Shark DNA Forensics: Applications and Impacts on Genetic Diversity

Luis Fernando Rodrigues-Filho1, Danillo Pinhal2, Davidson Sodre1 and Marcelo Vallinoto1,3 1Federal University of Para, Campus of Braganca, Institute of Coastal Studies (IECOS), Braganca, Para 2Department of Genetics, Biosciences Institute Sao Paulo State University (UNESP), Botucatu, SP 3CIBIO/UP, Research Center in Biodiversity and Genetic Resources Campus Agrario de Vairao, University of Porto 1,2Brazil 3Portugal


Introduction
Despite their worldwide distribution, an increasing conservation appeal and humans' great fascination with them, many aspects of the biology and life history of sharks remain enigmatic.Shark-focused investigations are important because these animals are prominent elements in the marine ecosystem, and the majority of species play pivotal roles as apex predators in the food web.
Over the past decades, several studies have shown the remarkable and accelerated depletion of the natural stocks of many sharks worldwide; population reductions that range from 50% to 89% have been documented (Kotas et al., 1995, Vooren, 1997;Musick et al., 2000;Baum et al., 2003;Baum & Myers, 2007;Dulvy et al., 2008).This decline has been explained to be a consequence of the extensive, unregulated exploitation of wild stocks by fisheries coupled with the restrictive biological characteristics that are intrinsic to the majority of shark species, such as slow growth, high longevity, late sexual maturity and relatively low fecundity (Branstetter, 1990).Moreover, the pressure that is experienced by shark populations is augmented by accidental capture (by-catch) during fisheries that traditionally exploit bony fish species (e.g., tuna and billfishes).
According to Dulvy et al. (2003), the major forces that drive the extinction of shark species are habitat destruction and exploitation by fisheries.Regarding the fisheries, it is well known that the majority of shark populations may not be able to recover after periods of high fishing pressure, which can lead to a permanent loss of genetic diversity in their wild populations (Stevens et al., 2000).
Currently, several coastal and oceanic sharks have reached the status of threatened species (i.e., vulnerable, endangered or critically endangered) according to data that were released by the Red List of threatened fauna of the International Union for Conservation of Nature (IUCN, 2011).Assorted events are responsible for the current adverse condition that has been reported for diverse shark species worldwide.These events include the lack of governmental policies that regulate and control fishing activity in several countries; the inefficiency in creating priority areas for conservation; the high commercial value of shark fins, which are utilised as food and for pharmaceutical and medical purposes; and the extremely negative stereotypes, which have been acquired over the years due to relatively rare instances of attacks on swimmers, divers and surfers.
A remarkable example of overexploitation involves the daggernose shark Isogomphodon oxyrhynchus (Muller & Henle, 1839) (Figure 1), which is a coastal species with a low reproductive capacity (Bigelow & Schroeder, 1948) and a fragmented distribution between Venezuela and Northern Brazil (Lessa, 1999).Although it is protected by law in Brazil (MMA-IBAMA, 2004), I. oxyrynchus continues to be illegally caught by fisheries and is currently listed as critically endangered in the Red List of IUCN.
Fig. 1.A daggernose shark Isogomphodon oxyrhynchus specimen from the Guiana coast, Atlantic.This photo is courtesy of Dr. Keiichi Matsuura.
In response to the recognised low sustainability of shark fishing on a global scale, the Food and Agriculture Organization (FAO) developed an international plan of action, and a few countries have implemented equivalent national plans for the management and conservation of shark stocks.The primary recommendation of these plans is the collection of shark-catch and trade data on a species-specific basis because different shark species display a broad array of unique biological characteristics, which ultimately reflects their variable susceptibility to exploitation by fisheries (Heist & Gould, 1999).For this reason, precise species identification is required to determine genetic stock structure and population subdivision (Morgan et al., 2011).
Overall, the biodiversity of sharks is high, although species-specific information that can be used to assist in sustainable resource exploitation is scarce (Ovenden et al., 2011).A major recurrent impediment that is encountered in species monitoring is the historical lack of long-term data on shark landings and commerce on a species-by-species basis; this absence virtually prevents proper inferences regarding population status and future trends (Bonfil, 1994;Castro et al., 1999).In addition, identification is prejudiced due to the illegal finning practice, in which the head and fins, which are key morphological characters for the identification of sharks, are removed at sea.Therefore, unidentifiable landed carcasses are generically recorded as "sharks", which prevents the tracking of the composition of species and detection of species that are protected by law in fisheries (Figure 2).Moreover, the difficulty of accurate species identification is exacerbated by the fact that many exploited sharks, such as those in the orders Carcharhiniformes and Lamniformes (Last & Stevens, 1994;Naylor & Marcus, 1994), are morphologically very similar.This makes the desirable taxonomic identification to the species level a challenge even for experienced taxonomists (Castro, 1993;Stevens & Wayt, 1998;Heist, 2004).
Based on this scenario, research projects that are devoted to the conservation of genetic diversity have sought simple, rapid and low-cost tools that can be immediately implemented.Diverse DNA-based techniques have been developed primarily to assist in the identification of species and the monitoring of shark fishing and trade (Lavery, 1992;Heist & Gold, 1999).These molecular approaches are a resourceful way to identify shark body parts, such as fins, carcass, meat, and processed shark subproducts, which are sold in forms such as extracts and pills (Sebastian et al., 2008;Wong et al., 2009).
Due to the emergence of forensic genetics, individuals from numerous species that were previously not easily identifiable by traditional taxonomy can now be accurately traced back to their geographical region and population of origin using diminutive amounts of DNA.Accordingly, the identification of shark species by molecular methods has contributed to control programs that are directed to fisheries management, trade monitoring and market surveillance, and it has especially facilitated the tracking of endangered and/or heavily exploited shark species (Pank et al., 2001;Clarke et al., 2006;Rodrigues-Filho et al., 2009).
In the present chapter, we review the most important molecular markers and the associated technologies that are employed to resolve species identities and their impacts on the genetic diversity of natural shark stocks.

Molecular markers and approaches for shark species identification
Current DNA technologies that have been developed for shark species identification are based on one-or two-step approaches that are applied to polymorphic regions of the nuclear genome, mitochondrial genome or both genomes combined.Among the most popular and well-established methodologies for the discovery and validation of informative molecular markers are polymerase chain reaction (PCR) and nucleotide sequencing.Fig. 2. Headless and finless shark carcasses that were traded in the fish market at Bragança, Pará, Brazil.

PCR and associated methodologies
PCR (Mullis & Faloona, 1987) has been extensively used for species identification due to its simplicity, specificity and sensitivity.Among molecular markers that have been studied via PCR, the 5S rDNA has gained prominence for species identification by providing consistent results and utilising fast, low-cost strategies (Asensio et al., 2001;Imsiridou et al., 2007).The 5S rDNA is characteristically organised into multiple tandemly arrayed repeats that are distributed on one or several chromosomes in the genome.Structurally, this nuclear ribosomal region consists of highly conserved 120-base pair (bp) coding sequences that are isolated from each other by a variable nontranscribed spacer (NTS) (Long & David, 1980).Specifically, 5S rDNA is a highly informative region for studies that use the PCR technique due to the following features: (a) the 5S rRNA gene is widely conserved even in distantly related species, which makes it less laborious to isolate the whole repeat from different species by PCR; (b) the repeats do not exceed the range of PCR amplification; and (c) the sequences can be isolated from low-quality DNA because they are small in size and are tandemly organised in a large number of copies (Martins & Wasko, 2004;Pinhal et al., 2009Pinhal et al., , 2011)).Although the 5S rRNA genes are conserved even among unrelated taxa, their NTSs show extensive variations in length and nucleotide composition among species; these properties make these sequences highly useful as species-specific genetic markers (Wasko et al., 2001;Martins & Wasko, 2004;Pinhal et al., 2009).Such differences in the NTS architecture are useful for the PCR amplification of species-specific 5S rDNA profiles that can be directly discriminated with agarose gels.
Diverse studies have demonstrated the potential of 5S rDNA as a genetic marker for identifying shark species.For example, Pinhal et al. (2008) utilised 5S rDNA to discriminate eight shark species (Carcharhinus leucas, C. acronotus, C. limbatus, C. obscurus, Galeocerdo cuvier, Sphyrna lewini, Isurus oxyrinchus and Alopias superciliosus) by producing differently sized amplicons by simple PCR and analysing them in agarose gels.Later, the same genetic marker was employed to discriminate two closely related species of the genus Rhizoprionodon (Rhizoprionodon porosus and R. lalandii); however, in this case, the diagnostic required an extra step of enzymatic digestion in addition to PCR (Pinhal et al., 2009).A representative diagnostic 5S rDNA profile of three distinct shark species is shown below (Figure 3).The multiplex PCR approach, which is an extension of traditional PCR, utilises a reaction in which multiple primers are simultaneously used for PCR; this permits the simultaneous amplification of various DNA fragments.This approach is used to generate amplicons that are of variable lengths between different species (i.e., unique DNA profiles), which will ultimately permit the identification of one species from another (Rasmussen & Morrissey, 2008;Carrier et al., 2010).Additional advantages of the multiplex approach include the ease of data interpretation, the feasibility of application for high-throughput screening and the overall low cost per analysed sample.Moreover, this approach does not require the sequencing of all of the samples or an extra step of enzymatic digestion to discriminate species (Magnussen et al., 2007).
The multiplex PCR technique that uses ITS 2 is the most well-established approach for the recovery of a shark's identity.For sharks, sets of species-specific primers (SSP) are used in conjunction with universal primers (positive controls) in a single reaction.Pank et al. (2001) developed SSPs for multiplex PCR using the ITS 2 region to discriminate two shark species, Carcharhinus obscurus and C. plumbeus.This work was shortly expanded by Shivji et al. (2002), who incorporated five additional species.The same approach was used by Chapman et al. (2003) and subsequently by Shivji et al. (2005) in samples with the generic name of "shark" that were obtained in Asian markets.Both of the studies proved that the meat and fins present among the samples traded were of the great white shark (Carcharodon carcharias), a species whose capture and trade is prohibited in many countries.In another study by the same group, Abercrombie et al. ( 2005) validated new SSPs to identify three large-body-sized species of hammerhead sharks (Sphyrna lewini, S. zygaena and S. mokarran), and their results confirmed the worldwide commerce of these species.Subsequently, Clarke et al. (2006) estimated the proportion of each taxon in the total shark fins and meat that was sold in Asian markets by using statistical approaches coupled with molecular diagnosis by multiplex PCR.Although the multiplex PCR approach was originally designed to identify large pelagic and oceanic sharks, it has also has been applied to genetically profile globally distributed coastal shark species that are intensively exploited by fisheries (Pinhal et al. in preparation).In this work, the authors designed and validated seven species-specific PCR primers in a multiplex format that could simultaneously discriminate body parts from the seven known sharpnose shark species (genus Rhizoprionodon).Unlike the majority of shark species, Rhizoprionodon species exhibit life-history characteristics (rapid growth, early maturity and annual reproduction) that suggest that they could be fished in a sustainable manner if an investment in monitoring, assessment and careful management was made.Therefore, the usefulness of the developed assay is noteworthy because the acquisition of catch and trade data for species with a large quantity of landed individuals, which is the case for sharpnose sharks, will require high-throughput profiling.Currently, approximately 35 ITS2-based SSPs are available for shark species identification, and SSPs for an additional two species are currently being validated (Nachtigall et al. in preparation).
Recently a new multiplex PCR-based tool that utilises real-time PCR (RT-qPCR) and highresolution melting (HRM) technologies to identify shark species was developed.Using a multiplex RT-HRM PCR assay, Moore et al. ( 2011) distinguished among three closelyrelated sharks (Carcharhinus tilstoni, C. limbatus and C. amblyrhynchoides) based on mutations in the mtDNA ND4 gene.Although promising, this technology still requires more sophisticated equipment, reagents and trained personnel before it can be implemented in routine species identification.

Nucleotide sequencing
DNA sequencing is a powerful and sensitive method for species identification, and it has gained prominence as a rapid, accessible methodology owing to technological advances and the decrease in the cost per sample (Wong et al., 2009).
When sequencing samples for species identification, it is important to correctly choose the genomic portion to be analysed.Distinct regions of the genome evolve at variable rates; therefore, to avoid errors in species diagnostics, the chosen portion should display high interspecific and low intraspecific variability (Bossier, 1999).
For example, Heist & Gold (1999) sequenced mitochondrial DNA (mtDNA) to discriminate among 11 species of Carcharhiniformes.Douady et al. (2003) conducted a study with a 2.4kb fragment of the mitochondrial genome, which extended from the 12S rRNA gene to the 16S rRNA gene.They examined the phylogenetic relationships among the orders of sharks, which demonstrated that this region may be useful for resolving relationships at lower taxonomic levels.Subsequently, Greig et al. (2005) extended on this analysis by discriminating 35 shark species in the North Atlantic based on the high intraspecific and low interspecific divergence of these mitochondrial gene sequences.
Recently, Rodrigues-Filho et al. ( 2009) utilised the same marker to discriminate shark species that are exploited by fisheries in Northern Brazil.They revealed the occurrence of 11 species among morphologically indistinguishable landed specimens (Figure 4).Among the species that were identified, the species Carcharhinus porosus and sharks of the genus Rhizoprionodon were the most frequently caught.The former species is not listed by IUCN; however, local C. porosus stocks have been depleted, which has caused this species to be included in the Brazilian Red List of endangered fauna (MMA, 2004).In addition, Rodrigues-Filho et al. ( 2009) used genetic screening to show that the previously abundant Isogomphodon oxyrhynchus was not present among species that were caught by fisheries.
A different strategy that is widely used to identify species is the sequencing of the DNA barcode region (Hebert et al., 2003).In this approach, the identification of a given species is based on a profile that is generated with an approximately 650-base pair fragment of the mitochondrial COI gene.Studies have shown that this portion of COI displays low intraspecific and high interspecific variability, which is important for the discrimination of species on the basis of permanent nucleotide differences that exist among them.An advantage of this approach is the availability of a global reference databank, which is the result of international efforts that were mediated by the Consortium for the Barcode of Life (CBOL; http://www.barcoding.si.edu).The primary databank is called BOLD (Barcode of Life Data System, http://www.barcodinglife.org/views/login.php)(Ratnasingham & Hebert, 2007), which has currently assembled approximately 100,000 barcoded species.BOLD, which is the host of the FISH-BOL databank (Fish Barcode of Life campaign, http://www.fishbol.org) is dedicated to the assembly of barcode sequences from marine and freshwater species of fishes.Currently, 556 species of sharks and rays have been barcoded; this represents approximately 50% of the total number of elasmobranches that have been described (FISH-BOL, accessed in July/2011).Some of the primordial studies that used DNA barcodes were performed by Ward et al. (2005), who sequenced the COI gene of 61 species and standardised this region for the identification of elasmobranches.Other studies that have used the DNA barcode approach have efficiently identified shark species (Ward et al., 2007;Moura et al., 2009;Ward et al., 2008;Holmes et al., 2009;Wong et al., 2009;Barbuto et al., 2010).In a wide screen, Ward et al. (2008) evaluated the variability of COI sequences from 210 species of Chondrichthyes (123 species of sharks) and discriminated 99% of the species that were screened.In addition, Holmes et al. (2009) identified and quantified the relative abundance of 20 shark species by analysing dried fins that were confiscated from illegal fisheries.Similarly, the DNA barcoding approach was also useful for the detection of fraudulent mislabelling in Italian fish markets, in which 80% of the commercialised sharks did not corresponded to the referred species that were sold (Barbuto et al., 2010).
Despite of the large number of studies that have demonstrated the potential of DNA barcode techniques for species identification, its use is controversial.Many authors argue that a single, short marker is not reliable as a taxonomic tool, and traditional systematics utilises a large number of characteristics to delimit species (Dasmahapatra & Mallet, 2006).In addition, the limitations of the DNA barcode approach include its inability to detect hybridisation (which originated from gene flow between distinct species) due to the maternal inheritance of mitochondrial DNA (Moritz & Cicero, 2004).Therefore, to gauge the confidence of the DNA barcode technique for species identification, scientists have recommended the examination of a large number of individuals, especially of globally distributed species, to confirm that the genetic divergence of COI is higher between species than within species (see Shivji, 2010).Fig. 4. Maximum likelihood tree of the mitochondrial genes 12S-16S that was used to discriminate shark samples (genus Carcharhinus).

Genetic diversity measured by molecular markers and implications for shark management
Among the large migratory fish species, sharks perhaps represent some of the more challenging taxa to trace demographically because they are rarely observed in the open ocean where they feed, breed and otherwise interact with conspecifics (Bradshaw et al., 2007).Despite the advances in mark-recapture and other tagging strategies that are used to estimate survival rates and long-range movements, these methods still have restrictions for deriving population estimates and exchange (Kohler & Turner, 2001), and they cannot provide a quantitative measure of the genetic diversity of the stocks.
Genetic techniques have been used with increasing frequency to inform conservation and management (Crandall et al., 2000;Allendorf et al., 2004), and they are especially useful for studying species for which ecology, demographics or behaviour limit the traditional field methods that are used to estimate the population trends.
The decreasing fish stocks and near extinction of several species will impact the economy and the whole environment.By measuring the variability of genetic stocks, it is possible to determine the real impact of fisheries on shark populations.
Because genetic diversity is correlated with population fitness (Reed & Frankham, 2003), understanding patterns of gene flow and dispersal is crucial for deriving how shark populations are distributed and if they constitute distinct genetic stocks.When this occurs populations are alleged to be strutucted.Therefore, the presence of genetic structure is indicative of low levels of migration among distinct stocks and indicates the need for the adoption of independent management measures for each stock (Schrey & Heist, 2003).This information is particularly useful for large free-living marine animals as sharks and can greatly assist for the conservation of wildlife in oceanic landscapes.
Sharks and other large marine fishes with a broad distribution frequently exhibit little population structure (Graves, 1998;Heist, 2004;Curley & Gillings, 2009).Exceptions to this pattern have been attributed to viviparous reproduction, sedentary behaviour and disjunct distributions, but they have been principally attributed to reproductive philopatry (Palumbi 1994;Pratt & Carrier, 2001;Waples et al., 2001;Keeney et al., 2003;Hueter et al., 2004;Keeney et al., 2005;Duncan et al., 2006;Karl et al., 2010).Philopatry is defined as the tendency of an individual to return to or stay in its home area, natal site, or another adopted locality; this is in opposition to nonreturning roaming behaviour or simple dispersal away from home areas (Hueter et al., 2004).Philopatric tendencies can be strong or weak for a given species, and special patterns of this behaviour can include natal philopatry (returning to the natal nursery area) and sex-specific philopatry (where one sex is more philopatric than the other, as in many male birds and female mammals) (Hueter et al., 2004).Females that exhibit philopatric behaviour return to coastal embayments that are called "nursery areas" to give birth (Pardini et al., 2001;Feldheim et al., 2002;Schrey & Heist, 2003).Nursery areas are usually located in shallow coastal waters and offer the advantages of low predation rates, prey abundance and spatial segregation from adults, which use geographically distant feeding areas, to neonates and juveniles (Morrissey & Gruber, 1993;Merson & Pratt, 2001).As a result, philopatric behaviour may clearly directly influence the levels of population structure and genetic diversity of a species, and molecular markers can be employed to detect such pattern.
Currently, a wide set of genetic markers has been utilised to infer population genetic structure in marine organisms (Baker et al., 1990;Baker et al., 1996;Amos et al., 1993;Innes et al., 1998).Due to high polymorphism and mutation rates (among other features), the molecular markers that are commonly used are those that involve the analysis of mitochondrial DNA and nuclear microsatellite loci.
In the mitochondrial genome, the non-coding control region (CR) is the molecular marker that is frequently utilised (Brown et al., 1985;Warn et al., 1986;Avise, 2000;Warn, 2000) due to its high evolutionary rates (Li, 1997;Warn, 2000).Specifically, the control region contains portions of its sequence in which the variability is even greater, which makes the region a highly informative target for population studies.
Studies that have used the CR as molecular marker for sharks are somewhat scarce in comparison to other marine species (for review, see Shivji et al., 2010).This implies incipient information regarding migration rates, dispersion movements, gene flow and the composition of genetic stocks for the majority of species.Indeed, knowledge of the genetic structure and diversity of shark populations is critical for maintaining species reproductive strength, resistance to diseases and the ability to adapt to environmental changes (Frankham et al., 2002).
Overall, most of the studies that have been performed on sharks have shown that distinct species possess lower genetic diversity when compared to other taxonomic groups (FAO).Nevertheless, despite the extensive exploitation by fisheries, some studies have reported that species still carrying high levels of genetic diversity (Pardini et al., 2001;Bowen et al., 2004;Keeney et al., 2005;Duncan et al., 2006;Martinez et al., 2006;Natoli et al., 2006) (Table 1).However, a few studies, such as the study that was published by Keeney et al. (2003), reported a low genetic diversity for the CR sequences from some populations of C. limbatus in the Atlantic coast.Other species that displayed low levels of diversity were Cetorhinus maximus, which is a worldwide distributed species that is highly susceptible exploitation by fisheries, and Sphyrna tiburo, which is heavily exploited in the Gulf of Mexico (Chapman et al., 2004;Hoelzel et al., 2006).

Species
Within the nuclear genome, microsatellites are among the most utilised markers.These sequences can correspond to than 20 alleles and carry heterozygosity above 95% (Heist, 2004).Other advantages of microsatellites are that they are diploid, codominant and analysable from small amounts of DNA; they also display high and constant mutational rates per generation (Jarne & Lagoda, 1996).The PCR allows access to different alleles at microsatellite loci and requires small amounts of DNA.
Recently, significant advances have been made in developing new statistical tools to analyse specific microsatellite loci (Pritchard et al., 2000).Such specificity in the analysis makes microsatellites powerful markers, and their applicability has been proven by studies in fish, such as in genetic mapping and paternity tests (Kocher et al., 1998;Keeney & Heist, 2003, Feldheim et al., 2004).Additionally, microsatellites are extensively used in studies that evaluate genetic variability among species and populations, evolutionary history, population structure and conservation and natural resource management (Bruford & Wayne, 1993;Warn, 2010).The variation in the number of repeated units in each microsatellite locus is the basis for studies that use these markers.
Currently, microsatellite markers are employed to study several shark species, and these markers can be useful for estimating genetic structure and gene flow in wild populations.In fact, DNA markers with different modes of inheritance, such as the maternally inherited mitochondrial DNA (mtDNA) and bi-paternally inherited nuclear microsatellites, are the markers that are most commonly used to infer the genetic diversity and structure of shark populations (e.g., gummy shark Mustelus antarcticus, Gardner & Ward 1998; great white shark Carcharodon carcharias, Pardini et al., 2001; blacktip sharks Carcharhinus limbatus, Keeney et al., 2003Keeney et al., , 2005; shortfin mako Isurus oxyrinchus, Schrey & Heist 2003; whale shark, Rhincodon typus, Castro et al., 2007;bull sharks Carcharhinus leucas, Karl et al., 2010).Several shark studies have used a combination of both markers, which has facilitated the obtainment of improved data regarding historical and contemporary events that drive population dynamics.

Conclusion
Oceans are a bountiful but not unlimited source of fishes that have been extensively harvested for decades.As a consequence, several top predators, such as the majority of shark species, are now endangered or live on the edge of extinction.Given the worldwide depletion of shark populations, which has been largely documented globally, there is an urgent need to increase the knowledge of the life history traits and other fundamental aspects of shark biology to promote conservation.
Therefore, several molecular technologies and methodologies that are summarised and discussed in the present chapter are extremely relevant to monitor the exploitation and prevent the loss of genetic diversity of sharks.It is important to note that molecular identification using distinct markers can be applied to virtually all living shark species to prevent extinction and to assist in their sustainable use as a natural resource.Furthermore, the correct monitoring of genetic stocks is extremely important to ensure the possibility of the full recovery of depleted stocks and the maintenance of genetic diversity.

Table 1 .
Genetic diversity of the mitochondrial control region from several shark species.