NOPP Progress Report (January 12, 2001)

ZooGene, a DNA sequence database for calanoid copepods and euphausiids:
an OBIS tool for uniform standards of species identification

Ann Bucklin, University of New Hampshire Durham, NH
Bruce W. Frost, University of Washington, Seattle, WA
Peter H. Wiebe, Woods Hole Oceanographic Institution, Woods Hole, MA
Michael J. Fogarty, NOAA/NMFS Northeast Fisheries Science Center, Woods Hole, MA

Funded under the auspices of the
National Ocean Partnership Program and the Census of Marine Life
by the National Science Foundation (Grant No. OCE-0003884)

Project web site:

Project Summary

An international partnership has been established to develop a zooplankton genomic (ZooGene) database of DNA type sequences for calanoid copepods and euphausiids. The ZooGene partnership includes four principal investigators and thirteen expert taxonomic consultants from seven countries. Zooplankton samples are being sorted from existing archival collections, obtained in coordination with planned oceanographic research efforts, and collected during National Marine Fisheries Service field surveys. The taxonomic experts are confirming species' identifications; DNA sequencing is being done at the University of New Hampshire and, in some cases, in other partners' laboratories. For each species, a DNA type sequence is being determined for a portion of the mitochondrial cytochrome oxidase I (mtCOI) gene; multiple mtCOI sequences will be included as necessary to reflect intraspecific variation. The ZooGene database is being designed, created, managed, maintained, and distributed as part of the proposed work; the data will be integrated into the Ocean Biogeographical Information System (OBIS). Operational and research uses the ZooGene database include: 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.

Education and outreach efforts include: training of graduate students at UNH and UW; a hands- on Molecular Systematics Workshop at UNH during Summer, 2002; exchanges between the P.I.s, international partners, and their colleagues and students; inclusion of ZooGene molecular and environmental data in the OBIS database, and others; creation and maintenance of a project web site; production of a CD with the ZooGene database and simple search tools; and development of a ZooGene curriculum module for high school students by the NH Sea Grant Marine Docents.

Objectives of the ZooGene Partnership

Work Completed

New ZooGene collections: ZooGene partners have collected and shipped to UNH comprehensive sets of samples for molecular analysis. Samples are in hand for the copepod genera: Paracalanus and Tortanus, collected and identified by Susumu Ohtsuka (Hiroshima University, Japan); Acartia, collected and identified by David McKinnon (Australian Institute of Marine Science, Australia), with additional collections planned by Janet Bradford-Grieve (National Institute of Water & Atmospheric Research, New Zealand), and Pseudocalanus, collected and identified by Bruce Frost (University of Washington, USA) and Fredrica Norrbin (Tromso University, Norway). Euphausiid samples are in hand from Shozo Sawamoto (Tokai University, Japan) and Jaime Frber Lorda (Centro de Investigacin Cientifica y de Educacin Superior, Mexico). Detailed planning is underway for ZooGene collections in association with Ecosystem Monitoring Surveys by the NOAA/NMFS NE Fisheries Science Center to assess spatial and temporal variation in gene frequencies in selected taxa.

DNA sequences of calanoid copepods: We have determined mtCOI type sequences for 34 species of six calanoid genera. These sequences have been submitted to GenBank and are the subject of two publications: Hill et al. (2001) and Bucklin et al. (in preparation). MtCOI sequences have proved to be diagnostic molecular systematic characters. In addition, the sequences are likely to resolve large-scale population genetic structure, racial and other subspecific differentiation, and cryptic species, based on preliminary analysis of three geographic populations of Calanus helgolandicus and two subspecies of C. pacificus (Hill et al., 2001; Bucklin et al., in preparation). MtCOI sequence variation resolved evolutionary relationships among most species, although the most distantly-related species were not well resolved.

DNA sequences of euphausiids: Determination of the mtCOI type sequences is complete for 14 species in eight genera of euphausiids. A manuscript summarizing this work has been accepted with revision for publication in the Journal of Marine Research. The sequences will be submitted to GenBank in the near future. MtCOI is a diagnostic molecular systematic character for euphausiid identification, and also resolves evolutionary relationships among congeneric species with statistical significance. These preliminary studies have confirmed that species of calanoid copepods and euphausiids - whether sibling species or species of different families - are readily distinguishable based on mtCOI sequence variation, which is thus a valid systematic character. Further, the levels and patterns of sequence variation are suitable for the design of rapid molecularly-based species' identification protocols, such as competitive species-specific PCR, which requires identification of unique and diagnostic sequence regions for each species (see Bucklin et al., 1997a, 1998a, 2000a).

Education and outreach: There are several types of education and outreach activities planned and underway for ZooGene:

Publications resulting from this award to date:

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 (in press)

Bucklin, A., S.B. Smolenack, J.J. Pierson, and P.H. Wiebe (2001) Population genetic diversity and structure of the euphausiid, Stylocheiron elongatum, in the Gulf Stream, with a molecular phylogeny of six Stylocheiron species. Journal of Marine Research (in revision)

Bucklin, A., L.D. Allen, B.M. Seppa, B.W. Frost, and J. Bradford Grieve (in preparation) Molecular systematic assessment of twenty-four calanoid copepod species of the Calanidae and Clausocalanidae using DNA sequences of mtCOI and nuclear 18S rRNA.