Population data on 15 STR loci using AmpF/STR Identifiler kit in a Malay population living in and around Kuala Lumpur, Malaysia.

Allele frequencies for 15 short tandem repeat (STR) loci D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA (AmpF/STR Identifiler PCR Amplification kit, PE Applied Biosystems) were obtained from a sample of 110 unrelated individuals from the Malay population living in and around Kuala Lumpur, Malaysia, and the characteristics of the population was compared with other East Asian populations.

Y-chromosomal binary haplogroups in the Japanese population and their relationship to 16 Y-STR polymorphisms.

We investigated Y chromosomal binary and STR polymorphisms in 263 unrelated male individuals from the Japanese population and further examined the relationships between the two separate types of data. Using 47 biallelic markers we distinguished 20 haplogroups, four of which (D2b1/-022457, O3/-002611*, O3/-LINE1 del, and O3/-021354*) were newly defined in this study. Most haplogroups in the Japanese population are found in one of the three major clades, C, D, or O. Among these, two major lineages, D2b and O2b, account for 66% of Japanese Y chromosomes. Haplotype diversity of binary markers was calculated at 86.3%. The addition of 16 Y-STR markers increased the number of haplotypes to 225, yielding a haplotype diversity of 99.40%. A comparison of binary haplogroups and Y-STR type revealed a close association between certain binary haplogroups and Y-STR allelic or conformational differences, such as those at the DXYS156Y, DYS390m, DYS392, DYS437, DYS438 and DYS388 loci. Based on our data on the relationships between binary and STR polymorphisms, we estimated the binary haplogroups of individuals from STR haplotypes and frequencies of binary haplogroups in other Japanese, Korean and Taiwanese Han populations. The present data will enable researchers to connect data from binary haplogrouping in anthropological studies and Y-STR typing in forensic studies in East Asian populations, especially those in and around Japan.

Variation of HLA class II genes in the Nganasan and Ket, two aboriginal Siberian populations.

Allelic frequencies at the three most polymorphic loci of the HLA class II region (DRB1, DQA1 and DQB1) were determined in the Nganasan and Ket, the remnants of the two most ancient groups in the Lower Yenisey River/Taimyr Peninsula region in northern Siberia. By single-stranded conformational polymorphism typing, verified by sequencing, 19 HLA-DRB1-DQA1-DQB1 haplotypes and 15 HLA-DRB1, seven DQA1 and 11 DQB1 alleles were found. The most frequent alleles were DRB1*1301 (23.5%), DQA1*0103 (29.4%), *0501/03/05 (29.4%), and DQB1*0301/09 (32.4%) in the Ket, and DRB1*0901 (25%), DQA1*0301 (39.6%), and DQB1*0301/09 (37.5%) in the Nganasan. The distribution patterns and comprehensive phylogenic analysis based on the haplotype frequencies of 17 Siberian populations suggest that the founders of both the Ket and the Nganasan came from Palaeolithic populations in the Altai-Sayan Upland.

Microsatellite variation in Japanese and Asian horses and their phylogenetic relationship using a European horse outgroup.

The genetic relationships of seven Japanese and four mainland-Asian horse populations, as well as two European horse populations, were estimated using data for 20 microsatellite loci. Mongolian horses showed the highest average heterozygosities (0.75-0.77) in all populations. Phylogenetic analysis showed the existence of three distinct clusters supported by high bootstrap values: the European cluster (Anglo-Arab and thoroughbreds), the Hokkaido-Kiso cluster, and the Mongolian cluster. The relationships of these clusters were consistent with their geographical distributions. Basing our assumptions on the phylogenetic tree and the genetic variation of horse populations, we suggest that Japanese horses originated from Mongolian horses migrating through the Korean Peninsula. The genetic relationship of Japanese horses corresponded to their geographical distribution. Microsatellite polymorphism data were shown to be useful for estimating the genetic relationships between Japanese horses and Asian horses.

Phylogeny of antigen-processing enzymes: cathepsins of a cephalochordate, an agnathan and a bony fish.

Cathepsins are enzymes that have been cleaving peptide bonds of lysosomal proteins probably since lysosomes appeared in early eucaryotes. When the adaptive system emerged in gnathostomes, cathepsins were recruited to produce peptides for loading onto the major histocompatibility complex class II molecules and for degrading the class II-associated invariant chain just before the loading. The circumstances under which this recruitment took place are unclear because the knowledge about vertebrate cathepsins is limited largely to mammals. To shed light on the recruitment, 10 amphioxus, one lamprey and one cichlid fish cathepsin cDNA clone were characterized and analysed phylogenetically. Disregarding cathepsin O, whose phylogenetic position is uncertain, the analysis confirms the existence of two old lines of descent, the B and the L lineages of cathepsins, which diverged from each other early in the evolution of eucaryotes. The B lineage encompasses cathepsins B, C and Z (X). The L lineage splits off sublineages encompassing cathepsins F and W before the plant-animal separation and cathepsin H early in the evolution of the metazoa. The remaining cathepsins belonging to the L lineage diverged from one another during the evolution of vertebrates: S, K and L before the emergence of bony fishes, and the group of rodent placentally expressed cathepsins [J (P), M, Q, R, 3, 6, 7 and 8] as well as the testis/ova-expressed cathepsins (testins) probably after the divergence of rodents from primates. The part possibly played by the adaptive immune system in some of these divergences is discussed.

Conservation of Mhc class III region synteny between zebrafish and human as determined by radiation hybrid mapping.

In the HLA, H2, and other mammalian MHC:, the class I and II loci are separated by the so-called class III region comprised of approximately 60 genes that are functionally and evolutionarily unrelated to the class I/II genes. To explore the origin of this island of unrelated loci in the middle of the MHC: 19 homologues of HLA class III genes, we identified 19 homologues of HLA class III genes as well as 21 additional non-class I/II HLA homologues in the zebrafish and mapped them by testing a panel of 94 zebrafish-hamster radiation hybrid cell lines. Six of the HLA class III and eight of the flanking homologues were found to be linked to the zebrafish class I (but not class II) loci in linkage group 19. The remaining homologous loci were found to be scattered over 14 zebrafish linkage groups. The linkage group 19 contains at least 25 genes (not counting the class I loci) that are also syntenic on human chromosome 6. This gene assembly presumably represents the pre-MHC: that existed before the class I/II genes arose. The pre-MHC: may not have contained the complement and other class III genes involved in immune response.

Mhc class II B gene evolution in East African cichlid fishes.

A distinctive feature of essential major histocompatibility complex (Mhc) loci is their polymorphism characterized by large genetic distances between alleles and long persistence times of allelic lineages. Since the lineages often span several successive speciations, we investigated the behavior of the Mhc alleles during or close to the speciation phase. We sequenced exon 2 of the class II B locus 4 from 232 East African cichlid fishes representing 32 related species. The divergence times of the (sub)species ranged from 6,000 to 8.4 million years. Two types of evolutionary analysis were used to elucidate the pattern of exon 2 sequence divergence. First, phylogenetic methods were applied to reconstruct the most likely evolutionary pathways leading from the last common ancestor of the set to the extant sequences, and to assess the probable mechanisms involved in allelic diversification. Second, pairwise comparisons of sequences were carried out to detect differences seemingly incompatible with origin by nonparallel point mutations. The analysis revealed point mutations to be the most important mechanism behind allelic divergences, with recombination playing only an auxiliary part. Comparison of sequences from related species revealed evidence of random allelic (lineage) losses apparently associated with speciation. Sharing of identical alleles could be demonstrated between species that diverged 2 million years ago. The phylogeny of the exon was incongruent with that of the flanking introns, indicating either a high degree of convergent evolution at the peptide-binding region-encoding sites, or intron homogenization.

The origin and age of haplochromine fishes in Lake Victoria, east Africa.

According to a widely held view, the more than 300 species of haplochromine cichlid fishes in Lake Victoria (LV), East Africa, originated from a single founder species in less than 12,000 years. This view, however, does not follow from the published geological and molecular evidence. The former does indeed suggest that the LV basin dried out less than 15,000 years ago, but it does not provide any information about the species that re-colonized the new lake or that remained in the rivers draining the area. The molecular evidence is inconclusive with respect to the origin of the LV haplochromines because cichlids from critical regions around LV were not adequately sampled; and as far as the age of the LV haplochromines is concerned, it in fact led to an estimate of 250,000-750,000 years old. In the present study, mitochondrial DNA (control region) variation was determined by heteroduplex and sequencing analyses of more than 670 specimens collected at widely distributed East African riverine and lacustrine localities. The analyses revealed the existence of seven haplogroups (I-VII) distinguishable by characteristic substitutions. All endemic LV samples tested fell into one of these haplogroups (V) which, however, was also found to be present at various other localities, both riverine and lacustrine, outside LV. Within this haplogroup, four subgroups (VA through VD) could be distinguished, two of which (VB and VC) were represented in LV and at other localities. The great majority of the LV haplochromine species could be classified as belonging to the VC subgroup, which was found only in LV and in the rivers draining into it. Hence, while the endemic haplochromine species of LV could not have originated from a single founding population, the lake does harbour a large species flock which probably arose in situ.

A molecular phylogenetic framework for the Ryukyu endemic rodents Tokudaia osimensis and Diplothrix legata.

Diplothrix legata and Tokudaia osimensis are rodent species endemic to the Ryukyu Islands (Okinawa area) of the southern part of Japan. To place them within a phylogenetic framework of Asian rodent species, we examined the molecular relationships among these species and Apodemus, Micromys, Mus, and Rattus, using an analysis of mitochondrial cytochrome b gene (1140 bp) and nuclear IRBP gene (782 bp) sequences. The results indicate that the four main murine genera and Tokudaia diverged at similar evolutionary times, namely at the time of the radiation of Murinae, 14 to 40 million years ago, depending on different concepts for the divergence times of Rattus and Mus. In contrast, Diplothrix separated from the Rattus group more recently. Our results indicate that D. legata and the Rattus group diverged at 20-30% of the divergence time of Rattus and Mus under the assumption of the molecular clock, suggesting that D. legata established its lineage at least by the beginning of the Pleistocene era. It is thus evident that the Okinawa area preserves rare indigenous species with various levels of genetic endemicity.

Correct and incorrect vertebrate phylogenies obtained by the entire mitochondrial DNA sequences.

Concatenated sequences of all protein-coding genes in mitochondria recovered a known phylogeny of 11 vertebrate species correctly with statistical significance. However, when it was rooted by lampreys or sea urchins, the root of the vertebrate tree was placed between the mammal cluster and the chicken-frog-fish cluster or between the mammal-chicken cluster and the frog-fish cluster, depending on the tree-making method used. Although the frog-fish or chicken-frog-fish cluster was biologically incorrect, it was again supported with a significantly high bootstrap value. In this study, we investigated the reasons why this happened. It has been suggested that an incorrect phylogeny may be constructed due to a change of amino acid composition in different lineages or due to homoplasies at sites with hydrophobic amino acids. However, our results indicated that these were not the causes of the incorrect rooting of the vertebrate tree. Rather, it was important to take into account an extensive rate variation across sites and different probabilities of substitution among different amino acids. The substitution rates for mitochondrial sequences vary considerably for different vertebrate lineages. In such a case, it is known to be important to use the model that reflects the actual substitution probability to obtain a correct tree topology. The correct rooting of the vertebrate tree was recovered when rate variation across sites was properly accounted for.

Tie trees generated by distance methods of phylogenetic reconstruction.

In examining genetic data in recent publications, Backeljau et al. showed cases in which two or more different trees (tie trees) were constructed from a single data set for the neighbor-joining (NJ) method and the unweighted pair group method with arithmetic mean (UPGMA). However, it is still unclear how often and under what conditions tie trees are generated. Therefore, I examined these problems by computer simulation. Examination of cases in which tie trees occur shows that tie trees can appear when no substitutions occur along some interior branch(es) on a tree. However, even when some substitutions occur along interior branches, tie trees can appear by chance if parallel or backward substitutions occur at some sites. The simulation results showed that tie trees occur relatively frequently for sequences with low divergence levels or with small numbers of sites. For such data, UPGMA sometimes produced tie trees quite frequently, whereas tie trees for the NJ method were generally rare. In the simulation, bootstrap values for clusters (tie clusters) that differed among tie trees were mostly low (< 60%). With a small probability, relatively high bootstrap values (at most 70%-80%) appeared for tie clusters. The bias of the bootstrap values caused by an input order of sequence can be avoided if one of the different paths in the cycles of making an NJ or UPGMA tree is chosen at random in each bootstrap replication.

Genetic distances and reconstruction of phylogenetic trees from microsatellite DNA.

Recently many investigators have used microsatellite DNA loci for studying the evolutionary relationships of closely related populations or species, and some authors proposed new genetic distance measures for this purpose. However, the efficiencies of these distance measures in obtaining the correct tree topology remains unclear. We therefore investigated the probability of obtaining the correct topology (PC) for these new distances as well as traditional distance measures by using computer simulation. We used both the infinite-allele model (IAM) and the stepwise mutation model (SMM), which seem to be appropriate for classical markers and microsatellite loci, respectively. The results show that in both the IAM and SMM CAVALLI-SFORZA and EDWARDS' chord distance (DC) and NEI et al.'s DA distance generally show higher PC values than other distance measures, whether the bottleneck effect exists or not. For estimating evolutionary times, however, NEI's standard distance and GOLDSTEIN et al.'s (delta mu)2 are more appropriate than other distances. Microsatellite DNA seems to be very useful for clarifying the evolutionary relationships of closely related populations.

Efficiencies of different genes and different tree-building methods in recovering a known vertebrate phylogeny.

The relative efficiencies of different protein-coding genes of the mitochondrial genome and different tree-building methods in recovering a known vertebrate phylogeny (two whale species, cow, rat, mouse, opossum, chicken, frog, and three bony fish species) was evaluated. The tree-building methods examined were the neighbor joining (NJ), minimum evolution (ME), maximum parsimony (MP), and maximum likelihood (ML), and both nucleotide sequences and deduced amino acid sequences were analyzed. Generally speaking, amino acid sequences were better than nucleotide sequences in obtaining the true tree (topology) or trees close to the true tree. However, when only first and second codon positions data were used, nucleotide sequences produced reasonably good trees. Among the 13 genes examined, Nd5 produced the true tree in all tree-building methods or algorithms for both amino acid and nucleotide sequence data. Genes Cytb and Nd4 also produced the correct tree in most tree-building algorithms when amino acid sequence data were used. By contrast, Co2, Nd1, and Nd41 showed a poor performance. In general, large genes produced better results, and when the entire set of genes was used, all tree-building methods generated the true tree. In each tree-building method, several distance measures or algorithms were used, but all these distance measures or algorithms produced essentially the same results. The ME method, in which many different topologies are examined, was no better than the NJ method, which generates a single final tree. Similarly, an ML method, in which many topologies are examined, was no better than the ML star decomposition algorithm that generates a single final tree. In ML the best substitution model chosen by using the Akaike information criterion produced no better results than simpler substitution models. These results question the utility of the currently used optimization principles in phylogenetic construction. Relatively simple methods such as the NJ and ML star decomposition algorithms seem to produce as good results as those obtained by more sophisticated methods. The efficiencies of the NJ, ME, MP, and ML methods in obtaining the correct tree were nearly the same when amino acid sequence data were used. The most important factor in constructing reliable phylogenetic trees seems to be the number of amino acids or nucleotides used.

The root of the phylogenetic tree of human populations.

Although African populations have been shown to be most divergent from any other human populations, it has been difficult to establish the root of the phylogenetic tree of human populations since the rate of evolutionary change may vary from population to population owing to the fluctuation of population size and other factors. However, the root can be determined by using the chimpanzee as an outgroup and by employing proper statistical methods. Using this strategy, we constructed phylogenetic trees of human populations for five different sets of gene frequency data. The data sets used were two sets of microsatellite loci data (25 and 8 loci, respectively), restriction fragment length polymorphism (RFLP) data (79 loci), protein polymorphism data (15 loci), and Alu insertion frequency data (4 loci). All these data sets showed that the root is located in the branch connecting African and non-African populations, and in the four data sets the root was established at a significant level. These results indicate that Africans are the first group of people that split from the rest of the human populations.

Phylogenetic test of the molecular clock and linearized trees.

To estimate approximate divergence times of species or species groups with molecular data, we have developed a method of constructing a linearized tree under the assumption of a molecular clock. We present two tests of the molecular clock for a given topology: two-cluster test and branch-length test. The two-cluster test examines the hypothesis of the molecular clock for the two lineages created by an interior node of the tree, whereas the branch-length test examines the deviation of the branch length between the tree root and a tip from the average length. Sequences evolving excessively fast or slow at a high significance level may be eliminated. A linearized tree will then be constructed for a given topology for the remaining sequences under the assumption of rate constancy. We have used these methods to analyze hominoid mitochondrial DNA and drosophilid Adh gene sequences.

Evolutionary history of the COII/tRNALys intergenic 9 base pair deletion in human mitochondrial DNAs from the Pacific.

Length changes in human mitochondrial DNA (mtDNA) are potentially useful markers for inferring the evolutionary history of populations. One such length change is a nine base pair (9-bp) deletion that is located in the intergenic region between the COII gene and the Lysine tRNA gene (COII/tRNALys intergenic region). This deletion has been used as a genetic marker to trace descent from peoples of East Asian origin. A geographic cline of the deletion frequency across modern Pacific Islander populations suggests that the deletion may be useful for tracing prehistoric Polynesian origins and affinities. Mitochondrial DNA sequence variation within two variable segments of the control region (CR) permits a number of inferences regarding the evolutionary history of the 9-bp deletion that cannot be determined from frequency data alone. We obtained CR sequences from 74 mtDNAs with the 9-bp deletion from Indonesia, coastal Papua New Guinea (PNG), and American Samoa. Phylogenetic and pairwise distribution analysis of these CR sequences pooled with previously published CR sequences reveals that the deletion arose independently in Africa and Asia and suggests possible multiple origins of the deletion in Asia. A clinal increase of the frequency of the 9-bp deletion across the three Pacific populations is associated with a decrease in CR sequence diversity, consistent with founder events. Furthermore, analysis of pairwise difference distributions indicates an expansion time of proto-Polynesians that began 5,500 yr ago from Southeast Asia. These results are consistent with the express train model of Polynesian origins.

Molecular phylogeny and divergence times of drosophilid species.

The phylogenetic relationships and divergence times of 39 drosophilid species were studied by using the coding region of the Adh gene. Four genera--Scaptodrosophila, Zaprionus, Drosophila, and Scaptomyza (from Hawaii)--and three Drosophila subgenera--Drosophila, Engiscaptomyza, and Sophophora--were included. After conducting statistical analyses of the nucleotide sequences of the Adh, Adhr (Adh-related gene), and nuclear rRNA genes and a 905-bp segment of mitochondrial DNA, we used Scaptodrosophila as the outgroup. The phylogenetic tree obtained showed that the first major division of drosophilid species occurs between subgenus Sophophora (genus Drosophila) and the group including subgenera Drosophila and Engiscaptomyza plus the genera Zaprionus and Scaptomyza. Subgenus Sophophora is then divided into D. willistoni and the clade of D. obscura and D. melanogaster species groups. In the other major drosophilid group, Zaprionus first separates from the other species, and then D. immigrans leaves the remaining group of species. This remaining group then splits into the D. repleta group and the Hawaiian drosophilid cluster (Hawaiian Drosophila, Engiscaptomyza, and Scaptomyza). Engiscaptomyza and Scaptomyza are tightly clustered. Each of the D. repleta, D. obscura, and D. melanogaster groups is monophyletic. The splitting of subgenera Drosophila and Sophophora apparently occurred about 40 Mya, whereas the D. repleta group and the Hawaiian drosophilid cluster separated about 32 Mya. By contrast, the splitting of Engiscaptomyza and Scaptomyza occurred only about 11 Mya, suggesting that Scaptomyza experienced a rapid morphological evolution. The D. obscura and D. melanogaster groups apparently diverged about 25 Mya. Many of the D. repleta group species studied here have two functional Adh genes (Adh-1 and Adh-2), and these duplicated genes can be explained by two duplication events.

Inconsistency of the maximum parsimony method when the rate of nucleotide substitution is constant.

The inconsistency of the maximum parsimony method is known to occur even when the rate of nucleotide substitution is constant. To understand why this inconsistency occurs, a mathematical study was conducted for the cases of five, six, and seven sequences. The results obtained indicate that this inconsistency occurs because the probability of occurrence of nucleotide configurations generated by one substitution on a short interior branch is often lower than that of configurations generated by more substitutions on other longer branches. The chance of occurrence of this event--or, the inconsistency of the maximum parsimony method--apparently increases as the number of sequences increases. The inconsistency may occur even when the extent of sequence divergence is relatively small.

Relative efficiencies of the maximum-likelihood, neighbor-joining, and maximum-parsimony methods when substitution rate varies with site.

The relative efficiencies of the maximum-likelihood (ML), neighbor-joining (NJ), and maximum-parsimony (MP) methods in obtaining the correct topology and in estimating the branch lengths for the case of four DNA sequences were studied by computer simulation, under the assumption either that there is variation in substitution rate among different nucleotide sites or that there is no variation. For the NJ method, several different distance measures (Jukes-Cantor, Kimura two-parameter, and gamma distances) were used, whereas for the ML method three different transition/transversion ratios (R) were used. For the MP method, both the standard unweighted parsimony and the dynamically weighted parsimony methods were used. The results obtained are as follows: (1) When the R value is high, dynamically weighted parsimony is more efficient than unweighted parsimony in obtaining the correct topology. (2) However, both weighted and unweighted parsimony methods are generally less efficient than the NJ and ML methods even in the case where the MP method gives a consistent tree. (3) When all the assumptions of the ML method are satisfied, this method is slightly more efficient than the NJ method. However, when the assumptions are not satisfied, the NJ method with gamma distances is slightly better in obtaining the correct topology than is the ML method. In general, the two methods show more or less the same performance. The NJ method may give a correct topology even when the distance measures used are not unbiased estimators of nucleotide substitutions. (4) Branch length estimates of a tree with the correct topology are affected more easily than topology by violation of the assumptions of the mathematical model used, for both the ML and the NJ methods. Under certain conditions, branch lengths are seriously overestimated or underestimated. The MP method often gives serious underestimates for certain branches. (5) Distance measures that generate the correct topology, with high probability, do not necessarily give good estimates of branch lengths. (6) The likelihood-ratio test and the confidence-limit test, in Felsenstein's DNAML, for examining the statistical of branch length estimates are quite sensitive to violation of the assumptions and are generally too liberal to be used for actual data. Rzhetsky and Nei's branch length test is less sensitive to violation of the assumptions than is Felsenstein's test. (7) When the extent of sequence divergence is < or = 5% and when > or = 1,000 nucleotides are used, all three methods show essentially the same efficiency in obtaining the correct topology and in estimating branch lengths.(ABSTRACT TRUNCATED AT 400 WORDS)