Minority cytotypes in European populations of the Gymnadenia conopsea complex (Orchidaceae) greatly increase intraspecific and intrapopulation diversity

Abstract

BACKGROUND AND AIMS:
Patterns of ploidy variation among and within populations can provide valuable insights into the evolutionary mechanisms shaping the dynamics of plant systems showing ploidy diversity. Whereas data on majority ploidies are, by definition, often sufficiently extensive, much less is known about the incidence and evolutionary role of minority cytotypes.
METHODS:
Ploidy and proportions of endoreplicated genome were determined using DAPI (4',6-diamidino-2-phenylindole) flow cytometry in 6150 Gymnadenia plants (fragrant orchids) collected from 141 populations in 17 European countries. All widely recognized European species, and several taxa of less certain taxonomic status were sampled within Gymnadenia conopsea sensu lato.
KEY RESULTS:
Most Gymnadenia populations were taxonomically and/or ploidy heterogeneous. Two majority (2x and 4x) and three minority (3x, 5x and 6x) cytotypes were identified. Evolution largely proceeded at the diploid level, whereas tetraploids were much more geographically and taxonomically restricted. Although minority ploidies constituted <2 % of the individuals sampled, they were found in 35 % of populations across the entire area investigated. The amount of nuclear DNA, together with the level of progressively partial endoreplication, separated all Gymnadenia species currently widely recognized in Europe.
CONCLUSIONS:
Despite their low frequency, minority cytotypes substantially increase intraspecific and intrapopulation ploidy diversity estimates for fragrant orchids. The cytogenetic structure of Gymnadenia populations is remarkably dynamic and shaped by multiple evolutionary mechanisms, including both the ongoing production of unreduced gametes and heteroploid hybridization. Overall, it is likely that the level of ploidy heterogeneity experienced by most plant species/populations is currently underestimated; intensive sampling is necessary to obtain a holistic picture.

Full text

Minority cytotypes in European populations of the Gymnadenia
conopsea complex (Orchidaceae) greatly increase intraspecific
and intrapopulation diversity
Pavel Tra
´vnı
´c
ˇek1, Jana Jersa
´kova
´2, Barbora Kuba
´tova
´3, Jana Krejc
ˇı
´kova
´1, Richard M. Bateman4,
Magdalena Luc
ˇanova
´1, Eva Krajnı
´kova
´3, Tamara Te
ˇs
ˇitelova
´2, Zuzana S
ˇtı
´pkova
´2, Jean-Pierre Amardeilh5,
Emilia Brzosko6, Edyta Jermakowicz6, Olivier Cabanne7, Walter Durka8, Peter Efimov9, Mikael Hedre
´n10,
Carlos E. Hermosilla11, Karel Kreutz12, Tiiu Kull13, Kadri Tali13, Olivier Marchand14, Manel Rey15,
Florian P. Schiestl15, Vladislav C
ˇurn3and Jan Suda1, *
1
Department of Botany, Faculty of Science, Charles University in Prague, CZ-128 01 Prague, Czech Republic and Institute of
Botany, Academy of Sciences of the Czech Republic, CZ-252 43 Pru
˚honice, Czech Republic,
2
Faculty of Science, University of
South Bohemia in C
ˇeske
´Bude
ˇjovice, CZ-370 05 C
ˇeske
´Bude
ˇjovice, Czech Republic,
3
Biotechnological Centre, Faculty of
Agriculture, University of South Bohemia in C
ˇeske
´Bude
ˇjovice, CZ-370 05 C
ˇeske
´Bude
ˇjovice, Czech Republic,
4
Royal Botanic
Gardens, Kew, Richmond, Surrey TW9 3AB, UK,
5
Socie
´te
´Franc¸aise d’Orchidophilie, FR-75019 Paris, France,
6
Institute of
Biology, University of Bialystok, S
´wierkowa 20B, PL-15-950 Bialystok, Poland,
7
19 le Bourg, FR-33330 St Pey d’Armens,
France,
8
Helmholtz Centre for Environmental Research– UFZ, Department of Community Ecology, Theodor-Lieser-Str. 4,
D-06120 Halle, Germany,
9
Herbarium, Komarov Botanical Institute of Russian Academy of Sciences, Prof. Popov str. 2, 197376
Saint-Petersburg, Russia,
10
Department of Biology, University of Lund, Solvegatan 37, SE-22362 Lund, Sweden,
11
c/ Francisco
Cantera 11 18izda, E-09200 Miranda de Ebro, Spain,
12
Oude Landgraaf 35a, NL-6373 Landgraaf, The Netherlands,
13
Department of Botany, Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, Kreutzwaldi
5, EE-51014 Tartu, Estonia,
14
21 rue de l’Indre, FR-36700 Cha
ˆtillon-sur-Indre, France and
15
Institute of Systematic Botany,
University of Zu
¨rich, Zollikerstrasse 107, CH-8008 Zu
¨rich, Switzerland
* For correspondence. E-mail suda@natur.cuni.cz
Received: 9 March 2012 Returned for revision: 10 May 2012 Accepted: 11 June 2012
†Background and Aims Patterns of ploidy variation among and within populations can provide valuable insights
into the evolutionary mechanisms shaping the dynamics of plant systems showing ploidy diversity. Whereas data
on majority ploidies are, by definition, often sufficiently extensive, much less is known about the incidence and
evolutionary role of minority cytotypes.
†Methods Ploidy and proportions of endoreplicated genome were determined using DAPI (4’,6-diamidino-2-
phenylindole) flow cytometry in 6150 Gymnadenia plants (fragrant orchids) collected from 141 populations in
17 European countries. All widely recognized European species, and several taxa of less certain taxonomic
status were sampled within Gymnadenia conopsea sensu lato.
†Key Results Most Gymnadenia populations were taxonomically and/or ploidy heterogeneous. Two majority (2x
and 4x) and three minority (3x,5xand 6x) cytotypes were identified. Evolution largely proceeded at the diploid
level, whereas tetraploids were much more geographically and taxonomically restricted. Although minority ploi-
dies constituted ,2 % of the individuals sampled, they were found in 35 % of populations across the entire area
investigated. The amount of nuclear DNA, together with the level of progressively partial endoreplication, sepa-
rated all Gymnadenia species currently widely recognized in Europe.
†Conclusions Despite their low frequency, minority cytotypes substantially increase intraspecific and intrapopu-
lation ploidy diversity estimates for fragrant orchids. The cytogenetic structure of Gymnadenia populations is re-
markably dynamic and shaped by multiple evolutionary mechanisms, including both the ongoing production of
unreduced gametes and heteroploid hybridization. Overall, it is likely that the level of ploidy heterogeneity
experienced by most plant species/populations is currently underestimated; intensive sampling is necessary to
obtain a holistic picture.
Key words: Coexistence, contact zone, cytogeography, flow cytometry, fragrant orchid, Gymnadenia,
Orchidaceae, hybridization, mixed-ploidy population, polyploidy, sympatry, unreduced gametes.
INTRODUCTION
Polyploidy (the multiplication of complete chromosome sets in
somatic cells above the diploid state) is a prominent and recur-
ring process in the evolution of eukaryotic organisms (Otto and
Whitton, 2000). Although polyploidy has been documented in
all major lineages of eukaryotes, land plants show the highest
incidence of polyploidy (Jiao et al.,2011). Karyological evi-
dence suggests that at least 70 and 95 % of angiosperms and
ferns, respectively, are polyploid (Masterson, 1994). Genomic
data also support the near ubiquity of polyploidy, traces of
#The Author 2012. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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ancient whole-genome duplication having been detected in vir-
tually all angiosperms (Soltis et al., 2009). The success of poly-
ploid plants can be related to different evolutionary transitions
that may alter their genetic composition, phenotypic plasticity
or ecological amplitude, and can ultimately lead to increased
vigour and competitive superiority over diploid ancestors
(Levin, 2002). Polyploid plants can combine genomes of two
or more parental species (allopolyploids) or arise from the
same parental species (autopolyploids). Whereas allopolyploids
have long been assumed to prevail in situ,recentdatasuggest
that the frequency of autopolyploids is much higher than previ-
ously considered and they play important evolutionary and eco-
logical roles in natural populations (Soltis et al.,2007;Parisod
et al.,2010). Autopolyploid derivates may originate through
somatic chromosome doubling, but it is the formation of unre-
duced gametes that drives the dynamics of their genesis
(Bretagnolle and Thompson, 1995;Ramsey and Schemske, 1998).
Although genome duplication is often associated with speci-
ation (Wood et al., 2009), ploidy variation is also observed
within traditionally delimited taxonomic species. This is espe-
cially true for autopolyploids, which more closely resemble
their diploid/lower ploid progenitors than do allopolyploids
and so are rarely recognized in formal classifications. For
example, chromosomal data for the Californian flora indicate
that approx. 13 % of the species listed are ploidy polymorphic
and several of them possess more than two different cytotypes
(Soltis et al., 2007). Based on a broad survey of species, Wood
et al. (2009) reported that 12–13 % of angiosperm species and
17 % of fern species are variable for ploidy. In general, ploidy
heterogeneity within species is likely to have been underesti-
mated and is predicted to continue to increase with more inten-
sive sampling. Indeed, ploidy screening across large spatial
scales and in a representative number of individuals per popu-
lation, made possible by the advent of flow cytometry (FCM),
has resulted in a substantial increase in the number of ploidy-
heterogeneous plant species recognized and in the number of
different cytotypes recorded per species (Kron et al., 2007).
Fragrant orchids of the Gymnadenia conopsea aggregate
constitute a highly ploidy-variable and taxonomically challenging
species complex native to temperate Europe and Asia. Besides the
karyological polymorphism (Marhold et al., 2005;Tra
´vnı
´c
ˇek
et al., 2011), members of the complex were also found to vary
in morphology (Dworschak, 2002;Marhold et al., 2005;Vo
¨th
and Sontag, 2006; R. Bateman et al., unpubl. res.), floral scent
biochemistry (Huber et al., 2005;Jersa
´kova
´et al., 2010), flower-
ing phenology (Soliva and Widmer, 1999;Gustafsson and Lo
¨nn,
2003) and preferred habitats (Dworschak, 2002). Investigations
into phenotypic and genetic variation have often revealed strong
genetic divergence among the recognized taxa but a lower level
of morphological differentiation (e.g. Scacchi and de Angelis,
1989;Soliva and Widmer, 1999;Bateman et al., 2003;
Gustafsson and Lo
¨nn, 2003;Stark et al., 2011; R. Bateman
et al., unpubl. res.). Taxonomic delimitation is further complicated
by weak pre-zygotic and post-zygotic barriers (Jersa
´kova
´et al.,
2010) that allow frequent formation of spontaneous hybrids at
both intrageneric and intergeneric levels (e.g. Hedre
´net al.,
2000;Lo
¨nn et al., 2006).
Setting aside the former genus Nigritella, recent classifica-
tions of Gymnadenia in Europe mostly recognize five major
taxa at different taxonomic levels, depending on the author’s
preferred concept. Most recent British authors have followed
Bateman et al. (2003) in recognizing all of these taxa as full
species, whereas the most influential Continental monographers
(e.g. Kreutz, 2004;Delforge, 2006) have treated most of
these taxa as varieties only. In addition to the widespread
G. conopsea (L.) R.Br. sensu stricto (s.s.), G. densiflora
(Wahlenb.) A.Dietr. and G. odoratissima (L.) Rich., G. frivaldii
Hampe ex Griseb. is a Balkan endemic only recently confirmed
as assignable to Gymnadenia (Bateman et al., 2006). Originally
described from a type locality in Cumbria, G. borealis (Druce)
R.M.Bateman, Pridgeon & M.W.Chase is regarded by some
authors as being confined to Britain and Ireland, though morpho-
logically identical plants also occur along the Scandinavian
mountain chain (Strann and Bjerke, 2010). Several local morpho-
types with a more questionable taxonomic status have also been
described, including the compact, late-flowering G. conopsea var.
friesica Schlechter from sand dunes on the Friesian Islands
(Schlechter, 1919;Kreutz and Dekker, 2000) and the slender
alpine ecotype referred to as G. conopsea var. alpina Rchb.f.
ex Beck (1893). Robust plants from the Pyrenees that resemble
the short-spurred G. odoratissima but have a spur about one-third
longer than the ovary have been recognized as var. pyrenaica
(Philippe) P.Delforge (2005). A substantially longer spur is also
supposedly diagnostic of G. odoratissima subsp. longicalcarata
C.E.Hermosilla & J.Sabando (1996) from northern Spain.
Several additional taxa from the Bavarian Alps were recent-
ly described on the basis of morphological observations
(Dworschak, 2002): G. graminea Dworschak, G. conopsea
subsp. serotina (Scho
¨nh.) Dworschak, G. splendida Dworschak
and G. vernalis Dworschak.
Our previous study (Tra
´vnı
´c
ˇek et al., 2011) provided new
insights into ploidy variation but only at population and re-
gional scales, being confined to the Czech Republic plus
Slovakia. We found a surprisingly high proportion of mixed-
ploidy populations, consisting of different combinations of
two majority and three minority cytotypes. In addition, unique
FCM profiles (i.e. different levels of progressively partial endo-
replication; see Discussion for detailed explanation) were
observed for G. conopsea s.s. and G. densiflora. The present
study builds on our previous research, aiming to assess ploidy
variation across much larger spatial scales and encompassing
all major European Gymnadenia species. Patterns of ploidy
variation, both among and within populations, can provide
useful insights into the evolutionary mechanisms that shape
the dynamics of these polyploid systems.
Specifically, we address the following questions. (1) Which
patterns of progressively partial endoreplication can be found
among the investigated plants? Is this variation geographically
or taxonomically structured? (2) Where is the geographical
centre of ploidy variation located? (3) How frequent are
mixed-ploidy populations? Do different Gymnadenia taxa
differ in this respect? (4) How common and how widespread
are minority cytotypes? Do they preferentially occur in popu-
lations with a particular composition of majority ploidies?
MATERIALS AND METHODS
Field sampling
Plantsampleswerecollectedin17Europeancountries
between 2004 and 2011, spanning the geographical range
Tra
´vnı
´c
ˇek et al. — Minority cytotypes in fragrant orchids978
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40 857′N–59 817′N and 06 801′W–30 830′E (for locality
details, see Supplementary Data Table S1) and totalling
6150 individuals from 141 populations. The number of local-
ities and individuals sampled for specific countries were as
follows: Austria, 9/318; Belgium, 1/26; Bulgaria, 3/36;
Estonia, 3/91; France, 20/958; Germany, 20/877; Italy, 10/
594; Macedonia, 1/6; The Netherlands, 1/48; Poland, 2/58;
Romania, 9/209; Russia, 7/130; Scotland, 19/600; Slovakia,
5/266; Spain, 1/13; Sweden, 15/1348; and Switzerland, 15/
572. Although taxonomic revision of the Gymnadenia conop-
sea aggregate was beyond the scope of this study, we aimed to
encompass most of the taxonomic and phenotypic diversity
recognized in Europe. In addition to traditionally accepted
species, we also sampled known localities for recently described
taxa of questionable taxonomic status (e.g. Dworschak, 2002;
Supplementary Data Table S1). Due to taxonomic uncertainties,
some plants from France with distinct FCM profiles and morph-
ology were not assigned to any particular taxon and instead are
provisionally named ‘French diploid’ and ‘French tetraploid’.
The taxonomic composition of our data set is summarized in
Table 1.
Whenever possible, leaf tissue from at least 50 individuals
was collected at each locality (the actual number of samples
per locality varied from one to 191; Supplementary Data
Table S1). The number of samples chosen per locality reflected
(1) population size; (2) taxonomic composition (more intensive
sampling in mixed-species populations); and (3) morphological/
phenological variation (more intensive sampling in populations
showing high phenotypic variation or supporting multiple var-
iants with contrasting flowering periods). Leaf tissue was
wrapped in moist paper towels, placed in plastic bags and trans-
ported rapidly to the FCM laboratory. Because one or more
Gymnadenia species rank among threatened plants in several
European countries, we preferred images to herbarium speci-
mens as vouchers. Plants were imaged at each locality
(Supplementary Data Fig. S2), and herbarium specimens (kept
in PRC or CBFS) were taken only from selected representative
sites (Supplementary Data Table S1). Because the majority of
diagnostic characters are located on floral parts, two flowers
per plant were collected at each locality and stored in 70 %
ethanol.
Flow cytometry
Relative fluorescence intensities of plant samples were
determined by DAPI (4’,6-diamidino-2-phenylindole) FCM
following the methodology detailed by Tra
´vnı
´c
ˇek et al.
(2011). Up to five individuals were processed together. Each
plant was re-analysed separately in cases of mixed-ploidy
samples or if the coefficient of variation of either the
unknown sample or the internal standard peaks exceeded
5%. Pisum sativum ‘Ctirad’ (2C ¼9.09 pg) was selected as
a primary reference standard, as it has a genome size close
to, but not overlapping, that of most Gymnadenia samples.
Vicia faba ‘Inovec’ served as a reference standard for measure-
ments of G. borealis; the relative nuclear DNA amount of
Vicia was calibrated against Pisum (3.14×greater; Suda
et al., 2007). Karyologically counted (2n¼40 and 2n¼80)
plants of G. conopsea from the Czech Republic were used as
reference points when interpreting the FCM results. Some
data, such as the incidence of individuals with putatively 50
somatic chromosomes among FCM-screened progeny of our ex-
perimental crosses (J. Jersa
´kova
´et al., unpubl. res.; see also
Tra
´vnı
´c
ˇek et al., 2011) may indicate that x¼10 is the basic
chromosome number in the G. conopsea aggregate.
Nonetheless, in line with the generally accepted view (e.g.
Marhold et al., 2005;Stark et al., 2011), we interpreted
here plants with 2n¼40 and 2n¼80 as diploids and tetra-
ploids, respectively, pending any stronger cytological evi-
dence for x¼10.
Statistical analyses
Flow cytometry data were analysed using the SAS 8.1 stat-
istical package (SAS Institute, Cary, NC, USA). Interspecific
differences in relative fluorescence intensities and proportions
of endoreplicated genome were tested by GLM (general linear
model) because of unbalanced data design, and Tukey’s pro-
cedure was applied to compare mean values.
Binomial multiple regression (LOGISTIC procedure in SAS)
was used to test whether polyploids (i.e. 3x–6x)ortetraploids
specifically are linked to geographical parameters of sampled
populations (latitude, longitude, altitude and their combinations;
Manzaneda et al.,2011). The presence/absence of polyploids or
TABLE 1. Flow cytometric results for five major European Gymnadenia species and two undetermined taxa from France
Species
Ploidy
level
Relative fluorescence intensity against
internal reference standard, Pisum
sativum (mean +s.d.)*
Proportion of replicated
genome (mean +s.d., %)*
No. of FCM
analyses
No. of
individuals
G. borealis 2x0.956 +0.017
c
53.7+1.7
e
139 599
G. conopsea (incl. subsp. serotina p.p.,
var. alpina,G. graminea,G. splendida
p.p., G. vernalis)
2x0.853 +0.021
f
58.1+1.9
c
496 2114
4x1.588 +0.029
b
60.7+2.3
b
161 528
G. densiflora (incl. G. conopsea subsp.
serotina p.p., G. conopsea var. friesica,
G. splendida p.p.)
2x0.748 +0.014
g
74.4+2.4
a
362 1538
G. frivaldii 2x0.857 +0.031
f
50.8+1.9
e
10 32
G. odoratissima 2x0.906 +0.019
e
56.8+1.8
cd
106 464
French diploid 2x0.923 +0.018
d
56.2+1.7
d
163 565
French tetraploid 4x1.673 +0.026
a
60.6+2.0
b
90 192
*Different letters indicate groups of taxa that are significantly different at a¼0.05.
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´vnı
´c
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tetraploids in populations fitted a binomial distribution, which
was therefore used with the logit link function as parameters
of the model.
RESULTS
Genome characteristics
Flow cytometric analysis of 6150 plants (Fig. 1) resulted in
five distinct groups of fluorescence intensities, corresponding
to diploids (5312 individuals; 86.4 %), triploids (94 indivi-
duals; 1.5 %), tetraploids (720 individuals; 11.7 %), penta-
ploids (17 individuals; 0.3 %) and hexaploids (seven
individuals; 0.1 %). Table 1shows FCM characteristics of
the majority (2xand 4x) ploidies for five species and two un-
determined Gymnadenia taxa. Two groups of tetraploids with
significantly different relative nuclear DNA contents were
found; one corresponded to G. conopsea s.s.(Tra
´vnı
´c
ˇek
et al., 2011), whereas the other was not assigned to any
species; it is provisionally referred to simply as ‘French tetra-
ploid’. Disregarding minority ploidies, all other species were
diploid. Their mean relative fluorescence intensities (setting
the value for the reference standard P. sativum to unity)
varied 1.278-fold, ranging from 0.748 in G. densiflora to
0.956 in G. borealis. With the exception of G. conopsea vs.
G. frivaldii, the remaining diploids possessed significantly dif-
ferent relative amounts of nuclear DNA (Table 1). The propor-
tions of endoreplicated genome also differed significantly
among several Gymnadenia taxa (Table 1). Gymnadenia
frivaldii was the species with the lowest level of progressively
partial endoreplication (50.8 % on average), whereas
G. densiflora showed the highest level (74.4 % on average).
Flow cytometric profiles (a combination of relative fluores-
cence values together with the proportion of endoreplicated
genome) therefore offer a reliable method of distinguishing
between all major Gymnadenia species recognized in the
more accurate of the recent European classifications.
Cytogeography and population structure
Half of the Gymnadenia populations sampled (71 of 141)
were deemed complex in terms of species composition, karyo-
logical variation or both (Table 2). Up to three different taxa
and five different cytotypes coexisted at a single site. In
total, we found 22 different species–majority ploidy combina-
tions (Table 2), and the frequent occurrence of one or more mi-
nority cytotypes further increased the intrapopulation
heterogeneity. Diploids and tetraploids were recorded in 133
and 25 populations, respectively; however, only 83 and four
populations, respectively, were homogeneous for ploidy. The
most common type of ploidy mixture involved sympatry of
diploids and triploids, suggesting regular formation of unre-
duced gametes. Some form of ploidy variation was observed
in 54 (38.3 %) populations; two, three and four different cyto-
types coexisted in 40, ten and three populations, respectively.
All five cytotypes grew together in population FR04 near
Sainte-Maure-de-Touraine in France (Supplementary Data
Table S1), which also maintained two coexisting taxa. In
total, more than two taxa were observed in nearly one-third
(41) of the populations analysed, the most common combination
being 2x G. conopsea,2xG.densifloraand 2x G. odoratissima
(ten populations), followed by sympatry of the two former
species (nine populations; Table 2).
160 A
B
C
D
120
II
II
II
II
III
III
III
III
I
I
I
I
S
S
S
S
IV
IV
80
40
0
280
210
140
70
0
0 200 400 600
Relative fluorescence
Number of nuclei
160
120
80
40
0
Number of nuclei
160
120
80
40
0
Number of nuclei Number of nuclei
800 1000
FIG. 1 . Representative flow cytometric histograms of the studied Gymnadenia
taxa (analysed together with the internal reference standard). Nuclei of both
the sample and standard were isolated, stained with DAPI and simultaneously
run on the flow cytometer. (A) Diploid G. borealis (loc. GB05) – ratios
between individual Gymnadenia peaks 1 : 1.54 : 2.63 : 4.81; (B) diploid
G. odoratissima (loc. IT05) – peak ratios 1 : 1.56 : 2.71 : 4.91; (C) French
diploid (loc. FR04) – peak ratios 1 : 1.58 : 2.78; (D) French tetraploid (loc.
FR 04) – peak ratios 1 : 1.58 : 2.78. I, II, III and IV, peaks of Gymnadenia
nuclei undergoing different numbers of partial endoreplication cycles. S, in-
ternal standard: Vicia faba in (A), Pisum sativum in (B– D).
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´vnı
´c
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Diploids were recorded in all 17 countries (Fig. 2A),
whereas tetraploids were restricted to just five of these coun-
tries: Austria, France, Germany, Romania and Switzerland
(Fig. 2B). The multiple regression analysis showed a signifi-
cant negative relationship between the incidence of polyploids
and latitude (
b
+s.d. ¼–0.294 +0.095; d.f. 1,133; P¼
0.0021). Tetraploids were strongly negatively associated with
latitude and also less strongly with altitude (
b
+s.d. ¼
–0.607 +0.188; d.f. 1,133; P¼0.0013 and
b
+s.d. ¼
–0.036 +0.018; d.f. 1,133; P¼0.0433, respectively).
Minority cytotypes
Minority ploidies constituted ,2 % of all samples, but they
were present in more than one-third (50 of 141) of our study
populations, distributed across the area investigated (Table 2,
Fig. 2C). Triploids, pentaploids and hexaploids occurred in
45, seven and four populations, respectively. Although it is dif-
ficult to determine the taxonomic identity of minority cyto-
types in multispecies populations, our data indicate that they
were formed in all widely recognized taxa (Table 2). Most
triploids were recorded in otherwise exclusively diploid popu-
lations (33 populations), although in 11 populations they co-
occurred with diploids and tetraploids. Significantly higher
proportions of triploid individuals occurred in mixed 2x–4x
populations than in otherwise uniform 2xpopulations
(Mann–Whitney U-test: 6.9 % vs 3.2%, n¼44, P¼0.0037
and 7.1 % vs 2.2%, n¼39, P,0.001, as assessed for, re-
spectively, all populations and only populations yielding
.30 analysed individuals). These observations suggest that,
in addition to the formation of unreduced gametes, interploidy
hybridization was also involved in the genesis of triploids.
This inference can also be reached from the proportion of
populations of different ploidy composition that harboured tri-
ploids; although triploids were present in 64.7 % of 2x–4x
populations, this proportion fell to 28.4 % if only 2xpopula-
tions were considered. Higher polyploids (5xand 6x) were
always associated with tetraploids, and in six out of nine of
these populations, diploids were also present.
DISCUSSION
This study represents by far the most comprehensive investiga-
tion of ploidy variation in the G. conopsea complex in terms of
taxonomic coverage, geographical scale and the number of
cytotyped plants.
Genome characteristics
Somatic tissues of at least some orchids are known to
undergo ‘progressively partial endoreplication’, a phenomenon
that was first described in Vanilla planifolia by Bory et al.
(2008). Unlike conventional whole-genome endoreplication,
which has been documented in plant species from a range of
families (Barow, 2006), only part of the genome is duplicated
during progressively partial endoreplication. Consequently, the
ratio between the first and second peaks in FCM histograms is
substantially less than 2:1. Previously (Tra
´vnı
´c
ˇek et al., 2011),
we observed differences in the proportion of endoreplicated
genome between the two Gymnadenia species native to the
TABLE 2. Taxonomic and ploidy composition of the 141 Gymnadenia populations investigated
No. of populations for a given taxonomic composition
harbouring minority cytotypes
Taxonomic composition (majority
ploidies)
Total no. of populations with the given taxonomic
composition 3x5x6x3x+5x5x+6x3x+6x3x+5x+6x
2C 34 9
2D 21 6
2O 2
2Sp 8 3
2B 19 1
2F 2 1
2C +2D 9 3
2C +2O 6 2
2C +2Sp 2 2
2D +2Sp 1
2O +2Sp 1
2C +2D +2O 10 6
2C +2D +2Sp 1
4C 7 1 1 1
4Sp 1 1
4C +2C 6 3 1 1
4C +2D 4 1 1
4C +2C +2D 2 1 1
4C +2C +2O 1 1
4C +2D +2O 1 1
4Sp +2Sp 2 1
4Sp +2Sp +2D 1 1
2B, 2x G. borealis; 2C, 2x G. conopsea; 2D, 2x G. densiflora;2F,2x G. frivaldii; 2O, 2xG. odoratissima; 2Sp, undetermined diploid from France; 4C, 4x
G. conopsea; 4Sp, undetermined tetraploid from France.
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´vnı
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Czech Republic and Slovakia, G. conopsea (mean value
58.5 %) and G. densiflora (mean value 74.7%). The present
study confirmed the validity of interspecific differences between
G. conopsea and G. densiflora across Europe (Table 1)and
revealed new species-specific profiles for G. borealis (53.7%of
endoreplicated genome) and G. frivaldii (50.8 % of endorepli-
cated genome). With the exception of G. frivaldii, there is a
negative relationship between the proportion of endoreplicated
genome and the total amount of nuclear DNA (Table 1). It is
therefore possible that the level of endoreplication has an adap-
tive role and contributes to shaping, either directly or indirectly,
optimal genome size and/or cell size (Gregory, 2005).
Genome characteristics of the less well known taxa (e.g.
Dworschak, 2002) were indistinguishable from those of the
major Gymnadenia species. Because their morphological de-
lineation also remains ambiguous, we have provisionally syno-
nymized G. conopsea var. alpina,G. graminea and G. vernalis
with the nominate variety of G. conopsea and G. conopsea var.
friesica with G. densiflora. On the basis of FCM results, indi-
viduals corresponding to G. conopsea subsp. serotina and
G. splendida sensu Dworschak (2002) were classified as
either G. conopsea or G. densiflora (Table 1).
Cytogeography and population structure
The results provided new insights into cytotype variation at
different spatial scales, from transcontinental to intrapopula-
tional. Five different ploidies (2x,3x,4x,5x, and 6x) were
60N
55N
50N
2C
2D
2O
2Sp
2B
2F
40N
15W 10W 5W 0 5E 10E 15E 20E 25E 30E
45N
A
FIG. 2 . Ploidy variation and taxonomic composition of 141 studied populations of the Gymnadenia conopsea complex in Europe. (A) Diploid populations (either
ploidy-uniform or with the presence of minority cytotypes). Intrapopulation taxonomic heterogeneity is indicated by mixed colours. (B) Tetraploid (squares) and
mixed 2x–4x(triangles) populations. Intrapopulation taxonomic heterogeneity is indicated by mixed colours. (C) Populations harbouring minority cytotypes (3x,
blue; 5x, yellow; 6x, red). The presence of both majority ploidies (2xand 4x) is illustrated by a circle, whereas triangles illustrate exclusive di- or tetraploid popula-
tions. Co-occurrence of different minority cytotypes is indicated by mixed colours. Arrows indicate populations in which an additional cytotype (most probably diploid)
is predicted (sympatry of 3x+4xor 4x+5x). Taxa abbreviations in (A) and (B): 2B, 2x G. borealis;2C,2x G. conopsea;2D,2x G. densiflora;2F,2x G. frivaldii;2O,
2x G. odoratissima; 2Sp, undetermined diploid from France; 4C, 4x G. conopsea; 4Sp, undetermined tetraploid from France.
Tra
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found among the present samples, reflecting our previous
smaller scale study confined to the Czech Republic and
Slovakia (Tra
´vnı
´c
ˇek et al., 2011). (Note that previously we
referred to these cytotypes as tetraploid, hexaploid, octoploid,
etc.; Tra
´vnı
´c
ˇek et al., 2011.) The evolution of the G. conopsea
complex proceeded mostly at the diploid level, which was
detected in all five recognized species plus one undetermined
taxon (Table 1, Fig. 2A, Supplementary Data Fig. S1A).
Tetraploids were more restricted, both taxonomically and spa-
tially. Although polyploidy is generally more frequent at
higher latitudes (Brochmann et al., 2004), the binomial mul-
tiple regression provided evidence that tetraploids (and poly-
ploids in general) in Gymnadenia tended to occur in
southern parts of the investigated area. The most common cat-
egory of tetraploids corresponded to G. conopsea; it extends lati-
tudinally from its centre of distribution in Central Europe at least
as far as France and Romania (Fig. 2B, Supplementary Data
Fig. S1B). France is also the home of tetraploids that possess
slightly larger amounts of nuclear DNA and were not assigned
by us to a particular pre-existing species. Potentially, they may
correspond to G. conopsea var. pyrenaica (a full species accord-
ing to Bourne
´rias and Prat, 2005), but for the present we refrain
from any taxonomic conclusion. Most published records of
tetraploid fragrant orchids have been made in Austria (Groll,
1965;Mrkvic
ˇka, 1993;Marhold et al., 2005;Stark et al.,
2011), Germany (Wegener, 1966;Stark et al.,2011) and the
Czech Republic and Slovakia (Marhold et al.,2005;
Tra
´vnı
´c
ˇek et al.,2011). More recently, Stark et al. (2011)
observed tetraploids at one locality in France, Heusser (1938)
having earlier reported this cytotype from Switzerland. Our
new discoveries from two sites in Romania (Fig. 2B,
Supplementary Data Fig. S1B), and published counts from
the Caucasus (Sokolovskaya and Strelkova, 1940)and
Armenia (Torosyan, 1990), demonstrate that tetraploids extend
from Central to Eastern Europe and further into Asia Minor.
In contrast, they appear to be absent from northern Europe, as
we did not find any tetraploid plants among samples from
Sweden, Estonia or Russia.
4C
4Sp
4C+2C
4C+2D
4Sp+2Sp
4C+2C+2D
4C+2C+2O
4C+2D+2O
4Sp+2D+2Sp
15W 10W 5W 0 5E 10E 15E 20E 25E 30E
60N
55N
50N
40N
45N
B
Fig. 2 Continued
Tra
´vnı
´c
ˇek et al. — Minority cytotypes in fragrant orchids 983
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Several species can co-occur at the sample locality, in par-
ticular when different microhabitats are present; we observed
two and three different Gymnadenia taxa at 28 and 13 sites, re-
spectively (Table 2). Mixed-species populations clearly pre-
vailed in G. odoratissima (90.5 %) and G. densiflora
(58.0 %), and were also common in G. conopsea (43.4%).
The coexistence of multiple species opens up obvious possibil-
ities for interspecific hybridization. We occasionally observed
morphotypes intermediate between 2x G. conopsea and 2x
G. odoratissima (e.g. localities IT04, IT06; Supplementary
Data Table S1). In addition, a few plants from mixed popula-
tions of G. conopsea and G. densiflora yielded unusual FCM
profiles that might indicate hybridization (e.g. population
AT07 from the Dachstein Mts.; Supplementary Data Table
S1). Such individuals were excluded from the present study
and will be subjected to further investigation using detailed
molecular techniques. Although only species-uniform popula-
tions of G. borealis and G. frivaldii were recorded in our study,
we regard this outcome as an artefact of sampling; only two
populations were available for G. frivaldii and all of our nu-
merous collections of G. borealis originated from Scotland.
Mixed sites of G. borealis and G. conopsea have been reported
from more southern parts of the UK (Campbell et al., 2007).
However, the only mixed-species populations in Britain and
Ireland detected by one of us during 35 years of fieldwork
involved G. borealis plus G. densiflora in central Scotland
and G. borealis plus G. conopsea s.s. in western Ireland
(R. Bateman et al., unpubl. res.).
Minority cytotypes
Large-scale population screenings, made possible by FCM,
have changed our perception of intraspecific and intrapopula-
tion ploidy heterogeneity (Kron et al., 2007;Suda et al.,
2007). Previously overlooked minority cytotypes (often occur-
ring at frequencies ,1 %), such as odd ploidy levels or high
polyploids, have recently been discovered in several plant
species; these include Parasenecio auriculata (0.4 % triploids;
2x+4x
3x
6x
5x
3x+5x
3x+6x
5x+6x
3x+5x+6x
2x/4x
15W 10W 5W 0 5E 10E 15E 20E 25E 30E
60N
55N
50N
40N
45N
C
Fig. 2 Continued
Tra
´vnı
´c
ˇek et al. — Minority cytotypes in fragrant orchids984
at Univerzita Karlova v Praze on September 21, 2012http://aob.oxfordjournals.org/Downloaded from

Nakagawa, 2006), Vicia cracca (0.1 % triploids; Tra
´vnı
´c
ˇek
et al., 2010), Actinidia chinensis (0.6 % pentaploids; Li
et al., 2010), Pilosella officinarum (0.3 % heptaploids; Mra
´z
et al., 2008) and Senecio carniolicus (0.1, 0.7, 0.1 and 0.1%
tri-, penta-, hepta- and nonaploids, respectively; Sonnleitner
et al., 2010).
Three minority cytotypes (3x,5xand 6x) with a cumulative
frequency of approx. 2.7 % have also been found in the
G. conopsea complex in the Czech Republic and Slovakia
(Tra
´vnı
´c
ˇek et al., 2011). The substantial extension of the
investigated area and much more intensive sampling in the
present study did not lead to the discovery of further minority
cytotypes. However, although the minority ploidies accounted
for only 1.9 % of all samples (118 out of 6150 individuals),
they markedly increased estimates of both intraspecific and
intrapopulation variation. Without minority cytotypes, only
one species (G. conopsea) and 17 out of 141 populations
(approx. 12 %) would be categorized as mixed ploidy. In
reality, however, ploidy variation (mostly caused by the inci-
dence of minority cytotypes) occurred in all recognized taxa
and in 54 (approx. 38 %) study populations (Table 2). The
number of populations with sympatric 2x+3xcytotypes was
almost double the number of populations where the two major-
ity ploidies (2x+4x) co-occurred (33 vs. 17). In addition, rare
triploids also occupied much wider ranges in Europe than their
more common tetraploid counterparts (cf. Fig. 2B, C; and
Supplementary Data Fig. S1B, C).
A recent survey of ploidy diversity in natural plant popula-
tions (Husband et al., 2012) revealed that although mixed-
ploidy sites occur commonly in some species (e.g. Burton
and Husband, 1999;Sonnleitner et al., 2010), this pattern
largely reflects the coexistence of two or more majority ploi-
dies. Gymnadenia is thus far unique in that it is the incidence
of rare minority cytotypes that largely drives intrapopulation
ploidy variation. One of the few plant systems known to
possess a similar population structure is the daisy Aster
amellus (Manda
´kova
´and Mu
¨nzbergova
´, 2006), which,
however, maintains a much lower proportion of populations
that show sympatry of a majority and a minority cytotype.
Conclusions
Although several chromosomal counts have been published
for the G. conopsea aggregate (e.g. Marhold et al., 2005, and
references therein), only large data sets such as that presented
here, requiring a sampling scheme that is both extensive (many
sites throughout the distribution range) and intensive (many
plants per site), can generate a genuinely holistic picture of
ploidy variation of complex systems and thereby provide
deeper insights into the population dynamics of the studied
systems. We have shown that most Gymnadenia populations
exhibit considerable cytogenetic (and, to a lesser degree, taxo-
nomic) heterogeneity, which should be considered in any
future research to avoid biases introduced by pooling data
from coexisting but nonetheless cytogenetically distinct popu-
lations. We suggest that ongoing production of unreduced
gametes in the majority (2xand 4x) cytotypes, together with
their hybridization in contact zones, led to the establishment
of the minority ploidies (3x,5xand 6x). All of the minority
cytotypes occur only at low frequencies. We assume that
they most probably always originate de novo and that their re-
productive potential is limited. Nonetheless, minority cyto-
types substantially increase intraspecific and intrapopulation
ploidy diversity estimates for fragrant orchids. Our ongoing re-
search aims to explore, using morphometric, molecular and ex-
perimental approaches, the evolutionary history of populations
with ploidy heterogeneity and mechanisms maintaining co-
occurring mixtures of cytotypes.
SUPPLEMENTARY DATA
Supplementary data are available online at www.aob.oxford-
journals.org and consist of the following: Table S1: locality
details and taxonomic/ploidy composition of 141 Gymnadenia
populations from 17 European countries. Figure S1: distribution
of Gymnadenia cytotypes in Europe based on a combination of
present and our previous (Tra
´vnı
´c
ˇek et al., 2011) data. Figure
S2: images of the investigated taxa of Gymnadenia.
ACKNOWLEDGEMENTS
We thank L. Berger, J. Cambece
`des, W. Dworschak,
O. Gerbaud, S. Gustafsson, J.-M. Lewin, W. Mohrmann,
D. Prat, M.-A. Selosse, M. S
ˇtech, T. Urfus and E. Vicherova
´
for their help in the field. We are grateful to the Nature
Conservation Agencies of Aargau, Grison, Ticino and Valais
cantons in Switzerland, National Park Schiermonnikoog,
Conservatoire Botanique National des Pyre
´ne
´es et de Midi-
Pyre
´ne
´es and Landesamt fu
¨r Umweltschutz Sachsen-Anhalt for
issuing collection permits. This study was supported by the
Czech Science Foundation (project 206/09/0843). Additional
support was provided by the Academy of Science of the
Czech Republic (long-term research development project no.
RVO 67985939) and institutional resources of the Ministry of
Education, Youth and Sports of the Czech Republic for the
support of science and research, the Grant Agency of
University of South Bohemia ( projects GAJU 145/2010/P
and GAJU 064/2010/Z), the Estonian Science Foundation
(grant 8584) and Herbarium TAA.
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