Integrating mitochondrial and nuclear genomic data to decipher the evolutionary history of Eubranchipus species in Japan.

Understanding the genetic diversity and evolutionary history of species is crucial for their conservation and management. In this study, we investigated the genetic diversity and phylogenetic relationships among Eubranchipus species occurring in Japan. Phylogenetic analyses revealed that nuclear and mitochondrial data yield incompatible results. In E. uchidai, nuclear data support the monophyly of the Shimokita area, while mitochondrial data indicate a clustering of Higashidori2 individuals with Hokkaido (Ishikari and Wakkanai) E. uchidai. Similar incongruences were observed in E. hatanakai, where nuclear data favor the monophyly of the Chokai area, while mitochondrial data cluster some Chokai pool 3 individuals with Aizu individuals. These incompatibilities might be caused by mitochondrial gene flow. The findings emphasize the importance of considering both nuclear and mitochondrial data during phylogenetic studies and provide valuable insights into the complex dynamics of migration and genetic exchange in Eubranchipus species.

Pseudogenization of the Hair-Related Genes PADI3 and S100A3 in Cetaceans and Hippopotamus amphibius.

Hair-related genes in mammals play important roles in the development and maintenance of hair and other keratinous structures in mammals. The peptidyl arginine deiminase 3 (PADI3) gene encodes an enzyme that catalyzes the conversion of arginine residues to citrulline. The S100 calcium binding protein A3 (S100A3) gene encodes a protein that is highly expressed in the hair cuticle and contains arginine residues that are converted to citrullines by PADI enzymes. In this study, we investigated the pseudogenization events of PADI3 and S100A3 in cetaceans and Hippopotamus amphibius. We found that PADI3 underwent three independent pseudogenization events during cetacean evolution, in baleen whales, toothed cetaceans other than Physeter catodon, and P. catodon. Notably, the entire PADI3 gene is absent in the baleen whales. Pseudogenization of S100A3 occurred independently in cetaceans and H. amphibius. Interestingly, we found that in cetaceans S100A3 underwent pseudogenization before PADI3, suggesting that differential selection pressures were acting on the two genes. Our findings provide valuable insights into the molecular evolution of these genes in cetaceans and hippopotamuses, highlighting their importance for understanding the evolution of hair-related genes.

Complete mitochondrial genomes of three fairy shrimps from snowmelt pools in Japan.

BACKGROUND: Fairy shrimps belong to order Anostraca, class Branchiopoda, subphylum Crustacea, and phylum Arthropoda. Three fairy shrimp species (Eubranchipus uchidai, E. asanumai, and E. hatanakai) that inhabit snowmelt pools are currently known in Japan. Whole mitochondrial genomes are useful genetic information for conducting phylogenetic analyses. Mitochondrial genome sequences for Branchiopoda members are gradually being collated. RESULTS: Six whole mitochondrial genomes from the three Eubranchipus species are presented here. Eubranchipus species share the anostracan pattern of gene arrangement in their mitochondrial genomes. The mitochondrial genomes of the Eubranchipus species have a higher GC content than those of other anostracans. Accelerated substitution rates in the lineage of Eubranchipus species were observed. CONCLUSION: This study is the first to obtain whole mitochondrial genomes for Far Eastern Eubranchipus species. We show that the nucleotide sequences of cytochrome oxidase subunit I and the 16S ribosomal RNA of E. asanumai presented in a previous study were nuclear mitochondrial DNA segments. Higher GC contents and accelerated substitution rates are specific characteristics of the mitochondrial genomes of Far Eastern Eubranchipus. The results will be useful for further investigations of the evolution of Anostraca as well as Branchiopoda.

Identification and Characterization of Genes for the Allo A Lectins in Japanese Rhinoceros Beetle (Trypoxylus dichotomus [Allomyrina dichotoma]).

The Japanese rhinoceros beetle (Trypoxylus dichotomus [Allomyrina dichotoma]) produces the lectins allo A-I and allo A-II, which have strong N-acetyllactosamine (Galß1-4GlcNAc)-binding activity. It has been suggested that the two lectins are formed from three subunits (α, ß, and γ), with allo A-I comprising α and γ subunits and allo A-II comprising ß and γ subunits. Here, we determined the cDNA sequences of these subunits using both conventional polymerase chain reaction (PCR)-cloning-sequencing and transcriptome-sequencing analyses. For the α and ß subunits, one gene (locus) for each was predicted, whereas for the γ subunit, two types of cDNA sequences were obtained, which we named γ1 and γ2. These two types probably have distinct loci. Average nucleotide sequence identities among the subunits ranged from 87.6% (between α and γ1) to 92.6% (between γ1 and γ2), suggesting that they form a gene family. Although no homology was found between the sequences of allo A and other known lectin proteins in a protein database search, some unknown proteins containing the DUF3421 domain were identified. Those DUF3421 domain-encoding proteins are upregulated in the insect larval midgut. Thus, we infer that allo A genes also play an important role in larvae and that their lectin activity may have been obtained collaterally.

Genetic diversity of two populations of the tufted puffin Fratercula cirrhata (Pallas, 1769).

The tufted puffin Fratercula cirrhata (Charadriiformes: Alcidae) is distributed throughout the boreal and low Arctic areas of the North Pacific, from California, USA to Hokkaido, Japan. Few studies have investigated the genetic diversity of this species. Therefore, we analyzed the genetic diversity of two captive populations using nucleotide sequences of two mitochondrial loci (COX1 and D-loop) and one nuclear locus (RHBG). We sequenced these loci for birds from Tokyo Sea Life Park (Kasai Rinkai Suizokuen), originally from Alaska, and birds from Aqua World Oarai, originally from far eastern Russia. We found five COX1 haplotypes and 17 D-loop haplotypes for the mitochondrial loci, and obtained 14 predicted haplotypes for the nuclear RHBG locus. The major haplotypes of all three loci occurred in individuals from both populations. Thus, there were no clear genetic differences between the populations with respect to these three loci. Although the breeding range of the tufted puffin covers the boreal and low Arctic from California to Hokkaido, our results suggest that the species has not genetically diverged within its breeding range.

Nucleotide sequencing of the HoxA gene cluster using Gorilla fosmid clones.

We sequenced the western gorilla (Gorilla gorilla) HoxA cluster region using seven fosmid clones, and found that the total tiling path sequence was 214,185 bp from the 5' non-genic region of HoxA1 to the 3' non-genic region of Evx1. We compared the nucleotide sequence with the gorilla genome sequence in the NCBI database, and the overall proportion of nucleotide difference was estimated to be 0.0005-0.0007. These estimates are lower than overall genomic polymorphism in gorillas.

Behavior of eukaryotic symbionts in large benthic foraminifers Calcarina gaudichaudii and Baculogypsina sphaerulata under exposure to wastewater.

Large benthic foraminifers (LBFs) are significant contributors to coral island formation in the Pacific Ocean. In recent years, the population of LBFs has decreased because of the increase in anthropogenic influences, such as wastewater (WW) discharge. To implement efficient mitigation measures, pollution tolerance in LBFs should be understood. However, the effects of WW on LBFs and their symbionts have not yet been demonstrated. This study examined the changes in the photosynthetic efficiency (Y[II]) of Calcarina gaudichaudii and Baculogypsina sphaerulata in response to WW by using a pulse-amplitude-modulation fluorometer. These LBFs were exposed to WW with different dilution levels for 22 days. The Y(II) values of the LBFs were found to deteriorate within 1-2 days. However, the Y(II) values both deteriorated and were enhanced in the experiments, thus indicating that WW contains both harmful and beneficial components. Baculogypsina sphaerulata showed an earlier response and greater sensitivity to WW and a higher epibiont infestation than C. gaudichaudii. This result can be attributed to the differences in the physiological and morphological responses of distinct LBFs. A sequencing analysis of 18S rDNA confirmed that the dominant eukaryotic symbionts in the two LBFs studied were Ochrophyta and Labyrinthulomycetes. These eukaryotic symbionts were released and attached as epibionts onto LBFs that were exposed to WW, thus leading to an increase in inactive LBFs. The Shannon-Weaver and Simpson diversity indices revealed that eukaryotic symbiont communities decreased in biodiversity after exposure to WW because of the abundance of algal symbionts. On the basis of these results, we conclude that WW, even with 10,000 × dilution, causes a decrease in active LBF populations owing to the release of eukaryotic symbionts, the decrease in biodiversity, and the infestation of epibionts even though Y(II) is temporarily enhanced. These responses are more significant in B. sphaerulata than in C. gaudichaudii.

Evolution of S100A3 and PAD3, two important genes for mammalian hair.

Hair is one of the defining characteristics of mammals. The hair shaft has a two-layer structure comprising the cortex, which is the inner layer and is composed of cortical cells, and the cuticle, which is the outermost layer. S100 calcium-binding protein A3 (S100A3) is expressed at high levels in the human hair cuticle. Arginine 51 of S100A3 protein is citrullinated specifically by peptidylarginine deiminase 3 (PAD3), and this citrullination is related to maturation of the cuticle. However, the detailed evolutionary processes of S100A3 and PAD3 during mammalian evolution are unknown. Here, we show that nonsynonymous changes in S100A3 accelerated in the common ancestral branch of mammals, probably as a result of positive selection that returned after the acquisition of hair cuticle-specific function in mammals. Later, pseudogenisation or nonfunctionalisation of S100A3 and PAD3 occurred in some species, such as the cetaceans. Our results show that positive selection and relaxation of the functional constraints of genes played important roles in the evolution of mammalian hair.

Brown hagfish from the northwest and east coasts of Honshu, Japan are genetically different.

The brown hagfish (Eptatretus atami) is one of several known hagfish species occurring in Japanese coastal waters. To date, there has been no research studying genetic polymorphisms in the species. In the present study, we analyzed differences in nucleotide sequences between two populations: one from Suruga Bay on the Pacific coast of Honshu, Japan, and the other from the Sea of Japan, off Akita on the northwest coast of Honshu. We sequenced part of the cytochrome oxidase subunit 1 gene (COX1) from the mitochondrial genome, and three G protein-coupled receptor genes from the nuclear genome. Phylogenetic networks of all four genes showed divergence between the two populations. Further, comparison of the COX1 data using a phylogenetic tree for a range of hagfish species indicated clear differences between the populations, suggesting that they differ at the species level. The numbers of their teeth, in particular of fused cusps (anterior/posterior multicusps), also supported these findings. Individuals of the Suruga Bay population had 3/3 fused cusps, as described for E. atami, whereas individuals of the Akita population had 3/2 fused cusps. These results suggest that the brown hagfish from the Sea of Japan, off the northwest coast of Honshu, is a distinct species from E. atami.

Impaired Healing of a Cutaneous Wound in an Inducible Nitric Oxide Synthase-Knockout Mouse.

Background. We investigated the effects of loss of inducible nitric oxide synthase (iNOS) on the healing process of cutaneous excisional injury by using iNOS-null (KO) mice. Population of granulation tissue-related cell types, that is, myofibroblasts and macrophages, growth factor expression, and reepithelialization were evaluated. Methods. KO and wild type (WT) mice of C57BL/6 background were used. Under general anesthesia two round full-thickness excision wounds of 5.0 mm in diameter were produced in dorsal skin. After specific intervals of healing, macroscopic observation, histology, immunohistochemistry, and real-time reverse transcription-polymerase chain reaction (RT-PCR) were employed to evaluate the healing process. Results. The loss of iNOS retards granulation tissue formation and reepithelialization in excision wound model in mice. Detailed analyses showed that myofibroblast appearance, macrophage infiltration, and mRNA expression of transforming growth factor b and of collagen 1α2 were all suppressed by lacking iNOS. Conclusions. iNOS is required in the process of cutaneous wound healing. Lacking iNOS retards macrophage invasion and its expression of fibrogenic components that might further impair fibrogenic behaviors of fibroblasts.

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_RHß, and Patr_RHγ). In the Patr_RHß locus, a short version of the gene, which lacked the middle part containing exons 4-8, was observed. The Patr_RHα and Patr_RHß genes were located on the locations corresponding to Hosa_RHD and Hosa_RHCE, respectively, and Patr_RHγ was in the immediate vicinity of Patr_RHß. Sequence comparisons revealed high sequence similarity between Patr_RHß 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.

Resistance phenotypes and genotypes among multiple-antimicrobial-resistant Salmonella enterica subspecies enterica serovar Choleraesuis strains isolated between 2008 and 2012 from slaughter pigs in Okinawa Prefecture, Japan.

A total of 349 Salmonella enterica subspecies enterica serovar Choleraesuis (S. Choleraesuis) strains, which were isolated between 2008 and 2012 from 349 pigs at two slaughterhouses in Okinawa Prefecture, Japan, were investigated for antimicrobial susceptibility and the presence of antimicrobial resistance genes. All isolates were resistant to at least four antimicrobial agents. The antimicrobial agents for which isolates showed a high incidence of resistance were as follows: ampicillin (100%) and streptomycin (100%), followed by gentamicin (99.7%), oxytetracycline (99.7%), sulfamethoxazole/trimethoprim (99.4%), nalidixic acid (40.1%) and oxolinic acid (40.1%). All isolates were sensitive to cefuroxime, ceftiofur, colistin, fosfomycin, enrofloxacin, orbifloxacin and danofloxacin. The predominant resistance phenotypes and genotypes were: resistance to ampicillin, streptomycin, gentamicin, oxytetracycline and sulfamethoxazole/trimethoprim (58.5%, 204/349) and blaTEM-strA-strB-aadA1-aadA2-aacC2-tet (B)-sul1-sul2-dhfrXII-dhfrXIII (36.1%, 126/349). The quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE of the quinolone-resistant isolates (n=12) showed amino acid substitutions of Ser-83→Phe or Asp-87→Tyr in GyrA and Ser-107→Ala in ParC. To our knowledge, this is the first report on the molecular characterization of antimicrobial resistance among S. Choleraesuis strains in Japan.

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 (Galß1-4GlcNAc) 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.

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.

Analysis of a Larger SNP Dataset from the HapMap Project Confirmed That the Modern Human A Allele of the ABO Blood Group Genes Is a Descendant of a Recombinant between B and O Alleles.

The human ABO blood group gene consists of three main alleles (A, B, and O) that encode a glycosyltransferase. The A and B alleles differ by two critical amino acids in exon 7, and the major O allele has a single nucleotide deletion (Δ261) in exon 6. Previous evolutionary studies have revealed that the A allele is the most ancient, B allele diverged from the A allele with two critical amino acid substitutions in exon 7, and the major O allele diverged from the A allele with Δ261 in exon 6. However, a recent phylogenetic network analysis study showed that the A allele of humans emerged through a recombination between the B and O alleles. In the previous study, a restricted dataset from only two populations was used. In this study, therefore, we used a large single nucleotide polymorphism (SNP) dataset from the HapMap Project. The results indicated that the A101-A201-O09 haplogroup was a recombinant lineage between the B and O haplotypes, containing the intact exon 6 from the B allele and the two critical A type sites in exon 7 from the major O allele. Its recombination point was assumed to be located just behind Δ261 in exon 6.

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 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.

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.

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.