Targeted, PCR-based gene disruption in cyanobacteria: inactivation of the polyhydroxyalkanoic acid synthase genes in Synechocystis sp. PCC6803

A PCR-based method is described for the efficient construction of targeted gene disruptions and gene fusions in the cyanobacterium Synechocystis sp. PCC6803. In a simple two-step PCR approach, a gene conversion cassette was synthesized targeting the polyhydroxyalkanoic acid (PHA) synthase genes. Upon transformation, PHA production in Synechocystis under normal as well as high production culture conditions was undetectable. The application of this method to the genetic inactivation of the phaE-C _Syn gene cluster demonstrates its potential for genetic engineering of cyanobacteria and the study of functional genomics in Synechocystis. Received: 3 March 2000 / Received revision: 1 June 2000 / Accepted: 9 June 2000     

SNP deserts of Asian cultivated rice: genomic regions under domestication

When performing a genome‐wide comparison between indica (93‐11) and japonica (Nipponbare), we find 8% of the genome, which have an extremely low SNP rate (< 1 SNP/kb). Inside these ‘SNP deserts’, experimentally confirmed genes show increased K_a/K_s that indicate adaptive selection. To further elucidate this connection, we survey the level and pattern of genetic variation in both cultivated and wild rice groups, using 155 noncoding regions located within SNP deserts. The results suggest that cultivated rice has greatly reduced genetic variation within SNP deserts as compared to either the nondesert or corresponding genomic regions in wild rice. Consistent with this reduction in genetic variation, we find a biased distribution of derived allele frequency in the cultivated group, indicative of positive selection. Furthermore, over half of the confirmed, domestication‐related genes are found within SNP deserts, also suggesting that SNP deserts are strongly related to domestication, and might be the key sites in the process of domestication.     

Genetic drift outweighs natural selection at toll‐like receptor (TLR) immunity loci in a re‐introduced population of a threatened species

During population establishment, genetic drift can be the key driver of changes in genetic diversity, particularly while the population is small. However, natural selection can also play a role in shaping diversity at functionally important loci. We used a well‐studied, re‐introduced population of the threatened Stewart Island robin (N = 722 pedigreed individuals) to determine whether selection shaped genetic diversity at innate immunity toll‐like receptor (TLR) genes, over a 9‐year period of population growth following establishment with 12 genetic founders. We found no evidence for selection operating with respect to TLR diversity on first‐year overwinter survival for the majority of loci, genotypes and alleles studied. However, survival of individuals with TLR4_BE genotype was significantly improved: these birds were less than half as likely to die prior to maturity compared with all other TLR4 genotypes. Furthermore, the population frequency of this genotype, at a two‐fold excess over Hardy–Weinberg expectation, was increased by nonrandom mating. Near‐complete sampling and full pedigree and reproductive data enabled us to eliminate other potential causes of these patterns including inbreeding, year effects, density dependence, selection on animals at earlier life history stages or genome‐level association of the TLR4_E allele with ‘good genes’. However, comparison of observed levels of gene diversity to predictions under simulated genetic drift revealed results consistent with neutral expectations for all loci, including TLR4. Although selection favoured TLR4_BE heterozygotes in this population, these effects were insufficient to outweigh genetic drift. This is the first empirical study to show that genetic drift can overwhelm natural selection in a wild population immediately following establishment.     

Peanut Resistance Gene Expression in Response to Aspergillus flavus Infection During Seed Germination

Aspergillus flavus produces potent mutagenic and carcinogenic polyketide‐derived secondary metabolites known as aflatoxins. Development of host plant resistance in peanut and other crops is the most environmentally friendly and cost‐effective method to eliminate the serious problem of aflatoxin contamination in grains. To confirm that putative peanut genes identified in a previous microarray study were involved in peanut resistance to A. flavus infection, 14 genes were selected for further investigation through real‐time PCR. The results revealed diverse patterns of gene expression during seed germination after A. flavus inoculation. Based on the expression levels and the relative‐expression patterns over a 7‐day period, the 14 host genes could be classified into six different groups belonging to three main biochemical and genetic defence processes of lipid metabolism, oxidative signalling and cell‐wall synthesis during counter‐attack. A network of gene expression patterns was activated in sequential order in response to A. flavus invasion in both resistant and susceptible peanut lines during seed germination. Understanding gene expression patterns in peanut will be useful to breeders and other scientists interested in incorporating genetic resources of resistance against A. flavus into peanut germplasm and/or commercial cultivars via conventional and/or molecular methods.     

Non‐endogenous ketocarotenoid accumulation in engineered Synechocystis sp. PCC 6803

The cyanobacterium Synechocystis sp. PCC 6803 is a model species commonly employed for biotechnological applications. It is naturally able to accumulate zeaxanthin (Zea) and echinenone (Ech), but not astaxanthin (Asx), which is the highest value carotenoid produced by microalgae, with a wide range of applications in pharmaceutical, cosmetics, food and feed industries. With the aim of finding an alternative and sustainable biological source for the production of Asx and other valuable hydroxylated and ketolated intermediates, the carotenoid biosynthetic pathway of Synechocystis sp. PCC 6803 has been engineered by introducing the 4,4′ β‐carotene oxygenase (CrtW) and 3,3′ β‐carotene hydroxylase (CrtZ) genes from Brevundimonas sp. SD‐212 under the control of a temperature‐inducible promoter. The expression of exogenous CrtZ led to an increased accumulation of Zea at the expense of Ech, while the expression of exogenous CrtW promoted the production of non‐endogenous canthaxanthin and an increase in the Ech content with a concomitant strong reduction of β‐carotene (β‐car). When both Brevundimonas sp. SD‐212 genes were coexpressed, significant amounts of non‐endogenous Asx were obtained accompanied by a strong decrease in β‐car content. Asx accumulation was higher (approximately 50% of total carotenoids) when CrtZ was cloned upstream of CrtW, but still significant (approximately 30%) when the position of genes was inverted. Therefore, the engineered strains constitute a useful tool for investigating the ketocarotenoid biosynthetic pathway in cyanobacteria and an excellent starting point for further optimisation and industrial exploitation of these organisms for the production of added‐value compounds.     

A simple method for isolation and construction of markerless cyanobacterial mutants defective in acyl-acyl carrier protein synthetase

Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) secrete free fatty acids (FFAs) into the external medium and hence have been used for the studies aimed at photosynthetic production of biofuels. While the wild-type strain of Synechocystis sp. PCC 6803 is highly sensitive to exogenously added linolenic acid, mutants defective in the aas gene are known to be resistant to the externally provided fatty acid. In this study, the wild-type Synechocystis cells were shown to be sensitive to lauric, oleic, and linoleic acids as well, and the resistance to these fatty acids was shown to be enhanced by inactivation of the aas gene. On the basis of these observations, we developed an efficient method to isolate aas-deficient mutants from cultures of Synechocystis cells by counter selection using linoleic acid or linolenic acid as the selective agent. A variety of aas mutations were found in about 70 % of the FFA-resistant mutants thus selected. Various aas mutants were isolated also from Synechococcus sp. PCC 7002, using lauric acid as a selective agent. Selection using FFAs was useful also for construction of markerless aas knockout mutants from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002. Thus, genetic engineering of FFA-producing cyanobacterial strains would be greatly facilitated by the use of the FFAs for counter selection.     

Adaptive molecular evolution of MC1R gene reveals the evidence for positive diversifying selection in indigenous goat populations

Detecting signatures of selection can provide a new insight into the mechanism of contemporary breeding and artificial selection and further reveal the causal genes associated to the phenotypic variation. However, the signatures of selection on genes entailing for profitable traits between Chinese commercial and indigenous goats have been poorly interpreted. We noticed footprints of positive selection at MC 1R gene containing SNP s genotyped in five Chinese native goat breeds. An experimental distribution of F _ST was built based on approximations of F _ST for each SNP across five breeds. We identified selection using the high F _ST outlier method and found that MC 1R candidate gene show evidence of positive selection. Furthermore, adaptive selection pressure on specific codons was determined using different codon based on maximum‐likelihood methods; signature of positive selection in mammalian MC 1R was explored in individual codons. Evolutionary analyses were inferred under maximum likelihood models, the HyPhy package implemented in the DATAMONKEY Web Server. The results of codon selection displayed positive diversifying selection at the sites were mainly involved in development of genetic variations in coat color in various mammalian species. Positive diversifying selection inferred with recent evolutionary changes in domesticated goat MC 1R provides new insights that the gene evolution may have been modulated by domestication events in goats.     

High degree of genetic differentiation in marine three‐spined sticklebacks (Gasterosteus aculeatus)

Populations of widespread marine organisms are typically characterized by a low degree of genetic differentiation in neutral genetic markers, but much less is known about differentiation in genes whose functional roles are associated with specific selection regimes. To uncover possible adaptive population divergence and heterogeneous genomic differentiation in marine three‐spined sticklebacks (Gasterosteus aculeatus), we used a candidate gene‐based genome‐scan approach to analyse variability in 138 microsatellite loci located within/close to (<6 kb) functionally important genes in samples collected from ten geographic locations. The degree of genetic differentiation in markers classified as neutral or under balancing selection—as determined with several outlier detection methods—was low (F_ST = 0.033 or 0.011, respectively), whereas average F_ST for directionally selected markers was significantly higher (F_ST = 0.097). Clustering analyses provided support for genomic and geographic heterogeneity in selection: six genetic clusters were identified based on allele frequency differences in the directionally selected loci, whereas four were identified with the neutral loci. Allelic variation in several loci exhibited significant associations with environmental variables, supporting the conjecture that temperature and salinity, but not optic conditions, are important drivers of adaptive divergence among populations. In general, these results suggest that in spite of the high degree of physical connectivity and gene flow as inferred from neutral marker genes, marine stickleback populations are strongly genetically structured in loci associated with functionally relevant genes.     

A POPULATION GENETIC THEORY OF CANALIZATION

Canalization is the suppression of phenotypic variation. Depending on the causes of phenotypic variation, one speaks either of genetic or environmental canalization. Genetic canalization describes insensitivity of a character to mutations, and the insensitivity to environmental factors is called environmental canalization. Genetic canalization is of interest because it influences the availability of heritable phenotypic variation to natural selection, and is thus potentially important in determining the pattern of phenotypic evolution. In this paper a number of population genetic models are considered of a quantitative character under stabilizing selection. The main purpose of this study is to define the population genetic conditions and constraints for the evolution of canalization. Environmental canalization is modeled as genotype specific environmental variance. It is shown that stabilizing selection favors genes that decrease environmental variance of quantitative characters. However, the theoretical limit of zero environmental variance has never been observed. Of the many ways to explain this fact, two are addressed by our model. It is shown that a “canalization limit” is reached if canalizing effects of mutations are correlated with direct effects on the same character. This canalization limit is predicted to be independent of the strength of stabilizing selection, which is inconsistent with recent experimental data (Sterns et al. 1995). The second model assumes that the canalizing genes have deleterious pleiotropic effects. If these deleterious effects are of the same magnitude as all the other mutations affecting fitness very strong stabilizing selection is required to allow the evolution of environmental canalization. Genetic canalization is modeled as an influence on the average effect of mutations at a locus of other genes. It is found that the selection for genetic canalization critically depends on the amount of genetic variation present in the population. The more genetic variation, the stronger the selection for canalizing effects. All factors that increase genetic variation favor the evolution of genetic canalization (large population size, high mutation rate, large number of genes). If genetic variation is maintained by mutation-selection balance, strong stabilizing selection can inhibit the evolution of genetic canalization. Strong stabilizing selection eliminates genetic variation to a level where selection for canalization does not work anymore. It is predicted that the most important characters (in terms of fitness) are not necessarily the most canalized ones, if they are under very strong stabilizing selection (k > 0.2V_e). The rate of decrease of mutational variance V_m is found to be less than 10% of the initial V_m. From this result it is concluded that characters with typical mutational variances of about 10^–3 V_e are in a metastable state where further evolution of genetic canalization is too slow to be of importance at a microevolutionary time scale. The implications for the explanation of macroevolutionary patterns are discussed.     

Genomewide signatures of selection in Epichloë reveal candidate genes for host specialization

Host specialization is a key process in ecological divergence and speciation of plant‐associated fungi. The underlying determinants of host specialization are generally poorly understood, especially in endophytes, which constitute one of the most abundant components of the plant microbiome. We addressed the genetic basis of host specialization in two sympatric subspecies of grass‐endophytic fungi from the Epichloë typhina complex: subsp. typhina and clarkii. The life cycle of these fungi entails unrestricted dispersal of gametes and sexual reproduction before infection of a new host, implying that the host imposes a selective barrier on viability of the progeny. We aimed to detect genes under divergent selection between subspecies, experiencing restricted gene flow due to adaptation to different hosts. Using pooled whole‐genome sequencing data, we combined F_ST and D_XY population statistics in genome scans and detected 57 outlier genes showing strong differentiation between the two subspecies. Genomewide analyses of nucleotide diversity (π), Tajima's D and dN/dS ratios indicated that these genes have evolved under positive selection. Genes encoding secreted proteins were enriched among the genes showing evidence of positive selection, suggesting that molecular plant–fungus interactions are strong drivers of endophyte divergence. We focused on five genes encoding secreted proteins, which were further sequenced in 28 additional isolates collected across Europe to assess genetic variation in a larger sample size. Signature of positive selection in these isolates and putative identification of pathogenic function supports our findings that these genes represent strong candidates for host specialization determinants in Epichloë endophytes. Our results highlight the role of secreted proteins as key determinants of host specialization.     

Positive selection of IL‐33 in adaptive immunity of domestic Chinese goats

The identification of the candidate genes that play key role in phenotypic variation in livestock populations can provide new information about evolution and positive selection. IL‐33 (71954) (Interleukin) gene is associated with the increased nematode resistance in small ruminants; however, the role of IL‐33 for the genetic control of different diseases in Chinese goat breeds is poorly described in scientific literature. Therefore, the current investigation was performed for the better understanding of the molecular evolution and the positive selection of single‐nucleotide polymorphism in IL‐33 gene. Fixation Index (F_ST)‐based method was used for the outlier loci determination and found that IL‐33 was present in outlier area with the provisional combined allocation of mean heterozygosity and F_ST. Positively selected IL‐33 gene was significantly, that is, p(Simul F_ST < sample F_ST = 0.98*) present in corresponding positive selection area. Hence, our study provided novel information about the nucleotide variations in IL‐33 gene and found to be nonsynonymous which may helpful for the genetic control of diseases by enhancing the immune system in local Chinese goat breeds as well as in other analyzed vertebrate species.     

Climatic adaptation and ecological divergence between two closely related pine species in Southeast China

Climate is one of the most important drivers for adaptive evolution in forest trees. Climatic selection contributes greatly to local adaptation and intraspecific differentiation, but this kind of selection could also have promoted interspecific divergence through ecological speciation. To test this hypothesis, we examined intra‐ and interspecific genetic variation at 25 climate‐related candidate genes and 12 reference loci in two closely related pine species, Pinus massoniana Lamb. and Pinus hwangshanensis Hisa, using population genetic and landscape genetic approaches. These two species occur in Southeast China but have contrasting ecological preferences in terms of several environmental variables, notably altitude, although hybrids form where their distributions overlap. One or more robust tests detected signals of recent and/or ancient selection at two‐thirds (17) of the 25 candidate genes, at varying evolutionary timescales, but only three of the 12 reference loci. The signals of recent selection were species specific, but signals of ancient selection were mostly shared by the two species likely because of the shared evolutionary history. F_ST outlier analysis identified six SNPs in five climate‐related candidate genes under divergent selection between the two species. In addition, a total of 24 candidate SNPs representing nine candidate genes showed significant correlation with altitudinal divergence in the two species based on the covariance matrix of population history derived from reference SNPs. Genetic differentiation between these two species was higher at the candidate genes than at the reference loci. Moreover, analysis using the isolation‐with‐migration model indicated that gene flow between the species has been more restricted for climate‐related candidate genes than the reference loci, in both directions. Taken together, our results suggest that species‐specific and divergent climatic selection at the candidate genes might have counteracted interspecific gene flow and played a key role in the ecological divergence of these two closely related pine species.     

Genome‐wide single‐generation signatures of local selection in the panmictic European eel

Next‐generation sequencing and the collection of genome‐wide data allow identifying adaptive variation and footprints of directional selection. Using a large SNP data set from 259 RAD‐sequenced European eel individuals (glass eels) from eight locations between 34 and 64^oN, we examined the patterns of genome‐wide genetic diversity across locations. We tested for local selection by searching for increased population differentiation using F_ST‐based outlier tests and by testing for significant associations between allele frequencies and environmental variables. The overall low genetic differentiation found (F_ST = 0.0007) indicates that most of the genome is homogenized by gene flow, providing further evidence for genomic panmixia in the European eel. The lack of genetic substructuring was consistent at both nuclear and mitochondrial SNPs. Using an extensive number of diagnostic SNPs, results showed a low occurrence of hybrids between European and American eel, mainly limited to Iceland (5.9%), although individuals with signatures of introgression several generations back in time were found in mainland Europe. Despite panmixia, a small set of SNPs showed high genetic differentiation consistent with single‐generation signatures of spatially varying selection acting on glass eels. After screening 50 354 SNPs, a total of 754 potentially locally selected SNPs were identified. Candidate genes for local selection constituted a wide array of functions, including calcium signalling, neuroactive ligand–receptor interaction and circadian rhythm. Remarkably, one of the candidate genes identified is PERIOD, possibly related to differences in local photoperiod associated with the >30° difference in latitude between locations. Genes under selection were spread across the genome, and there were no large regions of increased differentiation as expected when selection occurs within just a single generation due to panmixia. This supports the conclusion that most of the genome is homogenized by gene flow that removes any effects of diversifying selection from each new generation.     

Genome scan in the mosquito Aedes rusticus: population structure and detection of positive selection after insecticide treatment

Identification of genes involved in local adaptation is particularly challenging for species functioning as a network of interconnected populations undergoing frequent extinctions–recolonizations, because populations are submitted to contrasted evolutionary pressures. Using amplified fragment length polymorphism markers, population genetic structure of the mosquito Aedes rusticus was analysed in five geographical areas of the French Rhône‐Alpes region. We included a number of sites that were treated with the bio‐insecticide Bacillus thuringiensis israelensis (Bti) for more than 15 years. Analysis of molecular variance revealed that most of the genetic variability was found within populations (96%), with no significant variation among geographical areas, although variation among populations within areas (4%) was significant. The global genetic differentiation index F_ST was low (0.0366 ± 0.167). However, pairwise F_ST values were significant and no isolation‐by‐distance at the regional level was observed, suggesting a metapopulation structure in this species. Bti‐treatment had no effect on genetic structure and on within‐population genetic diversity. Potential signatures of positive selection associated with Bti‐treatment were detected for five loci, even though toxicological bioassays performed on field‐collected larvae showed no significant difference in mortality between Bti‐treated and nontreated sites. The difficulty of detecting moderate resistance in field‐collected larvae together with possible differential persistence of toxins in the environment may explain our inability to detect a toxicological response to Bti in treated sites. The evidence for positive selection occurring at several genomic regions suggests a first step towards Bti resistance in natural mosquito populations treated with this bio‐insecticide. Furthermore, this signal was detectable using genomic tools before any toxicological evidence for resistance could be identified.     

Functional analysis of the two homologous psbA gene copies in Synechocystis PCC 6714 and PCC 6803

The cyanobacteria Synechocystis 6803 and 6714 contain three genes (psbA) coding for the D₁ protein. This protein is an essential subunit of photosystem II (PSII) and is the target for herbicides. We have used herbicide-resistant mutants to study the role of the two homologous copies of the psbA genes in both strains (the third copy is not expressed). Several herbicide resistance mutations map within the psbAI gene in Synechocystis 6714 (G. Ajlani et al.), Plant Mol. Biol. 13 (1989): (469–479). We have looked for mutations in copy II. Results show that in Synechocystis 6714, only psbAI contains herbicide resistance mutations. Relative expression of psbAI and psbAII has been measured by analysing the proportions of resistant and sensitive D₁ in the thylakoid membranes of the mutants. In normal growth conditions, 95% resistant D₁ and 5% sensitive D₁ were found. In high light conditions, expression of psbAII was enhanced, producing 15% sensitive D₁. This enhancement is specifically due to high light and not to the decrease of D₁ concentration caused by photoinhibition. Copy I of Synechocystis 6714 corresponds to copy 2 of Synechocystis 6803 since it was always psbA2 which was recombined in Synechocystis 6803 transformants. PSII of the transformant strains was found to be 95% resistant to herbicides as in resistant mutants of Synechocystis 6714.     

On the heritability of blue‐green eggshell coloration

Abstract Avian blue‐green eggshell coloration has been proposed as a female signal of genetic or phenotypic quality to males. However, little is known about the relative importance of additive genetic and environmental effects as sources of eggshell colour variation in natural populations. Using 5 years of data and animal models, we explored these effects in a free‐living population of pied flycatchers. Permanent environmental and year effects were negligible, although year environmental variance (V_Year) was significant for all but one of the traits. However, we found high–moderate narrow‐sense heritabilities for some colour parameters. Within‐clutch colour variability showed the highest coefficient of additive genetic variation (i.e. evolvability). Previous evidence suggests that eggshell colour is sexually selected in this species, males enhancing parental effort in clutches with higher colour variability and peak values. Eggshell colour could be driven by good‐genes selection in pied flycatchers although further genetic studies should confirm this possibility.     

Genome‐wide scans to detect positive selection in Large White and Tongcheng pigs

Due to the direction, intensity, duration and consistency of genetic selection, especially recent artificial selection, the production performance of domestic pigs has been greatly changed. Therefore, we reasoned that there must be footprints or selection signatures that had been left during domestication. In this study, with porcine 60K BeadChip genotyping data from both commercial Large White and local Chinese Tongcheng pigs, we calculated the extended haplotype homozygosity values of the two breeds using the long‐range haplotype method to detect selection signatures. We found 34 candidate regions, including 61 known genes, from Large White pigs and 25 regions comprising 57 known genes from Tongcheng pigs. Many selection signatures were found on SSC1, SSC4, SSC7 and SSC14 regions in both populations. According to quantitative trait loci and network pathway analyses, most of the regions and genes were linked to growth, reproduction and immune responses. In addition, the average genetic differentiation coefficient F_ST was 0.254, which means that there had already been a significant differentiation between the breeds. The findings from this study can contribute to further research on molecular mechanisms of pig evolution and domestication and also provide valuable references for improvement of their breeding and cultivation.     

A haplotype method detects diverse scenarios of local adaptation from genomic sequence variation

Identifying genomic targets of population‐specific positive selection is a major goal in several areas of basic and applied biology. However, it is unclear how often such selection should act on new mutations versus standing genetic variation or recurrent mutation, and furthermore, favoured alleles may either become fixed or remain variable in the population. Very few population genetic statistics are sensitive to all of these modes of selection. Here, we introduce and evaluate the Comparative Haplotype Identity statistic (χ_MD), which assesses whether pairwise haplotype sharing at a locus in one population is unusually large compared with another population, relative to genomewide trends. Using simulations that emulate human and Drosophila genetic variation, we find that χ_MD is sensitive to a wide range of selection scenarios, and for some very challenging cases (e.g. partial soft sweeps), it outperforms other two‐population statistics. We also find that, as with F_ST, our haplotype approach has the ability to detect surprisingly ancient selective sweeps. Particularly for the scenarios resembling human variation, we find that χ_MD outperforms other frequency‐ and haplotype‐based statistics for soft and/or partial selective sweeps. Applying χ_MD and other between‐population statistics to published population genomic data from D. melanogaster, we find both shared and unique genes and functional categories identified by each statistic. The broad utility and computational simplicity of χ_MD will make it an especially valuable tool in the search for genes targeted by local adaptation.     

Rapid genome‐wide evolution in Brassica rapa populations following drought revealed by sequencing of ancestral and descendant gene pools

There is increasing evidence that evolution can occur rapidly in response to selection. Recent advances in sequencing suggest the possibility of documenting genetic changes as they occur in populations, thus uncovering the genetic basis of evolution, particularly if samples are available from both before and after selection. Here, we had a unique opportunity to directly assess genetic changes in natural populations following an evolutionary response to a fluctuation in climate. We analysed genome‐wide differences between ancestors and descendants of natural populations of Brassica rapa plants from two locations that rapidly evolved changes in multiple phenotypic traits, including flowering time, following a multiyear late‐season drought in California. These ancestor‐descendant comparisons revealed evolutionary shifts in allele frequencies in many genes. Some genes showing evolutionary shifts have functions related to drought stress and flowering time, consistent with an adaptive response to selection. Loci differentiated between ancestors and descendants (F_ST outliers) were generally different from those showing signatures of selection based on site frequency spectrum analysis (Tajima's D), indicating that the loci that evolved in response to the recent drought and those under historical selection were generally distinct. Very few genes showed similar evolutionary responses between two geographically distinct populations, suggesting independent genetic trajectories of evolution yielding parallel phenotypic changes. The results show that selection can result in rapid genome‐wide evolutionary shifts in allele frequencies in natural populations, and highlight the usefulness of combining resurrection experiments in natural populations with genomics for studying the genetic basis of adaptive evolution.     

Disentangling the effects of recombination,selection, and demography on the genetic variation at a major histocompatibility complex class II gene in the alpine chamois

The major histocompatibility complex (MHC) harbours some of the most polymorphic loci in vertebrate genomes. MHC genes are thought to be subject to some form of balancing selection, most likely pathogen‐mediated selection. Hence, MHC genes are excellent candidates for exploring adaptive processes. In this study, we investigated the genetic variation at exon 2 of the DRB class II MHC locus in 191 alpine chamois (Rupicapra rupicapra) from 10 populations in the eastern Alps of Italy. In particular, we were interested in distinguishing and estimating the relative impact of selective and demographic factors, while taking into account the confounding effect of recombination. The extremely high d_n/d_s ratio and the presence of trans‐species polymorphisms suggest that a strong long‐term balancing selection effect has been operating at this locus throughout the evolutionary history of this species. We analysed patterns of genetic variation within and between populations, and the mitochondrial D‐loop polymorphism patterns were analysed to provide a baseline indicator of the effects of demographic processes. These analyses showed that (i) the chamois experienced a demographic decline in the last 5000–30 000 years, most likely related to the postglacial elevation in temperature; (ii) this demographic process can explain the results of neutrality tests applied to MHC variation within populations, but cannot justify the much weaker divergence between populations implied by MHC as opposed to mitochondrial DNA; (iii) similar sets of divergent alleles are probably maintained with similar frequencies by balancing selection in different populations, and this mechanism is also operating in small isolated populations, which are strongly affected by drift.