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Population genetic diversity of the clonal plant Geum reptans (Rosaceae) in the Swiss Alps¹

Institute of Botany, University of Basel, Schoenbeinstr. 6, CH-4056 Basel, Switzerland

Received for publication January 13, 2004. Accepted for publication August 26, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
In the alpine landscape most plant populations are spatially isolated due to extreme patchiness and strong natural fragmentation. We used RAPD-PCR (randomly amplified polymorphic DNA polymerase chain reaction) for a study of the genetic diversity within and among 20 populations of Geum reptans, an outcrossing clonal plant species in the Swiss Alps. Populations were sampled at different altitudes, in early-, medium- and late-successional habitats (population origin) using a spatially hierarchical design, with distances among populations ranging from 0.2 to 208 km. Seed and pollen dispersibility was estimated by direct measurements. Seed dispersibility by wind was low with only 0.015% of the seeds flying over 100 m. Observed pollen flow was even more restricted. Molecular diversity within populations was irrespective of population origin (H_e = 0.22 ± 0.004) and similar to the average of other RAPD studies. Contrary to our expectation, populations were only moderately differentiated (G_st = 0.14). However, there was a clear spatial genetic structure and a positive relationship between pairwise genetic and geographic distances. Our results indicate considerable gene flow among populations within the same regional area, and we found no indication for genetic depletion during succession or in peripheral habitats. We conclude that, despite the high natural fragmentation and the importance of vegetative reproduction in this alpine plant, gene flow and repeated seedling recruitment during succession might be more frequent than commonly suggested.

Key Words: altitude • Geum reptans • molecular diversity • pioneer plant species • RAPD • Rosaceae • successional habitats

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Alpine plant life is characterized by habitats with steep environmental gradients, sharp boundaries, strong natural fragmentation, and high disturbance frequency. As growth conditions are impaired by hostile climatic conditions, establishment from seeds is restricted to safe sites (Urbanska and Schütz, 1986 ). A high proportion of alpine plants are characterized by clonal growth even in pioneer communities on glacier foreland, alpine screes, or moraines (Hartmann, 1957 ; Gray, 1993 ; Stöcklin and Bäumler, 1996 ; Klimes et al., 1997 ). Clonality and spatial isolation of populations may lower genetic diversity within and increase genetic separation among populations (Barrett and Kohn, 1991 ; McLellan et al., 1997 ; Gaudeul et al., 2000 ; Landergott et al., 2001 ; Cheon et al., 2002 ; Despres et al., 2002 ). So far, however, molecular studies in alpine plants mostly considered phylogeny or the migration of species from refuge sites after the ice ages, concentrating on the molecular patterns created by historical gene flow (e.g., Bauert et al., 1998 ; Holderegger et al., 2002 ; Stehlik, 2002 ; Tribsch et al., 2002 ). The consequence of the island nature of alpine vegetation for actual gene flow is fairly unknown. Direct measurements of pollen movement and seed dispersal tend to underestimate the importance of long-distance dispersal for the movement of genes (Ouborg et al., 1999 ; Cain et al., 2000 ). Thus, indirect measurements using molecular markers are needed for a better understanding of genetic diversity patterns in fragmented alpine environments. The genetic consequences of clonality in long-lived plants are also poorly known (McLellan et al., 1997 ).

Plant molecular studies have shown that fragmentation of habitats and small population size may negatively affect population genetic diversity (Ellstrand and Elam, 1993 ; Fenster and Dudash, 1994 ; Fischer and Matthies, 1998 ; Luijten et al., 2000 ; Paschke et al., 2002 ). The genetically less diverse populations have a reduced ability to buffer the effects of poor environmental conditions or competition (e.g., Fischer et al., 2000 ; Pluess and Stöcklin, 2004 ). The fragmented alpine landscape might particularly affect the partitioning of genetic diversity among populations and strong selection forces under the harsh environmental conditions might strengthen population differentiation. In general, populations of clonal plants exhibit considerable levels of genetic diversity (Ellstrand and Roose, 1987 ; Parker and Hamrick, 1992 ; Widen et al., 1994 ; Hamrick and Godt, 1997 ). It has been suggested that this is also true for long-lived clonal plants from alpine habitats (Steinger et al., 1996 ; Diggle et al., 1998 ; Holderegger et al., 2002 ). However, in a recent review by Till-Bottraud and Gaudeul (2002) , only two studies of alpine plants (Gugerli et al., 1999 ; Jones and Gliddon, 1999 ) were available with a suitable sampling design to study the pattern of genetic diversity within and among populations accurately, i.e., comprising at least 10 populations and 20 individuals per population.

We selected a clonal plant for our study because vegetative reproduction is one of the most noticeable adaptations to severe environments and nutrient poorness in cold habitats (Callaghan, 1988 ; Klimes et al., 1997 ). Clonal growth has benefits, like the ability to forage for resources, to support the establishment of offspring, or to minimize the mortality risk of a genet. On the other hand, there are costs, like the easy transmission of diseases or a reduced availability of resources for sexual reproduction (Jackson et al., 1985 ; Callaghan et al., 1992 ; Klimes et al., 1997 ). It is generally assumed that reproduction from seeds is infrequent in clonal plants, and this might be particularly true for plants from cold environments (see review in Eriksson, 1989 ). If no repeated seedling recruitment takes place after colonization, differences in the success of particular genets and random processes are expected to decrease genetic diversity over time (Soane and Watkinson, 1979 ; Watkinson and Powell, 1993 ). Moreover, clonal growth may act as an enhancer of genetic drift by reducing the effective size of local populations (Chung and Kang, 1996 ; Jones and Gliddon, 1999 ). However, simulation models have shown that even rare establishment from seeds is sufficient to maintain genetic diversity in long-lived clonal plants (Watkinson and Powell, 1993 ).

The assumption that clonal reproduction is necessarily correlated with a reduced level of reproduction by seeds must not always be true (Eriksson, 1989 ; Stöcklin and Bäumler, 1996 ). Well-established populations of clonal species may be an important source of seeds for the colonization of nearby patches or unoccupied habitats. Eriksson (1992) even suggested that clonal growth may have been selected more frequently in lineages with seeds adapted for long-distance dispersal. However, extremely localized dispersal is common in many plants (Freckleton and Watkinson, 2002 ) and short-range dispersal might be expected to dominate the colonization processes. Less isolated habitats are more likely to be colonized than are more isolated ones (Harrison et al., 2000 ), which may result in a spatial genetic structure with nearby populations being more related than more distant ones. Even in alpine species with good dispersal mechanisms, populations may tend to be genetically aggregated because potential habitats are isolated from each other and colonizations from nearby sites are more likely than random dispersal events.

Here, we focus on the genetic diversity within and among populations of the clonal pioneer Geum reptans L. This species occurs mainly on glacier forelands and is able to persist until later successional stages. Population increase of this species is rapid by vegetative offspring produced at the end of stolons. At the same time the plant produces plenty of seeds which are wind-dispersed by conspicuously elongated, feathery styles on the nutlets (Rusterholz et al., 1993 ). We studied the genetic structure of 20 populations in the Swiss Alps within a core area on two nearby glacier forelands, in a regional area surrounding this core area, and from three sites distant to the core area (Fig. 1). Furthermore, we measured gene flow via seeds and pollen directly in the field for a comparison with the molecular data. Populations occur on a gradient from early- to late-successional communities and at different altitudes. We hypothesize, that (1) the genetic diversity in populations of this clonal pioneer plant is high, but, because of the fragmentation of the alpine landscape, we expect populations to be genetically structured in space. (2) Genetic variation is higher in populations from early-successional communities than in populations of later successional stages due to high seedling recruitment in early succession and loss of genotypes due to competition pressure in later succession. (3) Populations from lowest and highest altitudes are genetically less diverse than populations from medium altitudes, because environmental constraints at the elevational distribution boundaries of a species are expected to reduce the number of successful genotypes (Lesica and Allendorf, 1995 , and references therein). (4) Based on direct estimates in the field, the potential of gene flow via seeds is larger than via pollen.

View larger version (30K): [in this window] [in a new window]   Fig. 1. Geographic distribution of the studied populations of the alpine plant Geum reptans in Switzerland. Populations are grouped into a core area (including SCL, SCE, GR4, GR2, and GR1), in a regional area (enlarged map section, including 17 populations) and three populations at great distance to the core area

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The plant species
The alpine pioneer Geum reptans L. (Rosaceae) is a hemicryptophytic outcrossing perennial plant. The distribution of the species extends from the Alps to the Carpathians, the Illyric mountains, and Macedonia at altitudes between 1950 and 3800 m above sea level (a.s.l.) (Hegi, 1995 ). Geum reptans preferentially occurs on virgin soils of early-successional habitats on glacier forelands, moraines, mountain ridges, and river beds with a low chalk content and persists in such sites until later successional herbaceous communities (Hegi, 1995 ), but is absent from closed grasslands (Rusterholz et al., 1993 ).

Individuals consist of 1–7 rosettes (rarely more). Age at first reproduction, measured by counting growth rings in the main root of small reproducing individuals, was 5 yr in late- and 10 yr in early-successional communities and maximum age, observed in very large individuals, was 30 yr (A. Pluess and J. Stöcklin, personal observation). Adults reproduce vegetatively by forming new rosettes at the end of aboveground stolons or sexually by seeds borne on a single-flowered stem. Buds are preformed in autumn and emerge in the following spring. Flowers are proterogynous, pollinated by inseccts and produce approximately 100 seeds (T. Weppler and J. Stöcklin, unpublished data). Viable seeds are only produced by outcrossing, indicating self-incompatibility (Rusterholz et al., 1993 ). After pollination, the style develops into an elongated feathery structure of up to 3 cm length, which facilitates dispersal by wind. Stolons grow to a length of up to 100 cm with a terminal rosette with adventive roots. At the end of the growing season, stolons wither and unrooted rosettes die.

Sampling design
We sampled all available populations (N = 5) in a core area of 0.55 x 2.8 km, which included the foreland of two glaciers (Scaletta glacier and Grialetsch glacier near Davos in the eastern part of the Swiss Alps), and all available populations in a regional area of 25 x 54 km (+12 populations; Fig. 1; Tables 1, 3). To include populations more distant to the core area, we sampled three additional populations: two populations from the Central Alps (Muttgletscher and Blauberg; 120 km distant from the core area) and one population in the Bernese Alps (Fluhseeli; 200 km distant from the core area). In all but one population, we collected tissue of young leaves from 20 randomly chosen individuals along a transect of 100 m in autumn 2001. To keep the risk of resampling the same clone low, spacing between individuals was at least 4 m. In the population at Flüela Schwarzhorn, only 16 individuals with a minimum spacing of 4 m were available. Leaf material was dried with silica gel and stored at room temperature until analysis.

RAPD-PCR
Of each individual, 20 mg dry leaf material was grounded (Retsch MM2, Retsch GmbH & Co KG, Haan, Germany) for the extraction of total DNA with a DNeasy plant mini kit (Qiagen GmbH, Hilden, Germany). DNA concentrations were determined by fluometry (Turner Design, Sunnyvale, California, USA) with PicoGreen dsDNA quantitation reagent (Molecular Probes Inc., Eugene, Oregon, USA). From 21 decamer primers (Kit P Operon Technologies Inc., Alameda, California, USA and M-6 Microsynth, Balgach, Switzerland), five were selected for the complete survey (OPP-8 [ACA TCG CCC A]; OPP-9 [GTG GTC CGC A]; OPP-17 [TGA CCC GCC T]; OPP-19 [GGG AAG GAC A]; M-6 [GTG GGC TGA C]) after a detailed preliminary screening with three individuals each from four populations. MgCl₂ concentration was optimized for each primer. Reproducibility of RAPD banding pattern was tested with timely repeated amplifications of the 12 individuals and confirmed with consistent amplifications. Furthermore one individual was used as a standard marker, beside the 1 kb ladder, for scoring bands and to confirm consistent amplifications during the whole study. Amplifications were carried out in 25-µL reaction mixture containing 3 ng of template DNA, 100 µM dNTPs, 0.2 µmol/L primer, 1x Taq Polymerase Buffer (Amersham Pharmacia Biotech, Piscataway, New Jersey, USA), additional 0.5 mmol/L MgCl for the primers OPP-17, OPP-19, and M-6, and 1 U Taq DNA Polymerase (Amersham Pharmacia Biotech, Piscataway, New Jersey, USA). To endorse consistency in the PCRs, we kept aliquots of a single master mix per two primers for all samples only adding primer, Taq Polymerase, and DNA before PCR. All PCRs were performed in the same thermal cycler (PTC-100, MJ Research, Inc., Watertown, Massachusetts, USA) programmed for 60 s at 93°C to denature the DNA followed by 34 cycles of 30 s at 92°C, 30 s at 37°C and 90 s at 72°C. Final extension lasted for 5 min at 72°C. Samples were kept at 4°C until further analysis. PCR products were separated on 1.6% agarose gels (Sea Kem LE agarose, BMA, Rockland, Maryland, USA) in 1x TAE (Tris/Acetat/EDTA) buffer in an electrical field (170 mV, 2.5 h). The banding patterns were made visible with ethidium bromide under UV light. The presence or absence of bands was scored for clear and reproducible bands with estimated fragment lengths of between 500 and 2000 base pairs. The final data set contained 386 individuals instead of 396 individuals due to the failure of the amplification in 10 individuals (Table 1).

RAPD analysis
Statistical analyses of RAPD banding patterns was based on the following assumptions: (1) RAPD fragments behave as diploid, dominant markers with alleles being either present (amplified) or absent (nonamplified); (2) comigrating fragments represent homologous loci; (3) polymorphic loci are inherited in a nuclear (Mendelian) fashion (Arafeh et al., 2002 ); and (4) populations are in Hardy-Weinberg equilibrium (HWE; F_is = 0). Even though the information on the heterozygosity of populations was lacking, HWE should not be violated, because a pollination experiment in G. reptans resulted in a very low seed set after self-pollination with no germinating seeds (Rusterholz et al., 1993 ). Assuming that the populations are in HWE, allele frequencies were estimated based on the square root of the frequency of the null (recessive) allele. Only polymorphic bands were taken into account. To avoid biased results, data analyses were restricted to bands whose observed frequencies were less than 1 – (3/N), where N is the mean number of sampled individuals per population (Lynch and Milligan, 1994 ).

The molecular diversity within populations was quantified as (1) Nei's expected heterozygosity (H_e), (2) the Shannon index (SI; Lewontin, 1972 ) and (3) the percentage of polymorphic bands (P_p). To quantify the variation of molecular diversity among populations, we calculated the coefficient of variation (CV) for H_e, SI, and P_p. H_e and SI were calculated with POPGENE (version 1.21; Yeh et al., 1997 ). The relations of the three molecular indices and the population sizes assessed were calculated as a nonparametric Spearman's Rho (r_s) correlation. The relation of altitude and the molecular indices was calculated as two-polynomial regression. We used JMP (version 3.1; 1995, SAS Institute, Cary, North Carolina, USA) for calculations of correlations.

The molecular differentiation between population pairs was quantified with the pairwise F_st, calculated with Arlequin (Schneider et al., 2000 ). The F-statistic was calculated across all bands. To test for isolation by distance (Slatkin, 1993 ), the genetic distance matrix (pairwise F_st values) and the geographic distance matrix was correlated (Mantel test, implemented in Arlequin). Significance levels were obtained after performing 10 100 and 10 000 random permutations for the pairwise genetic distances (F_st) and the Mantel test, respectively. Moreover, a UPGMA cluster analysis of pairwise Nei's unbiased genetic distances (Nei, 1978 ; TFPGA [tools for population genetic analysis]: Miller, 1997 ), and bootstrapping of 10 000 replicates, was calculated and displayed as a dendrogram to test for spatial separation.

Among-population differentiation was calculated using Nei's (1973) fixation index G_st with POPGENE and the fixation index F_st by calculating the molecular analysis of variance (AMOVA, implemented in Arlequin). G_st values are identical to F_st values if a locus consists of two alleles as applicable in RAPD marker analysis (Nybom and Bartish, 2000 ). Besides calculating G_st for the whole data set, it was calculated for the regional and the core areas separately. Furthermore, as genetic diversity correlates with the spatial scale of the distribution of populations (Nybom and Bartish, 2000 ), we calculated the fixation index for only those populations with 20, 10, or 2 km distance to each other. All fixation indices were calculated with polymorphic bands less frequent than 1 – (3/N) (Lynch and Milligan, 1994 ). To test for differences in the level of genetic diversity between populations from early-, medium-, and late-successional stages as well as low (2070–2140 m a.s.l.), medium (2330–2680 m a.s.l.), and high (2900–3080 m a.s.l.) elevation, we used AMOVA, which enables the extraction of variance components, beside the calculation of the fixation index. Euclidean squared distances among individuals were computed prior to the AMOVA and significance level for AMOVA was evaluated after 16 000 random permutations.

Directly observed gene flow
Gene flow occurs via pollen and seeds. (1) We directly measured pollen dispersal distances on the foreland of the Muttgletscher on two midsummer days with good weather conditions (24 July 2001 and 19 July 2002). The frequency of pollinator groups (flies, syrphids, and bumble bees) was estimated by counting flower visitations by insects during 6 h of observation. Dispersal distances of pollen were measured using fluorescent dye (Stockhouse, 1976 ; fluorescent dye from Radiant colour, Brussels, Belgium). Early in the morning, the anthers of four flowers were marked in an area of 10 x 25 m with different colored fluorescent dyes. Insects visited the flowers and dispersed the colorpowder during the day. With a UV torch, we searched the fluorescent powder after sunset and measured the distance to the initially marked flower. (2) Seed dispersal distances by wind were estimated with simulations using "Pappus" (Tackenberg, 2003 ). With this model, seed dispersal distances are calculated based on the terminal velocity of seeds, spatially explicit landscape data (from the core area), and the assessment of thermally induced turbulence and convection currents. Wind measurements took place on the Scaletta glacier foreland during the period of seed release in 2001. The wind measurements during the week with the best dispersal conditions were taken for a calculation of the dispersal spectra and estimates of the maximal dispersal distances. To consider the numbers of seeds dispersed, the absolute number of seeds produced by the population of G. reptans within the investigated area was estimated by measuring the density of flowering ramets in the area (for more details see O. Tackenberg and J. Stöcklin, unpublished data).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Using five primers, we detected 51 polymorphic RAPD bands, of which two were rejected due to their high frequency. Out of the 386 investigated individuals 384 RAPD phenotypes were found. Two of the populations from the highest altitude contained the same RAPD phenotype twice. None of the 49 scored bands was fixed at the level of populations.

Molecular diversity within populations
The molecular diversities of individual populations calculated from polymorphic RAPD bands are listed in Table 2: Nei's expected heterozygosity, H_e, ranged from 0.16 to 0.25 (CV = 8.7%) with a mean of 0.22 (SE = 0.004); Shannon indices (SI) of RAPD phenotypic diversity ranged from 0.24 to 0.37 (CV = 8.7%), with a mean of 0.33 (SE = 0.01) and percentage of polymorphic bands (P_p) within populations ranged from 49.0 to 81.6% (CV = 10.7%), with a mean of 71.3% (SE = 1.7).

Pairwise differentiation and spatial structure of the populations
Genetic differentiation (F_st) between population pairs ranged from 0.02 to 0.45 (mean = 0.15, SE = 0.01, CV = 62.3%). All but one of the 190 pairwise F_st values were significant (tested against 10 100 random permutations). The two early-successional populations of recently deglaciated sites from neighboring glacier forelands in the core area were genetically not differentiated (SCE and GR1, P = 0.06), even though the distance among them was 2.2 km. The genetic differentiation between the most distant population (FLS) and populations from the regional area ranged from 0.23 to 0.45 (mean = 0.37, SE = 0.01, CV = 10.5%).

The pairwise values of genetic differentiation (F_st) among the 20 populations correlated significantly with the spatial distances (Mantel test: R = 0.81, P < 0.001; Fig. 2).

View larger version (13K): [in this window] [in a new window]   Fig. 2. Matrix correlation of genetic (pairwise Fst values) and geographic distances among 20 populations of Geum reptans

View larger version (16K): [in this window] [in a new window]   Fig. 3. Dendrogram of the UPGMA cluster analysis based on Nei's (1978) unbiased measure of genetic distance. Populations of the core and the regional area (A), the two central populations (B) and the most western population (C) were separated (* indicate bootstrap values larger than 50%, based on 10 000 permutations)

Differentiation due to successional stage and altitude
Genetic variation was not significantly different among early-, medium- and late-successional populations. Moreover, successional stage in the AMOVA model did not explain any variation among populations (AMOVA, 15 populations; F_{among groups} = 0, P = 0.66). If populations are grouped into elevational classes (low, medium, and high), the percentage of variation explained by this grouping factor was low (AMOVA: 2.4%; P = 0.06). This corresponds to the lack of relationship of molecular variation and altitude reported above.

Direct estimates of pollen and seed dispersal distances
In total, we observed 435 flower visitations of pollinators on G. reptans within 6 h, i.e., 3.5 visitors per flower and per hour. Geum reptans is pollinated mainly by flies of different sizes (94%), followed by syrphids (4.6%), and bumble bees (1.4%). The traces of fluorescing dyes were found 50 times. The frequency of the observed dispersal distances decreased dramatically from 4 cm to 11.5 m with a single rare dispersal event over 30 m (Fig. 4). The seed dispersal spectra obtained from simulations with the model Pappus showed that most seeds (99.9%) are dispersed <10 m (Fig. 5). Long-distance seed dispersal with seeds dispersed over >100 m and 1000 m occurred in only 0.015% and 0.005% of all cases, respectively. Based on estimates of the yearly seed production on the Scaletta glacier foreland of >10 Mio seeds, on this particular glacier foreland 1580 and 520 seeds are dispersed over >100 m and 1000 m, respectively.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Frequency of pollinator flight distances grouped in intervals of 2 m (N = 50)

View larger version (15K): [in this window] [in a new window]   Fig. 5. Frequency of seeds dispersed by wind, grouped in intervals of 10 m. Assessment of seed dispersal distances were calculated with the model Pappus (Tackenberg, 2003 )

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Genetic diversity within populations of a clonal alpine species
The amplification of randomly selected gene loci (RAPD-PCR) is usually a more sensitive method to detect genetic variation in plant species compared to gene product level methods (e.g., isozymes) (Nybom and Bartish, 2000 ). RAPDs are sometimes questioned with respect to reproducibility and the biallelic nature of DNA bands. Heterozygotes cannot be separated from homozygotes and Hardy-Weinberg equilibrium has to be assumed for analysis. Our analysis is based on well reproducible RAPD banding patterns and because selfed seeds in G. reptans are nonviable (Rusterholz et al., 1993 ), selfing can be neglected.

Isozyme studies suggest that within and among populations genetic variation does not depend on either sexual and/or clonal reproduction (Hamrick and Godt, 1989 ). Furthermore, from the now available studies of alpine plants, there is little indication that plant species from high altitude have lower levels of genetic diversity compared to lowland plants (Holderegger et al., 2002 ; Till-Bottraud and Gaudeul, 2002 ). We can confirm both statements with our RAPD data of G. reptans: mean molecular variation within populations (H_e = 0.22 ± 0.004) was neither particularly high nor low and similar to the mean of 41 RAPD studies reviewed by Nybom and Bartish (2000) (H_e = 0.21 ± 0.12). The mean molecular variation reported in this review is probably somehow underestimated because sexually as well as clonally reproducing species were included. To avoid a biased estimate of genetic diversity in studies of clonal plants, each genotype should be collected only once (McLellan et al., 1997 ). In Cladium jamaicense, for example, overall genetic variation was underestimated if only genotypic diversity was considered (Ivey and Richards, 2001 ). But in Viola riviniana genotypic diversity and overall genetic diversity were almost identical (Auge et al., 2001 ). We tried to avoid sampling the same genetic individual twice and indeed 99.5% of all our samples were genetically different. This high resolution of RAPD phenotypes also indicates that single genets of G. reptans only exceptionally grow >4 m in diameter, as supposed when we chose the sampling design. Our results with G. reptans support the statement of Hamrick and Godt (1989) that, in general, clonal plants are genetically as diverse as nonclonal plants (Ellstrand and Roose, 1987 ; Widen et al., 1994 ). Genotypic diversity in the studied populations was high, as only exceptionally the same genotype was found twice.

Effects of successional and elevational gradients on genetic variation within populations
We assumed high initial recruitment from seeds when a population is founded after the retreat of a glacier, as several other authors did (e.g., MacDonald and Lieffers, 1991 ; Jelinski and Cheliak, 1992 ). Later, sexual recruitment might be low in clonal plants (Eriksson, 1989 ). It was also suggested that diversity should decline due to increased selection pressure during succession (Till-Bottraud and Gaudeul, 2002 ). In G. reptans a low genetic diversity might be expected particularly in late-successional habitats or in peripheral altitudinal habitats, because selection might be particularly strong at the distributional limits of a species. We found no evidence that population genetic variability in G. reptans is affected by environmental gradients. Changes in population genetic variability due to ecological conditions are rarely observed (Shimizu et al., 2002 ; Young et al., 2002 ; but see Gugerli et al., 1999 ; Auge et al., 2001 ; Li and Ge, 2001 ; Stenström et al., 2001 ; Bonnin et al., 2002 ). For example, in Parnassia palustris habitat type affected neither within-population genetic diversity nor genetic and phenotypic differentiation among populations (Bonnin et al., 2002 ) and in the alpine Saxifraga oppositifolia no effect of altitude on genetic population variability was detected (Gugerli et al., 1999 ). Along environmental gradients, several parameters such as individual age, overlap of generations, or recruitment frequency in climatically favorable years may change and influence intrapopulation genetic variation (Molau, 1997 ). But these parameters appear to have either no major or opposing effects on genetic variation in most cases studied so far, and this also holds for G. reptans. Long ramet life-span and potentially immortal genets may enhance the maintenance of genetic diversity in G. reptans, as observed in other studies of clonal plants (Lee and Chung, 1999 ; Brzosko et al., 2002 ). A high disturbance frequency in alpine habitats may allow repeated seedling establishment at least in favorable years in spite of the general harsh environmental conditions. Nevertheless, sexual reproduction alone does not guarantee the preservation of genetic variation. Various factors, such as drift, inbreeding, and strong selection, may result in genetic depletion (McLellan et al., 1997 ) if there is no gene flow from immigrating seeds or pollen. Our results suggest that the loss of genotypes during succession might be negligible and repeated seedling recruitment takes place irrespective of environmental conditions.

Population size may be critical for the maintenance of genetic variation (review by Frankham, 1996 ). In large populations, genetic drift is insignificant, but it becomes important in small populations and may be particularly pronounced after dramatic reduction in range size and fragmentation of habitats of a species (Srikwan and Woodruff, 2000 ). Alpine habitats are naturally fragmented, population sizes are expected to be stable, and, indeed, even though population sizes were very variable (CV = 61.7%), we found no effect of population size on genetic diversity in G. reptans.

Genetic differentiation and gene flow in the fragmented alpine environment
Due to high natural fragmentation and limited gene flow, we expected a comparably high genetic differentiation among populations in the alpine G. reptans. However, the observed estimate of genetic variation among all populations (G_st = 0.14) is not particularly high if compared with the mean reported in the review of 41 RAPD studies by Nybom and Bartish (2000) (G_st = 0.29 ± 0.21). In addition to the amount of gene flow and drift, G_st values are dependent on life history traits, colonization events, and the extent of the area under study, with a positive correlation of genetic differentiation and maximum spatial distance between population pairs (Nybom and Bartish, 2000 ). In our study, genetic differentiation increased from the core area, to the regional, to the whole area. However, the average spatial distances in our study are much lower than the mean distances among populations in most of the studies reviewed by Nybom and Bartish (2000) , which in part might explain the comparably low value of population differentiation in G. reptans. In Trollius europaeus, a similar estimate of genetic differentiation among populations in the Alps was observed, but in this case genetic and geographic distances were not correlated (Despres et al., 2002 ).

In our study, we observed considerable variation among pairwise population F_st values (ranging from 0.02 to 0.45), indicating random genetic drift. Even population pairs on the same glacier foreland were significantly differentiated, but to a lower degree than the more distant populations. This suggests that gene flow among populations of G. reptans is dependent on distance. The nonsignificant genetic difference between the two youngest populations in adjacent valleys may however indicate that younger populations are not necessarily founded by the nearest and elder populations from the same glacier forland, but by immigrants from more than one population in the same region. In G. reptans, we do not expect genetic bottlenecks in populations because our results indicate that gene flow is not restricted to single habitats (i.e., single glacier foreland) and because even early-successional populations were not genetically depleted. The observed gene flow via pollinators was very low, but the calculated seed dispersal distances may explain the results of the molecular study. Even if the simulation model predicts that only a small proportion of G. reptans seeds is dispersed over long distances, this may be sufficient for colonization and gene flow over considerable distances because of the large numbers of seeds produced every year. To infer the relative contribution of seed and pollen to gene flow, genetic population structure due to maternally inherited and nuclear genes should be compared (McCauley, 1995 ). However, we have not yet been successful in finding variation in maternally inherited genes in G. reptans with RFLP (random fragment length polymorphism).

Conclusion
Our results indicate that an alpine plant species may exhibit a similar level of genetic variation as lowland species. We observed no severe consequences of the highly fragmented habitats of Geum reptans on molecular genetic diversity and genetic differentiation among populations was not particularly high. In addition, clonal reproduction in this species has no severe consequences for population genetic variability and neither did successional age or elevation of the populations. Clearly, our results indicate that, at least within the same region, considerable gene flow is occurring among populations of G. reptans over larger distances, probably mainly by seed. Nevertheless, we observed a clear genetic structure according to the geographic distribution of the studied populations. Even populations within the same glacier foreland were significantly differentiated, but to a small degree, while genetic differentiation increased among more distant populations. We conclude that, in spite of the highly fragmented alpine landscape, random genetic drift is not the main factor determining population genetic structure of a species like G. reptans and that gene flow might be more important than commonly suggested.

	FOOTNOTES

 
¹ The authors thank Tina Weppler for helping collecting leaf material, the lab assistant Meral Turgay, and Oliver Tackenberg for calculating the frequency distribution of dispersal distances of seeds. This study was supported by the Swiss National Science Foundation grant 31-59271.99 and the Janggen-Poehn Foundation in St. Gallen, Switzerland.

² Current address: UCLA—EEB Dept., Box 951786, Los Angeles, California 90095-1786, USA; Andrea.Pluess{at}unibas.ch

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
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