DNA Barcoding Johannes Bergsten Swedish Museum of Natural History Department of Entomology E-mail: johannes.bergsten@nrm.se Biodiversity Informatics Course, 14-24 September, 2009 Swedish Museum of Natural History, Stockholm, Sweden Image credit: Barcoding institute of ontario

Johannes Bergsten

Swedish Museum of Natural History

Department of Entomology

E-mail: johannes.bergsten@nrm.se

How 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 assigning test 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? A way of identifying samples to species based on a short standardised gene-region Keywords: Identify Samples Species Gene Short Standardised

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 Barcoding identify specimens – a global identification system discover new species – aid and speed up the discovery of the remaining biodiversity

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 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

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

Examples Credit for slide: David E. Schindel What is the fillet served on your plate, on a market or in a package? What are the eggs or molt in the ballast water of ships? Are they non- native invasive species?

Further examples Illegally traded bushmeat, sharkfins, skins Do the products come from protected or banned-for-trade species?

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 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-illustrated This is where we stand today! Credit : David E. Schindel

Sequencing is getting cheap Credit for slide: David E. Schindel

The Vision Credit: iBOL Image credit: Barcoding institute of ontario

“ - 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 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.

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 Metazoans Cytochrome Oxidase subunit I Mitochondrial Easy to amplify Relatively fast evolving Credit: iBOL

Cytochrome Oxidase subunit I

Mitochondrial

Easy to amplify

Relatively fast evolving

The chosen genes for plants Plastid genes rbcL and matK form a 2-locus plant barcode

What are you waiting for? Credit: iBOL

BOLD - project managment

Projects

BOLD – identification engine

No match

Read Publication on BOLD

DNA Barcode standards The standards include three components: 1) Creation of a reserved keyword (” B A RCODE ” ) . NCBI and its collaborators will add the BARCODE ’ F l ag ’ 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);

The standards include three components: 1) Creation of a reserved keyword (” B A RCODE ” ) . NCBI and its collaborators will add the BARCODE ’ F l ag ’ 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 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.

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 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

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;

The 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

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 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.

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 . 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

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 . 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.

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

Linkout from Genbank to taxonomy databases

BOLD linkout from genbank

Trace archives

Recommended data elements

How to submit data

Will DNA Barcoding work? Image credit: Barcoding institute of ontario

The Barcoding gap Barcoding rest on the idea that between species genetic distance is larger, than within species variation. Genetic distance 1%

Differ by >an order of magnitude = Barcoding Gap Supporting data for the Barcoding Gap Critique: Well sampled? Ball et al (2005) 99.00% 18.10% 1.10% 1.9 0.032 80 2,500 Regional (N. Am.) World mayflies Hebert et al (2003) 100% 6.80% 0.25% 1.7 0.0022 200 91700 Local (Guelph) World Lepidopt. 3 sup fam Hebert et al (2004) 100% 7.93% 0.43% 2 0.028 260 9000 Regional (N. Am.) World Birds Barrett & Hebert (2005) 100% 16.40% 1.40% 3 0.0042 168 40,000 Local (Canada) World Spiders paper Id. success intersp div. intrasp var. ind/sp. Prop. sampled species Geographical sampling Distribution Organism

Sisterspecies vs congeners Sisterspecies vs congeners Sisterspecies 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) 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 Carex rostrata (flaskstarr) Carex vesicaria (blåsstarr) Pipistrellus pipistrellus (Pipistrell) Pipistrellus pygmaeus (dvärgfladdermus)

Overlap in cowries Meyer and Paulay, PLoS Biology (2006)

Overlap the reality

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

“ 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.”

How 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.”

” 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 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)

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 cristatus Aulonogyrus 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 P<0.01

Large inventories of the unknown