RESEARCH ARTICLE
Improving the Conservation of Mediterranean
Chondrichthyans: The ELASMOMED DNA
Barcode Reference Library
Alessia Cariani
1☯
, Silvia Messinetti
1☯¤
*, Alice Ferrari
1☯
, Marco Arculeo
2
, Juan J. Bonello
3
,
Leanne Bonnici
3
, Rita Cannas
4
, Pierluigi Carbonara
5
, Alessandro Cau
4
, Charis Charilaou
6
,
Najib El Ouamari
7
, Fabio Fiorentino
8
, Maria Cristina Follesa
4
, Germana Garofalo
8
,
Daniel Golani
9
, Ilaria Guarniero
10
, Robert Hanner
11
, Farid Hemida
12
, Omar Kada
7
,
Sabrina Lo Brutto
2
, Cecilia Mancusi
13
, Gabriel Morey
14
, Patrick J. Schembri
3
,
Fabrizio Serena
13
, Letizia Sion
15
, Marco Stagioni
1
, Angelo Tursi
15
, Nedo Vrgoc
16
,
Dirk Steinke
11
, Fausto Tinti
1
1 Department of Biological, Geological and Environmental Sciences, University of Bologna, Ravenna, Italy,
2 Department STEBICEF, University of Palermo, Palermo, Italy, 3 University of Malta, Msida, Malta,
4 Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy, 5 COISPA
Technology and Research, Bari, Italy, 6 Department of Fisheries and Marine Research, Ministry of
Agriculture, Rural Development and Environment, Nicosia, Republic of Cyprus, 7 Centre Re
´
gional de Institut
National Recherche Halieutique, Nador, Morocco, 8 Institute for Coastal Marine Environment (IAMC)
National Research Council (CNR), Mazara del Vallo, Italy, 9 Department of Evolution, Systematics and
Ecology, The Hebrew University of Jerusalem, Jerusalem, Israel, 10 Department DIMEVET, University of
Bologna, Ozzano dell’Emilia, Italy, 11 Centre for Biodiversity Genomics, Biodiversity Institute of Ontario,
University of Guelph, Guelph, Ontario, Canada, 12 Ecole Nationale Superieure des Sciences de la Mer et
d’Amenagement du Littoral, Campus Universitaire de Dely Ibrahim, Alger, Algeria, 13 Regional Agency for
Environmental Protection-Toscana (ARPAT), Livorno, Italy, 14 Asociacio
´
n Ondine, Palma de Mallorca,
Spain, 15 Department of Biology, University of Bari Aldo Moro, Bari, Italy, 16 Institute of Oceanography and
Fisheries, Split, Croatia
☯ These authors contributed equally to this work.
¤ Current address: Department of BioSciences, University of Milano, Milano, Italy
*
silvia.messinetti@unimi.it
Abstract
Cartilaginous fish are particularly vulnerable to anthropogenic stressors and environmental
change because of their K-selected reproductive strategy. Accurate data from scientific sur-
veys and landings are essential to assess conservation status and to develop robust protec-
tion and management plans. Currently available data are often incomplete or incorrect as a
result of inaccurate species identifications, due to a high level of morphological stasis, espe-
cially among closely related taxa. Moreover, several diagnostic characters clearly visible in
adult specimens are less evident in juveniles. Here we present results generated by the
ELASMOMED Consortium, a regional network aiming to sample and DNA-barcode the Medi-
terranean Chondrichthyans with the ultimate goal to provide a comprehensive DNA barcode
reference library. This library will support and improve the molecular taxonomy of this group
and the effectiveness of management and conservation measures. We successfully bar-
coded 882 individuals belonging to 42 species (17 sharks, 24 batoids and one chimaera),
including four endemic and several threatened ones. Morphological misidentifications were
found across most orders, further confirming the need for a comprehensive DNA barcoding
library as a valuable tool for the reliable identification of specimens in support of taxonomist
PLOS ONE | DOI:10.1371/journal.pone.0170244 January 20, 2017 1 / 21
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OPEN ACCESS
Citation: Cariani A, Messinetti S, Ferrari A, Arculeo
M, Bonello JJ, Bonnici L, et al. (2017) Improving
the Conservation of Mediterranean
Chondrichthyans: The ELASMOMED DNA Barcode
Reference Library. PLoS ONE 12(1): e0170244.
doi:10.1371/journal.pone.0170244
Editor: A. Peter Klimley, University of California
Davis, UNITED STATES
Received: August 25, 2016
Accepted: December 30, 2016
Published: January 20, 2017
Copyright: © 2017 Cariani et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: Sampling and
biological data as well as digital images of dorsal/
ventral sides (when recorded) are available in the
“ELASMOMED Consortium” project (Project Code:
ELAMO) accessible on the Barcode of Life Data
system (BOLD,
http://www.barcodinglife.org).
GenBank accession numbers for all barcoded
specimens are reported in
S3 Table.
Funding: This research work was supported by the
University of Bologna, the Canadian Centre for DNA
Barcoding, and the Biodiversity Institute of Ontario
who are reviewing current identification keys. Despite low intraspecific variation among their
barcode sequences and reduced samples size, five species showed preliminary evidence of
phylogeographic structure. Overall, the ELASMOMED initiative further emphasizes the key
role accurate DNA barcoding libraries play in establishing reliable diagnostic species specific
features in otherwise taxonomically problematic groups for biodiversity management and
conservation actions.
Introduction
To assess and conserve biodiversity, it is critical to correctly identify species that occur in a
given ecosystem in order to evaluate species richness and abundance. In recent years, methods
of identification based on morphology were gradually integrated with methods based on DNA
sequences, such as DNA barcoding thereby forming the so called molecular taxonomy. DNA
barcoding seeks to advance both specimens identification and species discovery through the
analysis of patterns of sequences divergence of a universal, standardized gene region. Many
studies have shown the effectiveness of the mitochondrial cytochrome c oxidase subunit I
(COI) gene as a universal barcode sequence for species identification in animal lineages [
1,2].
Molecular taxonomy overcomes some problems posed by traditional morphological identi-
fication such as homoplasy [
3] and phenotypic plasticity [4] of characters used for species
identification. Secondly, because often the adopted morphological keys are effective only for a
particular life stage, many individuals, especially in their juvenile phases, cannot be assigned to
species [5]. Finally, traditional taxonomy doesn’t allow the identification of cryptic species [6].
DNA barcoding has been fundamental in case studies related to immature specimens’ identifi-
cation (e.g. fish larvae, [
5,7]; amphibians and reptiles, [8,9]). In many cases it has been success-
fully employed to resolve species boundaries between morphologically conserved taxa (e.g.
tribe Bombini, [
10]).
In the marine realm, the conservation of similar morphological traits appears quite common
among sibling species [11–14] and it is often combined with the lack of visual communication
in many taxa, in favour of chemical [15] or electrical signals [16–18], both intrinsic conditions
to the definition of cryptic species. Among marine organisms, Chondrichthyans seem to have
experienced frequent cryptic speciation events across different taxa: large lantern sharks [
19],
skates [
20–22], blacktip sharks [23], hammerhead sharks [24] and guitarfish [25] might have
undergone isolating mechanisms which precluded mating between co-occurring species.
Despite controversies and criticisms [
26,27], cryptic species discovery and, in general, species
cataloguing are fundamental, as species represent the basic unit for the management, conserva-
tion, and legal protection of biodiversity and for the distribution of limited resources [
28–33].
To maximize the potential of molecular taxonomy is necessary to have solid and compre-
hensive DNA barcodes reference libraries. The Fish Barcode of Life campaign (Fish-BOL,
http://www.fishbol.org) is an initiative started in 2005 with the goal to barcode all fish species
[
34–36]. As of August 2016, 665 out of 1228 chondrichthyan species have been barcoded.
Fish-BOL’s efforts comprise of numerous projects covering low-level taxonomic groups and
several projects that have targeted specific regional chondrichthyan faunas [
37–43]. In addi-
tion, recent studies demonstrated the effectiveness of DNA barcodes in describing phylogeo-
graphic patterns in this class [
22,44,45].
One of those targeted Fish-BOL projects is the ELASMOMED Consortium, a regional net-
work active since 2009 involving fish biologists, fishery scientists, and molecular zoologists
from 15 research institutions, most of which also participate in the Mediterranean
Barcoding of Mediterranean Chondrichthyans
PLOS ONE | DOI:10.1371/journal.pone.0170244 January 20, 2017 2 / 21
both supported by the government of Canada
through Genome Canada and the Ontario
Genomics Institute. Rita Cannas, Maria Cristina
Follesa and Alessandro Cau were funded by the
Autonomous Region of Sardinia (RAS) grant n˚ L.
R.7 CRP-25321. Dirk Steinke was funded by the
Alfred P Sloan Foundation. These latter funders had
no role in study design, data collection and
analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
International Trawl Survey (MEDITS) scientific program[46]. The aims of this network are
the sampling and barcoding Chondrichthyans of the Mediterranean Sea.
The Mediterranean Sea is appraised as a global marine biodiversity hotspot and it is home
to 89 chondrichthyan species (sharks, skates, rays and chimaeras), which corresponds to about
7% of the global species diversity of the group [
47–51]. In detail, the Mediterranean Chon-
drichthyans include one chimaera, 49 sharks belonging to 17 families and 27 genera, and 39
batoids consisting of nine families with 16 genera. Five batoids are considered endemic: Leu-
coraja melitensis, Raja asterias, Raja radula, Raja polystigma, and Mobula mobular, although
the endemic status of the latter is uncertain as its separation from the widespread congener
Mobula japonica has been recently questioned [
52]. In parallel, a total of 86 Mediterranean
Chondrichthyan species (
S1 Table) was reviewed for the European Red List of Marine Fishes
[
53]. This list includes species threatened by extinction in European waters. Assessed taxa were
categorized as Critically Endangered (15), Endangered (13), Vulnerable (11), Near Threatened
(15), Least Concern (12) and Data Deficient (20).
Several exotic elasmobranchs have recently been recorded in the Mediterranean (
S1 Table),
however their particular status (vagrant, alien, or established) is still under debate [
48,54–57].
The presence of six species (Carcharhinus melanopterus, Dipturus batis complex, Torpedo alex-
andrinsis, Leucoraja fullonica, Raja africana), however, has not been confirmed [
50,58–61]. In
contrast, the presence of Dipturus nidarosiensis in the Mediterranean, a species formerly
reported only from the North-Eastern Atlantic, has been ascertained by recent studies [
58,62].
Chondrichthyans’ reproductive strategy makes them particularly vulnerable to anthropo-
genic stressors, such as the use of different fishing gears and the direct and indirect effects of
environmental changes and habitat fragmentation [
63]. For example, neritic species such as
Squatina spp. and Scyliorhinus stellaris are highly depleted as a consequence of the use of unse-
lective fishing gear [
64]. Available fisheries data for Chondrichthyes are incomplete and incor-
rect, because they are often recorded at higher taxonomic levels than species, with frequent
misidentifications of individuals [51,65,66]. In 2012, the General Fisheries Commission for the
Mediterranean (GFCM) issued Recommendation GFCM/36/2012/3 on fisheries management
measures for conservation of sharks and rays in the GFCM area stating that “cartilaginous
fish are kept on board, trans-shipped, landed and marketed at first sale in a way that species
are recognizable and identifiable and catches, incidental takings and, whenever appropriate,
releases by species can be monitored and recorded” [
67]. However, morphological identifica-
tion of cartilaginous fish remains difficult because of low levels of differentiation among spe-
cies across multiple taxa and several diagnostic taxonomic characters are clearly exhibited by
adult specimens but are less pronounced in juveniles [
22,51,65,66]. This can lead to erroneous
species attribution even among skate species that are not closely related, e.g. Leucoraja fullonica
and L. circularis. Such taxonomic uncertainties often occur within a larger group constituted
of Raja polystigma, R. montagui, R. asterias and R. brachyura [
22,66]. For sharks, similar diffi-
culties were reported for the congeneric species Squalus blainville and Squalus megalops
[
68,69] and for Mustelus mustelus and Mustelus punctulatus [70]. Finally, cryptic species (sensu
Bickford et al. [
6]) have frequently been reported in elasmobranchs as shown by molecular
studies of the common skate Dipturus batis, once one of the most abundant skate resources in
the North-Eastern Atlantic trawl fishery and today heavily depleted in most of its range [
63].
D. batis actually comprises two cryptic species, Dipturus cf intermedia and Dipturus cf flossada
[
20,71,72]. A genetic analysis of Eastern Atlantic and Mediterranean species of the genus Raja
revealed several recently diverged peripatric sibling species, such as Raja clavata and R. strae-
leni [73], R. polystigma and R. montagui [22,74], as well as the R. miraletus complex [75,76].
Here we report the establishment of DNA reference barcodes for 42 chondrichthyan
species, mostly collected as part of the MEDITS program, as an integrative tool to improve the
Barcoding of Mediterranean Chondrichthyans
PLOS ONE | DOI:10.1371/journal.pone.0170244 January 20, 2017 3 / 21
effectiveness of the above mentioned measures for conservation. This new library will: i) offer a
valuable tool for reliable identification of specimens and clarify the taxonomic status of important
cartilaginous fishes; ii) support taxonomists who are reviewing the current identification keys,
and iii) provide a robust scientific baseline for management and conservation actions, especially
in relation to endemic and endangered species. Finally, the comparison and integration of the
ELASMOMED dataset with other public barcode datasets will also allow the preliminary identifi-
cation of geographical population structure and help to determine candidate conservation units.
In general, the assembly of a comprehensive DNA barcode reference library for biological
species would mean to compile a biodiversity inventory on different scales and dimensions,
making molecular systematics a fundamental tool for the implementation of biodiversity mon-
itoring programmes worldwide. Several of such programmes already exploit the potential of
accurate DNA reference libraries by integrating environmental DNA analyses in their stan-
dard monitoring programmes [
77–79].
Materials and Methods
Sampling
Specimens used in this study were collected from Mediterranean individuals caught during sci-
entific research programs. No specific approval of this vertebrate work is required since the indi-
viduals sampled in this study were obtained from scientific and commercial fishing activities. A
total of 998 individuals were collected from several locations within six Mediterranean FAO
fishing divisions (
http://www.fao.org/fishery/area/Area37/en). Starting in 2009, dedicated sam-
pling was performed mainly in the framework of the MEDITS scientific surveys (
http://www.
sibm.it/SITO%20MEDITS/principaleprogramme.htm) or by contracted commercial fishermen
on designated cruises (
S2 Table). Additional samples were provided by each partner of the
ELASMOMED Consortium, from each Institute’s collections. Specimen and collection data, as
well as voucher digital images (when recorded) were uploaded to the “ELASMOMED Consor-
tium” project (Project Code: ELAMO) accessible through the Barcode of Life Data system
(BOLD,
http://www.barcodinglife.org, [80]). Individual fin clips or skeletal muscle tissue sam-
ples were collected and preserved in 96% ethanol and kept at -20˚C until laboratory analyses.
DNA extraction, amplification and sequencing
Laboratory work was jointly carried out by the Centre for Biodiversity Genomics (CBG) and
at the University of Bologna (UNIBO). At CBG 650bp of the mitochondrial COI region were
obtained by following standardized high-throughput protocols for DNA barcode amplification
and sequencing [
81]. At UNIBO the same COI fragment was amplified using the primer set
FishF2 and FishR2 following the protocol described in Ward et al. [
82]. Amplification prod-
ucts were checked on a 1.5% agarose gel. A commercial sequence service provider (Macrogen
Europe, Amsterdam, Netherlands) performed sequencing employing the same primers used
for the amplification. Trace files and sequence data were uploaded to BOLD and subsequently
submitted to GenBank (Accession numbers are provided in
S3 Table).
Specimens’ identification and spatial scale of barcode variation
Specimens were identified on board or in the lab using morphological taxonomic characters
according to guidelines provided in [
83]. A p-distance metric with pairwise deletion was used
for sequence comparisons [
84]. Genetic distances and Neighbour-joining (NJ) tree clustering
[
85] were obtained using MEGA version 6 [86]. Confidence in estimated relationships of NJ
tree topologies was evaluated by a bootstrap analysis with 1,000 replicates [87].
Barcoding of Mediterranean Chondrichthyans
PLOS ONE | DOI:10.1371/journal.pone.0170244 January 20, 2017 4 / 21
The mean and maximum intraspecific genetic distances and the mean distance to the Near-
est Neighbour (NN) were computed using the ‘Barcoding Gap Analysis’ tool on BOLD [
80].
Maximum intraspecific distance was plotted against the mean distance to the NN for each spe-
cies to infer the presence of a “barcode gap”, which is defined as a distinct gap between intra-
specific and interspecific variability [
84].
The Barcode Index Number (BIN) System clusters sequences using a Refined Single Link-
age algorithm to produce operational taxonomic units that closely correspond to species. BINs
were automatically assigned by BOLD and assessed using the ‘BIN Discordance Report’ analy-
sis tool [88]. This tool labels a BIN as “concordant” when it comprises sequences attributed to
the same species, and “discordant” when it comprises sequences of different species.
An arbitrary measure of taxonomic reliability was attributed to each barcoded taxon
according to the criteria proposed by Costa et al. [
89]. Representative barcode sequences for
each species were queried using the BOLD Identification Engine with the Species Level option.
Grades ranging from A (full concordance) to E (full discordance) were attributed according to
the following criteria:
• Grade A- External concordance: unambiguous species match with specimens from other
BOLD projects or published sequences. Monophyletic species with a maximum of 2%
(patristic) sequence divergence.
• Grade B- Internal concordance: species congruent within our dataset, where at least 3 speci-
mens of the same species are available, with a maximum of 2% (patristic) sequence diver-
gence. No matching sequences found through the BOLD-IDS.
• Grade C- Sub-optimal concordance (possible within species genetic structure): at least 3
specimens of the same species are available within the library and form a monophyletic clus-
ter; however intraspecific distance is greater than 2%; and/or the BOLD-IDS indicates
monophyletic nearest neighbour of the same species, with more than 2% patristic distance.
• Grade D- Insufficient Data: low number of specimens analysed (1 or 2 individuals) and no
matching sequence available in BOLD.
• Grade E- Discordant species assignments: sequences for a given species in our dataset did
not match with the same species in BOLD. The specimen may match with a different species
or may display paraphyly or polyphyly.
Because of the several Mediterranean geographical areas covered by ELASMOMED, we
tested for the presence of phylogeographic signal at regional level for species with barcode data
from multiple FAO divisions. Species-specific haplotype networks were created using Haplo-
viewer (
http://www.cibiv.at/~greg/haploviewer). Parsimony trees required for Haploviewer
were reconstructed with the dnapars of the PHYLIP package version 3.6 [
90,91].
Results
DNA barcodes could be recovered for 884 of the 998 individuals. Stop codons were recorded
only for two specimens: ELAMO028-15 (S. blainville) and ELAME1143-11 (Torpedo marmor-
ata), which were excluded from further analyses. The newly generated Mediterranean barcode
library ELASMOMED includes 42 species: 17 sharks, 24 skates/rays and one chimera, belong-
ing to eight orders and 18 families (
S2 Table). Overall nucleotide frequencies were 25.16% ade-
nine (A), 26.12% cytosine (C), 16.78% guanine (G) and 31.94% thymine (T), with an average
GC content of 42.90%.
Barcoding of Mediterranean Chondrichthyans
PLOS ONE | DOI:10.1371/journal.pone.0170244 January 20, 2017 5 / 21