1. Academia Sinica, 16 January 2007 DNA Barcoding: An Emerging Global Standard for Species Identification Consortium for the Barcode of Life National Museum of Natural History Smithsonian Institution http://www.barcoding.si.edu 202/633-0808; fax 202/633-2938

2. Academia Sinica, 16 January 2007 A DNA barcode is a short gene sequence taken from standardized portions of the genome, used to identify species

3. Academia Sinica, 16 January 2007 Characteristics of Barcode Regions Flanked by conserved regions Easy to amplify Low intraspecies variability Discontinuous variation between species Long enough to work in all groups Short enough for single reads

4. Academia Sinica, 16 January 2007 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

5. Academia Sinica, 16 January 2007 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

6. Academia Sinica, 16 January 2007 1. Databasing 2. Labeling 3. Imaging 4. Tissue sampling 5. DNA extraction 6. PCR 7. PCR check 8. Sequencing reaction 9. Sequencing cleanup 10. Sequencing 11. Trace editing & submission Analytical chain

7. Academia Sinica, 16 January 2007 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

8. Academia Sinica, 16 January 2007 Methods

9. Academia Sinica, 16 January 2007 Barcode of Life Database

10. Academia Sinica, 16 January 2007 1. Databasing 2. Labeling 3. Imaging 4. Tissue sampling 5. DNA extraction 6. PCR 7. PCR check 8. Sequencing reaction 9. Sequencing cleanup 10. Sequencing 11. Trace editing & submission Analytical chain

11. Academia Sinica, 16 January 2007 Current Norm: High throughput ABI 3100 capillary automated sequencer Large capacity PCR and sequencing reactions

12. Academia Sinica, 16 January 2007 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

13. Academia Sinica, 16 January 2007 Producing Barcode Data: 2008 Faster, more portable: Hundreds of samples per hour Integrated DNA microchipsTable-top microfluidic systems

14. Academia Sinica, 16 January 2007 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

15. Academia Sinica, 16 January 2007 Methods

16. Academia Sinica, 16 January 2007 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

17. Academia Sinica, 16 January 2007 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

18. Academia Sinica, 16 January 2007 Associating Life Stages, Processed Parts, Dimorphic Genders

19. Academia Sinica, 16 January 2007 Hypopygus lepturus Hoedeman 1962 Hypopygus lepturus Hoedeman 1962 Steatogenys elegans Steatogenys duidae Steatogenini until the early 90’s

20. Academia Sinica, 16 January 2007 Nijssen & Isbrüker 1972 Color patterns in Hypopygus

21. Academia Sinica, 16 January 2007 Hypopygus neblinae Mago-Leccia 1994 Hypopygus neblinae Mago-Leccia 1994 Hypopygus lepturus Hoedeman 1962 Hypopygus lepturus Hoedeman 1962 Steatogenys Steatogenini during the 90’s

22. Academia Sinica, 16 January 2007 Hypopygus neblinae Mago-Leccia 1994 Hypopygus neblinae Mago-Leccia 1994 Hypopygus lepturus Hoedeman 1962 Hypopygus lepturus Hoedeman 1962 Stegostenopos Triques 1997 Stegostenopos Triques 1997 Steatogenys Steatogenini during the 90’s / today

23. Academia Sinica, 16 January 2007 Steatogenys sp. Hypopygus lepturus Stegostenopos cryptogenes R. Bernhard, 2004 8a

24. Academia Sinica, 16 January 2007 A A C C D D Steatogenys H. lepturus RAG 1 MP/ML/Dist Stegostenopus Hypopygus neblinae

25. Academia Sinica, 16 January 2007 12S16S Strict of ML/MP/Dist A A C C E E D D H. neblinae Stegostenopus Steatogenys H. lepturus

26. Academia Sinica, 16 January 2007 D D E E H. lepturus 2792 2791 D-loop MP/ML/Dist

27. Academia Sinica, 16 January 2007 Eigenmannia sp. COI - BARCODE MP H. lepturus A A C C 2792 2791 D D E E H. neblinae Stegostenopus

28. Academia Sinica, 16 January 2007 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

29. Academia Sinica, 16 January 2007 Wider Impacts of Barcoding: 2008 Catalyzing interoperability of databases –Barcode data standards link sequences, specimens, species names and publications Improving the information infrastructure –Digital library initiative in taxonomy Renewing the mission of museums –DNA recovery from formalin-fixed specimens –Promoting the growth of DNA banks Expanding analytical toolbox for taxonomy

30. Academia Sinica, 16 January 2007 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

31. Academia Sinica, 16 January 2007

33. 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

34. Academia Sinica, 16 January 2007 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

35. Academia Sinica, 16 January 2007 CBOL Member Organizations June 2006: 120 Member Organizations, 40 countries

36. Academia Sinica, 16 January 2007 CBOL’s Working Groups Database: Designing/constructing the Barcode Section of GenBank DNA: Protocols for formalin-fixed and old museum specimens; Producing LIMS for dissemination Data Analysis: Beyond phenetic methods; population genetics perspective Plants: Identify gene region(s) for barcoding

37. Academia Sinica, 16 January 2007 Infrastructure of Taxonomy: Fragmented, Disconnected Collections and databases of specimens Compilations of taxonomic names Data repositories (characters, gene sequences, images, trees) Monographs Floristic and faunistic surveys/inventories Revisions The (undigitized) Taxonomic Literature

38. Academia Sinica, 16 January 2007 Barcode Records in INSDC Consensus results of Front Royal meeting –GBIF  ITIS  GRIN –NBII  Species2000  IPNI –ICZN  ZooRecord  OBIS Structured link to voucher specimen Species name selected from authority Online access to metadata Trace files and quality scores Minimum sequence length

39. Academia Sinica, 16 January 2007 Barcode Sequence Voucher Specimen Species Name Specimen Metadata Literature (link to content or citation) BARCODE records in GenBank 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

40. Academia Sinica, 16 January 2007 Digitizing Taxonomic Literature CBOL’s catalytic efforts: –Library-Laboratory meeting in London on electronic access to taxonomic literature –Led to formation of Biodiversity Heritage Library initiative –Proactive steps with PubMed to add taxonomic journals to online abstracts –Aggressive negotiation with publishers of barcoding papers

41. Academia Sinica, 16 January 2007 CBOL’s Working Groups Database: Designing/constructing the Barcode Section of GenBank DNA: Protocols for formalin-fixed and old museum specimens; Producing LIMS for dissemination Data Analysis: Beyond phenetic methods; population genetics perspective Plants: Identify gene region(s) for barcoding

42. Academia Sinica, 16 January 2007 The Barcode Assembly Line: 2006 Freshly collected specimens Frozen tissue Young museum specimens DNA Barcode Data

43. Academia Sinica, 16 January 2007 The Barcode Assembly Line: 2008 Opening the museum treasure-trove Freshly collected specimens Frozen tissue Young museum specimens DNA Barcode Data Formalin-fixed specimens Older museum specimens

44. Academia Sinica, 16 January 2007 CBOL Formalin Workshop Literature survey of DNA recovery protocols from formalin-fixed specimens Solicited proposal from National Research Council May 8-9 workshop in Washington Chemists, biochemists, biophysicists, biomedical researchers Create a new research agenda

45. Academia Sinica, 16 January 2007 CBOL’s Working Groups Database: Designing/constructing the Barcode Section of GenBank DNA: Protocols for formalin-fixed and old museum specimens; Producing LIMS for dissemination Data Analysis: Beyond phenetic methods; population genetics perspective Plants: Identify gene region(s) for barcoding

46. Academia Sinica, 16 January 2007 Data analysis protocols in 2008 A Bigger, Better Analytical Toolkit to handle the Barcode Data Explosion Collaboration of statisticians, computer scientists, population geneticists Sampling issues: –Sample size versus confidence level –Sample size in light of geography, gene flow Analytical tools and protocols: –Treatment of missing DNA site data –Identification versus species delimitation (classification versus clustering)

47. Academia Sinica, 16 January 2007 CBOL’s Working Groups Database: Designing/constructing the Barcode Section of GenBank DNA: Protocols for formalin-fixed and old museum specimens; Producing LIMS for dissemination Data Analysis: Beyond phenetic methods; population genetics perspective Plants: Identify barcode gene region(s) for land plants

48. Academia Sinica, 16 January 2007 Progress toward Plant Barcode Kress 2005 proposal for ITS and trnh-psbA Kew Garden receives Sloan/Moore Foundation support Phase 1 screens 100 genes across 50 sibling species pairs Phase 2 tests of matK, rpcoC1, rpoB, ndhJ, and accD Canadian proposal for rbcL CBOL protocols for approving barcode regions

49. Academia Sinica, 16 January 2007 Current and Planned CBOL Barcoding Projects FishBOL and All Birds Initiatives “Demonstrator Systems: by 2008: –Tephritid fruit flies (agricultural pests) –Mosquitoes (disease vectors) African Scale Insect Barcoding Initiative (planned at Cape Town Regional Meeting) Barcoding for Conservation Committee

50. Academia Sinica, 16 January 2007 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

51. Academia Sinica, 16 January 2007 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

52. Academia Sinica, 16 January 2007 Planned Outreach Regional meetings in: –Cape Town, South Africa, 7-8 April 2006, SANBI –Nairobi, Kenya, 18-19 October 2006, NMK –Sao Paolo, Brazil, February 2007, INPA –Southern/SE Asia, mid-2007 Second International Barcode Conference –Southeast Asia (?), September 2007 (?) Support from CBOL, host governments and international development agencies

53. Academia Sinica, 16 January 2007 Milestones for 2008 200720082006 Q1Q2Q3Q4Q1Q2Q3Q4Q1Q2 International Conference Development of Consensus Plant Barcode Region Data Analysis Protocols and S/W Formalin Study Advanced Lab Protocols 200K records500K records100K records Demonstrator System Launched Database: Data Analysis WG: DNA WG: Plant WG: Database WG: Extended DB Interoperability BoLI Data Portal Launched Campaigns: Regional Groups Operational First Data Releases 10K birds 30K fish Data Standards