Adenophora liliifolia: Condition of its Populations in Central Europe

Abstract

This study deals with populations of the European-South-Siberian geoelement Adenophora liliifolia (L.) A. DC. in the Czech Republic, Slovakia, Hungary, Romania, and Poland, where this species has its European periphery distribution. We studied the population size, genetic variability, site conditions, and vegetation units in which A. liliifolia grows. Recent and historical localities of A. liliifolia were ranked into six vegetation units of both forest and non-forest character. A phytosociological survey showed differences in the species composition among localities. Only a weak pattern of population genetic structure was observed (22% at the interpopulation level, AMOVA analysis), with moderate values for gene diversity (Hj = 0.141) and polymorphism (P = 27.6%). Neighbor-joining and Bayesian clusterings suggest a similar genetic background for most of the populations from Slovakia, the Czech Republic, and Poland, contrary to the populations from Hungary, Romania, as well as two populations from Central and South Slovakia. This might be explained by a relatively recent fragmentation of the A. liliifolia populations in Central Europe. Nevertheless, it seems that several populations in Romania, South Hungary, and Slovakia were isolated for a longer period of time and their genetic differentiation is more evident.

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© Polish Academy of Sciences and Jagiellonian University, Cracow 2016
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ACTA BIOLOGICA CRACOVIENSIA Series Botanica 58/2: 83–105, 2016
DOI: 10.1515/abcsb-2016-0018
ROMANA PRAUSOVÁ1a*, LUCIE MAREČKOVÁ2a, ADAM KAPLER3, L’UBOŠ MAJESKÝ2,
TÜNDE FARKAS4, ADRIAN INDREICA5, LENKA ŠAFÁŘOVÁ6 AND MILOSLAV KITNER2
1University of Hradec Králové, Faculty of Science, Department of Biology,
500 02 Hradec Králové, Czech Republic
2Palacký University in Olomouc, Faculty of Science, Department of Botany,
Šlechtitelů 27, 783 71 Olomouc-Holice, Czech Republic
3PAS Botanical Garden – Center for Biological Diversity Conservation in Powsin,
Prawdziwka 2, 02-973 Warsaw 76, Poland
4Aggteleki Nemzeti Park Igazgatóság, Tengerszem oldal 1, 3759 Jósvafő, Hungary
5Transilvania University of Brasov, Faculty of Forestry, Şirul Beethoven – 1,
500123 Braşov, Romania
6East Bohemian Museum in Pardubice, Zámek 2, 530 02 Pardubice, Czech Republic
Received June 16, 2016; revision accepted September 30, 2016
This study deals with populations of the European-South-Siberian geoelement Adenophora liliifolia (L.) A. DC. in
the Czech Republic, Slovakia, Hungary, Romania, and Poland, where this species has its European periphery distri-
bution. We studied the population size, genetic variability, site conditions, and vegetation units in which A. liliifolia
grows. Recent and historical localities of A. liliifolia were ranked into six vegetation units of both forest and non-for-
est character. A phytosociological survey showed differences in the species composition among localities. Only a weak
pattern of population structure was observed (only 22% of total genetic variation present at the interpopulation level,
AMOVA analysis), with moderate values for gene diversity (Hj = 0.141) and polymorphism (P = 27.6%). Neighbor-
joining and Bayesian clusterings suggest a similar genetic background for most of the populations from Slovakia, the
Czech Republic, and Poland, contrary to the populations from Hungary, Romania, as well as two populations from
Central and South Slovakia. This might be explained by a relatively recent fragmentation of the A. liliifolia popula-
tions in Central Europe. Nevertheless, it seems that several populations in Romania, South Hungary, and Slovakia
were isolated for a longer period of time and their genetic differentiation is more evident.
Keywords: AFLP, Campanulaceae, European periphery distribution, declining population, European-
South-Siberian geoelement, genetic variability, vegetation
* Corresponding author, email: romana.prausova@uhk.cz
a These two authors contributed equally to this work.
ADENOPHORA LILIIFOLIA:
CONDITION OF ITS POPULATIONS IN CENTRAL EUROPE
INTRODUCTION
The present-day flora of Central Europe reflects
its geographic position, varied geology and topog-
raphy, as well as climate and vegetation history,
and it is influenced by glacial cycles during the
Quaternary Period (Grulich, 2012; Kaplan, 2012).
Numerous species that are extinct in Central
Europe survived in Eastern Asia (e.g., Platycladus
orientalis (L.) Franco (Farjon and Filer, 2013)),
in Transcaucasia (e.g., Pterocarya pterocarpa
(Michx.) Kunth ex Iljinsk. (Denk et al., 2001)),
or in the Balkan Peninsula (e.g., Picea omorika
(Pančić) Purk. (Ravazzi, 2002)). Some species,
such as Ligularia sibirica (L.) Cass. (Šmídová et
al., 2011) or Pedicularis sudetica Willd. (Hendrych
and Hendrychová, 1988), became glacial or postgla-
cial relicts, but also new local endemics appeared,
e.g., Galium sudeticum Tausch, G. cracoviense
Ehrend., Cochlearia polonica E. Froelich (Cieślak
et al., 2007, 2010, 2015; Cieślak and Szeląg, 2009,
2010; Kolář et al., 2013, 2015) or Sorbus sudet-
ica (Tausch) Bluff et al. (Kaplan, 2012). Climatic
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Prausová et al.
84
changes during the Quaternary Period strongly
affected the species composition and the species
distribution in Europe (Szafer, 1946–47, 1954;
Kaplan, 2012). Since the Neolithic Period, humans
have become another important factor affecting the
regional floras (Szymura, 2012; Hejcman et al.,
2013; Roleček et al., 2014; Plieninger et al., 2015).
Many species, such as A. liliifolia (L.) A. DC,
changed their distribution and their recent pres-
ence in floras is highly influenced by human activity.
A. liliifolia is considered to represent the European-
South-Siberian geoelement, which tolerates extreme
continental climate with a short growing season,
warm but short summers, and long winters with
severe frosts (Kucharczyk, 2007; Kaplan, 2012;
Kucharczyk et al., 2014). The centre of A. liliifolia
distribution is in Western Asia-Southern Siberia,
and from there it extends to Mongolia and Western
China in the East, and to the North-West of Turkey,
and to South-, Eastern-, Central- Europe up to
Western Europe in the West (Tacik, 1971; Fedorov,
1978; Deyuan et al., 2011; Urgamal, 2014).
Although Smelansky et al. (2004) reported A. liliifo-
lia as a common species in the steppes and forest-
steppes in Southern Siberia, Boronnikova (2009)
reported a 25% decrease of populations in the Perm
region (Ural, Russia) during the last 15 years due
to agricultural activities in the territory. Also, in the
whole Central European region, A. liliifolia popula-
tions are declining not only in the number of locali-
ties, but also in the number of plants representing
a single population.
A. liliifolia is scattered across Europe and
forms isolated populations in Germany (Meusel
and Jäger, 1992; Castroviejo et al., 2010),
Austria, Switzerland (Moser, 1999), Italy, Czechia
(Martinovský, 1967; Kovanda, 2000), Poland
(Witkowski et al., 2003; Korzeniak and Nobis,
2004; Ciosek, 2006; Kapler et al., 2015), Slovakia
(Goliášová and Šípošová, 2008), Hungary (Farkas
and Vojtkó, 2012, 2013; Vojtkó, 2013), Croatia,
Bosnia and Herzegovina, Montenegro, Serbia
(Vladimirov et al., 2009; Vukojičić et al., 2011),
Romania (Jones et al., 2010; Indreica, 2011), and
Slovenia (Babij, 2004; Acetto, 2007). In Belarus,
the species was thought to be extinct (Kozlovskaja,
1978), but one population at Sporowski Zakaznik
was restored with plants multiplied in vitro and
cultivated in the Minsk Botanical Garden of the
Belarussian Academy of Sciences (Wiliams and
Gotin, 2012). The information about A. liliifolia
from France (Schnittler and Günther, 1999) and
Bulgaria (Dimitrov, 2002) is uncertain, as no her-
barium records from France and the current
Bulgarian territory exist.
A. liliifolia is protected in Europe according to
the Directive on the conservation of natural habi-
tats and of wild fauna and flora (92/43/EEC); it is
considered as a species of least concern (Bilz et al.,
2011); and it is threatened by vigorous shrubby
vegetation and by inappropriate forest manage-
ment (Anonymous, 2009). A. liliifolia is considered
a plant species of European Community interest,
whose conservation requires designation of special
areas of conservation. Moreover, it is an indicator
species of thermophilous forest hotspots, signaling
remnant pools of biodiversity (Kiedrzyński et al.,
2015). A typical habitat of A. liliifolia is the cop-
pice, which is a formerly widespread way of forest
management. However, changes in landscape man-
agement during the last two centuries caused the
extinction of this species because of the shady and
more eutrophicated high forests (often with coni-
fers) that replaced the coppices (Szymura, 2012;
Müllerová et al., 2015). Today A. liliifolia grows in
lowlands in small populations in remnants of for-
mer light oak forests, their ecotones, and adjacent
meadows. At higher altitudes it grows on the rocky
outcrops in beech forests (Moser, 1999; Dražil,
2002), and in the portions of riparian forests
receiving large quantites of sunlight (Siklósi, 1984;
Farkas and Vojtkó, 2012, 2013). These ecological
demands make A. liliifolia a suitable model species
for studying the changes and the impact of human
activities on populations of species with similar
characteristics.
In spite of the critical conservation status of
A. liliifolia in Europe, no large-scale population
genetic studies have been done so far. Only two
studies have investigated the population struc-
ture of A. liliifolia: Boronikova (2009) analyzed
four populations from the Ural region (Perm,
Russia), and Manole et al. (2015) described the
genetic diversity of one A. liliifolia population
from Romania. The present study aimed to pro-
vide overall information on the current condition
of A. liliifolia populations in Central Europe, and
thus to better know the factors threatening this spe-
cies and suggest appropriate management for the
current populations. We performed: 1) a survey and
comparison of A. liliifolia populations in Czechia,
Slovakia, Hungary, Romania, and Poland; as well as
2) a screening of the genetic variability and relation-
ship among the studied populations by means of
Amplified Fragment Length Polymorphism (AFLP).
MATERIALS AND METHODS
STUDY SPECIES
The lilyleaf ladybells Adenophora liliifolia is a her-
baceous perennial diploid (2n = 34) plant from
the Campanulaceae family with erect, leafed and
branched stems. The root is spindle-shaped or
branched. The plant usually grows to a height of
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Adenophora liliifolia in Central Europe
85
40–90 cm (Kovanda, 2000), although plants with
heights of 205 cm have been observed in Poland
(Ciosek, 2006). Basal leaves and leaves of young
plants are long petiolate, with cordate to rounded
and coarsely serrate blades. Stem leaves are ses-
sile, alternating with an elliptical to lanceolate,
serrate to entire blade with a wedge-shaped base.
Inflorescences are panicles or racemes, flowers
are fragrant. Calyx lobes are triangular, point-
ed, 3–4 mm long and finely serrate. The corolla
is bell-shaped, 12–20 mm long, pale blue, rarely
white. The pistil is twice as long when compared
to the corolla. The species flowers from late June
to August and is pollinated by insects. The fruits
are pear-shaped, curved, 8–12 mm long capsules,
opening with three holes at the base. The seeds
are flattened, reddish brown, from 2.0–2.5 mm
long and are spread by wind (Kovanda, 2000;
Kucharczyk et al., 2014). The precise ecological
demands of A. liliifolia require further studies.
According to Ellenberg et al. (1992), the ecological
demands are as follows: light (L) = 7; temperature
(T) = 6; continentality (C) = 6; moisture (F) = 6;
soil reaction (R) = 8; nutrients (N) = 2.
CHARACTERISTICS OF A. LILIIFOLIA POPULATIONS
AND THEIR LOCALITIES
Monitoring in Czechia, Slovakia, Hungary, and
Romania was performed according to the Natura
2000 methodology (Marhoul and Turoňová, 2008)
during July and August in 2012 or 2013. The
number of tufts and number of fertile and sterile
stems in each tuft were determined at each locality
in all the countries. The condition of the locality
and its changes were observed during the moni-
toring of populations in 2012–2013. The danger
of possible damage such as grazing, drying, grub-
bing out, damage from human activities, etc., was
qualitatively recorded. Morphological differences
including stem height, number of leaves per stem,
length and width of 3 leaves at the central part of
the stem, and number of branches and flowers in
an inflorescence were observed too. Information
about the Polish localities of Kisielany and Dąbrowa
originates from papers by Ciosek (2006) and Rapa
(2012). In total we studied 23 localities (all current
localities in Czechia, Slovakia; chosen representa-
tive and accesible localities in Romania, Hungary,
and Poland (Fig. 1). The characteristics of the stud-
ied localities are shown in Tab. 1 and Fig. 2, and
were summarized from published data ( Comitetul
de Stat al Geologiei – Institutul Geologic CSG-IG,
1968; Mihai, 1975; Miklós, 2002; AOPK ČR, 2005;
Káčer et al., 2005; Cháb et al., 2007; Tolasz, 2007;
European Soil Data Center, 2008–2015; Dövenyi,
2010; Climate Change Knowledge Portal, 2015;
IUSS Working Group WRB. 2015; One Geology –
Europe, 2015).
The localities were ordinated with Principal
Component Analysis (PCA). The climatic character-
istics (annual mean temperature, annual precipi-
tation), the altitude, affiliation with particular bio-
geographic regions in Europe, and forest/non-forest
character of vegetation were used as supplementary
data to assist with data interpretation. The calcula-
tions were done in the CANOCO 4.5 program (ter
Braak and Šmilauer, 2002).
Fig. 1. Map of the studied localities of A. liliifolia in Czechia, Slovakia, Poland, Hungary and Romania and its geographic
range (made by J. Gamrát in ArcGIS 10 program).
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TABLE 1. Site conditions of the studied localities (GPS coordinates indicate the approximate center of the locality)
Site Latitude
Longitude
Alt.
(m) Bedrock Soil
(acc. to WRB 2014) Habitat acc. o EH Habitat Directive
Czechia
Babínské
meadows
50°35’52”
14°07’36” 538 mesozoic marl, claystone eutrophic cambisol,
planosol, stagnosol Intermittently wet Molinia meadows
Bílichovské
valley
50°15’51”
13°53’57” 429 mesozoic marl, claystone cambisol Oak-hornbeam forest
Karlické valley 49°57’07”
14°15’24” 325 paleozoic limestone cambisol Oak-hornbeam forest
Karlštejn 49°57’35”
14°10’24” 400 paleozoic limestone cambisol, phaeozems Central European basiphilous
thermophilous oak forest
Vražba 50°20’05”
15°49’19” 330 mesozoic marl, claystone cambisol Oak-hornbeam forest
Hungary
Aggtelek 48°31’14”
20°33’08” 495 light steinalm limestones
modal cambisol,
chernozem,
kastanozem
Mountain hay meadows
Dabas 47°10’04”
19°16’03” 100
organic rich sediment,
lacustrine and paludal clay,
silt, calcareous mud, peat
histosol, planosol,
stagnosol Riparian mixed gallery forests
Füzér 48°33’42”
21°25’13” 520 rhyolite, andesite
stagnosol, fluvisol,
podsol, retisol,
phaeozem
Intermittently wet Molinia meadows
Kiskőrös 46°39’11”
19°16’29” 104
organic rich sediment,
lacustrine and paludal clay,
silt, calcareous mud, peat
histosol, planosol,
stagnosol Riparian mixed gallery forests
Ocsa 47°15’42”
19°15’35” 247
organic rich sediment,
lacustrine and paludal clay,
silt, calcareous mud, peat
histosol, planosol,
stagnosol Riparian mixed gallery forests
Regéc 48°26’19”
21°21’56” 680 andesite histosol, fluvisol,
podsol, andosol Intermittently wet Molinia meadows
Poland
Dąbrowa 50°45’55”
22°09’02” 200 outwash sands and gravels,
Lithotamnium limestone
haplic arenosol, haplic
luvisol Thermophilous oak forests
Kisielany 52°15’12”
22°12’26” 146 outwash sands and gravels,
clays stagnic retisol Thermophilous oak forests
Romania
Herculian 46°07’05”
25°42’38” 635 volcanic sediments andosols, chernozem,
kastanozem
Central Europ. basiphilous
thermophilous oak forest
Prejmer 45°43’59”
25°44’20” 518 quaternary sediments histosol, stagnosol,
fluvisol Intermittently wet Molinia meadows
Slovakia
Cigánka 48°45’49”
20°03’43” 825 dolomitical rocks podzolic cambisol Limestone beech forest
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Site Latitude
Longitude
Alt.
(m) Bedrock Soil
(acc. to WRB 2014) Habitat acc. o EH Habitat Directive
Kopanec 48°54’54”
20°17’16” 850 sandy and gravelly sediments cambisol, litosol rubble
rendzina Limestone beech forest
Michalovo 49°00’43”
19°45’05” 1136 dolomitical rocks cambisol, carbonate
litosol
Limestone beech forest, relict pine forest
on limestone
Pusté pole – E 48°53’05”
20°14’50” 914 sandy and gravelly sediments cambisol, litosol rubble
rendzina Limestone beech forest
Pusté pole – W 48°53’16”
20°13’44” 990 sandy and gravelly sediments cambisol, litosol rubble
rendzina Limestone beech forest
Silica 48°34’27”
20°33’12” 596 light steinalm limestones histosol, stagnosol,
fluvisol Intermittently wet Molinia meadows
Suchá Belá 48°57’18”
20°22’46” 680 sandy and gravelly sediments cambisol, litosol rubble
rendzina Limestone beech forest
Trsteník 48°48’36”
20°07’53” 860 sandy and gravelly sediments histosol, stagnosol,
fluvisol
Montane Alnus incana galeries,
Alder swamp wood
Fig. 2. Ordination diagram of the studied A. liliifolia localities based on PCA analysis. Czech Republic – circle, Slovakia
– square, Romania – up-triangle, Poland – diamond and Hungary – down-triangle. The first two axes explained 92% of
the total variability. Bio. Reg.: Cont., Carp., Pan – biogeographical region in Europe: Continental, Carpathian, Panonian.
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CHARACTERISTICS OF A. LILIIFOLIA HABITATS
The vegetation type in which A. liliifolia occurred
was assessed using 43 phytosociological relevés
from the surveyed population in Czechia (19 rel.),
Poland (6 rel.), Slovakia (10 rel.), and Romania
(8 rel.), 29 published relevés from Poland (7 rel.)
by Ciosek (2006), from Hungary (20 rel.) by Farkas
and Vojtkó (2012), and from Romania (2 rel.) by
Indreica (2011), and 32 relevés from the TURBOVEG
database for Czechia (Working Group for Vegetation
Science, 2011; 3 rel.) and Slovakia (Working Group
on Vegetation Research, 2012; 29 rel.). The nomen-
clature of the plant communities corresponds to
that used in Chytrý (2007, 2013). The cover and
the abundance of species was evaluated in the
9-grade Braun-Blanquet scale (Braun-Blanquet,
1964; Working Group for Vegetation Science, 2011).
A synoptic table was made in JUICE 7 (Tichý, 2011)
using the frequency percentage of a particular spe-
cies. Only diagnostic species with a fidelity ≥75%,
constant species with a frequency ≥70%, and domi-
nant species with a cover treshold ≥25% are shown
in Tab. 4. For each relevé, Ellenberg values for con-
tinentality (C), light (L), moisture (F), nutrient (N),
soil reaction (R), and temperature (T) were excerpt-
ed using JUICE 7 (Tichý, 2011). Ellenberg indicator
values were used to characterize the site conditions
of 6 determined vegetation units in the STATISTICA
12 program (StatSoft, 2015).
GENETIC ANALYSES
Plant material and DNA extraction. A total of
84 samples collected from 23 localities from five
European countries (Tab. 5) were used for the
genetic analyses. Each sample was represented
by two leaves taken from one stem of a random-
ly selected tuft at each locality, and immediately
preserved in plastic bags with silica gel until DNA
extraction could be performed. Genomic DNA was
extracted using a modified CTAB protocol (Doyle
and Doyle, 1987). The integrity and quality of the
extracted DNA was estimated using 1.5% agarose
gel. The DNA concentrations were determined
using a NanoDrop ND-1000 Spectrophotometer
(NanoDrop Technologies, Delaware, USA).
AFLP analysis. AFLP analysis was carried out
according to the procedure of Vos et al. (1995), with
modifications according to Kitner et al. (2008). In
total, eight selective primer combinations were
chosen to generate the AFLP profiles (Tab. 2).
The amplification products were separated on
6%, 0.4 mm-thick denaturing polyacrylamide gels
(0.5×TBE buffer) using a T-REX sequencing gel
electrophoresis apparatus (Thermo Scientific Owl
Separation Systems, Rochester, NY, USA). As a size
standard, 30-330-bp AFLP® DNA Ladder (Thermo
Fischer Scientific) was used. Silver staining was
used to detect the AFLP fragments after electropho-
retic separation.
TABLE 2. Primers and primer sets for preamplification and amplification reactions with the total number of scored (NB)
and polymorphic bands (NPB).
Preamplification Primers Sequence
EcoRI 5’ – G ACT GCG TAC CAA TTC A – 3’
MseI 5’ – G ATG AGT CCT GAG TAA C – 3’
Amplification Primer Sets Sequences NBNPB
Set A EcoRI primer E-GG / MseI primer M-AAC 41 31
Set B EcoRI primer E-CC / MseI primer M-AAC 43 38
Set C EcoRI primer E-CC / MseI primer M-AAT 49 44
Set D EcoRI primer E-CG / MseI primer M-AAC 35 29
Set E EcoRI primer E-CG / MseI primer M-AAT 42 36
Set F EcoRI primer E-TCG / MseI primer M-AACG 22 17
Set G EcoRI primer E-TCC / MseI primer M-AACG 26 20
Set H EcoRI primer E-TC / MseI primer M-AACCG 33 27
total 291 242
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DATA ANALYSIS
To check the reliability of the AFLP analysis, the
amplification for each primer combination with
the whole sample set and, additionally, the ampli-
fication of randomly chosen samples (from two to
three samples per each population) was repeated.
The AFLP profiles were checked visually, and only
clear and unambiguous bands were scored for their
presence (1) or absence (0). In the last step, the
results of scoring were compared and checked for
the number of markers, intensity of the markers,
and relative position of the markers. In the final
binary matrix only verified markers (present in the
original and repeated amplification) were used. The
error rate was calculated as the difference in the
total number and the number of fragments used
in the final matrix. In order to determine wheth-
er genetic subpopulations can be detected in the
analyzed sample set, the Bayesian approach was
used as implemented in STRUCTURE 2.2 (Falush
et al., 2007). Computation in STRUCTURE was set
up for the recessive allele model and the admix-
ture model with correlated allele frequencies. The
K was set to 1–10 with 10 replicate runs for each
K using the 1,000,000 MCMC iterations follow-
ing the period of 100,000 burn-in iterations. For
the graphical interpretation of clustering for the
appropriate K, STRUCTURE HARVESTER (Earl and von
Holdt, 2012), CLUMPP (Jacobsson and Rosenberg,
2007), and DISTRUCT (Rosenberg, 2004) software
were used. For the further visualization of the pop-
ulation genetic structure and relationships among
individuals a Neighbor-joining (NJ) dendrogram
was constructed [based on the Dice coefficient of
similarity, 1,000 bootstrap replicates (Felsenstein,
1985)] using FREETREE software (Pavlíček et al.,
1999), and the resulting tree was visualised and
arranged in FIGTREE v1.4.0 software (FIGTREE,
2015). The statistical indices for polymorphism
(P%) and Shannon’s Information Index (I) were
performed using GENALEX 6 software (Peakall and
Smouse, 2006). The number of private bands (NPB;
a band unique for a given population, but not for all
individual), and the number of fixed private bands
(NFPB; the number of bands common for all indi-
viduals within a single population) were calculat-
ed in FAMD 1.31 (Schlüter and Harris, 2006). The
ARLEQUIN 3.5 (Excoffier and Lischer, 2010) was used
for calculating the analysis of molecular variance
(AMOVA) to inspect the partitioning and significance
of the genetic variation distribution among and
within the analyzed populations. AFLPdat (Ehrich,
2006) was used for the calculation of DW or “fre-
quency-down-weighted marker” values according to
Schönswetter and Tribsch (2005). DW values were
used as a standardized measure of divergence and
identification of long-term isolation. For the calcu-
lation of DW values no adjustment for the number
of individiuals was made, and DW values were cal-
culated for all of the individuals within each popu-
lation. AFLP-SURV 1.0 (Vekemans, 2002) (square
root method) was used to assess the gene diver-
sity under Hardy-Weinberg genotypic proportions
(Hj), also called Nei’s gene diversity, the total gene
diversity (Ht), and fixation index (FST). Correlation
and regression analyses (to check the relationship
between the obtained indices, population sizes, i.e.,
numbers of tufts and generative ramets), were com-
puted in MS Excel add-in XLSTAT 2015 (Addinsoft,
2015), as well as the Mantel test to explore the
hypothesis of isolation by distance (IBD) by exam-
ining the correlation between the matrices repre-
senting Fst/(1-Fst) and the natural logarithm of geo-
graphic distance (ln d) for pairs of subpopulations
(10.000 permutations). The regression analysis was
also performed to provide the information about
the linkage of the geographic position of localities
(longitude) with polymorphism and gene diversity.
RESULTS
THE CURRENT CONDITION
OF A. LILIIFOLIA POPULATIONS
With respect to the population size in particu-
lar localities (number of tufts, number of fertile
and sterile stems, and average number of stems
in a tuft) the data are shown in Tab. 3. All of the
Slovak localities (except for Michalovo and Silica)
show a strong similarity based on a higher altitude,
higher average annual precipitation, and A. liliifolia
occurrence in forest vegetation units. Thus these
localities represent a distinct group among other
investigated localities (Fig. 2). The Romanian and
the majority of the Czech localities are similar to
one another. These localities represent non-forest
habitats, or forest ecotones with higher than aver-
age annual temperatures. The Karlické valley and
Vražba (CZ) are different, and their characteris-
tics are close to the localities in Poland (Kisielany,
Dąbrowa) and Hungary (Fűzér, Regéc). Two locali-
ties – Babínské meadows (CZ) and Silica (SK)
represent non-forest localities with higher average
annual temperatures in comparison with the other
localities.
VEGETATION IN A. LILIIFOLIA LOCALITIES
The linkage between A. liliifolia occurence and the
type of vegetation present in the locality can be
seen in the synoptic table (Tab. 4). According to the
analysis of all the recorded and published relevés
(Ciosek, 2006; Farkas and Vojtkó, 2012; Indreica,
2011), six vegetation units in recent and historic
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localities of A. liliifolia were distinguished: 1) inter-
mittently wet Molinia meadows (alliance Molinion
caeruleae W. Koch 1926), 2) thermophilous oak
forests (association Potentillo albae-Quercetum
Libb. 1933), also Kiskőrös (HU), which is located
in transition to alluvial forests, was ranked into
this unit, 3) Central European basiphilous thermo-
philous oak forests (alliance Quercion pubescen-
ti-petraeae Klika 1933 corr. Moravec in Beg. et
Theurill 1984), 4) oak-hornbeam forests (asso-
ciation Tilio cordatae-Carpinetum betuli Tracz.
1962), 5) limestone beech forests (association
TABLE 3. Threat categories according to Red Lists of particular countries (CR – critically endangered, EN – endangered,
VU – vulnerable), size of populations, morphological characteristics and number of species in phytosociological relevés
in locality.
Locality
Threat
in
country
Size of population Morphological characteristics Number
of species
in
a relevé
Tufts Stems
%
fertile
stems
Average
number of
stems/span
in one tuft
Average
hight
(cm)
Average
number
of
leaves
Average
ratio length/
width leaf
Average
number
of flowers/
infloresc.
Average
number of
branches/
infloresc.
Czechia
Babínské
meadows CR 20 44 72.7 2.2/1–15 43 19.95 3.6 8.13 2.88 41–53
Bílichovské
valley CR 26 23 13 1.4/1–7 64.44 36.56 3.5 24.67 4.33 21–29
Karlické valley CR 22 26 23.1 1.2/1–3 33.5 19.44 2.7 5.33 0 58–60
Karlštejn CR 22 63 84.1 2.9/1–6 91.78 36.37 3.2 46.89 9.68 38–81
Vražba CR 83 200 96.5 2.4/1–14 98.04 32.71 3.5 48.18 9.34 38–64
Hungary
Füzér EN 18 22 31.8 1.2/1–2 34.42 19.7 2.6 16 3.33 30–42
Regéc EN 38 62 17.7 1.6/1–4 26.08 15.97 2.3 4.9 0.5 16–49
Poland
Dąbrowa EN 53 76 73.7 1.4/1–8 missing data 39–58
Kisielany EN 1000 1500 66.7 1.5/1–11 148.7 48 3.9 54 12 41–56
Romania
Herculian VU 9 18 22.2 2/1–5 48.83 24.05 4.3 10.75 4.29 31–42
Prejmer VU 13 98 63.3 7.5/1–14 70.72 33.09 3.6 40.23 10.52 34–49
Slovakia
Cigánka EN 440 552 56.2 1.3/1–3 58.46 30.18 3.6 24.65 6.55 36–54
Kopanec EN 8 15 100 1.8/1–3 85.2 33 3.1 18.5 3.1 49–54
Michalovo EN 61 210 55.7 2.2/1–4 67.73 27.64 2.6 23.78 6.67 51–58
Pusté pole – E EN 5 7 100 1.4/1–2 75.8 31 3.5 20 2.5 27–36
Pusté pole – W EN 41 96 70.8 2.3/1–5 83.78 34.31 4.5 38.4 8.92 49–53
Silica EN 38 60 11.7 1.6/1–5 44.83 21.34 3.4 14.75 4.8 36–43
Suchá Belá EN 7 7 100 1/1–1 72.1 27 3.3 14.8 2.6 50–80
Trstenik EN 343 474 49.8 1.4/1–6 93.37 27.93 3.6 31 7.64 47–57
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TABLE 4. Synoptic table with 6 clusters using percentage frequency of species in the vegetation unit. Vegetation units:
1 – intermittently wet Molinia meadows, 2 – thermophilous oak forests (association Potentillo albae-Quercetum),
3 – Central European basiphilous thermophilous oak forests (alliance Quercion pubescenti-petraeae), 4 – oak-horn-
beam forests (association Tilio-Carpinetum), 5 – limestone beech forests (association Cephalanthero-Fagetum),
6 – mosaic of montane Alnus incana galleries and alder swamp wood on basic and neutral substrate included in Alnion
incanae alliance. Percentage of 70% and above in bold, except for unit 6 column, where percentage 100% in bold (only
2 relevés).
Vegetation unit 1 2 3456
Number of relevés 24 22 8 19 30 2
E3
Quercus robur 50 16
Carpinus betulus 32 42
Quercus virgiliana 63
Quercus petraea 16
Fagus sylvatica 26 50
Abies alba 16 57
Alnus incana 100
Salix pentandra 100
E2
Cornus mas 75 5
Viburnum lantana 75 11
Quercus petraea 21 55 13 74 3
Corylus avellana 4451374 13
Crataegus sp. 8 64 13 26
Frangula alnus 17 64 5 7
Alnus incana 100
Salix pentandra 100
E1
Adenophora liliifolia 100 100 100 100 100 100
Betonica officinalis 83 68 38 21
Molinia caerulea s.l. 83 18
Convallaria majalis 33 86 63 80
Lathyrus niger 477 50 53
Carex montana 25 77 5
Melittis melissophyllum 473 13 58 13
Melica nutans 64 25 89 57 50
Brachypodium sylvaticum 4413874
Carex digitata 414 74 57
Aegopodium podagraria 414 74 3
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Vegetation unit 1 2 3 4 5 6
Number of relevés 24 22 8 19 30 2
Asarum europaeum 584 23
Hepatica nobilis 589
Pulmonaria obscura 579
Calamagrostis varia 83 100
Galium schultesii 41 25 16 83
Pimpinella major 29 21 70
Laserpitium latifolium 45 80
Rubus saxatilis 14 87
Cirsium erisithales 77
Carduus crassifolius ssp. glaucus 70
Cruciata glabra 54 9 38 5 7 50
Ajuga reptans 25 59 58 3 100
Angelica sylvestris 841 513100
Astrantia major 54 55 37 40 100
Lathyrus pratensis 38 5 5 7 100
Trollius europaeus 17 14 3 100
Colchicum autumnale 38 5 50
Succisa pratensis 42 14 3 100
Carex umbrosa 13 9 3 50
Deschampsia cespitosa 45 21 100
Leontodon hispidus 33 17 50
Thalictrum aquilegiifolium 427 100
Listera ovata 97100
Tanacetum clusii 53 100
Gentiana asclepiadea 50 100
Cirsium oleraceum 37 100
Carex paniculata 17 100
Carex panicea 850
Equisetum palustre 8100
Gymnadenia conopsea 8100
Filipendula ulmaria 4100
Galium palustre 450
Knautia maxima 10 100
TABLE 4.
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93
Cephalanthero-Fagenion R. Tx. 1955), 6) mosaic
of montane Alnus incana galleries, montane fens
with Swertia perennis from the Caricion davalli-
anae Klika 1934 alliance and alder swamp wood
on basic and neutral substrate included in the
Alnenion glutinoso-incanae Oberd. 1953 alliance
(Tab. 4).
SITE CONDITIONS OF THE A. LILIIFOLIA LOCALITIES
Site conditions of the six distinguished vegetation
units with the presence of A. liliifolia were com-
pared using Ellenberg’s indicators (Ellenberg et
al., 1992) for nutrients, soil reaction, temperature,
light, moisture, and continentality (Fig. 3). When
comparing the ecological demands of A. liliifolia
according to Ellenberg et al. (1992) with the ecologi-
cal conditions calculated from the phytosociological
relevés using Ellenberg indicators (Fig. 3), we can
say that the current optimal vegetation units for this
species in Central Europe are Central European
basiphilous thermophilous oak forests (alliance
Quercion pubescenti-petraeae) and intermittently
wet Molinia meadows. The light-, temperature-,
moisture-, and soil reaction values calculated in
these vegetation units are the most similar to the
ecological demand of A. liliifolia. While intermittent-
ly wet Molinia meadows offer the most convenient
conditions with respect to light, continentality, and
moisture, Central European basiphilous thermo-
philous oak forests are most suitable for A. liliifolia
with respect to temperature and soil reaction. Low
moisture can be a limiting factor for this species in
Central European basiphilous thermophilous oak
forests. According to Ellenberg et al. (1992), the
optimal value for nutrients is 2, but in all localities,
this value was 3–6. Central European basiphilous
thermophilous oak forests, which are compara-
tively the most convenient vegetation unit concern-
ing nutrients, have the second widest amplitude
with respect to this factor. The worst conditions
for A. liliifolia were found in oak-hornbeam forests
(association Tilio cordatae-Carpinetum betuli),
limestone beech forests (association Cephalanthero
damassonii-Fagetum sylvaticae Oberdorfer 1957),
and mosaic of montane Alnus incana galleries and
alder swamp wood, mainly because of low light
intensity and temperature. In these habitats, A. lili-
ifolia can only grow thanks to disturbances and
management directed to an open forest.
Adenophora liliifolia shows a high morphologi-
cal variability related to the geological bedrock, soil,
moisture, and habitat in which it grows (Tab. 3).
The investigated localities showed differences in
species alpha-diversity. The most species-rich were
two Czech localities (Vražba and Karlštejn) and
three Slovak localities (Malý Sokol, Suchá Belá,
and Michalovo). The fewest number of species were
recorded in the Hungarian locality Regéc and in the
Czech locality Bílichovské valley (Tab. 3).
Vegetation unit 1 2 3456
Number of relevés 24 22 8 19 30 2
Centaurea pseudophrygia 3100
Carex davalliana 50
Carex flava 100
Valeriana simplicifolia 100
Juv.
Acer campestre 5 38 79
Alnus incana 100
E0
Hylocomium splendens 427100
Rhytidiadelphus sp. 17 100
Aulacomnium palustre 100
Climacium dendroides 100
TABLE 4.
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MANAGEMENT IN A. LILIIFOLIA LOCALITIES
Adenophora liliifolia populations in Czechia are
negatively influenced by many factors (e.g., over-
populated wild animals, inappropriate forest man-
agement, global eutrophication, expansive herbs,
young trees, parasitic insects, and fungi). All Czech
populations growing in forests, if not protected by
fences against grazing, were browsed by overpopu-
lated hoofed game. The level of grubbing out with
respect to the underground organs was found to
be increasing, especially in oak-hornbeam forests
located at Karlštejn in Český kras PLA (Protected
Landscape Area). The population in the Babinské
meadows was negatively influenced not only by
grazing and grubbing out, but also by expansion of
Calamagrostis epigejos (L.) Roth. Rubus L. expan-
sion also impacts A. liliifolia populations, mainly
in oak-hornbeam forests. Not only the Czech locali-
ties, but also Trsteník in the Muránska planina NP
(National park) in Slovakia and both of the Polish
localities studied are affected by inappropriate for-
est management. Several Czech localities (Vražba,
Bílichovské valley, and Karlštejn) have special man-
agement regulations protecting A. liliifolia against
grazing by fences and against competitive vascular
plants by cutting. Sheep pasturing occurs at the
Silická planina in Slovenský kras NP (Silica, SK),
and the A. liliifolia present at Silica is intensive-
ly grazed every year before it creates flowers and
seeds. The most stable habitat for A. liliifolia popu-
lations is the limestone beech forest in the local-
ity of Michalovo in Nízke Tatry NP. The plants grow
there in slightly shaded parts of the forest, often
on rocky bedrock. They are rarely influenced by
grazing, grubbing out, or by human activities, and
their seeds have enough space for germination. The
other localities (Slovenský raj NP, Muránska plan-
ina NP, Slovenský kras NP, Czech, Romanian, and
Hungarian localities) are affected by grazing and
grubbing out by wildlife.
GENETIC VARIABILITY
A total of 84 A. liliifolia plants from 23 popula-
tions were analyzed using eight AFLP primer com-
binations (Tab. 2), which generated 291 bands, of
which 242 were polymorphic (83.2%). Replication
of the analysis revealed high reliability of AFLP, with
an error rate of 2.4%. Statistical indices (Tab. 5)
were not computed for four localities which were
represented by one or two samples. The highest
values for Nei’s gene diversity (Hj) were observed
for samples from Kopanec (Hj = 0.171) and Pusté
pole-E (Hj = 166), both located in Slovakia. The
lowest value was observed for the Czech popula-
tion from Karlštejn (Hj = 105). We recorded only
a single fixed private band unique for populations
located in Silica (SK) which was present among all
of the sampled localities. The values for the DW
index ranged from 1.7 to 4.2. The highest indices
(DW = 4.2) were observed for the Slovak popula-
tions located at Suchá Belá and Pusté pole E, fol-
lowed by the Hungarian locality at Ocsa (DW = 3.9).
The lowest values were recorded for the Czech pop-
ulations at Karlštejn (DW = 1.8) and the Bílichovké
valley (DW = 1.7). The computation of Shannon’s
Information Index produced the lowest value for the
Fig. 3. Comparison of 6 vegetation units (in this order: 1. Molinion caeruleae, 2. Potentillo albae-Quercetum, 3. Quer-
cion pubescenti-petraeae, 4. Carpinion betuli, 5. Cephalanthero damassonii-Fagetum sylvaticae, 6. Alnenion gluti-
noso-incanae) based on used releves by means of Ellenberg indicator values (a) light, (b) temperature, (c) moisture,
(d) continentality, (e) soil reaction, (f) nutrients).
a
d
b
e
c
f
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TABLE 5. List of analysed samples with population genetic statistics (n, number of samples; NPB, number of private
bands; NFPB, number of fixed private bands; P%, polymorphism; I, Shannon‘s Information Index; Hj, Nei‘s gene diversity;
DW, frequency down — weighed marker value; SE, sum of errors)
Site ID of samples n NPB NFPB P% I (SE) Hj (SE) DW
Czechia 18 2 0 56.1 0.194 (0.013) 0.128 (0.009) 2.3
Babínské meadows 1–4 4 0 0 28.2 0.142 (0.014) 0.137 (0.010) 2.8
Bílichovské valley 5–8 4 0 0 25.9 0.131 (0.014) 0.127 (0.010) 1.7
Karlické valley 9–10 2 — — — — — —
Karlštejn 11–13 3 0 0 18.5 0.087 (0.012) 0.105 (0.009) 1.8
Vražba 14–18 5 2 0 32.4 0.154 (0.014) 0.137 (0.010) 2.8
Hungary 21 5 0 64.4 0.229 (0.013) 0.149 (0.009) 3.II
Aggtelek 19 1 — — — — — —
Dabas 20–21 2 — — — — — —
Füzér 22–26 5 0 0 28.2 0.148 (0.015) 0.137 (0.011) 3.0
Kiskőrös 27–31 5 0 0 31.7 0.148 (0.014) 0.132 (0.009) 3.3
Ocsa 32–34 3 0 0 24.2 0.122 (0.013) 0.148 (0.010) 3.9
Regéc 35–39 5 0 0 37.2 0.177 (0.014) 0.152 (0.010) 3.0
Poland 6 0 0 38.6 0.167 (0.014) 0.132 (0.009) 2.3
Dąbrowa 40 1 — — — — — —
Kisielany 41–45 5 0 0 36.2 0.168 (0.014) 0.141 (0.010) 2.6
Romania 10 1 0 48.2 0.181 (0.013) 0.128 (0.009) 3.1
Herculian 46–50 5 0 0 37.8 0.157 (0.014) 0.133 (0.010) 2.9
Prejmer 51–55 5 0 0 32.1 0.150 (0.014) 0.131 (0.009) 3.5
Slovakia 29 14 0 68.8 0.229 (0.013) 0.144 (0.009) 3.1
Cigánka 56–60 5 0 0 29.6 0.144 (0.014) 0.127 (0.010) 2.3
Kopanec 61–63 3 0 0 25.4 0.161 (0.015) 0.171 (0.011) 3.2
Michalovo 64–68 5 0 0 30.3 0.138 (0.013) 0.122 (0.009) 2.5
Pusté pole — E 69–71 3 0 0 26.5 0.146 (0.015) 0.166 (0.011) 4.2
Pusté pole — W 72–74 3 0 0 18.9 0.106 (0.013) 0.131 (0.010) 3.3
Silica 75–78 4 1 1 20.6 0.105 (0.013) 0.115 (0.010) 3.5
Suchá Belá 79–81 3 0 0 29.6 0.139 (0.014) 0.160 (0.011) 4.2
Trsteník 82–84 3 0 0 20.6 0.113 (0.013) 0.135 (0.011) 2.4
total mean (populations separately) 27.6 0.139 (0.014) 0.144 (0.009) 3.0
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Czech locality at Karlštejn (I = 0.087) and the high-
est value was computed for the Hungarian popula-
tion from Regéc (I = 0.177). The total gene diversity
(Ht) was 0.157.
POPULATION GENETIC STRUCTURE
The Neighbor-Joining clustering analysis divided
the A. liliifolia samples into seven main clades
(Groups A–G; Fig. 4), where clustering of the sam-
ples was not strictly associated with their geograph-
ical origin and only a weak bootstrap (i.e., values
below 15, not shown) support was recorded.
Nevertheless, a certain level of clustering which
might be linked with the geographical origin can
be observed on the NJ tree. All of the samples
from Slovakia appeared in three groups (A–C).
Group A represents samples solely from South
(Silica) and Central Slovakia. Group B consists
of samples from several Slovak localities and one
outlying Hungarian sample (Aggtelek). Two sam-
ples from Trsteník (SK) together with one sample
from Vražba (CZ) are located between Groups A
and B. Two samples from Poland, one sample from
Regéc (HU), and six samples from Czechia were
mixed with samples which originated mainly from
Michalovo and Cigánka (SK) and formed Group
C. Group D is separated from the previous groups
and is represented only by six Czech samples,
while the remaining 5 samples from Czechia are
located in Group E (4 samples) and F (one sample).
All of the samples from South Hungary, together
with two Romanian and two Polish samples are
present in Group E. Eight out of ten remaining
Romanian samples were present in Group F. The
samples from North Hungary (Füzér, Regéc) fell into
a separate group, Group G (Fig. 4). The Polish sam-
ples were spread through the NJ tree in groups C
(2 samples), E (3 samples), and F (one sample).
Further analysis of the population genetic
structure by Bayesian clustering implemented in
STRUCTURE suggested a subdivision into two or five
clusters (maximum value ∆K = 57.627 for K = 2
and ∆K = 16.419 for K = 5) (Fig. 5). Bayesian clus-
tering for K = 2 stressed the genetic differences in
the Slovakian populations from Slovenský kras NP
(Slovakian karst, Silica) and Pusté pole (W), while
all of the remaining samples were highly similar.
Subdivision into five groups (K = 5) basically reflects
the results of the NJ clustering: i) a unique group of
Fig. 4. Unrooted Neighbour-joining dendrogram (based on 291 AFLP markers and Dice similarity matrix) of 84
Adenophora liliifolia samples from five European countries. Putative groups are designed by capital letters A–G.
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97
Slovakian samples (roughly equal to Group A on the
NJ tree: ~NJGroup A); ii) the remaining Slovakian sam-
ples sharing a similar genetic background with some
of the Czech and Polish samples (~NJGroups B,C,D);
iii) separation of samples from North Hungary
(~NJGroup G); iiii) similarity of the South Hungarian
and Romanian samples with some samples from
Poland and Czechia (~NJGroups F,E) (Fig. 5).
The AMOVA computations revealed that 22%
of the total genetic variation represents differenc-
es among the populations, while 78% is related to
the genetic variation among plants within popula-
tions, with FST = 0.125. The Mantel test revealed
no significant correlation between geographical
distance and genetic distance or pairwise FST (i.e.,
lack of genetic isolation by distance; r = -0.141,
P = 0.1580), except slightly positive correlations
(not significant) of the geographic position of locali-
ties (longitude) with polymorphism (r = 0.466,
P = 0.217) or genetic diversity (r = 0.426,
P = 0.182).
DISCUSSION
POPULATIONS, SITE CONDITIONS, AND VEGETATION
Our survey of A. liliifolia populations in Central
Europe revealed the decline in number of its locali-
ties in all of the investigated countries. Comparison
of the present survey with historical data from
the 19th and early 20th century showed that in
the past A. liliifolia had occurred in 20 locali-
ties within the phytogeographical district of Czech
Thermophyticum, and in 6 localities within the
Mesophyticum. At present, the species occurs very
rarely in 5 localities of the Czech Thermophyticum
(Kovanda, 2000; Prausová and Truhlářová, 2009).
In Slovakia, the species is still present in both of
the phytogeographical districts of Pannonicum
and Carpaticum (Goliášová and Šípošová, 2008).
Currently, A. liliifolia grows only in about 10 locali-
ties in the Carpathians and their foothills, while his-
torical data describe about 30 former populations
in this territory. In Pannonicum A. liliifolia occurs
in the Slovenský kras NP (only one verified locality
near the Silica village). In Poland, this species was
previously found in circa 100 localities within all of
the phytogeographical provinces, but nowadays it
is only known to occur in approximately 21–22 of
them. Their location in the central and the north-
eastern part of the country represents the current
northern distribution border of A. liliifolia in Europe
(Pawłowska, 1972; Ciosek, 2006; Kucharczyk, 2007;
Piękoś-Mirkowa, 2008; Rapa, 2012; Kucharczyk et
al., 2014; Kapler et al., 2015). Similarly, in Romania,
Adenophora is only recorded in 6 out of 34 for-
mer localities and in two newly found localities in
Transylvania (Indreica, 2011). In Hungary, it is
recorded in 7 out of 30 former localities (Farkas and
Vojtkó, 2012, 2013). In the Balkan Peninsula, in the
former Socialist Federal Republic of Yugoslavia, the
species remains common in river valleys, but is sup-
posedly extinct in many localities (Acetto, 2007).
The highest number of A. liliifolia tufts per
population were observed in the Slovak localities in
Fig. 5. Results of the STRUCTURE analysis of 84 A. liliifolia samples showing results for K = 2 and K = 5. Each vertical
bar represents one individual with the color representing the probability of assignment to different clusters. The origin
of the populations is displayed below the graphics.
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Prausová et al.
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Muránska planina NP (Cigánka, Trsteník), followed
by the Czech locality of Vražba (Tab. 3). This is
probably related to the remoteness of these Slovak
localities from populated areas and to appropriate
management at the locality Vražba (CZ). A. liliifolia
shows a high morphological variability related to
the site conditions and habitat in which it grows.
The tallest individuals were found in oak-horn-
beam and beech forests, the smallest individuals
were found in meadows (Regéc, Babinské mead-
ows) and also in the Karlické valley which is influ-
enced by inappropriate forest management, where
young plants of A. liliifolia have been overgrown by
juvenile trees and shrubs. The number of stems in
a tuft varied from 1 (common at several localities)
to 20 (Prejmer, RO). A single stem occurrence was
most common in the Karlické valley (CZ), Fűzér
(HU), and at Suchá Belá (SK), where most of the
stems were sterile. The most species rich biotopes
were observed at the Czech localities Vražba and
Karlštejn, and the Slovak localities at Suchá Belá
and Michalovo.
According to the Ellenberg indicators
(Ellenberg et al., 1992), A. liliifolia is classified
as a heliophilous, thermophilous, and basiphil-
ous species requiring enough moisture and with
little to no demand for nitrogen. On the contrary,
many current localities (mainly Czech, Polish, and
several historical Slovak localities) have a higher
content of nutrients that support the develop-
ment and spread of nitrophilous species including
Aegopodium podagraria L., Urtica dioica L., and
Stachys sylvatica L. which have become impor-
tant competitors of A. liliifolia. It is assumed that
basiphilous and mesotrophic thermophilous oak
forests previously grew in these localities, but that
they changed into mesophilous oak-hornbeam for-
ests due to eutrophication (Müllerová et al., 2015)
and missing disturbances like pasturage. The soils
in the localities of Herculian (RO), Silica (SK), and
the Babinské meadows (CZ) were found to have the
highest pH of all of the studied localities because of
both calcareous substrate and the greatest nutrient
content resulting from a rapid humification pro-
cess. Our finding of high soil pH in most of the cur-
rent localities corresponds with other data about
the occurence of A. liliifolia on calcareous rocks
in beech and pine forests, and also in subalpine
grasslands in Slovenia (Babij, 2004; Acetto, 2007).
In Switzerland (Moser, 1999) and Slovakia (Dražil,
2002), the species grows in calciphilous beech for-
ests (association Cephalanthero damassonii-Fage-
tum sylvaticae). In Hungary (Siklósi, 1984; Farkas
and Vojtkó, 2012, 2013), A. liliifolia was found in
riparian forests (association Fraxino pannonicae-
Ulmetum glabrae Aszód 1935 corr. Soó 1963).
Roleček (2007) states that A. liliifolia belongs
to heliophilous species of subcontinental oak for-
ests which grow in climatically non-extreme sites.
It has a limited ability for long distance disper-
sal and successional changes from subcontinen-
tal oak forests to oak-hornbeam forest or shady
mixed oak forests do not facilitate its spreading.
It is thought that A. liliifolia could survive in light
oak coppices and grazed forests of lower eleva-
tions, and also in light forests at higher altitudes,
mainly on rocky outcrops, in erosion-prone sites,
areas influenced by the grazing of wild animals,
and thus generally in various forest ecotones. The
distribution of this species followed continually
changing light conditions in forests. Válek (Válek
in Rohlena and Dostál 1936) provided informa-
tion about hundreds of A. liliifolia individuals that
had reappeared in the locality of Vražba (Czechia)
at clearings in mixed forests containing spruce
after an attack of the moth Lymantria monacha L.
The previously shady forest was then replaced
temporarily by a non-forest or open forest area.
Roleček (2007) states that the best conditions for
this species are in the S Ural and in SW Siberia,
where the continental climate prevents broadleaf,
mesophilous trees and shrubs from extension and
where A. liliifolia grows in the hemiboreal forests of
Brachypodio pinnati-Betuletea pendulae Ermakov,
Korolyuk & Latchinsky 1991 (Ermakov et al., 1991;
Ermakov and Maltseva, 1999; Chytrý et al., 2012).
Central European forest habitats have changed a lot
since the Preboreal and Boreal period. Once light
Preboreal and Boreal forests were later massively
replaced by deciduous forests with more closed
canopies in Central Europe (Kaplan, 2012). Open
forests containing heliophilous trees like Pinus syl-
vestris L., Betula pendula Roth., and Larix decid-
ua Mill. supposedly occured in Central Europe and
SW Siberia about 9,500 years BC; they disappeared
from Central Europe due to climate changes and
human activities. According to Roleček (2007),
there are only fragments of relict hemiboreal for-
ests surviving in Central Europe today. Martynenko
(2009) designates the S Ural region as the eastern-
most part of the distribution range of thermophil-
ous oak forests of class Quercetea pubescentis
Doing Kraft ex Scamoni et Passarge 1959, namely
Lathyro-Quercion roboris Solomeshch et al. 1989
nom. inval. alliance. According to Roleček et al.
(2015), similar vegetation to that present in the
S Ural region appeared in Central Europe after
the expansion of oak during the Boreal period,
and in suitable places it could resist, although the
degree of climate oceanity increased and highly
competitive wood species expanded (Fagus syl-
vatica L., Carpinus betulus L.). This relict veg-
etation shelters rare species with disjunct distri-
bution, such as A. liliifolia, Veratrum nigrum L.,
and Dracocephalum ruyschiana L. Patches of light
oak or oak-pine forests and forest-meadow eco-
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Adenophora liliifolia in Central Europe
99
tones, which are suitable secondary habitats for
light-demanding, often basiphilous species, were
established by human activities in the Middle Ages.
Therefore, A. liliifolia survives in Central Europe
mainly in the light edges of forests and in intermit-
tently wet Molinia meadows (Roleček, 2007). The
current meadow vegetation of the A. liliifolia local-
ity in the Central Bohemian Uplands PLA is con-
sidered as a relict of forest-steppe vegetation which
prevailed in this area in the past and was later
influenced by prehistoric settlement, agriculture,
gradual overgrowing, and eutrophication. Despite
these changes, a high level of biodiversity and spe-
cies of the relict mesic sites vegetation remain here
(e.g., A. liliifolia, Potentilla alba L., Serratula tinc-
toria L.). Adenophora liliifolia was also observed
in the shrub association of alliances Berberidion
Br.-Bl. 1950 and Prunetalia spinosae R.Tx. 1952,
in Peucedano cervariae-Coryletum Kozł. 1925 em.
Medw.-Korn. 1952 scrub (Ciosek, 2006; Kovanda,
2000; Kapler et al., 2015), in sunny patches at
forest edges of Tilio cordatae-Carpinetum betuli
(Kapler et al., 2015), and in mesic meadows (asso-
ciation Anthyllido-Festucetum rubrae Soó, 1971)
(Farkas and Vojtkó, 2012, 2013). Hungarian relevés
from Regéc and Fűzér were identified as the asso-
ciation of Nardo-Molinietum hungaricae (Kovács
1962) Borhidi 2001 (Farkas and Vojtkó, 2013). In
Poland, A. liliifolia at its north-easternmost locali-
ties (Czarna Białostocka and Dobry Lasek) also
grows in a mosaic of ruderal and segetal communi-
ties, often with ecotones of thermophilous scrubs
and light spruce-pine-oakwoods (Kapler et al.,
2015).
GENETIC VARIABILITY
AND POPULATION GENETIC STRUCTURE
To compare the results of our genetic analysis
we searched for studies dedicated to plants with
an Euro-Sibirian distribution similar to A. liliifo-
lia. Two different species of Stipa L. (Poaceae) were
studied by the AFLP method, and populations from
their periphery in Central Europe were compared
with populations within their main distribution
area in Russia (Wagner et al., 2011, 2012). In both
cases, the authors did not find any relationship
between the size of the population (represented
by the number of plants) and the observed genetic
diversity, similar to the results of the present study.
Nevertheless, this statement has to be proved in
the future, due to the low number of analyzed indi-
viduals used in our study. In populations of Stipa
pennata L., the genetic diversity declined signifi-
cantly from the distribution’s center to its periph-
ery (Wagner et al., 2012). The same trend seems
also to be present for A. liliifolia, as we observed
only slight correlations between the geographi-
cal position of populations (increscent longitude),
polymorphism, and heterozygosity (althought not
significant). For populations of Stipa capillata L.,
low values of polymorphism were found both in
its distributional center (21.9%) and on its periph-
ery (20.0%), values that are close to the value of
polymorphism detected within the present study
(average P = 27.6% for the analyzed populations,
Tab. 5). Similar results (low polymorphism both
in the peripheral and the central populations) were
observed in the relict steppe species Iris aphylla L.
(Wróblewska, 2008). Another example of a spe-
cies with a similar Euro-Siberian distribution is
Ligularia sibirica L., whose populations from the
Czechia and Slovakia were analyzed by Šmídová et
al. (2011) using allozyme analysis. Similar to our
study, the results of the investigated populations
of L. sibirica showed high genetic diversity within
populations (80.8%) and a lower level of genetic
differentiation between populations (FST = 0.179).
Contrary to the results of our study, the genetic dis-
tance between populations correlated significantly
with the geographic distance, and there was also
a significant positive correlation between genetic
diversity and population size. However, Šmídová et
al. (2011) used codominant allozyme markers (trac-
ing variation in proteins), which generally detect
a lower level of genetic variation, contrary to the
dominant and highly variable AFLP markers uti-
lized in this study, which allow the direct examina-
tion of DNA sequence variation.
There are only a few studies which investigated
the genetic diversity of populations of Adenophora
spp., including two isozyme based studies by Ge et
al. (1999) and Chung and Epperson (1999); and
two more recent studies based on ISSR markers
by Boronnikova (2009) and Manole et al. (2015).
Boronnikova (2009) analyzed four A. liliifolia popu-
lations from the Perm region in Russia using ISSR
markers, and detected (similarly to our study)
a weak population genetic structure and high intra-
population variation (nearly 84.5% of the total vari-
ation). The expected heterozygosity values ranged
from 0.159 to 0.275, with a mean HE = 0.228.
These values are twice as great as the gene diver-
sity values detected in the present study. This can
be influence by a) the greater number of sampled
plants per population in the study by Boronnikova
(2009); and b) the fact that the investigated popula-
tions were geographically closer to the species’ cent-
er of distribution, thus possessing a greater degree
of genetic variability. A recent study by Manole et
al. (2015) investigated 12 mature specimens of
A. liliifolia from one Romanian population using
ISSR markers. Also in this study, a relatively high
intrapopulation genetic variation was observed as
measured by Shannon´s index of genotypic diver-
sity (0.812), contrary to our calculations (mean
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Prausová et al.
100
value of I = 0.139, which may be caused by the dif-
ferent type of molecular markers used and/or the
different sample size). Ge et al. (1999) investigat-
ed two Adenophora species in China – the endan-
gered A. lobophylla D.Y. Hong and its widespread
relative A. potaninii Korsh. For these two species,
the differentiation among the investigated popula-
tions was higher among A. potaninii populations
(FST = 0.155) than among A. lobophylla popula-
tions (FST = 0.071). The FST value obtained for
A. liliifolia was 0.125, which is quite close to the
results for A. potaninii. This result is probably due
to the similar geographical distances between the
most remote localities of A. potaninii and A. lili-
ifolia (ca 850 and 1000 km respectively), contrary
to the weak population genetic structure detected
among the A. lobophylla populations, which were
located at a maximum distance of 25 km from
each other. In Korea, with respect to the endemic
Hanabusaya asiatica Nakai (a genus closely relat-
ed to Adenophora), there is an apparent pattern of
isolation by distance among the assessed popula-
tions. Despite the fact that the allozyme differentia-
tion among the populations is low (CST = 0.132),
the species maintains a high allozyme diversity
(HES = 0.217) (Chung et al., 2001). In A. liliifolia
populations, we failed to find a significant corre-
lation between geographical distance and genetic
distance or pairwise FST.
Adenophora liliifolia is a species with prevail-
ing sexual reproduction and regular generation of
viable seeds (personal observation), which has a
scattered occurrence in Europe and inhabits dif-
ferent habitats (in terms of abiotic conditions, see
text above). The longest distance between the stud-
ied localities was more than 1000 km [between the
Babínské meadows (CZ) and Prejmer (RO)] and
our field observations showed morphological dif-
ferences between the localities (e.g., shape of the
leaves, presence or absence and different lengths
of the leaf petioles, color of the corolla). Based on
these observations, we initially expected to observe
a clearly resolved inter-population genetic structure;
however, this is not what was found.
The relatively high genetic diversity value
obtained and the results of the AMOVA analysis
showed that the majority of the genetic variation is
present within populations. This pattern of genetic
variability distribution may be due to vigorous sex-
ual reproduction, which dominates over vegetative
spreading within A. liliifolia populations (Manole et
al., 2015). Furthermore, the low number of unique
markers accompanied by the almost absolute
absence of fixed-private markers, low FST and DW
values, and weak inter-population genetic structure
suggest that the separation of the analyzed popula-
tions took place only sub-recently, because of the
short time for population differentiation by genetic
drift, which would result in the detection of a clear
population genetic structure. On the contrary, we
detected high overall variation and high similarity
of the sampled populations, suggesting frequent
gene-flow among populations. This, however, seems
unlikely, due to the considerable geographical dis-
tance between the sampled localities utilized in this
study. Our results rather suggest that there was
a large meta-population of A. liliifolia in the Central
European area, which has fragmented relatively
recently into the isolated populations present today.
Nevertheless, some populations in the
Pannonian Biogeographic Region (in Romania,
South Hungary, and Slovak populations in Silica
and Pusté pole (W) are genetically more different
from the other remaining populations, thus these
might have been isolated for a longer period of
time.
CONSERVATION OF A. LILIIFOLIA
The conservation of A. liliifolia strongly
depends on the specific management supporting its
seedlings, which are not vigorous enough to survive
without protection (e.g., by removing invasive or
nitrophilous species; see e.g., Ciosek, 2006; Manole
et al., 2015). Such management may be difficult
because of the presence of other protected species,
thus it should be planned with respect to the whole
locality, not only to a single species.
Recently a conservation program for A. liliifo-
lia was started in Czechia with the aim to find the
most successful and efficient way of management
for each of the present localities. It aims not only at
preserving the natural populations in situ but also
at developing appropriate techniques for ex situ
preservation (creation of a sterile tissue culture,
appropriate storage of seeds in seed banks, experi-
mental germination tests and cultivation in order to
identify the critical factors for seedling growth). The
other countries of Central Europe suggested a simi-
lar approach to protect this species, although with-
out the official funded conservation programs (e.g.,
Kucharczyk, 2007; Puchalski et al., 2014; Manole
et al., 2015).
CONCLUSION
This study provides overall information about the
present condition of Adenophora liliifolia popula-
tions in several countries located in Central Europe,
combining molecular data with the results of a phy-
tosociological survey. A. liliifolia was found in 6 veg-
etation units, where it prefers sunny places with
moist alkaline soil. The richest populations are
in the Polish locality of Kisielany and two Slovak
localities – Trsteník and Cigánka (both in Muránska
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Adenophora liliifolia in Central Europe
101
planina NP). The greatest numbers of species in
the phytosociological relevés were recorded in the
Czech localities of Vražba and Karlštejn and in
three Slovak localities – Malý Sokol, Suchá Belá,
and Michalovo. Light forests and their edges are
optimal biotopes for A. liliifolia. Eutrophication,
shading, overpopulated wild animals, and expan-
sive broad-leaved herbs are the main factors caus-
ing the decline of A. liliifolia populations in Central
Europe. Despite the fact that the majority of the
investigated populations (except for Slovakia) are
rather isolated and geographically distant from
each other, our results indicate high interpopula-
tion homogenity, typical for populations with exten-
sive gene flow. The lack of stronger interpopulation
differentiation can be explained by the relatively
recent fragmentation of a larger population due to
shrinking of suitable habitats, their disappearance,
or overall changes in landscape management. The
findings of the present study show that A. liliifolia
populations are not primarily threatened by loss
of genetic diversity, but are endangered by loss of
suitable habitats. Therefore, a specific management
strategy is necessary in most of the localities.
AUTHORS’ CONTRIBUTIONS
Prausová – phytosociological research and analysis
in Juice, monitoring of the Czech and Slovak popu-
lations; Marečková – monitoring of the Czech and
Slovak populations, genetic analysis; Kapler – phy-
tosociological research and monitoring of the Polish
populations; Majeský – genetic analysis; Farkas –
phytosociological research and monitoring of the
Hungarian populations; Indreica – phytosociological
research and monitoring of the Romanian popula-
tions; Šafářová – ecological statistical analysis in
Canoco; Kitner – genetic analysis.
ACKNOWLEDGEMENTS
The study was supported by the funds for Specific
research financed by the Ministry of Education of
the Czech Republic No. 2121/2011, 2122/2012,
2106/2013 and the Internal Grant Agency of
Palacký University (IGA 2015_001 and IGA
2016_001). The Ministries of Environment of the
Czech and the Slovak Republics granted us with
permission to collect and analyze samples of this
species and enter into its localities. We would like
to express our thanks to the staff of the investigated
protected areas in the Czech Republic and Slovakia,
namely to Roman Hamerský, Tomáš Tichý, Josef
Mottl, Peter Turis, Drahoš Blanár, Robert Šuvada,
Štefánia Bryndzová, for providing us with informa-
tion about the localities where A. liliifolia occurs.
We would also like to express our gratitude to Adam
Rapa and Marek Ciosek for providing us with sam-
ples from the Polish populations. We are grateful
to prof. Jerzy Puchalski for support of research on
the Polish A. liliifolia localities. We would like to
thank František Krahulec for valuable suggestions
to the manuscript and Jakub Gamrát for creating
of Fig. 1.
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