bermingham
history patterns. Mar Biol. 1990; 107:371–
E, Lamb T, Neigel JE et al. Intraspecific
81. https://doi.org/10.1007/BF01313418
phylogeography: The mitochondrial DNA
bridge between population genetics and • Bowen BW, Rocha LA, Toonen RJ,
systematics. Annu Rev Ecol Syst. 1987; Karl SA. The origins of tropical marine
18(1):489–522. https://doi.org/10.1146/ biodiversity. Trends Ecol Evol. 2013;
annurev.es.18.110187.002421 28(6):359–66. https://doi.org/10.1016/j.
tree.2013.01.018
• Benton MJ. Stems, nodes, crown clades,
and rank-free lists: is Linnaeus dead? Biol • Carlson J, Charvet P, Blanco-Parra M,
Rev Camb Philos Soc. 2000; 75(4):633–48. Briones Bell-lloch A, Cardenosa D,
https://doi.org/10.1111/j.1469-185x.2000. Derrick D et al. Hypanus americanus.
tb00055.x IUCN Red List Threat Species 2020. 2020a.
https://doi.org/10.2305/IUCN.UK.2020-3.
• Bickford D, Lohman DJ, Sodhi NS, Ng
RLTS.T181244884A104123787.en
PKL, Meier R, Winker K et al. Cryptic
species as a window on diversity and • Carlson J, Charvet P, Blanco-Parra M,
conservation. Trends Ecol Evol. 2007; Briones Bell-lloch A, Cardenosa D,
22(3):148–55. https://doi.org/10.1016/j. Derrick D et al. Hypanus guttatus. IUCN
tree.2006.11.004 Red List Threat Species 2020. 2020b.
https://doi.org/10.2305/IUCN.UK.2020-3.
• Boeseman M. Some preliminary notes
RLTS.T44592A104125629.en
on Surinam sting rays, including the
description of a new species. Zool Meded. • Carlson J, Charvet P, Blanco-Parra M,
1948; 30(2):31–47. Available from: https:// Briones Bell-lloch A, Cardenosa D,
repository.naturalis.nl/pub/318932 Derrick D et al. Hypanus say. IUCN Red
List Threat Species 2020. 2020c. https://
• Boore JL, Brown WM. Big trees from little
doi.org/10.2305/IUCN.UK.2020-3.RLTS.
genomes: mitochondrial gene order as a
T60159A3090316.en
phylogenetic tool. Curr Opin Genet Dev.
1998; 8(6):668–74. https://doi.org/10.1016/
s0959-437x(98)80035-x

• Carlson J, Charvet P, Blanco-Parra M, • Costa TLA, Thayer JA, Mendes LF.

Briones Bell-lloch A, Cardenosa D, Population characteristics, habitat and diet
Derrick D et al. Hypanus sabinus. IUCN of a recently discovered stingray Dasyatis
Red List Threat Species 2020. 2020d. marianae: Implications for conservation.
https://doi.org/10.2305/IUCN.UK.2020-3. J Fish Biol. 2015; 86(2):527–43. https://doi.
RLTS.T60158A124445557.en org/10.1111/jfb.12572
• Carstens BC, Pelletier TA, Reid • Craig MT, Hastings PA, Pondella DJ,
NM, Satler JD. How to fail at species Robertson DR, Rosales-Casián JA.
delimitation. Mol Ecol. 2013; 22(17):4369– Phylogeography of the flag cabrilla
83. https://doi.org/10.1111/mec.12413 Epinephelus labriformis (Serranidae):
Implications for the biogeography of the
• Ceretta BF, Fogliarini CO, Giglio VJ,
Tropical Eastern Pacific and the early
Maxwell MF, Waechter LS, Bender
stages of speciation in a marine shore fish.
MG. Testing the accuracy of biological
J Biogeogr. 2006; 33(6):969–79. https://doi.
attributes in predicting extinction risk.
org/10.1111/j.1365-2699.2006.01467.x
Perspect Ecol Conserv. 2020; 18(1):12–18.
https://doi.org/10.1016/j.pecon.2020.01.003 • Darriba D, Taboada GL, Doallo R,
Posada D. jModelTest 2: more models,
• Charvet P, Derrick D, Faria V, Motta F,
new heuristics and parallel computing.
Dulvy NK. Hypanus berthalutzae. IUCN
Nat Methods. 2012; 9(8):772. https://doi.
Red List Threat Species 2020. 2020. https://
org/10.1038/nmeth.2109
T181244306A181246271.en • Diniz-Filho JAF, Loyola RD, Raia P,
Mooers AO, Bini LM. Darwinian shortfalls
• Coates JD, Byrne M, Moritz C. Genetic
in biodiversity conservation. Trends Ecol
diversity and conservation units: dealing
Evol. 2013; 28(12):689–95. https://doi.
with the species-population continuum in
org/10.1016/j.tree.2013.09.003
the age of genomics. Front Ecol Evol. 2018;
6(165):1–13. https://doi.org/https://doi. • Dominguez E, Wheeler QD. Taxonomic
org/10.3389/fevo.2018.00165 stability is ignorance. Cladistics.
1997; 13(4):367–72. https://doi.
• Colborn J, Crabtree RE, Shaklee JB, E,
org/10.1111/j.1096-0031.1997.tb00325.x
Pfeiler E, Bowen BW. The evolutionary
enigma of bonefishes (Albula spp.): • Drummond AJ, Bouckaert RR.
cryptic species and ancient separations Bayesian evolutionary analysis with
in a globally distributed shorefish. BEAST. Bayesian Evol Anal with BEAST.
Evolution. 2001; 55(4):807–20. https://doi. 2015:1–249. https://doi.org/10.1017/
org/10.1554/0014-3820(2001)055[0807:teeo CBO9781139095112
ba]2.0.co;2
• Dubois A. Zoological nomenclature in the
• Compagno LJV, Roberts TR. Marine and century of extinctions: priority vs.‘usage’.
freshwater stingrays (Dasyatidae) of West Org Divers Evol. 2010; 10:259–74. https://
Africa, with description of a new species. doi.org/10.1007/s13127-010-0021-3
Proc Calif Acad Sci. 1984; 43(18):283–300.
• Dudgeon CL, Blower DC, Broderick D,
• Corcoran MJ, Wetherbee BM, Shivji MS, Giles JL, Holmes BJ, Kashiwagi T et al.
Potenski MD, Chapman DD, Harvey A review of the application of molecular
GM. Supplemental feeding for ecotourism genetics for fisheries management and
reverses diel activity and alters movement conservation of sharks and rays. J Fish
patterns and spatial distribution of the Biol. 2012; 80(5):1789–843. https://doi.
southern stingray, Dasyatis americana. org/10.1111/j.1095-8649.2012.03265.x
PLoS ONE. 2013; 8(3):e59235. https://doi.
• Ebert DA, White WT, Goldman KJ,
org/10.1371/journal.pone.0059235
Compagno LJV, Daly-Engel TS, Ward
• Corrigan S, Beheregaray LB. A recent RD. Resurrection and redescription of
shark radiation: Molecular phylogeny, Squalus suckleyi (Girard, 1854) from the
biogeography and speciation of North Pacific, with comments on the
wobbegong sharks (family: Orectolobidae). Squalus acanthias subgroup (Squaliformes:
Mol Phylogenet Evol. 2009; 52(1):205–16. Squalidae). Zootaxa. 2010; 2612(1):22–40.
https://doi.org/10.1016/j.ympev.2009.03.007 https://doi.org/10.11646/zootaxa.2612.1.2
• Costa TLA, Petean FF, Berbel-Filho WM, • Edgar RC. MUSCLE: multiple sequence
Solé-Cava AM, Mendes LF, Lima SMQ. alignment with high accuracy and high
Molecular testing of the São Francisco throughput. Nucleic Acids Res. 2004;
River as an ecological filter for the 32(5):1792–97. https://doi.org/10.1093/nar/
Brazilian large-eyed stingray Hypanus gkh340
marianae (Dasyatidae, Myliobatiformes).
Hydrobiologia. 2022; 849:2435–48. https://
doi.org/10.1007/s10750-022-04828-6

• Egge JJD, Simons AM. The challenge • Gomes UL, Rosa RS, Gadig OBF.
of truly cryptic diversity: diagnosis and Dasyatis marianae sp. n.: A new species
description of a new madtom catfish of stingray (Chondrichthyes: Dasyatidae)
(Ictaluridae: Noturus). Zool Scr. 2006; from the Southwestern Atlantic. Copeia.
35(6):581–95. https://doi.org/10.1111/j.1463- 2000; 2000(2):510–15. https://doi.
6409.2006.00247.x org/10.1643/0045-8511(2000)000[0510:DMS
NAN]2.0.CO;2
• Engel MS, Ceríaco LMP, Daniel GM,
Dellapé PM, Löb l, Marinov M et al. • Gonzalez C, Postaire B, Domingues RR,
The taxonomic impediment: a shortage Feldheim KA, Caballero S, Chapman D.
of taxonomists, not the lack of technical Phylogeography and population genetics of
approaches. Zool J Linn Soc. 2021; the cryptic bonnethead shark Sphyrna aff.
193(2):381–87. https://doi.org/10.1093/ tiburo in Brazil and the Caribbean inferred
zoolinnean/zlab072 from mtDNA markers. J Fish Biol. 2021;
99(6):1899–911. https://doi.org/10.1111/
• Fahmi, Tibbetts IR, Bennett MB,
jfb.14896
Dudgeon CL. Delimiting cryptic species
within the brown-banded bamboo shark, • Günther A. Catalogue of the fishes in
Chiloscyllium punctatum in the Indo- the British Museum. Catalogue of the
Australian region with mitochondrial Physostomi, containing the families
DNA and genome-wide SNP approaches. Gymnotidae, Symbranchidae, Muraenidae,
BMC Ecol Evol. 2021; 21:121. https://doi. Pegasidae, and of the Lophobranchii,
org/10.1186/s12862-021-01852-3 Plectognathi, Dipnoi, ...[thru] ...
Leptocardii, in the British Museum. V. 8.
• Flowers KI, Ajemian MJ, Bassos-Hull K,
1870.
Feldheim KA, Hueter RE, Papastamatiou
YP et al. A review of batoid philopatry, • Harrison RG. Animal mitochondrial
with implications for future research DNA as a genetic marker in population
and population management. Mar Ecol and evolutionary biology. Trends
Prog Ser. 2016; 562:251–61. https://doi. Ecol Evol. 1989; 4(1):6–11. https://doi.
org/10.3354/meps11963 org/10.1016/0169-5347(89)90006-2
• Fontenelle JP, Lovejoy NR, Kolmann • Hebert PDN, Gregory TR. The promise
MA, Marques FPL. Molecular phylogeny of DNA barcoding for taxonomy. Syst
for the Neotropical freshwater stingrays Biol. 2005; 54(5):852–59. https://doi.
(Myliobatiformes: Potamotrygoninae) org/10.1080/10635150500354886
reveals limitations of traditional taxonomy.
• Henderson AC, Reeve AJ, Jabado RW,
Biol J Linn Soc. 2021; 134(2):381–401.
Naylor GJP. Taxonomic assessment
https://doi.org/10.1093/biolinnean/blab090
of sharks, rays and guitarfishes
• Fowler HW. Notes on batoid fishes. Proc (Chondrichthyes: Elasmobranchii) from
Acad Nat Sci Philadelphia. 1910; 62:468–75. south-eastern Arabia, using the NADH
dehydrogenase subunit 2 (NADH2) gene.
• Fricke R, Eschmeyer WN, Van der
Zool J Linn Soc. 2016; 176(2):399–442.
Laan R. Eschmeyer’s catalog of fishes:
https://doi.org/10.1111/zoj.12309
genera, species, references [Internet].
San Francisco: California Academy of • Hildebrand SF, Schroeder WC. Fishes of
Science; 2023. Available from: http:// chesapeake bay. US Government Printing
researcharchive.calacademy.org/research/ Office; 1928.
ichthyology/catalog/fishcatmain.asp
• Hoezel AR. Where to now with the
• Fujisawa T, Barraclough TG. Delimiting evolutionarily significant unit? Trends Ecol
species using single-locus data and Evol. 2023; 38(12):1134–42. https://doi.org/
the generalized mixed yule coalescent https://doi.org/10.1016/j.tree.2023.07.005
approach: A revised method and
• Hoorn C, Wesselingh FP, Ter Steege H,
evaluation on simulated data sets. Syst
Bermudez MA, Mora A, Sevink J et al.
Biol. 2013; 62(5):707–24. https://doi.
Amazonia through time: Andean uplift,
org/10.1093/sysbio/syt033
climate change, landscape evolution, and
• Gaither MR, Bowen BW, Rocha LA, biodiversity. Science. 2010; 330(6006):927–
Briggs JC. Fishes that rule the world: 31. https://doi.org/10.1126/science.1194585
circumtropical distributions revisited.
• International Union for Conservation
Fish Fish. 2016; 17(3):664–79. https://doi.
of Nature (IUCN). The IUCN Red List of
org/10.1111/faf.12136
Threatened Species. Version 2020. 2020.
• Garman S. The Plagiostomia. (Sharks, Available from: https://www.iucnredlist.
Skates, and Rays). Cambridge, USA: org/
Memoirs of the Museum of Comparative
Zoology at Harvard College XXXVI; 1913.
• Garman S. New species of selachians in
the museum collection. Bull Mus Comp
Zool. 1880; 6(11):167–72.

• Iwasaki W, Fukunaga T, Isagozawa • Kumar S, Stecher G, Li M, Knyaz

R, Yamada K, Maeda Y, Satoh TP et C, Tamura K. MEGA X: Molecular
al. MitoFish and mitoannotator: A evolutionary genetics analysis across
mitochondrial genome database of fish computing platforms. Mol Biol Evol. 2018;
with an accurate and automatic annotation 35:1547–49. https://doi.org/10.1093/molbev/
pipeline. Mol Biol Evol. 2013; 30(11):2531– msy096
40. https://doi.org/10.1093/molbev/mst141
• Lanfear R, Frandsen PB, Wright AM,
• Jabado RW, Badji L, Chartrain E, Senfeld T, Calcott B. Partitionfinder 2:
De Bruyne G, Derrick D, Dia M et New methods for selecting partitioned
al. Fontitrygon margarita. IUCN Red models of evolution for molecular and
List Threat Species 2021. 2021a:e. morphological phylogenetic analyses. Mol
T161495A104172843. https://dx.doi. Biol Evol. 2017; 34(3):772–73. https://doi.
org/10.2305/IUCN.UK.2021-2.RLTS. org/10.1093/molbev/msw260
T161495A104172843.en
• Last PR, Naylor GJP, Manjaji-Matsumoto
• Jabado RW, Badji L, Chartrain E, De BM. A revised classification of the
Bruyne G, Derrick D, Dia M et al. family Dasyatidae (Chondrichthyes:
Fontitrygon margaritella. IUCN Red Myliobatiformes) based on new
List Threat Species 2021. 2021b:e. morphological and molecular insights.
T161520A124498844. https://dx.doi. Zootaxa. 2016a; 4139(3):345–68. https://doi.
org/10.2305/IUCN.UK.2021-2.RLTS. org/10.11646/zootaxa.4139.3.2
T161520A124498844.en
• Last PR, Naylor GJP, Séret B, White WT,
• Jabado RW, De Bruyne G, Derrick D, de Carvalho MR, Stehmann MFW. Rays
Doherty P, Diop M, Leurs GHL et al. of the World. Clayton South VIC: CSIRO
Hypanus rudis. IUCN Red List Threat Publishing; 2016b.
Species 2021. 2021c:e.T161620A124516434.
• Li C, Corrigan S, Yang L, Straube N,
10.2305/IUCN.UK.2021-2.RLTS.
Harris M, Hofreiter M et al. DNA
T161620A124516434.en
capture reveals transoceanic gene flow in
• Jabado RW, Chartrain E, De Bruyne endangered river sharks. Proc Natl Acad
G, Derrick D, Diop M, Doherty P et al. Sci. 2015; 112(43):13302–07. https://doi.
Fontitrygon ukpam. IUCN Red List Threat org/10.1073/pnas.1508735112
Species 2021. 2021d:e.T39414A104174049.
• Li C, Hofreiter M, Straube N, Corrigan
https://dx.doi.org/10.2305/IUCN.UK.2021-2.
S, Naylor GJP. Capturing protein-coding
RLTS.T39414A104174049.en
genes across highly divergent species.
• Jabado RW, Keith Diagne L, Sayer Biotechniques. 2013; 54(6):321–26. https://
C, Tamo A, Williams AB. Fontitrygon doi.org/10.2144/000114039
garouaensis. IUCN Red List Threat Species
• Lim KC, Lim PE, Chong VC, Loh KH.
2021. 2021e:e.T39406A104171509. https://
Molecular and morphological analyses
dx.doi.org/10.2305/IUCN.UK.2021-2.RLTS.
reveal phylogenetic relationships
T39406A104171509.en
of stingrays focusing on the family
• Kapli P, Lutteropp S, Zhang J, Kobert K, Dasyatidae (Myliobatiformes). PLoS ONE.
Pavlidis P, Stamatakis A et al. Multi-rate 2015; 10(4):1–21. https://doi.org/10.1371/
Poisson tree processes for single-locus journal.pone.0120518
species delimitation under maximum
• Marshall AD, Compagno LJV, Bennett
likelihood and Markov chain Monte Carlo.
MB. Redescription of the genus Manta
Bioinformatics. 2017; 33(11):1630–38.
with resurrection of Manta alfredi (Krefft,
https://doi.org/10.1093/bioinformatics/
1868) (Chondrichthyes; Myliobatoidei;
btx025
Mobulidae). Zootaxa. 2009; 28(2301):1–28.
• Kashiwagi T, Marshall AD, Bennett MB, https://doi.org/10.3161/000345409X484856
Ovenden JR. The genetic signature of
• Martin AP, Naylor GJP, Palumbi SR.
recent speciation in manta rays (Manta
Rates of mitochondrial DNA evolution in
alfredi and M. birostris). Mol Phylogenet
sharks are slow compared with mammals.
Evol. 2012; 64(1):212–18. https://doi.
Nature. 1992; 357(6374):153–55. https://doi.
org/10.1016/j.ympev.2012.03.020
org/10.1038/357153a0
• Kottillil S, Rao C, Bowen BW, Shanker
• Mendonça FF, Oliveira C, Burgess G,
K. Phylogeography of sharks and rays: a
Coelho R, Piercy A, Gadig OBF et al.
global review based on life history traits
Species delimitation in sharpnose sharks
and biogeographic partitions. PeerJ.
(genus Rhizoprionodon) in the western
2023; 11:e15396. https://doi.org/10.7717/
Atlantic Ocean using mitochondrial DNA.
peerj.15396
Conserv Genet. 2011; 12:193–200. https://
• Krueger F. Trim Galore. 2020. Available doi.org/10.1007/s10592-010-0132-6
from: https://github.com/FelixKrueger/
TrimGalore

• Miller MA, Pfeiffer W, Schwartz T. • Pavan-Kumar A, Singh S, Mishra A,

Creating the CIPRES Science gateway for Suman S, Gireesh-Babu P, Chaudhari A
inference of large phylogenetic trees. et al. Characterization of mitochondrial
Proc Gatew Comput Environ Work. New genome of Indian Ocean blue-spotted
Orleans, LA: 2010. p.1–08. maskray, Neotrygon indica and its
phylogenetic relationship within
• Moritz C. Defining “Evolutionary
Dasyatidae Family. Int J Biol Macromol.
Significant Units” for conservation. Trends
2022; 223:458–67. https://doi.org/10.1016/j.
Ecol Evol. 1994; 9(10):373–75. https://doi.
ijbiomac.2022.10.277
org/10.1016/0169-5347(94)90057-4
• Petean FF, Naylor GJP, Lima SMQ.
• Moritz C, Dowling TE, Brown WM.
Integrative taxonomy identifies a
Evolution of animal mitochondrial DNA:
new stingray species of the genus
Relevance for population biology and
Hypanus Rafinesque, 1818 (Dasyatidae,
systematics. Annu Rev Ecol Syst. 1987;
Myliobatiformes) from the Tropical
18(1):269–92. https://doi.org/10.1146/
Southwestern Atlantic. J Fish Biol. 2020;
annurev.es.18.110187.001413
97(4):1120–42. https://doi.org/10.1111/
• Naylor GJP, Caira JN, Jensen K, jfb.14483
Rosana KAM, White WT, Last PR. A
• Pollom R, Avalos C, Bizzarro J, Burgos-
DNA sequence-based approach to the
Vázquez MI, Cevallos A, Espinoza M
identification of shark and ray species and
et al. Hypanus longus. IUCN Red List
its implications for global elasmobranch
Threat Species 2020. 2020a. https://
diversity and parasitology. Bull Am
Museum Nat Hist. 2012; 2012(367):1–262.
T60157A124445324.en
https://doi.org/10.1206/754.1
• Pollom R, Barreto R, Charvet P, Faria V,
• Neilson ME, Stepien CA. Evolution and
Herman K, Marcante F et al. Hypanus
phylogeography of the tubenose goby
marianae. IUCN Red List Threat Species
genus Proterorhinus (Gobiidae: Teleostei):
2020. 2020b:e.T45925A104130004. https://
evidence for new cryptic species. Biol J
Linn Soc. 2009; 96(3):664–84. https://doi.
T45925A104130004.en
org/10.1111/j.1095-8312.2008.01135.x
• Pollom R, Bizzarro J, Burgos-Vázquez MI,
• Nie R, Vogler AP, Yang XK, Lin M.
Cevallos A, Velez-Zuazo X, Avalos C et al.
Higher-level phylogeny of longhorn
Hypanus dipterurus. IUCN Red List Threat
beetles (Coleoptera: Chrysomeloidea)
Species 2020. 2020c. https://doi.org/10.2305/
inferred from mitochondrial genomes.
IUCN.UK.2020-3.RLTS.T60152A80677563.en
Syst Entomol. 2021; 46(1):56–70. https://doi.
org/10.1111/syen.12447 • Pollom R, Charvet P, Barreto R, Faria V,
Herman K, Marcante F et al. Fontitrygon
• Nosil P, Harmon L. Niche dimensionality
colarensis. He IUCN Red List Threat Species
and ecological speciation. In: Butlin R,
2020. 2020d. 10.2305/IUCN.UK.2020-3.RLTS.
Bridle J, Schluter D, editors. Speciation
T60151A104170822.en
and Patterns of Diversity. Cambridge:
Cambridge University Press; 2009. • Pollom R, Charvet P, Faria V, Herman
p.127–54. https://doi.org/10.1017/ K, Lasso-Alcalá O, Marcante F et al.
CBO9780511815683.009 Fontitrygon geijskesi. IUCN Red List
Threat Species 2020. 2020e. https://
• Oliveira CDL, Oliveira CYB, Camilo JPG,
Batista VS. Demographic analysis reveals
T60153A104172152.en
a population decline of the longnose
stingray Hypanus guttatus in Northeastern • Potkamp G, Fransen CHJM. Speciation
Brazil. Reg Stud Mar Sci. 2021; 41:101554. with gene flow in marine systems. Contrib
https://doi.org/10.1016/j.rsma.2020.101554 to Zool. 2019; 88:133–72. https://doi.
org/10.1163/18759866-20191344
• Palacios-Barreto P, Mar-Silva AF,
Bayona-Vasquez NJ, Adams DH, Díaz- • Puillandre N, Brouillet S, Achaz G.
Jaimes P. Characterization of the complete ASAP: assemble species by automatic
mitochondrial genome of the brazilian partitioning. Mol Ecol Resour. 2021;
cownose ray Rhinoptera brasiliensis 21(2):609–20. https://doi.org/10.1111/1755-
(Myliobatiformes, Rhinopteridae) in the 0998.13281
western Atlantic and its phylogenetic
• Quattro JM, Driggers WBI, Grady JM,
implications. Mol Biol Rep. 2023;
Ulrich GF, Roberts MA. Sphyrna gilberti
50(5):4083–95. https://doi.org/10.1007/
sp. nov., a new hammerhead shark
s11033-023-08272-0
(Carcharhiniformes, Sphyrnidae) from the
western Atlantic Ocean. Zootaxa. 2013;
3702(2):159–78. https://doi.org/10.11646/
zootaxa.3702.2.5

• De Queiroz K. Species concepts • Saitou N, Nei M. The neighbor-joining

and species delimitation. Syst Biol. method: a new method for reconstructing
2007; 56(6):879–86. https://doi. phylogenetic trees. Mol Biol Evol. 1987;
org/10.1080/10635150701701083 4(4):406–25. https://doi.org/10.1093/
oxfordjournals.molbev.a040454
• Raposo MA, Kirwan GM, Lourenco
ACC, Sobral G, Bockmann FA, Stopiglia • Sales JBL, Oliveira CN, Santos WCR,
R. On the notions of taxonomic Rotundo MM, Ferreira Y, Ready J et al.
‘impediment’,‘gap’,‘inflation’and Phylogeography of eagle rays of the genus
‘anarchy’, and their effects on the field Aetobatus: Aetobatus narinari is restricted
of conservation. Syst Biodivers. 2020; to the continental western Atlantic Ocean.
19(3):296–311. https://doi.org/10.1080/1477 Hydrobiologia. 2019; 836:169–83. https://
2000.2020.1829157 doi.org/10.1007/s10750-019-3949-0
• Richards VP, DeBiasse MB, Shivji M. • Sandoval-Castillo J, Beheregaray LB.
Deep mitochondrial lineage divergence Oceanographic heterogeneity influences
among populations of the southern an ecological radiation in elasmobranchs. J
stingray (Hypanus americanus (Hildebrand Biogeogr. 2020; 47(7):1599–611. https://doi.
& Schroeder, 1928)) throughout the org/10.1111/jbi.13865
Southeastern United States and Caribbean.
• Santos HRS, Gomes UL, Charvet-Almeida
Mar Biodivers. 2019; 49:1627–34. https://
P. A new species of whiptail stingray
doi.org/10.1007/s12526-018-0930-5
of the genus Dasyatis Rafinesque, 1810
• Richards VP, Henning M, Witzell W, from the Southwestern Atlantic Ocean
Shivji MS. Species delineation and (Chondrichthyes: Myliobatiformes:
evolutionary history of the globally Dasyatidae). Zootaxa. 2004; 492(1):1–12.
distributed spotted eagle ray (Aetobatus https://doi.org/10.11646/zootaxa.492.1.1
narinari). J Hered. 2009; 100(3):273–83.
• Satoh TP, Miya M, Mabuchi K, Nishida
https://doi.org/10.1093/jhered/esp005
M. Structure and variation of the
• Rocha LA. Patterns of distribution and mitochondrial genome of fishes. BMC
processes of speciation in Brazilian reef Genomics. 2016; 17:719. https://doi.
fishes. J Biogeogr. 2003; 30(8):1161– org/10.1186/s12864-016-3054-y
71. https://doi.org/10.1046/j.1365-
• Schwanck TN, Schweinsberg M, Lampert
2699.2003.00900.x
KP, Guttridge TL, Tollrian R, O’Shea O.
• Rodrigues Filho LFS, Feitosa LM, Linking local movement and molecular
Nunes JLS, Palmeira ARO, Martins analysis to explore philopatry and
APB, Giarrizzo T et al. Molecular population connectivity of the southern
identification of ray species traded along stingray. J Fish Biol. 2020; 96(6):1475–88.
the Brazilian Amazon coast. Fish Res. https://doi.org/10.1111/jfb.14325
2020; 223:105407. https://doi.org/10.1016/j.
• Schwartz RS, Mueller RL. Branch
fishres.2019.105407
length estimation and divergence
• Rosenberger LJ. Phylogenetic dating: estimates of error in Bayesian
relationships within the stingray genus and maximum likelihood frameworks.
Dasyatis (Chondrichthyes: Dasyatidae). BMC Evol Biol. 2010; 10:5. https://doi.
Copeia. 2001; 2001(3):615–27. https://doi. org/10.1186/1471-2148-10-5
org/10.1643/0045-8511(2001)001[0615:PRW
• da Silva FS, Cruz ACR, Almeida Medeiros
TSG]2.0.CO;2
DB, Silva SP, Nunes MRT, Martins LC et
• Roux C, Fraïsse C, Romiguier J, Anciaux al. Mitochondrial genome sequencing and
Y, Galtier N, Bierne N. Shedding light phylogeny of Haemagogus albomaculatus,
on the grey zone of speciation along a Haemagogus leucocelaenus, Haemagogus
continuum of genomic divergence. PLoS spegazzinii, and Haemagogus tropicalis
Biol. 2016; 14(12):e2000234. https://doi. (Diptera: Culicidae). Sci Rep. 2020;
org/10.1371/journal.pbio.2000234 10:16948. https://doi.org/10.1038/s41598-
020-73790-x
• Rutledge KM. A new guitarfish of
the genus Pseudobatos (Batoidea: • Spalding MD, Fox HE, Allen GR,
Rhinobatidae) with key to the guitarfishes Davidson N, Ferdaña ZA, Finlayson M
of the Gulf of California. Copeia. 2019; et al. Marine ecoregions of the world: A
107(3):451–63. https://doi.org/10.1643/CI- bioregionalization of coastal and shelf
18-166 areas. Bioscience. 2007; 57(7):573–83.
https://doi.org/10.1641/B570707
• Ryder OA. Species conservation and
systematics – the dilemma of subspecies. • Springer VG. A revision of the
Trends Ecol Evol. 1986; 1(1):9–10. https:// carcharhinid shark genera Scoliodon,
doi.org/10.1016/0169-5347(86)90059-5 Loxodon, and Rhizoprionodon. Proc United
States Natl Mus. 1964; 115(3493):559–632.
• Sáez AG, Lozano E. Body doubles.
Nature. 2005; 433:111. https://doi.
org/10.1038/433111a

• Stamatakis A. RAxML Version 8: A tool for • White WT, Furumitsu K, Yamaguchi

phylogenetic analysis and post-analysis of A. A new species of eagle ray Aetobatus
large phylogenies. Bioinformatics. 2014; narutobiei from the northwest Pacific:
30(9):1312–13. https://doi.org/10.1093/ an example of the critical role taxonomy
bioinformatics/btu033 plays in fisheries and ecological sciences.
PLoS ONE. 2013; 8(12):e83785. https://doi.
• Struck TH, Feder JL, Bendiksby M,
org/10.1371/journal.pone.0083785
Birkeland S, Cerca J, Gusarov VI et al.
Finding evolutionary processes hidden • White WT, Corrigan S, Yang L,
in cryptic species. Trends Ecol Evol. 2018; Henderson AC, Bazinet AL, Swofford DL,
33(3):153–63. https://doi.org/10.1016/j. Naylor GJP. Phylogeny of the manta and
tree.2017.11.007 devilrays (Chondrichthyes: Mobulidae),
with an updated taxonomic arrangement
• Sukumaran J, Knowles LL. Multispecies
for the family. Zool J Linn Soc. 2018;
coalescent delimits structure, not species.
182(1):50–75. http://dx.doi.org/10.1093/
Proc Natl Acad Sci. 2017; 2016:201607921.
zoolinnean/zlx018
https://doi.org/10.1073/PNAS.1607921114
• Yokota L, Carvalho MR. Taxonomic and
• Tosetto EG, Bertrand A, Neumann-Leitão
morphological revision of butterfly rays of
S, Nogueira Júnior M. The Amazon River
the Gymnura micrura (Bloch & Schneider,
plume, a barrier to animal dispersal in the
1801) species complex, with the description
Western Tropical Atlantic. Sci Rep. 2022;
of two new species (Myliobatiformes:
12(1):1–12. https://doi.org/10.1038/s41598-
Gymnuridae). Zootaxa. 2017; 4332(1):1–74.
021-04165-z
https://doi.org/10.11646/zootaxa.4332.1.1
• Vilasboa A, Lamarca F, Solé-Cava AM,
• Zhang J, Kapli P, Pavlidis P, Stamatakis
Vianna M. Genetic evidence for cryptic
A. A general species delimitation method
species in the vulnerable spiny butterfly
with applications to phylogenetic
ray Gymnura altavela (Rajiformes:
placements. Bioinformatics. 2013;
Gymnuridae). J Mar Biol Assoc United
29(22):2869–76. https://doi.org/10.1093/
Kingdom. 2022; 102(5):345–49. https://doi.
bioinformatics/btt499
org/10.1017/S002531542200056X
• Waples RS. Pacific salmon, Oncorhynchus
spp., and the definition of “species” under
the Endangered Species Act. Mar Fish Rev.
1991; 53:11–22.
• Waples 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 conservations. Bethesda,
Maryland: American Fisheries Society;
1995. p.8–27.
• White 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 rays Borneo.
CSIRO Marine and Atmospheric Research;
2010. p.141–65.

AUTHORS’ CONTRIBUTION
Flávia F. Petean: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation,
Methodology, Project administration, Resources, Writing-original draft, Writing-review and editing.
Lei Yang: Data curation, Formal analysis, Investigation, Methodology, Resources, Writing-review and
editing.
Shannon Corrigan: Data curation, Formal analysis, Investigation, Methodology, Resources, Writing-
review and editing.
Sergio M. Q. Lima: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation,
Gavin J. P. Naylor: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation,
ETHICAL STATEMENT
The care and use of animals within Brazilian territory followed the Ministry of the Environment animal
welfare laws, guidelines, and policies as approved by Chico Mendes Institute for Biodiversity Conservation,
license SISBIO 54254–3.
This is an open access article under the terms of the

Creative Commons Attribution License, which permits COMPETING INTERESTS
use, distribution and reproduction in any medium,
provided the original work is properly cited.
The author declares no competing interests.
Distributed under
Creative Commons CC-BY 4.0
© 2024 The Authors.

Diversity and Distributions Published by SBI HOW TO CITE THIS ARTICLE
• Petean FF, Yang L, Corrigan S, Lima SMQ, Naylor GJP. How many lineages are there of the
stingrays genus Hypanus (Myliobatiformes: Dasyatidae) and why does it matter? Neotrop
Ichthyol. 2024; 22(1):e230046. https://doi.org/10.1590/1982-0224-2023-0046

Articulo

Uploaded by

Copyright:

Available Formats

Articulo

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Articulo

Uploaded by

Copyright:

Available Formats

Neotropical Ichthyology Original article https://doi.org/10.

How many lineages are there

Keywords: Atlantic Ocean, Conservation, Cryptic species, Diversification,

Neotropical Ichthyology, 22(1):e230046, 2024 1/31

As raias com ferrão do gênero Hypanus atualmente compreendem nove espécies

Palavras-chave: Conservação, Diversificação, Elasmobrânquios, Espécies

Speciation in marine environments is usually a complex process involving geographic

2/31 Neotropical Ichthyology, 22(1):e230046, 2024 ni.bio.br | scielo.br/ni

ni.bio.br | scielo.br/ni Neotropical Ichthyology, 22(1):e230046, 2024 3/31

4/31 Neotropical Ichthyology, 22(1):e230046, 2024 ni.bio.br | scielo.br/ni

MATERIAL AND METHODS

ni.bio.br | scielo.br/ni Neotropical Ichthyology, 22(1):e230046, 2024 5/31

Species N Sampled locality Distribution

EP: Hawaii (USA), California (USA) to northern Chile,

EP: Baja California (Mexico) to Ecuador, including the

EA: Mediterranean Sea, Madeira, and Canary Islands to

Indo-West Pacific, Hawaii (USA), Eastern Atlantic, and

WP: Solomon Islands, Red Sea, Indo-West Pacific: East

Red Sea, Indo-West Pacific: East and South Africa, east

Fontitrygon margarita (Günther, 1870) 1 Senegal EA: Senegal to Congo

6/31 Neotropical Ichthyology, 22(1):e230046, 2024 ni.bio.br | scielo.br/ni

Phylogenetic reconstructions. To study the relationships within the genus Hypanus,

ni.bio.br | scielo.br/ni Neotropical Ichthyology, 22(1):e230046, 2024 7/31

there is a reasonable sampling for analyses (ρ = 1) (Drummond, Bouckaert, 2015) in

Lineage delimitation methods. By analyzing the relationships among species, we

DNA Barcode analyses. The mitochondrial protein-coding gene region cytochrome

8/31 Neotropical Ichthyology, 22(1):e230046, 2024 ni.bio.br | scielo.br/ni

ni.bio.br | scielo.br/ni Neotropical Ichthyology, 22(1):e230046, 2024 9/31

Delimitation of lineages. Candidate species of both species complexes, H. guttatus

10/31 Neotropical Ichthyology, 22(1):e230046, 2024 ni.bio.br | scielo.br/ni

ni.bio.br | scielo.br/ni Neotropical Ichthyology, 22(1):e230046, 2024 11/31

12/31 Neotropical Ichthyology, 22(1):e230046, 2024 ni.bio.br | scielo.br/ni

TABLE 2 | Pairwise average distances of mitogenome within Hypanus species in %.

Species % of average divergence within each species

H. aff. americanus 0.070

H. aff. guttatus 0.262

H. aff. say 0.047

ni.bio.br | scielo.br/ni Neotropical Ichthyology, 22(1):e230046, 2024 13/31

DNA Barcoding. By analyzing the protein-coding region mt-co1 of 11 samples

H. geijskesi 4.6 0.03 (±0.03)

14/31 Neotropical Ichthyology, 22(1):e230046, 2024 ni.bio.br | scielo.br/ni

ni.bio.br | scielo.br/ni Neotropical Ichthyology, 22(1):e230046, 2024 15/31

% H. berthalutzae H. aff. guttatus H. guttatus

H. aff. guttatus 5.73 0.38 (±0.26)

H. guttatus 5.92 1.27 0.19 (±0.07)

16/31 Neotropical Ichthyology, 22(1):e230046, 2024 ni.bio.br | scielo.br/ni

% H. berthalutzae H. dipterurus H. sabinus H. say H. aff. say

H. dipterurus 10.76 1.43 (±0.29)

H. sabinus 15.61 13.47 0.19 (±0.11)

H. say 11.06 5.63 13.92 0.15 (±0.1)

H. aff. say 10.60 5.12 14.05 1.21 0

ni.bio.br | scielo.br/ni Neotropical Ichthyology, 22(1):e230046, 2024 17/31

18/31 Neotropical Ichthyology, 22(1):e230046, 2024 ni.bio.br | scielo.br/ni

H. berthalutzae 6.09 0.17 (±0.08)

H. americanus 7.18 1.83 0.04 (±0.02)

H. aff. americanus 6.68 1.69 0.50 0.00 (±0.00)

H. rudis 6.47 0.59 1.96 1.79 0.17 (± 0.11)

H. longus 5.51 1.48 2.34 2.17 1.29 0 (± 0.00)

Phylogenetic considerations. Hypanus was recovered as monophyletic by using