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- MATERIAL and METHODS
An effect of geographical barriers on the level of population structuring and ways of northwards colonization in the common shrew Sorex araneus was investigated by contrasting gene flow inferred by one Y-linked and eight autosomal microsatellites. A total of 102 shrews trapped at eight localities separated by mountain ridges of the Śnieżnik Massif (East Sudetes, Poland) were studied. The genetic structure of populations was estimated using the standard analysis of molecular variance based on F-statistic, as well as two clustering methods implemented in Structure and Geneland. In pair-wise population comparisons both FST and RST were estimated. A Mantel-test was used to investigate the patterns and causes of structuring. No significant correlation between genetic differentiation and geographical distance was found for autosomal loci and for the Y-linked locus. Significant genetic structuring was found in four out of six pairs of populations. Studying autosomal loci we found nonsignificant correlations between pair-wise matrices of FST and RST and the presence of the barrier. On the other hand, for the Y-linked locus these correlations were significant, both for FST and RST, suggesting reduced gene flow between populations for males. Patterns of genetic structuring in the common shrew of the Massif of Śnieżnik may suggest two possible ways of northwards colonization, which promoted genetic distinction of shrews migrating different routes.
The role of geographical barriers, such as mountain ridges, in processes of genetic isolation and speciation, is one of the fundamental aspects of evolutionary biology. Heterogeneous habitats may influence gene flow among populations, shaping their genetic structure. Unlimited and high gene flow prevents the fixation of alleles, slowing down local adaptations and as a consequence the process of speciation (Barton and Hewitt 1985). New polymorphisms, however, which can be brought about by gene flow, generates new gene combinations enriching the genetic potential of the population. Thus, geographically structured populations provide insight into the processes that affect their evolutionary ability (Balloux and Lugon-Moulin 2002).
The contribution of natural barriers to gene flow among populations of the common shrew Sorex araneus (Linnaeus, 1758) and their genetic structuring has mainly been investigated in mountain ranges. The influence of the alpine topography on speciation in Sorex araneus group and on the genetic differentiation of populations, living in isolated valleys, has been analysed in the French and Swiss Alps (Wyttenbach et al. 1999; Lugon-Moulin et al. 2000; Lugon-Moulin and Hausser 2002). Results of these studies indicate that high mountain ridges form geographical barriers, shaping the genetic structure of isolated populations (Lugon-Moulin et al. 2000).
In Poland, the possible effect of mountain ridges on gene flow levels among common shrew populations, has not yet been studied. To our knowledge, only the anthropogenic barrier – a railway embankment (Jadwiszczak et al. 2006) or aquatic barriers (Wierzbicki et al. 2011) and their influence on the exchange of genes between subpopulations inhabiting fragmented area has been investigated.
The Śnieżnik Massif is one of the places located in the mountains (East Sudetes, southwest Poland), where such processes can be studied. It consists of five main mountain ranges, dispersing from the one point – the top of Śnieżnik (Fig. 1). All ridges of the massif are separated by valleys with steep slopes. The Massif of Śnieżnik is distinctly different from the Alps, because of the presence of much lower mountain ridges (the highest peak is Śnieżnik – 1425 m a.s.l.). Therefore, the question arises if gene flow is unconstrained among geographically isolated populations of S. araneus given the topographic conditions of the Śnieżnik Massif.
Figure 1. Map of the study area showing topography of the Śnieżnik Massif and location of sampling sites (full circles, n = 8) separated by mountain ridges (thick lines); triangles indicate tops of mountains (meters).
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The aims of the present study were: 1) to investigate the influence of selected mountain ridges of the Śnieżnik Massif on gene flow between populations of S. araneus living in the neighbouring valleys, 2) to assess the level of genetic differentiation among populations under investigation, and 3) to suggest possible ways of northwards colonization. To achieve these objectives we used genetic markers: biparentally inherited (autosomal) microsatellites and a uniparentally inherited (Y-linked) microsatellite.
- Top of page
- MATERIAL and METHODS
The main goal of the present study was to investigate if landscape features of the Śnieżnik Massif, which is much lower than the Alps, affect gene flow among the common shrew populations and shape their genetic structuring.
The studied populations of the common shrew, revealed small but significant genetic differentiation among most of them (FST ranged from 0.010 to 0.076). These findings were congruent with results reported by Lugon-Moulin et al. (2000), who performed the pair-wise sample comparisons for the populations of S. araneus inhabiting the Swiss Alps. They estimated values of FST ranging from 0.007 to 0.120. When we analyzed the genetic differentiation between pairs of populations separated by mountain ridges, using FST- and RST-statistics, FST-values varied from 0.010 to 0.069 and RST-values ranging from –0.012 to 0.155 were found. Comparable values of FST were found in between-valley analysis in the Swiss Alps (FST: 0.013–0.083, RST: 0.018–0.148; Lugon-Moulin et al. 2000). In the Alps, significant genetic differentiation in five out of six pairs of valley-populations was found (Lugon-Moulin et al. 2000). Our results, obtained in much lower the Polish Sudetes, indicate significant genetic differentiation between the following pairs of samples: MI/MII, MI/MIV, J/MII, J/MIV and MIII/K. The first two pairs were separated by the ridge of 1124 m a.s.l. (Smrekowiec Mt), J/MII and J/MIV by the ridge of 1185 m a.s.l. (Wysoczka Mt) and the last pair by the ridge of 1153 m a.s.l. (Żmijowiec Mt). Significant genetic differentiation between MI and MII samples, separated by Smrekowiec Mt was also confirmed by RST-statistics (RST: 0.155, P < 0.05). This suggests that these two populations are isolated from each other.
Geographical distance did not influence genetic differentiation in the studied populations, as indicated by the Mantel-test. Neither for autosomal loci nor for the Y-linked locus significant correlation between genetic differentiation and geographical distance was found. These results suggest that in the common shrew populations inhabiting the Polish Sudetes, mountain ridges more than geographical distance, may prevent the dispersal of shrews. However, the pair-wise comparison of FST (Table 4) showed significant genetic differentiation between 14 out of 21 pairs of populations that were not separated by mountain ridges. These patterns were not consistent with divergence by distance. Cryptic barriers to dispersal appear to exist in the area of Śnieżnik Massif, allowing for small-scale genetic differentiation.
Environmental gradients such as vegetation or climate can function as cryptic barriers to gene flow (Yoshio et al. 2009). This may reduce migration between coexisting or nearby populations, leading to genetic divergence. According to Bergek and Björklund (2007) cryptic barriers to dispersal exist even in small, apparently homogenous environments.
In the Massif of Śnieżnik, the climate is very severe with long periods of snow cover and short vegetation. Milder climatic conditions and longer vegetation occur in the valleys that separate the ridges of the massif. Thus, factors such as distribution of vegetation and climatic conditions (there are no rivers and lakes in the massif which could be obstacles to gene flow) may have helped genetic structuring of the studied common shrew populations.
Analyzes of genetic differentiation between populations revealed, that two most remote populations (J and NM) which were separated by a few mountain ridges showed nonsignificant genetic differentiation (Table 4). In order to elucidate this we investigated the associations between topographic conditions of the Massif of Śnieżnik and the neighbouring Králicky Sneěžnik (situated in the Czech Republic), and values of FST estimated for each of the studied populations. The results of the study may suggest that shrews from the areas located south of Králicky Sneěžnik (these individuals may have created a founder population for further spread into Poland) migrated northwards, colonizing the studied area using two ways which bypassed the highest ridges (Fig. 2). First way of migration (“west route”) may have bypassed the Massif of Śnieżnik and the neighbouring ridges from the west, leading through J to MIII. Second way of migration (“east route”) may have led through the Płoszczyna Pass and NM towards MI, bypassing the massif from the east. This hypothesis explains 1) lack of significant genetic differentiation between J and NM (both populations originate from the common ancestors living in the area located south of Králicky Sneěžnik), 2) genetic similarity between NM and KA, KA and K, KA and MI (“east route” of migration), 3) genetic distinction of J and MIII (“west route” of migration) from NM, KA, K, MI (“east route” of migration). Significant genetic differentiation observed between MI and MIII, would indicate that these samples were taken from populations migrating using different ways. Geographic separation (shown in Fig. 4) of shrews migrating different routes (“east” and ”west”) may have promoted their genetic distinction.
In our study, values of FIS estimated over all populations and over all loci were very high and significantly different from zero (FIS= 0.19 and FIS= 0.18, respectively, P < 0.001). Five out of eight populations: J, KA, MI, MIII and MIV showed a high and significant heterozygote deficit (Table 2). Potential reasons explaining high values of FIS, i.e. inbreeding, selection or small sample size were probably not important in our study. Previous studies did not show inbreeding of S. araneus (Bengtsson and Frykman 1990). In the common shrew, an effect of inbreeding is reduced by a special strategy of females, which are highly promiscuous and regularly mate to close relatives (Stockley et al. 1993). The selection also does not seem a good explanation for a significant FIS in our study. All specimens represented the Drnholec chromosome race. It means that among studied individuals, there were no hybrids and possibly only the small number of Robertsonian heterozygotes. In the common shrew, selection against simple heterozygotes is very weak and negligible (Searle and Wójcik 1998). The deficit of heterozygotes, observed in our study, cannot rather be explained by the small sample sizes (number of individuals ranged from 7 to 27). It is noteworthy that a high FIS can be caused by more than one factor, and sometimes it is difficult to decide which factor is causative (Freeland 2008).
The findings on the relationship between genetic differentiation of the studied populations and presence of barriers were revealing. We found non-significant correlations between pair-wise matrices of FST and RST and the presence of the barrier. This indicates that the ridges of the Śnieżnik Massif have no significant impact on genetic structure of the populations of S. araneus. Low altitudes biotopes seem well connected. On the other hand, for the Y-linked locus, these correlations were significant, both for FST and RST, suggesting restricted male-mediated gene flow between the populations (FST= 0.379, P < 0.01; RST= 0.337, P < 0.01). Our findings are comparable with results previously reported by different authors, who observed sex-biased dispersal in the common shrew (Balloux et al. 2000; Lugon-Moulin and Hausser 2002; Andersson 2004; Yannic et al. 2008). However, in these studies, the reduced gene flow of males was observed in hybrid zones, and was interpreted as a classic example of Haldane's rule (male sterility in F1 hybrids). In our study, all specimens belonged to the same chromosome race (Drnholec), thus the Haldane rule cannot be applied. Thus, there must be another explanation for the barrier effect of relatively low mountain ranges to male-mediated gene flow.
Dispersal of individuals often goes along with high costs due to predation, natural death or reduced resources. One of the models describing patterns of dispersal is the Greenwood's model of competition for resources (Greenwood 1980). According to this model, philopatry (the ability to exploit resources and reproduce close to the birthplace) benefits the sex which is responsible for reproductive success. In monogamous species, males benefit more, while in polygynous species, females benefit. It is known that philopatry may promote genetic divergence among populations by limiting gene flow (Piertney et al. 1998).
Evidence for multiple paternity in the common shrew was reported by Searle (1990). This suggests that dispersal in the shrews is male-biased, in contrast to female- biased dispersal in monogamous species. Contrary to Searle's study, our results, suggest that in the Massif of Śnieżnik, females disperse more than males. A similar observation was reported by Fivaz et al. (2003) who studied postglacial recolonization of the Valais by the Sorex antinorii. The analysis of the Y-chromosome microsatellite showed a nearly complete absence of male gene flow between populations from the Simplon Pass and the St. Bernard Pass. The authors speculated that the long isolation of S. antinorii in Italy may have changed the mating behaviour of this species to monogamy, as observed in Crocidura russula (Favre et al. 1997).
The shrews inhabiting the Massif of Śnieżnik have not been isolated for a very long time (the Pleistocene glaciers did not reach this area, Bieroński et al. 2007). Thus it is unlikely that the limited male gene flow between studied localities can be attributed to the C. russula-like mating behaviour.
When we used autosomal loci to study genetic differentiation in both sexes separately we found that male's FST was nearly two-fold lower as compared with the female's one. This shows that males play a leading role in dispersal. Higher mobility of males in the genus Sorex was reported by Cantoni (1993) and Churchfield et al. (1995). According to these authors, females are considered to be territorial through most of their lifespan. However, the simple relation between FST and migration generally does not hold because of the non realistic assumptions of the underlying island model of migration (Balloux and Lugon-Moulin 2002).
One of the explanations for incongruence between autosomal and the Y-linked markers may be a high variance in male reproductive success. According to Lugon-Moulin and Hausser (2002), this variance may alter the genetic structure of Y-specific markers because of the Y- chromosome effective population size (NE). With a balanced sex ratio, the effective population size of the Y-chromosome is fourfold lower than for autosomes so that the effect of genetic drift is more important.
The estimated number of migrants per generation (Nm) does not reflect the geographical distance among the studied populations (Table 4). We did not find associations between remoteness of the populations and the migration rate among them. The number of migrants, however, seems to be affected by physical barriers.
The lowest number of migrants between the localities separated by Smrekowiec Mt may be the outcome of deforestation of the top parts of the mountain during ecological disaster. In consequence, unsuitable habitats (open fields and dry slopes) for the dispersal of the common shrew have come into being. The limited gene flow among localities separated by Smrekowiec Mt (MI/MII and MI/MIV), confirmed by statistically significant values of pair-wise FST (0.069, P < 0.05 and 0.061, P < 0.01, respectively) seems to support this hypothesis.
In conclusion, the present study revealed that most of the studied populations of the common shrew inhabiting the Massif of Śnieżnik showed little, but significant genetic structuring. We found that male- mediated exchange of genes between the populations was restricted by the ridges of the Polish Sudetes. Besides historical processes and life histories, heterogeneous habitats may also alter genetic structure of the populations.
Patterns of genetic structuring in the common shrew of the Massif of Śnieżnik may suggest two possible ways of northwards colonization, which promoted genetic distinction of shrews migrating different routes. If dispersal is geographically restricted, we can expect important implications for evolutionary processes in the isolated populations.