Adaptive and Slightly Deleterious Evolution in a Conifer, <Emphasis Type="Italic">Cryptomeria japonica</Emphasis>

In order to evaluate effects of the population structure and natural selection on organisms having long generation times, we surveyed DNA polymorphisms at five loci encoding 9-cis-epoxycarotenoid dioxygenase (NCED), ammonium transporter, calmodulin, aquaporin, and the second major allergen with polymethylgalacturonase enzyme activity in the pollen (Cryj2) in a conifer, Cryptomeria japonica. The average nucleotide diversity at silent sites across 12 loci including the previously analyzed seven loci was 0.0044. The population recombination rate (4Nr, where N and r are the effective population size and recombination rate per base per generation, respectively) was estimated as 0.00046 and a slow reduction in the population size was indicated, according to the maximum likelihood method implemented in LAMARC. At NCED, the McDonald-Kreitman (MK) test revealed an excess of replacement polymorphisms, suggesting contributions of slightly deleterious mutations. In contrast, the MK test revealed an excess of replacement divergence at Cryj2 and a maximum likelihood approach using the PAML package revealed that certain amino acid sites had a nonsynonymous/synonymous substitution rate ratio (omega) > 4.0, indicating adaptive evolution at this locus. The overall analysis of the 12 loci suggested that adaptive, neutral, and slightly deleterious evolution played important roles in the evolution of C. japonica.     

DNA variability and recombination rates at X-linked loci in humans.

We sequenced 11,365 bp from introns of seven X-linked genes in 10 humans, one chimpanzee, and one orangutan to (i) provide an average estimate of nucleotide diversity (pi) in humans, (ii) investigate whether there is variation in pi among loci, (iii) compare ratios of polymorphism to divergence among loci, and (iv) provide a preliminary test of the hypothesis that heterozygosity is positively correlated with the local rate of recombination. The average value for pi was low 0.063%, SE = 0.036%, about one order of magnitude smaller than for Drosophila melanogaster, the species for which the best data are available. Among loci, pi varied by over one order of magnitude. Statistical tests of neutrality based on ratios of polymorphism to divergence or based on the frequency spectrum of variation within humans failed to reject a neutral, equilibrium model. However, there was a positive correlation between heterozygosity and rate of recombination, suggesting that the joint effects of selection and linkage are important in shaping patterns of nucleotide variation in humans.     

Multilocus patterns of polymorphism and selection across the X chromosome of Caenorhabditis remanei

Natural selection and neutral processes such as demography, mutation, and gene conversion all contribute to patterns of polymorphism within genomes. Identifying the relative importance of these varied components in evolution provides the principal challenge for population genetics. To address this issue in the nematode Caenorhabditis remanei, I sampled nucleotide polymorphism at 40 loci across the X chromosome. The site-frequency spectrum for these loci provides no evidence for population size change, and one locus presents a candidate for linkage to a target of balancing selection. Selection for codon usage bias leads to the non-neutrality of synonymous sites, and despite its weak magnitude of effect (N(e)s approximately 0.1), is responsible for profound patterns of diversity and divergence in the C. remanei genome. Although gene conversion is evident for many loci, biased gene conversion is not identified as a significant evolutionary process in this sample. No consistent association is observed between synonymous-site diversity and linkage-disequilibrium-based estimators of the population recombination parameter, despite theoretical predictions about background selection or widespread genetic hitchhiking, but genetic map-based estimates of recombination are needed to rigorously test for a diversity-recombination relationship. Coalescent simulations also illustrate how a spurious correlation between diversity and linkage-disequilibrium-based estimators of recombination can occur, due in part to the presence of unbiased gene conversion. These results illustrate the influence that subtle natural selection can exert on polymorphism and divergence, in the form of codon usage bias, and demonstrate the potential of C. remanei for detecting natural selection from genomic scans of polymorphism.     

Multilocus patterns of nucleotide polymorphism and demographic change in Taxodium distichum (Cupressaceae) in the lower Mississippi River alluvial valley

? Premise of the Study: Studies of the geographic patterns of genetic variation can give important insights into the past population structure of species. Our study species, Taxodium distichum L. (bald-cypress), prefers riparian and wetland habitats and is widely distributed in southeastern North America and Mexico. We compared the genetic variation of T. distichum with that of its close relative, Cryptomeria japonica, which is endemic to Japan. ? Methods: Nucleotide polymorphisms of T. distichum in the lower Mississippi River alluvial valley, USA, were examined at 10 nuclear loci. ? Key Results: The average nucleotide diversity at silent sites, π(sil), across the 10 loci in T. distichum was higher than that of C. japonica (π(sil) = 0.00732 and 0.00322, respectively). In T. distichum, Tajima's D values were each negative at 9 out of 10 loci, which suggests a recent population expansion. Maximum-likelihood and Bayesian estimations of the exponential population growth rate (g) of T. distichum populations indicated that this species had expanded approximately at the rate of 1.7-1.0 × 10(-6) per year in the past. ? Conclusions: Taxodium distichum had significantly higher nucleotide variation than C. japonica, and its patterns of polymorphism contrasted strikingly with those of the latter, which previously has been inferred to have experienced a reduction in population size.     

The relationship of nucleotide polymorphism, recombination rate and selection in wild tomato species

We analyzed the effects of mating system and recombination rate on single nucleotide polymorphisms using 14 single-copy nuclear loci from single populations of five species of wild tomatoes (Solanum section Lycopersicon). The taxa investigated comprise two self-compatible (SC) and three self-incompatible (SI) species. The observed reduction in nucleotide diversity in the SC populations compared to the SI populations is much stronger than expected under the neutral effects of the mating system on effective population size. Importantly, outgroup sequences available for 11 of the 14 loci yield strong positive correlations between silent nucleotide diversity and silent divergence, indicative of marked among-locus differences in mutation rates and/or selective constraints. Furthermore, using a physical estimate of local recombination rates, we find that silent nucleotide diversity (but not divergence) is positively correlated with recombination rate in two of the SI species. However, this correlation is not nearly as strong as in other well-characterized species (in particular, Drosophila). We propose that nucleotide diversity in Lycopersicon is dominated mainly by differences in neutral mutation rates and/or selective constraints among loci, demographic processes (such as population subdivision), and background selection. In addition, we hypothesize that the soil seed bank plays an important role in the maintenance of the large genetic diversity in the SI species (in particular L. peruvianum).     

Evidence for positive selection at the pantophysin (Pan I) locus in walleye pollock, Theragra chalcogramma

Nucleotide polymorphism at the pantophysin (Pan I) locus in walleye pollock, Theragra chalcogramma, was examined using DNA sequence data. Two distinct allelic lineages were detected in pollock, resulting from three amino acid replacement mutations in the first intravesicular domain of the protein. The common Pan I allelic group, comprising 94% of the samples, was less polymorphic (pi = 0.005) than the uncommon group (pi = 0.008), and nucleotide diversity in both was higher than for two allelic lineages in the related Atlantic cod, Gadus morhua. Phylogenetic analyses of Pan I sequences from these two species did not clearly resolve orthology among allelic groups, in part because of recombination that has occurred between the two pollock lineages. Conventional tests of neutrality comparing polymorphisms within and between homologous regions of the Pan I locus in walleye pollock and Atlantic cod did not detect the effects of selection. This result is likely attributed to low levels of synonymous divergence among allelic lineages and a lack of mutation-drift equilibrium inferred from nucleotide mismatch frequency distributions. However, the ratio of nonsynonymous to synonymous substitutions per site (dN/dS) exceeded unity in two intravesicular domains of the protein and the influence of positive selection at multiple codon sites was strongly inferred through the use of maximum-likelihood analyses. In addition, the frequency spectrum of linked neutral variation showed indirect effects of adaptive hitchhiking in pollock resulting from a selective sweep of the common allelic lineage. Recombination between the two allelic classes may have prevented complete loss of the older, more polymorphic lineage. The results suggest that recurrent sweeps driven by positive selection is the principle mode of evolution at the Pan I locus in gadid fishes.     

Nucleotide variation at the runt locus in Drosophila melanogaster and Drosophila simulans.

Intra- and interspecific nucleotide variation for the major developmental gene runt in Drosophila was studied in D. melanogaster and D. simulans. The 1.5-kb protein-coding region and the 0.4-kb intron of the runt gene were sequenced for 11 alleles in each species. The D. melanogaster alleles originated from east Africa. Estimated parameters of intraspecific variation in D. melanogaster (exons: theta = 0.018, pi = 0.018; intron: theta = 0.014, pi = 0.014) and D. simulans (exons: theta = 0.007, pi = 0.005; intron: theta = 0.008, pi = 0.005) were below average for other X-linked genes, while divergence between species (exons: D = 0.094; intron: D = 0.069) fell within the normal range for both silent and replacement changes. This estimate for runt, along with published values for three other genes in regions of normal recombination, show east African D. melanogaster to be roughly twice as polymorphic as D. simulans. The majority of nucleotide variation, silent and replacement, in both species was found to be selectively neutral using various statistical tests (HKA, McDonald-Kreitman, Tajima, and Fu and Li tests). Monte Carlo simulations of the coalescent process significantly rejected a Wright-Fisher model with respect to an amino acid polymorphism and the distribution of polymorphic sites among the D. simulans lines. This indicated an old lineage and may reflect ancestral population substructuring in D. simulans.     

Nucleotide Variation at the Gpdh Locus in the Genus Drosophila

The Gpdh locus was sequenced in a broad range of Drosophila species. In contrast to the extreme evolutionary constraint seen at the amino acid level, the synonymous sites evolve at rates comparable to those of other genes. Gpdh nucleotide sequences were used to infer a phylogenetic tree, and the relationships among the species of the obscura group were examined in detail. A survey of nucleotide polymorphism within D. pseudoobscura revealed no amino acid variation in this species. Applying a modified McDonald-Kreitman test, the amino acid divergence between species in the obscura group does not appear to be excessive, implying that drift is adequate to explain the patterns of amino acid change at this locus. In addition, the level of polymorphism at the Gpdh locus in D. pseudoobscura is comparable to that found at other loci, as determined by a Hudson-Kreitman-Aguade test. Thus, the pattern of nucleotide variation within and between species at the Gpdh locus is consistent with a neutral model.     

Low nucleotide diversity at the pal1 locus in the widely distributed Pinus sylvestris.

Nucleotide polymorphism in Scots pine (Pinus sylvestris) was studied in the gene encoding phenylalanine ammonia-lyase (Pal, EC 4.3.1.5). Scots pine, like many other pine species, has a large current population size. The observed levels of inbreeding depression suggest that Scots pine may have a high mutation rate to deleterious alleles. Many Scots pine markers such as isozymes, RFLPs, and microsatellites are highly variable. These observations suggest that the levels of nucleotide variation should be higher than those in other plant species. A 2,045-bp fragment of the pal1 locus was sequenced from five megagametophytes each from a different individual from each of four populations, from northern and southern Finland, central Russia, and northern Spain. There were 12 segregating sites in the locus. The synonymous site overall nucleotide diversity was only 0.0049. In order to compare pal1 with other pine genes, sequence was obtained from two alleles of 11 other loci (total length 4,606 bp). For these, the synonymous nucleotide diversity was 0.0056. These estimates are lower than those from other plants. This is most likely because of a low mutation rate, as estimated from between-pine species synonymous site divergence. In other respects, Scots pine has the characteristics of a species with a large effective population. There was no linkage disequilibrium even between closely linked sites. This resulted in high haplotype diversity (14 different haplotypes among 20 sequences). This could also give rise to high per locus diversity at the protein level. Divergence between populations in the main range was low, whereas an isolated Spanish population had slightly lower diversity and higher divergence than the remaining populations.     

Using multilocus sequence data to assess population structure, natural selection, and linkage disequilibrium in wild tomatoes

We employed a multilocus approach to examine the effects of population subdivision and natural selection on DNA polymorphism in 2 closely related wild tomato species (Solanum peruvianum and Solanum chilense), using sequence data for 8 nuclear loci from populations across much of the species' range. Both species exhibit substantial levels of nucleotide variation. The species-wide level of silent nucleotide diversity is 18% higher in S. peruvianum (pi(sil) approximately 2.50%) than in S. chilense (pi(sil) approximately 2.12%). One of the loci deviates from neutral expectations, showing a clinal pattern of nucleotide diversity and haplotype structure in S. chilense. This geographic pattern of variation is suggestive of an incomplete (ongoing) selective sweep, but neutral explanations cannot be entirely dismissed. Both wild tomato species exhibit moderate levels of population differentiation (average F(ST) approximately 0.20). Interestingly, the pooled samples (across different demes) exhibit more negative Tajima's D and Fu and Li's D values; this marked excess of low-frequency polymorphism can only be explained by population (or range) expansion and is unlikely to be due to population structure per se. We thus propose that population structure and population/range expansion are among the most important evolutionary forces shaping patterns of nucleotide diversity within and among demes in these wild tomatoes. Patterns of population differentiation may also be impacted by soil seed banks and historical associations mediated by climatic cycles. Intragenic linkage disequilibrium (LD) decays very rapidly with physical distance, suggesting high recombination rates and effective population sizes in both species. The rapid decline of LD seems very promising for future association studies with the purpose of mapping functional variation in wild tomatoes.     

Nucleotide Variation and Conservation at the Dpp Locus, a Gene Controlling Early Development in Drosophila

A study of polymorphism and species divergence of the dpp gene of Drosophila has been made. Eighteen lines from a population of D. melanogaster were sequenced for 5200 bp of the Hin region of the gene, coding for the dpp polypeptide. A comparison was made with sequence from D. simulans. Ninety-six silent polymorphisms and three amino acid replacement polymorphisms were found. The overall silent polymorphism (0.0247) is low, but haplotype diversity (0.0066 for effectively silent sites and 0.0054 for all sites) is in the range found for enzyme loci. Amino acid variation is absent in the N-terminal signal peptide, the C-terminal TGF-β peptide and in the N-terminal half of the pro-protein region. At the nucleotide level there is strong conservation in the middle half of the large intron and in the 3' untranslated sequence of the last exon. The 3' untranslated conservation, which is perfect for 110 bp among all the divergent species, is unexplained. There is strong positive linkage disequilibrium among polymorphic sites, with stretches of apparent gene conversion among originally divergent sequences. The population apparently is a migration mixture of divergent clades.     

Nucleotide diversity and linkage disequilibrium in wild avocado (Persea americana Mill.)

Resequencing studies provide the ultimate resolution of genetic diversity because they identify all mutations in a gene that are present within the sampled individuals. We report a resequencing study of Persea americana, a subtropical tree species native to Meso- and Central America and the progenitor of cultivated avocado. The sample includes 21 wild accessions from Mexico, Costa Rica, Ecuador, and the Dominican Republic. Estimated levels of nucleotide polymorphism and linkage disequilibrium (LD) are obtained from fully resolved haplotype data from 4 nuclear loci that span 5960 nucleotide sites. Results show that, although avocado is a subtropical tree crop and a predominantly outcrossing plant, the overall level of genetic variation is not exceptionally high (nucleotide diversity at silent sites, pi(sil) = 0.0102) compared with available estimates from temperate plant species. Intralocus LD decays rapidly to half the initial value within about 1 kb. Estimates of recombination rate (based on the sequence data) show that the rate is not exceptionally high when compared with annual plants such as wild barley or maize. Interlocus LD is significant owing to substantial population structure induced by mixing of the 3 botanical races of avocado.     

Multiple polymorphic sites at the ITS and ATAN loci in cultured isolates of Perkinsus marinus

Sequence analysis of genomic DNA from the protozoan parasite Perkinsus marinus at two loci revealed genetic polymorphisms within and among different cultured isolates. Genomic DNA from 12 Perkinsus marinus isolates was amplified at the internal transcribed spacer region and at an anonymous locus previously identified to contain polymorphisms by restriction fragment length polymorphism analysis. Fourteen polymorphic nucleotide positions were identified at the internal transcribed spacer region; eight in internal transcribed spacer 1 and six in internal transcribed spacer 2. Thirteen polymorphic nucleotide sites were identified within the anonymous locus. In some instances, more than three different sequences were observed at both the internal transcribed spacer region and at the anonymous locus from a single clonal isolate, suggesting the possibility of recombination in cultured cells and/or strand jumping during the polymerase chain reaction. Intra-isolate sequence variation (3.46% for the anonymous locus and 3.08% for internal transcribed spacer 1) was in several cases as high as inter-isolate sequence variation, even in one isolate where recombination was not evident. High intra- and inter-isolate variation detected at both loci demonstrates the importance of determining the genetic variation of each locus prior to development of sequence-based molecular diagnostics.     

Testing for effects of recombination rate on nucleotide diversity in natural populations of Arabidopsis lyrata

We investigated DNA sequence diversity for loci on chromosomes 1 and 2 in six natural populations of Arabidopsis lyrata and tested for the role of natural selection in structuring genomewide patterns of variability, specifically examining the effects of recombination rate on levels of silent polymorphism. In contrast with theoretical predictions from models of genetic hitchhiking, maximum-likelihood-based analyses of diversity and divergence do not suggest reduction of diversity in the region of suppressed recombination near the centromere of chromosome 1, except in a single population from Russia, in which the pericentromeric region may have undergone a local selective sweep or demographic process that reduced variability. We discuss various possibilities that might explain why nucleotide diversity in most A. lyrata populations is not related to recombination rate, including genic recombination hotspots, and low gene density in the low recombination rate region.     

DNA variation at the rp49 gene region of Drosophila simulans: evolutionary inferences from an unusual haplotype structure.

An approximately 1.3-kb region including the rp49 gene plus its 5' and 3' flanking regions was sequenced in 24 lines of Drosophila simulans (10 from Spain and 14 from Mozambique). Fifty-four nucleotide and 8 length polymorphisms were detected. All nucleotide polymorphisms were silent: 52 in noncoding regions and 2 at synonymous sites in the coding region. Estimated silent nucleotide diversity was similar in both populations (pi = 0.016, for the total sample). Nucleotide variation revealed an unusual haplotype structure showing a subset of 11 sequences with a single polymorphism. This haplotype was present at intermediate frequencies in both the European and the African samples. The presence of such a major haplotype in a highly recombining region is incompatible with the neutral equilibrium model. This haplotype structure in both a derived and a putatively ancestral population can be most parsimoniously explained by positive selection. As the rate of recombination in the rp49 region is high, the target of selection should be close to or within the region studied.     

Historical Selection, Amino Acid Polymorphism and Lineage-Specific Divergence at the G6pd Locus in Drosophila Melanogaster and D. Simulans

The nucleotide diversity across 1705 bp of the G6pd gene is studied in 50 Drosophila melanogaster and 12 D. simulans lines. Our earlier report contrasted intraspecific polymorphism and interspecific differences at silent and replacement sites in these species. This report expands the number of European and African lines and examines the pattern of polymorphism with respect to the common A/B allozymes. In D. melanogaster the silent nucleotide diversity varies 2.8-fold across localities. The B allele sequences are two- to fourfold more variable than the derived A allele, and differences between allozymes are twice as among B alleles. There is strong linkage disequilibrium across the G6pd region. In both species the level of silent polymorphism increases from the 5' to 3' ends, while there is no comparable pattern in level of silent site divergence or fixation. The neutral model is not rejected in either species. Using D. yakuba as an outgroup, the D. melanogaster lineage shows a twofold greater rate of silent fixation, but less than half the rate of amino acid replacement. Lineage-specific differences in mutation fixation are inconsistent with neutral expectations and suggest the interaction of species-specific population size differences with both weakly advantageous and deleterious selection.     

Genetic variation versus recombination rate in a structured population of mice

The correlation between genetic variation and recombination rate was investigated in a structured mouse population. Nucleotide sequence data from 19 autosomal DNA loci from eight inbred strains of mouse (Mus musculus) sampled from three major subspecies were analyzed. The recombination rate was estimated from the comparison of genetic and physical map distances between markers flanking a 10-cM region of each locus. The strains were categorized into four groups (subpopulations) based on geography. By partitioning the genetic diversity into within-group and among-group variation, we detected a positive correlation between the recombination rate and nucleotide diversity within groups. The level of nucleotide differentiation among groups (G(ST)) showed a negative correlation with the rate of recombination. There was no significant correlation between recombination rate and nucleotide diversity when data from different subpopulations were pooled. No correlation was detected between recombination rate and nucleotide divergence of M. musculus and M. spicilegus. These patterns deviate from the strict neutral expectation under the constant nucleotide substitution rate, and they are likely to have been formed either by a hitchhiking effect of positively selected mutants or by background selection of deleterious mutants occurring in a subdivided population. Our series of comparisons show that because a real population always has some structure, incorporation of its information is important in detecting non-neutral evolution.     

Comparison of DNA and protein polymorphisms between humans and chimpanzees

To examine the nucleotide diversity at silent (synonymous + intron + untranslated) and non-silent (nonsynonymous) sites in chimpanzees and humans, genes at six nuclear loci from two chimpanzees were sequenced. The average silent diversity was 0.19%, which was significantly higher than that in humans (0.05%). This observation suggests a significantly larger effective population size and a higher extent of neutral polymorphism in chimpanzees than in humans. On the other hand, the non-silent nucleotide diversity is similar in both species, resulting in a larger fraction of neutral mutations at non-silent sites in humans than in chimpanzees. Other types of polymorphism data were collected from the literature or databases to examine whether or not they are consistent with the nuclear DNA sequence polymorphism observed here. The nucleotide diversity at both silent and non-silent sites in mitochondrial (mt) DNA genes was compatible with that of the nuclear genes. Microsatellite loci showed a similar high extent of heterozygosity in both species, perhaps due to the combined effect of a high mutation rate and a recent population expansion in humans. At protein loci, humans are more heterozygous than chimpanzees, and the estimated fraction of neutral alleles in humans (0.84) is much larger than that in chimpanzees (0.26). These data show that the neutral fraction in non-silent changes is relatively large in the human population. This difference may be due to a relaxation of the functional constraint against proteins in the human lineage. To evaluate this possibility, it will be necessary to examine nucleotide sequences in relation to the physiological or biochemical properties of proteins.     

DNA polymorphism, haplotype structure and balancing selection in the Leavenworthia PgiC locus.

A study of DNA polymorphism and divergence was conducted for the cytosolic phosphoglucose isomerase (PGI:E.C.5.3.1.9) gene of five species of the mustard genus Leavenworthia: Leavenworthia stylosa, L. alabamica, L. crassa, L. uniflora, and L. torulosa. Sequences of an internal 2.3-kb PgiC gene region spanning exons 6-16 were obtained from 14 L. stylosa plants from two natural populations and from one to several plants for each of the other species. The level of nucleotide polymorphism in L. stylosa PgiC gene was quite high (pi = 0.051, theta = 0.052). Although recombination is estimated to be high in this locus, extensive haplotype structure was observed for the entire 2.3-kb region. The L. stylosa sequences fall into at least two groups, distinguished by the presence of several indels and nucleotide substitutions, and one of the three charge change nucleotide replacements within the region sequenced correlates with the haplotypes. The differences between the haplotypes are older than between the species, and the haplotypes are still segregating in at least two of five species studied. There is no evidence of recent or ancient population subdivision that could maintain distinct haplotypes. The age of the haplotypes and the results of Kelly's Z(nS) and Wall's B and Q tests with recombination suggest that the haplotypes are maintained due to balancing selection at or near this locus.     

Contrasting evolutionary histories of two introns of the duchenne muscular dystrophy gene, Dmd, in humans

The Duchenne muscular dystrophy (Dmd) locus lies in a region of the X chromosome that experiences a high rate of recombination and is thus expected to be relatively unaffected by the effects of selection on nearby genes. To provide a picture of nucleotide variability at a high-recombination locus in humans, we sequenced 5. 4 kb from two introns of Dmd in a worldwide sample of 41 alleles from Africa, Asia, Europe, and the Americas. These same regions were also sequenced in one common chimpanzee and one orangutan. Dramatically different patterns of genetic variation were observed at these two introns, which are separated by >500 kb of DNA. Nucleotide diversity at intron 44 pi = 0.141% was more than four times higher than nucleotide diversity at intron 7 pi = 0.034% despite similar levels of divergence for these two regions. Intron 7 exhibited significant linkage disequilibrium extending over 10 kb and also showed a significant excess of rare polymorphisms. In contrast, intron 44 exhibited little linkage disequilibrium and no skew in the frequency distribution of segregating sites. Intron 7 was much more variable in Africa than in other continents, while intron 44 displayed similar levels of variability in different geographic regions. Comparison of intraspecific polymorphism to interspecific divergence using the HKA test revealed a significant reduction in variability at intron 7 relative to intron 44, and this effect was most pronounced in the non-African samples. These results are best explained by positive directional selection acting at or near intron 7 and demonstrate that even genes in regions of high recombination may be influenced by selection at linked sites.