This document discusses DNA barcoding, which is a method for identifying species using short gene sequences from standardized portions of the genome. It provides background on Carl Linnaeus who established the system of classifying organisms, and discusses how DNA barcoding can be used to establish reference libraries to identify unknown specimens by comparing DNA sequences. While not intended to replace traditional taxonomy, DNA barcoding is presented as a useful tool that can help create a 21st century research environment for taxonomy.

1. Using mtDNA to distinguish species By RoyaShariati ANU College of Archaeology and Anthropology Supervisor: Prof. Colin Groves

2. Carl Linnaeus 250 years ago Carl Linnaeus is best known for creating the system of classifying living organisms that became the international standard. “You say tomato, I say Lycopersiconesculentum. You say potato, I saySolanumtuberosum. But Carl Linnaeus was the real plant buff.” Often called the father of classification, Swedish naturalist Linnaeus established the familiar dual Latin names by which all creatures are now known.

3. DNA Barcoding A DNA barcode is a short gene sequence taken from standardized portions of the genome, used to identify species

4. An organism’s chromosome complement is its karyotype

6. tRNA The following genes encode tRNA: Transport chain rRNA Mitochondrial rRNA is encoded by MT-RNR1 (12S) and MT-RNR2 (16S).

7. D-Loop Small ribosomal RNA Large ribosomal RNA Cyt b ND1 ND6 COI COI ND5 L-strand ND2 H-strand ND4 COI ND4L COII ND3 ATPase subunit 8 COIII ATPase subunit 6 The Mitochondrial Genome

9. Comparison of mitochondrial gene orders among typical vertebrates, E. pelecanoides, and S. lavenbergi. Five blocks of gene regions (A, tRNAGlu–tRNAGln; B, ATP8-ND3; C, tRNALeu(CUN)–ND6; D, tRNAIle–tRNALys; and E, tRNAArg–tRNASer(AGY)gene regions) retain the typical vertebrate gene order with the exception of several tRNA genes (arrowheads). The partial mtDNA sequences for four gene junctions (vertical bar: ND1-ATP8, ND3-ND5, ND6-ND2, and ND4-cyt b regions), the gene order of which greatly differ from that of other typical vertebrates, were determined for four additional E. pelecanoides individuals (data available form DDBJ/EMBL/GenBank with accession numbers AB046475–AB046490)

10. Aremarkably short DNA sequence can contain more than enough information to resolve 10 or even 100 million species. For example, a 600-nucleotide segment of a protein-coding gene contains 200 nucleotides that are in the third position within a codon. At these sites, substitutions are (usually) selectively neutral and mutations accumulate through random drift. Even if a group of organisms was completely biased to either adenosine or thymine (or alternatively, to either guanosine or cytosine) at third nucleotide positions there would still be 2200 , or 1060 , possible sequences based on third-position nucleotides alone. DNA sequence analysis of a uniform target gene to enable species identification has been termed DNA bar-coding, by analogy with the Uniform Product Code barcodes on manufactured goods.

11. The Phylogenetic species concept tokogenetic versus phylogenetic relationships The phylogenetic species concept Speciation and phylogenetic relationships

12. Applied tool for identifying regulated species: Disease vectors, agricultural pests, invasive 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 Uses of DNA Barcodes

13. 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

14. Reactions to Barcoding: 2004 From ecologists and other users:“This is what we need! How soon can we get started?” From traditional taxonomists:“Species should be based on lots of characters, not just barcodes” From forward-looking taxonomists:“Using molecular data as species diagnostics isn’t new, but standardization and broad implementation are great!” From barcoding practitioners:“I had my doubts at the beginning, but it really works as a tool for identification (96% accurate in a recent mollusc paper) and it is at least as good as traditional approaches to discovering new species.”

15. 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 21st century research environment for taxonomy

16. 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 21st century research environment for taxonomy

17. BARCODE Data Standard Voucher Specimen Species Name Specimen Metadata Indices - Catalog of Life - GBIF/ECAT Nomenclators - Zoo Record - IPNI NameBank Publication links - New species GeoreferenceHabitatCharacter setsImagesBehaviorOther genes BarcodeSequence Trace files Primers Other Databases Literature(link to content or citation) PhylogeneticPop’n GeneticsEcological

18. Barcoding projects have four components: 1.The Specimen Collection: Desired Specimen must be collected for which we want to generate DNA Barcodes. 2.The Laboratory Analysis: Barcoding protocols as described in the Materials and Method Section can be followed to obtain DNA Barcode sequences from these collected specimens. 3. The Database: One of the most important components of the Barcode Initiative is the construction of a public library of species identifiers which could be used to assign unknown specimens to known species

19. 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

21. IDS – Identification System

22. DNA from identified voucher Create BARCODE reference record ID unknowns Refine taxonomy of group DNA from unidentified immature specimen Create BARCODE reference records Associate immatures with names DNA from identified adult voucher Repository of provisional vouchers ID unknowns Add names to vouchered immatures Refine taxonomy of group

23. Diagnosis of new species. Taxonomy is rapidly absorbing genetics into its panoply of approaches. Barcoding should be a useful addition to the existing tools for species identification, but it is not intended to replace them. In many groups, alpha taxonomy requires data from morphology, behavior, ecology, natural history, and geographic variation. These data can only be enhanced by complementary information regarding DNA sequences RESULT

25. http://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=An%20external%20file%20that%20holds%20a%20picture%2C%20illustration%2C%20etc.%0AObject%20name%20is%20zpq0040609290003.jpg%20%5BObject%20name%20is%20zpq0040609290003.jpg%5D&p=PMC3&id=1348015_zpq0040609290003.jpg

26. References 1. Fang SG, Wan QH, Fijihara N. 2002. Formalin removal from archival tissue by critical point drying. BioTechniques 33:604-611. 2. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology. 1994 3:294-299. 3. Hebert PDN, Cywinska A, Ball SL, deWaard JR. 2003a. Biological identifications through DNA barcodes. Proceedings of the Royal Society of London, Series B 270:313-322. 4. Hebert PDN, Ratnasingham S, deWaard JR. 2003b. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London, Series B