Phylogeography and Population Genetics of the Moor frog (Rana arvalis Nilsson) in Northern Europe

The ongoing climate change along with increasing levels of pollutants, diseases, habitat loss and fragmentation constitute global threats to the persistence of many populations, species and ecosystems. However, for the long-term persistence of local populations, one of the biggest threats is the intrinsic loss of genetic variation. In order to adapt to changes in the environment, organisms must have a sufficient supply of heritable variation in traits important for their fitness. With a loss of genetic variation, the risk of extinction will increase. For conservational practices, one should therefore understand the processes that shape the genetic population structure and also the broader (historical) phylogenetic patterning of the species in focus. In this thesis, microsatellite markers were applied to study genetic diversity and population differentiation of the protected moor frog (Rana arvalis) in Fennoscandia from both historical (evolutionary) and applied (conservation) perspectives. The results demonstrate that R. arvalis populations are highly structured over rather short geographic distances. Moreover, the results suggest that R. arvalis recolonized Fennoscandia from two directions after the last ice age. This has had implications for the genetic structuring and population differentiation, especially in the northernmost parts where the two lineages have met. Compared to more southern populations, the genetic variation decreases and the interpopulation differentiation increases dramatically towards north. This could be an outcome of serial population bottlenecking along the recolonization route. Also, current isolation and small population sizes increase the effect of drift, thus reinforcing the observed pattern. The same pattern can also be seen in island populations. However, though R. arvalis on the island of Gotland has lost most of its neutral genetic variability, our results indicate that the levels of additive genetic variation have remained high. This conforms to the conjecture that though neutral markers are widely used in conservation purposes, they may be quite uninformative about the levels of genetic variation in ecologically important traits. Finally, the evolutionary impact of the typical amphibian mating behaviour on genetic diversity was investigated. Given the short time available for larval development, it is important that mating takes place as early as possible. The genetic data and earlier capture-recapture data suggest that R. arvalis gather at mating grounds they are familiar with. However, by forming leks in random to relatedness, and having multiple paternities in single clutches, the risk of inbreeding may be minimized in this otherwise highly philopatric species.

Behavioural, population, and genetic processes affecting metapopulation dynamics of the Glanville fritillary butterfly

In my thesis I have been studying the effects of population fragmentation and extinction-recolonization dynamics on genetic and evolutionary processes in the Glanville fritillary butterfly (Melitaea cinxia). By conducting crosses within and among newly-colonized populations and using several fitness measures, I found a strong decrease in fitness following colonization by a few related individuals, and a strong negative relationship between parental relatedness and offspring fitness. Thereafter, I was interested in determining the number and relatedness of individuals colonizing new populations, which I did using a set of microsatellites I had previously developed for this species. Additionally, I am interested in the evolution of key life-history traits. By following the lifetime reproductive success of males emerging at different times in a semi-natural setup, I demonstrated that protandry is adaptive in males, and I was able to rule out, for M. cinxia, alternative incidental hypotheses evoked to explain the evolution of protandry in insects. Finally, in work I did together with Prof. Hanna Kokko, I am proposing bet-hedging as a new mechanism that could explain the evolution of polyandry in M. cinxia.

Ecological impacts of Phlebiopsis gigantea biocontrol treatment against Heterobasidion spp. as revealed by fungal community profiling and population analyses

Wood decay fungi belonging to the species complex Heterobasidion annosum sensu lato are among the most common and economically important species causing root rot and stem decay in conifers of the northern temperate regions. New infections by these pathogens can be suppressed by tree stump treatments using chemical or biological control agents. In Finland, the corticiaceous fungus Phlebiopsis gigantea has been formulated into a commercial biocontrol agent called Rotstop (Verdera Ltd.). This thesis addresses the ecological impacts of Rotstop biocontrol treatment on the mycoflora of conifer stumps. Locally, fungal communities within Rotstop-treated and untreated stumps were analyzed using a novel method based on DGGE profiling of small subunit ribosomal DNA fragments amplified directly from wood samples. Population analyses for P. gigantea and H. annosum s.l. were conducted to evaluate possible risks associated with local and/or global distribution of the Rotstop strain. Based on molecular community profiling by DGGE, we detected a few individual wood-inhabiting fungal species (OTUs) that seemed to have suffered or benefited from the Rotstop biocontrol treatment. The DGGE analyses also revealed fungal diversity not retrieved by cultivation and some fungal sequence types untypical for decomposing conifer wood. However, statistical analysis of DGGE community profiles obtained from Rotstop-treated and untreated conifer stumps revealed that the Rotstop treatment had not caused a statistically significant reduction in the species diversity of wood-inhabiting fungi within our experimental forest plots. Locally, ISSR genotyping of cultured P. gigantea strains showed that the Rotstop biocontrol strain was capable of surviving up to six years within treated Norway spruce stumps, while in Scots pine stumps it was sooner replaced by successor fungal species. In addition, the spread of resident P. gigantea strains into Rotstop-treated forest stands seemed effective in preventing the formation of genetically monomorphic populations in the short run. On a global scale, we detected a considerable level of genetic differentiation between the interfertile European and North American populations of P. gigantea. These results strongly suggest that local biocontrol strains should be used in order to prevent global spread of P. gigantea and hybrid formation between geographically isolated populations. The population analysis for H. annosum s.l. revealed a collection of Chinese fungal strains that showed a high degree of laboratory fertility with three different allopatric H. annosum s.l. taxa. However, based on the molecular markers, the Chinese strains could be clearly affiliated with the H. parviporum taxonomical cluster, which thus appears to have a continuous distribution range from Europe through southern Siberia to northern China. Keywords: Rotstop, wood decay, DGGE, ISSR fingerprinting, ribosomal DNA

Population dynamics of blue mussels in a variable environment at the edge of their range

Spatial and temporal variation in the abundance of species can often be ascribed to spatial and temporal variation in the surrounding environment. Knowledge of how biotic and abiotic factors operate over different spatial and temporal scales in determining distribution, abundance, and structure of populations lies at the heart of ecology. The major part of the current ecological theory stems from studies carried out in central parts of the distributional range of species, whereas knowledge of how marginal populations function is inadequate. Understanding how marginal populations, living at the edge of their range, function is however in a key position to advance ecology and evolutionary biology as scientific disciplines. My thesis focuses on the factors affecting dynamics of marginal populations of blue mussels (Mytilus edulis) living close to their tolerance limits with regard to salinity. The thesis aims to highlight the dynamics at the edge of the range and contrast these with dynamics in more central parts of the range in order to understand the potential interplay between the central and the marginal part in the focal system. The objectives of the thesis are approached by studies on: (1) factors affecting regional patterns of the species, (2) long-term temporal dynamics of the focal species spaced along a regional salinity gradient, (3) selective predation by increasing populations of roach (Rutilus rutilus) when feeding on their main food item, the blue mussel, (4) the primary and secondary effects of local wave exposure gradients and (5) the role of small-scale habitat heterogeneity as determinants of large-scale pattern. The thesis shows that populations of blue mussels are largely determined by large scale changes in sea water salinity, affecting mainly recruitment success and longevity of local populations. In opposite to the traditional view, the thesis strongly indicate that vertebrate predators strongly affect abundance and size structure of blue mussel populations, and that the role of these predators increases towards the margin where populations are increasingly top-down controlled. The thesis also indicates that the positive role of biogenic habitat modifiers increases towards the marginal areas, where populations of blue mussels are largely recruitment limited. Finally, the thesis shows that local blue mussel populations are strongly dependent on high water turbulence, and therefore, dense populations are constrained to offshore habitats. Finally, the thesis suggests that ongoing sedimentation of rocky shores is detrimental for the species, affecting recruitment success and post-recruit survival, pushing stable mussel beds towards offshore areas. Ongoing large scale changes in the Baltic Sea, especially dilution processes with attendant effects, are predicted to substantially contract the distributional range of the mussel, but also affect more central populations. The thesis shows that in order to understand the functioning of marginal populations, research should (1) strive for multi-scale approaches in order to link ecosystem patterns with ecosystem processes, and (2) challenge the prevailing tenets that origin from research carried out in central areas that may not be valid at the edge.

Causes and consequences of variation in nestling immune function

Defence against pathogens is a vital need of all living organisms that has led to the evolution of complex immune mechanisms. However, although immunocompetence the ability to resist pathogens and control infection has in recent decades become a focus for research in evolutionary ecology, the variation in immune function observed in natural populations is relatively little understood. This thesis examines sources of this variation (environmental, genetic and maternal effects) during the nestling stage and its fitness consequences in wild populations of passerines: the blue tit (Cyanistes caeruleus) and the collared flycatcher (Ficedula albicollis). A developing organism may face a dilemma as to whether to allocate limited resources to growth or to immune defences. The optimal level of investment in immunity is shaped inherently by specific requirements of the environment. If the probability of contracting infection is low, maintaining high growth rates even at the expense of immune function may be advantageous for nestlings, as body mass is usually a good predictor of post-fledging survival. In experiments with blue tits and haematophagous hen fleas (Ceratophyllus gallinae) using two methods, methionine supplementation (to manipulate nestlings resource allocation to cellular immune function) and food supplementation (to increase resource availability), I confirmed that there is a trade-off between growth and immunity and that the abundance of ectoparasites is an environmental factor affecting allocation of resources to immune function. A cross-fostering experiment also revealed that environmental heterogeneity in terms of abundance of ectoparasites may contribute to maintaining additive genetic variation in immunity and other traits. Animal model analysis of extensive data collected from the population of collared flycatchers on Gotland (Sweden) allowed examination of the narrow-sense heritability of PHA-response the most commonly used index of cellular immunocompetence in avian studies. PHA-response is not heritable in this population, but is subject to a non-heritable origin (presumably maternal) effect. However, experimental manipulation of yolk androgen levels indicates that the mechanism of the maternal effect in PHA-response is not in ovo deposition of androgens. The relationship between PHA-response and recruitment was studied for over 1300 collared flycatcher nestlings. Multivariate selection analysis shows that it is body mass, not PHA-response, that is under direct selection. PHA-response appears to be related to recruitment because of its positive relationship with body mass. These results imply that either PHA-response fails to capture the immune mechanisms that are relevant for defence against pathogens encountered by fledglings or that the selection pressure from parasites is not as strong as commonly assumed.

Conceptual and statistical modelling of environmental effects in population dynamics

Population dynamics are generally viewed as the result of intrinsic (purely density dependent) and extrinsic (environmental) processes. Both components, and potential interactions between those two, have to be modelled in order to understand and predict dynamics of natural populations; a topic that is of great importance in population management and conservation. This thesis focuses on modelling environmental effects in population dynamics and how effects of potentially relevant environmental variables can be statistically identified and quantified from time series data. Chapter I presents some useful models of multiplicative environmental effects for unstructured density dependent populations. The presented models can be written as standard multiple regression models that are easy to fit to data. Chapters II IV constitute empirical studies that statistically model environmental effects on population dynamics of several migratory bird species with different life history characteristics and migration strategies. In Chapter II, spruce cone crops are found to have a strong positive effect on the population growth of the great spotted woodpecker (Dendrocopos major), while cone crops of pine another important food resource for the species do not effectively explain population growth. The study compares rate- and ratio-dependent effects of cone availability, using state-space models that distinguish between process and observation error in the time series data. Chapter III shows how drought, in combination with settling behaviour during migration, produces asymmetric spatially synchronous patterns of population dynamics in North American ducks (genus Anas). Chapter IV investigates the dynamics of a Finnish population of skylark (Alauda arvensis), and point out effects of rainfall and habitat quality on population growth. Because the skylark time series and some of the environmental variables included show strong positive autocorrelation, the statistical significances are calculated using a Monte Carlo method, where random autocorrelated time series are generated. Chapter V is a simulation-based study, showing that ignoring observation error in analyses of population time series data can bias the estimated effects and measures of uncertainty, if the environmental variables are autocorrelated. It is concluded that the use of state-space models is an effective way to reach more accurate results. In summary, there are several biological assumptions and methodological issues that can affect the inferential outcome when estimating environmental effects from time series data, and that therefore need special attention. The functional form of the environmental effects and potential interactions between environment and population density are important to deal with. Other issues that should be considered are assumptions about density dependent regulation, modelling potential observation error, and when needed, accounting for spatial and/or temporal autocorrelation.

Human genetic variation in the Baltic Sea region: Features of population history and natural selection

In this thesis, the genetic variation of human populations from the Baltic Sea region was studied in order to elucidate population history as well as evolutionary adaptation in this region. The study provided novel understanding of how the complex population level processes of migration, genetic drift, and natural selection have shaped genetic variation in North European populations. Results from genome-wide, mitochondrial DNA and Y-chromosomal analyses suggested that the genetic background of the populations of the Baltic Sea region lies predominantly in Continental Europe, which is consistent with earlier studies and archaeological evidence. The late settlement of Fennoscandia after the Ice Age and the subsequent small population size have led to pronounced genetic drift, especially in Finland and Karelia but also in Sweden, evident especially in genome-wide and Y-chromosomal analyses. Consequently, these populations show striking genetic differentiation, as opposed to much more homogeneous pattern of variation in Central European populations. Additionally, the eastern side of the Baltic Sea was observed to have experienced eastern influence in the genome-wide data as well as in mitochondrial DNA and Y-chromosomal variation – consistent with linguistic connections. However, Slavic influence in the Baltic Sea populations appears minor on genetic level. While the genetic diversity of the Finnish population overall was low, genome-wide and Y-chromosomal results showed pronounced regional differences. The genetic distance between Western and Eastern Finland was larger than for many geographically distant population pairs, and provinces also showed genetic differences. This is probably mainly due to the late settlement of Eastern Finland and local isolation, although differences in ancestral migration waves may contribute to this, too. In contrast, mitochondrial DNA and Y-chromosomal analyses of the contemporary Swedish population revealed a much less pronounced population structure and a fusion of the traces of ancient admixture, genetic drift, and recent immigration. Genome-wide datasets also provide a resource for studying the adaptive evolution of human populations. This study revealed tens of loci with strong signs of recent positive selection in Northern Europe. These results provide interesting targets for future research on evolutionary adaptation, and may be important for understanding the background of disease-causing variants in human populations.

Development of salinity tolerance in rice by constitutive-overexpression of genes involved in the regulation of programmed cell death

Environmental factors contribute to over 70% of crop yield losses worldwide. Of these drought and salinity are the most significant causes of crop yield reduction. Rice is an important staple crop that feeds more than half of the world’s population. However among the agronomically important cereals rice is the most sensitive to salinity. In the present study we show that exogenous expression of anti-apoptotic genes from diverse origins, AtBAG4 (Arabidopsis), Hsp70 (Citrus tristeza virus) and p35 (Baculovirus), significantly improves salinity tolerance in rice at the whole plant level. Physiological, biochemical and agronomical analyses of transgenic rice expressing each of the anti-apoptotic genes subjected to salinity treatment demonstrated traits associated with tolerant varieties including, improved photosynthesis, membrane integrity, ion and ROS maintenance systems, growth rate, and yield components. Moreover, FTIR analysis showed that the chemical composition of salinity-treated transgenic plants is reminiscent of non-treated, unstressed controls. In contrast, wild type and vector control plants displayed hallmark features of stress, including pectin degradation upon subjection to salinity treatment. Interestingly, despite their diverse origins, transgenic plants expressing the anti-apoptotic genes assessed in this study displayed similar physiological and biochemical characteristics during salinity treatment thus providing further evidence that cell death pathways are conserved across broad evolutionary kingdoms. Our results reveal that anti-apoptotic genes facilitate maintenance of metabolic activity at the whole plant level to create favorable conditions for cellular survival. It is these conditions that are crucial and conducive to the plants ability to tolerate/adapt to extreme environments.

From actin monomers to bundles: The roles of twinfilin and α-actinin in Drosophila melanogaster development

The actin cytoskeleton is essential for a large variety of cell biological processes. Actin exists in either a monomeric or a filamentous form, and it is very important for many cellular functions that the local balance between these two actin populations is properly regulated. A large number of proteins participate in the regulation of actin dynamics in the cell, and twinfilin, one of the proteins examined in this thesis, belongs to this category. The second level of regulation involves proteins that crosslink or bundle actin filaments, thereby providing the cell with a certain shape. α-Actinin, the second protein studied, mainly acts as an actin crosslinking protein. Both proteins are conserved in organisms ranging from yeast to mammals. In this thesis, the roles of twinfilin and α-actinin in development were examined using Drosophila melanogaster as a model organism. Twinfilin is an actin monomer binding protein that is structurally related to cofilin. In vitro, twinfilin reduces actin polymerisation by sequestering actin monomers. The Drosophila twinfilin (twf) gene was identified and found to encode a protein functionally similar to yeast and mammalian twinfilins. A strong hypomorphic twf mutation was identified, and flies homozygous for this allele were viable and fertile. The adult twf mutant flies displayed reduced viability, a rough eye phenotype and severely malformed bristles. The shape of the adult bristle is determined by the actin bundles that are regularly spaced around the perimeter of the developing pupal bristles. Examination of the twf pupal bristles revealed an increased level of filamentous actin, which in turn resulted in splitting and displacement of the actin bundles. The bristle defect was rescued by twf overexpression in developing bristles. The Twinfilin protein was localised at sites of actin filament assembly, where it was required to limit actin polymerisation. A genetic interaction between twinfilin and twinstar (the gene encoding Cofilin) was detected, consistent with the model predicting that both proteins act to limit the amount of filamentous actin. α-Actinin has been implicated in several diverse cell biological processes. In Drosophila, the only function for α-actinin yet known is in the organisation of the muscle sarcomere. Muscle and non-muscle cells utilise different α-actinin isoforms, which in Drosophila are produced by alternative splicing of a single gene. In this work, novel α-actinin deletion alleles, including ActnΔ233, were generated, which specifically disrupted the transcript encoding the non-muscle α-actinin isoform. Nevertheless, ActnΔ233 homozygous mutant flies were viable and fertile with no obvious defects. By comparing α-actinin protein distribution in wild type and ActnΔ233 mutant animals, it could be concluded that non-muscle α-actinin is the only isoform expressed in young embryos, in the embryonic central nervous system and in various actin-rich structures of the ovarian germline cells. In the ActnΔ233 mutant, α-actinin was detected not only in muscle tissue, but also in embryonic epidermal cells and in certain follicle cell populations in the ovaries. The population of α-actinin protein present in non-muscle cells of the ActnΔ233 mutant is referred to as FC-α-actinin (Follicle Cell). The follicular epithelium in the Drosophila ovary is a well characterised model system for studies on patterning and morphogenesis. Therefore, α-actinin expression, regulation and function in this tissue were further analysed. Examination of the α-actinin localisation pattern revealed that the basal actin fibres of the main body follicle cells underwent an organised remodelling during the final stages of oogenesis. This involved the assembly of a transient adhesion site in the posterior of the cell, in which α-actinin and Enabled (Ena) accumulated. Follicle cells genetically manipulated to lack all α-actinin isoforms failed to remodel their cytoskeleton and translocate Ena to the posterior of the cell, while the actin fibres as such were not affected. Neither was epithelial morphogenesis disrupted. The reorganisation of the basal actin cytoskeleton was also disturbed following ectopic expression of Decapentaplegic (Dpp) or as a result of a heat shock. At late oogenesis, the main body follicle cells express both non-muscle α-actinin and FC-α-actinin, while the dorsal anterior follicle cells express only non-muscle α-actinin. The dorsal anterior cells are patterned by the Dpp and Epidermal growth factor receptor (EGFR) signalling pathways, and they will ultimately secrete the dorsal appendages of the egg. Experiments involving ectopic activation of EGFR and Dpp signalling showed that FC-α-actinin is negatively regulated by combined EGFR and Dpp signalling. Ubiquitous overexpression of the adult muscle-specific α-actinin isoform induced the formation of aberrant actin bundles in migrating follicle cells that did not normally express FC-α-actinin, provided that the EGFR signalling pathway was activated in the cells. Taken together, this work contributes new data to our knowledge of α-actinin function and regulation in Drosophila. The cytoskeletal remodelling shown to depend on α-actinin function provides the first evidence that α-actinin has a role in the organisation of the cytoskeleton in a non-muscle tissue. Furthermore, the cytoskeletal remodelling constitutes a previously undescribed morphogenetic event, which may provide us with a model system for in vivo studies on adhesion dynamics in Drosophila.