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Molecular species identification and population genetics of chondrichthyans in South Africa: current challenges, priorities and progress a

b

Aletta E Bester-van der Merwe & Katie S Gledhill a

Molecular Breeding and Biodiversity Group, Department of Genetics, Stellenbosch University, Stellenbosch, South Africa b

South African Shark Conservancy, Old Harbour Museum, Hermanus, South Africa Published online: 11 Aug 2015.

Click for updates To cite this article: Aletta E Bester-van der Merwe & Katie S Gledhill (2015): Molecular species identification and population genetics of chondrichthyans in South Africa: current challenges, priorities and progress, African Zoology To link to this article: http://dx.doi.org/10.1080/15627020.2015.1063408

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AFRICAN ZOOLOGY ISSN 1562-7020 EISSN 2224-073X http://dx.doi.org/10.1080/15627020.2015.1063408

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Molecular species identification and population genetics of chondrichthyans in South Africa: current challenges, priorities and progress Aletta E Bester-van der Merwe1* and Katie S Gledhill2 Molecular Breeding and Biodiversity Group, Department of Genetics, Stellenbosch University, Stellenbosch, South Africa South African Shark Conservancy, Old Harbour Museum, Hermanus, South Africa * Corresponding author, email: [email protected]

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Molecular genetic techniques, such as DNA barcoding and genotyping, are increasingly being used to assist with the conservation and management of chondrichthyans worldwide. Southern Africa is a shark biodiversity hotspot, with a large number of endemic species. According to the IUCN Red List, a quarter of South Africa’s chondrichthyans are threatened with extinction. South Africa’s commercial shark fisheries have increased over the last decade and there is a need to ensure sustainable utilisation and conservation of this fisheries resource. Here, we give an overview of the molecular techniques that are used to assist in the identification of species, cryptic speciation and possible interspecific hybridisation, as well as the assessment of population structure and reproductive behaviour of endemic and commercially important chondrichthyan species of southern Africa. We discuss the potential application of these techniques for management and conservation of several species affected by South African fisheries. Acquiring baseline barcode data of all chondrichthyans in southern African waters and assessing the population structure of exploited species on a local and greater regional scale are recommended as research priorities. Future prospects should also include high-throughput molecular marker development and investigation of intraspecies functional variation using next generation sequencing technology. Keywords: chondrichthyans, conservation, fisheries, genetics, sharks, southern Africa

Introduction The increasing use of and advances in molecular techniques have allowed for a more integrated approach to conservation management of exploited chondrichthyan species in recent years (Dudgeon et al. 2012). Molecular tools, including genetic markers and genotype data, provide the means to assess dispersal abilities, population connectivity and size, as well as reproductive strategies within species (Portnoy and Heist 2012). This provides an insight into the evolutionary and biological processes that shape species and populations. Advantages of molecular techniques are numerous and are increasingly applied to substantiate morphology-based species identification (reviewed in Hebert et al. 2003) and stock structure assessments (Ovenden et al. 2015). Molecular genetics are further used to identify the species composition of a fishery (Doukakis et al. 2011; Sembiring et al. 2015), identify which species are being traded, including threatened species (Chapman et al. 2003; Abercrombie et al. 2005), and to demonstrate seafood mislabelling or seafood fraud (Barbuto et al. 2010; Cawthorn et al. 2012). Genetic population structure within a species can be assessed on a local, regional or global scale with important management implications (Keeney et al. 2005; Dudgeon et al. 2009). With knowledge expanding and technology costs decreasing, advances and applications in the field of genetics in southern Africa are now more accessible to the developing region. In light of the global change and increased pressure on marine resources, failure to

incorporate genetic information into future management and conservation of chondrichthyans puts the unique regional biodiversity and future populations at risk. Chondrichthyans (sharks, rays and chimaeras) are a relatively small evolutionarily conservative group of cartilaginous fishes of ~1 200 species that have been present in the world’s oceans for at least 400 million years (Fowler et al. 2005). Globally, there is a growing concern for shark populations. Approximately 100 million sharks are harvested annually (Worm et al. 2013), with commercial fisheries, largely driven by the Asian shark fin market, contributing to a large proportion of this decline (Clarke et al. 2006, 2007; Dulvy et al. 2008). Based on the assessments from the International Union for the Conservation of Nature (IUCN) Red List of Threatened species (hereafter referred to as IUCN Redlist), approximately one-quarter of all chondrichthyans are threatened with extinction (Dulvy et al. 2014), making them one of the most vulnerable extant vertebrate groups. Life-history traits, such as slow growth rates, late age at maturity, low fecundity and small litter sizes, make it difficult for many chondrichthyan populations to recover from anthropogenic pressures (Musick et al. 2000; Stevens et al. 2000; Field et al. 2009). South-east Africa is recognised as a global shark biodiversity hotspot (Lucifora et al. 2011). An estimated 30% of species are endemic to South Africa and neighbouring countries, such as Namibia and Mozambique

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(Compagno 1999). In the latest comprehensive review, 185 species of chondrichthyans were recorded in South African waters and consist of 109 shark species, 68 ray species and eight chimaera species (Compagno et al. 2005). Approximately 20 additional species have since been described, redescribed or newly documented in South African waters, particularly rays and deep-dwelling species (e.g. Smale et al. 2002; Ebert and Compagno 2007, 2009; Aschliman et al. 2010; Kemper et al. 2010; Ebert et al. 2011; Ebert and Cailliet 2011; Ebert et al. 2015). Despite this large level of biodiversity and endemism, our knowledge of South African chondrichthyans is relatively scarce (Compagno 1999; Ebert and van Hees 2015). Furthermore, approximately one-third of the southern African species are classified as rare and are known from less than 10 records of each species (RSA 2014). A diverse range of habitats over a small spatial scale restricts distribution ranges within many species, resulting in low abundance and pronounced vulnerability to over-fishing (Turpie et al. 2000; Best et al. 2013). There is evidence of declines for many shark species in South Africa, based on fishery-dependent and -independent catch data (Dudley and Simpfendorfer 2006; da Silva and Bürgener 2007), while it is difficult to determine the impact that fisheries have on ray and skate species, as there is a lack of species-specific data (Attwood et al. 2011; Best et al. 2013). This is particularly concerning, since Dulvy et al. (2014) identified rays as among the most threatened of global chondrichthyan taxa. In line with global trends, approximately one-quarter of chondrichthyan species found in southern African waters are listed as threatened in the IUCN Red List (IUCN 2009; Dulvy et al. 2014). South African shark fisheries have gained a significant profile over the last decade with increased shark exports from South Africa expanding into the fintrade and shark fillet industry (da Silva and Bürgener 2007). To meet the high demand, a large number of sharks are caught in South Africa via directed and by-catch fisheries with a total shark catch estimated at 6 562 t y−1 (DAFF 2013). Approximately 49% of local chondrichthyan species are affected by nine fisheries in South Africa (da Silva et al. 2015). The main species that are affected by the demersal shark trade include smoothhound sharks (Mustelus mustelus Linnaeus, 1758), tope sharks (Galeorhinus galeus Linnaeus, 1758), bronze whaler sharks (Carcharhinus brachyurus Günther, 1870), dusky sharks (Carcharhinus obscurus Lesueur, 1818) and whitespotted smoothhound sharks (Mustelus palumbes Smith, 1957) (da Silva and Bürgener 2007). These, and the other shark species affected by the South African fisheries (estimated 2010 catch 1 t; DAFF 2013), are listed in Table 1, together with their respective IUCN Redlist status. The endemic ploughnose chimaera species, Callorhinchus capensis Duméril, 1865, is also listed since the total catch in 2010 was estimated to be approximately 900 t (DAFF 2013). In this paper, we aim to highlight the important gaps in genetic information of southern African chondrichthyan species, with particular reference to issues related to species identification, taxonomy and cryptic speciation, population structure and reproductive behaviour. We provide examples on how molecular genetics can be

Bester-van der Merwe and Gledhill

used to assist with the management and conservation of southern African sharks and discuss issues in light of the unique chondrichthyan biodiversity and fisheries challenges in terms of species and stock structure identification for a developing region. Species identification and related issues for shark fisheries management One of the most basic and fundamental, but arguably one of the most important, requirements for sound conservation and management is the correct identification of species. Effective planning and enforcement of management regulations is reliant on identifying harvested and non-harvested organisms and their products (Ovenden et al. 2015). Correct species identification is essential to define the species composition of a fishery, identify by-catch (whether retained or discarded), delineate distribution ranges and to assess fishery stocks. Correct species identification is challenging for fishers, managers, compliance officers and researchers alike, particularly for shark species with little phenotypic variation, such as carcharhinids (Tillett et al. 2012a). Of particular concern is that most fisheries do not undertake species identification in catch statistics and sharks are often grouped into generic categories (FAO 2011). Furthermore, sharks are primarily processed at sea where the main morphological characteristics used to identify sharks to the species level, such as heads and fins, are removed. Without correct species identification information, the impact that fisheries could have on individual species is largely unknown. Non species-specific reporting of chondrichthyans Generic reporting for elasmobranch catches in South African fisheries is a common practice. Dogfish Squalus spp., skates Raja spp., shysharks Haploblepharus spp. are all listed in generic groups (Attwood et al. 2011; Best et al. 2013). Processed spotted gully sharks (Triakis megalopterus Smith, 1839), tope sharks and two smoothhound species M. mustelus, M. palumbes are all referred to as gummy or hound sharks. Bronze whaler (or copper) sharks, dusky sharks and blacktip sharks (Carcharhinus limbatus Müller and Henle, 1839) are processed and all sold under the name bronze whalers (da Silva and Bürgener 2007). For chondrichthyans that are listed to the species level, there is also potential for misidentification in fisheries records. Whitespotted smoothhounds, dusky sharks and, although in small numbers, sandbar sharks (Carcharhinus plumbeus Nardo, 1827) have all been documented in fisheries in False Bay (Lamberth 2006; da Silva and Bürgener 2007). None of these species, however, were reported in fisheries survey and catch data, possibly due to misidentification as other species or generic fisheries reporting (Best et al. 2013). Furthermore, all Squalus species occurring along the southern African coast are lumped into Squalus spp. records including the two most common species, Squalus acutipinna (formerly S. megalops Macleay, 1881) and S. cf. mitsukurii, as well as an offshore species, Squalus acanthias Linnaeus, 1758. Many of the Squalus species have previously been

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Table 1: Shark species affected by South African fisheries: International Union for the Conservation of Nature (IUCN) Redlist status, where DD  Data Deficient, NT  Near Threatened, V  Vulnerable, E  Endangered, estimated catch of 2010 in tons and main fishery sectors impacting on the respective species (DAFF 2013). Species in bold denote sharks endemic to southern Africa. Trawl  inshore, offshore, midwater and prawn trawl; Linefish  traditional linefishery; Shark longline  demersal and pelagic shark longline; Hake longline  hake longline fishery; Tuna longline  tuna and swordfish pelagic longline (DAFF 2013)

Family Hexanchidae Carcharhinidae

Triakidae

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Sphyrnidae

Lamnidae Squalidae Scyliorhinidae

Callorhinchidae

Species Notorynchus cepedianus Carcharhinus brachyurus Carcharhinus obscurus Carcharhinus limbatus Prionace glauca Galeorhinus galeus Mustelus mustelus Mustelus palumbes Mustelus mosis Triakis megalopterus Sphyrna zygaena Sphyrna lewini Sphyrna mokarran Isurus oxyrinchus Squalus acutipinna Halaelurus natalensis Haploblepharus edwardsii Haploblepharus fuscus Haploblepharus pictus Holohalaelurus regani Poroderma africanum Poroderma pantherinum Scyliorhinus capensis Callorhinchus capensis

IUCN status DD NT VU NT NT VU VU DD DD NT VU EN EN VU DD DD NT VU LC LC NT DD NT LC

misidentified (Naylor et al. 2012) and the genus is under ongoing revision. Without species-specific data, population trends and accurate fisheries data cannot be assessed. Due to the difficulty in identifying chondrichthyans to the species level, particularly postharvest, genetic techniques can be used to assist in this pursuit. Seafood fraud and illegal trade Recently, there has been considerable interest and concern surrounding the mislabelling of fishery products, both globally and locally. Helyar et al. (2014), for example, reported on seafood mislabeling in the UK and highlighted the importance of genetic testing across the supply chain with the ultimate goal of achieving sustainability of exploited marine resources. A high incidence (31%) of mislabelling of South African commercial fish products and species substitution and fraud has been found (Cawthorn et al. 2012). Although this study did not detect any substitution with shark meat per se, according to the Southern Africa Sustainable Seafood Initiative (SASSI) fish retailers are increasingly selling unlabelled or mislabelled shark products due to its unpopularity with local consumers (Atkins 2010). Shortfin mako sharks, for example, are being renamed and sold as gummy, lemon fish or ocean fillets to increase its consumer appeal and to disguise allowable catch limits (Atkins 2010). DNA barcoding, for example, revealed a high incidence of seafood fraud in Italian commercial shark fillets (Barbuto et al. 2010). Lucrative Mustelus species were substituted with the less valuable

Estimated catch 2010 (t) 1–10 201–300 11–100 1–10 301–400 401–500 300–400 11–100 1–10 1–10 1–10 1–10 1–10 501–600 11–100 1–10 1–10 1–10 1–10 1–10 1–10 1–10 1–10 801–900

Main directed fisheries

Main by-catch fisheries

Shark longline, Linefish Shark longline, Linefish Shark longline, Linefish Shark longline Pelagic longline Shark longline, Linefish Shark longline, Linefish Shark longline, Linefish Shark longline, Linefish Shark longline, Linefish Shark longline Shark longline Shark longline Pelagic longline – – – – – – – – – St Joseph net

– Hake longline, Trawl Hake longline, Trawl – Tuna pole Hake longline, Trawl Hake longline, Trawl Hake longline, Trawl Hake longline, Trawl Hake longline, Trawl Linefish, Tuna longline Linefish, Tuna longline Linefish, Tuna longline Tuna pole Trawl, Hake longline Trawl, Hake longline Trawl, Shark longline Trawl Trawl Trawl, Hake longline Trawl, Shark longline Trawl, Shark longline Trawl Trawl

spiny dogfish (Squalus acanthias), shortfin mako (Isurus oxyrinchus Rafinesque, 1810) and blue shark (Prionace glauca Linnaeus, 1758) meat in 77% of samples tested. Similarly, in a small-scale study testing 20 commercial samples taken from different Spanish supermarkets, only 20% of ray species (Raja spp.) were correctly labelled (Lago et al. 2012). To date, no studies have been conducted to assess the accuracy of labelling of shark products in South Africa. Since most of the aforementioned species are prevalent and caught by fisheries along the southern African coast, simple but sensitive molecular tests will aid in monitoring local shark fisheries and enhance transparency on the domestic market. Despite a growing movement of regional, national and international regulations and management of shark species, there is evidence of continued trade in protected species. Approximately 5% of shark fillets sampled at markets, supermarkets, street vendors and in restaurants in Taiwan were identified as species listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) Appendix II, including two species of hammerhead Sphyrna spp., oceanic white tip (Carcharhinus longimanus Poey, 1861) and white shark (Carcharodon carcharias Linnaeus, 1758) (Liu et al. 2013). A high incidence (55% of all samples tested) of the critically endangered largetooth sawfish (Pristis pristis Linnaeus, 1758) were found in Brazilian fish markets (Melo Palmeira et al. 2013) and almost one-third of fish samples tested for species substitution in Italian fish markets included white

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shark (Filonzi et al. 2010). Of similar concern is the growing demand for the Asian shark fin market that is undoubtedly a major contributor to the global shark trade (Clarke et al. 2005, 2006). The number of fins obtained from Illegal, Unreported and Unregulated (IUU) sources is estimated to be three to four times the amount of reported catch data (Clarke et al. 2006). In southern Africa, illegal shark finning continues to be problematic, despite legislation prohibiting the activity (da Silva et al. 2015). Taxonomic identification and confusion There is confusion around the identification and taxonomy of some of the smaller, demersal sharks in South African waters, including Mustelus and Haploblepharus species. It has been proposed that there is potential for cryptic species and/or hybrids within these genera (Human 2007). Off the southern African coast, there are three Mustelus species found: Mustelus mustelus, M. palumbes and hardnose smoothhound (Mustelus mosis Hemprich and Ehrenberg, 1899) (Smale and Compagno 1997). Mustelus mustelus and M. palumbes are overlapping in distribution, whereas M. mosis rarely occurs on the east coast. Although M. palumbes is fairly easily distinguishable from M. mustelus, there is a lack of species-specific data and identification remains problematic, especially for juveniles. Fisheries, population and distribution data for these sharks may be confounded due to misidentification between the species. The identification and nomenclature of Mustelus species are also problematic globally (White and Last 2006, 2008; Farrell et al. 2009; Boomer et al. 2012; Velez-Zuazo et al. 2015). Based on a combination of microsatellites and mtDNA forensic markers, species misidentification of Mustelus spp. by a South African commercial fishing company was recently revealed. Fifty-nine hound shark samples were morphologically identified as G. galeus or ‘vaalhaai’ (the local Afrikaans common name for soupfin sharks) (Maduna 2014). Using cytochrome oxidase subunit 1 (CO1) barcoding analysis, only 25 were confirmed to be G. galeus, whereas 20 were revealed as M. mustelus and a further four as M. palumbes (Maduna 2014). This highlights the difficulty in distinguishing between these commercial species and how species-specific catch data can be subsequently misrepresented in commercial fisheries records. Further studies need to be conducted to determine the degree of this misidentification in all fisheries sectors in South Africa. Apart from identification issues that relate to known species, classification of cryptic or newly described species is complicated. A holistic approach combining genetic and morphometric data is essential. With such great elasmobranch diversity in the Southern Hemisphere, taxonomic problems exist for the smaller demersal and benthopelagic species with high prevalence of cryptic species complexes, as well as for many deep-sea species vulnerable to offshore fishing pressures (Straube et al. 2010; Dudgeon et al. 2012). Other than the Mustelus species, the family Scyliorhinidae (catsharks) is also considered as one of the most systematically troublesome taxonomic groups. A high degree of morphological conservatism is observed in the genus Haploblepharus (Human 2003, 2007). Four Haploblepharus species are found in South Africa. Three species are endemic to South Africa; puffadder

Bester-van der Merwe and Gledhill

shysharks (Haploblepharus edwardsii Schinz, 1822), brown shysharks (H. fuscus Smith, 1950) and Natal shysharks (H. kistnasamyi Human and Compagno, 2006), whereas the dark shyshark (H. pictus Müller and Henle, 1838) is also found in Namibia (Compagno et al. 2005). The poor choice of morphological characteristics, including colour patterns, in species identification keys (Human 2007) has potentially led to the over- and/or under-estimation of the abundance of these species and confusion over the distribution ranges. Juvenile specimens are difficult to identify and it is possible that hybridisation may occur between species of this genus, further complicating species identification (Human 2007). Correct identification of Haploblepharus species is of conservation importance due to differences in IUCN Redlist classifications of each of the species. All species are listed on the IUCN Redlist under the following categories: H. kistnasamyi – Critically Endangered; H. fuscus – Vulnerable; H. edwardsii – Near Threatened; and H. pictus – Least Concern (Table 1). There is a need for phylogenetic analysis and a revision of morphological characters to clarify the relationships between species of this group (Human et al. 2006). Molecular markers could be used to re-evaluate species separation in Haploblepharus and confirm whether the current morphological characters used in taxonomic keys should be reassessed. This will aid in the conservation and management of the southern African shyshark species. Molecular tools for species identification Currently, the most frequently used genetic tool in species identification is DNA barcoding, in which a standardised DNA fragment, typically mitochondrial DNA, is sequenced and compared against a reference database in order to identify or ‘fingerprint’ a particular species (Hebert et al. 2003). DNA barcoding of elasmobranchs is primarily based on a single fragment of the CO1 mitochondrial gene with a length of 648 bp. Results from a variety of animal taxa show that the CO1 gene has been effective in distinguishing between 95% of species (Hebert et al. 2003; Ward et al. 2005; Hajibabaei et al. 2006). DNA barcoding using the CO1 gene has been used to successfully identify fish species (Ward et al. 2005, 2008; Hubert et al. 2008; Steinke et al. 2009), including shark species (Holmes et al. 2009; Wong et al. 2009; Ward 2009; Moftah et al. 2011; Moura et al. 2015). The efficiency of the CO1 gene region for identification of many chondrichthyans at a species-specific level remains untested and currently there is some uncertainty about the use of this region as a universal DNA barcoding gene for chondrichthyans (Daley et al. 2012; Naylor et al. 2012). The limitation of this region was demonstrated in a recent study that evaluated the suitability of the CO1 region for species identification in three co-occurring Haploblepharus species, H. edwardsii, H. pictus and H. fuscus. The betweenspecies CO1 nucleotide variation was extremely low and could, for example, not discriminate the taxonomically identified H. fuscus specimens from the H. pictus specimens (Robbertze unpublished data). There are open-access public reference databases, such as the Barcode of Life Database (BOLD; http://www. boldsystems.org), FISH-BOL (http://www.fishbol.org) and GenBank (http://www.ncbi.nlm.nih.gov/genbank), for storing

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and matching genetic data. The BOLD and FISH-BOL databases are part of the International Consortium of the Barcode of Life initiative (http://www.ibol.org). The BOLD database consists of several recognised DNA barcoding genes with high-quality assurance requirements, whereas GenBank is a repository for all available DNA sequences. While using barcoding as a tool for identifying verified species is well accepted, it’s use in ‘discovering’ new species and assigning taxonomic placements based solely on genetic data remains controversial (DeSalle et al. 2005; DeSalle 2006). DNA barcoding should be a tool to accompany and complement taxonomy. Classical taxonomy screens numerous individuals from multiple localities across the range of a species to distinguish variation within a species from variation between species (DeSalle et al. 2005). This same approach needs to be applied for barcoding data. At present, global reference databases, such as BOLD or FISH-BOL, are incomplete and multiple entries exist primarily for cosmopolitan species. Multiple entries from different regions for each species will improve the accuracy of these databases (Dudgeon et al. 2012; Velez-Zuazo

Endemics regional

et al. 2015). Barcoding data are available for almost 70% of the 66 extant shark species found in southern African waters, with multiple entries in BOLD for the majority of the cosmopolitan species (Figure 1). However, less than 50% of southern African endemic species have been barcoded. The integrity of the barcode data available through online databases is dependent on the correct identification of the specimen that was sampled. Since 35% of the southern African species are considered rare (i.e. less than 10 records), taxonomic clarity through morphometric identification, photographic identification, detailed specimen and collection data to accompany the genetic data is essential to ensure the accuracy of reference barcode data. Barcode sequences should be accompanied by voucher specimens deposited in museums or, at the very least, voucher photographs considering the logistical challenges associated with the preservation and archival of large-bodied animals such as sharks (Doukakis et al. 2011). It is recommended that nucleotide sequence similarity searches against reference databases should be performed using the BOLD database (restricted to taxonomically validated specimens) in preference to

49%

Species barcoded Taxonomic records

66%

Chondrichthyans regional Carcharhiniformes global

78%

Orectolobiformes global

91%

Heterodontiformes global

75%

Lamniformes global

88%

Hexanchiformes global

100%

Squaliformes global

82%

Squatiniformes global

90%

Pristiformes global

55%

Pristiophoriformes global

60% 77%

Rajiformes global Torpediniformes global

85%

Chimaeriformes global

94%

0

100

200 300 NUMBER OF SPECIES

400

500

Figure 1: Percentage of recorded chondrichthyan species with DNA barcodes available in the Barcode of Life Database (BOLD). Southern African fauna and endemics (depicted as ‘regional’) are compared with the total number of DNA barcodes (‘global’) available for 12 of the 13 orders

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GenBank, in order to ensure success and accuracy of species identification (Helyar et al. 2014). Molecular species identification is not restricted to barcoding and several other markers and approaches have sufficient discriminatory power in chondrichthyans. Using a 1 044 bp fragment of the mitochondrial NADH2 gene, Naylor et al. (2012) identified 574 species of elasmobranchs (305 sharks and 269 batoids), representing 56 of the 57 described families. Similarly, multiplex-PCR (Farrell et al. 2009), restriction fragment length polymorphism (RFLPs), PCR-RFLPs (Mendonça et al. 2009) and amplified fragment length polymorphisms (AFLPs) (Zenger et al. 2006) have also been used in shark species identification. The South African National Plan of Action for the Conservation and Management of Sharks (hereafter referred to as SA NPOA-Sharks) has identified that there are gaps in the knowledge of the taxonomy and classification of South Africa’s shark species (DAFF 2013). The SA NPOA-Sharks has listed the reclassification of all rays, skates and deepwater shark species using morphometrics and genetics as an immediate priority (DAFF 2013). Acquiring baseline barcode data of all South African chondrichthyans should be a research and funding priority. Reference barcode data for all described South African species are urgently needed so that samples from an undetermined species can be compared to reference barcode databases. Barcoding the South African endemic species needs to be a priority, as less than half of the species have been barcoded. Similarly, molecular genetic tools can be used to identify species of conservation concern postharvest and in processed products available in the commercial market, including meat, fins and even in processed, multi-species products, such as shark cartilage pills (Chapman et al. 2003). Species-specific assays using multiplex PCR reactions incorporating species-specific primers targeting the nuclear ribosomal RNA internal transcribed spacer 2 (rRNA ITS2) and mitochondrial cytochrome b (cytb) have been developed to identify sharks of conservation concern in the shark fin market, such as white sharks (Chapman et al. 2003), three hammerhead species listed in CITES Appendix II (Abercrombie et al. 2005), and basking sharks (Cetorhinus maximus Gunnerus, 1765) (Magnussen et al. 2007). This is a rapid, low-cost technique that requires only PCR and distinguishes between multiple species simultaneously (Abercrombie et al. 2005). As such, this technique is suitable for use in developing countries, such as southern African nations, where financial restraints may limit large-volume sequencing. Evidence of cryptic speciation and hybridisation of elasmobranchs is emerging, further complicating taxonomic assessment and species identification. The first documented naturally occurring shark hybrids between two sympatric blacktip shark species of the genus Carcharhinus were recently confirmed, using a combination of morphological characteristics and DNA-based diagnostic markers (Morgan et al. 2012). Subsequently, the first wild hybrid individual of two Manta species (Walter et al. 2013) and hybridisation between two freshwater stingrays has been identified (Cruz et al. 2015). Two co-occurring Mustelus species, M. mustelus and M. punctulatus, in the Adriatic Sea

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also showed signs of hybridisation and/or gene introgression when tested for the nuclear codominant marker ITS2 to evaluate reproductive interactions (Di Francesco 2011). Taking into account the similar reproductive modes for at least two of the Mustelus species present in southern Africa (Boomer et al. 2012), hybridisation is a possibility. DNA-based identification together with morphological data, such as stretched total length (STL), pre-caudal length (PCL) and vertebral counts (Morgan et al. 2012), could verify the occurrence of hybrids or potential hybrid zones that need to be maintained and ideally managed separately from the parental species. Clarification of the taxonomy and identification of the Mustelus spp. and Haploblepharus spp. is imperative due to the commercial importance of Mustelus spp. in South Africa and the varying threatened statuses of these species. Identifying the presence of cryptic species or hybrids using a combination of morphological and genetic techniques will determine whether this is contributing to taxonomic confusion in these species (Dudgeon et al. 2012; Morgan et al. 2012). Population structure assessment for shark fisheries management Population genetic analysis at and below the species level forms an important basis for species conservation and sustainable exploitation through the identification of the number of stocks and management units within the geographical distribution of a target species (Ovenden et al. 2009). In fisheries, the stock concept is directly linked to the management of populations or units that are ecologically separated through space and time (Waples 1998). Correct identification of genetically differentiated stocks is vital for the proper management of exploited species and the preservation of their genetic diversity, and the genetic integrity of individual species. Despite the absence of obvious barriers to gene flow, most fish species do not comprise a single-continuously distributed, panmictic population of individuals (Hemmer-Hansen et al. 2007). In the past, the extent of genetic differentiation between shark populations was expected to be low due to their capacity to move extensively, however, several studies have shown different levels of population genetic subdivision over large and smaller spatial scales (e.g. Dudgeon et al. 2009; Ovenden et al. 2009; Feldheim et al. 2010; Geraghty et al. 2013). Population genetic structure can range from, for example, basking sharks that display little genetic differentiation over ocean basins globally (Hoelzel et al. 2006) to blacktip sharks that show significant structure over small, regional scales (Keeney et al. 2005; Sodré et al. 2012). At present, carcharhinids have the greatest representation in the phylogeographic literature, as many of these species are of economic importance in commercial, recreational and artisanal fisheries (Dudgeon et al. 2012). These species, however, may have been studied in greater detail due to the fact that it is easier to obtain access to a sufficient number of samples of the species that are encountered through fisheries. Along the southern African coast, the warm Agulhas Current and the cold Benguela Current have a strong influence on marine populations of the east and west

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coast of southern Africa, respectively (Hutchings et al. 2009). It has recently been proposed that the physiological features and dynamics between these two ocean currents, together with climate oscillation, are the major driving forces behind the historical and contemporary restriction of gene flow across the Indian/Atlantic boundary (Teske et al. 2013). Despite the uncertainty of the exact mechanism, this Indian/Atlantic boundary is evident in many globally distributed species as well as in a number of coastal fishes around southern Africa (Henriques et al. 2012, 2014), where the Benguela Current has shown to have a strong influence on the magnitude and directionality of gene flow. Signatures of historical gene flow across the boundary during warm interglacial periods (Peeters et al. 2004) and evidence for intensification of the Agulhas leakage due to climate change and a weaker Agulhas Current (Biastoch et al. 2008; van Sebille et al. 2009) could explain the lesser degree of genetic distinctness between the west and east coast populations for some species, as well as the presence of a transition/admixture zone for some. For example, the Benguela barrier does not appear to be a genetic barrier to temperate shark species, such as Carcharhinus brachyurus (Benavides et al. 2011) and Galeorhinus galeus (Chabot and Allen 2009). In a recent study, microsatellites revealed strong genetic differentiation between South-east Atlantic and South-west Indian Ocean Mustelus mustelus, indicating that for this smaller coastal shark, the Indian/Atlantic boundary (Teske et al. 2013) most likely restricts contemporary gene flow (Maduna 2014). Interestingly, mtDNA sequence data for the same species found relatively high levels of interoceanic gene flow and these differential genetic signals reflected by the two molecular marker types was attributed to either genderbiased dispersal or the more conservative evolutionary properties inherent to mitochondrial DNA markers as apposed to microsatellites (Maduna 2014). Reproductive philopatry and resultant gender-biased dispersal is another mechanism that can account for population structure, despite the absence of barriers to movement (Dudgeon et al. 2012; Chapman et al. 2015). Reproductive philopatry, where females show site-fidelity to nursery grounds, has been identified in numerous coastal, viviparous shark species using a combination of microsatellites and mtDNA sequence data (Feldheim et al. 2002; Keeney et al. 2005; Karl et al. 2011; Tillett et al. 2012b). Importantly, the spatial scale at which philopatry occurs varies between species, thereby further contributing to differential genetic structuring patterns observed amongst species. Identifying critical areas within a species’ distribution range, such as nursery sites, could make a strong case for giving these areas special attention (Mourier and Planes 2013; Feldheim et al. 2014). In South Africa, many coastal embayment or river mouth areas such as Storms River, Algoa Bay and Thukela River have been identified as nursery grounds for shark species (Smale 2002; Dicken 2008; Hussey et al. 2009). We need to gain a greater understanding of the behavioural dynamics of shark populations in these areas and how these processes may affect the genetic structure of a species, particularly for commercially important species, in order to identify areas of critical protection.

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Molecular tools for population structure and demographic assessment Several molecular marker types are available for assessing gene flow patterns of organisms with different life-history traits and dispersal capabilities. In sharks, genetic variation is known to be low, with mitochondrial sequence evolution estimated to be six to eight times slower than that of mammals (Martin et al. 1992). Despite this, a number of studies have been successful in assessing population differentiation in sharks using a portion of the mitochondrial control region (mtCR) (Keeney et al. 2005; Keeney and Heist 2006; Duncan et al. 2006; Chabot and Allen 2009; Sodré et al. 2012). Microsatellites, or simple sequence repeats (SSRs), have also been used and these highly variable short, tandem repeat DNA sequences have proven to be ideal for fine-scale stock structure investigations relevant to short-term fisheries management in sharks (Dudgeon et al. 2012). Although several species-specific microsatellite markers have been isolated and characterised for elasmobranch species (see Portnoy and Heist 2012) and cross-species transferability of microsatellites has been successful (Boomer and Stow 2010; Chabot and Nigenda 2011; Maduna et al. 2014), very few studies have assessed genetic structure of sharks and other elasmobranchs along the South African shoreline (Table 2). While a number of global or cross-oceanic studies of cosmopolitan species included samples from southern Africa, only a few studies sampled both the Atlantic and Indian coastlines of the region and could test, for example, the validity of the Benguela barrier as a barrier to dispersal. The effect of biogeographic barriers on the genetic structure of smaller, demersal and inshore-dwelling shark species is still largely unknown and warrants further investigation. Oceanographic features, such as ocean currents and thermal fronts, may have varying degrees of impact on species with different ecology and life-history traits. Preliminary investigation into the genetic structure of two demersal shark species targeted in regional fisheries, tope sharks and common smoothhounds, has revealed substantial differences in the degree of population differentiation (Bitalo et al. 2015). This highlights the importance of considering species-specific gene flow patterns in fisheries management practices and stock assessments. The Draft South African Shark Biodiversity Management Plan has identified the need for population genetic studies on sharks and has stated one of its objectives is ‘to improve knowledge of population delineation and genetic diversity of identified shark species’ (RSA 2014: 27). The South African coastline has recently been redefined as holding as many as nine marine bioregions, that incorporate both previously recognised coastal biogeographic regions, as well as newly demarcated offshore regions (Griffiths et al. 2010). Given the diversity of biogeographic regions and the many unique habitats that sharks inhabit along the South African coastline, such as the Langebaan Lagoon on the West Coast and the Breede and Umfolozi Rivers, it is imperative for the management and conservation of these species to understand whether these areas hold unique breeding and reproductive stocks (da Silva et al. 2013). Investigation

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Table 2: Studies involving population structure analysis of shark species targeted in South African fisheries. Information given includes the study area investigated, whether samples from southern Africa were included (SA samples), type and number of molecular markers used (mtDNA and nuclear), the population genetic structure found and references. mtCR  mitochondrial control region, ND2  nicotinamide adenine dehydrogenase subunit 2, ND4  nicotinamide adenine dehydrogenase subunit 4, SSRs  short sequence repeats or microsatellites

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Species

Study area

Carcharhinus brachyurus Global Carcharhinus obscurus Australia Carcharhinus limbatus North-western Atlantic Global Western Atlantic Prionace glauca Global Galeorhinus galeus Global South Africa Global Mustelus mustelus Southern Africa Triakis megalopterus Southern Africa Sphyrna lewini Global Australia Mexico Isurus oxyrinchus Global Global

SA samples included Yes No No Yes No – Yes Yes Yes Yes Yes Yes No No No Yes

Markers used mtDNA mtCR mtCR mtCR mtCR mtCR mtCR mtCR ND2 ND2 ND4 mtCR – ND4 mtCR – –

into the genetic stock structure of shark species, particularly species that are affected by fisheries, should form an integral part of the multidisciplinary approach to fisheries management. Table 2 shows a summary for the species for which this has been addressed on a regional scale and highlights the substantial gaps in the basic population genetic information for southern African sharks affected by fisheries. Similar to population structure inference, molecular markers and genetics have increasingly been used to assess reproductive biology and mating behaviour in sharks. Most notably is the investigation of polyandry and multiple paternity, using as few as four microsatellite markers. Multiple paternity has been identified in numerous shark species (reviewed in Byrne and Avise 2012); however, the benefits of multiple paternity in sharks is to a large extent still unclear (Daly-Engel et al. 2010; Portnoy and Heist 2012). No evidence of increased genetic diversity and fitness in multiple-sired litters of lemon sharks (Negaprion brevirostris Poey, 1868) were found when compared to singly sired litters (DiBattista et al. 2008). Karl (2008) demonstrated that multiple paternity might even lead to a reduction in effective population size (Ne) by increasing the variance in male reproductive success. Nonetheless, it is important to know whether reproductive behaviour is affected in commercially fished areas and how this behaviour varies across species and populations. Three species of commercial importance in South Africa, M. mustelus, S. lewini and C. obscurus, have been examined for the presence of multiple paternity (MP) using microsatellite panels optimised for each species (Rossouw unpublished data). Differences in frequencies of multiple paternity were observed between species and coincides with the variation in MP rates previously observed across species (Boomer et al. 2013). The differences in MP rates between populations of the same

Nuclear – 4 SSRs 8 SSRs – – 16 SSRs – 22 SSRs 22 SSRs 12 SSRs 6 SSRs 13 SSRs 8 SSRs 15 SSRs RFLPs 4 SSRs

Genetic structure Inter-oceanic Regional, only mtCR Regional, both markers Across Atlantic Regional, western Atlantic Across equator Inter-oceanic Low to absent Inter-oceanic Atlantic-Indian Atlantic-Indian Low to absent Absent Regional, both markers Absent Absent

Reference Benavides et al. (2011) Ovenden et al. (2009) Keeney et al. (2005) Keeney and Heist (2006) Sodré et al. (2012) Fitzpatrick (2012) Chabot and Allen (2009) Bitalo (unpublished data) Bitalo (unpublished data) Maduna (2014) Soekoe (unpublished data) Daly-Engel et al. (2012) Ovenden et al. (2011) Nance et al. (2011) Heist et al. (1996) Schrey and Heist (2003)

species should ideally also be assessed to elucidate the possible effect of fishing on mating behaviour in heavily fished versus non-fished areas. Furthermore, the identification of genetic bottlenecks and any other population size fluctuations due to anthropogenic pressures, such as overfishing, are of great importance to management and conservation of marine species. Populations that are currently large may have had small long-term Ne due to a genetic bottleneck or population size decline in the past (Turner et al. 2002). The identification of present populations with a reduced Ne is therefore essential for conserving a species’ genetic diversity. Estimating contemporary Ne for chondrichthyans is important, since many species are overexploited in fisheries globally (Musick et al. 2000). The effective population size could give an indication of both the breeding population size and the general health of a population (Portnoy et al. 2009). In particular, understanding the relationship or ratio between Ne and demographic census sizes can be informative for management decisions (Dudgeon et al. 2012). Estimates of Ne can be based on genetic or demographic data, but since demographic information (e.g. sex ratio, fluctuation in population size and pedigree data) is often unavailable for wild populations, current approaches to estimating effective population sizes are based on indirect methods using genetic information (Dudgeon et al. 2012). Most valuable to fisheries management is the development and utility of single time-point estimations of Ne, in which only a single sampling of a population is needed. Using molecular markers, the single time-point method has recently successfully been applied to estimate Ne in a number of elasmobranch species (Ahonen et al. 2009; Chapman et al. 2011; Blower et al. 2012). An emphasis should be placed on effective population size estimates of species that are particularly vulnerable to exploitation. This could enable the identification of species or populations that show critically

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lower population estimates compared with unexploited species or populations in protected areas. Next generation technologies and future investigations In the last decade, advances in molecular technologies, such as next generation sequencing (NGS) and high-throughput genotyping methods, have greatly facilitated the genetic characterisation in many non-model organisms (Ekblom and Galindo 2011). With such a diverse shark biodiversity in southern Africa, these technologies now also allow for individual species to be characterised on a genome-wide level at a lower cost. With the many NGS and high-throughput genotyping platforms now available, a larger number of molecular markers with a higher genome representation can be developed and genotyped in sharks. Single nucleotide polymorphisms (SNPs) are becoming increasingly popular for genetic applications due to their abundance in genomes and suitability for automation (Morin et al. 2009). Novel approaches towards identifying SNPs include the utilisation of these NGS data sets, followed by various SNP discovery bioinformatics pipelines, allowing increased throughput while reducing the cost (Etter et al. 2011). When studying organisms lacking a reference genome, genome subsampling can be attained using restriction-site associated DNA (RAD) sequencing, which creates short DNA fragments that are adjacent to recognition sites of specific restriction enzymes, resulting in RAD tag libraries (Etter et al. 2011). The SNPs are then detected by sequencing the nucleotides next to the restriction enzyme sites. The RAD sequencing approach accelerates SNP discovery and, in turn, can be used to genotype hundreds or thousands of SNP markers in several individuals across different populations (Etter et al. 2011; Davey et al. 2013). Similarly, larger numbers of SNPs can also be identified alongside microsatellites through next generation transcriptome sequencing (Helyar et al. 2012; Krück et al. 2013). Transcriptome sequencing enables characterisation of all transcriptional activity, coding and non-coding, and can therefore also be applied to study gene expression and functional variation (Marguerat and Bähler 2010). In sharks, this can be a powerful tool to investigate, for example, ecological adaptation of species to certain areas, such as freshwater or river areas. In addition, comparative transcriptomics can be useful in assessing important molecular features associated with physiological and behavioural differences within species, thereby examining various hydrological and temperature regimes as well as biogeographic zones across the southern Africa coastline. Conclusion A growing concern for declining shark populations, coupled with an increase in South Africa’s shark fisheries, means that shark populations and stocks need careful monitoring in order to achieve sustainable utilisation of this fisheries resource. Species- and population-specific information is imperative to reach this goal. Together with traditional fisheries management measures, genetic techniques can be used for a wide range of chondrichthyan conservation and management applications. Molecular tools can assist

in species identification by elucidating species composition of fisheries, identifying by-catch, and identifying fisheries products postharvest. Genetic data can be used for compliance and enforcement purposes, and to identify species of conservation concern, such as those protected by national legislation (e.g. the South African Marine Living Resources Act; RSA 1998) or international trade conventions (e.g. CITES). Evidence of mislabelling or seafood fraud, and fishing and trade in protected species, can be gathered and used in prosecution cases. In addition, variation in patterns of genetic structuring among different shark species has been demonstrated and genetics is increasingly being incorporated into stock structure assessment in fisheries. Our present knowledge of the genetic structure and connectivity of southern Africa’s shark populations on a national and regional scale is scarce, and only a few studies have evaluated population differentiation across known biogeographical barriers. More information is urgently needed, and both the South African NPOA-Sharks (DAFF 2013) and the South African Draft Shark Biodiversity Management Plan (RSA 2014) identify the need to employ genetics to assist with stock structure analysis. Historically, the great expense of genetic technologies has limited research in developing nations and the exciting transition towards NGS and genome-wide research will see greater development and application of genetic research on a regional scale. The declining costs in equipment and reagents, coupled with advances in knowledge, techniques and high-throughput technologies, is making the field more affordable and more tangible for scientists and managers from developing nations. The integration of genetic research, development and application will assist scientists, managers, government and compliance agencies in closing these gaps in current knowledge. This will contribute to an integrated approach to conservation and management of chondrichthyan populations in southern Africa and aid in the future sustainability of this fishery resource. Acknowledgements — We would like to acknowledge Simo Maduna, Daphne Bitalo and Charné Rossouw for laboratory work and for generating the genetic data that was used for the preliminary results and unpublished data mentioned in this study. We thank the Department of Agriculture Forestry and Fisheries, South African Shark Conservancy, the KwaZulu-Natal Sharks Board and the Port Elizabeth Museum, especially Charlene da Silva, Meaghen McCord, Malcolm Smale, Sabine Wintner and Tamzyn Zweig for collaboration and for providing valuable information and samples that made the theme of this manuscript possible. We thank the anonymous reviewers, whose constructive comments improved the final version of this manuscript. Financial support was provided by the the National Research Foundation, South Africa.

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Received 28 February 2015, accepted 9 June 2015 Associate Editor: Sandi Willows-Munro