Journal of Fish Biology (2010) 77, 1165–1172 doi:10.1111/j.1095-8649.2010.02770.x, available online at wileyonlinelibrary.com Genetic data show that Carcharhinus tilstoni is not confined to the tropics, highlighting the importance of a multifaceted approach to species identification J. J. Boomer*†, V. Peddemors‡ andA.J.Stow* *Department of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia and ‡Cronulla Fisheries Research Centre of Excellence, P.O. Box 21, Cronulla, NSW 2230, Australia (Received 26 June 2010, Accepted 9 August 2010) This study shows a range extension for the Australian blacktip shark Carcharhinus tilstoni,which was believed to be restricted to Australia’s tropical waters, of >1000 km into temperate waters, revealing its vulnerability to a wider commercial fishery. © 2010 The Authors Journal compilation © 2010 The Fisheries Society of the British Isles Key words: blacktip shark; Carcharhinus limbatus; mtDNA; shark fishery; species distribution. Sharks are proving to be especially vulnerable to anthropogenic pressures, in part this is a response to the K-selected natural-history traits that characterize many species along with pressure from targeted and non-targeted fishing activities (Stevens et al., 2000). In many cases, species distributions span state and international management boundaries, across which protection and management processes may vary for any given species (Last & Stevens, 2009). Therefore, effective conservation and manage­ ment require knowledge of species distributions. Despite the size and notoriety of sharks, distributions of some species remain uncertain due to limited opportunities for observation or difficulties with species identification. Sharks, especially those in the carcharhinid family, can be very difficult to identify to species level using morphological features (Chan et al., 2003). Distinguishing features for some species are few, and sharks caught by commercial fishers often have important features removed (e.g. head and teeth) before being landed ashore, making reliable identification very difficult (Chan et al., 2003; Last & Stevens, 2009). The fin trade exacerbates problems with identifying harvested sharks. A solution has been found with the development of DNA markers for species identification. Over the last decade, rapid improvements in DNA technology have seen a suite of markers developed which require only a small sample of tissue for reliable species identification (Chapman et al., 2003; Greig et al., 2005; Ward et al., 2008). The use of this technology has resulted in further taxonomic revision and improved †Author to whom correspondence should be addressed. Tel.: +61 2 9850 8143; fax: +61 2 9850 7972; email: [email protected] 1165 © 2010 The Authors Journal compilation © 2010 The Fisheries Society of the British Isles 1166 J. J. BOOMER ET AL . knowledge of shark species distributions (Gardner & Ward, 2002; Quattro et al., 2006; Corrigan et al., 2008). There are two species of shark referred to by the common name blacktip shark, the common blacktip shark Carcharhinus limbatus (Muller¨ & Henle) and the Australian blacktip shark Carcharhinus tilstoni (Whitley). These species are distinguished by vertebral counts and forensic methods (Lavery & Shaklee, 1991; Last & Stevens, 2009). Carcharhinus limbatus has a global distribution while C. tilstoni is believed to be restricted to the tropical waters off northern Australia (Fig. 1) where it occurs in sympatry with C. limbatus (Last & Stevens, 2009). Blacktip sharks are taken in commercial fisheries worldwide. It is generally accepted that C. limbatus is taken in most regions while in northern Australia both C. limbatus and C. tilstoni are taken by commercial fisheries (Ovenden et al., 2010). At present, no external morphological features are available to distinguish these species and therefore poor knowledge of catch rates compromises the development of sustainable fisheries. DNA technologies provide a solution to this problem (Shivji et al., 2002). Blacktip sharks are within the top five shark species commercially harvested in temperate waters of New South Wales (NSW) on the Australian east coast (Macbeth et al., 2009). Additionally, they are caught in this region by the shark beach meshing programme, which operates between September and May off 51 popular beaches between Newcastle and Wollongong (Green et al., 2009a). Historically, blacktip sharks occurring in NSW waters have been classified as C. limbatus (Green et al., 2009a). The species identity of blacktip sharks occurring in NSW, however, has Sydney N km 0 312·5 625 1250 1875 2500 Fig. 1. Currently recognized distribution of Carcharhinus tilstoni ( ), which is known from Thevenard Island (Western Australia) to Rockhampton (Queensland) along the continental shelf of tropical Australia (Last & Stevens, 2009). ( ), area from which samples for this study were collected. © 2010 The Authors Journal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 77, 1165–1172 RANGE EXTENSION OF CARCHARHINUS TILSTONI 1167 never been thoroughly investigated. In this study, genetic approaches were used to identify species of blacktip sharks occurring off Sydney, NSW, Australia. Tissue samples were collected from sharks caught in the NSW Shark Meshing (Bather Protection) Program (NSWDPI, 2009) (Fig. 1). DNA was extracted using a modified salting out technique (Sunnucks & Hales, 1996). Two mitochondrial genes (COI and ND4 ) were amplified. The cytochrome oxidase I (COI ) gene was ampli­ fied with the primers FishF1Mod 5� ACC AAC CAC AAA GAY ATY GGC AC 3� (modified from Ward et al., 2005) and FishR15� TAG ACT TCT GGG TGG CCA AAG AAT CA 3� (Ward et al., 2005). The sodium dehydrogenase subunit 4 (ND4 ) gene was amplified with primers designed in this study MaND4F 5� ACC MAA AGC YCA CGT WGA AGC 3� and MaND4R 5� TCT TGC TTG GAG TTG CAC CA 3�. These genes were selected for analysis because they have previously been successfully applied to distinguish C. limbatus and C. tilstoni (Ovenden et al., 2010). The COI gene was used to initially screen samples. A total of 54 samples identified by field observers as some form of carcharhinid shark were screened, 13 of these were identified as C. limbatus and five as C. tilstoni based on COI.Asa prior study suggested that C. limbatus and C. tilstoni may share haplotypes at the COI gene (Wong et al., 2009), these 18 samples were further investigated using the ND4 gene to confirm their species identity. For both genes, PCR reactions were in 15 μL volumes and contained ×1 PCR buffer, 2 mM MgCl2,0·2 mM of each deoxynucleoside triphosphate (dNTP), 0·5 μM of each primer, 1 U Taq DNA poly­ merase (Promega; www.promega.com) and 1 μL DNA template. Cycling conditions ◦ consisted of an initial denaturation step at 94 C for 3 min, followed by a touch­ ◦ down PCR with six cycles, decreasing the annealing temperature by 1 C per cycle. ◦ ◦ Denaturation was at 94 C (30 s), annealing at 60 to 55 C (1 min) and extension ◦ ◦ ◦ ◦ at 72 C (1 min). A cycle of 94 C(30s),55 C(30s) and72 C(1min)was ◦ then repeated 30 times, followed by a final extension of 72 C for 5 min. The PCR product was purified with EXOSAP-IT (USB; www.usbweb.com) and sequenced in an ABI 377 automated DNA sequencer. DNA sequences were assembled and aligned in MEGA version 4 (Tamura et al., 2007) with reference sequences for C. tilstoni and C. limbatus as well as reference sequences for the graceful shark Carcharhinus amblyrhynchoides (Whitley) and the bull shark Carcharhinus leucas (Muller¨ & Henle). Carcharhinus amblyrhynchoides has been shown to be very closely related to C. limbatus and was included in analyses to ensure this species was not present in the sample set (Ward et al., 2008; Ovenden et al., 2010). Reference sequences for each of these species were obtained for the COI region from GenBank (GenBank accession numbers: C. tilstoni, DQ108283; C. limbatus, EU39862; C. amblyrhynchoides, EF609307 and C. leucas, EF609311). The reference sequences for the ND4 gene for each species except C. leucas were obtained from Ovenden et al. (2010) (GenBank accession numbers: C. limbatus, GQ227272; C. tilstoni, GQ227268; C. amblyrhynchoides, GQ227276). The ND4 ref­ erence sequence for C. leucas was obtained as part of this study. The ND4 and COI sequences were concatenated for phylogenetic analysis giving a final sequence of 1353 base pairs (bp). Phylogenetic relationships were inferred using the neighbour­ joining (NJ) distance method implemented in MEGA 4 (Tamura et al., 2007) and the Bayesian method implemented in MrBayes 3.1 (Ronquist & Huelsebeck, 2003). For the NJ approach, the best model of nucleotide substitution was determined to be a Tamura–Nei model (TrN) using Akaike information criterion with the software © 2010 The Authors Journal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 77, 1165–1172 1168 J. J. BOOMER ET AL . FindModel (LANL, 2009), which is an internet-based application of the programme ModelTest (Posada & Crandall, 1998). Reliability of tree nodes for the NJ tree was assessed using 10 000 bootstrap replicates. The Bayesian approach to tree building was completed in MrBayes 3.1 (Ronquist & Huelsebeck, 2003) using a GTR + G substitution model. Four chains were run for 500 000 generations and a consensus tree was constructed. Carcharhinus leucas was used as the out-group for each of these phylogenetic analyses. Commercially harvested sharks, particularly species with slow growth and low fecundity such as the carcharhinids, are in dire need of well-developed management plans to slow population declines (Field et al., 2009). Of fundamental importance is the species identification. This study highlights the benefits of applying genetic approaches to species identification. The results show that the range of C. tilstoni extends into temperate waters, >1000 km further south than previously known, and that at least two species of blacktip sharks (C. limbatus and C. tilstoni ) occur in the temperate waters off Sydney, Australia. The topology of the phylogenetic trees clearly supports the presence of these two species using both the NJ and Bayesian approaches.