Grantee Research Project Results
Final Report: Intraspecies genetic diversity measures of environmental impacts.
EPA Grant Number: R826599Title: Intraspecies genetic diversity measures of environmental impacts.
Investigators: Krane, Daniel , Grasman, Keith A. , Burton, Jr., G. Allen
Institution: Wright State University - Main Campus
EPA Project Officer: Hahn, Intaek
Project Period: August 1, 1998 through July 31, 2002
Project Amount: $420,278
RFA: Ecological Indicators (1998) RFA Text | Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , Aquatic Ecosystems
Objective:
The objectives of this research project were to: (1) assess the suitability of randomly amplified polymorphic deoxyribonucleic acid (DNA)-polymerase chain reaction (RAPD-PCR) based measures of genetic diversity as an alternative to, or screening test for, U.S. Environmental Protection Agency (EPA) acute and chronic toxicity and bioaccumulation tests of ecological risk in a wide variety of resource types; and (2) characterize a set of organisms from a range of niches and trophic levels that are amenable to genetic diversity-based approaches that sensitively, rapidly, and inexpensively assay the impact of a wide range of environmental stressors. Hypotheses that have been addressed in this study include the following:
(1) RAPD-PCR based DNA profiles can provide reliable estimates of the genetic diversity of naturally occurring populations of a wide variety of organisms.
(2) Intraspecies estimates of genetic diversity based on multiple anonymous loci are correlated with currently used ecological indicators.
(3) Indices based on genetic diversity measurements from a site-specific suite of indicator species that span a variety of trophic levels and niches are more sensitive, robust, and reliable indicators of environmental risk than genetic diversity measures that rely upon a single organism.
(4) Measures of genetic diversity of local populations are less sensitive to spatial and seasonal influences than traditional methods of measuring environmental impacts.
(5) Potentially significant differences exist in the genetic diversity of organisms used in laboratory-based toxicity testing relative to those of naturally occurring populations.
(6) Genetic diversity measures are more effective guides to determining the overall impact of a wide range of environmental perturbations than any individual descriptor of physicochemical conditions, indigenous biodiversity estimates, toxicity, or bioaccumulation alone.
The genetic variability within a free-living population is directly related to its ability to adapt to its changing environment and to its ability to optimally and broadly utilize resources. Changes in a population's genetic diversity relative to that observed in a similar reference site have the potential to be reliable measures of the true impact of a wide variety of environmental insults. Taken as a whole, the studies develop and assess the utility of RAPD-PCR based measures of genetic diversity as a sensitive, rapid, and inexpensive alternative or supplement to traditional ecological indicators.
Summary/Accomplishments (Outputs/Outcomes):
Environmental insults diminish an ecosystem's ability to maintain productive and adaptable populations of organisms. We have analyzed the DNA profiles of naturally occurring populations of organisms within freshwater and terrestrial sites with varying degrees of exposure to stressors. We 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. Because a population's genetic diversity largely is responsible for its vigor and ability to adapt to stressors, these results suggest a generally applicable and sensitive means of directly assessing the impact of stressors upon individual species within an ecosystem.
Longstanding concern over the deteriorating quality of water resources within the United States has resulted in interventions through several far-reaching pieces of federal legislation. Emphasis on the maintenance of the biological integrity of the nation's waters has resulted in significant improvements in their vigor and economic productivity. Serious questions have been raised, however, regarding the appropriate means of assessing progress toward that goal. Toxicity and biologically based indices have proved 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. 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.
Although species diversity and population densities often return to normal levels shortly after remediation in both terrestrial and aquatic habitats, 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 long-term diminished genetic diversity can be profound. Free-living populations with high levels of genetic diversity use resources more broadly and efficiently, and selection by either natural or anthropogenic stressors is less likely to result in local extinction of genetically diverse populations.
As a direct result, short-term aquatic field studies using allozyme-based measures of genetic diversity appear to be more sensitive and reliable as a means of measuring environmental impacts than assays of more transiently variable features of populations, such as their densities or variances in their morphologies. Many currently used allozyme markers, however, are from loci that are under direct selection by anthropogenic stressors and may not reflect overall levels of genetic variability within a population. This, in conjunction with practical limitations in their sensitivity, has caused allozyme assays of genetic diversity to be poorly correlated with generally accepted biotic indices of water quality.
An approach that more generally and sensitively assesses the extent to which anthropogenic stressors have altered levels of overall genetic diversity in populations should be of great utility. RAPD-PCR 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. RAPD-PCR differs from conventional PCR in that it uses short (typically 10 nucleotides long) primers to amplify anonymous and often polymorphic loci from genomic DNA isolates. The resulting markers generally are free of selective constraint and can be: (1) readily resolved on agarose gels because of differences in their sizes; (2) reproducibly amplified; and (3) used to distinguish between even closely related individuals of the same species.
Methodology
All genomic DNA isolations used in the generation of RAPD-PCR profiles by our research group are made with "QIAquick" PCR purfication kits (manufactured by Qiagen). The resulting pellets are washed with 70 percent ethanol, dried, and resuspended in 50 µL of Tris EDTA (10 mM hydroxymethyl [Tris], pH = 8.3; 1 mM ethylene diamine tetraacetic acid [EDTA]). The quantities of DNA isolated for each sample are estimated by electrophoresis on a 1 percent agarose yield gel. Upon dilution to make DNA concentrations consistent between samples, all isolates were either immediately utilized or frozen at -20°C for later use.
RAPD-PCR profiles are generated routinely from total genomic DNA using the following conditions: final reaction volumes were 10 µL and contained 2 µL of diluted genomic DNA, 1.5 units of KlenTaq 1, 20 mM Tris (pH 8.0), 2.5 mM MgCl2, 16 mM (NH4)2SO4, 150 µg/mL bovine serum albumin, 0.2 µM of a single primer, and 60 µM dNTP. MJ Research thermocyclers (PTC-100 and Mini-cycler models) are used for amplifications for 45 cycles consisting of the following steps: 92°C for 1 minute, 36°C for 1 minute, and 68°C for 2 minutes. An additional extension at 68°C for 5 minutes follows the last round of amplification. All samples are held at 4°C until RAPD-PCR products can be resolved by gel electrophoresis. RAPD products then are electrophoresed in 2 percent agarose gels in Tris-borate-EDTA (TBE) buffer (10 mM Tris, pH 8.3; 10 mM boric acid, 1 mM EDTA) at 4°C. Gels were stained in ethidium bromide for 30 minutes and destained in water for 1 hour. Bands then can be visualized with an ultraviolet lamp and documented using a Gel Print 1000i imaging system (manufactured by BioPhotonics Corporation).
Scoring and Data Analysis. Each RAPD-DNA profile is routinely scored twice, independently, and the RAPD-PCR amplification of a sample is repeated in cases where scorings are 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 (f) 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. For example, pair-wise similarities generated in this way for crayfish 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 < 0.01) where pair-wise similarities ranged from 0.72 to 0.92 (x = 0.848; s = 0.044). Average pair-wise genetic similarities between crayfish at reference sites were consistently lower than those observed among individuals collected at otherwise similar impacted sites.
Biologically based indices, such as the Index of Biotic Integrity (IBI) and the Invertebrate Community Index (ICI), presently are the methods of choice for assessing the impact of anthropogenic stressors at aquatic sites. Whereas both of these indices reduce a large number of metrics (ranging from measures of population density to species diversity) to a final summary value, neither utilizes direct measures of the genetic diversity of organisms resident to a site. In addition to utilizing smaller quantities of sample material and being less expensive to perform, the average pair-wise genetic similarities generated with RAPD-PCR profiling generate a measure of genetic diversity that is significantly correlated (for crayfish, N = 144; r = -0.770; p<<0.001 and N = 144; r = -0.707; p<<0.001 for IBI and ICI, respectively) with these other accepted indicators of biotic integrity, unlike allozyme-based approaches.
A nonparametric measure, the Jonckheere test for ordered alternatives, also suggested that a monotonic trend (not necessarily linear) existed between diminished genetic diversity in crayfish and reported IBI values for all the sites included in this study (p = 0.0002). The probability value was determined based on an analysis of 5,000 random permutations of the data set with the null hypothesis stating that there is no difference among the mean pair-wise genetic similarities from the 8 sites used in this study (HO: µ1 = µ2 = µ3 = … = µn) and the alternative hypothesis that there is a monotonic trend based on a priori information (HA: µ1 = µ2 = µ3 = … = µn, where at least 1 of the inequalities is a strict inequality).
Similar work with snails (Physella gyrina) from the same sites used in the crayfish study indicates that a similar relationship between genetic diversity and environmental quality exists for populations of these nonmigratory organisms. Interestingly, damsel fly populations displayed an opposite trend. (Polluted sites had the highest genetic diversities perhaps because of local die-offs and a greater proportion of immigrant individuals included in the sampling.) Earthworms, a terrestrial organism collected from areas closely associated with the same sites (usually the banks of the rivers) had significantly less genetic diversity at polluted sites than at unaffected sites. Pill bugs collected from reclaimed and abandoned strip mines, however, showed an opposite trend. Genetic diversity of these populations was highest at the most disrupted sites.
Preliminary analyses of data from Hyallela azteca populations, collected at the same sites as well as from mining sites in Colorado, also displayed a decrease in genetic diversity that was positively correlated with decreased environmental quality. Even the lowest levels of genetic diversity observed in naturally occurring populations from highly impacted sites were still significantly greater than those observed in laboratory-reared strains provided by three different sources for use in environmental monitoring work. This observation is potentially significant for those drawing parallels between the health of resident species and species used for in situ monitoring efforts.
Prospects
Environmental stress is only one of many potential causes for reduction of standing variation within a population. Genetic diversity measures based on RAPD-PCR are unlikely to be able to distinguish between naturally occurring 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, studies of adult damsel fly populations actually show an increase in genetic diversity at polluted sites. Damsel flies are a 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, studies 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. Decreased predation and increased refuge availability at polluted sites allow them to maintain larger, more diverse populations than at unaffected sites.
Nonetheless, RAPD-PCR 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, RAPD-PCR 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.
Unlike the metrics that are considered in assigning IBI and ICI values, genetic diversity measures should be relatively insensitive to naturally occurring seasonal and habitat variability, as well as other complicating factors. Instead, genetic diversity measures directly assess the biological basis of a population's ability to adapt and respond to both natural and anthropogenic stressors and, as a result, to its vigor and economic potential. Because RAPD-PCR profiles simultaneously survey a relatively large set of random genetic markers that are very likely to be free from selection by stressors, they also are more likely to provide a truer estimate of a population's genetic diversity than other molecular-based systems. As a direct result, they should allow detection of both chronic and acute exposures to stressors within populations of organisms with low migration rates and provide earlier warnings of impending local and even global extinctions. The choice of organisms with long lifespans (such as trees), at high trophic levels, or with large geographic ranges (such as eagles) might also allow characterizations of habitat quality over long timespans and/or large geographic ranges. Unlike other biologically based indicators, genetic diversity approaches also have the additional advantage of being potentially useful in a wide variety of systems, ranging from freshwater to marine to terrestrial.
Just as measures such as IBI and ICI rely upon information derived from a variety of species, measures of genetic diversity also should be broad-based, as the genetic diversity of any single species can be influenced by a variety of natural factors such as competition and niche size. The similar trends, however, observed in our analyses of crayfish, snails (Physella gyrina), pill bugs (T. rathkei), earth worms, H. azteca, and damsel flies from the same sites used in this study indicate that measures of the genetic diversity of even a single, well-chosen, sentinel species are highly indicative of the extent to which a site has been impacted by anthropogenic stressors.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
Other project views: | All 35 publications | 2 publications in selected types | All 2 journal articles |
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Krane DE, Sternburg DC, Burton GA. Randomly amplified polymorphic DNA profile-based measures of genetic diversity in crayfish correlated with environmental impacts. Environmental Toxicology and Chemistry 1999;18(3):504-508. |
R826599 (Final) R826200 (Final) |
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Pilgrim EM, Roush SA, Krane DE. Combining DNA sequences and morphology in systematics: testing the validity of the dragonfly species Cordulegaster bilineata. Heredity 2002;89(3):184-190. |
R826599 (Final) |
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Supplemental Keywords:
water, watersheds, groundwater, land, soil, sediments, ecological effects, sensitive populations, animal, organism, population, stressor, genetic polymorphisms, ecosystem, indicators, terrestrial, aquatic, biology, monitoring, surveys, measurement methods, population genetics, genetic diversity, randomly amplified polymorphic deoxyribonucleic acid-polymerase chain reaction, RAPD-PCR, DNA., RFA, Geographic Area, Ecosystem Protection/Environmental Exposure & Risk, Ecosystem/Assessment/Indicators, exploratory research environmental biology, Ecosystem Protection, Chemical Mixtures - Environmental Exposure & Risk, State, Ecological Effects - Environmental Exposure & Risk, Monitoring/Modeling, Ecological Effects - Human Health, Ecological Indicators, ecological exposure, risk assessment, anthropogenic stresses, stressors, biodiversity, stream ecosystems, molecular detection, biotic integrity, ecosystem indicators, terrestrial, Ohio (OH), DNA, RAPD-PCR, water quality, snailsRelevant Websites:
Wright State University - College of Mathematics and Science - Department of Biological Sciences Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.