Grantee Research Project Results
Final Report: Ecosystem Monitoring via Genetic Diversity Surveys of Dandelions using VNTR multi-locus DNA probes
EPA Grant Number: R826602Title: Ecosystem Monitoring via Genetic Diversity Surveys of Dandelions using VNTR multi-locus DNA probes
Investigators: Rogstad, Steven , Keane, Brian
Institution: University of Cincinnati
EPA Project Officer: Hahn, Intaek
Project Period: July 14, 1998 through July 13, 2001 (Extended to July 13, 2003)
Project Amount: $291,045
RFA: Ecological Indicators (1998) RFA Text | Recipients Lists
Research Category: Aquatic Ecosystems , Ecological Indicators/Assessment/Restoration
Objective:
New methodologies in molecular genetics may provide novel types of ecological indicators for monitoring the integrity and sustainability of natural ecosystems that are affected by anthropogenic influences. One ideal ecological indicator would be an organism that grows in a wide variety of habitats that could easily be used to monitor for the presence of mutagens or anthropogenic factors that alter normal population genetics processes. We are investigating dandelions (Taraxacum officinale Weber; Asteraceae) as a potential model ecological indicator organism because: (1) dandelions have an extremely wide ecological amplitude, growing almost worldwide from sea-level to alpine biomes, and from the tropics to north-temperate habitats; (2) dandelions grow rapidly, and populations can easily be manipulated and monitored; (3) it has been documented that a variety of pollutants can be sequestered in dandelion tissues; (4) dandelion seeds are produced asexually rendering the detection of mutations easy; and (5) we have generated data using variable-number-tandem-repeat (VNTR) DNA probes (used in DNA fingerprinting) that suggest that mutation rates and population genetic diversity parameters can be successfully analyzed with dandelions. The specific hypothesis tested was that dandelions can be used as a sensitive ecological indicator species through comparative analyses of VNTR genetic markers because differences in mutation rates and/or population genetic structure can be detected between pollution impacted versus nonimpacted populations.
Summary/Accomplishments (Outputs/Outcomes):
Twenty nine study sites in the central United States were selected to span a range of pollution exposures as determined by analyses of PM10 (a measure of the average amount of airborne particulate matter ≤ 10 microns; or by a known history of intense pollution exposure (four sites). Soils and dandelion tissues from these sites were analyzed for eight metals (Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn). Examining the data across sites, a positive, statistically significant correlation exists between the mean annual PM10 and soil concentration of each metal, implying that airborne particulate matter is a good indicator of soil metal contamination. Four of the metals (Cr, Mn, Pb, and Zn) exhibited positive, statistically significant correlations between soil and fall leaf concentrations. Furthermore, sampling dandelion tissues collected from eight sites in both spring and fall revealed that leaf tissue metal concentrations increased for five of the metals over the growing season.
These results indicate that the amount of a metal measured in air or soil at a site does not always predict the amount sequestered in dandelion tissues. Other factors such as time of year, the specific metal, site seasonal climate history, substrate characteristics, and/or dandelion genotypes present at a site possibly interact with uptake.
We now have analyzed the transmission of 82,715 VNTR genetic markers in more than 120 parent-to-clonal offspring arrays (1,258 offspring) from 16 sites (from Colorado to Pennsylvania) spanning a range of metal exposures. Six VNTR probes were used for each individual, resulting in an average of 65.8 markers scored per offspring. Dandelions provide a rapid system for VNTR genetic marker mutation because the detection of mutations is facilitated by the asexual, clonal seed production of dandelions (agamospermy: the embryo arises from a 2n maternal cell) in which all markers in the seed-producing plant also should occur in the offspring. A genetic marker that differs in one offspring, therefore, is recognized easily and scored. Across sites, the average, single-event, parent-offspring, minisatellite marker transmission rate is 0.0067 (1 mutant marker per 149 markers transmitted), ranging from 0.002 to 0.015, a 7.5-fold difference. The average rate is among the highest rates of mutation for genetic markers found for plants, and eukaryotes in general.
In no case was a significant correlation detected between the single-event mutation rate and either PM10 or soil concentrations of any of the metals. However, single-event mutation rates were significantly (P<0.05) and positively correlated to increasing leaf tissue concentrations of four metals (Cr, Fe, Mn, and Ni) across sites (the other metals exhibited nonsignificant relationships). Multiple regression analysis suggests that a model including three metals (in order of importance to the model: Cr [P = 0.002], Fe [P = 0.02], and Ni [P = 0.005]; overall P for model = 0.001) may improve the ability to predict mutation rate relative to leaf metal concentrations in dandelions. Thus, we conclude that mutations at minisatellite loci may provide convenient biomarkers by which to assess the mutagen stressor risk present in environments. Whereas, the global distribution of the common dandelion, along with the taxon's ability to tolerate a wide range of environmental conditions, make this "species" a particularly attractive candidate to evaluate for its value as a biological monitor of environmental mutation stress, using parent-offspring clonal arrays to rapidly survey for differences in population minisatellite mutation rates may be feasible for most sexually apomictic (asexual) organisms. A manuscript describing this research is in press (see publication list).
Analysis of VNTR marker variation in populations (three large transects, and sampling at three polluted versus three relatively pristine sites) indicates that some dandelion clones are very widely distributed, although clones with narrow distributions (e.g., restricted to a site) also have been found (Rogstad, et al., 2002, in press). In related research, we have determined that unique dandelion clones (identified with VNTR DNA fingerprinting) differ in productivity characteristics when grown on contaminated versus pristine growth media. Specifically, dandelion plants from unpolluted sites are more productive than dandelions from polluted sites. This productivity difference is reversed when both types of plants are grown on unpolluted media. These results demonstrate that dandelion populations do differ at sites due to selection imposed by metal presence or absence (or correlated factors), and that there is a cost to being either metal-pollution tolerant or sensitive. These findings suggest that, in the search for plants to be used for phytoremediation, not all individuals or populations of a species will interact with metals in the same way. Populations on contaminated soils, or soils with high metal content, should be the best populations from which to sample lines to be used in phytoremediation studies. Just because a species has been reported to be a good metal accumulator does not mean that all populations or individuals will be similar in that characteristic. Manuscripts concerning this research have been submitted or are in preparation (see Ph.D. thesis by Collier, and Collier, et al., manuscripts in publications list).
In relation to the above investigations, we have conducted research to determine whether dandelions from polluted sites are metal-tolerant because they are metal excluders, or they sequester and tolerate metals in their tissues, and whether these characteristics differ among metal contamination sensitive versus tolerant individuals. Our results demonstrate that plants from polluted sites take up larger amounts of metals into their roots and shoots than plants from nonpolluted sites, when both are grown from seed on the same growth media polluted with a defined level of metals. We conclude that metal-tolerant dandelions are not metal excluders, but rather, are metal-sequestering tolerators, with plants from polluted sites sequestering and tolerating higher levels of metals than dandelions from nonpolluted sites. A manuscript describing these findings is in preparation (see Ph.D. thesis by Collier).
Assessing the genetic structure of natural populations differentially impacted by anthropogenic contaminants can be a useful tool for evaluating the population genetic consequences of exposure to pollution. Measures of genetic diversity at VNTR loci in six dandelion populations (three urban and three rural) were examined. Our results suggest that patterns of dandelion population diversity have been influenced by exposure to environmental contaminants. Mean genetic similarity among individuals within a population was significantly and positively correlated with increasing levels of airborne particulate matter (≤ µm, PM10) and soil concentrations of four metals (Cd, Fe, Ni, and Pb). In addition, comparison of mean genetic similarity between rural versus urban sites found mean genetic similarity to be always significantly higher at the urban, more metal-polluted sites. There was a significant negative correlation between the number of genotypes at a site and increasing amounts of PM10, concentrations of five soil metals (Cd, Cu, Fe, Ni, and Pb), and leaf tissue levels of Fe. There was a significant positive correlation between the extent of clonality at a site and levels of PM10 and soil concentrations of five metals (Cd, Cu, Fe, Ni, and Pb). Although this study does not directly establish a causal link between the specific contaminants detected at the study sites and differences in genetic diversity, these data are consistent with the hypothesis that pollution-induced or pollution-related selection has contributed in some fashion to the lower genetic diversity found at the urban sites. These results suggest that dandelion populations may be a sensitive biological indicator of pollution-induced genetic effects resulting from exposure to levels of pollution typical of that associated with industrialized and urban areas in the United States, and may have implications for phytoremediation and natural germplasm conservation.
In summary, a list of our main findings includes:
• PM10 is a good indicator of the degree of soil contamination for the metals examined.
• Dandelions are not effective biomonitors in the sense that their tissues sequester metals in proportion to the concentration of metals in the soil, except perhaps for extremely polluted sites as described by previous authors.
• Dandelions may be good ecosystem biomonitors in the sense that they reflect metal bioavailability (i.e., how the plant population at a site is interacting with the metals to sequester them).
• There is abundant genetic diversity of clones of dandelions, with some very widespread, common clones, with other clones being perhaps less common and more limited in distribution.
• Dandelion seed dispersal is extremely effective in distributing the clones, with measures of diversity being essentially the same for small (600-m length), medium (30-km length), and large (330-km length) random transects.
• Dandelions appear to be good mutation stressor biomonitors, indicating that as the tissue exposure to four metals, or possibly other related mutagen stressors, increases, mutation rates at a site increase (a 7.5-fold difference in mutation rates across sites was found).
• Dandelion population genetic diversity decreases at more polluted sites, primarily due to a higher representation of a few clones.
• Dandelion dispersal of clones appears to be very effective; the metals at a site (or related factors) can select for dandelions that differ in response to metal presence.
• There are costs associated with the pollution intolerance or tolerance.
• Dandelions appear to exist in the presence of soil metals by tolerating the metals in their tissues rather than excluding metals.
• Metal uptake by dandelions increases throughout one growing season.
• Dandelion lineages collected on polluted sites take up more soil metals (primarily into their roots) than dandelion lineages collected at more pristine sites.
The above findings, taken as a whole, suggest that analyses of dandelion populations for VNTR marker mutation and population genetic variation with further research should provide an easily utilized biomonitor to survey the ecological integrity and sustainability of a wide range of habitats across multiple spatial scales throughout the world.
Journal Articles on this Report : 13 Displayed | Download in RIS Format
Other project views: | All 27 publications | 15 publications in selected types | All 13 journal articles |
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Collier MH, Rogstad SH. Clonal variation in floral stage timing in the common dandelion Taraxacum officinale (Asteraceae). American Journal of Botany 2004;91(11):1828-1833. |
R826602 (Final) |
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Collier MH, Keane B, Rogstad SH. Productivity differences between dandelion (Taraxacum officinale; Asteraceae) clones from pollution impacted versus non-impacted soils. Plant and Soil 2010;329(1-2):173-183. |
R826602 (Final) |
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Keane B, Smith MK, Rogstad SH. Genetic variation in red raspberries (Rubus idaeus L.; Rosaceae) from sites differing in organic pollutants compared with synthetic tandem repeat DNA probes. Environmental Toxicology and Chemistry 1998;17(10):2027-2034. |
R826602 (Final) |
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Keane B, Pelikan S, Toth GP, Smith MK, Rogstad SH. Genetic diversity of Typha latifolia (Typhaceae) and the impact of pollutants examined with tandem-repetitive DNA probes. American Journal of Botany 1999;86(9):1226-1238. |
R826602 (1998) R826602 (1999) R826602 (2000) R826602 (2001) R826602 (Final) |
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Keane B, Lim HW, Rogstad SH. Re-probing DNA blots: wet is better than dry storage of uncharged nylon membranes after removing probes. Plant Molecular Biology Reporter 2000;18(1):17-21. |
R826602 (2001) R826602 (Final) |
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Keane B, Collier MH, Shann JR, Rogstad SH. Metal content of dandelion (Taraxacum officinale) leaves in relation to soil contamination and airborne particulate matter. Science of The Total Environment 2001;281(1-3):63-78. |
R826602 (2001) R826602 (Final) |
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Keane B, Collier MH, Rogstad SH. Pollution and genetic structure of North American populations of the common dandelion (Taraxacum officinale). Environmental Monitoring and Assessment 2005;105(1-3):341-357. |
R826602 (Final) |
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Kumar A, Rogstad SH. A hierarchical analysis of minisatellite DNA diversity in Gambel oak (Quercus gambelii Nutt.; Fagaceae). Molecular Ecology 1998;7(7):859-869. |
R826602 (Final) |
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Lim HW, Pelikan S, Rogstad SH. Genetic diversity among populations and size classes of buckeyes (Aesculus: Hippocastanaceae) examined with multilocus VNTR probes. Plant Systematics and Evolution 2002;230(3-4):125-141. |
R826602 (1998) R826602 (1999) R826602 (2000) R826602 (2001) R826602 (Final) |
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Rogstad SH, Keane B, Keiffer CH, Hebard F, Sisco P. DNA extraction from plants: the use of pectinase. Plant Molecular Biology Reporter 2001;19(4):353-359. |
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Rogstad SH, Keane B, Beresh J. Genetic variation across VNTR loci in central North American Taraxacum surveyed at different spatial scales. Plant Ecology 2002;161(1):111-121. |
R826602 (1999) R826602 (2000) R826602 (2001) R826602 (Final) |
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Rogstad SH. Plant DNA extraction using silica. Plant Molecular Biology Reporter 2003;21(4):463. |
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Rogstad SH, Keane B, Collier MH. Minisatellite DNA mutation rate in dandelions increases with leaf-tissue concentrations of Cr, Fe, Mn, and Ni. Environmental Toxicology and Chemistry 2003;22(9):2093-2099. |
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Supplemental Keywords:
air, soil, risk assessment, ecological effects, genetic polymorphisms, population responses, bioavailability, sensitive populations, mutagens, cumulative effects, particulates, integrated assessment, conservation, phytoremediation, bioremediation, environmental chemistry, monitoring, ecology, genetics, measurement methods, midwest, CO, IN, KY, OH, PA, Colorado, Indiana, Kentucky, Ohio, Pennsylvania, midwest., RFA, Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, Ecology, exploratory research environmental biology, Ecosystem/Assessment/Indicators, Ecosystem Protection, Ecological Effects - Environmental Exposure & Risk, Environmental Monitoring, Ecological Risk Assessment, Ecology and Ecosystems, Ecological Indicators, ecological exposure, risk assessment, anthropogenic stresses, dandelions, molecular genetics, genetic polymorphism, multiple spatial scales, survey, ecosystem indicators, assessment methods, DNA, genetic diversityRelevant Websites:
http://www.nrc.ca/cisti/journals/ispmb/reporter.html 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.