Download Genetic Diversity and Gene Flow Among Populations of Witheringia

Genetic diversity and gene flow among populations of Witheringia solanacea
Crystiana Tsujiura (’14) and Judy L. Stone
Department of Biology, Colby College, Waterville ME
Objective
Figure 4. Gel electrophoresis
of a sample of Monte Verde
DNA for a PCR with the CTF2 microsatellite
Figure 3. Illustration of a microsatellite. Green region indicates the
region being amplified, which varies in length among individuals.
Expected vs. Observed Heterozygosity at Low
Elevations
0.900
0.900
0.800
0.800
0.700
0.700
0.600
0.600
0.500
Ho
0.400
He
Frequency
To evaluate fitness consequences of population structure in the
Costa Rican plant Witheringia solanacea by looking at inbreeding
within populations and gene flow between populations.
Frequency
Expected vs. Observed Heterozygosity at High
Elevations
0.500
He
0.300
0.300
0.200
0.200
0.100
0.100
0.000
Ho
0.400
0.000
CA-E2
CT-A3
CT-B11
GATA-A6
CT-F2
CA-E2
Locus
Introduction
CT-B11
GATA-A6
CT-F2
Locus
Figure 6. Expected versus observed heterozygosity in high (left) and low (right) elevations by locus. Pairs of
bars with an asterisk are the populations where Ho < He, which meant there was inbreeding within the
population.
Allele Frequency for CA-E2
Allele Frequency for CT-A3
0.600
0.400
Pop1
0.200
Frequency
0.800
Frequency
Many plant species have self-incompatibility mechanisms, which prevent self-fertilization by
recognition and rejection of self pollen. Loss-of-function mutations in the biochemical
pathway that provide self-incompatibility can permit certain individuals within these species
to self-fertilize. Self-fertilization in an historically outcrossing species typically causes severe
inbreeding depression and can have dramatic impacts on population genetic structure.
CT-A3
Pop2
0.000
202
206
208
212
214
0.500
0.400
0.300
0.200
0.100
0.000
216
Pop1
Pop2
141
143
145
CA-E2
Alleles
147
149
151
160
CT-A3
Alleles
0.060
We are also interested in gene flow among populations. At low elevations, where there are
many pollinators, it may be beneficial for plants to be self-incompatible so that their progeny
will not suffer from inbreeding depression. But if seeds from these plants are carried to high
elevations, they will bring the presumably non-adaptive self-incompatibility gene there. By
comparing allele frequencies among populations, we can estimate how much gene flow
there is between them.
0.800
Pop1
Pop2
172
175
177
179
181
0.040
0.400
Pop1
0.200
Pop2
0.000
181
CT-B11
Locus
Allele Frequency for CT-F2
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000
Pop1
Pop2
286
288
290
292
185
193
201
209
213
220
GATA-A6
Alleles
294
296
298
CT-F2
Alleles
Number of Alleles per Locus
Effective Population Size
Observed Heterozygosity
Expected Heterozygosity
Inbreeding Coefficient
Elevation: > 1,280 m
0.600
Figure 5. Allele frequencies for each locus.
Population 1 corresponds to high elevation while
Population 2 corresponds to low elevation. We
expected a bell curve for frequencies (e.g. CT-A3
graph in the top right) for all loci, but some loci’s
frequencies did not exhibit this distribution pattern
(CT-B11, CA-E2).
High Elevation
5.60
2.52
0.48
0.58
0.20
Low Elevation
5.20
3.06
0.56
0.65
0.15
Table 1. Population genetic characteristics for both populations averaged across five loci.
Fst value
0.600
0.500
0.400
0.300
0.200
0.100
0.000
Figure 7. Genetic
differentiation between
the two populations as
estimated by each
locus. When Fst = 0,
there is complete
admixture. Fst = 1
means complete
isolation.
0.050
Allele Frequency for GATA-A6
Frequency
Frequency
Allele Frequency for CT-B11
Frequency
Our study species, Witheringia solanacea, contains both self-incompatible and selfcompatible individuals. It lives in a mountainous area of Costa Rica with great variation in
climate and suitability for pollinating bees. At higher elevations, plant populations are
smaller and there are fewer pollinators (Stone and Jenkins 2007). We expect that due to low
number of pollinators and smaller population size in populations at high elevation, there will
be greater levels of inbreeding in high elevation populations of Witheringia solanacea.
0.030
0.020
0.010
0.000
CA-E2
CT-A3
CT-B11
GATA-A6
CT-F2
Locus
Discussion
We found, as expected, that the high elevation populations had a higher inbreeding
coefficient than low elevation populations. We expected this because of the lower number
of pollinators and smaller population sizes at high elevations. Plants who can reproduce by
self-fertilization may have higher reproductive success in high elevation populations.
Our Fst values indicate that there is substantial gene flow between the high elevation
population and low elevation population. Therefore, genes for self-incompatibility will be
continually re-introduced into high elevation populations, even if natural selection there
would favor self-fertilization.
Elevation < 1,100 m
Results
Figure 1. Topographic map of the Monte Verde region in
Costa Rica. The three areas where we obtained our
samples from are indicated.
Figure 2. Witheringia solanacea in the wild.
Materials and Methods
We collected leaf tissue from W. solanacea at greater than 5 m spacing along roadsides
and trails. DNA from 50 of these plants was extracted. Populations were differentiated by
elevation, where high elevation plants were grouped as greater or equal to 1,280 meters
elevation, and low elevation plants were grouped as lesser or equal to 1100 meters
elevation (Figure 1). We then performed the polymerase chain reaction (PCR) with five
different microsatellite primers (Figure 3) in order to amplify short segments of DNA from
each plant. Successful amplification was confirmed using gel electrophoresis (Figure 4). We
then sent our fluorescently-tagged PCR product to the genetic analyzer to complete the
genotyping of each individual. Genalex, population genetics software for Excel, was used to
analyze our data (Peakall and Smouse, 2006).
The number of alleles per locus was greater in the high elevation population, while the
effective population size was greater in the low elevation population (Table 1). This
indicates that allele frequencies are more evenly distributed in the low elevation population.
The larger effective population size is consistent with the larger actual population size.
In both the high and low populations, observed heterozygosity (H0) was less than expected
heterozygosity (He), demonstrating that there is some inbreeding (Figure 6). The average
observed heterozygosity (H0) for the low elevation population was greater than for the high
elevation population. Likewise, our expected heterozygosity (He) was greater for the low
elevation population than for the high elevation population. Thus, the inbreeding coefficient
in the high elevation population was greater than the low elevation population (Table 1).
The genetic differentiation (Fst) values varied across loci, with the greatest value at the CTF2 locus, and the lowest value at the GATA-A6 locus (Figure 7). The mean Fst value was
0.022, with a standard error of 0.008.
References
Stone, JL, and E. G. Jenkins (2007). Pollinator abundance and pollen limitation of a
Solanaceous shurub at premontane and lower montane sites. Biotropica, in press.
Peakall, R., P.E. Smouse. (2006). Genalex 6: genetic analysis in Excel. Population genetic
software for teaching and research. Molecular Ecology Notes 6: 288-295.
Acknowledgements
We would like to thank Patti Easton for genotyping our samples.