Species Identification, Regulatory Agencies and DNA Barcoding David E. Schindel, Executive Secretary National Museum of Natural History Smithsonian Institution / ; fax 202/

Genomics Subgenomics Current Systematic Studies Microbes - 16S Plants - RBCL Animals - COI

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

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Specimens Characters Distributions of Character Variation Taxonomic Decision- Making Socioeconomic Decisions Specimens Concerns/ Regulations The Practice of Taxonomy Taxonomists The Uses of Taxonomy

Taxonomic Processes Specimens Taxon Concepts Characters Formal Taxa

Growth of Biodiversity Databases Authority files of taxonomic names Museum databases of associated data Biodiversity Heritage Library

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

Non-COI regions for other taxa Land plants: –Chloroplast matK and rbcL approved Nov 09 –Non-coding plastid and nuclear regions being explored Fungi and protists: –CBOL Working Groups convened –Recommendations expected in 2010

How Barcoding Works First, build a barcode reference library: –Well-identified specimen –Tissue subsample –DNA extraction, PCR amplification –DNA sequencing –Data submission to GenBank Second, use it to identify unknowns: –Any unidentified juvenile, adult, fragment, product –Tissue sample, DNA, sequencing –Comparison with sequences in reference library

Associating Life Stages, Processed Parts, Dimorphic Genders

How Barcoding is Done From specimen to sequence to species Voucher Specimen DNA extractionCO1 geneDNA sequencing Trace file Database of Barcode Records Collecting ND3ND3 COIIICOIII ND2ND2 ND1ND1

NBII, 25 February 2009 GenBank, EMBL, and DDBJ Global, Open Access to Barcode Data

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

Emerging Norm: Table-top Labs Faster, more portable: Hundreds of samples per hour Integrated DNA microchipsTable-top microfluidic systems

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

Consortium for the Barcode of Life (CBOL) Established May 2004 with Sloan Foundation grant Secretariat opens at Smithsonian, September 2004 Now in its third two-year funding period Workshops, Working Groups, networking, representation/marketing Now an international affiliation of 200+ members in 50+ countries: –Natural history museums, biodiversity organizations –Users: e.g., government agencies –Private sector biotech companies, database providers

CBOL Member Organizations: Member organizations, 50 countries 35+ Member organizations from 20+ developing countries

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.

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, March 2010

CBOL’s Global Projects Fish Barcode of Life (FISH-BOL) –30,000 marine/freshwater species by 2010 All Birds Barcoding Initiative (ABBI) –10,000 species by 2010 Tephritid fruit flies –2,000 pest/beneficial species and relatives by 2008 Mosquitoes –3,300 species by 2008 Endangered species Trees of the world

Adoption by Regulators Food and Drug Administration –Reference barcodes for commercial fish NOAA/NMFS –$100K for Gulf of Maine pilot project –FISH-BOL workshop with agencies, Taipei, Sept 2007 Federal Aviation Administration – $500K for birds Environmental Protection Agency –$250K pilot test, water quality bioassessment FAO International Plant Protection Commission –Proposal for Diagnostic Protocols for fruit flies CITES, National Agencies, Conservation NGOs –International Steering Committee, identifying pilot projects

Why barcodes are short Low cost and simplicity –Single capillary reads adequate –Avoid post-sequencing IT needed for pyroseq Adequate for taxonomy –Miminizes uniformative sequence length –Limiting supply rate of identified specimens –Technology more accessible to small labs Regulatory/inspection applications easier

Barcoding and Metagenomics Lots of interest for ecology, less for regulation Pyrosequencing of environmental mixtures –Construct species list from mixture –Predator-prey relations from gut contents, feces –Food-web reconstruction Relies on high accuracy (some species differ by only a few percent) Not cost-effective for single specimens

What barcode providers have and are producing 770,000 records from ~100,000 species –Agricultural pests –Water quality indicators –Disease vectors –Endangered species –Commercial species of food, feed, commodities 5 million records from 500K species by 2015

International Barcode of Life Project (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 Their Parasitoids WG 1.6Pollinators WG 1.7Freshwater Bio-Surveillance WG 1.8Marine Bio-Surveillance WG 1.9Terrestrial Bio-Surveillance WG 1.10 Polar Life

What barcode providers want High PCR and sequencing success rates Bigger window into older, compromised samples Better software integration to eliminate bottlenecks Smaller labs/developing countries: –Lower equipment and maintenance costs –Simplification for techs with less training –Install anywhere without lab renovations –Willing to accept slower throughput

What barcode users want Answers to specific questions: –Is this thing on this list of species or not? –Is this thing a member of this genus/family? –Which of the species on this list is this thing? –What species is this thing? Production-scale capabilities: –Hundreds to thousands of installations –Lower but constant throughput –Rapid turnaround –The right price-point and limited life cycle costs

What barcode users would do with the reference libraries Inspection stations at every port and international airport for: –Agricultural pest control –Illegal trade in endangered species –Violations of trade quotas Regular Federal and State water quality surveys Federal, State and local food inspection Public health monitoring and diagnoses

NBII, 25 February 2009 GenBank, EMBL, and DDBJ Global, Open Access to Barcode Data

NBII, 25 February 2009 Linkout from GenBank to BOLD

NBII, 25 February 2009 Linkout from GenBank to Taxonomy

NBII, 25 February 2009 Link from GenBank to Museums