### SUMMARY OF DISCUSSION, WORKING GROUP II: ZOOPLANKTON GENETICS

Discussion Leaders: Dennis Hedgecock and Michael Lynch

The GLOBEC initiative seeks fundamental information about basic mechanisms that determine the abundance and distribution of zooplankton populations, including holoplankton, meroplankton and ichthyoplankton. Understanding the processes that cause present-day variability of these populations about their average values should assist in the prediction of population responses to global environmental change. Obtaining fundamental information about mechanisms depends critically on the ability to characterize zooplankton field samples according to taxonomic and population genetic criteria. The following information was discussed as background in formulating the draft "Request for Proposals" ("GLOBEC Call for Research Proposals Concerning Genetic Identification and Characterization of Zooplankton Populations", Appendix III of this report).

Prospects for Real-Time, Automated Taxonomic Identification of Plankton Samples. An "idealized" schema for sampling zooplankton that had been developed earlier by some members of the GLOBEC Steering Committee was presented as a starting point for discussion in this working group. This schema depicted in-line coupling of a plankton pump to reaction vessels for molecular probing and then to flow cytometers for sample sorting. The output would be taxonomically sorted in real time, providing intact and possibly even living specimens for physiological analysis.

It was recognized that this was a hypothetical set-up employed to stimulate discussion, and the working group quickly agreed that such a schema could not be achieved in the foreseeable future. Although molecular methods are indeed powerful tools for systematic biology and taxonomy, they do not yield results in "real-time" and they are destructive. Two hours is a more realistic minimum time required for characterization of a specimen by current molecular methods. DNA probing, for example, requires at least three basic steps: (1) extraction of DNA, or making permeable the intact specimen; (2) hybridization of the probe; and (3) washing under stringent conditions to reduce background or non-specific probe binding. The biochemistry involved in these steps constrains shortening of the time required. Moreover, for organisms as small as zooplankton, the whole organism would generally be consumed in this process, making it unavailable for physiological studies. This is not to say that molecular methods cannot play an important role in GLOBEC studies. Characterizing species-specific markers through studies of molecular systematics will provide a foundation for unambiguous identification and enumeration of zooplankton species, and assessing intraspecific variation in molecular markers will allow characterization of conspecific zooplankton populations on spatial/temporal scales appropriate to physical oceanographic features or processes that may influence population mixing or recruitment success. However, it must be recognized that current molecular methods are likely to provide only retrospective information on plankton composition, either in parallel subsamples taken at a station or on specimens already used for physiological analysis.

A "Spread and Probe" Strategy for Zooplankton Identification. The working group considered two alternative strategies for determining the species and genotypic compositions of zooplankton samples: quantitative analysis of bulk samples vs. qualitative analysis of individuals. Quantification of molecular species in bulk samples is difficult even in simple systems such as mammalian cell cultures; quantification of species in a batch sample of mixed zooplankton would be fraught with error. The alternative strategy of spreading individuals out in two dimensions for molecular probing would probably prove more reliable for species or genotype characterization.

Size Limits. The small sizes of zooplankton should not prove to be a limitation for molecular technology. Conventional analysis of restriction fragment length polymorphisms in DNA (RFLPs, detected by restriction enzyme digestion of sample DNA, electrophoretic separation of resulting fragments, transfer of fragments to a membrane, and hybridization of membrane-bound fragments to a radiolabeled probe) requires nanogram quantities of high molecular weight DNA but have been applied successfully to species identification of large fish eggs. The polymerase chain reaction (PCR), on the other hand, can amplify a particular segment of DNA by a factor of several million or even a billion; enough DNA can be obtained from as little tissue as a single cell for analysis or characterization. Moreover, DNA can be extracted and amplified from alcohol-, and in some cases formalin-preserved, material. Thus, molecular studies could be done retrospectively in samples preserved during a cruise or on samples in historical zooplankton collections.

Adapt Existing Molecular Methods to Zooplankton; Obtain Basic Population Data. The working group felt that most methods of molecular biology probably could be adapted to zooplankton studies. To bring the power of molecular biology to bear on the problems of taxonomic identification and population genetic analysis of zooplankton, at least in the retrospective sense described above, we do not need to develop new technology. It will, however, be critical for GLOBEC to insure the support of the basic laboratory bench work required to adapt existing molecular methods to marine zooplankton.

Following this, the working group identified a great need for basic information on the amount of molecular genetic variation within and between populations and species of marine zooplankton. Present knowledge does not allow for the design of taxon-specific probes or markers for most of the organisms likely to be important in GLOBEC studies, nor does it allow an evaluation of which molecular techniques are likely to be the most useful in a particular context. A range of existing molecular techniques -- allozyme electrophoresis, RFLP analysis, sequence-specific oligonucleotide probing of PCR products, DNA fingerprinting, and immunofluorescent probes -- are available to discriminate morphologically similar species and to analyze population genetic structure. These techniques differ greatly in ease and efficiency of application and yield information of relevance in proportion to the amount of underlying variation in the particular species being investigated. The working group recommended that GLOBEC support basic studies of molecular genetic variation, using the full range of existing molecular methods, so that decisions regarding the most appropriate method could be based on informational content and sampling efficiency.

Scope of Sampling. An additional problem identified by the working group concerned the necessary scope of sampling for population genetic analysis. In order to understand the capacity of animal populations to adapt to environmental change in an area, one must understand the genetic diversity and structure of the total species population. Limiting genetic or physiological studies to only one local population (e.g., Georges Bank) might not allow prediction of the capacity of that organism (e.g., cod) to remain in that area under a regime of changing climate; the local stock might simply go extinct and be replaced by a stock with a different genetic composition from another area. A shift in gene frequencies in the local population under study might be difficult to interpret without background information on this species from a much broader geographical area.

Genetic Basis of Adaptation to Global Climate Change. An additional area of genetic research was felt to be important to the overall aims of GLOBEC -- studies of the genetic bases of variation in phenotypes likely to determine the responses of zooplankton populations to climate change. These studies primarily would utilize the methods of quantitative genetics rather than molecular biology. Two categories of phenotypic traits that are likely to be important in adapting to environmental change and that might be influenced directly by factors such as warmer temperature are: (1) characters relevant to recruitment ( e.g., duration of pelagic larval phase, competency for metamorphosis, response to environmental cues for settlement); and (2) juvenile and adult physiological, behavioral or life-history traits (e.g., age- or size-specific fecundity).

New Technology. While prospects for automated real-time plankton sorting by existing molecular methods appear dim, breakthroughs in biotechnology are difficult to anticipate. Research on the development of new technology for rapid and accurate identification of preserved, intact, or live zooplankton ought to be encouraged in a call for proposals. Such new technology also might utilize physical methods, such as optical image analysis, perhaps in combination with molecular labeling of intact zooplankton. Any research that might reduce existing or new biotechnologies to shipboard practice also should be solicited.