Barcoding and Border Biosecurity: Identifying Cyprinid Fishes in the Aquarium Trade

et al. (2012) Barcoding and Border Biosecurity: Identifying Cyprinid Fishes in the Aquarium Trade. PLoS ONE 7(1): e28381. doi:10.1371/journal.pone.0028381 Barcoding and Border Biosecurity: Identifying Cyprinid Fishes in the Aquarium Trade Rupert A. Collins 0 Karen F. Armstrong 0 Rudolf Meier 0 Youguang Yi 0 Samuel D. J. Brown 0 Robert H. Cruickshank 0 Suzanne Keeling 0 Colin Johnston 0 Dirk Steinke, Biodiversity Insitute of Ontario - University of Guelph, Canada 0 1 Bio-Protection Research Centre, Lincoln University , Canterbury , New Zealand , 2 Department of Biological Sciences and University Scholars Programme, National University of Singapore , Singapore, Singapore , 3 Department of Ecology, Faculty of Agriculture and Life Sciences, Lincoln University , Canterbury , New Zealand , 4 Animal Health Laboratory, Investigation and Diagnostic Centre, Ministry of Agriculture and Forestry , Upper Hutt , New Zealand Background: Poorly regulated international trade in ornamental fishes poses risks to both biodiversity and economic activity via invasive alien species and exotic pathogens. Border security officials need robust tools to confirm identifications, often requiring hard-to-obtain taxonomic literature and expertise. DNA barcoding offers a potentially attractive tool for quarantine inspection, but has yet to be scrutinised for aquarium fishes. Here, we present a barcoding approach for ornamental cyprinid fishes by: (1) expanding current barcode reference libraries; (2) assessing barcode congruence with morphological identifications under numerous scenarios (e.g. inclusion of GenBank data, presence of singleton species, choice of analytical method); and (3) providing supplementary information to identify difficult species. Methodology/Principal Findings: We sampled 172 ornamental cyprinid fish species from the international trade, and provide data for 91 species currently unrepresented in reference libraries (GenBank/Bold). DNA barcodes were found to be highly congruent with our morphological assignments, achieving success rates of 90-99%, depending on the method used (neighbour-joining monophyly, bootstrap, nearest neighbour, GMYC, percent threshold). Inclusion of data from GenBank (additional 157 spp.) resulted in a more comprehensive library, but at a cost to success rate due to the increased number of singleton species. In addition to DNA barcodes, our study also provides supporting data in the form of specimen images, morphological characters, taxonomic bibliography, preserved vouchers, and nuclear rhodopsin sequences. Using this nuclear rhodopsin data we also uncovered evidence of interspecific hybridisation, and highlighted unrecognised diversity within popular aquarium species, including the endangered Indian barb Puntius denisonii. Conclusions/Significance: We demonstrate that DNA barcoding provides a highly effective biosecurity tool for rapidly identifying ornamental fishes. In cases where DNA barcodes are unable to offer an identification, we improve on previous studies by consolidating supplementary information from multiple data sources, and empower biosecurity agencies to confidently identify high-risk fishes in the aquarium trade. - Funding: This work was supported by a Ministry of Agriculture and Forestry Biosecurity New Zealand scholarship for RAC; grant R377-000-040-112 by the Ministry of Education (Singapore) for RM and YY. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Globalisation in the form of international trade breaches biogeographical as well as administrative boundaries, enabling organisms to colonise regions beyond their contemporaneous natural ranges [1]. The impacts of invasive alien species are well documented as a leading cause of global biodiversity decline and economic loss [2,3], and particularly as a driving force in the biotic homogenisation and degradation of freshwater ecosystems [46]. Biosecurity challenges exist in effectively monitoring and managing the complex pathways involved [1,7,8], with a key issue for risk assessment being the identification of traded biological materials to species [911]. Effective cataloguing of both potential propagules (all traded species) and known invasive alien species, can inform risk analyses and facilitate pre- or post-border control measures (i.e., import restrictions and quarantine). In circumstances where species cannot be diagnosed easily by morphology and/or only certain life history stages can be identified, standardised molecular protocols for species identification are important for biosecurity [911]. However, these techniques still require further testing and reference libraries need to be expanded to encompass more species. The ornamental aquatic industry is among the worlds largest transporters of live animals and plants, with an annual trade volume estimated at US$1525 billion [12,13]. Data from the United States implicates the industry as the primary transport vector in 37 of 59 fish introductions [6]. In Singaporea global aquarium fish trading hubat least 14 invasive ornamental fish species were reported to be resident in reservoirs in 1993 [14]. The risks presented by this industry are not, however, limited to traded invasive fishes. Associated pathogenic organisms such as protozoa, bacteria and viruses are equally undesirable, with exotic pathogens known to cause harm to native species [15], industrial food aquaculture [1618], and also the ornamental fish trade [13]. Compounding this, some pathogens can be vectored by carrier hosts with no clinical signs of disease [13,15,18], and host-taxon specific pathogens may also require special quarantine measures [13,18]. Aquarium fishes are both wild caught, and captive bred at aquaculture facilities, with over one billion fishes traded through more than 100 countries in 2000 [18]. In the case of freshwater fishes, 90% of the trade volume is in a relatively small number of popular species sourced from commercial farms [19], while more diverse wild caught exports contribute the remainder. A complex supply chain exists for these ornamental fishes, and before they arrive at a retailer they may have passed though a series of regional and international distribution centres where consignments can be consolidated, reconsolidated and subdivided [13]. This potentially increases the number of access points for undesirable organisms to enter each shipment [13], as well as opportunities for mislabelling. While statistics are available on total volumes sold, little quantitative data exist on the number and composition of species involved in the aquarium trade, but it has been estimated that up to 5,300 species have been available at some point [20]. The industry in aquatic ornamentals for the aquarium hobby is a dynamic business, with new and undescribed species frequently appe (...truncated)