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

Species Identification, Species Identification, Regulatory Agencies and Regulatory Agencies and

DNA BarcodingDNA Barcoding

David E. Schindel, Executive SecretaryDavid E. Schindel, Executive SecretaryNational Museum of Natural HistoryNational Museum of Natural History

Smithsonian InstitutionSmithsonian Institution

[email protected];; http://www.barcoding.si.edu202/633-0812; fax 202/633-2938202/633-0812; fax 202/633-2938

Genomics

Subgenomics

Current SystematicStudies

Microbes - 16SPlants - RBCLAnimals - COI

Species Identification MattersSpecies Identification MattersBasic research on evolution, ecologyBasic research on evolution, ecologyEndangered/protected species Endangered/protected species Agricultural pests/beneficial speciesAgricultural pests/beneficial speciesDisease vectors/pathogensDisease vectors/pathogensInvasive species (e.g., in ballast water)Invasive species (e.g., in ballast water)Environmental quality indicatorsEnvironmental quality indicatorsManaging for sustainable harvestingManaging for sustainable harvestingConsumer protection, ensuring food qualityConsumer protection, ensuring food qualityFidelity of seedbanks, culture collectionsFidelity of seedbanks, culture collections

5

Specimens

Characters

Distributions of Character Variation

Taxonomic Decision-Making

Socioeconomic Decisions

Specimens

Concerns/ Regulations

The Practice of Taxonomy

Taxonom

ists

The Uses of Taxonomy

Taxonomic ProcessesTaxonomic Processes

Specimens

Taxon Concepts

Characters

Formal Taxa

Growth of Biodiversity DatabasesGrowth of Biodiversity Databases

Journal Publication

Species Name

Voucher Specimen

Authority files of taxonomic

names

Museum databases of

associated data

Biodiversity Heritage Library

A DNA barcode is a A DNA barcode is a short gene sequence short gene sequence

taken from taken from standardized portions standardized portions

of the genome,of the genome, used to identify species used to identify species

An Internal ID System for All Animals

Typical Animal Cell

Mitochondrion

DNA

mtDNA

D-Loop

ND5

H-strand

ND4

ND4L

ND3COIII

L-strand

ND6

ND2

ND1

COII

Small ribosomal RNA

ATPase subunit 8

ATPase subunit 6

Cytochrome b

COICOI

The Mitochondrial Genome

Non-COI regions for other taxaNon-COI regions for other taxa

Land plants:Land plants:– Chloroplast Chloroplast matKmatK and and rbcLrbcL approved Nov 09 approved Nov 09– Non-coding plastid and nuclear regions being Non-coding plastid and nuclear regions being

exploredexplored

Fungi and protists:Fungi and protists:– CBOL Working Groups convenedCBOL Working Groups convened– Recommendations expected in 2010Recommendations expected in 2010

How Barcoding WorksHow Barcoding WorksFirst, build a barcode reference library:First, build a barcode reference library:– Well-identified specimenWell-identified specimen– Tissue subsampleTissue subsample– DNA extraction, PCR amplificationDNA extraction, PCR amplification– DNA sequencingDNA sequencing– Data submission to GenBankData submission to GenBank

Second, use it to identify unknowns:Second, use it to identify unknowns:– Any unidentified juvenile, adult, fragment, productAny unidentified juvenile, adult, fragment, product– Tissue sample, DNA, sequencingTissue sample, DNA, sequencing– Comparison with sequences in reference libraryComparison 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 extraction CO1 gene DNA sequencing Trace file

Database of Barcode Records

Collecting

ND3

COIII

ND2

ND1

NBII, 25 February 2009NBII, 25 February 2009

GenBank, EMBL, and DDBJGenBank, EMBL, and DDBJGlobal, Open Access to Barcode DataGlobal, Open Access to Barcode Data

http://www.insdc.org/

Current Norm: High throughputLarge labs, hundreds of samples per day

ABI 3100 capillary

automated sequencer

Large capacity PCR and

sequencing reactions

Emerging Norm: Table-top LabsEmerging Norm: Table-top Labs Faster, more portable: Hundreds of samples per hour Faster, more portable: Hundreds of samples per hour

Integrated DNA microchips Table-top microfluidic systems

Producing Barcode Data: 201?Producing Barcode Data: 201?Barcode data anywhere, instantlyBarcode data anywhere, instantly

Data in seconds to Data in seconds to minutesminutes

Pennies per Pennies per samplesample

Link to reference Link to reference databasedatabase

A taxonomic GPSA taxonomic GPS

Usable by non-Usable by non-specialistsspecialists

• 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 Consortium for the Barcode of Life (CBOL)Barcode of Life (CBOL)

Established May 2004 with Sloan Foundation grantEstablished May 2004 with Sloan Foundation grantSecretariat opens at Smithsonian, September 2004Secretariat opens at Smithsonian, September 2004Now in its third two-year funding periodNow in its third two-year funding periodWorkshops, Working Groups, networking, Workshops, Working Groups, networking, representation/marketingrepresentation/marketingNow an international affiliation of 200+ members in Now an international affiliation of 200+ members in 50+ countries:50+ countries:– Natural history museums, biodiversity organizationsNatural history museums, biodiversity organizations– Users: e.g., government agenciesUsers: e.g., government agencies– Private sector biotech companies, database providersPrivate sector biotech companies, database providers

CBOL Member Organizations: 2009

• 200+ 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

GeoreferenceHabitat

Character setsImages

BehaviorOther genes

Trace filesOther

DatabasesPhylogenetic

Pop’n GeneticsEcological

Primers

Databases - Provisional sp.

Outreach ActivitiesOutreach ActivitiesCape Town, South Africa, April 2006, SANBICape Town, South Africa, April 2006, SANBI– Scale insects in African agricultureScale insects in African agriculture

Nairobi, Kenya, October 2006 Nairobi, Kenya, October 2006 – Commercial fisheries in Rift Valley lakesCommercial fisheries in Rift Valley lakes

Brazil, March 2007Brazil, March 2007– Hardwood tree speciesHardwood tree species– Endangered mammals, reptiles, amphibiansEndangered mammals, reptiles, amphibians

Taiwan, September 2007Taiwan, September 2007

Nigeria, October 2008Nigeria, October 2008

Beijing, May 2009Beijing, May 2009

India, March 2010India, March 2010

CBOL’s Global ProjectsCBOL’s Global ProjectsFish Barcode of Life (FISH-BOL)Fish Barcode of Life (FISH-BOL)– 30,000 marine/freshwater species by 201030,000 marine/freshwater species by 2010

All Birds Barcoding Initiative (ABBI)All Birds Barcoding Initiative (ABBI)– 10,000 species by 201010,000 species by 2010

Tephritid fruit fliesTephritid fruit flies– 2,000 pest/beneficial species and relatives by 20082,000 pest/beneficial species and relatives by 2008

MosquitoesMosquitoes– 3,300 species by 20083,300 species by 2008

Endangered speciesEndangered speciesTrees of the worldTrees of the world

Adoption by RegulatorsAdoption by Regulators

Food and Drug Administration Food and Drug Administration – Reference barcodes for commercial fishReference barcodes for commercial fish

NOAA/NMFSNOAA/NMFS– $100K for Gulf of Maine pilot project$100K for Gulf of Maine pilot project– FISH-BOL workshop with agencies, Taipei, Sept 2007FISH-BOL workshop with agencies, Taipei, Sept 2007

Federal Aviation Administration – $500K for birdsFederal Aviation Administration – $500K for birdsEnvironmental Protection AgencyEnvironmental Protection Agency– $250K pilot test, water quality bioassessment$250K pilot test, water quality bioassessment

FAO International Plant Protection CommissionFAO International Plant Protection Commission– Proposal for Diagnostic Protocols for fruit flies Proposal for Diagnostic Protocols for fruit flies

CITES, National Agencies, Conservation NGOsCITES, National Agencies, Conservation NGOs– International Steering Committee, identifying pilot projectsInternational Steering Committee, identifying pilot projects

Why barcodes are shortWhy barcodes are short

Low cost and simplicityLow cost and simplicity– Single capillary reads adequateSingle capillary reads adequate– Avoid post-sequencing IT needed for pyroseqAvoid post-sequencing IT needed for pyroseq

Adequate for taxonomyAdequate for taxonomy– Miminizes uniformative sequence lengthMiminizes uniformative sequence length– Limiting supply rate of identified specimensLimiting supply rate of identified specimens– Technology more accessible to small labsTechnology more accessible to small labs

Regulatory/inspection applications easier Regulatory/inspection applications easier

Barcoding and MetagenomicsBarcoding and Metagenomics

Lots of interest for ecology, less for regulationLots of interest for ecology, less for regulation

Pyrosequencing of environmental mixturesPyrosequencing of environmental mixtures– Construct species list from mixtureConstruct species list from mixture– Predator-prey relations from gut contents, fecesPredator-prey relations from gut contents, feces– Food-web reconstructionFood-web reconstruction

Relies on high accuracy (some species differ Relies on high accuracy (some species differ by only a few percent)by only a few percent)

Not cost-effective for single specimensNot cost-effective for single specimens

What barcode providers have What barcode providers have and are producingand are producing

770,000 records from ~100,000 species770,000 records from ~100,000 species– Agricultural pestsAgricultural pests– Water quality indicatorsWater quality indicators– Disease vectorsDisease vectors– Endangered speciesEndangered species– Commercial species of food, feed, commoditiesCommercial species of food, feed, commodities

5 million records from 500K species by 20155 million records from 500K species by 2015

International Barcode of Life Project (iBOL) International Barcode of Life Project (iBOL) Theme 1 – DNA Barcode LibraryTheme 1 – DNA Barcode Library

WG 1.1 VertebratesWG 1.2 Land PlantsWG 1.3 FungiWG 1.4 Human Pathogens and ZoonosesWG 1.5 Agricultural and Forestry Pest and Their

ParasitoidsWG 1.6 PollinatorsWG 1.7 Freshwater Bio-SurveillanceWG 1.8 Marine Bio-SurveillanceWG 1.9 Terrestrial Bio-SurveillanceWG 1.10 Polar Life

What barcode providers wantWhat barcode providers wantHigh PCR and sequencing success ratesHigh PCR and sequencing success rates

Bigger window into older, compromised Bigger window into older, compromised samplessamples

Better software integration to eliminate Better software integration to eliminate bottlenecksbottlenecks

Smaller labs/developing countries:Smaller labs/developing countries:– Lower equipment and maintenance costsLower equipment and maintenance costs– Simplification for techs with less trainingSimplification for techs with less training– Install anywhere without lab renovationsInstall anywhere without lab renovations– Willing to accept slower throughputWilling to accept slower throughput

What barcode users wantWhat barcode users wantAnswers to specific questions:Answers to specific questions:– Is this thing on this list of species or not?Is this thing on this list of species or not?– Is this thing a member of this genus/family?Is this thing a member of this genus/family?– Which of the species on this list is this thing?Which of the species on this list is this thing?– What species is this thing?What species is this thing?

Production-scale capabilities:Production-scale capabilities:– Hundreds to thousands of installationsHundreds to thousands of installations– Lower but constant throughputLower but constant throughput– Rapid turnaroundRapid turnaround– The right price-point and limited life cycle costsThe right price-point and limited life cycle costs

What barcode users would do What barcode users would do with the reference librarieswith the reference libraries

Inspection stations at every port and international Inspection stations at every port and international airport for:airport for:

– Agricultural pest controlAgricultural pest control

– Illegal trade in endangered speciesIllegal trade in endangered species

– Violations of trade quotasViolations of trade quotas

Regular Federal and State water quality surveysRegular Federal and State water quality surveys

Federal, State and local food inspectionFederal, State and local food inspection

Public health monitoring and diagnosesPublic health monitoring and diagnoses

NBII, 25 February 2009NBII, 25 February 2009

GenBank, EMBL, and DDBJGenBank, EMBL, and DDBJGlobal, Open Access to Barcode DataGlobal, Open Access to Barcode Data

http://www.insdc.org/

NBII, 25 February 2009NBII, 25 February 2009

Linkout from GenBank to BOLD

NBII, 25 February 2009NBII, 25 February 2009

Linkout from GenBank to Taxonomy

NBII, 25 February 2009NBII, 25 February 2009

Link from GenBank to Museums