Ann Bucklin University of Connecticut, USA

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Barcoding for CoML Assessing Zooplankton
Diversity
Ann Bucklin University of Connecticut,
USA Photos by R.R. Hopcroft University of
Alaska, USA and L.P. Madin Woods Hole Oceanogr.
Inst., USA
DNA Barcoding Symposium May 15, 2006
Netherlands Royal Academy of Arts Sciences,
Amsterdam
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CoML Ocean Realm Field Projects

• 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

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Why Barcode Zooplankton?

• 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.

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Barcoding Goals for CMarZ

• 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

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CMarZ Cooperating Projects
During 2005 2007, more than 25 CMarZ
cooperating projects are contributing to a global
survey of zooplankton. Morphological and
molecular taxonomic analysis of samples is
coordinated across the CMarZ Network.
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CMarZ Ocean Exploration Cruise
April 2006, Peter Wiebe (WHOI, USA) led CMarZ
Steering Group members and students from 14
countries on a cruise to collect zooplankton and
fish from the deepest waters of the NW Atlantic.
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CMarZ Ocean Exploration Cruise
Deep-sea sampling to gt5000m used a 10-m2 trawl
rigged with fine-mesh nets. Rare, small,
deep-living zooplankton were captured and
returned to the surface in good shape for
taxonomic analysis and DNA sequencing.
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CMarZ Ocean Exploration Cruise
UConn Team DNA set up a DNA sequencing
laboratory on board the RV Ron Brown. DNA
barcodes were determined at sea for 87 species
and 220 individuals.
Brian Ortman, Rob Jennings, Paola Batta Lona,
Ebru Unal, Leo Blanco Bercial, Jason Beaudet
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CMarZ Ocean Exploration Cruise
Species Indiv. Species Group Collect DN
A Known ------------------------------------------
--------------------------------------------------
---------- Protozoa Foraminifera 19 1,541 49
Ciliata 23 -- 450 Ctenophora 22 31 90
Cnidaria Hydromedusae 33 63 842 Siphonophora 7
0 103 160   Scyphozoa 7 12 161
Crustacea Amphipoda 31 47 400   Copepoda 138 1
90 2,000     Euphausiidae 14 20 86   Ostracoda
58 100 169    Other Crustacea 18 23 --
Mollusca Gastropoda 44 107 144   Other
Mollusca 27 17 -- Others   Larvacea 12 26 64  
Nemertea 1 3 99   Polychaeta 3 4 110   Tha
liacea 14 18 45 ---------------------------------
--------------------------------------------------
-------------------  Total (all
groups) 502 768 6,800
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Barcoding Ostracods (Crustacea)
Expert taxonomist Martin Angel (SOC/UK)
identified 58 of 140 known N. Atlantic ostracod
species during the CMarZ cruise. At-sea
discovery of 6 new species, with more new species
likely with continued analysis. Among the first
DNA barcodes for this important but
underappreciated group of crustacean zooplankton.
Martin Angel (center) at sea
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Barcoding Ostracods (Crustacea)
Ostracod species differed in mtCOI sequence by
15 to 38 for 19 species. At UConn, barcodes
will be determined for all 58 ostracod species.
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Barcoding Euphausiids (Crustacea)
Peter Wiebe (center) at sea
Fourteen of 86 euphausiid species were identified
at sea by Peter Wiebe (WHOI, USA). 50
euphausiids including 19 species of Euphausia
have been barcoded to date. Barcodes can
identify species and may reveal cryptic species
within widespread species.
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Barcoding Planktonic Molluscs
At sea, Russell Hopcroft (Univ. Alaska, USA) and
Dhugal Lindsay (JAMSTEC, Japan) identified 71
species of planktonic molluscs, including 44
gastropods and 13 cephalopods. Among 33 known
species of shelled pteropods, 20 were identified
at sea from the cruise samples.
Russ Hopcroft and Dhugal Lindsay at sea
Species Species Gastropods
Identified Known Euthecosome (shelled)
pteropods 20 33 Gymnosome (naked) pteropods
5 51 Pseudothecosome pteropod
1 7 Heteropods 17 29 Nudibranch 1 6
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Barcoding Planktonic Gastropods
MtCOI barcodes for 10 species of gastropods
(pteropods and heteropods) differed by 14 to
35. All remaining species will be barcoded at
UConn.
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Barcoding Medusozoans (Cnidaria)
Francesc Pages at sea
Francesc Pages (ICM, Spain) and Larry Madin
(WHOI, USA), identified 110 species of Cnidarians
at sea, including 70 of 160 species of
siphonophores. Barcoding is done by Brian Ortman
(UConn/USA).
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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.
• Barcoding of Arctic zooplankton diversity
  including gt400 species, with 150 hydromedusae
  and 150 copepods is within reach.

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Future Applications of Barcodes

• Zooplankton barcode database requires coordinated
  efforts by CMarZ taxonomists and geneticists,
  working taxon-by-taxon and region-by-region.
• Barcode database will lead to rapid, automatable
  DNA-based protocols for species identification of
  zooplankton.
• DNA microarrays or chips will be designed with
  detection sequences for species of a geographic
  region or zooplankton group.
• Environmental sequencing of unsorted
  zooplankton samples will speed development of
  species-identification protocols.

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Results and Conclusions

• 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
• Zooplankton are indicators of ecosystem health
• Zooplankton species diversity is base-line for
  assessing mans effects on the ocean climate,
  pH, pollution, and commercial fisheries.

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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
(robert.jennings_at_uconn.edu). Got data? DNA
barcodes for zooplankton should be submitted to
1. BARCODE section of GenBank
(www.ncbi.nlm.nih.gov/BankIt/barcode) 2.
Barcode of Life Database (BOLD see
www.barcodinglife.org.
UCONN address for specimen shipment Ann
BucklinDepartment of Marine Sciences University
of Connecticut - Avery Point 1080 Shennecossett
Road, Groton, CT 06340 USA Email
ann.bucklin_at_uconn.edu
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Acknowledgements CMarZ Steering Group Members
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
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Acknowledgements UConn Team DNA
Rob Jennings (CMarZ-USA Project Manager /
Postdoc) 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.) Jason Beaudet not shown (Univ.
Conn. Lab Manager)
UConn Team DNA on the RV Ron Brown April 2006