Utah State University – 29 Nov 2006 DNA Barcoding: An Emerging Global Standard for Species Identification Consortium for the Barcode of Life National Museum of Natural History Smithsonian Institution 202/ ; fax 202/

Utah State University – 29 Nov 2006 A DNA barcode is a short gene sequence taken from standardized portions of the genome, used to identify species

The Mitochondrial Genome Cyt b D-Loop ND5 H-strand ND4 ND4L ND3 CO III CO I L-strand ND6 CO I ND2 ND1 CO II Small ribosomal RNA Large ribosomal RNA ATPase subunit 8 ATPase subunit 6

Utah State University – 29 Nov 2006 Uses of DNA Barcodes Applied tool for identifying regulated species: Disease vectors, agricultural pests, invasives Environmental indicators, protected species Using minimal samples, damaged specimens, gut contents, droppings Research tool for improving species-level taxonomy: Associating all life history stages, genders Testing species boundaries, finding new variants “Triage” tool for flagging potential new species: Undescribed and cryptic species

Utah State University – 29 Nov 2006 Using DNA Barcodes Establish reference library of barcodes from identified voucher specimens If necessary, revise species limits Then: –Identify unknowns by searching against reference sequences –Look for matches (mismatches) against ‘library on a chip’ –Before long: Analyze relative abundance in multi-species samples

Utah State University – 29 Nov 2006 Reactions to Barcoding: 2004 From ecologists and other users: “This is what we need! How soon can we get started?” From traditional taxonomists: “Species should be based on lots of characters, not just barcodes” From forward-looking taxonomists: “Using molecular data as species diagnostics isn’t new, but standardization and broad implementation are great!” From barcoding practitioners: “I had my doubts at the beginning, but it really works as a tool for identification (96% accurate in a recent mollusc paper) and it is at least as good as traditional approaches to discovering new species.”

Utah State University – 29 Nov 2006 What DNA Barcoding is NOT Barcoding is not DNA taxonomy; no single gene (or character) is adequate Barcoding is not Tree of Life; barcode clusters are not phylogenetic trees Barcoding is not just COI; standardizing on one region has benefits and limits Molecules in taxonomy is not new; but large-scale and standardization are new Barcoding can help to create a 21 st century research environment for taxonomy

Utah State University – 29 Nov 2006

What DNA Barcoding is NOT Barcoding is not DNA taxonomy; no single gene (or character) is adequate Barcoding is not Tree of Life; barcode clusters are not phylogenetic trees Barcoding is not just COI; standardizing on one region has benefits and limits Molecules in taxonomy is not new; but large-scale and standardization are new BUT…Barcoding can help to create a 21 st century research environment for taxonomy

Barcode Sequence Voucher Specimen Species Name Specimen Metadata Literature (link to content or citation) BARCODE Data Standard Indices - Catalog 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

Current Norm: High throughput ABI 3100 capillary automated sequencer Large capacity PCR and sequencing reactions

Utah State University – 29 Nov 2006 Fresh/FrozenMuseum Tissue Sampling $0.41 DNA Extraction$0.34$2.00 PCR Amplification$0.24$0.48 PCR Product Check$0.35$0.70 Cycle Sequencing$1.04$2.08 Sequencing Cleanup$0.32$0.64 Sequence$0.40$0.80 Total:$3.10$7.11 Cost of Reagents and Disposables

Producing Barcode Data: 2008 Faster, more portable: Hundreds of samples per hour Integrated DNA microchipsTable-top microfluidic systems

Utah State University – 29 Nov 2006 Reference vs. Micro-Barcodes BARCODE reference records –Adhere to data standards –Bidirectional reads, 500+ bp long –Linked to voucher, species name Query barcode records –Used in BLAST or other searches –Often single pass reads –Often very short – 100+ bp for good IDs –Can cost less than $2, take less than 6 hours

Utah State University – 29 Nov 2006 Producing Barcode Data: 2010? Barcode data anywhere, instantly Data in seconds to minutes Pennies per sample Link to reference database A taxonomic GPS Usable by non- specialists

Utah State University – 29 Nov 2006 Consortium for the Barcode of Life (CBOL) First barcoding publications in 2002 Cold Spring Harbor planning workshops in 2003 Sloan Foundation grant, launch in May 2004 Secretariat opens at Smithsonian, September 2004 First international conference February 2005 Now an international affiliation of: –130+ Members Org’s, 40 countries, 6 continents –Natural history museums, biodiversity organizations –Users: e.g., government agencies –Private sector biotech companies, database providers

CBOL Member Organizations June 2006: 120 Member Organizations, 40 countries

Utah State University – 29 Nov 2006 CBOL’s Underlying Principles Standardization, cost-effectiveness Minimalism (scientific) – short sequence Global participation Tangible, realistic goals, near-term results Respond to applied user communities Minimalisim (organizational) – Coordination/Facilitation of Bottom-Up activities, build on existing activities, avoid new ones

Utah State University – 29 Nov 2006 CBOL’s Working Groups Database: Designing/constructing the Barcode Section of GenBank DNA: Protocols for formalin-fixed and old museum specimens; advice to new labs Data Analysis: Beyond phenetic methods; population genetics perspective Plants: Identify gene region(s) for barcoding

Utah State University – 29 Nov 2006 Outreach to Developing Countries Regional meetings in: –Cape Town, South Africa, 7-8 April 2006, SANBI –Nairobi, Kenya, October 2006, NMK –Sao Paolo, Brazil, February 2007, INPA –Southern Asia, 2 nd quarter 2007 Second International Barcode Conference –Singapore, June 2007 Support from CBOL, host governments and international development agencies

Utah State University – 29 Nov 2006 Goals of Regional Meetings Raise awareness Explore potential applications in the region Assess greatest needs and opportunities in the region Identify highest priorities, construct national and regional action plans Start intra-regional networks and intercontinental partnerships

Utah State University – 29 Nov 2006 Current and Planned Projects Four Working Groups FishBOL and All Birds Initiatives Developing “Demonstrator Systems by 2008 African Scale Insect Barcoding Initiative (planned at Cape Town Regional Meeting) International Network for Barcoding Invasive and Pest Species (INBIPS) Forming a Conservation Committee

Utah State University – 29 Nov 2006 Demonstrator Development GOAL: By June 2008, create an operational system for global ID of an important group Mosquitoes (disease vectors) –Barcode 85% (2932) of 3449 known species –Minimum 5 specimens per species –Reliance on archival collections Tephritid fruit flies (agricultural pests) –Barcode 2000 species of 4500, 5 per species –All pests and beneficials, with closest relatives –Invasive non-destructive sampling of existing collections

Utah State University – 29 Nov 2006 ABBI and FISH-BOL Global initiatives to create reference library Enable users to adopt barcode ID systems All-species barcode database will: –Strengthen specimen/species data –Improve collections, tissue/DNA resources –Attract users to barcoding for specimen IDs Regional Working Groups Small Steering Committee and CBOL

Utah State University – 29 Nov 2006 BoLD Data System Developed/hosted by Univ. Guelph Workbench for most barcode projects Laboratory Information Management System (LIMS) for assembling data Management and Analysis System Identification system for matching unknowns to reference records Uploading to GenBank

IDS – Identification System

Linkage to Mapping System

Utah State University – 29 Nov 2006 Launching CBOL Projects Assembling Steering Committee –Users –Taxonomists, collection curators –Service providers (BoLD, analytical labs) Plan for scope, timetable, logistics Pilot tests of primers, PCR amplification Assemble pipeline of specimens to lab

DNA from identified voucher Create BARCODE reference record ID unknownsRefine taxonomy of group DNA from identified adult voucher Create BARCODE reference records Associate immatures with names ID unknowns Refine taxonomy of group DNA from unidentified immature specimen Repository of provisional vouchers Add names to vouchered immatures