Genetic differentiation in Alsace and Wallonia
Globally, the genetic differentiation between populations in both Alsace and Wallonia is limited or follow natural, geographical or historical isolation (River Meuse in Wallonia, or the southern part of the Ardennes). Indeed, as no or only weak isolation by distance was detected (opposite to the results in UK; Pernetta et al., 2011; see below), it is likely that other processes are influencing the genetic differentiation between populations, such as some landscape elements (e.g., rivers) or historical events (e.g., climate fluctuations) that we could not detect with this study.
In some specific cases, populations are more differentiated than expected based on the genetic pattern observed in both regions. In Alsace, the south-westernmost group (cluster 3, Fig. 1A) seems to be isolated from the others, according to GENELAND and presenting a significant pairwise FST with all other sampling. Also, it has a negative FIS value (-0.048), which, though not significant, indicates a propensity to outbreeding. This result could be caused by a reduction of the size of this population, due to a recent isolation event, and thus conduction to some genetic drift. Indeed, this sampling site is located west and north of two main highways. These elements might represent recent physical barriers to gene flow. Similarly, other sub-populations included in cluster 1 separated by major highways or by large areas of crop fields also show high and significant FST values (e.g., highways between pop4 and pop8; crop fields between pop4 and pop5; see Table 2). Therefore, it would then be possible that populations of C. austriaca located at the southwest of Alsace are isolated from the rest due to the fragmentation caused by highways. It has been shown that average sized and small species of snakes tend to avoid crossing roads (Andrews and Gibbons 2005) or are killed when trying to cross roads (Bonnet et al. 1999). Our results tend to suggest that it should be the case for C. austriaca, as highways could indeed constitute a strong barrier to gene flow if no underpasses are found along large sections of such roads.
In Wallonia, a significant but weak (r = 0.033) signal of isolation by distance was detected. Indeed, it could be related to the global structure detected with GENELAND, with the occurrence of four clusters, three of them representing well-separated regions [southern region (cluster 6); central region (cluster 4) and the edge of the central region (cluster 5)]. Even if some signal of IBD could be detected within clusters 5 and 6, it was significant only for cluster 5. Moreover, the grouping realised with GENELAND is not based on distances as sampling sites of clusters 4 and 5 are sometimes very close. This splitting is probably more related to historical reasons. For instance, the group6 gathered all the populations from the southern part of the Ardennes, where the species is not very common as habitats are cold and mainly composed of forests. We can hypothesise the differentiation between clusters 4 and 5 is resulting from the occurrence of the Meuse River. Indeed, all but one (sampling site 28) sampling sites from the group5 is on the north-western part of the Meuse River. Within group4, all sampling site except sampling site 3 are on the shore or south-eastern part of this river. The Meuse River is the largest river in Belgium; it probably acted as a barrier to the movement of C. austriaca for several centuries. Cluster 7, that gathered individuals from the single sampling site 21, does not present particular geographic barriers with other populations of the group5 that could explain its genetic differentiation. Local monitoring in the sampling site 21 highlighted a strong increase of individuals during the last years, with the lack of smooth snake 30 years ago (Graitson et al. 2012). We can consequently hypothesise that this population undergone a strong founder effect with the colonisation by a very limited number of individuals only 2–3 generations ago, which could explain the significant FIS value. Contrary to what is observed in Alsace, the difference between the groups does not seem to be explained by a barrier effect induced by motorways, which are nevertheless present in the sampled area, but more by geographical elements. We believe that the hilly terrain in Wallonia offers more possibilities to cross the motorways through underpasses.
Such a low genetic differentiation was unexpected, as strong differentiation was detected in several species of snakes with similar ecology requirements, even within putative interconnected habitat: for example, ecologically interconnected populations of Nerodia sipedon, an aquatic colubrid from North America, which has a similar home range as C. austriaca (between 1 and 4 ha), showed a marked genetic differentiation, maybe resulting from a high degree of philopatry (Prosser et al. 1999). For species with different life-history traits (‘sit and wait‘ predators and strict capital breeders) but similar in size and shape, a high genetic structure was also observed within three viperids: Vipera berus in Western Europe (Ursenbacher et al. 2009), Vipera ursinii in Southern France (Ferchaud et al. 2011), and Sistrurus catenatus in the north of the United States and South of Canada (Gibbs et al. 1997). On the other hand, the genetic structure of a terrestrial elapid, Hoplocephalus bungaroides, in Australia, demonstrated a low genetic structure (Dubey et al. 2011), like for C. austriaca in Alsace. So we can assume that, if the barriers are limited, the smooth snake keeps populations interconnected over long distances, due to underestimated individual movement, but also a larger and more diffuse presence of the species in between recognised populations (see below).
Comparison between regions
Our study reveals a contrasting pattern of genetic structure among the same species in different studied regions, one located more to the core of the range, and the other to the edge of the range. Moreover, Pernetta et al. (2011) have demonstrated that isolation by distance is marked for smooth snake between population patches distributed in a small forest area in Southern England (highest distance between two patches <6km; Pernetta et al., 2011). In this case, isolation by distance might rather be the result of the low dispersal capacities of the species, rather than the fragmentation of habitat, as the authors mentioned the occurrence of suitable habitats that could be used as corridors between population patches (Pernetta et al. 2011). Moreover, the density is probably lower in Southern England, conducting to a higher genetic drift and thus higher FST values. This situation is rather different from what was observed in Alsace and in Wallonia. In Alsace where the overall FST values were similar (0.075 vs. 0.078 in England), but where the sampled area was much wider (maximum distance between sampled sites ≈ 85 km in Alsace vs. <6 km in England) (Pernetta et al. 2011). In Wallonia, the FST value is higher (0.114), but over an even larger distance (maximum distance between sampled sites ≈ 125 km) and with the occurrence of at least four genetic groups. It is to note that the FST values between the three studies did not result from the exact same set of genetic markers (2/8 were similar for the three studies; 3/8 between Alsace or Wallonia and England), but the genetic diversity, number of alleles and allelic richness are similar between all loci, suggesting that the use of different markers would have only a limited impact on the comparison. Also, the effect of isolation by distance was significant in Southern England (r=0.511, p<0.05), whereas no effect was detected in Alsace and only a weak significant signal in Wallonia (fig. 2). We expected to find a stronger effect of isolation in both Alsace and Wallonia due to the larger distances between populations if a similar genetic pattern as in England has been detected, which was not the case. This observed discrepancy obtained at a different scale should lead to further studies at the same spatial scale and with the same set of microsatellites in order to avoid artefacts due to large variation in distance between populations. However, the comparison of Pernetta et al. (2011) and this project clearly suggests that, within a species, genetic structure can strongly vary between habitats or regions. Such differences of genetic structure and diversity between regions have already been demonstrated in other groups, with a more marked genetic structure and lower diversity at the edge in comparison to the core of the distribution (Munwes et al. 2010; Dudaniec et al. 2012; Meeus et al. 2012; Ursenbacher et al. 2015). Therefore, it would be interesting to investigate the genetic structure of populations of Coronella austriaca in other parts of its distribution limits and in similar habitats (i.e., lowlands), in Scandinavia or in Western France for example, to detect if the dispersal behaviour varies in function of the positions within the distribution of the species or more due to local geographical elements.
Moreover, the studied populations in both regions are still large enough and rather widespread to avoid a strong genetic drift, as shown by the similar level of genetic diversity and limited FST values (Table 1 and 2), contrary to the populations studied in England (Pernetta et al. 2011). Though our sampling pattern shows populations that are geographically separated (fig. 1), our results suggest that the dispersal capacities are underrated for this species. Preliminary capture-recapture data suggest that smooth snakes are rather philopatric in Alsace (J.P. Vacher, unpublished data), which is in accordance to what is known in the literature for this species and other temperate snakes (Völkl and Käsewieter 2003; Pattishall and Cundall 2008; Pernetta et al. 2011). Still, further studies on dispersal would be necessary to assess this question (Keogh et al. 2007), as most information on movement behaviour within this species concern movement within the home range and not actual dispersal (Clobert et al. 2012).
Smooth snakes have been found in a rather wide range of habitats and microhabitats in Alsace (Thiriet and Vacher 2010) and in Wallonia (Jacob et al. 2007; Graitson et al. 2020), such as dry grassland, heathlands within forests, peat bogs, rocky elements along roads, tracks, railways and dykes, river embankments, stone walls in vineyards, old buildings, and even in gardens. Even though the smooth snake is an elusive species and
we certainly tend to underestimate population sizes, our results also suggest that dispersal might also be underestimated, at least for some individuals (males or juveniles) and in mainland Europe, even if, locally, when the density is low or at the edge of the distribution area, the diversification could be strong. We think that further studies on its ecology, distribution, and population dynamics should be carried out to better understand the use of the landscape, the importance of dispersal on the population dynamics of this species, as well as the position in the distribution area or the density on its genetic diversification.