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(American Journal of Botany. 2008;95:1254-1261.) doi: 10.3732/ajb.2007159 © 2008 Botanical Society of America, Inc.

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Comparison of clonal diversity in mountain and Piedmont populations of Trillium cuneatum (Melanthiaceae-Trilliaceae), a forest understory species¹

² Biology Department, Appalachian State University, Boone, North Carolina 28607 USA ³ Plant Biology Department, University of Georgia, Athens, Georgia 30602 USA ⁴ Department of Ecology, Evolution & Natural Resources, Rutgers University, 14 College Farm Road, New Brunswick, New Jersey 08901-8551 USA

Received for publication 22 May 2007. Accepted for publication 14 July 2008.

ABSTRACT

The balance between clonal and sexual reproduction can vary widely among plant populations, and the extent of clonality may be influenced by the combined effects of historical land use and variation in environmental conditions. We investigated patterns of clonal spread in five Trillium cuneatum populations, two in the Appalachian Mountains characterized by mesic, cooler conditions, and three at lower elevations experiencing warmer, drier conditions and greater disturbance. Using a new measure of the genet effective number and innovative orthogonal contrast methods, we quantified genet structure, contrasting clonal growth in the mountains with that in the Piedmont. Asexual propagation was more common in the Piedmont, where 25% of the sampled ramets were clonally derived, but was much less frequent in the mountains (7% clonal replicates). Hierarchical partitioning of variation in genet diversity showed that the majority (75.8%) of the variation resulted from more vegetative replication in the Piedmont. Most of the remaining variation (21.6%) was attributable to differences between urban and rural Piedmont populations, and a small, statistically nonsignificant fraction of the variation (2.6%) was due to interpopulation differences within the mountains. Higher frequency of cloning may enhance both genetic and demographic population viability in fragmented Piedmont habitats.

Key Words: clonal structure • genet diversity • heterogeneous environments • orthogonal contrasts • Southeastern USA • vegetative reproduction • Trillium

Clonal reproduction can have important consequences for ecological and evolutionary processes, particularly in response to heterogeneous environmental conditions. Cloning has been suggested as particularly advantageous in less favorable and geographically marginal environments (Callaghan, 1988; Eckert, 2002; Honnay and Bossuyt, 2005) and in habitats with seasonal fluctuations in environmental conditions and/or continual disturbance (Suzuki and Stuefer, 1998). Clonal reproduction can be viewed as an alternative life cycle trait that prolongs population survival in the absence of successful sexual reproduction. (Honnay and Bossuyt, 2005). Many long-lived, temperate woodland herbs are rhizomatous. These species proliferate by producing genetically identical ramets, frequently associated with sporadic sexual recruitment (e.g., Cook, 1979; Whigham, 1974; Eckert, 1999). The relative importance of sexual vs. clonal recruitment varies not only among species, but among populations within species, yet there are very few studies that quantify intraspecific variation between the two modes of reproduction (Eckert, 2002).

Although there might be a long term evolutionary cost, if prolonged clonal growth leads to the extinction of sexuality (e.g., Dorken et al., 2004), there are compensating benefits. Besides providing a buffer against environmental and demographic stochasticity, clonally propagating populations may retard the loss of genetic diversity, thus maintaining evolutionary potential for adaptation to spatiotemporal habitat heterogeneity (Orive, 1993; Young et al., 1996; Honnay and Bossuyt, 2005). Vegetative spread affects the spatial distribution of phenotypic and genetic variation within populations, ultimately shaping their evolution (Eckert, 1999). Not only does natural selection act on local phenotypes, but clonal growth patterns may have complex consequences for plant mating patterns (Handel, 1985; Vuorisalo et al., 1997). Effective population size affects the rate of genetic drift, levels of inbreeding, and the ability of populations to respond to environmental change. Failure to account for vegetative spread can lead to substantial overestimates of the effective population size, N_e (Chambers, 1995; Chung et al., 2004).

This study investigates patterns of clonal reproduction within five populations of a rhizomatous woodland species, Trillium cuneatum Raf. (Trilliaceae sensu Dahlgren et al. [1985], or the Melanthiaceae sensu APG [1998]), a long-lived spring ephemeral that often carpets the floors of mature mesic deciduous forests, both in large continuous mountainous habitats and fragmented remnants in the Piedmont in the eastern USA. This investigation was motivated by a larger study of patterns of genetic diversity within and among natural populations. Standard population genetics theory predicts reduced genetic diversity in small, fragmented populations, but we have not seen erosion of genetic variation in the fragmented Piedmont habitats of this species (Gonzales and Hamrick, 2005). These results combined with field observations of spatial clusters, including physically connected branching rhizomes (Fig. 1), typically absent in the mountains, suggest that clonality may reduce the negative effects of forest fragmentation on population genetic diversity.

View larger version (118K): [in this window] [in a new window]   Fig. 1. Photograph of branching rhizome of Trillium cuneatum. The oldest portion of a rhizome decays with age, while the two branches continue to grow, eventually giving rise to independent, disconnected ramets.

MATERIALS AND METHODS

Study species
Trillium cuneatum is native to deciduous forests of SE North America (Fig. 2). Plants require 10 or more years to reach reproductive maturity (Ohara, 1989), and even then, successful fruit production is infrequent. However, once established from seeds, individuals may persist for many decades. In March and April, a plant produces a single flower (rarely two), pollinated by weakly flying insects such as fruit flies, Drosophila spp. (Drosophilidae) (E. Gonzales, personal observation; D. Promislow, University of Georgia, personal communication) or occasionally by blow flies (Calliphoridae). Preliminary greenhouse pollination experiments suggest that T. cuneatum is a predominantly outcrossing species with a leaky self-incompatible system (E. Gonzales, unpublished data). This finding is in accord with previously published studies that indicate low levels of inbreeding in natural populations of T. cuneatum (Gonzales and Hamrick, 2005; Gonzales et al., 2006). Ohara (1989, p. 12) reported that T. cuneatum "reproduce exclusively by seeds," but elsewhere in the same report, he mentions a rare possibility for clonal reproduction. Vegetative spread has been documented for T. cuneatum under cultivation (Jacobs and Jacobs, 1997), and similar vegetative spread by rhizomes transplanted into the greenhouse from natural populations has been observed (E. Gonzales, unpublished data).

View larger version (34K): [in this window] [in a new window]   Fig. 2. Geographic distribution of Trillium cuneatum populations, indicated by the dark-gray area. The two mountain populations (black dots; GRM: 34°51'N, 84°39'W, JKMF: 35°21'N 83°56'W) are located in the southern Appalachian Mountains at 1000–1200 m a.s.l. The three Piedmont populations (white dots; ATL1: 33°47'N 84°22'W, ATL: 33°48'N 84°19'W, TMF: 34°05'N 83°51'W) are found at elevations of 280–300 m a.s.l.

Two Piedmont populations (ATL1, ATL2) occupying small forest fragments, completely surrounded by urban development, and subjected to ongoing human disturbance, were located in Atlanta, Georgia. Although these populations occur in small remnants of mature, secondary forest, the Trillium populations themselves are not small; each site contains several thousand ramets. The third Piedmont population, Thompson Mill Experimental Forest (TMF) is less affected by anthropogenic disturbance, though it is subject to heavy deer browsing. Plants occur mostly along the margins of this mature, secondary growth deciduous forest (130 ha), surrounded by pastures. The two sites in the Appalachian Mountains of northern Georgia and western North Carolina occupy cooler, more mesic habitats; they consist of tens of thousands of individuals, spread over hundreds of hectares of continuous hardwood forest. The Grassy Mountain (GRM) population is in relatively undisturbed Chattahoochee National Forest, Georgia. The wilderness area of the Joyce Kilmer Memorial Forest (JKMF), part of the Nantahala National Forest in western North Carolina, contains the fifth population.

Sampling design
We employed a nested design where populations serve as replicates within population type (i.e., region), and plots serve as replicates within populations. However, our sampling design is not completely balanced, because some plots were originally established to study contemporary pollen dispersal, in which only flowering ramets were sampled (Table 1). We established 12 plots in these five populations, sampling a total of 2617 plants (Table 1). Each population is represented by at least two rectangular sampling plots (minimum size: 10 x 16 m, maximum size: 18 x 26 m). Plot size varied, due to different plant densities. Plots were separated by at least 100 m within the Piedmont populations, and by larger distances (>300 m) in the mountain sites. Spatial (x, y) coordinates were recorded for all plants within each plot. All mapped plants were sampled for allozyme analyses. Voucher specimens are available from the University of Georgia Herbarium (GA) (accessions 241236-9, 241246-47, 241249-51, and 241253-55).

Genetic resolution
The ability to resolve clonal ramets is a function of the number of polymorphic loci and population level genetic diversity at each locus. The level of assayable genetic variation determines the overall mismatch probability (i.e., the likelihood by Mendelian chance of a second sample having a different diploid genotype from a focal individual). We calculated the multilocus mismatch probability as the complement to the probability of identity P_I (adapted from the single-locus formula in Paetkau and Strobeck, 1994, p. 490), using independently determined allele frequencies based on sample sizes of 48 randomly chosen ramets (Gonzales and Hamrick, 2005; E. Gonzales and J. Hamrick, unpublished manuscript, available from E. Gonzales) from each population and assuming multiple-locus Hardy–Weinberg equilibrium. For any given population and for the l-th locus with J alleles, the probability of identity is

(1A)

where p_i and p_j represent allele frequencies for the locus. Multiplying Eq. 1A across L loci provides a multilocus Mendelian match rate (probability of identity P_I), the probability of drawing two sexually produced individuals at random with the same genotype,

(1B)

The probability of a match provides an overall measure of the potential usefulness of the genetic battery in distinguishing among sexually produced individuals. In our study, the probability of identity in each of our five populations was less than 0.01, resulting in a mismatch probability >0.99. While very high, the P_I values are all less than 1; accordingly, we should find a low proportion of matched pairs, even from sexual reproduction. When, however, matching genotypes are tightly clumped, we considered them to be clonally derived.

We recorded the total number of sampled ramets (N) and the number of unique multilocus genotypes (G). We estimated clonal size (n) as the number of proximal ramets with identical multilocus genotypes. We measured spatial clonal spread (d) as the maximum physical distance between ramets of the same genet. We treated genetic matches as ramets belonging to the same clone if the maximum distance between them was less than 50 cm, but treated genotypic matches of more distant ramets (>50 cm apart) as independent sexual genets. This arbitrary (and generous) 50-cm threshold was based on field observations of excavated clonal clusters (Fig. 1) and observed rhizome growth rates (E. Gonzales, personal observation). The numbers of all distinguishable genotypes (G) and number of genets adjusted for recurrences of identical genotypes based on the 50-cm criterion (G_Z) are available from Table 1.

Genet richness and diversity
We measured genet richness as the number of genets (G_Z), divided by the number of samples in each plot (N). We measured genet diversity (D) as the complement to Simpson's index, corrected for finite sample size (Simpson, 1949),

(2)

where n_g is the number of replicates of the g-th genet and N is the total sample size of the plot. Values of D range between 0 and 1, with higher values representing greater genet diversity and lower numerical dominance of any particular genet in the sample.

We defined a frequency-weighted measure, G_Ze, the multilocus genet diversity available within the population, as

(3)

This G_Ze index, which we label the effective genet number, ranges from one (in a population containing one genet) to infinity (where each individual is a separate, sexually produced genet). The index G_Ze can be viewed as an equivalent number of equally replicated genets that would yield the value of D encountered in the data from the population. Additionally, we computed hierarchical F statistics, according to Weir and Cockerham (1984), using the program ARLEQUIN, version 3.01 (Excoffier et al., 2005). See Supplemental Data accompanying the online version of this article for these values (Appendix S1, S2).

Orthogonal contrasts
Based on our nested design, we constructed a set of 11 orthogonal (independent) contrasts to evaluate differences in genet diversity between geographic regions, among populations within regions, and among plots within locations. (For the table of orthogonal coefficients, see Appendix S3 in online Supplemental Data.) Orthogonal contrasts are particularly appropriate for answering a specific set of a priori questions about the habitats under examination. The first contrast compares regional genet diversity between mountain (JKMF and GRM) and Piedmont (TMF, ATL1 and ATL2) populations. The second compares the two mountain (JKMF and GRM) populations. The third compares the rural (heavily deer-browsed) Piedmont population (TMF) with the two urban (human-disturbed) sites (ATL1 and ATL2). The fourth contrast compares the two urban sites, ATL1 with ATL2. The remaining seven contrasts are comparisons of plots within each population, a measure of local replication variation within specific habitats (Appendix S3).

Our test criterion of choice is a modification of t tests described by Keefe and Bergersen (1977, second equation, p. 690), who suggested that to compare two Simpson indices, D_i and D_j, one should use a measure of approximate t test form:

(4)

where and are estimated variances of D_i and D_j, respectively. We have constructed linear combinations of the D values of different plots for each contrast:

(5A)

(5B)

where the w values represent orthogonal contrast coefficients (For more information, see online Appendix S3.). For example, the first contrast, the comparison of the four mountain plots with the eight Piedmont plots, uses

(6)

The corresponding estimated variances for these particular linear combinations of D-values are (for this same mountain [MN] vs. Piedmont [PD] contrast):

(7A)

(7B)

where the estimated variance for any particular plot (e.g., the k-th) was computed as in Nei (1987, eq. 8.12), but in our terminology

(8)

N_k is the sample size of the k-th plot, D_k is defined as in Eq. 2, and p_kg is the parametric frequency of the g-th genet in the k-th plot. We use a sample-size-corrected estimate for the summation term,

(9)

where n_kg is the number of ramets of the g-th genet in the k-th plot.

Because sample sizes (and degrees of freedom) are large, each single contrast t value is comparable to a normally distributed z variable. We note that a squared normal deviate is ² (df = 1), and the ²values from orthogonal contrasts are additive. With additivity, we can partition the total variation in genet diversity among plots into independent components that are a priori sensible for the problem, allowing us to highlight the major factors contributing to differences in genet diversity. We conducted 11 independent tests, and to avoid inflating type I error rates, we assessed statistical significance via sequential Bonferroni correction, with an = 0.05 experiment-wide error rate (Holm, 1979; Rice, 1989).

RESULTS

Overall quantification of clonality
We found clonality in all T. cuneatum populations, though a high proportion of the ramets were produced sexually; 60.4–92.9% had unique multilocus genotypes. Clonal clusters were spatially restricted; the overall average distance of vegetative spread was 13 cm. We detected vegetative reproduction in the Piedmont habitats more frequently than in the mountain habitats. We also saw more plot-to-plot variation within the Piedmont populations, while clonal reproduction was quite uniform, both within and between mountain populations. Mean genet richness was greater in the mountains (G_Z[MN] ÷ N_MN = 0.934, range 0.919–0.954), than in the Piedmont (G_Z[PD] ÷ N_PD = 0.751, range 0.620 – 0.861) (P = 0.004, Mann–Whitney test). The greater range of variation in genet richness in the Piedmont reflects greater variation in the frequency of clonal reproduction (Table 1).

Regional differences
The two geographic regions also differed substantially in the number of ramets per genet and the frequency of vegetative replication (Fig. 3). Mountain populations rarely contained a genet composed of more than two ramets, though we recorded one genet with five ramets. In Piedmont populations, however, we encountered clonal genets more frequently, and the clonal clusters were composed of more ramets, with a maximum of n_PD = 23 ramets/genet (Table 1). However, Piedmont clusters were somewhat more spatially restricted (mean d_PD = 12.2 cm, SD = 5.10 cm) than were those in the mountains (mean d_MN = 13.9 cm, SD = 3.11 cm).

View larger version (13K): [in this window] [in a new window]   Fig. 3. Differences in Trillium cuneatum clonal growth between the two geographic regions (southern Appalachian Mountains and Piedmont). All samples within each region were pooled for comparison (NMN = 938, NPD = 1681). Plants in the Piedmont spread vegetatively with greater frequency, forming clumps, while clonal reproduction in the mountains is rare, so that large clumps of genetically identical ramets are absent.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Partitioning of population genet diversity (D) indicates that most of the differences are between regions (Mountains vs. Piedmont). There are statistically significant differences among populations located in rural Georgia (affected by deer disturbance) vs. Atlanta (affected by human disturbance). Remaining variation between mountain populations is not statistically significant, and variation between the two Atlanta populations is zero.

Although we encountered a trend toward greater plot-to-plot variation within the Piedmont, relative to the mountains, these differences remained nonsignificant in three of five comparisons, after sequential Bonferroni correction (Table 2). Tests that showed significant differences were: ATL2-1 (plants of all life stages included) vs. ATL2-3 (only flowering plants sampled) (P < 0.01) and one comparing complete plots within ATL1 (P < 0.04).

Hierarchical partitioning of variation in genet diversity between and within regions, based on ²values (Table 2), demonstrated that the majority (75.8%) of the variation is from differences in clonality between the Piedmont and mountain regions. Most of the remaining variation (21.6%) was due to urban vs. rural differences in the Piedmont, while no significant divergence of genet diversity was observed between the two urban populations. A small, statistically nonsignificant fraction (2.6%) was attributable to population differences within the mountain region.

DISCUSSION

The major objective of this study was to provide a quantitative assessment of among-population variation in Trillium cuneatum clonality in different habitats. Our results revealed clonal reproduction in all five sites, but relatively high levels of genet richness (G ÷ N = 0.60–0.93) indicate continuing recruitment of sexually produced genets in all populations. Our observations are consistent with evidence showing that populations of clonal species often consist of multiple genets (e.g., Ellstrand and Roose, 1987; Kudoh et al., 1999; Brzosko et al., 2002; Franks et al., 2004). The interesting point is that the relative balance of vegetative spread and sexual reproduction in T. cuneatum populations varied with habitat type. These results also explain a previous report of T. cuneatum as a nonclonal species by Ohara (1989), who examined a mountain population (approximate elevation 1100 m a.s.l.) and did not observe clonal spread.

Although spatial clustering of identical multilocus genotypes could be caused by predominant self-pollination combined with lack of seed dispersal, rather than clonal spread, this scenario cannot explain our results. Previous investigations demonstrated that natural populations of this largely outcrossing species undergo low levels of inbreeding (Gonzales and Hamrick, 2005; Gonzales et al., 2006; see also online Appendix S2 with this study). Interestingly, Piedmont populations with spatial clustering of identical genotypes have slightly lower levels of inbreeding than do the less clonal mountain populations. This observation is further corroborated by our pollen dispersal study (Gonzales et al., 2006); we reported greater contemporary pollen movement, higher effective number of pollen donors/fruit, and higher fruit production in the same Piedmont populations, though seedling recruitment was lower (14% of genets were seedlings in the Piedmont, 22% of genets in the mountains; E. Gonzales, unpublished data).

Regional differences
We detected statistically significant regional differences in clonal reproduction between Piedmont and mountain populations. Asexual reproduction was more common in the Piedmont, where 25% of the sampled ramets were clonally derived, while vegetative spread was much less frequent in the mountain sites (7% clonal replicates). Relatively higher clonal recruitment may be a result of environmental factors impeding seedling establishment. Indeed, we observed less seedling recruitment in the Piedmont, in spite of higher fruit and seed set (Gonzales et al., 2006). Although decreased sexual recruitment probably results from different ecological conditions (producing increased seed desiccation and competition with ground covering invasives such as Hedera helix and Lonicera japonica) (Jacobs and Jacobs, 1997; E Gonzales, personal observation), this is not sufficient to explain regional differences in vegetative reproduction. Whereas genets with more than two ramets are rarely encountered in mountain populations, clones form multiramet clumps in the Piedmont (Fig. 3).

The discrepancy in reproductive strategies is even more evident from comparisons of effective genet number, G_Ze(MN) = 1405 vs. G_Ze(PD) = 277. It would require nearly five times as many equally replicated genets (i.e., clones of equal size n) in Piedmont populations to achieve the same level of genet diversity encountered in mountain populations. Such differences can have long-term implications for effective population size in populations with mixed reproductive strategies. Although estimates of effective population size were not the prime focus of this paper, our results agree with theoretical predictions that high rates of asexual reproduction should lower the N_e to N ratio and lower effective population size (Orive, 1993; Balloux et al., 2003).

Differences within regions
Only a small (<3%), portion of the variation in D was attributable to divergence between the two mountain populations, but there is considerable heterogeneity in genet diversity between the rural Piedmont population and the urban Piedmont sites (22% of the total ², P < 0.001). Additionally, one Piedmont plot (ATL1-1) is unusual; distances greater than 50 cm separate a disproportionate number of identical genotypes. In fact, we identified matching genotypes separated by distances up to 8.5 m. Although we have no historical information on this plot, it seems probable that physical disturbance (by humans or foraging animals) may have scattered rhizomes in this area. Lacking detailed historical information, we cannot resolve the situation compellingly, but we view the spatial pattern of ATL1-1 as "atypical." The other significant contrast within urban locations is between the "flowering" plot (ATL2-3) and plots where all plants were sampled. It is not surprising that a disproportionate number of clonal ramets were nonflowering. A clonal ramet is often small while physically connected to its "maternal" plant, forming a branched rhizome and giving rise to several aboveground stems. A clonal rhizome does not support more than a few flowers during a single reproductive bout. By comparison, however, the "flowering" vs. "all" plots contrast in the mountains is small and nonsignificant, probably because vegetative spread in the mountains is so infrequent that excluding nonflowering ramets makes essentially no difference.

Ecological and evolutionary implications
Trillium cuneatum occupies deciduous forests of the southeastern United States, where there is a strong elevational and climatic gradient, from cool, moist conditions in the mountains to drier and warmer conditions in the Piedmont. Species distributed across this climatic gradient and exposed to temporal abiotic heterogeneity may have evolved adaptations to their local environments (Etterson, 2004; Jacquemyn et al., 2005). Piedmont populations experience more environmental stress (e.g., heat, drought, seasonal flooding, human disturbance, heavy deer browsing), while mountain habitats are more mesic with cooler temperatures, fewer droughts, and considerably less anthropogenic disturbance. Similarly, R. Pulliam, University of Georgia, (personal communication) in a study of a sympatric forest herbaceous perennial species, Polygonatum biflorum, detected significantly more vegetative reproduction in his Piedmont sites than in the more mesic, cooler, and more stable mountain sites. Divergent environmental conditions may have contributed to life-history differentiation between mountain and Piedmont populations of T. cuneatum.

Several studies have investigated clonal reproduction in natural populations of perennial herbaceous species (e.g., Cheplick and Gutierrez, 2000; Brzosko et al., 2002), but little is known about the processes that determine intraspecific variation in asexual reproduction, either within or among sites. In some species, vegetative spread is associated with stressful environmental conditions and is interpreted as an important adaptation for survival under unfavorable conditions (e.g., Tybjerg and Vestergaard, 1992; Klime et al., 1997). Jacquemyn et al. (2005) investigated variation in clonal diversity of a forest understory herb, Paris quadrifolia, a close relative of Trillium. Consistent with our results, they found fewer genotypes and clumped ramets in dry, relatively unproductive sites, whereas moist sites contained more genotypes, fewer clumped plants, and higher genet diversity. Kudoh et al. (1999) investigated genet structure in gap and closed canopy habitats and attributed a significant role to light availability and forest canopy disturbance in shaping clonal structure of a forest understory herb, Uvularia perfoliata. There are also biotic factors that may affect the propensity to clone. For instance, Marshall (1990) and Evans and Cain (1995) have documented reduced vegetative growth under interspecific competition, presumably due to resource limitations. The two urban Piedmont populations have fewer Trillium congeners and other herbaceous species, while the forest understory in the rural Piedmont forest has intermediate richness of herbaceous flora, and both mountain populations are species rich (including five Trillium congeners), supporting the argument that inter-specific competition may reduce vegetative spread in T. cuneatum populations.

Differences in clonal reproduction may also reflect different land-use histories and habitat changes, whether natural or human-induced. European settlers largely deforested the Piedmont, converting previously contiguous ecosystems into an archipelago-like landscape with small natural habitats embedded in an agricultural and urban matrix. Forest habitats were exposed to edge effects, further magnifying environmental differences between Piedmont and mountain habitats, where disturbance is less severe. While cloning is infrequent in relatively undisturbed, and species-rich mountain habitats, the rural Piedmont population (TMF) subjected to deer browsing has intermediate levels of clonal reproduction. In contrast, both Atlanta populations are species depauperate, subjected to severe edge effects, ongoing human trampling and trash, as well as pressure from encroaching invasive vines and shrubs. These urban populations have the greatest frequency of clonal spread, lower genet richness and diversity, and largest clone size. Although we cannot clearly differentiate between the confounding anthropogenic and natural factors affecting T. cuneatum clonality, greater disturbance appears to be associated with increased vegetative spread.

Despite the overall large sample sizes representing each habitat type analyzed in our study, some interpretive caution is in order, due to the relatively small number of populations sampled within each region. Taken at face value, the results demonstrate that there are large differences in clonal reproduction between the two physiographic regions. Our results are consistent with other studies (e.g., Jacquemyn et al., 2005; Pluess and Stocklin, 2005) suggesting that the greater fluctuations and environmental conditions that characterize the Piedmont region, further magnified by edge effects due to fragmentation, probably contribute to its higher clonal frequency and the trend toward greater plot-to-plot and location-to-location variation in genet diversity.

Data collected in natural populations provide no direct evidence for the phenotypic plasticity or evolutionary adaptation hypotheses; our study was not designed to investigate processes causing vegetative spread. Both genetic differences in reproductive mode and/or plasticity could account for our observations. However, we have observed that rhizomes replanted from Piedmont populations into the greenhouse produced clonal ramets, whereas rhizomes transplanted from the mountains did not branch, suggesting potential variation in cloning ability. We show that all populations contain plants capable of vegetative reproduction, but environmental pressures, exacerbated by recent anthropogenic disturbance in Piedmont habitats, may have increased the frequency of genotypes that do so more readily. A clear separation of heritable and environmentally plastic differences will require experimental manipulation of multiple genets from different source populations. Regardless of whether population differences in clonal reproduction represent environmentally plastic or long-term evolutionary responses, they contribute to the organism’s ability to maintain both genetic and demographic viability. Such adaptation would function as fortuitous in fragmented populations that occur primarily in the Piedmont. This may explain, in part, why the fragmented and highly disturbed Piedmont populations have maintained levels of genetic diversity equivalent to the more continuous mountain populations (Gonzales and Hamrick, 2005; Gonzales and Hamrick, unpublished manuscript).

FOOTNOTES

¹ The authors wish to thank R. Pulliam for helpful comments on the manuscript and for sharing his unpublished weather data. M. Arnold, J. Avise, S. M. Chang and J. J. Robledo reviewed previous versions of this manuscript. They also greatly appreciate field and laboratory assistance by C. Deen, L. Kappa, S. Conrad, and K. and T. Bercikova. This research was supported by funds from the Department of Plant Biology Palfrey Fund to E.G., by NSF-DEB0211526 to J.L.H. and by NSF-GRS0327147 to J.L.H. and E.G. P.E.S. was funded by NJAES/USDA-17111 and by NSF-DEB-0211430.

⁵ Author for correspondence: (e-mail: gonzaleseb{at}appstate.edu)

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