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Nowadays, because of the constant increase in the capture and trade of sharks all over the world and reports of several species already showing important signs of over-exploitation, the establishment of registration mechanisms, evaluation and fishery control become urgent. Morphological identification of captured sharks is very difficult, and sometimes impossible, due to the removal of the animals’ parts. At this point, techniques of genetic identification through the molecular markers are considered essential tools for fishery monitoring. In this study, we present a method of multiplex-PCR, based on the gene Cytochrome Oxidase I, with species-specific primers developed for simultaneous identification of nine species of lamniform and carcharhiniform sharks, some of which are worldwide distributed. KeywordsSharks identification-Trade monitoring-Molecular markers-Cytochrome oxidase I-Multiplex-PCR
Genetic identification of lamniform and carcharhiniform sharks
using multiplex-PCR
F. F. Mendonc¸a
D. T. Hashimoto
B. De-Franco
F. Porto-Foresti
O. B. F. Gadig
C. Oliveira
F. Foresti
Received: 13 October 2009 / Accepted: 18 October 2009 / Published online: 2 November 2009
Ó Springer Science+Business Media B.V. 2009
Abstract Nowadays, because of the constant increase in
the capture and trade of sharks all over the world and
reports of several species already showing important signs
of over-exploitation, the establishment of registration
mechanisms, evaluation and fishery control become urgent.
Morphological identification of captured sharks is very
difficult, and sometimes impossible, due to the removal of
the animals’ parts. At this point, techniques of genetic
identification through the molecular markers are consid-
ered essential tools for fishery monitoring. In this study, we
present a method of multiplex-PCR, based on the gene
Cytochrome Oxidase I, with species-specific primers
developed for simultaneous identification of nine species of
lamniform and carcharhiniform sharks, some of which are
worldwide distributed.
Keywords Sharks identification Trade monitoring
Molecular markers Cytochrome oxidase I
Historically, the consumption of shark meat was kept
almost restricted to the riverine populations until some
decades ago. However, with the reduction of the traditional
fishery stocks, this resource became more diffuse and
appreciated. The great demand for shark fins by the Asian
kitchen is the most important reason for the depletion of
these animal populations on a large scale. Over the last two
decades, there has been an increasing concern about the
vulnerability of the sharks to fishery exploitation (Camhi
et al. 1998; Castro et al. 1999), as well as about the doc-
umentation of finning (removal of sharks fins and the dis-
card of the carcass at sea). These facts have contributed to
the development of several studies in order to provide a
more adequate management of the shark fishery in many
parts of the world, where it has reached unbearable levels,
causing several species to vanish (Camhi 1999).
Considering that each species responds independently to
environmental pressures, it is essential to know in a more
refined way, the composition of shark captures per species
and their relative abundance, in order to establish recovery
plans for these populations and the consequent ordering of
these activities (Lessa et al. 1999). However, one of the
main obstacles in obtaining data about the capture and
trade of sharks is the difficulty in identifying many species
by using the traditional taxonomic tools (Stevens 2004),
since morphological and meristic criteria are lost during the
carcass processing (from which the head and fins are
removed) or due to the low practicability in field studies
that demand fast morphological identification of numerous
samples. Moreover, economically important sharks in some
particular species of the families Lamnidae and Carcha-
rhinidae can exhibit a conservative external morphology,
without clear differences, resulting in great difficulty in
F. F. Mendonc¸a (&) B. De-Franco C. Oliveira F. Foresti
rio de Biologia e Gene
tica de Peixes, Departamento
de Morfologia, Instituto de Biocie
ncias de Botucatu,
Universidade Estadual Paulista—UNESP, Distrito de Rubia
nior, s/n, Botucatu, SP CEP 18618-000, Brazil
D. T. Hashimoto F. Porto-Foresti
rio de Gene
tica de Peixes, Departamento de Biologia,
Faculdade de Cie
ncias, Universidade Estadual
Paulista—UNESP, Bauru, SP CEP 17033-360, Brazil
O. B. F. Gadig
Campus Experimental do Litoral Paulista, Universidade Estadual
Paulista—UNESP, Pc¸a. Infante Dom Henrique, s/n, Sa
o Vicente,
SP CEP 11330-900, Brazil
Conservation Genet Resour (2010) 2:31–35
DOI 10.1007/s12686-009-9131-7
identifying the species correctly (Bonfil 1994; Castro et al.
1999). In order to put the actions of conservation and
management of sharks in practice, there is an urgent need
to minimize these problems.
For more than 30 years, several different molecular
markers have been used with taxonomical purpose among
the numerous organisms groups (Ward et al. 2005). In
relation to sharks, for instance, the available molecular
markers for species identification developed by Pank et al.
(2001), Shivji et al. (2002), Chapman et al. (2003), Nielsen
(2004), Abercrombie et al. (2005) use the differences
among the nucleotide bases of the ITS2 spacer of ribo-
somal genes. Yet, Blanco et al. (2008) use sequences of the
Cytochrome b gene. Considering the large range of dif-
ferent techniques that can be used for species identification,
exploiting several genomic regions, Hebert et al. (2003)
suggested that a single genetic sequence would be enough
to differentiate all, or at least most of the animal species
and proposed the utilization of mitochondrial DNA Cyto-
chrome Oxidase subunit I (COI) for a global bioidentifi-
cation system for animals, and the consequent description
of each species in a barcode sequence.
Also seeking the unification of a world information
system dedicated to the genetic taxonomic identification,
the present study describes a methodological resource to
characterize nine species of Lamniform and Carcharhini-
form sharks, usually exploited by commercial fishery,
developed from the exclusive characteristics of each spe-
cies and expressed in the COI gene sequences.
Materials and methods
Sample characterization
Among the species for which the genetic identification
methods were developed, three belong to Lamniformes
(Alopias superciliosus, Alopias vulpinus, and Isurus oxy-
rinchus) and six belong to Carcharhiniformes (Prionace
glauca, Galeocerdo cuvier, Carcharhinus falciformis,
Rhizoprionodon lalandii, Rhizoprionodon porosus, and
Sphyrna lewini). Other 16 shark species: Isistius brasili-
ensis, Squatina argentina, Squatina guggenheim, Gingly-
mostoma cirratum, Lamna nasus, Galeorhinus galeus,
Mustelus higmani, Mustelus schmitti, Schroederichthys sp.,
Scyliorhinus sp., Carcharhinus acronotus, Carcharhinus
leucas, Carcharhinus obscurus, Carcharhinus plumbeus,
Carcharhinus porosus, and Sphyrna tudes were jointly
analyzed for the nucleotide diversity evaluation among the
species, verification of false positive in multiplex-PCR, and
later generation of new identification primers.
The shark samples were obtained from fishery unloading
along the Brazilian coast. The lamniform were captured in
the Southeastern coast of Brazil, in a region close to the
State of Sa
o Paulo. Among the carcharhiniform, we
obtained specimens of P. glauca and G. cuvier from the
Southeastern and Northeastern coast. The samples of the
C. falciformes species are from the Northern coast and
the specimens of R. lalandii, R. porosus and S. lewini were
captured in a vast area including the Southern, Southeastern
and Northeastern coast of Brazil. The samples of the other
16 species were obtained in the Brazilian Southeastern
region, except the species G. cirratum and M. higmani that
are from the Northeastern region and the specimens of
C. porosus, from the Northern region. The sharks were
identified according to Gadig (2001). The Carcharhinus
genus was additionally identified based on Garrick (1982).
After the taxonomic identification, tissue samples were
collected for molecular analyses.
DNA extraction, amplification through PCR
and sequencing
The genomic DNA was extracted from epithelial cells, using
the saline extraction method described by Aljanabi and
Martinez (1997). Amplification reactions of the Cytochrome
Oxidase gene subunit I (COI) were carried out in PCR
thermal cycler using 25 ll of solution 0.8 mM of dNTP,
1.5 mM of MgCl2, enzyme buffer Taq DNA polymerase
(Tris–HCl 20 mM pH 8.4 and KCl 50 mM), 1 unit of
enzyme Taq Polymerase (Invitrogen) and 0.5 mM ng of
primers, using the primers F1 5
and R1 5
, described by Ward et al.
(2005). Each amplification cycle through PCR was basically
formed by denaturation at 95°C for 30 s, hybridization at
50°C for 30 s and extension at 68°C for 2 min, with 35
repetitions. The amplified DNA segments were visualized
on agarose gel at 2%, stained with ethidium bromide, under
ultraviolet light.
The sequences of the COI gene were obtained using ABI
Prism 377 (Perking-Elmer) with the kit DYEnamicTM ET
Terminator Cycle Sequencing (Amersham Biosciences),
and were then manually analyzed and lined using the
program CLUSTAW—Macvector 65 (1998) for identifi-
cation of polymorphic sites among the species.
From the nucleotide composition of the COI gene, whose
characteristics were exclusive, the polymorphic sites
among the species were identified, and the species- specific
primers designed thereafter. The amplification reactions
were carried out including the F1 primer (Forward) used as
positive control for the reaction, the R1 primer (Reverse),
all the 9 species-specific primers for identification and, in
32 Conservation Genet Resour (2010) 2:31–35
each reaction tube, the DNA of one of the species. Besides
the samples of the 9 species that had primers of identifi-
cation developed, PCR reactions were carried out under the
same conditions for the other 16 species of shark, in order
to detect other possible false positive. All the reactions
were carried out using PCR thermal cycler in 25 llof
solution with 0.8 mM of dNTP, 1.5 mM of MgCl2,
enzyme buffer Taq DNA polymerase (Tris–HCl 20 mM
pH 8.4 and KCl 50 mM), 1 unit of enzyme Taq Polymerase
(Invitrogen) and 0.5 mM of each primer. Each amplifica-
tion cycle through PCR was basically formed by denatur-
ation at 95°C for 30 s, hybridization at 50°C for 30 s and
extension at 68°C for 2 min, with 35 repetitions. The
primers developed for each species, the number of ana-
lyzed samples and the estimated size of the amplified DNA
segments are presented in Table 1.
From the identification of 590 nucleotide bases of the gene
COI of the 25 shark species possible to be analyzed, we
observed an average nucleotide divergence of 17.8%.
Among the lamniform shark species, the nucleotide
divergence was as high as 17.4%, and the divergence
among the species of Alopias, 12.1%. Among all the car-
charhiniform species, the genetic divergence was estimated
at 10.8%. The nucleotide difference among the species of
Carcharhinus was 5.1%, whereas among the species of
Rhizoprionodon it was 3.2%.
The species-specific primers were gradually placed
along the sequences of the Cytochrome Oxidase I gene.
Thus, in the PCR reactions each synthesized primer gen-
erates a fragment of a distinct size, presenting diagnostic
bands for each species after electrophoresis. This reaction
containing also the primer COI F1 yielded a 650 bp sec-
ondary band that was used as a reaction positive control.
During the tests we confirmed the efficiency of the primers
in individual reactions including only the specific primer
for a single species, the primers F1 and R1 and the DNA of
the target species. In these reactions, we observed the
amplification of the diagnostic size fragment and the
positive control fragment for the reaction in all the ana-
lyzed samples showing high functionality. In the multiplex-
PCR reactions including all the nine identification primers
species-specific, besides the primers F1 and R1 and the
DNA of only one of each species, we observed a high
stringency reaction for all the 406 shark samples presented
in Table 1. The amplified diagnostic fragments for each
species were used for reaction control (Fig. 1). During the
multiplex-PCR analyses, in order to evaluate the possible
occurrence of false positives using the samples from the
other 16 shark species, only the amplification of the posi-
tive control bands occurred, confirming the primers
Even though the numbers of species that can be identified
using multiplex-PCR presented in this work is discreet, the
studied species correspond to an extremely exploited group
and represent a great portion of the world captures. From
the commercial point of view, lamniform and carcharhin-
iform sharks are very important. The first represent an
important percentage of the captures of large epipelagic
oceanic sharks in the world and also in Brazil where,
mainly Isurus oxyrinchus stand out among the species
captured by the longline fleet, jumping from 13 tons in
1975, to 138 tons in 1990, based on the boats operating in
the Southeast and South of Brazil (Costa et al. 1996).
Among the carcharhiniform, the families Carcharhinidae
and Sphyrnidae are commercially the most important, with
emphasis on Prionace glauca (Carcharhinidae), which is
the most captured shark species by the longline fleet in
these environments, chiefly in Brazil (Hazin and Lessa
Table 1 Developed primers,
size of the bands generated on
agarose, number of samples per
species (n) and COI GenBank
access numbers
Size of the amplified fragments
in base pairs (bp)
Species Primers Fragment (bp) n GenBank
Isurus oxyrinchus CTTCCACTTGGCTGGGTATCTCG 280 30 FJ895090
Alopias vulpinus CCTCAGCTGGAGTTGAAGCC 410 18 FJ895092
Alopias superciliosus GGTTATACCCGTAATAATTGGG 530 34 FJ895091
Galeocerdo cuvier ACTACATTCTTTGATCCAGCG 50 22 FJ895097
Prionace glauca TCCAGTTCTTGCAGCAGGT 105 80 FJ895098
Carcharhinus falciformis GATCTATTCTTGTAACCACG 145 16 FJ895094
Rhizoprionodon porosus CCCATTAGCTAGTAATA 360 110 FJ895096
Sphyrna lewini GGCCTTCCCACGAATAAAC 480 43 FJ895093
Rhizoprionodon lalandii TCAACCTGGATCTCTTTTAGGT 610 90 FJ895095
Conservation Genet Resour (2010) 2:31–35 33
2005), and the small sharks of the genus Rhizoprionodon
that represent more than 50% of the small sharks captured
in the coastal area by the artisanal fleet (Motta et al. 2005).
The hammerhead sharks (Sphyrnidae) are also important
fishery resources in the ocean areas, while large sharks are
target fishery for longliner boats and driftnets (Amorin
et al. 2002), as opposed to fishery in the coastal area,
especially in the Southeast and South of Brazil (Vooren
et al. 2005), where young fish populations are their target.
The efficiency of the COI gene for fish identification has
been strongly supported by several papers (Ward et al.
2005; Hubert et al. 2008; Valdez-Moreno et al. 2009).
Mendonc¸a et al. (2009) analyzed nucleotide sequences of
the same gene in 18 shark species observing a very sig-
nificant divergence, even among species of the same genus,
however, maintaining a strong level of similarity in the
same taxon, where they observed average divergences of
1.2%. The present results agree with those previous studies
showing the existence of high genetic divergence among
shark species, including those from the same genus, but a
high conservation level among individuals of the same
species. The viability of multiplex-PCR application for
identification of shark species with high functionality and
specificity is also supported by the fact that no polymorphic
sign was detected among the binding sites of the species-
specific primers used in this work.
This work makes use of multiplex-PCR techniques
using the COI gene as a safe method to characterize the
global fishery exploitation. Furthermore, considering the
increase in worldwide trade in shark products, low cost
protocols like those developed in the present study also
represent a certification method, adding value to the fish
Acknowledgments The authors thank Fundac¸a
o de Amparo a
Pesquisa no Estado de Sa
o Paulo (FAPESP) and Conselho Nacional
de Desenvolvimento Cientı
fico e Tecnolo
gico (CNPq), for their
financial support.
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Fig. 1 Band pattern produced by multiplex-PCR reaction using the
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(base pairs), P. glauca 105 bp, C. falciformes 145 bp, I. oxyrinchus
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M molecular weight marker 50 bp
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... catch rate, annual landings and bycatch/discard level) stems from misidentified species or elasmobranchs that have been discarded at sea, and because fisheries report only retained (landed) catches (Lack and Sant 2009;FAO 2014). For multi-species fisheries, species identification during port inspections is highly challenging if using traditional morphological or taxonomic tools, as carcasses are usually processed at sea, where key distinguishing morphological features such as heads and fins of specimens are often removed (Abercrombie et al. 2005;Mendonça et al. 2010;Gulak et al. 2017). Additionally, morphological features are frequently similar between species-such as for carcharhinids like the common blacktip C. limbatus and the Australian blacktip C. tilstoni (Tillett et al. 2012)-making discriminant species identification difficult. ...
... Molecular-based methods have regularly been used over the last decade as alternatives to morphological identification (Amaral et al. 2017). These molecular techniques include DNA barcoding and sequence-based identification methods (Ward et al. 2005;Blanco et al. 2008) as well as polymerase chain reaction (PCR) multiplex methods (Farrell et al. 2009;Mendonça et al. 2010). DNA barcoding entails using universal primers targeting a short, standardised gene region (~650 bp for animal species) of the mitochondrial gene encoding cytochrome c oxidase subunit I (COI) (Hebert et al. 2003). ...
Full-text available
In recent decades, a combination of increasing demand and economic globalisation has created a global market for elasmobranch products, especially the highly prized shark fins for Asian markets. Morphological species identification, as well as traditional cytochrome c oxidase subunit I (COI) barcoding of shark fins and other products, become challenging when in a processed state (such as dried or bleached shark fins). Here a mini-barcoding multiplex assay was applied to determine the species of origin in case studies from southern Africa involving confiscated shark fins in different states of processing. This highlights that the illegal shark fin trade in southern Africa to a large extent comprises threatened species. Matching of sequences of the confiscated fins against public databases revealed several threatened species, including the CITES-listed species Carcharodon carcharias, Carcharhinus longimanus, Isurus oxyrinchus, Rhynchobatus djiddensis and Sphyrna lewini. The findings highlight the need for improved trade monitoring, such as to eliminate illegal trade in shark fins, which can in part be achieved through more widespread genetic sampling of internationally traded products. However, a major limitation to DNA barcoding in general lies in the lack of curated voucher specimens available on public databases. To facilitate the application of molecular methods in a more comprehensive evaluation of elasmobranch trade regionally, a concerted effort to create reliable curated sequence data is recommended.
... An aggregate of 150 specimens of C. longimanus was collected from 5 locations along the Indian coast (mainly peninsular Indian region; Lakshadweep, Kochi, Kollam, Trivandrum, and TamilNadu) ( Fig. 1) (30 samples from each location). DNA was extracted following standard protocols (Sambrook and Russell 2001) and partial control region sequences amplified using the primers (Primer (F-5′CTCCCAAAGCCAAG ATTCTG 3′), Primer (R-5′GGCTTAGCAAGATGTCTTGG G 3′)) (Mendonça et al. 2010) with slight modifications in the reverse primer sequence (Online Resource Table S4). The 25 μl PCR reaction mixture consisted of dNTPs (200 μM each of dNTPs), MgCl 2 (1.5 mM), 1x buffer (10 mM Tris-HCl pH 8.4, and 50 mM KCl), Oligonucleotides (0.2 μM), 1 unit of Taq DNA polymerase, and 50 ng of template DNA. ...
Sharks are undergoing population declines worldwide and it is imperative to devise conservation and management strategies to prevent their extinction. Oceanic whitetip sharks are large pelagic sharks distributed circumglobally and recent IUCN assessments classified them as “critically endangered.” Considering their vulnerability, we investigated the intraspecific diversity and genetic stock structure of oceanic whitetip shark, Carcharhinus longimanus, along the Indian coast using mitochondrial control region sequences so that viable management guidelines can be formulated in the Indian Ocean region. Population genetic analyses revealed a lack of significant genetic differentiation along the Indian coast indicating substantial gene flow and connectivity among populations. Comparisons of data of the present study with that of Atlantic Ocean regions indicated significant connectivity and gene flow between Indian and East Atlantic regions and a lack of connectivity between Indian and West Atlantic Ocean regions. Oceanic whitetip sharks have substantial capacity for oceanic migration resulting in the mixing of gene pools. Despite these capabilities, overfishing is one of the major drivers of population decline worldwide, resulting in severe fragmentation of populations. Based on the results of the present study, this species can be managed as a single stock along the Indian coast. Further co-management measures along with countries bordering East Atlantic coast can also be devised. Management should consider a complete or seasonal ban of the fishery in addition to restrictions in gear types.
... Morphological identification is hampered by on-board processing because of the common practice of cutting off the head, fins, and tail of elasmobranchs, whose bodies are dumped in the sea, to reduce the space required for storage and to preserve their meat longer (Bonfil, 1994;Franco, Mendonça, Oliveira, & Foresti, 2012;Holmes, Steinke, & Ward, 2009). Molecular tools are now being applied for the improved identification of landed species offered to consumers (Chuang, Hung, Chang, Huang, & Shiao, 2016;Domingues, Amorim, & Hilsdorf, 2013;Mendonça et al., 2010;Ribeiro et al., 2012), in the assessment of fraudulent labelling (Bornatowski et al., 2013;Carvalho, Palhares, Drummond, & Frigo, 2015) and in the identification of species threatened with extinction (Bunholi et al., 2018;Feitosa et al., 2018;Ferrette et al., 2019). However, in spite of their strength, genetic analyses are expensive and take time, which limits their usefulness for the inspection of species at the time of the fish landing. ...
Statistical fisheries data are usually obtained during landings, through rapid fish classification and their categorization under the trade names given to species or groups of species. However, species classification is often difficult, particularly concerning elasmobranchs whose fins and heads have been removed, leading to labelling errors. The aim of this paper is to identify the ray species composition landed in south‐east Brazil, providing identification strategies to support a plan for more efficient labelling and management. Samples were obtained from artisanal fleet landings, between November 2012 and May 2014. For taxonomic identification of whole or processed animals ( n = 279, belonging to 10 species), morphological, metric, and molecular techniques were employed. The common name used by fishermen was more related to the location where they lived than the fishing gear used. Morphometric analyses resulted in pectoral fin to length and weight conversion equations of whole individuals for each species, and 10 variables were found to be diagnostic of each genus. A genetic identification, based on sequencing of the mitochondrial genes cytochrome b and cytochrome oxidase I, was applied to verify morphological identification. A dichotomous key that allowed ray identification at the species level from pectoral fin morphology was developed. The approach was field tested and deemed adequate, leading to a robust monitoring strategy for estimating the biomass of specific landed rays.
... This makes it possible to identify unknown DNA samples to a registered species based on comparison to a reference library. Consequently, over the last few years, several studies have aimed to create a Chondrichthyes DNA database and tools for their identification in order to evaluate their biodiversity (Mendonça et al. 2009(Mendonça et al. , 2010Ribeiro et al. 2012;Ward et al. 2005Ward et al. , 2008Ward et al. , 2009Wong et al. 2009;Cerutti-Pereyra et al. 2012). Moreover, this technique has been applied for shark fin species identification worldwide (Fields et al. 2015Holmes et al. 2009;Liu et al. 2013;Sembiring et al. 2015;Shivji et al. 2002;Steinke et al. 2017). ...
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Sharks developed life history traits that make them susceptible to overfishing. This is, in turn, a risk for extinction, and several species are affected. The high price of shark fins in the international trade has triggered the widespread capture of sharks at unsustainable levels, prompting illegal and unethical practices, such as finning. To address these concerns, the present study aimed to identify species composition using molecular techniques based on DNA barcoding and DNA polymorphism on samples taken from illegal shark fin seizures conducted by the Federal Environmental Agency of Brazil. A species-specific DNA-based identification from three finning seizures in Brazil found at least 20 species from 747 shark fins, some of which were identified as endangered and protected under Brazilian legislation, while others were representative of restricted catches, according to Appendix II of CITES. In the seizure from Belém, 338 fins were identified as belonging to at least 19 different species, while in the seizure from Natal 211, fins belonging to at least 8 different species were identified. Furthermore, 198 fins from Cananéia were identified through PCR-Multiplex as belonging to Isurus oxyrinchus. These results raise concerns about the environmental and socioeconomic effects of finning on developing countries. Furthermore, this study represents the first finning evaluation from Brazil in the Southwest Atlantic, highlighting the importance of developing policies aimed toward restricting and regulating the shark trade and detecting IUU fisheries and illegal trade of endangered species, mainly in developing countries, where fisheries management, surveillance, and species-specific fisheries catch data are often sporatic.
... In the last few years, several studies in Brazil have developed and implemented molecular markers to identified elasmobranchs [25,26,89,90]. However, overall, a few studies have been focused on batoids [28,77,91]. ...
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Today, elasmobranchs are one the most threatened vertebrate groups worldwide. In fact, at least 90% of elasmobranch species are listed in the International Union for Conservation of Nature (IUCN) Red List, while more than 40% are data-deficient. Although these vertebrates are mainly affected by unsustainable fishery activities, bycatch is also one of the major threats to sharks and batoids worldwide, and represents a challenge for both sustainable fishery management and for biodiversity and conservational efforts. Thus, in this study, DNA barcode methodology was used to identify the bycatch composition of batoid species from small-scale industrial fisheries in the southwest Atlantic and artisanal fisheries from southeast Brazil. A total of 228 individuals belonging to four Chondrichthyes orders, seven families, and at least 17 distinct batoid species were sequenced; among these individuals, 131 belonged to species protected in Brazil, 101 to globally threatened species, and some to species with trade restrictions provided by Appendix II of the Convention on International Trade in Endangered Species (CITES). These results highlight the impacts on marine biodiversity of bycatch by small-scale industrial and unmanaged artisanal fisheries from the southwest Atlantic, and support the implementation of DNA-based methodologies for species-specific identification in data-poor fisheries as a powerful tool for improving the quality of fisheries' catch statistics and for keeping precise bycatch records.
... In Brazil, molecular techniques have been used to identify species of the genus Squatina (Falcão et al., 2014) and guitarfish (De Franco et al., 2012). In addition, studies have used molecular markers to identify several species of Elasmobranchs (Mendonça et al., 2010;Ribeiro et al., 2012;Domingues et al., 2013), as well as identify fraudulent labeling of different species of fish (Carvalho et al., 2015) and species threatened with extinction (Rodrigues et al., 2016). ...
Morphological identification in the field can be extremely difficult considering fragmentation of species for trade or high similarity between congeneric species. In this context, the shark group belonging to the genus Squatina is composed of three species distributed in the southern part of the western Atlantic. These three species are classified in the IUCN Red List as endangered, and they are currently protected under Brazilian law, which prohibits fishing and trade. Molecular genetic tools are now used for practical taxonomic identification, particularly in cases where morphological observation is prevented, e.g., during fish processing. Consequently, DNA barcoding was used in the present study to track potential crimes against the landing and trade of endangered species along the São Paulo coastline, in particular Squatina guggenheim (n=75) and S. occulta (n=5), as well as the Brazilian guitarfish Pseudobatos horkelii (n=5). DNA barcoding revealed the continuous fishing and trafficking of these protected species, thus giving clear evidence that the current conservation models and methods of monitoring are not working.
... Identification of bio-economically important sharks during port inspections is very difficult (or even impossible) when using traditional taxonomic tools because of carcass processing at sea, where the head and fins are removed (Abercrombie, Clarke, & Shivji, 2005;Akhilesh et al., 2014;Stevens, 2004). During processing morphological and meristic criteria which are pivotal to the accurate identification of specimens are lost (Mendonça et al., 2010;da Silva & Bürgener, 2007). ...
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The common smoothhound (Mustelus mustelus) is the topmost bio-economically and recreationally important shark species in southern Africa, western Africa and Mediterranean Sea. Here, we used the Illumina HiSeq™ 2000 next-generation sequencing (NGS) technology to develop novel microsatellite markers for Mustelus mustelus. Two microsatellite multiplex panels were constructed from 11 polymorphic loci and characterised in two populations of Mustelus mustelus representative of its South African distribution. The markers were then tested for cross-species utility in Galeorhinus galeus, Mustelus palumbes and Triakis megalopterus, three other demersal coastal sharks also subjected to recreational and/or commercial fishery pressures in South Africa. We assessed genetic diversity (NA, AR, HO, HE and PIC) and differentiation (FST and Dest) for each species and also examined the potential use of these markers in species assignment. In each of the four species, all 11 microsatellites were variable with up to a mean NA of 8, AR up to 7.5, HE and PIC as high as 0.842. We were able to reject genetic homogeneity for all species investigated here except for T. megalopterus. We found that the panel of the microsatellite markers developed in this study could discriminate between the study species, particularly for those that are morphologically very similar. Our study provides molecular tools to address ecological and evolutionary questions vital to the conservation and management of these locally and globally exploited shark species.
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The dalatiid genus Isistius Gill (1864) has three valid species currently recognized in the literature: Isistius brasiliensis Quoy & Gaimard (1824), I. plutodus Garrick & Springer (1964), and I. labialis Meng, Zhu & Li (1985). The most common species, I. brasiliensis, has a wide geographic distribution and is found in subtemperate and tropical seas circumglobally. A comparative analysis of specimens from different localities throughout its range, however, had never been undertaken. In the present paper, the morphological variation of this species along its entire distribution has been thoroughly analyzed, corroborating that it represents a single widespread species and that I. labialis is its junior synonym. The other congeneric species, I. plutodus, is known from only a few specimens and is also distributed worldwide. A detailed comparative analysis of available material of I. plutodus was conducted verifying its validity as a single widespread species. The present study analyzed in detail the external morphology (coloration, dentition, dermal denticles), internal morphology (skeleton, musculature), lateral-line canals, and morphometric and meristic characters of species of Isistius in order to better define the genus and its included valid species.
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Here, we report trading of endangered shark species in a world hotspot for elasmobranch conservation in Brazil. Data on shark fisheries are scarce in Brazil, although the northern and northeastern regions have the highest indices of shark bycatch. Harvest is made primarily with processed carcasses lacking head and fins, which hampers reliable species identification and law enforcement on illegal catches. We used partial sequences of two mitochondrial genes (COI and/or NADH2) to identify 17 shark species from 427 samples being harvested and marketed on the northern coast of Brazil. Nine species (53%) are listed under some extinction threat category according to Brazilian law and international authorities (IUCN-International Union for Conservation of Nature; CITES-Convention on International Trade of Endangered Species of Wild Fauna and Flora). The number increases to 13 (76%) if we also consider the Near Threatened category. Hammerhead sharks are under threat worldwide, and composed 18.7% of samples, with Sphyrna mokarran being the fourth most common species among samples. As illegal trade of threatened shark species is a worldwide conservation problem, molecular identification of processed meat or specimens lacking diagnostic body parts is a highly effective tool for species identification and law enforcement.
We developed a multiplex PCR DNA marker for quick and easy identification of the AAGG-genome timopheevii lineage, including Triticum timopheevii , Triticum araraticum and hexaploid Triticum zhukovskyi (AAA m A m GG), and the AABB-genome emmer wheat lineage, including Triticum durum , Triticum dicoccum and Triticum dicoccoides . Distinguishing between tetraploid AAGG- and AABB-genome wheat species based on morphology is known to be difficult. This multiplex PCR system is based on the simultaneous PCR amplification of two chloroplast regions, matK and rbcL . The matK region molecularly distinguishes the two lineages, whereas the rbcL region is a positive control amplicon. We also examined whether the simple sequence repeat is a fixed mutation within species, using genetic resources in the collection of KOMUGI, Kyoto University, which comprises accessioned species collected across diverse geographical areas. The multiplex PCR marker distinguished AAGG from AABB species with complete accuracy.
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The historical series from 1971 to 2000 of Chondrichthye s caught by longliners based in Santos City, Sao Paulo State, Brazil, have been reviewed and the fishery biology of some of the species has been studied. The Brazilian Santos fleet started in 1965/66 with two boats, increasing gradually until a maximum of 20 boats, in 1998 and 19 in 1999. In 2000 comprised 14 vessels (13 national and 1 leased) operating in the area 17-35o S, 27-52o W. Since 1977 yields of sharks have shown an increasing trend, reaching about 60% of the longline total catch in 1993. Since the beginning of this fishery, most of shark's meat had market. Nevertheless only few shark species had high commercial value, so some of them were discarded, mainly blue shark. After 1977, shark meat market started increasing and the fishermen began to gradually bring all hooked sharks including blue shark. Also the price of shark fins brought as bycatch had six times increase in the last ten years. Blue shark (Prince glauca) was the main species and responsible for about 30% of the Santos longliners catch. The 33 shark and 2 ray species identified belong to the following families: Alopiidae, Carcharhinidae, Hexanchidae, Lamnidae, Megachasmidae, Odontaspididae, Pseudocarchariidae, Sphyrnidae, Dasyatidae and Mobulidae. Yield, fishing effort and biological aspects of some species are presented.
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A very simple, fast, universally applicable and reproducible method to extract high quality megabase genomic DNA from different organisms is decribed. We applied the same method to extract high quality complex genomic DNA from different tissues (wheat, barley, potato, beans, pear and almond leaves as well as fungi, insects and shrimps' fresh tissue) without any modification. The method does not require expensive and environmentally hazardous reagents and equipment. It can be performed even in low technology laboratories. The amount of tissue required by this method is ∼50–100 mg. The quantity and the quality of the DNA extracted by this method is high enough to perform hundreds of PCR-based reactions and also to be used in other DNA manipulation techniques such as restriction digestion, Southern blot and cloning.
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Although much biological research depends upon species diagnoses, taxonomic expertise is collapsing. We are convinced that the sole prospect for a sustainable identification capability lies in the construction of systems that employ DNA sequences as taxon 'barcodes'. We establish that the mitochondrial gene cytochrome c oxidase I (COI) can serve as the core of a global bioidentification system for animals. First, we demonstrate that COI profiles, derived from the low-density sampling of higher taxonomic categories, ordinarily assign newly analysed taxa to the appropriate phylum or order. Second, we demonstrate that species-level assignments can be obtained by creating comprehensive COI profiles. A model COI profile, based upon the analysis of a single individual from each of 200 closely allied species of lepidopterans, was 100% successful in correctly identifying subsequent specimens. When fully developed, a COI identification system will provide a reliable, cost-effective and accessible solution to the current problem of species identification. Its assembly will also generate important new insights into the diversification of life and the rules of molecular evolution.
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The great white shark, Carcharodon carcharias, is the most widely protected elasmobranch in the world, and is classified as Vulnerable by the IUCN and listed on Appendix III of CITES. Monitoring of trade in white shark products and enforcement of harvest and trade prohibitions is problematic, however, in large part due to difficulties in identifying marketed shark parts (e.g., dried fins, meat and processed carcasses) to species level. To address these conservation and management problems, we have developed a rapid, molecular diagnostic assay based on species-specific PCR primer design for accurate identification of white shark body parts, including dried fins. The assay is novel in several respects: It employs a multiplex PCR assay utilizing both nuclear (ribosomal internal transcribed spacer 2) and mitochondrial (cytochrome b) loci simultaneously to achieve a highly robust measure of diagnostic accuracy; it is very sensitive, detecting the presence of white shark DNA in a mixture of genomic DNAs from up to ten different commercially fished shark species pooled together in a single PCR tube; and it successfully identifies white shark DNA from globally distributed animals. In addition to its utility for white shark trade monitoring and conservation applications, this highly streamlined, bi-organelle, multiplex PCR assay may prove useful as a general model for the design of genetic assays aimed at detecting body parts from other protected and threatened species.
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SUMMARY This document presents a synopsis of the available fishery and biological information for blue shark caught off Brazil. Fishery information in terms of distribution of the catches by the longline fleet and in terms of trends in CPUE are presented. In addition, the document provides information on reproductive biology and growth. Based on the estimated growth rates and catches, estimates of total mortality are provided.
Although the Brazilian sharpnose shark, Rhizoprionodon lalandii (Muller and Henle, 1839), is an inshore species widely distributed in the Western Atlantic from Panama to Uruguay, there is little available information on its biology. During a longterm study of small coastal sharks caught by gill net fisheries in southeastern Brazil (PROJETO CAC¸A˜ O), 3643 specimens of R. lalandii were examined, comprising 61.3% of the total sharks, and including all sizes classes, from 30 to 78.5 cm TL, and weights from 100 to 2950 g. The length–weight relationships were not significantly different between sexes. Overall sex ratio favoured the males slightly at the rate of 1.3:1. Sex ratios, however, did differ significantly between season and size classes. This species occurred in this area all year long. Three seasonal size-class occurrence patterns were recognized: (1) between October and March, the juveniles were more frequent; (2) from April to July, adults were most common; and (3) from August to September, neonates were most numerically abundant. Such patterns were associated with reproductive tactics that may reduce intra-specific and inter-specific competition with hammerhead shark neonates (Sphyrna lewini), probably result in reduced natural mortality of the offspring during their first few months.