bs_bs_banner Botanical Journal of the Linnean Society, 2014, ••, ••–••. With 1 figure Ploidy in the alpine sedge Kobresia pygmaea (Cyperaceae) and related species: combined application of chromosome counts, new microsatellite markers and flow cytometry ELKE SEEBER1*, GRIT WINTERFELD2, ISABELL HENSEN2,3, TIMOTHY F. SHARBEL4, WALTER DURKA5, JIANQUAN LIU6, YONG-PING YANG7 and KARSTEN WESCHE1,3 1Botany Department, Senckenberg Museum of Natural History Goerlitz, PO Box 300 154, D-02806 Goerlitz, Germany 2Department of Geobotany and Botanical Garden, Martin-Luther-University Halle-Wittenberg, Am Kirchtor 1, D-06108 Halle (Saale), Germany 3German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Deutscher Platz 5e, D-04103 Leipzig, Germany 4Apomixis Research Group, Leibniz Institute of Plant Genetics and Crop Plant Research–IPK, Corrensstraße 3, D-06466 Stadt Seeland, OT Gatersleben, Germany 5Department of Community Ecology, Helmholtz Centre for Environmental Research–UFZ, Theodor-Lieser-Straße 4, D-06120 Halle (Saale), Germany 6State Key Laboratory of Grassland Agro-ecosystem, School of Life Sciences, Lanzhou University, Lanzhou, Gansu 730000, China 7Kunming Institute of Botany, Chinese Academy of Sciences, Heilongtan 650204, Kunming, China Received 26 October 2013; revised 12 May 2014; accepted for publication 29 May 2014 Polyploidy is a fundamental mechanism in evolution, but is hard to detect in taxa with agmatoploidy or aneuploidy. We tested whether a combination of chromosome counting, microsatellite analyses and flow cytometric measure- ments represents a suitable approach for the detection of basic chromosome numbers and ploidy in Kobresia (Cyperaceae). Chromosome counting resulted in 2n = 64 for Kobresia pygmaea and K. cercostachys,2n = 58 and 64 for K. myosuroides, and 2n = 72 for K. simpliciuscula. We characterized eight microsatellite loci for K. pygmaea, which gave a maximum of four alleles per individual. Cross-species amplification was tested in 26 congeneric species and, on average, six of eight loci amplified successfully. Using flow cytometry, we confirmed tetraploidy in K. pygmaea. Basic chromosome numbers and ploidy were inferred from chromosome counts and the maximum number of alleles per locus. We consider the basic numbers as x = 16 and 18, with irregularities derived from agmatoploidy and aneuploidy. Across all Kobresia taxa, ploidy ranged from diploid up to heptaploid. The combination of chromosome counts and microsatellite analyses is an ideal method for the determination of basic chromosome numbers and for inferring ploidy, and flow cytometry is a suitable tool for the identification of deviating cytotypes. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, ••, ••–••. ADDITIONAL KEYWORDS: 454 sequencing – basic chromosome number – cross-amplification – Kobresia pygmaea ecosystem – next-generation sequencing – palaeopolyploidy – Tibetan Plateau. *Corresponding author. E-mail: [email protected] © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, ••, ••–•• 1 2 E. SEEBER ET AL. INTRODUCTION dominant mechanism across Cyperaceae as a conse- quence of the peculiar pollen formation in the family Polyploidy is a major force in plant evolution and (Heilborn, 1939). One exception based on chromosome occurs frequently across a number of taxa (e.g. Soltis & counts is a possible allopolyploid origin for K. nepa- Soltis, 1999; Soltis, Soltis & Tate, 2004). Wood et al. lensis from ancestors such as K. myosuroides (Vill.) (2009) estimated that 35% of extant vascular plant Fiori and K. esenbeckii (Kunth) Noltie (Yano et al., genera are polyploid, and that 15% of speciation events 2011). in angiosperms were facilitated by a duplication of the Three methods are frequently used to determine chromosome set. The multiplication of entire genomes ploidy: chromosome counting, microsatellite analyses is usually regarded as being associated with higher and flow cytometric measurements. The traditional tolerance to environmental stress (e.g. Grant, 1981; approach, using direct analysis of metaphase chromo- Goldman et al., 2004), because polyploid taxa are somes, provides the actual chromosome number. known to be relatively more abundant than diploids in However, in Cyperaceae, interpretations of counts are arctic and alpine environments (Stebbins, 1985; difficult because of agmatoploidy and aneuploidy. A Hijmans et al., 2007). Polyploidization therefore plays fundamental constraint is the availability of growing a pivotal role in the diversification of plants, especially tissue, especially in rare species or those growing in mountain habitats (Burnier et al., 2009). under conditions that are not easily reproduced. Several genera of the family Cyperaceae are con- Second, nuclear microsatellite markers (simple sidered to be polyploids (Roalson, 2008), although sequence repeats, SSRs) can successfully be used in ploidy is hard to verify in this group because species ecological studies in diploid and polyploid species have variable chromosome numbers (Hipp, Rothrock (Pfeiffer et al., 2011; Ritz & Wissemann, 2011; Avolio & Roalson, 2009b, and references therein) as a result et al., 2012). Their co-dominant expression enables of agmatoploidy (the fission and fusion of chromo- the detection of heterozygotes and allows for the somes, Davies, 1956; Luceño & Castroviejo, 1991) and linking of the maximum number of alleles found in aneuploidy (the decrease or increase by single chro- one individual to ploidy (Besnard et al., 2008; Stark mosomes, Wolfe, 2001). Members of the family Cyper- et al., 2011) and, where the chromosome number aceae are of ecological importance as they are major of the species is known, to the basic chromosome components of many ecosystems (Leck & Schütz, number. This method requires only a few milligrams 2005), especially in arctic and alpine habitats, arous- of dried plant material, but the statistical probability ing a new interest in the biology of the group. Sedges of complete heterozygotes decreases with increasing show large species diversity and some are also eco- ploidy, and there are numerous genera in which nomically important (especially species of Cyperus L. suitable SSR loci are not yet available. The use of and related taxa). microsatellite markers developed for other taxa is In the present study, we investigated the basic potentially possible, but cross-amplification rates are chromosome number and ploidy in the sedge genus typically low, even between congeneric species, and Kobresia Willdenow. This genus comprises c.60 they are greatly reduced in monocots relative to eud- species (Zhang, 2001), which are widespread and icots (Peakall et al., 1998; Barbará et al., 2007). In locally dominant in the Northern Hemisphere, with addition, the probability of null alleles increases the highest species diversity occurring in alpine as a result of mutation accumulation in the primer regions of the Himalayas and adjacent mountain binding sequences, and subsequent partial heterozy- ranges (Dahlgren, Clifford & Yeo, 1985; Zhang, Liang gotes introduce ambiguity (Erler, Stoneking & & Dai, 1995). Few cytological studies are available, Kayser, 2004). Therefore, it is critical to ensure reli- mainly because of the limited access to growing able amplification in interspecific tests when micros- tissue. Chromosome counts are rarely reported and atellites are to be used to assess ploidy. In any case, vary from 2n =32 in K. schoenoides Boeckeler to such methodological constraints can be minimized by 2n = c. 122 in K. nepalensis (Nees) Kük. (Poghosian, using a sufficient number of samples and reliable loci. Narinian & Voskanian, 1971; Hoshino, Rajbhandari & Third, the ploidy of species can be determined from Ohba, 2000). Basic chromosome numbers of the genus the DNA contents. Doubling of the chromosome remain under debate, with estimates ranging from numbers usually leads to an increase or doubling of dibasic with x = 26 and 36, to tribasic with values the DNA contents. Recently, such changes have of x = 8, 9 and 13 (Darlington & Wylie, 1955; Mehra & become easy to determine using flow cytometry, in Sachdeva, 1976). No study has yet specifically which only small amounts of fresh/dried tissue or examined the origin of ploidy in Kobresia, but studies seeds suffice (Suda & Trávnícˇek, 2006). However, in the closely related Carex L. revealed that auto- DNA content appraisal using flow cytometry does not polyploidy rather than allopolyploidy dominates reveal ploidy directly, and reference samples with (Heilborn, 1939). This is also regarded as the pre- known ploidy are required (e.g. Pellicer et al., 2012). © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, ••, ••–•• PLOIDY IN THE GENUS KOBRESIA 3 In this study, we chose K. pygmaea C.B.Clarke ex CHROMOSOME NUMBERS Hook.f. for the initial establishment of microsatellite Chromosome counts were conducted in one individual protocols. This species is the most ecologically and each of K. pygmaea, K. cercostachys and K. sim- economically important representative of the genus, pliciuscula and two individuals of K. myosuroides. 2 covering more than 450 000 km of the Tibetan Excised root tips were treated in iced water for 20 h Plateau and adjacent areas (Miehe et al., 2008, to accumulate metaphases, and then fixed in absolute 2011). It is the dominant species in the second ethanol–glacial acetic acid (3 : 1) for 3 h and stored largest alpine ecosystem and forms the basis for