Mitochondrial DNA, October 2011; 22(S1): 27–36 FULL LENGTH RESEARCH PAPER DNA barcoding of billfishes ROBERT HANNER1, ROBIN FLOYD1,*, ANDREA BERNARD2,†, BRUCE B. COLLETTE3,‡,& MAHMOOD SHIVJI2,{ 1Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone Road East, Guelph, ON, Canada N1G 2W1, 2Guy Harvey Research Institute, Oceanographic Center, Nova Southeastern University, 8000 N. Ocean Drive, Dania Beach, FL 33004, USA, and 3National Marine Fisheries Service Systematics Laboratory, National Museum of Natural History, MRC-0153, Smithsonian Institution, Washington, DC 20560, USA (Received 6 October 2010; revised 16 May 2011; accepted 10 June 2011) Abstract DNA barcoding is a method promising fast and accurate identification of animal species based on the sequencing of the mitochondrial c oxidase subunit (COI) gene. In this study, we explore the prospects for DNA barcoding in one particular fish group, the billfishes (suborder Xiphioidei—swordfish, marlins, spearfishes, and sailfish). We sequenced the mitochondrial COI gene from 296 individuals from the 10 currently recognized species of billfishes, and combined these data with a further 57 sequences from previously published projects. We also sequenced the rhodopsin gene from a subset of 72 individuals to allow comparison of mitochondrial results against a nuclear marker. Five of the 10 species are readily distinguishable by COI barcodes. Of the rest, the striped marlin (Kajikia audax) and white marlin (K. albida) show highly similar sequences and are not unambiguously distinguishable by barcodes alone, likewise are the three spearfishes Tetrapturus angustirostris, T. belone, and T. pfluegeri. We discuss the taxonomic status of these species groups in light of our and other data, molecular and morphological. Keywords: COI, rhodopsin, mitochondrial DNA, species delimitation Introduction life stage assessments (Victor et al. 2009), and scientific research generally (Yancy et al. 2008). Fish In the continuing quest to catalog and monitor the species identification has traditionally been carried out biodiversity of the world’s oceans, molecular methods based on the examination of specimen morphology; are assuming an increasing prominence. Fishes however, in many cases in which species identifi- constitute the largest vertebrate group on the planet, cations are needed, morphological features may not be with approximately 30,000 species (Nelson 2006), available (e.g. when food inspectors wish to determine and fisheries and related industries are vital to the whether a piece of fillet sold at a market is correctly economies of many nations providing a significant named), or not yet developed (e.g. in which fish eggs fraction of global human food needs. Accurate and or larvae need to be assigned to species). In such cases reliable species identifications of fish and fish tissues in which morphological information is lacking, are important in many situations, including the molecules (DNA, RNA, and proteins) may serve as detection of market substitutions (in which fish is unique identifiers to discriminate among species sold under an incorrect species name, either to (Arnot et al. 1993; Floyd et al. 2002). increase profit or to conceal illegal catches) (Wong and In the past, a variety of molecular techniques Hanner 2008; GAO 2009), monitoring of fisheries have been utilized to properly discriminate among landings for stock assessments and management, early species, including protein electrophoresis, nucleic acid Correspondence: R. Hanner, Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone Road East, Guelph, ON, Canada N1G 2W1. Phone: 519-824-4120 x53479. Fax: 519-767-1656. E-mail: [email protected] ISSN 1940-1736 print/ISSN 1940-1744 online q 2011 Informa UK, Ltd. DOI: 10.3109/19401736.2011.596833 28 R. Hanner et al. ‘fingerprinting’ methods such as restriction fragment All billfish species are of commercial value with length polymorphism (RFLP) and Amplification particular importance in some Asian markets (Hsieh fragment length polymorphism (AFLP), and direct et al. 2005; Gentner 2007). Furthermore, all species sequencing of specific genomic regions (Hsieh et al. are also highly desirable targets in recreational 2005, 2007). These methods have generally been of fisheries; just in the USA, for example, expenditures small scale, developed on a case-by-case basis by by recreational billfish anglers are estimated to exceed different research groups and applicable only to small $2 billion annually (SEFSC/NMFS 2004). Commer- groups of species. In contrast, DNA barcoding is a cial long-line fisheries cover nearly the entire natural sequencing-based approach that attempts to establish distributions of the species, whereas sport fishing a single global system of both laboratory protocols and grounds are more restricted. Areas where the two data management, which should ultimately be zones overlap have been subject to conflicts over applicable to all animals (Hebert et al. 2003a). fishing rights (Nakamura 1985). Populations of all In animals, DNA barcoding is based on sequencing billfish species investigated have declined in recent a ,658 base-pair fragment of the mitochondrial gene, years due to high levels of human exploitation, and cytochrome c oxidase subunit 1 (COI; Hebert et al. international fishery management organizations have 2003a,b). This signature sequence can be compared highlighted the need for more fisheries and basic against a database of known sequences from reference biological information, including better tracking of specimens to obtain a species identification (Ekrem landings by species and identification of spawning et al. 2007). Because this method depends on habitats, to aid global billfish management and molecular approaches rather than on morphology, conservation (Gentner 2007; Mora et al. 2009). it may be applied to organisms of any life stage from Although billfishes are both targeted in specific egg to adult. It also aims to employ standardized international fisheries and caught extensively as by- protocols that may be applied to a wide range of catch in pelagic fisheries, few countries keep accurate organisms by individuals possessing a minimum track of their landings, and even when they do, often amount of technical expertise and without the need group species together (e.g. sailfish and spearfishes) in for extensive knowledge of traditional morphological their landings records (SEFSC/NMFS 2004; Gentner taxonomy. Sequence and specimen data are stored 2007). Monitoring landings by species even for adult and made available in the Barcode of Life Data fish is further complicated by the difficulties in System (BOLD) database (Ratnasingham and Hebert identifying processed fishes that end up as similar 2007), an online collaborative workbench used to looking carcasses or smaller body parts in the trade construct and curate a global barcode reference pipeline (McDowell and Graves 2002; SEFSC/NMFS sequence library. Under the organizing framework of 2004; Hsieh et al. 2005). Delineation of billfish the International Barcode of Life (iBOL) project, spawning habitat is typically accomplished by con- global campaigns are under way to barcode the ducting oceanographic surveys to find and monitor world’s biodiversity, including Fish Barcode of Life the temporal and spatial distributions of individuals (FISH-BOL), the campaign to barcode fishes across life-stages, including eggs, larvae, and adults (Costa and Carvalho 2007; Ward et al. 2009). Here, (Kawakami et al. 2010; Richardson et al. 2010). Yet we discuss the prospects for DNA barcoding in identification of billfish early life stages (eggs and one particular fish group, the billfishes (suborder larvae) based on morphological characters is often Xiphioidei). problematic (SEFSC/NMFS 2004; Hyde et al. 2005). The billfishes are a group of ray-finned fishes (class These species identification difficulties in the Actinopterygii) comprising two extant families: the context of the ecological and fishery importance of monotypic Xiphiidae (swordfish, Xiphias gladius) and billfishes have led to the development of a variety of Istiophoridae (marlins, spearfishes, and sailfish) genetic approaches to facilitate the accurate identifi- (Collette et al. 2006). All are large, active apex cation of their body parts and early life stages. predators characterized by an elongate, sword-like, or Approaches developed to date include restriction spear-like snout. All species are dioecious (having fragment length polymorphism analysis of polymerase separate sexes) and females attain larger sizes than chain reaction (PCR) amplified loci, multiplex PCR, males, but otherwise do not display strong sexual and direct sequencing of either the mitochondrial 16S dimorphism (Collette, 2010). They are among the rRNA or cytochrome b loci (McDowell and Graves largest and fastest swimming bony fishes, and are 2002; Hsieh et al. 2005; Hyde et al. 2005; Luthy et al. capable of trans-oceanic movements (Sedberry and 2005; Richardson et al. 2007; Kawakami et al. 2010). Loefer 2001). Billfishes are wide ranging, inhabiting These approaches successfully discriminate among both tropical and temperate waters, and, seasonally, the subset of billfish species tested in each study; also cold waters (to which they migrate during however, none of these approaches have been applied summer months for feeding); spawning of most to all known billfish species. In particular, the white species occurs in tropical and subtropical waters marlin and striped