Genetic variation and genotypic diversity in Epipactis helleborine

Genetic variation and genotypic diversity in Epipactis helleborine

Plant Syst. Evol. (2004)
DOI 10.1007/s00606-004-0140-4
Genetic variation and genotypic diversity in Epipactis helleborine
populations from NE Poland
E. Brzosko, A. Wróblewska, and I. Tałałaj
University of Białystok, Institute of Biology, Department of Botany, Białystok, Poland
Received April 28, 2003; accepted February 9, 2004
Published online: August 17, 2004
Ó Springer-Verlag 2004
Abstract. Enzyme electrophoreses were used to
estimate genetic variation in five populations of
Epipactis helleborine from two National Parks
(Biebrza and Wigry) in northeast Poland. It has
been proved that populations from these two regions
differed in genetic structure, with the populations
belonging to the Biebrza group having a higher level
of genetic variation than those from Wigry. The
number of polymorphic loci (P) ranged from 18.2%
to 40.9% and the mean number of alleles per
polymorphic locus (Ap) from 2.25 to 2.80. Although
the observed heterozygosity (Ho) was lower than the
expected one (He) in each population, the values of
Ho and He were 2–3 times lower in the populations
from Wigry than in those from Biebrza. Excluding
the LUK population with the smallest genotypic
diversity, the majority of 345 distinct multilocus
genotypes occurred only once, sporadically 2–4
times in each population and their frequency ranged
from 0.2% to 3.7%. Moreover, factors shaping
genetic structure of E. helleborine and their intensity
varied in the populations studied. Reproduction
from seeds seems to influence greatly the genetic
variation of the populations from the Biebrza
National Park, while an assortative mating between
related individuals or population size appears to be
more important in the case of the populations from
the Wigry National Park.
Key words: Allozymes, gene flow, genotypes,
Epipactis helleborine, orchids.
Introduction
A main goal of both evolutionary and conservation biology is to know about population
genetic structure and understanding its causes
and consequences. There is now much evidence
that the major determinants of the amounts of
genetic variability in plant species are: historical aspects, human activity, narrow habitat
preference, natural selection and gene flow
among populations, drift, founder effect, geographical distribution, biological properties of
given species (especially mating system) (Karron et al. 1988; Case 1993, 1994; Chung 1995).
The importance of the above mentioned factors
differs between species and populations.
In the case of rare plants, such as orchid
species, size of populations and the problem
of isolation is often very important in shaping
the genetic structure of populations. Species
with more continuously distributed populations should experience more gene flow than
species with discrete, isolated populations
and, therefore, have relatively lower variation
among the populations (Ellstrand and Elam
1993, Hamrick and Godt 1989, Chung 1996).
Moreover, geographically restricted species
tend to have lower levels of genetic variation
within populations (Karron 1987, Hamrick
E. Brzosko et al.: Genetic variation in Epipactis helleborine
and Godt 1989, Loveless and Hamrick 1984),
although some of them possess relatively high
level of genetic variation (Gitzendanner and
Soltis 2000).
The general distribution area of E. helleborine extends from Europe to North Africa,
southern Siberia and the Himalayas (Hultén
and Fries 1986, Tuulik 1998). The species was
introduced from Europe to North America
over 100 years ago and is now common in
eastern Canada and north-eastern USA (Light
and MacConaill 1991). It occurs in an
extensive range of forest habitats, mainly in
damp coniferous and mixed forests. Sometimes
E. helleborine occurs in dry habitats – at
roadsides, in dry boreal forests or even on
coastal dunes and in urban sites (Adamowski
and Conti 1991, Tuulik 1998, Ehlers and
Pedersen 2000). Though E. helleborine as an
orchid species is under protection in Poland, it
is relatively common in comparison with other
orchids and occupies more differentiated habitats, both natural and changed by human
activity (Adamowski 1998, Adamowski and
Conti 1991). E. helleborine is a perennial herb
which flowers from the middle to the end of
July. E. helleborine is an entomophilous species
pollinated by social wasps (Piper and Waite
1988, Ehlers and Pedersen 2000), but some
authors suggest that self-pollination may occur
(Waite et al. 1991, Squirrell et al. 2001).
Individuals are often multistemmed (Squirrell
et al. 2001; personal observations), indicating
vegetative spread occurring in this species.
This paper is a part of wider studies
concerning genetic variation of island populations of orchids in north-east Poland (Brzosko
et al. 2002a, b; Brzosko and Wróblewska 2003a,
b). It is interesting to compare the genetic
structure of populations existing in sites, which
differ in many characters (topography, type of
vegetation). The purposes of this paper were 1.
to assess the levels of genetic variation and
genotypic diversity within E. helleborine populations from northeast Poland and compare
them with the results from others countries, 2. to
investigate genetic differentiation among populations from one geographic region and between
regions (Biebrza National Park and Wigry
National Park).
E. helleborine, in contrast to other orchid
species, was genetically studied in a few
countries and it is known as a species with
high levels of genetic variation (Harris and
Abbott 1997, Hollingsworth and Dickson
1997, Ehlers and Pedersen 2000, Pedersen and
Ehlers 2000, Squirrell et al. 2001). Due to the
relatively rich data referring genetic variation,
E. helleborine may be used as a model species to
explain both genetic and evolutionary processes within its geographic range.
Material and methods
Study sites and sampling procedures. Five populations of E. helleborine, three from the Biebrza
National Park and two from the Wigry National
Park, have been investigated. Both protected areas
are localized in north-east Poland and are separated by c. 100 km (Fig. 1). The Biebrza National
Park is known as the largest complex of peatbogs
of the country, while the Wigry National Park is
mostly covered by forest communities, being under
the domination of semi-natural, mixed forests (over
60%). One of the characteristic elements of the
Wigry National Park is the presence of small lakes
and peatbogs, often localized among large forests.
Two populations were investigated in the Wigry
National Park. The distance between them is about
3 km. The LUK population, occupying c. 1700 m2,
is situated on the slope of a hill at the coast of Białe
Lake where Corylus avellana dominates. The LUB
population exists at the margin of mixed pine forest
(with domination of Pinus sylvestris) close to a
didactic asphalt pathway and its total area is c.
120 m2. The island character of E. helleborine
populations from Biebrza is the result of their
discrete distribution and localization on mineral
islands among peatbogs. The three E. helleborine
populations from the Biebrza National Park, being
2–4 km apart, were situated on islands named
Zabudnik (ZAB), Oparzelisko (OPA) and Długi
Gra̧d (DLUG). These mineral elevations are about
1 m above peat level and their areas range from
5000 to 7500 m2.
In ZAB only a very small piece of the oaklinden-hornbeam community exists, and the remaining area is covered by shrubs and reed grass
E. Brzosko et al.: Genetic variation in Epipactis helleborine
Fig. 1. Localities of Epipactis helleborine populations
studied in the Biebrza and Wigry National Parks
patches. OPA island is covered by birch trees, 15–
20 years old and the herb layer is typical for a
hornbeam forest. DLUG is dominated by shrubs of
small lime and hazel.
In 2002 samples for genetic analyses were
collected. A total of 342 leaf samples collected from
three populations of E. helleborine from Biebrza and
218 samples from two populations from Wigry were
used to estimate of genetic variation by means of
allozyme. In the smallest populations (LUB and
DLUG) samples from all found, not damaged
individuals were taken. In the other three populations (ZAB, OPA, LUK) individuals are dispersed
over the large area, therefore fragments of leaves
(from all living and not damaged individuals) were
collected from the permanent plots where long-term
demographic investigations are conducted.
An experiment which aimed at investigating
self-pollination was carried out. Fifteen inflorescences were covered with cotton net to evaluate
seed production without pollinators (autogamy).
These inflorescences were covered before the
flowers were open and the cotton net remained in
place until the fruiting time.
Allozyme electrophoresis. About 50 mg of leaf
sample per individual was homogenized in 30 ll of
extraction buffer at pH 7.5 containing polyvinylpolypyrolidone (PVPP) and 2-mercaptoethanol (Hollingsworth et al. 1995). Only in preparation of the
extract for the PRX (peroxidase) enzyme system we
homogenized the tissue with water (Szweykowski
and Odrzykowski 1990). The following 15 enzyme
systems were examined: alcohol dehydrogenase
(ADH: E.C. 1.1.1.1), diaphorase (DIA: E.C.
1.6.99), glutamate dehydrogenase (GDH: E.C.
1.4.1.2), glutamate oxaloacetate transaminase
(GOT: E.C. 2.6.1.1), phosphoglucose isomerase
(GPI: E.C. 5.3.1.9), isocitrate dehydrogenase
(IDH: E.C. 1.1.1.41), 6-phosphogluconate dehydrogenase (6PGD: E.C. 1.1.1.49), phosphoglucomutase
(PGM: E.C. 5.4.2.2), peroxidase (PRX: E.C.
1.11.1.7), malate dehydrogenase NADP+ (ME:
E.C. 1.1.1.40), malate dehydrogenase NAD+
(MDH: E.C. 1.1.1.37), mannose phosphate isomerase (MPI: E.C. 5.3.1.8), shikimic dehydrogenase
(SKD: E.C. 1.1.1.25), superoxide dismutase (SOD:
E.C. 1.15.1.1), triose-phosphate isomerase (TPI:
E.C. 5.3.1.1). GDH, GOT, PRX were resolved on a
10% Lithium-borate horizontal starch gel at pH 8.2/
8.3 with a voltage of 330V, while DIA, IDH, MDH,
ME, MPI, 6PGD, SOD, SKD, TPI were resolved on
a 10% Histidine-citrate buffer at pH 7.0/7.0 with
170V (Soltis and Soltis 1989). The other enzyme
systems: ADH, GPI, PGM were screened using
Titan III cellulose acetate plates (Helena Laboratories, Beaumont, TX), which were resolved onto
Tris-glicine buffer at pH 8.6 (Richardson et al. 1986)
and run at 200V for 40 minutes. The enzyme staining
recipes were based on Soltis and Soltis (1989) and
Richardson et al. (1986) with modifications.
Genetic variation and genotypic diversity. The
following measures of diversity were calculated
using TFPGA software (Miller 1997): percent of
polymorphic loci (P), the mean number of alleles
per polymorphic locus (Ap), 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 HWE with a Markov Chain
algorithm. When multiple tests were performed, a
Bonferroni correction was applied.
E. Brzosko et al.: Genetic variation in Epipactis helleborine
Fixation indices, FIS (an inbreeding coefficient)
and FST (an indicator of the degree of differentiation among populations within regions only) were
calculated based on Weir and Cockerham (1984)
estimators, using the program FSTAT 2.9.3.2
(Goudet 2002). Positive or negative significance of
FIS was tested using 1000 randomization (default
parameter in FSTAT) for each locus and across
loci for each population. Theoretical number of
migrants entering every population per generation
(Nm) was estimated using the following formula:
Nm ¼ ð1 FST Þ=4 FST (Wright 1951). In addition,
Nei’s (1978) unbiased genetic identity was calculated among the populations studied.
To test genetic differentiation between Biebrza
and Wigry regions, among populations between
these two regions and among populations within
regions a hierarchical analysis Fxy was performed
using the program ARLEQUIN 2.000 (Schneider
et al. 2000). A hierarchical F-statistic was also
chosen to estimate the number of migrants (Nm)
among populations between regions.
The parameters of within-population genotypic
diversity were also estimated. All sampled ramets
were sorted by a multilocus genotype based on the
nine polymorphic loci. Each of the detected distinct
multilocus genotypes 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
P corrected for the finite sample
size, D ¼ 1 ½fni ðni 1Þ=½NðN 1Þg, where ni
is the number of ramets of the ith genet and N is the
total number of ramets sampled (Pielou 1969). A
genet size was defined as the number of ramets per
genet in the population samples.
PCA analysis. We investigated the genetic
relationship within and among populations using
Principial Component Analyses (PCA). We performed the PCA analyses based on the allozyme gene
frequency data by application PCAgen 1.2. software
(Goudet). An important characteristic of this
program is that it tests the significance of the total
inertia as well as individual PCA axes inertia by using
a randomization procedure (Manly 1997). Therefore, it allows to avoid interpreting of nonsignificant
axis. To test the significance of individual axis
inertia, 1000 randomizations of genotypes were
performed. The genotypes were permutated among
the samples and a PCA was realized on each
permutated data set. The proportion of values larger
than or equal to the observed one was an unbiased
estimate of the P-value of the test (Goudet).
Results
Demographic properties. The five E. helleborine populations studied differed in size. The
number of individuals on the permanent plots
in the OPA and ZAB populations were 221
and 198, it was evaluated that the total number
of individuals in all populations was about two
times larger. In the LUK there were 400
individuals observed on the permanent plot
and it applied to 3/4 of all individuals. The
LUB and DLUG populations were the smallest and consisted of 39 and 108 individuals,
respectively. The proportion of flowering
individuals varied among the study populations ranging from 4.5% in the LUK to 71.1%
in the LUB population.
In the pollinator-exclusion experiment only
3 fruits were set on 15 covered inflorescences
(295 flowers).
Genetic variation. For 15 enzyme systems,
22 loci were resolved in all E. helleborine
populations. Nine polymorphic loci were detected (Got-1, Gpi-2, Idh-1, Idh-2, Mdh-1, Mdh2, Pgm, Skd and Tpi-2). Monomorphic loci
included: Adh, Dia-1, Dia-2, Gdh, Got-2, Gpi-1,
Me, Mpi, 6Pgd, Prx-1, Prx-2, Sod, Tpi-1.
Numbers of polymorphic loci were higher in
populations of E. helleborine from the Biebrza
National Park (9 in OPA and DLUG and 8 in
ZAB) than in populations from the Wigry
National Park (6 in LUK and 4 in LUB;
Appendix 1). The mean number of alleles per
polymorphic locus (Ap) ranged from 2.25 in the
LUB population to 2.80 in the OPA population, while the mean number of alleles per locus
(A) varied from 1.22 to 1.72. Both parameters
were higher in the populations from the Biebrza
than from the Wigry group (Table 1). The
proportions of polymorphic loci were also
higher in populations from Biebrza and ranged
E. Brzosko et al.: Genetic variation in Epipactis helleborine
from 18.2% in the LUB population to 40.9% in
the OPA population. The observed heterozygosity (Ho) was lower than the expected one
(He) in each population. The values of Ho and
He were two to three times lower in populations
from Wigry than in populations from Biebrza
(Table 1). The Mann-Whitney U-test showed
significant differences between Biebrza and
Wigry groups in Ho ðU ¼ 156:5; p < 0:05Þ and
in He ðU ¼ 131:0; p < 0:01Þ. Most of variable
loci were in H-W expectation. The Tpi-2 locus
was at non-equilibrium in all populations, and
Idh-1 locus was in H-W equilibrium only in the
DLUG population. On the other hand Pgm,
Got-1, Mdh-1 and Idh-2 loci were in HWE in all
populations, in which they were detected.
Generally, significant departures from HWE
(FIS>0) were observed in 17 out of 36 tests. In
16 cases (out of 17) an overabundance of
homozygotes was detected (FIS ranged from
0.101 to 1; Table 2), while for the Skd locus in
the OPA population an excess of heterozygotes
was found (FIS ¼ 0:443).
Genetic identity (I) among populations
ranged from 0.826 to 0.964. Moreover, higher
genetic identity values (I) between populations
from Biebrza than between populations from
the Wigry National Park were found (Table 3).
The estimated levels of gene flow between
populations within each region varied from
1.72 to 5.56 (Table 3). It was reflected in FST
values between population pairs. The greatest
Table 1. Genetic variation and genotypic diversity in five Epipactis helleborine populations in NE Poland.
N – number of ramets sampled, P – percent of polymorphic loci (%), A – mean number of alleles per locus,
Ap – mean number of alleles per polymorphic locus, Ho – observed heterozygosity, He – expected heterozygosity, G – number of genotypes, G/N – clonal diversity, D – Simpson’s diversity index
OPA
ZAB
DLUG
LUK
LUB
N
P
Ap
A
Ho
He
G
G/N
D
201
108
33
192
26
40.9
36.4
40.9
27.3
18.2
2.80
2.75
2.55
2.33
2.25
1.72
1.63
1.63
1.37
1.22
0.158
0.091
0.103
0.053
0.049
0.164
0.119
0.154
0.081
0.058
194
87
32
29
18
0.97
0.81
0.97
0.15
0.70
0.99
0.99
0.99
0.90
0.97
Table 2. Fixation index (FIS) for five Epipactis helleborine populations in NE Poland. The values were
significantly different from expectation under Hardy-Weinberg equilibrium when: *P < 0.05 **P < 0.01
and ***P < 0.001
Loci
Biebrza
Wigry
OPA
ZAB
DLUG
LUB
LUK
overall
Got-1
Gpi-2
Pgm-2
Idh-1
Idh-2
Skd-2
Mdh-1
Mdh-2
Tpi-2
)0.007
0.280**
)0.141
0.301***
)0.095
)0.443***
0.068
0.096
0.106**
)0.006
0.074
0.066
0.681***
–
)0.019
0.109
0.101*
0.842***
0.183
0.232*
0.197
0.458**
)0.016
)0.067
0.448*
)0.066
1.000***
–
)0.112
0.153
1.000***
–
–
–
0.791***
–
–
0.314**
)0.018
0.721***
–
–
0.194*
)0.018
0.832***
0.034
0.158*
0.051
0.632***
)0.022
)0.109
0.164
0.181*
0.556***
overall
0.018**
0.205*
0.263*
0.203*
0.225**
0.183*
E. Brzosko et al.: Genetic variation in Epipactis helleborine
FST were observed among populations belonging to the Biebrza group and the LUB
population (Table 3).
The results of the hierarchical F-statistic
indicate that differences exist among populations between regions (FST) and among populations within regions (FSC). FCT value was
low and not significant indicating the lack of
genetic differentiation between Biebrza and
Wigry regions (Table 4). The migration rate
among populations between regions (Nm)
gave a value of 0.89 migrants per generation.
The majority of the variation was partitioned
within populations (76.30%), with smaller
amounts among populations (18.28%) and
between regions (5.43%; Table 4).
Genotypic diversity. Among five E. helleborine populations studied, 345 different multilocus genotypes were detected. The highest
number of different multilocus genotypes (194
Table 3. Genetic differentiation among five Epipactis helleborine populations in NE Poland. I –
Nei’s unbiased genetic identity, FST – an indicator
of the degree of differentiation among populations,
Nm – mean number of migrants per generation,
*P<0.05
I
FST
Nm
Biebrza
OPA-ZAB
OPA-DLUG
ZAB-DLUG
0.923
0.964
0.957
0.13*
0.04*
0.08*
1.72
5.66
2.88
Wigry
LUK-LUB
0.832
0.11*
1.94
among 201 samples analysed) was detected in
the OPA population, whereas in the LUK
population with similar sample size (192) only
29 genotypes were found. The most common
genotype in this population was represented by
41 shoots (21.4%) and three others by 23–24
shoots (12.0–12.5%). Excluding the LUK
population, the majority of genotypes occurred only once, sporadically 2–4 times in
each population and their frequency ranged
from 0.2% to 3.7%. Most of distinct multilocus genotypes were unique for a given
population. Only 12 of them were common
for 2 or 3 populations and the frequency of
these genotypes was very low.
Genotypic diversity measured by G/N was
the highest in the three populations from
Biebrza, especially in the OPA and DLUG
populations. In contrast, the LUK population
only showed a 15% chance of a new E.
helleborine individual being a new genotype.
The values of the genotype diversity index (D)
were very similarly ranging from 0.9 in the
LUK population to almost 1 in the three
populations from the Biebrza National Park
(OPA, ZAB, DLUG; Table 1).
Principial Component Analyses. A twodimensional representation of PCA analysis
computed from allele frequencies of allozymes
revealed clearly separated groups of the
Biebrza and Wigry populations (Fig. 2). The
first two components extracted 72% (p ¼ 0.02)
and 17% (p ¼ 0.90) of the total variance
among populations. The ordination diagrams
for within-population genetic structure also
Table 4. Hierarchical F-statistics for Epipactis helleborine populations from the Biebrza and Wigry regions.
Significance tests are based on 1000 permutations, ***P < 0.001
Source of
variation
d.f.
Sum of
squares
Variance
components
Percentage
of variation
Between regions
Among populations within regions
Within populations
1
3
987
126.670
122.564
1175.609
0.08490
0.28589
1.19351
5.43
18.28
76.30
Between regions
Among populations between regions
Among populations within regions
Fct = 0.05 n.s
Fst = 0.22***
Fsc = 0.19***
E. Brzosko et al.: Genetic variation in Epipactis helleborine
showed distinct patterns for the Biebrza and
Wigry populations. Individuals from two
Biebrza populations (ZAB and OPA) formed
partly overlapping discrete small groups with
single individuals additionally occupying
different and distant positions in the ordination diagram (Fig. 2). In the third (DLUG)
population individuals did not form groups
but were dispersed over the whole PCA
diagram plot. The groups of individuals, in
two Wigry populations, were clearly separated
from each other as they did not overlap.
Discussion
Populations of E. helleborine from northeast
Poland differed in levels of genetic variation
from populations of this species from other
countries (Harris and Abbott 1997, Hollingsworth and Dickson 1997, Ehlers and Pedersen
2000, Pedersen and Ehlers 2000, Squirrell et al.
2001). Polish populations of E. helleborine had
lower proportion of polymorphic loci (P) and
number of alleles per locus (A) than those
analyzed by Squirrell et al. (2001) and Ehlers
Fig. 2. Principal Component Analyses within and among Epipactis helleborine populations from Biebrza and
Wigry regions; significant value for axis
E. Brzosko et al.: Genetic variation in Epipactis helleborine
and Pedersen (2000). E. helleborine populations from northeast Poland showed more
similarities with respect to P and A parameters
with populations investigated by Harris and
Abbott (1997) and Hollingsworth and Dickson
(1997) in England and Scotland. Heterozygosity, both observed and expected, was lower in
the Polish populations than in populations
from other geographic regions (Table 5). We
can not compare our data with those from
other papers precisely due to differences in the
number of investigated populations, enzyme
systems and loci.
Among the major factors shaping genetic
structure of E. helleborine populations from
Poland, are the type of reproduction and
breeding system, properties which are often
reflected in the genetic structure of plant
populations. The distribution of genotypes in
the Biebrza populations suggests that most
individuals present here were the result of
sexual reproduction as vegetative spread only
occurred sporadically. We expected that shoots
appearing almost in the same place on one field
could belong to one genetic individual. It was
surprising that neighbouring shoots, with distances of few cm between them were distinct
multilocus genotypes. Autogamy did not reduce the level of genetic variation in the
populations from Biebrza, because among a
few covered inflorescences in the pollinatorexclusion experiment only 3 fruits were
recorded. Thus, our observations confirm
observations by Ehlers et al. (2002) that
E. helleborine is a mainly outcrossing species,
although Waite et al. (1991) and Squirrell et al.
(2001) reported that autogamy and/or geitonogamy may occur in this species. We cannot
exclude that autogamy is present in the LUK
population, where ants often penetrating flowers of E. helleborine could transfer pollen within
one inflorescence, resulting in high proportions
of fruit set. In the case of the LUK population
we can suppose, as the other authors, that
presence of autogamy may be reflected in the
positive significant FIS values. Since we obtained the positive significant FIS values in
other populations, where self-pollination is less
probable, we suggest other factors than inbreeding depression could more influence FIS
values.
A lower level of genetic variation in the
LUK may be explained by vegetative reproduction. The presence of vegetative reproduction could result in a small number of distinct
multilocus genotypes in the LUK populations.
This can be illustrated by a comparison
between OPA and LUK populations. The sizes
of both populations were similar (201 and 192
shoots) but the number of distinct multilocus
Table 5. Genetic variation within populations of Epipactis helleborine in Europe and in North America.
Np – number of population studied, S/L – number of enzymatic systems/number of all investigated loci, P –
percent of polymorphic loci, A – number of alleles per locus, (Ap) – number of alleles per polymorphic
locus, Ho – observed heterozygosity, He – expected heterozygosity
Source
Harris and Abbott (1997) England,
Scotland
Hollingsworth and Dickson (1997)
England, Scotland
Ehlers and Pedersen (2000) Denmark
Squirrell et al. (2001) Europe
(Belgium, Denmark, England,
France, Germany, Scotland,
Switzerland)
North America (Canada)
Brzosko, Wróblewska, Tałałaj Poland
Np
S/L
P
A (Ap)
Ho
He
2
9/17
27.0–40.0
1.33–1.40
–
–
13
8/13
23.1–38.5
1.20–1.60
0.062–0.207
0.055–0.204
12
35
9/25
6/9
73.6
55.0
(2.62)
1.77
0.256
–
0.274
0.230
12
5
15/22
58.0
32.7
1.90
1.72 (2.53)
–
0.091
0.232
0.115
E. Brzosko et al.: Genetic variation in Epipactis helleborine
genotypes was quite different (194 vs. 29).
Individuals of this species are sometimes multistemmed (Squirrell et al. 2001) and the habitat,
in which the LUK population is localized,
seems to be suitable for vegetative spread (a
low cover of other species). The fact that
ramets possessing the same multilocus genotype often appeared at short distances might
also support this hypothesis. The influence of
vegetative spread on the genetic structure of
plant populations is documented in many
papers (Ellstrand and Roose 1987, Chung
1995, Brzosko et al. 2002a). The next explanation for the low level of genetic variation and
significant positive FIS value in the LUK
population could be the lowest proportion of
flowering plants in comparison with the other
study populations. Reduced level of flowering
individuals may decrease the amount of outcrossing possible among individuals. Similarly
to the LUK population, the suggestion that a
lower level of genetic variation is a consequence
of assortative mating between related individuals can also be used in the case of the LUB
population with the smallest number of individuals. However, the size of the LUB
population seems to be the most important
factor in shaping its genetic variation. This
population was the smallest one and was
characterized by the lowest level of genetic
variation. The size of LUB is probably below
the minimum effective population’s size capable to retain sufficient allelic richness (Frankel
et al. 1995). Other authors (Sun 1996, Hollingsworth and Dickson 1997, Wong and Sun 1999)
also claimed that polymorphism and allelic
diversity were mostly affected by population
size. A lower level of genetic variation found in
the LUB population may be the result of a
founder effect. A small number of polymorphic
loci and completely inbreeding in the Idh-1
locus may also suggest a relatively recent origin
of the LUB population. Holderegger and
Stehlik (1999) reported that inbreeding depression may also occur in small populations of
almost completely outbred species because
many crossing events are likely to be
among the relatives. PCA analyses indicated
that individuals both in the LUK and LUB
populations occurred noticeably in not
overlapping groups of genotypes occupying
two spectra of PCA plots. Those genotypes
represented small, distinct kinship groups
opposite to the homogeneous distribution
of genotypes within the populations from
Biebrza.
We suggest that one of the factors shaping
genetic structure of E. helleborine populations
is their history strictly connected with the
history of investigated areas. On the basis of
the similarities with respect to the estimates of
genetic variation among the populations
of E. helleborine from Biebrza and the levels
of differentiation among them we can suppose
that these populations probably share a
common ancestral population. It is possible
that a rapid colonization from a single source
population could produce populations that
were genetically similar. Alternatively, the
present E. helleborine populations from Biebrza constitute the rest of one coherent, ancient
population, which was divided into several
ones because of the environmental fragmentation (creation of mineral islands separated by
peatbogs). Despite the similarity rooted in the
common source, there are differences between
the populations, which could be explained by a
separate development in time. One of them is a
different composition of distinct and unique
multilocus genotypes. It could be suggested
that after environmental isolation, both pollen
and seed dispersal occurred rather within
populations than between them. Ehlers and
Pedersen (2000) have also demonstrated that
gene flow within E. helleborine occurs on a very
local scale. The influence of historical factors
in shaping the genetic structure of orchid
species has been noticed in other species
studied in the Biebrza valley, Cypripedium
calceolus (Brzosko et al. 2002a, b), Listera
ovata (Brzosko and Wróblewska 2003a) and
Cephalanthera rubra (Brzosko and Wróblewska 2003b) and by other authors (Karron
et al. 1988; Case 1993, 1994; Chung 1995;
Bingham and Ranker 2000; Edwards and
Sharitz 2000; Wallace and Case 2000).
E. Brzosko et al.: Genetic variation in Epipactis helleborine
The second distinct group with lower levels
of genetic variation consists of two populations from the Wigry National Park. In
contrast to the Biebrza National Park, where
vegetation has a natural character, in the
Wigry National Park except from natural
communities there is a large area covered by
anthropogenically changed vegetation. The
populations of E. helleborine exist in the
places, which were deforested about 200–
300 years ago to be used for agricultural
purposes. When the agricultural use came to
its end, the areas had already been forested.
Now pine forests, dominated by Pinus sylvestris (with maximum age c. 90 years), cover this
territory. The above-mentioned history of the
area seems to be strictly connected with the
history of the LUK population. Its lower level
of genetic variation in comparison to populations from Biebrza (except for factors deliberated above) can be explained by a much
shorter period of time of development.
The history of the second LUB population
from the Wigry National Park is somewhat
different. It is localized at the margin of the
didactic asphalt path, which was build after the
formation of the Wigry National Park. It is
possible that during building of the asphalt
road a fragment of forest was destroyed and the
colonization of the LUB population took place
there. If it is true, this population is the
youngest, which may explain why it has the
smallest size and the lowest level of genetic
variation. Hollingsworth and Dickson (1997)
have also found a reduced level of variation
when recently colonized (urban) versus established (rural) sites of E. helleborine were
compared. E. helleborine as a species with large
colonizing abilities is known from many habitats changed due to human activity (Adamowski and Conti 1991, Adamowski 1998,
Hollingsworth and Dickson 1997, Squirrell
et al. 2001). E. helleborine in the Wigry
National Park could also find favourable
conditions for its development in a destroyed
fragment of the forest. Larger values of FST
between the LUB population and others might
indicate that the LUB population has origi-
nated from a quite different source, for
example, from seeds present in the material
used to build the road. We can exclude that this
population originated from the nearest LUK
population. One of the evidences may be the
fact that in the Tpi-2 locus exclusively dd
homozygotes were found in the LUB population, while in the LUK population almost
100% of cc homozygotes were noted. The
presence and frequencies of other alleles confirmed this situation.
On the other hand, our results indicated the
lack of significant differences in gene flow
between both Biebrza and Wigry regions.
Similarly as Ehlers and Pedersen (2000), we
conclude that rather historical than recent gene
flow between two regions could be more
extensive and any equilibrium in genetic
differentiation between them has not been
established yet.
Orchid seeds play a more important role
than pollen in gene flow between populations,
thus seed dispersal (at distances about 5–
10 km) influences more genetic structure in
orchid species (Peakall and Beattie 1991,
Rasmussen 1995). In E. helleborine both pollen
and seed dispersal are greatly restricted and
occur at a local scale (Ehlers and Pedersen
2000). On the basis of this information and our
results, we can suppose that gene exchange
appears mainly within populations and between populations belonging to a given geographic region (Biebrza or Wigry region) due
to short distances between these populations.
In this way we can explain the highest Nm
values (5.56) between the nearest OPA and
DLUG. A possibility of pollen and seed
dispersal among areas results from a different
character of the vegetation. Populations from
the Biebrza National Park are localized on the
mineral island separated by an open area.
Therefore, easier transport of propagules can
occur when compared to the population from
Wigry region, where propagation in dense
forests is more difficult. Although our deliberation concerning historical aspects of genetic
variation and differentiation of E. helleborine
populations from Poland has a somewhat
E. Brzosko et al.: Genetic variation in Epipactis helleborine
speculative character, we can not exclude such
scenarios. Many authors, on the basis of
similar data, also accentuate the importance
of historical factors in shaping the genetic
structure of plant populations (Karron et al.
1988; Case 1993, 1994; Chung 1995; Bingham
and Ranker 2000; Edwards and Sharitz 2000;
Wallace and Case 2000).
Appendix 1.
Appendix 1. Allele frequencies for nine polymorphic loci in five Epipactis helleborine populations in NE Poland
Loci &
alleles
Biebrza
Wigry
OPA ZAB DLUG LUB LUK Overall
Got-1 a
b
c
Gpi-2 a
b
c
Pgm-2 a
b
c
Idh-1 a
b
Idh-2 a
b
Skd-1 a
b
Mdh-1 a
b
Mdh-2 a
b
c
Tpi-2 a
b
c
d
e
0.910
0.083
0.007
0.647
0.129
0.223
0.018
0.673
0.309
0.679
0.320
0.089
0.910
0.320
0.679
0.507
0.493
0.025
0.834
0.140
0.050
0.179
0.496
0.230
0.043
0.986
0.009
0.005
0.289
0.275
0.436
0.005
0.408
0.587
0.849
0.151
0.000
1.000
0.023
0.977
0.216
0.784
0.059
0.679
0.261
0.009
0.000
0.784
0.179
0.027
0.879
0.030
0.090
0.379
0.258
0.364
0.015
0.621
0.363
0.742
0.258
0.030
0.969
0.076
0.924
0.590
0.409
0.181
0.712
0.106
0.030
0.000
0.576
0.394
0.000
1.000
0.000
0.000
0.211
0.173
0.616
0.000
0.769
0.231
0.692
0.308
0.000
1.000
0.000
1.000
0.000
1.000
0.269
0.731
0.000
0.000
0.000
0.000
1.000
0.000
1.000
0.000
0.000
0.000
0.031
0.969
0.000
0.655
0.345
0.972
0.028
0.000
1.000
0.000
1.000
0.124
0.876
0.000
0.657
0.343
0.000
0.003
0.984
0.008
0.005
0.955
0.025
0.020
0.305
0.173
0.522
0.008
0.625
0.367
0.787
0.213
0.024
0.976
0.084
0.916
0.287
0.713
0.107
0.723
0.170
0.018
0.036
0.568
0.362
0.015
We are grateful to Bodil K. Ehlers and Peter H.
Hollingsworth, who shared information referring
the protocols and recipes for electrophoretical
analysis. We would also thank Maciek Romański
for helping us locate Epipactis helleborine popula-
tions in the Wigry National Park. This work was
supported by the Polish State Committee for
Scientific Research (KBN grant No 6P04C 10121).
References
Adamowski W. (1998) Colonisation success of
orchids in disturbed habitats. In: Falińska K.
(ed.) Plant population biology and vegetation
processes. W. Szafer Institute of Botany, Polish
Academy of Sciences, Kraków, pp. 167–169.
Adamowski W., Conti F. (1991) Mass occurrence
of orchids in poplar plantations near Czeremcha
village as an example of apophytism. Phytocoenosis 3: 259–267.
Bingham R. A., Ranker T. A. (2000) Genetic
diversity in alpine and foothill populations of
Campanula rotundifolia (Campanulaceae). Int. J.
Pl. Sci. 161(3): 403–411.
Brzosko E., Ratkiewicz M., Wróblewska A.
(2002a) Allozyme differentiation and genetic
structure of Lady’s slipper island populations
(Cypripedium calceolus) in NE Poland. Bot. J.
Linn. Soc. 138: 433–440.
Brzosko E., Wróblewska A., Ratkiewicz M.
(2002b) Spatial genetic structure and clonal
diversity of island populations of Lady’s slipper
(Cypripedium calceolus) from the Biebrza National Park (northeast Poland). Mol. Ecol. 11:
2499–2509.
Brzosko E., Wróblewska A. (2003a) Low allozymic
variation in two island populations of Listera
ovata (Orchidaceae). Ann. Bot. Fenn. 40: 309–
315.
Brzosko E., Wróblewska A. (2003b) Genetic
variation and clonal diversity in island Cephalanthera rubra populations from the Biebrza
National Park. Bot. J. Linn. Soc. 143(1): 99–
108.
Case M. A. (1993) High levels of allozyme variation
within Cypripedium calceolus (Orchidaceae) and
low levels of divergence among its varieties. Syst.
Bot. 18(4): 663–677.
Case M. A. (1994) Extensive variation in the levels
of genetic diversity and degree of relatedness
among five species of Cypripedium (Orchidaceae). Amer. J. Bot. 81(2): 175–184.
Chung M. G. (1995) Low levels of genetic diversity
within populations of Hosta clausa (Liliaceae).
Pl. Spec. Biol. 9: 177–182.
Chung M. G. (1996) Spatial genetic structure
among Korean populations of Hosta minor and
E. Brzosko et al.: Genetic variation in Epipactis helleborine
H. capitata (Liliaceae). Bot. Bull. Acad. Sin. 37:
25–30.
Edwards A. L., Sharitz R. (2000) Population
genetic of two rare perennials in isolated
wetlands: Sagittaria isoetiformis and S. teres
(Alismataceae). Amer. J. Bot. 87(8): 1147–
1158.
Ehlers B. K., Olesen J. M., Ågren J. (2002) Floral
morphology and reproductive success in the
orchid Epipactis helleborine: regional and local
across-habitat variation. Plant Syst. Evol. 236:
19–32.
Ehlers B. K., Pedersen H. Æ. (2000) Genetic
variation in three species of Epipactis (Orchidaceae): geographic scale and evolutionary inferences. Biol. J. Linn. Soc. 69: 411–430.
Ellstrand N. C., Elam D. R. (1993) Population
genetic consequences of small population size.
Implications for plant conservation. Annual
Rev. Ecol. Syst. 24: 217–242.
Ellstrand N. C., Roose M. L. (1987) Patterns of
genotypic diversity in clonal plant species. Amer.
J. Bot. 74(1): 123–131.
Frankel O. H., Brown A. H. D., Burdon J. J. (1995)
The conservation of plant biodiversity. Cambridge University Press, Cambridge.
Gitzendanner M. A., Soltis S. (2000) Patterns of
genetic variation in rare and widespread plant
congeners. Amer. J. Bot. 87: 783–792.
Goudet J. PCA-GEN, ver. 1.2. Institute of
Ecology, Biology Building, UNIL, CH-1015
Lausanne, Switzerland. Computer Software distributed by the author.
Goudet J. FSTAT ver. 2.9.3.2. (2002) A program to
estimate and test gene diversities and fixation
indices. Available from http://www.unil.ch/izea/
softwares/fstat.html.
Hamrick J. L., Godt M. J. W. (1989) Allozyme
diversity in plant species. In: Brown A. H. D.,
Clegg M. T., Kahler A. L., Weir B. S. (eds.)
Plant population genetics, breeding, and genetic
resources. Sinauer Associates, Sunderland, Massachusetts, pp. 43–63.
Harris S. A., Abbott R. J. (1997) Isozyme analysis
of the reported origin of a new hybrid orchid
species, Epipactis youngiana (Young’s helleborine), in the British Isles. Heredity 79: 401–407.
Holderegger R., Stehlik I. (1999) Sibmating in a
small, isolated population of the dioecious plant
species Mercurialis ovata. Biochem. Syst. Ecol.
27: 681–685.
Hollingsworth P. M., Gornall R. J., Preston C. D.
(1995) Genetic variability in British populations
of Potamogeton coloratus (Potamogetonaceae).
Plant Syst. Evol. 197: 71–85.
Hollingsworth P. M., Dickson J. H. (1997) Genetic
variation in rural and urban populations of
Epipactis helleborine (Orchidaceae) in Britain.
Bot. J. Linn. Soc. 123: 321–331.
Hultén E., Fries M. (1986) Atlas of north European
plants north of the Tropic of Cancer. Koeltz
Scientific Books, Königstein.
Karron J. D. (1987) A comparison of levels of
genetic polymorphism and self-compatibility in
geographically restricted and widespread plant
congeners. Evol. Ecol. 1: 47–58.
Karron J. D., Linhart Y. B., Chaulk C. A.,
Robertson C. A. (1988) Genetic structure of
populations of geographically restricted and
widespread species of Astragalus (Fabaceae).
Amer. J. Bot. 75(8): 1114–1119.
Light M. H. S., MacConaill M. (1991) Patterns of
appearance in Epipactis helleborine (L.) Crantz.
In: Wells T. C. E., Willems J. H. (eds.)
Population ecology of terrestrial orchids. SPB
Academic Publishing, The Hague, pp. 77–87.
Loveless M. D., Hamrick J. L. (1984) Ecological
determinants of genetic structure in plant populations. Ann. Rev. Ecol. Syst. 15: 65–95.
Manly B. F. J. (1997) Randomization, Bootstrap
and Monte Carlo. Methods in biology, 2nd edn.
Chapman and Hall, London.
Miller M. P. (1997) Tools for population genetic
analyses (TFPGA) 1.3: A Windows program for
the analysis of allozyme and molecular population genetic data. Computer Software distributed by author.
Nei M. (1978) Estimation of average heterozygosity
and genetic distance from a small number of
individuals. Genetics 89: 583–590.
Peakall R., Beattie A. J. (1991) The genetic
consequences of worker ant pollination in a
self-compatible, clonal orchid. Evolution 45(8):
1837–1848.
Pedersen H. Æ., Ehlers B. K. (2000) Local
evolution of obligate autogamy in Epipactis
helleborine subsp. neerlandica (Orchidaceae).
Plant Syst. Evol. 223: 173–183.
Pielou E. C. (1969) An introduction to mathematical ecology. Wiley Interscience, New York.
Piper J. G., Waite S. (1988) The gender role of
broad leaved Helleborine, Epipactis helleborine
E. Brzosko et al.: Genetic variation in Epipactis helleborine
(L.) Crantz (Orchidaceae). Funct. Ecol. 2: 35–
40.
Rasmussen H. N. (1995) Terrestrial Orchids from
seed to Mycotrophic plant. Cambridge University Press, Cambridge.
Richardson B. J., Adams M., Baverstock P. R.
(1986) Allozyme electrophoresis. Academic
Press, United Kingdom.
Schneider S., Roessli D., Excoffier L. (2000)
ARLEQUIN ver. 2.000. A software for population genetics data analysis. Available from
http://www.anthro.unige.ch/arlequin.
Soltis D. E., Soltis P. S. (1989) Isozymes in plant
biology. Dioscorides Press, Portland, Oregon.
Squirrell J., Hollingsworth P. M., Bateman R.,
Dickson J. H., Light M. H. S., MacConaill M.,
Tebbitt M. C. (2001) Partitioning and diversity
of nuclear and organelle markers in native and
introduced populations of Epipactis helleborine
(Orchidaceae). Amer. J. Bot. 88: 1409–1418.
Sun M. (1996) Effects of population size, mating
system, and evolutionary origin on genetic
diversity in Spiranthes sinenis and S. hongkongensis. Conserv. Biol. 10: 785–795.
Szweykowski J., Odrzykowski I. (1990) Chemical
differentiation of Aneura pinguis (L.) Dum.
(Hepaticae, Aneuraceae) in Poland and some
comments on application of enzymatic markers
in bryology. Bryophytes. Their chemistry and
taxonomy, pp. 437–448.
Tuulik T. (1998) Hiiumaa orhideed. Biosfääri
Kaitseala Hiiumaa Keskus. Pirrujaak 5, pp. 134.
Waite S., Hopkins N., Hitchings S. (1991) Levels of
pollinia export, import and fruit set among
plants of Anacamptis pyramidalis, Dactylorhiza
fuchsii and Epipactis helleborine. In: Wells T. C.
E., Willems J. H. (eds.) Population ecology of
terrestrial orchids. SPB Academic Publishing,
The Hague, pp. 103–110.
Wallace L. E., Case M. A. (2000) Contrasting
allozyme diversity between northern and southern populations of Cypripedium parviflorum
(Orchidaceae): Implications for pleistocene refugia and taxonomic boundaries. Syst. Bot.
25(2): 281–296.
Weir B., Cockerham C. (1984) Estimating
F-statistics for the analyses of population structure. Evolution 38: 1358–1370.
Wong K. C., Sun M. (1999) Reproductive
biology and conservation genetics of Goodyera
procera (Orchidaceae). Amer. J. Bot. 86(10):
1406–1413.
Wright S. (1951) The genetical structure of
populations. Ann. Eugenics 15: 323–354.
Addresses of the authors: Emilia Brzosko
(e-mail: [email protected]), Ada Wróblewska
(e-mail: [email protected]), Izabela Tałałaj
(e-mail: [email protected]), University of Białystok, Institute of Biology, Department of Botany,
Świerkowa 20b, 15-950 Białystok, Poland.