The current distribution areas of plants were shaped by various environmental changes during and after the glaciation period. Rapid fluctuations in environmental conditions shifted the boundaries of colonized areas (Hewitt 1996) and caused the extreme fragmentation and disjunct distribution of many plant species (Jermakowicz et al. 2017). Plant populations that occur at the edge of their distributional range or, in the case of disjunct distribution, which is caused by habitat rarity, are prone to environmental and biotic stressors. Plants that are scattered and isolated are more susceptible to variability in climate, habitat and biogenetic factors (Kołos et al. 2015). This applies to isolated remnants of e.g. Arctic-Alpine species, resulting from shifts in their distribution range during glacial and inter-glacial periods. However, it also applies to boreal-alpine species that survived climate change during the Holocene and now only occur in a few places in Central Europe (Svenning et al. 2008, Sundberg 2014, Kołos et al. 2015, Jermakowicz et al. 2017). In recent decades, the conservation value of relict populations has been emphasized as they are adapted to stressful ecological conditions, which could be important for future distribution range shifts owing to climate change (Vogler and Reisch 2013).

In a rapidly changing environment, many plant species have become rare due to habitat loss and fragmentation and are restricted to very small, isolated populations with an increased risk of local or regional extinction (Jermakowicz et al. 2017). Apart from immediate extinction, the most severe consequence of habitat fragmentation is the loss of genetic diversity. This occurs due to random genetic drift, increased inbreeding, and reduced gene flow between populations. This leads to a loss of offspring and of population fitness and at the same time reduces adaptability to environmental changes (Willi et al. 2006).

Long distances between isolated populations further reduce pollen transfer and limit pollination (Wilcock and Neiland 2002). Many authors (Scobie and Wilcock 2009, Gaudeul et al. 2019, Kikowska et al. 2022) have reported a decline or even complete failure of sexual reproduction in small populations of rare plant species. All these reproduction problems are even greater in species that rely on cross-pollination (Wróblewska 2013). A typical example is the twinflower, Linnaea borealis L. (Caprifoliaceae), a circumboreal plant species that is already in severe decline throughout Europe (except in its boreal range in Scandinavia). However, its geographical distribution ranges across the Northern Hemisphere, occurring from Scotland and northern Europe through Russia to Siberia, northern Asia to Kamchatka and Japan, northern China and Mongolia, and from Alaska and Canada to Greenland (Alm 2006). This clonal, self-incompatible plant is a shade-tolerant, evergreen dwarf shrub. In the natural Swedish populations, L. borealis reproduces vegetatively, mainly by stolons, and forms large clones up to 10 m long (Eriksson 1988, Niva 2003). In Scotland, low seed production is attributed to reproductive isolation caused by the lack of availability of compatible mates within populations and limited pollen dispersal between them (Ross and Roi 1990, Wilcock and Neiland 2002, Scobie and Wilcock 2009, Wiberg et al. 2016).

In the Alps, L. borealis is considered a relict species with some localities in France, Switzerland, Italy (Fornaciari 1964, Hegi 1966, Wilhalm 2010), Austria and Slovenia (the southeasternmost locality) (Wraber 1963). According to the literature, there should be three almost neighbouring sites in the Eastern-Southeastern Alps (Ernet in Franz 2011): in the Austrian part of Carinthia (1) and Styria (2), and in the Slovenian part of Styria (3). However, the species is still present at two sites (Slovenian and Austrian parts of Styria, but the third site (Carinthia (AT)) has not been confirmed since 1992 (Ernet and Franz 2011). All these sites are of great importance from a biogeographical point of view and raise a number of research questions related to the survival of L. borealis in this disjunct southeasternmost distribution. In this respect, the L. borealis can be considered a model species for the study of glacial relicts in the Alps. In addition, various conservation measures have already been implemented to guard against any further loss of its habitat and genetic diversity (Wróblewska 2013, Wiberg et al. 2016).

Our aim was therefore to investigate the genetic variability within and between two isolated (but spatially close) populations of L. borealis from the Eastern-Southeastern Alps (Julian Alps (SI) and Gurktaler Alps (AT)) and to compare this with the genetic variability of three populations from the northern range area (Sweden, Scotland and Russia). We used nine previously described microsatellite loci (A’Hara et al. 2011) to assess genetic variability. In this way, we attempted (1) to determine the extent to which the two isolated populations are genetically distinct and (2) to answer the question of whether the L. borealis of the Eastern-Southeastern Alps is a postglacial colonizer or a glacial relict. Our findings will provide essential information for the successful conservation strategies for the survival of this plant species in situ over a longer period of time, especially from the aspect of climate change.

Molecular analysis: Microsatellite analysis revealed 86 alleles at 9 microsatellite loci. The number of alleles detected per locus (Tab. 1) ranged from 6 (locus D110 and D118) to 16 (locus B119), with an average of 10 alleles per locus.

Tab . 1. Allele sizes (bp) and allele frequencies (in parentheses) of the 73 Linnaea borealis genotypes at nine microsatellite loci (A5, A102, D110a, D7, D110, D118, A112, B119, C105).

Analysis based on nine microsatellite loci, revealed very low genetic variability within the two isolated Eastern-Southeastern Alpine populations of L. borealis (SI and AT). This was to be expected as only a low number of specimens (individuals) were recorded at each location (sampling site) and therefore only a limited number of distinguishable genets were available. A low number of individuals could also be because of the small size of the remnant. In Slovenia the site of the population is around 0.2 ha, in Austria it is even smaller. However, a very high genetic variability was found between the two populations (SI and AT), even though they are, compared to other sampled populations, relatively closely positioned. This indicates a very low or no dispersal ability; nevertheless, the absence of suitable habitats should not be underestimated. This leads to the conclusion that the two spatially and reproductively isolated Eastern-Southeastern Alpine populations most likely represent glacial relicts, such a pattern having been observed in previous studies of glacial relict plants (e.g. Hensen et al. 2010, Jermakowicz et al. 2017). These researchers explained the high genetic variability between populations by consecutive bottlenecks and long-term reproductive isolation. In addition, Vogler and Reisch (2013) claimed that genetic variability may be high in the case of different populations of glacial relicts and low in the case of postglacial colonization. Low genetic diversity within the population might be also explained as a shift in mating system from outcrossing to selfing (Surina et al. 2023).

In a comprehensive study by Wróblewska (2013), a phylogeographic structure derived from plastid DNA and a moderate genome-wide diversity estimated from AFLP markers were found for L. borealis in Eurasia. Six haplotypes were identified in an area from Scotland to Altai and from Norway to Italy. However, Wróblewska (2013) concluded that although half of the populations studied were highly isolated, they still showed similar levels of genetic diversity across the geographic range. Furthermore, she stated that she had found no support for the hypothesis that a bottleneck and/or inbreeding. factors affecting genetic diversity, were associated with habitat fragmentation. The case of L. borealis clearly shows that it was not habitat fragmentation, but rapidly changed climatic conditions after the last glaciation that caused the narrow isolation of the species leading to glacial relict populations, like the one in Slovenia and Austria. Regarding the geographical distribution of Alpine populations, she only sampled in the Western Alps (Aosta, Italy; Les Allues, France) and the Central Alps (Zernez, Switzerland). However, according to Meusel and Jäger (2017), its distribution is not that rare. East of the line Innsbruck–Wipp Valley–Brenner Pass–Eisack Valley–Etsch Valley there are only scattered small populations, especially in the Hohe Tauern mountain range. From there, L. borealis has not been found in the rest of the Eastern Alps, with the exception of three localities on the south-eastern borders of the Alps (Ernet and Franz 2011). This could be the reason why four out of six haplotypes occur at the above-mentioned locations in the Western and Central Alps.

In any case, the warming after the end of the last glaciation led to a loss of suitable habitats, resulting in a more or less strong fragmentation of the Alpine population, which eventually led – at least in the Eastern Alps – to only a few isolated patches remaining in specific habitat conditions related to elevation, northern exposure or particular situations in terms of substrate conditions. Ernet and Franz (2011) mentioned the boulder-strewn slopes where cold air typically emerges from under the scree in summer. This is exactly the characteristic of the Austrian location in the Gurktaler Alps, but is even more pronounced at the Slovenian site, where at the extremely low elevation of only 495 m a.s.l., from holes in the rubble ice-cold air blows during the growing season. There is no direct sunlight on the steep northern slopes, which also increases soil and air humidity. According to Wraber (1963), therefore, no zonal beech forest has developed at this location, but a community dominated by Rhododendron hirsutum L., Pinus sylvestris L. and Larix decidua Miller.

To conclude, it is well known that the most severe negative impact of fragmentation is caused by genetic drift, increased inbreeding, and reduced gene flow among populations (Scobie and Wilcock 2009, Wiberg et al. 2016). This led to a reduced number of offspring and a reduction in population fitness (Scobie and Wilcock 2009). In the case of L. borealis which is a self-incompatible plant, the effect of isolation on reproductive success is even greater. For successful fertilization, self-incompatible plants like L. borealis require cross-pollination with a compatible mate, which does not belong to the same clone. It has been reported that in extreme cases where all remaining plants have the same alleles for genetic control of pollen-pistil interactions seed set can be reduced to zero (Demauro 1993). However, in small populations of many clonal, self-incompatible plants such as Rubus saxatilis L., Aster furcatus E.S.Burgess, ­Calystegia collina (Greene) Brummitt, Arnica montana L., Ranunculus reptans L. and Maianthemum bifolium (L.) F.W.Schmidt (Scobie and Wilcock 2009), the limited availability of partner plants is increasingly being identified as the cause of reproductive failure, the plants perhaps adapting to turning to selfing.

Assuming that the two isolated populations of L. borealis in the Eastern-Southeastern Alps are glacial relicts with considerably reduced genetic variability, further research on their viability and reproductive mating system is needed to ensure better conservation success.

We thank the Slovenian Environment Agency in Ljubljana and the Styrian Environment and Spatial Planning office in Graz for issuing the sampling permits. Many thanks, to Professor Sara Cousins (Stockholm) for sampling the specimens from Bollnäs in Central Sweden. We are grateful to Tea Kelc (who helped us with DNA isolation and microsatellite analysis) and Anže Žerdoner Čalasan (sampling the specimens from the valley of the River Aktru, Russia). We also thank Stuart A’Hara for providing us with isolated DNA samples from Scotland, Great Britain (GB).

This work was supported by the Slovenian Research Agency (Research program P1-0164, P1-0403 and P6-0372) and project J7-60129 “Development of Research Infrastructure for the International Competitiveness of the Slovenian RRI Space – RI-SI-LifeWatch”, co-financed by the Republic of Slovenia, Ministry of Education, Science and Sport and the European Union from the European Regional Development Fund.

All authors contributed to the study conception and design. All authors read and approved the final manuscript.

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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