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One important goal of population genetics is to understand the relative importance of different evolutionary processes for shaping variation in natural populations. Here, I use multilocus data to show that natural selection on both synonymous and nonsynonymous mutations plays an important role in shaping levels of synonymous polymorphism in European aspen (Populus tremula). Previous studies have documented a preferential fixation of synonymous mutations encoding preferred codons in P. tremula. The results presented here show that this has resulted in an increase in codon bias in P. tremula, consistent with stronger selection acting on synonymous codon usage. In addition, positive selection on nonsynonymous mutations appears to be common in P. tremula, with approximately 30% of all mutations having been fixed by positive selection. In addition, the recurrent fixation of beneficial mutations also reduces standing levels of polymorphism as evidenced by a significantly negative relationship between the rate of protein evolution synonymous site diversity and silent site diversity. Finally, I use approximate Bayesian methods to estimate the strength of selection acting on beneficial substitutions. These calculations show that recurrent hitchhiking reduces polymorphism by, on average, 30%. The product of strength of selection acting on beneficial mutations and the rate by which these occur across the genome (2N(e)lambdas) equals 1.54x10( - 7), which is in line with estimates from Drosophila where recurrent hitchhiking has also been shown to have significant effects on standing levels of polymorphism.

This article documents the addition of 238 microsatellite marker loci and 72 pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Adelges tsugae, Artemisia tridentata, Astroides calycularis, Azorella selago, Botryllus schlosseri, Botrylloides violaceus, Cardiocrinum cordatum var. glehnii, Campylopterus curvipennis, Colocasia esculenta, Cynomys ludovicianus, Cynomys leucurus, Cynomys gunnisoni, Epinephelus coioides, Eunicella singularis, Gammarus pulex, Homoeosoma nebulella, Hyla squirella, Lateolabrax japonicus, Mastomys erythroleucus, Pararge aegeria, Pardosa sierra, Phoenicopterus ruber ruber and Silene latifolia. These loci were cross-tested on the following species: Adelges abietis, Adelges cooleyi, Adelges piceae, Pineus pini, Pineus strobi, Tubastrea micrantha, three other Tubastrea species, Botrylloides fuscus, Botrylloides simodensis, Campylopterus hemileucurus, Campylopterus rufus, Campylopterus largipennis, Campylopterus villaviscensio, Phaethornis longuemareus, Florisuga mellivora, Lampornis amethystinus, Amazilia cyanocephala, Archilochus colubris, Epinephelus lanceolatus, Epinephelus fuscoguttatus, Symbiodinium temperate-A clade, Gammarus fossarum, Gammarus roeselii, Dikerogammarus villosus and Limnomysis benedeni. This article also documents the addition of 72 sequencing primer pairs and 52 allele specific primers for Neophocaena phocaenoides., (© 2009 Blackwell Publishing Ltd.)

Two haplotypes of the Vibrio cholerae quorum-sensing system regulator hapR are described: hapR1, common among nonpandemic, non-O1, non-O139 strains, and hapR2, associated with pandemic O1 and O139 and epidemic O37 V. cholerae strains. The hapR2 has evolved under strong natural selection, implying that its fixation was influenced by conditions that led to cholera pandemics.

Background: Evolution of synonymous codon usage is thought to be determined by a balance between mutation, genetic drift and natural selection on translational efficiency. However, natural selection on codon usage is considered to be a weak evolutionary force and selection on codon usage is expected to be strongest in species with large effective population sizes., Results: I examined the evolution of synonymous codons using EST data from five species of Populus. Data on relative synonymous codon usage in genes with high and low gene expression were used to identify 25 codons from 18 different amino acids that were deemed to be preferred codons across all five species. All five species show significant correlations between codon bias and gene expression, independent of base composition, thus indicating that translational selection has shaped synonymous codon usage. Using a set of 158 orthologous genes I detected an excess of unpreferred to preferred (U --> P) mutations in two lineages, P. tremula and P. deltoides. Maximum likelihood estimates of the strength of selection acting on synonymous codons was also significantly greater than zero in P. tremula, with the ML estimate of 4Nes = 0.720., Conclusion: The data is consistent with weak selection on preferred codons in all five species. There is also evidence suggesting that selection on synonymous codons has increased in P. tremula. Although the reasons for the increase in selection on codon usage in the P. tremula lineage are not clear, one possible explanation is an increase in the effective population size in P. tremula.

I have studied nucleotide polymorphism and linkage disequilibrium using multilocus data from 77 fragments, with an average length of fragments of 550 bp, in the deciduous tree Populus tremula (Salicaceae). The frequency spectrum across loci showed a modest excess of mutations segregating at low frequency and a marked excess of high-frequency derived mutations at silent sites, relative to neutral expectations. These excesses were also seen at replacement sites, but were not so pronounced for high-frequency derived mutations. There was a marked excess of low-frequency mutations at replacement sites, likely indicating deleterious amino acid-changing mutations that segregate at low frequencies in P. tremula. I used approximate Bayesian computation (ABC) to evaluate a number of different demographic scenarios and to estimate parameters for the best-fitting model. The data were found to be consistent with a historical reduction in the effective population size of P. tremula through a bottleneck. The timing inferred for this bottleneck is largely consistent with geological data and with data from several other long-lived plant species. The results show that P. tremula harbors substantial levels of nucleotide polymorphism with the posterior mode of the scaled mutation rate, theta = 0.0177 across loci. The ABC analyses also provided an estimate of the scaled recombination rate that indicates that recombination rates in P. tremula are likely to be 2-10 times higher than the mutation rate. This study reinforces the notion that linkage disequilibrium is low and decays to negligible levels within a few hundred base pairs in P. tremula.

We investigated the utility of association mapping to dissect the genetic basis of naturally occurring variation in bud phenology in European aspen (Populus tremula). With this aim, we surveyed nucleotide polymorphism in 13 fragments spanning an 80-kb region surrounding the phytochrome B2 (phyB2) locus. Although polymorphism varies substantially across the phyB2 region, we detected no signs for deviations from neutral expectations. We also identified a total of 41 single nucleotide polymorphisms (SNPs) that were subsequently scored in a mapping population consisting of 120 trees. We identified two nonsynonymous SNPs in the phytochrome B2 gene that were independently associated with variation in the timing of bud set and that explained between 1.5 and 5% of the observed phenotypic variation in bud set. Earlier studies have shown that the frequencies of both these SNPs vary clinally with latitude. Linkage disequilibrium across the region was low, suggesting that the SNPs we identified are strong candidates for being causally linked to variation in bud set in our mapping populations. One of the SNPs (T608N) is located in the "hinge region," close to the chromophore binding site of the phyB2 protein. The other SNP (L1078P) is located in a region supposed to mediate downstream signaling from the phyB2 locus. The lack of population structure, combined with low levels of linkage disequilibrium, suggests that association mapping is a fruitful method for dissecting naturally occurring variation in Populus tremula.

High-throughput DNA sequencing and genotyping technologies have enabled a new generation of research in plant genetics where combined quantitative and population genetic approaches can be used to better understand the relationship between naturally occurring genotypic and phenotypic diversity. Forest trees are highly amenable to such studies because of their combined undomesticated and partially domesticated state. Forest geneticists are using association genetics to dissect complex adaptive traits and discover the underlying genes. In parallel, they are using resequencing of candidate genes and modern population genetics methods to discover genes under natural selection. This combined approach is identifying the most important genes that determine patterns of complex trait adaptation observed in many tree populations.

A correct timing of growth cessation and dormancy induction represents a critical ecological and evolutionary trade-off between survival and growth in most forest trees (Rehfeldt et al. 1999; Horvath et al. 2003; Howe et al. 2003). We have studied the deciduous tree European Aspen (Populus tremula) across a latitudinal gradient and compared genetic differentiation in phenology traits with molecular markers. Trees from 12 different areas covering 10 latitudinal degrees were cloned and planted in two common gardens. Several phenology traits showed strong genetic differentiation and clinal variation across the latitudinal gradient, with Q(ST) values generally exceeding 0.5. This is in stark contrast to genetic differentiation at several classes of genetic markers (18 neutral SSRs, 7 SSRs located close to phenology candidate genes and 50 SNPs from five phenology candidate genes) that all showed F(ST) values around 0.015. We thus find strong evidence for adaptive divergence in phenology traits across the latitudinal gradient. However, the strong population structure seen at the quantitative traits is not reflected in underlying candidate genes. This result fit theoretical expectations that suggest that genetic differentiation at candidate loci is better described by F(ST) at neutral loci rather than by Q(ST) at the quantitative traits themselves.

The prevailing wisdom of the plant mitochondrial genome is that it has very low substitution rates, thus it is generally assumed that nucleotide diversity within species will also be low. However, recent evidence suggests plant mitochondrial genes may harbor variable and sometimes high levels of within-species polymorphism, a result attributed to variance in the influence of selection. However, insufficient attention has been paid to the effect of among-gene variation in mutation rate on varying levels of polymorphism across loci. We measured levels of polymorphism in seven mitochondrial gene regions across a geographically wide sample of the plant Silene vulgaris to investigate whether individual mitochondrial genes accumulate polymorphisms equally. We found that genes vary significantly in polymorphism. Tests based on coalescence theory show that the genes vary significantly in their scaled mutation rate, which, in the absence of differences among genes in effective population size, suggests these genes vary in their underlying mutation rate. Further evidence that among-gene variance in polymorphism is due to variation in the underlying mutation rate comes from a significant positive relationship between the number of segregating sites and silent site divergence from an outgroup. Contrary to recent studies, we found unconvincing evidence of recombination in the mitochondrial genome, and generally confirm the standard model of plant mitochondria characterized by low substitution rates and no recombination. We also show no evidence of significant variation in the strength or direction of selection among genes; this result may be expected if there is no recombination. The present study provides some of the most thorough data on plant mitochondrial polymorphism, and provides compelling evidence for mutation rate variation among genes. The study also demonstrates the difficulty in establishing a null model of mitochondrial genome polymorphism, and thus the difficulty, in the absence of a comparative approach, in testing the assumption that low substitution rates in plant mitochondria lead to low polymorphism.

Codon bias is generally thought to be determined by a balance between mutation, genetic drift, and natural selection on translational efficiency. However, natural selection on codon usage is considered to be a weak evolutionary force and selection on codon usage is expected to be strongest in species with large effective population sizes. In this paper, I study associations between codon usage, gene expression, and molecular evolution at synonymous and nonsynonymous sites in the long-lived, woody perennial plant Populus tremula (Salicaceae). Using expression data for 558 genes derived from expressed sequence tags (EST) libraries from 19 different tissues and developmental stages, I study how gene expression levels within single tissues as well as across tissues affect codon usage and rates sequence evolution at synonymous and nonsynonymous sites. I show that gene expression have direct effects on both codon usage and the level of selective constraint of proteins in P. tremula, although in different ways. Codon usage genes is primarily determined by how highly expressed a genes is, whereas rates of sequence evolution are primarily determined by how widely expressed genes are. In addition to the effects of gene expression, protein length appear to be an important factor influencing virtually all aspects of molecular evolution in P. tremula.

Maximum likelihood models of codon substitutions were used to analyze the molecular evolution of a Kunitz trypsin inhibitor (KTI) gene family in Populus and Salix. The methods support previous assertions that the KTI genes comprise a rapidly evolving gene family. Models that allow for codon specific estimates of the ratio of nonsynonymous to synonymous substitutions (omega) among sites detect positive Darwinian selection at several sites in the KTI protein. In addition, branch-specific maximum likelihood models show that there is significant heterogeneity in omega among branches of the KTI phylogeny. In particular, omega is substantially higher following duplication than speciation. There is also evidence for significant rate heterogeneity following gene duplication, suggesting different evolutionary rates in newly arisen gene duplicates. The results indicate uneven evolutionary rates both between sites in the KTI protein and among different lineages in the KTI phylogeny, which is incompatible with a neutral model of sequence evolution.

The initiation of growth cessation and dormancy represents a critical ecological and evolutionary trade-off between survival and growth in most forest trees. The most important environmental cue regulating the initiation of dormancy is a shortening of the photoperiod and phytochrome genes have been implicated in short-day-induced bud set and growth cessation in Populus. We characterized patterns of DNA sequence variation at the putative candidate gene phyB2 in 4 populations of European aspen (Populus tremula) and scored single-nucleotide polymorphisms in an additional 12 populations collected along a latitudinal gradient in Sweden. We also measured bud set from a subset of these trees in a growth chamber experiment. Buds set showed significant clinal variation with latitude, explaining approximately 90% of the population variation in bud set. A sliding-window scan of phyB2 identified six putative regions with enhanced population differentiation and four SNPs showed significant clinal variation. The clinal variation at individual SNPs is suggestive of an adaptive response in phyB2 to local photoperiodic conditions. Three of four SNPs showing clinal variation were located in regions with excessive genetic differentiation, demonstrating that searching for regions of high genetic differentiation can be useful for identifying sites putatively involved in local adaptation.

Floral traits endowing high reproductive fitness can also affect the probability of plants contracting sexually transmitted diseases. We explore how variations in floral traits influence the fitness of Silene dioica females in their interactions with pollinators carrying pollen or spores of the sterilizing anther-smut fungus Microbotryum violaceum. We collected healthy and infected plants in a highly diseased population and grew them under conditions that 'cure' infected individuals, and used standard regression methods to detect natural selection on floral traits. Narrow-sense heritabilities, coefficients of additive genetic variation (CV(A)) and genetic correlations among traits were estimated from paternal half-sib groups. Pollinator preferences imposed strong direct and directional selection on traits affecting female attractiveness and pollen-/spore-capturing abilities. Levels of additive genetic variance were high in these traits, suggesting that rapid responses to selection are possible. By considering our results in the light of spatial and temporal heterogeneity resulting from the colonization dynamics typical for this species, we suggest that the conflicting selective effects of pollen/spore loads lead to the maintenance of genetic variation in these traits.

Plants defend themselves against the attack of natural enemies by using an array of both constitutively expressed and induced defenses. Long-lived woody perennials are overrepresented among plant species that show strong induced defense responses, whereas annual plants and crop species are underrepresented. However, most studies of plant defense genes have been performed on annual or short-lived perennial weeds or crop species. Here I use molecular population genetic methods to survey six wound-inducible protease inhibitors (PIs) in a long-lived woody, perennial plant species, the European aspen (Populus tremula), to evaluate the likelihood of either recurrent selective sweeps or balancing selection maintaining amino acid polymorphisms in these genes. The results show that none of the six PI genes have reduced diversities at synonymous sites, as would be expected in the presence of recurrent selective sweeps. However, several genes show some evidence of nonneutral evolution such as enhanced linkage disequilibrium and a large number of high-frequency-derived mutations. A group of at least four Kunitz trypsin inhibitor genes appear to have experienced elevated levels of nonsynonymous substitutions, indicating allelic turnover on an evolutionary timescale. One gene, TI1, has enhanced levels of intraspecific polymorphism at nonsynonymous sites and also has an unusual haplotype structure characterized by two divergent haplotypes occurring at roughly equal frequencies in the sample. One haplotype has very low levels of intraallelic nucleotide diversity, whereas the other haplotype has levels of diversity comparable to other genes in P. tremula. Patterns of sequence diversity at TI1 do not fit a simple model of either balancing selection or recurrent selective sweeps. This suggests that selection at TI1 is more complex, possibly involving allelic cycling.

Populus is an important model organism in forest biology, but levels of nucleotide polymorphisms and linkage disequilibrium have never been investigated in natural populations. Here I present a study on levels of nucleotide polymorphism, haplotype structure, and population subdivision in five nuclear genes in the European aspen Populus tremula. Results show substantial levels of genetic variation. Levels of silent site polymorphisms, pi(s), averaged 0.016 across the five genes. Linkage disequilibrium was generally low, extending only a few hundred base pairs, suggesting that rates of recombination are high in this obligate outcrossing species. Significant genetic differentiation was found at all five genes, with an average estimate of F(ST) = 0.116. Levels of polymorphism in P. tremula are 2- to 10-fold higher than those in other woody, long-lived perennial plants, such as Pinus and Cryptomeria. The high levels of nucleotide polymorphism and low linkage disequilibrium suggest that it may be possible to map functional variation to very fine scales in P. tremula using association-mapping approaches.

The Hudson-Kreitman-Aguade (HKA) test is based on the prediction from the neutral theory that levels of polymorphism within a species and the divergence between two closely related species should be correlated. Population subdivision has been shown to alter both the amounts of polymorphism segregating within species and the rate of divergence between species, meaning that genomic regions with different population structures also differ in their divergence to polymorphism ratios. Population subdivision may hence hamper the utility of the HKA test for detecting deviations from the standard neutral model, especially for organelle genomes that often have different patterns of population structure compared with nuclear genes. In this paper, I show that population subdivision inflates the number of instances where the HKA test detects deviations from the neutral model. Using coalescent simulations I show that this bias is most apparent when population subdivision is strong and differs substantially between the loci included. However, if divergence time is large and population structure substantial even changes in the levels of polymorphism and divergence associated with differences in the effective population size between two loci is enough to substantially alter the number of significant outcomes of the HKA test. A dataset on cytoplasmic diversity in Sileine vulgaris and S. latifolia (Ingvarsson & Taylor, 2002) is also reanalysed. The previous study had shown a marked excess of intraspecific polymorphism in both species. However, when effects of population subdivision were removed, ad hoc, levels of intraspecific polymorphism were no longer significantly different from neutral expectations, suggesting that population subdivision contributed to the observed excess of intraspecific polymorphism seen in both species of Silene.

Insertions, deletions, and inversions in the chloroplast genome of higher plants have been shown to be extremely useful for resolving phylogenetic relationships both between closely related taxa and among more basal lineages. Introns and intergenic spacers from the chloroplast genome are now increasingly used for phylogenetic and population genetic studies of populations from a single species, and it is therefore interesting to know whether indels can provide useful data and hence increase the power of intraspecific studies. Here, we show that indels in three cpDNA intergenic spacers and one cpDNA intron for two species of Silene evolve at slightly higher rates than base pair substitutions. Repeat indels appear to have the highest rate of evolution and are thus more prone to homoplasy. We show that coded indel data have high information content for phylogenetic analysis, and indels thus provide useful information to infer phylogenetic relationships at the intraspecific level.

Segregation distortion is increasingly recognized as a potentially powerful evolutionary force. This runs counter to the perception that non-Mendelian genes are rare genetic curiosities, a view that seems to be supported by the near ubiquity of the Mendelian system of inheritance. There are several reasons why segregation distortion may be more important than is evidenced by known empirical examples. One possibility is that the types of segregation distorters we have found are only a subset of a broader range of non-Mendelian systems, many of which go undetected. In this paper, we review what is known about the sex-linked meiotic drive system in the plant, Silene latifolia, and present some data on the mechanism of segregation distortion. We outline the general features that segregation distorters in plants and animals have in common. In some cases, such as the paucity of systems that directly alter meiotic segregation, there are likely to be inherent constraints on the range of systems that can possibly occur. Other generalities, however, support the notion that many forms of meiotic drive are possible, and that the known examples of segregation distortion are likely to be only subset of those that can possibly occur. Non-Mendelian genes may therefore have greater evolutionary importance than their current abundance in nature would suggest.

Partial self-fertilization is common in higher plants. Mating system variation is known to have important consequences for how genetic variation is distributed within and among populations. Selfing is known to reduce effective population size, and inbreeding species are therefore expected to have lower levels of genetic variation than comparable outcrossing taxa. However, several recent empirical studies have shown that reductions in genetic diversity within populations of inbreeding species are far greater than the expected reductions based on the reduced effective population size. Two different processes have been argued to cause these patterns, selective sweeps (or hitchhiking) and background selection. Both are expected to be most effective in reducing genetic variation in regions of low recombination rates. Selfing is known to reduce the effective recombination rate, and inbreeding taxa are thus thought to be particularly vulnerable to the effects of hitchhiking or background selection. Here I propose a third explanation for the lower-than-expected levels of genetic diversity within populations of selfing species; recurrent extinctions and recolonizations of local populations, also known as metapopulation dynamics. I show that selfing in a metapopulation setting can result in large reductions in genetic diversity within populations, far greater than expected based the lower effective population size inbreeding species is expected to have. The reason for this depends on an interaction between selfing and pollen migration.

The population structure of the mycophagous beetle Phalacrus substriatus is characterized by many small, local populations interconnected by migration over a small spatial scale (10 × 75 m 2 ). Each local P. substriatus population has a relatively short expected persistence time, but persistence of the species occurs due to a balance between frequent local extinctions and recolonizations. This nonequilibrium population structure can have profound effects on how the genetic variation is structured between and within populations. Theoretical models have stated that the genetic differentiation among local populations will be enhanced relative to an island model at equilibrium if the number of colonizers is less than approximately twice the number of migrants among local populations. To study these effects, a set of 50 local P. substriatus populations were surveyed over a four-year period to record any naturally occurring extinctions and recolonizations. The per population colonization and extinction rate were 0.237 and 0.275, respectively. Mark-recapture techniques were used to estimate a number of demographic parameters: local population size (N = 11.1), migration rate (m∘=0.366), number of colonizers (k = 4.0), and the probability of common origin of colonizers (φ = 0.5). The theoretically predicted level of differentiation among local populations (measured as Wright's F ST ) was 0.070. Genetic data obtained from an electrophoretic survey of seven polymorphic loci gave an estimated degree of differentiation of 0.077. There was thus a good agreement between the empirical results and the theoretical predictions. Young populations (θ∘young=0.090) had significantly higher levels of differentiation than old, more established populations (θ∘old=0.059). The extinction-recolonization dynamics resulted in an overall increase in the genetic differentiation among local populations by c. 40%. The global effective population size was also reduced by c. 55%. The results give clear evidence to how nonequilibrium processes shape the genetic structure of populations., (© 1997 The Society for the Study of Evolution.)

I investigated the effects of delayed population growth on the genetic differentiation among populations subjected to local extinction and recolonization, for two different migration functions; (1) a constant migration rate, and (2) a constant number of migrants. A delayed period of population growth reduces the size of the newly founded populations for one or several generations. Whether this increases differentiation among local populations depends on the actual pattern of migration. With a constant migration rate, fewer migrants move into small populations than into large, thus providing ample opportunity for drift to act within a population. A prolonged period of population growth thus makes the conditions for enhanced differentiation between local populations less restrictive and also inflates the actual levels of differentiation. The effect depends on the relative magnitudes of k e , the effective number of colonizers and k, the actual number of colonizers. When there is a constant number of migrants into a population per generation, migration into small populations is increased. This increase of migration in small populations counteracts the effects of genetic drift due to small population size. It increases the rate by which populations approach equilibrium, as small populations are swamped by migrants from larger populations closer to genetic equilibrium, and overall levels of differentiation are thus reduced. I also discuss situations for which the results of this paper are relevant., (© 1997 The Society for the Study of Evolution.)