To Örebro University

The New World Vulture [Coragyps] occidentalis (L. Miller, 1909) is one of many species that were extinct by the end of the Pleistocene. To understand its evolutionary history we sequenced the genome of a 14,000 year old [Coragyps] occidentalis found associated with megaherbivores in the Peruvian Andes. occidentalis has been viewed as the ancestor, or possibly sister, to the extant Black Vulture Coragyps atratus, but genomic data shows occidentalis to be deeply nested within the South American clade of atratus. Coragyps atratus inhabits lowlands, but the fossil record indicates that occidentalis mostly occupied high elevations. Our results suggest that occidentalis evolved from a population of atratus in southwestern South America that colonized the High Andes 300 to 400 kya. The morphological and morphometric differences between occidentalis and atratus may thus be explained by ecological diversification following from the natural selection imposed by this new and extreme, high elevation environment. The sudden evolution of a population with significantly larger body size and different anatomical proportions than atratus thus constitutes an example of punctuated evolution.

The moose (Alces alces) is the most intensely managed game species in Fennoscandia; approximately one-third of the population, ca. 160,000 animals, is harvested annually. Despite the species' biological and socioeconomic importance, there are knowledge gaps with respect to its intraspecific diversity and genetic structure. Recent studies of moose in neighboring countries report 2 genetic groups in Finland, 3 in Norway with one of them suggested to be of ancient origin, and no indications of bottlenecks. To delineate the spatial genetic landscape of the Swedish moose, we used allozyme variability from over 20,000 georeferenced moose collected all over Sweden in combination with 12 microsatellites (n = 1,200) and mitochondrial DNA (mtDNA) sequences (n = 44). We combined individual-based and traditional statistical approaches with coalescence-based simulations. The results indicate a complex history with bottlenecks and recent expansions that is consistent with historical records. Swedish moose are separated into 2 major genetic groups, a northern and a southern one, where the southern group is further divided into 3 subgroups. The 2 main subpopulations are moderately differentiated (F-ST = 0.1; R-ST = 0.07) and separated by sharp genetic discontinuities occurring over a relatively narrow transition zone in central Sweden that coincides with a similar, previously reported transition zone in Norway. This differentiation is not reflected in mtDNA variation, where no significant divergence was observed. Together with the F-ST andR(ST) similarities, this suggests that the 2 major subpopulations in Sweden reflect divergence shaped after the postglacial recolonization of Scandinavia. Neighborhood size assessments indicate that gene flow is relatively restricted with an estimated average dispersal distance of 3.5-11.1 km, and spatial autocorrelograms suggest that genetic similarity decreases almost linearly over space resulting in continuous genetic clines within major subgroups. Management areas largely coincide with genetic clusters, simplifying the integration of genetic information into management.

The Scandinavian brown bear went through a major decline in population size approximately 100 years ago, due to intense hunting. After being protected, the population subsequently recovered and today numbers in the thousands. The genetic diversity in the contemporary population has been investigated in considerable detail, and it has been shown that the population consists of several subpopulations that display relatively high levels of genetic variation. However, previous studies have been unable to resolve the degree to which the demographic bottleneck impacted the contemporary genetic structure and diversity. In this study, we used mitochondrial and microsatellite DNA markers from pre- and postbottleneck Scandinavian brown bear samples to investigate the effect of the bottleneck. Simulation and multivariate analysis suggested the same genetic structure for the historical and modern samples, which are clustered into three subpopulations in southern, central and northern Scandinavia. However, the southern subpopulation appears to have gone through a marked change in allele frequencies. When comparing the mitochondrial DNA diversity in the whole population, we found a major decline in haplotype numbers across the bottleneck. However, the loss of autosomal genetic diversity was less pronounced, although a significant decline in allelic richness was observed in the southern subpopulation. Approximate Bayesian computations provided clear support for a decline in effective population size during the bottleneck, in both the southern and northern subpopulations. These results have implications for the future management of the Scandinavian brown bear because they indicate a recent loss in genetic diversity and also that the current genetic structure may have been caused by historical ecological processes rather than recent anthropogenic persecution. 

Both long-term environmental changes such as those driven by the glacial cycles and more recent anthropogenic impacts have had major effects on the past demography in wild organisms. Within species, these changes are reflected in the amount and distribution of neutral genetic variation. In this thesis, mitochondrial and microsatellite DNA was analysed to investigate how environmental and anthropogenic factors have affected genetic diversity and structure in four ecologically different animal species. Paper I describes the post-glacial recolonisation history of the speckled-wood butterfly (Pararge aegeria) in Northern Europe. A decrease in genetic diversity with latitude and a marked population structure were uncovered, consistent with a hypothesis of repeated founder events during the postglacial recolonisation. Moreover, Approximate Bayesian Computation analyses indicate that the univoltine populations in Scandinavia and Finland originate from recolonisations along two routes, one on each side of the Baltic. Paper II aimed to investigate how past sea-level rises affected the population history of the convict surgeonfish (Acanthurus triostegus) in the Indo-Pacific. Assessment of the species’ demographic history suggested a population expansion that occurred approximately at the end of the last glaciation. Moreover, the results demonstrated an overall lack of phylogeographic structure, probably due to the high dispersal rates associated with the species’ pelagic larval stage. Populations at the species’ eastern range margin were significantly differentiated from other populations, which likely is a consequence of their geographic isolation. In Paper III, we assessed the effect of human impact on the genetic variation of European moose (Alces alces) in Sweden. Genetic analyses revealed a spatial structure with two genetic clusters, one in northern and one in southern Sweden, which were separated by a narrow transition zone. Moreover, demographic inference suggested a recent population bottleneck. The inferred timing of this bottleneck coincided with a known reduction in population size in the 19^th and early 20^th century due to high hunting pressure. In Paper IV, we examined the effect of an indirect but well-described human impact, via environmental toxic chemicals (PCBs), on the genetic variation of Eurasian otters (Lutra lutra) in Sweden. Genetic clustering assignment revealed differentiation between otters in northern and southern Sweden, but also in the Stockholm region. ABC analyses indicated a decrease in effective population size in both northern and southern Sweden. Moreover, comparative analyses of historical and contemporary samples demonstrated a more severe decline in genetic diversity in southern Sweden compared to northern Sweden, in agreement with the levels of PCBs found.

The Eurasian otter (Lutra lutra) population in Sweden went through a drastic decline in population size between the 1950s and 1980s, caused mostly by anthropogenic factors such as high hunting pressure and the introduction of environmental toxic chemicals into the otter’s habitats. However, after the bans of PCBs and DDT in the 1970s, the population began to recover in the 1990s. This study compares microsatellite data across twelve loci from historical and contemporary otter samples to investigate whether there has been a change in population structure and genetic diversity across time in various locations throughout Sweden. The results suggest that otters in the south were more severely affected by the bottleneck, demonstrated by a decline in genetic diversity and a shift in genetic composition. In contrast, the genetic composition in otters from northern Sweden remained mostly unchanged, both in terms of population structure and diversity. This suggests that the decline was not uniform across the country. Moreover, our analyses of historical samples provide an overview of the level of genetic variation and population structure that existed prior to the bottleneck, which may be helpful for the future management and conservation of the species.

The post-glacial recolonisation of northern Europe has left distinct signatures in the genomes of many organisms, both due to random demographic processes and divergent natural selection. However, information on differences in genetic variation in conjunction with patterns of local adaptations along latitudinal gradients is often lacking. In this study, we examine genetic diversity and population structure in the speckled wood butterfly Pararge aegeria in northern Europe to investigate the species post-glacial recolonisation history and discuss how this may have affected its life-history evolution. We collected 209 samples and analysed genetic variation in nine microsatellite loci. The results demonstrated a more pronounced population structure in northern Europe compared with populations further south, as well as an overall decrease in genetic diversity with latitude, likely due to founder effects during the recolonisation process. Coalescent simulations coupled with approximate Bayesian computation suggested that central Scandinavia was colonised from the south, rather than from the east. In contrast to further south, populations at the northern range margin are univoltine expressing only one generation per year. This suggests either that univoltinism evolved independently on each side of the Baltic Sea, or that bivoltinism evolved in the south after northern Europe was recolonised.

Wilson disease (WND) is an autosomal recessive condition that results in accumulation of copper in the liver and brain when a membrane bound copper transporter, ATP7B, is defective. ATP7B is expressed in hepatic, brain and kidney cells, and a defect can lead to liver, neurological and renal damage in WND patients. Presentation is variable with a broad range of age of onset and symptoms, and not all biochemical signs used in diagnosis are found in every patient. Therefore, diagnosis by mutation analysis is particularly important. To date, there are approximately 380 probable disease-causing variants in ATP7B, 33 of which are splice site variants that are predicted to affect splicing, based on their location. Few of these splice site variants have been analyzed in vivo. Some exonic variations also have the potential to affect splicing. The aim of this project was to use minigenes for transcript analysis. We have chosen exon 8 as our focus and have cloned a wild-type three-exon minigene into a mammalian expression vector. After transfection, extracted RNA was analyzed by reverse transcription PCR and accurate splicing was detected. This minigene will facilitate the analysis of the numerous potential splice variants identified in exon 8 of ATP7B, with the advantage that patient cell lines are not required for each variant.