DNA Barcoding Amy Driskell Laboratories of Analytical Biology National Museum of Natural History Smithsonian Institution, Wash. DC

Outline Barcoding in general Uses & Examples Barcoding Bocas Algae Data, Analysis, etc.

What is a DNA barcode? A fingerprint for identification of everything A short, easily and universally amplifiable, and reasonably variable piece of DNA

Requirements For “Barcode” label in GenBank, for “Reference” in BOLD Sequence is from a vouchered specimen - Re-identify Voucher meta-information required: GPS coordinates, photographs (in situ, in some cases), collector and identifier data - Re-collect DNA sequence is high quality (minimum length, minimum coverage, minimum “quality scores” - Compare Raw data (chromatograms) publicly available - Re-analyze

Organizations Consortium for the Barcode of Life (CBOL) (www.barcoding.si.edu) Barcode of Life Database (BOLD) (www.barcodinglife.org) International Barcode of Life (iBOL) (www.ibolprojec.org) FishBOL, All Birds Barcoding Initiative, MarBOL, etc. etc.

Barcode “Markers” Animals: Mitochondrial cytochrome oxidase subunit I (CO1/COX1) ~ 650 bp BUT, not variable in cnidarians, 16S rDNA historically more informative in many groups (e.g. frogs, some crustaceans). Second marker possible. Fungi: Nuclear internal transcribed spacer (ITS) Non-coding gene and difficult to align. But long history in fungal studies, large existing databases, CO1 contains introns.

Barcode “Markers” Red & Brown Algae: CO1 Green Plants: Not decided Not nearly as “universally” amplifiable as in animals, requires many different primers Green Plants: Not decided SI botanists promote rBCL and the trnH-psbB spacer Others prefer for rBCL and matK All are chloroplast genes Green algae? Still not clear

DNA Barcoding is an imperfect science Evolutionary History: Hybridization (plastid genomes) Pseudogenes Heteroplasmy or multiple copies (nuclear markers) Lineage sorting, recent speciation Slow rates of sequence divergence Practical Difficulties Lack of universality Co-amplification Incomplete sampling Lack of taxonomic experts

Barcode of Life Database (BOLD)

Today’s Statistics BOLD GenBank Total: 570,000 Species: 62,000 “Reference”: 234,000 “Ref” species: 13,774 GenBank “Barcode” keyword: 37,000

Algal Barcodes in BOLD

SI’s Barcoding Philosophy Collaborate - taxonomic experts, students Train - students, interns, other researchers Assist - lab setup, protocol development, collecting Archive - a “lending library” of high quality, well-vouchered DNAs Explore biodiversity in collaboration with taxonomists and phylogeneticists.

Biologically Interesting Uses Biodiversity Exploration Discovery of sibling species Quick assessment of local diversity “DNA assisted alpha taxonomy” Ecological & Behavioral Studies Habitat assessment Stomach content analysis

L.A.B. Examples LAB & Bocas taxonomic workshops CeDAMAR Antarctic deep sea surveys Caribbean Fish Central American Frogs Biocode Moorea

Practical, Fundable Uses Method of identification, particularly useful to non-experts Import/export and pest control Fisheries management Water and habitat quality assessment Partial or fragmentary sample analysis Associating different life stages

L.A.B. Examples USAF/FAA “Bird Strike” Project Larval Fish ID US Food & Drug Agency US Enviromental Protection Agency

Barcoding Bocas Algae Collections 2007, 2008, 2009 ~1000 specimens Red Algae: ~250 specimens sequenced Lab protocol and primer development

The Process of Barcoding Extraction: methods, machines, timing

The Process of Barcoding 2. Amplification - Polymerase Chain Reaction -Primers

The Process of Barcoding 3. Sequencing

The Process of Barcoding Submission and organization of metadata Quality control, data processing Accuracy checking

Nov. 9-13, 2009 Website: dnabarcodes2009.org