Chapter 19
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Genetic Diversity Provides a Useful Measure of Environmental Impacts Dan E. Krane Department of Biological Sciences, Wright State University, Dayton, OH 45435-0001
Environmental insults diminish an ecosystem's ability to maintain productive and adaptable populations of organisms. We have analyzed the D N A profiles of naturally occurring populations of organisms within freshwater and terrestrial sites with varying degrees of exposure to stressors and find that changes in the underlying genetic diversity of these populations are significantly correlated with the extent to which they have been exposed to anthropogenic stressors. Since it is a population's genetic diversity that is largely responsible for its vigor and ability to adapt to subsequent stressors, these results suggest a generally applicable and sensitive means of directly assessing the impact of stressors upon individual species within an ecosystem.
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© 2002 American Chemical Society Lipnick et al.; Chemicals in the Environment ACS Symposium Series; American Chemical Society: Washington, DC, 2002.
341 Long-standing concern over the deteriorating quality of water resources within the United States has resulted in intervention though several far-reaching pieces of federal legislation (1). Emphasis on the maintenance of the biological integrity of the nation's waters has resulted in significant improvements in their vigor and economic productivity (2) though serious questions have been raised regarding the appropriate means of assessing progress toward that goal (3). Toxicity and biologically based indices have proven to be among the most sensitive and useful measures of environmental impacts through their direct assessment of the extent to which water resource systems can harbor adaptable biological communities (4). These indices on the biochemical, individual, population and community levels have demonstrated that both acute and chronic exposures to stressors can exert selective pressures upon organisms and that the bioavailability of pollutants is strongly correlated with decreased survival and reproductive success within populations (5). While species diversity and population densities often return to normal levels shortly after remediation in both terrestrial and acquatic habitats (6), the population bottlenecks imposed by anthropogenic stressors can result in significant reductions in genetic diversity that are only slowly restored in nature through the processes of migration and mutation. The consequences of longterm diminished genetic diversity can be profound. Free-living populations with high levels of genetic diversity utilize resources more broadly and efficiently (7), and selection by either natural or anthropogenic stressors is less likely to result in local extinction of genetically diverse populations (8). As a direct result, short-term acquatic field studies using allozyme-based measures of genetic diversity appear to be more sensitive and reliable means of measuring environmental impacts than assays of more transiently variable features of populations such as their densities or variances in their morphologies (9). However, many currently used allozyme markers are from loci that are under direct selection by anthropogenic stressors (10) and may not reflect overall levels of genetic variability within a population ( / / ) . This, in conjunction with practical limitations in their sensitivity (12), has caused allozyme assays of genetic diversity to be poorly correlated with generally accepted biotic indices of water quality (13). A n approach that more generally and sensitively assesses the extent that anthropogenic stressors have altered levels of overall genetic diversity in populations should be of great utility. R A P D - P C R (random amplified polymorphic D N A polymerase chain reaction) (14) is a good candidate method for the assessment of population health in that it has been found to generate very sensitive measures of genetic relatedness within populations of organisms (15). R A P D - P C R differs from conventional PCR in that it utilizes short (typically 10 nucleotide long) primers to amplify anonymous and often polymorphic loci (16) from genomic D N A isolates (17). The resulting markers are generally free of selective constraint (18) and can be: 1) readily resolved on agarose gels due to differences in their sizes; 2) reproducibly amplified; and 3) used to distinguish between even closely related individuals of the same species (19).
Lipnick et al.; Chemicals in the Environment ACS Symposium Series; American Chemical Society: Washington, DC, 2002.
342 Preliminary results
Site selection To test the usefulness of RAPD-PCR-based measures of genetic diversity as an augmentation of existing measurements of the effect of environmental impacts, we have used relatively small numbers of primers to generate sets of D N A profiles such as those shown in Figure 1. The R A P D - P C R profiles shown in Figure 1 have been made using the genomic D N A of representative populations (N=18) of native crayfish (Orcorectes rustics) collected from three impacted stream systems with closely associated unaffected reference sites within Ohio. Each of the three streams investigated have well documented histories of exposure to anthropogenic stressors and depressed aquatic communities. Specifically, the Little Scioto River at Marion, Ohio is subject to severe sediment contamination from polycyclic aromatic hydrocarbons and receives metal and organic inputs from combined sewer overflows, municipal and industrial effluents, and agricultural runoff. The reference site upstream of the affected region of the river however is free of contamination. Similarly, Dick's Creek at Middletown, Ohio receives several discharges from a large steel plant and non-point source inputs from neighboring hazardous waste landfills. A nearby reference stream (Elk Creek) of similar order provided a suitable reference comparison. Lastly, the impacted sites of the Ottawa River at Lima, Ohio receives refinery and municipal waste water effluents, combined sewer overflows, and agricultural runoff unlike the immediately upstream reference site that has a good fish and benthic community.
Methodology A l l genomic D N A isolations used in the generation of R A P D - P C R profiles by our research group are made with QIAquick PCR purfication kits (Qiagen). The resulting pellets are washed with 70% ethanol, dried and resuspended in 50 \xL of T E [lOmM TRIS (pU 8.3), 1 m M E D T A ] . The quantities of D N A isolated for each sample are estimated by electrophoresis on a 1% agarose yield gel. Upon dilution to make D N A concentrations consistent between samples, all isolates are either immediately utilized or frozen at -20° for later use. R A P D - P C R profiles are routinely generated from total genomic D N A using the following conditions: final reaction volumes were 10 and contained 2 μΐ, of diluted genomic D N A , 1.5 units of KT1 KlenTaq (Wayne Barnes, Washington University, St. Louis, MO), 20 m M TRIS, pU 8.0, 2.5 m M M g C l , 16 m M (NH4)2S04, 150 μg/mL bovine serum albumin, 0.2 μΜ of a single primer (i.e. A-01: 5 ' - C A G G C C C T T C - 3 ' or A-02: 5 ' - T G A T C C C T G G - 3 ' ) and 2
Lipnick et al.; Chemicals in the Environment ACS Symposium Series; American Chemical Society: Washington, DC, 2002.
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Figure 1. RAPD-PCR profiles of crayfish collected from an impacted site and one of its associated reference sites. PCR products generated using the A01 primer (Operon Technology) on genomic DNA isolated from six crayfish collected at a reference site (IB! value: 36) on the Ottawa River (lanes 2-7), and from six crayfish collected at a downstream impacted site (IBI value: 21) on the same river (lanes 9-15). Molecular size markers are included in lanes I, 8 and 16 and are indicated at left.
Lipnick et al.; Chemicals in the Environment ACS Symposium Series; American Chemical Society: Washington, DC, 2002.
344 60 μ Μ dNTP. M J Research thermocyclers (PTC-100 and Mini-cycler models) are used for amplifications for 45 cycles consisting of the following steps: 92° for 1 minute, 36° for 1 minute, 68° for 2 minutes. A n additional extension at 68° for 5 minutes follows the last round of amplification. A l l samples are held at 4° until R A P D - P C R products can be resolved by gel electrophoresis. R A P D products are then electrophoresed in 2% agarose gels in T B E buffer (10 m M TRIS, pH 8.3, 10 m M boric acid, 1 m M E D T A ) at 4°. Gels are stained in ethidium bromide for one half hour and c'estained in water for one hour. Bands can then be visualized with a U V lamp and documented using a Gel Print lOOOi imaging system (BioPhotonics Corp.).
Scoring and data analysis Each R A P D - D N A profile is routinely scored twice, independently, and the R A P D - P C R amplification of a sample is repeated in cases where scorings were not in complete agreement. A measure of the genetic similarity of individual organisms within a population to others collected at the same site was obtained by determining the fraction (/) of markers it shared with other individuals from the same site using the following equation:
where mxy is the number of bands any two samples share and mx and my are the number of bands amplified from each organism (20). For example, pairwise similarities generated in this way forcrayfish populations ranged from 0.62 to 0.86 (x=0.739; s=0.056) for reference sites and were significantly less than those observed at impacted sites (p = μ3 = ... = μ ) and the alternative hypothesis that there is a monotonie trend based on a priori information ( H : μι > \ii > μ3 > ... > Μη, where at least one of the inequalities is a strict inequality). On-going work with snails (Physella gyrina) from the same sites used in the crayfish study indicate that a similar inverse relationship between genetic diversity and environmental quality exists for populations of these nonmigratory organisms. =
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Prospects Environmental stress is just one of many potential causes for reduction of standing variation within a population and genetic diversity measures based on R A P D - P C R are unlikely to be able to distinguish between naturally occuring and anthropogenic stressors. In addition, a reduction of genetic diversity is not necessarily the only selective response to anthropogenic stressors. Trends contrary to those seen for crayfish and snails provide practical examples of the consequences of these other effects. For instance, preliminary studies of adult damsel fly populations actually show an increase in genetic diversity at polluted sites. Damsel flies are popular ecoindicator species because of the sensitivity of their aquatic larvae to sediment contamination. Migrant adults from surrounding unaffected sights appear to dominate sampling at polluted sites and result in genetically diverse populations whose progeny are likely to not survive development. Similarly, other on-going studies within the laboratory on pill bug populations also suggest an increase in genetic diversity that is correlated with anthropogenic stressor levels. Pill bugs are known to be unusually resistant to a wide range of pollutants, and decreased prédation and increased refuge availability at polluted sites allows them to maintain larger, more diverse populations than at unaffected sites. Nonetheless, R A P D - P C R appears to be a sufficiently sensitive measure of population health in at least four different species to detect significant differences in genetic diversity between sites affected by common anthropogenic stressors and very similar but unaffected reference sites. As such, R A P D - P C R based measures of a population's genetic diversity have the potential to be the basis of a valuable alternative or augmentation to conventional assessments of environmental insults.
Lipnick et al.; Chemicals in the Environment ACS Symposium Series; American Chemical Society: Washington, DC, 2002.
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genetic similarity Figure 2. Index of Biotic Integrity (IBI) values are inversely correlated with RAPD-PCR based measures of genetic similarity. Average pairwise similarity of each organism relative to all others collected at its site are plotted against the independently obtained IBI value (21) for that site (N=144, r=0.770, p«0.001) and a solid line shows the best fit linear regression. Crayfish collected from the impacted and reference Ottawa River sites are displayed as triangles (A) those collected from sites along the Little Scioto River are displayed as circles ( O) and those collected from Elk Creek and its reference stream, Dick's Creek, are shown as squares (Π). Large X's correspond to the mean pairwise genetic similarity of crayfish at each site (N=8, r=-0.804, p
