VIRGINIA ENDANGERED PLANT AND INSECT SPECIES
PROJECT PROPOSALS FOR 2003 SECTION 6 FUNDING CONSIDERATION
Principle investigator: Alan B. Griffith, Ph.D.
Address: Department of Biological Sciences
Jepson Hall
Mary Washington College
1301 College Avenue
Fredericksburg, VA 22401
Telephone:540-654-1422
Fax:540-654-1081
Email:
VIRGINIA ENDANGERED PLANT AND INSECT SPECIES
PROJECT PROPOSALS FOR 2003 SECTION 6 FUNDING CONSIDERATION
PROJECT TITLE:
Chloroplast DNA variability of Aeschynomene virginica, a rare wetland plant
NEED:
Aeschynomene virginica (Fabaceae) is a federally threatened plant (U.S. Fish and Wildlife Service 1992) that grows in freshwater tidal wetlands on the east coast of the United States. Populations are found from southern New Jersey to central North Carolina, but the majority of populations are found in Virginia (U.S. Fish and Wildlife Service 1995). Populations typically grow in patches along stream banks, often in areas of decreased standing vegetation (Griffith and Forseth 2003). Population size varies from less than 10 to as many as 1000 stems in different populations during a season (U.S. Fish and Wildlife Service 1995). Year to year population size often varies by a factor of 10 at a given site (U.S. Fish and Wildlife Service 1995, A. B. Griffith personal observation, The Nature Conservancy 2000, J. Patt personal communication).
One of the recovery tasks for A. virginica is to “determine seed dispersal and banking capabilities” (U.S. Fish and Wildlife Service 1995). This task further states the need to investigate how long and how far seeds of this species float (U.S. Fish and Wildlife Service 1995). One study (Griffith and Forseth 2002) estimated the maximum potential dispersal distance for A. virginica seeds. But, this study did not track secondary seed dispersal and the long term fate of individual seeds.
While the fate of dispersing seeds is difficult to physically track, molecular markers have been successfully used to measure seed dispersal (Shapcott 2000, McCauley 1995), the relative importance of pollen movement and seed movement in population genetic structure (McCauley 1997, Comes and Abbott 1999, Tarayre et al. 1997), and the relative significance of dispersal and genetic drift in population genetic structure (Shapcott 2000). In particular, the distribution of chloroplast DNA (cpDNA) can provide much information about seed dispersal within and among populations of plants (Forcioli et al. 1998, McCauley 1995). Chloroplast DNA is maternally inherited in most angiosperms (Mogensen 1996, McCauley 1995) and hence disperses via seed and not pollen.
Seed dispersal and genetic drift act, in concert, to shape the genetic differentiation of populations. Seed dispersal among populations tends to have a homogenizing effect on the genetic differentiation of populations (Ellstrand and Elam 1993). If genes flow equally between any two populations in a group of populations, then genetic variability among populations will decrease relative to the genetic variability within each population. Separated small populations also undergo genetic drift or random changes at genetic loci. Genetic drift has the opposite effect to seed dispersal on genetic differentiation among small populations. It increases the among population genetic differentiation relative to the within population genetic differentiation (Ellstrand and Elam 1993). The amount of dispersal relative to genetic drift then determines the overall genetic differentiation between populations.
Population genetic structure can also answer general conservation questions. Genetic variability of rare plant populations impacts the potential for the species to adapt and persist, in the long term (Huenneke 1991). Levels of genetic variability may determine a plant’s ability to withstand biotic and abiotic variation in the short and long term (Ellstrand and Elam 1993). Thus, studying genetic variability of a rare plant can give information about potential genetic threats to the species.
Hence, population genetic structure, measured with molecular markers, can provide important ecological and conservation information for the management of this threatened species. Yet, little is known about the population genetics of A. virginica except for work by Carulli and Fairbrothers (1988). Using allozymes, they showed that A. virginica does not hybridize with congeners (A. indica and A. rudis) and that there is very little allozyme variability among populations of this species.
OBJECTIVE:
The lack of allozyme variability was not unusual (Carulli and Fairbrothers 1988), but it left open questions about A. virginica’s population genetic structure, seed dispersal, and genetic drift. In the fall 2003, I will sample multiple A. virginica populations in 7 river systems across the known distribution of the plant. During the summer of 2004, DNA will be extracted from approximately 400 plant samples collected. Using polymerase chain reaction (PCR), chloroplast DNA (cpDNA) will be amplified from each sample and then digested in one or more restriction enzymes. Restriction enzyme digestion of cpDNA may result in variable DNA banding patterns for different plant samples. These banding patterns can then be analyzed to compare the within river system and the among river system genetic variability. This is analogous to comparisons of the within population and among population genetic variability discussed above.
EXPECTED RESULTS AND BENEFITS:
We hypothesize that most genetic variability of these plant populations will be found among populations in different river systems. Seeds float and disperse in the streams along which populations grow and potential dispersal distance is limited by the distance seeds can float during a tidal cycle (Griffith and Forseth 2002). There are no known seed vectors that may carry seeds across the land barriers between river systems. Hence, seed dispersal among populations within a river system should homogenize genetic variability within a river system. Genetic drift should create random genetic variation among populations in different river systems and thus increase genetic variability among river systems relative to populations within a river system.
While the life history of A. virginica is well known, many details of this plant’s ecological requirements are unknown (U.S. Fish and Wildlife Service 1995). Managers need to know basic ecological requirements like dispersal ability (i.e. dispersal rates and distances) to understand habitat requirements. This research will directly address a recovery task for A. virginica and therefore will directly benefit the conservation of this plant. The relative distribution of genetic variability within and among rivers will tell us the relative importance of seed dispersal and genetic drift in shaping the population genetic structure of A. virginica. High rates of seed dispersal between populations within a river system would suggest populations of A. virginica are connected demographically (Griffith and Forseth 2002) and strengthen suggestions that A. virginica lives in a metapopulation (Griffith and Forseth 2002, U.S. Fish and Wildlife Service 1995). If these patchy populations are a metapopulation, it will be crucial to protect current and potential population sites (Hanski and Simberloff 1997).
Another practical benefit of this research will include information about the genetic health of populations. For example, results that show a significant amount of genetic variability within populations would suggest a genetically healthy plant population. That is to say, high within population genetic variation would suggest populations could withstand environmental variability (Ellstrand and Elam 1993).
Last, this research could improve the management of the genetic stock of this rare plant. The hypothesized population genetic structure of A. virginica is a homogeneous distribution of cpDNA within a river system and genetic divergence among rivers. To maintain and protect this hypothesized genetic variability, plant tissues from across the range of the species would have to be protected. Any ex situ stocks of seeds would have to sample broadly across the distribution of populations to maintain this type of genetic variability (Hogbin and Peakall 1999). This distribution of genetic variability would also suggest that seeds should not be transported among river systems to re-establish populations of A. virginica. Mixing the divergent genetic stock would homogenize any existing genetic variability.
APPROACH:
Complete genomic DNA will be extracted from 100 mg of leaf tissue from each plant sample. This tissue may be frozen leaf tissue stored at -80 °C or fresh leaf tissue. Standard polymerase chain reaction (PCR) techniques will be used to amplify short segments of non-coding regions of cpDNA (Demesure et al. 1995, Dumolin-Lapègue et al. 1997). We will employ 3 to 4 universal PCR primer pairs that amplify different segments of cpDNA. Each cpDNA segment from each plant sample will be cut using restriction endonucleases. These segments of cpDNA are short enough (i.e. 1400 bp to 3500 bp) to warrant digestion with restriction endonucleases that recognize unique 4 nucleotide sequences (Demesure et al. 1995, Petit et al. 1997, Taberlet et al. 1991). Additions or deletions of base pairs at restriction sites along a cpDNA sequence will result in different banding patterns for different samples (Dumolin-Lapègue 1997, Byrne and Moran 1994). We will analyze and interpret different banding patterns as genetic variability in A. virginica samples.
The objective of my current research is to develop procedures and protocols for DNA extraction, PCR amplification with universal primers, and restriction enzyme digestion of amplified cpDNA that are optimized for A. virginica. I grew seeds of A. virginica in a field garden on the Cumberland Marsh Preserve, New Kent Co., VA. The Cumberland Marsh Preserve supports one of the largest A. virginica populations (U.S. Fish and Wildlife Service 1995) and has been the site of recent ecological research on this species (Griffith and Forseth 2002, Griffith and Forseth 2003, Griffith 2002). I germinated these field grown seeds in a greenhouse at Mary Washington College and extracted DNA from fresh, young leaves. Figure 1 shows PCR product of cpDNA on 1% agarose gel from five plant samples. The PCR primer pair used was trn K1 – trn K2 (Demesure et al. 1995). Figure 2 shows banding patterns on 2% agarose gel from the
Figure 1: cpDNA from PCR with primer pair tK1 - tK2. Lane 1 is
lambda size marker. Lane 7 is a water blank. All DNA was stained
with ethidium bromide and visualized under UV light.
digestion of one plant sample by different restriction enzymes. These procedures and protocols will apply directly to the plant tissue collected for this proposed research.
LOCATION:
All molecular work will be done at Mary Washington College, Fredericksburg, VA. Plant materials will be sampled from NJ, MD, VA, and NC.
Figure 2. Example banding pattern of cpDNA cut with restriction
enzymes. Lanes 1 and 8 are 100-bp ladder. Lanes 2 - 6 are different
restriction enzymes. Lane 7 is a control of undigested cpDNA. All
DNA was stained with ethidium bromide and visualized under UV light.
ESTIMATED COST: (itemize)
Year 1:Year 2:
Student researcher
On campus room
and board1200
Stipend2000
FICA (7.65% of stipend) 153
Supplies
DNA extractions1551
PCR primers 42
PCR supplies 200
Pipet tips 220
Gloves 85
TOTAL $5451
References:
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Carulli, J. P. and D. E. Fairbrothers 1988. Allozyme variation in three eastern United State species of Aeschynomene (Fabaceae), including the rare A. virginica. Systematic Botany 13: 559 - 566.
Comes, H. P. and R. J. Abbott. 1999. Population genetic structure and gene flow across arid versus mesic environments: a comparative study of two parapatric Senecio species from the near east. Evolution 53: 36 – 54.
Demesure, B., N. Sodzi, and R. J. Petit. 1995. A set of universal primers for amplifications of polymorphic non-coding regions of mitochondrial and chloroplast DNA in plants. Molecular Ecology 4: 129 – 131.
Dumolin-Lapègue, S. M-H Pemonge, and R. J. Petit. 1997. An enlarged set of consensus primers for the study of organelle DNA in plants. Moelcular Ecology 6: 393 – 397.
Ellstrand, N. C. and D. R. Elam. 1993. Population genetic consequences of small population size: implications for plant conservation. Annual Review of Ecology and Systematics 24: 217 – 242.
Forcioli, D., P. Saumitou-Laprade, M. Valero, P. Vernet, and J. Cuguen. 1998. Distribution of chloroplast DNA diversity within and among populations in gynodioecious Beta vulgaris ssp. maritima (Chenopodiaceae). Molecular Ecology 7:1193 – 1204.
Griffith, A. B. 2002. The population dynamics of Aeschynomene virginica, a rare, wetland annual. Dissertation. University of Maryland.
Griffith, A. B. and I. N. Forseth. 2002. Primary and secondary dispersal of a rare, tidal wetland annual, Aeschynomene virginica. Wetlands 22: 696 – 704.
Griffith, A. B. and I. N. Forseth. 2003. Establishment and reproduction of Aeschynomene virginica (L.) (Fabaceae) a rare, annual, wetland species in relation to vegetation removal and water level. Plant Ecology 167: 117 -125.
Hanski, I. A. , and D. Simberloff. 1997. The metapopulation approach, its history, conceptual domain, and application to conservation. Pages 5-26 in I. A. Hanski and M. E. Gilpin, eds. Metapopulation Biology: Ecology, Genetics, and Evolution. Academic Press, NY.
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Tarayre, M., P. Saumitou-Laprade, J. Cuguen, D. Couvet, and J. D. Thompson. 1997. The spatial genetic structure of cytoplasmic (cpDNA) and nuclear (allozyme) markers within and among populations of the gynodioecious Thymus vulgaris (Labiatae) in southern France. American Journal of Botany. 84: 1675 – 1684.
The Nature Conservancy. 2000. Results of monitoring studies for the sensitive joint-vetch (Aeschynomene virginica): 2000 final report. The Nature Conservancy, VAFO Technical Report 00-2.
U.S. Fish and Wildlife Service. 1992. Endangered and threatened wildlife and plants; determination of threatened status for the sensitive joint-vetch (Aeschynomene virginica). Federal Register 57: 21569 – 21574.
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