Genetic variability and conservation of the endangered Pannonian root vole in fragmented habitats of an agricultural landscape

The distribution of the endangered glacial relict subspecies, the Pannonian root vole Alexandromys oeconomus mehelyi Éhik, 1928, is restricted to scattered localities in south-western Slovakia, which belong to the north-eastern zone of its range. Human-induced changes and fragmentation of the landscape have led to the gradual loss of suitable habitats and threaten its long-term survival. The study area in the Danubian Lowland is characterised by small habitat fragments and temporal fluctuations of the habitat area. Root voles were sampled at nine sites to study the level of genetic variability and structure of local subpopulations by scoring 13 microsatellite loci in 69 individuals. Genetic differentiation varied amongst local populations and we did not find a significant isolation-by-distance pattern. Bayesian clustering analysis suggested that dispersal effectively prevents marked genetic subdivision between studied habitat fragments. Significant pairwise differentiation between some subpopulations, however, may be the result of putatively suppressed gene flow. Low genetic diversity in the recent populations probably reflects the isolated location of the study area in the agricultural landscape, suggesting that long-term survival may not be assured. In order to maintain genetic diversity, it is essential to preserve (or even restore) habitats and ensure the possibility of gene flow; habitat protection is, therefore, recommended. Continuous assessment is necessary for effective conservation management and to predict the long-term survival chances of the Pannonian root vole in the study area. Nature Conservation 43: 167–191 (2021) doi: 10.3897/natureconservation.43.58798 https://natureconservation.pensoft.net Copyright Krisztina A. Kelemen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE Launched to accelerate biodiversity conservation A peer-reviewed open-access journal


Introduction
The root vole Alexandromys (Microtus) oeconomus (Pallas, 1776) is the only extant Holarctic species of the Microtini tribe with twenty-five known subspecies distributed in the Northern Hemisphere (Pardiñas et al. 2017). The widespread species is categorised as 'Least Concern' in the IUCN Red List of Threatened Species (Linzey et al. 2016).
As a result of glacial and postglacial changes in its distribution range, the root vole is currently represented by three subspecies in Western and Central Europe (Musser and Carleton 2005;Pardiñas et al. 2017), A. oeconomus stimmingi (Nehring, 1899) (Nehring 1899), A. oeconomus arenicola (de Sélys-Longchamps, 1841) (de Sélys-Longchamps 1841) and A. oeconomus mehelyi Éhik, 1928 (Éhik 1928), all of which belong to the Central European phylogroup (Brunhoff et al. 2003). As a protected species of the European fauna, the root vole is included in Appendix III of the Bern Convention (Council of Europe 1982). Two subspecies, A. o. mehelyi and A. o. arenicola, are endangered and need a feasible conservation action plan with special management strategy to maintain populations (Pardiñas et al. 2017). The Pannonian root vole A. o. mehelyi, an isolated subspecies present in the Carpathian Basin, occurs in the southern part of Slovakia in the Danubian Lowland (Miklós et al. 2014;Ambros et al. 2016), locally in north-eastern Austria (Thissen et al. 2015a) and in some isolated localities in Hungary (Horváth and Herczeg 2013;Lanszki et al. 2015;Kalmár and Riezing 2017). This subspecies is considered to be a glacial relict in Central-Europe (Brunhoff et al. 2003) with populations inhabiting refugial areas including freshwater marshes, swamps, floodplains or watersides (Baláž and Fraňová 2013;Kalmár and Riezing 2017). Based on intensive research in the past, all studies defined the suitable habitats of the Pannonian root vole as wet sites with dense vegetation cover, typically dominated by sedge (Carex sp.) or hummocks of sedge and common reed (Phragmites australis) mosaics (Kratochvíl and Rosický 1955;Stollmann and Ambros 1998;Hulejová Sládkovičová et al. 2016). Due to its special habitat requirements and rarity, apart from some larger known populations, it is common that merely one or a few specimens are found sporadically or only indirect evidence indicates its occurrence (Miklós et al. 2011;Purger 2014;Thissen et al. 2015a, b). The subspecies is sensitive and exhibits rapid response to natural and anthropogenic disturbances, especially to their additive effects and it was shown that individuals could cross barriers to reach suitable areas (Horváth and Herczeg 2013). Habitat loss, fragmentation and degradation due to changes in land-use, like drainage or mowing, are amongst the wellknown risk factors that threaten the Panonian root vole (Thissen et al. 2015a;Gubányi et al. 2009). As this rare, habitat specialist subspecies is present exclusively in scattered populations in these remnant habitats, it appears amongst the priority species of communities in Annexes II and IV of the Habitats Directive (European Commission 1992).
The distribution of Pannonian root vole in the territory of Slovakia is determined by the change of the landscape and its natural conditions in the postglacial era. Over the past 130 years, the Danubian Lowland in southern Slovakia has experienced significant landscape modifications, such as extensive flood protection interventions and construction of a channel network in agricultural lands. As a consequence, habitats preferred by the root vole are currently fragmented and isolated by agricultural lands in southern Slovakia (Ambros et al. 2016). Furthermore, flooding events, the oscillation of ground water level and dry periods have a substantial effect on the connectivity between remnant patches of suitable habitat, determining gene flow amongst them. Landscape fragmentation and loss of habitats represent a significant risk factor in the Danubian Lowland for this rare subspecies which occurs only in scattered populations in south-western Slovakia, thus, the Pannonian root vole is strictly protected in the country (Miklós et al. 2014;Ambros et al. 2016).
Habitat loss and fragmentation caused by landscape modification affect wildlife populations worldwide (Wilson et al. 2016). The negative impact of edge effects, higher sensitivity to environmental and demographic stochasticity and the change of different characteristics of small, fragmented populations in reduced habitats may lead to increased risk of local extinction (Willi and Hoffmann 2009). Simultaneously, as the isolation of remnant patches becomes larger, dispersal and thus gene flow amongst local populations may become hampered (Storfer et al. 2010). Consequently, subdivided and isolated populations undergo changes in their genetic structure and variability (Lino et al. 2019).
The response of small mammalian populations to habitat fragmentation has been widely studied (e.g. Kozakiewicz et al. 1999;Mortelliti et al. 2010). Wetland and marshland associated species are particularly threatened by the negative effects of fragmentation (Rushton et al. 2000;Pita et al. 2010), as the available habitats are already fragmented and isolated to varying degrees as a result of human activities, such as infrastructure development, water management or agricultural drainage (Čížková et al. 2013). The consequences of habitat fragmentation on genetic diversity in subdivided small mammal populations have been in the focus of comprehensive research (e.g. Gaines et al. 1997;Fietz et al. 2014). A number of papers aimed to study Microtine rodents as appropriate model organisms (Redeker et al. 2006;Marchi et al. 2013) in the investigation of habitat fragmentation which poses substantial threat to endemic and relict vole species or subspecies that are especially sensitive to its negative effects (Buzan et al. 2010;Pita et al. 2014). Rare species and subspecies, including voles inhabiting wetlands, have been the subject of studies that investigated the genetic characteristics of populations occurring in fragmented or isolated habitats in the Nearctic (List et al. 2010;Neuwald 2010;Parmenter et al. 2015) and Palearctic regions (Van De Zande et al. 2000;Telfer et al. 2003;Centeno-Cuadros et al. 2011) and, based on the levels of genetic diversity and revealed genetic patterns, many authors stressed implications or proposed measures for conservation.
To ensure the viability of root vole populations or subpopulations, it is reasonable to study genetic variability and genetic structure as factors affecting the adaptive traits and future persistence of populations. Wetland habitats experienced fragmentation and significant shrinkage in south-western Slovakia and only a few habitat fragments are left for root voles. In the present survey, we used microsatellite analyses to study the genetic diversity and structure of Pannonian root vole subpopulations inhabiting remnant habitat fragments in the Danubian Lowland, where the greatest threat to their long-term survival is habitat loss, fragmentation and degradation (Gubányi et al. 2009;Horváth et al. 2012;Horváth and Herczeg 2013;Miklós et al. 2014;Thissen et al. 2015a;Ambros et al. 2016). Understanding the spatio-temporal dynamics and population genetic patterns of this subspecies could be valuable for developing and improving an appropriate and effective conservation action plan and to derive recommendations for the conservation management of this endangered subspecies.

Study area and samples
Located in the south-western part of Slovakia, the study area lies in the Danubian Plain (Podunajská rovina), part of the Danubian Lowland, belonging to the Pannonian biogeographical region. The landscape was formed by the tributaries of the Danube (Little Danube and others) and by the southern Váh, Nitra and Žitava Rivers. Soil properties and climatic conditions make this region ideal for agriculture. In the area, wheat, sugar beets, sweet corn, vegetables, fruits and tobacco are grown. In the late 19 th century, the study area was located in the centre of wet meadows interlaced by meanders of Žitava River, as shown by the Third Military Survey of the Habsburg Empire (1869-1887) (2018). Over the past 130 years, extensive flood protection measures and construction of the channel network fragmented the area of natural meanders of the Žitava River, which changed the direction and hydrodynamics of their flow and led to the aridification of the surrounding ecosystems. By 1926, the original wet meadows had dried up and a network of meanders had formed into a compact wetland habitat crossed by two channels. The consequences of these interventions were still visible in 1964, but the size of wetland habitats had become further reduced by 1990 (Topographic Maps of Czechoslovakia 2018). Today, remnants of the original meander system exist only as a few patches, where the Pannonian root vole still survives. The extension, shape and connectivity of recent wetland habitats scattered across the agricultural landscape are determined by water levels and temporary floods. In the summer and winter of 2010, the extreme precipitation raised water levels and caused the flooding of the Danube (Slovak Hydrometeorological Institute 2011). The last highest water level rise of the river was recorded in spring 2013 (Jakubcová et al. 2014;Matoková and Smrtík 2014), which also influenced our study area. The localities included in this study are situated in the area interlaced by three channels between Patince, Chotín and Marcelová Villages (Fig. 1).
The research was carried out in different types of habitat fragments like waterlogged areas overgrown by Carex spp. and Phragmites spp. at the edges of channels, oxbow lakes and remnants of former tributaries intersecting large areas of agricultural lands. Animals were captured with live traps baited with apple and cereals. One line transect of 25 traps with 10 m intervals was established for five consecutive nights in each trapping site. Trapping was conducted three times a year (spring, summer, autumn), from 2014 to 2017. Traps were inspected once a day. Upon capture, each vole was investigated for body weight, age and reproductive status before release. At the first capture, the tail tip of each vole was clipped for genetic analyses. The clipped tail tips were put immediately in 96% ethanol and preserved at -85 °C in the laboratory until DNA extraction. Trapping and sampling methods were realised in agreement with the rules of State Nature Protection of Slovak Republic "Species and habitats monitoring of European importance within the Habitats Directive and the Birds Directive" project. Due to the rarity and endangered status of the Pannonian root vole, sample sizes were low in some trapping transects and, therefore, a total of 69 tissue samples from nine sites were used in molecular analyses. suitable habitat fragments appearing occasionally, depending on water levels; 3) total area of all habitat fragments measured in hectares; 4) average size of habitat fragments (ha); and 5) overall connectivity of our region of interest. Suitable habitat fragments were delimited based on the subspecies' known habitat preference for humid, densely vegetated areas (see Introduction), which markedly differed from the vegetation of agricultural parcels in the study area. The size of particular habitat fragments (ha) was calculated in QGIS software 3.4.12-Madeira (QGIS Development Team 2019). To calculate the connectivity of the whole area (total size: 2873.8 ha), the equivalent connectivity (EC) of PC (probability of connectivity) probabilistic index was used (Saura et al. 2011a, b), where Euclidian edge-to-edge distances with 700 metres' maximum dispersal distance threshold parameter (5% probability for a species to exceed 700 m) were set as a connection between habitat fragments. This distance was set as the average dispersal distance observed on the root vole by Steen (1994). All connectivity indices were calculated in Conefor 2.6 (Saura and Torné 2009). The maps of habitat fragments were created as digitalised topographic maps of Google Earth in map scale 1:5000. Individuals from the same locality were treated as a putative subpopulation (hereafter referred to as subpopulation).

Changes in habitat fragment patterns and estimation of subpopulation sizes
Due to different trapping efforts at each study location, the size of each subpopulation was evaluated as the relative abundance (rA) of individuals captured at the location per 100 trap-nights (C/100TN), based on data without recaptures. The number of captured specimens (N) was recalculated into the transformed rA index assuming a random (Poisson) distribution (rA = (-ln (1-N/100) 100) of small mammals to remove the saturation effect caused by single traps (Caughley 1977). Relative abundance values were averaged over trapping occasions.
We tested the correlation between average rA of Pannonian root vole and average habitat fragment size using Pearson correlation analysis, where both data were logtransformed due to non-normal distribution.

DNA extraction
DNA extractions were performed using commercial Isolate II Genomic DNA Kit (Bioline) according to the manufacturer's protocol with the following modifications: during pre-lysis, samples were incubated overnight at room temperature; after adding preheated Elution Buffer G (70 °C), the elution step covered the incubation of samples at room temperature for 30 minutes and after that at 70 °C for 5 minutes before elution.

Analysis of genetic markers and genotyping
The thirteen microsatellite loci included in the analyses were developed for Microtus arvalis: Mar003, Mar016, Mar049, Mar063, Mar076 (Walser and Heckel 2008)  Multiplex PCR reactions were performed in 12 μl volumes containing 2 μl (~80 ng) of DNA and a volume of 10 μl of the following mixture: 3.9 μl of KAPA2G Fast Multiplex Mix (KAPA Biosystems), 0.8 μl of BSA, 0.5 μl (concentration of 10 pm/ng) of each primer and RNase-free water to fill the volume to 10 μl. Amplification of DNA was carried out using peqSTAR 96X Universal thermal cycler (Peqlab).
To amplify microsatellites in Set 1A, Set 1B and Set 2 the PCR reaction consisted of the initial step at 94 °C for 7 minutes, 30 cycles including: denaturation at 94 °C for 1 minute, annealing at 55 °C in case of Set 2 and 60 °C in case of Set 1A and Set 1B for 2 minutes and extension at 72 °C for 90 seconds, followed by a final step at 72 °C for 10 minutes. Microsatellites in Set 3 were amplified in a PCR reaction including the following steps: 7 minutes at 95 °C, 35 cycles of 30 seconds at 94 °C, 1 minute at 57 °C and 90 seconds at 72 °C and after the cycles a final step of 10 minutes at 72 °C. To prepare the genotyping procedure 1 μl PCR product of each sample was mixed with 12 μl formamide and 0.3 μl GeneScan 500-LIZ size standard (Applied Biosystems). After a denaturation step of 5 minutes at 95 °C, a cooling step was implemented. Genotyping was carried out using ABI PRISM 310 Genetic Analyser (Applied Biosystems) and microsatellite genotypes were examined using GeneMapper software v.4.0 (Life Technologies).

Clustering analysis of samples
We successfully genotyped 69 individual samples and the amplification success varied amongst markers (94.2-100%). The presence of null alleles may cause significant heterozygote deficit and deviation from the HWE. We therefore estimated the proportion of null alleles (NA) at each locus in each subpopulation using the programme FREE-NA (Chapuis and Estoup 2007). All genotypic distributions were in accordance with HWE expectations for all loci and, as we did not detect null alleles based on FREENA, consequently, all loci were included in the analyses.
Bayesian clustering of microsatellite genotypes was performed using STRUCTURE v.2.3.2 (Pritchard et al. 2000). Ten independent runs were performed for each value of K ranging from one to ten under a model assuming admixture and correlated allele frequencies (Falush et al. 2003). Each run comprised a burn-in period of 100,000 replications followed by a run length of 1,000,000 Markov Chain Monte Carlo (MCMC) iterations. The results of replicated runs for each value of K from one to ten were combined using STRUCTURE HARVESTER v.0.6.94 (Earl and vonHoldt 2012) and the optimal value of K was assessed by the inspection of log-likelihood values and according to the ΔK method developed by Evanno et al. (2005). Twenty independent runs were conducted with K fixed at the estimated optimal number of clusters where a burn-in of 100,000 and 1,000,000 MCMC iterations were used. The outputs of replicated runs were combined using the Greedy algorithm in CLUMPP v.1.1.2 (Jakobsson and Rosenberg 2007) and DISTRUCT v.1.1 (Rosenberg 2004) was used to visualise cluster assignments.

Genetic variability in subpopulations
The mean number of alleles (A), observed (H O ) and expected (H E ) heterozygosity (Nei 1978) and inbreeding coefficients (F IS ) were calculated using GENETIX v.4.05.2 (Belkhir et al. 2004). The allelic richness (AR) was estimated using the rarefaction procedure for the lowest sample size (n = 10) in the programme FSTAT v.2.9.4 (Goudet 2003). Deviation of subpopulations from Hardy-Weinberg equilibrium (HWE) was calculated with exact tests assessing heterozygote deficiency and excess in GENEPOP v.4.2 (Rousset 2008). The basic level of significance was set to P = 0.05 and for multiple comparisons, we applied a Bonferroni procedure compensating for the risk of an inflating type 1 error.

Genetic variation between subpopulations
The programme FREENA was used to estimate global F ST , by performing 10,000 permutations. In addition, a Monte Carlo test of likelihood ratio G-statistic (Goudet et al. 1996) was performed using package hierfstat v.0.04-22 (Goudet and Jombart 2015) in R v.3.5.0 (R Core Team 2018) to test the presence of genetic structuring. Estimations of pairwise F ST were implemented in FSTAT v.2.9.4 according to Weir and Cockerham (1984). Significant differences of F ST estimators from zero were tested using 100,000 permutations without the assumption of Hardy-Weinberg equilibrium and significance level was set to P = 0.05 and adjusted using the Bonferroni correction. In addition, isolation by distance was tested by assessing the correlation between the geographical distance matrix (given in kilometres) and pairwise F ST /(1-F ST ) estimates using Mantel's Test with 10,000 permutations in the programme ISOLDE in GENEPOP v.4.2. The GENETIX v.4.05.2 package was used to investigate genetic relationships amongst all genotyped individuals of the nine subpopulations by factorial correspondence analysis (FCA), a method that identifies the linear combination of variables (allele frequencies at different loci) that captures the most variation between observations (individuals or populations) and visualises genetic relationships in 2D space determined by FCA axes. Analysis of Molecular Variance (AMOVA) (Excoffier et al. 1992) was implemented in GENALEX v.6.5 Smouse 2006, 2012) to test the proportion of genetic variance amongst individuals and subpopulations. Statistical significance of the variance components was assessed with 999 permutations.

Habitat fragment patterns and subpopulation sizes
Between 2004 and 2019, we identified 26 permanent habitat fragments as suitable habitats for root voles, in 14 of which their presence were confirmed. We have also identified several temporarily suitable fragments, the number of which varied season-ally and annually. All measured characteristics of potential habitats (number of fragments and temporary fragments, total area of all habitat fragments, mean fragment size and connectivity of the whole area) changed over the six time periods of study (see Table 1). In general, connectivity had higher values in periods when the number and size of habitat fragments were higher. In March 2004 and March 2011, the number of temporary fragments increased, filling the matrix of agricultural land between permanent fragments with new suitable habitat (see Fig. 2). The highest values of parameters were observed in 2011, except for the average fragment size. After this period, the total area of habitat fragments, the number of temporary fragments and the connectivity decreased and permanent fragments were thus more isolated. Values of parameters remained relatively similar in the following years. Partial changes in habitat fragment characteristics and connectivity are detectable in seasonal comparison between March and August 2017, with higher values in spring. In a detailed view of the study sites in Fig. 2, it can be seen that, while in August 2017, sites SK3, SK4, SK5 and SK6 were relatively small and isolated from each other, in March 2004, 2011 and 2017, the same sites were larger and only a channel interrupted their direct connection.
The average occupancy of fragmented habitats by the Pannonian root vole varied spatially (Fig. 3). While some fragments had a high relative population size, others had very low. The analysis did not confirm a positive correaltion between average rA of Pannonian root vole and average size of fragmented habitats (r = 0.15, N = 9, P = 0.74).

Clustering analysis
Genetic structuring inferred from STRUCTURE analysis is presented in Fig. 4 for K values ranging from 2 to 5. The ΔK method indicated that the optimal number of clusters was 5. However, this method can evaluate ΔK only for K > 1 and the inspection of log-likelihood scores revealed that K = 1 had a relatively high likelihood, similar to other values of K. Generally, assignment probabilities were roughly symmetric, which indicated that none of the clustering analyses captured the real population structre. Notably, when K was fixed at 5, SK1 individuals sampled in 2014 (individuals 3, 6, 7 and 8) and an additional individual from SK5 (individual 26 sampled in 2015) had relatively high assignment probabilities to the same cluster and the same for individuals 57 and 60 sampled in SK9 in 2017. However, not all individuals sampled in 2014 and 2015 were assigned to this cluster (indicated with grey in Fig. 4).

Hardy-Weinberg equilibrium and genetic diversity in subpopulations
Clustering analysis did not give a strong evidence of structuring; therefore, measures of genetic diversity were calculated for all subpopulations separately and for all samples pooled together. Genetic diversity and HWE were not calculated for locations SK3, SK4 and SK7 due to their small sample size (N = 3). The number of alleles per locus in subpopulations ranged from 2 (locus Moe4) to 16 (locus Moe7), and the mean number of alleles per locus (A) ranged from 4.31 to 5.62 (Table 2). Allelic richness (AR) across subpopulations ranged from 4.22 to 5.46, being the highest in the SK1 subpopulation and lowest in the SK8 subpopulation. H O (range between 0.562 and 0.669) was the highest in subpopulations SK8 and SK9, while the lowest value was shown for SK5. In contrast, H E (range between 0.624 and 0.657) showed another pattern, with the highest value for SK1 and the lowest value observed for SK8. Two subpopulations, SK8 and SK9 had heterozygosity excess, based on observed and expected heterozygosity values. Subpopulations SK1, SK2, SK5 and SK6 showed significant heterozygote deficiency on the basis of F IS (significant positive values). Subpopulations SK1, SK2 and SK5 deviated from HWE according to heterozygote deficiency exact tests in GENEPOP (P = 0.05) after Bonferroni correction   ( Table 2). Heterozygote excess exact tests were not significant in any of the cases (data not shown). Mean number of alleles and expected heterozygosity indicated higher genetic diversity and the exact test showed a significant deficiency of heterozygotes when all samples were pooled together.

Genetic differentiation between subpopulations
Subpopulations SK3, SK4 and SK7 were not included in F ST analyses because of their small sample size (N = 3). The global F ST for six subpopulation samples was 0.025 (95% CI: 0.01-0.041). The overall G-test was significant (P < 0.001), indicating genetic structuring amongst locations. Pairwise F ST values were relatively low, although variable. The highest F ST values were observed for SK8 and SK9. Pairwise genetic differentiation was not significant in most of the comparisons, except in cases where one subpopulation of the pair was always SK6, SK8 or SK9 (Table 3). Significant F ST was observed between SK1 and SK8, but when the individuals of SK1 sampled in 2014 were excluded from pairwise F ST analyses, this difference became non-significant (F ST = 0.048). Microsatellite-based genetic distances did not correlate with geographical distances between subpopulations (a = 0.0254, b ~ 0, P = 0.6), thus spatial separation (IBD) was not confirmed. The FCA plot, based on individual genotypes, clearly separated SK1 along the first factorial axis (explaining 20.1% of variation) from all other subpopulations. The second axis (explaining 17.9% of variation) mainly separated the individuals from SK1 and SK9, while individuals from SK6 showed only a weak segregation  ( Fig. 5A). Subopulation SK8 was separated along the third axis (explaining 14.6% of variation, Fig. 5B).
In the Analysis of Molecular Variance, significant genetic variation was attributed to the differences between subpopulations (4.4%, P < 0.001) and most of the variability occured within subpopulations (95.6%).

Discussion
Our results show that genetic variation and differentiation in subpopulations of the Pannonian root vole is in good agreement with connectivity between habitat fragments, with temporary fragments playing an important role in vole migration between flood events.

Habitat connectivity
The number, size and shape of habitat fragments in the studied region varies in time as the result of exogenous factors (precipitation, surface water levels, agricultural activities). These dynamic changes have an effect on fragment connectivity, suggesting that connectivity was positively influenced by the number of habitat fragments. As we have also noted, permanent habitat fragments, relatively distant from each other at one time, can change size and shape and become neighbouring habitats at another time. In addition, the temporary fragments can play the role of stepping stones during vole movements. Thus, despite the constant presence of habitat fragments and channel-side vegetation, fragment connectivity can vary seasonally and yearly, as can change the possibility of individuals' replacement between the studied subpopulations. In Norway, root voles increased dispersal distance as a response to fragmentation, but it was less affected by connectivity (Bjørnstad et al. 1998). Detailed data on the dispersal ability of the Pannonian root vole are still missing, although they have been shown to be able to cross barriers when the quality of original habitats deteriorated and they may follow stepping stones when searching for new habitats (Horváth and Herczeg 2013). Moreover, Kratochvíl and Rosický (1955) observed that Pannonian root voles in sedge (Carex sp.) survived on hummocks when the water level increased (50-70 cm). When the water level decreased, voles spread across the lower parts of these hummocks, while some individuals dispersed to new suitable fragments. In view of this, a possible explanation for the observed genetic patterns could be that the animals found shelter on hummocks during floods in 2010 and probably 2013, thus opening the way for admixture. Later, when water levels fell, presumably admixed individuals resettled lower areas.

Genetic differentiation of the Pannonian root vole
Levels of genetic differentiation between the habitat fragments varied, but were mostly non-significant and we found no support for isolation by distance between subpopula-tions. Bayesian clustering in STRUCTURE did not reveal pronounced genetic structuring, indicated by approximately equal assignment probabilities to different clusters in all cases of K from 2 to 5. This result suggested that dispersal effectively prevents marked genetic subdivision between studied habitat fragments, which can be additionally confirmed by the lack of isolation by distance between fragments. Given the small geographical scale and landscape pattern of the study area, we would expect gene flow between localities to maintain very low or no differentiation between subpopulations. In a study conducted in the Netherlands, Mauritzen et al. (1999) concluded that ditches are likely to favour linear movements of root voles and may enhance connectivity in an agricultural landscape. Most habitat fragments sampled in our study were directly connected by reed-and sedge-lined channels that are likely to serve as dispersal corridors and permanent or temporary habitat fragments in the study area also likely to facilitate dispersal (Ambros et al. 2016). Although the longest geographical distance along the channels separating our sampling sites from their nearest neighbours was nearly 6 km, as for SK1 and SK2, the presence of root vole was confirmed in other permanent habitat fragments between these two sampling sites (which could not be included in our study due to the small number of samples) and most of the habitat fragments sampled were relatively close to each other. Movement distances of male root voles can exceed several hundred metres in a short time or more than two kilometres within a few days (Steen 1994;Andreassen et al. 1996).
Consistent with the changing possibility of individuals' replacement between the studied fragments, AMOVA results also showed a low, but significant, percentage of variability between subpopulations. In addition, signs of genetic differentiation were detected between subpopulations SK8, SK9 and SK6, based on significant pairwise F ST values and the FCA analysis confirmed the separation of these samples. Results may therefore indicate that dispersal is not unhindered between all subpopulations and root vole individuals in the network of studied habitat fragments may not be viewed as a panmictic population.

Genetic diversity of the Pannonian root vole
In a detailed study, Hulejová  already noted that, despite the high genetic variability of the subspecies, local populations of Pannonian root vole may show signs of genetic depletion. Although our analyses were limited by small sample sizes due to the rarity of the Pannonian root vole, our results indicate lower genetic diversity in subpopulations (in terms of allelic richness, mean number of alleles per locus and expected heterozygosity) than what was measured in other populations from south-western Slovakia (Hulejová . Similarly, reduced genetic diversity (based on the mean number of alleles per locus and the expected heterozygosity) was observed compared to the populations of another isolated Western European root vole subspecies, namely A. o. arenicola (Van de Zande et al. 2000). Expected heterozygosity and mean number of alleles in the total studied population indicated decreased variability compared to other Slovakian populations (Hulejová . Low genetic diversity of the studied root vole population putatively reflects a pronounced geographical isolation of the surviving population and the decrease in effective population size that has likely occurred. In addition, flood events may have reduced the size of the vole population (Kratochvíl and Rosický 1955), which may have created a bottleneck, although its influence on the genetic diversity of the studied population cannot be clearly confirmed on the basis of our results alone. The reduced genetic diversity is in accordance with the view of Hulejová  who suggested that the Danube River and associated branch network could facilitate vole dispersal, but local populations embedded in the agricultural landscape and further from the Danube are likely to remain isolated. Moreover, based on the evaluation of the distribution of Pannonian root vole, our study area is close to the eastern boundary of the subspecies' range (Gubányi et al. 2009;Ambros et al. 2016) and populations occurring here are likely isolated by the distance from other populations to the west.
Subpopulations SK8 and SK9 tend to have lower levels of allelic richness, which is consistent with the possibly lower probability of dispersal through the agricultural land matrix compared to other sites. However, we did not find significant deviation from the Hardy-Weinberg equilibrium in these subpopulations. We observed deviations from the Hardy-Weinberg equilibrium in SK1, SK2 and SK5 and significant positive F IS coefficients in the same subpopulations, together with SK6, which may result from the social structure of root voles. Matriline-based groups in root vole populations (Tast 1966) give the possibility of sampling kin individuals, which may result in departures from the Hardy-Weinberg equilibrium (Aars et al. 2006). On the other hand, Stewart et al. (1999) argue that genetic composition (and departures from the Hardy-Weinberg equilibrium) of water vole (Arvicola amphibius) colonies is under the influence of yearly fluctuation and suggested that genetic drift is a plausible cause resulting in that pattern. They interpreted high levels of genetic diversity as a sign of gene flow between colonies. Similarly, despite the relatively large number of analysed individuals, Pilot et al. (2010) observed yearly changes in departures from the Hardy-Weinberg equilibrium in a root vole population in Poland; nevertheless, a high level of genetic diversity was maintained over the years. In addition, they found no genetic differentiation between years regardless of changes in density, kin structure and deviations from HWE, indicating that genetic composition is stable over time in large, demographically-stable populations. In contrast, studies of water vole metapopulations revealed temporal genetic differentiation, but genetic variability remained high as a result of gene flow (Stewart et al. 1999;Aars et al. 2006).

Possible migration from Danube River
Regarding individuals 3, 6, 7 and 8 (sampled in 2014 and 2015) in SK1, highlighted by clustering analyses, their high assignment probability to a separate cluster may reflect their distinct origin. Habitat fragment SK1 is the closest to the Danube River amongst the studied fragments and the floods in 2010 or 2013 potentially facilitated dispersal from further areas and the aforementioned individuals might be immigrants or their descendants. Other individuals that had relatively high assignment probabilities to the same cluster were captured in SK5 (individual 26 from 2015) and SK9 (individual 57 and 60). These can be found at a few kilometres distance from SK1, but given the small spatial scale, it is not unlikely that these specimens may be the offspring of dispersing individuals. Alternatively, it is also possible that genetic drift over time changed the genetic composition of subpopulations in the fragmented landscape; hence, some (but not all) samples collected in 2014 and 2015 were highlighted by STRUCTURE clustering. However, we are not able to declare which possibility is more plausible without genotyping individuals from other areas and without temporal analysis of samples.
The genetic diversity of the local subpopulations in the study area is relatively low; although the studied subpopulations probably have connections with each other due to the effect of extensive floods and the network of fragments and channels in the agricultural landscape, their reduced genetic variability is detectable compared to the pooled genetic variability of other populations of Pannonian root vole occurring closer to the more uninterrupted marshlands in Szigetköz, Hanság and Neusiedlersee Regions (Hulejová . Based on the pattern of remnant habitat fragments, it is likely that root vole subpopulations in our study area form a metapopulation system and the observed genetic patterns do not contradict this possibility. However, studies on extinction and recolonisation patterns and migration rates would be essential to support this view (Gaggiotti 2004;van der Merwe et al. 2016).

Conclusions and management implications for conservation
Landscape changes and habitat destruction resulted in the fragmented distribution of root vole habitats in the study area and fluctuating surface water levels induce considerable changes in habitat size, quality and connectivity to this day. Only one fragment (SK2) in our study area is protected as a Special Protection Area. However, for the long-term persistence of root vole populations, it would be critical to ensure legal protection of habitats. The importance of protected core areas has been demonstrated for water vole metapopulations (MacPherson and Bright 2011) and mainland or source population demography has been shown to shape habitat use of root voles (Glorvigen et al. 2013). Based on the low differentiation, it seems unlikely that the studied vole subpopulations formed a mainland-island type metapopulation system (Stewart et al. 1999), but the signs of admixture highlight the vital role of habitat fragments and gene flow in the maintenance of genetic diversity. On the other hand, Van de Zande et al. (2000) proposed that weak differentiation may be an indicator of progressive isolation of local populations and accordingly, the prevention of fragmentation should be considered in the conservation management of A. o. arenicola. It has been suggested for other vole species and subspecies that persistent gene flow between habitat fragments is crucial for the maintenance of genetic variability (Telfer et al. 2003). Therefore, it is necessary to protect not only habitat fragments with current occurrences of the Pannonian root vole, but also other potentially-suitable habitat fragments, to form a habitat network and to ensure the possibility of gene flow (Neuwald 2010). Water levels in the study area and, as we demonstrated, total size of permanent habitat fragments and number of temporary habitats are in dynamic change, which further emphasises the contribution of these fragments to the survival of the subspecies in the agricultural landscape (Kalmár and Riezing 2017).
It is expected that the overall genetic diversity of the subpopulations will decrease as a result of their small size and isolated location in the agricultural matrix. This implies that the restoration of habitats and corridors is indispensable for the long-term preservation of diversity, as has been stressed earlier (Thissen et al. 2015a;Hulejová Sládkovičová et al. 2018). As a habitat specialist, the Pannonian root vole is sensitive to habitat quality changes, which are likely to be the key factors determining habitat use (Glorvigen et al. 2013;Horváth and Herczeg 2013). Thus, it is important to preserve optimal water level conditions for the root vole, for example, by controlled construction of infrastructure that may cause aridification and the disappearance of the subspecies (List et al. 2010;Kalivodová et al. 2018). For this reason, it is necessary to take the needs of the subspecies into consideration when infrastructural projects are planned and managers should be involved in landscape and land-use planning. Finally, long-term monitoring is recommended to provide further information about population and subpopulation parameters (Hayes et al. 2017), genetic diversity and structure (Proença-Ferreira et al. 2019) and on the changes of habitats (Martensen et al. 2017) to elaborate an effective conservation management action plan that ensures the long-term survival of the Pannonian root vole.