We found that continuous grasslands and verges harboured a similar species pool (Fig. 1). The reason for this is twofold. As verges were formed during the past centuries in parallel with the increasing agricultural activities, they were able to preserve a large subset of the regional species pool similar to that harboured by the continuous grasslands. Similar species pools also suggest that verges and continuous grasslands might have a functional spatial connection even in fragmented landscapes (i.e. at certain locations they have connection points), allowing the dispersal of species between the two habitat types. This connection is supported by the linear structure of the verges; they mesh the landscape and therefore they provide corridors for the dispersion of plants. Given the fact that verges are in close contact with roads, dispersal of plants is also supported by the various means of transportation and mowing machines used for cutting the verges (Fekete et al. 2018). In addition, in agricultural landscapes human vectors can transport grassland specialist species on the clothes of field workers and on agricultural machinery (Auffret and Cousins 2013). However, humans can also act as dispersal vectors for invasive and weed species (Valkó et al. 2020). By decreasing dispersal limitations, all the above processes can considerably increase the similarity of the species composition between continuous grasslands and verges.
As shown by the results of the t-SNE and the PERMANOVA analyses, the species composition of the vegetation on the kurgans differed considerably from that of the vegetation of the continuous grasslands and verges. This might be due to their ancient origin; secondary grasslands on kurgans were formed millennia ago, before the large-scale landscape transformations. After the emergence of the kurgans they were likely occupied by the species that were present in the historical species pool since they were surrounded by grassland habitats and even the soil used for their construction contained the propagules of grassland species (Lisetskii et al. 2016). However, the special environmental conditions provided by the shape of the kurgans should have acted as an environmental filter for plant establishment, allowing only a certain subset of species to establish on the kurgans after their construction (Lisetskii et al. 2016; Szava-Kovats et al. 2012). This compositional difference was conserved by the special hill shape of the kurgans in subsequent millennia, both by environmental filtering, and by preventing certain human disturbances such as ploughing or construction.
Shannon diversity and species richness
Differences in the Shannon diversity and richness of the studied species groups among the three habitat types can be explained by the marked differences in their origin, the shape and size of the habitats, the applied management regimes, and the level of habitat heterogeneity typical of them. Despite their small area, lack of management and the neighbouring unfavourable landscape matrix (i.e. ploughlands), the millennia-old grassland islands maintained by kurgans were able to preserve a high number of specialists. Patch-like grassland fragments embedded in agricultural fields are generally characterised by drier soil conditions than the neighbouring areas, and this provides a favourable habitat for stress-tolerant dry grassland specialists even without management (Lindborg et al. 2014). In the case of kurgans this effect is enhanced by the special hill shape, which results in an enhanced flow of precipitation on the slopes and an increased distance between the topsoil layer and the groundwater (Lisetskii et al. 2016). Due to their steep slopes human disturbances (e.g. ploughing) have been suppressed on kurgans, which has further increased the chance for the maintenance of specialists (Deák et al. 2016). Furthermore, despite their small size the increased level of microhabitat diversity preserved on the heterogeneous surface of kurgans allows the co-existence of specialist species with slightly different habitat requirements (Lisetskii et al. 2016; Deák et al. 2016). In contrast, the species richness of generalists was low on kurgans. The main reason for this is that generalists typically occur under less stressful habitat conditions than those provided by kurgans. However, we found high species richness and cover of weeds on kurgans, indicating the vulnerability of these grassland fragments to the mass effect of incoming seed rain from the neighbouring ploughlands (Deák et al. 2018).
Due to the small or missing core area, specialists in the verges are especially exposed to the negative effects (e.g. chemical load, seed rain of weeds and soil disturbances) originating from the neighbouring matrix (roads and ploughlands) and to local disturbances (trampling, frequent mowing and application of pesticides), leading to the stochastic extinction of specialists from certain habitat patches (Tikka et al. 2000). In contrast with kurgans and continuous grasslands, the small level of environmental heterogeneity in verges cannot counterbalance these negative changes (Tikka et al. 2000). Due to the dense seed bank, high dispersal ability and high regeneration potential of weeds and generalists, they have a higher chance to re-establish in the disturbed patches than specialists; thus, in the long term, their populations dominate these habitats (Cousins 2006). Even though linear landscape elements have the potential to act as green corridors for grassland specialists given their connections to continuous grasslands (Bátori et al. 2016, 2020), they may also provide corridors for generalist and weed species, especially in transformed landscapes (Fekete et al. 2018), where their spread is enhanced by transportation and by mowing machinery (Tikka et al. 2000).
Species accumulation curves
Despite their small size and isolated state, the steepest accumulation curves calculated for the total species pool were found on kurgans, which might be the result of the high topographic heterogeneity and microhabitat diversity (that are also responsible for their high species richness) (Fig. 3). As was found by Polyakova et al. (2016) in steppe habitats and Filibeck et al. (2019) in limestone grasslands, variability in the micro-relief and unevenness of the surface support the existence of numerous microhabitats within short distances, providing various niche spaces available for several taxa. These adjacent microhabitats are characterised by different species pools; thus, spatial variability of species combinations increases considerably even within small spatial scales of a few metres.
As was found in the case of midfield islets (Lindborg et al. 2014) and coastal grasslands (Dupre and Diekmann 2001), abandonment did not flatten the accumulation curve, in fact it even made it steeper. The reason for this pattern is likely the presence of harsh environmental conditions, such as drought stress in the case of midfield islets and kurgans and salt stress in the case of coastal habitats, preventing the monodominance of a few strong competitors and providing patchy species occurrence patterns in the community. Regarding the total species pool, the steepness of the curves was similar in continuous grasslands and verges; however, mechanisms sustaining these patterns were likely different. In continuous grasslands the extensive and often selective cattle grazing and trampling suppress the abundant competitor species, and allow the establishment of subordinate species with poorer competitive abilities (Polyakova et al. 2016). In this way, grazing enhances the patchiness of the vegetation, and thus the steepness of the species accumulation curve. In verges, the high level of disturbance resulting in open patches might be the main factor that maintains a steepness comparable to continuous grasslands.
On the level of specialist species, kurgans and continuous grasslands were characterised by a similarly high steepness in the accumulation curve, suggesting that the number and spatial variability of species of high conservation importance are the highest in these habitats. In continuous grasslands the traditional method of land use is responsible for this pattern. Probably the most interesting finding of our study is that kurgans—despite their abandonment, small size and isolated state—showed a comparable conservation value to continuous grasslands; because they have preserved a considerable proportion of the historical habitat-specific species pool. The preservation of specialist species is also supported by the harsh environment and their heterogeneous topography. In verges, the accumulation curve was flatter for the specialists due to the high level of disturbance by frequent mowing and trampling, eliminating several disturbance-sensitive species. Another reason for this pattern might be the increased level of nutrients received from the neighbouring agricultural land. As was observed in grasslands in Italy and Germany (Chiarucci et al. 2006), increased nutrient levels might suppress specialists and flatten the species accumulation curves. Although kurgans were characterised by steep accumulation slopes for specialists, they also had the steepest slopes for generalists and weeds, suggesting that these important refuges are highly endangered. The reason for the above patterns is the presence of several disturbed microhabitats (ploughing and chemical infiltration at the foot of the kurgan, the presence of fox burrows, and microsites with high litter accumulation) on the kurgans that can be occupied by generalist and weed species (see also Godó et al. 2018).
We found several indicator species for the habitat types studied, which may reflect the history, management and landscape context of these habitats. The presence of grazing-tolerant specialists (such as Centaurea scabiosa s.l., Thymus glabrescens, Koeleria cristata and Dianthus pontederae) in continuous grasslands indicated the century-long grazing management applied. Kurgans were characterised by some tall-growing specialists (e.g. Elymus hispidus, Agropyron cristatum and Thalictrum minus), and some protected species (e.g. Inula germanica, Phlomis tuberosa and Carduus hamulosus) that are usually missing from the continuous grasslands of the region. Although most of these species could have been typical of the continuous grasslands in the past, their populations have almost completely disappeared from the landscape due to agricultural intensification and melioration works (Biró et al. 2018). The presence of these specialists underlines the high conservation importance of kurgans, which often hold the remnant populations of many species that are endangered due to intensive land use (Deák et al. 2020).
The shared indicator species between continuous grasslands and kurgans were mostly specialists (Cruciata pedemontana, Verbascum phoeniceum and Stipa capillata) and generalist species typical to grasslands (Alopecurus pratensis, Veronica verna, Hypericum perforatum and Cerastium semidecandrum) reflecting the common historical species pool. Whilst continuous grasslands held weeds typical of extensive pastures, the abandoned kurgans were characterised by weeds typical of oldfields and ploughlands. The presence of Lepidium draba indicates soil disturbance by foxes, which prefer kurgans embedded in ploughlands for burrowing (Godó et al. 2018). Other arable weeds such as Lathyrus tuberosus, Vicia spp. and Bromus arvensis were likely established on the kurgans from the neighbouring ploughlands and oldfields (Sudnik-Wójcikowska et al. 2011). The invasion of the terrestrial reed (Phragmites communis) indicates the long-term abandonment of the grasslands on kurgans. The unique species composition of kurgans is also indicated by the high (99%) coverage of indicator species.
Verges harboured many generalist and weed indicator species, reflecting the species pool of the contemporary intensively used landscape. Indicator generalist species of verges (Verbascum chaixii, Equisetum ramosissimum, Bromus commutatus and Silene vulgaris) and shared generalists between continuous grasslands and verges (Festuca rupicola, Achillea collina, Eryngium campestre, Plantago lanceolata, Centaurea pannonica, Agrimonia eupatoria and Knautia arvensis) are adapted to frequent disturbances such as biomass removal and trampling, which are typical in mown verges affected by continuous human presence. Due to the high level and frequency of disturbances, only a few specialist species such as Peucedanum alsaticum and Aster sedifolius could establish in the verges. These species are able to colonize open disturbed soil surfaces due to their good dispersal and establishment ability. Bromus inermis was typical both on kurgans and in verges, as the effective clonal spread of this species is a successful strategy both in disturbed and environmentally heterogeneous habitats (Rosenthal and Lederbogen 2008). The presence of woody indicator species (Rubus caesius and Prunus spinosa) was also typical on the kurgans and in the verges. On kurgans their presence is a consequence of the abandonment, while in verges they are typical because of the high disturbance levels, and their effective dispersal is supported by birds and transportation on the adjacent roads (Suárez-Esteban et al. 2013).
We found that cPD values calculated for the total species pool were lower on kurgans than in continuous grasslands, which might be attributed to the high number of generalist species in continuous grasslands which increased the sum of the branch lengths (Fig. 4). A possible reason for this pattern is that the earliest terrestrial plant species may have been generalists and cosmopolitans (Steemans et al. 2009), which then underwent a rapid diversification, and thus are located on different branches of the phylogenetic tree. The high cPD values calculated for the total species pool in verges might also be attributed to the high number of generalists and also to the high number of weeds. We found that in the case of specialists, kurgans represented a similarly high phylogenetic diversity to that of continuous grasslands due to the presence of phylogenetically distinct specialist species and species groups. This suggests that both kurgans and continuous grasslands have been able to preserve a considerable amount of evolutionary history across the specialist species, and hence should be the focus of conservation (Barak et al. 2017). The low cPD values of specialists in verges are due to the low species richness of specialists. In verges the species pool was likely filtered by the special habitat characteristics, to which only a narrow group of species (likely with the same evolutionary history) could adapt.
The high phylogenetic diversity of specialists represented by the branch lengths in the continuous grasslands is due to the maintenance of the original species pool preserving the original community structure, whilst in case of kurgans it is likely the outcome of the high level of environmental heterogeneity that supports the co-existence of several specialist species with different evolutionary histories (Kassen 2002). The increased taxonomic and phylogenetic diversity of specialists on kurgans provides flexibility under changing environmental conditions, such as climate fluctuations or small-scale disturbances (Szabó et al. 2019). High diversity of species and the presence of adaptation abilities coded on the gene level provide a higher chance for an adaptive community-level response both for local and larger scale habitat changes. In a taxonomically and phylogenetically more diverse community, there is a higher chance that at least some of the species can survive in a changing environment (Bátori et al. 2019). The low cPD scores for specialist species in verges may be attributed to the high level of disturbance. As previous studies have shown, both secondary origin and disturbance by frequent mowing can considerably decrease the phylogenetic diversity in grasslands (Barak et al. 2017; Turley and Brudvig 2016). Due to dispersal constrains and the presence of abiotic filters, secondary origin can mitigate the immigration of specialist species that otherwise could inhabit the grassland fragment. The increased frequency of mowing can decrease the level of competition, and may result in the selection of close-relative disturbance-tolerant taxa (Grime 1979; Helmus et al. 2010).
High AED values of specialists on kurgans and in continuous grasslands suggest that abundances of specialists are phylogenetically more clustered in these habitats, meaning that species belonging to fewer numbers of clades dominate in these habitats compared to verges. Thus, abundances of indicator specialists are not evenly distributed across the phylogenetic tree. Linking AED to IndVal scores further clarifies this pattern: most of the indicator species representing an evolutionarily unique set of the given community’s specialists were present on kurgans (11) and in continuous grasslands (8); their number was lower in verges (6) (Fig. 5). The phylogenetic imbalance observed in continuous grasslands and on kurgans might be attributed to the special habitat conditions to which specialists are adapted. In the case of the continuous grasslands, specialists are especially clustered towards the Lamiaceae family reflecting the long-term moderate level of grazing applied in these habitats. In case of the kurgans, specialists are represented by the tussock-forming species of Poaceae, which are adapted to dry kurgan microhabitats. Specialists belonging to the Asteraceae family and to the clade consisting of the Lamiaceae and Scrophulariaceae families represent a species pool adapted to dry, but relatively nutrient rich microhabitats, and can tolerate the effects of abandonment typical of kurgan habitats. We did not observe such clustering in the case of verges, which might be due to their relatively young age and the high level of disturbance. These circumstances allowed a smaller portion of the historical species pool of specialists to occur in verges, and also, due to the frequent and often stochastic disturbances evolutionary adaptations are mostly related to re-colonisation (i.e. good dispersal ability) and toleration of disturbance.