Rob Jennings – University of Connecticut, USA Photos by R.R. Hopcroft – University of Alaska, USA L.P. Madin – Woods Hole Oceanographic Institution, USA.

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Rob Jennings – University of Connecticut, USA Photos by R.R. Hopcroft – University of Alaska, USA L.P. Madin – Woods Hole Oceanographic Institution, USA N. Copley – Woods Hole Oceanographic Institution, USA CMarZ Steering Group Meeting 6 November – Ocean Research Institute, University of Tokyo Barcoding for CoML: Assessing Zooplankton Diversity

 Natural Geography in Shore Areas – NaGISA  Gulf of Maine Area Program – GOMA  Pacific Ocean Shelf Tracking – POST  Census of Diversity of Abyssal Marine Life – CeDAMar  Tagging of Pacific Pelagics – TOPP  Patterns and Processes of Ecosystems in the Northern  Mid-Atlantic – MAR-ECO  Biogeography of Chemosynthetic Ecosystems – ChEss  Arctic Ocean Diversity – ArcOD  International Census of Marine Microbes – ICOMM  Census of Marine Zooplankton - CMarZ  Global Census of Marine Life on Seamounts – CenSeam  Global Census of Coral Reef Ecosystems – CREEFS  Continental Margin Ecosystems on a Worldwide Scale  – CoMargE  Census of Antarctic Marine Life – CAML CoML Ocean Realm Field Projects

What is DNA Barcoding?  Definition: Derivation of short DNA sequence(s) that enables species identification or recognition in a particular domain of life (e.g., eucaryotes).  Focus to date: For animals, a 658 base-pair fragment of the mitochondrial gene, cytochrome oxidase subunit I (mtCOI).  Barcode of Life Initiative (BOLI) will resolve barcodes for named species and use a barcoding approach to assess undescribed biological diversity.

What isn’t DNA Barcoding?  It is not intended to, in any way, supplant or invalidate existing taxonomic practice.  It is not DNA taxonomy; it does not equate species identity, formally or informally, with a particular DNA sequence.  It is not intended to duplicate or compete with efforts to resolve deep phylogeny (e.g., Assembling the Tree of Life, ATOL).

 DNA is particularly useful to study animal plankton, because the organisms are frequently rare, fragile, and/or small.  Evolutionarily-conserved body plans for some groups (e.g., copepods) makes morphological identification difficult and mistakes likely.  Many species are widespread or circumglobal; DNA can be used to evaluate taxonomic significance of geographic variation.  DNA-based species identification will speed analysis of samples for known species.  Zooplankton will test barcode protocols, since 15 animal groups (phyla) are represented. Why Barcode Zooplankton?

Link morphological / molecular systematic analysis for global zooplankton assemblage DNA barcode ~7,000 described species in 15 phyla Submit DNA, specimen & collection data: - Barcode section of GenBank - CMarZ database with environmental data - Searchable from OBIS portal Reveal cryptic species within circumglobal species by population genetic analysis Discover new species by sampling biodiversity hotspots, unexplored ocean regions, deep sea Assess zooplankton diversity by ‘environmental sequencing’ of unsorted samples Develop automatable DNA chip-based approaches and protocols to identify and quantify species Barcoding Goals for CMarZ

Barcoding Ostracods (Crustacea) Ostracod species differed in mtCOI sequence by 15% to 38% for 19 species (15 more completed). Barcoding for Ostracods: Good species identification Cryptic variation within species

Barcoding Euphausiids (Crustacea) Fourteen of 86 euphausiid species were identified by Peter Wiebe. 50 euphausiids  including 19 species of Euphausia  have been barcoded to date. Barcoding for Euphausiids: Good at species identification Can reveal cryptic species

Barcoding Planktonic Gastropods MtCOI barcodes for 17 species of gastropods (pteropods and heteropods) differed by 1.4% to 44%. Barcoding for Gastropods: Good at species identification Less consistent species phylogeny

Barcoding Medusozoans (Cnidaria) Barcoding is done by Brian Ortman (UConn/USA). 13 species of siphonophores greatly expanded, much of siphonophore diversity (160 spp.)

Barcoding Arctic Zooplankton  ArcOD (Arctic Ocean Diversity) sends identified specimens of Arctic zooplankton for barcoding by CMarZ.  Comprehensive DNA database of ~210 species of the Central Arctic assemblage is underway.  DNA obtained from ~50 species already, comprising several dates and locations; work in progress at UConn

 Barcoding cruises  “DNA sensitive” collection protocols- anyone can collect  Standardized and web-available sampling and processing protocols  Taxonomic training  Molecular training  Increased ability to use preserved (e.g. formalin) samples  Distributed network of barcoding centers  Local taxonomic expertise  Care of specimens  Capacity-building CMarZ Barcoding Structure and Operations

CMarZ Barcoding Centers Existing barcoding centers UConn, USA  Oviedo, Spain  ORI, Japan Emergent barcoding centers Qingdao, China  Goa, India

 Global-scale analysis of zooplankton species diversity; integrated morphological and molecular analysis.  DNA barcodes for all ~6,800 known species of 15 phyla in the zooplankton.  Species discovery by CMarZ will double number of zooplankton species - sampling new ocean regions (deep sea) - cryptic species within circumglobal species Results and Conclusions

Got specimens? CMarZ encourages and challenges the CoML community to provide identified specimens of holozooplankton groups for barcoding. A DNA barcode (700 base-pair region of mtCOI with replicates / metadata) will be determined for appropriately preserved (i.e., frozen or in alcohol) and identified holozooplankton specimens. Contact Rob Jennings Got data? DNA barcodes for zooplankton should be submitted to: 1. BARCODE section of GenBank ( 2. Barcode of Life Database (BOLD; see UCONN address for specimen shipment: Ann Bucklin Department of Marine Sciences University of Connecticut - Avery Point 1080 Shennecossett Road, Groton, CT USA

Demetrio Boltovskoy (Arg.) Janet Bradford-Grieve (NZ) Ann Bucklin (USA) Colomban de Vargas (France) Ruben Escribano (Chile) Steven Haddock (USA) Steve Hay (UK) Russell R. Hopcroft (USA) Ahmet Kideys (Turkey) Laurence P. Madin (USA) Webjørn Melle (Norway) Vijayalakshmi Nair (India) Shuhei Nishida (Japan) Mark D. Ohman (USA) Francesc Pagés (Spain) Annelies Pierrot-Bults (Netherlands) Chris Reid (UK) Sigrid Schiel (Germany) Sun Song (China) Erik Thuesen (USA) Hans Verheye (South Africa) Peter Wiebe (USA) CoML Zooplankton Workshop Portsmouth, NH USA, March 2004 Acknowledgements CMarZ Steering Group Members

Brian Ortman (Univ. Conn. Ph.D. student) Leo Blanco Bercial (Univ. Oviedo, Spain) Ebru Unal (Univ. Conn. Ph.D. student) Paola Batta Lona (Univ. Conn. Res. Assist.) Lisa Nigro – not shown (Univ. Conn. Lab Manager) CJ Sweetman – not shown (Univ. Conn. Guest Researcher) UConn Team DNA on the RV Ron Brown April 2006 Acknowledgements UConn Team DNA