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:
agriffit@mwc.edu
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
1 2 3 4 5 6 7
cpDNA bands
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.
1
2
3
4
5
6
7
8
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 board
1200
Stipend
2000
FICA (7.65% of stipend) 153
Supplies
DNA extractions
1551
PCR primers
42
PCR supplies
200
Pipet tips
220
Gloves
85
TOTAL
$5451
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Carulli, J. P. and D. E. Fairbrothers 1988. Allozyme variation in three eastern United
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Comes, H. P. and R. J. Abbott. 1999. Population genetic structure and gene flow across
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