Development and characterization of microsatellite markers in Zanthoxylum ailanthoides (Rutaceae).

Nine microsatellite markers were developed for Zanthoxylum ailanthoides, a typical pioneer tree. Averaged over the nine loci, the number of alleles per locus was 5.1. The observed and expected heterozygosities ranged from 0.233 to 0.833 and from 0.314 to 0.823, with averages of 0.606 and 0.641, respectively. No loci showed significant departures from Hardy-Weinberg equilibrium or linkage equilibrium after Bonferroni correction (P > 0.05). These markers will be useful for parentage analyses and studies of population genetic structure in the species.

High population differentiation and unusual haplotype structure in a shade-intolerant pioneer tree species, Zanthoxylum ailanthoides (Rutaceae) revealed by analysis of DNA polymorphism at four nuclear loci.

Differences in demographic history, life-history traits, and breeding systems affect nucleotide variation patterns. It is expected that shade-intolerant pioneer tree species have different patterns of genetic polymorphism and population structure than climax species. We studied patterns of nucleotide polymorphism at four putative starch pathway loci (agpSA, agpSB, agpL, and GBSSI) in Zanthoxylum ailanthoides, a shade-intolerant pioneer tree species that occupies forest gaps in warm-temperate forests of East Asia. Genetic diversity was lower within each population than among populations, and differentiation among populations was high across the loci (F(ST) = 0.32-0.64), as expected from the insect-pollinated breeding system and the metapopulation structure of this pioneer species. Numbers of haplotypes were smaller than those expected from the observed numbers of segregating sites. Single haplotypes accounted for more than 47% of all the sampled genes at the respective loci. These variation patterns were incompatible with neutral predictions for populations of a finite island model. Complex population dynamics, such as bottleneck and/or admixture, in the history of this pioneer tree species might have resulted in the observed patterns of genetic variation and population structure, which are different from those of climax wind-pollinated tree species, such as conifers. In contrast to the other loci investigated in this study, agpL showed nearly no variation in Z. ailanthoides (one singleton only), but there was some extent of variation in a closely related species, Zanthoxylum schinifolium. This suggests possibly a recent selective sweep at or near the locus in Z. ailanthoides.

PERMANENT GENETIC RESOURCES: Isolation and characterization of microsatellite loci in the cichlid fish in Lake Victoria, Haplochromis chilotes.

Twelve short tandem repeat markers were successfully isolated from a cichlid, Haplochromis chilotes, in Lake Victoria, and characterized in Haplochromis pyrrhocephalus. The microsatellite regions of these markers were found to have between two and 48 alleles with heterozygosity ranging from 0.07 to 0.97. No loci showed significant departures from the Hardy-Weinberg or linkage equilibrium after the Bonferroni correction (P > 0.05). Cross-species amplification in other cichlids of Lake Victoria, Haplochromis laparogramma, Lithochromis rubripinnis, L. rufus and Haplochromis sp. 'rockkribensis', was successful.

Nucleotide sequence analyses of human complement 6 (C6) gene suggest balancing selection.

The sixth complement component (C6) has a common charge polymorphism, C6A and C6B, with similar gene frequencies in all major populations. In addition, C6B2 is also found in Japanese populations at a frequency of about 6%. Sequence analyses of the coding region of three human and ape C6 alleles indicated four nonsynonymous and three synonymous changes in C6*B2 relative to C6*A, suggesting that a recombination event occurred between C6*B2 and C6*A to give rise to C6*B. Sequence variation in a 3.86 kb region encompassing exon 3, where the causal base change of the common C6 polymorphism is found, indicated that several single nucleotide polymorphisms (SNPs) were in extensive linkage disequilibrium (LD), with little differentiation among populations. Sliding window estimates of two test statistics for neutrality revealed significant values in a subregion where the replacement coding polymorphism resides, in all three human populations. These results raise the possibility that the two common C6 alleles in human populations are maintained by balancing selection.

Population differences in DNA sequence variation and linkage disequilibrium at the PON1 gene.

Polymorphisms of the promoter region (-108C/T) and the coding region (192Q/R) of the paraoxonase 1 gene (PON1) showed differences in association with cardiovascular disease risk in various populations. To characterize the genetic variation underlying these important polymorphisms, we examined DNA sequence variation both in a 1.3-kb promoter region 16.5 kb from codon 192, and in a 1.7-kb region centered on the 192Q/R polymorphic site of the coding region of PON1, in 30 Africans, 30 Europeans and 64 Japanese. We found 10 polymorphic sites and 11 haplotypes in the 1.3-kb promoter region and 10 biallelic polymorphic sites and 10 haplotypes in the 1.7-kb region. From the PON1 sequences of chimpanzees and an orangutan, the ancestral type of codon 192 was found to be R. The number of pairs of polymorphic sites between the promoter and 1.7-kb regions that were in significant linkage disequilibrium was much higher in a Japanese population than in African and European populations. In addition, the pairs of polymorphic sites in linkage disequilibrium differed among the three populations. These results suggest that some of the population differences in association with risk for coronary heart disease can be explained by population differences in haplotype frequency of PON1 haplotypes.

Contrasting patterns of polymorphisms at the ABO-secretor gene (FUT2) and plasma alpha(1,3)fucosyltransferase gene (FUT6) in human populations.

The coding sequences ( approximately 1 kb) of FUT2 [ABO-Secretor type alpha(1,2)fucosyltransferase] and of FUT6 [plasma alpha(1,3)fucosyltransferase] were analyzed for allelic polymorphism by direct sequencing in five populations. The nucleotide diversities of FUT2 estimated from pairwise sequence differences were 0.0045, 0.0042, 0.0042, 0.0009, and 0.0008 in Africans, European-Africans, Iranians, Chinese, and Japanese, respectively. The nucleotide diversities of FUT6 were 0.0024, 0.0016, 0.0015, 0.0017, and 0.0020 in Africans, European-Africans, Iranians, Chinese, and Japanese, respectively. At FUT2, excesses in pairwise sequence differences compared to the number of polymorphic sites as indicated by a significantly positive Tajima's D were observed in European-Africans and in Iranians. The data do not fit expectations of the equilibrium neutral model with an infinite number of sites. On the other hand, Tajima's D's at FUT6 in each of the five populations and at FUT2 in Africans, Chinese, and Japanese were not significantly different from zero. F(ST) between the Asians and the others measured at FUT2 was higher than at FUT6. These results suggest that natural selection was responsible for the generation of the FUT2 polymorphism in European-Africans and in Iranians.

Phylogenetic relationships in Taxodiaceae and Cupressaceae sensu stricto based on matK gene, chlL gene, trnL-trnF IGS region, and trnL intron sequences.

Nucleotide sequences from four chloroplast genes, the matK, chlL, intergenic spacer (IGS) region between trnL and trnF, and an intron of trnL, were determined from all species of Taxodiaceae and five species of Cupressaceae sensu stricto (s.s.). Phylogenetic trees were constructed using the maximum parsimony and the neighbor-joining methods with Cunninghamia as an outgroup. These analyses provided greater resolution of relationships among genera and higher bootstrap supports for clades compared to previous analyses. Results indicate that Taiwania diverged first, and then Athrotaxis diverged from the remaining genera. Metasequoia, Sequoia, and Sequoiadendron form a clade. Taxodium and Glyptostrobus form a clade, which is the sister to Cryptomeria. Cupressaceae s.s. are derived from within Taxodiaceae, being the most closely related to the Cryptomeria/Taxodium/Glyptostrobus clade. These relationships are consistent with previous morphological groupings and the analyses of molecular data. In addition, we found acceleration of evolutionary rates in Cupressaceae s.s. Possible causes for the acceleration are discussed.

Evidence for recent invasion of the medaka fish genome by the Tol2 transposable element.

Tol2 is a transposable element of the terminal-inverted-repeat class, residing in the genome of the medaka fish Oryzias latipes. The genus Oryzias contains more than 10 species for which phylogenetic relationships have previously been estimated. To infer the history of Tol2 in this genus we performed genomic Southern blots and PCR analyses of 10 of the species. It was revealed that Tol2 occurs in 2 of the 10 species (O. curvinotus and O. latipes) and that the length and the restriction map structure of Tol2 are identical in the two cases. Further, sequencing analysis revealed an extremely low level of divergence compared with that in a nuclear gene. These results suggest recent incorporation of Tol2 into one or both of the two species, implying horizontal transfer of Tol2 from one species to the other or into them both from a common source.

Molecular evolution in a multisite nearly neutral mutation model.

A simple nearly neutral mutation model of protein evolution was studied using computer simulation assuming a constant population size. In this model, a gene consists of a finite number of codons and there is no recombination within a gene. Each codon has two replacement and one silent sites. The fitness of a gene was determined multiplicatively by amino acids specified by codons (the independent multicodon model). Nucleotide diversity at replacement sites decreases as selection becomes stronger. A reduction of nucleotide diversity at silent sites also occurs as selection intensifies but the magnitude of the reduction is not a monotone function of the intensity of selection. The dispersion index is close to one. The average value of Tajima's and Fu and Li's statistics are negative and their absolute values increases as selection intensifies. However, their powers of detecting selection under the present model were not high unless the number of sites is large or mutation rate is high. The MK test was shown to detect intermediate selection fairly well. For comparison, the house-of-cards model was also investigated and its behavior was shown to be more sensitive to changes of population size than that of the independent multicodon model. The relevance of the present model for explaining protein evolution was discussed comparing its prediction and recent DNA data.

DNA evolution under weak selection.

Some DNA data show patterns of variation not expected under the neutral theory. Here, the independent multicodon (IMC) model, a nearly neutral mutation model assuming no interaction among codons, was studied when population size changes using computer simulation. Patterns of variation expected under the model were investigated using statistics for the neutrality tests. The average dispersion index is more than one when population size changes slowly but it never becomes large. The diversity at linked silent site decreases when the strength of selection is intermediate and the reduction is larger when population size changes slowly. Tajima's (1989. Genetics 123, 585-595) D is generally negative. Rejections by the Tajima's test occur more frequently if population size changes quickly but the effect of selection is confounded with the size change itself in this case. If we apply the test of McDonald and Kreitman (1991. Nature 351, 652-654), the rejection is always in the direction of excess replacement polymorphisms. The rejection probability decreases as the rate of population size changes decreases. These results show that the predictions of the IMC model are consistent with the pattern observed in mitochondrial DNA data but not consistent with some data of nuclear DNA. Interaction among codons or variable selection would be necessary to explain such cases.

Ancient origin of the null allele se(428) of the human ABO-secretor locus (FUT2).

In human populations, a null allele having several nucleotide differences from the wild-type allele is segregating at the FUT2 locus (the ABO-Secretor locus) encoding alpha(1,2)fucosyltransferase. To estimate the age of the most recent common ancestor (MRCA) of these two alleles, we sequenced FUT2 homologues from chimpanzee, gorilla, orangutan, and green monkey. Since we did not detect acceleration or any heterogeneity in the substitution rate at this locus among these species, the age of the MRCA was estimated to be around 3 MYA, assuming the divergence time of human and chimpanzee to be 5 MYA. We developed a simple test to examine whether or not the old age of the MRCA of the FUT2 is consistent with that expected for two divergent neutral alleles sampled from a random mating population. An application of the test to the data at FUT2 indicated that the age of the MRCA is too old to be explained by the simple neutral assumptions, although our test depends on accurate estimation of the divergence time of human and chimpanzee in units of twice the human population size. Various possibilities including balancing selection are discussed to explain this old age of the MRCA.

Evolution of multigene families by gene duplication. A haploid model.

Evolution of multigene families by gene duplication and subsequent diversification is analyzed assuming a haploid model without interchromosomal crossing over. Chromosomes with more different genes are assumed to have higher fitness. Advantageous and deleterious mutations and duplication/deletion also affect the evolution, as in previous studies. In addition, negative selection on the total number of genes (copy number selection) is incorporated in the model. First, a Markov chain approximation is used to obtain formulas for the average numbers of different alleles, genes without pseudogene mutations, and pseudogenes assuming that mutation rates and duplication/deletion rates are all very small. Computer simulation shows that the approximation works well if the products of population size with mutation and duplication/deletion rates are all small compared to 1. However, as they become large, the approximation underestimates gene numbers, especially the number of pseudogenes. Based on the approximation, the following was found: (1) Gene redundancy measured by the average number of redundant genes decreases as advantageous selection becomes stronger. (2) The number of different genes can be approximately described by a linear pure-birth process and thus has a coefficient of variation around 1. (3) The birth rate is an increasing function of population size without copy number selection, but not necessarily so otherwise. (4) Copy number selection drastically decreases the number of pseudogenes. Available data of mutation rates and duplication/deletion rates suggest much faster increases of gene numbers than those observed in the evolution of currently existing multigene families. Various explanations for this discrepancy are discussed based on our approximate analysis.

Molecular phylogeny of Dipetrocarpaceae in Southeast Asia based on nucleotide sequences of matK, trnL intron, and trnL-trnF intergenic spacer region in chloroplast DNA.

To obtain a refined molecular phylogeny of dipterocarp species in Southeast Asia, nucleotide sequences of matK, the intron of trnL, and intergenic spacer region between trnL and trnF in chloroplast DNA were determined in 16 species throughout 10 genera. In the resultant trees Southeast Asian dipterocarp species were divided into two clusters. One cluster consisted of Anisoptera, Vatica, Cotylelobium, and Upuna, all with the base chromosome number of x = 11. The second cluster consisted of Hopea, Shorea, Neobalanocarpus, Dryobalanops, Parashorea, and Dipterocarpus, mostly with the base chromosome number of x = 7. Dipterocarpus was the only genus that had the base chromosome number x = 11 in the latter cluster. This result suggests that the chromosome number changed from x = 11 to x = 7 after Dipterocarpus branched in the latter cluster. Other evolutionary changes of morphological characters are also discussed.

Bottleneck effect on evolutionary rate in the nearly neutral mutation model.

Variances of evolutionary rates among lineages in some proteins are larger than those expected from simple Poisson processes. This phenomenon is called overdispersion of the molecular clock. If population size N is constant, the overdispersion is observed only in a limited range of 2N sigma under the nearly neutral mutation model, where sigma represents the standard deviation of selection coefficients of new mutants. In this paper, we investigated effects of changing population size on the evolutionary rate by computer simulations assuming the nearly neutral mutation model. The size was changed cyclically between two numbers, N1 and N2 (N1 > N2), in the simulations. The overdispersion is observed if 2N2 sigma is less than two and the state of reduced size (bottleneck state) continues for more than approximately 0.1/u generations, where u is the mutation rate. The overdispersion results mainly because the average fitnesses of only a portion of populations go down when the population size is reduced and only in these populations subsequent advantageous substitutions occur after the population size becomes large. Since the fitness reduction after the bottleneck is stochastic, acceleration of the evolutionary rate does not necessarily occur uniformly among loci. From these results, we argue that the nearly neutral mutation model is a candidate mechanism to explain the overdispersed molecular clock.

Molecular evolution of the Amy multigenes in the subgenus Sophophora of Drosophila.

Molecular evolution of the Amy multigenes in Drosophila was investigated using PCR amplification. Twenty-five partial Amy sequences from 13 species belonging mainly to the subgenus Sophophora were determine, and a molecular phylogeny of the Amy genes in Drosophila was constructed, together with published Amy sequences. Clustering of species are mostly consistent with the traditional classification and that inferred from other genes. From sequence divergence between PCR products, several species, including D. elegans and D. fuyamai, were suggested to have multiple copies of the Amy genes. The loss of an intron took place at least three times after the Sophophora radiation. In order to investigate the mechanism of sequence evolution, the numbers of amino acid replacement and synonymous substitutions in five lineages were estimated. The heterogeneity in the relative numbers of synonymous and replacement substitutions among the lineages was found. Possible roles of selection in the sequence evolution of the Amy gene are discussed.

Molecular analysis of the intergenic region of the duplicated Amy genes of Drosophila melanogaster and Drosophila teissieri.

The intergenic regions between the duplicated amylase coding regions (Amy) of D. melanogaster and D. teissieri were sequenced. Their lengths in D. melanogaster and D. teissieri were 4,536 bp and 4,621 bp, respectively. Since homology between the upstream regions of the two duplicated genes was found up to 450 bp from the initiation codon of the Amy genes, the ancestral Amy coding region duplicated together with at least 450 bp of the 5'-flanking region as one unit. Comparison of the regions between the two species revealed that the level of divergence was very heterogeneous. Although the mean level of the nucleotide difference in this region was 0.107, no nucleotide substitution was found in four subregions whose sizes were more than 100 bp. Since the probability of these four subregions being completely conserved between D. melanogaster and D. teissieri was very low, these subregions were considered to have relatively important roles in evolution. Large insertions and deletions were not observed in this region but small ones were observed all over the region except for an about 1-kb subregion. This 1-kb region corresponded to an open reading frame encoding a protein which had some sequence identity with the proteins of the serine protease inhibitor superfamily (serpin). Since we could find a transcript of this gene and the synonymous substitution rate was higher than the replacement substitution rate, we suggest that this gene encodes an active serpin in Drosophila.

A population genetic study of the evolution of SINEs. II. Sequence evolution under the master copy model.

A transient population genetic model of SINE (short interspersed repetitive element) evolution assuming the master copy model is theoretically investigated. Means and variances of consensus frequency of nucleotides, nucleotide homozygosity, and the number of shared differences that are considered to have caused by mutations occurring in the master copy lineages are computed. All quantities investigated are shown to be monotone functions of the duration of the expansion period. Thus, they can be used to estimate the expansion period although their sampling variances are generally large. Using the theoretical results, the Sb subfamily of human Alu sequences is analyzed. First, the expansion period is estimated from the observed mean and variance of homozygosity. The expansion period is shown to be short compared to the time since the end of the expansion of the subfamily. However, the observed number of the shared differences is more than twice that expected under the master copy model with the estimated expansion period. Alternative models including that with multiple master copy loci to explain this observation are discussed.

Molecular evolution of the 5'-flanking regions of the duplicated Amy genes in Drosophila melanogaster species subgroup.

The nucleotide sequences of the 5'-flanking regions of the duplicated Amy genes in eight sibling species belonging to the melanogaster species subgroup are analyzed. In Drosophila melanogaster, a region of about 450 bp immediately upstream of the translation initiation site of the two paralogous genes (the proximal and distal genes) has sequence similarities. However, we could not detect any significant sequence similarity in the region more upstream than -450. This result indicates that the coding regions of the ancestral Amy gene were duplicated together with 450 bp of the 5'-flanking region as one unit. Multiple alignment of these 450-bp sequences in the proximal and distal genes of all eight species revealed a mosaic pattern of highly conserved and divergent regions. The conserved regions included almost all the putative regulatory elements identified in previous analyses of the sequences. A phylogenetic analysis of the aligned sequences shows that these 450-bp sequences are clustered into the proximal and the distal groups. As a whole, the divergence between groups in this region is very large in contrast to that in the coding regions. Based on the divergence between groups, the 450-bp region is divided into two subregions. We found that the ratios of the divergence between groups to that within groups differ in the two subregions. From these observations, we discuss a possibility of positive selection acting on the subregion immediately upstream of the Amy coding region to cause divergence of regulatory elements of the paralogous genes.