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The mitogenomic phylogeny of the Elasmobranchii (Chondrichthyes)

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 Mitochondrial DNA Part A
DNA Mapping, Sequencing, and Analysis

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The mitogenomic phylogeny of the Elasmobranchii

(Chondrichthyes)

Cesar R. L. Amaral, Filipe Pereira, Dayse A. Silva, António Amorim & Elizeu F.
de Carvalho

To cite this article: Cesar R. L. Amaral, Filipe Pereira, Dayse A. Silva, António Amorim & Elizeu
F. de Carvalho (2017): The mitogenomic phylogeny of the Elasmobranchii (Chondrichthyes),
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 MITOCHONDRIAL DNA PART A, 2017
https://doi.org/10.1080/24701394.2017.1376052

RESEARCH ARTICLE

The mitogenomic phylogeny of the Elasmobranchii (Chondrichthyes)

nio Amorimc,d,e and Elizeu F. de Carvalhoa
Cesar R. L. Amarala, Filipe Pereirab, Dayse A. Silvaa, Anto
a
Departamento de Ecologia, Laborato
rio de Diagn
osticos por DNA, Instituto de Biologia Roberto Alc^antara Gomes, Universidade do Estado
do Rio de Janeiro, Rio de Janeiro, Brazil; bInterdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto,
Porto, Portugal; ci3S – Instituto de Investigaç~ao e Inovaç~ao em Sa

ude, Universidade do Porto, Porto, Portugal; dInstituto de Patologia e
Imunologia Molecular da Universidade do Porto (IPATIMUP), Porto, Portugal; eFaculdade de Ci^encias, Universidade do Porto, Porto, Portugal

ABSTRACT ARTICLE HISTORY

Here we present a mitogenomic perspective on the evolution of sharks and rays, being a first glance Received 29 April 2017
on the complete mitochondrial history of such an old and diversified group of vertebrates. The Accepted 1 September 2017
Elasmobranchii is a diverse subclass of Chondrichthyes, or cartilaginous fish, with about 1200 species of
ocean- and freshwater-dwelling fishes spread all over the world’s seas, including some of the ocean’s
KEYWORDS
largest fishes. The group dates back about 400 million years near the Devonian–Silurian boundary, Elasmobranchii; phylogeny;
being nowadays represented by several derivative lineages, mainly related to Mesozoic forms. Although
Downloaded by [186.205.1.125] at 03:59 20 September 2017

systematics; sharks; rays;

considered of ecological, commercial and conservation importance, the phylogeny of this old group is mitogenome
poorly studied and still under debate. Here we apply a molecular systematic approach on 82 complete
mitochondrial genomes to investigate the phylogeny of the Elasmobranchii. By using maximum likeli-
hood (ML) and Bayesian analyses, we found a clear separation within the shark clade between the
Galeomorphii and the Squalomorphii, as well as sister taxa relationships between the Carcharhiniformes
and the Lamniformes. Moreover, we found that Pristoidei clusters within the Rhinobatoidei, having
been recovered as the sister taxon of the Rhinobatos genus in a clade which also includes the basal
Zapteryx. Our results also reject the Hypnosqualea hypothesis, which proposes that the Batoidea should
be placed within the Selachii.

Introduction monophyletic groups: batoids and sharks (Regan 1906; White

The Elasmobranchii is considered one of the most ancient and
successful vertebrate lineages (Corrigan and Beheregaray
2009) dating back about 400 million years, near the
Devonian–Silurian boundary (Corrigan and Beheregaray 2009;
Heinicke et al. 2009). This group is the most diverse clade of
large predatory fishes with about 1200 species spread all over
the world’s seas (V
elez-Zuazo and Agnarsson 2011), including
ocean- and freshwater-dwelling fishes, such as sharks, skates
and rays. These species play a crucial ecological role by acting
as primary predators (Myers and Worm 2003; Myers et al.
2007; Schindler et al. 2008; Vaudo and Heithaus 2009; V
elez-
Zuazo and Agnarsson 2011) and occupying an important pos-
ition in marine ecosystems (Libralato et al. 2005).
The Elasmobranchii is an overexploited group, as a conse-
quence of unsustainable fishing activities, and a resultant
population decline can be foreseen. Fished for their meat
and fins, several species are considered under high threat
and are now facing extinction (Robbins et al. 2006; Dulvy
et al. 2008; Ferretti et al. 2008), with about 93% of nominal
species included on the IUCN Red List (Vi
e et al. 2008, www.
iucnredlist.org).
As summarized in Table 1, the first taxonomic classifica-
tions considered Elasmobranchii to be subdivided into two
1937; Holmgren 1941). Based on morphology, Compagno
(1973, 1977), followed by Carvalho (1996), divided the
Elasmobranchii into the Galeomorphii, the Squatinomorphii,
the Squalomorphii and the Batoidea (Table 1). It has been
suggested that batoids should be regarded as a derived clade
within the shark’s phylogeny, closely related to saw sharks
and angel sharks, hence proposing the superorder
Hypnosqualea, which was based on cladistic methodology
and followed by several authors (Shirai 1992a, 1992b, 1996;
Carvalho and Maisey 1996).
As expected, the development of molecular techniques
also impacted studies of the Elasmobranchii. The first molecu-
lar studies (Stock 1992; Dunn and Morrissey 1995; Kitamura
et al. 1996; Arnason et al. 2001) were restricted to a few spe-
cies and focused uniquely on partial gene analyses of both
mitochondrial and nuclear genes (Stock 1992; Dunn and
Morrissey 1995; Kitamura et al. 1996; Arnason et al. 2001;
Douady et al. 2003; V
elez-Zuazo and Agnarsson 2011). These
pioneering works did not support the Hypnosqualea hypoth-
esis (Stock 1992; Dunn and Morrissey 1995; Kitamura et al.
1996; Arnason et al. 2001). More recently, the extensive
molecular phylogeny of Douady et al. (2003) was based on a
fragment of mitochondrial 12S, 16S and transfer RNA valine

CONTACT Cesar R. L. Amaral crlamaral@yahoo.com.br Departamento de Ecologia, Laborat
orio de Diagn
osticos por DNA, Instituto de Biologia Roberto
Alc^antara Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil
Supplemental data for this article can be accessed here.
ß 2017 Informa UK Limited, trading as Taylor & Francis Group
 2 C. R. L. AMARAL ET AL.

Table 1. Historical classification of Elasmobranchii.

References
Holmgren (1941), Regan (1906), White (1937)
Compagno (1973, 1977), Carvalho (1996), Carvalho
and Maisey (1996), Shirai (1992a, 1992b, 1996)
Stock (1992), Dunn and Morrissey (1995), Kitamura
et al. (1996), Arnason et al. (2001)
Douady et al. (2003)
V
elez-Zuazo and Agnarsson (2011)
Method
Morphology
Morphology, cladistics
Molecular systematics
Molecular systematics
Molecular systematics
Main findings
Elasmobranchii subdivided into two monophyletic groups:
batoids and sharks
Elasmobranchii subdivided into Galeomorphii, (including
Carcharhiniformes, Heterodontiformes, Lamniformes and
Orectolobiformes); Squalomorphii (including the
Hexanchiformes, Pristiophoriformes and Squaliformes);
Batoidea (including the Myliobatiformes, Pristiformes,
Rajiformes, Rhinobatiformes and Torpediniformes)
Batoids represent a derived clade within the shark's phyl-
ogeny – Hypnosqualea hypothesis.
Restricted to only a few species; Hypnosqualea hypothesis
not supported
Sharks and batoids are monophyletic; Hypnosqualea
hypothesis not supported; Squatina and pristiophoriforms
as squalomorphs
Sharks and batoids are monophyletic; Hypnosqualea
hypothesis not supported; Squatina and pristiophoriforms
as squalomorphs

genes (2400 nucleotides) from over 20 Elasmobranchii. This sorted into transitional and transversional against the K-80
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taxonomic grouping led to the conclusion that batoids are
not recovered within the sharks, that sharks are monophy-
letic, thus rejecting the Hypnosqualea clade, and that
Squatina and pristiophoriforms are squalomorphs (Table 1).
Using four mitochondrial and one nuclear gene, V
elez-Zuazo
and Agnarsson (2011) also observed the same relationships,
with sharks being recovered as monophyletic and batoids not
recovered within them, consequently rejecting the
Hypnosqualea hypothesis (Table 1).
Over the last few years, although several complete mito-
chondrial genomes from Elasmobranchii have been pub-
lished, a mitogenomic approach to the phylogeny of the
Elasmobranchii was not yet employed. Therefore, we herein
provide a comprehensive mitogenomic phylogenetic investi-
gation of the Elasmobranchii using maximum likelihood (ML)
and Bayesian analyses.
Methods
A total of 82 complete mitochondrial sequences were
retrieved from the NCBI database covering the major groups
of Elasmobranchii (Table 2), including three outgroup sequen-
ces from two species (Chimaera monstrosa and Callorhinchus
callorynchus). In order to avoid misidentification of the
sequences, the COI barcoding region of all sequences was
extracted and the identity of the whole mitochondrial
sequences was cross-validated on both NCBI and BOLD data-
bases, and with sequences with voucher specimens when
available. Sequence substitution saturation was evaluated
using the Data Analysis in Molecular Biology and Evolution
(DAMBE) software (v.5.6.7) (Xia and Xie 2001) (Figure 1). The
resultant Bayesian and ML trees can be observed in Figures
2–5. All sequences were aligned using the default parameters
of KALIGN (Lassmann and Sonnhammer 2005) and MUSCLE
(Edgar 2004) running on XSEDE (Extreme Science and
Engineering Discovery Environment – https://www.xsede.org/)
at CIPRES Science Gateway (https://www.phylo.org).
For the saturation plot, codon positions 1–2 and 3 were
separately evaluated. Nucleotide differences were plotted and
distance. The loss of phylogenetic signal was assessed follow-
ing Xia et al. (2003) and Xia and Lemey (2009). ML analyses
were performed with PHYML 3.0 (Guindon et al. 2010) and
MEGA v.6 (Tamura et al. 2013) with bootstrap (
1000) as a
supporting measure. All trees were built with four substitu-
tion rate categories and the remaining parameters of the sub-
stitution model as estimated by the program. We used a
BioNJ starting tree with a NNI tree topology search and opti-
mized tree topology and branch length. Bootstrapping was
performed with 100 trials. The GTR þ G þ I was estimated as
best model of nucleotide substitution using jModelTest
v.0.1.1 (Guindon and Gascuel 2003; Posada 2008) based on
the Akaike criterion. In order to evaluate the similarity of the
results using all three codon positions (only the first and
second codon positions, or only the third codon position), ML
trees were pairwise compared using the overall topological
score, as provided by the Compare2Trees software, following
Nye et al. (2005).
Bayesian analyses were conducted with MrBayes2 within
the XSEDE environment (CIPRES Science Gateway). The
Metropolis-coupled Markov chain Monte Carlo process was
set so that four independent chains ran simultaneously for
10,000,000 generations, each starting from a random tree. In
all trees, we used the GTR þ G þ I model with gamma-distrib-
uted rate variation across sites approximated by four discrete
categories and the program’s default prior probabilities on
model parameters.
Results
The alignment included 82 complete mitochondrial sequen-
ces from distinct species of Elasmobranchii with a total length
of 16.7 kb. We started by evaluating the level of saturation
for all codon positions, for the codon positions 1 þ 2 or only
position 3 separately. Although some saturation could be
observed for asymmetrical topologies with more than 32
OTUs, the Iss was always significantly lower than the Iss.c for
symmetrical topologies, suggesting little saturation and good
signal-to-noise ratio in the database (Table 3).

Table 2. List of mitochondrial sequences and GenBank accession numbers used in the present
study.
Aetobatus flagellum – KF482070
Alopias pelagicus – KF412639
Alopias superciliosus – KC757415
Amblyraja radiata – AF106038
Anoxypristis cuspidata – KP233202
Atlantoraja castelnaui – KM507724
Callorhinchus callorynchus – HM147135
Callorhinchus capensis – HM147136
Callorhinchus milii – HM147137
Carcharhinus acronotus – KF728380
Carcharhinus amblyrhynchoides – KF956523
Carcharhinus falciformis – KF801102
Carcharhinus leucas – KF646785
Carcharhinus longimanus – KM434158
Carcharhinus macloti – KJ865755
Carcharhinus melanopterus – KJ720818
Carcharhinus obscurus – KC470543
Carcharhinus plumbeus – KJ740750
Carcharhinus sorrah – KF612341
Carcharias taurus – KF569943
Carcharodon carcharias – KC914387
Cetorhinus maximus – KF597303
Chiloscyllium griseum – JQ434458
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Chiloscyllium plagiosum – FJ853422
Chiloscyllium punctatum – JQ082337
Chimaera monstrosa – NC003136
Cirrhigaleus australis – KJ128289
Dasyatis akajei – KC526959
Dasyatis bennetti – KC196067
Dasyatis microphthalmus – KF840390
Dasyatis zugei – JX524174
Dipturus kwangtungensis – KF318309
Galeocerdo cuvier – KF111728
Glyphis garricki – KF646786
Glyphis glyphis – KF006312
Gymnura poecilura – KJ617038
Heterodontus francisci – NC003137
Heterodontus zebra – KC845548
Hexanchus griseus – KF894491
Himantura gerrardi – KP091437
Himantura granulata – KF751650
Hongeo koreana – KC914433
Isurus oxyrinchus – KF361861–1
Figure 1. Substitution saturation plot. The x-axis ‘K80 distance’ is based on the K80 substitution model and is expected to increase linearly with divergence time.
The vertical axis is for the observed proportion of transitions (s) and transversions (v), respectively.
MITOCHONDRIAL DNA PART A 3
Isurus paucus – KJ616742
Lamna ditropis – KF962053
Leucoraja erinacea – JQ034406
Megachasma pelagios – KC702506
Mitsukurina owstoni – EU528659
Mobula eregoodootenkee – KM361353
Mobula japanica – JX392983
Mustelus griseus – KF889325
Mustelus manazo – AB015962
Narcine entemedor – KM386678
Neotrygon kuhlii – KC992792
Okamejei kenojei – AY525783
Orectolobus japonicus – KF111729
Pavoraja nitida – KJ741403
Plesiobatis daviesi – AY597334
Potamotrygon motoro – KF709642
Prionace glauca – KF356249
Pristiophorus japonicus – NC024110
Pristis clavata – KF381507
Pseudocarcharias kamoharai – KM575726
Pteroplatytrygon violacea – KJ641617
Raja pulchra – KF623030
Raja rhina – KC914434
Rhincodon typus – KC633221
Rhinobatos hynnicephalus – KF534708
Rhinobatos schlegelii – KJ140136
Scoliodon macrorhynchos – JQ693102
Scyliorhinus canicula – NC001950
Sphyrna zygaena – KM489157
Squalus acanthias – NC002012
Squatina formosa – KM084865
Squatina japonica – KJ619663
Squatina nebulosa – KM264313
Taeniura lymma – KM881715
Taeniura meyeni – JX827260
Triaenodon obesus – KJ748376
Zapteryx exasperata – KM370325
Zearaja chilensis – KF648508
 4 C. R. L. AMARAL ET AL.
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Figure 2. ML mitogenomic phylogeny of the Elasmobranchii including all codon positions.
Additionally, all topologies were highly convergent inde-
pendent of using different codon positions (Figures 2–4),
with the exception of the phylogenetic position recovered for
the Heterodontiformes, the Mitsukurinidae and the species
Scoliodon macrorhynchos. The overall topological similarity
between the ML tree including all codon positions (Figure 2)
and the tree including only the first and second codon posi-
tions (Figure 3) was 91.8%, while the similarity between the
ML tree including all codon positions (Figure 2) and the tree
including only the third codon position (Figure 4) was 93.4%
(Supplementary File 1).
All phylogenetic analyses (Figures 2–5) suggest that
Elasmobranchii, Batoidea and the Selachii are monophyletic,
while Selachii and Batoidea clades are sister taxa. The
Batoidea suborder was found, including the group formed by
the Rajoidei and Torpedinoidei, the group formed by the
Myliobatoidei, the group formed by the basal Zapteryx and
the sequential group formed by the Pristoidei and the genus
Rhinobatos. The Torpedinoidei was recovered as a sister
group of the Rajoidei clade, albeit with weak support. The
Rajoidei was uniquely represented by the Rajidae members
and was recovered with a basal group formed by the species

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Figure 3. ML mitogenomic phylogeny of the Elasmobranchii based only on the first and second codon positions.
Atlantoraja castelnaui and Pavoraja nitida, followed by a
group formed by the species Amblyraja radiata and Leucoraja
erinacea as the sister taxa of the remaining rajids. Within the
Myliobatoidei, the Myliobatidae was recovered as paraphyletic
in terms of the basal position recovered for Aetobatus flagel-
lum. The Dasyatidae was recovered as monophyletic and
closely related to its sister family, the Potamotrygonidae. The
genus Dasyatis was recovered as paraphyletic with Dasyatis
microphthalmus in a variable position, but always related to
the group formed by the species Dasyatis akajei, D. bennetti,
D. zugei, Pteroplatytrygon violacea and Taeniura meyeni. The
genus Taeniura was recovered as paraphyletic with both
MITOCHONDRIAL DNA PART A 5
species recovered in a variable position. The Himanturidae,
herein represented by Himantura gerrardi and H. granulata,
were found as nested in all analyses, basal to the group
formed by Dasyatidae and Potamotrygonidae. Finally, the
Gymnuridae was found scattered over the tree, being a sister
taxon of Potamotrygonidae in the ML results or sister taxon
of Plesiobatidae in the Bayesian trees.
The Selachii was recovered as split between
Squalomorphii and Galeomorphii, in agreement with consen-
sus. The Squalomorphii was recovered with the basal
Hexanchiformes, being sequentially followed by the
Squatiniformes as sister taxa of the group formed by the
 6 C. R. L. AMARAL ET AL.
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Figure 4. ML mitogenomic phylogeny of the Elasmobranchii based only on the third codon position.
Squaliformes and Pristiophoriformes, while the Galeomorphii
was recovered with the Heterodontiformes in a basal pos-
ition, being followed by the Orectolobiformes and, sequen-
tially, by the group formed by the Carcharhiniformes and
Lamniformes. However, the Heterodontiformes was found as
a sequential sister taxa between the Orectolobiformes and
the Carcharhiniformes in the analysis using codon positions 1
and 2 (Figure 3).
Within the Orectolobiformes, the Orectolobidae was recov-
ered in a basal position related to the group formed by the
Rhincodontidae (Rhincodon) and the Hemiscylliidae. The
Carcharhiniformes presented the Scyliorhinidae in a basal
position in relation to the paraphyletic Carcharhinidae. The
Carcharhinidae was found as paraphyletic based on the
phylogenetic position observed for the species Scoliodon
macrorhynchos, which was recovered in a sequential basal
position following Scyliorhinus canicula. This result is also cor-
roborated by the phylogenetic position of Galeocerdo cuvier,
which was recovered deeply nested within the
Carcharhinidae joining a polytomy with the Triakidae and
Sphyrnidae. Scoliodon macrorhynchos was recovered within
the Carcharhinidae plus Sphyrnidae group when using only
codon positions 1 þ 2 (Figure 3).
Within the Lamniformes, two groups were consistently
recovered, one formed by basal Odontaspididae and the
group formed by Cetorhinidae and Laminidae, while the
 MITOCHONDRIAL DNA PART A 7
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Figure 5. Bayesian mitogenomic phylogeny of the Elasmobranchii, a majority rule tree coloured by orders. Posterior probabilities represented above the nodes.

Table 3. Test for substitution saturation for (A) all codon positions, (B) codon positions 1 þ 2 and (C) codon position 3.
NrOTU Iss Iss.cSym T DF P Iss.cAsym T DF P
A. 4 0.449 0.860 72.496 14644 0.0000 0.848 70.299 14644 0.0000
8 0.479 0.845 48.241 14644 0.0000 0.761 37.157 14644 0.0000
16 0.524 0.854 34.271 14644 0.0000 0.676 15.773 14644 0.0000
32 0.570 0.82 21.122 14644 0.0000 0.575 0.429 14644 0.6678
B. 4 0.444 0.858 59.382 9705 0.0000 0.846 57.715 9705 0.0000
8 0.469 0.845 40.242 9705 0.0000 0.762 31.351 9705 0.0000
16 0.514 0.850 28.392 9705 0.0000 0.676 13.626 9705 0.0000
32 0.561 0.818 17.671 9705 0.0000 0.572 0.755 9705 0.4501
C. 4 0.407 0.851 49.826 5667 0.0000 0.843 48.94 5667 0.0000
8 0.435 0.846 34.604 5667 0.0000 0.768 27.972 5667 0.0000
16 0.470 0.835 24.332 5667 0.0000 0.681 14.085 5667 0.0000
32 0.509 0.812 16.502 5667 0.0000 0.568 3.179 5667 0.0015
NrOTU: number of operational taxonomic units; Iss: index of substitution saturation; Iss.cSym: index of substitution saturation for symmetric topologies; Iss.cAsym:
index of substitution saturation for asymmetric topologies.
T, DF and P are the test parameters for mean comparisons.
 8 C. R. L. AMARAL ET AL.
second group of Lamniformes includes Alopiidae as sister
taxa of the group formed by the Megachasmidae and the
Pseudocarcharidae. Finally, the Mitsukurinidae (Mitsukurina
owstoni) was found to vary from basal sister taxa of the
group formed by the Alopiidae, Megachasmidae and
Pseudocarcharidae in the ML results to a basal phylogenetic
position in relation to all the analyzed Lamniformes in the
Bayesian results. It was also recovered as a sister taxon of
Odontaspididae when using only codon positions 1 þ 2
(Figure 3).
Discussion
Both ML (Figures 2–4) and Bayesian (Figure 5) phylogenies
support the hypothesis of Selachii and Batoidea clades as sis-
ter taxa, thus rejecting the Hypnosqualea hypothesis of
Compagno (1977), Carvalho (1996), Carvalho and Maisey
(1996), and Shirai (1992a, 1992b, 1996). This result corrobo-
rates recent molecular analyses (Douady et al. (2003),
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Winchell et al. (2004), V
elez-Zuazo and Agnarsson (2011), and
Naylor et al. (2012). The Hypnosqualea hypothesis (Shirai
1992a, 1992b) proposes that the Batoidea should be placed
within the Selachii based on morphological characters from
the skull, shoulder girdle and impaired fin musculature (Shirai
1992a, 1996). However, molecular analyses provide a different
scenario (Douady et al. 2003; Winchell et al. 2004), suggesting
that these morphological characters could be regarded as
homoplasious, or even as convergent adaptations to benthic
life, a point herein reinforced with our mitogenomic analyses.
Several phylogenies are available for Batoidea, and they
mostly agree on dividing the group into five major clades:
skates (Rajoidei), electric rays (Torpedinoidei), sawfishes
(Pristoidei), stingrays (Myliobatoidei) and the so-called guitar-
fishes, a paraphyletic group of batoids (McEachran and
Aschliman 2004; Compagno 2005; Aschliman et al. 2012;
Naylor et al. 2012). However, consensus has not been reached
regarding the relationships among these groups, with several
studies, including those supported by both morphological
and molecular data, producing contradictory results (e.g.
McEachran et al. 1996; Shirai 1996; Lovejoy et al. 1998; Dunn
et al. 2003; McEachran and Aschliman 2004; Human et al.
2006; Toffoli et al. 2008; V
elez-Zuazo and Agnarsson 2011).
Our results suggest the existence of a basal division
among the group formed by the Rajoidei and Torpedinoidei
and the group formed by the Myliobatoidei and the
Rhinobatoidei. Previous morphological studies found a close
relationship between skates and stingrays based on a highly
depressed disk supported by pectoral radials and the reduced
development of the tail, caudal and dorsal fins (Nishida 1990;
Shirai 1996). However, McEachran and Aschliman (2004)
believed that these characters were independently acquired
by these groups, a view also supported by the study of
Aschliman et al. (2012) and corroborated by our results.
Gait
an-Espitia et al. (2016) recently produced the most
complete phylogeny of the Torpediniformes, based on six
mitogenomes and including a species from the genera
Torpedo, Typhlonarke and four species of Narcine. The authors
recovered a sister taxa relationship between the
Torpediniformes and the thornback rays of the family
Platyrhinidae, with the Rajiformes being recovered in a low
support node as the basal sister taxa of Pristiformes and
Myliobatiformes. The observed divergence between our
results and those presented by Gait
an-Espitia et al. (2016)
seems to be related to the distinct species coverage of both
studies and the low support of some internal nodes, at least
for the Torpediniformes relationships. Although our database
does not encompass species from the Platyrhinidae,
we should also observe that the Torpediniformes sister
taxa relationships observed in our results were recovered
in a low support node, as well as the division observed
between Torpediniformes þ Rajiformes and Pristiformes þ
Myliobatiformes.
The Pristoidei was recovered deeply nested within the
guitarfish group formed by the basal Zapteryx and
Rhinobatos. Based on their Bayesian topology, Aschliman
et al. (2012) showed the paraphyly of the guitarfishes, recov-
ering two distinct groups. The first group, including Zapteryx
and Trygonorrhina, was sequentially followed by the second
group, including the Pristoidei, which was deeply nested
between the basal Rhinobatos and the group formed by the
genera Rhyncobatus and Rhina. Our results do not corrobor-
ate this view since we found the guitarfishes in a monophy-
letic group, with the Pristoidei deeply nested within it, thus
rejecting guitarfishes (Rhinobatoidei) without the inclusion of
the Pristoidei as a derived monophyletic group. Naylor et al.
(2012) recovered the same topology with the basal Zapteryx
followed by a group which includes the Zanobatidae,
Rhinobatidae, Pristidae and Rhynchobatidae, called
Rhinopristiformes, a view herein corroborated based on our
mitogenomic results.
As discussed by Aschliman et al. (2012), sawfishes
(Pristoidei) and saw sharks (Pristiophoriformes) seem to have
independently achieved a highly elongated rostrum bearing
lateral spines. They also pointed out that these structures
have influenced morphological studies which suggested that
saw sharks and angel sharks (Squatiniformes) are successive
sister groups to Batoidea in that sawfishes are the sister taxa
to all remaining extant batoids (e.g. Shirai 1996; Kriwet and
Benton 2004), therefore implying a gradation between squa-
loid-like sharks and batoids with progressive dorsoventral flat-
tening and the development of the saw. However, as
observed by the authors, if the rostral saws are treated as
homologous, the derived position of sawfishes in the molecu-
lar phylogeny implies at least seven independent losses of
this structure, a view corroborated by our results regarding
the phylogenetic position recovered for the Pristoidei in our
mitogenomic Bayesian approach. Saw sharks are strongly sup-
ported as squaloid-like sharks by several studies, such as
those of Shirai (1992b, 1996), Carvalho (1996) and Maisey
et al. (2004), and also the lack of the eight well-accepted
batoid synapomorphies (McEachran et al. 1996). However,
independent of the similarity of these structures, Kriwet and
Benton (2004) additionally pointed out that they present key
differences in canal architecture and in the articulation of
spines, thus dispelling their homology.
Based on morphology and some molecular studies,
Myliobatidae is often recovered as a monophyletic group,
although herein it is suggested as paraphyletic due to the

basal position recovered for Aetobatus flagellum. This conflict
is most likely due to misidentification, since Aetobatus is a
basal and well-diagnosed genus, constantly recovered within
the Myliobatidae from both morphology and molecular analy-
ses (Naylor et al. 2012).
The Dasyatidae was recovered as monophyletic and sister
family of Potamotrygonidae although recent results such as
Naylor et al. (2012) and Lim et al. (2015) point to a paraphy-
letic condition for the family. We should also observe that
our analyses did not include representatives from the
Urotrygonidae, the accepted sister family of the
Potamotrygonidae, and the relationships herein observed
between the Potamotrygonidae and Dasyatidae seems to be
obviously influenced by the limited taxonomic sampling. In
addition, the Himanturidae was also suggested as a valid
family, basal to the group formed by the Dasyatidae and the
Potamotrygonidae, therefore corroborating the recent pro-
posal of Lim et al. (2015). Finally, we should observe that the
support for the deep internodes of the suborder
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Myliobatoidei is far from being robust; therefore, the present
topologies could be greatly influenced by the low number of
species included in the phylogenies. In this sense, an
extended sampling of the suborder Myliobatoidei would
greatly enhance the phylogenetic resolution of this group.
Regarding the phylogenetic relationships within the
Selachii clade, our results do agree with recent studies
(Compagno 1977; Shirai 1992a, 1992b, 1996; Carvalho 1996;
Carvalho and Maisey 1996; Douady et al. 2003; Winchell et al.
2004; Naylor et al. 2005; Human et al. 2006; Heinicke et al.
2009) which recovered the eight well-known shark orders div-
ided into two distinct clades, the Squalomorphii and the
Galeomorphii, a result in agreement with morphological char-
acters (Compagno 1977). Regarding the Squalomorphii, our
results are similar to those observed by V
elez-Zuazo and
Agnarsson (2011) with the Hexanchiformes sequentially fol-
lowed by the Squatiniformes as sister taxa of the group
formed by the Squaliformes and Pristiophoriformes. These
results do not corroborate the close relationships between the
Squatiniformes and the Squaliformes, as already suggested by
Mallatt and Winchell (2007) and Heinicke et al. (2009), nor do
they corroborate the close relationships between the
Squatiniformes and Pristiophoriformes, as suggested by
Douady et al. (2003), Naylor et al. (2005) and Human et al.
(2006). Our results do also agree with Tanaka et al. (2013)
about the inclusion of the Hexanchiformes within the
Squalomorphii. Tanaka et al. (2013) discussed the evolution of
the Hexanchiformes deep sea sharks using mitogenomes. The
authors analyzed the phylogenetic relationships between the
Hexanchiformes and some species from the Squaliformes,
Orectolobiformes, Lamniformes, Carcharhiniformes and some
batoids. However, the observed sister taxa relationships
between Squaliformes and Hexanchiformes presented in their
ML phylogeny are likely to be related to the limited
Squalomorphii species coverage of the study.
The Galeomorphii was recovered exhibiting the
Heterodontiformes in a basal position, followed by the
Orectolobiformes and sequentially by the group formed by
the Carcharhiniformes and Lamniformes. The phylogenetic
position of the Heterodontiformes, as sister taxon of the
MITOCHONDRIAL DNA PART A 9
other galeomorphs, has already been indicated by several
authors (cf. Holmgren 1940, 1941; Compagno 1973; Maisey
1983; Carvalho 1996; Naylor et al. 2005; Heinicke et al.
2009). In this sense, the phylogenetic position recovered
for the Heterodontiformes does not corroborate a close
relationship with the squalimorphs, as suggested by the
studies of Mallatt and Winchell (2007) and Human et al.
(2006).
For the relationships between the Carcharhiniformes and
the Lamniformes, our results are broadly similar to those
observed in recent studies (V
elez-Zuazo and Agnarsson 2011),
although they reinforce the relationships between both clades,
as already suggested by Naylor et al. (2005) and Heinicke et al.
(2009). Our analyses consistently recovered the relationships
between Carcharhiniformes and Lamniformes. Nevertheless,
on the basis of molecular data advocated for a group formed
by Lamniformes and Orectolobiformes, Winchell et al. (2004)
related this group to the Isurida group of White (1936). The
Isurida group was proposed by White (1936) based on mor-
phological similarities associated with the vertebral column,
the intestinal valves and the absence of nictitating membrane,
a view not corroborated by the study of Compagno (1973),
which considered these as homoplasious characters. Of the six
morphological characters supporting the clade which joins
together the Carcharhiniformes and the Lamniformes,
Winchell et al. (2004) pointed out that one unique character,
tripodal rostrum, is not homoplasious, arguing that the
remaining five characters could be treated as convergent
adaptations for pelagic carnivory, an opinion not corroborated
here based on the phylogenetic sister taxon position recov-
ered to both Carcharhiniformes and Lamniformes.
Our results for the galeomorphs recovered the
Heterodontiformes, herein represented uniquely by the mono-
phyletic genus Heterodontus, in a basal position followed by
the Orectolobiformes, thus contradicting previous hypotheses
which suggest Heterodontus and Chlamydoselachus as basal
neoselachians, or even non-neoselachian sharks (Regan 1906;
Goodrich 1909; Allis 1923; Gudger and Smith 1933; White
1937). In addition, sequence saturation could have influenced
the basal position observed for the Heterodontiformes since
this phylogenetic position was not corroborated when third
codon information was discarded, thereby placing the
Heterodontiformes clade between the Orectolobiformes and
the Carcharhiniformes. The order Orectolobiformes includes
the basal Orectolobidae and the group formed by the
Hemiscylliidae and Rhincodontidae, sequentially followed by
the group formed by the sister taxa Carcharhiniformes and
Lamniformes.
Within the Carcharhiniformes, the basal Scyliorhinidae was
followed by a paraphyletic group which includes a polytomy
with the Triakidae, the carcharhinids Scoliodon macrorhynchos
and Galeocerdo cuvier, and the group formed by the
Sphyrnidae and the remaining Carcharhinidae. However,
when discarding the third codon position, the carcharhinid
Scoliodon macrorhynchos was recovered together with
Galeocerdo cuvier within the group formed by the
Carcharhinidae þ Sphyrnidae. Ve
lez-Zuazo and Agnarsson
(2011) recovered Scoliodon as the sister genus of
Rhizoprionodon while Naylor et al. (2012) recovered Scoliodon
 10 C. R. L. AMARAL ET AL.
deeply nested within the Carcharhinidae, as the sister genus
of Loxodon in a group with Rhizoprionodon as the basal
genus. In this sense, as our results do not include the genera
Loxodon and Rhizoprionodon, we believe that the phylogen-
etic position herein observed for Scoliodon should be influ-
enced by the taxonomic sampling.
These results reinforce the previous observations based on
morphology in which scyliorhinids represent the basal lineage
followed by triakids and the clade formed by the derived
carcharhinids and sphyrnids (White 1937; Compagno 1973,
1988). Recent phylogenetic analyses diverge about the phylo-
genetic status of Sphyrnidae. Using mitochondrial and
nuclear genes, V
elez-Zuazo and Agnarsson (2011) observed
the same topology, with the Sphyrnidae embedded within
the Carcharhinidae, rather than a sister taxon relationship.
However, based on partial mitochondrial sequences, Naylor
et al. (2012) obtained the same results for the basal
Galeocerdo and the group formed by Carcharhinidae and
Sphyrnidae. However, they also pointed out that Galeocerdo
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does not belong to the Caracharhinidae, thus reinforcing a
sister taxon relationship for both clades, as supported by the
study of Lopez et al. (2006) and unpublished nuclear data.
Regarding the exposed above and the low support values we
obtained in this particular region of the topology, we believe
further investigation is still required to enhance the resolution
on these particular nodes. However, we should observe that
disregarding both Galeocerdo and Scoliodon, the resultant
topology points to the well-accepted sister family relation-
ships between both Carcharhinidae and Sphyrnidae.
In regard to the Lamniformes, the Mitsukurinidae was
recovered in a variable basal position, depending on the type
of analysis performed. Previous morphological and molecular
studies place Mitsukurina basal to all other Lamniformes
(Shirai 1996; Naylor et al. 1997; Compagno 1999; Martin 1999;
Martin and Burg 2002; Martin et al. 2002), a result reinforced
by our Bayesian results, but conflicting with the basal pos-
ition observed in relation to the group formed by the
Alopiidae, Megachasmidae and Pseudocarcharidae. The group
formed by the monophyletic Alopiidae was recovered as a
sister taxon of the group formed by the Megachasmidae and
the Pseudocarcharidae. For the Laminidae, our results do
agree with those presented by Compagno (1990) and V
elez-
Zuazo and Agnarsson (2011) regarding the monophyly of the
Lamnidae, although they do not corroborate the phylogen-
etic reconstruction, as established by these authors, since we
recovered the white shark Carcharodon carcharias as basal to
the group formed by the genera Lamna and Isurus.
Conclusions
This manuscript presents a mitogenomic perspective on the
evolutionary relationships within the Elasmobranchii clade.
Although recent phylogenies commonly include both mito-
chondrial and nuclear genes, a mitogenome analysis includ-
ing several representatives of the entire Elasmobranchii clade
was still not tested until now.
By reinforcing the sister taxon relationships between the
Batoidea and the Selachii clades, our results refute the
Hypnosqualea hypothesis. In addition, within the Batoidea,
the Pristoidei was recovered deeply nested in a group with
the genus Rhinobatos and the basal Zapteryx, therefore reject-
ing the Rhinobatoidei without the inclusion of Pristoidei as a
derived batoid clade. This result corroborates the proposal of
the Rhinopristiformes of Naylor et al. (2012).
Within the Selachii, our results suggest a main division
of sharks, the Galeomorphii and the Squalomorphii, with
the Squatiniformes being recovered as basal sister taxa
of the group formed by the Squaliformes and the
Pristiophoriformes. The Galeomorphii was recovered with the
basal Heterodontiformes followed by the Carcharhiniformes,
recovered as the sister taxa of the Lamniformes.
Finally, although based on several species representing
the major groups of sharks and rays, it should be stressed
that the taxonomic sampling plays a crucial role here. Some
uncertainties observed in our results could be related to the
absence of proper taxonomic sampling in the particular
region of the topology. To overcome this issue, we believe in
a short period, with the advance of the massive sequencing
techniques, the availability of mitogenomes will enhance the
phylogenetic resolution along the entire phylogeny.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study was supported by Coordenaç~ao de Aperfeiçoamento de
Pessoal de N
ıvel Superior (CAPES), Fundaç~ao Carlos Chagas Filho de
Amparo a Pesquisa do Estado do Rio de Janeiro, FEDER-Fundo Europeu
de Desenvolvimento Regional funds through the COMPETE 2020-
Operacional Programme for Competitiveness and Internationalisation
(POCI), Portugal 2020, and by Portuguese funds through FCT-Fundaç~ao
para a Ci^encia e a Tecnologia/Minist
erio da Ci^encia, Tecnologia e
Inovaç~ao in the framework of the project “Institute for Research and
Innovation in Health Sciences” (POCI-01-0145-FEDER-007274). FP was sup-
ported by Investigator FCT (IF/01356/2012) and MARINFO (NORTE-01-
0145-FEDER-000031).
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