ZooGene, a DNA sequence database for calanoid copepods and euphausiids:

 an OBIS tool for uniform standards of species identification


P.I. Ann Bucklin

Ocean Process Analysis Laboratory

University of New Hampshire, Durham, NH 03824

phone: (603) 862-0122   fax: (603) 862-0243   email: ann.bucklin@unh.edu


Co-P.I. Peter H. Wiebe

Department of Biology

Woods Hole Oceanographic Institution, Woods Hole, MA 02543

phone: (508) 268-2313   fax: (508) 457-2169   email: pwiebe@whoi.edu


Co-P.I. Bruce W. Frost

School of Oceanography

University of Washington, Seattle, WA 98195

phone: (206) 543‑7186   fax: (206) 543‑6073   email: frost@ocean.washington.edu


Co-P.I. Michael J. Fogarty

Northeast Fisheries Science Center

National Marine Fisheries Service, Woods Hole, MA 02543

phone: (508) 495-2386   fax: (508) 495-2258   email: fogarty@usglobec.org


NSF Grant No. OCE-0003884





An international partnership will be established to develop a zooplankton genomic (ZooGene) database of  DNA type sequences for calanoid copepods and euphausiids.  The ZooGene database will provide molecular systematic information that will be integrated with taxonomic and biogeographical data [as one of the Ocean Biogeographical Information System (OBIS) federation of databases], with molecular data [via entry-level links to the National Center for Biotechnology Information (NCBI) molecular database GenBank], and with environmental and oceanographic data [via the U.S. GLOBEC Data and Information Management System].  The ZooGene database will have many potential uses, including: 1) uniform standards of species’ identification, 2) evaluation of the taxonomic significance of geographic variation within widespread species, 3) identification of cryptic species,  4) accurate estimation of species’ diversity, 5) determination of evolutionary relationships among species, and 6) design of rapid molecularly-based species’ identification protocols. 




Objectives of the ZooGene partnership include: 1) establishment of a nexus of taxonomic experts for calanoid copepods and euphausiids, with focused effort on selected genera and regions; 2) determination of mtCOI type sequences for 200 species of calanoid copepods and 86 species of euphausiids, based on verified identification of species by taxonomic authorities; 3) design, creation, management of the ZooGene database, a searchable web-based database integrated with the GenBank DNA sequence database and OBIS, as a tool for uniform species identification; and 4) design and implementation of a ZooGene curriculum module for high school students




The ZooGene partnership includes four lead investigators and thirteen expert taxonomic consultants from seven countries.  Zooplankton samples are sorted from existing archival collections, obtained in coordination with planned oceanographic research efforts, and/or collected during National Marine Fisheries Service field surveys.  The taxonomic experts confirm species’ identifications; DNA sequencing is done at the University of New Hampshire and, in some cases, in other partners’ laboratories.  For each species, a DNA type sequence is determined for a portion of the mitochondrial cytochrome oxidase I (mtCOI) gene; multiple mtCOI sequences are included as necessary to reflect intraspecific variation.  For selected species, a portion of the slowly-evolving, nuclear small-subunit (18S) rRNA is sequenced to examine evolutionary relationships among genera and families.  The ZooGene database is designed, created, managed, maintained, and distributed as part of the proposed work; the data will be integrated into the Ocean Biogeographical Information System (OBIS).




New ZooGene collections: ZooGene partners have collected and shipped to UNH comprehensive sets of samples for molecular analysis using ships of opportunity.  Samples are in hand for many copepod genera, based on samples collected and identified by Susumu Ohtsuka (Hiroshima University, Japan); David McKinnon (Australian Institute of Marine Science); Janet Bradford-Grieve (National Institute of Water & Atmospheric Research, New Zealand); Bruce Frost (University of Washington); Peter Wiebe and Nancy Copley (Woods Hole Oceanographic Institution); and Fredrica Norrbin (Tromsoe University).  Euphausiid samples are in hand from Shozo Sawamoto (Tokai University, Japan) and Jaime Färber Lorda (Centro de Investigación Cientifica y de Educación Superior, Mexico).  ZooGene samples have been collected in association with Ecosystem Monitoring Surveys by the NOAA/NMFS Northeast Fisheries Science Center (NEFSC), and are being used to assess spatial and temporal variation in gene frequencies in selected taxa.  New collections are now planned for the subtropical NW Atlantic by Peter Wiebe (WHOI).


DNA type sequences:   To date, considering only the molecular analyses done in A. Bucklin’s laboratory at UNH, the ZooGene project has determined and submitted to GenBank 150 DNA sequences for 81 species of calanoid copepods and 44 DNA sequences for 18 species of euphausiids.  These numbers are increasing rapidly with ongoing work at UNH and elswhere.  MtCOI sequences have proven to be diagnostic molecular systematic characters for both copepods and euphausiids.  The sequences are also useful to reconstruct phylogenetic relationships among congeneric species, resolve large-scale population genetic structure and taxonomically-significant geographic variation, and may help reveal cryptic species (Hill et al., 2001; Bucklin et al., 2002).




Rapid molecular protocols for species identification: We have designed molecular protocols based on species-specific PCR that can discriminate cryptic, co-occurring copepod species: multiplexed SS-PCR protocols are available for two species of Pseudocalanus (Bucklin et al., 1999, 2001); four species of Calanus (Bucklin et al., 1999; Hill et al., 2001; Fig. 1); and two species of Metridia (Bucklin et al, in preparation).



Figure 1.  Diagram showing steps in the molecular identification of copepods, including A) homogenization of the copepod in a microcentrifuge tube, B) multiplexed PCR reaction with all species-specific reagents, and C) results of the PCR reaction displayed on an agarose gel following electrophoresis.  The species can be identified by the migration distance of the product band in the gel.  For more details, see Hill et al., 2001.


Phylogeographic analyses: Cosmopolitan species of copepods and euphausiids have received particular attention under ZooGene.  Samples are taken across each species’ range, in order to evaluate evidence for geographic isolation and genetic differentiation within the species.  MtCOI has provided preliminary evidence of population genetic structure within some geographically widespread species (Bucklin et al., 2002).

Phylogenetic reconstructions: Evolutionary relationships among congeneric copepod and euphausiid species are typically well-resolved by mtCOI sequence variation.  MtCOI gene trees were largely concordant with morphological phylogenetic analyses for species of both copepods (Bucklin et al., 2002) and euphausiids (Bucklin, in preparation).  Phylogenetic relationships among genera and families of calanoid copepods and euphausiids have been examined using 18S rRNA sequences, which provide accurate resolution (Bucklin et al., 2002, unpublished; Grabbert et al., in preparation; Fig. 2), and are useful for comparison with morphological analyses (Bradford-Grieve et al., unpublished). 




Figure 2.   Gene tree showing evolutionary relationships of selected calanoid copepod species, based on a 660 base-pair sequence of the nuclear 18S rRNA gene.  This analysis clustered together species and genera of the same family, as expected from the alpha taxonomy of the group; the evolutionary relationships among the families will require comparison with morphological analyses which have not been completed yet.  Data from Bucklin et al (2002) and S. Grabbert (Univ. Oldenburg; pers. comm.).

The ZooGene database: The ZooGene database is accessible at http://www.ZooGene.org.  The database includes detailed collection information for the individual copepod or euphausiid used to determine the mtCOI type sequence and 18S rRNA sequence.  Also included are the DNA sequences; links to the GenBank database will be implemented shortly (via the GenBank accession number); and to environmental data in the U.S. GLOBEC Data and Information Management System (via the collection location).  The database is linked to and searchable from the Ocean Biogeographical Information System (OBIS) portal/server (http://www.iobis.org/OBISPortal).




We anticipate that biologists, ecologists, and systematists will increasingly use DNA sequences as additional characters for taxonomic identification.  The molecular information may be used to ensure uniform standards of species identification, evaluate the taxonomic significance of geographic variation, reveal cryptic species, accurately estimate species diversity, determine evolutionary relationships among species, develop rapid molecularly-based species identification protocols, and identify morphologically-indistinguishable species at any life-stage.  Despite the challenges, it is important that ecologists and oceanographers identify species accurately.  Zooplankton species are of considerable importance in biogeochemical cycles, because of their numerical abundance, biomass predominance, and/or critical position in coastal and ocean trophic webs.




Quality of Life

ZooGene will allow rapid and accurate identification of planktonic species and estimation of marine biodiversity, which are essential for understanding, assessing, and predicting ecosystem health, and facilitating wise use of marine resources and sound coastal zone planning.


Science Education and Communication


·                      We are organizing a comprehensive systematic and molecular systematic workshop for calanoid copepods and euphausiids to be held at the completion of ZooGene activities.  The workshop will be a forum for ZooGene partners and students, and will facilitate preparation of collaborative manuscripts and renewal proposals.


·                      At the University of Washington, a graduate student (Mikelle Rasmussen) is nearing completion of the Masters’ degree:  Genetic variation among subspecies of the planktonic copepod Calanus pacificus: A ZooGene Application. University of Washington, Seattle.


·                      An international student exchange was conducted between UNH and the Alfred Wegener Institute in Germany.  Sabine Grabbert, a PhD student of Dr. Sigrid Schiel, spent 6 months in A. Bucklin’s laboratory at UNH, completing genetic analysis of copepods that will be included in her doctoral dissertation for the University of Oldenburg (Germany).


·                      A ZooGene molecular kit and curriculum module were designed and implemented for use in New Hampshire high schools.  An informative brochure for teachers was produced, and a one-day work-shop for 5 teachers was held in August, 2000.  A NH Sea Grant Marine Docent is taking the lead in this effort, which is now being tested in local high schools.


·                      A project web site, http://www.ZooGene.org, has been established.  The web site provides general information on the partnership, as well as technical information to guide colleagues in collecting and preserving zooplankton for molecular analysis.



Bucklin, A., R.S. Hill, M. Guarnieri, A.M. Bentley, and S. Kaartvedt (1999)  Taxonomic and systematic assessment of planktonic copepods using mitochondrial COI sequence variation and competitive, species-specific PCR.  Hydrobiol. 401:239-254.


Bucklin, A., M. Guarnieri, D.J. McGillicuddy, and R.S. Hill (2001)  Spring evolution of Pseudocalanus spp. abundance on Georges Bank based on molecular discrimination of P. moultoni and P. newmani.  Deep-Sea Res. 48: 589-609.




Bucklin, A. (2000)  Methods for Population Genetic Analysis of Zooplankton.  Chapter 11 in: The Zooplankton Methodology Manual, International Council for the Exploration of the Sea. Academic press, London.  Pp. 533-570.


Hill, R.S., L.D. Allen, and A. Bucklin (2001)  Multiplexed species-specific PCR protocol to discriminate four N. Atlantic Calanus species, with a mtCOI gene tree for ten Calanus species.  Marine Biology 139: 279-287.


Bucklin, A., P.H. Wiebe, S. B. Smolenack, N.J. Copley, and M.E. Clarke (2002)  Integrated biochemical, molecular genetic, and bioacoustical analysis of mesoscale variability of the euphausiid Nematoscelis difficilis in the California Current. Deep-Sea Res. 49: 437-462.


Bucklin, A.,  B.W. Frost,  J. Bradford-Grieve, L.D. Allen and N.J. Copley (2003) Molecular systematic assessment of thirty-four calanoid copepod species of the Calanidae and Clausocalanidae using DNA sequences of mtCOI and nuclear 18S rRNA.  Mar. Biol. 142: 333-343.


Caudill, C.C. and A. Bucklin. Population genetic diversity of Acartia tonsa: evidence for cryptic speciation.  Hydrobiol. (In revision)