| Title |
Investigators | Department | Objectives | Approach Keywords | Progress Reports | Impact Statements | Publications | |
Project * COL00180 | |
| Title | *Genetics of invasive Centaurea species in Colorado |
| Investigator(s) | Hufbauer, RA; |
| Department | Bioagricultural Sciences and Pest Mgmt. |
| Objectives | The long term goals of this proposed research program are to locate the origins of two invasive weeds and examine the genetic and phenotypic effects of their introductions. Our ultimate aims are to improve our understanding of the basic principles governing the spread of invasive weeds, and to lay the essential groundwork necessary for targeting coevolved, effective biological control agents for introduction. We are examining levels of genetic variation of North American populations of spotted and diffuse knapweeds (Centaurea maculosa and C. diffusa) using DNA markers. Our initial work is focusing on ISSRs (see Approach), and we are developing three other marker systems for this project: chloroplast DNA markers useful for exploring geographic variation among populations, and two anonymous nuclear marker systems (AFLP and microsatellites) for finer-scale information on levels of genetic variation, potential interbreeding within populations of each species, and hybridization among the two species. We choose to pursue this research on both spotted and diffuse knapweeds simultaneously because they have multiple biological control agents in common, and there is new evidence (Ochsmann 2000) that these species may hybridize in Europe. By measuring the extent of genetic and phenotypic diversity both within and between native and introduced plant populations we can gain a better understanding of how genetic and phenotypic traits influence the invasion of exotic plants into rangelands and wilderness areas. The specific objectives of this research are to: (1) Examine the levels of genetic variation of North American C. maculosa and C. diffusa using molecular markers developed from chloroplast DNA and from anonymous nuclear sites (ISSRs). Are populations genetically diverse or depauperate? What are the relationships of North American populations? What is the genetic composition of Colorado populations in particular? (2) Compare morphology and phenology of introduced and native plants in a common garden. It has been hypothesized that the introduction and invasion processes select for more vigorous and weedy plant genotypes than are present in native populations (Blossey and Notzold 1995; Blossey and Kamil 1996). We propose to test this hypothesis by determining whether introduced North American populations of these knapweeds grow faster and larger, and flower sooner than the native Eurasian populations from which they are derived. |
| Approach | Collections: Diffuse and spotted knapweed seed or tissue samples are being collected from both North America and Europe. Where possible, we have collected (or will collect) seeds because they are easy to store for travel and shipment and requires no liquid nitrogen or chemical preservatives. Sample size may depend in part upon the collection location: in the native range these plants are often difficult to find, and populations are sparse and individuals widespread (Jensen 2000). The goal will be to sample a minimum of 30 plants per population and two populations per region. Molecular Data Collection: Genomic DNA will be extracted from samples using the DNeasy Plant Mini Kit from Qiagen. Amplification of ISSRs will typically be performed in single primer reactions. Separation on agarose gels and visualization will be as described above. The digital images will be analyzed with the BioMax1D image analysis software (Eastman Kodak Company). Only bright and reproducible bands will be scored. Molecular Data Analysis: The phylogeographic relationships will be derived with parsimony analysis. The data will be scored as unordered multi-state characters and the most-parsimonious tree(s) will be inferred using the heuristic search option of Paup* Version 4.0 (Swofford 1999). The total diversity, diversity within populations, and the level of differentiation among populations (GST) and their standard errors will be estimated. Differentiation among populations will be further investigated using UPGMA analysis, to infer phenograms from pairwise genetic distances between populations (PHYLIP, Felsenstein 1995). Karyotyping: We will determine the ploidy level of the plants used in the genetic analysis using standard cytological techniques (e.g. Ownbey 1963; Radford et al. 1974). Ecological work Seeds will be germinated for comparisons of plant growth and development in greenhouse studies. Three introduced North American populations of each species and three hybrid populations (if found) will be compared to three native Eurasian populations of each species plus hybrids. The populations will be chosen based upon the genetic data. The introduced populations will be chosen to represent the variability found in the neutral molecular data. The native Eurasian populations chosen will be the three most closely related to the North American populations in their molecular genetic profiles. From each of these 18 populations, three individuals from each of five parents will be grown (total of 270 plants). We will measure maximum photosynthetic rates every two weeks, time to first flowering, and number of flowers per seed head. After a three-month growing period, we will harvest the plants and separate the tissue into below and above ground portions. The roots and shoots will be dried and weighed to compare the final biomass of the plants. |
| Keywords | Centaurea, spotted knapweed, diffuse knapweed, knapweed, rangeland, invasion, weed origins, weed, genetics, hybridization, population genetics, biological control |
| Progress Reports | |
| 2002 | Most weedy plants in Colorado's natural ecosystems, rangelands, and parks are exotic invaders. Invasive weeds reduce forage for wild animals and livestock, can increase fuels and therefore frequency and intensity of burns, and alter ecosystem function (e.g. nutrient cycling). To manage invasive plants effectively, whether through herbicides, biological control, or altering competitive regimes, we must understand the fundamental processes involved in their colonization to and spread in novel environments. The knapweeds (Centaurea spp.) are particularly troublesome invasive plants in Colorado because of their negative chemical effects (allelopathy) on other plants. In addition, multiple species are found in Colorado and among those species are pairs that are known to hybridize. We are studying two of these species: diffuse and spotted knapweed (Centaurea diffusa and C. maculosa). We are using genetic tools to explore their population structure. We are studying the frequency and importance of hybridization in the invasion of these species. In addition we are exploring the ecological effects of ploidy levels (diploid vs. tetraploid) on their invasiveness, in particular, their ability to have allelopathic effects on each other and native vegetation. Our ultimate aim is to improve their control through biological, chemical, and cultural methods. Our research is of relevance to state and national land managers and ranchers faced with controlling these noxious weeds. This year we have focused on 1) gathering samples for ecological and genetic study, 2) determining ploidy levels of introduced populations, 3) determining competitive and allelopathic effects of these weeds on each other and on plants used for restoration. We have found that the introduced populations contain a surprising mixture of diploid and tetraploid individuals. We are in the process of determining their relative competitiveness and allelopathic potential. Although the allelopathic chemical produced by spotted knapweed has a greater effect on diffuse knapweed than the reverse, competition experiments show that diffuse knapweed is less negatively affected by growing with spotted than vise versa. There is significant genetic differentiation of populations both plants within the intermountain west, and hybrid individuals appear to be common. |
| 2003 | We are exploring the genetics of invasive diffuse and spotted knapweed in Colorado, and their hybrids. Chloroplast DNA sequence data show that the populations of spotted knapweed in Colorado are most similar to those in the Ukraine area, and not as closely related to those from France, Germany, Switzerland, or Greece. We have developed new markers that can be used to definitively identify hybrids. Some people think that most of what we call "diffuse knapweed" may actually be of hybrid origin. Now that we have these markers, we will start assaying Colorado populations to determine their status. |
| 2004 | Exotic, invasive plants threaten Colorado agriculture, rangeland, and wildlands. To manage invasive plants effectively, whether through herbicides, biological control, or altering competitive regimes, we must understand the fundamental processes involved in their success in novel environments. Several species of knapweed (genus Centaurea) have invaded Colorado. We are studying two of these species which are known to hybridize with each other: diffuse and spotted knapweed (Centaurea diffusa and C. maculosa). We are using genetic tools to explore their population structure. We are studying the frequency and importance of hybridization in the invasion of these species. In addition we are exploring the ecological effects of ploidy levels (diploid vs .tetraploid) on their invasiveness, in particular, their ability to have allelopathic effects on each other and native vegetation. Our ultimate aim is to improve their control through biological, chemical, and cultural methods, and to provide guidelines regarding hybrids. If they are less aggressive than the parent species, managers can spend valuable time focusing on the parents, while if they are more aggressive, then managers should target them for control. This year we have focused on 1) expanded our study of ploidy levels of introduced populations, 2) evaluated the potential for allelopathy in greenhouse experiments, 3) improved methods for the documentation of production of root exudates that may be allelopathic, 4) compared production of root exudates from native and introduced populations of spotted knapweed, and 5) ongoing population genetic research to evaluate origins of these weeds. We have found that all hybrid individuals appear to be diploid, as are 97% of diffuse knapweed. Spotted knapweed is 55% diploid, with some populations comprising almost entirely tetraploids, and some 100% diploids. In the greenhouse experiments, we found that activated carbon, which often is used to absorb allelochemicals to distinguish between allelopathy and competition, by itself increase plant growth several fold for five species of grasses. As part of a collaboration, we developed more efficient methods for extracting catechin from soil and media. Using these methods, we compared output of potential allelochemicals from populations from the native and introduced range. We found a trend for higher production in North American individuals. Overall, with the more efficient extraction methods, production of root exudates was an order of magnitude less than reported in the literature. We found that catechin disappears from soils within 24 hours, suggesting that this chemical will not prevent the revegetation of previously infested sites. Population genetic analyses suggest a cryptic invasion of a species closely related and morphologically similar to spotted knapweed. |
| 2005 | Exotic, invasive plants threaten Colorado agriculture, rangeland, and wildlands. To manage invasive plants effectively, whether through herbicides, biological control, or altering competitive regimes, we must understand the fundamental processes involved in their success in novel environments. Several species of knapweed (genus Centaurea) have invaded Colorado. We are studying two of these species which are known to hybridize with each other: diffuse and spotted knapweed (Centaurea diffusa and C. maculosa). We are using genetic tools to explore their population structure. We are studying the frequency and importance of hybridization in the invasion of these species. Our ultimate aim is to improve their control through biological, chemical, and cultural methods, and to provide guidelines regarding hybrids. If they are less aggressive than the parent species, managers can spend valuable time focusing on the parents, while if they are more aggressive, then managers should target them for control. This year we have focused on 1) ongoing population genetic research to evaluate origins of these weeds (Marrs et al. in review), 2) documenting the frequency of hybridization in the field, 3) creating hybrids in the laboratory, and 4) developing genetic markers with which to identify hybrids. Population genetic analyses suggest a cryptic invasion of a species closely related and morphologically similar to spotted knapweed. Hybrids are extremely common in populations of C. diffusa, and less common in populations of C. maculosa. Experimental crosses of C. diffusa and C. maculosa have lead to the production of viable seed. We are currently analyzing whether those seeds are true hybrids. We are conducting that analysis using newly developed amplified fragment length polymorphism (AFLP) markers, as well as microsatellite markers. |
| 2006 | Exotic, invasive plants threaten Colorado agriculture, rangeland, and wildlands. To manage invasive plants effectively, whether through herbicides, biological control, or altering competitive regimes, we must understand the fundamental processes involved in their success in novel environments. Several species of knapweed (genus Centaurea) have invaded Colorado. We are studying two of these species that are thought to hybridize with each other: Centaurea diffusa (diffuse knapweed) and C. stoebe (spotted knapweed, also known as C. maculosa). Additionally, both species are thought to be allelopathic. We are using genetic tools to explore the introduction of and hybridization between these species. Additionally, we are examining whether production of the putative allelochemical, catechin, leads to build up of the chemical in the soil to toxic levels. Our ultimate aim is to improve the control of these noxious weeds through biological, chemical, and cultural methods, and to provide guidelines regarding hybrids. If hybrids are less aggressive than the parent species, managers can spend valuable time focusing on the parents, while if they are more aggressive, then managers should target them for control. This year we have focused on 1) ongoing population genetic research to evaluate the origins of these weeds and the genetic consequences of their invasion, 2) documenting the frequency of hybridization in the field in Europe to compare with previous data from the US, 3) determining the ploidy of US plants, 4) developing genetic markers with which to identify hybrids, and 5) evaluating levels of catechin in soils from spotted knapweed infestations. Population genetic analyses suggest a cryptic invasion of a species closely related and morphologically similar to spotted knapweed. Hybridization appears to be as common in some regions of Europe as in populations of C. diffusa in the US. Analysis of ploidy levels confirm that US C. diffusa is diploid and C. stoebe is tetraploid. This strongly suggests that the hybrid genotypes found in the US were imported from Europe, and that there is not on-going hybridization here. F1 hybrids from a cross of US C. diffusa with diploid European C. stoebe stoebe have been confirmed genetically to be hybrids, and are being used to backcross into C. diffusa. Finally, we found catechin at only trace levels in soils from knapweed infestations, and wetting the soils degraded the chemical. We hypothesize that rainfall will prevent build-up of catechin to toxic levels. |
| Impact | |
| 2002 | The magnitude of the problem posed by diffuse and spotted knapweed is vast in the state of Colorado where these plants infest over 146,000 acres. With a cost of control at approximately $40 per acre, managing these plants with traditional methods is simply not feasible. The expected impacts of this research are: (1) Economic. The information we gain about the ability of different species of grasses used for restoration to withstand the allelochemicals exuded by the plants should streamline and speed restoration efforts. In addition, over the longer term, this research will improve biological control through importation of new, better adapted biocontrol agents. (2) Scientific. We will provide our international colleagues who are actively searching for more effective biological control agents with a focal area to collect prospective agents, and (3) Environmental. By fostering biological control and restoration efforts, we will facilitate the control of these noxious invasive environmental weeds. |
| 2003 | The information on origins of North American knapweeds is being used by colleagues with the USDA ARS facility overseas to target searches for new biological control agents. |
| 2004 | The research this year has produced some exciting results. The finding that the putative allelochemical catechin disappears quickly from soils should make restoration efforts simpler. Finding what appears to be a distinct species may have a dramatic economic impact if it spreads. Areas where the distinct samples came from are being targeted for eradication in hopes of preventing such an invasion. Scientifically, the methodology developed for working with the putative allelochemicals, and the finding that carbon alone can facilitate plant growth should facilitate future work on allelopathy. |
| 2005 | The publication from last years work makes it clear that restoration and revegetation efforts are not likely to be hindered by allelochemicals. Documenting the whereabouts of hybrids is the first step in their eventual control. Future common-garden experiments will determine whether hybrids are susceptible to existing biological control agents and herbicides. |
| 2006 | Land managers have been quite concerned by the appearance of hybrid genotypes. Our findings strongly suggest that hybridization is not on-going in the US. If European diploid spotted knapweed were inadvertently introduced, then hybridization within the US could occur, and may prove damaging. These species are the focus of intense study, and multiple labs have imported seed from the native range to understand this invasion better. Those importations should be kept under extremely tight control, given that many of the collections are likely of the diploid subspecies that is not yet loose in the US. The levels of catechin that we found suggest that restoration efforts will not be hindered by residual chemicals in the soil. On the contrary, a single rainfall may be all that is needed to reduce the compound from trace levels to undetectable. |
| Publications | |
| 2002 |
Bais, H. P, T. S. Walker, F. R. Stermitz, R. A. Hufbauer, and J. M. Vivanco. 2002. Enantiomeric dependent phytotoxic and antimicrobial activity of (+/-)-catechin; a rhizosecreted racemic mixture from Centaurea maculosa (spotted knapweed). Plant Physiology. 128:1173-1179. Marrs, RA, RA Hufbauer, SE Carney, L Smith. 2002. Population Structure and Ploidy Level in North American Spotted Knapweed, Centaurea maculosa. Proceedings, Western Society of Weed Science, Salt Lake City, Utah. |
| 2003 |
Hufbauer, RA, RA Marrs, AK Jackson, R Sforza, HP Bais, JM Vivanco and SE Carney. 2004 Population structure, ploidy levels and allelopathy of spotted and diffuse knapweed in North America and Eurasia. Proceedings of the XI International Symposium on Biological Control of Weeds, JM Cullen, Editor. Commonwealth Scientific and Industrial Research Organization (CSIRO) , Melbourne, Australia. |
| 2004 |
Hufbauer, RA, Marrs,RA, Jackson,AK, Sforza,R, Bais,HP, Vivanco,JM and Carney,SE. 2004. Population structure, ploidy levels and allelopathy of spotted and diffuse knapweed Pp. 121-126 in North America and Eurasia. Proceedings of the XI International Symposium on Biological Control of Weeds, Cullen,JM, Briese,DT, Kriticos,DJ, Lonsdale,WM, Morin,L, Scott,JK eds. CSIRO Entomology, Canberra, Australia. |
| 2005 |
Blair, A.C., Hanson, B.G., Brunk, G.R., Marrs, R.A., Westra, P., Nissen, S.J., and Hufbauer, R .A. 2005. New techniques and findings in the study of a candidate allelochemical implicated in invasion success. Ecology Letters. 8:1039-1047. |
| 2006 |
Blair AC, SJ Nissen, GR Brunk, RA Hufbauer. 2006. Lack of evidence for an ecological role of the putative allelochemical (+/-)-catechin in spotted knapweed invasion success. Journal of Chemical Ecology. 32:2327-2331. Marrs, RA, RA Hufbauer, SM Bogdanowicz, R Sforza. 2006. Nine polymorphic microsatellite markers in Centaurea stoebe L. [subspecies C. s. stoebe and C. s. micranthos (S. G. Gmelin ex Gugler) Hayek] and C. diffusa Lam (Asteraceae). Molecular Ecology Notes. 6:897-899. |