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Inbreeding depression (ID) has since long been recognized as a significant factor in evolutionary biology. It is mainly the consequence of (partially) recessive deleterious mutations maintained by mutation-selection balance in large random mating populations. When population size is reduced, recessive alleles are increasingly found in homozygous condition due to drift and inbreeding and become more prone to selection. Particularly at slow rates of drift and inbreeding, selection will be more effective in purging such alleles, thereby reducing the amount of ID. Here we test assumptions of the efficiency of purging in relation to the inbreeding rate and the experimental conditions for four traits in D. melanogaster. We investigated the magnitude of ID for lines that were inbred to a similar level, F ≈ 0.50, reached either by three generations of full-sib mating (fast inbreeding), or by 12 consecutive generations with a small population size (slow inbreeding). This was done on two different food media. We observed significant ID for egg-to-adult viability and heat shock mortality, but only for egg-to-adult viability a significant part of the expressed inbreeding depression was effectively purged under slow inbreeding. For other traits like developmental time and starvation resistance, however, adaptation to the experimental and environmental conditions during inbreeding might affect the likelihood of purging to occur or being detected. We discuss factors that can affect the efficiency of purging and why empirical evidence for purging may be ambiguous.

Genetic variation that is expressed only under specific environmental conditions can contribute to additional adverse effects of inbreeding if environmental conditions change. We present a proteomic characterization of a conditional lethal found in an inbred line of Drosophila melanogaster. The lethal effect is apparent as a large increase in early mortality at the restrictive temperature (29 °C) as opposed to normal survival at the permissive temperature (20 °C). The increased mortality in response to the restrictive temperature is probably caused by a single recessive major locus. A quantitative trait locus (QTL) region segregating variation affecting the lethal effect has been identified, allowing for a separation of primary/causal effects and secondary consequences in the proteome expression patterns observed. In this study, the proteomic response to the restrictive temperature in the lethal-line (L-line) was compared with the response in an inbred-control-line (IC-line) and an outbred-control-line (OC-line). Quantitative protein changes were detected using isobaric tags for relative and absolute quantitation (iTRAQ) two-dimensional liquid chromatography-tandem mass spectrometry. In all, 45 proteins were found to be significantly differently regulated in response to the restrictive temperature in the L-line as compared with the IC-line. No proteins were significantly differently regulated between the IC-line and the OC-line, verifying that differential protein regulation was specific to a genetic defect in the L-line. Proteins associated with oxidative phosphorylation and mitochondria were significantly overrepresented within the list of differentially expressed proteins. Proteins related to muscle contraction were also found to be differentially expressed in the L-line in response to the restrictive temperature, supporting phenotypic observations of moribund muscle hyper-contraction Genetic variation that is expressed only under specific environmental conditions can contribute to additional adverse effects of inbreeding if environmental conditions change. We present a proteomic characterization of a conditional lethal found in an inbred line of Drosophila melanogaster. The lethal effect is apparent as a large increase in early mortality at the restrictive temperature (29 °C) as opposed to normal survival at the permissive temperature (20 °C). The increased mortality in response to the restrictive temperature is probably caused by a single recessive major locus. A quantitative trait locus (QTL) region segregating variation affecting the lethal effect has been identified, allowing for a separation of primary/causal effects and secondary consequences in the proteome expression patterns observed. In this study, the proteomic response to the restrictive temperature in the lethal-line (L-line) was compared with the response in an inbred-control-line (IC-line) and an outbred-control-line (OC-line). Quantitative protein changes were detected using isobaric tags for relative and absolute quantitation (iTRAQ) two-dimensional liquid chromatography-tandem mass spectrometry. In all, 45 proteins were found to be significantly differently regulated in response to the restrictive temperature in the L-line as compared with the IC-line. No proteins were significantly differently regulated between the IC-line and the OC-line, verifying that differential protein regulation was specific to a genetic defect in the L-line. Proteins associated with oxidative phosphorylation and mitochondria were significantly overrepresented within the list of differentially expressed proteins. Proteins related to muscle contraction were also found to be differentially expressed in the L-line in response to the restrictive temperature, supporting phenotypic observations of moribund muscle hyper-contraction.

The European bison (Bison bonasus) has recovered successfully after a severe bottleneck about 90 years ago. Pedigree analysis indicates that over 80% of the genes in the contemporary population descend from just 2 founder individuals and the pedigree-based inbreeding coefficient averages almost 0.5, while microsatellite heterozygosity is around 0.3. This paper presents a comparison of the effectiveness of 17 microsatellite and 960 SNP markers in paternity and identity analysis in the Lowland line of European bison ('pure' European bison). Identity analysis revealed that 16% of the microsatellite-based genotypes were common to more than one individual while a smaller set of SNP-based genotypes were all unique, even. Microsatellite-based paternity analysis did not succeed due to low marker heterozygosity. Using SNP markers, simulations suggest that 80-90 randomly selected loci would be sufficient to ensure successful paternity analysis, or just 50-60 of the most heterozygous loci. For the purpose of standardizing future parentage and identity analysis, a panel of 50-60 SNPs characterized by high heterozygosity and an even distribution in the genome could be selected. This panel of selected markers could be typed using e.g. Veracode (Illumina) or SNPlex (ABI) technologies to provide an efficient and reliable method for parentage and identity analysis in European bison. Customizing off-the-shelf SNP marker panels created for domestic species can provide powerful, low-cost tools for genetic analysis in related species Udgivelsesdato: October The European bison (Bison bonasus) has recovered successfully after a severe bottleneck about 90 years ago. Pedigree analysis indicates that over 80% of the genes in the contemporary population descend from just 2 founder individuals and the pedigree-based inbreeding coefficient averages almost 0.5, while microsatellite heterozygosity is around 0.3. This paper presents a comparison of the effectiveness of 17 microsatellite and 960 SNP markers in paternity and identity analysis in the Lowland line of European bison ('pure' European bison). Identity analysis revealed that 16% of the microsatellite-based genotypes were common to more than one individual while a smaller set of SNP-based genotypes were all unique, even. Microsatellite-based paternity analysis did not succeed due to low marker heterozygosity. Using SNP markers, simulations suggest that 80-90 randomly selected loci would be sufficient to ensure successful paternity analysis, or just 50-60 of the most heterozygous loci. For the purpose of standardizing future parentage and identity analysis, a panel of 50-60 SNPs characterized by high heterozygosity and an even distribution in the genome could be selected. This panel of selected markers could be typed using e.g. Veracode (Illumina) or SNPlex (ABI) technologies to provide an efficient and reliable method for parentage and identity analysis in European bison. Customizing off-the-shelf SNP marker panels created for domestic species can provide powerful, low-cost tools for genetic analysis in related species

Species colonizing new areas disjunct from their original habitat may be subject to novel selection pressures, and exhibit adaptive genetic changes. However, if colonization occurs through a small number of founders, the genetic composition of the colonized population may differ from that of the original population simply due to genetic drift. Disentangling the effects of founder drift and selection after colonization is crucial to understanding the adaptive process. Drosophila buzzatii colonized Australia some 600-700 generations ago, and spread rapidly over a wide geographical range. Genetic variation for 15 microsatellite loci in each of nine populations in eastern Australia was used to estimate the size of the bottleneck, and to determine if any of these microsatellites marked genomic regions subject to recent selection. We estimate that on its introduction to Australia, D. buzzatii went through a moderate bottleneck (approximately 30-40 founders). Linkage disequilibrium was common, both intrachromosomal and between loci on different chromosomes. Of the 15 loci, 2 showed evidence of selection, one exhibiting local adaptation in different populations and the other balancing selection. We conclude that linkage disequilibria may be far more common in natural populations than is generally assumed, and the loci apparently affected by selection may well be marking selection in large genome regions including many loci that are not necessarily closely linked.

Despite the recent discovery of significant genetic structuring in a large number of marine organisms, the evolutionary significance of these often minute genetic differences are still poorly understood. To elucidate the adaptive relevance of low genetic differentiation among marine fish populations, we studied expression differences of osmoregulatory and stress genes in genetically weakly differentiated populations of the European flounder (Platichthys flesus), distributed across a natural salinity gradient. Flounders were maintained in a long-term reciprocal transplantation experiment mimicking natural salinities in the North Sea and the Baltic Sea. Applying real-time quantitative PCR and microarray analysis we studied expression of four candidate genes (hsp70, angiotensinogen, Na/K-ATPase-alpha and 5-aminolevulinic acid synthase (ALAS)) in gill, kidney and liver tissues. Genes involved in osmoregulative processes (Na/K-ATPases-alpha and angiotensinogen) showed highly plastic but similar expression in the two populations dependent on environmental salinity. However, we observed a unique sixfold up-regulation of hsp70 in kidney tissue of flounder from the North Sea following long-term acclimation to Baltic salinities. Similarly, significant differences between North Sea and Baltic flounders in expression of ALAS in response to different salinities were found in gill and liver tissue. These findings strongly suggest that gene expression in flounders is shaped by adaptation to local environmental conditions. This identification of adaptive differences in high gene flow marine organisms adds a new dimension to our current understanding of evolutionary processes in the sea and is of paramount importance for identification, protection and sustainable management of marine biodiversity.

A half-sib analysis was used to investigate genetic variation for three morphological traits (thorax length, wing length and sternopleural bristle number) and two life-history traits (developmental time and larva-to-adult viability) in Drosophila melanogaster reared at a standard (25 degrees C) and a low stressful (13 degrees C) temperature. Both phenotypic and environmental variation showed a significant increase under stressful conditions in all traits. For estimates of genetic variation, no statistically significant differences were found between the two environments. Narrow heritabilities tended to be higher at 13 degrees C for sternopleural bristle number and viability and at 25 degrees C for wing length and developmental time, whereas thorax length did not show any trend. However, the pattern of genetic variances and evolvability indices (coefficient of genetic variation and evolvability), considered in the context of literature evidence, indicated the possibility of an increase in additive genetic variation for the morphological traits and viability and in nonadditive genetic variation for developmental time. The data suggest that the effect of stressful temperature may be trait-specific and this warns against generalizations about the behaviour of genetic variation under extreme conditions.

One hundred and twenty-four specimens of the harbour porpoise, Phocoena phocoena, occurring in inner Danish waters (IDW), the North Sea and West Greenland were analysed to study subdivision into genetically differentiated subpopulations using PCR-amplified DNA-microsatellites and isozyme markers. Three polymorphic microsatellites, 415/416, 417/418 and Igf-I (insulin-like growth factor I) were detected with nine, eight and 15 alleles, respectively, and from a former study two polymorphic isozymes, Mpi-1 and Pgm, with three and two alleles, respectively, were used in the analysis. Overall deviations from the expected Hardy-Weinberg distribution were only observed in the total sample and at a single locus in the North Sea-summer sample and at two loci in the West Greenland sample. Whenever this occurred a surplus of homozygotes was observed, suggesting a Wahlund effect, a null allele or nonrandom mating. The analysis of the genetical population structure showed that harbour porpoises from West Greenland, the North Sea and IDW were three geographically, genetically differentiated populations even though connected through some degree of gene flow. A tendency for females to be more stationary than males was suggested. Furthermore, the population structure suggested a closer relationship between IDW and the North Sea.

The effect of inbreeding on survival after a short-term heat shock was tested for two age groups of the cactophilic fruit fly, Drosophila buzzatii, reared under nonstress conditions. Four inbreeding levels (F = 0, F = 0.25, F = 0.375, F = 0.5) were generated by outcrossing or full-sib mating. All flies were conditioned at 36.5 degrees C for 75 min prior to exposure to stress, to activate the synthesis of heat-shock proteins. These proteins are known to protect cells against stress damage. The younger group of flies were exposed to a thermal stress of 40.7 degrees C for 88 min, 103 min, or 118 min and the older flies to the same temperature only for 88 min or 103 min, as the survival of older flies after heat stress was much lower than that of the younger flies. Survival after heat shock declined with increased inbreeding in both age groups. For the younger flies, the slope of the regression line, F, on survival was lower at higher stress levels. For the older flies, inbreeding effects were similar at both stress levels. Mortality without stress also differed significantly among inbreeding groups, mainly because of a large difference between the F = 0.5 group and all others.

Genetic variation for selfing rate and the dependence of selfing rate on mating history in Brassica napus (rape seed)

For a deterministic population genetic model, we have shown that partial selfing can be an optimal mating strategy. The only necessary assumption is that inbreeding depression in consecutive generations of selfing is described by a decreasing exponential function. The model gave an estimate of the expected selfing rate in a population with mixed mating and a known inbreeding depression function. Calculations were done for both a one-locus and a co-dominant two-locus model. There were unstable or stable polymorphic equilibria that were dependent on the selfing rate of the heterozygotes. The model was shown to be in good agreement with data on inbreeding depression and selfing rates from the literature.

Little is known about local adaptations in marine fishes since population genetic surveys in these species have typically not applied genetic markers subject to selection. In this study, we used a candidate gene approach to investigate adaptive population divergence in the European flounder (Platichthys flesus L.) throughout the northeastern Atlantic. We contrasted patterns of genetic variation in a presumably neutral microsatellite baseline to patterns from a heat-shock cognate protein gene, Hsc70. Using two different neutrality tests we found that the microsatellite data set most likely represented a neutral baseline. In contrast, Hsc70 strongly deviated from neutral expectations. Importantly, when estimating standardized levels of population divergence (F(ST)'), we also found a large discrepancy in the patterns of structuring in the two data sets. Thus, samples grouped according to geographical or historical proximity with regards to microsatellites, but according to environmental similarities with regards to Hsc70. The differences between the data sets were particularly pronounced in pairwise comparisons involving populations in the western and central Baltic Sea. For instance, the genetic differentiation between geographically close Baltic Sea and North Sea populations was found to be 0.02 and 0.45 for microsatellites and Hsc70 respectively. Our results strongly suggest adaptive population divergence and indicate local adaptations at the DNA level in a background of high levels of gene flow, typically found in many marine fish species. Furthermore, this study highlights the usefulness of the candidate gene approach for demonstrating local selection in non-model organisms such as most marine fishes.

Variation in 19 traits possibly relevant for thermal adaptation was studied in 11 populations of Drosophila buzzatii collected in southeast Australia. Using stepwise multiple regression, the variation was compared to variation in geographic coordinates and to a set of climatic variables estimated for each collection site. For 13 of the traits, a significant part of the variation was explained by climatic variables and/or geographic coordinates, suggesting directional selection for adaptation to the environment in the majority of traits studied. In 10 of the traits, both geographic coordinates and climatic variables explained significant proportions of the variation, with R2 ranging from 0.075 to 0.58. Although larvae, pupae and adults of D. buzzatii share a common habitat, the measured traits were not correlated across life stages and gender. Also, there seemed to be special conditions in marginal populations near species borders, giving rise to nonlinear relations with latitude. Climate apparently does influence the adaptive evolution of the traits studied, but they also are affected by other factors that vary with latitude, longitude and distance to coast. These results highlight the complex challenges imposed by the environment on the adaptive process.

Here we studied three phenotypic traits in Drosophila buzzatii that are strongly effected by temperature, and are expected to be closely associated with fitness in nature. The traits measured were thermal threshold of male sterility, time for males to gain fertility when reared at a sterility-inducing temperature and transferred to 25 degrees C on eclosion and survival after development. The last two traits were measured under four temperature regimes, constant 12 degrees C, 25 degrees C, 31 degrees C, and fluctuating 25 degrees C (18 h) and 38 degrees C (6 h). We looked for genetic variation in these traits and relations among them in four lines of D. buzzatii originating from Argentina and Tenerife. The thermal threshold of heat-induced male sterility was found to lie within the range of 30.0-31.0 degrees C. When measuring the time for males to gain fertility, males reared at a nonstressful temperature (25 degrees C) were fertile 58-67 h after emergence with only minor differences among lines. When reared constant 31 degrees C, males were fertile 174-225 h after hatching. The Argentinean lines were significantly faster in recovering from sterility than were the lines from Tenerife. When reared in a fluctuating temperature regime, differences among lines increased, dividing the lines into three significantly different groups, with a sterility period of 135-215 h. When reared at 12 degrees C from the pupal stage, males were fertile after 106-130 h with significant difference in the variance but not in the mean duration of sterility. Significant differences in viability were found among development temperatures, but not among lines, and viability and the duration of sterility seem to be genetically independent.

Latitudinal variation in thorax and wing size traits was studied in wild-caught flies of the cactophilic Drosophila species, D. aldrichi and D. buzzatii, and their laboratory-reared progeny. The flies originated from five populations in Queensland, Australia, spanning an 800-km transect. The laboratory flies were reared at controlled densities and three temperatures, 20, 25, and 30 degrees C. We measured the same traits for the laboratory-reared flies as for the wild-caught flies, plus developmental time and viability. Latitudinal variation in wild-caught flies of both species followed a similar pattern for all linear size traits, with size generally increasing from north to south, but with flies from one intermediate locality markedly smaller. A drier environment at this locality and weather conditions immediately prior to collection, most likely explain the reduced size. Laboratory-reared D. aldrichi from this locality also were smaller than those from other localities, and had the fastest developmental time and highest viability. In laboratory-reared flies, body size traits did not show any clear trend to increase with latitude. The patterns of change with latitude were different between species, with D. aldrichi more similar in pattern to that of the natural populations. D. aldrichi had comparatively higher coefficients of variation in the laboratory-reared flies and lower viability at all temperatures. However, fluctuating asymmetry was lower in D. aldrichi in both wild-caught and laboratory-reared flies. The differences among populations of D. aldrichi for all traits were much larger than for D. buzzatii. As these differences in the laboratory-reared flies are expected to be largely genetic, they most likely reflect adaptation to specific (unknown) environmental factors that do not show linear latitudinal variation on the geographical scale studied.

Although exposure to environmental stress is common in most populations, and the physiological effects of stress on individuals are well studied, the evolutionary importance of stress to populations is not well understood. To address multitrait responses to environmental change and potential constraints on character evolution, we analysed, in 100 isofemale lines of Drosophila buzzatii, the genetic relationships between resistance to a short heat shock and several life-history traits: survival in benign conditions, larval developmental time, fecundity and longevity. Estimates of heritability of larval thermotolerance were low, but significant, and all life-history traits varied significantly among isofemale lines. Several of these traits covaried significantly. Most correlations indicated positive life-history relationships, but males and females from lines where female fecundity was higher developed more slowly in the absence of stress, which is a negative life-history relationship. The stress reduced or negated many trait associations, and showed one additional relationship; more larvae from lines that developed fast at 25 degrees C survived to adult after stress than did larvae from slow developing lines. These shifts in fitness relationships, when a single stress bout is applied, suggest that even small increases in environmental stress can have profound effects on evolutionary relationships among life-history traits.