2007 SOUTHEASTERN NATURALIST 6(2):359–364
Clonal Diversity in Differently Aged Patches of the Dune
Grass Uniola paniculata
Stephen P. Bush1,* and Marcio E. Stelato1,2
Abstract - Uniola paniculata (sea oats) is a perennial, clone-forming dune grass of
coastal beaches in the southeastern United States. We used random amplified polymorphic
DNA (RAPD) markers to compare clonal diversity in younger and older
patches of U. paniculata. Older patches were found to contain a significantly higher
level of clonal diversity, suggesting that, in some cases, U. paniculata populations
may increase in clonal diversity over time. The high level of clonal diversity found in
the older patches provides further evidence for the important role of sexual reproduction
in maintaining diversity in U. paniculata.
Introduction
Uniola paniculata L. (sea oats), the dominant grass on dunes of the
Atlantic and Gulf coasts of the southeastern United States, plays a critical
ecological role in the formation and stabilization of coastal sand dunes. Sea
oats is among the initial colonizers that invade bare sands of coastal beaches
to initiate dune building. Colonizing plants then trap wind-blown sand and,
through subsequent episodes of sand trapping followed by dune grass
growth, dunes are formed (Wagner 1964, Woodhouse et al. 1968). Uniola
paniculata also helps to stabilize established dunes through an extensive
underground network of roots and rhizomes.
In facilitating sand dune formation and stabilization, populations of perennial,
clone-forming sea oat plants persist for many years. Clonal plants are
very common in the early stages of vegetative succession (Prach and Pysek
1994). For example, clonal taxa are important colonizers of man-made disturbed
sites in Europe (Prach and Pysek 1994, 1999), of accreting shorelines
of the Great Lakes in the United States (Cowles 1899), and of habitat exposed
by glacial retreat (Stocklin and Baumler 1996). Through lateral growth, clonal
plants are able to rapidly colonize newly exposed habitats in early successional
sites (Macdonald and Lieffers 1991, Prach and Pysek 1994). Clonal
growth likely influences the population genetic structure of colonizing species.
At one extreme, the growth of vigorous clones may produce populations
with low levels of clonal diversity (Ellstrand and Roose 1987). Thus, even
when populations are founded by a wide variety of propagules, extensive
vegetative growth by better-adapted clones may reduce clonal diversity
(Ellstrand and Roose 1987, Macdonald and Lieffers 1991). In the predominantly
clone-forming Ammophila breviligulata Fern. (dune grass), Laing
(1967) proposed that populations are established in brief periods by an initial
number of individuals. Once established, clonal diversity is likely to decrease,
1Department of Biology, Coastal Carolina University, Conway, SC 29526. 2Current
address - Dakocytomation, 6392 Via Real, Carpinteria, CA 93013. *Corresponding
author - bush@coastal.edu.
360 Southeastern Naturalist Vol. 6, No. 2
so that a few large clones dominate the dune vegetation (Laing 1967). In sea
oats, vegetative reproduction via rhizomatous growth is widespread (Wagner
1964, Woodhouse et al. 1976), while recruitment of sea oat seedlings is
infrequently observed (Wagner 1964, Franks 2003). Therefore, clonal diversity
in sea oats was hypothesized to be low (Franks et al. 2004).
After a habitat is colonized, environmental conditions may become more
favorable for sexual recruitment, and clonal plants may, therefore, maintain
levels of variation similar to those found in predominantly sexual species
(Macdonald and Lieffers 1991). For example, in a recent study of succession
in coastal dunes inhabited partly by sea oats, seedling establishment was
found to be more likely in vegetated areas (Franks 2003). Seeds were more
abundant under established dune plants than in unvegetated sands, and
more seeds emerged and survived when growing under established plants
than in open areas (Franks 2003). Seeds likely accumulate as established
plants trap wind-blown propagules, and dune vegetation then increases the
survival of seedlings by protecting against excessive sand movement that
may either bury or expose emerging seedlings growing in open areas (Franks
2003). Therefore, although clonal growth may increase the frequency of
some genotypes, it may also increase sexual recruitment by providing favorable
conditions for seedling establishment (Franks 2003).
A recent study of clonal diversity in eight patches of sea oats found that
diversity differed greatly among sampling sites. The authors suggested
that both vegetative growth and sexual recruitment are important factors in
maintaining genetic diversity in sea oats (Franks et al. 2004). The persistence
of diversity in many patches of sea oats despite clonal growth is not
atypical of clonal plants. Numerous predominantly clonal species have been
investigated, and surveys indicate that populations typically contain a diversity
of clones (Ellstrand and Roose 1987, Widen et al. 1994), and that the
distribution of genetic diversity within and among clonal plant populations
is similar to that found in predominantly sexually reproducing species
(Hamrick and Godt 1996).
We hypothesized that clonal diversity may be higher in older patches of sea
oats due to the facilitation of seedling recruitment by established plants. We
used random amplified polymorphic DNA (RAPD) markers to investigate
clonal diversity in differently aged patches of sea oats, comparing recently
vegetated beaches to patches found on large, established dunes. RAPDs are
neutral markers that are unlikely to be altered by mutations (Van de Ven and
McNicol 1995). Thus, RAPDs are typically conserved among the ramets of a
genet (Bush and Mulcahy 1999, Eckert et al. 2003, Kjolner et al. 2004), and
have been utilized in numerous studies of clonal plants.
Methods
Patches of sea oats were sampled on Waites Island (33°50'43"N,
78°35'12"W), an undeveloped barrier island on the North Carolina/South
Carolina state line. The foredunes on Waites Island were destroyed by Hurricane
Hugo; however, large dunes towards the interior of the island survived
the hurricane. Therefore, using beach profiles of Waites Island taken before
2007 S.P. Bush and M.E. Stelato 361
and after the hurricane, differently aged patches of sea oats were located and
sampled. These included two younger patches (aged less than seven years)
that were situated on foredunes that had developed since the hurricane, and
two older patches located on dunes present prior to the hurricane. While the
exact age of these older patches could not be determined, their large size
suggested an age considerably greater than seven years. All four patches were
less than 1.5 km inland from the shoreline. In each patch, rectangular grids
were sampled by collecting leaf tissue from the closest ramet at each 1.0-m
interval throughout the grid. Samples were placed on ice upon collection, and
later stored at -80 °C until DNA extraction.
DNA was isolated using a standard CTAB protocol (Berntazky and
Tanksley1986), and then further purified using the NucleiClean kit (Sigma,
St. Louis, MO). The DNA concentration of each sample was next quantified
using a DyNA Quant 200 Flourometer (Hoefer, San Francisco, CA). Polymerase
chain reactions were prepared in 12.5-ml volumes that contained: 0.5
units Taq polymerase (Promega, Madison, WI); 1.25 l 10X buffer; 0.25
mM each of dNTP, 0.5-l 20-mM MgCl2, and 0.5-l 10-mM primer; and
12.5 ng of template DNA. Each reaction mixture was then covered with a
drop of mineral oil and amplified with the polymerase chain reaction. Each
of the three initial polymerase chain-reaction cycles consisted of 1 min at
94 °C, 1 min at 35 °C, and 1 min at 72 °C. The remaining 42 cycles were: 5
sec at 94 °C, 1 min at 35 °C, and 2 min at 72 °C. Polymerase chain-reaction
products were subjected to electrophoresis on 1.5% agarose gels and then
stained with ethidium bromide and photographed.
To identify informative polymerase chain-reaction primers for sea oats,
two plants were initially screened with 100 primers of random sequence, and
seven that yielded clear, polymorphic bands were selected for the study
(Operon primers OP-H5 and OP-G18, and University of British Columbia
primers UBC-215, UBC-226, UBC-227, UBC-353, and UBC-363). All 57
individuals were treated with these seven primers, and all polymerase chain
reactions were performed in duplicate. Twelve bands, scored as either
present or absent, were tallied in all individuals. The probability that two
ramets of different genets could, by chance, share the same multilocus
RAPD band phenotype, or the genotype probability (Bush and Mulcahy
1999, Sydes and Peakall 1998), was calculated as:
Pgen = pi,
where pi is, for the genotype at each locus, the observed frequency of the
locus genotype among all 57 plants sampled.
Standard measures of clonal diversity were calculated for each patch,
including G/N (the number of genets/ramet) and the genotypic diversity
(Simpson’s D), which is:
D = 1 - {[ni(ni - 1)]/N(N - 1)]},
where ni is the number of individuals of genet i and N equals the total
number of ramets within the patch. Potential differences in clonal diversity
among the differently aged patches was also assessed using a chi-square test.
362 Southeastern Naturalist Vol. 6, No. 2
Results
The seven random primers yielded 12 loci that were polymorphic in sea
oats. Eight of the twelve loci were polymorphic in two or more patches,
although no loci were variable in patch 1 (Table 1). The overall proportion of
genets distinguishable, or G/N, was 0.40, as 23 multilocus genotypes were
identified out of 57 plants sampled (Table 1). The Pgen values, which represent
the chance that two different genets could randomly share the same multilocus
phenotype, were all less than 5% (mean = 0.74%). Therefore, as each
multilocus genotype was also restricted to a single patch, plants sharing the
same multilocus phenotype could confidently be assigned to the same genet.
Diversity varied greatly among patches, and older patches contained a
higher level of clonal diversity. Little to no diversity was detected in the two
younger-aged patches. Only three genets were distinguished among the 27
ramets sampled in the two young patches (G/N = 0.11), and the mean patch
genotypic diversity (D) was 0.23. The two old patches were much more
diverse, containing a total of 21 genets out of 30 ramets genotyped (G/N =
0.7), with a mean D value of 0.95. The mean number of genets was significantly
higher in older patches than in younger patches (chi-square = 14.5,
d.f. = 3, p < 0.005). Genotypic diversity among all patches was 0.59.
Discussion
A previous study of sea oats examined both geographical genetic variation
and fine-scale clonal diversity (Franks et al. 2004). The estimates of withinpopulation
genetic diversity in U. paniculata were generally comparable to
species with similar life-history traits. However, genetic differentiation among
sea oat populations was higher than most outcrossing species, indicating
moderate restriction of gene flow. Clonal diversity differed widely among the
eight patches studied, as the proportion of genotypes distinguishable with a
patch (G/N) ranged from 0.033 to 0.263. The causes of this variation could not
be determined; however, it was suggested that episodic sexual recruitment was
likely a contributing factor (Franks et al. 2004).
Our estimate of genotypic diversity was similar to that found in the previous
study of sea oats (0.60; Franks et al. 2004). However, overall clonal diversity
among all of the plants that we sampled (G/N = 0.4) was considerably higher
Table 1. Clonal diversity in patches of Uniola paniculata. The percent loci polymorphic in each
patch (P), and the number of ramets sampled (N), the number of genets (or clones) identified
(G), the proportion of genets distinguishable (G/N), and Simpson’s genotypic diversity (D) are
reported for each patch, as well as their mean values in the old and young patches.
P N G G/N D
Young patches
1 0 14.0 1.0 0.07 0.00
2 8 13.0 2.0 0.15 0.46
Mean 13.5 1.5 0.11 0.23
Old patches
3 92 15.0 9.0 0.60 0.99
4 67 15.0 12.0 0.80 0.91
Mean 15.0 10.5 0.70 0.95
2007 S.P. Bush and M.E. Stelato 363
than in the previous study (G/N = 0.11). Furthermore, the average level of
clonal diversity found in the two older patches sampled here (mean G/N = 0.70)
was far greater than has been found previously in any patch of sea oats. Among
studies of clonal plants, the number of loci examined is positively correlated
with the number of clones identified (Ellstrand and Roose 1987). Although the
greater number of polymorphic loci that we utilized may have slightly increased
the level of clonal resolution, it is unlikely to have resulted in the
observed four-fold increase in diversity. Instead, our findings likely reflect
actual differences in population diversity.
Given the limited number of individuals and patches sampled, the conclusions
that can be made regarding diversity and population age in sea oats are
somewhat limited. Processes that are independent of population age may
have, by chance, contributed to the differences in clonal diversity among
younger and older patches. Nevertheless, the higher number of clones in older
patches suggests that, in some cases, sea oat populations may increase in
clonal diversity over time. An initial surge of vegetative reproduction in sea
oats may then be followed by an accumulation of different clones over time.
Genetic diversity in newly formed populations is influenced by the level of
genetic diversity among the initial colonists, as well as by subsequent rates
of vegetative and sexual reproduction. It is likely that the colonization patterns
of sea oat seedlings and plant fragments on newly exposed sands varies.
Instances of rapid colonization of bare sands by sea oat seedlings have been
observed (Ehrenfeld 1990), although the survival of sea oat seedlings in open
areas has also been found to be lower than on vegetated dunes (Franks 2003).
Sea oats are also capable of regeneration via rhizome fragments that are
detached from established plants during storms (Miller et al. 2003). These
fragments are capable of surviving transport via ocean currents, and may then
resume growth when redeposited on a beach. Following colonization by either
seed or rhizome fragments, rapid clonal growth may be favored by the foredune
environment. Sand deposition, which stimulates rhizomatous growth in sea
oats (Wagner 1964), occurs at a high rate on vegetated dunes near the shoreline.
Therefore, whatever the source of colonization, clonal growth likely enables
sea oats to spread quickly over previously unvegetated areas. Young populations
like those sampled in this study may then contain little clonal diversity.
Seedling establishment is likely to increase after sea oat populations
become established, as vegetated dunes create conditions that are suitable for
sexual recruits. The high level of clonal diversity found in the older sampled
patches, suggestive of either sustained or episodic seedling growth, provides
further evidence for the fundamental role of sexual reproduction in maintaining
diversity in sea oats. Genetic diversity is critical for the continued adaptation of
populations to changing environments like the maturing sand dunes. Thus,
whether frequent or uncommon, sexual recruitment likely generates new
genotypes that are important for the persistence of sea oat populations.
Literature Cited
Berntazky, R., and S.D. Tanksley. 1986. Genetics of actin-related sequences in
tomato. Theoretical and Applied Genetics 72:314–321.
364 Southeastern Naturalist Vol. 6, No. 2
Bush, S.P., and D.L. Mulcahy. 1999. The effects of regeneration by fragmentation
upon clonal diversity in the tropical forest shrub Poikilacanthus macranthus:
RAPD results. Molecular Ecology 8:865–870.
Cowles, H.C. 1899. The ecological relations of the vegetation on the sand dunes of
Lake Michigan. Botanical Gazette 27:95–117, 167–202, 281–308, 361–391.
Eckert, E.G., K. Lui, K. Bronson, P. Corradini, and A. Bruneau. 2003. Population
genetic consequences of extreme variation in sexual and clonal reproduction in
an aquatic plant. Molecular Biology 12:331–344.
Ehrenfeld, J.G. 1990. Dynamics and processes of barrier island vegetation. Reviews
in Aquatic Sciences 2:437–480.
Ellstrand, N.C., and M.L. Roose. 1987. Patterns of genotypic diversity in clonal
plant species. American Journal of Botany 74:123–131.
Franks, S.J. 2003. Facilitation in multiple life-history stages: Evidence for nucleated
succession in coastal plants. Plant Ecology 168:1–11.
Franks, S.J., C.L. Richards, E. Gonzales, J.E. Cousins, and J.L. Hamrick. 2004. Multiscale
genetic analysis of Uniola paniculata (Poaceae): A coastal species with a
linear, fragmented distribution. American Journal of Botany 91:1345–1351.
Hamrick, J.L., and M.J.W. Godt. 1996. Effects of life history traits on genetic
diversity in plant species. Philosophical Transactions of the Royal Society of
London, Series B 351:1291–1298.
Kjolner, S., S.M. Sastad, P. Taberlet, and C. Brochmann. 2004. Amplified fragment
length polymorphism versus random amplified polymorphic DNA markers:
Clonal diversity in Saxifraga cernua. Molecular Ecology 13:81–86.
Laing, C.C. 1967. The ecology of Ammophila breviligulata II. Genetic change as a
factor in population decline on stable dunes. American Midland Naturalist
77:495–500.
Macdonald, S.E., and V.C. Lieffers. 1991. Population variation, outcrossing, and
colonization of disturbed areas by Calamagrostis canadensis: Evidence from
allozyme analysis. American Journal of Botany 1991:1123–1129.
Miller, D.L., L. Yager, M. Thetford, and M. Schneider. 2003. Potential use of
Uniola paniculata rhizome fragments for dune restoration. Restoration Ecology
11:359–369.
Prach, K., and P. Pysek. 1994. Clonal plants: What is their role in succession? Folia
Geobotanica Phytotaxonomica 29:307–320.
Prach, K., and P. Pysek. 1999. How do species dominating in succession differ from
others? Journal of Vegetation Science 10:383–392.
Stocklin, J., and E. Baumler. 1996. Seed rain, seedling establishment, and clonal
growth strategies on a glacier foreland. Journal of Vegetation Science 7:45–56.
Sydes, M.A., and R. Peakall. 1998. Extensive clonality in the endangered shrub
Haloragodendron lucasii (Holoragaceae) revealed by allozymes and RAPDs.
Molecular Ecology 7:87–93.
Van de Ven, W.T.G., and R.J. McNicol. 1995. The use of RAPD markers for the
identification of Sitka spruce (Picea sitchensis) clones. Heredity 75:126–132.
Wagner, R.H. 1964. The ecology of Uniola paniculata L. in the dune-strand habitat
of North Carolina. Ecological Monographs 34:79–96.
Widen, B., N. Cronberg, and M. Widen. 1994. Genotypic diversity, molecular
markers and spatial distribution of genets in clonal plants: A literature survey.
Folia Geobotanica Phytotaxonomica 29:245–263.
Woodhouse, W.W., E.D. Seneca, and A.W. Cooper. 1968. Use of sea oats for dune
stabilization in the southeast. Shore and Beach 36:15–21.
Woodhouse, W.W., E.D. Seneca, and S.W. Broome. 1976. Ten years of development
of man-initiated coastal barrier dunes in North Carolina. North Carolina Agricultural
Experiment Station. Bulletin No. 435.