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. (): 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, 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 previ- ously 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;

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 within or between populations of a single species (Ramsey & Schemske, 1998) are often genetically and morphologically The Balkan Peninsula and the adjacent Carpathians are similar to their parents (Levin, 1983, 2002; Parisod & al., 2010). centres of European biodiversity (Turrill, 1929; Davis & al., Recent molecular studies, which often made use of methodo- 1994; Kryštufek & Reed, 2004). In the past ten years several logical advances in flow cytometry such as the availability of phylogeographic studies have explored diversification patterns protocols optimised for the use of dried material (Suda on the Balkan Peninsula (e.g., Park & al., 2006; Liber & al., & Trávníček, 2006a, b), indicate a much higher frequency of 2008; Stefanović & al., 2008; Kučera & al., 2010; Surina & al., autopolyploidy than previously assumed (Husband & Sabara, 2011) and in the Carpathians (e.g., Mráz & al., 2007; Puşcaş 2004; Kron & al., 2007; Kolář & al., 2009; Bardy & al., 2010). & al., 2008; Ronikier & al., 2008). However, only a few (e.g., Despite the widely recognised importance of polyploidisation Frajman & Oxelman, 2007; Csergö & al., 2009) have exam- for plant diversification, very little is known about its contri- ined the biogeographic connections between these two areas, bution to the high diversity on the Balkan Peninsula. previously acknowledged on the basis of chorological patterns The Sesleria Scop. (, Seslerieae) comprises (e.g., Meusel & al., 1965; Ronikier, 2011). ca. 28 mostly European species (Deyl, 1980), with a centre of A major force in plant evolution is polyploidy (e.g., Wendel, diversity on the Balkan Peninsula. It is taxonomically intricate 2000; De Bodt & al., 2005; Soltis & al., 2009; Van de Peer which may relate to the high incidence of polyploidy in the & al., 2009; Jiao & al., 2011), which fosters adaptation to new genus, as the majority of taxa are tetra- and/or octoploid (2n ecological niches and confers reproductive isolation, ultimately = 4x = 28, 2n = 8x = 56, e.g., Strgar, 1979; Lysak & Doležel, leading to speciation (Otto & Whitton, 2000; Comai, 2005; but 1998; Petrova, 2000). One of the polyploid species is S. rigida see Mayrose & al., 2011). As much as 15% of angiosperm spe- Heuff. ex Rchb. sensu Fl. Eur. (Deyl, 1980; for simplicity termed ciation events are associated with an increase in ploidy (Wood “S. rigida s.l.” in the following). So far only tetraploids were & al., 2009). Whereas allopolyploids usually differ conspicu- reported for this taxon (Ujhelyi, 1959, 1960; Deyl, 1980; Petrova, ously from their diploid progenitors in their genomic constitu- 2000), even though Deyl (1946) assumed that octoploids are tion and morphology, autopolyploids that arise from the crosses present in some morphologically deviating populations.

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Sesleria rigida s.l. belongs to S. sect. Calcariae Deyl, and and synthesize these data with AFLPs in order to test if oc- together with S. tenuifolia Schrad. s.l., S. insularis Sommier toploids originated by auto- or allopolyploidy. Third, we search and S. taygetea Hayek constitutes the “turma” (= swarm) Rigida for congruence between the genetic lineages and morpholog- (Deyl 1946), whose centre of origin was suggested to be located ically distinct groups and characterise well-supported groups on the western Balkan Peninsula (Deyl, 1946). Taxa from this with respect to their morphology, chromosome number and species group have narrow, pruinose leaves which are most distribution. Finally, we provide a taxonomic treatment and an often convolute or flat. Their thin spikes are composed of large identification key for all taxa investigated. 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.), Materials and methods less than 13 veins in the tiller leaves and usually hairy leaves (vs. S. insularis) and lack of white bracts subtending the spikes Plant material. — As various taxa were described and later (vs. S. taygetea). It is distributed in Romania, Bulgaria, Greece, considered at different ranks, we assigned the investigated pop- Serbia and Bosnia (Deyl, 1946; Tatić, 1976); a doubtful and ulations to the rankless entities “rigida“, “degenii“, “pancicii“, highly disjunct record from Croatia (Sekulić & al., 1988) could “achtarovii“, “filifolia“ and “serbica“ based on their morphol- not be confirmed in the course of the present study (Nevena ogy and distribution (Fig. 1; Appendix 1). Our study includes Kuzmanović, field obs.). The species thus spans several moun- all described constituents of S. rigida s.l. mentioned in the In- tain ranges of the Balkan Peninsula (Dinaric Alps, Balkan Mts. troduction with the exception of S. haynaldiana, for which no [Stara planina], Rhodope Mts.) as well as the Romanian Car- type material could be located. Based on the protologue (Schur, pathians. It has a wide altitudinal distribution from lowlands 1856), where no locus classicus is specified, the identity of this to the alpine vegetation belt, growing mostly on carbonates, taxon, which was regarded as conspecific with S. rigida s.str. by more rarely on serpentines. Intraspecific morphological and Deyl (1946, 1980) and Valdés & Scholz (2011), remains unclear. ecological variation of S. rigida s.l. is immense and several Molecular and genome size analyses are based on leaf intraspecific taxa have been described (e.g., S. rigida var. de- material of five to ten individuals per population and up to 25 genii Deyl, var. pancicii Deyl, subsp. achtarovii (Deyl) Deyl). populations per taxon (in total 218 individuals from 45 popu- Different authors described new species belonging to this lations). Leaf material for molecular studies was collected in group which were later either synonymised with S. rigida (e.g., the field and desiccated in silica gel. For chromosome counts S. filifolia Hoppe, S. haynaldiana Schur) or considered only as living were collected and grown in the Botanical garden ecotypes, such as S. serbica (Adam.) Ujhelyi from serpentine “Jevremovac“ in Belgrade. For morphometric analysis plant areas in western Serbia and eastern Bosnia and Herzegovina material of ten to fifteen individuals (453 leaves and 341 stems (Deyl, 1980). Taxonomic value, delimitation and distribution with spikes) was collected from 21 populations. These analyses of most of these taxa are not clear and they are mostly consid- were performed on dissected plant organs preserved in 50% ered conspecific with S. rigida (Deyl, 1946, 1980; Tatić, 1976; ethanol (leaves) or in 1 : 1 glycerol : ethanol solution (stems with Diklić & Nikolić, 1986). Deyl (1980) in Flora Europaea rec- spikes). Voucher specimens are deposited at BEOU. Voucher ognised only a single species, S. rigida with two subspecies, numbers and collecting details are given in Appendix 1. subsp. rigida and subsp. achtarovii ; all other names were Chromosome counts. — Chromosome numbers were de- neglected or considered synonyms. In most recently published termined for five individuals per population (20 populations in national Floras Deyl’s (1980) concept with two subspecies total). Root tips obtained from plants in pots were pre-treated is being followed (e.g., Tatić, 1976; Diklić & Nikolić, 1986; with 0.002 M 8-hydroxyquinoline for 24 h at 4°C. After fixation Gustavsson, 1991; Delipavlov & al., 2003; Assyov & Petrova, in cold Carnoy fixative (ethanol/acetic acid 3 : 1) for at least 2006; Ciocarlan, 2009). Some authors (e.g., Sekulić & al., 1988; 24–48 h, hydrolysis was performed in 1N HCl for 14 min at Stevanović & al., 1995, 2003; Valdés & Scholz, 2011), following 60°C. Staining was performed in Schiff’s reagent as described Ujhelyi (1959), recognised S. serbica as an independent species, by Feulgen & Rossenbeck (1924), followed by squashing in a and this was also supported by a recent leaf anatomical study drop of acetic carmine. After freezing at −80°C, cover slips (Kuzmanović & al., 2012). Also S. filifolia is treated at spe- were removed, preparations were dried at least 24 h and then cific rank by some authors (Gergelyi & Beldie, 1972; Assyov mounted in Euparal. Chromosome plates were observed under & Petrova, 2006; Ciocarlan, 2009), and S. haynaldiana as a a Leica DMLS light microscope (Leica Microsystems, Wetzlar, subspecies of S. rigida (Gergelyi & Beldie, 1972). Germany) and photographs were taken with a Leica DCF 295 Taking the intricate taxonomy of S. rigida s.l. into consid- camera (Leica Microsystems). eration, our main goal is to evaluate the taxonomic status of Estimation of relative and absolute genome size. — Flow its constituents using molecular and morphological characters. cytometry (FCM) of 4′,6-diamidino-2-phenylindole (DAPI)- The first aim is to reconstruct the relationships among popu- stained nuclei was used to estimate the relative DNA content of lations based on amplified fragment length polymorphisms five to ten silica gel-dried samples per population following the (AFLPs; Vos & al., 1995) and to test whether the intraspecific procedure described in Suda & Trávníček (2006a). After incu- taxa constitute distinct genetic entities. Second, we screen all bation for 5 min at room temperature, the relative fluorescence genetically investigated populations for their ploidy level using intensity of 3000 particles was recorded using a Partec PA II flow cytometry combined with several chromosome counts flow cytometer (Partec, Münster, Germany) equipped with an

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HBO mercury arc lamp. After initial trials, up to three samples 1 cm²) was processed following the procedure described by were pooled. If the coefficient of variation (CV) of the G0/G1 Kolář & al. (2012). After incubation for 5 min at room tem- peak of a sample exceeded the 5% threshold, the analysis was perature, the relative fluorescence intensity of 5000 particles discarded and the sample re-measured. Pisum sativum ‘Kleine was recorded using a Partec PA II flow cytometer equipped Rheinländerin’ (2C = 8.84 pg, Greilhuber & Ebert, 1994) was with an HBO mercury arc lamp. Vicia faba ‘Inovec’ (2C = selected as primary reference standard (Doležel & al., 1998). 26.90 pg) served as an internal standard as the peaks of Pisum To extrapolate the ploidy level of all individuals, the relative sativum ‘Kleine Rheinländerin’ partly overlapped with those genome size was correlated with chromosome numbers de- of tetraploid Sesleria samples. termined for selected individuals from several populations, AFLP analysis. — Total genomic DNA was extracted from representative for the whole genome size range. ca. 10 mg of dried tissue with the DNeasy 96 plant kit (Qiagen, FCM of propidium iodide (PI)-stained nuclei was used Hilden, Germany) following the manufacturer’s protocol. AFLP to estimate the absolute genome size of two individuals each fingerprinting was performed for 45 populations with usually from six populations (Appendix 1). Fresh green leaf tissue (ca. five individuals per population. The AFLP procedure followed

Fig. 1. Sampled populations of Sesleria rigida s.l. and the dis- tantly 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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Table 1. Characters used for the morphometric analysis of Sesleria rigida s.l. Vos & al. (1995) with the modifica- Wilks’ tions described in Schönswetter & al. Lambda F-remove P (2009). In addition, 0.25 U of polymer- Habitus ase were used in the preselective and Plant height 0.027 0.841 0.472 selective amplifications (0.4 U for the Length of leaf sheath 0.028 4.741 0.003 NED-labelled primer combination). Fifteen selective primer combinations Length of tiller leaf 0.028 3.240 0.022 have been initially screened. The three Number of culm leaves 0.029 8.039 0.000 final selective primer combinations for Distance between uppermost node and spike base 0.028 2.399 0.067 the selective PCR (fluorescent dye in Spike length 0.028 2.680 0.047 brackets) were EcoRI (6-FAM)-ACT / Spike width 0.028 1.971 0.118 MseI-CAC, EcoRI (VIC)-ACG / MseI- Number of spikelets per spike 0.028 1.395 0.244 CAC, and EcoRI (NED)-ACC / MseI- Cross section of tiller leaves CAC. Purification and visualisation of Width of leaf blade 0.029 11.580 0.000 PCR products were done as described Distance between middle rib and point where leaf blade is thickest 0.029 10.404 0.000 in Rebernig & al. (2010). Twenty-eight samples were used as replicates be- Thickness of leaf blades in zone of central rib 0.040 64.834 0.000 tween PCR batches to test the repro- Largest thickness of leaf blades 0.030 13.509 0.000 ducibility of AFLP fingerprinting. Width of central rib 0.030 15.877 0.000 Electropherograms were analysed Length of trichomes on abaxial side 0.027 0.567 0.637 with Peak Scanner version 1.0 (Applied Length of trichomes on adaxial side 0.029 7.182 0.000 Biosystems, Foster City, California, Height of central vascular bundle 0.027 1.000 0.393 U.S.A.) using default peak detection Width of central vascular bundle 0.027 0.137 0.938 parameters except light peak smooth- Height of largest lateral vascular bundle 0.028 2.122 0.097 ing. The minimum fluorescent thresh- Width of largest lateral vascular bundle 0.030 17.124 0.000 old was set to 50 relative fluorescence units. Automated binning and scoring Height of sclerenchyma strand of central vascular bundle 0.032 25.442 0.000 was performed using RawGeno v.2.0 Height of sclerenchyma strand/girder in widest zone 0.028 2.483 0.060 (Arrigo & al., 2009), a package for the Sclerenchyma surface 0.030 12.262 0.000 software R (R Development Core Team, Surface of leaf blades 0.030 13.961 0.000 2010), with the following settings: scor- Number of sclerenchyma strands on adaxial side 0.028 1.503 0.213 ing range = 50–500 bp, minimum in- Number of sclerenchyma strands on abaxial side 0.029 8.780 0.000 tensity = 50 relative fluorescence units Number of sclerenchyma girders on adaxial side 0.030 14.646 0.000 (rfu), minimum bin width = 1 bp, and Number of sclerenchyma girders on abaxial side 0.036 44.271 0.000 maximum bin width = 1.5 bp. Fragments with a reproducibility lower than 80% Number of major vascular bundles 0.028 5.014 0.002 based on sample-replicate comparisons Number of minor vascular bundles 0.030 17.012 0.000 were eliminated. The error rate (Bonin Dimension of bulliform cells 0.028 2.953 0.032 & al., 2004) was calculated as the ra- Spikelets and flowers tio of mismatches (scoring of 0 vs. 1) Length of lower glume 0.028 6.101 0.000 over phenotypic comparisons in AFLP Width of lower glume 0.028 1.566 0.197 profiles of replicated individuals. Frag- Length of upper glume 0.028 4.569 0.004 ments present/absent in only one indi- Width of upper glume 0.028 3.594 0.014 vidual were removed from the dataset. Length of lemma of lower flower 0.028 3.493 0.016 A neighbor-joining (NJ) analysis based on the matrix of Nei-Li genetic Width of lemma of lower flower 0.028 3.610 0.013 distances (Nei & Li, 1979) was con- Length of palea of lower flower 0.027 1.006 0.390 ducted and bootstrapped (1000 pseu- Width of palea of lower flower 0.028 2.413 0.066 do-replicates) with TREECON v.1.3b Length of lemma of upper flower 0.027 0.045 0.987 (Van de Peer & De Wachter, 1997). Width of lemma of upper flower 0.027 1.264 0.286 The tree was rooted with S. taygetea. Length of palea of upper flower 0.027 0.756 0.519 A principal coordinate analysis (PCoA) Width of palea of upper flower 0.028 1.659 0.175 based on a matrix of Jaccard distances Discriminant Function Analysis Summary. Details of the anatomical leaf characters are given among individuals (excluding S. tayge- in Kuzmanović & al. (2009). Wilks’ lambda is a multivariate generalisation of the univariate tea) was calculated using the modules F-distribution; F-remove represents a measure of the extent to which a variable makes a unique “SimQual”, “Dcenter” and “Eigen” contribution to the prediction of a group membership. P-values < 0.05 are printed in bold. from NTSYS-pc v.2.0 (Rohlf, 1997).

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Morphometric analyses. — Analysis of 26 anatomical leaf correlations exceeding this threshold were found. The hypoth- characters was performed on cross-sections of tiller leaves esis of morphological separation of four groups of populations as described by Kuzmanović & al. (2009). An additional 23 recognised by AFLPs was tested using canonical discriminant characters of external morphology were analysed following analysis (CDA) conducted on the resulting data matrix compris- combined and adjusted methods used for L. (Auquier, ing 42 characters after standardisation to zero mean and unit 1974; Lakušić, 1999; Foggi & al., 1999, 2006) and Sesleria variance. Characters included in the CDA are listed in Table 1. (Alegro, 2007; Di Pietro, 2007). The measurements were per- The CDA was performed with 4 a priori defined groups × 453 formed using Leica Qwin (Leica Microsystem) and Digimizer individuals × 42 characters. Discriminant function analysis Image Analysis software (MedCalc Software, Belgium). (Table 1) was done to estimate the contribution of individ- Seven invariable characters were excluded. Further charac- ual characters to overall discrimination. Canonical scores for ter reduction was envisaged by computing pairwise Spearman each case were calculated with the aim to measure distances correlations and retaining only one out of character pairs with between individuals, and a scatterplot of canonical scores (see absolute values of correlation coefficients exceeding 0.9, but no 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

Chromosome numbers and genome size. — Chromosome numbers were determined for 100 individuals from 20 popula- tions (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, re- spectively, and were excluded from further analyses. The initial error rate (according to Bonin & al., 2004) before the exclusion of unreliable characters was 3%. Fig. 2. Mitotic chromosomes of Sesleria rigida s.l. A, population S275 The NJ analysis revealed four clusters with high boot- (2n = 28); B, population S468 (2n = 56). — Scale bar = 20 μm. strap support (BS) between 96% and 100% (Fig. 3). The most

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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“rigida” “pancicii” “filifolia”

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divergent cluster (BS 100%) corresponds to “serbica“ from ser- height of sclerenchyma strand of the central vascular bundle, pentine areas in western Serbia and eastern Bosnia. A further width of largest lateral vascular bundle, number of minor vas- cluster (BS 100%) contains accessions of “achtarovii“ found cular bundles, width of the central rib, number of sclerenchyma in the Rhodopi Mts. (Bulgaria and northeastern Greece). Two girders on the adaxial side, surface of leaf blades and largest well-supported (BS 100) sister clusters include “rigida“ and thickness of leaf blades. The analysis showed that morpholog- “degenii“ from the Romanian Carpathians (BS 100%) as well ical characters with the strongest influence on discrimination as “rigida“, “filifolia“ and “pancicii“ from eastern and north- are number of culm leaves, length of leaf sheath, length of tiller eastern Serbia, western and northern Bulgaria and Munţii Ba- leaf and length of spike within habit characters, and length of natului in Romania (BS 96%). lower glume, length of upper glume, width of lemma of lower The PCoA (excluding the outgroup S. taygetea) resulted flower, width of upper glume and length of lemma of lower in separation of four groups (Fig. 4) matching perfectly those flower within spikelets and flower characters (Table 1). 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 Discussion of “rigida“ from eastern Serbia are not genetically differenti- ated from tetraploids from the same region (Figs. 3–4). Our multifaceted study employing genomic data (AFLPs), Morphometric data. — The CDA largely supported the genome size, chromosome counts and morphometry applied to hypothesis of morphological separation of four genetic groups a broad sample of populations revealed a clear structure within identified by AFLPs (Fig. 5). Discriminant function analy- the taxonomically intricate Sesleria rigida s.l. This complex, sis showed that anatomical characters (cross section of tiller distributed on the Balkan Peninsula and the Carpathians, in- leaves) contribute dominantly to the overall discrimination cludes six taxa, whose taxonomic value has been controversial (Table 1). Furthermore, the contribution of characters of habitus in the past (see Introduction). The AFLP data covering most is slightly higher than the contribution of characters of spikelets taxa throughout their geographic ranges revealed four allopat- and flowers. Most of the discrimination of anatomical parame- rically distributed genetically divergent entities (Figs. 3–4), ters is contributed by thickness of leaf blades in the zone of the which failed to form a monophyletic group in a genus-wide central rib, number of sclerenchyma girders on the abaxial side, AFLP study (N. Kuzmanović & P. Schönswetter, unpub.). The

Fig. 4. Principal coordinate 5 analysis (PCoA) of AFLP data “achtarovii” of Sesleria rigida s.l. First and “degenii” second factors explain 5.7% “filifolia” 1 and 3.2% of the total variation, “pancicii” 4 1 respectively. The taxonomic cod- “rigida” 4x ing of individuals is according “rigida” 8x to Figs. 1 and 3. The inset shows “serbica” the geographic distribution of the 2 four identified groups, which are cross-referenced with the ordina- 3 tion plot with numbers. 2 4

1 4

2 Factor 2

0 -3 .00 1 2 3 3 4 5 Factor 1

-2

-3

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first (cluster 1, Fig. 3; PCoA group 1, Fig. 4) includes popula- Serbia and the last (cluster 4; PCoA group 4) contains popu- tions of S. rigida (“rigida” and “degenii”) from the Romanian lations of S. achtarovii from the Rhodopi Mts. in Bulgaria, Carpathians, the second (cluster 2; PCoA group 2) comprises northeastern Greece and the island Thassos. Similar results populations of S. filifolia (“filifolia” and “pancicii”) from the were obtained by Comanescu (2011), albeit based on a less Munţii Banatului in Romania and the Balkan Mts. in eastern comprehensive sample, and with the main aim of resolving the and northeastern Serbia and western and northern Bulgaria, taxonomic status of S. filifolia in Romania. the third (cluster 3; PCoA group 3) consists of populations of The canonical discriminant analysis of the morphometric S. serbica from serpentine areas in eastern Bosnia and western 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 charac- 4 ters. The four a priori defined groups are the genetic groups identified in Figs. 3 and 4. In 2 (A) the labelling is in accordance with the genetic groups, whereas

in (B) the labelling follows Deyl 2

(1946, 1980). The informal enti- t

o 0

o ties “degenii” and “pancicii” were

R not included as no material was available for morphometric study. -2

-4 Sesleria achtarovii Sesleria filifolia Sesleria rigida A Sesleria serbica -6 -6 -4 -2 0 2 4 6

Root 1

6

4

2

2

t

o 0

o

R

-2

-4 “achtarovii” “filifolia” “rigida” 4x “rigida” 8x B “serbica” -6 -6 -4 -2 0 2 4 6

Root 1

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are also morphologically distinct. Four taxa can thus be rec- important component of temperate heaths (Erico-Seslerietum ognised: S. achtarovii, S. filifolia, S. rigida and S. serbica, community-type; Lakušić & al., 2005). The other three spe- which are presented below in the Taxonomic Treatment. As cies are strictly calcicole and grow in rock crevices (Asple- the populations originally assigned to “degenii” and “pancicii” nietea trichomanis), rocky grasslands (Festuco-Brometea) were neither genetically nor morphologically differentiated, we and subalpine and alpine pastures (Festuco-Seslerietea or synonymise S. rigida var. degenii and S. rigida var. pancicii Daphno-Festucetea) between 100 and 2140 m a.s.l., and also with S. rigida s.str. and S. filifolia, respectively. All four species in beech forests (Seslerio rigidae-Fagetum of the class Quer- in the revised circumscription are allopatrically distributed and co-Fagetea) and Scots pine forests (Seslerio rigidae-Pinetum their distribution ranges are placed within different phytoge- sylvestris of the class Erico-Pinetea) (Lakušić & al., 2005; Kar- ographic regions and provinces (Meusel & Jäger, 1992; Jäger akiev & Tzonev, 2011; Comanescu, 2011). & Welk 2003). Whereas S. rigida belongs to the Carpathian, Previously, only tetraploids were known in S. rigida s.l. the other three species belong to the different provinces of (Ujhelyi, 1959, 1960; Deyl, 1980; Petrova, 2000). Our genome the sub-Mediterranean region. Sesleria filifolia is restricted size measurements and chromosome counts (Appendix 1; to the Moesian (i.e., Balkan), S. serbica to the Illyrian and Figs. 2–3) have revealed some octoploid (2n = 56) populations S. achtarovii to the Macedonian-Thracian province, with one of S. filifolia­ , mostly from southeastern and eastern Serbia, isolated population on the island of Thassos in the Aegean whereas tetraploids are evenly distributed throughout the province of the Macaronesian-Mediterranean region. studied area in all analysed taxa. The octoploid populations of The geographically isolated (Fig. 1) S. achtarovii and S. filifolia are genetically indistinguishable from neighbour- S. serbica are also genetically most distant (Fig. 3), whereas ing tetraploid populations. This is supported by the neighbour S. filifolia and S. rigida with distribution areas separated by joining analysis (Fig. 3) as well as by Bayesian cluster analyses minimally 20 km are also genetically closer and form a highly conducted with STUCTURE v.2.3 (Pritchard & al., 2000) under supported clade. Their distribution areas come into close con- different parameterisations (with and without admixture, allele tact in the Iron Gates region along the Danube in Serbia and frequencies correlated or not) suggesting a single unstructured Romania. This area has traditionally been considered a likely gene pool as most likely solution (Peter Schönswetter, unpub.). migration corridor between the Carpathians and the Dinaric The lack of genetic structure within S. filifolia strongly suggests Mts., and this is supported also by phylogeographic studies a recent autopolyploid origin of the octoploid cytotype. Based (e.g., Frajman & Oxelman, 2007; Ronikier, 2011). Our results on the disjunct distribution of octoploids we hypothesize that support this hypothesis, but it is not clear whether both taxa they have independently originated at least twice. Autopoly- came here into secondary contact after extending their distri- ploidy is much more common in plants then traditionally as- bution from two isolated refugia, or if the Carpathian S. rigida sumed. Initially considered an evolutionary dead-end (Clausen originated from the Balkan S. filifolia in the course of a north- & al., 1945; Stebbins, 1971), it was later shown that genome eastern range expansion. The Danube river valley was obvi- multiplication per se may represent an evolutionary advantage, ously no impassable barrier for the constituents of S. rigida e.g., by enforcing successful range expansion and ecological s.l. as two populations (S317, S372) of S. filifolia were sampled radiation as demonstrated in different natural autopolyploids north of the Danube in Romania. The distribution of S. filifolia, (Manton, 1937; Soltis & al., 2007; Parisod & al., 2010). extending from southeastern and eastern Serbia to the central No cytotype mixtures were found even when expanding Stara planina in Bulgaria but missing from the adjacent Rho- our ploidy level screening to ten individuals per population dopi Mts. (Fig. 1) is seen also in the regional distributions of in contact zones of the two cytotypes (Appendix 1). In the several other plant species such as Aconitum burnatii Gáyer, same line, geographically close octoploid and tetraploid pop- Allium victorialis L., Anthemis carpatica Waldst. & Kit. ex ulations are ecologically and altitudinally differentiated. For Willd., Campanula transsilvanica Schur ex Andrae, Carex instance, although the horizontal distance between tetraploids brevicollis DC., Centaurea napulifera Rochel, Dryas octo- from the Gornjak gorge (population S234) and octoploids from petala L.and Jacobaea abrotanifolia (L.) Moench (Senecio Mt. Vukan (population S466) is less than 2 km, tetraploids abrotanifolius L.) (Assyov & Petrova, 2006). On the other hand, occur exclusively in rock crevices (Asplenietea trichomanes) the Rhodopi Mts. share some taxa with adjacent mountains in in a gorge at ca. 200 m a.s.l., whereas octoploids inhabit rocky Greece (e.g., Haberlea rhodopensis Friv., Petkovia orphanidea grasslands (Festuco-Brometea) on wind-exposed ridges at ca. (Boiss.) Stef., Saxifraga ferdinandi-coburgi Kellerer & Sünd., 700 m a.s.l. Tolerance of plants with higher ploidy level to hab- Saxifraga stribrnyi (Velen.) Podp., Scabiosa rhodopensis Stoj. itats with stronger competition (Hülber & al., 2009) is also re- & Stef., Polygala rhodopea Janch.), a distribution pattern seen flected in their overall size. Already Deyl (1946) hypothesised also in S. achtarovii. that plants from the eastern Serbian Mt. Basara that are larger Ecologically, S. serbica shows the highest specificity. It is in all parts have a higher ploidy level. Herbarium vouchers an obligate serpentinophyte that inhabits submontane and mon- sampled in populations from which cytological information is tane rocky grasslands (Festuco-Brometea) from the thermophil- available confirmed that octoploids are generally more robust ous oak to the cryophilous spruce belt and subalpine pastures than tetraploids. Particularly in leaves of octoploid individuals (Festuco-Seslerietea). At the same time, this species dominates all size characters are larger than in tetraploids (Kuzmanović the understory of specific pine forests (Seslerio-Pinetum nigrae & al., 2009). The absence of any hexaploids, despite screen- and Seslerio-Pinetum sylvestris community-type) and is an ing a large number of individuals, suggests that octoploids are

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reproductively isolated from tetraploids, even when they occur Sesleria achtarovii Deyl in Opera. Bot. Čech. 3: 193. 1946 ≡ in geographic proximity. The observed ecological differen- S. rigida subsp. achtarovii (Deyl) Deyl in Bot. J. Linn. tiation may play a role in the apparent isolation (e.g., Lysak Soc. 76: 364. 1978 – Lectotype (designated here): & Doležel, 1998; Petrova, 2000). [Bulgaria] in saxosis calcareis mt. Rhodope centralis, Similar to other grass genera (e.g., Festuca, Helictotrichon Kuru-dere ad Stanimaka, 23 Mar 1914, I. Mrkvička s.n. Besser, Puccinellia Parl., Stipa L.), our results clearly indicate (SOM no. 100252!). that leaf morphology and especially anatomy play a primary Note. – We examined several specimens of original mate- role in morphological discrimination of the constituents of rial (SOM no. 100252!, SOM no. 4441!, PRC no. PRC519968!, S. rigida s.l. On the basis of leaf shape (convolute vs. flat), W no. 1892-0009528!), and decided to select the sheet from presence of indumentum on the adaxial side of leaves (hairy Kuru-Dere (SOM no. 100252) as lectotype. Of all the speci- vs. glabrous), type of the subepidermal sclerenchyma bands mens cited in the protologue it is the most representative, and (continuous vs. interrupted), number of major and minor vas- has several well developed plants. cular bundles, as well as the number of sclerenchyma girders, Description. – Caespitose perennial. Culm (12.7)16.5– it is relatively straightforward to distinguish the four species. 33.0(42.3) cm tall. Spike (13)15–20(25) × (4)5–7(8) mm, whit- Additionally, characters of overall habit, such as shape of tufts ish, with (9)12–18(28) spikelets; spikelets on short pedicels, (stoloniferous vs. compact) and spikes (lax and interrupted with 2–3 flowers. Tiller leaves (6)11–26(37) cm long, flat or vs. dense and compact), further facilitate their identification. 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 Taxonomic treatment vascular bundles, number of sclerenchyma girders varies from (6)8–12(14) on adaxial side of leaves and (4)6–9(12) on abaxial Sesleria rigida s.l. comprises densely caespitose perennials side of leaves, subepidermal sclerenchyma discontinuous. without reticulate basal leaf sheaths. Leaves are 0.6–2.1 mm Chromosome number. – 2n = 4x = 28 wide, plicate, sometimes pruinose above, and have fewer than Ecology. – Rock crevices (Asplenietea trichomanis) and 13 major vascular bundles. The uppermost stem leaf is 0.2– subalpine and alpine pastures (Daphno-Festucetea) on car- 2.5 cm long. The spike is cylindrical, 3–5 times longer than wide, bonate bedrock (limestone, marble); 450–2000 m a.s.l. bluish or whitish, without prominent white bracts subtending the Distribution. – Rhodope Mts. in southern Bulgaria and inflorescence. Spikelets are laterally compressed and bear 2–3 northeastern Greece. florets. Glumes are unequal and membranous with a single vein. The lemma is sparsely pubescent to glabrous between the veins, Sesleria filifolia Hoppe in Flora 17: 384. 1834 – Lectotype membranous, five-veined, with usually five aristulate teeth at (designated here): [Romania], in rupibus calcareis the apex, the middle tooth being the longest. The palea is as long ad Krassova [Caraşova], Apr 1831, J. Heuffel s.n. (BP no. as or shorter than the lemma and is two-veined. 19625!, only the two right-hand plants). Based on the cytological, genetic and morphometric results = Sesleria rigida var. pancicii Deyl in Opera. Bot. Čech. 3: presented here, we recognise four previously described species 191. 1946 – Lectotype (designated here): Serbia, auf within the studied group, i.e., S. achtarovii, S. filifolia, S. rigida Kalkfelsen Debeli lug, Majdan pek, Apr 1879, J. Pavlović and S. serbica. s.n. (PR no. 161815!). Note on lectotypification of S. filifolia. – On the herbarium Key to the species of Sesleria rigida s.l. sheet four plants from two different collections are mounted. Only the two right-hand plants clearly from Krassova are se- In the morphological descriptions, value ranges correspond lected as the lectotype. to the mean ± standard deviation, with the minimal and maxi- Note on lectotypification of S. rigida var. pancicii. – In mal values in brackets. Morphological descriptions are reduced PR, we found four specimens from Serbia (PR no. 161815!, no. and include only diagnostic morphological characters. 161821!) and Bulgaria (PR no. 161816!, no. 161817!) that were labelled as Sesleria rigida Heuff. var. pancicii by Deyl. We have 1. Tuft stoloniferous, spike elongated and lax, 4–5 times excluded the Bulgarian specimens as potential types, as (1) the longer than wide, growing on serpentine ...... S. serbica plant from Zemen collected by Achtaroff (PR no. 161817!) is not 1. Tuft compact, spike dense, 3 times longer than wide, grow- cited in “Specimina ut sequuntur vidi” (Deyl 1946: 187–188), ing on carbonate ...... 2 which implies that Deyl did not have this material at hand at 2. Leaf transverse section with continuous stratum of sube- the time when he described this variety, and (2) the plant from pidermal sclerenchyma ...... S. rigida Persenka Balkan in Bulgaria collected by Podpèra (PR no. 2. Leaf transverse section without continuous stratum of sub- 161816!) belongs to S. achtarovii (Deyl 1946: 193). Since the epidermal sclerenchyma ...... 3 specimen from Debeli lug collected by Pavlović (PR no. 161815!) 3. Leaves convolute, rarely flat, densely hairy on adaxial is more representative than the specimen from Niška Banja col- side ...... S. filifolia lected by Ilić (PR no. 161821!), we select the former as lectotype. 3. Leaves flat, rarely convolute, sparsely hairy on adaxial Description. – Caespitose perennial. Culm (13.1)24.7– side ...... S. achtarovii 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, Note. – Ujhelyi (1959), when raising S. rigida var. ser- with 2–3 flowers. Tiller leaves (1.3)12.4–25.4(37.5) cm long, bica to the species level, incorrectly cited a specimen from BP convolute, rarely flat, densely hairy on adaxial side, rarely as the “holotype”: “Bosnien: Gostovic-Gebiet auf Serpentin. pruinose above; transverse sections of tiller leaf (0.59)0.76– Leg. W. Ludwig, Mai 1955” (BP no. 0734231!). However, as 1.13(1.67) mm × (0.15)0.19–0.25(0.33) mm, with (3)4–6(7) major he clearly based Sesleria serbica on Adamović’s S. rigida var. vascular bundles and (3)5–8(11) minor vascular bundles, num- serbica, the two names must be taken as homotypic. We ex- ber of sclerenchyma girders varies from (5)6–9(12) on adaxial amined several specimens of original material (e.g., specimens side of leaf and (4)6–8(10) on abaxial side of leaf, subepidermal collected by L. Adamović in the vicinity of Gornji Milanovac sclerenchyma not continuous. in May 1893 [BP no. 593596!, WU no. 0042093!, GZU no. Chromosome number. – 2n = 4x = 28 and 2n = 8x = 56. 259577!] and in April 1896 [W no. 1897-0006747!]), and se- Ecology. – Rock crevices (Asplenietea trichomanis), rocky lected the specimen from PRC as lectotype. The lectotype is grasslands (Festuco-Brometea) and subalpine and alpine pastures quite representative of the species, and it is very well preserved (Festuco-Seslerietea), on carbonate bedrock; 90–1830 m a.s.l. with several plants on the sheet. Distribution. – Munţii Banatului in Romania and Balkan Description. – Stoloniferous perennial. Culm (15.7)23.8– Mts. in eastern Serbia and western and northern Bulgaria. 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 Sesleria rigida Heuff. ex Rchb., Fl. Germ. Excurs.: 1403. 1831 – prominent pedicels, with 2–3 flowers. Tiller leaves (4.5)13.4– Lectotype (designated here): [Romania], in cacumine 33.4(47.2) cm long, convolute, hairy on adaxial and abaxial montis Domuglett (“3000 ped”), ad Thermas Herculis, side; transverse sections of tiller leaves (0.60)0.83–1.09(1.42) Apr [without indication of year], J. Heuffel s.n. (W no. × (0.18)0.21–0.26(0.29) mm, with (3)4–6(7) major vascular 1889-0204591!). bundles and (3)5–7(12) minor vascular bundles, number of = Sesleria rigida var. degenii Deyl in Opera. Bot. Čech. 3: 190. sclerenchyma girders on adaxial side of leaves (6)8–11(14) and 1946 – Lectotype (designated here): [Romania], in on abaxial side of leaves (5)8–11(14), with tendency to form saxosis ad cacuminen montis Domugled supra thermas continuous subepidermal sclerenchyma. Herkulis, 7 May 1894, A. de Degen s.n. (BP no. 19851!). Chromosome number. – 2n = 4x = 28. Note. – The specimen selected as the lectotype of Ses- Ecology. – Rocky grasslands (Festuco-Brometea), forests leria rigida var. degenii was seen by M. Deyl, and collected (Erico-Pinetea) and subalpine pastures (Festuco-Seslerietea), prior to the publication of the protologue. Thus we consider exclusively on serpentine bedrock; 250–1900 m a.s.l. it likely represents a part of the original material. Although Distribution. – Dinaric Mts. of eastern Bosnia and western Deyl’s undated annotation label lists only the name S. rigida, Serbia. 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 Acknowledgements Deyl appears to have only added annotations to specimens at PR after describing new taxa in his monograph. This work was financed by the European Commission in the Description. – Caespitose perennial. Culm (6.9)17.3– SEE-ERA.NET PLUS framework (project “Evolution, biodiversity 35.6(45.8) cm tall. Spike (11)14–23(30) mm × (4)6–8(10) mm, and conservation of indigenous plant species of the Balkan Peninsula” whitish, with (9)13–21(32) spikelets; spikelets on short pedi- to P.S.) and the Ministry of Education and Science of the Republic cels, with 2–3 flowers. Tiller leaves (2.0)9.3–28.2(39.7) cm of Serbia (grant 173030 to D.L.). The Austrian Agency for Interna- long, flat or convolute, pruinose above; transverse sections tional Cooperation in Education & Research (OeAD; financed by the of tiller leaves (0.72)1.03–1.56(2.09) × (0.15)0.20–0.28(0.34) Austrian Federal Ministry of Science and Research) granted a one- mm, with (3)5–8(11) major vascular bundles and (4)5–11(21) month visit at the Institute of Botany of the University of Innsbruck minor vascular bundles, sclerenchyma girders on adaxial side (ICM-2012-02562) to N.K. Support from the Royal Botanic Gardens of leaf (4)8–15(22), on abaxial side (4)8–15(20), with continuous Kew and the European SYNTHESYS programme (Project No. GB- subepidermal sclerenchyma band. TAF-334) granted to P.C. is also acknowledged. We thank M. Magauer, Chromosome number. – 2n = 4x = 28. D. Pirkebner and M. Winkler for conducting the laboratory work in Ecology. – Rock crevices (Asplenietea trichomanis), rocky an excellent way. We are grateful to P.D. Schlorhaufer and his col- grasslands (Festuco-Brometea), pastures (Festuco-Seslerietea) leagues from the Botanical Garden of the University of Innsbruck for and forests (Querco-Fagetea, Erico-Pinetea), on carbonate bed- successfully cultivating our living collection of Sesleria. We gratefully rock; 160–2140 m a.s.l. acknowledge the herbarium curators L. Pignotti (W), W. Till (WU), Distribution. – Carpathians in Romania. Z. Barina (BP), L. Somlyay (BP), O. Šida (PRC), J. Štepanek (PR) and S. Bancheva (SOM) for help with searching for type specimens Sesleria serbica (Adamović) Ujhelyi in Feddes Repert. Spec. of the investigated taxa. Our colleagues S. Vukojičić, M. Niketić, Nov. Regni Veg. 62: 64. 1959 ≡ S. rigida var. serbica Ad- G. Tomović, M. Plećaš (Belgrade), V. Ranđelović, B. Zlatković, I. Fusi- amović in Allg. Bot. Z. Syst. 2: 120. 1896 – Lectotype janović (Niš), D. Uzunov, Ch. Gusev (Sofia) and S. Redžić (Sarajevo) (designated here): [Serbia], in saxosis ad Gornji Milan- helped with field work and collecting of the plant samples. We are ovac, Apr 1893, L. Adamović s.n. (PRC no. PRC 451931!). grateful to B. Foggi (Florence) for help with nomenclatural problems.

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Finally, the two anonymous reviewers, the subject editor P. Comes, the Di Pietro, R. 2007. Taxonomic features of Sesleria calabrica (Poaceae), editor-in-chief J. Kadereit and the nomenclature editors Gerry Moore a neglected species from southern Italy. Folia Geobot. 42: 289–313. http://dx.doi.org/10.1007/BF02806468 and James C. Lendemer provided very helpful suggestions to improve Diklić, N. & Nikolić, V. 1986. Sesleria rigida Heuff. var. serbica previous versions of the manuscript. Adamović. P. 248 in: Sarić, M.R. & Diklić, N. (eds.), Flora SR Srbije, vol. 10. Belgrade: Srpska akademija nauka i umetnosti. Doležel, J., Greilhuber, J., Lucretti, S., Meister, M., Lysák, M.A., Literature cited Nardi, L. & Obermayer, R. 1998. Plant genome size estimation by flow cytometry: Inter-laboratory comparison. Ann. Bot. (Oxford) Alegro, A. 2007. Sistematika i rasprostranjenost kompleksa Sesleria 82(Suppl. A): 17–26. juncifolia na području Dinarida [Systematics and distribution of http://dx.doi.org/10.1006/anbo.1998.0730 Sesleria juncifolia complex in the Dinaric area]. Dissertation, Uni- Feulgen, R. & Rossenbeck, H. 1924. Mikroskopisch-chemischer Nach- versity of Zagreb, Croatia. [in Croatian with English summary] weis einer Nukleinsäure vom Typus der Thymonukleinsäure und Arrigo, N., Tuszynski, J.W., Ehrich, D., Gerdes, T. & Alvarez, N. die darauf beruhende elektive Färbung von Zellkernen in mi­kro­ 2009. Evaluating the impact of scoring parameters on the structure skopischen Präparaten. Hoppe-Seyler’s Z. Physiol. Chem. 135: of intra-specific genetic variation using RawGeno, and R package 203–248. http://dx.doi.org/10.1515/bchm2.1924.135.5-6.203 for automating AFLP scoring. B. M. C. Bioinf. 10: 33. Foggi, B., Rossi, G. & Signorini, M.A. 1999. The Festuca violacea http://dx.doi.org/10.1186/1471-2105-10-33 aggregate (Poaceae) in the Alps and Apennines (Central-Southern Assyov, B. & Petrova, A. (eds.) 2006. Conspectus of the Bulgarian Europe). Canad. J. Bot. 77: 989–1013. vascular flora: Distribution maps and floristic elements, 3rd, rev. Foggi, B., Gherardi, M.E., Signorini, M.A., Rossi, G. & Bruschi, P. and enl. ed. Sofia: Bulgarian Biodiversity Foundation. 2006. Festuca inops De Not. and Festuca gracilior (Hack.) Auquier, P. 1974. Biosystématique, taxonomie et nomenclature du Markgr.-Dannenb. (Poaceae): Are they two different species? Bot. groupe de Festuca ovina L. s.l. (Poaceae) en Belgique et dans J. Linn. Soc. 151: 239–258. quelques régions voisines. Dissertation, University of Liège, Bel- http://dx.doi.org/10.1111/j.1095-8339.2006.00496.x gium. Frajman, B. & Oxelman, B. 2007. Reticulate phylogenetics and phyto- Bardy, E.K., Albach, C.D., Schneeweiss, M.G., Fischer, A.M. & geographical structure of Heliosperma (Sileneae, Caryophyllaceae) Schönswetter, P. 2010. Disentangling phylogeography, polyploid inferred from chloroplast and nuclear DNA sequences. Molec. evolution and taxonomy of a woodland herb (Veronica chamae- Phylogen. Evol. 43: 140–155. drys group, Plantaginaceae s.l.) in southeastern Europe. Molec. http://dx.doi.org/10.1016/j.ympev.2006.11.003 Phylogen. Evol. 57: 771–786. Gergelyi, I. & Beldie, A. 1972. Sesleria. P. 215 in: Savulescu, T. (ed.), http://dx.doi.org/10.1016/j.ympev.2010.06.025 Flora României, vol. 12. Bucharest: Editura Academiei Române. Bonin, A., Bellemain, E., Eidesen, P.B., Pompanon, F., Brochmann, Greilhuber, J. & Ebert, I. 1994. Genome size variation in Pisum sa- C. & Taberlet, P. 2004. How to track and assess genotyping errors tivum. Genome 37: 646–655. http://dx.doi.org/10.1139/g94-092 in population genetic studies. Molec. Ecol. 13: 3261–3273. Gustavsson, L.A. 1991. Sesleria Scop. Pp. 777–783 in: Strid, A. & Kit http://dx.doi.org/10.1111/j.1365-294X.2004.02346.x Tan (eds.), Mountain flora of Greece, vol. 2. Edinburgh: Edinburgh Ciocarlan, V. 2009. Flora ilustrată a României: Pteridophyta et Sper- University Press. matophyta. Bucharest: Edit. Ceres. Hülber, K., Sonnleitner, M., Flatscher, R., Berger, A., Dobrovsky, Clausen, J., Keck, D.D. & Hiesey, W.M. 1945. Plant evolution through R., Niessner, S., Nigl, T., Schneeweiss, G.M., Kubešová, M., amphiploidy and autoploidy, with examples from the Madiinae. Rauchová, J., Suda, J. & Schönswetter, P. 2009. Ecological seg- Washington, D.C.: Carnegie Institution of Washington. regation drives fine scale cytotype distribution of Senecio carnio- Comai, L. 2005. The advantages and disadvantages of being polyploid. licus (Asteraceae) in the Eastern Alps. Preslia 81: 309–319. Nat. Rev. Genet. 6: 836–846. Husband, B.C. & Sabara, H.A. 2004. Reproductive isolation between http://dx.doi.org/10.1038/nrg1711 autotetraploids and their diploid progenitors in fireweed Chamer- Comanescu, P. 2011. Genul Sesleria în Flora României: Taxonomie, ion angustifolium (Onagraceae). New Phytol. 161: 703–713. morfo-anatomie, ecologie, biosistematică [Genus Sesleria in Flora http://dx.doi.org/10.1046/j.1469-8137.2004.00998.x of Romania: Taxonomy, morpho-anatomy, biosystematics]. Dis- Jäger, E.J. & Welk, E. 2003. Pflanzengeographische Gliederung Euro- sertation, University of Bucharest, Romania. [in Romanian] pas. Pp. 79–86 in: Bohn, U., Neuhäusl, R., Gollub, G., Hettwer, Csergö, A.M., Schönswetter, P., Gyöngyvér, M., Deák, T., Boşcaiu, C., Neuhäuslová, Z., Raus, T., Schlüter, H. & Weber, H. (eds.), N. & Höhn, M. 2009. Genetic structure of peripheral, island-like Karte der natürlichen Vegetation Europas = Map of the natural populations: A case study of Saponaria bellidifolia Sm. (Caryo- vegetation of Europe. Maßstab / Scale 1 : 2 500 000. Münster: Land- phyllaceae) from the southeastern Carpathians. Pl. Syst. Evol. 278: wirtschaftsverlag. 33–41. http://dx.doi.org/10.1007/s00606-008-0129-5 Jiao, Y., Wickett, N.J., Ayyampalayam, S., Chanderbali, A.S., Land- Davis, S.D., Heywood, V.H. & Hamilton, A.C. 1994. Centres of plant herr, L., Ralph, P.E., Tomsho, L.P., Hu, Y., Liang, H., Soltis, diversity, vol. 1, Europe, Africa, southwest Asia and the Middle P.S., Soltis, D.E., Clifton, S.W., Schlarbaum, S.E., Schuster, East. Gland: International Union for the Conservation of Nature S.C., Ma, H., Leebens-Mack, J. & dePamphilis, C.W. 2011. and Natural Resources. Ancestral polyploidy in seed plants and angiosperms. Nature 473: De Bodt, S., Maere, S. & Van de Peer, Y. 2005. Genome duplication 97–100. http://dx.doi.org/10.1038/nature09916 and the origin of angiosperms. Trends Ecol. Evol. 20: 591–597. Karakiev, T. & Tzonev, R. 2011. Saxifrago ferdinandi-coburgi-Ses- http://dx.doi.org/10.1016/j.tree.2005.07.008 lerietum actarovii—A new association from the subalpine belt Delipavlov, D., Cheshmedzhiev, I., Popova, M., Tersiyski, D. & of the Slavianka (Orvilos) Mts. (Bulgaria). Hacquetia 10: 49–71. Kovachev, I. 2003. Key to the Bulgarian plants. Plovdiv: Agrar- http://dx.doi.org/10.2478/v10028-011-0004-8 ian University Press. [in Bulgarian] Kolář, F., Štech, M., Trávníček, P., Rauchová, J., Urfus, T., Vít, P., Deyl, M. 1946. Study of the genus Sesleria. Opera Bot. Čech. 3: 1–246. Kubešová, M. & Suda, J. 2009. Towards resolving the Knau- Deyl, M. 1980. Sesleria Scop. Pp. 173–177 in: Tutin, T.G., Heywood, tia arvensis agg. (Dipsacaceae) puzzle: Primary and secondary V.H., Burges, N.A., Moore, D.M., Valentine, D.H., Walters, S.M. & contact zones and ploidy segregation at landscape and microgeo- Webb, D.A. (eds.), Flora Europea, vol. 5, Alismataceae to Orchid­ graphic scales. Ann. Bot. (Oxford) 103: 963–974. aceae. Cambridge: Cambridge University Press. http://dx.doi.org/10.1093/aob/mcp016

Version of Record (identical to print version). 469 Kuzmanović & al. • Differentiation of Sesleria rigida sensu Fl. Eur. TAXON 62 (3) • June 2013: 458–472

Kolář, F., Lučanová, M., Těšitel, J., Loureiro, J. & Suda, J. 2012. Otto, S.P. & Whitton, J. 2000. Polyploid incidence and evolution. Glycerol-treated nuclear suspensions—An efficient preservation Annual Rev. Genet. 34: 401–437. method for flow cytometric analysis of plant samples. Chromosome http://dx.doi.org/10.1146/annurev.genet.34.1.401 Res. 20: 303–315. http://dx.doi.org/10.1007/s10577-012-9277-0 Parisod, C., Holderegger, R. & Brochmann, C. 2010. Evolutionary Kron, P., Suda, J. & Husband, B.C. 2007. Applications of flow cy- consequences of autopolyploidy. New Phytol. 186: 5–17. tometry to evolutionary and population biology. Annual Rev. Ecol. http://dx.doi.org/10.1111/j.1469-8137.2009.03142.x Evol. Syst. 38: 847–876. Park, J.M., Kovačić, S., Liber, Z., Eddie, W.M.M. & Schneeweiss, http://dx.doi.org/10.1146/annurev.ecolsys.38.091206.095504 G.M. 2006. Phylogeny and biogeography of isophyllous species Kryštufek, B. & Reed, J.M. 2004. Pattern and process in Balkan bio- of Campanula (Campanulaceae) in the Mediterranean area. Syst. diversity—An overview. Pp. 1–8 in: Griffiths, H.I., Kryštufek, Bot. 31: 862–880. http://dx.doi.org/10.1600/036364406779695924 B. & Reed, J.M. (eds.), Balkan biodiversity. Dordrecht: Kluwer Petrova, A. 2000. Karyological study of some species of Sesleria Academic Publishers. (Po­aceae) growing in Bulgaria. Bot. Chron. (Patras) 13: 133–140. http://dx.doi.org/10.1007/978-1-4020-2854-0_1 Pritchard, K.J., Stephens, M. & Donnelly, P. 2000. Inference of Kučera, J., Marhold, K. & Lihová, J. 2010. Cardamine maritima population structure using multilocus genotype data. Genetics group (Brassicaceae) in the amphi-Adriatic area: A hotspot of 155: 945–959. species diversity revealed by DNA sequences and morphological Puşcaş, M., Choler, P., Tribsch, A., Gielly, L., Rioux, D., Gaudeul, variation. Taxon 59: 148–164. M. & Taberlet, P. 2008. Post-glacial history of the dominant al- Kuzmanović, N., Šinžar-Sekulić, J. & Lakušić, D. 2009. Leaf anat- pine sedge Carex curvula in the European Alpine System inferred omy of the Sesleria rigida Heuffel ex Reichenb. (Poaceae) in Ser- from nuclear and chloroplast markers. Molec. Ecol. 17: 2417–2429. bia. Bot. Serbica 33: 51–67. http://dx.doi.org/10.1111/j.1365-294X.2008.03751.x Kuzmanović, N., Šinžar-Sekulić, J. & Lakušić, D. 2012. Ecologically R Development Core Team 2010. R: A language and environment determined variation in leaf anatomical traits of Sesleria rigida for statistical computing, version 2.13.1. Vienna: R Foundation Heuffel ex Reichenb. (Poaceae) in Serbia – Multivariate morpho- for Statistical Computing. http://www.r-project.org/foundation/ metric evidence. Folia Geobot. 47: 41–57. Ramsey, J. & Schemske, D.W. 1998. Pathways, mechanisms, and rates http://dx.doi.org/10.1007/s12224-011-9104-y of polyploid formation in flowering plants. Annual Rev. Ecol. Syst. Lakušić, D. 1999. Ekološka i morfološka diferencijacija uskolisnih 29: 467–501. http://dx.doi.org/10.1146/annurev.ecolsys.29.1.467 vijuka (Festuca L. subgen. Festuca) na prostoru Durmitora [Eco- Rebernig, C.A., Schneeweiss, G.M., Bardy, K.E., Schönswetter, P., logical and morphological differentiation of narrow-leaved fescue Villasenor, J.L., Obermayer, R., Stuessy, T.F. & Weiss-Schnee- (Festuca L. subgen. Festuca) in the area of Mt. Durmitor]. Dis- weiss, H. 2010. Multiple Pleistocene refugia and Holocene range sertation, University of Belgrade, Serbia. [in Serbian with English expansion of an abundant southwestern American desert plant summary] species (Melampodium leucanthum, Asteraceae). Molec. Ecol. Lakušić, D., Blaženčić, J., Ranđelović, V., Butorac, B., Vukojičić, 19: 3421–3443. http://dx.doi.org/10.1111/j.1365-294X.2010.04754.x S., Zlatković, B., Jovanović, S. & Šinžar-Sekulić, J. 2005. Fito- Rohlf, F.J. 1997. NTSYS-pc: Numerical taxonomy and multivariate cenoze Srbije—Baza podataka [Phytocoenosis of Serbia—Data analysis system, version 2.0.2. Setauket, New York: Exeter Software. base]. Institute of Botany and Botanical Garden Jevremovac, Fac- Ronikier, M. 2011. Biogeography of high-mountain plants in the Car- ulty of Biology, University of Belgrade. http://habitat.bio.bg.ac.rs/ pathians: An emerging phylogeographical perspective. Taxon 60: stanista_srbije.htm 373–389. Levin, D.A. 1983. Polyploidy and novelty in flowering plants. Amer. Ronikier, M., Cieślak, E. & Korbecka, G. 2008. High genetic dif- Naturalist 122: 1–25. http://dx.doi.org/10.1086/284115 ferentiation in the alpine plant Campanula alpina Jacq. (Cam- Levin, D.A. 2002. The role of chromosomal change in plant evolution. panulaceae): Evidence for glacial survival in several Carpathian Oxford: Oxford University Press. regions and long-term isolation between the Carpathians and the Liber, Z., Kovačić, S., Nikolić, T., Likić, S. & Rusak, G. 2008. Rela- Alps. Molec. Ecol. 17: 1763–1775. tions between western Balkan endemic Campanula L. (Campanul­ http://dx.doi.org/10.1111/j.1365-294X.2008.03664.x aceae) lineages: Evidence from chloroplast DNA. Pl. Biosyst. 142: Schönswetter, P., Solstad, H., García, P.E. & Elven, R. 2009. A com- 40–50. http://dx.doi.org/10.1080/11263500701872283 bined molecular and morphological approach to the taxonomically Lysak, M.A. & Doležel, J. 1998. Estimation of nuclear DNA content intricate European mountain plant Papaver alpinum s.l. (Papav- in Sesleria (Poaceae). Caryologia 51: 123–132. eraceae)—taxa or informal phylogeographical groups? Taxon 58: http://dx.doi.org/10.1080/00087114.1998.10589127 1326–1343. Manton, I. 1937. The problem of Biscutella laevigata L. II. The evi- Schur, P.J.F. 1856. Ueber siebenbürgische Seslerien. Verh. K.K. Zool.- dence from meiosis. Ann. Bot. (Oxford) 51: 439–465. Bot. Ges. Wien 6: 191–214. Mayrose, I., Zhan, S.H., Rothfels, C.J., Magnuson-Ford, K., Barker, Sekulić, B., Lovrić, A.Ž. & Rac, M. 1988. Značajna flora i fitocenoze M.S., Rieseberg, L.H. & Otto, S.P. 2011. Recently formed poly- ultramafitnih stijena Banije i odnos prema ostalim balkanskim ploid plants diversify at lower rates. Science 333: 1257. ofiolitima [Important flora and phytocoenosis of ultramaphitic http://dx.doi.org/10.1126/science.1207205 rocks of Banija and their relationship to other Balkan ophiolits]. Meusel, H. & Jäger, E.J. 1992. Vergleichende Chorologie der zentra- Pp. 349–356 in: Slišković, T. (ed.), Zbornik referata naučnog skupa leuropäischen Flora, vol. 3. Jena: Fischer. Minerali, stijene, izumrli i živi svijet Bosne i Hercegovine [Pro- Meusel, H., Jäger, E. & Weinert, E. 1965. Vergleichende Chorologie ceedings of the Symposium Minerals, rocks, extinct and living der zentraleuropäischen Flora, vol. 1. Jena: Fischer. world of Bosnia and Herzegovina]. Sarajevo: Zemaljski muzej Mráz, P., Gaudeul, M., Rioux, D., Gielly, L., Choler, P., Taberlet, Bosne i Hercegovine. [in Croatian] P. & Intrabiodiv Consortium 2007. Genetic structure of Hypo- Soltis, D.E., Soltis, P.S., Schemske, D.W, Hancock, J.F., Thomp- chaeris uniflora (Asteraceae) suggests vicariance in the Carpath- son, J.N., Husband, B.C. & Judd, W.S. 2007. Autopolyploidy ians and rapid post-glacial colonization of the Alps from an eastern in angiosperms: Have we grossly underestimated the number of Alpine refugium. J. Biogeogr. 34: 2100–2114. species? Taxon 56: 13–30. http://dx.doi.org/10.1111/j.1365-2699.2007.01765.x Soltis, D.E., Albert, V.A., Leebens-Mack, J., Bell, C.D., Paterson, Nei, M. & Li, W.H. 1979. Mathematical model for studying genetic vari- A.H., Zheng, C., Sankoff, D., dePamphilis, C.W., Wall, P.K. ation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. & Soltis, P.S. 2009. Polyploidy and angiosperm diversification. U.S.A. 76: 5269–5273. http://dx.doi.org/10.1073/pnas.76.10.5269 Amer. J. Bot. 96: 336–348. http://dx.doi.org/10.3732/ajb.0800079

470 Version of Record (identical to print version). TAXON 62 (3) • June 2013: 458–472 Kuzmanović & al. • Differentiation of Sesleria rigida sensu Fl. Eur.

StatSoft 1996. STATISTICA (data analysis software system), version serpyllifolius and E. tenuifolius, Campanulaceae) within the Balkan 5.1. Tulsa: StatSoft Inc. www.statsoft.com refugium. J. Biogeogr. 38: 1381–1393. Stebbins, G.L. 1971. Chromosomal evolution in higher plants. London: http://dx.doi.org/10.1111/j.1365-2699.2011.02493.x Edward Arnold. Tatić, B. 1976. Sesleria Scop. Pp. 325–330 in: Josifović, M. (ed.), Flora Stefanović, S., Lakušić, D., Kuzmina, M., Međedović, S., Kit Tan SR Srbije, vol. 8. Belgrade: Srpska akademija nauka i umetnosti. & Stevanović, V. 2008. Molecular phylogeny of Edraianthus Turrill, W.B. 1929. The plant-life of the Balkan peninsula: A phytogeo- (Grassy Bells; Campanulaceae) based on non-coding plastid DNA graphical study. Oxford: Clarendon Press. sequences. Taxon 57: 452–475. Ujhelyi, J. 1959. Species Sesleriae generis novae. Feddes Repert. 62: Stevanović, V., Jovanović, S., Lakušić, D. & Niketić, M. 1995. 59–70. http://dx.doi.org/10.1002/fedr.19590620108 Diverzitet vaskularne flore Jugoslavije sa pregledom vrsta od Ujhelyi, J. 1960. Weitere zytotaxonomische Beiträge zur Kenntnis der međunarodnog značaja [Diversity of vascular plants of Yugosla- Gattung Sesleria. Bot. Közlem. 48: 278–280. via, with review of internationally significant species]. Pp. 183–217 Valdés, B. & Scholz, H. Last updated January 2011. Sesleria serbica in: Stevanović, V. & Vasić, V. (eds.), Biodiverzitet Jugoslavije sa (Adamović) Ujhelyi. Euro+Med Plantbase – the information re- pregledom vrsta od međunarodnog značaja [Biodiversity of Yugo- source for Euro-Mediterranean plant diversity. http://ww2.bgbm slavia with an overview of species of global importance]. Belgrade: .org/EuroPlusMed/PTaxonDetail.asp?NameId=142052&PTRe Ecolibri, Biološki fakultet. [in Serbian] fFk=7100000 (accessed 4. Nov. 2012). Stevanović, V., Kit, T. & Iatrou, G. 2003. Distribution of the endemic Van de Peer, Y. & De Wachter, R. 1997. Construction of evolution- Balkan flora on serpentine I. – Obligate serpentine endemics. Pl. ary distance trees with TREECON for Windows: Accounting for Syst. Evol. 242: 149–170. variation in nucleotide substitution rate among sites. Computer http://dx.doi.org/10.1007/s00606-003-0044-8 Applic. Biosci. 13: 227–230. Strgar, V. 1979. Kromosomska števila treh balkanskih seslerij. Biol. Van de Peer, Y., Fawcett, J.A., Proost, S., Sterck, L. & Vandepoele, Vestn. 27: 71–74. [in Slovenian with German summary] K. 2009. The flowering world: A tale of duplications. Trends Pl. Suda, J. & Travniček, P. 2006a. Estimation of relative nuclear DNA Sci. 14: 680–688. http://dx.doi.org/10.1016/j.tplants.2009.09.001 content in dehydrated plant tissues by flow cytometry. Unit 7.30, Vos, P., Hogers, R., Bleeker, M., Reijans, M., Van de Lee, T., Hornes, 7.30.1–7.30.14 in: Robinson, J.P., Darzynkiewicz, Z., Dobrucki, J., M., Frijters, A., Pot, J., Peleman, J., Kuiper, M. & Zabeau, M. Hyun, W., Nolan, J., Orfao, A. & Rabinovitch, P. (eds.), Current 1995. AFLP: A new technique for DNA fingerprinting. Nucl. Acids protocols in cytometry. New York: Wiley. Res. 23: 4407–4414. http://dx.doi.org/10.1093/nar/23.21.4407 Suda, J. & Travniček, P. 2006b. Reliable DNA ploidy determination in Wendel, J.F. 2000. Genome evolution in polyploids. Pl. Molec. Biol. dehydrated tissues of vascular plants by DAPI flow cytometry–new 42: 225–249. http://dx.doi.org/10.1023/A:1006392424384 prospects for plant research. Cytometry 69A: 273–280. Wood, T.E., Takebayashi, N., Barker, M.S., Mayrose, I., Greenspoon, http://dx.doi.org/10.1002/cyto.a.20253 P.B. & Rieseberg, L.H. 2009. The frequency of polyploid specia- Surina, B., Schönswetter, P. & Schneeweiss, G.M. 2011. Quaternary tion in vascular plants. Proc. Natl. Acad. Sci. U.S.A. 106: 13875– range dynamics of ecologically divergent species (Edraianthus 13879. http://dx.doi.org/10.1073/pnas.0811575106

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. Ploidy Relative Absolute Taxon ID Locality NGS level 2n GS ± SD GS NAFLP M No. Collector(s) S. achtarovii achtarovii S333 Bulgaria, Asenovgrad, Kuru Dere 5 4x 0.700 ± 0.005 5 x 31961 Vukojičić, S. & al. achtarovii S335 Bulgaria, Trigrad, Trigradsko ždrelo, 5 4x 28 0.678 ± 0.005 5 x 31973 Vukojičić, S. & al. near waterfall achtarovii S350 Greece, Makedonia, Pangaeon 5 4x 0.701 ± 0.003 3 x 31559 Lakušić, D., Lakušić, B. achtarovii S351 Greece, Aegian islands, Tassos, 5 4x 0.699 ± 0.004 4 x 31558 Lakušić, D., Lakušić, B. Ipsarion S. filifolia filifolia S317 Romania, Dubova, Mali Kazan 5 4x 28 0.667 ± 0.015 3 x 31515 Kuzmanović, N., Comanescu, P. filifolia S372 Romania, Carasova 5 4x 0.646 ± 0.007 5 32107 Kuzmanović, N., Plećaš, M. pancicii S245 Bulgaria, Zemen, Zemenski prolom 10 4x 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 gorge 6 4x 28 0.624 ± 0.007 5 21166 Lakušić, D., Lakušić, B. rigida S228 Serbia, Malinik, canyon of Lazareva 10 4x 28 0.659 ± 0.018 5 x 27098 Lakušić, D., Kuzmanović, N. river rigida S230 Serbia, Niš, Jelašnica gorge 10 4x 28 0.649 ± 0.017 5 x 27444 Kuzmanović, N. rigida S234 Serbia, Gornjak gorge 10 4x 28 0.692 ± 0.001 5 30320 Lakušić, D. rigida S236 Serbia, Kučevo, Pek gorge 10 4x 0.670 ± 0.012 8.584 5 x 30322 Lakušić, D.

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Appendix 1. Continued. Ploidy Relative Absolute Taxon ID Locality NGS level 2n GS ± SD GS NAFLP M No. Collector(s) S. filifolia (continued) rigida S237 Serbia, Paraćin, Izvor, gorge of 10 4x 0.660 ± 0.015 5 30323 Vukojičić, S. river Grza rigida S238 Serbia, Pirot, Rsovci 10 8x 56 1.312 ± 0.008 5 x 30324 Zlatković, B. rigida S239 Serbia, Bor, Stol 5 4x 28 0.666 ± 0.003 5 30325 Fusijanović, I. rigida S240 Serbia, Bor, Rgotski kamen 10 4x 0.681 ± 0.009 5 30326 Fusijanović, I. rigida S241 Serbia, Sićevo gorge, monastery 10 8x 1.305 ± 0.015 5 30327 Ranđelović, V. Sv. Petka rigida S246 Bulgaria, Mt. Konjevska, Viden 10 4x 0.664 ± 0.007 5 x 30495 Lakušić, D. & al. rigida S247 Serbia, Vidlič, Visoka stena 10 8x 56 1.325 ± 0.029 5 30497 Lakušić, D. & al. rigida S250 Serbia, Mt. , Trem 10 8x 56 1.292 ± 0.004 5 x 30546 Kuzmanović, N. & al. 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, 5 4x 0.664 ± 0.006 5 32002 Vukojičić, S., Kuzmanović, N., Jušković, M. rigida S402 Bulgaria, Veliko Trnovo, Jantra river 5 4x 0.661 ± 0.023 5 33234 Niketić, M., Tomović, G. gorge (Kandiljka hill) rigida S403 Bulgaria, Mts. Central Stara Planina, 5 4x 0.661 ± 0.009 5 33275 Niketić, M., Tomović, G. Šipčenska Planina (Ispolin peak) 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 10 8x 56 1.345 ± 0.017 16.895 5 34169 Lakušić, D. & al. kamen S. rigida degenii S353 Romania, Domogled 5 4x 28 0.682 ± 0.001 5 x 31538 Lakušić, D. & al. rigida S242 Romania, Deva, Craciunesti 10 4x 28 0.669 ± 0.013 8.872 5 x 30329 Kuzmanovic, N., Comanescu, P. rigida S243 Romania, Baia Mare, Remeti 10 4x 28 0.668 ± 0.011 5 x 30333 Kuzmanovic, N., Comanescu, P. rigida S244 Romania, Braşov, Timpa 10 4x 28 0.690 ± 0.010 9.208 4 x 30337 Kuzmanovic, N., Comanescu, P. rigida S319 Romania, Turda, Cheile Turzii 5 4x 28 0.642 ± 0.010 8.917 3 x 31517 Kuzmanovic, N., Comanescu, P. rigida S352 Romania, Domogled, Prolaz 5 4x 0.664 ± 0.009 5 31537 Lakušić, D. & al. S. serbica serbica S269 Serbia, , Zlatiborsko jezero 10 4x 0.643 ± 0.003 5 28741 Kuzmanović, N. serbica S272 Serbia, Divčibare, Ljuti krš 10 4x 0.650 ± 0.007 4 x 30308 Kuzmanović, N. serbica S273 Serbia, , Šargan 10 4x 0.637 ± 0.010 4 x 30309 Lakušić, D. serbica S274 Serbia, Ibar valley, Maglić old town 10 4x 0.645 ± 0.011 5 30310 Lakušić, D. serbica S275 Serbia, Mali Vujan, Banjska vrata 10 4x 28 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, 5 4x 0.635 ± 0.007 4 32105 Niketić, M., Tomović, G. Gostovićka river (Sv. Pantelej) serbica S374 Serbia, Rogozna, Izbice-Negotinac 5 4x 0.631 ± 0.004 4 32150 Niketić, M., Tomović, G. (mouth of Seoski potok) serbica S422 Serbia, Mt. , Treska 5 4x 28 0.666 ± 0.007 5 33674 Kuzmanović, N. & al. serbica S436 Serbia, Mt. Ozren, Kamarišta 5 4x 0.669 ± 0.004 5 33875 Niketić, M., Tomović, G. S. taygetea taygetea S362 Greece, Peloponnisos, Taigetos, 3 4x 0.758 ± 0.000 5 31851 Lakušić, D. & al. Langada canyon

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