$¢PhytoKeys PhytoKeys 246: 295-314 (2024) DOI: 10.3897/phytokeys.246.118796 Research Article Glacial history of Saxifraga wahlenbergii (Saxifragaceae) in the context of refugial areas in the Western Carpathians Elzbieta CieSlak'®, Michat Ronikier’®, Magdalena Szczepaniak'® 1 W. Szafer Institute of Botany, Polish Academy of Sciences, Lubicz 46, PL-31-512 Krakow, Poland Corresponding author: Elzbieta Cieslak (e.cieslak@botany. pl!) OPEN Qaceess Academic editor: Pamela S. Soltis Received: 14 January 2024 Accepted: 14 August 2024 Published: 20 September 2024 Citation: Cieslak E, Ronikier M, Szczepaniak M (2024) Glacial history of Saxifraga wahlenbergii (Saxifragaceae) in the context of refugial areas in the Western Carpathians. PhytoKeys 246: 295-314. https://doi.org/10.3897/ phytokeys.246.118796 Copyright: © Elzbieta Cieslak et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Abstract Despite the wealth of data available for mountain phylogeography, local-scale studies focused on narrow endemic species remain rare. Yet, knowledge of the genetic structure of such species biogeographically linked to a restricted area is of particular importance to understand the history of the local flora and its diversity patterns. Here, we aim to contribute to the phylogeographical overview of the Western Carpathians with a genetic study of Saxifraga wahlenbergii, one of the most characteristic endemic species of this region. We sampled populations from all discrete parts of the species’ distribution range to apply sequencing of selected non-coding cpDNA and nuclear ribosomal DNA (ITS) regions, as well as Amplified Fragment Length Polymorphism (AFLP) fingerprinting. First, while ITS sequences showed weak diversification, the genetic structure based on cpDNA sequences revealed two well-differentiated groups of haplotypes. One of them is restricted to the main center of the distribution range in the Tatra Mountains (Mts), while the second group included a series of closely related haplotypes, which in most cases were unique for particular isolated groups of populations in peripheral mountain ranges and in the south-eastern part of the Tatra Mts. AFLP fingerprinting also revealed a pattern of divergence among populations, while only partly corroborating the division observed in cpDNA. Taking into account all the data, the pattern of genetic structure, supported by the high levels of unique genetic markers in populations, may reflect the historical isolation of populations in several local refugia during the last glacial period. Not only the center of the range in the Tatra Mts, but also other, neighboring massifs (Mala Fatra, Nizke Tatry, Choéské vrchy, Muranska planina), where populations are char- acterized by separate plastid DNA haplotypes, could have acted as separate refugia. Key words: AFLP, haplotype, high mountain plant, narrow endemic, phylogeography, refugial areas, Tatra Mts Introduction Mountainous systems of temperate Europe are biodiversity hotspots, due, among others, to their high floristic richness, including endemic species that contribute to the natural uniqueness of a given biogeographical unit. The evo- lution of mountain species is strongly related to historical environmental and climatic factors, including the Pleistocene climatic fluctuations that caused alternating glacial and interglacial periods (Hewitt 2000, 2004; Kadereit et al. 295 Elzbieta Cieslak et al.: Glacial history of Saxifraga wahlenbergii 2004). Phylogeographic analysis of various mountain species allowed us to discover genetic patterns and, to some extent, understand mechanisms and dynamics of processes involved in the formation of local floristic hotspots within large mountain massifs. The results have made it possible to determine the scenarios of events and processes related to the survival of species during glaciations, including the location of refugial areas and recolonization routes to the areas currently occupied by these species. The revealed convergence of genetic diversity patterns between mountain species indicates that similar factors had a prevailing role in shaping the history of the flora in different areas, especially with regard to the history of Quaternary glaciations (e.g., Koch et al. 2003; Kropf et al. 2008; Lowe and Allendorf 2010; Ronikier 2011; Schmitt 2017; Sramkova-Fuxova et al. 2017; Koneéné et al. 2019). Endemic species are a special element of the biodiversity of mountain areas. In this group, species with highly limited local ranges are of particular impor- tance, as they most often reflect evolution in specific microclimatic isolation in geographically small and isolated areas (Kochjarova et al. 2006; Cieslak et al. 2007, 2021; Pittet et al. 2020). Their emergence confirms the role of local ecological niche systems (including topography, climate, and bedrock factors) in shaping the regional mountain flora during historical evolutionary processes. In mountain ranges, establishing an understanding of such local systems is crucial to understanding their contemporary distribution and biodiversity. Con- sequently, it will allow the determination of the response range of these unique, often isolated, local habitat systems to past and ongoing climate fluctuations, such as the Pleistocene glacial/interglacial sequences (Nagy et al. 2003; Ste- hlik 2003; Kadereit et al. 2004; Tribsch 2004; Schmitt 2009; Gentili et al. 2015). It has been confirmed that the Western Carpathians are an important, inde- pendent center of endemism with both Pan-Carpathian species and a group of endemic species specific to this region (Pawtowski 1970; Kliment 1999; Piekos-Mirkowa and Mirek 2003; Mraz and Ronikier 2016). Their location close to the northern ice sheet during glacial periods, heterogeneous environments constituting a mosaic of habitats, and microtopographically diverse environ- ments on a regional scale were crucial to the establishment of an important evolutionary center in this region of the Carpathians (Paclova 1977). Recent- ly, phylogeographic analyses of Cochlearia tatrae, the endemic species of the Tatra Mts, showed a significant level of intra-specific variability with several geographically arranged genetic groups in this small mountain range (Cieslak et al. 2021). This pattern may reflect the isolation of the populations in several micro-refugia, indicating that the systems of local factors in the Tatra Mts and its outskirts were crucial in formation of the genetic structure of this species. In this study, we address Saxifraga wahlenbergii Ball, one of the most char- acteristic endemic species of the Western Carpathians, with wide ecological preferences, including a large elevational range and limited bedrock restriction. It occurs in several massifs with its range center in the Tatra Mts (Piekos-Mir- kowa et al. 1996). Recent phylogenetic study based on nrDNA and cpDNA re- gions revealed a complex hybrid origin of S. wahlenbergii with unidirectional introgression and different parental contributions observed in extant genotypes (Tkach et al. 2019). The maternal parent has been shown to belong to the West Eurasian lineage of alpine taxa grouped in the subsection Androsaceae, most likely the widespread S. androsacea. The putative paternal parent was probably PhytoKeys 246: 295-314 (2024), DOI: 10.3897/phytokeys.246.118796 096 Elzbieta Cieslak et al.: Glacial history of Saxifraga wahlenbergii S. adscendens, which belongs to a distantly related subsection Tridactylites. Contribution from both groups was confirmed by a next-generation sequenc- ing (NGS) analysis of within-individual ITS variation (Tkach et al. 2019). The supported topological incongruencies between phylogenies reconstructed from nuclear and plastid DNA regions, as previously found (Tkach et al. 2015; Gerschwitz-Eidt and Kadereit 2020), may suggest that interspecific transfer of adaptive traits through hybridization may have played an important role in the evolution of Saxifraga sect. Saxifraga. In general, hybrid speciation events involv- ing polyploidization are common in the genus Saxifraga and have played an im- portant role in the diversification of this large genus (e.g., Ebersbach et al. 2020). Interestingly, in the framework of the phylogenetic analysis, regional genetic variation was found in the populations of S. wahlenbergii including two distinct cpDNA clades, which was a direct motivation to undertake a more detailed analy- sis of the species’ phylogeographical structure. While unequivocally dating the hy- brid origin of S. wahlenbergii could not be assessed, it could theoretically precede the Pleistocene and several arguments pointed to a possibly ancient rather than recent age of this species (Tkach et al. 2019), hence also its geographical range. In the region of the Western Carpathians, with its high topographic and hab- itat heterogeneity, S. wahlenbergii, a plant with a rather wide altitudinal range, could survive in the massifs where it occurs today, over the Pleistocene cli- matic oscillations, following the altitudinal shifts or persistence in long-term, non-glaciated microrefugia. However, as cold (glacial) periods have led to a sig- nificant increase in habitats suitable for alpine plants in the lower parts of the Carpathians (Ronikier 2011), the present range could also result from a recent (Last Glacial Maximum) migration to peripheral massifs. The main objective of this study is to determine the range-wide genetic structure of S. wahlenbergii, to provide insight into its glacial history. For this purpose, an extended sequence analysis of selected nuclear and plastid DNA regions complementing data from Tkach et al. (2019) and supplemented by population genotyping with Amplified Fragment Length Polymorphism method (Vos et al. 1995; Kirschner et al. 2021), were used. The study was based on the analysis of S. wahlenbergii populations from the area of their highest density in the Tatra Mts and those from all neighboring mountain massifs, where it occurs less abundantly (sometimes as isolated populations). Genetic diversity and divergence were analyzed to identify potential distinct lineages and areas of genetic discontinuities of species. Based on the above data, an attempt was made to resolve whether the con- temporary distribution results from long-term survival in several isolated areas (local refugia) and thus is a relic of ancient events or whether the species re- cently spread from a single refugium (likely located in the Tatra Mts — central part of the range). Materials and methods Study species Saxifraga wahlenbergii Ball (sect. Saxifraga; Saxifragaceae) is an endemic pe- rennial species of the Western Carpathians (in Poland and Slovakia). Its range includes the massifs of Tatra Mts, Mala Fatra Mts, Chocské vrchy Mts, Nizke PhytoKeys 246: 295-314 (2024), DOI: 10.3897/phytokeys.246.118796 997 Elzbieta Cieslak et al.: Glacial history of Saxifraga wahlenbergii Tatry Mts, and Muranska planina. However, it is a common species only in the Tatra Mts (the highest and environmentally most complex massif of the West- ern Carpathians) — it is abundant at higher altitudes above the tree line (up to 2540 m a.s.l.) and also descends to lower elevations, e.g., along streams (880 m a.s.l.) (Pawtowska 1966). The species grows on both limestone and granitic substrates, with preference for limestones. Throughout its range, it is found mainly on moist edges of limestone and granite screes, in the shade of rocks, on ledges and in rock crevices or on the edge of forests. It is a charac- teristic species of the Saxifragetum wahlenbergii community (Matuszkiewicz 2005) (described as Saxifragetum perdurantis Pawtowski and Stecki 1927). This community is considered an endemic community of the limestone Tatra Mts, Mala Fatra Mts and Choéské vrchy Mts (Matuszkiewicz 2005). Species is a hexaploid with a chromosome number of 2n = 66 (x = 11) (Skalifska 1963). Population sampling Plant material of Saxifraga wahlenbergii was sampled in natural populations, spanning the entire natural distribution area of this species in the Western Carpathians. Populations were assigned to regional geographical units, which were further assigned into predefined groups: the Western Tatra Mts, the Eastern Tatra Mts and those outside of the Tatra Mts, including localities from: the Mala Fatra Mts, Choéské vrchy Mts, Nizke Tatry Mts and Muranska planina (Kondracki 1989) (Fig. 1A and Table 1 for location details). The num- ber of samples per population varied and depended on population size. Spe- cial attention was paid to include samples from all isolated massifs where the species occurs. In total, our dataset comprised 57 individuals collected from 11 populations of S. wahlenbergii. Leaves from each individual were placed in a tube or bag with silica gel immediately after collecting and stored at room temperature until the DNA isolation. Herbarium material (vouchers) was collected only from large populations, due to conservation reasons and deposited in the Herbarium of W. Szafer Institute of Botany, Polish Academy of Sciences in Krakow (KRAM). Laboratory analysis The total genomic DNA was isolated from 5-10 mg of dried leaf tissue of col- lected samples using the DNeasy Plant Mini Kit system (Qiagen, Hilden, Germa- ny) according to the manufacturer's protocol (final elution step was carried out using 2x50 uL of elution buffer). DNA quality and concentration were estimated against A-DNA on 1% agarose gel stained with ethidium bromide. The purified DNA isolates were the basis of DNA sequencing and AFLP analyses. Samples from the Muranska planina population (S11) were collected later than the core sample set and they could only be used in the sequencing analysis. The non-coding chloroplast DNA regions (cpDNA) — rps16-trnK and rp/32- trnL (Shaw et al. 2007) - and nuclear ribosomal DNA region (nrDNA) — ITS (ITS1A-ITS4, White et al. 1990; Blattner 1999) — were used for DNA sequencing. Primers were selected and analyzed according to the protocols described in detail by Tkach et al. (2015, 2019). PhytoKeys 246: 295-314 (2024), DOI: 10.3897/phytokeys.246.118796 998 Elzbieta Cieslak et al.: Glacial history of Saxifraga wahlenbergii Qs 7 em Tatra Mts 2 Tatra Mts sto 4 Yt ie wt , 49°00'N » Sta border between Poland and Slovakia - -— - — border of the Tatra Mountains border between physiographical units $1-S11 localities Figure 1. Location of studied populations of Saxifraga wahlenbergii and their genetic variability based on DNA sequence data A distribution of 11 populations of S. wahlenbergii and haplotypes and ribotypes in the populations B haplotype network based on the combined chloroplast regions: rps16-trnK and rp/32-trnL C ribotype network based on ITS region. Networks obtained from TCS based on a 95% connection limit. The relative sizes of circles in networks are proportional to haplotype and ribotype frequencies. For population acronyms see Table 1. AFLP analysis was performed according to Vos et al. (1995) as described in detail by Cieslak et al. (2007). For high-quality AFLP profiles, we tested selective primer combinations using four individuals from distant popula- tions. All samples were analyzed using three selective primers combinations that yielded clear, unambiguous, and polymorphic profiles — EcoRI-AAG/Msel- CTG, EcoRI-ACT/Msel-CAG and EcoRI-AGA/Msel-CAC. Genotyping reproduc- ibility was tested by including duplicates for each population (Bonin et al. 2004). Amplification products were separated with an internal size standard (GeneScan ROX-500) on the ABI Prism 3100 Avant automated sequencer us- ing POP-4 polymer (Applied Biosystems, Foster City, CA, USA). Obtained AFLP marker sets were imported to Genographer Software (v. 1.6.0; J. Benham, Montana State University, 1998-2001) (Benham et al. 1999), which was used to score the fragments in the range of 50-500 bp. AFLP fragments for primer combination were saved as present (1) or absent (0) for a binary data matrix (see Suppl. material 1). PhytoKeys 246: 295-314 (2024), DOI: 10.3897/phytokeys.246.118796 999 Elzbieta Cieslak et al.: Glacial history of Saxifraga wahlenbergii Table 1. Localities of populations of Saxifraga wahlenbergii used in the study and parameters of their genetic variability based on AFLP, nrDNA (ITS) and cpDNA sequences. N,/N, — population sampling for AFLP analysis and DNA sequenc- ing; P/% — number and percentage of polymorphic markers; He — mean (+SD) Nei’s gene diversity; / - mean (+SD) Shan- non’s Index; DW — frequency down-weighted marker values; R — ribotypes (variants of ITS of nrDNA) and H — haplotypes (variants of cpDNA) in population (the number of individuals representing a particular ribotype or haplotype is given in parentheses). Country code: PL - Poland, SK - Slovakia. Collectors code: AD - Anna Delimat, AR — Anna Ronikier, MR — Michat Ronikier, RL - Roman Letz, PM — Patrik Mraz, PT — Peter Turis. ; AFLP ITS cpDNA Code Locality N./N, P/% He I DW R(No.) H(No.) Western Tatra Mts (Tatry Zachodnie, Zapadné Tatry) Si PL, Dolina Chochotowska valley, 1370 m a.s.l., 5/2 | 59/27.31 0.10 0.15 leh R1(2) H1(2) A9°14'N, 19°48'E (AD) (40.18) | (+0.26) S2 PL, between the Gaborowa Przetecz pass and A/2 | 61/28.24 0.12 0.17 20.06 R2(2) H1(1) H3(1) Bystra Przetecz pass, ~1930 ma.s.l., 49°12'N, (+0.19) (+0.28) 19°49'E (RL, PM) S3 PL, Przetecz pod Kopa Kondracka pass, 1500 m 5/2 | 61/28.24 0.11 0.16 25.07 R1(2) H1(1) H4(1) a.s.l., 49°14'N, 19°55'E (AD) (40.18) | (40.26) S4__| PL, Piekietko (Piekto) valley, 1640 ma.s..,49°14'N, | 9/2 |91/42.10, 0.15 0.23 | 58.57. R2(1) R3(1)/ ~—«H1(2) 19°56'E (AD) (40.20) | (+0.28) Eastern Tatra Mts (High Tatra Mts, Tatry Wysokie, Vysoké Tatry) S5 PL, N slopes of the pass Zawrat, 2100 m a.s.l., 4/2 | 79/36.57 0.14 0.20 25.46 | R2(1) R3(1) | H1(1) H2(1) A9°13'N, 20°01'E (MR) (40.19) | (40.28) S6 _| PL, Mieguszowiecki Szczyt Czarny Mt., 2220 m 7/2 | 83/38.43 0.14 0.20 36.37 R1(2) | H1(1)H7(1) a.l.s.,49°11'N, 20°03'E (AD) (40.20) | (+0.28) 57 SK, Hruby vrch Mt, ~ 2350 m a.s.I.,49°10'N, 20°01'E | 8/2 | 85/39.35 0.14 0.21 40.09 R3(2) H7(2) (MR) (40.19) | (40.28) Mala Fatra Mts S8 _| SK, Velky Rozsutec Mt., 1550 ma.s.l., 49°14'N, 5/2 | 76/35.19| 0.13 0.19 | 26.90| R1(1)R5(1)| 10 (2) 19°06'E (MR, AR) (40.19) | (40.27) Choéské vrchy Mts S9__| SK, Velky Choé Mt., 1600 ma.s.l., 49°09'N, 19°20 | 4/2 60/27.78 0.10 0.15 | 23.36 R1(1) R4(1) | H5(1) H6(1) (MR) (40.18) | (+0.26) Nizke Tatry Mts S10 | SK, Sind Mt., 1422 ma.s.l., 49°00'N, 19°33'E (RL,PT) | 4/2 | 61/28.24' 0.11 0.27. (29.54) R1(2) | H8(1) H9(1) (40.19) | (40.27) Spissko-gemersky kras S11__| SK, Muranska planina, Vel’ka Stozka, 1242 ma.s.l., | -/2 = i 7 — |R1(1)R2(1)| H11 (2) 48°46'N, 19°58'E (PT) cpDNA and ITS of nrDNA data analysis Analyses of cpDNA and ITS of nrDNA regions were performed separately. For- ward and reverse DNA sequences data were automatically assembled and aligned based on ClustalW algorithm (Thompson et al. 1994; Larkin et al. 2007) using the Geneious Pro 6.0.2 program (Drummond et al. 2011). The obtained sequences are deposited in GenBank with accession numbers: rps16-trnK — 0Q706232-53; rp/32-trnL -— 0Q706254-73, OR682717-18 and ITS1A-ITS4 - 0Q678158-79 (see Suppl. material 2). Gene diversity (h) and nucleotide diversity (™) were calculated based on cpDNA and ITS sequence variation for the total sample of Saxifraga wahlenber- gii and for predefined region groups within the species range using the DNAsp 5.0 program (Librado and Rozas 2009). To detect past demographic expansion, evidence for possible selection and/or genetic bottlenecks, Tajima’s D neutral- ity test was implemented (Tajima 1989). Indels were treated as single poly- morphic sites. The relationships between nrDNA ITS ribotypes (R) and cpDNA PhytoKeys 246: 295-314 (2024), DOI: 10.3897/phytokeys.246.118796 300 Elzbieta Cieslak et al.: Glacial history of Saxifraga wahlenbergii haplotypes (H) were separately analyzed using the statistical parsimony (SP) algorithm (Templeton et al. 1992) as implemented in TCS v1.2 (Clement et al. 2000); coding indels longer than 1 bp were treated as single characters. Statis- tical parsimony networks and the maximum number of mutational steps were obtained with 95% connection limit approach. Phylogenetic reconstruction us- ing Bayesian inference was accomplished with MrBayes 3.2.7a program (Ron- quist et al. 2012). The analysis was completed for four chains, parameter values of nst=6 and rates=invgamma, 50 million generations with sampling trees every 100 generations. 25% of the initial trees were discarded and the remaining 75% were used to build majority consensus tree and to calculate Bayesian posterior probabilities. The tree was visualized using FigTree 1.4.2 (Rambaut 2014). AFLP data analysis The genetic diversity of Saxifraga wahlenbergii at species and within-species level (populations) was assessed on the basis of binary AFLP data matrix by calculating the genetic parameters, including the number (P) and percentage of polymorphic markers (%), Nei’s gene diversity (He), Shannon’s information index (/) and gene flow (Nm) using POPGENE v. 1.32 software (Yeh et al. 1999). In order to identify long-term isolated and genetically unique populations, fre- quency down-weighed marker values (DW; Sch6nswetter and Tribsch 2005) were calculated using R-script AFLPdat (Ehrich 2006). The relationships among individuals and populations were analyzed by a Principal Coordinates Analysis (PCoA) based on the Nei-Li genetic distance matrix computed in FAMD v. 1.25 (Schliiter and Harris 2006) and by a split network (Neighbor-Net) also based on the Nei-Li coefficient with branch support estimated by bootstrapping with 1000 replicates, implemented in SPLITStree4 (Huson and Bryant 2006). Further, the model-based Bayesian clustering procedure in STRUCTURE Vv. 2.3.4 (Pritchard et al. 2000) was used to determine the genetic structure of populations. The anal- ysis was performed by setting the number of populations (K) from 2 to 12. The burn-in steps and the number of replicates were 10,000 and 50,000 for each K, respectively. All runs were repeated 100 times at each K and the optimal K value was selected as a point of a marked change in the envelope slope (kink of the curve) of InP(D) as a function of K. Genetic population structure was in- vestigated by a hierarchical analysis of molecular variance (AMOVA), and rela- tionships between populations from different parts of the species’ range were assessed based on pairwise genetic divergence (F,,) for all populations, both implemented in Arlequin v. 3.5 (Excoffier and Lischer 2010). Results cpDNA and ITS of nrDNA variation The sequences of cpDNA and ITS of nrDNA regions were obtained from 22 individuals from eleven populations of Saxifraga wahlenbergii (Table 1). The alignments of rps16-trnK and rp/32-trnL regions of cpDNA were 796 bp and 655 bp in length, respectively (concatenated cpDNA alignment — 1451 bp in length). 15 variable sites were found — 4 singleton variable sites and 11 parsimony informative sites, which represented transitions (C-T, 3 A-G) and PhytoKeys 246: 295-314 (2024), DOI: 10.3897/phytokeys.246.118796 301 Elzbieta Cieslak et al.: Glacial history of Saxifraga wahlenbergii transversions (3 G-T, 2 A-T and 2 A-C). In the rp/32-trnL region a thirty-one-nu- cleotide insertion/deletion was also identified. Eleven haplotypes (H1—H11; Fig. 1A, B) determined by these polymorphisms were revealed. Each population harbored one or two cpDNA haplotypes, mostly specific for individual populations and/or mountain ranges. H1-H4 and H7 haplotypes oc- curred only in the Tatra Mts, with the most frequent H1 haplotype present in almost all Tatra populations (six out of seven populations) (Fig. 1A, B, Tables 1, 2). On the other hand, H8—H11 haplotypes were detected exclusively in popu- lations from the isolated locations outside the Tatra Mts. Accordingly, the net- work of cpDNA haplotypes based on statistical parsimony analysis revealed two main groups separated by nine mutations and corresponding to the Tatra Mts versus other mountain ranges (Fig. 1A, B). In the Tatra Mts group, haplotypes displayed a star-like pattern, with H1 as the dominant haplotype, widespread across the Western Tatra Mts and the Eastern Tatra Mts. In the second group from outside the Tatra Mts, haplotypes occurred with comparable frequency. The phylogenetic tree, as inferred through Bayesian analysis, revealed two dis- tinct groups — individuals from the Tatra Mts form a sister genetic group to the group including all other individuals. A certain level of divergence was observed only among individuals from the Eastern Tatra Mts in both clades (Fig. 2). Overall, S. wahlenbergii displays a moderate gene diversity (h = 0.81 +0.06) and nucleotide diversity (mt = 0.0036 +0.0003) of cpDNA. At the local level of predefined regional groups, the highest gene diversity (h = 0.86 +0.11) was found in the group of populations outside of the Tatra Mts (i.e., in Mala Fatra Mts, Choéské vrchy Mts, Nizke Tatry Mts and Muranska planina) with low nu- cleotide diversity (m = 0.0013 +0.0003; Table 2). Within the Tatra Mts, popu- lations of S. wahlenbergii from the eastern part were characterized by higher gene (h = 0.73 +0.16) and nucleotide diversity (mt = 0.0036 +0.0004) than popu- lations from the western part of this mountain range (h = 0.46 +0.20; 1 = 0.0003 +0.0007, respectively). Testing deviation from neutrality (Tajima’s D) revealed no significant indications for departure from neutrality within regions (P > 0.05). The obtained ITS alignment was 730 bp long, with very low sequence diver- sity. Only four indels (three poly-A and one poly-G stretches) were found and on this basis five ribotypes were established (R1—R5) (Fig. 1A, C). Only R1 ribotype was widespread and shared by seven populations within the species’ range. The following ribotypes were specific for geographical regions: R3 for the Tatra Mts, R4 for the Mala Fatra Mts and R5 for the Choéské vrchy Mts (Tables 1, 2). The network analysis indicated close links between five ribotypes, and internal divisions into groups (Fig. 1C). Table 2. Genetic diversity of nDNA and cpDNA sequences of Saxifraga wahlenbergii calculated for a priori delimitation of regional groups. R — ribotypes (variants of nrDNA ITS) and H — haplotypes (variants of cpDNA); h — mean (+SD) gene diversity; 1 — mean (+SD) nucleotide diversity; D — Tajima’s D statistic value; *non-significant at the 5% level (P > 0.05). ITS cpDNA Regional groups R h Tt D H h Tt D Western Tatra Mts R1,R2,R3 0.00 0.00 | 0.00 H1, H3, H4 0.46 (+0.20) | 0.0003 (+0.0007) | -1.31* Eastern Tatra Mts R1,R2,R3 | 0.00 0.00 0.00 Hil, H2,-A7 0.73 (+0.16) | 0.0036 (+0.0004) | 1.80* Tatra Mts (as a whole) R1,R2,R3 | 0.00 | 0.00 0.00 H1, H2, H3, H4, H7 0.66 (+0.12) | 0.0024 (+0.0008) | -0.15* Outside of the Tatra Mts | R1,R2,R4,R5 0.00 | 0.00 0.00 H5, H6, H8, H9,H10,H11 | 0.86 (40.11) | 0.0013 (40.0003) | -0.33* PhytoKeys 246: 295-314 (2024), DOI: 10.3897/phytokeys.246.118796 302 Elzbieta Cieslak et al.: Glacial history of Saxifraga wahlenbergii | SEES ZZ an © 0) = if?) | vrchy Mts Eastern Tatra Mts Nizke Tatry Mts Muranska planina Western Tatra Mts Eastern Tatra Mts Figure 2. MrBayes tree based on the combined plastid ros16-trnK and rp/32-trnL re- gions of Saxifraga wahlenbergii (reduced dataset; see text for details). Numbers at nodes, in the order shown, correspond to posterior probabilities estimated in MrBayes. For population acronyms see Table 1. AFLP variation The AFLP analysis yielded 213 DNA markers, of which 181 (84.98%) were polymorphic for 55 individuals from ten populations of Saxifraga wahlenbergii (Table 1). Reproducibility of obtained AFLP band profiles was ~96%. The num- ber of polymorphic markers in populations ranged from 59 (S1) to 91 (S4), with a mean of 83 markers (+20.29) per individual. There were no identical AFLP phenotypes among studied individuals. Only in populations from the Velky Choé Mt. (S9, Choéské vrchy Mts) and Sina Mt. (S10, Nizke Tatry Mts) one pri- vate marker in each was identified. At the species level, Nei’s gene diversity (He) was 0.16 (+0.17) and ranged from 0.10 (S1) to 0.15 (S4), with an average value of 0.12 (+0.02). The frequen- cy of down-weighted markers (DW) was similar across most populations and ranged from 20 to 30, with much higher values in populations S4 (59), S7 (40) and S6 (36) (Table 1). The further analysis of PCoA performed on the entire dataset revealed that S. wahlenbergii populations are not clearly genetically divergent and form partially overlapping groups. In general, the population’s scatter is characterized by the west-east gradient across the distribution range of S. wahlenbergii. In 1-3 axes arrangement, the populations from the disjunct parts of the range (populations: $1, S8, S9 and S10) are opposite to the high- est locations of Hruby vrch Mt. and Mieguszowiecki Szczyt Czarny Mt. (the PhytoKeys 246: 295-314 (2024), DOI: 10.3897/phytokeys.246.118796 303 Elzbieta Cieslak et al.: Glacial history of Saxifraga wahlenbergii Eastern Tatra Mts). In the central part of plot, individuals from population of the Western Tatra Mts (S3, S4) and Mala Fatra Mts were located. The first three factors of the PCoA accounted for 35.39% of the total variation in the dataset (Fig. 3A). The Neighbor-Net diagram demonstrated two groups, each consisting of clusters representing single, spatially isolated populations, but with differ- ent bootstrap support. The first group included those with higher bootstrap values, such as Nizke Tatry Mts (98%), Dolina Chochotowska valley (96%), Gaborowa Przetecz pass (85%), Velky Choé Mt. (62%) and Mala Fatra Mts (61%). The second group consisted of populations with very low support (Fig. 3B). It is characteristic that individuals of S. wahlenbergii from the higher altitudes of the Eastern Tatra Mts were closely related with each oth- er and genetically more distant from individuals from slightly lower altitudes in the Western Tatra Mts. Analysis of genetic variation (AMOVA) showed that a major part of S. wahlen- bergii variation is attributed to the within-population level — 72.09%, in relation to among-population variation — 27.91% (F,, = 0.28, P < 0.001). The same pat- tern can be found when analyzing geographical groups (Table 3, see Suppl. material 3). AMOVA also confirmed low but statistically significant genetic dif- ferences between regions (F,, = 0.05, P< 0.01). Based on the comparison of F,,, values between pairs of populations, a gradation in differentiation between the populations of S. wahlenbergii from individual range regions was found. The population from Nizke Tatry Mts is the most distinct one, while the population from Mala Fatra Mts shows greater similarity with those from the Tatra Mts compared to the other parts of the range (see Suppl. materials 4, 5). Within the Tatra Mts, populations from the Eastern Tatra Mts presented greater genetic affinity with each other than with populations from the Western Tatra Mts. The greater mean diversity of the population in the Western Tatra Mts is due to the significant distinctiveness of the population from the Dolina Chochotowska valley, where the F,, values between other populations range from 0.36 to 0.49 (see Suppl. material 4). AFLP data indicates a low level of gene flow between populations of S. wahlenbergii (Nm = 0.66). In the STRUCTURE analysis of AFLP data, the stable and optimal number of population groups was selected based on the kink in the envelope of InP(D) values. As can be seen from Fig. 4A, B, a clear change in the slope was found for K = 3. In order to further justify the reasons for such a choice, similar analyses were performed both for K = 2 and 3. For K = 2, the population from Dolina Chochotowska valley, represented a near- ly homogeneous and clearly separated genetic group whereas populations from Eastern Tatra Mts, Nizke Tatry Mts and remaining populations from the Western Tatra Mts formed the second one. The populations from Mala Fatra Mts and Velky Choc Mts displayed genetic admixture with a significant con- tribution of both genetic pools. For K = 3, population from the Dolina Cho- chotowska valley is even more genetically distinct, while all the populations from the Eastern Tatra Mts, Gaborowa Przetecz pass in the Western Tatra Mts and Nizke Tatry Mts are similar to each other. Remaining populations from the Western Tatra Mts, as well as from Mala Fatra Mts and Vel'ky Choc Mts, represent the third group. PhytoKeys 246: 295-314 (2024), DOI: 10.3897/phytokeys.246.118796 304 Elzbieta Cieslak et al.: Glacial history of Saxifraga wahlenbergii Tatra Mts @ —S1, Dolina Chocholowska valley Hl —S2, Gaborowa Przetecz pass X -—S3, Przetecz pod Kopa Kondracka pass @ - $4, Piekietko valley WV -S65, Przetecz Zawrat pass € -S6, Mieguszowiecki Szczyt Czarny Mt. A -S7, Hruby vrch Mt. Mala Fatra Mts @® -S8, Mala Fatra Mts Choéské vrchy Mts @ -S9, Velky Chod Mt. Nizke Tatry Mts — $10, Sina Mt. PC3 (8.86% PC2 (10.90%) PC1 (15.63%) Figure 3. Phylogeographic structure within of Saxifraga wahlenbergii based on AFLP dataset (55 individuals from 10 populations) A Principal Component Analysis diagram, ordination at 1 vs 2 vs 3 axes B Neighbor-Net diagram with the bootstrap values derived from an analysis of 2,000 replicates above 50% has been given. Both diagrams were prepared based on the Nei-Li coefficient. For population acronyms see Table 1. Table 3. AMOVA analysis based on AFLP data for the populations of Saxifraga wahlenbergii calculated for all populations and a priori delimitation of regional groups. Significance tests based on 1023 permutations, ***P < 0.001, **P < 0.01. Source of variation d.f. Among populations 9 Within populations 45 Total 54 Among regional groups — Western Tatra Mts vs. Eastern 2 (High) Tatra Mts vs. outside of the Tatra Mts Among populations 7 Within population 45 Total 54 PhytoKeys 246: 295-314 (2024), DOI: 10.3897/phytokeys.246.118796 Sums of Squares 487.480 784.738 1272.218 145.588 341.892 784.738 1272.218 Variance components 6.753 17.439 24.192 1.206 9.872 17.439 24.517 % Eee Total F statistics variance 27.91%*** F,, ='0.28 72.09 4.92** Fide = 0.05 23.9 5%*** Fee =0.25 71.13 Fae = 0.29 305 Elzbieta Cieslak et al.: Glacial history of Saxifraga wahlenbergii A Mala Fatra Mts Choéské vrchy Mts Nizke Tatry Mts Western Tatra Mts Eastern Tatra Mts InP(D) $8 $9 S10 $1 $4 $3 $2 S65 S6 S7 Figure 4. A The histograms representing the assignment of 55 individuals of Saxifraga wahlenbergii to different clusters by Bayesian spatial clustering (STRUCTURE software). Each vertical bar corresponds to an individual, highlighted in gray for clarity, contrasting with the cluster assignments, respectively, at K = 2 and K = 3 Bin P(Data) values in function of K are shown. For population acronyms see Table 1. Discussion Phylogenetic analyses of the Saxifraga section indicated the hybrid origin of Saxifraga wahlenbergii and allowed us to estimate the possible oldest age of its hybrid origin to the late Neogene (4.7 Ma). These analyses also provided insights into its internal diversity (Tkach et al. 2019). Our analysis confirmed two distinct groups of plastid haplotypes corresponding to the geographical locations of populations and revealed additional local phylogeographical struc- tures. This distribution of genetic lineages on a small spatial scale, character- istic of the Tatra Mts, was also observed in the endemic species Cochlearia tatrae (Koch et al. 2003; Cieslak et al. 2021). This spatial pattern, along with the presence of unique haplotypes in populations, implies survival in local refugia with limited contact among them. In mountain conditions, the process of isolation by distance contributed to historical interruption of gene flow between populations, leading to geo- graphically driven groups of populations (e.g., Tribsch and Sch6nswetter 2003; Schonswetter et al. 2005; Kropf et al. 2006; Christe et al. 2014; Melicharkova et al. 2019). Consequently, contemporary populations represent more or less distinct units in the landscape, potentially facilitated by the periglacial environ- ment. This environment consisted of a mosaic of habitats that allowed the sur- vival of various population groups (Birks and Willis 2008; Provan and Bennett 2008). The accumulation of intraspecific diversity in S. wahlenbergii, particu- larly within the cpDNA, among populations from different regions, such as the Mala Fatra Mts, Chocéské vrchy Mts, Nizke Tatry Mts, and Muranska planina, points to the scenario that these regions may have acted as distinct glacial re- fugia for high mountain species in the Western Carpathians. Analyses of AFLP data highlight a weakly resolved but distinct position of spatially isolated popu- lations and groups, further supporting the above interpretation. In the Tatra Mts, subalpine populations of S. wahlenbergii are more closely related to those geographically closest from the same mountain range than to their subalpine counterparts from other mountain ranges. This suggests that the source area of their recolonization could have been populations from low elevations, such as the extant population from the Dolina Chochotowska valley, a site which remained outside the glaciation area (Ktapyta et al. 2016; Ktapy- ta and Zasadni 2017-2018). In the Tatra Mts, which are clearly different from their periphery, a high genetic diversity of haplotypes is observed. Apart from PhytoKeys 246: 295-314 (2024), DOI: 10.3897/phytokeys.246.118796 306 Elzbieta Cieslak et al.: Glacial history of Saxifraga wahlenbergii several haplotypes from both cpDNA groups, the presence of R2 and R3 ITS variants is restricted to populations from the Tatras range. The genetic struc- ture of S. wahlenbergii in this area was likely influenced by the topographically complex environment and historical conditions in the glacial periods. The Tatra Mts, unlike most of the Western Carpathians, were strongly, albe- it unevenly, glaciated during the Pleistocene glaciations (Zasadni and Ktapyta 2014; Zasadni et al. 2022). Due to the occurrence of extensive glaciers in the valleys (Ktapyta et al. 2016), available local glacial refugia were physically iso- lated. Within the mountains, refugia were generally distributed along steep, un- covered rocky crests at the highest altitudes and lower crests below the snowl- ine. Large areas with a mosaic of habitats potentially suitable for high-mountain plants were also available in adjacent low-altitude locations along the entire range. Consequently, survival in lower, periglacial habitats appears appropriate for S. wahlenbergii. During cold glacial periods, these could have been places both at the foot and along the glacial moraines, in the glacier ablation zones, as well as in the areas occupied by steppe-tundra. Survival in these areas could have been possible thanks to the ability of species to live in a wide range of habitat conditions, such as moist edges of limestone and granite scree, in the shade of rocks, on shelves and in rock crevices. On the other hand, low values of F,, (the lowest in relation to the compared pairs of populations) observed in the AFLP data from the Tatra Mts can be the result of the maintenance of gene flow between populations during recoloni- zation of this area after the last glaciation and may counteract incipient differ- entiation processes, thereby avoiding bottlenecks, genetic drift, and the loss of genetic diversity. Characteristically, the highest values of the F,, were noted between populations from the areas with the highest altitudes, namely the Ta- tra Mts and the Nizke Tatry Mts. These results suggest that mountain ridges acted as a stronger barrier for gene flow more effectively than the elevation differences between subalpine and lower-lying areas within the same ranges. In addition, the genetic structure of S. wahlenbergii, a relic mountain plant spe- cies, certainly reflects processes acting in different time periods. Populations that survived when environmental conditions became unfavorable could retain genetic variability. Becoming a source of remigration in new conditions, they could also host new local mutation fixations. Therefore, it can be assumed that both Quaternary climatic oscillations and ecological divergence have played a role in shaping the distribution and divergence patterns observed in S. wahlen- bergii. Similarly, in the species complex Alyssum montanum-A. repens, a clear elevational shift was identified, indicating that differential ecological adapta- tion occurred in the respective mountain areas (Melicharkova et al. 2019). It should be emphasized that these findings are consistent with previous results of phylogeographical analyses (Ronikier et al. 2012; Cieslak et al. 2021), which showed the Tatra Mountains as an important, independent area within the Western Carpathians, where the local structure of species was formed. These patterns indicate that the Tatra Mts also served as a refugium or a system of microrefugia, likely due to their high topographical and habitat diversity. How- ever, the genetic position of populations from remaining massifs, especially the series of massif-specific cpDNA haplotypes, also indicates their role sup- porting the S. wahlenbergii populations, forming a system of discrete parts of the range likely over a longer temporal scale. In the case of an allopolyploid PhytoKeys 246: 295-314 (2024), DOI: 10.3897/phytokeys.246.118796 307 Elzbieta Cieslak et al.: Glacial history of Saxifraga wahlenbergii species of hybrid origin it cannot also be ruled out that a polytopic hybrid origin may have played a role in the two main intraspecific lineages observed (Meli- charkova et al. 2019), which have later undergone an internal diversification in the isolated mountain environment. Acknowledgments We would like to thank the Editor and Reviewer for their useful comments and suggestions; Tony Dixon for improving our English; Anna Delimat, Anna Ronikier, Roman Letz, Patrik Mraz and Peter Turis for their valuable help in sampling. Collecting permits were granted by Tatrzanski Park Narodowy, Poland (no. Bot-203) and by Ministerstvo Zivotného Prostredia Slovenskej Republiky (Rozhodnutie MZP SR no. 6188/2017-6.3 from 13.12.2017). Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Funding This study was funded from the statutory funds of the W. Szafer Institute of Botany, Polish Academy of Sciences. Author contributions Elzbieta CieSlak: Research concept and design, Data analysis and interpretation, Writing the article, Critical revision of the article, Final approval of the article. Michat Ronikier: Research concept and design, Collection and/or assembly of data, Critical revision of the article, Final approval of the article. Magdalena Szczepaniak: Data analysis and in- terpretation, Critical revision of the article, Final approval of the article. Author ORCIDs Elzbieta CieSlak © https://orcid.org/0000-0002-6267-9333 Michat Ronikier © https://orcid.org/0000-0001-7652-6787 Magdalena Szczepaniak © https://orcid.org/0000-0002-7483-3932 Data availability All of the data that support the findings of this study are available in the main text or Supplementary Information. References Benham J, Jeung JU, Jasieniuk M, Kanazin V, Blake T (1999) Genographer: A graphical tool for automated fluorescent AFLP and microsatellite analysis. Journal of Agricul- tural Genomics 4: 1-3. Birks HJB, Willis KJ (2008) Alpines, trees, and refugia in Europe. Plant Ecology & Diversity 1(2): 147-160. https://doi.org/10.1080/17550870802349146 PhytoKeys 246: 295-314 (2024), DOI: 10.3897/phytokeys.246.118796 308 Elzbieta Cieslak et al.: Glacial history of Saxifraga wahlenbergii Blattner FR (1999) Direct amplification of the entire ITS region from poorly preserved plant material using recombinant PCR. 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Link: https://doi.org/10.3897/phytokeys.246.118796.suppl1 Supplementary material 2 The datasets of nrDNA and cpDNA of Saxifraga wahlenbergii Authors: Elzbieta Cieslak, Michat Ronikier, Magdalena Szczepaniak Data type: docx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/phytokeys.246.118796.suppl2 PhytoKeys 246: 295-314 (2024), DOI: 10.3897/phytokeys.246.118796 313 Elzbieta Cieslak et al.: Glacial history of Saxifraga wahlenbergii Supplementary material 3 AMOVA analysis based on AFLP data for populations of Saxifraga wahlenbergii calculated with a priori delimitation of regional groups Authors: Elzbieta CieSlak, Michat Ronikier, Magdalena Szczepaniak Data type: docx Explanation note: Significance tests based on 1023 permutations; *P < 0.001. Regional grouping of populations see in Materials and methods. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/phytokeys.246.118796.suppl3 Supplementary material 4 Pairwise genetic divergence (F,.) across 10 populations of Saxifraga wahlenbergii based on AFLP data Authors: Elzbieta Cieslak, Michat Ronikier, Magdalena Szczepaniak Data type: docx Explanation note: Significance tests based on 1023 permutations; P < 0.001. For popu- lation acronyms see Table 1. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/phytokeys.246.118796.suppl4 Supplementary material 5 Spatial arrangement of varying divergences (F,,) among populations within distribution range of Saxifraga wahlenbergii Authors: Elzbieta Cieslak, Michat Ronikier, Magdalena Szczepaniak Data type: pdf Explanation note: Above the line, the average F., values are given; the line colors corre- spond to scale in the right corner of the map. For population acronyms see Table 1. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/phytokeys.246.118796.suppI5 PhytoKeys 246: 295-314 (2024), DOI: 10.3897/phytokeys.246.118796 314