The Pilosella alpicola group (Asteraceae) comprises four vicariant species of perennial herbs distributed disjunctly in subalpine areas of Europe (the Alps, Carpathians, and Balkan mountains). In this study, we followed the generic circumscriptions outlined by Bräutigam and Greuter (2007) in our treatment of the P. alpicola group. Our recent studies (Šingliarová et al., 2011a, b) showed that although the taxa of this group are very closely related and diverged relatively recently, they are morphologically well defined and display contrasting cytogeographic patterns and variation in breeding systems. Two species (P. ullepitschii (Błocki) Szeląg from the Carpathians and P. serbica (F. W. Schultz & Sch. Bip.) Szeląg from Serbia) are exclusively diploid and outcrossing. The Balkan taxon P. rhodopea (Griseb.) Szeląg represents a unique diploidpolyploid complex with up to five cytotypes (diploids to hexaploids) occurring mostly in mixed-ploidy populations. All P. rhodopea cytotypes are outcrossing, although polyploids exhibit severely reduced fertility (Šingliarová et al., 2011b). The alpine P. alpicola F. W. Schultz & Sch. Bip. (s. str.) has two allopatric cytotypes (tetraploids and pentaploids) with presumably polytopic allopolyploid origins and agamospermic mode of reproduction (Šingliarová et al., 2011a, b). All species of the P. alpicola group are rather rare, and their populations are often small and endangered by human activities. Recently, the conservation status of all taxa of the group was evaluated and their inclusion in relevant national Red Lists was proposed (Šingliarová et al., 2013). Interesting distributional patterns and a variety of evolutionary mechanisms playing a role in the differentiation of the P. alpicola group suggest it as a promising model system for studying plant speciation, adaptation, and recent polyploidization.
Microsatellite markers have already been developed for the related P. officinarum F. W. Schultz & Sch. Bip. (Zini and Komjanc, 2007) and the closely related genus Hieracium L. (s. str.) (Jönsson et al., 2010). The cross-amplification of the P. officinarum markers in other Pilosella Hill species and Hieracium s. str. gave unsatisfactory results (P. Trávníček, personal communication; Jönsson et al., 2010). Low cross-amplification success reported so far for the developed markers in Hieracium s. str. and Pilosella led us to deduce their poor transferability also to the P. alpicola group; therefore, a new set of markers is developed here. In addition, other Pilosella, Hieracium s. str., and Andryala L. taxa were used here to evaluate the potential cross-amplification of the developed markers in other phylogenetic lineages within the subtribe Hieraciinae (Fehrer et al., 2007; Krak and Mráz, 2008).
METHODS AND RESULTS
Total genomic DNA was extracted from fresh or silica gel–dried leaf material using the DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. DNA from one diploid individual of P. rhodopea was used for 454 sequencing. Genomic library preparation and 1/4 run on the Roche GS FLX platform was performed at GATC Biotech (Konstanz, Germany). MSATCOMMANDER version 0.8.2 (Faircloth, 2008) was used to identify reads containing di-, tri-, tetra-, penta-, and hexanucleotide repeats and to design primers. Primers were successfully designed to 179 microsatellite-containing reads.
Sixty primer pairs were selected and tested for amplification in three diploid individuals, each representing a different species of the studied group. Each reaction contained l× PCR buffer with KC1 (Fermentas, St. Leon, Germany), 2.5 mM MgCl2, 0.2 mM of each dNTP, 0.2 µM of both primers, 0.5 units of Taq DNA polymerase (Fermentas), and 20 ng of DNA. The cycling conditions were: 3 min at 95°C; followed by 35 cycles of 95°C for 30 s, locus-specific annealing temperature (see Table 1) for 30 s, and 72°C for 1 min; and a final extension at 72°C for 10 min. PCR products were checked on 2.5% agarose gels. Twenty-three primer pairs that amplified in the three tested species of the P. alpicola group were further applied to the complete set of 31 diploid individuals, originating from five distinct and geographically well-separated natural populations (Appendix 1). In this step, the amplification protocol was modified as described in Schuelke (2000) to facilitate fluorescent labeling of the PCR products by 6-FAM (Applied Biosystems, Foster City, California, USA). One microliter of each PCR product was mixed with 0.1 µL of GeneScan 500 LIZ internal size standard (Applied Biosystems) and 12 µL of Hi-Di formami de (Applied Biosystems) and electrophoresed using ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). Allele calling was performed using GeneMarker version 2.4.0 (SoftGenetics, State College, Pennsylvania, USA). Six primer pairs amplified three or four alleles in the majority of samples, indicating possible duplications of these loci; these were therefore discarded. One locus, Palpi21, was invariable. Two other loci, Palpi25 and Palpi35, showed multiple peaks in four samples belonging to populations COZ, DOD, and MEN (see Appendix 1 for population localities). For these particular samples, the allele lengths were scored as missing data, and the markers were retained for genetic diversity estimates. For the summary information on the 16 polymorphic loci see Table 1.
Characteristics of 17 newly developed microsatellite markers for the Pilosella alpicola group. Locus Palpi21 is monomorphic; the 16 other loci are polymorphic.
The genetic diversity of these 16 loci was then estimated. Number of alleles per locus, expected and observed heterozygosity, and polymorphic information content were calculated based on allele frequencies for each population as well as for the complete data set using CERVUS version 3.0.3 (Field Genetics, London, United Kingdom). All markers showed a relatively high degree of polymorphism (Table 2). The number of detected alleles ranged from seven for Palpi1 to 16 for Palpi18, Palpi34, and Palpi37 (average 11.4). The observed heterozygosity ranged from 0.45 at Palpi32 to 0.84 at Palpi49 (average 0.64). The values of expected heterozygosity varied from 0.72 at Palpi1 to 0.92 at Palpi34 (average 0.85). The values of the polymorphic information content varied from 0.67 at Palpi1 to 0.89 at Palpi34 (average 0.81). The level of variability within the tested populations was comparable for the populations of P. rhodopea, P. serbica, and one population of P. ullepitschii (population MEN). The other population of P. ullepitschii (SMARE) showed lower values for all estimated parameters (see Table 2 for details).
Genetic diversity parameters estimated for 16 polymorphic microsatellite loci for each analyzed population and the complete data set of the Pilosella alpicola group.
To evaluate the potential utility of the newly developed markers in other phylogenetic lineages of the Asteraceae subtribe Hieraciinae (Fehrer et al., 2007; Krak and Mráz, 2008), cross-amplification experiments were performed in one individual of P. echioides F. W. Schultz & Sch. Bip., P. officinarum, Hieracium umbellatum L., H. stelligerum Froel., Andryala laevitomentosa (Nyár. ex Sennikov) Greuter, and A. integrifolia L. The results of cross-amplification tests are summarized in Table 1. Higher cross-amplification success was reached within the genus Pilosella, where 10 markers could be cross-amplified in P. echioides and five in P. officinarum. Only one marker (Palpi29) was cross-amplified in all tested taxa. No other marker gave a positive result in the three Hieracium s. str. species and A. laevitomentosa. In A. integrifolia, cross-amplification of Palpi49 was positive as well.
In this study, we have developed 17 novel microsatellite markers for the P. alpicola group. Although only a relatively small sample set was used to evaluate their variability, 16 markers showed high levels of polymorphism. The transferability of these markers to two additional Pilosella species and to the closely related genera Andryala and Hieracium s. str. is restricted. Five markers were cross-amplified in P. officinarum and 10 in P. echioides, suggesting high species specificity of these markers. The cross-amplification in the other Hieraciinae genera was even less successful. Only one marker amplified in all four species and an additional one in A. integrifolia.
 The authors thank S. Píšová for technical assistance and J. Chrtek and J. Zahradníček for plants for the cross-amplification experiments. The study was financially supported by the Research and Development Support Agency (grant no. APVV-0320-10 to K.M.), by the long-term research development project no. RVO 67985939 (to P.V. and K.K.), and by the project ITMS6240120014 (Centre of Excellence for Biodiversity and Land-Use Conservation), supported by the Research and Development Operational Program funded by the European Regional Development Fund (ERDE).