johannes bergsten dna barcoding
DESCRIPTION
Johannes Bergsten lecture on Thursday, Sept 17, 2009, for the Biodiversity Informatics Course, a Swedish Taxonomy Initiative (Svenska Artprojektet) course at the Swedish Natural History Museum, Stockholm, supported by the Swedish Species Service (ArtDatabanken) and the Swedish GBIF node.TRANSCRIPT
DNA BarcodingDNA Barcoding
Johannes BergstenSwedish Museum of Natural History
Department of EntomologyE-mail: [email protected]
Biodiversity Informatics Course, 14-24 September, 2009Swedish Museum of Natural History, Stockholm, Sweden
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How it all started in 2003How it all started in 2003
Propose a CO1-based (~650bp of the 5’ end)global identification system of animals, and show the success (96.4-100%) of assigningtest specimens to the correct phyla, order and species(Lepidoptera from Guelph) through a CO1-profile.
98% of congeneric species in 11 animal phyla showed>2% sequence divergence in CO1
What is DNA Barcoding?What is DNA Barcoding?
• A way of identifying samples to species based on a short standardised gene-region
• Keywords:
• Identify
• Samples
• Species
• Gene
• Short
• Standardised
2 main uses of DNA Barcoding2 main uses of DNA Barcoding
• identify specimens – a global identification system
• discover new species – aid and speed up the discovery of the remaining biodiversity
Credit for slide: Paul Hebert
Why DNA Barcoding?-the applications
Why DNA Barcoding?-the applications
• Identification of all life stages, eggs, larvae, nymphs, pupa, adults• Identification of fragments or products of organisms• Identification of stomach contents, trace ecological food-chains• Identification of cryptic look-alike species• Food control• Customs control• Invasive species control• Disease vector control• Police • Agriculture• Forestry• Conservation• Education• Etc
ExamplesExamples
Credit for slide: David E. Schindel
What is the filletserved on your plate, on a market or in a package?
What are the eggsor molt in the ballast waterof ships? Are they non-native invasive species?
Further examplesFurther examples
Illegally traded bushmeat, sharkfins, skins
Do the products comefrom protected or banned-for-tradespecies?
Why DNA Barcoding?The biodiversity-taxonomy crisis
Why DNA Barcoding?The biodiversity-taxonomy crisis
• The Biodiversity crisis
• We have yet to discover and describe maybe 90% of the biodiversity
• Humans are responsible for a mass extinction that is going fast!
• Traditional taxonomy is too slow!
• Taxonomic expertise is vanishing and training new taxonomists is too expensive
• Democratizing taxonomic knowledge
The crisis-illustratedThe crisis-illustrated
This is where we stand today! Cre
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Sequencing is getting cheapSequencing is getting cheap
Credit for slide: David E. Schindel
The VisionThe Vision
Credit: iBOL
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“- Mum is this a grizzly bear or a black bear?”
“- Well Johnnie why don’t you go poke your barcoder
into it and find out.”
(Cameron et al Syst. Biol: 2006)
Criticism
The Barcoding Movement The Barcoding Movement
• CBOL: a consortium of 200 member institutions/organizations from 50 countries that promote and standardize DNA Barcoding
• iBOL: an alliance of 16 nations trying to get the big bucks to do the job.
The chosen gene for MetazoansThe chosen gene for Metazoans
• Cytochrome Oxidase subunit I• Mitochondrial• Easy to amplify• Relatively fast
evolving
Credit: iBOL
The chosen genes for plantsThe chosen genes for plants
Plastid genes rbcL and matK form a 2-locus plant barcode
What are you waiting for?What are you waiting for?
Credit: iBOL
BOLD - project managmentBOLD - project managment
ProjectsProjects
BOLD – identification engineBOLD – identification engine
No matchNo match
Read Publication on BOLDRead Publication on BOLD
DNA Barcode standardsDNA Barcode standards
• The standards include three components: 1) Creation of a reserved keyword (”BARCODE”). NCBI and its collaborators will add the BARCODE ’Flag’ to new submissions that meet the standards established in consultation with CBOL. Data records that meet these criteria will be known as BARCODE records in INSDC (BRIs);
Required data elementsRequired data elements
• 2) Required data elements.
• To provide the user community with reliable, retrievable and verifiable information concerning the barcode sequence itself, the specimen from which it was obtained, and the species name that was applied by the submitter.
Data on the specimenData on the specimen
• a) Include a link to a voucher specimen using a structured field* specified by CBOL and NCBI, and to the metadata associated with that specimen and contained in the public database of the voucher specimen’s repository.
• b) Include a link to a documented species name found in one of the sources specified by CBOL and NCBI;
• c) Include Country-Code, using the controlled vocabulary used by GenBank;
*(institution|collection|item) e.g. NHRS:ENT-LEPI:AA008745
The Barcode regionThe Barcode region
• d) Come from a gene region accepted by CBOL as an effective barcode. Initially, only cytochrome c oxidase 1 is approved as a barcode region, defined relative to the mouse mitochondrial genome as the 648 bp region that starts at position 58 and stops at position 705.
• (For plants matK and rbcL is expected to get the same status very soon)
• CBOL has procedures for applying for other generegions to be given barcode status
Quality of sequenceQuality of sequence
• e) Include at least 500 contiguous unambiguous base-pairs from bidirectional sequencing within the approved barcode region. However, if requested, GenBank could assign the BARCODE flag to records with shorter sequences
• f) Include no more than 1% ambiguous sites for the entire submitted sequence;
• g) Include the name of the gene region used; • h) Be associated with trace file submitted to the NCBI Trace Archive or
the Ensembl Trace Server;• i) Include the sequences of all forward and reverse primers used. For
records in which the contiguous sequence was assembled from more than one amplicon or when a cocktail of multiple primers was used for amplification, multiple sets of primer pairs must be provided. In addition, submission of the names of the forward and reverse primers with the primer sequences is strongly recommended.
Strongly recommended data elements.
Strongly recommended data elements.
• Strongly recommended data elements. The following data elements have been added to the INSDC at CBOL’s request for validation of the voucher specimen, and will be strongly recommended but not required:
• j) Latitude and longitude;
• k) Name of the identifier;
• l) Name of the collector;
• m) Date of collection
Governance rules.Governance rules.
• 3) Governance rules. The INSDC provides an archive of records that can only be changed by the submitter. In the case of BRIs, the following modifications are implemented:
• CBOL can allow <500bp sequences to get barcode status (e.g. types, extinct spp.)
• CBOL maintains a process by which alternative generegions can attain barcode status
• BRIs submitted via BOLD are jointly submitted by the researcher and BOLD and can be edited by both.
• CBOL can recommend the BARCODE status to be removed from sequences submitted to INSDC by an individual researcher.
• A system for attaching third-party comments, criticism and suggested corrections to BRIs will be installed.
Credit for slide: David E. Schindel
Voucher repository linkout from genbank
Voucher repository linkout from genbank
Linkout from Genbank to taxonomy databases
Linkout from Genbank to taxonomy databases
BOLD linkout from genbankBOLD linkout from genbank
Trace archivesTrace archives
Recommended data elementsRecommended data elements
How to submit dataHow to submit data
Will DNA Barcoding work?Will DNA Barcoding work?
Image credit: Barcoding institute of ontario
Barcoding rest on the idea that between species genetic distance is larger, than within species variation.
Genetic distance
The Barcoding gapThe Barcoding gap
1%
OrganismDistribution
Geographical sampling species
sampled Prop.
ind/sp.
intrasp var.
intersp div.
Id. success paper
Spiders WorldLocal (Canada) 40,000 168
0.0042 3 1.40% 16.40% 100%
Barrett & Hebert (2005)
Birds WorldRegional (N. Am.) 9000 260 0.028 2 0.43% 7.93% 100%
Hebert et al (2004)
Lepidopt. 3 sup fam World
Local (Guelph) 91700 200
0.0022 1.7 0.25% 6.80% 100%
Hebert et al (2003)
mayflies WorldRegional (N. Am.) 2,500 80 0.032 1.9 1.10% 18.10% 99.00%
Ball et al (2005)
Differ by >an order of magnitude= Barcoding Gap
Supporting data for the Barcoding Gap
Critique:Well sampled?
Sisterspecies vs congenersSisterspecies vs congeners
Panthera leo (lejon)
Panthera tigris (tiger)Motacilla flava (gulärla) Motacilla alba (sädesärla)Carabus nitens (guldlöpare) Carabus coriaceus (läderlöpare)Salix herbacea (dvärgvide)
Salix caprea (sälg)
Sisterspecies vs congeners
Agabus elongatus
A. congener A. lapponicus
A. thomsoni
A. moestus
A. levanderi
A. clypealis
A. pseudoclypealis
Sylvia minula (ökenärtsångare)
Sylvia curucca (ärtsångare)Eupeodes luniger
Eupeodes latilunulatus
Sisterspecies vs congeners
Carex rostrata (flaskstarr)Carex vesicaria (blåsstarr)
Pipistrellus pipistrellus (Pipistrell)
Pipistrellus pygmaeus (dvärgfladdermus)
Overlap in cowriesOverlap in cowries
Meyer and Paulay, PLoS Biology (2006)
Overlap the realityOverlap the reality
How DNA barcodes should not be used
How DNA barcodes should not be used
• “It is expected that DNA barcodes will contribute to the discovery and formal recognition of new species. However, DNA barcodes should not be used as the sole criterion for description of new species, which instead require analysis of diverse data, including morphology, ecology, and behavior, as well as genetics.”
From draft conference report: Taxonomy, DNA, and the Barcode of Life, 2003
How not to be usedHow not to be used
• ”We were interested to see whether Xus exemplaris would be considered a species under standard DNA barcoding protocol”
• ”Using the DNA Barcoding protocol…..therefore under a 3% threshold and a 10x mean intraspecific threshold Xus exemplaris would be considered a good species.
• ”However if we use the smallest among-species divergence as recomended by Meier et al (2008) Xus exemplaris would not be considered a good species under the protocol.”
Barcodes are very useful for species discovery
Barcodes are very useful for species discovery
• For poorly known groups DNA delimitation can be a good starting point for species discovery
• There are alternatives to an artifical 1, 2 or 3% sequence divergence as a threshold
• E.g. GMYC General Mixed Yule Coalescence method (Pons et al, 2006)
Aulonogyrus cristatusAulonogyrus goudoti
Gyrinus madagascariensis
Dineutes subspinosus
Dineutes sinuosipennis
Dineutes proximus
Gyrinus ignitus
Orectogyrus cyanicollis
Orectogyrus pallidocinctus
Orectogyrus vestitus
Orectogyrus sedilloti
GMYC model (Pons et al, 2006)
Andasibe
Ranomafana
Mont. D’Ambre
Antsabe
likelihood
574
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578
580
582
584
586
588
590
592
-1 4 9 14 19 24 29 34 39 44 49
likelihood
P<0.01
Large inventories of the unknown