Genetic diversity in natural populations of noble crayfish (Astacus astacus L.) in north-western Poland on the basis of combined SSR and AFLP data

Discussion

The analysis of genetic diversity among and within natural populations of noble crayfish in northwestern Poland was conducted using two types of genetic markers, SSR and AFLP. Both revealed significant genetic differentiation in the five analysed populations. One of the most important factors influencing genetic diversity of the crayfish populations from the Bytów Lakeland is a diverse topography shaped by the action of glaciers (Lindner & Marks, 2008). Lakes located in close proximity (three km) but separated by a deep valley (e.g., Kwisno and Sęki) host crayfish populations regarded as genetically distinct (F_ST = 0.211; Table 4). In comparison, large interconnected water bodies in Finland enable uninterrupted crayfish movement which led to genetic homogenization and an overall loss of genetic diversity.

Generally open lakes facilitate movement and hence new combinations of alleles in the offspring; unless there is selection pressure on populations towards homo- or heterozygosity, the genetic balance is thus maintained (Alaranta et al., 2006). The two adjacent lakes in our study, Kw and Se, also belong to different catchments, that of the Wieprza River flowing north and entering the Baltic Sea and that of the Brda River flowing south as a tributary to the Vistula River. Kwisno is an example of a water body which might have lost its connection to the Wieprza River as a result of a lowered water level after the last glaciation (Lindner & Marks, 2008). The crayfish population in this isolated lake is characterised by an F_IS coefficient (heterozygote excess) of –0.285, a low allelic richness value (2.77) and a statistically significant deviation from the HWE in the genotype distribution (Table 3). All indices suggest that the crayfish population from Lake Kwisno has a small reproductive size with only few breeders likely to contribute to the next generation. Reasons for heterozygote excess in crayfish populations remain poorly understood, although several mechanisms potentially account for negative F_IS coefficients. These include a low number of mating individuals, selection pressure towards heterozygotic individuals, and asexual reproduction (Stoeckel et al., 2006). Gross et al. (2013) reported negative F_IS values for several crayfish populations from Sweden (Ljungan, Måssjön, Skiringen), Finland (Linkullasjön, Mikkolanjärvi) and Estonia (Laugi). In contrast to the populations studied here, all mean F_IS values calculated for populations in those countries were positive (Table 3).

Except for evidence obtained from topography analysis, genetic results confirmed that populations from Kwisno and Graniczne Lakes had the lowest level of genetic differentiation (F_ST = 0.04). Lake Graniczne has a permanent connection to the Pokrzywna River, a left-bank tributary of the Wieprza River. Therefore, it is conceivable that populations could move between the two lakes in the past and thus interbreed. However, over the years, Lake Kwisno and the Wieprza River became disconnected and a subpopulation could emerge as a result of a founder effect, a mechanism that has been widely described also for other species (Bernatchez & Wilson, 1998). F_ST values support the notion that Kwisno emerged as a part of the Lake Graniczne-Wieprza River population (Figs. 2 and 4, Table 4), whereas the reduction in genetic diversity revealed by values of allelic richness, F_IS and Apr are in line with the assumption that a founder event occurred in the past (Table 3). Furthermore, interviews with local inhabitants suggest that the population of Lake Kwisno experienced extensive illegal exploitation, which could have further reduced diversity of the genetic pool (Śmietana, 2013). A similar situation was identified for Lakes Sęki and Babinki in the Brda River catchment. Lake Sęki, similar to Lake Kwisno, is an isolated body of water. The crayfish population inhabiting this lake is characterised by a negative F_IS coefficient of −0.420 (Table 3). An analysis of the diverse topography of the Bytów Lakeland (Śmietana, 2013) juxtaposed with the present genetic results reveal that in the past the population from Lake Sęki lost the connection with the populations in the Brda River catchment, including the population in Lake Babinki. The crayfish from Lake Babinki belong to a large and non-isolated population that shows no signs of disruptive circumstances in the past that could have changed the distribution of genotypes and allele frequencies (Table 3). Instead, a high degree of similarity between the populations of the two lakes (F_ST = 0.057) suggests common ancestors, further supporting the above conclusions derived from the use of other methods (Figs. 2–4).

Among all results of the present study, those on Lake Biwakowe are the most interesting. The crayfish population of this lake is characterised by the highest diversity of all populations investigated here (Figs. 2, 4, 5 and 6). In contrast, genetic variability of the populations in Lakes Seki and Kwisno was considerably reduced, as shown by a low F_IS value of −0.410 (Table 3). Differences in genetic diversity (F_ST) between the investigated populations was greatest between Lake Biwakowe and the lakes of the Brda catchment. This outcome is most likely the result of geographic isolation, which has probably existed since the Bytów Lakeland was formed when the glacier receded after the last glaciation (Willisa & Van Andelb, 2004; Marks, 2012). Significant differences between populations can indeed result from landscape characteristics, as described for the genetic divergence of populations of Pacific jumping mice (Zapus trinotatus; Vignieri, 2005), tiger salamanders (Ambystoma tigrinum melanostictum; Spear et al., 2005) or long-toed salamanders (Ambystoma macrodactulym; Giordano, Ridenhour & Storfer, 2007). Results obtained here for noble crayfish from the Bytów Lakeland provide another such example for populations of a threatened species.

Significant genetic separation of the five investigated populations is also confirmed by the results of AMOVA on the microsatellite loci, showing that the diversity among populations corresponds to 18% and 82% of the total and intrapopulation variability, respectively. Variability at this level was also confirmed by the AFLP results. Additionally, an apparent grouping of populations was confirmed by the STRUCTURE analysis, which designated four separate clades (K = 4) among the five populations (Fig. 4). These results indicate genetic differentiation of the noble crayfish population in Lakes Kwisno and Graniczne from those in Lakes Babinki and Sęki. The geographically isolated population of Lake Biwakowe, which has no surface water connection with the other four lakes, proved to be genetically distinct from all others. This finding is in agreement with possible routes of crayfish movement, since Lakes Kwisno and Graniczne belong to the same catchment of the Wieprza River, whereas Lakes Babinki and Sęki are part of the Brda River catchment.

The findings of the preent study add information to the results of a large-scale survey about the diversity, recolonization routes and structure of noble crayfish populations in Europe (Gross et al., 2013). Gross et al. (2013) determined the lowest mean A_R value for Finnish (2.0) and the highest value for German (4.2) populations. The populations of the Bytów Lakeland investigated here had an intermediate value (3.3). Similarly, the average H_O or H_E values of 0.470 and 0.380, respectively, in the present study ranged between the highest and lowest average values for the German (H_O = 0.533, H_E = 0.598) and Finnish (H_O = 0.212, H_E = 0.228) populations. Strong within-country genetic differentiation of noble crayfish was observed in Estonia, probably due to limited gene fiow among populations in different catchments as well as to genetic drift (Gross et al., 2013). Notably, this level of within-region variation was restricted to Estonia, where both Baltic and Black Sea genotypes were observed (Gross et al., 2013). A similar situation might exist in Poland where populations of different catchments show strong genetic differentiation. Given its geographical position, Poland might be a region where the southern and northern genotypes identified by Gross et al. (2013) co-occur as well. This makes the area particularly interesting and important to determine the genetic structure of noble crayfish populations. The indices obtained in the present study confirm the northern direction of crayfish re-colonisation along with decreasing genetic diversity, as is commonly observed also for other species (Taberlet et al., 1998; Hewitt, 1999). However, for an unambiguous reconstruction of crayfish colonisation routes after glaciation, an analysis of peri-Baltic populations is required, based on a large number of microsatellite markers (or SNP) and in combination with mitogenome analyses.

An assessment of the size of noble crayfish populations in selected lakes of Pomerania by standard methods (i.e., capture per unit effort, CPUE) (Stuecheli, 1991; Dorn & Wojdak, 2005) showed that crayfish were most abundant in Lake Graniczne and occurred at only slightly lower densities in Lake Sęki (Śmietana, 2013). However, while fluctuations in population size were the lowest in Lakes Kwisno and Babinki, the greatest reduction in the population size was observed in Lake Sęki, a small lake which had probably been subject to extensive illegal exploitation (Śmietana, 2013). Strong exploitation pressure might also explain the considerable decrease in crayfish genetic diversity in that lake.

Research in the Bytów Lakeland over the past 20 years has shown that noble crayfish may be an indicator of aquatic habitat quality (Śmietana, 2013). Numbers have exponentially declined in the area, apparently due to habitat deterioration by eutrophication, poaching, eel stocking and the invasion of non-indigenous crayfish species carrying the crayfish plague. At present, the most favourable habitats for the species in Pomerania occur in isolated areas at higher altitude with cooler and more humid climate (Śmietana, 2013). The location of the lakes where noble crayfish were caught for the present study reflects geomorphological changes in the Bytów Lakeland since the last glaciation approximately 10,000 years ago (Marks, 2012). The area is characterized by high landscape diversity with numerous moraine uplands rising to ridges above 200 m a.s.l. and deep subglacial gullies partly filled with lakes and river valleys (Raczkowski & Sroka, 2010). Lakes in the area currently inhabited by noble crayfish are characterised by forest cover in the catchments close to 85% (Śmietana, 2013). This and the results presented here indicate that anthropogenic disturbance have a notable influence on crayfish populations in the area (Śmietana, 2013). Lake Graniczne situated at 27.9 km from the nearest human settlement had the highest allelic richness and Lake Biwakowe (5.7 km distance) had the lowest, while the three other crayfish populations in Lakes Babinki (18.8 km), Sęki (11.4 km) and Kwisno (16.8 km) were characterised by intermediate values (Table 3). Poaching, eel stocking, the crayfish plague transmitted by non-indigenous crayfish species, all of which are related to human activities, apparently had the greatest impact (Śmietana, 2013). This and the evidence presented here suggest that noble crayfish should be valued for nature conservation in Pomerania as much as the animal species listed in the Habitats Directive of the European Commission (1992).

Conclusions

The five crayfish populations investigated in the present study have been constantly monitored for two decades. Results of these monitoring efforts indicate that conservation of the populations requires active protection measures such as restocking with offspring obtained especially through aquaculture breeding programmes of crayfish with known genetic make-up. DNA markers facilitated the genetic characterisation of selected remaining natural populations of A. astacus in norhwestern Poland. Given their diversity, the last populations of noble crayfish in the Bytów Lakeland analysed in the present study have great value as a live genetic bank for such breeding and restitution purposes. As the results presented here show, genetic analyses of the populations are also valuable to inform about the genetic diversity, post-glacial re-colonisation routes and population structure of noble crayfish populations in Europe. However, additional comprehensive analyses are needed to assess the genetic structure, origin and vulnerability of the remaining natural populations in norhwestern Poland.

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