Download Genetic, cytological and morphological differentiation within the

Kuzmanović & al. • Differentiation of Sesleria rigida sensu Fl. Eur.
TAXON 62 (3) • June 2013: 458–472
Genetic, cytological and morphological differentiation within
the Balkan-Carpathian Sesleria rigida sensu Fl. Eur. (Poaceae):
A taxonomically intricate tetraploid-octoploid complex
Nevena Kuzmanović,1 Petronela Comanescu,2 Božo Frajman,3 Maja Lazarević,1 Ovidiu Paun,4
Peter Schönswetter3 & Dmitar Lakušić1
1 Institute of Botany and Botanical Garden, Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, Serbia
2 Botanical Garden “Dimitrie Brandza”, Sos. Cotroceni 32, 060101 Bucharest, Romania
3 Institute of Botany, University of Innsbruck, Sternwartestraße 15, 6020 Innsbruck, Austria
4 Department of Systematic and Evolutionary Botany, University of Vienna, Rennweg 14, 1030 Vienna, Austria
Authors for correspondence: Nevena Kuzmanović, [email protected]; Peter Schönswetter, [email protected]
Abstract Reconstruction of relationships among populations of the morphologically polymorphic and taxonomically intricate
Sesleria rigida sensu Fl. Eur. based on Amplified Fragment Length Polymorphisms (AFLPs) revealed four clearly differentiated
genetic groups that did only partly follow recent taxonomic concepts, but were strictly allopatric. While some of the previously described taxa constitute distinct genetic entities, others have no taxonomic value. Synthesizing our AFLP data with
ploidy-level information obtained from all genetically investigated individuals as well as with chromosome counts revealed
that tetraploid individuals prevail, while octoploids occur only within S. filifolia. Lack of AFLP divergence between tetra- and
octoploids suggests an autopolyploid origin of the latter. The genetic differentiation pattern was reflected by morphological
differentiation, allowing for a taxonomic revision of the constituents of S. rigida sensu Fl. Eur. resulting in recognition of the
four species S. achtarovii, S. filifolia, S. rigida, and S. serbica.
Keywords AFLP; autopolyploidy; flow cytometry; morphometrics; Sesleria rigida; taxonomy
Supplementary Material The Electronic Supplement (Fig. S1) is available in the Supplementary Data section of the online
version of this article (http://www.ingentaconnect.com/content/iapt/tax).
Received: 12 Nov. 2012; revision received: 11 Mar. 2013; accepted: 27 Apr. 2013
Introduction
The Balkan Peninsula and the adjacent Carpathians are
centres of European biodiversity (Turrill, 1929; Davis & al.,
1994; Kryštufek & Reed, 2004). In the past ten years several
phylogeographic studies have explored diversification patterns
on the Balkan Peninsula (e.g., Park & al., 2006; Liber & al.,
2008; Stefanović & al., 2008; Kučera & al., 2010; Surina & al.,
2011) and in the Carpathians (e.g., Mráz & al., 2007; Puşcaş
& al., 2008; Ronikier & al., 2008). However, only a few (e.g.,
Frajman & Oxelman, 2007; Csergö & al., 2009) have examined the biogeographic connections between these two areas,
previously acknowledged on the basis of chorological patterns
(e.g., Meusel & al., 1965; Ronikier, 2011).
A major force in plant evolution is polyploidy (e.g., Wendel,
2000; De Bodt & al., 2005; Soltis & al., 2009; Van de Peer
& al., 2009; Jiao & al., 2011), which fosters adaptation to new
ecological niches and confers reproductive isolation, ultimately
leading to speciation (Otto & Whitton, 2000; Comai, 2005; but
see Mayrose & al., 2011). As much as 15% of angiosperm speciation events are associated with an increase in ploidy (Wood
& al., 2009). Whereas allopolyploids usually differ conspicuously from their diploid progenitors in their genomic constitution and morphology, autopolyploids that arise from the crosses
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within or between populations of a single species (Ramsey
& Schemske, 1998) are often genetically and morphologically
similar to their parents (Levin, 1983, 2002; Parisod & al., 2010).
Recent molecular studies, which often made use of methodological advances in flow cytometry such as the availability of
protocols optimised for the use of dried plant material (Suda
& Trávníček, 2006a, b), indicate a much higher frequency of
autopolyploidy than previously assumed (Husband & Sabara,
2004; Kron & al., 2007; Kolář & al., 2009; Bardy & al., 2010).
Despite the widely recognised importance of polyploidisation
for plant diversification, very little is known about its contribution to the high diversity on the Balkan Peninsula.
The genus Sesleria Scop. (Pooideae, Seslerieae) comprises
ca. 28 mostly European species (Deyl, 1980), with a centre of
diversity on the Balkan Peninsula. It is taxonomically intricate
which may relate to the high incidence of polyploidy in the
genus, as the majority of taxa are tetra- and/or octoploid (2n
= 4x = 28, 2n = 8x = 56, e.g., Strgar, 1979; Lysak & Doležel,
1998; Petrova, 2000). One of the polyploid species is S. rigida
Heuff. ex Rchb. sensu Fl. Eur. (Deyl, 1980; for simplicity termed
“S. rigida s.l.” in the following). So far only tetraploids were
reported for this taxon (Ujhelyi, 1959, 1960; Deyl, 1980; Petrova,
2000), even though Deyl (1946) assumed that octoploids are
present in some morphologically deviating populations.
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Kuzmanović & al. • Differentiation of Sesleria rigida sensu Fl. Eur.
Sesleria rigida s.l. belongs to S. sect. Calcariae Deyl, and
together with S. tenuifolia Schrad. s.l., S. insularis Sommier
and S. taygetea Hayek constitutes the “turma” (= swarm) Rigida
(Deyl 1946), whose centre of origin was suggested to be located
on the western Balkan Peninsula (Deyl, 1946). Taxa from this
species group have narrow, pruinose leaves which are most
often convolute or flat. Their thin spikes are composed of large
spikelets with elongated glumes and shortly awned lemmas.
Sesleria rigida s.l. differs from the other species of Rigida by
the absence of reticulate basal leaf sheaths (vs. S. tenuifolia s.l.),
less than 13 veins in the tiller leaves and usually hairy leaves
(vs. S. insularis) and lack of white bracts subtending the spikes
(vs. S. taygetea). It is distributed in Romania, Bulgaria, Greece,
Serbia and Bosnia (Deyl, 1946; Tatić, 1976); a doubtful and
highly disjunct record from Croatia (Sekulić & al., 1988) could
not be confirmed in the course of the present study (Nevena
Kuzmanović, field obs.). The species thus spans several mountain ranges of the Balkan Peninsula (Dinaric Alps, Balkan Mts.
[Stara planina], Rhodope Mts.) as well as the Romanian Carpathians. It has a wide altitudinal distribution from lowlands
to the alpine vegetation belt, growing mostly on carbonates,
more rarely on serpentines. Intraspecific morphological and
ecological variation of S. rigida s.l. is immense and several
intraspecific taxa have been described (e.g., S. rigida var. degenii Deyl, var. pancicii Deyl, subsp. achtarovii (Deyl) Deyl).
Different authors described new species belonging to this
group which were later either synonymised with S. rigida (e.g.,
S. filifolia Hoppe, S. haynaldiana Schur) or considered only as
ecotypes, such as S. serbica (Adam.) Ujhelyi from serpentine
areas in western Serbia and eastern Bosnia and Herzegovina
(Deyl, 1980). Taxonomic value, delimitation and distribution
of most of these taxa are not clear and they are mostly considered conspecific with S. rigida (Deyl, 1946, 1980; Tatić, 1976;
Diklić & Nikolić, 1986). Deyl (1980) in Flora Europaea recognised only a single species, S. rigida with two subspecies,
subsp. rigida and subsp. achtarovii ; all other names were
neglected or considered synonyms. In most recently published
national Floras Deyl’s (1980) concept with two subspecies
is being followed (e.g., Tatić, 1976; Diklić & Nikolić, 1986;
Gustavsson, 1991; Delipavlov & al., 2003; Assyov & Petrova,
2006; Ciocarlan, 2009). Some authors (e.g., Sekulić & al., 1988;
Stevanović & al., 1995, 2003; Valdés & Scholz, 2011), following
Ujhelyi (1959), recognised S. serbica as an independent species,
and this was also supported by a recent leaf anatomical study
(Kuzmanović & al., 2012). Also S. filifolia is treated at specific rank by some authors (Gergelyi & Beldie, 1972; Assyov
& Petrova, 2006; Ciocarlan, 2009), and S. haynaldiana as a
subspecies of S. rigida (Gergelyi & Beldie, 1972).
Taking the intricate taxonomy of S. rigida s.l. into consideration, our main goal is to evaluate the taxonomic status of
its constituents using molecular and morphological characters.
The first aim is to reconstruct the relationships among populations based on amplified fragment length polymorphisms
(AFLPs; Vos & al., 1995) and to test whether the intraspecific
taxa constitute distinct genetic entities. Second, we screen all
genetically investigated populations for their ploidy level using
flow cytometry combined with several chromosome counts
and synthesize these data with AFLPs in order to test if octoploids originated by auto- or allopolyploidy. Third, we search
for congruence between the genetic lineages and morphologically distinct groups and characterise well-supported groups
with respect to their morphology, chromosome number and
distribution. Finally, we provide a taxonomic treatment and an
identification key for all taxa investigated.
Materials and methods
Plant material. — As various taxa were described and later
considered at different ranks, we assigned the investigated populations to the rankless entities “rigida“, “degenii“, “pancicii“,
“achtarovii“, “filifolia“ and “serbica“ based on their morphology and distribution (Fig. 1; Appendix 1). Our study includes
all described constituents of S. rigida s.l. mentioned in the Introduction with the exception of S. haynaldiana, for which no
type material could be located. Based on the protologue (Schur,
1856), where no locus classicus is specified, the identity of this
taxon, which was regarded as conspecific with S. rigida s.str. by
Deyl (1946, 1980) and Valdés & Scholz (2011), remains unclear.
Molecular and genome size analyses are based on leaf
material of five to ten individuals per population and up to 25
populations per taxon (in total 218 individuals from 45 populations). Leaf material for molecular studies was collected in
the field and desiccated in silica gel. For chromosome counts
living plants were collected and grown in the Botanical garden
“Jevremovac“ in Belgrade. For morphometric analysis plant
material of ten to fifteen individuals (453 leaves and 341 stems
with spikes) was collected from 21 populations. These analyses
were performed on dissected plant organs preserved in 50%
ethanol (leaves) or in 1 : 1 glycerol : ethanol solution (stems with
spikes). Voucher specimens are deposited at BEOU. Voucher
numbers and collecting details are given in Appendix 1.
Chromosome counts. — Chromosome numbers were determined for five individuals per population (20 populations in
total). Root tips obtained from plants in pots were pre-treated
with 0.002 M 8-hydroxyquinoline for 24 h at 4°C. After fixation
in cold Carnoy fixative (ethanol/acetic acid 3 : 1) for at least
24–48 h, hydrolysis was performed in 1N HCl for 14 min at
60°C. Staining was performed in Schiff’s reagent as described
by Feulgen & Rossenbeck (1924), followed by squashing in a
drop of acetic carmine. After freezing at −80°C, cover slips
were removed, preparations were dried at least 24 h and then
mounted in Euparal. Chromosome plates were observed under
a Leica DMLS light microscope (Leica Microsystems, Wetzlar,
Germany) and photographs were taken with a Leica DCF 295
camera (Leica Microsystems).
Estimation of relative and absolute genome size. — Flow
cytometry (FCM) of 4′,6-diamidino-2-phenylindole (DAPI)stained nuclei was used to estimate the relative DNA content of
five to ten silica gel-dried samples per population following the
procedure described in Suda & Trávníček (2006a). After incubation for 5 min at room temperature, the relative fluorescence
intensity of 3000 particles was recorded using a Partec PA II
flow cytometer (Partec, Münster, Germany) equipped with an
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TAXON 62 (3) • June 2013: 458–472
HBO mercury arc lamp. After initial trials, up to three samples
were pooled. If the coefficient of variation (CV) of the G0/G1
peak of a sample exceeded the 5% threshold, the analysis was
discarded and the sample re-measured. Pisum sativum ‘Kleine
Rheinländerin’ (2C = 8.84 pg, Greilhuber & Ebert, 1994) was
selected as primary reference standard (Doležel & al., 1998).
To extrapolate the ploidy level of all individuals, the relative
genome size was correlated with chromosome numbers determined for selected individuals from several populations,
representative for the whole genome size range.
FCM of propidium iodide (PI)-stained nuclei was used
to estimate the absolute genome size of two individuals each
from six populations (Appendix 1). Fresh green leaf tissue (ca.
1 cm²) was processed following the procedure described by
Kolář & al. (2012). After incubation for 5 min at room temperature, the relative fluorescence intensity of 5000 particles
was recorded using a Partec PA II flow cytometer equipped
with an HBO mercury arc lamp. Vicia faba ‘Inovec’ (2C =
26.90 pg) served as an internal standard as the peaks of Pisum
sativum ‘Kleine Rheinländerin’ partly overlapped with those
of tetraploid Sesleria samples.
AFLP analysis. — Total genomic DNA was extracted from
ca. 10 mg of dried tissue with the DNeasy 96 plant kit (Qiagen,
Hilden, Germany) following the manufacturer’s protocol. AFLP
fingerprinting was performed for 45 populations with usually
five individuals per population. The AFLP procedure followed
Fig. 1. Sampled populations of
Sesleria rigida s.l. and the distantly related S. taygetea used as
outgroup in the neighbour-joining
analysis presented in Fig. 2.
Octoploid populations of “rigida”
are marked with a white margin;
all other populations exclusively
contained tetraploid accessions.
Population numbers correspond
to Appendix 1, the taxonomic
assignment follows Deyl (1946,
1980).
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Kuzmanović & al. • Differentiation of Sesleria rigida sensu Fl. Eur.
Wilks’
Lambda F-remove P
Habitus
Plant height
0.027
0.841
0.472
Length of leaf sheath
0.028
4.741
0.003
Length of tiller leaf
0.028
3.240
0.022
Number of culm leaves
0.029
8.039
0.000
Distance between uppermost node and spike base
0.028
2.399
0.067
Spike length
0.028
2.680
0.047
Spike width
0.028
1.971
0.118
Number of spikelets per spike
0.028
1.395
0.244
Cross section of tiller leaves
Width of leaf blade
0.029
11.580
0.000
Distance between middle rib and point where leaf blade is thickest 0.029
10.404
0.000
Thickness of leaf blades in zone of central rib
0.040
64.834
0.000
Largest thickness of leaf blades
0.030
13.509
0.000
Width of central rib
0.030
15.877
0.000
Length of trichomes on abaxial side
0.027
0.567
0.637
Length of trichomes on adaxial side
0.029
7.182
0.000
Height of central vascular bundle
0.027
1.000
0.393
Width of central vascular bundle
0.027
0.137
0.938
Height of largest lateral vascular bundle
0.028
2.122
0.097
Width of largest lateral vascular bundle
0.030
17.124
0.000
Height of sclerenchyma strand of central vascular bundle
0.032
25.442
0.000
Height of sclerenchyma strand/girder in widest zone
0.028
2.483
0.060
Sclerenchyma surface
0.030
12.262
0.000
Surface of leaf blades
0.030
13.961
0.000
Number of sclerenchyma strands on adaxial side
0.028
1.503
0.213
Number of sclerenchyma strands on abaxial side
0.029
8.780
0.000
Number of sclerenchyma girders on adaxial side
0.030
14.646
0.000
Number of sclerenchyma girders on abaxial side
0.036
44.271
0.000
Number of major vascular bundles
0.028
5.014
0.002
Number of minor vascular bundles
0.030
17.012
0.000
Dimension of bulliform cells
0.028
2.953
0.032
Spikelets and flowers
Length of lower glume
0.028
6.101
0.000
Width of lower glume
0.028
1.566
0.197
Length of upper glume
0.028
4.569
0.004
Width of upper glume
0.028
3.594
0.014
Length of lemma of lower flower
0.028
3.493
0.016
Width of lemma of lower flower
0.028
3.610
0.013
Length of palea of lower flower
0.027
1.006
0.390
Width of palea of lower flower
0.028
2.413
0.066
Length of lemma of upper flower
0.027
0.045
0.987
Width of lemma of upper flower
0.027
1.264
0.286
Length of palea of upper flower
0.027
0.756
0.519
Width of palea of upper flower
0.028
1.659
0.175
Discriminant Function Analysis Summary. Details of the anatomical leaf characters are given
in Kuzmanović & al. (2009). Wilks’ lambda is a multivariate generalisation of the univariate
F-distribution; F-remove represents a measure of the extent to which a variable makes a unique
contribution to the prediction of a group membership. P-values < 0.05 are printed in bold.
Vos & al. (1995) with the modifications described in Schönswetter & al.
(2009). In addition, 0.25 U of polymerase were used in the preselective and
selective amplifications (0.4 U for the
NED-labelled primer combination).
Fifteen selective primer combinations
have been initially screened. The three
final selective primer combinations for
the selective PCR (fluorescent dye in
brackets) were EcoRI (6-FAM)-ACT /
MseI-CAC, EcoRI (VIC)-ACG / MseICAC, and EcoRI (NED)-ACC / MseICAC. Purification and visualisation of
PCR products were done as described
in Rebernig & al. (2010). Twenty-eight
samples were used as replicates between PCR batches to test the reproducibility of AFLP fingerprinting.
Electropherograms were analysed
with Peak Scanner version 1.0 (Applied
Biosystems, Foster City, California,
U.S.A.) using default peak detection
parameters except light peak smoothing. The minimum fluorescent threshold was set to 50 relative fluorescence
units. Automated binning and scoring
was performed using RawGeno v.2.0
(Arrigo & al., 2009), a package for the
software R (R Development Core Team,
2010), with the following settings: scoring range = 50–500 bp, minimum intensity = 50 relative fluorescence units
(rfu), minimum bin width = 1 bp, and
maximum bin width = 1.5 bp. Fragments
with a reproducibility lower than 80%
based on sample-replicate comparisons
were eliminated. The error rate (Bonin
& al., 2004) was calculated as the ratio of mismatches (scoring of 0 vs. 1)
over phenotypic comparisons in AFLP
profiles of replicated individuals. Fragments present/absent in only one individual were removed from the dataset.
A neighbor-joining (NJ) analysis
based on the matrix of Nei-Li genetic
distances (Nei & Li, 1979) was conducted and bootstrapped (1000 pseudo-replicates) with TREECON v.1.3b
(Van de Peer & De Wachter, 1997).
The tree was rooted with S. taygetea.
A principal coordinate analysis (PCoA)
based on a matrix of Jaccard distances
among individuals (excluding S. taygetea) was calculated using the modules
“SimQual”, “Dcenter” and “Eigen”
from NTSYS-pc v.2.0 (Rohlf, 1997).
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Table 1. Characters used for the morphometric analysis of Sesleria rigida s.l.
Kuzmanović & al. • Differentiation of Sesleria rigida sensu Fl. Eur.
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Morphometric analyses. — Analysis of 26 anatomical leaf
characters was performed on cross-sections of tiller leaves
as described by Kuzmanović & al. (2009). An additional 23
characters of external morphology were analysed following
combined and adjusted methods used for Festuca L. (Auquier,
1974; Lakušić, 1999; Foggi & al., 1999, 2006) and Sesleria
(Alegro, 2007; Di Pietro, 2007). The measurements were performed using Leica Qwin (Leica Microsystem) and Digimizer
Image Analysis software (MedCalc Software, Belgium).
Seven invariable characters were excluded. Further character reduction was envisaged by computing pairwise Spearman
correlations and retaining only one out of character pairs with
absolute values of correlation coefficients exceeding 0.9, but no
correlations exceeding this threshold were found. The hypothesis of morphological separation of four groups of populations
recognised by AFLPs was tested using canonical discriminant
analysis (CDA) conducted on the resulting data matrix comprising 42 characters after standardisation to zero mean and unit
variance. Characters included in the CDA are listed in Table 1.
The CDA was performed with 4 a priori defined groups × 453
individuals × 42 characters. Discriminant function analysis
(Table 1) was done to estimate the contribution of individual characters to overall discrimination. Canonical scores for
each case were calculated with the aim to measure distances
between individuals, and a scatterplot of canonical scores (see
Fig. 5 below) was made to visualise the relationship between
a priori defined groups. Statistical analyses were performed
using Statistica v.5.1 (StatSoft, 1996).
Results
Fig. 3. Neighbour-joining dendrogram of Sesleria rigida s.l. based on a matrix of Nei-Li distances (Nei & Li 1979) among AFLP phenotypes. The
tree is rooted with S. taygetea. Octoploid individuals of “rigida” are shown with thicker branches; all other accessions are tetraploid. Numbers
1 to 4 at the main branches correspond to the main clusters identified by the PCoA (Fig. 4). Bootstrap values (based on 1000 replicates) greater
than 50% are given for branches with ≥ 3 terminals. The taxonomic assignment following Deyl (1946, 1980) is given in the middle panel, the
new taxonomic concept emerging from the present study is given in the right panel. Symbols correspond to Figs. 1 and 4; symbols at the tips
of branches are only given when deviating from the majority assignment in the middle panel. Population numbers given at the tips of branches
correspond to Appendix 1.
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◄
Fig. 2. Mitotic chromosomes of Sesleria rigida s.l. A, population S275
(2n = 28); B, population S468 (2n = 56). — Scale bar = 20 μm.
Chromosome numbers and genome size. — Chromosome
numbers were determined for 100 individuals from 20 populations (Appendix 1). Two ploidy levels were found, tetraploids
with 2n = 4x = 28 in fifteen populations and octoploids with
2n = 8x = 56 in five populations (Fig. 1).
Chromosomes were 2.05–5.99 µm long in tetraploids and
1.75–5.35 µm in octoploids. Several chromosomes in both
ploidy levels were bearing satellites. Their number is variable,
from two to six in tetraploids and up to ten in octoploids. They
are usually situated on large metacentric chromosomes and
smaller acrocentric or subtelocentric chromosomes (Fig. 2).
In several cases chromatic particles were visible that could be
chromosome fragments, detached satellites or B-chromosomes.
Flow cytometry analyses mostly yielded high-resolution
histograms. The relative genome size (RGS) ranged from 0.62
to 1.35, whereas the absolute genome size was between 8.58 and
16.90 pg. According to RGS all analyzed individuals were clearly
separated into tetraploids (86.4% of the samples) with RGS from
0.62 to 0.76, and octoploids (13.6%) with RGS from 1.29 to 1.35
(Appendix 1; Electr. Suppl.: Fig. S1). Neither odd-ploidy level
cytotypes nor mixed-ploidy populations were detected.
AFLP data. — A total of 2580 AFLP fragments were scored
in 218 individuals from which high-quality, reproducible
AFLP-fingerprints were obtained. A total of 171 and 6 bands
were found to be present or absent in only one individual, respectively, and were excluded from further analyses. The initial
error rate (according to Bonin & al., 2004) before the exclusion
of unreliable characters was 3%.
The NJ analysis revealed four clusters with high bootstrap support (BS) between 96% and 100% (Fig. 3). The most
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“rigida”
“pancicii”
“filifolia”
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divergent cluster (BS 100%) corresponds to “serbica“ from serpentine areas in western Serbia and eastern Bosnia. A further
cluster (BS 100%) contains accessions of “achtarovii“ found
in the Rhodopi Mts. (Bulgaria and northeastern Greece). Two
well-supported (BS 100) sister clusters include “rigida“ and
“degenii“ from the Romanian Carpathians (BS 100%) as well
as “rigida“, “filifolia“ and “pancicii“ from eastern and northeastern Serbia, western and northern Bulgaria and Munţii Banatului in Romania (BS 96%).
The PCoA (excluding the outgroup S. taygetea) resulted
in separation of four groups (Fig. 4) matching perfectly those
revealed by the NJ analysis. The genetic groups only partly
follow taxonomic boundaries but are strictly allopatric (Fig. 4).
Both NJ and PCoA analyses showed that octoploid accessions
of “rigida“ from eastern Serbia are not genetically differentiated from tetraploids from the same region (Figs. 3–4).
Morphometric data. — The CDA largely supported the
hypothesis of morphological separation of four genetic groups
identified by AFLPs (Fig. 5). Discriminant function analysis showed that anatomical characters (cross section of tiller
leaves) contribute dominantly to the overall discrimination
(Table 1). Furthermore, the contribution of characters of habitus
is slightly higher than the contribution of characters of spikelets
and flowers. Most of the discrimination of anatomical parameters is contributed by thickness of leaf blades in the zone of the
central rib, number of sclerenchyma girders on the abaxial side,
height of sclerenchyma strand of the central vascular bundle,
width of largest lateral vascular bundle, number of minor vascular bundles, width of the central rib, number of sclerenchyma
girders on the adaxial side, surface of leaf blades and largest
thickness of leaf blades. The analysis showed that morphological characters with the strongest influence on discrimination
are number of culm leaves, length of leaf sheath, length of tiller
leaf and length of spike within habit characters, and length of
lower glume, length of upper glume, width of lemma of lower
flower, width of upper glume and length of lemma of lower
flower within spikelets and flower characters (Table 1).
Fig. 4. Principal coordinate
analysis (PCoA) of AFLP data
of Sesleria rigida s.l. First and
second factors explain 5.7%
and 3.2% of the total variation,
respectively. The taxonomic coding of individuals is according
to Figs. 1 and 3. The inset shows
the geographic distribution of the
four identified groups, which are
cross-referenced with the ordination plot with numbers.
Discussion
Our multifaceted study employing genomic data (AFLPs),
genome size, chromosome counts and morphometry applied to
a broad sample of populations revealed a clear structure within
the taxonomically intricate Sesleria rigida s.l. This complex,
distributed on the Balkan Peninsula and the Carpathians, includes six taxa, whose taxonomic value has been controversial
in the past (see Introduction). The AFLP data covering most
taxa throughout their geographic ranges revealed four allopatrically distributed genetically divergent entities (Figs. 3–4),
which failed to form a monophyletic group in a genus-wide
AFLP study (N. Kuzmanović & P. Schönswetter, unpub.). The
5
“achtarovii”
“degenii”
1
“filifolia”
1
4
“pancicii”
“rigida” 4x
“rigida” 8x
“serbica”
2
3
2
-3
Factor 2
2
4
4
1
0
.00
1
Factor 1
-2
-3
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3
3
4
5
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first (cluster 1, Fig. 3; PCoA group 1, Fig. 4) includes populations of S. rigida (“rigida” and “degenii”) from the Romanian
Carpathians, the second (cluster 2; PCoA group 2) comprises
populations of S. filifolia (“filifolia” and “pancicii”) from the
Munţii Banatului in Romania and the Balkan Mts. in eastern
and northeastern Serbia and western and northern Bulgaria,
the third (cluster 3; PCoA group 3) consists of populations of
S. serbica from serpentine areas in eastern Bosnia and western
Serbia and the last (cluster 4; PCoA group 4) contains populations of S. achtarovii from the Rhodopi Mts. in Bulgaria,
northeastern Greece and the island Thassos. Similar results
were obtained by Comanescu (2011), albeit based on a less
comprehensive sample, and with the main aim of resolving the
taxonomic status of S. filifolia in Romania.
The canonical discriminant analysis of the morphometric
dataset (Fig. 5) showed that the genetically defined groups
6
Fig. 5. Canonical discriminant
analysis (CDA) of Sesleria rigida
s.l. based on 453 individuals and
42 morpho-anatomical characters. The four a priori defined
groups are the genetic groups
identified in Figs. 3 and 4. In
(A) the labelling is in accordance
with the genetic groups, whereas
in (B) the labelling follows Deyl
(1946, 1980). The informal entities “degenii” and “pancicii” were
not included as no material was
available for morphometric study.
4
Root 2
2
0
-2
-4
-6
Sesleria achtarovii
Sesleria filifolia
Sesleria rigida
Sesleria serbica
A
-6
-4
-2
0
2
4
6
Root 1
6
4
Root 2
2
0
-2
“achtarovii”
“filifolia”
“rigida” 4x
“rigida” 8x
“serbica”
-4
-6
B
-6
-4
-2
0
2
4
6
Root 1
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TAXON 62 (3) • June 2013: 458–472
are also morphologically distinct. Four taxa can thus be recognised: S. achtarovii, S. filifolia, S. rigida and S. serbica,
which are presented below in the Taxonomic Treatment. As
the populations originally assigned to “degenii” and “pancicii”
were neither genetically nor morphologically differentiated, we
synonymise S. rigida var. degenii and S. rigida var. pancicii
with S. rigida s.str. and S. filifolia, respectively. All four species
in the revised circumscription are allopatrically distributed and
their distribution ranges are placed within different phytogeographic regions and provinces (Meusel & Jäger, 1992; Jäger
& Welk 2003). Whereas S. rigida belongs to the Carpathian,
the other three species belong to the different provinces of
the sub-Mediterranean region. Sesleria filifolia is restricted
to the Moesian (i.e., Balkan), S. serbica to the Illyrian and
S. achtarovii to the Macedonian-Thracian province, with one
isolated population on the island of Thassos in the Aegean
province of the Macaronesian-Mediterranean region.
The geographically isolated (Fig. 1) S. achtarovii and
S. serbica are also genetically most distant (Fig. 3), whereas
S. filifolia and S. rigida with distribution areas separated by
minimally 20 km are also genetically closer and form a highly
supported clade. Their distribution areas come into close contact in the Iron Gates region along the Danube in Serbia and
Romania. This area has traditionally been considered a likely
migration corridor between the Carpathians and the Dinaric
Mts., and this is supported also by phylogeographic studies
(e.g., Frajman & Oxelman, 2007; Ronikier, 2011). Our results
support this hypothesis, but it is not clear whether both taxa
came here into secondary contact after extending their distribution from two isolated refugia, or if the Carpathian S. rigida
originated from the Balkan S. filifolia in the course of a northeastern range expansion. The Danube river valley was obviously no impassable barrier for the constituents of S. rigida
s.l. as two populations (S317, S372) of S. filifolia were sampled
north of the Danube in Romania. The distribution of S. filifolia,
extending from southeastern and eastern Serbia to the central
Stara planina in Bulgaria but missing from the adjacent Rhodopi Mts. (Fig. 1) is seen also in the regional distributions of
several other plant species such as Aconitum burnatii Gáyer,
Allium victorialis L., Anthemis carpatica Waldst. & Kit. ex
Willd., Campanula transsilvanica Schur ex Andrae, Carex
brevicollis DC., Centaurea napulifera Rochel, Dryas octopetala L.and Jacobaea abrotanifolia (L.) Moench (Senecio
abrotanifolius L.) (Assyov & Petrova, 2006). On the other hand,
the Rhodopi Mts. share some taxa with adjacent mountains in
Greece (e.g., Haberlea rhodopensis Friv., Petkovia orphanidea
(Boiss.) Stef., Saxifraga ferdinandi-coburgi Kellerer & Sünd.,
Saxifraga stribrnyi (Velen.) Podp., Scabiosa rhodopensis Stoj.
& Stef., Polygala rhodopea Janch.), a distribution pattern seen
also in S. achtarovii.
Ecologically, S. serbica shows the highest specificity. It is
an obligate serpentinophyte that inhabits submontane and montane rocky grasslands (Festuco-Brometea) from the thermophilous oak to the cryophilous spruce belt and subalpine pastures
(Festuco-Seslerietea). At the same time, this species dominates
the understory of specific pine forests (Seslerio-Pinetum nigrae
and Seslerio-Pinetum sylvestris community-type) and is an
important component of temperate heaths (Erico-Seslerietum
community-type; Lakušić & al., 2005). The other three species are strictly calcicole and grow in rock crevices (Asplenietea trichomanis), rocky grasslands (Festuco-Brometea)
and subalpine and alpine pastures (Festuco-Seslerietea or
Daphno-Festucetea) between 100 and 2140 m a.s.l., and also
in beech forests (Seslerio rigidae-Fagetum of the class Querco-Fagetea) and Scots pine forests (Seslerio rigidae-Pinetum
sylvestris of the class Erico-Pinetea) (Lakušić & al., 2005; Karakiev & Tzonev, 2011; Comanescu, 2011).
Previously, only tetraploids were known in S. rigida s.l.
(Ujhelyi, 1959, 1960; Deyl, 1980; Petrova, 2000). Our genome
size measurements and chromosome counts (Appendix 1;
Figs. 2–3) have revealed some octoploid (2n = 56) populations
of S. fili­folia, mostly from southeastern and eastern Serbia,
whereas tetraploids are evenly distributed throughout the
studied area in all analysed taxa. The octoploid populations of
S. filifolia are genetically indistinguishable from neighbouring tetraploid populations. This is supported by the neighbour
joining analysis (Fig. 3) as well as by Bayesian cluster analyses
conducted with STUCTURE v.2.3 (Pritchard & al., 2000) under
different parameterisations (with and without admixture, allele
frequencies correlated or not) suggesting a single unstructured
gene pool as most likely solution (Peter Schönswetter, unpub.).
The lack of genetic structure within S. filifolia strongly suggests
a recent autopolyploid origin of the octoploid cytotype. Based
on the disjunct distribution of octoploids we hypothesize that
they have independently originated at least twice. Autopolyploidy is much more common in plants then traditionally assumed. Initially considered an evolutionary dead-end (Clausen
& al., 1945; Stebbins, 1971), it was later shown that genome
multiplication per se may represent an evolutionary advantage,
e.g., by enforcing successful range expansion and ecological
radiation as demonstrated in different natural autopolyploids
(Manton, 1937; Soltis & al., 2007; Parisod & al., 2010).
No cytotype mixtures were found even when expanding
our ploidy level screening to ten individuals per population
in contact zones of the two cytotypes (Appendix 1). In the
same line, geographically close octoploid and tetraploid populations are ecologically and altitudinally differentiated. For
instance, although the horizontal distance between tetraploids
from the Gornjak gorge (population S234) and octoploids from
Mt. Vukan (population S466) is less than 2 km, tetraploids
occur exclusively in rock crevices (Asplenietea trichomanes)
in a gorge at ca. 200 m a.s.l., whereas octoploids inhabit rocky
grasslands (Festuco-Brometea) on wind-exposed ridges at ca.
700 m a.s.l. Tolerance of plants with higher ploidy level to habitats with stronger competition (Hülber & al., 2009) is also reflected in their overall size. Already Deyl (1946) hypothesised
that plants from the eastern Serbian Mt. Basara that are larger
in all parts have a higher ploidy level. Herbarium vouchers
sampled in populations from which cytological information is
available confirmed that octoploids are generally more robust
than tetraploids. Particularly in leaves of octoploid individuals
all size characters are larger than in tetraploids (Kuzmanović
& al., 2009). The absence of any hexaploids, despite screening a large number of individuals, suggests that octoploids are
466
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Kuzmanović & al. • Differentiation of Sesleria rigida sensu Fl. Eur.
reproductively isolated from tetraploids, even when they occur
in geographic proximity. The observed ecological differentiation may play a role in the apparent isolation (e.g., Lysak
& Doležel, 1998; Petrova, 2000).
Similar to other grass genera (e.g., Festuca, Helictotrichon
Besser, Puccinellia Parl., Stipa L.), our results clearly indicate
that leaf morphology and especially anatomy play a primary
role in morphological discrimination of the constituents of
S. rigida s.l. On the basis of leaf shape (convolute vs. flat),
presence of indumentum on the adaxial side of leaves (hairy
vs. glabrous), type of the subepidermal sclerenchyma bands
(continuous vs. interrupted), number of major and minor vascular bundles, as well as the number of sclerenchyma girders,
it is relatively straightforward to distinguish the four species.
Additionally, characters of overall habit, such as shape of tufts
(stoloniferous vs. compact) and spikes (lax and interrupted
vs. dense and compact), further facilitate their identification.
Sesleria achtarovii Deyl in Opera. Bot. Čech. 3: 193. 1946 ≡
S. rigida subsp. achtarovii (Deyl) Deyl in Bot. J. Linn.
Soc. 76: 364. 1978 – Lectotype (designated here):
[Bulgaria] in saxosis calcareis mt. Rhodope centralis,
Kuru-dere ad Stanimaka, 23 Mar 1914, I. Mrkvička s.n.
(SOM no. 100252!).
Note. – We examined several specimens of original material (SOM no. 100252!, SOM no. 4441!, PRC no. PRC519968!,
W no. 1892-0009528!), and decided to select the sheet from
Kuru-Dere (SOM no. 100252) as lectotype. Of all the specimens cited in the protologue it is the most representative, and
has several well developed plants.
Description. – Caespitose perennial. Culm (12.7)16.5–
33.0(42.3) cm tall. Spike (13)15–20(25) × (4)5–7(8) mm, whitish, with (9)12–18(28) spikelets; spikelets on short pedicels,
with 2–3 flowers. Tiller leaves (6)11–26(37) cm long, flat or
convolute, sparsely hairy on adaxial side; transverse sections of
tiller leaves (0.87)0.97–1.31(1.63) × (0.15)0.18–0.23(0.27) mm,
with 5–6(7) major vascular bundles and (6)7–10(13) minor
vascular bundles, number of sclerenchyma girders varies from
(6)8–12(14) on adaxial side of leaves and (4)6–9(12) on abaxial
side of leaves, subepidermal sclerenchyma discontinuous.
Chromosome number. – 2n = 4x = 28
Ecology. – Rock crevices (Asplenietea trichomanis) and
subalpine and alpine pastures (Daphno-Festucetea) on carbonate bedrock (limestone, marble); 450–2000 m a.s.l.
Distribution. – Rhodope Mts. in southern Bulgaria and
northeastern Greece.
Taxonomic treatment
Sesleria rigida s.l. comprises densely caespitose perennials
without reticulate basal leaf sheaths. Leaves are 0.6–2.1 mm
wide, plicate, sometimes pruinose above, and have fewer than
13 major vascular bundles. The uppermost stem leaf is 0.2–
2.5 cm long. The spike is cylindrical, 3–5 times longer than wide,
bluish or whitish, without prominent white bracts subtending the
inflorescence. Spikelets are laterally compressed and bear 2–3
florets. Glumes are unequal and membranous with a single vein.
The lemma is sparsely pubescent to glabrous between the veins,
membranous, five-veined, with usually five aristulate teeth at
the apex, the middle tooth being the longest. The palea is as long
as or shorter than the lemma and is two-veined.
Based on the cytological, genetic and morphometric results
presented here, we recognise four previously described species
within the studied group, i.e., S. achtarovii, S. filifolia, S. rigida
and S. serbica.
Key to the species of Sesleria rigida s.l.
In the morphological descriptions, value ranges correspond
to the mean ± standard deviation, with the minimal and maximal values in brackets. Morphological descriptions are reduced
and include only diagnostic morphological characters.
1. Tuft stoloniferous, spike elongated and lax, 4–5 times
longer than wide, growing on serpentine ....... S. serbica
1. Tuft compact, spike dense, 3 times longer than wide, growing on carbonate . .............................................. 2
2. Leaf transverse section with continuous stratum of subepidermal sclerenchyma .............................. S. rigida
2. Leaf transverse section without continuous stratum of subepidermal sclerenchyma ...................................... 3
3. Leaves convolute, rarely flat, densely hairy on adaxial
side ................................................... S. filifolia
3. Leaves flat, rarely convolute, sparsely hairy on adaxial
side ................................................ S. achtarovii
Sesleria filifolia Hoppe in Flora 17: 384. 1834 – Lectotype
(designated here): [Romania], in rupibus calcareis
ad Krassova [Caraşova], Apr 1831, J. Heuffel s.n. (BP no.
19625!, only the two right-hand plants).
= Sesleria rigida var. pancicii Deyl in Opera. Bot. Čech. 3:
191. 1946 – Lectotype (designated here): Serbia, auf
Kalkfelsen Debeli lug, Majdan pek, Apr 1879, J. Pavlović
s.n. (PR no. 161815!).
Note on lectotypification of S. filifolia. – On the herbarium
sheet four plants from two different collections are mounted.
Only the two right-hand plants clearly from Krassova are selected as the lectotype.
Note on lectotypification of S. rigida var. pancicii. – In
PR, we found four specimens from Serbia (PR no. 161815!, no.
161821!) and Bulgaria (PR no. 161816!, no. 161817!) that were
labelled as Sesleria rigida Heuff. var. pancicii by Deyl. We have
excluded the Bulgarian specimens as potential types, as (1) the
plant from Zemen collected by Achtaroff (PR no. 161817!) is not
cited in “Specimina ut sequuntur vidi” (Deyl 1946: 187–188),
which implies that Deyl did not have this material at hand at
the time when he described this variety, and (2) the plant from
Persenka Balkan in Bulgaria collected by Podpèra (PR no.
161816!) belongs to S. achtarovii (Deyl 1946: 193). Since the
specimen from Debeli lug collected by Pavlović (PR no. 161815!)
is more representative than the specimen from Niška Banja collected by Ilić (PR no. 161821!), we select the former as lectotype.
Description. – Caespitose perennial. Culm (13.1)24.7–
42.6(65.3) cm tall. Spike (10)16–23(33) mm × (4)5–8(10) mm,
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bluish, with (5)11–19(27) spikelets; spikelets on short pedicels,
with 2–3 flowers. Tiller leaves (1.3)12.4–25.4(37.5) cm long,
convolute, rarely flat, densely hairy on adaxial side, rarely
pruinose above; transverse sections of tiller leaf (0.59)0.76–
1.13(1.67) mm × (0.15)0.19–0.25(0.33) mm, with (3)4–6(7) major
vascular bundles and (3)5–8(11) minor vascular bundles, number of sclerenchyma girders varies from (5)6–9(12) on adaxial
side of leaf and (4)6–8(10) on abaxial side of leaf, subepidermal
sclerenchyma not continuous.
Chromosome number. – 2n = 4x = 28 and 2n = 8x = 56.
Ecology. – Rock crevices (Asplenietea trichomanis), rocky
grasslands (Festuco-Brometea) and subalpine and alpine pastures
(Festuco-Seslerietea), on carbonate bedrock; 90–1830 m a.s.l.
Distribution. – Munţii Banatului in Romania and Balkan
Mts. in eastern Serbia and western and northern Bulgaria.
Note. – Ujhelyi (1959), when raising S. rigida var. serbica to the species level, incorrectly cited a specimen from BP
as the “holotype”: “Bosnien: Gostovic-Gebiet auf Serpentin.
Leg. W. Ludwig, Mai 1955” (BP no. 0734231!). However, as
he clearly based Sesleria serbica on Adamović’s S. rigida var.
serbica, the two names must be taken as homotypic. We examined several specimens of original material (e.g., specimens
collected by L. Adamović in the vicinity of Gornji Milanovac
in May 1893 [BP no. 593596!, WU no. 0042093!, GZU no.
259577!] and in April 1896 [W no. 1897-0006747!]), and selected the specimen from PRC as lectotype. The lectotype is
quite representative of the species, and it is very well preserved
with several plants on the sheet.
Description. – Stoloniferous perennial. Culm (15.7)23.8–
42.6(55.9) cm tall. Spike elongated and lax, (16)19–30(44) mm ×
(4)5–7(8) mm, bluish; with (7)11–16(20) spikelets. Spikelets on
prominent pedicels, with 2–3 flowers. Tiller leaves (4.5)13.4–
33.4(47.2) cm long, convolute, hairy on adaxial and abaxial
side; transverse sections of tiller leaves (0.60)0.83–1.09(1.42)
× (0.18)0.21–0.26(0.29) mm, with (3)4–6(7) major vascular
bundles and (3)5–7(12) minor vascular bundles, number of
sclerenchyma girders on adaxial side of leaves (6)8–11(14) and
on abaxial side of leaves (5)8–11(14), with tendency to form
continuous subepidermal sclerenchyma.
Chromosome number. – 2n = 4x = 28.
Ecology. – Rocky grasslands (Festuco-Brometea), forests
(Erico-Pinetea) and subalpine pastures (Festuco-Seslerietea),
exclusively on serpentine bedrock; 250–1900 m a.s.l.
Distribution. – Dinaric Mts. of eastern Bosnia and western
Serbia.
Sesleria rigida Heuff. ex Rchb., Fl. Germ. Excurs.: 1403. 1831 –
Lectotype (designated here): [Romania], in cacumine
montis Domuglett (“3000 ped”), ad Thermas Herculis,
Apr [without indication of year], J. Heuffel s.n. (W no.
1889-0204591!).
= Sesleria rigida var. degenii Deyl in Opera. Bot. Čech. 3: 190.
1946 – Lectotype (designated here): [Romania], in
saxosis ad cacuminen montis Domugled supra thermas
Herkulis, 7 May 1894, A. de Degen s.n. (BP no. 19851!).
Note. – The specimen selected as the lectotype of Sesleria rigida var. degenii was seen by M. Deyl, and collected
prior to the publication of the protologue. Thus we consider
it likely represents a part of the original material. Although
Deyl’s undated annotation label lists only the name S. rigida,
we believe that it was used by him to describe the new variety
and simply not reannotated because it was located outside of
his home institution (PR). This is supported by the fact that
Deyl appears to have only added annotations to specimens at
PR after describing new taxa in his monograph.
Description. – Caespitose perennial. Culm (6.9)17.3–
35.6(45.8) cm tall. Spike (11)14–23(30) mm × (4)6–8(10) mm,
whitish, with (9)13–21(32) spikelets; spikelets on short pedicels, with 2–3 flowers. Tiller leaves (2.0)9.3–28.2(39.7) cm
long, flat or convolute, pruinose above; transverse sections
of tiller leaves (0.72)1.03–1.56(2.09) × (0.15)0.20–0.28(0.34)
mm, with (3)5–8(11) major vascular bundles and (4)5–11(21)
minor vascular bundles, sclerenchyma girders on adaxial side
of leaf (4)8–15(22), on abaxial side (4)8–15(20), with continuous
subepidermal sclerenchyma band.
Chromosome number. – 2n = 4x = 28.
Ecology. – Rock crevices (Asplenietea trichomanis), rocky
grasslands (Festuco-Brometea), pastures (Festuco-Seslerietea)
and forests (Querco-Fagetea, Erico-Pinetea), on carbonate bedrock; 160–2140 m a.s.l.
Distribution. – Carpathians in Romania.
Sesleria serbica (Adamović) Ujhelyi in Feddes Repert. Spec.
Nov. Regni Veg. 62: 64. 1959 ≡ S. rigida var. serbica Adamović in Allg. Bot. Z. Syst. 2: 120. 1896 – Lectotype
(designated here): [Serbia], in saxosis ad Gornji Milanovac, Apr 1893, L. Adamović s.n. (PRC no. PRC 451931!).
468
Acknowledgements
This work was financed by the European Commission in the
SEE-ERA.NET PLUS framework (project “Evolution, biodiversity
and conservation of indigenous plant species of the Balkan Peninsula”
to P.S.) and the Ministry of Education and Science of the Republic
of Serbia (grant 173030 to D.L.). The Austrian Agency for International Cooperation in Education & Research (OeAD; financed by the
Austrian Federal Ministry of Science and Research) granted a onemonth visit at the Institute of Botany of the University of Innsbruck
(ICM-2012-02562) to N.K. Support from the Royal Botanic Gardens
Kew and the European SYNTHESYS programme (Project No. GBTAF-334) granted to P.C. is also acknowledged. We thank M. Magauer,
D. Pirkebner and M. Winkler for conducting the laboratory work in
an excellent way. We are grateful to P.D. Schlorhaufer and his colleagues from the Botanical Garden of the University of Innsbruck for
successfully cultivating our living collection of Sesleria. We gratefully
acknowledge the herbarium curators L. Pignotti (W), W. Till (WU),
Z. Barina (BP), L. Somlyay (BP), O. Šida (PRC), J. Štepanek (PR)
and S. Bancheva (SOM) for help with searching for type specimens
of the investigated taxa. Our colleagues S. Vukojičić, M. Niketić,
G. Tomović, M. Plećaš (Belgrade), V. Ranđelović, B. Zlatković, I. Fusijanović (Niš), D. Uzunov, Ch. Gusev (Sofia) and S. Redžić (Sarajevo)
helped with field work and collecting of the plant samples. We are
grateful to B. Foggi (Florence) for help with nomenclatural problems.
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TAXON 62 (3) • June 2013: 458–472
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and James C. Lendemer provided very helpful suggestions to improve
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Appendix 1. The names in bold represent four species recognized in our study, the rankless entities used in this study (see Materials and Methods) are in roman
letters. The columns are as follows: Population code (ID), sampling locality, number of individuals used for relative genome size measurements (NGS), ploidy
level, chromosome number (2n), relative and absolute genome size (GS) of the investigated populations of Sesleria rigida s.l., number of individuals used for
AFLP analysis (NAFLP), population used for morphometrics (M), number of herbarium voucher stored in the herbarium BEOU (No.), collector information.
Taxon
ID
Ploidy
NGS level
Locality
2n
Relative
GS ± SD
Absolute
GS NAFLP M
No.
Collector(s)
S. achtarovii
0.700 ± 0.005
5
x
31961 Vukojičić, S. & al.
0.678 ± 0.005
5
x
31973 Vukojičić, S. & al.
4x
0.701 ± 0.003
3
x
31559 Lakušić, D., Lakušić, B.
4x
0.699 ± 0.004
4
x
31558 Lakušić, D., Lakušić, B.
x
31515 Kuzmanović, N., Comanescu, P.
achtarovii
S333 Bulgaria, Asenovgrad, Kuru Dere
5
4x
achtarovii
S335 Bulgaria, Trigrad, Trigradsko ždrelo,
near waterfall
5
4x
achtarovii
S350 Greece, Makedonia, Pangaeon
5
achtarovii
S351 Greece, Aegian islands, Tassos,
Ipsarion
5
28
S. filifolia
filifolia
S317 Romania, Dubova, Mali Kazan
5
4x
filifolia
S372 Romania, Carasova
5
4x
pancicii
S245 Bulgaria, Zemen, Zemenski prolom
10
4x
28
0.667 ± 0.015
3
0.646 ± 0.007
5
32107 Kuzmanović, N., Plećaš, M.
28
0.662 ± 0.015
5
30442 Lakušić, D. & al.
pancicii
S439 Serbia, Debeli Lug, Valja Fundata
5
4x
0.667 ± 0.006
3
33936 Kuzmanović, N.
rigida
S225 Serbia, Golubac, Golubac gorge
6
4x
28
0.624 ± 0.007
5
21166 Lakušić, D., Lakušić, B.
rigida
S228 Serbia, Malinik, canyon of Lazareva 10
river
4x
28
0.659 ± 0.018
5
x
27098 Lakušić, D., Kuzmanović, N.
rigida
S230 Serbia, Niš, Jelašnica gorge
4x
28
0.649 ± 0.017
5
x
27444 Kuzmanović, N.
28
x
30322 Lakušić, D.
10
rigida
S234 Serbia, Gornjak gorge
10
4x
rigida
S236 Serbia, Kučevo, Pek gorge
10
4x
0.692 ± 0.001
0.670 ± 0.012
30320 Lakušić, D.
5
8.584
Version of Record (identical to print version).
5
471
Kuzmanović & al. • Differentiation of Sesleria rigida sensu Fl. Eur.
TAXON 62 (3) • June 2013: 458–472
Appendix 1. Continued.
Taxon
ID
Ploidy
NGS level
Locality
2n
Relative
GS ± SD
Absolute
GS NAFLP M
No.
Collector(s)
S. filifolia (continued)
rigida
S237 Serbia, Paraćin, Izvor, gorge of
river Grza
10
4x
rigida
S238 Serbia, Pirot, Rsovci
10
8x
rigida
S239 Serbia, Bor, Stol
5
4x
rigida
S240 Serbia, Bor, Rgotski kamen
10
rigida
S241 Serbia, Sićevo gorge, monastery
Sv. Petka
rigida
S246 Bulgaria, Mt. Konjevska, Viden
30323 Vukojičić, S.
0.660 ± 0.015
5
56
1.312 ± 0.008
5
28
0.666 ± 0.003
5
30325 Fusijanović, I.
4x
0.681 ± 0.009
5
30326 Fusijanović, I.
10
8x
1.305 ± 0.015
5
30327 Ranđelović, V.
10
4x
0.664 ± 0.007
5
x
30324 Zlatković, B.
x
30495 Lakušić, D. & al.
x
30546 Kuzmanović, N. & al.
30497 Lakušić, D. & al.
rigida
S247 Serbia, Vidlič, Visoka stena
10
8x
56
1.325 ± 0.029
5
rigida
S250 Serbia, Mt. Suva planina, Trem
10
8x
56
1.292 ± 0.004
5
rigida
S354 Serbia, Mt. Rtanj
5
4x
0.651 ± 0.005
5
32062 Lakušić, D. & al.
rigida
S355 Serbia, Mt. Rudina
5
4x
0.651 ± 0.010
4
32003 Ranđelović, V.
rigida
S356 Serbia, Soko Banja, Soko Grad
5
4x
0.664 ± 0.006
5
32002 Vukojičić, S., Kuzmanović, N.,
Jušković, M.
rigida
S402 Bulgaria, Veliko Trnovo, Jantra river
gorge (Kandiljka hill)
5
4x
0.661 ± 0.023
5
33234 Niketić, M., Tomović, G.
rigida
S403 Bulgaria, Mts. Central Stara Planina, 5
Šipčenska Planina (Ispolin peak)
4x
0.661 ± 0.009
5
33275 Niketić, M., Tomović, G.
rigida
S466 Serbia, Mt. Mali Vukan
10
8x
56
1.321 ± 0.011
16.649
4
34165 Lakušić, D. & al.
rigida
S468 Serbia, Mt. Suva planina, Sokolov
kamen
10
8x
56
1.345 ± 0.017
16.895
5
34169 Lakušić, D. & al.
degenii
S353 Romania, Domogled
5
4x
28
0.682 ± 0.001
rigida
S242 Romania, Deva, Craciunesti
10
4x
28
0.669 ± 0.013
rigida
S243 Romania, Baia Mare, Remeti
10
4x
28
0.668 ± 0.011
rigida
S244 Romania, Braşov, Timpa
10
4x
28
0.690 ± 0.010
rigida
S319 Romania, Turda, Cheile Turzii
5
4x
28
0.642 ± 0.010
rigida
S352 Romania, Domogled, Prolaz
5
4x
0.664 ± 0.009
5
serbica
S269 Serbia, Zlatibor, Zlatiborsko jezero
10
4x
0.643 ± 0.003
5
serbica
S272 Serbia, Divčibare, Ljuti krš
10
4x
0.650 ± 0.007
4
x
serbica
S273 Serbia, Mokra Gora, Šargan
10
4x
0.637 ± 0.010
4
x
serbica
S274 Serbia, Ibar valley, Maglić old town
10
4x
0.645 ± 0.011
5
S. rigida
5
x
31538 Lakušić, D. & al.
5
x
30329 Kuzmanovic, N., Comanescu, P.
5
x
30333 Kuzmanovic, N., Comanescu, P.
9.208
4
x
30337 Kuzmanovic, N., Comanescu, P.
8.917
3
x
31517 Kuzmanovic, N., Comanescu, P.
8.872
31537 Lakušić, D. & al.
S. serbica
28741 Kuzmanović, N.
30308 Kuzmanović, N.
30309 Lakušić, D.
30310 Lakušić, D.
serbica
S275 Serbia, Mali Vujan, Banjska vrata
10
4x
0.651 ± 0.008
5
30311 Lakušić, D.
serbica
S361 Serbia, Mt. Studena, Krčanik
5
4x
0.643 ± 0.002
4
31552 Kuzmanović, N.
serbica
S373 Bosnia & Herzegovina, Zavidovići,
Gostovićka river (Sv. Pantelej)
5
4x
0.635 ± 0.007
4
32105 Niketić, M., Tomović, G.
serbica
S374 Serbia, Rogozna, Izbice-Negotinac
(mouth of Seoski potok)
5
4x
0.631 ± 0.004
4
32150 Niketić, M., Tomović, G.
serbica
S422 Serbia, Mt. Kopaonik, Treska
5
4x
serbica
S436 Serbia, Mt. Ozren, Kamarišta
5
4x
S362 Greece, Peloponnisos, Taigetos,
Langada canyon
3
4x
28
28
0.666 ± 0.007
5
33674 Kuzmanović, N. & al.
0.669 ± 0.004
5
33875 Niketić, M., Tomović, G.
S. taygetea
taygetea
472
0.758 ± 0.000
Version of Record (identical to print version).
5
31851 Lakušić, D. & al.