bs_bs_banner Botanical Journal of the Linnean Society, 2015, 179, 126–143. With 6 figures Patterns of cytotype distribution and genome size variation in the genus Sesleria Scop. (Poaceae) MAJA LAZAREVIĆ1, NEVENA KUZMANOVIĆ1, DMITAR LAKUŠIĆ1, ANTUN ALEGRO2, PETER SCHÖNSWETTER3* and BOŽO FRAJMAN3 Department of Plant Ecology, Institute of Botany and Botanical Garden, Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, Serbia 2 Department of Botany, Faculty of Science, University of Zagreb, Marulićev trg 20/II, 10000 Zagreb, Croatia 3 Institute of Botany, University of Innsbruck, Sternwartestraße 15, 6020 Innsbruck, Austria Received 26 March 2015; revised 15 May 2015; accepted for publication 29 May 2015 Polyploidization has played an important role in the diversification of the genus Sesleria (Poaceae), which comprises c. 48 species and subspecies mostly distributed in Europe. The genus’ centre of diversity clearly is the Balkan Peninsula, harbouring about 80% of the species, half of which are endemic to this area. We employed chromosome counts, measurements of absolute genome size and determination of relative DNA-content for 460 populations belonging to 43 species of Sesleria. Our main aim was to provide essential baseline data for future molecular genetic reconstructions of the genus’ evolutionary history. Relative genome size allowed for a mostly clear separation of four ploidy levels. The most frequent and widespread cytotypes are tetraploids followed by octoploids, while di- and dodecaploids were only found in a few species. We present first chromosome numbers for the tetraploid species S. doerfleri, S. phleoides, S. skipetarum and S. tuzsonii as well as for diploid S. ovata. Based on relative and partly also on absolute genome size measurements, ploidy level was determined in tetraploid S. rhodopaea and S. voronovii for the first time, and new cytotypes were identified in S. interrupta, S. kalnikensis and S. wettsteinii (tetraploids), S. caerulea, S. klasterskyi, S. latifolia, S. tenerrima, S. ujhelyii and S. vaginalis (octoploids), and S. albanica and S. vaginalis (dodecaploids). While most Sesleria species are ploidy-uniform, several comprise two or even, in the case of S. vaginalis, three ploidy levels. Genome downsizing after polyploidization was confirmed by significant negative correlation between ploidy level and monoploid genome size. Finally, we found a significant increase in monoploid relative genome size towards the margin of the genus’ distribution area, which may be triggered by increased activity of transposable element in populations exposed to environmental or genomic stress. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 126–143. ADDITIONAL KEYWORDS: Balkan Peninsula – chromosome number – polyploidy INTRODUCTION The Balkan Peninsula is one of the centres of biodiversity in Europe (Turrill, 1929; Davis, Heywood & Hamilton, 1994; Kryštufek & Reed, 2004; Thompson, 2005; Hewitt, 2011; Nieto Feliner, 2014) harbouring c. 8000 native plant species and subspecies of which *Corresponding author. E-mail: peter.schoenswetter@uibk.ac.at 126 2600–2700 are endemic (Stevanović, 2005; Lubarda et al., 2014). Explanations for such diversity lie in the area’s topographic, geological and climatic diversity, lack of extensive Pleistocene glaciations, as well as in its geographical position on the crossroads between Asia Minor and Europe (Polunin, 1980; Kryštufek & Reed, 2004; Thompson, 2005; Nieto Feliner, 2014). In spite of its enormous diversity, the Balkan Peninsula has only recently become the focus of studies exploring the evolution of its biota employing molecular and cytogenetic methods (e.g. Frajman & Oxelman, 2007; © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 126–143 Downloaded from https://academic.oup.com/botlinnean/article/179/1/126/2416562 by guest on 08 July 2022 1 GENOME SIZE IN SESLERIA due to the high morpho-anatomical variability and weak differentiation of many species, as well as the high frequency of hybridization (Deyl, 1946). In addition, several discriminating characters are under the strong influence of environmental factors (Lysak & Doležel, 1998). Sesleria ovata A.Kern. and S. sphaerocephala Ard. (including S. leucocephala DC. according to current taxonomic concepts; see Plant material) were initially segregated as separate genera, Psilathera Link. and Sesleriella Deyl, respectively (Deyl, 1946), but later included in Sesleria (Deyl, 1980). The latter concept was followed in several national floras (e.g. Martinčič et al., 2007; Fischer, Oswald & Adler, 2008) and adopted in Euro+Med PlantBase (Valdés & Scholz, 2009). We accordingly included S. ovata and S. sphaerocephala in Sesleria in the present study although this concept has not yet been tested by comprehensive molecular phylogenies. Furthermore, based on morphological characters complemented by anatomy and ecology, the core of Sesleria (i.e. excluding Psilathera and Sesleriella) was traditionally divided into the two sections sect. Argenteae and sect. Sesleria (syn. sect. Calcariae) (Deyl, 1946). Section Argenteae comprises species from low and middle altitudes, with a long and acute upper-most culm leaf, cylindrical inflorescence and narrow lanceolate glumes. Section Sesleria contains species distributed from lowlands to the alpine belt, mostly characterized by short and broad or acuminate upper-most culm leaf, globose to subelliptical inflorescence and ovate to ovate– lanceolate glumes (Deyl, 1946). The first information about chromosome numbers in Sesleria was provided by Avdulov (1928, 1931); the most comprehensive investigations were conducted later by Ujhelyi (1938, 1959, 1960), Ujhelyi & Felföldy (1948) and Strgar (1979, 1981, 1982). Only a few taxa were reported to be diploid with 2n = 2x = 14 (Ujhelyi, 1960; Favarger & Huynh, 1964; Favarger, 1965; Kožuharov & Petrova, 1991; Petrova, 2000), most others being tetra- (2n = 4x = 28) and/or octoploid (2n = 8x = 56), whereas dodecaploids (2n = 12x = 84) are only known from S. calabrica (Deyl) Di Pietro from the southern Apennine Peninsula (Di Pietro, D’Amato & Trombetta, 2005; Trombetta et al., 2005; Di Pietro, 2007). A few hexaploid counts (2n = 6x = 42) probably indicate occasional hybrids between tetraand octoploid individuals (Kožuharov & Petrova, 1991; Petrova, 2000; Trombetta et al., 2005). Heteroploidy, i.e. the presence of two or more ploidy cytotypes (hereafter ‘cytotypes’) within a taxon, is frequent in Sesleria. Some heteroploid taxa were divided into homoploid subspecies, e.g. tetraploid S. heufleriana Schur subsp. heufleriana and octoploid S. heufleriana subsp. hungarica (Ujhelyi) Deyl (Deyl, 1980), but in most cases infraspecific taxonomy does © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 126–143 Downloaded from https://academic.oup.com/botlinnean/article/179/1/126/2416562 by guest on 08 July 2022 Frajman, Eggens & Oxelman, 2009; Frajman & Schneeweiss, 2009; Siljak-Yakovlev et al., 2010; Kuzmanović et al., 2013; Lakušić et al., 2013; Niketić et al., 2013; Pustahija et al., 2013; Coppi et al., 2014; Kutnjak et al., 2014; Surina et al., 2014). Polyploidization (whole genome multiplication) is one of the major evolutionary forces in plant evolution (Otto & Whitton, 2000; Wood et al., 2009; Madlung, 2013), and it has been suggested that almost all flowering plant species are of polyploid origin (Soltis et al., 2009; Jiao et al., 2011). Polyploidy has also played an important role in the evolution and speciation of grasses (Poaceae), with estimations that 20% (Wood et al., 2009) to 44% (DeWet, 1986) of this family’s species are of polyploid origin or include polyploid cytotypes. Different ploidy levels can be found among as well as within species, both sympatrically or allopatrically, triggering questions about evolutionary consequences of this phenomenon (e.g. Lumaret et al., 1987; Marhold et al., 2010; Sonnleitner et al., 2010; Thompson, Husband & Maherali, 2014). It is widely recognized that polyploidization can lead to relatively rapid changes in different aspects of a plant’s life (Levin, 2002), such as cell or body size, growth rate, phenology, physiology, gene expression and symbiotic interactions (e.g. Chen & Ni, 2006; Beaulieu et al., 2007, 2008; Zhang, Hu & Yao, 2010; Tĕšitelová et al., 2013; Weiss-Schneeweiss et al., 2013). Furthermore, polyploidization can lead to shifts in ecological tolerance, thus changing habitat preferences of species (Levin, 2002; Rieseberg & Willis, 2007; Leitch & Leitch, 2008; Parisod, Holderegger & Brochmann, 2010). An additional phenomenon related to polyploidization is genome downsizing (Verma & Rees, 1974; Leitch & Bennett, 2004), which reduces the negative effect of increased genome size (GS) in polyploids by fostering a more economic use of energy, nutrients and space at the cell level (Cavalier-Smith, 2005). Several mechanisms are involved in genome downsizing, such as unequal crossing over, higher rate of deletions than insertions and selection against transposable elements (Bennetzen, Ma & Devos, 2005). Polyploidization has played an important role in the diversification of Sesleria Scop. (Poaceae, Pooideae, Seslerieae), which comprises c. 48 species and subspecies distributed in Europe, with only a few taxa extending to north-western Africa and western Asia (Deyl, 1946, 1980; Valdés & Scholz, 2009). The Alps were suggested to be the primary centre of the genus’ diversification as the presumably most closely related genus Oreochloa Link is found in this region (Deyl, 1946). However, the Balkan Peninsula clearly is the centre of diversity of Sesleria, harbouring about 80% of the species, half of which are endemic (Deyl, 1980; Alegro, 2007). Sesleria is taxonomically complicated 127 128 M. LAZAREVIĆ ET AL. MATERIAL AND METHODS PLANT MATERIAL Plant material for GS and chromosome number estimation was collected in the field from 460 populations belonging to 43 species (Table 1) and 11 infraspecific taxa (Supporting Information, Appendix S1). Nomenclature and the taxonomic concepts used were based on Flora Europaea (Deyl, 1980) and Euro+Med PlantBase (Valdés & Scholz, 2009) with the following exceptions. For the S. juncifolia complex we adopted the concepts proposed by Strgar (1981), Alegro (2007), Di Pietro (2007) and Di Pietro et al. (2013), i.e. recognition of S. apennina Ujhelyi, S. albanica Ujhelyi, S. calabrica, S. kalnikensis Jáv., S. interrupta Vis., S. juncifolia Suffren. and S. ujhelyii Strgar. In the S. rigida complex we followed Kuzmanović et al. (2013, 2015) in recognizing S. achtarovii Deyl, S. filifolia Hoppe and S. rigida Heuff. ex Rchb. We subsumed S. leucocephala under S. sphaerocephala following recent floras covering the distribution areas of both taxa (e.g. Aeschimann et al., 2005). The circumscription of sect. Argenteae and sect. Sesleria follows Deyl (1946). We identified the taxa using the monograph ‘Study of the genus Sesleria’ (Deyl, 1946) and Flora Europaea (Deyl, 1980) as well as several national and regional floras (Pignatti, 1982; Davis, 1985; Aeschimann et al., 2005; Ciocarlan, 2009), with the exception of the following taxa (literature used for determination is given in parentheses): S. albanica and S. skipetarum Ujhelyi (Ujhelyi, 1959), S. ujhelyii and S. interrupta (Strgar, 1981), S. insularis Sommier subsp. barbaricina Arrigoni (Arrigoni, 1983), S. nitida Ten. subsp. sicula Brullo & Giusso (Brullo & Giusso Del Galdo, 2006), S. apennina, S. calabrica and S. juncifolia (Di Pietro, 2007), S. pichiana Foggi & al. (Foggi, Rossi & Pignotti, 2007), S. rhodopaea Tashev & Dimitrov (Tashev & Dimitrov, 2012), as well as S. achtarovii and S. serbica (Adam.) Ujhelyi (Kuzmanović et al., 2013). The sampling spanned almost the entire distribution area of Sesleria from Ireland to Armenia and from Sweden to Crete (Fig. 1). The number of populations sampled per species (1–54) was roughly proportional to the size of the species’ distribution areas. Vouchers were deposited in the herbaria BEOU, IB, KRAM, NHMR and ZA (abbreviations according to Thiers, 2015). Collecting details are given in Appendix S1 and further details can be retrieved from http://www.uibk.ac.at/botany/balkbiodiv/. For relative genome size determination, desiccated, silica geldried leaf material was used (Appendix S1). For absolute genome size measurements and chromosome counts, living plants collected in the field or grown from seeds in the Botanical Garden ‘Jevremovac’ in Belgrade or the Botanical Garden of the University of Innsbruck were used. For 36 populations, relative and absolute genome size as well as chromosome numbers were determined and the correlation between these data sets was evaluated. CHROMOSOME COUNTS Chromosome numbers were determined for five individuals each from 74 populations, covering all ploidy levels. Root tips were pre-treated with 0.002 M © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 126–143 Downloaded from https://academic.oup.com/botlinnean/article/179/1/126/2416562 by guest on 08 July 2022 not reflect ploidy levels, for instance in S. coerulans Friv. (comprising tetra- and octoploids; Ujhelyi, 1959; Kožuharov & Petrova, 1991; Petrova, 2000), S. comosa Velen. (di-, tetra- and octoploids; Ujhelyi, 1959; Andreev, 1979; Kožuharov & Petrova, 1991; Petrova, 2000), the S. juncifolia species complex (tetra-, hexa- and octoploids; Avdulov, 1928; Ujhelyi, 1959, 1960; Strgar, 1981; Di Pietro et al., 2005), S. latifolia (Adamović) Degen (tetra- and hexaploids; Ujhelyi, 1959, 1960; Kožuharov & Petrova, 1991; Petrova, 2000 under S. robusta Schott & al.) and S. robusta (tetra- and octoploids; Ujhelyi, 1959; Strid & Franzén, 1981). Compared with chromosome number estimations, data on DNA-content in Sesleria are scarce; they are available for S. caerulea (L.) Ard., S. heufleriana, S. sadleriana Janka and S. tatrae (Degen) Deyl from Western and Central Europe (Lysak & Doležel, 1998; Lysák et al., 2000; Budzáková et al., 2014), for S. latifolia and S. robusta from the Balkans (Siljak-Yakovlev et al., 2010), as well as for the Balkan–Carpathian S. rigida complex comprising four well-differentiated species (Kuzmanović et al., 2013). Here, we employed chromosome counts, measurements of absolute GS from propidium iodide (PI)-stained nuclei of fresh leaf material, and determination of relative DNA-content from 4′,6diamidino-2-phenylindole (DAPI)-stained nuclei of dried tissue for 460 populations belonging to 43 species of Sesleria sampled throughout most of their distributions. The main aim of our study was to explore variation of chromosome number, ploidy level and GS to identify patterns that may aid in reconstructing the genus’ evolutionary history. Specifically, (1) we explored variation of cytotypes within species and (2) investigated geographical patterns of cytotype distributions as well as possible ecological correlates. Furthermore, (3) we analysed whether GS allows reliable inference of ploidy levels in Sesleria, (4) tested for the occurrence of genome downsizing after polyploidization and (5) analysed cytotype and GS variation among and within the four major infrageneric entities (sect. Argenteae, sect. Sesleria, S. ovata and S. sphaerocephala). GENOME SIZE IN SESLERIA 129 Table 1. Summary results of flow cytometric analyses in Sesleria, using DAPI (relative DNA-content, RGS, given in arbitrary units) and PI (absolute DNA-content, AGS, given in pg) fluorochromes, as well as the determined chromosome number (with the number of examined populations given as a superscript) along with literature data; superscripts following species names refer to references for chromosome numbers not confirmed in our study given in the footnotes mean monoploid RGS 2x 2x 0.41–0.44 (0.43 ± 0.01) 0.48–0.54 (0.51 ± 0.02) 0.21 0.25 4x 0.70–0.73 (0.71 ± 0.02) S. argentea (Savi) Savi 4x S. autumnalis (Scop.) F.W. Schultz N measured for RGS (individuals/ populations) AGS (pg) min–max (mean ± SD) N measured for AGS (ind/pop) Chromosome number determined 45/6 57/7 5.89–6.01 (5.95 ± 0.09) – 2/1 – 141 141 – 14 (Ujhelyi, 1960; Favarger & Huynh, 1964; Favarger, 1965) 0.18 13/3 – – 281 0.65–0.72 (0.70 ± 0.02) 0.17 59/10 8.95–9.00 (8.97 ± 0.03) 3/2 281 4x 0.60–0.76 (0.69 ± 0.03) 0.17 158/31 8.53–8.63 (8.58 ± 0.04) 5/3 284 S. doerfleri Hayek 4x 0.78–0.85 (0.81 ± 0.02) 0.20 17/3 4/2 282 S. italica (Pamp.) Ujhelyi S. latifolia (Adamović) Degen† 4x 0.72–0.74 (0.73 ± 0.01) 0.18 14/3 10.40–10.59 (10.49 ± 0.08) 8.99–9.24 (9.13 ± 0.10) 28 (Kožuharov & Petrova, 1991; Petrova, 2000) 28 (Avdulov, 1931; Ujhelyi, 1960; Guinochet & Logeois, 1962; Löve & Kjellqvist, 1973) 28 (Avdulov, 1931; Ujhelyi, 1959; Di Pietro, 2005) – 5/3 281 28 (Foggi et al., 2007) 28 (Ujhelyi, 1959, 1960; Petrova, 2000) – 28 (Ujhelyi, 1960) 28 (Ujhelyi, 1959; Strid & Franzén, 1981) 56 (Ujhelyi, 1959; Strgar, 1979) – Taxon* S. ovata A.Kern. S. sphaerocephala Ard. (incl. S. leucocephala DC.) Sesleria sect. Argenteae S. alba Sm. S. nitida Ten.‡ S. robusta Schott & al. S. rhodopaea Tashev & Dimitrov S. skipetarum Ujhelyi S. tuzsonii Ujhelyi S. vaginalis Boiss. & Orph. S. voronovii Deyl S. wettsteinii Dörfl. & Hayek§ Sesleria sect. Sesleria S. achtarovii Deyl S. albanica Ujhelyi¶ S. angustifolia (Hack. & Beck) Deyl S. apennina Ujhelyi S. bielzii Schur S. caerulea (L.) Ard. S. calabrica (Deyl) Di Pietro Ploidy level 4x 0.64–0.76 (0.70 ± 0.02) 0.17 109/22 8.69–8.89 (8.78 ± 0.08) 5/3 284 8x 4x 4x 1.37–1.39 (1.38 ± 0.01) 0.65–0.75 (0.71 ± 0.02) 0.54–0.79 (0.70 ± 0.03) 0.17 0.18 0.18 10/2 119/22 190/29 – – 8.91–9.00 (8.94 ± 0.04) – – 4/4 – – 285 8x 1.44–1.49 (1.46 ± 0.02) 0.18 8/1 – – – 4x 0.67–0.68 (0.68 ± 0.01) 0.17 2/1 – – – 4x 4x 4x 0.69–0.73 (0.71 ± 0.01) 0.70–0.72 (0.71 ± 0.01) 0.74–0.75 (0.75 ± 0.01) 0.18 0.18 0.19 28/4 8/1 5/1 9.03–9.28 (9.16 ± 0.13) 8.80–8.88 (8.84 ± 0.06) – 3/2 2/1 – 282 281 – 8x 12x 4x 4x 1.40–1.48 2.01–2.03 0.74–0.75 0.69–0.74 (1.45 ± 0.04) (2.02 ± 0.01) (0.75 ± 0.01) (0.72 ± 0.01) 0.18 0.17 0.19 0.18 10/2 5/1 5/1 52/8 – – – 9.18–9.31 (9.24 ± 0.05) – – – 4/3 – – – 284 4x 0.67–0.70 (0.69 ± 0.01) 0.17 30/6 – – 282 12x 8x 1.91–1.99 (1.96 ± 0.03) 1.21–1.31 (1.26 ± 0.03) 0.16 0.16 15/3 28/6 – 2/1 841 562 8x 8x 4x 1.27–1.40 (1.33 ± 0.04) 1.32–1.37 (1.36 ± 0.02) 0.67–0.75 (0.71 ± 0.02) 1.17 1.17 0.18 35/6 4/2 107/22 – 16.47–16.58 (16.53 ± 0.08) 17.62 – 9.36 1/1 – 1/1 561 561 281 8x 12x 1.28–1.36 (1.32 ± 0.03) 1.70–2.03 (1.91 ± 0.12) 0.16 0.16 15/2 43/8 – – – – – – © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 126–143 Chromosome number from literature – – 28 (Baden, 1983; Gustavsson, 1991) – – – – 28 (Ujhelyi, 1960; Petrova, 2000; Kuzmanović et al., 2013) – 56 (Strgar, 1981) 56 (Ujhelyi, 1960) 56 (Ujhelyi, 1959) 28 (Kattermann, 1930; Ujhelyi & Felföldy, 1948; Bielecki, 1955; Ujhelyi, 1960; Parreaux, 1971; Murín & Májovský, 1978; Lysák, Číhalíková & Doležel, 1997; Lysak & Doležel, 1998; Lövkvist & Hultgård, 1999; Petrova, 2000; Probatova & Seledets, 2008; Budzáková et al., 2014) – 84 (Di Pietro et al., 2005; Trombetta et al., 2005; Di Pietro, 2007) Downloaded from https://academic.oup.com/botlinnean/article/179/1/126/2416562 by guest on 08 July 2022 RGS min–max (mean ± SD) 130 M. LAZAREVIĆ ET AL. Table 1. Continued Ploidy level Taxon* RGS min–max (mean ± SD) mean monoploid RGS N measured for RGS (individuals/ populations) AGS (pg) min–max (mean ± SD) N measured for AGS (ind/pop) Chromosome number determined – – – Chromosome number from literature 4x 0.75–0.77 (0.76 ± 0.01) 0.19 5/1 S. comosa Velen.†† 8x 4x 1.34–1.50 (1.43 ± 0.05) 0.74–0.79 (0.76 ± 0.02) 0.18 0.19 26/5 30/5 – 9.86 – 1/1 561 281 8x 1.43–1.48 (1.45 ± 0.02) 0.18 16/3 2/1 561 4x 0.57–0.70 (0.66 ± 0.02) 0.17 205/27 18.57–19.48 (19.02 ± 0.64) 8.58–8.59 (8.59 ± 0.01) 2/2 288 8x 1.26–1.38 (1.32 ± 0.03) 0.16 89/10 4/2 568 4x 0.70–0.78 (0.76 ± 0.03) 0.19 26/4 16.64–16.91 (16.77 ± 0.14) 9.65–9.73 (9.69 ± 0.04) 3/2 281 8x 1.39–1.42 (1.40 ± 0.02) 0.17 10/2 – – – 4x 0.70–0.74 (0.72 ± 0.01) 0.18 22/5 – – – S. interrupta Vis. 4x 8x 0.61–0.66 (0.63 ± 0.01) 1.12–1.31 (1.25 ± 0.03) 0.16 0.16 162/22 218/32 5/3 10/5 281 562 S. juncifolia Suffren§§ S. kalnikensis Jáv. 8x 4x 8x 1.15–1.33 (1.28 ± 0.04) 0.61–0.70 (0.67 ± 0.04) 1.27–1.32 (1.30 ± 0.02) 0.16 0.17 0.16 189/33 10/2 21/5 8.05–8.25 (8.15 ± 0.08) 15.78–16.54 (16.19 ± 0.29) 16.32 – – 1/1 – – 561 – – S. klasterskyi Deyl¶¶ 8x 0.17 5/1 – – 561 S. korabensis (Kumm. & Jávorka) Deyl S. phleoides Steven ex Roem. & Schult. S. pichiana Foggi & al. S. rigida Heuff. ex Rchb. 8x 1.39 −1.39 (1.39 ± 0.00) 1.29–1.41 (1.34 ± 0.03) 0.17 33/6 – – – 4x 0.84–0.90 (0.87 ± 0.02) 0.22 20/4 – – 281 56 (Strgar, 1979; Strid & Franzén, 1981) – 8x 1.31–1.40 (1.36 ± 0.03) 0.17 25/5 4/2 561 56 (Foggi et al., 2007) 4x 0.64–0.70 (0.67 ± 0.02) 0.17 58/9 17.37–18.12 (17.72 ± 0.36) 8.87–9.21 (9.00 ± 0.18) 3/3 287 S. sadleriana Janka 8x 1.29–1.43 (1.37 ± 0.05) 0.17 30/6 – – – S. serbica (Adam.) Ujhelyi 4x 0.53–0.69 (0.64 ± 0.03) 0.16 160/20 – – 281 S. tatrae (Degen) Deyl 8x 1.30–1.46 (1.40 ± 0.06) 0.17 24/4 – – – S. taygetea Hayek S. tenerrima (Fritsch) Hayek 4x 4x 0.76–0.80 (0.77 ± 0.02) 0.71–0.75 (0.72 ± 0.01) 0.19 0.18 8/2 15/3 – – – – – 282 S. ujhelyii Strgar 8x 4x 1.31–1.41 (1.35 ± 0.03) 0.59–0.65 (0.62 ± 0.01) 0.17 0.16 33/6 66/9 – 8.07–8.18 (8.11 ± 0.04) – 8/4 – 281 S. uliginosa Opiz 8x 4x 1.16–1.20 (1.18 ± 0.03) 0.54–0.74 (0.70 ± 0.04) 0.15 0.17 2/1 69/14 – 9.20 – 1/1 – 281 28 (Strgar, 1979; Petrova, 2000; Kuzmanović et al., 2013) 56 (Ujhelyi & Felföldy, 1948; Lysák et al., 1997; Lysak & Doležel, 1998) 28 (Ujhelyi, 1959; Kuzmanović et al., 2013) 56 (Rychlewski, 1955; Ujhelyi, 1959; Lysak & Doležel, 1998; Budzáková et al., 2014) 28 (Ujhelyi, 1960) 28 (Ujhelyi, 1960; Gustavsson, 1991) – 28 (Strgar, 1981; Strgar, 1982) – 28 (Bielecki, 1955; Lövkvist & Hultgård, 1999; Krahulcová, 2003) S. filifolia Hoppe S. heufleriana Schur S. insularis Sommier ‡‡ 28 (Kožuharov & Petrova, 1991; Petrova, 2000) 56 (Ujhelyi, 1959) 28 (Ujhelyi, 1959; Andreev, 1979) 56 (Andreev, 1979) 28 (Strgar, 1979; Kuzmanović et al., 2013) 56 (Kuzmanović et al., 2013) 28 (Ujhelyi, 1959; Murín & Májovský, 1976; Lysak & Doležel, 1998) 56 (Ujhelyi, 1959; Lysak & Doležel, 1998) 28 (Litardière, 1949; Cardona, 1976; Trombetta et al., 2005; Foggi et al., 2007) – 56 (Strgar, 1981) 56 (Strgar, 1981) – 56 (Ujhelyi, 1960; Strgar, 1981) – *References: †Kožuharov & Petrova (1991), Petrova (2000): 2n = 6x = 42.; ‡Trombetta et al. (2005): 2n = 6x = 42.; §Ujhelyi (1959): 2n = 8x = 56.; ¶Ujhelyi (1959): 2n = 4x = 28.; Kožuharov & Petrova (1991), Petrova (2000): 2n = 2x = 14.; ‡‡Trombetta et al. (2005): 2n = 8x = 56.; §§Avdulov (1931), Ujhelyi (1960): 2n = 4x = 28; Avdulov (1928): 2n = 6x = 42.; ¶¶Ujhelyi (1959): 2n = 4x = 28. †† © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 126–143 Downloaded from https://academic.oup.com/botlinnean/article/179/1/126/2416562 by guest on 08 July 2022 S. coerulans Friv. GENOME SIZE IN SESLERIA 131 Downloaded from https://academic.oup.com/botlinnean/article/179/1/126/2416562 by guest on 08 July 2022 Figure 1. Sampled populations of 43 species of Sesleria. Population identifiers correspond to Appendix S1. A, ploidy levels of all 460 sampled populations; B, S. ovata, S. sphaerocephala and some species from sect. Argenteae; C, additional species from sect. Argenteae; D–F, species from sect. Sesleria. Shading of symbols in B–F indicates ploidy level: light grey, diploids; white, tetraploids; black, octoploids; dark grey, dodecaploids. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 126–143 132 M. LAZAREVIĆ ET AL. 8-hydroxyquinoline for 24 h at 4 °C, fixed in cold Carnoy fixative (ethanol/glacial acetic acid 3:1) for at least 24–48 h, and hydrolysed in 1 M HCl at 60 °C for 14 min. After staining in Schiff’s reagent (Feulgen & Rossenbeck, 1924), root tips were squashed in a drop of acetic carmine. Chromosome plates were observed under a Leica DMLS light microscope (Leica Microsystems) and photographs were taken with a Leica DCF 295 camera (Leica Microsystems). OF RELATIVE (AGS) (RGS) AND ABSOLUTE GENOME SIZE Flow cytometry (FCM) of DAPI-stained nuclei was used for the estimation of relative DNA-content (Suda & Travníček, 2006) from silica gel-dried samples. Pools of 1–3 individuals totalling 1–19 individuals (mean six) from the same population were analysed; analyses were preceded by initial tests proving that a single individual with deviating ploidy could be detected if co-analysed with two individuals of another ploidy. This approach does not affect the reliability of RGS estimates (Suda et al., 2007; Sonnleitner et al., 2010; Trávníček et al., 2011; Kolář et al., 2013). Similar amounts of desiccated sample and fresh internal reference standard were co-chopped with a razor blade in a Petri dish containing 0.5 mL of ice-cold Otto I buffer (0.1 M citric acid, 0.5% Tween 20). Pisum sativum cv. Kleine Rheinländerin (2C = 8.84 pg, Greilhuber & Ebert, 1994) was selected as primary reference standard (Doležel et al., 1998). After filtration through a 42-μm nylon mesh, samples were stained for 10 min at room temperature in a solution containing 1 mL of Otto II buffer (0.4 M Na2HPO4.12H2O), 2-mercaptoethanol and DAPI at a final concentration of 4 μg mL−1. The relative fluorescence intensity of 3000 particles was recorded using a Partec PA II flow cytometer (Partec) equipped with an HBO mercury arc lamp after incubation for 5 min at room temperature. 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. AGS was estimated for one to two individuals each (three in the case of S331) from 54 populations following the procedure described by Kolář et al. (2012). PI (50 μg mL−1) served as DNA-binding fluorochrome. The relative fluorescence intensity of 5000 particles was recorded. Vicia faba cv. Inovec (2C = 26.90 pg; Doležel et al., 1998) was used as internal standard because the peaks produced by Pisum sativum cv. Kleine Rheinländerin partly overlapped with those of tetraploid Sesleria samples. Measurements of the same plant were repeated on three different days to ensure reliability of the STATISTICAL ANALYSES After testing normality of data distribution using Shapiro–Wilk’s W test, Spearman rank order correlations as implemented in Statistica 5.1 (StatSoft, 1996) were calculated to assess the relationship between RGS and AGS measured from the same populations, as well as between ploidy level and monoploid GS. The same analysis was used to test the relationship between average monoploid GS of a population and the geographical distance of that population from an arbitrarily defined coordinate positioned in an area with high frequency of small monoploid GS on the western Balkan Peninsula (43°30′N, 18°00′E; distances determined with ArcGIS 10, ESRI, 2011). Analyses were performed for the whole data set as well as for tetraploids and octoploids separately. Kruskal–Wallis tests followed by Mann–Whitney pairwise comparisons and Bonferroni corrections of P values conducted with PAST (Hammer, Harper & Ryan, 2001) were used to test for differences in monoploid RGS among ploidy levels, and among different bedrock types (assignment based on field observations). The same analysis was applied to test for significant differences in monoploid RGS among S. ovata, S. sphaerocephala, sect. Argenteae and sect. Sesleria, as well as among cytotypes within and across sect. Argenteae and sect. Sesleria. RESULTS CHROMOSOME NUMBERS Chromosome numbers were determined for 74 populations and 32 species (Table 1). Four ploidy levels were recorded. Diploids (2n = 2x = 14) were detected in two populations, tetraploids (2n = 4x = 28) in 52 populations, octoploids (2n = 8x = 56) in 19 populations and dodecaploids (2n = 12x = 84) in a single population (Fig. 2). Chromosome numbers were determined for the first time for S. doerfleri Hayek, S. phleoides Steven ex Roem. & Schult., S. skipetarum and S. tuzsonii Ujhelyi, which are tetraploids with 2n = 28 chromosomes, and S. ovata, which is diploid with 2n = 14 chromosomes. Chromosomes are moderate in size, being largest in diploids (2.59–6.63 μm) and smallest in dodecaploids (1.82–4.19 μm). A variable number of chromosomes bear satellites, from two in, for example, S. filifolia (population S232) to eight in dodecaploids (e.g. S. albanica, S375). They are usually situated on large metacentric chromosomes and smaller acrocentric or subtelocentric chromosomes (Fig. 2). In many © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 126–143 Downloaded from https://academic.oup.com/botlinnean/article/179/1/126/2416562 by guest on 08 July 2022 ESTIMATION results. The three values were averaged; analyses were repeated if the standard deviation exceeded the threshold of 0.2. GENOME SIZE IN SESLERIA 133 individuals chromatic particles, either detached satellites or B-chromosomes, were visible. DNA PLOIDY LEVELS AND GENOME SIZE RGS and AGS were strongly correlated (rs = 0.934, P < 0.001). All individuals analysed for RGS were clearly separated into four ploidy levels: DNA-diploids (3.1%), DNA-tetraploids (63.6%), DNA-octoploids (30.7%) and DNA-dodecaploids (2.6%). We use the prefix ‘DNA-’ to acknowledge that for most populations analysed no chromosome counts are available. For simplicity, the prefix is omitted hereafter. Ploidy levels clearly differed in genome size except for diploid S. sphaerocephala, which overlapped with a few tetraploid accessions (Table 1; Fig. 3). Alongside the five species for which we present the first chromosome counts (see above under Chromosome numbers), we established tetraploidy in S. rhodopea and S. voronovii Deyl for the first time, albeit based on RGS only. New ploidy levels were established for ten taxa: tetraploids in S. interrupta, S. kalnikensis and S. wettsteinii Dörfl. & Hayek, octoploids in S. caerulea, S. klasterskyi Deyl, S. latifolia, S. tenerrima (Fritsch) Hayek and S. ujhelyii, octo- and dodecaploids in S. vaginalis Boiss. & Orph., and dodecaploids in S. albanica (Table 1). No mixed-ploidy populations were detected and 31 out of 43 species exhibited a single cytotype. Two ploidy levels, tetraploids and octoploids, were detected in S. caerulea, S. coerulans, S. comosa, S. filifolia, S. heufleriana, S. kalnikensis, S. latifolia, S. robusta, S. tenerrima, S. interrupta and S. ujhelyii. Only in S. vaginalis were three ploidy levels discovered (tetraploids, octoploids, dodecaploids; Table 1). RGS (expressed as a ratio of its fluorescence to that for the internal standard Pisum sativum) in diploids varied from 0.41 in S. ovata (S068) to 0.54 in S. sphaerocephala (S026; 1.32-fold); AGS was 5.89 pg in S. ovata (S092). In tetraploids RGS ranged from 0.53 in S. serbica (S271) to 0.90 in S. phleoides (S520; 1.70-fold variation), whereas AGS ranged from 8.05 pg in S. interrupta (S282) to 10.59 pg in S. doerfleri (S039; 1.31-fold). RGS in octoploids varied from 1.12 in S. interrupta (S279) to 1.50 in S. coerulans (S213; 1.34-fold), whereas AGS ranged from 15.78 pg in S. interrupta (S290) to 19.48 pg in S. comosa (S003; 1.23-fold). In dodecaploids, minimal RGS (1.70) was measured in S. calabrica (S483) and maximal RGS (2.03) was determined both in S. calabrica (S444) and in S. vaginalis (S411), a 1.19fold variation; AGS was not measured due to lack of fresh leaf material. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 126–143 Downloaded from https://academic.oup.com/botlinnean/article/179/1/126/2416562 by guest on 08 July 2022 Figure 2. Mitotic chromosomes of Sesleria species. A, S. sphaerocephala subsp. leucocephala S025 (2n = 2x = 14); B, S. ovata S092 (2n = 2x = 14); C, S. filifolia S232 (2n = 4x = 28); D, S. robusta S261 (2n = 4x = 28); E, S. pichiana S064 (2n = 8x = 56); F, S. albanica S375 (2n = 12x = 84). Arrows indicate satellites and detached chromosome fragments. Details of the sampling localities are given in Appendix S1. All scale bars are 10 μm. 134 M. LAZAREVIĆ ET AL. Monoploid GS ranged from 0.15 in S. ujhelyii (several populations) to 0.27 in S. sphaerocephala (S026) for RGS and from 1.98 pg in octoploid S. interrupta (S321) to 2.98 pg in diploid S. ovata (S092) for AGS (Table 1, Fig. 3). Ploidy level and monoploid GS were significantly negatively correlated for both RGS (rs = –0.501, P < 0.001) and AGS (rs = −0.432, P < 0.001). In heteroploid species comprising tetraploid and octoploid cytotypes downsizing of the monoploid GS of 0.5–7.6% was observed (Appendix S2). The weakest downsizing (0.5%) was observed in S. filifolia. Slightly higher values were observed in S. interrupta (1.05%) and S. latifolia (1.20%). On the other hand, there is pronounced downsizing (7.6%) between tetraploid S. heufleriana subsp. heufleriana and octoploid S. heufleriana subsp. hungarica. The same trend was seen in S. vaginalis, whose octo- and dodecaploid cytotypes had 3.2 and 9.8% smaller monoploid RGS, respectively, than tetraploids. The only species with increase in RGS with higher ploidy level is S. robusta, where the single octoploid population S217 had a 3.9% higher monoploid RGS than tetraploids. GS VARIATION ACROSS INFRAGENERIC ENTITIES AND CYTOTYPES The exclusively diploid S. ovata and S. sphaerocephala had an average RGS of 0.41 (1.07-fold variation) and 0.51 (1.12-fold variation), respectively. Sect. Argenteae and sect. Sesleria both comprise tetra-, octo- and dodecaploids. In tetraploids of sect. Argenteae the smallest RGS of 0.54 was recorded in population S254 of S. robusta, and the highest (0.85) in population S039 of S. doerfleri, amounting to a 1.57-fold variation; in octoploids the RGS varied from 1.37 in population S398 of S. latifolia to 1.49 in population S217 of S. robusta (1.09-fold variation). Only population S411 of S. vaginalis was dodecaploid with an RGS of 2.02. Larger variation was detected in sect. Sesleria within both tetraploids and octoploids. The minimal RGS for tetraploids within this section and the entire genus was determined in population S271 of S. serbica (0.53). Similarly, the highest RGS among tetraploids of this section and the whole genus was detected in population S520 of S. phleoides (0.90; 1.70-fold variation). Among octoploids, the lowest RGS (1.12) was measured in population S279 of S. interrupta and the highest (1.50) in population S213 of S. coerulans (1.34-fold variation). In dodecaploids from sect. Sesleria both minimal (1.70; population S483) and maximal (2.03; population S444) values were found in S. calabrica from southern Italy (1.19-fold variation). A comparison of monoploid RGS across sect. Argenteae, sect. Sesleria, S. ovata and S. sphaerocephala confirmed clear differences (H = 392.3, P < 0.001), sect. Sesleria having the smallest and S. sphaerocephala having the largest monoploid RGS (Fig. 4A). Monoploid RGS is significantly different between tetraploids and octoploids from section Argenteae (which do not differ from each other) on the one hand and tetraploids and octoploids from section © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 126–143 Downloaded from https://academic.oup.com/botlinnean/article/179/1/126/2416562 by guest on 08 July 2022 Figure 3. Variation of relative genome size and monoploid relative genome size (inset) in Sesleria. Populations for which the chromosome number was established are either in grey (sections Argenteae and Sesleria) or marked with arrows (S. ovata and S. sphaerocephala). GENOME SIZE IN SESLERIA 135 Sesleria (which do significantly differ from each other) on the other hand (H = 347.4, P < 0.001; Fig. 4B). Dodecaploids from sect. Argenteae are significantly different only from octoploids from the same section (P < 0.05) while dodecaploids from sect. Sesleria have a smaller monoploid RGS than most other cytotypes (P < 0.001) except for octoploids from sect. Sesleria and dodecaploids from sect. Argenteae. CYTOTYPE DISTRIBUTIONS ACROSS THE GENUS’ RANGE The four cytotypes encountered in Sesleria have largely overlapping distributions (Fig. 1A). Whereas diploids are confined to the eastern and south-eastern Alps, tetraploids are present throughout the genus’ range. Octoploids are widespread and especially frequent from the Apennine and Balkan Peninsulas to the Western Carpathians. Finally, dodecaploids are restricted to the southern Apennine and Balkan Peninsulas. The predominantly tetraploid sect. Argenteae comprises only a few octoploid populations of S. latifolia, S. robusta and S. vaginalis, as well as a single dodecaploid population of S. vaginalis (S411; Fig. 1B, C). All these populations are located in the southern Balkan Peninsula (Peloponnesus and Scardo-Pindhic mountains in north-western Greece, southern Albania and north-western Macedonia). In sect. Sesleria the distribution of cytotypes is more complex. Whereas tetraploids predominate in the Alps (Fig. 1D), in the Apennine Peninsula octoploids have a wider distribution than tetraploids. In the Balkans tetraploids are more frequent in the central part of the peninsula (Fig. 1D–F). Dodecaploids have been recorded on both sides of the Adriatic Sea in the southern Apennine Peninsula and the mountains of south-western Albania. CORRELATION OF CYTOTYPE AND GS WITH GEOGRAPHY AND BEDROCK TYPE Populations with small monoploid RGS are mostly centred in southern Bosnia and Herzegovina and northern Montenegro, whereas taxa with larger monoploid RGS tend to occupy the periphery of the genus’ range (Fig. 5). Thus, there was a significant correlation between monoploid RGS and the geographical distance from a coordinate arbitrarily positioned in the area with the smallest RGS both for the complete data set (rs = 0.490, P < 0.001) and for tetraploids (rs = 0.492, P < 0.001) and octoploids (rs = 0.606, P < 0.001) separately. Sect. Argenteae and sect. Sesleria occupy three bedrock types – carbonate, silicate and serpentinite. In both sections populations from silicate bedrocks are characterized by a significantly higher monoploid RGS as compared with populations from serpentinite and carbonate bedrocks that have similar values (sect. Argenteae: H = 23.9, P < 0.001; sect. Sesleria: H = 85.1, P < 0.001; Fig. 6). DISCUSSION PLOIDY LEVELS AND CHROMOSOME NUMBERS To provide essential baseline data for future molecular genetic reconstructions of the evolutionary history of Sesleria, an intricate, species-rich grass genus © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 126–143 Downloaded from https://academic.oup.com/botlinnean/article/179/1/126/2416562 by guest on 08 July 2022 Figure 4. Variation of monoploid relative genome size in Sesleria. A, variation across the four major infrageneric entities; B, variation across cytotypes in sect. Argenteae and sect. Sesleria. Means not significantly different at P ≤ 0.01 are indicated by the same letter. Boxes define 25 and 75 percentiles; horizontal lines indicate medians; whiskers span the 5–95 percentiles; circles indicate outliers. 136 M. LAZAREVIĆ ET AL. Figure 6. Variation of monoploid relative genome size in Sesleria sect. Argenteae and sect. Sesleria depending on bedrock type. Means not significantly different at P ≤ 0.01 within a section are indicated by the same letter. Boxes define the 25 and 75 percentiles; horizontal lines indicate medians; whiskers span the 5–95 percentiles; circles indicate outliers. centred in the Balkan Peninsula, we here present chromosome counts from 74 populations and AGS and RGS data from 54 and 460 populations, respectively (Figs 1–3, Table 1) and contrast our data with previously published accounts. RGS allowed for a clear separation of four ploidy levels; the single exception is diploid S. sphaerocephala, which overlaps with a few tetraploid populations from other species (Fig. 3). The most frequent and widespread cytotype is tetraploid followed by octoploids, while di- and dodecaploids were found only in two and three species, respectively (Fig. 1; Table 1), highlighting the importance of polyploidization for the diversification of Sesleria. Wood et al. (2009) considered only 12 of the 27 species of Sesleria as polyploid, which is strongly contradicted by our data and previously published accounts. Tetraploids are geographically most widespread, whereas octoploids can only be found on the Apennine and Balkan Peninsulas and on the southern margin of the Alps as well as in the Carpathians and the Sudetes © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 126–143 Downloaded from https://academic.oup.com/botlinnean/article/179/1/126/2416562 by guest on 08 July 2022 Figure 5. Geographical distribution of monoploid relative genome size in Sesleria depicted as circles of varying size. A, tetraploids; B, octoploids. Diploids and dodecaploids are not shown due to their narrow distribution and thus low number of sampled populations. GENOME SIZE IN SESLERIA While 31 of the 43 investigated Sesleria species are ploidy-uniform, several comprise multiple ploidy levels. For instance, in S. coerulans most accessions from alpine grasslands were octoploid except for a tetraploid population from a peatbog on Mt Vitoša (S721; Fig. 1D, Appendix S1). Both ploidy levels were previously recorded (Table 1). In S. comosa tetraploids were found in the Scardo-Pindhic ranges and octoploids in the Dinaric Mountains; both cytotypes overlap on Mt Gjeravica/Ðeravica (Fig. 1D, Appendix S1). Three ploidy levels (di-, tetra- and octoploids) were previously indicated for this species in Bulgaria (Ujhelyi, 1959; Andreev, 1979; Petrova, 2000; see above for a discussion on diploids). In the widespread S. robusta, only tetraploids were found except for a single octoploid population in north-western Macedonia (S217; Fig. 1C, Appendix S1). Previously, tetraploids were reported from Mt Olympus in Greece (Strid & Franzén, 1981) and octoploids from Albania (Ujhelyi, 1959). Occurrence of hexaploids on the Bulgarian Stara planina range (Kožuharov & Petrova, 1991; Petrova, 2000) is doubtful as the species probably does not occur there (Deyl, 1980) and most probably pertain to S. latifolia. Sesleria tenerrima from the southern Balkan Peninsula was considered tetraploid based on material from Mt Timphi in Greece (Ujhelyi, 1960). We only found tetraploid individuals further north on Mt Nemërçkë on the border between Albania and Greece, whereas all other analysed populations were octoploid (Fig. 1D). In the intricate S. juncifolia species complex, octo- and dodecaploids are present in the Apennine Peninsula, while tetra-, octo- and dodecaploids exist in the Balkans (Fig. 1E, F). Tetraploids were previously reported only for S. albanica (Ujhelyi, 1959) and S. ujhelyii (Strgar, 1981, 1982). However, our results show that tetraploids are also present in S. interrupta and S. kalnikensis (Fig. 1E, F). By contrast, only dodecaploids were found in populations of S. albanica, rendering this species the second dodecaploid alongside the Italian S. calabrica (Di Pietro, 2007). The establishment and restricted distribution of S. calabrica was explained by its adaptation to local edaphic and climatic factors and by geographical isolation of the Pollino-Orsomarso massif (Di Pietro et al., 2005). Based on geographical considerations the two dodecaploid taxa probably originated independently, but molecular data are necessary to test this hypothesis. Sesleria vaginalis is the only species comprising three ploidy levels. Populations from southern Greece were octoploid, whereas on Mt Nemërçkë tetraploids and dodecaploids occur (Fig. 1C). The origin of dodecaploids is difficult to explain, but probably involved several polyploidisation events. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 126–143 Downloaded from https://academic.oup.com/botlinnean/article/179/1/126/2416562 by guest on 08 July 2022 (Fig. 1A). Both peninsulas probably served as Pleistocene refugia for Sesleria, as was shown previously for many other animal and plant species (Kryštufek & Reed, 2004; Thompson, 2005; Hewitt, 2011; Kutnjak et al., 2014; Nieto Feliner, 2014; Surina et al., 2014). We could not confirm the existence of hexaploids (Avdulov, 1928; Kožuharov & Petrova, 1991; Petrova, 2000; Trombetta et al., 2005) in spite of determining genome size of 2774 individuals. Although Ujhelyi (1960) doubted their existence, hexaploids were recently discovered in central Italy (Trombetta et al., 2005; R. Di Pietro, pers. comm.). On the same line, we did not encounter diploids outside the Alps. Diploidy was reported for S. comosa from high altitudes of the Rila and Pirin mountain ranges (Kožuharov & Petrova, 1991; Petrova, 2000; correct determination of material from Pirin confirmed by D. Lakušić and N. Kuzmanović), but this was not supported by our data as population S532 from Rila was clearly tetraploid as is population S533 from nearby Mt Vitoša. Additionally, in spite of extensive sampling in similar habitats in Greece and Macedonia we failed to detect any diploids, lending support to Ujhelyi (1959) and Andreev (1979), who reported only tetraploids and octoploids from Bulgaria. Evidently, further research is needed to reject or corroborate the presence of diploids on the Balkan Peninsula, which strongly bears on the evolutionary history of the entire genus. We here present the first chromosome counts for several taxa (Fig. 2, Table 1): tetraploid are S. doerfleri endemic to Crete, S. phleoides from the Caucasus and Asia Minor, S. skipetarum from Albania and S. tuzsonii from the Apennine Peninsula, while S. ovata from the Alps is diploid. Sesleria rhodopaea and S. voronovii were identified as tetraploid based on GS measurements. Furthermore, new ploidy levels were detected in ten taxa: tetraploids were found in S. interrupta throughout the Balkan Peninsula (Fig. 1F), in S. kalnikensis (populations S115 and S518 from the central part of the species’ range; Fig. 1E) and in S. wettsteinii from Albania, Kosovo and Montenegro (Fig. 1C); octoploids were identified in S. caerulea in populations S006 and S419 from the Julian Alps (Fig. 1D), in S. klasterskyi from Pirin in Bulgaria (S341; Fig. 1D), in S. latifolia in populations S397 and S398 from Greece (Fig. 1B), in S. tenerrima, in which they actually represent the most widespread cytotype (Fig. 1D), and in S. ujhelyii (S330; Fig. 1F). Both octo- (S366, S367) and dodecaploids (S411) were detected in S. vaginalis in Peloponnesus and Albania, respectively (Fig. 1C). Finally, all analysed populations of S. albanica (S375, S376 and S377) are dodecaploid, contrasting with previous reports of tetraploidy (Ujhelyi, 1959). A summary of new and previously published chromosome counts and ploidy levels is presented in Table 1. 137 138 M. LAZAREVIĆ ET AL. GENOME DOWNSIZING AND GEOGRAPHICAL VARIATION IN MONOPLOID GS 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 no. 173030 to D.L.). We thank the collectors Z. Barina, S. Bogdanović, R. Di Pietro, C. Dobeš, R. Flatscher, F. Kolář, M. Niketić, C. Pachschwöll, G. Pflugbeil, V. Rand̄elović, M. Ronikier, G. M. Schneeweiss, L. Schratt-Ehrendorfer, B. Surina, V. Šegota, M. Thulin, G. Tomović, A. Tribsch, S. Vukojičić, A. Tashev, R. P. Wagensommer and B. Zlatković for samples and M. Magauer and D. Pirkebner for conducting the laboratory work. 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. R. Di Pietro shared information about hexaploid S. nitida. A. Petrova provided us with a photograph of the voucher specimen for putatively diploid S. comosa from Pirin and B. Pernfuß with a photograph of S. ovata chromosomes. Valuable comments of © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 126–143 Downloaded from https://academic.oup.com/botlinnean/article/179/1/126/2416562 by guest on 08 July 2022 Genome downsizing after polyploidization in Sesleria was confirmed by the significant negative correlation between ploidy level and monoploid GS, which is a general trend in angiosperms (Kellogg & Bennetzen, 2004; Leitch & Bennett, 2004; Weiss-Schneeweiss, Greilhuber & Schneeweiss, 2006). Octoploid individuals of S. filifolia, which are genetically deeply nested within tetraploids and probably of recent autopolyploid origin (Kuzmanović et al., 2013), have a reduced GS suggesting that downsizing is acting rapidly. The only exception was in S. robusta where the monoploid RGS of the octoploid population was 3.91% higher than that of tetraploids. An increase of monoploid GS in polyploids in comparison with diploid progenitors is less well documented and perhaps less common (e.g. Hordeum, Jakob, Meister & Blattner, 2004; Nicotiana, Leitch et al., 2008; Ramonda serbica, Siljak-Yakovlev et al., 2008; Melampodium, WeissSchneeweiss et al., 2012). We identified a significant correlation between monoploid RGS and geographical distance from an arbitrary coordinate positioned in an area with high frequency of small monoploid RGS on the western Balkan Peninsula. As reported in previously studied Knautia (Caprifoliaceae; Frajman et al., in press), also populations in the centre of Sesleria’s range have smaller, while those on the margins have larger monoploid RGS (Fig. 5). Different, yet unidentified, ecological and geographical factors could have contributed to this pattern, as correlation between GS and abiotic factors was previously found (e.g. Price, Chambers & Bachmann, 1981; Castro-Jimenez et al., 1989; Knight, Molinari & Petrov, 2005; Šmarda et al., 2008; Dušková et al., 2010). For instance, Sesleria taxa growing on silicate bedrock have larger monoploid RGS than those from limestone and serpentinites (Fig. 6; see below). Thus, small RGS in the middle Western Balkans could relate to substrate type as silicate bedrocks are uncommon in that area. Alternatively, proliferation of transposable elements, which make up a large part of grass genomes (e.g. Bennetzen, 2000; Gaut, 2002; Kidwell, 2002; Šmarda et al., 2008), could be triggered in peripheral populations by environmental or genomic stresses, such as inbreeding or hybridization (Wessler, 1996; Levin, 2002), which has been suggested for several Sesleria species (Deyl, 1946; Ujhelyi & Felföldy, 1948; Strgar, 1981; Di Pietro, 2007). Populations on the margins of a genus’ range often have narrower ecological niches (Brussard, 1984) and activation of transposable elements could be a mechanism of adaptation to environmental conditions (Kalendar et al., 2000; Garnatje et al., 2004). However, the absence of a resolved phy- logeny prevents the analysis of the monoploid RGS within a phylogenetic framework and hence conclusions about the direction of changes in GS. Sesleria species inhabiting serpentinite and limestone substrates exhibit smaller monoploid RGS than silicicolous taxa (Fig. 6), corroborating previous analyses of Balkan serpentinophytes (Pustahija et al., 2013). Serpentinite habitats are characterized by a low Ca/Mg ratio, low concentrations of macronutrients and high levels of potentially toxic heavy metals (Brooks, 1987), while the availability of phosphor to plants is impaired in soils derived from calcareous bedrock (Mengel & Kirkby, 1987). In addition, soils on serpentinite and limestone are usually warmer and drier than those on silicate bedrock (Riter-Studnička, 1963; Brooks, 1987). Thus, these results support the hypothesis of advantage of smaller genomes in stressful conditions caused by high temperatures, water stress, presence of heavy metals or impairment of nutrient uptake (Price et al., 1981; Castro-Jimenez et al., 1989; Knight et al., 2005; Šmarda et al., 2008; Vidic et al., 2009; Pustahija et al., 2013). 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Detailed information about collected Sesleria populations, including population identifiers (ID) and voucher numbers (ID Herbarium), origin of the samples with geographical coordinates and altitude, habitat ecology and substrate type, as well as detailed results including determined chromosome numbers and flow cytometric analyses, using DAPI (relative DNA-content, RGS, given in arbitrary units) and PI (absolute DNA-content, AGS, given in pg) fluorochromes (mean ± SD), with calculated values of monoploid genome size based on both RGS and AGS. Appendix S2. Variation of monoploid relative genome size (1Cx) between tetra-, octo- and dodecaploids in Sesleria species including two or three cytotypes. Difference between monoploid genome size of different ploidy levels given as a percentage.