POLISH JOURNAL OF ECOLOGY (Pol. J. Ecol.) Regular research paper Alina STACHURSKA-SWAKOŃ 1 *, Elżbieta CIEŚLAK 2, Michał RONIKIER 2 1 Institute of Botany, Jagiellonian University, Kopernika

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POLISH JOURNAL OF ECOLOGY (Pol. J. Ecol.) Regular research paper Alina STACHURSKA-SWAKOŃ 1 *, Elżbieta CIEŚLAK 2, Michał RONIKIER 2 1 Institute of Botany, Jagiellonian University, Kopernika 27, Kraków, Poland, * (corresponding author) 2 Institute of Botany, Polish Academy of Sciences, Lubicz 46, Kraków, Poland GENETIC VARIABILITY OF SMALL ISOLATED POPULATIONS OF CICERBITA ALPINA (L.) WALLR. (ASTERACEAE) IN THE BESKID MAŁY MTS (SOUTHERN POLAND) ABSTRACT: The populations of Cicerbita alpina in the Beskid Mały Mts. (Western Carpathians, Poland) are the northernmost, spatially isolated localities of this subalpine tall-herb species in the Carpathians. The genetic structure of these populations was studied using the AFLP method. The analysis also included the populations of the larger, more population-abundant parts of the distribution range to the north (Scandinavia) and to the south (the Tatra Mts., Western Carpathians). The genetic similarity of the Beskid Mały populations with those from the Tatra Mts. and Scandinavia was relatively low and the populations formed geographically distinct genetic groups. The populations from the Beskid Mały Mts. were characterised by lower genetic variation, as well as the lowest degree of genetic differentiation (Nei and Shannon s coefficients), compared with those from the Tatra Mts. and Scandinavia. Our results indicate a relatively long period of isolation of the Beskid Mały Mts. populations; there is no evidence for recent dispersal or extant gene flow with populations from other regions. The differences among the populations also testify to fixation of genes in isolated areas, probably traced back to the founder individuals. KEY WORDS: Cicerbita alpina, geographical isolation, molecular variation, small population size, AFLP, tall-herb, Carpathians 1. INTRODUCTION The most frequent cause of the emergence of geographically isolated populations is fragmentation of habitats, stemming either from climatic changes (measured over long periods of time) or from anthropogenic activities (Huenneke 1991, Kornaś and Medwecka-Kornaś 2002). When there is a barrier to the gene flow between an isolated population and other populations in a continuous range of distribution, a closed population (Mayr 1974) emerges where some ecological and genetic processes occur, such as: genetic drift, inbreeding resulting in inbreeding depression, and increased levels of homozygosity, resulting in decreased genetic variation in a given population (Ellstrand and Elam 1993, Mitka 1997, Allendorf and Luikart 2007). The results of demographic or genetic studies of processes occurring in isolated populations provide important information for both taxonomic and evolutionary considerations. They allow tracing factors which may have affected the current level of genetic variation and differentiation in these populations and have influenced the current shape of the distribution range of a given taxon. Such collective data can journal 26.indb :20:26 280 Alina Stachurska-Swakoń et al. also have important practical implications, particularly when they concern rare and endangered species (Barrett and Kohn 1991, Treuren et al. 1993, Stewart and Nilsen 1995, S chaal and Leverich 1996, Cieślak et al. 2006, 2007a, 2007b, Caujape-Castells et al. 2008). Cicerbita alpina (L.) Wallr. (Alpine sowthistle) is a European mountain species. It grows in disjunct distribution areas including the Pyrenees, the Alps, the Carpathians, the Sudety Mts., the Balkans, as well as Scandinavia and Scotland (Hultén and Fries 1986, Marren et al. 1986, Meusel and Jäger 1992, Alexander 2006). It is a characteristic species for tall-herb communities of the Adenostylion alliariae Br.-Bl alliance, occurring principally in the subalpine altitudinal zone (Pawłowski 1977, Oberdorfer 1983, Matuszkiewicz 2005, Stachurska- Swakoń 2009). It is always associated with fertile and wet habitats, so it may also be found outside typical tall-herb associations in wet forests, in source areas of rivers, and along the banks of streams (B enum 1958, Tacik 1972, Oberdorfer 1983, Lid 1987). In the Carpathians it rarely grows below 700 m a.s.l. It is a perennial species, insect-pollinated and wind-propagated. It is diploid with 2n = 18 (Skalińska and Pogan 1966, Doležalová et al. 2002). Alpine sow-thistle forms small and dispersed populations in several mountain massifs of the Western Carpathians (Z ając and Z ając 2001). It also occurs as a very rare element of the flora in the Beskid Mały Mts. This mountain range is one of the northernmost parts of the Western Carpathians and, in consequence, these local populations of Alpine sow-thistle represent the northernmost localities in the Carpathians. The Beskid Mały Mts. have a somewhat isolated situation, surrounded by lower ground (the Silesian foreland, the Skawa river valley, the Biała river valley, the Kotlina Żywiecka basin and Pasmo Pewelskie hills). The highest peak in the Beskid Mały Mts. is only slightly above 900 m a.s.l. Despite these low elevations and lack of natural upper montane and subalpine zones, several high-mountain species occur there, as well as upper-montane-zone spruce forest regarded as native (Myczkowski 1958). The alpine species recorded in the Beskid Mały Mts. include Diphasiastrum alpinum (L.) Holub, Mutellina purpurea (Poir.) Thell., and Solidago virgaurea L. subsp. alpestris (Waldst. & Kit.) Rchb., and subalpine species Athyrium distentifolium Tausch ex Opiz, Cicerbita alpina, Doronicum austriacum Jacq., Gnaphalium norvegicum Gunnerus, Poa chaixii Vill. s. str., Ranunculus platanifolius L., Senecio subalpinus W.D.J. Koch and Veratrum lobelianum Bernh. (Pelc 1958, Kotońska 1991, W. Bartoszek, unpblished data). When considering the origin of the high-mountain flora of the Beskid Mały Mts. it is also interesting to note that the floras of the nearby ranges of the Beskidy Mts. lack alpine species despite sometimes higher altitudes. The nearest localities of such species are those within the Babia Góra Mt. (the highest massif of the Beskidy Mts. with 1725 m a.s.l.) Also interesting is the fact that all localities of these plants are concentrated in the eastern part of the Beskid Mały Mts. The present study is the first part of a larger-scale analysis of the genetic structure of C. alpina in its distribution range. Here, we examine the genetic variability of small and isolated populations at the northern edge of the Carpathian distribution of the species. Additionally, these populations are compared with those occurring in the areas where Alpine sow-thistle forms large and abundant populations: the Tatra Mts. (Western Carpathians) and Scandinavia. The specific objectives of this study were as follows: 1) to determine the levels of genetic variation and differentiation in the isolated populations of Cicerbita alpina in the Beskid Mały Mts. compared to the populations from more population-rich parts of the distribution range of the species (the Tatra Mts. and Scandinavia); 2) to estimate the degree of genetic isolation of the populations in the Beskid Mały Mts. and probability of extant gene exchange with other regions; 3) to attempt to ascertain whether the origin of the Beskid Mały populations may be connected with the recent long-distance dispersal processes from the Tatra Mts., being a regional centre of alpine flora, or rather with historical colonisation followed by longer isolation. journal 26.indb :20:26 Genetic variability of Cicerbita alpina in the Beskid Mały Mts MATERIALS AND METHODS 2.1. The study populations Two groups of populations were selected for this study: isolated populations from the Beskid Mały Mts., and populations from the larger and more population-rich parts of the distribution range (Fig. 1). The area of Beskid Mały Mts. was represented by two populations (Table 1). The first was growing in a source area of a stream on the northern slope of the Jaworzyna Mt. at 846 m a.s.l. This population consists of ca one hundred flowering shoots. Apart from Cicerbita alpina there were other subalpine tall-herb species growing there, including Ranunculus platanifolius, Senecio subalpinus, and Veratrum lobelianum. Its floristic composition represents a greatly impoverished tall-herb community and at present this locality is overgrown with raspberries. The second population was situated in the northern part of the Beskid Mały Mts., near the locality of Rzyki. It was formed by a group of sixteen flowering shoots growing in the lower montane forest zone, in a forest growing on an escarpment along a stream, at 567 m a.s.l. Here, the Alpine sow-thistle did not constitute part of any community. The distance between these two localities is about 5 km in a straight line. The populations representing the more continuous parts of the distribution range include those from the Tatra Mts. (4 populations) and Scandinavia (2 populations) (Table 1). The plant material from the Tatra populations was sampled in the subalpine zone, all in typical tall-herb communities. The plant material from the Scandinavian populations was sampled in open birch forests. All these populations are very rich in individuals, they consist of hundreds or thousands flowering shoots Material collection, DNA extraction and AFLP analysis Samples of 45 individuals were collected: 5 6 individuals per population. The individuals were randomly selected to represent variability in the populations. Single young and healthy leaves (1 2 leaves per plant) were cautiously removed and placed in hermetic plastic bags with silica gel for quick drying. Dried samples were stored in hermetic plastic boxes at room temperature. Total DNA was extracted from ca 15 mg of dried plant material. DNA extraction and washing was performed using a DNeasy Plant Mini-Kit system (Quiagen), according to the protocol provided by the manufacturer. The concentration and quality of the extracted Fig. 1. Location of sampled populations of Cicerbita alpina. NS Norway, Sør Varanger, NT Norway, Tynset, PM Poland, Beskid Mały I, PN Poland, Beskid Mały II, SS Slovakia, High Tatra I, Štrbské Pleso, SZ Slovakia, High Tatra II, Kriváň, TG Poland, High Tatra I, Hala Gąsienicowa, TM Poland, High Tatra II, Morskie Oko. See Table 1 for details. journal 26.indb :20:26 282 Alina Stachurska-Swakoń et al. DNA were estimated against DNA on 1% agarose gel stained with ethidium bromide. The AFLP analysis was carried out following the methods of Vos et al. (1995) with modifications described in Ronikier et al. (2008) Data analysis AFLP fragments were manually scored using GENESCAN analysis software (version 3.7, Applied Biosystems) and GENOG- RAPHER (version 1.6). Several statistics were calculated for each population to detect genetic variation at population and region levels: the total number of AFLP bands, percentage of polymorphic markers, number of discriminating markers, number of private markers, Nei s gene diversity in populations (Nei 1987) and Shannon s diversity index (H Sh = Σp i lnp i, where p i is the relative frequency of the i th fragment in dataset). Discriminating markers denote fragments which are present in all analysed samples of a respective population and are absent elsewhere, and private markers denote unique fragments for respective populations but not necessarily common for all of its samples (Cieślak et al. 2007b). Also, the genetic similarities between populations as Nei s Original Measures of Genetic Identity were used to estimate the genetic affinities between the study populations (Nei 1978). Statistical analysis were performed with the POPGENE version 1.32 software (Yeh et al. 1997). The AFLP phenotypes were used to detect the relationships between the individuals using principal coordinate analysis (PCoA) based on Euclidean distances computed with the MVSP version 3.1 software (Kovach 2007). The same data were used in clustering methods with UPGMA and neighbour-joining tree (both Nei and Li s similarity coefficient). MVSP version 3.1 software (Kovach 2007) and TREECON 1.3b software (Van dr Peer and De Wachter 1994) were used for these analyses. Support for resulting groups was calculated from 1000 bootstrap replicates. The data were also analysed with the Bayesian inference model in BAPS 4.14 (Corander and Marttinen 2006) using 70 runs with maximum K set to 8. Results of the mixture analysis were further used in the admixture analysis. Analysis of molecular variance (AMO- VA), with the partition of the total genetic variance into different levels (within populations, among populations, and among groups of populations) were carried out using ARLE- QUIN 2.0 (S chneider et al. 2000). 3. RESULTS A total of 207 DNA markers, including 56% of polymorphic bands were found in the study populations of Cicerbita alpina. The polymorphic bands ranged from 10% to 36% within the populations. The lowest Table 1. Sampled populations of Cicerbita alpina with some genetic characteristics. N number of individuals investigated in AFLP, % Pol percentage of polymorphic bands, M D discriminating markers, H Nei Nei s gene diversity, H Sh Shannon s diversity index. Acronym Sampling locality coordinates Altitude (m a.s.l.) N %Pol M D H Nei H Sh NS Norway, Sør Varanger N 62 10, E NT Norway, Tynset N 69 50, E PM Poland, Beskid Mały I N 49 47, E PN Poland, Beskid Mały II N 49 46, E SS Slovakia, High Tatra I N 49 07, E Štrbské Pleso SZ Slovakia, High Tatra II N 49 09, E Kriváň TG Poland, High Tatra I N 49 14, E Hala Gąsienicowa TM Poland, High Tatra II Morskie Oko N 49 11, E journal 26.indb :20:27 Genetic variability of Cicerbita alpina in the Beskid Mały Mts. 283 Fig. 2. PCoA scatter diagram of Cicerbita alpina individuals based on Euclidean distances of AFLP phenotypes. Abbreviations of population names as in Table 1 and Fig. 1. polymorphism characterised the populations from the Beskid Mały Mts. (10% for PM and PN sites). The highest percentages of polymorphic bands were noted in the Tatra Mts. populations. For this group of populations, 35% of polymorphism was found on average, ranging from 32 to 37%. Thus, these two Carpathian groups were characterised by a marked difference in polymorphism between them, while the within-group values were relatively even. In contrast, high withingroup differentiation of polymorphism was noted in the case of the Scandinavian population group 19% for NT site and 33% for NS site (Table 1). Population-specific discriminating markers were very rare. Only single such bands were found in the populations from each of the geographical groups: in Scandinavia (NS site), the Tatra Mts. (SZ site), and the Beskid Mały Mts. (PN site; see Table 1). No private markers were found. The values of Nei s and Shannon coefficients for the populations from the Beskid and Tatra mountains consistently showed the same tendency: the lowest values were found for the Beskid Mały Mts. populations where they amounted, respectively, to and for PM site, and and for PN site. Much higher values were determined for the Tatra Mts. populations, where they fluctuated within the range (0.12 on average) and (0.18 on average), respectively. Significant differentiation in the values of these coefficients was found among the Scandinavian populations, where they amounted to: 0.06 (NT site) and 0.14 (NS site) for Nei s coefficient and 0.10 (NT site) and 0.20 (NS site) for Shannon coefficient (Table 1). The distribution diagram prepared after the PCoA analysis and illustrating the degree of genetic differentiation of Cicerbita alpina, indicated three distinctly separated and geographically-driven genetic groups. One group was composed of populations from the Tatra Mts., the second from the Beskid Mały Mts., and the third from Scandinavia. The most homogeneous, compact group was composed of individuals from the Tatra Mts. populations. In the case of populations from the Beskid Mały Mts., and from Scandinavia, internal divisions appeared in each of these groups. This tendency was particularly noticeable between the populations of the Beskid Mały Mts. (Fig. 2). This division was further confirmed by the Bayesian analysis of population structure (Fig. 3). Interestingly, the result of this analysis indicated the existence of four genetic groups (with 100% probability), two of which were specific to the populations from the Beskid Mts., whereas the two remaining groups included all individuals from the Tatra Mts. and Scandinavia, respectively. journal 26.indb :20:27 284 Alina Stachurska-Swakoń et al. Table 2. Nei s Original Measures of Genetic Identity for sampled populations of Cicerbita alpina. Abbreviations of population names as in Table 1 and Fig. 1. NS NT PM PN SS SZ TG TM NS **** NT **** PM **** PN **** SS **** SZ **** TG **** 0.95 TM **** Fig. 3. Diagram presenting the results of a Bayesian analysis of population structure of Cicerbita alpina. Four variants of grey shading represent the four genetic groups detected by mixture analysis: S Scandinavian populations, B Beskid Mały Mts. populations, T Tatra Mts. populations. Abbreviations of population names as in Table 1 and Fig. 1. The clustering of individuals revealed by the UPGMA dendrogram fully supported the image of genetic differentiation obtained by the PCoA method (Fig. 4). The bootstrap values for the clusters were, however, very low (below 30). The results of this analysis also indicated that, from among the group of the populations studied, the most similar were the individuals of the Beskid Mts. populations, for which the similarity coefficient for PM site ranges from 0.98 to 0.96, but still with each of the populations being a separate group. The result for the Scandinavian populations was similar, with each population representing a separate group, although the individuals were characterised by a higher genetic variation than in the populations from the Beskid Mały Mts. The highest levels of genetic variation were represented by the populations from the Tatra Mts. In this case, the similarity coefficient fluctuated from 0.97 (for individuals from TM1 and TM3 sites) to None of the populations in this geographical group appeared evidently to be a separate group. This picture of genetic variation was further supported by the values of Nei s coefficient based on genetic similarities between populations (Table 2). The Tatra Mts. populations were highly similar to each other. For the following pairs of the Tatra populations: TG and TM, TG and SZ, TG and SS, SZ and SS, this coefficient had a high value of The Scandinavian populations were relatively less similar to each other, with the value of the similarity coefficient equal The populations from the Beskid Mały Mts. have the lowest level of similarity between themselves, as well as towards the other populations studied. The coefficient between PM and PN populations was The lowest value of the coefficient (0.83) characterised the pair PN NS populations. The similarity between the populations from the Beskid Mały and those from the Tatra Mts. fluctuated within a range. journal 26.indb :20:27 Genetic variability of Cicerbita alpina in the Beskid Mały Mts. 285 The results of the variance analysis indicate a markedly higher proportion of intrapopulation variation (60%), compared with the differences between the groups of populations (the divisions into groups corresponded to the structure obtained in the PCoA analysis and cluster analysis, as well as with the geographical locations of the populations under study). This result indicated lack of deep differentiation among the study groups. 4. DISCUSSION Our results indicate very low genetic variation in the isolated populations of Cicerbita alpina from the Beskid Mały Mts. Both the share of polymorphic bands and the genetic coefficients had very low values in the Beskid Mały Mts. populations, compared with the populations from the population-richer regions examined (the
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