DNA Barcoding of Pacific Invasive and Pest Species Pacific Science Congress Kuala Lumpur David E. Schindel, Executive Secretary National Museum of Natural History Smithsonian Institution / ; fax 202/

Today’s Goals Share information on barcoding, invasives Share information on projects and organizations in Pacific Discuss potential regional cooperation; and Discuss possible formation of a PSA Working Group on DNA barcoding of invasive/pest species: –Participants (individuals, labs, institutes, agencies) –Activities (training, workshops, collecting, producing data) –Deliverables (data, publications, websites)

Existing Activities National quarantine agencies (NPPOs) Regional agencies and initiatives (RPPOs, Quads, QBOL) Global Initiatives (IPPC, CABI, GISP) BioNET INTERNATIONAL LOOP PaciNET PBIF: Pacific Node of GBIF

The DNA Barcoding Initiative Barcoding is becoming a global standard for species identification Rapidly expanding by region, taxa, applications The Barcoding Initiative is global with participants in 50+ countries CBD, IPPC, Global Taxonomy Initiative, Census of Marine Life, others involved Government agencies: USDA, FDA, NOAA

Species Identification Matters Basic research on evolution, ecology Invasive species (e.g., in ballast water) Agricultural pests/beneficial species Endangered/protected species Disease vectors/pathogens Environmental quality indicators Managing for sustainable harvesting Consumer protection, ensuring food quality Fidelity of seedbanks, culture collections

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A DNA barcode is a short gene sequence taken from standardized portions of the genome, used to identify species

An Internal ID System for All Animals Typical Animal Cell Mitochondrion DNA mtDNA D-Loop ND5 H-strand ND4 ND4L ND3 CO III L-strand ND6 ND2 ND1 CO II Small ribosomal RNA ATPase subunit 8 ATPase subunit 6 Cytochrome b CO I The Mitochondrial Genome

Associating Life Stages, Processed Parts, Dimorphic Genders

Non-COI regions for other taxa Land plants: –Chloroplast matK and rbcL approved Nov 09 –70-75% resolving ability, higher in angiosperms –Non-coding plastid and nuclear regions being explored Fungi: –CBOL Working Group met this week in Amsterdam –Agreed to recommend ITS; 72% effective Protists: – CBOL Working Group July meeting, Berlin

How Barcoding Works PHASE 1: Build a barcode reference library: –Well-identified specimen –Tissue subsample –DNA extraction, PCR amplification –DNA sequencing –Data submission to GenBank PHASE 2: Identify unknowns: –Any unidentified juvenile, adult, fragment, product –Tissue sample, DNA, sequencing –Comparison with sequences in reference library

Current Norm: High throughput Large labs, hundreds of samples per day ABI 3100 capillary automated sequencer Large capacity PCR and sequencing reactions

● US$ K purchase ● 2-3 hours processing time ● samples per day ● US$3-5 per sample

Technology Development Partnership Goal The DNA Sequencing Lab of 2013?

NBII, 25 February 2009 BOLD System Workbench in Canada

USER /GenBank Key Mirroring Update Channel Private Records BARCODE Record Flow Chart

Barcode Sequence Voucher Specimen Species Name Specimen Metadata Literature (link to content or citation) BARCODE Records in INSDC Indices - Catalogue of Life - GBIF/ECAT Nomenclators - Zoo Record - IPNI - NameBank Publication links - New species Georeference Habitat Character sets Images Behavior Other genes Trace files Other Databases Phylogenetic Pop’n Genetics Ecological Primers Databases - Provisional sp.

Linkout from GenBank to BOLD

ISBER: 13 May 2009 Linkout from GenBank to Taxonomy

ISBER: 13 May 2009 Link from GenBank to Museums

Darwin Core Triplet Structured Link to Vouchers Institutional Acronym Collection Code Catalog ID ::

Structured Link to Vouchers NHMLEP :: personalDHJanzenSRNP12345 ::

NCBI’s Biorepository List Compiled from Index Herbariorum, literature sources, GenBank submissions 6,936 records 1,177 records with non-unique acronyms 517 homonymous acronyms 374 shared by two records 143 shared by three records

AMNH Icelandic Institute of Natural History, Akureyri DivisionAkureyriIceland AMNHAmerican Museum of Natural HistoryNew YorkUSA UNLUniversidad Autónoma de Nuevo León Monterrey, Nuevo LeónMexico UNLUniversity of Nebraska State MuseumLincoln, NebraskaUSA UNL Centro de Estratigrafia e Paleobiologia da Universidade Nova de LisboaMonte de CaparicaPortugal ZMKZoological Musem, KristianiaOsloNorway ZMKZoologisches Museum der Universität KielKielGermany ZMKZoological Museum, CopenhagenCopenhagenDenmark

CBOL/GBIF/NCBI Registry of Biorepositories

31 Malaysian Biorepositories Recorded 10 Confirmed, 21 Unconfirmed

Producing Barcode Data: 201? Barcode data anywhere, instantly Data in seconds to minutes Pennies per sample Link to reference database A taxonomic GPS Usable by non- specialists

Promote barcoding as a global standard Build participation Working Groups BARCODE standard International Conferences Increase production of public BARCODE records Networks, Projects, Organizations Barcode of Life Community 1,264,000 specimens already barcoded from 104,500 species

The International Barcode of Life Project (iBOL) 5 Million specimens, 500,000 species in 5 years $150 million with core funding from Genome Canada iBOL website, University of Guelph, Ontario:

iBOL Theme 1: DNA Barcode Library WG 1.1Vertebrates WG 1.2Land Plants WG 1.3Fungi WG 1.4Human Pathogens and Zoonoses WG 1.5Agricultural and Forestry Pest and Parasitoids WG 1.6Pollinators WG 1.7Freshwater Bio-Surveillance WG 1.8Marine Bio-Surveillance WG 1.9Terrestrial Bio-Surveillance WG 1.10 Polar Life

200+ Member organizations, 50 countries 35+ Member organizations from 20+ developing countries Consortium for the Barcode of Life (CBOL)CBOL Member Organizations: 2010

Outreach Activities Cape Town, South Africa, April 2006, SANBI –Scale insects in African agriculture Nairobi, Kenya, October 2006 –Commercial fisheries in Rift Valley lakes Brazil, March 2007 –Hardwood tree species –Endangered mammals, reptiles, amphibians Taiwan, September 2007 Nigeria, October 2008 Beijing, May 2009 India, November 2010

Adoption by Regulators International Plant Protection Commission –CBOL and APHIS to host Diagnostic Protocol Panel meeting, July 2010 Federal Aviation Administration – $500K for birds Environmental Protection Agency –$250K pilot test, water quality bioassessment Food and Drug Administration –Reference barcodes for commercial fish NOAA/NMFS –$100K for Gulf of Maine pilot project CITES, National Agencies, Conservation NGOs

Conclusions Barcoding is a cost-effective system for rapid identification Barcode reference libraries are being constructed for several endangered groups CBOL and iBOL provide a global network of specialists capable of constructing barcode reference libraries on selected groups Partnerships with national and regional groups and regulatory agencies are the critical missing components