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Annals of Botany 131: 71–86, 2023
https://doi.org/10.1093/aob/mcab123, available online at www.academic.oup.com/aob
PART OF A SPECIAL ISSUE ON POLYPLOIDY IN ECOLOGY AND EVOLUTION
Morphological and environmental differentiation as prezygotic
reproductivebarriers between parapatric and allopatric
Campanularotundifoliaagg. cytotypes
KristýnaŠemberová1,2,*, MarekSvitok3,4, KarolMarhold1,5, JanSuda† and RoswithaE.Schmickl1,2,
1Faculty of Science, Department of Botany, Charles University, Benátská 2, 12843, Prague, Czech Republic, 2Czech Academy of
Sciences, Institute of Botany, Department of Evolutionary Plant Biology, Zámek 1, 25243, Průhonice, Czech Republic, 3Faculty of
Ecology and Environmental Sciences, Technical University in Zvolen, T. G.Masaryka 24, 96001, Zvolen, Slovakia, 4Faculty of Science,
Department of Ecosystem Biology, University of South Bohemia, Branišovská 1760, 37005, ČeskéBudějovice, Czech Republic, and
5Institute of Botany, Plant Science and Biodiversity Centre, Slovak Academy of Sciences, Dúbravská cesta 9, 84523 Bratislava, Slovakia
* For correspondence. E-mail Kristyna.semberova@seznam.cz
†Deceased.
Received: 21 April 2021 Returned for revision: 24 August 21 Editorial decision: 20 September 2021 Accepted: 21 September 2021
Electronically published: 4 October 2021
• Background and Aims Reproductive isolation and local establishment are necessary for plant speciation.
Polyploidy, the possession of more than two complete chromosome sets, creates a strong postzygotic reproductive
barrier between diploid and tetraploid cytotypes. However, this barrier weakens between polyploids (e.g. tetraploids
and hexaploids). Reproductive isolation may be enhanced by cytotype morphological and environmental differen-
tiation. Moreover, morphological adaptations to local conditions contribute to plant establishment. However, the
relative contributions of ploidy level and the environment to morphology have generally been neglected. Thus, the
extent of morphological variation driven by ploidy level and the environment was modelled for diploid, tetraploid
and hexaploid cytotypes of Campanula rotundifolia agg. Cytotype distribution was updated, and morphological and
environmental differentiation was tested in the presence and absence of natural contact zones.
• Methods Cytotype distribution was assessed from 231 localities in Central Europe, including 48 localities
with known chromosome counts, using ow cytometry. Differentiation in environmental niche and morphology
was tested for cytotype pairs using discriminant analyses. Astructural equation model was used to explore the
synergies between cytotype, environment and morphology.
• Key Results Tremendous discrepancies were revealed between the reported and detected cytotype distri-
bution. Neither mixed-ploidy populations nor interploidy hybrids were detected in the contact zones. Diploids
had the broadest environmental niche, while hexaploids had the smallest and specialized niche. Hexaploids and
spatially isolated cytotype pairs differed morphologically, including allopatric tetraploids. While leaf and shoot
morphology were inuenced by environmental conditions and polyploidy, ower morphology depended exclu-
sively on the cytotype.
• Conclusions Reproductive isolation mechanisms vary between cytotypes. While diploids and polyploids are
isolated postzygotically, the environmental niche shift is essential between higher polyploids. The impact of poly-
ploidy and the environment on plant morphology implies the adaptive potential of polyploids, while the exclusive
relationship between ower morphology and cytotype highlights the role of polyploidy in reproductive isolation.
Key words: Campanula rotundifolia agg., polyploidy, cytotype distribution, reproductive isolation, contact zone,
diploid, tetraploid, hexaploid, morphological differentiation, environmental niche shift, parapatry, allopatry.
INTRODUCTION
Polyploidy, the possession of more than two complete chromo-
some sets gained through whole-genome duplication (WGD), is
present in all living angiosperms (Jiao etal., 2011) and is widely
considered one of the most important and common modes
of plant speciation (Weiss-Schneeweiss et al., 2013; Wendel,
2015; Landis etal., 2018). Understanding the indisputable evo-
lutionary role of polyploidy and the ubiquity of polyploids in
nature (Soltis and Burleigh, 2009; Mandáková etal., 2017) is
challenging because newly formed polyploids as a minority
cytotype face frequency-dependent selection that can lead to
their extinction (Levin, 1975). The complex physiological pro-
cesses needed for the successful survival and establishment
of polyploids and the necessity of reproductive isolation from
their diploid progenitors thus raised the hypothesis of poly-
ploids as dead ends (Soltis et al., 2009; Mayrose etal., 2015;
Levin, 2019). Reproductive barriers are crucial to reduce the
risk of insufcient fertilization (López-Jurado etal., 2019) and/
or the production of unt hybrids (Hopkins, 2013). Polyploidy
itself instantly creates an effective postzygotic reproductive
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Šemberová etal. — Drivers of polyploid morphology and reproductive isolation
72
barrier between diploids and related polyploids (tetraploids)
by producing undeveloped, less viable or sterile triploid off-
spring (triploid block; Köhler etal., 2010; Hülber etal., 2015).
However, the triploid block is probably not sufcient to prevent
ineffective hybridization, resulting in partially fertile triploids
(triploid bridge; Mandáková et al., 2013; Suda and Herben,
2013; Lafon-Placette et al., 2017; Mao et al., 2020) or tetra-
ploids formed by fusions of reduced and unreduced gametes
(Sutherland and Galloway, 2016). Therefore, prezygotic repro-
ductive barriers are important because they promote assortative
mating within each cytotype (Kolář etal., 2017; Castro etal.,
2020).
Altering genome dosage, WGD immediately impacts cell
size (nucleotypic effect; Levin, 1983) and plant phenotype
(Gigas effect; Ramsey and Schemske, 1998; Simón-Porcar
etal., 2017). The morphology often changes predictably, espe-
cially in synthetic neopolyploids, which may, however, differ
morphologically from polyploids already established in natural
populations under selection (Husband et al., 2016). Altered
oral reproductive traits often do not have an impact on pol-
linator behaviour or plant self-incompatibility (Porturas etal.,
2019; Castro et al., 2020). Thus, cytotype spatial segregation
often accompanied by environmental niche differentiation ex-
plains most patterns of reproductive isolation (López-Jurado
etal., 2019; Castro etal., 2020).
Polyploid niche evolution is limited by available niche space
(Brochmann etal., 2004) and may act not merely at the ploidy
level but also at different lineages within the same cytotype
(López-Jurado et al., 2019). Despite a possible lack of niche
differentiation between diploids and polyploids due to phylo-
genetic niche conservatism (Glennon et al., 2014) or their re-
cent origin and ongoing gene ow (Wos et al., 2019), niche
expansion is frequent for triploids and tetraploids. As the rst
polyploids formed, they ll the niche space unoccupied by
diploids, thus avoiding competition and interploidy mating.
For subsequent polyploids, however, environmental divergence
may be necessary to nd available niches, and the remaining
niche space is often at the extremes of environmental gradients.
Higher ploidies may then encounter constraints to the range due
to limitations in their environmental tolerances (Laport et al.,
2013; Muñoz-Pajares etal., 2017; López-Jurado etal., 2019).
When compared to their diploid or lower polyploid ancestors,
polyploids show niche contraction because they are locally
adapted to narrower and marginal niches in specic habitats
(Sonnleitner etal., 2016; López-Jurado etal., 2019).
Extreme climatic conditions and environmental stress may
also enhance the formation of unreduced gametes (Ramsey
and Schemske, 1998; Wilson etal., 2020), leading to recurrent
polyploid formation. Local adaptations of multiple lineages
within the same cytotype (Parisod and Besnard, 2007; Castro
etal., 2018) often lead to a wider polyploid distribution (López-
Jurado et al., 2019; Castro etal., 2020; Wilson et al., 2020).
Subfunctionalization and neofunctionalization of the dupli-
cated genes (Moore and Purugganan, 2005; Flagel and Wendel,
2009) may be essential for local adaptation and allow for col-
onization of new habitats, which may lead to invasiveness (te
Beest et al., 2012).
Successful polyploid establishment often requires more than
just size-related changes in plant phenotype (Porturas et al.,
2019). Phenotypic changes driven by the local environment
promote higher competitive abilities or habitat displacement
(Balao et al., 2011; Laport et al., 2017). However, they also
mask the morphological changes induced by polyploidization.
Distinguishing the effect of polyploidization from the effects of
selection and local adaptation that follow the reproductive iso-
lation of polyploids from their diploid progenitors is challen-
ging and crucial for studies on plants from natural populations.
Heteroploid taxa provide useful insight into the evolution of
different ploidy levels (Kolář etal., 2017) and allow the assess-
ment of whether differentiation in environmental requirements
and morphology proportionally increases with increasing
ploidy level, following the hypotheses of niche expansion
and the Gigas effect, or is individual for each cytotype. Since
ploidy level and cytotype spatial isolation may inuence the
strength of the prezygotic reproductive barriers, we compared
cytotype pairs based on their ploidy level and geographical dis-
tribution. We hypothesize that cytotypes in contact (sympatry,
parapatry) have higher morphological and ecological differenti-
ation driven by selection for assortative mating (reinforcement;
Hopkins, 2013). On the other hand, morphological and environ-
mental differentiation between populations with limited contact
(parapatry or allopatry) is hypothesized to be driven by local
ecological conditions in differentareas.
Three cytotypes (2x, 4x, 6x) from a polyploid complex of
Campanula rotundifolia agg. co-occur at different levels
of contact in Central Europe (the Bohemian Massif, the
Pannonian Basin and the Western Carpathians; Mráz, 2005).
Acontact zone is either present within a population (sympatry,
mixed-ploidy populations of 2x+4x and 4x+6x; Kovanda, 1967,
1970a, 2002) or between uniform-ploidy populations of each
cytotype (parapatry; Kovanda, 1966; Mráz, 2005; Rauchová,
2007; Šemberová, 2013). The known cytotype distribution
pattern shows a longitudinal shift from diploids in the west
(the Bohemian Massif) to tetraploids in the east (2x–4x mixed
ploidy populations in the Bohemian Massif and a continuous
range of tetraploids in the Pannonian Basin and the Western
Carpathians) (see rst gure in Supplementary Data Material
S1). Hexaploids are reported mainly from 4x–6x mixed ploidy
and a few uniform ploidy populations almost exclusively in the
Pannonian Basin (Gadella, 1964; Kovanda, 1967, 1970a, b,
1983, 2002; Mráz, 2005).
We aimed: (1) to revise and complement existing data on the
distribution of 2x, 4x and 6x cytotypes; (2) to detect differences
and variation in environmental requirements and morphological
differentiation between parapatric and allopatric cytotype pairs
and to test whether the changes proportionally increase with
each ploidy level, following the hypotheses of niche expansion
and the Gigas effect, or are individual for each ploidy level; (3)
to quantify the extent of morphological variability accounted
for by the local environmental conditions and cytotype effects;
and (4) to infer the contribution of environmental and morpho-
logical differentiation to prezygotic reproductive barriers be-
tween the three cytotypes.
MATERIALS ANDMETHODS
Study group and studyarea
The three cytotypes (2n=2x=34 chromosomes, 2n=4x=68
chromosomes, and 2n=6x = 102 chromosomes) of
Campanula rotundifolia agg. (harebells, Campanulaceae, also
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Šemberová etal. — Drivers of polyploid morphology and reproductive isolation 73
referred to as Campanula sect. Heterophylla sensu Mansion
etal., 2012) are widely distributed in Central Europe, an area
where mixed-ploidy, as well as uniform-ploidy, populations
are described (Supplementary Data Material S1; Gadella,
1964; Kovanda, 1967, 1970a, 1977, 1983, 2002; Mráz,
2005; Rauchová, 2007). While diploids were identied in the
Bohemian Massif (western part of the Czech Republic), tetra-
ploids inhabit the broadest geographical range consisting of
two disjunct areas, one in the Bohemian Massif and the other
in the Western Carpathians (north-eastern part of the Czech
Republic, central and eastern Slovakia), with overlap into
the northernmost part of the Pannonian Basin (south-eastern
part of the Czech Republic, north-eastern Austria, southern
Slovakia). Hexaploids are found in the north-western part
of the Pannonian Basin and the adjacent part of the Western
Carpathians.
Central European harebells are usually considered auto-
polyploids (Kovanda, 1970b; Laane et al., 1983; Sutherland
and Galloway, 2018) with complicated interspecic relation-
ships, unresolved phylogeny (Mansion etal., 2012; but see also
Nierbauer et al., 2017; Sutherland etal., 2018; Wilson et al.,
2020) and unclear taxon delimitation (Kovačić, 2004), often
based on ploidy-related changes in size-affected phenotypic
traits (stomata, pollen). However, the trend of size increase
is not beyond doubt (Kovanda, 2002; Rauchová, 2007), and
inferring polyploid origins from morphology only or combined
with niche or distributional range has pitfalls in merging evo-
lutionarily independent lineages (Segraves et al., 1999; Doyle
and Sherman-Broyles, 2017). Thus, similar to works in other
polyploid complexes (Sonnleitner etal., 2016; Hanušová etal.,
2019; López-Jurado et al., 2019), we studied cytotype envir-
onmental and morphological differentiation while omitting
taxonomic entities (diploids and tetraploids in the Bohemian
Massif are represented by C. rotundifolia L. and C. gentilis
Kovanda, tetraploids from the Western Carpathian and hexa-
ploids by C.moravica (Spitzner) Kovanda; Supplementary Data
Fig. S1). In the study region, diploid and tetraploid cytotypes
are also present in endemic taxa. These taxa were omitted
from the study because of their restricted distribution and strict
ecological requirements [subalpine tetraploids: C. bohemica
Hruby: Krkonoše Mts, C. gelida Kovanda: Jeseníky Mts,
C. rotundifolia subsp. sudetica (Hruby) Sóo: Krkonoše
Mts and Jeseníky Mts, C. tatrae Borbás: Tatry Mts, and
diploids: C.serrata (Kit. ex. Schult.) Hendrych: Carpathian Mts,
C.cochleariifolia Lam.: alpine zone in most European moun-
tains and diploid C.xylocarpa Kovanda from Slovakian karst].
Sampling
Plant material from 231 localities was sampled between
2012 and 2017 (Fig. 1; Supplementary Data Table S1), with
a particular focus on localities with published chromosome
counts and mixed ploidy localities (Supplementary Data
Material S1; Gadella, 1964; Kovanda, 1967, 1970a, 1977,
1983, 2002; Mráz, 2005; Rauchová, 2007, Šemberová, 2013).
At each locality, a GPS coordinate was saved, and plants for
further analyses were randomly sampled to cover the entire
morphological and habitat spectra (e.g. plant height, number of
owers, shaded vs. light stands) at a minimum of 15-m spans
to avoid clones (Stevens etal., 2012). On average, 14±18 in-
dividuals and 34±19 individuals were sampled on localities
without and with previously published chromosome counts, re-
spectively (Table S1 and Supplemetary Material S1). Selected
mixed-ploidy localities were subsequently studied to obtain
higher resolution data regarding cytotype spatial distribution.
DNA ploidy (relative genome size) estimation
All individuals were analyzed by ow cytometry (FCM) to
estimate the ploidy level following the best practice recommen-
dation (Sliwinska et al., 2021). Sample preparation followed a
simplied two-step procedure (Doležel etal., 2007): an appro-
priate amount of leaf tissue for both Campanula and internal
standard (Bellis perennis, 2C=3.38 pg, Schönswetter et al.,
2007b) was chopped together by a razor blade in 0.5mL of
ice-cold Otto Ibuffer (0.1 citric acid, 0.5% Tween 20)in
a plastic Petri dish. The suspension of nuclei was ltered
through a 42-µm nylon mesh. For ploidy level estimation,
1 mL of staining solution, containing Otto II buffer (0.4
Na2HPO4·12H2O), 4 µg·mL−1 4′,6-diamidino-2-phenylindole
(DAPI) and 25µL·mL−1 β-mercaptoethanol, was added to the
suspension of nuclei. The solution of stained nuclei was ana-
lysed using a Partec CyFlow ML cytometer (Partec GmbH,
Münster, Germany) equipped with a 365-nm UV-LED as a
source of UV light for DAPI excitation. The uorescence in-
tensity of at least 3000 particles was recorded for further data
processing. Up to 10 individuals were pooled, and samples
with a CV (coefcient of variation) >3% were re-analysed.
Altogether, 6011 individuals were screened for DNA ploidy
level.
Environmentaldata
The environmental niches of the cytotypes were dened
using 36 variables (Supplementary Data Table S2). The data
were acquired from climatic and topographic GIS layers pre-
processed by GeoModel Solar (Bratislava, Slovakia), developer
and operator of the SolarGIS service. Air temperatures at 2m
were derived from the Climate Forecast System Reanalysis
(National Centres for Environmental Prediction, USA) for the
period from 1990 to 2009. The data were spatially enhanced to
30arc-sec resolution by disaggregation based on the correlation
between terrain altitude and temperature. Precipitation data
were processed from the database of the Global Precipitation
Climatology Centre project (Schneider et al., 2014) for the
period from 1951 to 2000. Source data resolution was in-
creased to 2arc-minutes by disaggregation based on the focal
correlation of precipitation with SRTM 30 elevation data and
cloudiness (clear-sky index) derived from the SolarGIS data-
base. Topographic data (altitude, slope and aspect) were
obtained from the terrain elevation model (The Shuttle Radar
Topography Mission data – SRTM3) at 15arc-sec resolution.
Photosynthetically active radiation (PAR) was calculated by
the SolarGIS model for the period 1994–2013 at 15 arc-sec
resolution.
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Šemberová etal. — Drivers of polyploid morphology and reproductive isolation
74
For environmental modelling, long-term yearly and monthly
averages of air temperatures and precipitation were used. As a
measure of climatic variability, the average annual standard de-
viations of temperatures and precipitation were calculated. The
aspect values were linearized and rescaled to range from 0 to
4 (0=south, 1=south-east and south-west, 2=east and west,
3=north-east and north-west, 4=north). Finally, information
on geological bedrock was added from online maps (https://
mapy.geology.cz/geocr50/, http://apl.geology.sk/gm50js/), the
intensity of human inuence was estimated using personal
observations in the eld (human-inuenced vs. natural), and
habitat type was ranked from rocks (0) through grasslands (1)
to forests (2).
Plant morphologicaldata
A subset of 1157 individuals was selected from the sam-
pled material based on the presence of 19 primary charac-
ters (Supplementary Data Material S2) chosen according
to previous studies (Kovanda, 1970b; Rauchová, 2007).
Morphological characteristics were measured on material sam-
pled in July–August 2012 and 2013 using a digital calliper,
and the outer part of the ovary was checked for the presence
or absence of the papilla by using an Olympus SZ51 stereo-
microscope with a magnication of 8–10× (Olympus Corp.,
Tokyo, Japan).
Data analysis
Environmental niche shifts and morphological differentiation
among the cytotypes were assessed using discriminant analysis.
However, a high number of environmental variables (36) and
morphological variables (29) and strong correlations among
them (environmental data: Pearson’s r = −0.937–0.998, mor-
phological data: r=−0.745–0.885) pose severe limitations on
the use of traditional methods. Under these circumstances, clas-
sical linear discriminant analysis can result in a solution with
unstable coefcients or even in the inability to determine the
optimal discriminant function (Wehrens, 2011). An ideal way
of solving this problem would be to consider only a subset of
uncorrelated proximal variables (i.e. those directly inuencing
the distribution of the cytotypes or describing morphological
differentiation). Unfortunately, we had little prior knowledge of
the direct effects of habitat characteristics on the cytotype dis-
tribution or the biologically meaningful morphological differ-
ences between the cytotypes. Moreover, there is no guarantee
12°0'0''E
48°0'0''N 49°0'0''N 50°0'0''N 51°0'0''N
13°0'0''E
N2x
C. gentilis
C. moravica
C. rotundifolia
4x 5x
Western Carpathians
Bohemian Massif Pannonian Basin
02550 100 150 200
km
Sources: Esri, USGS, NOAA
6x
14°0'0''E 15°0'0''E 16°0'0''E 17°0'0''E 18°0'0''E 19°0'0''E 20°0'0''E 21°0'0''E 22°0'0''E 23°0'0''E
F. . Cytotype distribution of Campanula rotundifolia agg. in the investigated area of Central Europe inferred from ow cytometry analyses of individuals
from 231 localities in the Czech Republic, Slovakia and Austria. Black dots mark the populations used for morphometric analysis. Geographical features were
adapted from Mráz (2005) and simplied.
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Šemberová etal. — Drivers of polyploid morphology and reproductive isolation 75
that individual proximal variables would bear independent
(uncorrelated) information (Jiménez-Valverde et al., 2009).
Another possibility would be to exclude correlated environ-
mental and morphological variables prior to the analysis. De
Marco and Nóbrega (2018) advise against subjective dropping
of variables and advocate the use of latent variable methods
such as principal component analysis (PCA). The latent vari-
able approach is easily implemented and commonly used in en-
vironmental niche modelling (e.g. Broennimann et al., 2012;
De Marco and Nóbrega, 2018, and references therein) and
multivariate morphometrics (e.g. Blackith and Reyment, 1971;
Claude, 2008). We adopted a partial least square (PLS), super-
vised latent variable method that has an advantage over trad-
itional PCA since it takes the dependent variable into account
when dening ordination scores and loadings, whereas PCA
captures variance in predictors only. The general idea of PLS
is to construct a few latent variables while maximizing the co-
variance between a predictor and response matrices. PLS in dis-
crimination analysis provides a dimension reduction technique
that nds the optimal group separation while being guided
explicitly by between-group variability (Barker and Rayens,
2003). However, structured noise may complicate the inter-
pretability of PLS models, particularly when there are many
components (Eriksson et al., 2006). To remove such undesir-
able systematic variation in the data, we employed PLS with
orthogonal projection to latent structures (OPLS) (Trygg and
Wold, 2002). The objective of OPLS in discriminant analysis
(OPLS-DA) is to divide the systematic variation in the pre-
dictor matrix into two parts: the predictive part, which models
the relationships between variables and groups, and the orthog-
onal part, which captures the systematic variation in variables
that is linearly unrelated to group separation, which generally
leads to improved model interpretability.
The OPLS-DA models of environmental and morpho-
logical differences among the cytotypes were built with two
predictive components to efciently separate the three ploidy
levels. The environmental and morphological data were
standardized to z-scores to equalize the weight of variables in
the analysis. Since the PLS methods are prone to overtting,
we used cross-validation to select the optimal number of com-
ponents and to assess the model performance on independent
data (Westerhuis etal., 2008). We iteratively tted OPLS-DA
models with increasing complexity (up to 10 components),
trained them on all the data except for one observation at a
time and made predictions for those data points left out of the
training sets (leave-one-out cross-validation). In both environ-
mental and morphological datasets, models with more than two
components provided little or no additional predictive power
(Supplementary Data Fig. S2). Moreover, the relative import-
ance of the components was assessed using randomization tests
where variance explained by the components was compared
with its null distribution generated from data randomly reshuf-
ed 1000 times (Manly, 2006; Westerhuis etal., 2008). To fa-
cilitate interpretation of the results, OPLS-DA score plots with
95% condence ellipses were displayed. Variable weights for
the signicant components were plotted to assess the relative
inuence of environmental and morphological characteristics
on the discrimination of ploidy levels. Using an orthogonal pro-
jection to latent structures, these weights are primarily related
to differences between cytotypes and their interpretation is
quite straightforward (Wold etal., 2001).
Environmental niche breadth and morphological variation
were compared among the three cytotypes using distance-based
tests of homogeneity of multivariate dispersions (Anderson,
2006). Environmental heterogeneity was quantied by calcu-
lating pairwise Euclidean distances among sampling sites from
the matrix of standardized habitat characteristics. For morpho-
logical analyses, characters measured across multiple individ-
uals were averaged within sites to cope with autocorrelation
in the repeated measurements. The spread of sites from their
medians in principal component space was used as a measure
of heterogeneity. Equality of multivariate dispersion was tested
using a randomization test based on 1000 permutations of the
least absolute deviation residuals.
In addition to the overall comparison, cytotypes were
divided into subsets based on their geographical distribution,
and the differences in environmental niche and morphological
traits were compared between parapatric and allopatric popu-
lations. In parapatry, we compared 2x–4x from the Bohemian
Massif, 6x–4x from the Western Carpathians and 2x–6x from
the Bohemian Massif and the Pannonian Basin. In allopatry, we
compared 2x–4x from the Western Carpathians, 6x–4x from the
Bohemian Massif and 4x–4x from the Bohemian Massif and
the Western Carpathians. In selected sympatric populations,
detailed cytotype distribution was displayed to assess cytotype
microhabitat preferences.
Plant morphology is affected by both cytotype and environ-
ment, and the environment may also relate to cytotype distribu-
tion. The evidence for potential synergies was explored using a
causal network of piecewise structural equation models (SEMs;
Shipley, 2009). To gain more insight, the environmental data
were split into two subsets specifying local habitat conditions
and climate (Supplementary Data Table S2), and the morpho-
logical data were split into three subsets consisting of variables
characterizing the morphology of the leaf, ower and shoot
(Supplemetary Material S2). These multivariate datasets were
standardized and subjected to PCA to reduce their dimen-
sionality. The rst components for each subset accounted for
24–75% of the variance in the datasets and were used in the
SEM to represent original multivariate data. The variables were
arranged in a directed acyclic graph, and a series of regression
models was tted to integrate plant morphology with ploidy
levels and environment. Linear mixed models (LMMs; Pinheiro
and Bates, 2000) were used to test the effect of cytotype and
environment on plant morphology while accounting for mul-
tiple plants measured at the same sites. The structure of the
LMMs involved random intercepts of sites and xed effects of
cytotype, habitat and climate. The models were screened for
normality, homoscedasticity and spatial autocorrelation using
standard diagnostic plots of residuals and spline correlograms
(Bjørnstad and Falck, 2001). The LMMs for leaf and shoot
morphology showed heterogeneous error variances, and thus
the original models were reformulated by including an expo-
nential variance function structure to x the heteroscedasticity.
No other violations of model assumptions were detected. To
investigate the inuence of the environment on the distribution
of cytotypes, we tted the ordinal outcomes of ploidy levels by
using a cumulative logit mixed model with the Gauss–Hermite
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Šemberová etal. — Drivers of polyploid morphology and reproductive isolation
76
quadrature approximation (Tutz and Hennevogl, 1996).
Likelihood-ratio tests were used to assess the overall signi-
cance of the models and the signicance of individual terms.
Marginal determination coefcients (R2m) were calculated to
quantify the proportion of the total variance explained by the
models (Nakagawa et al., 2017). The relative contribution of
individual variables was assessed using semi partial marginal
determination coefcients (sR2m) derived from commonality
analysis (Ray‐Mukherjee etal., 2014). Correlative paths in the
SEM were estimated using the Pearson correlation coefcient.
All analyses were performed in R language version 3.6.0
(R Core Team, 2019) using the libraries ellipse (Murdoch and
Chow, 2018), ggplot2 (Wickham, 2016), lme (Pinheiro et al.,
2019), ordinal (Christensen, 2019), ncf (Bjørnstad, 2019), per-
formance (Lüdecke et al., 2020), pls (Mevik etal., 2019) and
vegan (Oksanen etal., 2019).
RESULTS
Cytotype spatial distribution, mixed-ploidy populations and
potential DNA aneuploids
A survey of 6011 individuals from 231 localities in Central
Europe analysed by ow cytometry revealed three main ploidy
levels (2x, 4x, 6x; Fig. 1, Supplementary Data Table S1),
one odd ploidy level (5x in the locality Dreveník; site ID 14,
Supplementary Data Material S3 and Supplementary Data
Fig. S3) and several irregularities in the relative uorescence
suggesting differences in relative genome size and interpreted
as potential DNA aneuploids (for all three ploidy levels from
17 localities; six, three and eight localities for 2x, 4x and 6x
potential DNA aneuploids, respectively; Supplementary Data
Material S3). Potential DNA-aneuploids were detected in sam-
ples where up to 10 plants were pooled, with the CV varying
from 1.18 to 2.14%.
Cytotypes were spatially clustered with parapatric distri-
bution (Fig. 1, Supplementary Data Material S1). Hexaploids
exclusively dominate the Pannonian Basin with a warm and
continental climate and the westernmost part of the Western
Carpathians (site IDs: 32, 33). Hexaploids share a long con-
tact zone with tetraploids at the borders between the Western
Carpathians and the Pannonian Basin, and another large area
of tetraploids was found in the Bohemian Massif (i.e. Southern
and Central Bohemia). Additional populations of tetraploids
were scattered in South Moravia where the Bohemian Massif,
the Pannonian Basin and the Western Carpathians are in contact
(site IDs: 57, 66, 90, 97, 101)and in relict-like habitats such as
serpentinite outcrops and rocky river valleys within the diploid
range (Western and Central Bohemia: site IDs: 128, 129, 139,
273, 274, 469)and the hexaploid range (Southern Slovakia, site
ID: 30). Diploids are widespread but almost exclusively in the
Bohemian Massif, where they occupy a large spectrum of habi-
tats (ruderal roadsides, pastures, meadows, forest edges, rocks
and castleruins).
A comparison of our ow cytometry data with previ-
ously published chromosome counts from the same locality
showed discrepancies in ploidy level in 38% of populations
(Supplementary Data Material S1; Gadella, 1964; Kovanda,
1967, 1970a, 1977, 1983, 2002), including mixed-ploidy popu-
lations (Kovanda, 1967, 1970a). All revised mixed-ploidy
populations were found to be of uniform ploidy, with only
the higher ploidy present (Supplementary Data Material S1).
However, eight mixed-ploidy populations (2x–4x, 4x–5x–6x,
2x–6x) were detected elsewhere, with higher ploidies always
a minority except for the 2x–6x mixed-ploidy population with
an almost equal proportion of 2x and 6x (Fig. 1, Supplementary
Data Table S1 and Fig. S3).
Environmental niche breadth and niche divergence among
cytotypes
OPLS-DA revealed signicant shifts in environmental niches
among the cytotypes (Table 1). The cytotypes were separated
along with the rst predictive component that signicantly ac-
counted for almost 52% of the variability in the dataset (Fig.
2A). The second component was statistically non-signicant.
The rst discriminant function represents the main climatic
gradient, the contrast between calcareous and siliceous bed-
rocks and, to some extent, the intensity of human pressure (Fig.
3; Supplementary Data Table S3). Hexaploids prefer hotter
and drier habitats with calcareous bedrock, and diploids oc-
cupy sites with lower temperatures and higher humidity often
situated on siliceous bedrock with higher anthropic impact.
Tetraploids typically dwell in intermediate conditions, which
is also apparent from their central position in the ordination
space of the OPLS-DA (Fig. 2A). Considering pairwise dif-
ferences, the environmental niche of hexaploids differed sig-
nicantly from both diploids (variance explained by the rst
component = 57.7 %, P = 0.027) and tetraploids (61.2 %,
P= 0.002). The niches of diploids and tetraploids overlapped
considerably and were statistically indistinguishable, either be-
tween parapatric or between allopatric cytotype pairs (Table 1).
The environmental niche breadth differed signicantly
(F=11.2, P<0.001), and the environmental space occupied
by the cytotypes decreased not gradually but in the following
direction: 4x>2x>6x (Table 1). The same trend was recorded
for parapatric and allopatric pairwise comparisons, although
niche breadth between parapatric 2x–4x in the Bohemian
Massif and the allopatric tetraploids did not differ signicantly.
Morphological differences and variation among cytotypes
While cytotype morphological variation did not increase
with increasing ploidy levels, their morphology differed sig-
nicantly (Table 1). The cytotypes were gradually separated
along with the rst OPLS-DA component, which signicantly
accounted for more than 22% of the variability in the mor-
phological dataset (Figs 2B and 3; Supplementary Data Table
S4). Diploid plants differed signicantly from both tetra-
ploids (variance explained by the rst component= 19.9%,
P<0.001) and hexaploids (25.7%, P < 0.001) in vegetative
characters. The latter cytotypes also differed, mainly in genera-
tive characters, although marginally non-signicantly (14.5%,
P=0.072). The density of the papillae on the ovary, the ratio
of the length/width of the leaves in the middle and upper parts
of the stem, the length of the leaves in the middle part of the
stem, and other characteristics continually increased from dip-
loids to hexaploids, while the width of the leaves in the middle
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Šemberová etal. — Drivers of polyploid morphology and reproductive isolation 77
and upper parts of the stem and the lament length continually
decreased (Fig. 3; Supplementary Data Table S4). Comparisons
of parapatric and allopatric cytotype pairs generally corrob-
orated these results; however, two specic ndings emerged
(Table 1; Supplementary Data Table S4). First, allopatric tetra-
ploids differed signicantly in generative characters (shorter
laments and anthers and smaller ratios of the corolla length
to calyx or corolla lobe lengths). Second, there were no differ-
ences between parapatric 2x–4x in the Bohemian Massif and
6x–4x from the Western Carpathians.
Environmental and ploidy level effects on morphology
Structural equation modelling showed that cytotype distri-
bution was inuenced by local habitat conditions (e.g. bed-
rock, PAR and altitude) rather than climatic characteristics,
although both groups of environmental features were positively
correlated (Fig. 4). Ploidy level signicantly affects the shape
of leaves (F=22.1, P<0.001), owers (F=19.3, P<0.001)
and, to a lesser extent, the overall plant (F=6.1, P=0.002).
Environmental conditions signicantly inuenced leaf morph-
ology (habitat effect: F = 39.4, P < 0.001; climate effect:
F = 10.9, P = 0.001) and shoot morphology (climate effect:
F=7.5, P=0.007), while the shape of generative organs was
inuenced only by ploidylevel.
DISCUSSION
The current cytotype distribution
Central Europe is acknowledged as a potential region of origin
of Campanula rotundifolia agg. (Sutherland et al., 2018;
Wilson etal., 2020) because it may host the original diploids.
From here, further spread of the complex was facilitated by
polyploidization (Sutherland and Galloway, 2018). However,
the longitudinal pattern of a ploidy increase from Central
Europe to Western Europe and North America (Sutherland
etal., 2018) is not supported in this study. Available chromo-
some counts (Böcher, 1936; Podlech, 1965) limited knowledge
of the full diploid distribution, and the presence of polyploids
in Central Europe was omitted, partly due to the intricate tax-
onomy of this group. The presented data favour the hypothesis
of the polytopic origin of polyploids on a small (e.g. tetra-
ploids from the Western Carpathians, the Bohemian Massif and
autotetraploids in mixed-ploidy populations, Fig. 1) as well as
a large geographical scale [e.g. hexaploids in the Pannonian
Basin; Fig. 1, in Northern Italy (Fenaroli et al., 2013) and in
Britain and Ireland (Wilson et al., 2020), tetraploids in the
Western Carpathians/the Bohemian Massif; Fig. 1, in Britain
(Wilson et al., 2020) and North America (Sutherland et al.,
2018)].
In contrast to previous cytotype distributions based on pub-
lished chromosome counts, cytotypes are geographically
clustered (Fig. 1, Supplementary Data Material S1). Diploids
have a distribution centre in the Bohemian Massif and are
otherwise rare or endemic in Europe and North America, ex-
cept for North Scandinavia (Laane et al., 1983). The largest
discordance was found for the tetraploid cytotype detected in
T . Summary of the tests for differences in environmental niche shifts, environmental niche breadth, morphology and morphological variability among three cytotypes
(2x, 4x and 6x) of Campanula rotundifolia agg. in Central Europe (tetraploids are divided into two groups based on their geographical occurrence: Western Carpathians=WC,
Bohemian Massif=BM). Tests of niche shifts and morphological differences are based on OPLS-DA, and the table shows variance explained by the predictive components
(comp 1, comp 2)along with probabilities in parentheses. Tests of differences in niche breadth and morphological variability are based on the homogeneity of multivariate
dispersion, and the table shows F statistics (F) and respective probabilities in parentheses. Results signicant at α=5% are highlighted in bold. Pairwise comparisons of the
signicant results are given in the contrast column. The type of cytotype spatial isolation (parapatry, allopatry) is indicated for pairwise comparisons.
Model Contact zone Environmental niche shift Environmental niche breadth Morphological differences Morphological
variability
Comp 1 (%) Comp 2 (%) Contrast F Contrast Comp 1 (%) Comp 2 (%) Contrast F Contrast
All (2x vs. 4x vs. 6x) 52 (0.027) 15 (0.517) 2x ≠ 6x, 4x ≠ 6x11.2 (< 0.001) 6x<2x<4x22 (< 0.001) 15 (0.181) 2x ≠ 6x, 2x ≠ 4x0.4 (0.691) –
2x vs. 4x BM para 35 (0.475) – – < 0.1 (0.888) – 14 (0.331) – – 0.7 (0.417) –
2x vs. 4x WC allo 31 (0.555) – – 5.7 (0.018) 2x<4x WC 22 (< 0.001) – 2x ≠ 4x WC 0.8 (0.370) –
6x vs. 4x BM allo 52 (0.029) – 6x ≠ 4x BM 11.5 (0.001) 6x<4x BM 20 (0.014) – 6x ≠ 4x BM 1.2 (0.287) –
6x vs. 4x WC para 65 (0.003) – 6x ≠ 4x WC 14.6 (< 0.001) 6x<4x WC 11 (0.466) – – 0.6 (0.457) –
2x vs. 6xpara 58 (0.027) – 2x ≠ 6x10.2 (0.002) 6x<2x26 (< 0.001) – 2x ≠ 6x0.3 (0.577) –
4x WC vs. 4x BM allo 31 (0.485) – – 2.7 (0.108) – 19 (0.012) – 4x WC ≠ 4x BM 1.9 (0.174) –
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Šemberová etal. — Drivers of polyploid morphology and reproductive isolation
78
all regions (the Bohemian Massif, the Pannonian Basin and
the Western Carpathians). Populations from the Pannonian
Basin (populations with site IDs: 57, 66, 90, 97, 101)were not
studied for morphological and environmental differentiation
because of their low number and uncertain origin. These popu-
lations nested in the contact zone of the diploid and hexaploid
cytotypes may represent autopolyploids that successfully estab-
lished uniform ploidy populations. Alternatively, these popula-
tions may have remained from a formerly large tetraploid area
that was now found to be hexaploid. However, none of these
populations was included in studies involving chromosome
counts (Supplementary Data Material S1). These tetraploids
could also represent hybrids between diploids and hexaploids
that may rarely be formed (Meeus etal., 2020) despite the rela-
tively high postzygotic reproductive barrier between 2x and 6x
cytotypes (Scott etal., 1998). Additional tetraploid populations
were scattered within the diploid area, mostly on serpentinite
outcrops (Fig. 1). The stressful nature of serpentinite envir-
onments (Brady et al., 2005) may lead to higher production
of unreduced gametes (Ramsey and Schemske, 2002; Wilson
etal., 2020) or may serve as a refugium for rare cytotypes, thus
bypassing minority-cytotype exclusion (Kolář et al., 2012).
Arange expansion was detected for the hexaploid cytotype. It
is almost exclusive in the Pannonian Basin, where hexaploids
were mostly reported either from mixed-ploidy or formerly
tetraploid populations (Gadella, 1964; Kovanda, 1967, 1970a,
1977, 1983; Mráz, 2005; Supplementary Data Material S1).
Contact zones similar to those between 2x–6x and 6x–4x that
roughly follow the borders of the studied regional features or
between the Czech Republic and Slovakia were also detected
for other ploidy-variable taxa (Mráz et al., 2008; Trávníček
etal., 2010; Kobrlová etal., 2016; Macková etal., 2020).
Mixed-ploidy population dynamics and potential DNA aneuploids
Flow cytometry detected a shift in the frequency and lo-
cation of mixed-ploidy populations (Supplementary Data
Material S1). This approach screened the actual ploidy vari-
ation in the population, while previous studies used chromo-
some counts on seedlings from seeds collected at the locality
(Gadella, 1964; Kovanda, 1966, 1970a, b; Rauchová, 2007).
The possible establishment and survival of such seedlings may
differ in the eld and experimental cultivation (Sutherland
and Galloway, 2016; Meeus etal., 2020). Experimental 2x–4x
crosses yielded an almost equal frequency of 3x and 4x offspring
(Sutherland and Galloway, 2016) However, in simulated 2x–
4x open-pollinated contact zones, the ratio of homoploid and
heteroploid crosses varied depending on cytotype frequency
and pollinator preferences for the rare cytotype (Sutherland
et al., 2020). Neither 3x nor 4x hybrids are likely to con-
tribute to the interploidy gene ow in mixed-ploidy popula-
tions (Sutherland and Galloway, 2021). In simulated 2x–4x
contact zones, the persistence of minority cytotypes was fa-
cilitated by pollinator preferences for the rare ploidy level and
near-complete postzygotic reproductive isolation. The lack of
intermediate cytotypes in mixed-ploidy populations (2x–4x,
2x–6x) or the 2x–4x contact zone supports the hypotheses of
a strong triploid block and no secondary contact zone (Scott
etal., 1998; Sutherland and Galloway, 2016; Lafon-Placette
6
2x (79%)
Cytotype
4x (58%)
6x (28%)
2x (73%)
Cytotype
4x (69%)
6x (44%)
3
0
–3
–6
–10 –5 05
Component 1 (51.6%,
P
= 0.027)
Component 2 (14.8%, P = 0.517)
6
3
0
–3
–6
Component 2 (15.5%, P = 0.181)
10 15 –8 –4 04
Component 1 (22.1%,
P
< 0.001)
8
Environmental niche shifts
Classification accuracy = 64.6%
Morphological differences
Classification accuracy = 65.1%
AB
F. . OPLS-DA score plots of predictive components with 95% condence ellipses showing differences in environmental niches (A) and morphology (B)
among three major cytotypes of Campanula rotundifolia agg. The relative proportion of variance explained by the components is displayed in parentheses along
with probabilities (P). Cross-validated classication accuracies and sensitivities are given as percentages for each model and cytotype, respectively.
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Šemberová etal. — Drivers of polyploid morphology and reproductive isolation 79
etal., 2017; Castro et al., 2020) where cytotype spatial sep-
aration may be enhanced via reinforcement (Sutherland and
Galloway, 2021). Mixed-ploidy (2x–4x) localities within the
diploid range thus probably represent a primary contact zone
of 2x and auto-4x, mediated by a high production of diploid
unreduced gametes (Castro et al., 2018) and low viability of
possibly formed triploids. The lack of tetraploids, pentaploids
or other cytotypes in the 2x–6x mixed-ploidy population sug-
gests strong endosperm barriers between these two cytotypes
(Scott etal., 1998; Meeus etal., 2020), supported by altitudinal
cytotype separation (Schönswetter etal., 2007a). An almost
similar proportion of diploids and hexaploids suggests it is a
secondary contact zone. Unlike 2x–4x experimental crosses of
Campanula rotundifolia agg. that yielded relatively balanced
frequencies of 3x and 4x offspring, the 4x–6x crosses yielded
almost exclusively pentaploids (Sutherland and Galloway,
2016). However, in simulated 4x–6x open-pollinated contact
zones, the pollinator preferences for the rare cytotype altered
the ratio of homoploid and heteroploid crosses with regard
to cytotype frequency (Sutherland et al., 2020). In contrast
to simulated 2x–4x contact zones, in simulated 4x–6x contact
zones, the lack of postzygotic reproductive barriers and fre-
quent formation of viable and fertile 5x may lead to asymmetric
gene ow depending on cytotype frequency and introgression
(Sutherland etal., 2020; Sutherland and Galloway, 2021). In
contrast to the results of Sutherland etal. (2020), Wilson etal.
(2020) detected a variable one-sided barrier favouring the per-
sistence of tetraploids in 4x–6x mixed-ploidy populations in
Britain. We found pentaploids and hexaploids within a pre-
dominantly tetraploid population (Fig. S3 and Supplementary
Data Material S3), similar to the pattern in Britain (Wilson
et al., 2020), Germany (Nierbauer et al., 2017) and North
America (Sutherland and Galloway, 2018). These hexaploids
are probably autopolyploids and form pentaploids via a 4x–
6x backcross, and pentaploids may further participate in the
heteroploid hybridization (Laport etal., 2016; Wilson et al.,
2020; Peskoller etal., 2021; Sutherland and Galloway, 2021).
At this locality, higher ploidies were found near tourist paths,
Environmental niche shifts Morphological differences
T11
AB
T8
T7
T6
Tyear
T4
T5
T9
T3
T10
Tsd
T2
T12
T1
Slope
Bedroc
k: heavy metal-rich
Psd
Aspect
Habitat
Bedrock: vlucanite
P6
P11
P10
P12
P4
P2
P5
P1
P3
P7
P9
P8
–0.2 0.0 0.2 –0.2 0.0 0.2
Pyear
Altitude
Bedrock: siliceous
Anthropic influence
PARsd
PARyear
Bedrock calcaneous
PA P
LMLONG/LMW
ULLONG/ULW
LMLOMG
STEM
ANTH
LREST
CLOBW
KLOBLONG
ULLONG
STEM/INFL
LDOWN
KLOBW
KLOBLONG/KLOBW
HAIRS
CLONG/KLOBLONG
INFL
BRANCH
CLONG/CLOBLONG
LDOWN(LDOWN+LREST)
FIL
ULW
LMW
Loading weights of the first predictive components
FLOWER
CLOBLONG/KLOBW
CLOBLONG/CLOBW
CLOBLONG
G
CLONG
F. . Va r iable weights for the rst (signicant) predictive components of OPLS-DAs (Fig. 2) performed on environmental conditions (A) and morphological
characteristics (B) of Campanula rotundifolia agg. cytotypes. For abbreviations of variable names, see Supplementary Data Material S2 and Table S3.
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Šemberová etal. — Drivers of polyploid morphology and reproductive isolation
80
which implies a role of human-induced stress in the higher
production of unreduced gametes (Wilson etal., 2020).
Potential DNA aneuploids were found in all cytotypes,
including pentaploids (Supplementary Data Material S3).
Chromosome counts for 4x C.gentilis with 68 euploid chromo-
somes detected a variation of 67, 70, 73 and 75 chromosomes
for aneuploids with a respective deviation of the DAPI ratio
in ow cytometry analyses (Rauchová, 2007). Similar devi-
ations were observed in Britain at sites with heavy metal soil
for tetraploids and autohexaploids (Wilson et al., 2020). This
nding further supports the role of environmental stress in the
production of unreduced gametes (Ramsey and Schemske,
1998). However, Rauchová (2007) also counted an individual
with a DAPI ratio corresponding to a potential DNA aneuploid
as a tetraploid with 68 euploid chromosomes, which suggests
intraspecic genome size variation. A mix of potential DNA
aneuploids and individuals with different genome sizes was
also observed for the studied individuals (Supplementary Data
Material S3).
A shift in cytotype distribution
The shift between published and observed ploidy distribution
favouring higher ploidy levels in mixed- and uniform-ploidy
populations (Supplementary Data Material S1) suggests that
polyploids are well established and have higher colonization
success. However, the time needed to successfully replace the
lower ploidy detected by earlier chromosome counting would
be relatively short (M. Kovanda performed his studies on
ULLLONG/ULW
PARsd
PARyear
Bedrock: calc.
Bedrock: silic.
Bedrock: vulc.
Altitude
T3
P8
T9
P7
T10
T11
T4
Tyear
Anthro
Aspect
Leaf
morphology Habitat
–0.550
–0.466
–0.439
–0.401
0.382
0.364
0.345
0.098
–0.102
–0.210
–0.209
0.208
–0.208
0.207
–0.204
–0.203
–0.203
–0.502
–0.485
–0.397
0.179
0.145
–0.430
–0.394
–0.383
–0.368
–0.335
–0.265
–0.233
0.539
0.527
0.501
–0.298
–0.091
–0.076
0.194
0.188
–0.199
LMLONG/LMW
LMLONG
R2 = 25.2%
P < 0.001
Cytotype
Climate
R2 = 37.2%
P < 0.001
Flower
morphology
R2 = 15.1%
P < 0.001
Shoot
morphology
R2 = 8.1%
P < 0.001
sR2 = 2.3%
sR2 < 0.1%
P = 0.001
P = 0.839
sR2 = 2.4%
sR2 < 0.1%
P = 0.231
sR2 < 0.1%
P = 0.288
r = 0.66
P < 0.001
sR2 = 11.0%
P < 0.001
sR2 = 4.4%
P = 0.006
sR2 = 2.9%
P < 0.001
P = 0.051
sR2 = 10.9%
P < 0.001
sR2 = 22.7%
P < 0.001
sR2 = 3.1%
P < 0.001
r = 0.16
P < 0.001
r = 0.71
P < 0.001
r = 0.71
P < 0.001
ULLONG
LMW
ULW
CLONG
CLOBW
G
CLOBLONG
KLOBLONG
ANTH
KLOBL/KLOBL
CLOBL/KLOBL
VET
INFL
FLOWER
STEM/INFL
STEM
LRES
HAIRS
LDOWN/(LD + LR)
F. . Structural equation model linking environmental characteristics (habitat and climate) with the distribution of cytotypes, leaf morphology, ower
morphology and shoot morphology. Arrows represent signicant (solid lines) and non-signicant (dashed lines) relationships between variables. Unidirectional
arrows denote direct effects of one variable on another, and their size is proportional to semi-partial marginal determination coefcients (sR2). Bidirectional
arrows denote parwise correlations between variables, and their size is proportional to Pearson correlation coefcients (r). For each response variable, vari-
ance explained by predictors (marginal determination coefcients – R2) and statistical signicance of whole models (P) are displayed. Morphological and
environmental characteristics are latent variables derived as rst components from principal component analyses on the original data. The most important ori-
ginal variables (rectangles) are shown along with their loadings on the latent morphological and environmental variables. For abbreviations of variable names,
see Supplementary Data Material S2 and Table S3.
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Šemberová etal. — Drivers of polyploid morphology and reproductive isolation 81
Campanula rotundifolia agg. 50years ago), and in simulated
contact zones of Campanula rotundifolia agg., the cytotypes
produced progeny of the same ploidy level more frequently than
expected under random mating (Sutherland etal., 2020; Wilson
etal., 2020). Thus, the likelihood of counting a seedling of a
different ploidy than that of the mother plant is relatively low,
especially in 4x or 4x–6x populations. Moreover, Wilson etal.
(2020) showed that under open pollination, tetraploid mothers
produced almost exclusively tetraploid offspring, while hexa-
ploid progeny was more variable, including many pentaploids
and aneuploids.
Although our sampling for the ploidy level estimates by ow
cytometry did not cover the entire population, two cytotypes
were often detected in populations from which only 11 individ-
uals were sampled (Supplementary Data Table S1). Localities
with published chromosome counts were sampled more thor-
oughly (30 individuals at a minimum; Supplementary Data
Material S1). Nevertheless, the previously published cytotypes
still may have been undetected in some undersampled
populations.
Several studies on other plant species also show large dis-
crepancies between published and observed ploidy variation in
a similar pattern (exclusive occurrence of higher ploidy levels)
for populations counted by M.Kovanda, and they assigned these
differences to errors or difculties in chromosome counting or
to sampling artefacts (Laane et al., 1983; Weiss et al., 2002;
Rauchová, 2007; Vít etal., 2012; Macková etal., 2020; Wilson
et al., 2020). This raises awareness that the original data do
not reect the actual cytogeography. However, these data
are still kept in online karyological databases (e.g. Marhold
et al., 2007: database available at http://www.chromosomes.
sav.sk; Rice et al., 2015: database available at http://ccdb.tau.
ac.il), which may bias further metanalyses using these sources
(Porturas etal., 2019; Rice etal., 2019). Therefore, the use of
published data without verifying the cytotype distribution in the
eld with high-throughput methods, such as ow cytometry, is
highly questionable. More cytogeographical studies are thus
needed because they very often discover unexpected vari-
ability, either at the local (Chumová et al., 2015; Trávníček
etal., 2012; Caperta etal., 2017; Nierbauer etal., 2017; Wilson
etal., 2020) or at broad geographical scales (Krejčíková etal.,
2013; Čertner etal., 2017; Nierbauer etal., 2017; Paule etal.,
2017; Hanušová etal., 2019; Rejlová etal., 2019). New data on
ploidy variation provide valuable insights into the evolutionary
mechanisms shaping the gene ow dynamics of polyploids
and, consequently, speciation. Minority cytotypes can help to
detect interploidy hybridization through a triploid or a penta-
ploid bridge (Mandáková et al., 2013; Peskoller et al., 2021)
with consequences for conservation genetics (Nierbauer etal.,
2017; Macková etal., 2018), to detect the dynamic formation
of unreduced gametes and neopolyploids (Wilson etal., 2020;
Sutherland and Galloway, 2021) and reveal cryptic variation
that may result in speciation events (Flatscher et al., 2015).
Describing cytotypes as different species has its pitfalls such as
in the case of Campanula gentilis and C.moravica. These two
taxa were delimited from C.rotundifolia agg. mainly because
of their different chromosome numbers (C. moravica) or the
presence of mixed-ploidy populations (C.gentilis), and minute
morphological differences related to WGD. The morphological
differentiation was later doubted (Kovanda, 2002; Rauchová,
2007; Šemberová, 2013), leaving geographical distribution and
chromosome number the only reliable identiers of C.gentilis
and C. moravica, respectively. A shift in cytotype distribu-
tion is also of relevance to the loci classici of C.gentilis and
C.moravica, which implies a need for a detailed taxonomical
revision. Taxonomic complexity, unclear geographical distribu-
tion and low morphological differentiation of individual spe-
cies led us to omit the species names in this study.
Cytotype morphological and environmental differentiation as
prezygotic reproductive barriers
Higher morphological variation, often created by polyploidy,
was not conrmed in higher polyploids (6x) despite the trend
toward larger size observed in some traits, especially leaf length
and width. Similarly, morphological differentiation was more
pronounced between diploids and polyploids than between
higher ploidies (Table 1, Fig. 2B; Supplementary Data Table
S4; Porturas etal., 2019). Only cytotype pairs involving hexa-
ploids and those that were spatially isolated differed morpho-
logically. Morphological changes induced by WGD may vary
within the same cytotype (Laport and Ramsey, 2015; López-
Jurado etal., 2019), suggesting that allopatric tetraploids prob-
ably represent lineages independently formed via recurrent
formation. The lack of an environmental niche shift suggests
that adaptations to local microclimatic or microhabitat condi-
tions mirrored by the different geomorphological histories and
ora of the two regions (Kaplan, 2012; Mráz and Ronikier,
2016) played a role in the tetraploid morphological differen-
tiation (Castro et al., 2018; López-Jurado et al., 2019; Wilson
et al., 2020). The lower postzygotic reproductive barrier and
higher gene ow between higher ploidy levels (Sutherland and
Galloway, 2021) would need to be compensated for by a strong
prezygotic reproductive isolation (e.g. by altering phenology or
pollinator preferences). However, in simulated contact zones
of C. rotundifolia agg., pollinators did not prefer a specic
cytotype but they overvisited the rare one (Sutherland et al.,
2020). This contrasts with studies comparing plant–pollinator
interactions along an elevational gradient where differences in
plant morphology correlated with altitude and a related shift
in pollinator size (Maad etal., 2013). In addition, differences
in phenology were detected between plants from different
European countries (Preite etal., 2015) but not on a smaller re-
gional scale, among the plants from Britain and Ireland (Wilson
etal., 2020). While diploids from across Europe owered later
than some European tetraploids (Gadella, 1964), diploids and
tetraploids in the Bohemian Massif started owering earlier than
hexaploids and tetraploids in the Pannonian Basin and Western
Carpathians, suggesting a longitudinal shift in phenology.
The narrowest environmental niche breadth and the niche
shift from all other cytotypes suggest niche specialization of
the hexaploid cytotype and support the niche-lling hypoth-
esis (López-Jurado etal., 2019). In contrast, tetraploids had the
widest niche, similar to the pattern of niche expansion observed
in other polyploid complexes (Karunarathne et al., 2018;
López-Jurado etal., 2019; Molina-Henao and Hopkins, 2019).
However, this was true only when tetraploids from the Bohemian
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Šemberová etal. — Drivers of polyploid morphology and reproductive isolation
82
Massif and the Western Carpathians were merged in the ana-
lysis. The environmental niche breadth of tetraploids from each
area (either Bohemian Massif or Western Carpathians) was
still broader than that of hexaploids but narrower than that of
diploids (Table 1). Diploids had the broadest niche. While poly-
ploids may benet from gene neofunctionalization serving as
pre-adaptation to a broader distribution, diploids, as the original
and rst cytotype, had the longest time to spread and adapt to
the local environment.
The lack of niche shift (Table 1) between diploids and tetra-
ploids, either in parapatry or in allopatry, may indicate a partly
sufcient postzygotic reproductive barrier because no triploids
were detected in the eld. The triploid block may be bypassed
by the tetraploid progeny (Sutherland and Galloway, 2016), and
ongoing gene ow could explain the lack of morphological dif-
ferentiation. However, no gene ow (Sutherland and Galloway,
2021) and no mixed-ploidy populations were found in the con-
tact zone between diploids and tetraploids in the Bohemian
Massif (Fig. 1), suggesting low viability of triploid and tetra-
ploid hybrids. In contrast, the lack of pentaploid hybrids be-
tween parapatric tetraploids and hexaploids despite the lack
of morphological differentiation may imply that the environ-
mental niche shift is an efcient prezygotic reproductive barrier
promoting assortative mating (Husband and Schemske, 2000;
Sonnleitner etal., 2010; Castro etal., 2020). However, the en-
vironmental niche shift turned out not to be affected by the level
of cytotype spatial isolation or strength of the postzygotic re-
productive isolation because it was detected only for cytotype
pairs, including hexaploids, irrespective of parapatry or
allopatry. Thus, it is probably not due to reinforcement but ra-
ther reects the niche specialization of the hexaploid cytotype
according to the niche lling hypothesis (López-Jurado et al.,
2019).
The strong postzygotic reproductive isolation in 2x–4x
contact zones results in neotetraploids and unt triploids not
participating in heteroploid gene ow. Isolated cytotypes thus
may, via reinforcement or minority cytotype exclusion, undergo
independent evolution (Sutherland and Galloway, 2021). In con-
trast, lower postzygotic isolation of polyploids allows the for-
mation of viable and fertile pentaploids participating in 4x–6x
gene ow (Wilson etal., 2020; Sutherland and Galloway, 2021).
Even if slowing the divergence (Sutherland and Galloway,
2021), pentaploids may represent an important bridge be-
tween the two cytotypes, allowing polyploids to benet from
a larger shared gene pool. We did not detect morphological
or phenological differentiation between parapatric polyploids
(4x Western Carpathians –6x) that could imply reinforcement.
Despite the lack of selection for assortative mating, no mixed-
ploidy populations or pentaploids were detected in the contact
zone. Environmental niche shifts driven by the specialization of
the hexaploids may thus serve as an efcient prezygotic repro-
ductive barrier.
Consequences of climate, environment and ploidy on plant
morphology
WGD immediately manifests in plant phenotype (Laport
and Ramsey, 2015), phenology (Simón-Porcar et al., 2017)
and reproductive mode (Pannell et al., 2004; Meeus et al,
2020). However, reproductive isolation is more pronounced
in established polyploids than in synthetic neopolyploids
(Husband et al., 2016; Porturas et al., 2019), which suggests
that local adaptation, environmental niche shifts (López-Jurado
et al., 2019) and reinforcement enhancing assortative mating
(Kirkpatrick, 2000; Hopkins, 2013) could act as prezygotic
reproductive barriers. The interactions between climate, en-
vironment and ploidy level on cytotype morphology and dis-
tribution are complex (Fig. 4). Despite niche specialization of
hexaploids, the cytotype distribution was more inuenced by
local habitat conditions (e.g. bedrock, PAR and altitude) than
climate. Leaf and shoot morphology were inuenced by ploidy
and environmental conditions, which mirrors the intricate taxo-
nomic complexity and endemism of Campanula rotundifolia
agg. (Podlech, 1965; Kovanda, 2002; Kovačić, 2004; Mansion
etal., 2012). Concerning local adaptations, the morphology of
leaves and the overall plant may provide the best adaptive po-
tential (Laport and Ramsey, 2015) without affecting pollinators
or thus plant reproduction. Therefore, despite the tendency to-
wards larger size in some traits, the longer and narrower leaves
of hexaploids may be a xeromorphic adaptation to the drier
and warmer Pannonian Basin, while diploids with wider and
shorter leaves prefer the humid and colder Bohemian Massif
(Figs 1 and 3). In contrast, generative traits were inuenced
by ploidy level only and differed among the higher polyploids
(Fig. 3). Through oral morphology, a shift in pollinator prefer-
ences or phenology may imply reinforcement and serve as one
of the prezygotic reproductive barriers (Hopkins, 2013; Laport
and Ramsey, 2015), although this is not immediately apparent
(Castro etal., 2020).
CONCLUSION
The discrepancies revealed between published and revised
ploidy variation imply a need for high-throughput methods
complementing chromosome counting. Environmental niche
specialization of hexaploids of Campanula rotundifolia agg.
may ensure their reproductive isolation from parapatric tetra-
ploids under the lack of a strong postzygotic reproductive
barrier (Sutherland and Galloway, 2016). In contrast, morpho-
logical differentiation was detected almost exclusively for spa-
tially isolated cytotypes, including allopatric tetraploids, which
implies their independent origin (Wei et al., 2017; López-
Jurado etal., 2019).
The absence of hybrids despite the lack of morphological
and environmental differentiation between parapatric diploids
and tetraploids emphasizes the strength of the triploid block.
However, ongoing gene ow towards tetraploids potentially oc-
curs due to the formation of tetraploids via unreduced gametes
(Sutherland and Galloway, 2016), which could explain the lack
of morphological differentiation between parapatric diploids
and tetraploids.
Cytotype distribution was inuenced by local habitat
conditions, and the level of cytotype spatial isolation had
no impact on reproductive isolation. The triploid block and
environmental niche shifts are the main mechanisms of re-
productive isolation, driven both by the ploidy level and en-
vironment. Polyploidy is the only variable inuencing ower
shape, which emphasizes the role of WGD as a prezygotic
reproductive barrier (Kennedy et al., 2006). The lack of
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Šemberová etal. — Drivers of polyploid morphology and reproductive isolation 83
pollinator delity between newly in silico created polyploids
and their diploid ancestors (Porturas etal., 2019) and between
evolutionarily young cytotypes of Campanula rotundifolia
agg. (Roquet et al., 2009; Sutherland et al., 2020) implies
a need for longer adaptive coevolution for pollinators to
effectively distinguish between cytotypes. The synergy be-
tween polyploidy, environment and morphology is complex
and varies between different ploidy levels and their level of
spatial isolation.
SUPPLEMENTARYDATA
Supplementary data are available online at https://academic.
oup.com/aob and consist of the following. Material S1: FCM
estimated ploidy level in localities with known chromosome
counts. Material S2: Morphological variables. Material S3:
Relative genome size and potential DNA aneuploids. Figure S1:
Photos of Campanula rotundifolia agg. Figure S2: Validation
plots for the OPLS-DA. Figure S3: Amixed-ploidy population.
Table S1: Localities. Table S2: Environmental variables. Table
S3: Summary of environmental variables. Table S4: Summary
of morphological variables.
ACKNOWLEDGEMENTS
The authors are grateful to Filip Kolář, Martin Čertner, Jana
Nosková, Kristýna Hanušová, Kateřina Hanušová, Kristýna
Hlavatá, Monika Pavlíková, Ludmila Rejlová, Romana
Urfusová, Jana Rauchová, Jana Mořkovská, Adam Knotek
(Faculty of Science, Charles University), Radek Štencl
(Nature Conservation Agency of the Czech Republic) and
Petr Petřík (Institute of Botany, CAS) for additional sam-
pling and Dušan Senko (Slovak Academy of Sciences) for
help with assessing the environmental variables. Pavel
Trávníček, Zuzana Chumová (Institute of Botany, CAS) and
two anonymous reviewers provided constructive comments
that improved the manuscript. The English text was edited by
the American Journal Experts (www.aje.com). JS, KŠ and RS
designed the study; KŠ conducted sampling, ow cytometry
analyses and morphological measurements; KŠ and MS ana-
lysed the data, and KŠ and MS wrote the initial draft of the
manuscript. KŠ, MS, KM and RS contributed to the nal ver-
sion of the manuscript. The paper is dedicated to the memory
of Jan Suda.
FUNDING
The study was supported by Charles University [project GA
UK No. 670213] to KŠ. KŠ and RS were supported by the
long-term research project of the Czech Academy of Sciences,
Institute of Botany (RVO 67985939). MS was supported by
the Operational Programme Integrated Infrastructure (OPII),
funded by the ERDF [ITMS 313011T721].
CONFLICT OF INTEREST
The authors declare no conict of interest.
LITERATURECITED
AndersonMJM. 2006. Distance‐based tests for homogeneity of multivariate
dispersions. Biometrics 62: 245–253.
BalaoF, HerreraJ, TalaveraS. 2011. Phenotypic consequences at the micro-
evolutionary scale of polyploidy and genome size dynamics. Journal of
Evolutionary Biology 192: 256–265.
BarkerM, RayensW. 2003. Partial least squares for discrimination. Journal
of Chemometrics 17: 166–173.
teBeestM, LeRouxJJ, RichardsonDM, etal. 2012. The more the better?
The role of polyploidy in facilitating plant invasions. Annals of Botany
109: 19–45.
BjørnstadON. 2019. ncf: spatial covariance functions. R package version 1.2-
8. https://ento.psu.edu/directory/onb1.
BjørnstadON, FalckW. 2001. Nonparametric spatial covariance functions:
estimation and testing. Environmental and Ecological Statistics 8: 53–70.
Blackith RE, Reyment RA. 1971. Multivariate morphometrics. London:
Academic Press.
BöcherTW. 1936. Cytological studies on Campanula rotundifolia. Hereditas
22: 269–277.
BradyKU, KruckebergAR, BradshawHD. 2005. Evolutionary ecology of
plant adaptation to serpentine soils. Annual Review of Ecology, Evolution,
and Systematics 36: 243–266.
Brochmann C, Brysting AK, AlsosIG, et al. 2004. Polyploidy in arctic
plants. Biological Journal of the Linnean Society 82: 521–536.
BroennimannO, FitzpatrickMC, PearmanPB, etal. 2012. Measuring eco-
logical niche overlap from occurrence and spatial environmental data.
Global Ecology and Biogeography 21: 481–497.
Caperta AD, Castro S, Loureiro J, et al. 2017. Biogeographical, eco-
logical and ploidy variation in related asexual and sexual Limonium taxa
(Plumbaginaceae). Botanical Journal of the Linnean Society 183: 75–93.
CastroM, CastroS, FigueiredoA, HusbandBC, LoureiroJ. 2018. Complex
cytogeographical patterns reveal a dynamic tetraploid–octoploid contact
zone. AoB Plants 10: 1–18.
CastroM, LoureiroJ, FigueiredoA, Serrano M, HusbandBC, CastroS.
2020. Different patterns of ecological divergence between two tetraploids
and their diploid counterpart in a parapatric linear coastal distribution
polyploid complex. Frontiers in Plant Science 11: 315.
CastroM, LoureiroJ, HusbandBC, CastroS. 2020. The role of multiple
reproductive barriers: strong post-pollination interactions govern cytotype
isolation in a tetraploid–octoploid contact zone. Annals of Botany 126:
991–1003.
ČertnerM, FenclováE, KúrP, etal. 2017. Evolutionary dynamics of mixed-
ploidy populations in an annual herb: dispersal, local persistence and re-
current origins of polyploids. Annals of Botany 120: 303–315.
Christensen RHB. 2019. ordinal - regression models for ordinal data. R
package version 2019.4-25. https://github.com/runehaubo/ordinal
Chumová Z, Krejčíková J, Mandáková T, Sud J, Trávníček P. 2015.
Evolutionary and taxonomic implications of variation in nuclear genome
size: lesson from the grass genus Anthoxanthum (Poaceae). Plos One 10:
e0133748. doi:10.1371/journal.pone.0133748.
Claude J. 2008. Morphometrics with R. New York: Springer Science &
Business Media.
DeMarcoP, NóbregaCC. 2018. Evaluating collinearity effects on species
distribution models: an approach based on virtual species simulation.
PLoS One 13: e0202403.
DoleželJ, GreilhuberJ, SudaJ. 2007. Estimation of nuclear DNA content in
plants using ow cytometry. Nature Protocols 2: 2233–2244.
DoyleJJ, Sherman-BroylesS. 2017. Double trouble: taxonomy and denition
of polyploidy. New Phytologist 213: 487–493.
ErikssonL, JohanssonE, Kettaneh-WoldN, TryggJ, WikströmC, WoldS.
2006. Multi- and megavariate data analysis. Part II. Advanced applica-
tions and method extensions. Umea: Umetrics Academy.
FenaroliF, PistarinoA, PeruzziL, CellineseN. 2013. Campanula martinii
(Campanulaceae), a new species from northern Italy. Phytotaxa 111:
27–38.
FlagelLE, WendelJF. 2009. Gene duplication and evolutionary novelty in
plants. New Phytologist 183: 557–564.
FlatscherR, GarcíaPE, HülberK, etal. 2015. Underestimated diversity in
one of the world’s best studied mountain ranges: the polyploid complex
of Senecio carniolicus (Asteraceae) contains four species in the European
Alps. Phytotaxa 213: 1–21.
Downloaded from https://academic.oup.com/aob/article/131/1/71/6374997 by guest on 28 March 2023
Šemberová etal. — Drivers of polyploid morphology and reproductive isolation
84
GadellaTWJ. 1964. Cytotaxonomic studies in the genus Campanula. Wentia
11: 1–104.
GlennonKL, RitchieME, SegravesKA. 2014. Evidence for shared broad-
scale climatic niches of diploid and polyploid plants. Ecology Letters 17:
574–582.
HanušováK, Čertner M, Urfus T, et al. 2019. Widespread co-occurrence
of multiple ploidy levels in fragile ferns (Cystopteris fragilis complex;
Cystopteridaceae) probably stems from similar ecology of cytotypes, their
efcient dispersal and inter-ploidy hybridization. Annals of Botany 123:
845–855.
HopkinsR. 2013. Reinforcement in plants. New Phytologist 197: 1095–1103.
HülberK, SonnleitnerM, SudaJ, etal. 2015. Ecological differentiation, lack
of hybrids involving diploids, and asymmetric gene ow between poly-
ploids in narrow contact zones of Senecio carniolicus (syn. Jacobaea
carniolica, Asteraceae). Ecology and Evolution 5: 1224–1234.
Husband BC, BaldwinSJ, Sabara HA. 2016. Direct vs. indirect effects
of whole-genome duplication on prezygotic isolation in chamerion
angustifolium: Implications for rapid speciation. American Journal of
Botany 103: 1259–1271.
HusbandBC, SchemskeDW. 2000. Ecological mechanisms of reproductive
isolation between diploid and tetraploid Chamerion angustifolium.
Journal of Ecology 88: 689–701.
JiaoY, WickettNJ, AyyampalayamS, et al. 2011. Ancestral polyploidy in
seed plants and angiosperms. Nature 473: 97–100.
Jiménez-ValverdeA, NakazawaYJ, Lira-NoriegaA, PetersonTownsendA.
2009. Environmental correlation structure and ecological niche model
projections. Biodiversity Informatics 6: 28–35.
KaplanZ. 2012. Flora and phytogeography of the Czech Republic. Preslia
84: 505–573.
Karunarathne P, Schedler M, Martínez EJ, Hon AI, Novichkova A,
HojsgaardD. 2018. Intraspecic ecological niche divergence and repro-
ductive shifts foster cytotype displacement and provide ecological oppor-
tunity to polyploids. Annals of Botany 121: 1183–1196.
Kennedy BF, Sabara HA, Haydon D, Husband BC. 2006. Pollinator-
mediated assortative mating in mixed ploidy populations of Chamerion
angustifolium (Onagraceae). Oecologia 150: 398–408.
KirkpatrickM. 2000. Reinforcement and divergence under assortative mating.
Proceedings of the Royal Society B: Biological Sciences 267: 1649–1655.
Kobrlová L, Hroneš M, Koutecký P, Štech M, Trávníček B. 2016.
Symphytum tuberosum complex in central Europe: cytogeography, morph-
ology, ecology and taxonomy. Preslia 88: 77–112.
KöhlerC, MittelstenScheidO, ErilovaA. 2010. The impact of the triploid
block on the origin and evolution of polyploid plants. Trends in Genetics
26: 142–148.
KolářF, ČertnerM, SudaJ, SchönswetterP, HusbandBC. 2017. Mixed-
ploidy species: progress and opportunities in polyploid research. Trends in
Plant Science 22: 1041–1055.
KolářF, FérT, ŠtechM, et al. 2012. Bringing together evolution on serpen-
tine and polyploidy: spatiotemporal history of the diploid-tetraploid com-
plex of Knautia arvensis (Dipsacaceae). PLoS One 7: e39988.
KovačićS. 2004. The genus Campanula L.(Campanulaceae) in Croatia, circum-
Adriatic and west Balkan region. Acta Botanica Croatica 63: 171–202.
KovandaM. 1966. Some chromosome counts in the Campanula rotundifolia
Complex II. Folia Geobotanica & Phytotaxonomica 1: 268–273.
Kovanda M. 1967. Polyploidie a variabilita v komplexu Campanula
rotundifolia L. PhD Thesis, Charles University, Czech Republic.
KovandaM. 1970a. Polyploidy and variation in the Campanula rotundifolia
complex. Part I.(general). Rozpravy Československé Akademie Věd 80:
5–95.
KovandaM. 1970b. Polyploidy and variation in the Campanula rotundifolia
complex. Part II. (Taxonomic) 1. Revision of the groups Saxicolae,
Lanceolatae and Alpicolae in Czechoslovakia and adjacent regions. Folia
Geobotanica et Phytotaxonomica 5: 171–208.
KovandaM. 1977. Polyploidy and variation in the Campanula rotundifolia
complex. Part II. (Taxonomic) 2. Revision of the groups Vulgares
and Scheuchzerianae in Czechoslovakia and adjacent regions. Folia
Geobotanica et Phytotaxonomica 12: 23–89.
KovandaM. 1983. Chromosome numbers in selected Angiosperms. Preslia
55: 193–205.
KovandaM. 2002. A range extension for Campanula moravica. Thaiszia 12:
179–181.
KrejčíkováJ, SudováR, LučanováM, etal. 2013. High ploidy diversity and
distinct patterns of cytotype distribution in a widespread species of Oxalis
in the Greater Cape Floristic Region. Annals of Botany 111: 641–649.
Laane MM, Croff BE, Wahlstrøm R. 1983. Cytotype distribution in the
Campanula rotundifolia complex in Norway, and cyto-morphological
characteristics of diploid and tetraploid groups. Hereditas 99: 21–48.
Lafon-PlacetteC, JohannessenIM, HornslienKS, etal. 2017. Endosperm-
based hybridization barriers explain the pattern of gene ow be-
tween Arabidopsis lyrata and Arabidopsis arenosa in Central Europe.
Proceedings of the National Academy of Sciences 114: E1027–E1035.
LandisJB, Soltis DE, LiZ, et al. 2018. Impact of whole-genome duplica-
tion events on diversication rates in angiosperms. American Journal of
Botany 105: 348–363.
Laport RG, Hatem L, MinckleyRL, Ramsey J. 2013. Ecological niche
modeling implicates climatic adaptation, competitive exclusion, and niche
conservatism among Larrea tridentata cytotypes in North American de-
serts. The Journal of the Torrey Botanical Society 140: 349–363.
LaportRG, MinckleyRL, RamseyJ. 2016. Ecological distributions, pheno-
logical isolation, and genetic structure in sympatric and parapatric popu-
lations of the Larrea tridentata polyploid complex. American Journal of
Botany 103: 1358–1374.
Laport RG, Muhia P, Kennell C, Hasan H. 2017. Assessing water use
variation among the cytotypes of the autopolyploid southwestern desert
Creosotebush (Larrea tridentata [DC.] Coville: Zygophyllaceae).
Madroño 64: 32–42.
LaportRG, RamseyJ. 2015. Morphometric analysis of the North American
creosote bush (Larrea tridentata, Zygophyllaceae) and the microspatial
distribution of its chromosome races. Plant Systematics and Evolution
301: 1581–1599.
LevinDA. 1975. Minority cytotype exclusion in local plant populations. Taxon
24: 35–43.
LevinDA. 1983. Polyploidy and novelty in owering plants. The American
Naturalist 122: 1–25.
Levin DA. 2019. Why polyploid exceptionalism is not accompanied by re-
duced extinction rates. Plant Systematics and Evolution 305: 1–11.
López-JuradoJ, Mateos-NaranjoE, Balao F. 2019. Niche divergence and
limits to expansion in the high polyploid Dianthus broteri complex. New
Phytologist 222: 1076–1087.
LüdeckeD, MakowskiD, WaggonerP. 2020. Performance: assessment of
regression models performance. R package version 0.4.4. https://easystats.
github.io/performance/.
Maad J, Armbruster WS, Fenster CB. 2013. Floral size variation in
Campanula rotundifolia (Campanulaceae) along altitudinal gradients:
patterns and possible selective mechanisms. Nordic Journal of Botany 31:
361–371.
MackováL, NoskováJ, ĎurišováĽ, UrfusT. 2020. Insights into the cytotype
and reproductive puzzle of Cotoneaster integerrimus in the Western
Carpathians. Plant Systematics and Evolution 306: 1–14.
MackováL, VítP, UrfusT. 2018. Crop-to-wild hybridization in cherries—
empirical evidence from Prunus fruticosa. Evolutionary Applications 11:
1748–1759.
MandákováT, KovaříkA, Zozomová-LihováJ, etal. 2013. The more the
merrier: recent hybridization and polyploidy in Cardamine. The Plant Cell
25: 3280–3295.
MandákováT, PouchM, HarmanováK, ZhanSH, MayroseI, LysakMA.
2017. Multispeed genome diploidization and diversication after an an-
cient allopolyploidization. Molecular Ecology 26: 6445–6462.
Manly BFJ. 2006. Randomization, bootstrap and Monte Carlo methods in
biology. Boca Raton: CRC Press.
MansionG, ParollyG, CrowlAA, etal. 2012. How to handle speciose clades?
Mass taxon-sampling as a strategy towards illuminating the natural his-
tory of Campanula (Campanuloideae). PLoS One 7: e50076. doi:10.1371/
journal.pone.0050076.
MaoY, GabelA, NakelT, etal. 2020. Selective egg cell polyspermy bypasses
the triploid block. eLife 9: 1–15.
Marhold K, Mártonfi P, Mereďa P, Mráz P, eds. 2007. Chromosome
number survey of the ferns and flowering plants of Slovakia.
Bratislava: VEDA.
MayroseI, ZhanSH, RothfelsCJ, etal. 2015. Methods for studying poly-
ploid diversication and the dead end hypothesis: a reply to Soltis etal.
(2014). New Phytologist 206: 27–35.
Downloaded from https://academic.oup.com/aob/article/131/1/71/6374997 by guest on 28 March 2023
Šemberová etal. — Drivers of polyploid morphology and reproductive isolation 85
MeeusS, ŠemberováK, DeStorme N, GeelenD, Vallejo-MarínM. 2020.
Effect of whole-genome duplication on the evolutionary rescue of sterile
hybrid monkeyowers. Plant Communications 1: 100093.
MevikB-H, WehrensR, LilandKH. 2019. pls: partial least squares and prin-
cipal component regression. R package version 2.7-1. https://github.com/
khliland/pls.
Molina-HenaoYF, HopkinsR. 2019. Autopolyploid lineage shows climatic
niche expansion but not divergence in Arabidopsis arenosa. American
Journal of Botany 106: 61–70.
MooreRC, PuruggananMD. 2005. The evolutionary dynamics of plant du-
plicate genes. Current Opinion in Plant Biology 8: 122–128.
Mráz P. 2005. Chromosome number and DNA ploidy level reports from
Central Europe - 1. Biologia, Bratislava 60: 99–103.
MrázP, RonikierM. 2016. Biogeography of the Carpathians: evolutionary
and spatial facets of biodiversity. Biological Journal of the Linnean
Society 119: 528–559.
Mráz P, Šingliarová B, Urfus T, Krahulec F. 2008. Cytogeography of
Pilosella ofcinarum (Compositae): altitudinal and longitudinal differ-
ences in ploidy level distribution in the Czech Republic and Slovakia and
the general pattern in Europe. Annals of Botany 101: 59–71.
Muñoz-PajaresAJ, PerfecttiF, LoureiroJ, etal. 2017. Niche differences
may explain the geographic distribution of cytotypes in Erysimum
mediohispanicum. Plant Biology 20: 139–147.
Murdoch D, Chow ED. 2018. ellipse: functions for drawing ellipses and
ellipse-like condence regions. R package version 0.4.1. https://CRAN.R-
project.org/package=ellipse.
NakagawaS, Johnson PCD, Schielzeth H. 2017. The coefcient of deter-
mination R2 and intra-class correlation coefcient from generalized linear
mixed-effects models revisited and expanded. Journal of the Royal Society
Interface 14: 20170213.
NierbauerKU, PauleJ, ZizkaG. 2017. Heteroploid reticulate evolution and
taxonomic status of an endemic species with bicentric geographical distri-
bution. AoB Plants 9: plx002.
OksanenJ, BlanchetFG, FriendlyM, etal. 2019. vegan: community ecology
package. R package version 2.5-4. https://github.com/vegandevs/vegan.
PannellJR, ObbardDJ, BuggsRJA. 2004. Polyploidy and the sexual system:
what can we learn from Mercurialis annua? Biological Journal of the
Linnean Society 82: 547–560.
Parisod C, Besnard G. 2007. Glacial in situ survival in the Western Alps
and polytopic autopolyploidy in Biscutella laevigata L.(Brassicaceae).
Molecular Ecology 16: 2755–2767.
Paule J, WagnerND, WeisingK, ZizkaG. 2017. Ecological range shift
in the polyploid members of the South American genus Fosterella
(Bromeliaceae). Annals of Botany 120: 233–243.
PeskollerA, SilbernaglL, HülberK, SonnleitnerM, SchönswetterP. 2021.
Do pentaploid hybrids mediate gene ow between tetraploid Senecio
disjunctus and hexaploid S. carniolicus s. str. (S. carniolicus aggre-
gate, Asteraceae)? Alpine Botany131, 151–160. https://doi.org/10.1007/
s00035-021-00254-x.
PinheiroJ, BatesD. 2000. Mixed-effects models in S and S-PLUS. New York:
Springer Science & Business Media.
PinheiroJ, BatesD, DebRoyS, SarkarD, TeamRC. 2019. nlme: linear and
nonlinear mixed effects models. R package version 3.1-139. https://svn.r-
project.org/R-packages/trunk/nlme/.
PodlechD. 1965. Revision der europäischen und nordafrikanischen Vertreter
der Subsect. Heterophylla (Wit.) Fed. der Gattung Campanula L. Feddes
Repertorium 71: 50–187.
PorturasLD, AnnebergTJ, CuréAE, WangS, AlthoffDM, SegravesKA.
2019. A meta-analysis of whole genome duplication and the effects on
owering traits in plants. American Journal of Botany 106: 469–476.
PreiteV, StöcklinJ, ArmbrusterGFJ, Scheepens JF. 2015. Adaptation of
owering phenology and tness-related traits across environmental gra-
dients in the widespread Campanula rotundifolia. Evolutionary Ecology
29: 249–267.
R Core Team. 2019. R: a language and environment for statistical computing.
Vienna: R Foundation for Statistical Computing.
RamseyJ, Schemske DW. 1998. Pathways, mechanisms, and rates of poly-
ploid formation in owering plants. Annual Review of Ecology and
Systematics 29: 467–501.
RamseyJ, SchemskeDW. 2002. Neopolyploidy in owering plants. Annual
Review of Ecology and Systematics 33: 589–639.
RauchováJ. 2007. Karyologická, fenetická a genetická diferenciace českého
subendemického taxonu Campanula gentilis Kovanda. MSc Thesis,
Charles University, Czech Republic.
Ray‐MukherjeeJ, NimonK, MukherjeeS, etal. 2014. Using commonality
analysis in multiple regressions: a tool to decompose regression effects
in the face of multicollinearity. Methods in Ecology and Evolution 5:
320–328.
Rejlová L, Chrtek J, Trávníček P, Lučanová M, VítP, UrfusT. 2019.
Polyploid evolution: the ultimate way to grasp the nettle. PLoS One 14:
1–24.
RiceA, Glick L, Abadi S, etal. 2015. The Chromosome Counts Database
(CCDB) - a community resource of plant chromosome numbers. New
Phytologist 206: 19–26.
RiceA, Šmarda P, NovosolovM, et al. 2019. The global biogeography of
polyploid plants. Nature Ecology and Evolution 3: 265–273.
RoquetC, SanmartínI, Garcia-JacasN, etal. 2009. Reconstructing the his-
tory of Campanulaceae with a Bayesian approach to molecular dating and
dispersal–vicariance analyses. Molecular Phylogenetics and Evolution 52:
575–587.
SchneiderU, BeckerA, FingerP, Meyer-ChristofferA, ZieseM, RudolfB.
2014. GPCC’s new land surface precipitation climatology based on
quality-controlled in situ data and its role in quantifying the global water
cycle. Theoretical and Applied Climatology 115: 15–40.
Schönswetter P, Lachmayer M, Lettner C, et al. 2007a. Sympatric dip-
loid and hexaploid cytotypes of Senecio carniolicus (Asteraceae) in the
Eastern Alps are separated along an altitudinal gradient. Journal of Plant
Research 120: 721–725.
Schönswetter P, SudaJ, PoppM, Weiss-Schneeweiss H, Brochmann C.
2007b. Circumpolar phylogeography of Juncus biglumis (Juncaceae) in-
ferred from AFLP ngerprints, cpDNA sequences, nuclear DNA content
and chromosome numbers. Molecular Phylogenetics and Evolution 42:
92–103.
ScottRJ, SpielmanM, BaileyH, DickinsonHG. 1998. Parent-of-origin ef-
fects on seed development in Arabidopsis thaliana. Development 125:
3329–3341.
SegravesKA, ThompsonJN, SoltisPS, SoltisDE. 1999. Multiple origins
of polyploidy and the geographic structure of Heuchera grossulariifolia.
Molecular Ecology 8: 253–262.
ŠemberováK. 2013. Population cytotype structure and phenotypic variation in
Campanula moravica. MSc Thesis, Charles University, Czech Republic.
ShipleyB. 2009. Conrmatory path analysis in a generalized multilevel con-
text. Ecology 90: 363–368.
Sliwinska E, LoureiroJ, LeitchIJ, ŠmardaP, BainardJ, BurešP,
et al. 2021. Application-based guidelines for best practices in plant
flow cytometry. Cytometry A. doi: 10.1002/cyto.a.24499.
Simón-Porcar VI, Silva JL, Meeus S, Higgins JD, Vallejo-Marín M.
2017. Recent autopolyploidization in a naturalized population of
Mimulus guttatus (Phrymaceae). Botanical Journal of the Linnean
Society 20: 1–19.
SoltisDE, AlbertVA, Leebens-MackJ, etal. 2009. Polyploidy and angio-
sperm diversication. American Journal of Botany 96: 336–348.
SoltisDE, BurleighGJ. 2009. Surviving the K-T mass extinction: new per-
spectives of polyploidization in angiosperms. Proceedings of the National
Academy of Sciences of the United States of America 106: 5455–5456.
Sonnleitner M, Flatscher R, Escobar García P, et al. 2010. Distribution
and habitat segregation on different spatial scales among diploid, tetra-
ploid and hexaploid cytotypes of Senecio carniolicus (Asteraceae) in the
Eastern Alps. Annals of Botany 106: 967–977.
Sonnleitner M, Hülber K, Flatscher R, et al. 2016. Ecological differenti-
ation of diploid and polyploid cytotypes of Senecio carniolicus sensu lato
(Asteraceae) is stronger in areas of sympatry. Annals of Botany 117: 269–276.
StevensCJ, WilsonJ, McAllisterHA. 2012. Biological Flora of the British
Isles: Campanula rotundifolia. Journal of Ecology 100: 821–839.
Suda J, Herben T. 2013. Ploidy frequencies in plants with ploidy hetero-
geneity: tting a general gametic model to empirical population data.
Proceedings of the Royal Society B: Biological Sciences 280: 20122387.
SutherlandBL, Galloway LF. 2016. Postzygotic isolation varies by ploidy
level within a polyploid complex. New Phytologist 213: 404–412.
SutherlandBL, GallowayLF. 2018. Effects of glaciation and whole genome
duplication on the distribution of the Campanula rotundifolia polyploid
complex. American Journal of Botany 105: 1760–1770.
Downloaded from https://academic.oup.com/aob/article/131/1/71/6374997 by guest on 28 March 2023
Šemberová etal. — Drivers of polyploid morphology and reproductive isolation
86
SutherlandBL, GallowayLF. 2021. Variation in heteroploid reproduction and
gene ow across a polyploid complex: one size does not t all. Ecology
and Evolution 11: 9676–9688.
SutherlandBL, Miranda-KatzT, GallowayLF. 2020. Strength in numbers?
Cytotype frequency mediates effect of reproductive barriers in mixed-
ploidy arrays. Evolution 74: 2281–2292.
SutherlandBL, QuarlesBM, GallowayLF. 2018. Intercontinental dispersal
and whole-genome duplication contribute to loss of self-incompatibility in
a polyploid complex. American Journal of Botany 105: 1–8.
TrávníčekP, EliášováA, SudaJ. 2010. The distribution of cytotypes of Vicia
cracca in Central Europe: the changes that have occurred over the last four
decades. Preslia 82: 149–163.
Trávníček P, Jersáková J, Kubátová B, et al. 2012. Minority cytotypes
in European populations of the Gymnadenia conopsea complex
(Orchidaceae) greatly increase intraspecic and intrapopulation diversity.
Annals of Botany 110: 977–986.
TryggJ, WoldS. 2002. Orthogonal projections to latent structures (O-PLS).
Journal of Chemometrics 16: 119–128.
TutzG, HennevoglW. 1996. Random effects in ordinal regression models.
Computational Statistics & Data Analysis 22: 537–557.
VítP, Lepší M, Lepší P. 2012. There is no diploid apomict among Czech
Sorbus species: a biosystematic revision of S. eximia and discovery of
S.barrandienica. Preslia 84: 71–96.
WehrensR. 2011. Chemometrics with R. Berlin: Springer-Verlag.
WeiN, TennessenJA, ListonA, Ashman TL. 2017. Present-day sympatry
belies the evolutionary origin of a high-order polyploid. New Phytologist
216: 279–290.
WeissH, DobešC, SchneeweissGM, GreimlerJ. 2002. Occurrence of tetra-
ploid and hexaploid cytotypes between and within populations in Dianthus
sect. Plumaria (Caryophyllaceae). New Phytologist 156: 85–94.
Weiss-Schneeweiss H, Emadzade K, Jang TS, Schneeweiss GM. 2013.
Evolutionary consequences, constraints and potential of polyploidy in
plants. Cytogenetic and Genome Research 140: 137–150.
Wendel JF. 2015. The wondrous cycles of polyploidy in plants. American
Journal of Botany 102: 1753–1756.
WesterhuisJA, HoefslootHC, SmitS, et al. 2008. Assessment of PLSDA
cross validation. Metabolomics 4: 81–89.
WickhamH. 2016. ggplot2: elegant graphics for data analysis. New York:
Springer.
WilsonJ, PerryA, ShepherdJR, Durán-CastilloM, JeffreeCE, CaversS.
2020. Invasion, isolation and evolution shape population genetic structure
in Campanula rotundifolia. AoB Plants 12: 1–14.
WoldS, Sjöström M, Eriksson L. 2001. PLS-regression: a basic tool of
chemometrics. Chemometrics and Intelligent Laboratory Systems 58:
109–130.
WosG, Mořkovská J, BohutínskáM, etal. 2019. Role of ploidy in colon-
ization of alpine habitats in natural populations of Arabidopsis arenosa.
Annals of Botany 124: 255–268.
Downloaded from https://academic.oup.com/aob/article/131/1/71/6374997 by guest on 28 March 2023
