172 ACTA BOT. CROAT. 77 (2), 2018 Acta Bot. Croat. 77 (2), 172–180, 2018 CODEN: ABCRA 25 DOI: 10.2478/botcro-2018-0019 ISSN 0365-0588 eISSN 1847-8476 Genetic variability and distance between Lactuca serriola L. populations from Sweden and Slovenia assessed by SSR and AFLP markers Michaela Jemelková1, Miloslav Kitner1, Eva Křístková1, Ivana Doležalová2, Aleš Lebeda1* 1 Palacký University in Olomouc, Faculty of Science, Department of Botany, Šlechtitelů 27, 783 71 Olomouc, Czech Republic 2 Department of Genetic Resources for Vegetables, Medicinal, and Special Plants of Crop Research Institute in Olomouc, Šlechtitelů 29, 783 71 Olomouc, Czech Republic Abstract – The study involved 121 samples of the common weed, Lactuca serriola L. (prickly lettuce), represent- ing 53 populations from Sweden and Slovenia. The seed materials, originating from different habitats, were re- generated and taxonomically validated at the Department of Botany, Palacký University in Olomouc, Czech Re- public. The morphological characterizations of the collected plant materials classified all 121 samples as L. serriola f. serriola; one sample was heterogeneous, and also present was L. serriola f. integrifolia. Differences in the amount and distribution of the genetic variations between the two regions were analyzed using 257 ampli- fied fragment length polymorphism (AFLP) and 7 microsatellite (SSRs) markers. Bayesian clustering and Neigh- bor-Network were used for visualization of the differences among the samples by country. Under the Bayesian approach, the best partitioning (according to the most frequent signals) was resolved into three groups. While the absence of an admixture or low admixture was detected in the Slovenian samples, and the majority of the Swedish samples, a significant admixture was detected in the profiles of five Swedish samples collected near Malmö, which bore unique morphological features of their rosette leaves. The Neighbor-Network analysis divid- ed the samples into 6 groups, each consisting of samples coming from a particular country. Reflection of mor- phology and eco-geographical conditions in genetic variation are also discussed. Key words: biogeography, Dinaric Alps and the Pannonian Plain, DNA polymorphism, ecology, habitats, mor- phological variation, prickly lettuce, Scandinavia * Corresponding author, e-mail: ales.lebeda@upol.cz Introduction Prickly lettuce (Lactuca serriola L., Asteraceae) is the most common species in the genus Lactuca L. (Feráková 1977), and has a circumglobal distribution (Lebeda et al. 2004). It is an annual or winter-annual therophyte (Fer- áková 1977), and an ‘r’strategist (Tilman 1988). Its evolution has trended towards a short life cycle, strong self-fertiliza- tion ability, good adaptation for wind dispersal, and quick germination (Frietema de Vries 1992, Lebeda et al. 2001). L. serriola is a drought-tolerant species (Werk and Ehleringer 1986), mainly growing in sunny microhabitats within an- thropogenic habitats such as roadsides, railways, dumps, and urban areas (Feráková 1977, Lebeda et al. 2001, 2004); it is considered a good colonist of a wide spectrum of dif- ferent habitats with different degrees of invasivity. Prickly lettuce is of Euro-Asian origin, also being native in North Africa (Feráková 1977). It has primarily spread in the Med- iterranean and the Near East (de Vries 1997, Lebeda et al. 2007a), and is considered an archaeophyte dependent on a culture from the northern part of central Europe (Meusel and Jäger 1992). The species belongs to a group of Mediter- ranean ruderal plants that have enlarged their distribution area during the last few centuries (Landolt 2001). The northern boundary of the European distribution area runs near latitude 65 °N through Finland, and 55 °N through Great Britain (Feráková 1977). The expanding dis- tribution of this species is accomplished by the transport of reproductive propagules, achenes. The ripened achenes with attached pappus are primarily dispersed by the wind, prob- GENETIC VARIABILITY OF LACTUCA SERRIOLA POPULATIONS ACTA BOT. CROAT. 77 (2), 2018 173 ably also by water (Weaver and Downs 2003). The spread of this species is also closely related with human activities, which primarily produce an increase in their transport (Leb- eda et al. 2001). Prickly lettuce has drastically increased its geographical range, invading many European, (North-) American, and Australian regions during the last 50-60 years (de Vries 1996, Lebeda et al. 2001, 2004); recently L. serriola has spread as an invasive weed throughout Europe (Lebeda et al. 2004, 2007b, D’Andrea et al. 2009), including Scandinavia (Rydberg 2013). Its synanthropic distribution has also been recorded from Australia, including Tasma- nia and New Zealand (Burbinge and Gray 1970, Webb et al. 1988), as well as Taiwan (Wang and Chen 2010), North America, southern Africa, and Argentina (Strausbaugh and Core 1978, Zohary 1991, Zuloaga and Morrone 1999). The study by Alexander (2010) supported a genetic basis for the differences in the elevation limits of L. serriola populations between two parts of its native and introduced ranges. Two primary morphological forms are recognized with- in L. serriola L. based on cauline leaf-shape variability; the pinnatifid-leaved form L. serriola L. f. serriola, and the un- lobed-leaved form L. serriola L. f. integrifolia (S.F. Gray) S.D. Prince et R. N. Carter. The serriola form is recorded as the most frequent species, occurring at a very high den- sity in Europe; the form integrifolia is not so common, and has been recorded in e.g., Switzerland, Italy, France, west- ern Germany, the Netherlands, and is prevalent in the UK (Lebeda et al. 2001, 2004, 2007a, b). Lactuca serriola is the best known wild species of the genus Lactuca, the geographic distribution, morphologi- cal, and phenological variations of which have been inten- sively studied (Lebeda et al. 2004, 2007a, Alexander 2010). L. serriola is also an important genetic resource for new re- sistance to diseases and pests (Lebeda et al. 2014), abiotic factors, as well as for genes responsible for physiological and quality characters (Lebeda et al. 2007a). Prickly let- tuce has been used in commercial lettuce breeding for more than 80 years (Lebeda et al. 2007a), especially as a source of race-specific resistance genes against lettuce downy mildew (Bremia lactucae Regel) (Parra et al. 2016). It has also been used over the last decade in various molecular studies to characterize genetic variation and diversity in both germ- plasm collections and natural populations (e.g. Koopman et al. 2001, Kitner et al. 2008, 2015). The most commonly used methods for the analysis of DNA polymorphism include amplified fragment length polymorphism (AFLP; Vos et al. 1995), and microsatellites (simple sequence repeats, SSRs); Simko (2009) contribut- ed significantly to the development of these for the genus Lactuca, and in particular for L. serriola Riar et al. (2011). These markers have been successfully applied in Lactuca re- search, addressing e.g., the distribution of the genetic varia- tion of prickly lettuce across Europe (Lebeda et al. 2009a), distribution of genetic variation in natural populations of L. serriola, L. saligna, and L. aculeata in Israel (Kitner et al. 2015), or analyses of gene flow from crops to their wild rela- tives (Uwimana et al. 2012). Southern / central Sweden is the northern limit of L. serriola distribution in Europe; Slovenia represents an area between the Central European and Mediterranean / Bal- kan distributions (Feráková 1977). The two areas differ in their climatic, ecogeographic, and ecologic conditions. In Slovenia, prickly lettuce is distributed throughout the en- tire territory, from the lowlands to the mountain regions (Martinčič and Sušnik 1984), and it most often grows in as- sociation with Stellarietea mediae – annual weed commu- nities species (Šilc and Košir 2006). In Sweden, L. serriola populations are found in southeastern areas, and mostly grow on surfaces and among stones in dry and sunny ex- posures (Doležalová et al. 2001). The genetic structure of populations represented by prickly lettuce plants growing at a specific time in a par- ticular site could emerge in at least four different ways: i) achenes can survive in a soil seed bank for 1 to 3 years (Marks and Prince 1982); at the moment of soil distur- bance, the seeds can germinate, and these plants bear/rep- resent “old” genotypes for a given population; ii) plants can grow from achenes newly transported to a particular local- ity by wind, humans, or other transport mechanisms, with such plants bearing “new” genotypes; iii) plants can grow on permanently disturbed soil from generation to genera- tion, and such plants represent a “modified” genotype re- sulting from continuous evolution under local conditions; iv) “hybrid” plants may appear after natural hybridization between different plant species within the genus Lactuca. The main purpose of this research was to describe the differences in genetic variability and population genetic structures between populations of prickly lettuce (Lactu- ca serriola) coming from two different and distant biogeo- graphic areas of the species’ distribution in Europe. Materials and methods Plant materials A set of 121 samples of L. serriola L. plants, represent- ing 53 populations, was collected by the authors in Swe- den (47 samples) and Slovenia (74 samples) during 2000 (Doležalová et al. 2001). The collected seed samples were regenerated in a greenhouse at the Department of Botany (Palacký University in Olomouc, Czech Republic). Dur- ing regeneration, the plants were described morphologi- cally according to Doležalová et al. (2002), and the taxo- nomic status of each sample was verified (Feráková 1977, Doležalová et al. 2002). From each plant two mature leaves were used for DNA extraction (i.e., 121 samples). Data from the individual samples are provided in On-line Suppl. Tab. 1., with the geographic positions of the collection sites giv- en in Fig. 1. DNA extraction, SSR, and AFLP analyses Total genomic DNA was extracted from 100 mg of fresh leaf tissue using the CTAB method (Kump and Javornik 1996), with minor modifications. After DNA extraction, the JEMELKOVÁ M., KITNER M., KŘÍSTKOVÁ E., DOLEŽALOVÁ I., LEBEDA A. 174 ACTA BOT. CROAT. 77 (2), 2018 quality of the DNA was inspected by 1.5% agarose gel elec- trophoresis, and the concentration measured on a Nano- Drop ND-1000 Spectrophotometer (NanoDrop Technolo- gies, Delaware, USA). For microsatellite genotyping, seven SSR loci were used: SML-002, SML-019, SML-045, SML-055 (Simko 2009), as well as WSULs-18, WSULs-75, and WSULs-163 (Riar et al. 2011). The primer pairs were selected according to their high diversity indices in previously published papers (Sim- ko 2009, Riar et al. 2011); however, randomly without any previous knowledge of their chromosome positions. Am- plification of the SSRs was performed according to Jemel- ková et al. (2015). The length of the SSR alleles was scored based on their migration relative to the molecular weight size markers 30-330bp AFLP® DNA ladder (Invitrogen, Carlsbad, California, USA). The AFLP analyses were car- ried out according to the protocol of Vos et al. (1995), with modifications, and the AFLP fragment detection according to Kitner et al. (2008, 2012). Five selective primer combina- tions, with two to three selective nucleotides, were chosen to generate the AFLP profiles (Tab. 2). The PCR products were separating on a 6%, 0.4 mm thick denaturating polyacrylamide gel using a T-REX se- quencing gel electrophoresis apparatus (Thermo Scientific Owl Separation Systems, Rochester, NY, USA). Tab. 1. Microsatellite (SSR) loci used to assess genetic variability in Lactuca sativa L. and L. serriola L.; NA – number of alleles; PIC – allelic polymorphic information content. Marker Reference NA Allele size (bp) PIC (%) SML-002 Simko (2009) 6 168-207 0.594 SML-019 Simko (2009) 2 163-164 0.599 SML-045 Simko (2009) 4 229-238 0.838 SML-055 Simko (2009) 5 221-240 1.072 WSULs-18 Riar et al. (2011) 4 208-235 0.494 WSULs-75 Riar et al. (2011) 4 161-206 0.684 WSULs-163 Riar et al. (2011) 7 183-197 1.052 Fig. 1. Collecting sites of the 121 samples Lactuca serriola in Swe- den and Slovenia. Colors of spots correspond to the results of Bayesian clustering presented in Fig. 2. Tab. 2. Amplified fragment length polymorphism (AFLP) primer sets for amplification reactions with the total number of scored and polymorphic fragments in the Lactuca serriola samples; NF – total number of fragments; NPOL – number of polymorphic frag- ments; P(%) – percentage of polymorphic fragments. Primer combination NF NPOL P(%) E - AGC, M - CTG 45 37 82.2 E - AGC, M - CAAC 49 36 73.5 E - AGC, M - CAAT 72 54 75.0 E - ACC, M - CAAC 43 35 81.4 E - ACC, M - CAAT 48 30 62.5 Total 257 192 Mean 51.4 38.4 74.9 Data scoring Microsatellite profiles were scored based on the length of the PCR product. The allele frequencies, percentage of polymorphic loci (P%), number of private alleles (PA), ob- served and expected heterozygosity (HO and HE) were all performed using GenAlEx 6 software (Peakall and Smouse 2012). The mean number of alleles per locus (A) was cal- culated manually. The relative discriminatory value of each microsatellite locus was estimated by the polymorphic in- formation content (PIC), which measures the information content as a function of a marker system´s ability to dis- tinguish between genotypes (Powell et al. 1996). The num- ber of different genotypes (NG), number of samples with a heterozygous constitution (NHET), and maximal number of heterozygous loci (NHETmax) were calculated manually. AFLP profiles were checked visually, and only clear and unambiguous bands were scored for their presence (1) or absence (0) across all samples. For AFLP data, the number of private bands (PA), the proportion of polymorphic loci (P%) and gene diversity (HE) were calculated using GenAl- Ex 6 software (Peakall and Smouse 2012). To evaluate the population genetic structure, a Bayesian clustering approach was used as implemented in Structure GENETIC VARIABILITY OF LACTUCA SERRIOLA POPULATIONS ACTA BOT. CROAT. 77 (2), 2018 175 2.3.4 (Falush et al. 2007). Structure attempts to assign indi- viduals to clusters/groups/populations on the basis of their genotypes, while simultaneously estimating population al- lele frequencies. This allows one to compute the likelihood of a given genotype having originated in a predefined num- ber (K) of clusters. In the simplest, ‘no-admixture’ model, it assumes that each individual belongs to a single cluster. In the more general ‘admixture model’ it estimates admixture proportions for each individual, allowing one to identify admixed individuals represented by a proportional mixture of two or more signals characteristic for the various clusters. In our analyses, SSR co-dominant data were transferred in- to binary data based on the presence/absence of a particular allele, and merged with the AFLP binary data; the samples were then ordered according to the increasing latitude of the sampling site within a particular country. An admix- ture model was used, with correlated allele frequencies. K was set at 1–10, and the highest K value was identified as the run with the highest likelihood value, as recommended by Pritchard et al. (2000). In addition, K values were aver- aged across 10 replicate runs for each K (100 000 burn-in iteration followed by 1 000 000 MCMC iterations). For the graphical interpretation of clustering for the appropriate K, Structure Harvester (Earl and von Holdt 2012), Clumpp (Jacobsson and Rosenberg 2007), and Distruct (Rosenberg 2004) software packages were used. The optimal K value was selected according to Evanno et al. (2005), who sug- gested the use of the ∆K value for identifying the correct number of clusters. To visualize the genetic relationships within and among the analyzed samples, a Neighbor-Network based on Dice´s similarity coefficient (D) was constructed in SplitsTree 4 (Huson and Bryant 2006). The Nexus input file for Split- sTree 4 was exported from GenAlEx. Also, for this pur- pose, the SSR data were transformed into a binary matrix and merged with the AFLP binary data. The reliability and robustness of the network were tested by bootstrap analysis with 1.000 bootstrap replicates. Results Taxonomic verification of L. serriola For all 121 plants, the taxonomic status of Lactuca serri- ola f. serriola according to Feráková (1977) was confirmed. Moreover, in one sample (no. 205_00, Bostahusen, Sweden) the plants were morphologically heterogeneous; with di- vided stem leaves belonging to L. serriola f. serriola, plants with entire stem leaves that ranged toward L. serriola f. inte- grifolia. In our analyses, this sample was split into two sub- samples 205_00A (f. serriola) and 205_00B (f. integrifolia) and treated (analyzed) separately. Genetic polymorphism The seven polymorphic SSR loci produced a total of 32 alleles across the 121 individual L. serriola plants. The num- ber of alleles per locus ranged from 2 to 7, with an aver- age of 4.57 alleles per locus (Tab. 1). The allele sizes varied from 161 to 240 bp. The mean PIC per SSR polymorphic allele was 0.762, within a range of 0.494 to 1.072. Null al- leles only appeared in two accessions from Slovenia (13_00 and 22_00) at the locus SML-055. Private alleles (PA) were present within both sam- pled regions (Tab. 3). The L. serriola samples from Swe- den possessed 5 unique alleles: 193 bp, 204 bp, and 207 bp for locus SML-002, 221 bp for locus SML-055 (i.e., 221 bpSML-055), and 188 bpWSULs-163. The samples from Slovenia possessed eight unique alleles: 172 bp, 198 bp for locus SML-002, 238 bpSML-045, 228 bpSML-055; 217 bp and 235 bp for locus WSULs-18, and lastly 183 bp and 195 bp for lo- cus WSULs-163. The observed and expected heterozygosity (HO and HE) ranged from 0.036 to 0.054 (mean 0.045), and from 0.341 to 0.432 (mean 0.387), respectively. The proportion of polymorphic loci (P%) was higher in the Slovenian (84.4%) than in the Swedish samples (75%). Based on SSR data, in all, 51 different genotypes (NG) were recognized (Sweden = 17; Slovenia = 34) (On-line Suppl. Tabs. 2,3). Genotype G3 was the most common in the samples from Sweden (36.2%), while genotype G29 represented 32.4% of the Slo- venian samples (On-line Suppl. Tabs. 2,3). We recorded 17 Slovenian samples that had at least one heterozygous lo- cus (NHET = 17), in contrast to eight samples from Sweden (On-line Suppl. Tabs. 2,3). Three samples from Slovenia and one sample from Sweden bore the maximum number of heterozygous loci (NHETmax = 3) observed from among all analyzed samples. In total, five primer combinations, with two to three se- lective bases, were applied for AFLP genotyping (Tab. 2), resulting in 257 unambiguously scored fragments. Detailed overall statistics calculated for each primer combination used are presented in Table 2. The number of private bands (PA) ranged from 19 (Slovenian samples) to 20 (Swedish samples). The expected heterozygosity (HE) ranged from 0.130 to 0.149 (mean HE = 0.140) (Tab. 3), and the propor- tion of polymorphic loci (P%) in the L. serriola samples ranged from 44.8% (Swedish population) to 52.9% (Slo- venian population). The genetic variability indices for all populations are summarized in Table 3. Cluster analysis of molecular data Based on seven microsatellite and 257 AFLP markers, Bayesian clustering and construction of a Neighbor-Net- work were used for visualization of the putative relation- ships among the analyzed individuals. Under the Bayesian approach implemented in Structure, the best partition into three clusters (K = 3, Fig. 2) was resolved (∆K = 214.73; St. dev. LnP(K) = 6.07); they are represented by the green (G- cluster), red (R-cluster), and blue (B-cluster) color signals in Figure 2. In general, a relatively low admixture was detect- ed in the Slovenian samples, which were clearly identified as genotypes from the G- or B-cluster. While the B-cluster can be considered as characteristic for L. serriola genotypes JEMELKOVÁ M., KITNER M., KŘÍSTKOVÁ E., DOLEŽALOVÁ I., LEBEDA A. 176 ACTA BOT. CROAT. 77 (2), 2018 from the southern part of Central Europe and the northern Balkans (representing ca. 1/3 of the Slovenian samples), the G-cluster represents the genotype largely dispersed across Europe, contributing significantly to the genotypic compo- sition of the Swedish populations. The signal characteristic for genotypes from the R-cluster was nearly absent in the Slovenian samples, but was recorded in each sample from Sweden; and 48.9% of the Swedish samples fell into the R- cluster with no admixture signal (Fig. 2). For 19 samples, the signal from the R-cluster contributes up to 30% of a par- ticular genotype, and is accompanied with an admixture of the G signal, which prevails in the Slovenian samples (Fig. 2). Further, we observed a nearly equal admixture of signals from all three clusters in five samples collected in southern Sweden near Malmö. The Neighbor-Network analysis divided the analyzed samples into 6 groups (A-F; Fig. 3), each consisting of sam- ples coming from a separate country. The results fit the re- sults of the Bayesian clustering in terms of assigning indi- viduals from a separate country to the revealed clusters (R-, G-, B-). The samples from Sweden were placed into the A, C, and D groups. While individuals placed in Group C rep- resent the genotype from the R-cluster, Group D is formed by samples with the G-cluster prevailing. Finally, Group A is formed by five samples 215_00, 217_00, 218_00, 219_00, and 220_00, having a strong admixture signal from all three Structure clusters. These samples represent populations no. 16 and 17 from collecting sites close to Malmö (On-line Suppl. Tab. 1). The samples from Slovenia were split into three groups: a majority of the samples fell in groups B and E, both representing the G-cluster in Fig. 2. Samples orig- inating from Slovenian localities below 46°14'34" lat. fell into a separate Group F, which represents genotypes from a unique B-cluster (Fig. 2). It is interesting, that all three Fig. 2. Results of Bayesian clustering based on the microsatellite (SSR) and amplified fragment length polymorphism (AFLP) data of 121 Lactuca serriola samples from Sweden (SWE) and Slovenia (SLO), ordered according to the increasing latitude of the sampling site within a specific country. Each individual is represented by a horizontal line partitioned into segments of different color, the lengths of which indicate the posterior probability of membership in each group as identified by Structure. Fig. 3. Neighbor-Network cluster analysis of 121 samples Lactuca serriola from Sweden and Slovenia, based on SSR and AFLP analy- sis. Resulting groups are highlighted by coloring that corresponds to the results of Bayesian clustering presented in Fig. 2. GENETIC VARIABILITY OF LACTUCA SERRIOLA POPULATIONS ACTA BOT. CROAT. 77 (2), 2018 177 “G-cluster” groups from both countries are in the center of the Neighbor-Network, which resemble their characteris- tics closely. On the other hand, Group C (SWE, R-cluster) and Group F (SLO, B-cluster) are placed on opposite sides of the network. Discussion Verification of the taxonomic status of the plants showed that Lactuca serriola f. serriola is predominant in both countries. In the entire territory of Slovenia only L. serriola f. serriola was recorded, which is in agreement with previous observations in Central Europe (Lebeda et al. 2001, 2004, 2007b). Within one sample from southern Swe- den (Bostahusen, sample 205_00), apart from L. serriola f. serriola plants, there were plants identified as L. serriola f. integrifolia. All remaining samples from Sweden were rep- resented only by L. serriola f. serriola. It is evident that both populations are very taxonomically homogeneous on the subspecific level. The very rare occurrence of L. serriola f. integrifolia in southern Sweden could be caused by the re- peated introduction (e.g., through truck or ship transporta- tion) of this form from the Netherlands or UK, where it is prevalent (Lebeda et al. 2007a, b). However, from our pre- vious results (Doležalová et al. 2001) it is evident, that this variety is not spreading into northern Scandinavia, where the northern limit of the European distribution for this spe- cies is (Feráková 1977). These conclusions are supported by recent observations in Sweden made by Rydberg (2013). Al- so, in Norway only L. serriola f. serriola has been recorded (Lebeda 2013, unpubl. results). The leaf shape (i.e., the division of the leaf blade), can be interpreted as an ecological adaptation of the plant to differ- ent factors, including a means of leaf thermoregulation in arid or hot environments, or in reaction to hydraulic con- straints (Nicotra et al. 2011). Doležalová et al. (2009) also confirmed the differences in the morphology of rosette and cauline leaves of Swedish and Slovenian L. serriola samples. The cauline leaves of Swedish L. serriola plants were longer and wider; plants from Slovenia had longer and narrower rosette leaves (divided) (Doležalová et al. 2009). The width and length of cauline leaves (divided) correlate with the lati- tude, which could be explained as adaptations of the plants to drought. Drier areas of lower latitudes are increasingly represented by plants with smaller leaves. Regarding alti- tude, a negative correlation with the length and width of the leaves was found (Doležalová et al. 2009), which could mean they are adapting to ecologically worse conditions at higher elevations. The occurrence of L. serriola f. integ- rifolia in temperate areas without a dry season (but with a warm summer) in the UK, western part of Germany, Ben- elux, and France (Peel et al. 2007) supports the theory of the ecological adaptation of leaves presented by Nicotra et al. (2011). The areas in Sweden where lettuce samples were collected belong to the cold climate type, without a dry sea- son or warm summer (Peel et al. 2007). Similarly significa- nt differences in morphological parameters of achenes of L. serriola from Slovenia and Sweden were found between populations within countries and between samples within population (Křístková et al. 2014). The higher phenotypic and genetic variability of the Slo- venian samples can be explained by the more favorable cli- matic and ecological conditions in the country (see Peel et al. 2007). L. serriola is distributed throughout the entire country, and movement of diaspores among the surround- ing countries is feasible (Lebeda et al. 2004). This is in op- position to Sweden, where the distribution is limited to the southern part (Doležalová et al. 2001), with very limited migration from the surrounding countries. In general, plant species occurring almost in and/or near the center of their diversity, with suitable environmental and ecological con- ditions, display more genetic/phenotypic variability. Con- versely, at the edge of the distribution area, where less fa- vorable conditions exist, the selection prioritizes stable and well-adapted genotypes. Our results on genetic variabili- ty are in relationship to the general principles of diversity and allele distribution formulated by Vavilov (1950). Kuang et al. (2008) suggested that eastern Turkey and Armenia, along with the surrounding regions, might be the center of diversity of L. serriola (and possibly its center of origin). L. serriola might have spread from its center of origin first to the Mediterranean basin and then to Central and West- ern Europe after the glaciers retreated in the Upper-Pleisto- cene / Holocene period (Kuang et al. 2008). Recent climatic changes and anthropogenic disturbances contributed sub- stantially to the rapid spread of L. serriola into new areas (D´Andrea et al. 2009, Rydberg 2013), as well as increasing the genetic diversity of their populations in the central parts of their natural distribution areas (Lebeda et al. 2009a, van de Wiel et al. 2010, Kitner et al. 2015). This phenomenon Tab. 3. Summary data based on 7 SSR and 257 amplified fragment length polymorphism (AFLP) loci of 121 Lactuca serriola samples from Sweden and Slovenia in recent study: N – sample size; PASSR – private microsatellite alleles; PAAFLP – private AFLP bands; A – mean number of alleles per locus; P(%) – percentage of polymorphic loci; observed Ho and expected He heterozygosity; SE – standard error. Country N Microsatellite (SSR) data AFLP data PASSR A P(%) HO HE ± SE PAAFLP P(%) HE ±SE Sweden 47 5 3.42 75.0 0.036 0.341 ± 0.065 20 44.8 0.130 ± 0.011 Slovenia 74 8 3.86 84.4 0.054 0.432 ± 0.049 19 52.9 0.149 ± 0.011 JEMELKOVÁ M., KITNER M., KŘÍSTKOVÁ E., DOLEŽALOVÁ I., LEBEDA A. 178 ACTA BOT. CROAT. 77 (2), 2018 was also clearly demonstrated in the genetic diversity of the Central European population of L. serriola (van de Wiel et al. 2010), as well as the resistance of the same population to Bremia lactucae. While the Czech Republic has the greatest diversity of resistance phenotypes, the lowest was recorded in the UK (Lebeda et al. 2008, Petrželová and Lebeda 2011). The results of our study on genetic variability are in good agreement with the different climatic conditions in Sweden and Slovenia. From the viewpoint of genetic vari- ation, the results have proven the existence of L. serriola genotypes characteristic for each country. These clearly dif- fer from one another, as is evident from Bayesian cluster- ing and Neighbor-Network analysis, where the R-cluster characteristic for the Swedish samples (Group C), and the B-cluster (Group F) unique for Slovenian samples were dis- tinguished (Figs. 2, 3). A number of samples from both countries were characterized by genotypes characteristic for the G-cluster, which might represent a common geno- type resulting from the rapid spread of L. serriola in Central Europe (Lebeda et al. 2001, 2007b, D´Andrea et al. 2009). We have not recorded a prevailing microsatellite genotype for the samples representing this G-cluster, and no linkage to the latitude or altitude of the sampled sites. The same phenomenon was described by Lebeda et al. (2009a), dem- onstrating that some L. serriola populations (e.g., Scan- dinavian, British, some Mediterranean) are quite isolat- ed genetically from the heterogeneous Central and West European populations. Genetic analysis (PCR-RFLP and SSR markers) on 101 populations of L. serriola from sev- enteen countries of Western and Central Europe made by D´Andrea et al. (2017) revealed a strong genetic differentia- tion between populations, and high inbreeding coefficients within populations. A clear geographical pattern of isola- tion by increasing distance was found; however, only a weak pattern of correlation between genetic diversity and geo- graphical distance was found on the continental scale. The greatest amount of genetic diversity was characterized in Central Europe, while populations from the western Med- iterranean (Spain and Portugal), southern Italy, Great Brit- ain, the Alps, and southern Scandinavia generally possessed lower gene diversities (D´Andrea et al. 2017). Discrepan- cies were present in Scandinavia with some polymorphic populations, and a monomorphic one. Further, in a recent study, higher genetic variability in the Slovenian samples was observed in terms of the recorded genetic variability indices (Tab. 3) and the higher number of SSR genotypes (SWENG = 17/SLONG = 34) (On-line Suppl. Tabs. 2,3). The level of genetic variation within and between populations can al- so result from intraspecific crossing. Although autogamy is the predominant breeding system within the genus Lactuca L., especially in the marginal parts of the distribution area (Feráková 1977); in the center of the distribution, a high- er occurrence of allogamy was estimated (Stebbins 1957). Lindquist (1960) proved experimentally that all species be- longing to the “serriola” group were self-fertile. L. serriola is primarily a self-pollinated species; however, not only in- termediate plants between the two L. serriola forms, but also interspecific hybrids of L. serriola can be detected in natural populations (Zohary 1990, Křístková et al. 2012). The main differences between the samples from Sweden and Slovenia can be characterized by the presence of geno- types characteristic for the R- or B-cluster, determined by Bayesian clustering (Fig. 2), each unique (with a few ex- ceptions) to a given country. The signal from the R-cluster was present in all Swedish samples and prevails in 48.9% of them. These samples formed Group C on the Neighbor- Network (Fig. 3), 65.2% of them represent the SSR geno- type G3, with a completely homozygous character at all loci, and originating from localities at a higher latitude (On-line Suppl. Tab. 2). A rather interesting characteristic of five L. serriola sam- ples was found in a group of plants collected near Malmö. These samples forming Group A on the Neighbor-Net- work, are represented by a significant admixture signal on the Bayesian diagram, and also bore unique morphologi- cal features of their rosette leaves. The apical parts of the rosette leaves in samples 215_00, 217_00, 218_00, 219_00, and 220_00 were not divided, forming a long apex; the re- maining two-thirds of the leaves were slightly divided (pin- nately lobed). Surprisingly, specific DNA patterns fit better to specific phenotypes of the rosette leaves than to pheno- types of the cauline leaves. This is in contrast to the generally accepted view that morphological traits of the cauline leaves have a more significant taxonomic value than do the rosette leaves. The city of Malmö is an international harbor in the region, and it is possible to explain the exceptional pheno- typic characteristics of these samples by the human-mod- erated introduction of non-indigenous genotypes into the southern parts of northern Europe, with subsequent natural hybridization with indigenous L. serriola genotypes. The B- cluster in Slovenian samples showed, with a few exceptions, a continuity with samples from a lower latitude; 96% of these samples are represented by the completely homozygous mi- crosatellite genotype G29 (On-line Suppl. Tab. 3). This study provides interesting insights into the genetic variability of L. serriola populations originating from com- pletely different eco-geographical areas. Specifically those from Slovenia, near the Mediterranean, a world diversity hotspot (Myers et al. 2000), the center of the greatest diver- sity of the genus Lactuca (Lebeda et al. 2009b); addition- ally, those from Sweden, a region at the northern border of L. serriola European distribution (Feráková 1977, Lebeda et al. 2004). This study showed that L. serriola populations originating from various eco-geographical conditions dif- fer significantly in their genetic background, which is also reflected in the geographic patterns of their phenotypic fea- tures. To obtain more comprehensive information on the genetic structure and variations of this species, it would be interesting to analyze: i) more populations with more indi- viduals from Sweden, and for a comparative study ii) addi- tional samples originating from areas with greater contrast- ing ecological conditions. GENETIC VARIABILITY OF LACTUCA SERRIOLA POPULATIONS ACTA BOT. 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