Progress 02/01/03 to 09/30/06
Outputs
Agricultural impacts of invasive species can be far reaching. Invasive weeds dominate landscapes in many cases, and are expensive and difficult to eradicate. Their presence in arable areas can reduce the agricultural value of land directly, as well as having further cascade effects on the agricultural environment, such as requiring inputs of pesticides, which may have non-target effects and financial implications which in turn influence sustainability. Invasive weeds may reduce the availability of grazing lands, reduce water flow from catchments and compete severely with crops. This research on dispersal and population genetics of invasive weeds and management implications concentrates on developing a model describing the dispersal patterns of three invasive species in Hawaii, and to extend this model to develop a management decision system for selecting appropriate management approaches to invasive weeds in Hawaii. We have used molecular biology as a tool to
investigate invasiveness of these species, by quantifying their genetic diversity and channels of dispersal. One of these species, the highly aggressive fountain grass that is notoriously as one of Hawaii's worst invaders, is the subject in a major population genetics study. This work has revealed a new but hitherto overlooked mechanism promoting invasiveness. From his genetics results, we have proved that fountain grass collected from various locations around the world share a single genotype (exactly the same DNA makeup, i.e. clone) and that this species' ability to tolerate various environmental conditions (known as phenotypic plasticity) contributes to its success in disturbed habitats in Hawai'i, California and Africa. To our knowledge this is the first time this phenomenon has been observed over such a large geographical scale. This has implications for the ornamental industry that often creates clones for nursery plants - we assume they are safe because they have low genetic
diversity, but is this true considering the fountain grass phenomenon? In collaborative work with the Hawaii Department of Agriculture we were able to identify the source population of another invasive species, fireweed (Senecio madagascariensis), and this information will be used in an ongoing biological control project to locate effective control organisms. Fireweed is of major concern to the beef ranching industry since it is toxic to livestock. We are currently assessing patterns of dispersal for this weed in the Hawaiian Islands making use of various DNA markers. We also are conducting genetic research on Miconia calvescens throughout the Pacific (Hawaii, Moorea, New Caledonia, Raiatea, Tahiti and Nuku Hiva). Miconia is especially problematic in Tahiti where it has taken over 65% of all vegetation and the climate similarities between Tahiti and Hawaii makes a genetic analysis a very important tool to establish the likelihood that Hawaii's Miconia problem can reach the same
devastating levels as in Tahiti. Similar genetic makeup should be alarming to management authorities. The importance of genetic diversity in invasion success for this species is another aspect we are examining.
Impacts
The attributes that make some organisms more likely to become invasive than others remain for the most part elusive and are vital to the prediction of biological invasions (BI) and the management of already-established invasions. Our population genetic and ecological data for one study system (P. setaceum) showed that this highly aggressive invader consists of a single clone on a global geographic scale. Our findings show extreme levels of phenotypic plasticity (differential response of clones under different environmental conditions) to counteract the actions of natural selection/local adaptation and thus the need for genetic diversity and thus that phenotypic plasticity might be a major contribution to BI. Further examination of invasive species may be found that this is a widespread mechanism, but hitherto overlooked, enhancing invasiveness of species. We furthermore show the use of molecular markers to identify otherwise impossible relationships (e.g. Senecio
madagascariensis complex) yielding important information on the origin of introduced species and the secondary routes that brought them to their new locations. This will greatly improve the success rate of biological control programs and help to identify sources of "invasive species contamination" such as import ports etc. Molecular markers can trace dispersal indirectly that is otherwise normally hard to quantify in plants. These patterns can be correlated to environmental conditions to predict regions most likely to be impacted by invaders and to prioritize certain areas for management.
Publications
- Le Roux, J. J., Wieczorek, A. M., Ramadan, M. M. and Tran, C. T. (2006) The native provenance of invasive fireweed (Senecio madagascariensis Poir.) in the Hawaiian islands as inferred from phylogenetic analysis. Diversity and Distributions 12, 694-702.
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Progress 10/01/04 to 09/30/05
Outputs
The aim of this project is tract dispersal for actively and passively dispersed invader plant species in Hawaii. Passively wind-dispersed fountain grass (Pennisetum setaceum) was chosen as one candidate species because it is well naturalized over the Hawaiian Islands. For this species 25 microsatellite markers were developed. Microsatellites are known to be informative for population genetic studies that aim at quantifying dispersal. Initial screening for polymorphism revealed that none of the 25 markers were polymorphic for Hawaiian populations of P. setaceum. The same results were obtained when screening included individuals from California, South Africa, Namibia and Egypt donated by the Hawaii Department of Agriculture (HDOA). It appears that P. setaceum encompasses a single genotype or clone. To assess if any geographical variation exists, a phylogeographical analysis was conducted making use of the hypervariable DNA sequences. Phylogenetic analysis aims to infer
evolutionary relationships among and between individual taxa. All P. setaceum species shared 100% DNA sequence homology irrespective of geographical origin. We assessed the role of phenotypic plasticity in the survival of different populations of P. setaceum. Various growth studies (nitrogen, total nutrient, moisture and pH gradients) were used. We measured responses to each of these gradients as the amount of biomass produced after a certain growth period. These results were congruent with all previous results in that no variation in phenotypic plasticity could be detected irrespective of geographical origin. These results are now implemented to seek target specific herbicides for P. setaceum, since no genetic variation will provide little chance to build up resistance once such a herbicide has been identified. A second species, Senecio madagascariensis or commonly known as fireweed, was identified for this study. This species is a significant threat to the ranching industry in
Hawaii and has managed to naturalize in many habitats on the islands. Twelve polymorphic loci were developed and characterized for fireweed. Initial analysis proves that high amounts of genetic variation exist despite the recentness of its introduction to the Hawaiian Islands. This could indicate multiple introductions of fireweed in Hawaii. HDOA donated DNA from the native ranges (Madagascar and South Africa) that will be included in the final analysis. From a DNA sequence phylogeny we infer that the Hawaiian populations are most likely from the east coast of South Africa. This is valuable information to improve the ongoing biological control project by HDOA in identifying target specific natural enemies. For our actively (animal) dispersed species, Miconia calvescens was chosen. The extreme destruction caused by this species in Tahiti is of great concern in Hawaii having very similar climate conditions to Tahiti. Eleven microsatellite containing loci were developed for M.
calvescens. Initial analysis showed that populations are characterized by low amounts of genetic variation and that these populations might be experiencing genetic bottlenecks.
Impacts
These results are now being implemented to seek target specific herbicides for P. setaceum, since no genetic variation will provide little chance to build up resistance once such a herbicide has been identified.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
P. setaceum (Fountain grass): We designed 26 primer pairs that specifically amplified microsatellite-containing loci. These primers were tested for polymorphism within and among populations. No genetic variation was observed for any of these primers at very large geographical scales (different continents). We concluded that very little to possibly no genetic variation exists within this species. We subsequently did a phylogeographical analyses in order to compare relatedness of P. setaceum populations from various locations globally where it is considered invasive and its native range. We also accessed phenotypic plasticity (intraspecific variation in response to environmental conditions) for P. setaceum by comparing the performance (fitness) of various populations under certain greenhouse conditions. We specifically looked at the influence of nitrogen and the availability of water on fitness. We subsequently proved that phenotypic plasticity plays a major role in the
invasive potential of fountain grass. Senecio madagascariensis (Fireweed): Specimens of S. madagascariensis were collected on Maui for initial DNA extractions and construction of genomic libraries. Fragments containing di- and tri-nucleotide repeat units [(GC)n] [(AAC)n] were enriched for and subsequently cloned. These clones were screened for the presence of microsatellite sequences. Results were good with very high numbers of positive clones for both the di- and tri-nucleotide inserts. A total of 250 clones were selected for further investigation. DNA from positive clones for fireweed was extracted; cloned loci amplified and sequenced to reveal microsatellite containing sequences. Based on the sequencing results 35 insert fragments were selected for primer design. Sequences chosen were mainly due to the presence of uninterrupted microsatellite sequences and long flanking regions enabling primer design. These primers were subsequently optimized for optimal temperatures and when
necessary for optimal MgCl2 concentration to ensure good amplification. All of the 25 loci were screened for polymorphism (genetic variation) making use of Gene chips. Out of these, 13 loci showed varied degrees of variability. These were labeled with flourophores and depending on the sizes and labels used, we optimized loci for multiplex PCR (amplifying more then one locus in the same reaction). We were successful in the construction of two multiplex PCR reactions, each containing 6 different loci. We collected populations of S. madagascariensis from Maui and Hawaii. The Hawaii department of Agriculture also donated many samples from the native range of S. madagascariensis (South Africa and Madagascar). Future work includes more extensive collections within the Hawaiian archipelago, followed by genotyping of the two multiplexes. Miconia calvescens: The same procedure that was described above was used to design primers for 57 microsatellite-containing loci. These are currently
optimized as previously described. We should have the multiplexes ready for genotyping in the near future. Populations have been collected on Hawaii and Kauai.
Impacts
Biological invasions are a serious threat to biodiversity and agriculture. Over 86% of Hawaiian vascular plants species are endemic. Dispersal of genes (which represents the effects of dispersal of seeds) can provide information about invasiveness. Given the importance of understanding dispersal patterns in managing invasive species, this project addresses the population genetic consequences of different dispersal modes, to invasiveness. Studies of genetic variation may help to predict the potential for populations of invasive species to evolve in response to management practices (e.g. evolution of resistance to herbicides or biological control agents). A decision system to select primary management strategies when faced with limited resources for the management of invasive weeds will be proposed. Dispersal is an aspect that should be addressed when managing the invasion. Therefore, the project aims to propose management protocols that are based on the expected
dispersal patterns of invasive species in Hawaii. We have constructed and screened genomic libraries for both species (M. calvescens and P. setaceum), designed and optimized primers for PCR amplification of microsatellites, and screened for polymorphism. We have obtained extensive material from locations where fountain grass is non-invasive (for population genetic comparison) as well as from locations where fountain grass is a weed (Hawai'i, California). End-users of these results and products will be people involved in development of pest management programs for invasive weeds and regulators and quarantine agencies.
Publications
- No publications reported this period
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Progress 10/01/02 to 09/30/03
Outputs
A graduate student was hired, starting February 2003. Both M. calvescens and P. setaceum were collected on Oahu, for construction of genomic libraries. Genomic DNA from one sample of each spp. was digested and enriched for Microsatellite sequences. Fragments containing dinucleotide repeat units [(GC)n] were selected. For both Miconia and fountain grass these fragments were cloned into PUC19 and transformed into E. coli. Before screening of the subsequent libraries the same was done for trinucleotide fragments [(AAC)n] in order to construct libraries. Probes were designed and labeled for hybridization. We used the DIG (Digoxigenin) system for labeling of probes. Colonies from previously constructed libraries were screened with the labeled probes for the presence of microsatellite sequences. A total of 150 clones from each species were selected for further investigation. DNA from all positive clones for fountain grass was extracted and amplified using flanking region
primers (M13 Forward and Reverse). Dinucleotide inserts, clones f1 - f94, were amplified, giving rise to fragments between 300 and 700 base pairs long. For the trinucleotide inserts, clones f95 - f148, a total of 54 inserts were amplified ranging from 200 to 600 base pairs in size. For sequencing, fragments that were bigger than or about 300 base pairs were selected. These amplified fragments were cleaned up using Exonuclease I and Shrimp Alkalinephosphotase. Using these as templates, the forward primer (M13 Forward) was used to sequence the fragment throughout its length on a ABI 3100 DNA sequencer. Fragment sizes ensured for long enough flanking regions to subsequently design primers and manipulate the expected size of PCR products from the newly designed primers. Initially a total of 47 clones encompassing both di- and tri-nucleotide inserts were sequenced. Based on the Microsatellite sequence and flanking region we decided on designing primers for loci. Based on the sequencing
results, 25 insert fragments were selected for primer design. Sequences were chosen based on presence of uninterrupted microsatellite sequences and long flanking regions enabling primer design. Raw sequence data was edited using the program, Chromas. We used the program Fast PCR for designing the primers. Once the primers were optimized, amplification of the sequence of interest was done for twelve individuals from different populations and islands. These preliminary results proved that out of the 13 primer sets screened, 4 sets showed variability. In order to determine the reproductive nature of invasive fountain grass, seeds from parent plants were collected for two populations. These seeds were germinated under greenhouse conditions. Subsequently ten seedlings were collected for each parent and subjected to DNA extraction. DNA was extracted for all the samples obtained from Africa. For the Hawaii populations, extractions were done for the Lanikai populations and one of the Kona
populations. A total of 300 DNA extractions are done. Objective 2 cannot be addressed until Objective 1 has been completed.
Impacts
Biological invasions are a serious threat to biodiversity and agriculture. Over 86% of Hawaiian vascular plants species are endemic. Dispersal of genes (which represents the effects of dispersal of seeds) can provide information about invasiveness. Given the importance of understanding dispersal patterns in managing invasive species, this project addresses the population genetic consequences of different dispersal modes, to invasiveness. Studies of genetic variation may help to predict the potential for populations of invasive species to evolve in response to management practices (e.g. evolution of resistance to herbicides or biological control agents). A decision system to select primary management strategies when faced with limited resources for the management of invasive weeds will be proposed. Dispersal is an aspect that should be addressed when managing the invasion. Therefore, the project aims to propose management protocols that are based on the expected
dispersal patterns of invasive species in Hawaii. We have constructed and screened genomic libraries for both species (M. calvescens and P. setaceum), designed and optimized primers for PCR amplification of microsatellites, and screened for polymorphism. We have obtained extensive material from locations where fountain grass is non-invasive (for population genetic comparison) as well as from locations where fountain grass is a weed (Hawai'i, California). End-users of these results and products will be people involved in development of pest management programs for invasive weeds and regulators and quarantine agencies.
Publications
- No publications reported this period
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