Genome size, chromosome counts and distribution of Homogyne alpina (Asteraceae) in the Slovak Carpathians

: Homogyne alpina (L.) Cass. represents the only species of the genus Homogyne Cass. in Slovakia. This study characterizes H. alpina in terms of chromosome counts, genome size, reproduction mode and distribution in Slovakia. Three known cytotypes of the species are known, of which the cytotype 2n = 160 is represented in the Slovak mountains and has not been documented from other countries yet. Flow cytometric analyses showed the genome size 2C = 21.30 pg for petioles and 2C = 21.82 pg for seeds. Sexual reproduction mode was confirmed by flow cytometric screening method based on embryo to endosperm genome size ratio. The centre of the species distribution is in high mountains of the Slovak Carpathians, where it is frequent in the altitude above 1000 m a. s. l. from mountane to alpine vegetation zone. The full list of the data of its distribution and the distribution map are also presented.

Another feature worth attention is the genome size. At the time of our analyses, one study reporting the genome size of H. alpina (Garcia et al. 2013) has already been published. Another study (Šmarda et al. 2019) was published during the evaluation of our data and writing the manuscript. Both studies claimed that they analysed plants of the cytotype 2n = 120, however, they did not count the chromosomes in their studies and only used data published in the database. Therefore, we wanted to compare our data for genome size with previously published ones in relation to the ploidy levels.
Apomixis, asexual reproduction by seeds, can be found in Asteraceae, with welldocumented examples in the genera as Taraxacum F. H. Wigg., Hieracium L., Pilosella Hill, etc. (Noyes 2007). The genus Homogyne within the Senecioneae tribe belongs to the strongly supported Petasites-clade together with the genera Endocellion Turcz. ex Heder, Petasites Mill. and Tussilago L. (Pelser et al. 2007;Steffen et al. 2016). Czapik (1996) stated that the genus Petasites is aposporous, however did not report any supporting data. Previous studies do not suggest the evidence of apomixis in Petasites, and we did not reveal such claim in the literature (Toman 1972;Cherniawsky & Bayer 1998). On the contrary, Homogyne alpina itself within 2n = 140 was documented as a sexual species (Urbańska 1956). Therefore, we decided to test if other cytotypes, potentially distributed in Slovakia reproduce in the same way.
The detailed distribution of the species in Slovakia has not been studied so far. This fact led us to sumarize all available distribution data, visualize it on the map of distribution, and also uncover the minimal and maximal altitude, where the species occurs.
Studying selected populations from the territory of Slovakia, this work summarizes (1) the chromosome counts, (2) the results of the measurements of genome size, (3) the evidence of sexual reproduction mode and (4) the distribution of Homogyne alpina in Slovakia, because the distribution range of the species in Slovakia has not been previously described in detail.

Plant material
Living plants for flow cytometry were collected during summers and autumns 2014 and 2015 at four localities in the Stolické vrchy Mts. and at two localities in the Volovské vrchy Mts. The plants were potted in their natural soil and cultivated in the Botanical Garden of P. J. Šafárik University in Košice. Seeds for flow-cytometric and karyological analyses were collected in summer 2015 from two localities in the Stolické vrchy Mts., one locality in the Západné Tatry Mts. and one locality in the Nízke Tatry Mts. Until the analyses were performed, the seeds were stored in refrigerator at the temperature 4°C. The herbarium specimens of the plants used for the flow-cytometric analyses are deposited in KO (Herbarium of the Botanical Garden of P. J. Šafárik University, Košice, Slovakia). Complete data on the collections and the herbarium specimens are given in the Appendix 1.

Karyological methods
For karyological analysis seedlings and root meristems of the cultivated plants from the selected localities were used (for details see Appendix 1). For the pretreatment, the root tips were transferred to 0.002 M aqueous solution of 8-hydroxyquinoline at the temperature of 4°C for 16 hours. Then the root tips were fixed for at least 1 hour in acetic ethanol (glacial acetic acid and 96 % ethanol in the ratio 1: 3), washed in distilled water and hydrolyzed for 3 minutes in 1N HCL at 60°C, then washed in distilled water. The meristems were squashed using a cellophane technique (Murín 1960) and stained in 7 % Giemsa stain solution in Sörensen phosphate buffer for 3 hours. The slides were then washed in distilled water, dried, and observed in a drop of immersion oil. Selected c-metaphase plates were photographed (using a Leica DM 2500 microscope equipped with camera DFC 290 HD and software Leica application suite version 3.5.0, Switzerland) and number of chromosomes was determined.

Flow cytometry
Flow cytometric analyses were performed both on petioles from living plants and on seeds. The samples were prepared by a two-step procedure, consisting of nuclear isolation and staining steps, using propidium iodide as DNA intercalator (Doležel & Göhde 1995;Loureiro et al. 2007). A method referred to as internal standardization was used (Doležel et al. 2007). To keep recommended maximum differences between standard and sample (Suda 2004) we used the internal reference standard: Vicia faba subsp. faba var. equina 'Inovec' (2C DNA content = 26.9 pg, Doležel et al. 1992). The seeds of the standard were acquired from the Institute of Experimental Botany, Olomouc, Czech Republic and grown in the Laboratory of Taxonomy at the Institute of Biological and Ecological Sciences of P. J. Šafárik University in Košice, Slovakia. For the determination of genome size three plants from each population were collected and one petiole per plant was used. To minimize possible deviations caused by the flow cytometer or sample preparation every plant was measured 3 times on 3 different days (Greilhuber & Obermayer 1997). These values were averaged for every plant extra, making 3 genome size values for each locality, 18 in total. For the detection of reproductive pathway and the estimation of genome size seeds from five plants per locality were used. Ten seeds of Homogyne alpina were used for each analysis and chopped together with the standard. Four samples from the locality Čuntava and five samples from each of the 3 remaining localities were measured once, making 19 measurements in total. Particular methodology of preparing the samples and their evaluation was identical with our previously published work (Koprivý et al. 2019). Statistical difference between population means of genome size values was tested with analysis of numerical variance (ANOVA). Genome size values obtained after analyses of petioles and seeds were then statistically evaluated with t test (or Mann-Whitney test, if assumptions for t test were not met). Prior to statistical analyses, normality and homoscedasticity of data were verified. Data analyses were done in Past ver. 3. 10 (Hammer et al. 2001).

Species distribution mapping
The study of the distribution of the species was based on the field work (2014)(2015)(2016)(2017)(2018)(2019)(2020)(2021), the revision of voucher specimens deposited in the public herbaria in Central Europe: BP, BRA, BRNM, BRNU, KO, MOP, MPS, NI, POP, PR, PRC, SAV, SLO, SMB, SMBB, SNV, TM, ZAM, ZV and W (acronyms according to Thiers (2020+) and Vozárová & Sutorý (2001) for the small local collections), the published literature records (cited in the Appendix 3) and the data from the database Comprehensive information and monitoring system (CIMS), available online at www.biomonitoring.sk (which are cited as "Bio"). A grid map for the distribution of Homogyne alpina in Slovakia was designed in the ArcGis program, version 9.2. The phytogeographical division follows Futák (1984).

Chromosome number, genome size and reproduction mode
One chromosome number record from Slovakia has previously been known: 2n = 160 from the locality Stinská in the Bukovské vrchy Mts. (Uhríková 1970). Based on our karyological analyses we approximately confirmed this number, 2n = ca 160 from Stolica Mt. We could not determine the exact number because several chromosomes overlapped (Fig. 1). Considering all the data summarized by Chromosome Counts Database, three different cytotypes of the species are reported. Our result most likely corresponds with the cytotype 2n = 160 determined by Uhríková (1970), which has been the only report for this cytotype so far. Thus, from the territory of Slovakia only the individuals with 2n = 160 are known. The cytotype 2n = 140 was described by Langlet (1936) for the first time. However, this result was only an approximate one due to high number of chromosome sets and problems with accurate counting. This chromosome number was later roughly confirmed by the embryological study of Urbańska (1956), who analysed plant material from the Polish side of the Tatry Mts., and by Pashuk (1987) from the Ukrainian Chornohora Mts. The third cytotype represents 2n = 120, with records from Austria (Kempiak in Lepper 1970;Favarger in Löve 1971), Italy (Löve & Löve 1982 or Bulgaria (Kuzmanov et al. 1986).
Some of the previously mentioned works (eg. Langlet 1936; Urbańska 1956) concede that the chromosome numbers are only approximate, since the chromosomes did not nicely stand apart and some chromosomes fully or partially overlapped. While preparing the slides, sometimes even the chromosomes from two different cells could mix, which also leads to inaccuracies.
To date, two studies providing the results of genome size of H. alpina have been known. In both studies the authors believed that they analysed plants with cytotype 2n = 120, although they dit not perform karyological analyses and only relied on the literature data. The samples originated from Spain (Garcia et al. 2013) Doležel et al. 2003) (Fig. 2, Tab. 1) from the petioles and 2C = 21.82 ± 0.47 pg (21 340 Mbp) from the seeds (Fig. 3). Our genome size values for petioles and seeds are the very first data for H. alpina of this ploidy level. At this point, comparison of our data with the previous works revealed diametrically different values for genome size. There is also difference in genome size of cca 0.9 pg between these two studies. Several reasons may be considered to be responsible for reported variation: a) it is uncertain if Garcia et al. (2013) and Šmarda et al. (2019) really analysed plants with 2n=120; b) different methods, reference standards, buffers may cause variation in estimated genome size; c) other genomic factors as genome downsizing. Considering the classification of genome sizes of Angiosperms by Leitch et al. (1998) and Soltis et al. (2003), our results for the genome size of H. alpina are assigned as the intermediate ones (3.5 < 1C < 14 pg).
As in our previous study (Koprivý et al. 2019) we planned to determine the genome size values for the leaves and the seeds and then compare the results. However, the analysis of the leaves has not provided proper data probably due to high content of secondary metabolites. Instead of leaves, petioles were used, which provided reliable results supported by high-class histograms. The genome size values from all analysed populations showed only little differences. The minimal value (20.94 pg) for petioles was determined for the sample from Kojšovská hoľa Mt., while the highest one (21.72 pg) from Volovec Mt. Assumptions of ANOVA are not violated, and we noticed marginal but statisticaly significant differences in genome size between populations (ANOVA, F5,12 = 4.221, p < 0.05). It results from slight deviation of plants from locality Volovec Mt., which in average have larger genome sizes compared to plants from Čuntava (2.22 % difference) and Kojšovská hoľa Mt. (1.84 %). These differences may be caused by small population sample and it deserves further attention. Better sampling of the populations from surrounding of Volovec Mt., Čuntava and Kojšovská hoľa Mt. will be needed to confirm or disprove detected differences.
According to Sliwinska et al. (2005) and Kolarčik et al. (2018) seeds are not only suitable for determining the genome size, but in some cases are even better material for FCM analysis than leaves. For seeds, the minimal value (21.19 pg) together with maximal one (22.85 pg) were revealed in the samples from Žiarska dolina valley. Data of genome size measurements from both petioles and seeds are available only for two populations, Čuntava and Stolica Mt. In both cases, statistically significant differences were observed: in Stolica Mt., t test, t = -2.77, p = 0.04 (2.93 % difference), and in Čuntava, Mann-Whitney test, z = -2.087, p = 0.04 (4.21 % difference). Statistical significance (p values) are only marginal, which once again indicates that for definitive confirmation of differences in genome size between petioles and seeds better sampling will be needed.

Locality
Type of analysis Simple comparison of the mean genome size values for the seeds and the petioles showed that the values for seeds are in average by 2.4 % (0.52 pg) higher. Several reasons may cause this variation in the genome size values. Kolarčik et al. (2018) mentioned that cytosolic compounds present in FCM samples may affect genome size estimates in Onosma sp. Along with this, they reported that staining duration may also have an effect on an estimate of this parameter in cases where fluorescence dye incorporation into DNA may be slowed down, as shorter staining time may result into inaccurate lower values of DNA content. Likewise, other aspects, such as different chromatine structure in cells of embryonic stage, i.e. nuclear size reduction during the seed maturation and nuclear architecture reorganization in the time of seedling establishment (Baluška 1990;Biradar & Rayburn 1994;van Zanten et al. 2011;Bourbousse et al. 2015), can lead to different binding of fluorescence stains to DNA and diverse values of 2C DNA obtained by FCM analyses. However, this requires further research. We suppose that the difference of circa 2 pg between the minimal and the maximal value is too little to indicate different ploidy levels among the populations. Ergo we conclude that the individuals from all of the populations have the same chromosome number, 2n ≈ 160. Even the individuals from the Západné Tatry Mts. (locality Žiarska dolina valley) and the Nízke Tatry Mts. (locality Kološňa) showed no significant variance compared to the values from other localities from the Stolické and the Volovské vrchy Mts. This suggests that the populations on the northern, Polish side of the Tatry Mts. (2n = 140, Urbańska 1956) and those on the southern, Slovak side may be of different ploidy levels. However, due to the lack of precise data, much more detailed study aiming at chromosome counts and genome size determination will be needed to support this hypothesis.
The ratio of ploidy level of embryo and endosperm was 2:3 in all cases, which corresponds to the sexual type of reproduction (for complete data see Appendix 2). The detailed embryological study of H. alpina by Urbańska (1956) identified the presence of pollen tubes in several sacs, which also contained typically developed embryos surrounded by endosperm. Hence, we verify this reproduction mode using flow cytometry.

Distribution in Slovakia
Homogyne alpina occurs in most of the mountains of the Slovak Carpathians, frequently at altitudes over 1000 m, in the submountain, mountain, subalpine and alpine vegetation zones. The occurrence of the species was documented in 25 phyto-  geographical districts and subdistricts of the Slovak flora and its distribution in the territory of Slovakia is shown in Fig. 4. The species is most frequent in the area of Eucarpaticum (the Malá Fatra Mts., the Veľká Fatra Mts., the Nízke Tatry Mts. and the Tatry Mts.), Beschidicum occidentale (the Západné Beskydy Mts.) and Beschidicum orientale (the Spišské vrchy Mts.), many records are also from the Slovenské Rudohorie Mts., the Muránska planina Mts., the Slovenský raj Mts. and the Čergov Mts., where H. alpina occurs in beech and spruce forests and in mountain meadows. The species grows in a wide range of altitudes, the maximum reaches at ca 2500 m in the Vysoké Tatry Mts. (Kotlový štít Mt.; for details see Appendix 3) and the lowermost occurrence ever recorded, very surprisingly, lies near the Meľov hill in Ondavská vrchovina Mts., where Chrtek & Křísa in 1976 found the species at the altitude of about 320 m. The species grows very rarely in north-eastern Slovakia due to maximum altitudes under 1000 m. Low altitudes do not allow the species to colonize permanently the hill areas of the Ondavská and Laborecká vrchovina. In the Slanské vrchy Mts. there are some hills over 1000 m, but only a single occurrence was recorded here at the altitude of 800 m. Rather isolated occurrences are situated in the easternmost Slovakian mountains (the Bukovské vrchy Mts. and the Vihorlat Mts.). Farther eastwards the species' range continues in the high hills towards the Ukrainian Eastern Carpathians (Chopik & Fedoronchuk 2015). The earliest known report from the territory of Slovakia dates back to 1851; the species was collected by János Fábry at the Muránsky hrad castle and the herbarium specimen is deposited in the collection of BRA (see Appendix 3).

Appendix 3. List of revised herbarium specimens, published and unpublished records of Homogyne alpina
For herbarium specimens the collector, year of collection and herbarium acronym (and evidence number if exists) are given; herbarium acronyms follow Thiers (2020+). The references for the published records from the sources not listed in the References chapter are given in an abridged form including the page of a particular Homogyne alpina record. For unpublished manuscripts and field records the year is given, followed by the name(s) of its author(s). The records are arranged following the phytogeographical division of Slovakia by Futák (1984) and assigned to the quadrants (in parentheses) of the CEBA grid template according Niklfeld (1971). Altitude (where available) is presented by a number followed by "m", which is abbreviation of m a. s. l. Abbreviations: N (north), S (south), W (west), E (east) and their combinations, NR -Nature reserve, NNR -National Nature Reserve. Mt., on the right side of the tourist pathway from sedlo za Hromovým saddle to Chleb Mt., 1548m, 49°11'25.8"N, 19°03'28"E (Šibíková et al. Thaiszia -J. Bot. 19: 16, 2009 Mt., S slope, 1600 m, 49°11'32.3"N, 19°03'41.4"E (6880a).