United States Environmental Protection Agency Office of Research and Development National Exposure Research Laboratory Research Abstract Government Performance Results Act (GPRA) Goal #4 Annual Performance Measure #249 Significant Research Findings: Report: "Genetic Diversity of Stream Fish in the Mid-Atlantic Assessment Area: Implications for Ecological Monitoring and Assessment Programs" Scientific Measures of genetic diversity within species have been suggested as potentially Problem and powerful indicators of ecological condition. Genetic diversity is a key component Policy Issues of biodiversity. Since it is shaped by environmental forces and helps determine future population risks, genetic diversity measures are potentially both prospective and retrospective indicators of the condition of populations. While measures of genetic diversity within species have great potential, they have not been thoroughly analyzed as tools for regional monitoring and assessment programs. This report summarizes extensive research by the EPA to gauge the utility of genetic approaches as one component of an ecological monitoring and assessment program. The assessment was performed in the Mid-Atlantic Integrated Assessment (MAIA) area. Three specific questions were addressed in this study: (1) How accurate and precise is field taxonomic identification of stream fish? (2) Does population genetic structure exist within recognized stream fish species within the MAIA region? (3) Do environmental factors affect levels of stream fish genetic diversity within the MAIA region? Research White sucker (Catostomus commersoni), creek chub (Semotilus atromaculatus), Approach fallfish (,S'. corporalis), and central stoneroller (Campostoma cmomalum) were collected in 1997-1999 in conjunction with the EPA's Environmental Monitoring and Assessment Program (EMAP) and DNA was extracted for genetic analyses. The cytochrome b gene of the mitochondrial genome was sequenced for representative samples from 104 white suckers, 337 creek chub, 13 fallfish, and 82 central stonerollers. Microsatellite variation was measured at 9 microsatellite loci for 644 white suckers from 40 sites, 3 microsatellite loci for 1,240 creek chubs from 70 sites, and 8 loci for 1,409 central stonerollers from 69 sites. Average heterozygosity of microsatellite loci was calculated and compared to multivariate descriptors of the environment at each site. Results and Results of the DNA sequence analysis indicated that four white suckers had Impact mitochondrial sequences characteristic of other species (minimum 96 percent accuracy). In addition, two fallfish samples had mitochondrial sequences characteristic of other fishes (minimum 82 percent accuracy). Actual accuracy of identification may have been higher if mitochondria from other species were introduced into these species through hybridization. No samples of creek chub or ------- central stoneroller appeared to have been misidentified. However, each of these species demonstrated great sequence divergence among distinct mitochondrial clades. The degree of genetic divergence (7-11 percent) is characteristic of species-level differences, suggesting the presence of several morphologically cryptic species or, minimally, subspecies. Thus, while misidentification of samples based on morphological analysis was relatively low, "under identification" of samples (the inability to detect morphologically similar but genetically distinct groups) was high and potentially problematic for ecological analyses. Greater use of genetic methods to verify morphological identifications is recommended. Little genetic differentiation was observed in either mitochondrial or microsatellite DNA for white suckers, suggesting that this species can be considered a single genetic unit within the entire MAIA region. Microsatellite variation confirmed the mitochondrial DNA analysis that differentiated creek chubs within the MAIA region into two distinct genetic groups. Microsatellite variation also confirmed the mitochondrial DNA analysis for central stonerollers by differentiating four genetic groups within the MAIA region. The large differences in microsatellite allele distributions and the concordance with mitochondrial DNA results confirmed that these genetic groups represent probable subspecies or higher-order taxonomic units. While white suckers, creek chub, and central stonerollers demonstrated markedly different population structure, there were some similarities in population boundaries. Genetic diversity was found to be significantly correlated with environmental characteristics for all species studied. An unexpected but very important finding was that the different subgroups of creek chub and central stoneroller appeared to respond to different environmental dimensions. This may indicate differential sensitivities of different genetic groups within morphologically defined species or simply reflect environmental differences between distinct geographic regions. Since these differences would be undetected in a typical fish community assessment, these results could have profound implications for fish and invertebrate monitoring and assessment programs. The work is of primary importance to the EPA's EMAP and the Regional Environmental Monitoring and Assessment Program (REMAP), similar State and Tribal environmental assessment programs, and the EPA's Office of Water and EPA's Office of Prevention, Pesticides, and Toxic Substances. This research was conducted primarily by a team of NERL staff scientists. Significant collaborators include the ORD's EMAP program and EPA Region 3. Contractor assistance in collecting some samples and performing molecular marker analyses was provided by Sobran, Inc. Preliminary research findings were presented at the scientific conferences for the Society for Environmental Toxicology and Chemistry (2001) and the American Society of Ichthyology and Herpetology (2002). Future Research Our research results to date indicate that measures of genetic diversity are informative for assessment of ecological condition because they help define ecological units and appear to be responsive to environmental stresses. However, measures of genetic diversity may have still greater importance as measures of Research Collaboration and Research Products ------- population vulnerability. This is because the capacity for populations to respond (adapt) to environmental change is proportional to the amount of genetic diversity within the populations. Planned research will focus on experimental determination of the importance of genetic diversity for buffering aquatic populations against environmental variability and predicting population vulnerability. A second research focus planned is to evaluate the benefits of integrating genetic diversity measures, landscape indicators, and population modeling tools into a single ecological assessment. The research is premised on the idea that genetic and demographic changes in population are linked and both should be tied to landscape-level changes. This is a collaborative effort between three NERL divisions: the Ecological Exposure Research Division (EERD), the Ecosystems Research Division (ERD), and the Environmental Sciences Division (ESD). A third research focus is the development of efficient methods for incorporating DNA sequence information into ecological assessments as a taxonomic identification tool. This work is currently targeted at development of rapid and cost-effective methods for assessing stream macroinvertebrates. Species-level identification of many macroinvertebrates using traditional methods is difficult and imprecise. Finally, ongoing research is aimed at evaluating the utility of long-term genetic monitoring of fish populations as an indicator of trends in the condition of aquatic ecosystems. One effort represents a collaboration with the Department of Fisheries and Oceans, Canada, and utilizes a 25-year DNA archive of fish populations experimentally exposed to a variety of stressors. A new effort will evaluate tissue contaminants and genetic profiles of Sierra Nevada fish and amphibians that were sampled from museum collections. Contacts for Additional Information Questions and inquiries concerning this research abstract can be directed to: Mark Bagley, Ph.D. U.S. Environmental Protection Agency National Exposure Research Laboratory (MD-642) Cincinnati, OH 45268 Phone: 513/569-7455 E-mail: bagley.mark@epa.gov ------- |