The processes underlying divergent selection and genetic adaptation have been on the evolutionary biologists agenda for a long time. In this study we used the three-spined stickleback (Gasterosteus aculeatus) study system, a perfect system to study the evolution of similar traits in different lineages exposed to similar environmental conditions. Lind and Grahn (2011) have found directional selection caused by pulp mill effluent on populations of three-spined stickleback along the Swedish coast. In their study, they identified 21 AFLP- outlier loci indicated to be under selection. Here we converted some of these anonymous AFLP loci into sequenced markers and aligned them to the stickleback genome. Four out of five loci, aligned within or close to coding regions, on chromosome I, chromosome VIII, chromosome XIX and chromosome XX. One of the locus, located on chromosome VIII, have been identified to be under selection for fresh water adaption in other studies, including Baltic Sea stickleback populations (Mäkinen et al. 2008a,b). We believe that this is feasibly method that can be used as a starting point for identification of genes and genomic regions possible involved in adaptation, both for model and non-model organisms. 

The last century, humans have been altering almost all natural environments at an accelerating rate, including the Baltic Sea that has highly eutrophicated areas and many coastal industries such as Pulp-mills. For animals living in a habitat that changes there are basically two alternatives, either to cope with the change or become locally extinct. This thesis aims to investigate if recent anthropogenic disturbance in the Baltic Sea can affect natural populations on a genetic level through natural selection.

First, we found a fine-scale genetic structure in three-spine sticklebacks (Gasterosteus aculeatus) populations along the Swedish coast (paper I), indicating limited gene-flow between populations in geographic proximity. Different genetic markers, specifically Amplified Fragment Lenght Polymorpism (AFLP, and microsatellites,  gave different results, highlighting the heterogeneous character of genomes which demonstrates that it is important to choose a genetic marker that is relevant for the question at hand. With a population genomic approach, and a multilocus genetic marker (AFLP), we detected convergent evolution in genotype composition in stickleback populations living in environments affected by pulp-mill effluent (paper II) and in highly eutrophicated environments (paper III), compared to adjacent reference populations. We found loci, in both studies (paper II, III), that were different from a neutral distribution and thus probably under divergent selection for the habitat differences investigated. The selective effect from pulp-mill effluents were more pronounced, but the two different habitats had mutual characters (AFLP loci). In paper IV, we converted five anonymous AFLP loci to sequenced markers and aligned them to the stickleback genome. Four out of five loci aligned within, or close to, coding regions on chromosome I, chromosome VIII, chromosome XIX and chromosome XX. One of the loci, located on chromosome VIII and identified as under divergent selection in both paper II and III, has been identified in other studies as to be under selection for fresh water adaptation, including Baltic Sea stickleback populations.

In conclusion, anthropogenic alterations of natural environments can have evolutionary consequences, probably adaptive, for the animals living there and the evolutionary response exhibited by natural populations can be very fast.

Contamination can cause a rapid environmental change which may require populations to respond with evolutionary changes. To evaluate the effects of pulp mill effluents on population genetics, we sampled three-spined sticklebacks (Gasterosteus aculeatus) near four pulp mills and four adjacent reference sites and analyzed Amplified Fragment Length Polymorphism (AFLP) to compare genetic variability. A fine scale genetic structure was detected and samples from polluted sites separated from reference sites in multidimensional scaling plots (P < 0.005, 1000 permutations) and locus-by-locus Analysis of Molecular Variance (AMOVA) further confirmed that habitats are significantly separated (F(ST) = 0.021, P < 0.01, 1023 permutations). The amount of genetic variation between populations did not differ between habitats, and populations from both habitats had similar levels of heterozygosity (polluted sites Nei's Hs = 0.11, reference sites Nei's Hs = 0.11). Still, pairwise F(ST): s between three, out of four, pairs of polluted-reference sites were significant. A F(ST)-outlier analysis showed that 21 (8.4%) loci were statistically different from a neutral distribution at the P < 0.05 level and therefore indicated to be under divergent selection. When removing 13 F(ST)-outlier loci, significant at the P < 0.01 level, differentiation between habitats disappeared in a multidimensional scaling plot. In conclusion, pulp mill effluence has acted as a selective agent on natural populations of G. aculeatus, causing a convergence in genotype composition change at multiple sites in an open environment.

Anthropogenic activities are causing change in natural habitats at an accelerating rate and affecting populations by altered selection pressures. One example is human-induced eutrophication in the Baltic Sea, were behaviour alterations are well documented in three-spined sticklebacks (Gasterosteus aculeatus). Here we have used 204 variable Amplified Fragment Length Polymorphism (AFLP) markers to investigate genetic differences between a set of ten hierarchal sampled populations of sticklebacks, five populations inhabiting eutrophicated habitats and five from control populations, in total 292 individuals. We found significant genetic variation that could be attributed to habitat (4.3% AMOVA). A combination of F_ST outlier analysis and classification analysis revealed seven AFLP-loci likely to be affected by divergent selection by eutrophication. Four of these seven loci have earlier been identified as under selection in stickleback populations living in pulp-mill effluents suggesting some similar selective factors between eutrophication and pulp-mill effluent effected habitats. 

There are three basic types of population structures in marine environments; populations that are distinct, with a continuous change and without any differentiation. In each type the population units are characterized by groups of individuals with panmixia within groups and site fidelity to a limited geographic area. Earlier studies of the population genetic structure on sticklebacks in the Baltic Sea have shown none or only little structure. We have sampled 8 sites (253 individuals) along Sweden’s coast to estimate the genetic structure, using five microsatellites and 173 Amplified Fragment Length Polymorphism (AFLP) markers and detected a fine scale genetic structure (AFLP F_ST= 25%, microsatellites F_ST = 2.7%). With AFLPs the observed variation followed isolation by distance model (but not with microsatellites). Even sites separated by only 2 km of water are significantly separated. Both Bayesian clustering analysis and Capscale separated populations and identified populations from Gulf of Bothnia (4 psu) and from the west coast (20 psu) as genetically distinctly different from Baltic populations (about 7-8 psu).  In conclusion, gene flow is limited between sampled sites, and since no geographic barriers can be distinguished the population structure is likely caused by the sticklebacks’ behavior. Hence, we have probably sampled either stationary populations of marine sticklebacks, or homing sticklebacks. In this study AFLP and microsatellites did not give congruent results; with AFLPs we got high separation, and genetic variation followed isolation by distance model and supported the continuous change type of population structure.