Takashi Kitano

Mitochondrial DNA sequence phylogeny of 4 populations of the widely distributed cynomolgus macaque (Macaca fascicularis fascicularis).

We studied the mitochondrial DNA (mtDNA) polymorphism of 304 Macaca fascicularis fascicularis (M. f. fascicularis) individuals, representative of 4 cynomolgus macaque populations (Indochina, Indonesia, Philippines, and Mauritius). By sequencing a 590-bp fragment in the hypervariable II region of the D-loop region, we defined 70 haplotypes. The homologous region was also characterized in 22 Chinese Macaca mulatta and 2 Macaca sylvanus. The phylogenetic analysis confirms the monophyly of M. f. fascicularis and defines 2 haplotype groups inside the M. f. fascicularis clade: one insular, encompassing 6 Philippines, 2 Mauritius, and 31 Indonesian haplotypes, the other continental that contains all Indochinese and 6 Indonesian haplotypes. Continental and insular group divergence time was estimated to be approximately 10(6) years before present (BP). Among Indonesian haplotypes, some have a continental origin. This suggests either direct migration from mainland to Indonesia or that remnant lineages from an ancient population genetically close to the mainland (i.e., in the Sunda Shelf, <550 000 years BP) were subsequently brought southward to Indonesia. The low nucleotide diversity in the Philippines population suggests a bottleneck following colonization by Indonesian individuals, around 110 000 years BP. mtDNA and further observations of nuclear genetic data corroborate the mixed origin (Indonesian/continental) hypothesis of Mauritius individuals and a population bottleneck.

Mitochondrial DNA analysis of Hokkaido Jomon skeletons: Remnants of archaic maternal lineages at the southwestern edge of former Beringia

To clarify the colonizing process of East/Northeast Asia as well as the peopling of the Americas, identifying the genetic characteristics of Paleolithic Siberians is indispensable. However, no genetic information on the Paleolithic Siberians has hitherto been reported. In the present study, we analyzed ancient DNA recovered from Jomon skeletons excavated from the northernmost island of Japan, Hokkaido, which was connected with southern Siberia in the Paleolithic period. Both the control and coding regions of their mitochondrial DNA (mtDNA) were analyzed in detail, and we confidently assigned 54 mtDNAs to relevant haplogroups. Haplogroups N9b, D4h2, G1b, and M7a were observed in these individuals, with N9b being the predominant one. The fact that all these haplogroups, except M7a, were observed with relatively high frequencies in the southeastern Siberians, but were absent in southeastern Asian populations, implies that most of the Hokkaido Jomon people were direct descendants of Paleolithic Siberians. The coalescence time of N9b (ca. 22,000 years) was before or during the last glacial maximum, implying that the initial trigger for the Jomon migration in Hokkaido was increased glaciations during this period. Interestingly, Hokkaido Jomons lack specific haplogroups that are prevailing in present-day native Siberians, implying that diffusion of these haplogroups in Siberia might have been after the beginning of the Jomon era, about 15,000 years before present.

Molecular evolution of the oxytocin-oxytocin receptor system in eutherians.

Oxytocin (OXT) is a nine-amino-acid peptide hormone that is mainly released at the times of uterine contractions during parturition and milk ejection during lactation, whereas a similar peptide hormone, arginine vasopressin, primarily exerts direct antidiuretic action on the kidney and causes vasoconstriction of the peripheral vessels. The genes coding for these peptides are tandemly located on the same chromosome. A tandem duplication occurring in the common ancestor of jawed vertebrates has been proposed as responsible. In contrast to the two peptide hormones, only one oxytocin receptor (OXTR) but three arginine vasopressin receptors (AVPR1A, AVPR1B, and AVPR2) are known; these receptors probably arose from two rounds of genome duplication in the common ancestor of vertebrates. In this study, we addressed the molecular evolution of the OXT-OXTR system in eutherians. Our analyses suggest that an amino acid change from isoleucine to lysine on the eighth site (I8L) of the peptide, which corresponded to a change from mesotocin to OXT, had occurred during the common ancestral lineage of eutherians. At around the same time that the emergence of OXT occurred, functional constraints on the OXT receptor (pre-OXTR) might have relaxed, and a series of nonsynonymous substitutions might have accumulated. Only a few of these nonsynonymous substitutions might have contributed to reestablishing the molecular relationship between the OXT ligand and its receptor, after which functional constraints on the OXTR were reinstated. Since the OXT-OXTR system plays an important role in eutherians, the evolution of the OXT-OXTR system was probably an essential component of the genesis of the eutherian signature.

The Functional A Allele Was Resurrected via Recombination in the Human ABO Blood Group Gene

Functional A and B alleles are distinguished at two critical sites in exon 7 of the human ABO blood group gene. The most frequent nonfunctional O alleles have one-base deletion in exon 6 producing a frameshift, and it has the A type signature in two critical sites in exon 7. Previous studies indicated that B and O alleles were derived from A allele in human lineage. In this study, we conducted a phylogenetic network analysis using six representative haplotypes: A101, A201, B101, O01, O02, and O09. The result indicated that the A allele, possibly once extinct in the human lineage a long time ago, was resurrected by a recombination between B and O alleles less than 300,000 years ago.

Phylogenetic positions of RH blood group-related genes in cyclostomes.

The RH gene family in vertebrates consists of four major genes (RH, RHAG, RHBG, and RHCG). They are thought to have emerged in the common ancestor of vertebrates after two rounds of whole genome duplication (2R-WGD). To analyze the detailed phylogenetic relationships within the RH gene family, we determined three types of cDNA sequence that belong to the RH gene family in lamprey (Lethenteron reissneri) and designated them as RHBG-like, RHCG-like1, and RHCG-like2. Phylogenetic analyses clearly showed that RHCG-like1 and RHCG-like2 genes, which were probably duplicated in the lamprey lineage, are orthologs of gnathostome RHCG. In contrast, the clear phylogenetic position of the RHBG-like gene could not be obtained. Probably some convergent events for cyclostome RHBG-like genes prevented the accurate identification of their phylogenetic positions.

Evolution of the RH gene family in vertebrates revealed by brown hagfish (Eptatretus atami) genome sequences

In vertebrates, there are four major genes in the RH (Rhesus) gene family, RH, RHAG, RHBG, and RHCG. These genes are thought to have been formed by the two rounds of whole-genome duplication (2R-WGD) in the common ancestor of all vertebrates. In our previous work, where we analyzed details of the gene duplications process of this gene family, three nucleotide sequences belonging to this family were identified in Far Eastern brook lamprey (Lethenteron reissneri), and the phylogenetic positions of the genes were determined. Lampreys, along with hagfishes, are cyclostomata (jawless fishes), which is a sister group of gnathostomata (jawed vertebrates). Although those results suggested that one gene was orthologous to the gnathostome RHCG genes, we did not identify clear orthologues for other genes. In this study, therefore, we identified three novel cDNA sequences that belong to the RH gene family using de novo transcriptome analysis of another cyclostome: the brown hagfish (Eptatretus atami). We also determined the nucleotide sequences for the RHBG and RHCG genes in a red stingray (Dasyatis akajei), which belongs to the cartilaginous fishes. The phylogenetic tree showed that two brown hagfish genes, which were probably duplicated in the cyclostome lineage, formed a cluster with the gnathostome RHAG genes, whereas another brown hagfish gene formed a cluster with the gnathostome RHCG genes. We estimated that the RH genes had a higher evolutionary rate than the RHAG, RHBG, and RHCG genes. Interestingly, in the RHBG genes, only the bird lineage showed a higher rate of nonsynonymous substitutions. It is likely that this higher rate was caused by a state of relaxed functional constraints rather than positive selection nor by pseudogenization.

No Distinction of Orthology/Paralogy between Human and Chimpanzee Rh Blood Group Genes

On human (Homo sapiens) chromosome 1, there is a tandem duplication encompassing Rh blood group genes (Hosa_RHD and Hosa_RHCE). This duplication occurred in the common ancestor of humans, chimpanzees (Pan troglodytes), and gorillas, after splitting from their common ancestor with orangutans. Although several studies have been conducted on ape Rh blood group genes, the clear genome structures of the gene clusters remain unknown. Here, we determined the genome structure of the gene cluster of chimpanzee Rh genes by sequencing five BAC (Bacterial Artificial Chromosome) clones derived from chimpanzees. We characterized three complete loci (Patr_RHα, Patr_RHβ, and Patr_RHγ). In the Patr_RHβ locus, a short version of the gene, which lacked the middle part containing exons 4–8, was observed. The Patr_RHα and Patr_RHβ genes were located on the locations corresponding to Hosa_RHD and Hosa_RHCE, respectively, and Patr_RHγ was in the immediate vicinity of Patr_RHβ. Sequence comparisons revealed high sequence similarity between Patr_RHβ and Hosa_RHCE, while the chimpanzee Rh gene closest to Hosa_RHD was not Patr_RHα but rather Patr_RHγ. The results suggest that rearrangements and gene conversions frequently occurred between these genes and that the classic orthology/paralogy dichotomy no longer holds between human and chimpanzee Rh blood group genes.

The PNarec method for detection of ancient recombinations through phylogenetic network analysis

Recombinations are known to disrupt bifurcating tree structure of gene genealogies. Although recently occurred recombinations are easily detectable by using conventional methods, recombinations may have occurred at any time. We devised a new method for detecting ancient recombinations through phylogenetic network analysis, and detected five ancient recombinations in gibbon ABO blood group genes [Kitano et al., 2009. Mol. Phylogenet. Evol., 51, 465-471]. We present applications of this method, now named as PNarec, to various virus sequences as well as HLA genes.

Application of Phylogenetic Network

A phylogenetic network is a generalization of the concept of a phylogenetic tree. In contrast to a phylogenetic tree, a phylogenetic network can show incompatible phylogenetic information on a single diagram using reticulations. Phylogenetic network methods would be helpful to analyze genes with complex evolutionary histories such as recombination, hybridization, and gene conversion. In this study, phylogenetic network analyses using mtDNAs of hominoids, OXTR genes of hominoids, and ABO genes of gibbons (three different data sets) are described with examples. Furthermore, how a recombination event is illustrated in a phylogenetic network is explained using hypothetical data. The phylogenetic network revealed the following pattern: a recombinant allele had a short external branch and was located on a diagonal with the outgroup allele and the two parental alleles were located on another diagonal with long external branches.

An N-acetyllactosamine-specific lectin, PFA, isolated from a moth (Phalera flavescens), structurally resembles an invertebrate-type lysozyme.

PFA (Phalera flavescens agglutinin) lectin purified from larvae of the lobster moth (P. flavescens) shows a strong binding ability specific to the N-acetyllactosamine (Galβ1-4GlcNAc) site. We determined the genomic and cDNA sequences of the PFA gene, which consists of five exons and spans approximately 5 kb of a genomic region. Surprisingly, the amino acid sequence (149 amino acids) was similar to invertebrate-type lysozymes and related proteins. The predicted tertiary structure of the PFA protein was similar to the lysozymes of clams such as the common orient clam (Meretrix lusoria) and Japanese littleneck (Venerupis philippinarum (Tapes japonica)). The PFA, however, lacks a catalytically essential amino acid, an Asp (D), which is one of the two important amino acids (Glu (E) and D) express the function of lysozyme. As a result, lysozyme activity assays indicated that PFA does not have lysozyme activity. Results suggest that the PFA gene evolved from a lysozyme gene through the loss of lysozyme activity sites and the acquisition of lectin activity during evolution of the genus Phalera.