Molecular Ecology (2002) 11, 2499–2509
Spatial genetic structure and clonal diversity of island
populations of lady’s slipper (Cypripedium calceolus) from
the Biebrza National Park (northeast Poland)
Blackwell Science, Ltd
E . B R Z O S K O , A . W R Ó B L E W S K A and M . R A T K I E W I C Z
University of Bialystok, Institute of Biology, ¤wierkowa 20B, 15–950 Bialystok, Poland
Abstract
Three populations of the rare and endangered plant species Cypripedium calceolus were
included in a study of genetic diversity and spatial genetic structure in the Biebrza National
Park, northeast Poland. Analysis of 11 allozyme loci indicate that the populations of this
species contained high genetic variability (P = 45.5%, A = 1.73). On the other hand, the
genetic differentiation (FST = 0.014, P < 0.05) among C. calceolus populations was very low
when compared to other species with similar life history characteristics. The observed high
rate of gene flow (Nm = 18) may suggest that the populations studied derived from each
other in the recent past. Five polymorphic allozyme markers identified 109 multilocus
genotypes in three populations and the majority of them (67%) were population-specific.
One of the populations studied, characterized by particularly extensive vegetative reproduction, showed the lowest clonal diversity (G/N = 0.15) and heterozygosity (HO = 0.111)
values and the highest FIS (0.380), when compared to other two populations (G/N = 0.26–
0.27, HO = 0.166–178, FIS = 0.024–0.055). This may indicate that clonal reproduction has an
important influence on the genetic structure of C. calceolus populations. The longevity of
genets, the out-crossing breeding system and the presence of recruitment from seeds are
factors maintaining genetic diversity in C. calceolus.
Keywords: allozymes, clonality, Cypripedium calceolus, endangered plant, genetic diversity, island
populations
Received 15 April 2002; revision received 7 August 2002; accepted 7 August 2002
Introduction
One of the main methodical problems in plant demography are difficulties with discrimination of individuals
in clonal plants. Recently, molecular methods (allozyme or
DNA analyses) have been frequently used to remedy these
difficulties (Ellstrand & Roose 1987; Chung & Epperson
1999; Pornon et al. 2000). Genetic methods are now widely
implemented in the explanation of genetic diversity among
and within populations, as well as conservation and
recovery of endangered plant species (Godt & Hamrick
1999; Gitzendanner & Soltis 2000). The use of genetic
markers also allows identifying genetic individuals (genet),
which may be composed of many independent units
(ramets, rosettes, etc.). The integration of a demographic
and genetic approach, however, is still rare in recent
Correspondence: E. Brzosko. Fax: (+ 48) 85 745 73 02; E-mail:
[email protected]
© 2002 Blackwell Science Ltd
literature, so are the studies on the relationships between
demographic and genetic units in plant populations
(Chung 1995; Pornon et al. 2000). The parameters
estimating clonal diversity in populations have been
calculated without information on population size or area
(Chung & Epperson 1999). The genetic/demographic
approach is particularly important in clonal plants, where
except of sexual reproduction, replication of genotypes by
vegetative reproduction and spread takes place. There are
different views concerning the effect of clonality on the
genetic structure of populations of clonal species. Some
studies document a lower level of genetic variation of
clonal, than nonclonal plants. This is attributed to clonal
reproduction, which may reduce the level of heterozygosity within population (Pornon et al. 2000). Genetic
uniformity can also be observed in species that completely
lack sexual reproduction or form extensive clones (Dietz &
Steinlein 2001). Conversely, studies on plant allozyme
variation in populations of clonally reproducing plants
2500 E . B R Z O S K O , A . W R Ó B L E W S K A and M . R A T K I E W I C Z
show that the level of their genetic variation is not
necessarily reduced (Ellstrand & Roose 1987).
Conclusions related to genetic variation of rare and
endangered species are also unequivocal, even though
one of the most important characteristics of rare species,
often quoted in the literature, is a low level of its withinpopulation genetic diversity (Karron 1991). Rare species
maintain low levels of genetic variation because their
populations are small and isolated, as the gene flow among
such populations is usually considerably restricted. On the
other hand, Gitzendanner & Soltis (2000) pointed out that
the level of genetic diversity of rare species may be at the
same or even higher level than that of their more common
congeners (see also Bijlsma et al. 1991; Chung 1995).
The maintenance of genetic diversity is very important
for the long-term survival of species (Frankel & Soulé
1981), because the loss of variation may significantly limit
the adaptability of the population to changing environments (Menges 1990; Bijlsma et al. 1991).
Here we present the results of electrophoretic analyses of
an extremely rare and endangered clonal species, C. calceolus L. from fragmented landscape in northeast Poland. We
aimed to: (i) document the level of genetic variation within
and among island populations of C. calceolus, and determine the factors contributing to the maintenance of genetic
variation; (ii) investigate whether populations are multiclonal, and how are genets spatially distributed within
populations; and (iii) determine how the spatial genetic
structure of populations of clonal C. calceolus is shaped by
clonal propagation and sexual reproduction.
C. calceolus is a particularly well-suited species for this
type of study, because it reproduces both sexually and
vegetatively, and is regarded as a rare and endangered
plant in Europe (Kull 1999). It is a diploid, herbaceous
perennial and is assumed to be an out-crosser, pollinated by
small bees with highly restricted ranges (Stoutamire 1967;
Nilsson 1979; Cribb 1997). It may also produce new ramets
from a horizontal rhizome underground. C. calceolus can
occur as single shoot (ramet) or more often as aggregation
of shoots (clump) of different size (even up to 70 shoots,
Brzosko 2002). Aggregations (clumps) are units, which are
well distinguished in space. On the other hand, distances
between shoots within aggregations are very small and it is
not possible to detect individuals in genetic sense (genets)
in the field. C. calceolus is mainly a boreal species which
occurs in shady deciduous and mixed woodland (rarely
in full sunlight), predominantly on calcareous soils
(Werpachowski & Brzosko 1994; Brzosko & Werpachowski
1998; Kull 1999). In Poland lady’s slipper occurs in sparsely
distributed sites (⁄wieboda 1976; Wika & Bernacki
1984; Fig. 1). This is primarily due to clear cutting of
forests, which changes light conditions and water balance
(Zaverucha et al. 1983; Wika & Bernacki 1984). Populations
of lady’s slipper have almost completely disappeared in
the vicinity of large cities in Poland due to over-collection
(Lucka 1950). Its largest populations are situated in the
Biebrza National Park (northeast Poland), which is the
biggest protected complex of swamps in the Central and
Western Europe, covering an area of 59 233 ha (Okruszko
1991). These populations are situated on mineral islands
among peat bogs and are subjected to spatial environmental isolation.
Materials and methods
Study sites and sampling
Our study on Cypripedium calceolus populations was
carried out in the Biebrza National Park. There are a few
hundred mineral islands among the widespread peat
bogs of the Biebrza river valley. Populations of lady’s
splipper were found on six of these islands. Three of the
mineral islands: Zabudnik (ZAB), Oparzelisko (OPA) and
Pogorzaly (POG) were chosen for the study (Fig. 1). The
islands differ in size, shape and vegetation cover (Brzosko
& Werpachowski 1998). C. calceolus plants present on a
given mineral island were regarded as the members of one
populations.
The populations of lady’s slipper were of different size
and during 13 years of study (1989–2001) considerable
fluctuations in populations’ size were observed (Brzosko
2002). The largest was the ZAB population, which in the
Fig. 1 Distribution of Cypripedium calceolus in Poland (redrawn from Polish Red
Data Book, Kaflmierczakowa & Zarzycki
2001) and localities of three Cypripedium
calceolus populations studied.
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 2499–2509
C L O N A L D I V E R S I T Y I N C Y P R I P E D I U M C A L C E O L U S 2501
year of the maximum abundance consisted of almost one
thousand ramets grouped in 159 clumps (Brzosko &
Werpachowski 1998; Brzosko 2002). The sizes of the two
other populations (OPA and POG) were similar, and in the
years of the greatest abundance each of them comprised
around 600 ramets (Brzosko & Werpachowski 1998; Brzosko
2002). The samples for allozyme study were collected in
1997 from three C. calceolus populations (ZAB, OPA and
POG). In our first study concerning genetic structure of
C. calceolus populations we analysed 431 shoots (Brzosko
et al. 2002). Most of the samples were collected as a single
shoot from different clumps. Samples collected in this way
did not allow for assessing clonal diversity and answer the
question as to whether the clump is one or a few individuals
in a genetic sense. Therefore, in this study, we increased
sample size in such a way that all ramets belonging to a
given clump were analysed. The total number of ramets
sampled was 722. In ZAB population we collected samples
from 302 ramets (out of 472 present), which belonged to 36
clumps; in OPA and POG populations we collected 202
ramets (out of 249 present) from 49 clumps, and 218 ramets
(out of 335) from 42 clumps, respectively.
Allozyme analysis
For each ramet sampled, one fresh leaf tip (approximately
2 cm long) was removed and placed into 1,5-mL tube. The
samples were then frozen in liquid nitrogen. Leaf tissue
was grounded in an extraction buffer with 2-mercapthoethanol
(Szweykowski & Odrzykowski 1990). Electrophoresis of
GOT enzyme system was carried out on horizontal 10%
starch gels. The other enzymes were screened using cellulose
acetate plates (Helena Laboratories, Beaumont, TX). Eleven
enzyme loci were assayed: malate dehydrogenase (Mdh);
alcohol dehydrogenase (Adh); esterase (Est); glutamate
dehydrogenase (Gdh); shikimic dehydrogenase (Skd);
phosphoglucose isomerase (Pgi); glutamate oxalate transaminase (Got); isocitric dehydrogenase-1 and -2 (Idh-1, Idh-2);
phosphoglucomutase (Pgm) and 6-phosphogluconate
dehydrogenase (6Pgd). The electrophoretic buffer system
tris-borate (Soltis & Soltis 1983) was used to resolve Got;
Est; tris-glicyne was used for 6Pgd, Pgm, Adh, Gdh; and
tris-citrate was used for Idh, Skd, Mdh, Pgi (Richardson et al.
1986). A locus was considered polymorphic if more than
one allele was detected.
Genetic variation and clonal diversity
The following measures of diversity were calculated using
tfpga software (Miller 1997): per cent polymorphic loci (P), the
mean number of alleles per locus (A), the average observed
(HO) and expected (HE) heterozygosity. Deviations from
Hardy–Weinberg equilibrium (HWE) at each polymorphic
locus in every population were tested using an exact test of
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 2499–2509
HWE with a Markov Chain algorithm. When multiple tests
were performed, a Bonferroni correction was applied.
Tests for linkage disequilibrium (LD) between loci for
each population were performed by generating exact probabilities of type-I error for the null hypothesis that a pair of
loci is unlinked. The test was performed for all locus pairs
within each population and over all populations with the
help of genepop version 3.2a (Raymond & Rousset 1995).
Fixation indices, FIS (inbreeding within individual in
population; inbreeding coefficient) and FST (an indicator of
the degree of differentiation among populations) were calculated based on Weir & Cockerham (1984) estimators ƒ
and θ, respectively, using the program fstat (Goudet
1995). Loci that showed LD in the entire data set were
excluded from FST calculations. Permuting over loci was
performed using 1000 replicates to generate 95% C. I. for
FIS and FST. The theoretical number of migrants entering
every population per generation (Nm) was estimated using
the formula Nm = (1 − FST)/4 FST (Wright 1951).
The program bottleneck (Cornuet & Luikart 1997) was
used to test for a recent reduction of effective population
size because alleles are generally lost faster than heterozygosity (Hedrick et al. 1986), and recently bottlenecked
populations will display an excess of heterozygosity relative
to that expected on the number of alleles.
As lady’s slipper reproduces both vegetatively and sexually, we also estimated within-population clonal diversity. All sampled ramets were sorted by multilocus
genotype based on the five polymorphic loci. Each of the
detected distinct multilocus genotype was assumed to be a
distinct genet. Two different measures of clonal diversity
were used in our study. The first was G/N, where G is the
number of genets and N is the number of ramets sampled.
G/N is the probability that the next ramet sampled will be
a different genotype. The second measure of genet diversity is the Simpson’s index corrected for the finite sample
size, D = 1 – Σ[{ni(ni − 1)]/[N(N − 1)}], where ni is the number
of ramets of the ith genet and N is the total number of
ramets sampled (Pielou 1969). Genet size was defined as
the number of ramets per genet in the population samples.
To investigate spatial genet structure we compared the
distribution of different multilocus genotypes in three
populations studied. Moran’s index was calculated using
the autocorg version 2.1 program (Hardy & Vekemans
1999). We used two-way anova (STATISTICA StatSoft 1997)
to evaluate the effect of distance and population affiliation
on Moran’s I index.
Results
Genetic structure within and between populations
Of the 11 loci examined, six (Adh, Est, Gdh, Mdh, Pgi and
Skd) were monomorphic in all populations studied, and
2502 E . B R Z O S K O , A . W R Ó B L E W S K A and M . R A T K I E W I C Z
the other five (Got, Idh-1, Idh-2, 6Pgd and Pgm) were
polymorphic for the species and in every population.
There were slight differences in allele frequencies between
populations (Table 1). Eleven resolved loci gave a total of
19 alleles with the mean number of 1.73 alleles per locus
(A). The proportion of polymorphic loci (P) was the same
in every population and equal 45.5%. The average
observed and expected heterozygosity values for the total
lady’s slipper data set over 11 loci were 0.151 and 0.178,
respectively. The average HE for the populations under
study ranged from 0.175 to 0.181. On the other hand, the
differences HO among populations were more pronounced
(Table 2). In ZAB population, characterized by the biggest
clumps (on average, 5.5 ramets per clump), we noted the
lowest value of HO. In OPA population, where number of
ramets per clump was the lowest (3.1), HO value was the
highest (Table 2).
Large deviations from HWE exist in all three populations (Fisher’s method, global test, P < 0.001). The Hardy–
Weinberg proportions were not found for Got in ZAB, OPA
and POG populations, for 6Pgd in ZAB and OPA populations, for Pgm in POG and for Idh-1 in ZAB and POG
populations. Idh-2 locus was in HWE in every population.
Table 1 Allele frequencies for five polymorphic loci in three
Cypripedium calceolus populations
Locus
Alleles
Populations
ZAB
OPA
POG
Got
a
b
c
d
e
a
b
a
b
a
b
a
b
0.018
0.165
0.024
0.193
0.600
0.206
0.794
0.600
0.400
0.582
0.418
0.047
0.953
0.029
0.159
0.071
0.306
0.435
0.194
0.806
0.694
0.306
0.706
0.294
0.082
0.918
0.053
0.143
0.043
0.271
0.490
0.101
0.899
0.575
0.425
0.559
0.441
0.053
0.947
6Pgd
Pgm
Idh-1
Idh-2
Table 2 Allozyme variation and clonal diversity within three populations of Cypripedium calceolus. N-number of ramets sampled, HEexpected heterozygosity, HO-observed heterozygosity, G-number
of distinct multilocus genotypes, G/N- clonal diversity, D-multilocus
genotypic diversity index
Populations
N
HE
HO
G
G/N
D
ZAB
OPA
POG
290
202
218
0.179
0.181
0.175
0.111
0.178
0.166
43
55
56
0.15
0.27
0.26
0.94
0.98
0.97
The overabundance of homozygotes was the most common
form of deviations from Hardy–Weinberg equilibrium. FIS
value in ZAB population (0.380) was significantly higher
than in other populations (0.024 and 0.055 in OPA and
POG, respectively).
The amount of genotypic linkage disequilibria (LD)
showed differences among populations, being the largest
in ZAB population. We found significant LD between the
following pairs of loci: Got-Pgm, Got-Idh-1 and 6Pgd-Idh-2
in ZAB population, Idh-1-Idh-2 in POG population and
6Pgd-Idh-2, Idh-1-Idh-2 in the entire data set. No genotypic
disequilibria were found in the OPA population, where
reproduction from seeds was most intense.
The genetic differentiation, calculated over four loci
(Got, Idh-1, 6Pgd and Pgm), among three studied populations
was small (FST = 0.014), albeit statistically significant (P <
0.05), after permuting genotypes within total using fstat.
The pairwise FST estimates revealed larger genetic differentiation between geographically closer OPA and POG (FST =
0.020) than between ZAB and POG (FST = 0.005) and ZAB
and OPA (FST = 0.017). The estimated level of effective gene
flow among populations studied was very high (Nm = 18).
Almost identical FST and Nm values were obtained when
Idh-2 locus was included in the analysis.
Tests for genetic signatures of a recent population bottleneck using the program bottleneck did not reveal any evidence for a population bottleneck in all samples studied.
Clonal diversity and spatial genet structure
The clumps of Cypripedium calceolus, discriminated in
space, consisted of different number of genets, as defined
by multilocus genotypes at five polymorphic loci. The
average genet numbers per clump were 2.7 ± 2.3 (ZAB),
2.1 ± 1.4 (OPA) and 2.6 ± 1.4 (POG). The maximum
number of genets per clump (seven) was found in ZAB
population. Different clumps of the same size consisted
of different number of genets. For example, one clump
of 22 ramets was a single genet, while another one, with
identical number of ramets was made up of six genets.
Despite such a big variation, we found a significant
positive correlation between the number of ramets per
clump and the number of genets per clump in all the
populations studied (r = 0.72–0.78, P < 0.05). The number
of ramets per genet also varied considerably. The genet
size ranged from 2.7 ramets per genet in OPA and POG
populations (SD ± 3.2 and ± 3.8, respectively) to 4.9 ± 8.5 in
ZAB population. The largest genets (38 and 39 ramets)
were observed in ZAB population, where the clumps were
the biggest. Thus, the clump size was influenced by the
genet number in a clump, but primarily by the genet size
(the number of ramets in a genet).
Using five polymorphic loci, we separated 109 different
multilocus genotypes in three C. calceolus populations. The
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 2499–2509
C L O N A L D I V E R S I T Y I N C Y P R I P E D I U M C A L C E O L U S 2503
majority of genotypes (67%) were population-specific.
Only 10 out of 109 genotypes were present in all populations. These common genotypes made up about 18% of all
genotypes in OPA and POG, and 23% of the genotypes in
ZAB. The number of multilocus genotypes ranged from
43 to 56 in different populations (Table 2). Within a given
population, around 50% of genotypes were populationspecific. The other genotypes were common for different
combinations of populations pairs (Fig. 2).
Clonal diversity (D) was similar in all three populations
(0.94–0.98). The probability of finding a new genet (G/N)
was higher in OPA and POG populations (0.27 and 0.26)
than in ZAB population (0.15). The clonal diversity value,
quantified as G/N ratio, was significantly lower in ZAB
than in the other two populations (chi-square test, P
< 0.01). Most multilocus genotypes (75.2%) occurred only
once or twice within each population. Only 2.7% multilocus genotypes were observed more than 10 times in the
whole data set. The most abundant genotypes were
present in at least two populations.
Patterns of spatial structure were population-specific. In
ZAB and OPA populations the clumps of lady’s slipper
were distributed throughout the whole area of mineral
elevations. On the other hand, the clumps in POG population were located on the edge of this mineral island
(Fig. 3 a,b,c). Different distances separated both clumps
and genets in space. The distances between distinct genets
within a given clump were very small (a few cm) and it was
not possible to distinguish them in space. Examination of
the distance between pairs of plants from different clumps
sharing the same genotype allowed us to infer the potential
for clonal spread. The distances among multilocus genotypes from different clumps ranged from 0.4 m to 13.7 m in
ZAB, from 0.8 m to 63 m in OPA, and from 1 m to 343 m in
POG populations. We noted that the probability of finding
the same multilocus genotype is independent of the distance
(Moran’s I index was not significant; two-way anova,
F = 0.628–1.340; d.f. = 9–24; P > 0.1).
Discussion
Genetic structure of populations
Fig. 2 The frequency of multilocus genotypes (common and
unique) in the three Cypripedium calceolus populations.
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 2499–2509
Genetic variation within Cypripedium calceolus populations
from the Biebrza valley found in this study is relatively
high. Although the sample size was increased almost twofold when compared to the previous study (Brzosko et al.
2002), the values of genetic variability (P and A) remained
the same, while HO and HE values slightly changed.
The percentage of polymorphic loci was 45.5% in all the
populations, and mean number of alleles per locus was
1.73. Our estimates of genetic variation are in good
agreement with the results reported by Hamrick & Godt
(1989) for all plants and with the estimates reported for
rare species (Karron 1991; Gitzendanner & Soltis 2000).
The values of P and A reported by Hamrick & Godt (1989)
for all plants are slightly higher than in C. calceolus populations from the Biebrza valley, while the level of HO is
lower than in our study. The higher values of genetic
variability parameters (P and A), in comparison to our
results, were noted by these authors for animal-pollinated
plants, but lower for species belonging to long-lived
perennials.
2504 E . B R Z O S K O , A . W R Ó B L E W S K A and M . R A T K I E W I C Z
Fig. 3 Spatial structure of clumps and
genets in ZAB (a), OPA (b) and POG (c)
populations. Genets, which belong to
the same clump are delimited by circle.
Each symbol in a given clump indicates
different genotype. Genotypes unique for
one population are marked by triangles in
ZAB, diamonds in OPA, squares in POG.
Genets, which have identical multilocus
genotypes are connected by lines.
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 2499–2509
C L O N A L D I V E R S I T Y I N C Y P R I P E D I U M C A L C E O L U S 2505
Fig. 3 Continued
In some species belonging to the Cypripedium genus
lower values of genetic variation for C. reginae (Case 1994),
for C. acaule (Bornbush et al. 1994), for C. parviflorum var.
parviflorum (Wallace & Case 2000) or completely lack of
variation for C. arietinum populations (Bornbuch et al.
1994; Case 1994) were found. Conversely, Wallace & Case
(2000) found a very high level of polymorphism (P =
81.8%), high number of alleles per locus (A = 2.4–2.5),
and high expected heterozygosity (HE = 0.22 – 0.29) in the
American populations of northern C. parviflorum var.
pubescens and C. parviflorum var. makasin. In seven out of
eight Estonian populations of this species (Kull & Paaver
1997) the level of HO were much higher (HO = 0.40–0.53)
than in the Biebrza valley populations (HO = 0.151). It
seems, that the genetic variation within Cypripedium genus,
and in different populations of a given species belonging
to this genus can vary substantially (Case et al. 1998). This
was also noted for other orchids (Scacchi et al. 1991;
Ackerman & Ward 1999; Wong & Sun 1999; Gustafsson
2000).
Genetic differences among populations of C. calceolus
were smaller than the genetic variation within populations. The low FST value (0.014) in C. calceolus with high
gene flow (average Nm = 18) may explain the maintenance
of little genetic divergence among populations (Young
et al. 1996). Because some loci were not in Hardy-Weiberg
proportions and showed linkage disequillibria, the interpretation according FST and number of migrants must be
taken with caution. However, the exclusion of Idh-2 locus
that was in linkage disequillibrium with other loci did not
change FST and Nm values. The gene flow may occur over
long distances by seeds rather than pollen dispersal in
orchid species (Peakall & Beattie 1991). It should be noted,
however, that this value of Nm represents historical average levels of gene flow and may not represent present-day
levels. The observed high rate of gene flow among C. calceolus populations in the Biebrza valley may suggest that
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 2499–2509
the populations studied are not at the genetic equilibrium
under the present levels of gene flow, with populations
derived from each other in the recent past. Rapid and
recent fragmentation of a big population should result in
populations being more similar genetically than if populations were isolated for longer periods of time.
Fragmented populations may be exposed to reduced
contemporary gene flow and subsequent loss of allelic variation owing to the long distances between neighbouring
localities (Frankel & Soulé 1981; Gustaffson 2000). In our
study, we did not detect the loss of genetic variation in the
lady’s slipper populations, although they were environmentally isolated. Tests for genetic signatures of a recent
population bottleneck, using the program bottleneck did
not reveal any evidence for a recent population bottleneck
in all the samples studied. However, fixation indices (FIS)
showed significant positive deviation from HWE, which
was the highest in ZAB population. The high and significant FIS value in ZAB (0.380), where the vegetative reproduction was more intense than in two other populations
(Brzosko 2002), may indicate increased level of inbreeding.
Deviations from HWE suggest that populations may be
genetically substructured (sampling different population
patches that differ in allele frequency would probably
generate high values of the positive fixation index;
Wahlund 1928).
The amount of genotypic linkage disequilibria also
suggests that two out of three populations (ZAB and POG)
are inbreeding and not panmictic. The populations could
have been established most recently through multiple
colonization. In combination with the rare recruitment
from seeds seen today (i.e. most reproduction may be
vegetative; Brzosko 2002) this indicates that recombination
has not been sufficient to break down the patterns of
linkage disequilibria in the populations acquired during
postglacial migration. Only in the OPA population, due to
more intense sexual reproduction than in the other two
2506 E . B R Z O S K O , A . W R Ó B L E W S K A and M . R A T K I E W I C Z
populations and the lowest vegetative reproduction, the
LD was broken down.
If C. calceolus populations are not panmictic, and sexual
reproduction only takes place occasionally and to some
extent among closely related individuals with possibly low
success of recruitment from seeds, the genotypic variation
found must be the result of gene flow in the past.
Clonal diversity
We found 109 different genotypes in three populations.
The majority of them (67%) were unique for a single population and 9.2% were common for all populations. To our
knowledge, only one example exists (Anemone nemorosa, a
tetraploid species) with the number of genotypes higher
than in our study (among them 95% were unique for a
single population, Stehlik & Holderegger 2000). Thus, the
number of genotypes in three C. calceolus populations seems
to be relatively high. In other studies genotypic variability
was lower, especially in Haloragodendron lucasii, where only
six multilocus genotypes were found (Sydes & Peakall 1998)
and in orchid Microtis parviflora, where seven genotypes in
five populations were observed (Peakall & Beattie 1991).
Relatively high numbers of multilocus genotypes in populations and a fairly large number of genotypes shared
between pairs of populations studied indicate that populations were established by more than one seed and/or that
sexually derived individuals have become established
after colonization. On the other hand, around 50% of
genotypes detected in every population were populationspecific, which may indicate that populations have
diverged in respect to genotypic composition. Thus, the
differences in multilocus genotypic composition among
C. calceouls populations may suggest that the divergence
is larger than one would expect from conventional FST
estimate. If there are only slight differences in allele
frequencies between given populations, the genotypic
distribution of these populations at a single locus will be
very similar. However, this does not hold when multiple
locus genotypes are taken into account. The frequency of
multilocus genotypes in given populations becomes more
different the more loci are considered (Allendorf et al.
2001), as even only minor shifts in allele frequencies among
populations are observed.
Small populations tend to have fewer multilocus genotypes and lower genotypic diversities than large populations (Murawski & Hamrick 1990). In our study all
populations had similar values of those parameters
(number of genotypes and Simpson’s index), irrespective
of their sizes. Clonal diversities measured by Simpson’s
index (D = 0.94–0.98) were higher in lady’s slipper populations than the mean value (D = 0.62) reported for multiclonal populations (Ellstrand & Roose 1987). High levels of
clonal diversity were also noted for many other plant spe-
cies (e.g. Hosta clausa, Chung 1995; Elliottia racemosa, Godt
& Hamrick 1999; Allium tricoccum var. burdickii, Vasseur
2001). The values of another measure of clonal diversity (G/
N) index were equal to 0.26 and 0.27, for POG and OPA
populations, respectively. They were higher than the mean
(0.17) reported by Ellstrand & Roose (1987) for the clonal
species, while G/N index in ZAB population was lower
(0.15). Thus, the probability of origination of a new genotype in ZAB population was almost two times lower than
in the other two populations. We suggest, that low clonal
diversity (G/N index) and the lowest heterozygosity
(HO = 0.111) in ZAB population was a consequence of particularly intensive vegetative reproduction (Brzosko 2002).
This is supported by the biggest clump and genet sizes
noted here (mean 5.5 ramets per clump in ZAB vs. 3.1 and
4.5 in OPA and POG populations, respectively; and 4.9
ramets per genets in ZAB vs. 2.7 in OPA and POG). Other
authors also suggested that vegetative reproduction might
lower the levels of clonal and genetic diversity (Chung
1995; Sydes & Peakall 1998).
Generally, despite widespread clonal reproduction, we
observed relatively high genetic variation and clonal diversity. Although, the recruitment is very low in populations
studied, it seems sufficient to maintain genetic variability.
The mean number of juvenile individuals found during
11 years of study was 3.5%, 2.7% and 7.2% in ZAB, POG
and OPA populations, respectively. New individuals usually appeared within or in vicinity of the clumps. This may
result in clumps usually consisting of many genets. For
example, in one of the permanent plots studied in 1997, 14
juvenile individuals appeared, six of which were located in
the area covered by the clumps, six others at the distance of
20–30 cm from the clumps and only two juveniles were
located at the distance of 0.5–1 m (Brzosko 2002). Successfully established seedlings are thus likely to be located near
to its mother plant (Murawski & Hamrick 1990). Many
authors agree that even a low rate of seedling recruitment
might be sufficient to maintain or even increase local
genetic diversity (e.g. Soane & Watkinson 1979; Stehlik &
Holderegger 2000).
Spatial genet structure in the populations
Within plant populations, individuals with identical
multilocus genotypes are located significantly closer than
nonidentical ones (Murawski & Hamrick 1990; Chung &
Epperson 1999; Kudoh et al. 1999). In contrast, we noted
that the probability of finding the same multilocus
genotype is independent of the distance. C. calceolus
individuals with identical multilocus genotypes can be
found 0.4 m to 343 m apart. In the POG population the
distance between identical genotypes averaged 155 m
and was significantly larger than in the ZAB and OPA
populations (6.4 m and 22.3 m, respectively). This suggests
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 2499–2509
C L O N A L D I V E R S I T Y I N C Y P R I P E D I U M C A L C E O L U S 2507
that the clones may expand on an extensive area. Several
identical genotype pairs were separated by long distances
(even over 300 m) in both POG and OPA populations.
This indicates that clones may undergo secondary displacements by various factors, such as activity of animals
(especially elks, Brzosko, personal observation), and
humans (mineral islands were battlegrounds during
the Second World War). The origin of the most spatially
separated genotypes calls for alternative explanations.
Long distances between identical multilocus genotypes
may indicate that they originated from different seeds by
chance. The probability that two identical multilocus
genotypes can originate by sexual reproduction may be
very high in the case of some genotypes, especially in the
presence of linkage disequilibria. Due to lack of HWE in
all the populations it was not possible to estimate the
probability of obtaining two or more identical genotypes
arising from sexual reproduction. In the experiment, with
the exclusion of pollinators, autogamy was not observed
(Brzosko, unpublished). However, this experiment was
carried out in ZAB population only and the sample size
was small when compared to population size. Thus,
autogamy may occur in POG & OPA. Ortiz-Barney &
Ackerman (1999) wrote that in some populations of
Encyclia cochleata self-pollination occurs and in others it is
absent. Moreover, the experiment was carried out in such
a way, that flowers on the same shoot were only observed.
It is possible that gene flow by pollen between different
shoots belonging to the same genet can occur. As orchids
seeds are minute, numerous and wind-dispersed if autogamy does occur, genetically identical seeds may spread
and many plants with the same multilocus genotypes may
appear even at long distances. Detailed breeding studies
are needed to confirm these hypotheses.
The field measurements of the distances between
identical genotypes allowed us to estimate the minimum
age of the clones. The annual increment of rhizomes in
lady’s slipper is about 1–1.5 cm (Kull 1999; Brzosko &
Werpachowski, personal observations). Thus, the age
of the clones ranged from about ten years to more than
30 000 years for the most widespread ones. This is an
underestimation because rhizome propagation does not
proceed along a straight line, but has a zigzag character (Kull 1999). However, it is rather improbable that
clones are over 30 000 years old. The history of mineral
islands is younger — they originated after the last glaciation
about 13 000 years ago (Ber 2000).
Conservation
The longevity of genets, the presence of recruitment and
the outcrossing breeding system are factors maintaining genetic diversity in the lady’s slipper. Knowledge of
the numbers and size of genets within populations is
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 2499–2509
important for studies on clonal plants, especially on rare
and endangered species (Esselman et al. 1999). The
occurrence of clonality in endangered plants can have
several important implications for their conservation. In
many cases clonal growth slows the loss of genetic
diversity within populations (Cook 1983). The author also
reported that plants with independent ramets could
spread the risk of mortality among ramets, thus reducing
the probability of genets’ death. This study, however, has
demonstrated that the extent of clonality in lady’s slipper
and presumably in other plants can not be accurately
estimated by field observations and has emphasized the
importance of genetic methods. Genetic studies are particularly desirable in the case of endangered plants species
with a large potential for clonality.
Acknowledgements
We are grateful to Prof M. Gêbczy~ski for the comments on the
earlier version of this manuscript and Prof M. Konarzewski for
improving our English. We also thank an anonymous reviewer for
valuable suggestions. This work was supported by Polish Scientific Committee (KBN grant no. 6P04C 10121).
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Emilia’s Brzosko main interests are in the understanding of demographic processes in populations of orchids. Ada Wróblewska is a
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