CSIRO PUBLISHING www.publish.csiro.au/journals/mfr Marine and Freshwater Research, 2010, 61, 253–262 Towards better management of Australia’s shark fishery: genetic analyses reveal unexpected ratios of cryptic blacktip species Carcharhinus tilstoni and C. limbatus J. R. OvendenA,C, J. A. T. MorganA, T. KashiwagiA, D. BroderickA and J. SaliniB AMolecular Fisheries Laboratory, Queensland Primary Industries and Fisheries, PO Box 6097, St Lucia, QLD 4069, Australia. BCSIRO Marine Laboratories, Cleveland, QLD 4163, Australia. CCorresponding author. Email: [email protected] Abstract. The common blacktip shark (Carcharhinus limbatus) and the Australian blacktip shark (C. tilstoni) are morphologically similar species that co-occur in subtropical and tropical Australia. In striking contrast to what has been previously reported, we demonstrate that the common blacktip shark is not rare in northern Australia but occurs in approximately equal frequencies with the Australian blacktip shark. Management of shark resources in northern Australia needs to take account of this new information. Species identification was performed using nucleotide sequences of the control, NADH dehydrogenase subunit 4 (ND4) and cytochrome oxidase I (COI) regions in the mitochondrial genome. The proportion of overall genetic variation (FST) between the two species was small (0.042, P < 0.01) based on allele frequencies at five microsatellite loci. We confirm that a third blacktip species (C. amblyrhynchoides, graceful shark) is closely related to C. tilstoni and C. limbatus and can be distinguished from them on the basis of mtDNA sequences from two gene regions. The Australian blacktip shark (C. tilstoni) was not encountered among 20 samples from central Indonesia that were later confirmed to be common blacktip and graceful sharks. Fisheries regulators urgently need new information on life history, population structure and morphological characters for species identification of blacktip shark species in Australia. Additional keywords: blacktip shark, COI, control region, cytochrome oxidase I, fisheries, Indonesia, NADH dehydrogenase subunit 4, ND4, species identification. Introduction The Australian blacktip shark (C. tilstoni) was described by Species are the basic unit for the sustainable management and Whitley (1950) and co-occurs in northern Australia with the conservation of biodiversity (Lindenmayer and Burgman 2005; common blacktip shark (C. limbatus). Carcharhinus tilstoni is King 2007). Species taxonomy is important because it shapes endemic to northern Australia and C. limbatus is found in sub- public recognition of biodiversity (Lindenmayer and Burgman tropical and tropical waters worldwide (Last and Stevens 2009). 2005), which drives the processes of ecosystem and biologi- There are no known external morphological characters for dis- cal resource management in the public and private sector. In tinguishing the species (Compagno et al. 2005), but among fisheries science, species are largely distinguished morphologi- specimens examined to date, C. tilstoni has 84–91 pre-caudal cally with the expectation of taxonomic congruence across other vertebrae while C. limbatus has 94–101 (Last and Stevens data types. DNA-based data can be used to test morpholog- 2009). Phylogenetic analyses by Lavery (1992) using allozyme ically defined species and can provide new tools for species characters and by Ward et al. (2008) using DNA characters identification. grouped C. tilstoni and C. limbatus with the graceful shark There are 322 chondrichthyan species currently recognised (C. amblyrhynchoides). in Australia (182 sharks, 125 rays and 15 chimaeras or ghost- Blacktip (C. tilstoni and C. limbatus) and other shark species sharks), which represents about one-third of worldwide chon- support a commercial fishery across northern Australia. Car- drichthyan biodiversity. Endemicity in Australia is high (51%) charhinus limbatus is also part of the elasmobranch fishery in (Last and Stevens 2009). Whaler sharks (genus Carcharhinus) south-eastern Asia (White et al. 2006). Like most marine preda- are a worldwide assemblage of over 30 commercially impor- tory species, sharks are vulnerable to overexploitation. They tant species that have largely coastal distributions (Compagno have naturally low abundance, fecundity that is orders of mag- et al. 2005). Twenty-one species occur in Australian waters, pre- nitude below most marine finfish species and a susceptibility dominantly in the tropical north, and two species (Carcharhinus to capture by gill-nets and long-lines (Last and Stevens 2009). fitzroyensis and C. tilstoni) are endemic to Australia (Last and State and national authorities manage the exploitation of black- Stevens 2009). tip sharks in northern Australia to ensure that the stock sizes © CSIRO 2010 10.1071/MF09151 1323-1650/10/020253 254 Marine and Freshwater Research J. R. Ovenden et al. 0 Irian Jaya Ϫ 5 Indonesia Papua New Guinea Java Bali Arafura Sea Ϫ10 East Timor Torres Strait Timor Sea Coral Sea Gulf of Darwin Carpentaria Ϫ15 N W E * Broome Townsville * Ϫ 20 S Australia 1000 km Ϫ25 Western Australia Northern Territory Queensland 105 110 115 120 125 130 135 140 145 Fig. 1. Collection locations for Carcharhinus tilstoni, C. limbatus and C. amblyrhynchoides from Western Australia, Northern Territory, Queensland and Indonesia. remain at sustainable levels (Salini et al. 2006). Carcharhinus in a co-occurring species (C. sorrah) from a previous study limbatus is believed to be relatively rare in northern Australia. (Ovenden et al. 2009). It was reported as occurring in a 1 : 300 ratio with C. tilstoni (Stevens and Wiley 1986) based on 987 C. tilstoni and 20 Materials and methods C. limbatus samples collected from northern Australian waters in 1982 to 1985 for an allozyme genetic study (Lavery and Sample collection Shaklee 1991). Last and Stevens (2009) stated that C. limbatus To assist the development of DNA-based species-identification is a minor component of the commercial harvest in northern methods, reference tissue samples were taken from four C. Australia. limbatus individuals and four C. tilstoni individuals from the The present study arose from a population genetic analysis north-western Australian coast. Species identification was con- of Australian blacktip sharks, where samples were taken along firmed in the field by pre-caudal vertebral counts made by the northern Australian coastline under the assumption that the R. Pillans and J. Stevens (CSIRO). Counts for C. limbatus ranged majority of blacktip shark would be C. tilstoni. However, pilot from 97 to 100 and were within the accepted range for this analyses showed the presence of an additional species, which was species. Counts for C. tilstoni samples were 80 to 85, slightly identified as C. limbatus. Using these blacktip shark samples, we below the recorded range of 84 to 91 (Last and Stevens 2009). test the assumption that C. limbatus is rare in northern Australia Populations from the west, north and east coasts of Australia using mitochondrial DNA (mtDNA) nucleotide sequence for (Fig. 1) were sampled to test species composition over a broad species identification. Distinctiveness of each species was tested spatial scale. Observers and fisheries biologists collected tis- by phylogenetic analysis of mtDNA sequence data in compar- sue samples from blacktip sharks that they presumed to be ison to Australian and Indonesian samples of C. limbatus and C. tilstoni. Pre-caudal vertebral counts were not performed to C. amblyrhynchoides, and appropriate outgroups. Furthermore, confirm species identification, except on reference samples. as evolutionary theory predicts that interspecific divergence Carcharhinus amblyrhynchoides and C. limbatus samples generally should exceed intraspecific divergence, the propor- were collected from Indonesian markets (Fig. 1) for compari- tion of overall genetic variation due to species-level distinc- son with Australian samples. The provenance of the Indonesian tion between C. tilstoni and C. limbatus was estimated using samples was within 300 km of markets based on interviews microsatellite loci and compared with intraspecific divergence with vendors. Approximately 200 mg of white muscle tissue Carcharhinus limbatus is abundant in northern Australia Marine and Freshwater Research 255 excluding skin was dissected from all specimens and preserved USA). Approximately 20 ng of DNA was used in standard in 1 mL of a 20% dimethyl sulfoxide solution (DMSO) in 5 M ABI Dye Terminator sequencing reactions and capillary gel NaCl. Samples were stored at room temperature in the field and separated on an ABI3130XL (Applied Biosystems, Mulgrave, −70◦C in the laboratory. Australia) sequencer. Sequence data was edited and aligned with Sequencher ver. 4.7 (Gene Codes, Ann Arbor, MI, USA) and Genomic DNA extraction ClustalX (Thompson et al. 1997). Identical mtDNA sequences were described as haplotypes. Haplotype sequences for C. lim- From each sample, 10–50 mg of tissue was digested in 500 µL batus were compared with mtDNA control region haplotypes of a suspension of 10% Chelex-100 (w/v; Biorad Laboratories assigned by Keeney and Heist (2006) using GenBank accession Inc, Sydney, Australia) in TE buffer (5 mM Tris-Cl pH 8.0 with numbers AY208861–73 and AY766123–46. 0.5 mM EDTA). Proteinase K (100 ng) was added and the tissue Phylogenetic analysis of mitochondrial DNA sequences was was digested to completion at 55◦C for at least 1 h on a shaking used to examine the pattern of similarity, which would test platform.