Genetic Structure of the Ancestral Population of Modern Humans

Neutral DNA polymorphisms from an 8-kb segment of the dystrophin gene, previously ascertained in a worldwide sample (n= 250 chromosomes), were used to characterize the population ancestral to the present-day human groups. The ancestral state of each polymorphic site was determined by comparing human variants with their orthologous sites in the great apes. The ``age before fixation' of the underlying mutations was estimated from the frequencies of the new alleles and analyzed in the context of these polymorphisms' distribution among 13 populations from Africa, Europe, Asia, New Guinea, and the Americas (n= 860 chromosomes in total). Seventeen polymorphisms older tan 100,000–200,000 years, which contributed ∼90% to the overall nucleotide diversity, were common to all human groups. Polymorphisms endemic to human groups or continentally restricted were younger than 100,000–200,000 years. Africans (six populations) with 13 such sites stood out from the rest of the world (seven populations), where only 2 population-specific variants were observed. The similarity of the frequencies of the old polymorphisms in Africans and non-Africans suggested a similar profile of genetic variability in the population before the modern human's divergence. This ancestral population was characterized by an effective size of about 10,000 as estimated from the nucleotide diversity; this size may describe the number of breeding individuals over a long time during the Middle Pleistocene or reflect a speciation bottleneck from an initially larger population at the end of this period. Received: 3 February 1998 / Accepted: 9 February 1998     

Predominance of one T-cell antigen receptor BV haplotype in African populations

 The human T-cell antigen receptor (TCR) is the counter-receptor for the HLA/peptide complex displayed on the surface of antigen-presenting cells. It confers antigen specificity on T lymphocytes and therefore plays a central role in pathogen recognition and host response. The most frequently used form of the TCR is a heterodimer composed of variable α and β chains. We investigated allele frequencies for four variable-region gene segments of the β chain (2S1, 3S1, 8S3, and 15S1) in 146 Caucasians and 165 Africans. The results reveal significant unexpected differences between the two populations for allele frequencies, phenotypes, genotypes, and haplotypes. Among Caucasians, there are 43 phenotypes, whereas there are 31 among the Africans studied. There are 17 haplotypes in the Caucasian sample but only 10 in Africans. This loss of diversity is largely due to the high frequency of one haplotype in the African sample which represents 65% of the informative chromosomes. At least one copy of this haplotype is present in 90% of informative individuals. As a result, 29% of Africans are homozygous for the common haplotype. Less genetic diversity at TCRBV is unexpected, since Africans usually show greater genetic diversity than other ethnic groups. For example, there are approximately twice as many HLA haplotypes in Africans compared to Caucasians. Homozygosity is also unexpected because it reduces the number of TCR variants available to recognize HLA pathogen-derived peptide complexes. Received: 23 September 1999 / Revised: 2 November 1999     

The association between HLA-A alleles and an Alu dimorphism near HLA-G

The AluYb8 sequences are a subfamily of short interspersed Alu retroelements that have been amplified within the human genome during recent evolutionary time and are useful polymorphic markers for studies on the origin of human populations. We have identified a new member of the Yb8 subfamily, AluyHG, located between the HLA-H and -G genes and 88-kb telomeric of the highly polymorphic HLA-A gene within the alpha block of the major histocompatibility complex (MHC). The AluyHG element was characterised with a view to examining the association between AluyHG and HLA-A polymorphism and reconstructing the history of the MHC alpha block. A specific primer pair was designed for a simple PCR assay to detect the absence or presence (dimorphism) of the AluyHG element within the DNA samples prepared from a panel of 46 homozygous cell-lines containing complete or recombinant ancestral haplotypes (AH) of diverse ethnic origin and 92 Caucasoid and Asian subjects on which HLA-A typing was available. The AluyHG insertion was most strongly associated with HLA-A2 and, to a lesser degree with HLA-A1, -A3, -A11, and A-19. The gene frequency of the AluyHG insertion for 146 Caucasians and 94 Chinese-Han was 0.30 and 0.32 and there was no significant difference between the observed and expected frequencies. The results of the association studies and the phylogenetic analysis of HLA-A alleles suggest that the AluyHG sequence was integrated within the progenitor of HLA-A2, but has been transferred by recombination to other human ancestral populations. In this regard, the dimorphic AluyHG element is an important diagnostic marker for HLA association studies and could help in elucidating the evolution and functions of the MHC alpha block and polymorphism within and between ancestral haplotypes. Received: 7 December 2000 / Accepted: 28 February 2001     

Absence of Protein Polymorphism in the Ras Genes of Drosophila melanogaster

Sequence analysis of 27 alleles of each of the three Ras-related genes in Drosophila melanogaster indicates that they all have low levels of polymorphism but may experience slightly different evolutionary pressures. No amino acid replacement substitutions were indicated in any of the sequences, or in the sibling species D. simulans and D. mauritiana. The Dras1 gene, which is the major ras homologue in Drosophila, has less within-species variation in D. melanogaster relative to the amount of divergence from the sibling species than does Dras2, although the contrast was not significant by the HKA test. Dras2 appears to be maintaining two classes of haplotype in D. melanogaster, one of which is closer to the alleles observed in the sibling species, suggesting that this is not likely to be a pseudogene despite the absence of a mutant phenotype. Although differences in level of expression may affect the function of the genes, it is concluded that genetic variation in the Ras signal transduction pathways cannot be attributed to catalytic variation in the Ras proteins. Received: 5 November 1998 / Accepted: 26 March 1999     

Ancient Mitochondrial DNA Reveals the Origin of Sus scrofa from Rebun Island, Japan

The Kabukai A site (5 to 8C A.D.) of the Okhotsk cultural area is on Rebun Island, a small island near the coast, north–northwest of Hokkaido, Japan. Specimens of Sus scrofa, called the Sakhalin pig, were discovered in five cultural layers at the Kabukai A site. Ancient DNA was extracted from the remains of 42 Sakhalin pig bones. Thirty-nine nucleotide sequences of the 574-bp mitochondrial DNA control region, estimated to have originated from at least 21 individuals, were amplified and analyzed phylogenetically. Nine distinct haplotypes (A1, A2, A3, B1, B2, C1, C2, D1, and D2) from this site were classified into four haplotype groups (A, B, C, and D) by parsimonious network analysis. Phylogenetic analysis of 9 ancient and 55 modern haplotypes indicated that the population of Sakhalin pigs at the Kabukai A site belonged to two distinct clusters; haplotype groups A and B formed a cluster comprised only of themselves, and haplotype groups C and D belonged to the cluster of one of the two genetic groups of Japanese wild boars uniquely distributed in the western part of Japan, including one northeast Mongolian wild boar. Analysis of the haplotype distribution among three archaeological sites and their historical transitions among the five layers reflecting the cultural periods at the Kabukai A site suggests that the Sakhalin pig populations were introduced from Sakhalin island and the Amur River basin in the northeastern Eurasian continent together with some cultural influences. Received: 18 April 2000 / Accepted: 24 November 2000     

Evolutionary Rate Acceleration of Cytochrome c Oxidase Subunit I in Simian Primates

We present an analysis of the evolutionary rates of the cytochrome c oxidase subunit I genes of primates and other mammals. Five primate genes were sequenced, and this information was combined with published data from other species. The sequences from simian primates show approximately twofold increases in their nonsynonymous substitution rate compared to those from other primates and other mammals. The species range and the overall magnitude of this rate increase are similar to those previously identified for the cytochrome c oxidase subunit II and cytochrome b genes. Received: 22 July 1999 / Accepted: 21 February 2000     

Evaluation of Sequence Variation and Selection in the Bindin Locus of the Red Sea Urchin, Strongylocentrotus franciscanus

Recent evidence suggests that gamete recognition proteins may be subjected to directed evolutionary pressure that enhances sequence variability. We evaluated whether diversity enhancing selection is operating on a marine invertebrate fertilization protein by examining the intraspecific DNA sequence variation of a 273-base pair region located at the 5′ end of the sperm bindin locus in 134 adult red sea urchins (Strongylocentrotus franciscanus). Bindin is a sperm recognition protein that mediates species-specific gamete interactions in sea urchins. The region of the bindin locus examined was found to be polymorphic with 14 alleles. Mean pairwise comparison of the 14 alleles indicates moderate sequence diversity (p-distance = 1.06). No evidence of diversity enhancing selection was found. It was not possible to reject the null hypothesis that the sequence variation observed in S. franciscanus bindin is a result of neutral evolution. Statistical evaluation of expected proportions of replacement and silent nucleotide substitutions, observed versus expected proportions of radical replacement substitutions, and conformance to the McDonald and Kreitman test of neutral evolution all indicate that random mutation followed by genetic drift created the polymorphisms observed in bindin. Observed frequencies were also highly similar to results expected for a neutrally evolving locus, suggesting that the polymorphism observed in the 5′ region of S. franciscanus bindin is a result of neutral evolution. Received: 19 June 1998 / Accepted: 2 August 2000     

The Behavior of a Daphnia pulex Transposable Element in Cyclically and Obligately Parthenogenetic Populations

Using Southern blot analysis, we have characterized restriction fragment patterns of a transposable element, Pokey, in obligately and cyclically parthenogenetic populations of the cladoceran crustacean Daphnia pulex. We show that the element is most likely active in cyclically parthenogenetic populations but is, for the most part, inactive in obligate parthenogens. This result is consistent with theory suggesting that transposable element dynamics are likely to change with a change in reproductive mode. Such changes could have important consequences for the long-term evolutionary potential of obligate parthenogens and may also be informative with regard to the underlying mechanisms that regulate transposable element frequencies in sexual organisms. Received: 29 August 2000 / Accepted: 1 March 2001     

Prehistoric Introduction of Domestic Pigs onto the Okinawa Islands: Ancient Mitochondrial DNA Evidence

Ancient DNAs of Sus scrofa specimens excavated from archaeological sites on the Okinawa islands were examined to clarify the genetic relationships among prehistoric Sus scrofa, modern wild boars and domestic pigs inhabiting the Ryukyu archipelago, the Japanese islands, and the Asian continent. We extracted remain DNA from 161 bone specimens excavated from 12 archaeological sites on the Okinawa islands and successfully amplified mitochondrial DNA control region fragments from 33 of 161 specimens. Pairwise difference between prehistoric and modern S. scrofa nucleotide sequences showed that haplotypes of the East Asian domestic pig lineage were found from archaeological specimens together with Ryukyu wild boars native to the Ryukyu archipelago. Phylogenetic analysis of 14 ancient sequences (11 haplotypes; 574 bp) indicated that S. scrofa specimens from two Yayoi-Heian sites (Kitahara and Ara shellmiddens) and two Recent Times sites (Wakuta Kiln and Kiyuna sites) are grouped with modern East Asian domestic pigs. Sus scrofa specimens from Shimizu shellmidden (Yayoi-Heian Period) were very closely related to modern Sus scrofa riukiuanus but had a unique nucleotide insertion, indicating that the population is genetically distinct from the lineage of modern Ryukyu wild boars. This genetic evidence suggests that domestic pigs from the Asian continent were introduced to the Okinawa islands in the early Yayoi-Heian period (1700–2000 BP), or earlier.     

Low Diversity and Divergence in the fil1 Gene Family of Antirrhinum (Scrophulariaceae)

Detailed nucleotide diversity studies revealed that the fil1 gene of Antirrhinum, which has been reported to be single copy, is a member of a gene family composed of at least five genes. In four Antirrhinum majus populations with different mating systems and one A. graniticum population, diversity within populations is very low. Divergence among Antirrhinum species and between Antirrhinum and Digitalis is also low. For three of these genes we also obtained sequences from a more divergent member of the Scrophulariaceae, Verbascum nigrum. Compared with Antirrhinum, little divergence is again observed. These results, together with similar data obtained previously for five cycloidea genes, suggest either that these gene families (or the Antirrhinum genome) are unusually constrained or that there is a low rate of substitution in these lineages. Using a sample of 52 genes, based on two measures of codon usage (ENC and GC3 content), we show that cyc and fil1 are among the least biased Antirrhinum genes, so that their low diversity is not due to extreme codon bias. Received: 20 June 2000 / Accepted: 25 October 2000     

Genotype frequency and F ST analysis of polymorphisms in immunoregulatory genes in Chinese and Caucasian populations

Selection and genetic drift can create genetic differences between populations. Cytokines and chemokines play an important role in both hematopoietic development and the inflammatory response. We compared the genotype frequencies of 45 SNPs in 30 cytokine and chemokine genes in two healthy Chinese populations and one Caucasian population. Several SNPs in IL4 had substantial genetic differentiation between the Chinese and Caucasian populations (F _ST ~0.40), and displayed a strikingly different haplotype distribution. To further characterize common genetic variation in worldwide populations at the IL4 locus, we genotyped 9 SNPs at the IL4 gene in the Human Diversity Panel’s (N = 1056) individuals from 52 world geographic regions. We observed low haplotype diversity, yet strikingly different haplotype frequencies between non-African populations, which may indicate different selective pressures on the IL4 gene in different parts of the world. SNPs in CSF2, IL6, IL10, CTLA4, and CX3CR1 showed moderate genetic differentiation between the Chinese and Caucasian populations (0.15 < F _ST < 0.25). These results suggest that there is substantial genetic diversity in immune genes and exploration of SNP associations with immune-related diseases that vary in incidence across these two populations may be warranted.     

Replication Orientation Affects the Rate and Direction of Bacterial Gene Evolution

In many bacterial genomes, the leading and lagging strands have different skews in base composition; for example, an excess of guanosine compared to cytosine on the leading strand. We find that Chlamydia genes that have switched their orientation relative to the direction of replication, for example by inversion, acquire the skew of their new ``host' strand. In contrast to most evolutionary processes, which have unpredictable effects on the sequence of a gene, replication-related skews reflect a directional evolutionary force that causes predictable changes in the base composition of switched genes, resulting in increased DNA and amino acid sequence divergence. Received: 27 April 2000 / Accepted: 1 August 2000     

Origin and evolution of GBV-C/hepatitis G virus and relationships with ancient human migrations

The GB virus C/hepatitis G virus (GBV-C/HGV) is a newly identified human RNA virus, belonging to the Flaviviridae family. Persistent infection by GBV-C/HGV is common in humans, and genetically divergent isolates have been identified in different parts of the world. Due to the absence of a real pathogenic role of GBV-C/HGV in liver disease and its extremely low mutation rate, this virus is a potential marker to trace prehistoric links between human populations. In this study, origin and evolution of GBV-C/HGV were examined using a set of fully sequenced strains of worldwide origin. A first phylogenetic analysis, addressed to the short (255 nucleotides) NS5A overlapping coding region by the neighbor-joining method, suggested an ancient African origin of GBV-C/HGV. This notion was confirmed when the same analysis was applied to the genomic regions showing the lowest rate of synonymous substitutions, covering one-fourth (2184 nucleotides) of the total coding potential of the virus genome. By using a multivariate statistical method and extending the analysis to the complete coding region, fine details of the evolutionary history of GBV-C/HGV were further elucidated. By this approach, isolates from Southeast Asia appeared to be the most closely related to those of African origin, consistent with a major route of ancient human migrations from Africa to southeastern parts of the Asian continent. Received: 26 October 2000 / Accepted: 28 February 2001     

mtDNA Sequence Diversity of Orangutans from the Islands of Borneo and Sumatra

A comparison of mitochondrial DNA sequences was undertaken for two genes among orangutans from Borneo and Sumatra. The distribution of haplotypes among 42 individuals for NADH dehydrogenease subunit 3 and 39 individuals for cytochrome B was used to infer population structure. The haplotypes among all Bornean orangutans form a cluster of closely related individuals for both genes, with two distinct haplotypes occupying different regions of the island. Sumatran haplotypes fall into three distinct, and highly diverged, groups. Strikingly, one of the Sumatran haplotypes shares sequence identity with the most widespread Bornean haplotype. This haplotype distribution is considered in the context of the highly complex geological history for the area around the Malay Archipelago. Alternating periods of geographic isolation and reunion, resulting from glacially induced land bridge formation, presented substantial opportunity for population dispersal between periodically isolated demes. We present a paleodispersal model that is consistent with genetic, geological, paleoecological, and fossil data. The disparity of sequences present in orangutan populations argues against a simple Sumatra–Borneo dichotomy. Our evidence, and that of others, suggests that the Sumatran population alone contains the remnants of at least three separate lineages. Received: 3 February 1999 / Accepted: 2 August 2000     

Current Intraspecific Dynamics of Sequence Evolution Differs from Long-Term Trends and Can Account for the AT-Richness of Honeybee Mitochondrial DNA

The very high AT content of hymenopteran mtDNA has warranted speculation about nucleotide substitution processes in this group. Here we investigate the pattern of honeybee, Apis mellifera, mtDNA nucleotide polymorphisms inferred from phylogeny in terms of differences between the ATPase6, COI, COII, COIII, cytochrome b, and ND2 genes and strand asymmetry in mutation rates. The observed transition/transversion ratios and the distribution of nonsynonymous substitutions between regions differed significantly. The pattern of differences between genes leading to these heterogeneities (the ATPase6 and COIII genes group apart from the rest) differed markedly from that predicted on the basis of long-term evolutionary change and may indicate differences between current and long-term dynamics of sequence evolution. Also, there is strong strand asymmetry in substitutions, which probably results in a mutability of G and C sufficiently high to account for the AT-richness of honeybee mtDNA. Received: 21 October 1998 / Accepted: 27 January 1999     

Molecular Evolution of Nitrogen Fixation: The Evolutionary History of the nifD, nifK, nifE, and nifN Genes

The pairs of nitrogen fixation genes nifDK and nifEN encode for the α and β subunits of nitrogenase and for the two subunits of the NifNE protein complex, involved in the biosynthesis of the FeMo cofactor, respectively. Comparative analysis of the amino acid sequences of the four NifD, NifK, NifE, and NifN in several archaeal and bacterial diazotrophs showed extensive sequence similarity between them, suggesting that their encoding genes constitute a novel paralogous gene family. We propose a two-step model to reconstruct the possible evolutionary history of the four genes. Accordingly, an ancestor gene gave rise, by an in-tandem paralogous duplication event followed by divergence, to an ancestral bicistronic operon; the latter, in turn, underwent a paralogous operon duplication event followed by evolutionary divergence leading to the ancestors of the present-day nifDK and nifEN operons. Both these paralogous duplication events very likely predated the appearance of the last universal common ancestor. The possible role of the ancestral gene and operon in nitrogen fixation is also discussed. Received: 21 June 1999 / Accepted: 1 March 2000     

Genetic Evidence for Gonochoristic Reproduction in Gynogenetic Silver Crucian Carp (Carassius auratus gibelio Bloch) as Revealed by RAPD Assays

Sex evolution has been a debating focus in evolutionary genetics. In lower vertebrates of reptiles, amphibians, and fish, a species or a bioform reproduces either sexually or asexually but never both. A few species were found to consist of all females in fish. These all-female species can propagate by asexual reproduction modes, such as gynogenesis and hybridogenesis. However, the coexistence of sexuality and asexuality in a single species was recently noted only in a cyprinid fish silver crucian carp, Carassius auratus gibelio. This fish had been demonstrated to be capable of gynogenesis stimulated by sperm from other related species. Surprisingly, natural populations of this fish consist of a minor but significant portion (approx. 20%) of males. As different clones with specific phenotypic and genetic characteristics have been found, and RAPD markers specific to each clone have recently been identified, this fish offers many advantages for analyzing whether or not genetic recombination occurs between different clones. In this study, artificial propagation was performed in clone F and clone D. Ovulated eggs from clone F were divided into two parts and respectively inseminated with sperm from a clone D male and from a red common carp (Cyprinus carpio) male. The control clone D individuals were selected from gynogenetic offspring of clone D activated by sperm of red common carp. The phenotype and sex ratio in the experimental groups were also observed. Using RAPD molecular markers, which allow for reliable discrimination and genetic analysis of different clones, we have revealed direct molecular evidence for gonochoristic reproduction in the gynogenetic silver crucian carp and confirmed a previous hypothesis that the silver crucian carp might reproduce both gynogenetically and gonochoristically. Therefore, we conclude that the silver crucian carp possesses two reproductive modes, i.e., gynogenetic and gonochoristic reproduction. The response mechanism of two reproductive development modes may be the first discovery in vertebrates. Additionally, we discuss the evolutionary implication between gynogenetic and gonochoristic reproduction modes and the contribution of the minor proportion of males to genetic flexibility in the gynogenetic silver crucian carp. Received: 5 January 2000 / Accepted: 3 August 2000     

Molecular Evolution of Calmodulin and Calmodulin-Like Genes in the Cephalochordate Branchiostoma

Calmodulin is a calcium-binding EF-hand protein that is an activator of many enzymes as well as ion pumps and channels. Due to its multiple targets and its central role in the cell, understanding the evolutionary history of calmodulin genes should provide insights into the origin of genetic complexity in eukaryotes. We have previously isolated and characterized a calmodulin gene from the early-diverging chordate Branchiostoma lanceolatum (CaM1). In this paper, we report the existence of a second calmodulin gene (CaM2) as well as two CaM-like genomic fragments (CaML-2, CaML-3) in B. lanceolatum and a CaM2 and three CaM-like genes (CaML-1, CaML-2, CaML-3) in B. floridae. The CaM-like genes were isolated using low-stringency PCR. Surprisingly, the nucleotide sequences of the B. lanceolatum CaM1 and CaM2 cDNAs differ by 19.3%. Moreover, the CaM2 protein differs at two positions from the amino acid sequence of CaM1; the latter is identical to calmodulins in Drosophila melanogaster, the mollusc Aplysia californica, and the tunicate Halocynthia roretzi. The two B. lanceolatum CaM-like genes are more closely related to the CaM2 than to the CaM1 gene. This relationship is supported by the phylogenetic analyses and the identical exon/intron organization of these three genes, a relationship unique among animal CaM sequences. These data demonstrate the existence of a CaM multigene family in the cephalochordate Branchiostoma, which may have evolved independently from the multigene family in vertebrates. Received: 2 November 1999 / Accepted: 25 April 2000     

Evolution of Microsatellite Alleles in Four Species of Mice (Genus Apodemus)

Microsatellite length variation was investigated at a highly variable microsatellite locus in four species of Apodemus. Information obtained from microsatellite allele sequences was contrasted with allele sizes, which included 18 electromorphs. Additional analysis of a 400-bp unique sequence in the flanking region identified 26 different haplotype sequences or ``true' alleles in the sample. Three molecular mechanisms, namely, (1) addition/deletion of repeats, (2) substitutions and indels in the flanking region, and (3) mutations interrupting the repeat, contributed to the generation of allelic variation. Size homoplasy can be inferred for alleles within populations, from different populations of the same species, and from different species. We propose that microsatellite flanking sequences may be informative markers for investigating mutation processes in microsatellite repeats as well as phylogenetic relationships among alleles, populations, and species. Received: 3 November 1999 / Accepted: 2 May 2000     

Comparative Genomics of Mitochondrial DNA in Drosophila simulans

The current study compares the nucleotide variation among 22 complete mitochondrial genomes of the three distinct Drosophila simulans haplotypes with intron 1 of the alcohol dehydrogenase-related locus. This is the first study to investigate the sequence variation of multiple complete mitochondrial genomes within distinct mitochondrial haplotypes of a single species. Patterns of variation suggest distinct forces are influencing the evolution of mitochondrial DNA (mtDNA) and autosomal DNA in D. simulans. First, there is little variation within each mtDNA haplotype but strong differentiation among them. In contrast, there is no support for differentiation of the mitochondrial haplotypes at the autosomal locus. Second, there is a significant deficiency of mitochondrial variation in each haplotype relative to the autosomal locus. Third, the ratio of nonsynonymous to synonymous substitutions is not equal in all branches of the well-resolved phylogeny. There is an excess of nonsynonymous substitutions relative to synonymous substitutions within each D. simulans haplotype. This result is similar to that previously observed within the mtDNA of distinct species. A single evolutionary force may be causally linked to the observed patterns of mtDNA variation—a rickettsia-like microorganism, Wolbachia pipientis, which is known to directly influence mitochondrial evolution but have a less direct influence on autosomal loci. Received: 16 September 1999 / Accepted: 14 March 2000