Population structure and evolution in the ant Plagiolepis pygmaea and its two social parasites Plagiolepis xene and Plagiolepis grassei

Social behaviour affects dispersal of animals and is an important modifier of genetic population structures. The female sex is often philopatric, which maintains coancestry within the breeding groups and promotes cooperative behaviours. This enables also inclusive fitness returns from altruism and explains why some individuals sacrifice personal reproduction for the good of others in social insects such as ants. However, reduced dispersal and population substructuring at the level of colonies may also entail inbreeding, loss of genetic diversity, and vulnerability. In addition, the most vulnerable ants are species that are evolved to parasitize colonies of other ants, and which compromise between abilities to disperse and the efficiency to parasitize the host. On the other hand, certain social organisations of ant colonies may facilitate a species to disperse outside its natural range and become a pest. Altogether, knowledge on genetic structuring of ant populations, as well as the evolution of their life histories can contribute to conservation biology and population management. The aim of this thesis was to investigate population structures and phylogenetic evolution of the ant Plagiolepis pygmaea and its two obligatory, workerless social parasites (inquilines) P. xene and P. grassei with genetic markers and DNA sequence data. The results support the general assumption that populations of inquiline parasites are highly fragmented and genetically vulnerable. Comparison of the two parasites suggests that differences in their relative abundance may follow from their interaction with the host, i.e. how well the species is adapted to reproduce in the host colonies. The results also indicate that the most recent free living ancestor to these two parasite species is their common host. This is considered to provide evidence for the controversial issue of sympatric speciation. Further, given that the level of adaptations to parasitic life history depends on the evolutionary time since the free-living ancestor, the results establish a link between species rarity and its evolutionary age. The populations of the host species P. pygmaea displayed significantly reduced dispersal both among the females (queens) and males, and high levels of inbreeding which may enhance worker altruism. In addition, the queens were found to mate with multiple males. Given the high relatedness between the queens and their mates, this occurs probably for non-genetic reasons, e.g. without benefits associated in genetically more diverse offspring. The results hence caution that the contribution of non-genetic factors to the prevailing mating patterns and genetic population structures should not be underestimated.

Language change as an evolutionary process

In the thesis it is discussed in what ways concepts and methodology developed in evolutionary biology can be applied to the explanation and research of language change. The parallel nature of the mechanisms of biological evolution and language change is explored along with the history of the exchange of ideas between these two disciplines. Against this background computational methods developed in evolutionary biology are taken into consideration in terms of their applicability to the study of historical relationships between languages. Different phylogenetic methods are explained in common terminology, avoiding the technical language of statistics. The thesis is on one hand a synthesis of earlier scientific discussion, and on the other an attempt to map out the problems of earlier approaches in addition to finding new guidelines in the study of language change on their basis. Primarily literature about the connections between evolutionary biology and language change, along with research articles describing applications of phylogenetic methods into language change have been used as source material. The thesis starts out by describing the initial development of the disciplines of evolutionary biology and historical linguistics, a process which right from the beginning can be seen to have involved an exchange of ideas concerning the mechanisms of language change and biological evolution. The historical discussion lays the foundation for the handling of the generalised account of selection developed during the recent few decades. This account is aimed for creating a theoretical framework capable of explaining both biological evolution and cultural change as selection processes acting on self-replicating entities. This thesis focusses on the capacity of the generalised account of selection to describe language change as a process of this kind. In biology, the mechanisms of evolution are seen to form populations of genetically related organisms through time. One of the central questions explored in this thesis is whether selection theory makes it possible to picture languages are forming populations of a similar kind, and what a perspective like this can offer to the understanding of language in general. In historical linguistics, the comparative method and other, complementing methods have been traditionally used to study the development of languages from a common ancestral language. Computational, quantitative methods have not become widely used as part of the central methodology of historical linguistics. After the fading of a limited popularity enjoyed by the lexicostatistical method since the 1950s, only in the recent years have also the computational methods of phylogenetic inference used in evolutionary biology been applied to the study of early language history. In this thesis the possibilities offered by the traditional methodology of historical linguistics and the new phylogenetic methods are compared. The methods are approached through the ways in which they have been applied to the Indo-European languages, which is the most thoroughly investigated language family using both the traditional and the phylogenetic methods. The problems of these applications along with the optimal form of the linguistic data used in these methods are explored in the thesis. The mechanisms of biological evolution are seen in the thesis as parallel in a limited sense to the mechanisms of language change, however sufficiently so that the development of a generalised account of selection is deemed as possibly fruiful for understanding language change. These similarities are also seen to support the validity of using phylogenetic methods in the study of language history, although the use of linguistic data and the models of language change employed by these models are seen to await further development.

Karl Popper's Philosophy of Science - And the Evolution of the Popperian Worlds

This doctoral thesis in theoretical philosophy is a systematic analysis of Karl Popper's philosophy of science and its relation to his theory of three worlds. The general aim is to study Popper's philosophy of science and to show that Popper's theory of three worlds was a restatement of his earlier positions. As a result, a new reading of Popper's philosophy and development is offered and the theory of three worlds is analysed in a new manner. It is suggested that the theory of three worlds is not purely an ontological theory, but has a profound epistemological motivation. In Part One, Popper's epistemology and philosophy of science is analysed. It is claimed that Popper's thinking was bifurcated: he held two profound positions without noticing the tension between them. Popper adopted the position called the theorist around 1930 and focused on the logical structure of scientific theories. In Logik der Forschung (1935), he attempted to build a logic of science on the grounds that scientific theories may be regarded as universal statements which are not verifiable but can be falsified. Later, Popper emphasized another position, called here the processionalist. Popper focused on the study of science as a process and held that a) philosophy of science should study the growth of knowledge and that b) all cognitive processes are constitutive. Moreover, the constitutive idea that we see the world in the searchlight of our theories was combined with the biological insight that knowledge grows by trial and error. In Part Two, the theory of three worlds is analysed systematically. The theory is discussed as a cluster of theories which originate from Popper's attempt to solve some internal problems in his thinking. Popper adhered to realism and wished to reconcile the theorist and the processionalist. He also stressed the real and active nature of the human mind, and the possibility of objective knowledge. Finally, he wished to create a scientific world view.

Characterization of the mitochondrial active-site serine protein LACTB: filaments in the mitochondrial intermembrane space

Mitochondria have evolved from endosymbiotic alpha-proteobacteria. During the endosymbiotic process early eukaryotes dumped the major component of the bacterial cell wall, the peptidoglycan layer. Peptidoglycan is synthesized and maintained by active-site serine enzymes belonging to the penicillin-binding protein and the β-lactamase superfamily. Mammals harbor a protein named LACTB that shares sequence similarity with bacterial penicillin-binding proteins and β-lactamases. Since eukaryotes lack the synthesis machinery for peptidoglycan, the physiological role of LACTB is intriguing. Recently, LACTB has been validated in vivo to be causative for obesity, suggesting that LACTB is implicated in metabolic processes. The aim of this study was to investigate the phylogeny, structure, biochemistry and cell biology of LACTB in order to elucidate its physiological function. Phylogenetic analysis revealed that LACTB has evolved from penicillin binding-proteins present in the bacterial periplasmic space. A structural model of LACTB indicates that LACTB shares characteristic features common to all penicillin-binding proteins and β-lactamases. Recombinat LACTB protein expressed in E. coli was recovered in significant quantities. Biochemical and cell biology studies showed that LACTB is a soluble protein localized in the mitochondrial intermembrane space. Further analysis showed that LACTB preprotein underwent proteolytic processing disclosing an N-terminal tetrapeptide motif also found in a set of cell death-inducing proteins. Electron microscopy structural studies revealed that LACTB can polymerize to form stable filaments with lengths ranging from twenty to several hundred nanometers. These data suggest that LACTB filaments define a distinct microdomain in the intermembrane space. A possible role of LACTB filaments is proposed in the intramitochondrial membrane organization and microcompartmentation. The implications of these findings offer novel insight into the evolution of mitochondria. Further studies of the LACTB function might provide a tool to treat mitochondria-related metabolic diseases.

Human Torque teno virus: epidemiology, cell biology and immunology

Torque teno virus (TTV) was discovered in 1997 in the serum of a Japanese patient who had a post-transfusion hepatitis of unknown etiology. It is a small virus containing a circular single-stranded DNA genome which is unique among human viruses. Within a few years after its discovery, the TTVs were noted to form a large family of viruses with numerous genotypes. TTV is highly prevalent among the general population throughout the world, and persistent infections and co-infections with several genotypes occur frequently. However, the pathogenicity and the mechanism for the sustained occurrence of the virus in blood are at present unclear. To determine the prevalence of TTV in Finland, we set up PCR methods and examined the sera of asymptomatic subjects for the presence of TTV DNA and for genotype-6 DNA. TTV was found to be highly prevalent also in Finland; 85% of adults harbored TTV in their blood, and 4% were infected with genotype-6. In addition, TTV DNA was detected in a number of different tissues, with no tissue-type or symptom specificity. Most cell-biological events during TTV infections are at the moment unknown. Replicating TTV DNA has, however, been detected in liver and the hematopoietic compartment, and three mRNAs are known to be generated. To characterize TTV cell biology in more detail, we cloned in full length the genome of TTV genotype 6. We showed that in human kidney-derived cells TTV produces altogether six proteins with distinct subcellular localizations. TTV mRNA transcription was detected in all cell lines transfected with the full-length clone, and TTV DNA replicated in several of them, including those of erythroid, kidney, and hepatic origin. Furthermore, the viral DNA replication was shown to utilize the cellular DNA polymerases. Diagnoses of TTV infections have been based almost solely on PCR, whereas serological tests, measuring antibody responses, would give more information on many aspects of these infections. To investigate the TTV immunology in more detail, we produced all six TTV proteins for use as antigens in serological tests. We detected in human sera IgM and IgG antibodies to occur simultaneously with TTV DNA, and observed appearance of TTV DNA regardless of pre-existing antibodies, and disappearance of TTV DNA after antibody appearance. The genotype-6 nucleotide sequence remained stable for years within the infected subjects, suggesting that some mechanism other than mutations is used by this minute virus to evade our immune system and to establish chronic infections in immunocompetent subjects.

Molecular genetics and evolution of Puumala hantavirus

Puumala virus (PUUV) is the causative agent of nephropathia epidemica (NE), a mild form of hemorrhagic fever with renal syndrome. Finland has the highest documented incidence of NE with around 1000 cases diagnosed annually. PUUV is also found in other Scandinavian countries, Central Europe and the European part of Russia. PUUV belongs to the genus Hantavirus in the family Bunyaviridae. Hantaviruses are rodent-borne viruses each carried by a specific host that is persistently and asymptomatically infected by the virus. PUUV is carried by the bank voles (Myodes glareolus, previously known as Clethrionomys glareolus). Hantaviruses have co-evolved with their carrier rodents for millions of years and these host animals are the evolutionary scene of hantaviruses. In this study, PUUV sequences were recovered from bank voles captured in Denmark and Russian Karelia to study the evolution of PUUV in Scandinavia. Phylogenetic analysis of these strains showed a geographical clustering of genetic variants following the presumable migration pattern of bank voles during the recolonization of Scandinavia after the last ice age approximately 10 000 years ago. The currently known PUUV genome sequences were subjected to in-depth phylogenetic analyses and the results showed that genetic drift seems to be the major mechanism of PUUV evolution. In general, PUUV seems to evolve quite slowly following a molecular clock. We also found evidence for recombination in the evolution of some genetic lineages of PUUV. Viral microevolution was studied in controlled virus transmission in colonized bank voles and changes in quasispecies dynamics were recorded as the virus was transmitted from one animal to another. We witnessed PUUV evolution in vivo, as one synonymous mutation became repeatedly fixed in the viral genome during the experiment. The detailed knowledge on the PUUV diversity was used to establish new sensitive and specific detection methods for this virus. Direct viral invasion of the hypophysis was demonstrated for the first time in a lethal case of NE. PUUV detection was done by immunohistochemistry, in situ hybridization and RT-nested-PCR of the autopsy tissue samples.

Molecular biology of progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1)

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessively inherited disorder characterized by age of onset at 6-15 years, stimulus-sensitive myoclonus, tonic-clonic epileptic seizures and a progressive course. Mutations in the cystatin B (CSTB) gene underlie EPM1. The most common mutation underlying EPM1 is a dodecamer repeat expansion in the promoter region of CSTB. In addition, nine other mutations have been identified. CSTB, a cysteine protease inhibitor, is a ubiquitously expressed inhibitor of cathepsins, but its physiological function is unknown. The purpose of this study was to investigate CSTB gene expression and CSTB protein function in normal and pathological conditions. The basal CSTB promoter was mapped and characterized using different promoter-luciferase gene constructs. The binding activity of transcription factors to one ARE half, five Sp1 and four AP1 sites in the CSTB promoter was demonstrated. The CSTB promoter activity was clearly decreased using a CSTB promoter with "premutation" repeat expansions and in individuals with alike expansions. The expression of CSTB mRNA and protein was markedly reduced in patient cells. The endogenous CSTB protein localized to the nucleus, cytoplasm and lysosomes, and in differentiated cells merely to the cytoplasm. This suggests that the subcellular distribution of CSTB is dependent on the differentation status of the cells. The proteins representing patient missense mutations failed to associate with lysosomes, implying the importance of the lysosomal association for the proper physiological function of CSTB. Several alternatively spliced CSTB isoforms were identified. Of these CSTB2 was widely expressed with very low levels whereas the other alternatively spliced forms seemed to have limited tissue expression. In patients CSTB2 expression was reduced similarly to that of CSTB. The physiological relevance of CSTB alternative splicing remains unknown. The mouse Cstb transcript was shown to be present in all embryonic stages and adult tissues examined. The expression was highest at embryonic day 7 and in thymus, as well as in postnatal brain in the cortex, caudate putamen, thalamus, hippocampus, and in the Purkinje cell layer of the cerebellum. Our data implies that CSTB expression is tightly temporally and spatially regulated. The data presented in my thesis lay the basis for further understanding of the role of CSTB in health and disease.

Molecular biology and functions of epilysin (MMP-28)

Epilysin (MMP-28) is the most recently identified member of the matrix metalloproteinase (MMP) family of extracellular proteases. Together these enzymes are capable of degrading almost all components of the extracellular matrix (ECM) and are thus involved in important biological processes such as development, wound healing and immune functions, but also in pathological processes such as tumor invasion, metastasis and arthritis. MMPs do not act solely by degrading the ECM. They also regulate cell behavior by releasing growth factors and biologically active peptides from the ECM, by modulating cell surface receptors and adhesion molecules and by regulating the activity of many important mediators in inflammatory pathways. The aim of this study was to define the unique role of epilysin within the MMP-family, to elucidate how and when it is expressed and how its catalytic activity is regulated. To gain information on its essential functions and substrates, the specific aim was to characterize how epilysin affects the phenotype of epithelial cells, where it is biologically expressed. During the course of the study we found that the epilysin promoter contains a well conserved GT-box that is essential for the basic expression of this gene. Transcription factors Sp1 and Sp3 bind this sequence and could hence regulate both the basic and cell type and differentiation stage specific expression of epilysin. We cloned mouse epilysin cDNA and found that epilysin is well conserved between human and mouse genomes and that epilysin is glycosylated and activated by furin. Similarly to in human tissues, epilysin is normally expressed in a number of mouse tissues. The expression pattern differs from most other MMPs, which are expressed only in response to injury or inflammation and in pathological processes like cancer. These findings implicate that epilysin could be involved in tissue homeostasis, perhaps fine-tuning the phenotype of epithelial cells according to signals from the ECM. In view of these results, it was unexpected to find that epilysin can induce a stable epithelial to mesenchymal transition (EMT) when overexpressed in epithelial lung carcinoma cells. Transforming growth factor b (TGF-b) was recognized as a crucial mediator of this process, which was characterized by the loss of E-cadherin mediated cell-cell adhesion, elevated expression of gelatinase B and MT1-MMP and increased cell migration and invasion into collagen I gels. We also observed that epilysin is bound to the surface of epithelial cells and that this interaction is lost upon cell transformation and is susceptible to degradation by membrane type-1-MMP (MT1-MMP). The wide expression of epilysin under physiological conditions implicates that its effects on epithelial cell phenotype in vivo are not as dramatic as seen in our in vitro cell system. Nevertheless, current results indicate a possible interaction between epilysin and TGF-b also under physiological circumstances, where epilysin activity may not induce EMT but, instead, trigger less permanent changes in TGF-b signaling and cell motility. Epilysin may thus play an important role in TGF-b regulated events such as wound healing and inflammation, processes where involvement of epilysin has been indicated.

Evolution and detection of cyanobacterial hepatotoxin synthetase genes

Mass occurrences (blooms) of cyanobacteria are common in aquatic environments worldwide. These blooms are often toxic, due to the presence of hepatotoxins or neurotoxins. The most common cyanobacterial toxins are hepatotoxins: microcystins and nodularins. In freshwaters, the main producers of microcystins are Microcystis, Anabaena, and Planktothrix. Nodularins are produced by strains of Nodularia spumigena in brackish waters. Toxic and nontoxic strains of cyanobacteria co-occur and cannot be differentiated by conventional microscopy. Molecular biological methods based on microcystin and nodularin synthetase genes enable detection of potentially hepatotoxic cyanobacteria. In the present study, molecular detection methods for hepatotoxin-producing cyanobacteria were developed, based on microcystin synthetase gene E (mcyE) and the orthologous nodularin synthetase gene F (ndaF) sequences. General primers were designed to amplify the mcyE/ndaF gene region from microcystin-producing Anabaena, Microcystis, Planktothrix, and Nostoc, and nodularin-producing Nodularia strains. The sequences were used for phylogenetic analyses to study how cyanobacterial mcy genes have evolved. The results showed that mcy genes and microcystin are very old and were already present in the ancestor of many modern cyanobacterial genera. The results also suggested that the sporadic distribution of biosynthetic genes in modern cyanobacteria is caused by repeated gene losses in the more derived lineages of cyanobacteria and not by horizontal gene transfer. Phylogenetic analysis also proposed that nda genes evolved from mcy genes. The frequency and composition of the microcystin producers in 70 lakes in Finland were studied by conventional polymerase chain reaction (PCR). Potential microcystin producers were detected in 84% of the lakes, using general mcyE primers, and in 91% of the lakes with the three genus-specific mcyE primers. Potential microcystin-producing Microcystis were detected in 70%, Planktothrix in 63%, and Anabaena in 37% of the lakes. The presence and co-occurrence of potential microcystin producers were more frequent in eutrophic lakes, where the total phosphorus concentration was high. The PCR results could also be associated with various environmental factors by correlation and regression analyses. In these analyses, the total nitrogen concentration and pH were both associated with the presence of multiple microcystin-producing genera and partly explained the probability of occurrence of mcyE genes. In general, the results showed that higher nutrient concentrations increased the occurrence of potential microcystin producers and the risk for toxic bloom formation. Genus-specific probe pairs for microcystin-producing Anabaena, Microcystis, Planktothrix, and Nostoc, and nodularin-producing Nodularia were designed to be used in a DNA-chip assay. The DNA-chip can be used to simultaneously detect all these potential microcystin/nodularin producers in environmental water samples. The probe pairs detected the mcyE/ndaF genes specifically and sensitively when tested with cyanobacterial strains. In addition, potential microcystin/nodularin producers were identified in lake and Baltic Sea samples by the DNA-chip almost as sensitively as by quantitative real-time PCR (qPCR), which was used to validate the DNA-chip results. Further improvement of the DNA-chip assay was achieved by optimization of the PCR, the first step in the assay. Analysis of the mcy and nda gene clusters from various hepatotoxin-producing cyanobacteria was rewarding; it revealed that the genes were ancient. In addition, new methods detecting all the main producers of hepatotoxins could be developed. Interestingly, potential microcystin-producing cyanobacterial strains of Microcystis, Planktothrix, and Anabaena, co-occurred especially in eutrophic and hypertrophic lakes. Protecting waters from eutrophication and restoration of lakes may thus decrease the prevalence of toxic cyanobacteria and the frequency of toxic blooms.

Magmatic evolution of topaz-bearing granite stocks within the Wiborg rapakivi granite batholith

This study brings new insights into the magmatic evolution of natural F-enriched peraluminous granitic systems. The Artjärvi, Sääskjärvi and Kymi granite stocks within the 1.64 Ga Wiborg rapakivi granite batholith have been investigated by petrographic, geochemical, experimental and melt inclusion methods. These stocks represent late-stage leucocratic and weakly peraluminous intrusive phases typical of rapakivi granites worldwide. The Artjärvi and Sääskjärvi stocks are multiphase intrusions in which the most evolved phase is topaz granite. The Kymi stock contains topaz throughout and has a well-developed zoned structure, from the rim to the center: stockscheider pegmatite equigranular topaz granite porphyritic topaz granite. Geochemically the topaz granites are enriched in F, Li, Be, Ga, Rb, Sn and Nb and depleted in Mg, Fe, Ti, Ba, Sr, Zr and Eu. The anomalous geochemistry and mineralogy of the topaz granites are essentially magmatic in origin; postmagmatic reactions have only slightly modified the compositions. The Kymi equigranular topaz granite shows the most evolved character, and the topaz granites at Artjärvi and Sääskjärvi resemble the less evolved porphyritic topaz granite of the Kymi stock. Stockscheiders are found at the roof contacts of the Artjärvi and Kymi stocks. The stockscheider at Artjärvi is composed of biotite-rich schlieren and pegmatite layers parallel to the contact. The schlieren layering is considered to have formed by velocity-gradient sorting mechanism parallel to the flow, which led to the accumulation of mafic minerals along the upper contact of the topaz granite. Cooling and contraction of the topaz granite formed fractures parallel to the roof contact and residual pegmatite magmas were injected along the fractures and formed the pegmatite layers. The zoned structure of the Kymi stock is the result of intrusion of highly evolved residual melt from deeper parts of the magma chamber along the fractured contact between the porphyritic granite crystal mush and country rock. The equigranular topaz granite and marginal pegmatite (stockscheider) crystallized from this evolved melt. Phase relations of the Kymi equigranular topaz granite have been investigated utilizing crystallization experiments at 100 to 500 MPa as a function of water activity and F content. Fluorite and topaz can crystallize as liquidus phases in F-rich peraluminous systems, but the F content of the melt should exceed 2.5 - 3.0 wt % to facilitate crystallization of topaz. In peraluminous F-bearing melts containing more than 1 wt % F, topaz and muscovite are expected to be the first F-bearing phases to crystallize at high pressure, whereas fluorite and topaz should crystallize first at low pressure. Overall, the saturation of fluorite and topaz follows the reaction: CaAl2Si2O8 (plagioclase) + 2[AlF3]melt = CaF2 (fluorite) + 2Al2SiO4F2 (topaz). The obtained partition coefficient for F between biotite and glass D(F)Bt/glass is 1.89 to 0.80 (average 1.29) and can be used as an empirical fluormeter to determine the F content of coexisting melts. In order to study the magmatic evolution of the Kymi stock, crystallized melt inclusions in quartz and topaz grains in the porphyritic and the equigranular topaz granites and the marginal pegmatite were rehomogenized and analyzed. The homogenization conditions for the melt inclusions from the granites were 700 °C, 300 MPa, and 24 h, and for melt inclusions from the pegmatite, 700 °C, 100 MPa, and 24/96 h. The majority of the melt inclusions is chemically similar to the bulk rocks (excluding H2O content), but a few melt inclusions in the equigranular granite show clearly higher F and low K2O contents (on average 11.6 wt % F, 0.65 wt % K2O). The melt inclusion compositions indicate coexistence of two melt fractions, a prevailing peraluminous and a very volatile-rich, possibly peralkaline. Combined petrological, experimental and melt inclusion studies of the Kymi equigranular topaz granite indicate that plagioclase was the liquidus phase at nearly water-saturated (fluid-saturated) conditions and that the F content of the melt was at least 2 wt %. The early crystallization of biotite and the presence of muscovite in crystallization experiments at 200 MPa contrasts with the late-stage crystallization of biotite and the absence of muscovite in the equigranular granite, indicating that crystallization pressure may have been lower than 200 MPa for the granite.