Molecular identification of museumspecimens

Contents

• Definition of DNA barcoding.– Species comparison.

• Barcoding gap.– Barcoding gap visualised.– Geographical barcoding

‘gaps’.• Consortium for the Barcode Of

Life.– Ongoing DNA barcoding

projects• Barcoding in plants• Importance of databases.

– BOLD in more detail.– Link to Genbank– Databasing in JEMU

• Barcoding workflow text– visualised.

• Prerequisites for DNA barcoding.

• Proposed uses of DNA barcoding.

Definition of DNA barcoding

• BARCODE OF LIFE: A short DNA sequence, from a uniform locality on the genome, used for identifying species. – DNA barcodes are compared with other DNA barcodes.

• Consortium for the Barcode of Life, CBOL.

Barcoding gap

• A higher inter specific variation compared to intra specific variation.• Pair wise comparison of sequences from different species. • and a comparison of sequences of the same species covering their

geographic range of occurrence.• Comparison with the Kimura-2-parameter model (K2P), estimated

with equal base composition and observed transition/transversionratio (ti/tv ratio).

Barcoding gap visualised

‘Gap’ between inter/intra specific variation

Barett en Hebert 2005 Hebert et al. 2004

Barcoding gap visualised(I) Intraspecific distance >2%; interspecific distance <2%: concordant with currentTaxonomy.(II) Intraspecific distance >2%; interspecific distance, >2%: probable composite species (i.e., candidate for taxonomic split)(III) Intraspecific distance <2%; interspecific distance, <2%:recent divergence, hybridization, or synonymy (IV) Intraspecific distance, <2%; interspecific distance >2%: probable misidentification of specimen.

Hebert et al. 2004

Geograpical barcoding gaps!

• Africa!!!• South East Asia• Middle East

Consortium for the Barcode Of Life

• An international collaboration of natural history museums, herbaria, biological repositories, and biodiversity inventory sites, together with academic and commercial experts in genomics, taxonomy, electronics, and computer science �RMCA & RBINS also take part.

• Spearheaded by University of Guelph, Canada and the Canadian government.

• Aims:– Compile DNA barcodes

– Make them available in a database, with all relevant information– Discover new species

– Promote development of portable devices for species identification!

Ongoing DNA barcoding projects

Barcoding all lepidopteran species --- http://www.lepbarcoding.org/progress.php9282 species barcoded, 97813 barcodes/ 36% of species barcoded

Fish barcoding of life --- http://www.fishbol.org/barcoded 4209 species, 14% of fish species

Baroding life on the (north) pole --- http://www.polarbarcoding.org/2560 species of which 693 have been barcoded or 27%, with barcodes belonging to 12798

All birds barcoding initiative --- http://www.barcodingbirds.org/2098 species barcoded, or 21% of all birds, with barcodes belonging to 11289 specimens

Tephritid barcoding initiative --- http://projects.bebif.be/fruitfly/index.htmlbarcoding: ongoing

And more…

Barcoding in plants

• For animals usually COI, for plants still undecided, but goodcandidates from the chloroplast such as matK, rpoC1, atpF/H

• Mtgenome difficult in plants

• Evolution speed, slow, with notable exceptions (Bakker et al. 2006)

• Conclusion, discuss with researcher and JEMU and use multiple genes

Animal mt DNA

Plant mt DNA

Importance of databases

• Generating data (DNA barcodes) is ‘easy’.

• Managing a lot of data correctly is not.

• �Barcoding of life data system. – A workbench

http://www.barcodinglife.org/views/login.php

BOLD in more detailProject overview

Individual specimen info

Detailed location info

Link to GenbankTo submit ‘barcodes’ as ‘barcodes’ in Genbank a set of criteria has to be met

Whenever possible alsoobtain the ‘recommended’ fields

Databasing in JEMU

• Properties

– Transient: specimens and/or tissue.

– Permanent?: DNA extracts/pcrproducts.

• Barcodes (real ones this time)

– Link to spreadsheet like data

Barcoding workflow

*Museum voucher specimen (compulsory) � Obtain the accompanying data .� determining which part is used for DNA extraction: feathers, toes,

abdomens, finclips, leaves, etc…� voucher the selected part extra, e.g. abdomen foto’s of fruitflies

(optional).� select markers, for animals usually COI part cds. � extraction of DNA in JEMU labs � amplify marker (pcr) � sequence marker.� use sequence in downstream analysis, e.g. phylogenies,

phylogeography.

Museum/herbarium/other specimens

Feather(shaft)

Talk to specialist/curator

Extra vouchering

Feather photo

DNA extraction

DNA extract

Decision on specimenpart & DNA marker

Amplifying marker

Pcr product

Sequencing

Sequence(s)

Analysis and research

Determining species

Publications in… nature, PNAS etc..

?

When is DNA barcoding difficult orimpossible

• Recent speciation/ Incomplete lineage sorting (e.g. tephritidfruitflies).

• Hybrydisation. (Hebert et al. 2003)• Relocated mt genes to the nucleus. (Daley et al. 2002)• Crossing over of genes, e.g. moss genes to plants. (Bergthorsson et

al. 2003)

Prerequisites for DNA barcoding

• Good taxonomy/taxonomists• Good coverage of specimens

– RBINS & RMCA & NBGB• Good acces to collections� for instance via synthesys

http://www.synthesys.info/• Good cooperations with other musea/institutions• Good contacts across the world• An informative marker, COI does not work for ‘young’ evolutionary

lineages.

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Proposed uses of DNA barcoding

• Identification of CITES species http://www.cites.org/• Identification of trade samples, especially processed ones,

(ivory/caviar) (Wasser et al. 1989 & Comstock et al 2003)• Identification of disease vectors (mosquitos, mice/rats)• Speeding up decision making on quarantine markers• Monitoring invasive species• Determining species richness in nature reserves• Species determination irrespective of life stage/size

– Fruitflies– Algae

• All the above, faster and in larger quatities empowering governments and consumers

ACTHANKGTGYOUCTAFORGTAYOURGCTATTENTION

Questions?

Sources• http://www.donaldheald.com/pictures/15835.

jpg (greater bird of paradise/paradisaeaapoda picture)

• http://www.grete-david.co.uk/4613.html (Balistarling/Leocopsar rothchildi picture)

• CBOL: ten reasons for barcoding• S. Ratnasingham and P. D. N. Hebert:

Barcode of life datasystem. Molecular Ecology Notes (2007)7, 355–364

• http://media.2theadvocate.com/ (1st sheet dead birds picture)

• http://evolgen.biol.metro-u.ac.jp/MEGA/manual/Contents.html (online MEGA manual)

• R. D. M. Page & E. C. Holmes: Molecularevolution a phhylogenetic approach

• Robert D. Ward, Tyler S. Zemlak, Bronwyn H. Innes, Peter R. Last and Paul D. N. Hebert: DNA barccoding australia’s fish species, Phil. Trans. R. Soc. B (2005) 360, 1847–1857

• K.E. Comstock, E. A. Ostrander & S. K. Wasser. Conservation biology, p 1840-1843, 17(6) 2003, amplyfying nucelar and mitochondrial DNA from african elephantivory: a tool for monitoring the ivory trade.

• S. K. Wasser, C. Mailand, R. Booth, B. Mutayoba, Emily Kisamo, B. Clark, and M. Stephens. PNAS 4228–4233 6, 2007, 104 (10). Using DNA to track the origin of the largest ivory seizure since the 1989 trade ban.

• J. H. Robins, M. Hingston, E. Matisoo-Smith and H. A. Ross. Identifying Rattus species using mitochondrial DNA. Molecular Ecology Notes (2007)7, 717–729.

• M. Hajibabaei, G. A. C. Singer, E. L. Clare and P. D. N. Hebert. BMC Biology 2007, 5:24. Design and applicability of DNA arrays and DNA barcodes in biodiversity monitoring.

• D. O. Daley, K. L. Adams, R. Clifton, S. Qualmann, A. H. Millar, J. D. Palmer, E. Pratjeand J. Whelan. The Plant Journal (2002) 30(1), 11±21. Gene transfer from mitoch-ondrion to nucleus: novel mechanisms for gene activation from Cox2

• P. D. N. Hebert, S. Ratnasingham and J. R. deWaard. Barcoding animal life: cytochromec oxidase subunit 1 divergences among closely related species. Proc. R. Soc. Lond. B (Suppl.) 2003.

• U. Bergthorsson, K. L. Adams, B. Thomason & Jeffrey D. Palmer. Widespread horizontal transfer of mitochondrial genes in flowering plants. NATURE, VOL 424, 10 JULY 2003, p. 197-201.

• R. D. H. Barrett and P. D. N. Hebert. Identifying spiders through DNA barcodes. Can. J. Zool. 83: 481–491 (2005)

• P. D. N. Hebert, M. Y. Stoeckle, T. S. Zemlak, C. M. Francis. Identification of Birds through DNA Barcodes. PLoS Biology www.plosbiology.org October 2004 Volume 2, Issue 10.

• Other recomended reading :� R. D. M. Page & E. C. Holmes:

Molecular evolution a phhylogenetic approach

� Griffiths et al. An introductionto genetic analysis.