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(American Journal of Botany. 2001;88:1409-1418.)
© 2001 Botanical Society of America, Inc.

Population Biology

Partitioning and diversity of nuclear and organelle markers in native and introduced populations of Epipactis helleborine (Orchidaceae)1

Jane Squirrell2, Peter M. Hollingsworth3,8, Richard M. Bateman4, James H. Dickson2, Marilyn H. S. Light5, Michael MacConaill6 and Mark C. Tebbitt7

2Division of Environmental and Evolutionary Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK 3Royal Botanic Garden, 20A Inverleith Row, Edinburgh, EH3 5LR, UK 4Botany Department, Natural History Museum, Cromwell Road, London SW7 5BD, UK 5The Professional Training Service, University of Ottawa, Ottawa, Ontario, K1N 6N5 Canada 6174 rue Jolicoeur, Hull, Québec, J8Z 1C9 Canada 7Brooklyn Botanic Garden, 1000 Washington Avenue, Brooklyn, New York 11225-1099 USA

Received for publication June 30, 2000. Accepted for publication January 25, 2001.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Variability of allozymes (1170 individuals, 47 populations) and chloroplast DNA (692 individuals, 29 populations) was examined in native European and introduced North American populations of Epipactis helleborine (Orchidaceae). At the species level, the percentage of allozyme loci that were polymorphic (P99) was 67%, with a mean of 2.11 alleles (A) per locus, and an expected heterozygosity (Hexp) of 0.294. At the population level, mean P99 = 56%, mean A = 1.81, and mean Hexp = 0.231. Although field observations suggest that self-pollination occurs frequently, populations had a genetic structure consistent with Hardy-Weinberg expectations and random mating (mean FIS = 0.002). There was significant deviation from panmixia associated with population differentiation (mean FST = 0.206). The distribution of two chloroplast haplotypes showed that 15 of the 29 populations were polymorphic. Using both nuclear and organelle FST estimates, a pollen to seed flow ratio of 1.43 : 1 was calculated. This is very low compared with published estimates for other plant groups, consistent with the high dispersability of orchid seeds. Finally, there was no evidence for a genetic bottleneck associated with the introduction of E. helleborine to North America.

Key Words: allozymes • chloroplast DNA • colonization • founder effects • FST • pollen to seed flow ratio


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
From the earliest days of human travel, the ranges of many plant species have been dramatically altered by the intentional or unintentional movement of living material (e.g., Elton, 1958 ; Cronk and Fuller, 1995 ). Horticulture, agriculture, forestry, medicine, and conservation have all benefited from plant introductions, but have also had to deal with their adverse effects when species escape and spread from their introduction sites. What makes some introduced species troublesome invasives while others remain benign is far from understood (Williamson and Fitter, 1996 ; Williamson, 1999 ). Although many generalizations can be made relating to species attributes and their behavior in their native range, in general our predictive ability is poor and the behavioral dynamics of any one given introduction need to be assessed on a case-by-case basis (Gray, 1986 ; Williamson and Fitter, 1996 ; Williamson, 1999 ).

In addition to our poor predictive capacity for the consequences of a given introduction on its local environment, it is also difficult to assess the effects that the introduction may have on the species itself. Introductions from one region to another will almost certainly involve a population bottleneck as the introduced individuals will only represent a subset of the total native population (Brown and Marshall, 1981 ; Barrett and Shore, 1989 ; Barrett and Husband, 1990 ). Theory predicts that introduced species will show lower levels of intrapopulation diversity and higher levels of population differentiation than their native counterparts (Brown and Marshall, 1981 ). However, on a case by case basis, empirical data show that the magnitude of these changes varies greatly. Studies showing comparative genetic depauperacy include Capsella bursa-pastoris (L.) Medik. (Brassicaceae) in North America, where it is introduced, vs. Europe, where it is native (Neuffer, 1996 ; Neuffer and Hurka, 1999 ). Likewise, lower levels of clonal diversity were observed in the introduced Australian range of Chondrilla juncea L. (Compositae) compared with its native Eurasian range (Chaboudez, 1994 ). By contrast, there was no evidence of a genetic bottleneck associated with the introduction of Apera spica-venti (L.) P. Beauv. (Poaceae) to Canada from Europe (Warwick, Thompson, and Black, 1987 ). In Bromus tectorum L. (Poaceae), while overall there were fewer alleles per locus, and fewer polymorphic loci in the introduced American range compared with the native Eurasian range, within individual introduced populations levels of allelic diversity and polymorphic loci were higher (Novak and Mack, 1993 ). Clearly the attributes of individual species need consideration. One key factor relates to the breeding system. Outcrossing species can be less sensitive than selfing species to loss of genetic variation during colonization (Brown and Marshall, 1981 ; Barrett and Richardson, 1986 ). Theoretical work also suggests that the size, composition, and dynamics of the founding population(s) are important determinants of the population genetic consequences of introductions (Nei, Maruyama, and Chakraborty, 1975 ; Barrett and Richardson, 1986 ; Barrett and Shore, 1989 ). If the founder population contains few individuals, is genetically depauperate, and remains small in size for some generations after the introduction, the effects in theory will be most pronounced. If, however, there are multiple founder populations, and/or the founding population(s) are large, diverse, and expand rapidly after introduction, these effects may be minimal. Indeed, given sufficiently variable founders, higher intrapopulation diversity and lower interpopulation differentiation can occur in introduced relative to native ranges especially if the introduced range has a greater habitat density and continuity.

Although there are numerous interesting studies investigating patterns of genetic diversity in introduced populations (e.g., Thébaud and Abbott, 1995 ; Weber and Schmid, 1998 ), there are still relatively few detailed studies comparing levels of genetic diversity in native vs. introduced populations (Neuffer and Hurka, 1999 ). This knowledge gap largely reflects the logistical difficulties of collecting material over the large spatial scales that such work usually entails. Nevertheless, such studies are necessary if we are to gain an increased understanding of the population dynamics of biological invasions. In addition, large spatial-scale population genetic analyses are of interest in their own right. Population processes that may not be evident from local studies can become apparent at broader scales, stressing the importance of range-wide investigations looking beyond the local population as the study unit (Barrett and Pannell, 1999 ). This is particularly pertinent for breeding system evolution, and there are some well-documented cases showing variation in mating patterns across the ranges of individual species, particularly in relation to colonization events (Husband and Barrett, 1991 ; Eckert, Manicacci, and Barrett, 1996 ; Barrett and Pannell, 1999 ).

To investigate the genetic consequences of introduction and to estimate population structure and breeding system behavior over a large spatial scale, we have studied European and North American populations of Epipactis helleborine (L.) Crantz (Broad-leaved Helleborine). This is a diploid, perennial, multiflowered, often multistemmed, wasp-pollinated, self-compatible, hermaphrodite terrestrial orchid (Jones, 1974 ; Tanaka and Kamemoto, 1974 ; Light and MacConaill, 1991, 1998 ; Proctor, Yeo, and Lack, 1996 ). It is native to Europe and Asia but also naturalized in North America (Judd, 1971 ; Light and MacConaill, 1991 ). Unusually for an orchid, it frequently occurs as a colonist in urban areas (Hollingsworth and Dickson, 1997 ; Dickson, Macpherson and Watson, 2000 ). The first record of E. helleborine in North America was of a group of flowering plants growing on a wooded hillside near Syracuse, New York in 1879 (Day, 1882 ; Correll, 1950 ). Shortly after it was recorded from near Toronto in 1890 and Montreal in 1892 (Mousley, 1927 ; Judd, 1971 ). It has since spread rapidly as far west as California (Brenan, 1983 ). In certain areas it is considered a troublesome weed of lawns and gardens, and it is the only introduced orchid species to have successfully invaded undisturbed woodlands in Canada (J. Eckenwalder, University of Toronto, personal communication 2000).

Our interest in this species is part of a broader project on the genus. Epipactis contains some 25–40 species and has a predominantly Eurasian distribution (Richards, 1982 ; Delforge, 1995 ). Some of the species are widespread, taxonomically uncontroversial putative outcrossers. However, within the genus there is also a complex of putatatively self-pollinating taxa, apparently closely related to E. helleborine (Richards, 1982 ; Delforge, 1995 ). Many of these are extremely localized in their distribution and occur in small populations, and thus have attracted conservation interest, albeit surrounded by taxonomic controversy (Richards and Porter, 1982 ; Anonymous, 1995 ). Epipactis helleborine has been implicated in the origin of many of these localized taxa, and hence knowledge of the population genetic structure of this species is a necessary prerequisite for understanding the processes responsible for the diversification and taxonomic complexity of the genus. In the current study we have used allozymes and chloroplast DNA markers to study the amounts and distribution of genetic variation in native and introduced populations of this species.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Plant materials
Plants were sampled from populations in England, Scotland, France, Belgium, Denmark, Germany, Switzerland, and Canada (Table 1). A total of 1170 individuals from 47 populations was used for the isozyme analyses. A total of 692 individuals from 29 populations was used for the cpDNA analyses.


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  		Table 1. Intrapopulation allozyme diversity indices for Epipactis helleborine

 
Isozyme analyses
Allozyme polymorphism was assessed using standard horizontal starch gel electrophoresis (Wendel and Weeden, 1989 ) using extracts from single bracts. Seven enzyme systems were investigated: Aat, Idh, Mdh, 6Pgd, Pgi, Pgm, and Skd. Electrophoretic conditions and staining and buffer recipes are as described by Hollingsworth, Preston, and Gornall (1995) and Hollingsworth and Dickson (1997) . In the absence of progeny arrays, interpretation of zymograms in terms of loci and alleles was based on expectations of quaternary structure, conserved number of isozyme loci, and subcellular compartmentalization (Gottlieb, 1981, 1982 ; Weeden and Wendel, 1989 ).

Chloroplast analyses
DNA was extracted from single bracts using a protocol modified from Doyle and Doyle (1990) . Using a ground glass rod attached to a domestic power drill, samples were homogenized in 2 x cetyltrimethylammonium bromide (CTAB) buffer (preheated to 65°C), with 0.2% ß-mercaptoethanol along with a pinch of polyvinylpolypyrrolidone (PVPP) and acid-washed sand. The samples were incubated at 65°C for 60 min. An equal volume of 24 : 1 chloroform/isoamyl alcohol was then added and the samples spun for 10 min at 13 000 rpm in a microfuge. The supernatant was removed and the chloroform/isoamyl alcohol step repeated. Following centrifugation, the supernatant was removed, and the DNA precipitated by the addition of two-thirds volume freezer-cold isopropanol. To collect the pellet, the samples were centrifuged at 13 000 rpm for 10 min. The isopropanol was then poured off, and after air drying for 20 min, the DNA was resuspended in 300 µL of tris-ethylenediaminetetraacetic acid (TE).

A 10-base pair (bp) duplication in the trnL intron of E. helleborine was discovered by sequencing 15 individuals from widely separated British populations (M. L. Hollingsworth, unpublished data). The distribution of this duplication within and among populations was investigated using a polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) approach. The trnL intron was amplified using primers "C" and "D" designed by Taberlet et al. (1991) . The PCR cocktail (25 µL) contained: template DNA (3 µL), 100 µmol/L of each deoxynucleotide (dNTP), 0.15 µmol/L of each primer, 1 unit Taq polymerase (Bioline, London, UK), 1.25 mmol/L MgCl2, and 2.5 µL reaction buffer. Polymerase chain reaction was performed using the following profile: one cycle of 94°C for 4 min followed by 30 cycles of 30 sec at 94°C, 30 sec at 55°C, and 1 min at 72°C and finally one cycle at 72°C for 10 min. A restriction digest was designed to screen for the presence or absence of the 10-bp duplication. Using the restriction enzyme Mbo1, the presence or absence of the duplication resulted in either a 59-bp or 69-bp fragment. To visualize this difference, the samples were run on 3% agarose gels in 1x tris borate EDTA (TBE) buffer at 150 V for ~1 h.

Data analyses
For the allozyme data the proportion of polymorphic loci (P99), the mean number of alleles per locus (A), the mean number of alleles per polymorphic locus (Ap), and expected heterozgosity (Hexp) were calculated using GDA 1d15 (Lewis and Zaykin, 2000) . Allele frequencies were calculated using Arelequin 1.1 (Schneider et al., 1997 ). Individual population level fixation indicies (f) were estimated using FSTAT 2.8 (Goudet, 1999 ), and the significance of heterozygote excess or deficit tested by randomizing (10 000 replicates) alleles among individuals within populations. The significance values were corrected by the sequential Bonferroni technique (Rice, 1989 ).

Wright's (1951) F statistics were used to investigate the partitioning of genetic variability among individuals and populations. Calculations were made using an analysis of variance approach (Weir and Cockerham, 1984 ) using Arelequin 1.1 (Schneider et al., 1997 ). The mean correlation of alleles within individuals within populations, FIS, was estimated as a measure of inbreeding within populations (in the notation of Weir and Cockerham = f). The mean correlation of alleles of different individuals in the same population, FST, averaged over all populations, was estimated as a measure of population differentiation (in the notation of Weir and Cockerham = {theta}). The correlation of alleles within individuals over all populations, FIT, was estimated to assess deviations from panmixia attributable to both nonrandom mating and population differentiation (in the notation of Weir and Cockerham = F). To test the significance of these values, permutation tests using >10 000 permutations were performed.

To test for differentiation between introduced (North American) and native (European) populations, a hierarchical Fxy analysis was performed using Arelequin 1.1 (Schneider et al., 1997 ). The levels in our hierarchy were: 1, populations relative to the total; 2, populations relative to their continents; and 3, continents relative to the total data set.

For the chloroplast data, the proportion of populations that are polymorphic (PP) for cpDNA, and the relative frequency of the two haplotypes were calculated. To assess the amount of population differentiation, FST was estimated using Arelequin 1.1 (Schneider et al., 1997 ). Significance was tested by permuting (with >10 000 permutations) haplotypes among populations. A hierarchical Fxy analysis was performed following the protocol applied to the allozyme data.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Isozyme analyses
Of the seven isozyme systems screened, six produced reliable and consistent banding profiles. Only Skd was inconsistent and hence omitted from further analyses. Nine scorable putative loci were resolved from the remaining six enzyme systems: Aat-1, Aat-2, Idh, Mdh-1, Mdh-2, 6Pgd, Pgi-1, Pgi-2, and Pgm. At the species level, 78% of loci were polymorphic (Mdh-1, Mdh-2, Idh, Pgi-2, Aat-2, 6Pgd, Pgm), although the variation in 6Pgd involved a very low frequency rare allele. Thus, P99 was 67% with a mean of 2.11 alleles per locus. The total expected heterozygosity was 0.294. Estimates of genetic diversity at the population level are presented in Tables 1 and 2. (Details of allele frequencies for individual loci are available from the corresponding author on request.) Within populations, on average, 56% of the loci were polymorphic (P99), and there was a mean of 1.81 alleles per locus (A), and a mean expected heterozygosity (Hexp) of 0.231. Excluding four populations with significant missing data (see Tables 1 and 2), a Mann-Whitney U test showed a significant difference in the total number of alleles from the six loci that showed widespread polymorphism (Mdh-1, Mdh-2, Idh, Pgi-2, Aat-2, Pgm) between European and North American populations (U = 268.0, P < 0.01). The introduced North American populations were more diverse than the native European populations. Similarly, values of A (Mann-Whitney U = 278.5, P < 0.01) and Ap (Mann-Whitney U = 258, P < 0.05) were significantly greater in North American populations than in European populations. There was no significant different between native and introduced populations for P99 and Hexp.

To investigate the partitioning of genetic diversity among individuals and populations we estimated FIS, FST, and FIT (Table 3). Mean estimates of FIS over all loci for all populations (0.002), for North American populations (0.060), and for European populations (–0.022) were not significantly different from 0, consistent with random mating. There was, however, a significant deviation from panmixia attributable to differentiation among populations (mean FST estimates = 0.206 [all populations]; 0.200 [European populations]; 0.090 [North American populations]).


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  		Table 3. Mean allozyme F-statistic estimates for Epipactis helleborine

 
To investigate hierarchical population structure, FXY estimates were calculated for populations within continents, populations among continents, and among continents (Table 4A). There was significant structure at each level (Table 4A). The majority of the variation was partitioned within populations (72.19%) with smaller amounts between populations (15.97%) and between continents (11.12%) (Table 4B).


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  		Table 4. Hierarchical allozyme population structure in Epipactis helleborine. (A) Hierarchical allozyme F-statistic estimates with two levels of subdivision (population and continent). The two continents considered are North America and Europe. (B) Percentage of allozyme variability partitioned at different hierarchical levels

 
Geographical structure was evident for alleles at the Idh locus (Table 5). In Britain, allele a was the most common, present at a mean frequency of 0.96, with 65% of populations fixed for this allele. A second allele (b) was present in some British populations, although at a low mean frequency (0.04). In continental Europe, a third allele (c) was detected in 27% of the populations we examined (mean allele frequencies for continental Europe, allele a = 0.94, b = 0.02, c = 0.04). In North America allele c was found in ten out of the 12 populations (83%) (mean allele frequencies for North America, allele a = 0.76, b = 0.10, c = 0.14).


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  		Table 5. Frequency of three Idh alleles with populations of Epipactis helleborine

 
Chloroplast analyses
Of the 29 populations examined for cpDNA polymorphism, 15 (52%) were polymorphic (Table 6). The distribution of this polymorphism, was, however, uneven. In Europe, only four of the 17 populations were polymorphic (24%), whereas in North America, 11 of the 12 populations (92%) were polymorphic. The mean frequencies of the two haplotypes (1 = presence, 2 = absence of the 10-bp trnL duplication) were 1 = 0.82, 2 = 0.18 (all populations); 1 = 0.93, 2 = 0.07 (Europe); 1 = 0.66, 2 = 0.34 (North America). A Mann-Whitney U test showed a significant difference in the diversity of haplotypes between European and North American populations (U = 187.5, P < 0.05). The introduced North American populations were more diverse than the native European populations.


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  		Table 6. Chloroplast DNA polymorphism in Epipactis helleborine

 
Estimates of FST were 0.471 (all populations), 0.506 (European populations), and 0.367 (North American populations) (Table 7). In terms of hierarchical population structure, 48.5% of the variation was partitioned within populations, 34.5% among populations, and 17% among continents (Table 8).


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  		Table 7. Mean cpDNA FST estimates for Epipactis helleborine

 

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  		Table 8. Hierarchical cpDNA population structure in Epipactis helleborine. (A) Hierarchical cpDNA F-statistic estimates with two levels of subdivision (population and continent). The two continents considered are North America and Europe. (B) Percentage of variability partitioned at different hierarchical levels

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Mating system behavior within populations
Baker (1955) noted that for colonizers, being a self-compatible hermaphrodite can be advantageous. Only one individual, rather than two, represents the minimum unit necessary for forming a sexually reproducing colony. This association between colonization and self-fertilization has subsequently been documented in many groups (e.g., Barrett and Shore, 1987 ; Husband and Barrett, 1991 ; see also Pannell and Barrett, 1998 , and references therein). However, for those colonizers that are predominantly outcrossing, but are self-compatible hermaphrodites, selection pressures for changes in mating patterns will be lower. Even for single founders, all that is required to commence the establishment of a new population is some occasional geitonogamous or autogamous pollination. Providing the population increases in size rapidly after founding, this can overcome the shortage of mates associated with colonization.

In E. helleborine, despite the transition between cross- and self-pollination being apparently labile (Richards, 1982 ), no difference in the breeding system was detected between native and introduced populations. Although occasionally some loci in some populations showed a significant excess of heterozygotes or homozygotes, mean FIS values were not significantly different from zero in either introduced or native populations. Similar results were obtained in studies focusing on native populations of this species (Hollingsworth and Dickson, 1997 ; Ehlers and Pedersen, 2000 ). From a population genetic perspective, cross-pollination (apparently most effective by wasps) appears to be equally efficient in both Europe (Richards, 1982 ; Proctor, Yeo, and Lack, 1996 ) and North America (Judd, 1971 ). This contrasts interestingly with observations made on pollinator behavior; numerous between-flower within-spike visits have been observed by pollinating wasps that might be expected to promote geitonogamy (Light and MacConail, 1998 ). In addition, some of the introduced populations in this study have shown what appears to be facultative autogamy (M. H. S. Light, University of Ottawa, unpublished data). At the Gatineau Park site 3 (North America), when plants flowering in drought conditions are subjected to showers and high relative humidity, the pollinia on some plants have been observed to "flip" longitudinally through 180° and come to rest on the stigmatic surface, resulting in a transfer of pollen to stigma within the same flower. However, neither this type of autogamy nor wasp-mediated geitonogamy seems to have translated into any measurable deviation from Hardy-Weinberg expectations within populations (Tables 1 and 3). It may be that inbreeding depression favors the recruitment of outcrossed progeny, but as yet we have no data to address this issue.

Genetic variation in the introduced vs. the native range
In terms of levels of variation, there is no evidence of a genetic bottleneck associated with the introduction of E. helleborine to North America. Allozymes and cpDNA RFLPs showed equivalent or higher levels of intrapopulation genetic diversity and lower levels of interpopulation differentiation in introduced relative to native populations. Although this runs contrary to theoretical predictions, the population genetic structure of E. helleborine provides a plausible explanation for this result.

The majority of genetic variability is held within rather than among populations of E. helleborine, and average observed heterozygosity is high (Table 1). Not many individuals would need to be sampled to capture the allelic diversity (from the loci measured here) of the European populations. Given that there are two cpDNA haplotypes and four isozyme loci with three alleles (Idh, Mdh-2, Aat-2, Pgi-2) in the introduced range, it is clear that more than one individual plant was introduced to North America. However, given the high observed levels of heterozygosity in native populations, such variability could be captured within a small number of plants (e.g., <5). Providing the North American colonist population(s) increased in size rapidly after their introduction, significant loss of allelic diversity by drift could have been prevented. The fact that the introduced populations often contain large numbers of individuals and that, at least in the study area, the populations occur frequently has presumably created conditions amenable to frequent genetic communication between populations and the maintenance of high levels of intrapopulation diversity.

Evidence for genetic structure within the introduced range is weak. The most marked population differentiation is the apparent fixation of chloroplast haplotype 2 in the University of Toronto population. This haplotype is not fixed in any other population and occurs at a lower frequency than haplotype 1 (Table 6). The University of Toronto plants also showed lower allozyme polymorphism than other introduced populations (Tables 1 and 2). However, compared to the other introduced sites examined in this study (woodlands, parks, and gardens), this population is somewhat atypical. It consists of a small courtyard on the university campus, surrounded on all sides by buildings. The presence of only one chloroplast haplotype and a maximum of two alleles at each of the isozyme loci (Table 2) is consistent with a single (or at least very few) founders. Similar population structure has been documented in urban populations growing in very small habitat patches in the native range (Hollingsworth and Dickson, 1997 ).


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  		Table 2. Number of alleles recovered from each polymorphic locus in each population of Epipactis helleborine

 
As well as providing information on amounts and partitioning of diversity in the introduced range, our data can also be used to infer the nature of the potential source population(s) of the introduced material. The Idh-C allele has not been recovered from the UK despite the large sample of 544 plants scored here from 23 widely distributed populations (Table 5). In contrast, our sample of 227 scored plants from 12 populations in continental Europe recovered this allele from three populations. This allele is present in 10 out of the 12 introduced North American populations. It would thus seem reasonable to suggest that at least some of the introduced material did not stem from the UK. Whether the introduced material all came from a single continental European population or from multiple populations (from this area or elsewhere) is unclear. The partitioning of variability predominantly within, rather than between, populations limits our ability to test this.

Estimates of pollen vs. seed flow
It would seem intuitive that for E. helleborine (as for many other orchids), the small dust-like and potentially highly dispersible seeds, rather than pollen, are the primary agent of interpopulation communication (Nielsen and Siegismund, 1999 ; Chung and Chung, 2000 ). The packaging of pollen into pollinia results in the male gametes being presented as a large assemblage, potentially not well suited to long-distance dispersal. Orchids in some respects represent the antithesis of our general perceptions of male and female reproductive biology with a small, highly dispersable female function, and an (effectively) large, potentially less well-dispersable male function. Of course, long-distance pollinator flights could result in successful gene flow, but the extent to which this happens relative to seed flow is unknown. That long-distance dispersal does occur by seed is evident from the colonization ability of orchids (see Arditti and Ghani, 2000) : pollen alone cannot form new colonies.

Recent theoretical advances in the combined analysis of nuclear and organelle genomes allow inferences to be made regarding the relative amounts of gene flow by pollen vs. seed, by comparing FST values from biparentally inherited nuclear markers with uniparentally inherited organelle markers (Petit, Kremer, and Wagner, 1993 ; Ennos, 1994 ; Ennos et al., 1999 ). We emphasize the point here that FST estimates are not measures of gene flow per se. Rather, they are summaries of the partitioning of genetic diversity within a species (Whitlock and McCauley, 1999 ). Distinction between historical and current gene flow is not possible, and translation of FST estimates into measures of gene flow via the familiar equation FST = 1/(4Nm + 1), rest upon assumptions such as drift-migration equilibrium, a low mutation rate relative to the migration rate, and an absence of selection on the study loci (Whitlock and McCauley, 1999 ). However, bearing these factors in mind, FST estimates themselves represent useful summaries of population differentiation (Whitlock and McCauley, 1999 ). When these estimates are made for markers with different modes of inheritance, some inference of the relative contributions of pollen and seed to gene flow can be made, albeit in a somewhat vague temporal context.

Thus, Ennos (1994) elegantly showed that in a hermaphrodite species, the pollen-to-seed-flow ratio could be estimated as:

(1)
where FST(B) = FST for biparentally inherited nuclear markers, FIS(B) = FIS for biparentally inherited nuclear markers, and FST(M) = FST for maternally inherited markers. Following Ennos (1994) , given that our allozyme (nuclear marker) FIS values were not significantly different from zero, the equation reduces to

(2)
In most angiosperms cpDNA is predominantly maternally inherited (Ennos et al., 1999 ), although we have not yet raised sufficient progeny from controlled crosses to test this directly in E. helleborine. Our calculations presented here are based on the assumption that maternal inheritance is the rule in E. helleborine, although we stress that this is currently untested.

Our estimate of the pollen to seed flow ratio is extremely low (1.43 : 1). We are not aware of any comparable figures published for orchids, but it is noteworthy that this pollen to seed flow ratio is lower than any reported from other plant families (Fig. 1). We stress that the ratio for E. helleborine should not be interpreted literally. When most gene flow is by pollen rather than by seeds (as for example in wind pollinated tree species with poorly dispersable seeds) there is a large discrepancy between FST(M) and FST(B). This gives a system in which the relative proportions of each can be assessed (within an order of magnitude or so). When gene flow is predominantly by seed, which contains both nuclear and organelle genomes, FST(B) and FST(M) become more similar, and a literal interpretation of the ratio becomes less relevant (Ennos, 1994 ). In the case of E. helleborine, the ratio could simply be interpreted as being consistent with interpopulation gene flow being predominantly by seed. It would be informative to obtain similar estimates for other orchid species covering a range of different distributional and reproductive attributes.



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  		Fig. 1. Available estimates of pollen to seed flow ratios. The values in the figure were obtained or calculated from the following references and references therein. 1Epipactis helleborine (L.) Crantz, this study; 2Argania spinosa (L.) Skeels (El Mousadik and Petit, 1996 ); 3Hordeum spontaneum Koch. (Ennos, 1994 ); 4Senecio gallicus Vill. (Comes and Abbott, 1998 ); 5Eucalyptus nitens Maiden (Byrne, Parrish and Moran, 1998 ); 6Thymus vulgaris L. (Tarayre et al., 1997 ); 7Pinus sylvestris L. (Scotland) (Sinclair, Morman, and Ennos, 1998 ); 8Alnus glutinosa (L.) Gaertn. (King and Ferris, 1998 ); 9Pinus contorta Douglas ex Loudon, (Ennos, 1994 ); 10Phacelia dubia (L.) Fern. (Levy and Neal, 1999 ); 11Fagus sylvatica L. (El-Mousadik and Petit, 1996 ); 12Pinus sylvestris L. (Spain) (Sinclair, Morman, and Ennos, 1999 ); 13Pinus flexilis James (Latta and Mitton, 1997 ); 14Quercus L. spp. (Ennos, 1994 ); 15Quercus robur L. (El-Mousadik and Petit, 1996 ); Quercus petraea (Matt.) Liebl. (El-Mousadik and Petit, 1996 ); Fagus crenata Blume (Tomaru et al., 1997, 1998 ). See also Ennos et al. (1999) and Newton et al. (1999) . Figure Abbreviations: H, herb; T, tree; I, insect-pollinated; S, self-pollinated; W, wind-pollinated or wind-dispersed seed; A, animal-dispersed seed; G, gravity-dispersed seed; Wa, water-dispersed seed.

 
Speculation on the introduction of E. helleborine to North America
While the genetic data were not helpful in discriminating between single and multiple introductions of E. helleborine to North America, it is worth considering whether the process was likely to be active or passive. There are three potential hypotheses. First, the introduction could be a simple range expansion by transatlantic wind-blown seed. Second, there could have been a deliberate introduction. Third, the introduction could have been accidental human transport of material. Clearly the first (natural colonization) cannot be excluded with certainty, although we consider it unlikely. The orchids' first appearance was some considerable distance inland, in an area, and at a time, of frequent human movement between Europe and North America, which points towards human involvement. Doyon and Cayoutte (1966) discussing the spread of orchids in Québec speculated that E. helleborine was planted in colonial gardens and had perhaps escaped from cultivation. Correll (1950) stated that "In European folklore, this plant is valued as a remedy for gout." However, an examination of early writings by Dioscorides (Wellman, 1958 ), Theophrastus (Hort, 1916 ) and Pliny (Jones, 1956a, b) revealed much laxity in the use of the words epipactis, helleborine, hellebore, and helleborus to describe plants of reputed medicinal value and none of the descriptions match E. helleborine. Both Pliny and Dioscorides named a small herb having vestigial leaves as epipactis, "also known as elleborine," useful for treating liver ailments and poisons. It is unlikely that this description is of what we now know as E. helleborine. One potential source of this confusion is the white hellebore, Veratrum album L. (Melanthiaceae), which E. helleborine resembles vegetatively. Veratrum album was formerly used in the treatment of chronic diseases that proved challenging to ordinary remedies, including gout (Stillé, 1860) .

If a deliberate introduction of E. helleborine via colonial herbal gardens was not the source of the introduced populations, how did a European orchid come to be established in North America, ~250 miles (~400 km) inland from the Atlantic Ocean? Accidental human transport seems a plausible explanation. It is possible that seeds or plants of E. helleborine were unwittingly transported along with the human and cargo traffic from Europe. Plant material in the soils carried with imported trees is one obvious vector, although there is a myriad of other possible alternatives. The extent to which different mycorrhizal associates have been used in the introduced range, compared with the native range, is unknown, but clearly suitable associates are available. Subsequent to the plants' local establishment, the highly dispersible seeds and the availability of effective wasp pollinators have proved capable of facilitating its subsequent remarkable spread.


   FOOTNOTES
 
1 The authors thank Michelle Hollingsworth, John Richards, Keith Watson, Richard Ennos, and Bodil Ehlers for many useful thoughts and ideas; Spencer Barrett and William Cole for laboratory space and very helpful discussion in Toronto; two anonymous referees for constructive comments; and P. Delforge, A. Gévaudan, D. W. Kapteyn den Boumeester, J.-M. Lewin, M. Lowe, H. Mathe, M. Rohmer, F. Tausch, W. Timpe, D. M. Turner Ettlinger, and M. Vauthey for field assistance. This work was primarily funded by the NERC Taxonomy Initiative grant GST/02/833. In addition PMH acknowledges the George Forrest Fellowship for funding work carried out in Canada. Back

8 Author for reprint requests. Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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