Environmental effects of thermal and radioactive discharges from nuclear power plants in the boreal brackish-water conditions of the northern Baltic Sea

During recent decades, thermal and radioactive discharges from nuclear power plants into the aquatic environment have become the subject of lively debate as an ecological concern. The target of this thesis was to summarize the large quantity of results obtained in extensive monitoring programmes and studies carried out in recipient sea areas off the Finnish nuclear power plants at Loviisa and Olkiluoto during more than four decades. The Loviisa NPP is located on the coast of the Gulf of Finland and Olkiluoto NPP on that of the Bothnian Sea. The state of the Gulf of Finland is clearly more eutrophic; the nutrient concentrations in the surface water are about 1½ 2 times higher at Loviisa than at Olkiluoto, and the total phosphorus concentrations still increased in both areas (even doubled at Loviisa) between the early 1970s and 2000. Thus, it is a challenge to distinguish the local effects of thermal discharges from the general eutrophication process of the Gulf of Finland. The salinity is generally low in the brackish-water conditions of the northern Baltic Sea, being however about 1 higher at Olkiluoto than at Loviisa (the salinity of surface water varying at the latter from near to 0 in early spring to 4 6 in late autumn). Thus, many marine and fresh-water organisms live in the Loviisa area close to their limit of existence, which makes the biota sensitive to any additional stress. The characteristics of the discharge areas of the two sites differ from each other in many respects: the discharge area at Loviisa is a semi-enclosed bay in the inner archipelago, where the exchange of water is limited, while the discharge area at Olkiluoto is more open, and the exchange of water with the open Bothnian Sea is more effective. The effects of the cooling water discharged from the power plants on the temperatures in the sea were most obvious in winter. The formation of a permanent ice cover in the discharge areas has been delayed in early winter, and the break-up of the ice occurs earlier in spring. The prolonging of the growing season and the disturbance of the overwintering time, in conditions where the biota has adjusted to a distinct rest period in winter, have been the most significant biological effects of the thermal pollution. The soft-bottom macrofauna at Loviisa has deteriorated to the point of almost total extinction at many sampling stations during the past 40 years. A similar decline has been reported for the whole eastern Gulf of Finland. However, the local eutrophication process seems to have contributed into the decline of the zoobenthos in the discharge area at Loviisa. Thermal discharges have increased the production of organic matter, which again has led to more organic bottom deposits. These have in turn increased the tendency of the isolated deeps to a depletion of oxygen, and this has further caused strong remobilization of phosphorus from the bottom sediments. Phytoplankton primary production and primary production capacity doubled in the whole area between the late 1960s and the late 1990s, but started to decrease a little at the beginning of this century. The focus of the production shifted from spring to mid- and late summer. The general rise in the level of primary production was mainly due to the increase in nutrient concentrations over the whole Gulf of Finland, but the thermal discharge contributed to a stronger increase of production in the discharge area compared to that in the intake area. The eutrophication of littoral vegetation in the discharge area has been the most obvious, unambiguous and significant biological effect of the heated water. Myriophyllum spicatum, Potamogeton perfoliatus and Potamogeton pectinatus, and vigorous growths of numerous filamentous algae as their epiphytes have strongly increased in the vicinity of the cooling water outlet, where they have formed dense populations in the littoral zone in late summer. However, the strongest increase of phytobenthos has extended only to a distance of about 1 km from the outlet, i.e., the changes in vegetation have been largest in those areas that remain ice-free in winter. Similar trends were also discernible at Olkiluoto, but to a clearly smaller extent, which was due to the definitely weaker level of background eutrophy and nutrient concentrations in the Bothnian Sea, and the differing local hydrographical and biological factors prevailing in the Olkiluoto area. The level of primary production has also increased at Olkiluoto, but has remained at a clearly lower level than at Loviisa. In spite of the analogous changes observed in the macrozoobenthos, the benthic fauna has remained strong and diversified in the Olkiluoto area. Small amounts of local discharge nuclides were regularly detected in environmental samples taken from the discharge areas: tritium in seawater samples, and activation products, such as 60Co, 58Co, 54Mn, 110mAg, 51Cr, in suspended particulate matter, bottom sediments and in several indicator organisms (e.g., periphyton and Fucus vesiculosus) that effectively accumulate radioactive substances from the medium. The tritium discharges and the consequent detection frequency and concentrations of tritium in seawater were higher at Loviisa, but the concentrations of the activation products were higher at Olkiluoto, where traces of local discharge nuclides were also observed over a clearly wider area, due to the better exchange of water than at Loviisa, where local discharge nuclides were only detected outside Hästholmsfjärden Bay quite rarely and in smaller amounts. At the farthest, an insignificant trace amount (0.2 Bq kg-1 d.w.) of 60Co originating from Olkiluoto was detected in Fucus at a distance of 137 km from the power plant. Discharge nuclides from the local nuclear power plants were almost exclusively detected at the lower trophic levels of the ecosystems. Traces of local discharge nuclides were very seldom detected in fish, and even then only in very low quantities. As a consequence of the reduced discharges, the concentrations of local discharge nuclides in the environment have decreased noticeably in recent years at both Loviisa and Olkiluoto. Although the concentrations in environmental samples, and above all, the discharge data, are presented as seemingly large numbers, the radiation doses caused by them to the population and to the biota are very low, practically insignificant. The effects of the thermal discharges have been more significant, at least to the wildlife in the discharge areas of the cooling water, although the area of impact has been relatively small. The results show that the nutrient level and the exchange of water in the discharge area of a nuclear power plant are of crucial importance.

Archaea in the Mycorrhizosphere of Boreal Forest Trees

Archaea were long thought to be a group of ancient bacteria, which mainly lived in extreme environments. Due to the development of DNA sequencing methods and molecular phylogenetic analyses, it was shown that the living organisms are in fact divided into three domains; the Archaea, Bacteria and the Eucarya. Since the beginning of the previous decade, it was shown that archaea generally inhabit moderate environments and that these non-extremophilic archaea are more ubiquitous than the extremophiles. Group 1 of non-extreme archaea affiliate with the phylum Crenarchaeota. The most commonly found soil archaea belong to the subgroup 1.1b. However, the Crenarchaeota found in the Fennoscandian boreal forest soil belong to the subgroup 1.1c. The organic top layer of the boreal forest soil, the humus, is dominated by ectomycorrhizal fungal hyphae. These colonise virtually all tree fine root tips in the humus layer and have been shown to harbour distinct bacterial populations different from those in the humus. The archaea have also been shown to colonise both boreal forest humus and the rhizospheres of plants. In this work, studies on the archaeal communities in the ectomycorrhizospheres of boreal forest trees were conducted in microcosms. Archaea belonging to the group 1.1c Crenarchaeota and Euryarchaeota of the genera Halobacterium and Methanolobus were detected. The archaea generally colonised fungal habitats, such as ectomycorrhizas and external mycelia, rather than the non-mycorrhizal fine roots of trees. The species of ectomycorrhizal fungus had a great impact on the archaeal community composition. A stable euryarchaeotal community was detected especially in the mycorrhizas, of most of the tested Scots pine colonising ectomycorrhizal fungi. The Crenarchaeota appeared more sporadically in these habitats, but had a greater diversity than the Euryarchaeota. P. involutus mycorrhizas had a higher diversity of 1.1c Crenarchaeota than the other ectomycorrhizal fungi. The detection level of archaea in the roots of boreal trees was generally low although archaea have been shown to associate with roots of different plants. However, alder showed a high diversity of 1.1c Crenarchaeota, exceeding that of any of the tested mycorrhizas. The archaeal 16S rRNA genes detected from the non-mycorrhizal roots were different from those of the P. involutus mycorrhizas. In the phylogenetic analyses, the archaeal 16S rRNA gene sequences obtained from non-mycorrhizal fine roots fell in a separate cluster within the group 1.1c Crenarchaeota than those from the mycorrhizas. When the roots of the differrent tree species were colonised by P. involutus, the diversity and frequency of the archaeal populations of the different tree species were more similar to each other. Both Cren- and Euryarchaeota were enriched in cultures to which C-1 substrates were added. The 1.1c Crenarchaeota grew anaerobically in mineral medium with CH4 and CO2 as the only available C sources, and in yeast extract media with CO2 and CH4 or H2. The crenarchaeotal diversity was higher in aerobic cultures on mineral medium with CH4 or CH3OH than in the anaerobic cultures. Ecological functions of the mycorrhizal 1.1c Crenarchaeota in both anaerobic and aerobic cycling of C-1 compounds were indicated. The phylogenetic analyses did not divide the detected Crenarchaeota into anaerobic and aerobic groups. This may suggest that the mycorrhizospheric crenarchaeotal communities consist of closely related groups of anaerobic and aerobic 1.1c Crenarchaeota, or the 1.1c Crenarchaeota may be facultatively anaerobic. Halobacteria were enriched in non-saline anaerobic yeast extract medium cultures in which CH4 was either added or produced, but were not detected in the aerobic cultures. They may potentially be involved in anaerobic CH4 cycling in ectomycorrhizas. The CH4 production of the mycorrhizal samples was over 10 times higher than for humus devoid of mycorrhizal hyphae, indicating a high CH4 production potential of the mycorrhizal metanogenic community. Autofluorescent methanogenic archaea were detected by microscopy and 16S rRNA gene sequences of the genus Methanolobus were obtained. The archaeal community depended on both tree species and the type of ectomycorrhizal fungus colonising the roots and the Cren- and Euryarchaeota may have different ecological functions in the different parts of the boreal forest tree rhizosphere and mycorrhizosphere. By employing the results of this study, it may be possible to isolate both 1.1c Crenarchaeota as well as non-halophilic halobacteria and aerotolerant methanogens from mycorrhizospheres. These archaea may be used as indicators for change in the boreal forest soil ecosystem due to different factors, such as exploitations of forests and the rise in global temperature. More information about the microbial populations with apparently low cell numbers but significant ecological impacts, such as the boreal forest soil methanogens, may be of crucial importance to counteract human impacts on such globally important ecosystems as the boreal forests.

Polypore assemblages in boreal old-growth forests, and associated Coleoptera

This thesis examines assemblages of wood-decaying fungi in Finnish old-growth forests, and patterns of species interactions between fruit bodies of wood-rotting Basidiomycetes and associated Coleoptera. The present work is a summary of four original publications and a manuscript, which are based on empirical observations and deal with the prevalence of polypores in old-growth forests, and fungicolous Coleoptera. The study area consists of eleven old-growth, mostly spruce- and pine-dominated, protected forests rich in dead wood in northern and southeastern Finland. Supplementary data on fungus beetle interactions were collected in southern Finland and the Åland Islands. 11251 observations of fruit bodies from 153 polypore species were made in 789 forest compartments. Almost a half of the polypore species demonstrated a distinct northern or southeastern trend of prevalence. Polypores with a northern prevalence profile were in extreme cases totally absent from the Southeast, although almost uniformly present in the North. These were Onnia leporina, Climacocystis borealis, Antrodiella pallasii, Skeletocutis chrysella, Oligoporus parvus, Skeletocutis lilacina, and Junghuhnia collabens. Species with higher prevalence in the southeastern sites were Bjerkandera adusta, Inonotus radiatus, Trichaptum pargamenum, Antrodia macra, and Phellinus punctatus. 198 (86%) species of Finnish polypores were examined for associated Coleoptera. Adult beetles were collected from polypore basidiocarps in the wild, while their larvae were reared to adulthood in the lab. Spatial and temporal parallels between the properties of polypore fruit body and the species composition of Coleoptera in fungus beetle interactions were discussed. New data on the biology of individual species of fungivorous Coleoptera were collected. 116 species (50% of Finnish polypore mycota) were found to host adults and/or larvae of 179 species from 20 Coleoptera families. Many new fungus beetle interactions were found among the 614 species pairs; these included 491 polypore fruit body adult Coleoptera species co-occurrences, and 122 fruit body larva interrelations. 82 (41%) polypore species were neither visited nor colonized by Coleoptera. The total number of polyporicolous beetles in Finland is expected to reach 300 species.

Effects of management and landscape structure on biodiversity in boreal agricultural farmland

Intensified agricultural practises introduced after the Second World War are identified as a major cause of global biodiversity declines. In several European countries agri-environment support schemes have been introduced to counteract the ongoing biodiversity declines. Farmers participating in agri-environment schemes are financially compensated for decreasing the intensity of farming practises leading to smaller yields and lower income. The Finnish agri-environment support scheme is composed of a set of measures, such as widened field margins along main ditches (obligatory measure), management of features increasing landscape diversity, management of semi-natural grasslands, and organic farming (special agreement measures). The magnitude of the benefits for biodiversity depends on landscape context and the properties of individual schemes. In this thesis I studied whether one agri-environment scheme, organic farming, is beneficial for species diversity and abundance of diurnal lepidopterans, bumblebees, carabid beetles and arable weeds. I found that organic farming did not enhance species richness of selected insect taxa, although bumblebee species richness tended to be higher in organic farms. Abundance of lepidopterans and bumblebees was not enhanced by organic farming, but carabid beetle abundance was higher in mixed farms with both cereal crop production and animal husbandry. Both species richness and abundance of arable weeds were higher in organic farms. My second objective was to study how landscape structure shapes farmland butterfly communities. I found that the percentage of habitat specialists and species with poor dispersal abilities in butterfly assemblages decreased with increasing arable field cover, leading to a dramatic decrease in butterfly beta diversity. In field boundaries local species richness of butterflies was linearly related to landscape species richness in geographic regions with high arable field cover, indicating that butterfly species richness in field boundaries is more limited by landscape factors than local habitat factors. In study landscapes containing semi-natural grasslands the relationship decelerated at high landscape species richness, suggesting that local species richness of butterflies in field boundaries is limited by habitat factors (demanding habitat specialists that occurred in semi-natural grasslands were absent in field margins). My results suggest that management options in field margins will affect mainly generalists, and species with good dispersal abilities, in landscapes with high arable field cover. Habitat specialists and species with poor dispersal abilities may benefit of management options if these are applied in the vicinity of source populations.

Farmland birds and habitat heterogeneity in intensively cultivated boreal agricultural landscapes

The biodiversity of farmland ecosystems has decreased remarkably during the latter half of the 20th century, and this development is due to intensive farming with its various environmental effects. In the countries of the EU the Common Agricultural Policy (CAP) is the main determinant affecting farmland biodiversity, since the agricultural policy defines guidelines of agricultural practices. In addition to policies promoting intensive farming, CAP also includes national agri-environment schemes (AES), in which a part of subsidies paid to farmers is directed to acts that are presumed to promote environmental protection and biodiversity. In order to shape AES into relevant and powerful tools for biodiversity protection, detailed studies on the effects of agriculture on species and species assemblages are needed. In my thesis I investigated the importance of habitat heterogeneity and effects of different habitat and landscape characteristics on farmland bird abundance and diversity in typical cereal cultivation-dominated southern Finnish agricultural environments. The extensive data used were collected by territory mapping. My two main study species were the drastically declined ortolan bunting (Emberiza hortulana) and the phenomenally increased tree sparrow (Passer montanus); in addition I studied assemblages of 20 species breeding in open arable and edge/bush habitats. In light of my results I discuss whether the Finnish AES take into account the habitat needs of farmland birds, and I provide suggestions for improvement of the future AES. My results show that heterogeneity of both uncultivated and cultivated habitats increases abundance and species richness among farmland birds, but in this respect the amount and diversity of uncultivated habitats are essential. Ditches in particular are a keystone structure for farmland birds in boreal landscapes. Ditches lined by trees or bushes increased ortolan bunting abundance. Loss of that kind of ditches (and clearance of forest and bush patches), reduced breeding ortolan buntings, mainly by decreasing availability of song-posts that are important for the breeding groups of the species. Heterogeneity of uncultivated habitats, most importantly open ditches and the habitat patch richness, increased densities and species richnesses of species assemblages of open arable and edge/bush habitats. Human impact (winter-feeding, nest-boxes) affected favourably the tree sparrow s rapid range expansion in southern Finland, but any habitat types had no significant effects. At the moment the Finnish agri-environmental policy does not conserve farmland ditches as a habitat type. Instead, sub-surface drainage is financially promoted. This is a fatal mistake as far as farmland biodiversity is concerned. In addition to the maintenance of ditches, at least the following aspects should be included more than is done previously in the measures of the future AES: 1) promotion of diverse crop rotation (especially by promoting animal husbandry), 2) maintenance of tree and bush vegetation in islets and along ditches, 3) promotion of organic farming.

Aerobic carbon-cycle related microbial communities in boreal peatlands: responses to water-level drawdown

Boreal peatlands represent a considerable portion of the global carbon (C) pool. Water-level drawdown (WLD) causes peatland drying and induces a vegetation change, which affects the decomposition of soil organic matter and the release of greenhouse gases (CO2 and CH4). The objective of this thesis was to study the microbial communities related to the C cycle and their response to WLD in two boreal peatlands. Both sampling depth and site type had a strong impact on all microbial communities. In general, bacteria dominated the deeper layers of the nutrient-rich fen and the wettest surfaces of the nutrient-poor bog sites, whereas fungi seemed more abundant in the drier surfaces of the bog. WLD clearly affected the microbial communities but the effect was dependent on site type. The fungal and methane-oxidizing bacteria (MOB) community composition changed at all sites but the actinobacterial community response was apparent only in the fen after WLD. Microbial communities became more similar among sites after long-term WLD. Litter quality had a large impact on community composition, whereas the effects of site type and WLD were relatively minor. The decomposition rate of fresh organic matter was influenced slightly by actinobacteria, but not at all by fungi. Field respiration measurements in the northern fen indicated that WLD accelerates the decomposition of soil organic matter. In addition, a correlation between activity and certain fungal sequences indicated that community composition affects the decomposition of older organic matter in deeper peat layers. WLD had a negative impact on CH4 oxidation, especially in the oligotrophic fen. Fungal sequences were matched to taxa capable of utilizing a broad range of substrates. Most of the actinobacterial sequences could not be matched to characterized taxa in reference databases. This thesis represents the first investigation of microbial communities and their response to WLD among a variety of boreal peatland habitats. The results indicate that microbial community responses to WLD are complex but dependent on peatland type, litter quality, depth, and variable among microbes.

Microbial activities in boreal soils: Biodegradation of organic contaminants at low temperature and ammonia oxidation

This thesis deals with the response of biodegradation of selected anthropogenic organic contaminants and natural autochthonous organic matter to low temperature in boreal surface soils. Furthermore, the thesis describes activity, diversity and population size of autotrophic ammonia-oxidizing bacteria (AOB) in a boreal soil used for landfarming of oil-refinery wastes, and presents a new approach, in which the particular AOB were enriched and cultivated in situ from the landfarming soil onto cation exchange membranes. This thesis demonstrates that rhizosphere fraction of natural forest humus soil and agricultural clay loam soil from Helsinki Metropolitan area were capable of degrading of low to moderate concentrations (0.2 50 µg cm-3) of PCP, phenanthrene and 2,4,5-TCP at temperatures realistic to boreal climate (-2.5 to +15 °C). At the low temperatures, the biodegradation of PCP, phenanthrene and 2,4,5-TCP was more effective (Q10-values from 1.6 to 7.6) in the rhizosphere fraction of the forest soil than in the agricultural soil. Q10-values of endogenous soil respiration (carbon dioxide evolution) and selected hydrolytic enzyme activities (acetate-esterase, butyrate-esterase and β-glucosidase) in acid coniferous forest soil were 1.6 to 2.8 at temperatures from -3 to +30 °C. The results indicated that the temperature dependence of decomposition of natural autochthonous soil organic matter in the studied coniferous forest was only moderate. The numbers of AOB in the landfarming (sandy clay loam) soil were determined with quantitative polymerase chain reaction (real-time PCR) and with Most Probable Number (MPN) methods, and potential ammonium oxidation activity was measured with the chlorate inhibition technique. The results indicated presence of large and active AOB populations in the heavily oil-contaminated and urea-fertilised landfarming soil. Assessment of the populations of AOB with denaturing gradient gel electrophoresis (DGGE) profiling and sequence analysis of PCR-amplified 16S rRNA genes showed that Nitrosospira-like AOB in clusters 2 and 3 were predominant in the oily landfarming soil. This observation was supported by fluorescence in situ hybridization (FISH) analysis of the AOB grown on the soil-incubated cation-exchange membranes. The results of this thesis expand the suggested importance of Nitrosospira-like AOB in terrestrial environments to include chronically oil-contaminated soils.

The role of lakes in carbon cycling in boreal catchments

Lakes are an important component of ecosystem carbon cycle through both organic carbon sequestration and carbon dioxide and methane emissions, although they cover only a small fraction of the Earth's surface area. Lake sediments are considered to be one of rather perma-nent sinks of carbon in boreal regions and furthermore, freshwater ecosystems process large amounts of carbon originating from terrestrial sources. These carbon fluxes are highly uncer-tain especially in the changing climate. -- The present study provides a large-scale view on carbon sources and fluxes in boreal lakes situated in different landscapes. We present carbon concentrations in water, pools in lake se-diments, and carbon gas (CO2 and CH4) fluxes from lakes. The study is based on spatially extensive and randomly selected Nordic Lake Survey (NLS) database with 874 lakes. The large database allows the identification of the various factors (lake size, climate, and catchment land use) determining lake water carbon concentrations, pools and gas fluxes in different types of lakes along a latitudinal gradient from 60oN to 69oN. Lakes in different landscapes vary in their carbon quantity and quality. Carbon (C) content (total organic and inorganic carbon) in lakes is highest in agriculture and peatland dominated areas. In peatland rich areas organic carbon dominated in lakes but in agricultural areas both organic and inorganic C concentrations were high. Total inorganic carbon in the lake water was strongly dependent on the bedrock and soil quality in the catchment, especially in areas where human influence in the catchment is low. In inhabited areas both agriculture and habitation in the catchment increase lake TIC concentrations, since in the disturbed soils both weathering and leaching are presumably more efficient than in pristine areas. TOC concentrations in lakes were related to either catchment sources, mainly peatlands, or to retention in the upper watercourses. Retention as a regulator of the TOC concentrations dominated in southern Finland, whereas the peatland sources were important in northern Finland. The homogeneous land use in the north and the restricted catchment sources of TOC contribute to the close relationship between peatlands and the TOC concentrations in the northern lakes. In southern Finland the more favorable climate for degradation and the multiple sources of TOC in the mixed land use highlight the importance of retention. Carbon processing was intensive in the small lakes. Both CO2 emission and the Holocene C pool in sediments per square meter of the lake area were highest in the smallest lakes. How-ever, because the total area of the small lakes on the areal level is limited, the large lakes are important units in C processing in the landscape. Both CO2 and CH4 concentrations and emissions were high in eutrophic lakes. High availability of nutrients and the fresh organic matter enhance degradation in these lakes. Eutrophic lakes are often small and shallow, enabling high contact between the water column and the sediment. At the landscape level, the lakes in agricultural areas are often eutrophic due to fertile soils and fertilization of the catchments, and therefore they also showed the highest CO2 and CH4 concentrations. Export from the catchments and in-lake degradation were suggested to be equally important sources of CO2 and CH4 in fall when the lake water column was intensively mixed and the transport of sub-stances from the catchment was high due to the rainy season. In the stagnant periods, especially in the winter, in-lake degradation as a gas source was highlighted due to minimal mixing and limited transport of C from the catchment. The strong relationship between the annual CO2 level of lakes and the annual precipitation suggests that climate change can have a major impact on C cycling in the catchments. Increase in precipitation enhances DOC export from the catchments and leads to increasing greenhouse gas emissions from lakes. The total annual CO2 emission from Finnish lakes was estimated to be 1400 Gg C a-1. The total lake sediment C pool in Finland was estimated to be 0.62 Pg, giving an annual sink in Finnish lakes of 65 Gg C a-1.

The ecology of the mountain hare (Lepus timidus L.) in managed boreal forests: habitat associations at different forest scales

Aims of this thesis This study is part of a larger hare project in Finland, which provides answers to basic ecological questions regarding the mountain hare. This study of the ecology of the mountain hare focuses in particular on different levels of managed boreal forest. The feeding habits and intensity of mountain hares in winter are explored, and the connections between mountain hares versus the forest structure are also studied (e.g. habitat use and the importance of different forest layers for hares). The use of the environment by hares at the landscape level was examined (forest patch structures), and the home ranges of mountain hares were studied. Finally, the productivity and survival rate of mountain hare populations were also studied (discussion e.g. predator effects on hare populations). Conclusions Feeding intensity seemed to be highest in the spring-winter, when home ranges were also largest. Favourable food species are covered by snow in winter and the mobility of hares is highest during late winter. A shortage of suitable food species may be problematic for hares, especially during the winter period. In this study mountain hares preferred a dense shrub layer at local level and deciduous and mixed tree forest over coniferous forest at the landscape level. Food and shelter are vital for hares and the preference for particular habitats may also affect the population dynamics of the mountain hare. It would be possible to improve the quality of food and shelter or at least prevent the most negative habitat changes through forest management. At a local level it is also possible to add supplementary food for hares through the winter period. The intensive clearing of young sapling stands and especially the removal of deciduous shrubs and trees reduces the quality of habitats for the mountain hare. Mountain hares primarily live in forest habitat and it is possible that changes in the forest structure play a crucial role in mountain hare habitat preference. Ecological knowledge of the mountain hare is vital to create habitat structure more suitable for the species. More deciduous trees should be saved in managing forests and the mechanical clearing of the shrub layer should be done carefully.

Microbial communities in Pb contaminated boreal forest soil

Lead contamination in the environment is of particular concern, as it is a known toxin. Until recently, however, much less attention has been given to the local contamination caused by activities at shooting ranges compared to large-scale industrial contamination. In Finland, more than 500 tons of Pb is produced each year for shotgun ammunition. The contaminant threatens various organisms, ground water and the health of human populations. However, the forest at shooting ranges usually shows no visible sign of stress compared to nearby clean environments. The aboveground biota normally reflects the belowground ecosystem. Thus, the soil microbial communities appear to bear strong resistance to contamination, despite the influence of lead. The studies forming this thesis investigated a shooting range site at Hälvälä in Southern Finland, which is heavily contaminated by lead pellets. Previously it was experimentally shown that the growth of grasses and degradation of litter are retarded. Measurements of acute toxicity of the contaminated soil or soil extracts gave conflicting results, as enchytraeid worms used as toxicity reporters were strongly affected, while reporter bacteria showed no or very minor decreases in viability. Measurements using sensitive inducible luminescent reporter bacteria suggested that the bioavailability of lead in the soil is indeed low, and this notion was supported by the very low water extractability of the lead. Nevertheless, the frequency of lead-resistant cultivable bacteria was elevated based on the isolation of cultivable strains. The bacterial and fungal diversity in heavily lead contaminated shooting sectors were compared with those of pristine sections of the shooting range area. The bacterial 16S rRNA gene and fungal ITS rRNA gene were amplified, cloned and sequenced using total DNA extracted from the soil humus layer as the template. Altogether, 917 sequenced bacterial clones and 649 sequenced fungal clones revealed a high soil microbial diversity. No effect of lead contamination was found on bacterial richness or diversity, while fungal richness and diversity significantly differed between lead contaminated and clean control areas. However, even in the case of fungi, genera that were deemed sensitive were not totally absent from the contaminated area: only their relative frequency was significantly reduced. Some operational taxonomic units (OTUs) assigned to Basidiomycota were clearly affected, and were much rarer in the lead contaminated areas. The studies of this thesis surveyed EcM sporocarps, analyzed morphotyped EcM root tips by direct sequencing, and 454-pyrosequenced fungal communities in in-growth bags. A total of 32 EcM fungi that formed conspicuous sporocarps, 27 EcM fungal OTUs from 294 root tips, and 116 EcM fungal OTUs from a total of 8 194 ITS2 454 sequences were recorded. The ordination analyses by non-parametric multidimensional scaling (NMS) indicated that Pb enrichment induced a shift in the EcM community composition. This was visible as indicative trends in the sporocarp and root tip datasets, but explicitly clear in the communities observed in the in-growth bags. The compositional shift in the EcM community was mainly attributable to an increase in the frequencies of OTUs assigned to the genus Thelephora, and to a decrease in the OTUs assigned to Pseudotomentella, Suillus and Tylospora in Pb-contaminated areas when compared to the control. The enrichment of Thelephora in contaminated areas was also observed when examining the total fungal communities in soil using DNA cloning and sequencing technology. While the compositional shifts are clear, their functional consequences for the dominant trees or soil ecosystem remain undetermined. The results indicate that at the Hälvälä shooting range, lead influences the fungal communities but not the bacterial communities. The forest ecosystem shows apparent functional redundancy, since no significant effects were seen on forest trees. Recently, by means of 454 pyrosequencing , the amount of sequences in a single analysis run can be up to one million. It has been applied in microbial ecology studies to characterize microbial communities. The handling of sequence data with traditional programs is becoming difficult and exceedingly time consuming, and novel tools are needed to handle the vast amounts of data being generated. The field of microbial ecology has recently benefited from the availability of a number of tools for describing and comparing microbial communities using robust statistical methods. However, although these programs provide methods for rapid calculation, it has become necessary to make them more amenable to larger datasets and numbers of samples from pyrosequencing. As part of this thesis, a new program was developed, MuSSA (Multi-Sample Sequence Analyser), to handle sequence data from novel high-throughput sequencing approaches in microbial community analyses. The greatest advantage of the program is that large volumes of sequence data can be manipulated, and general OTU series with a frequency value can be calculated among a large number of samples.