Characterization of Mhc genes in a multigenerational family of ring-necked pheasants

Little is known about the major histocompatibility (Mhc) genes of birds in different taxonomic groups or about how Mhc genes may be organized in avian species divergent by evolution or habitat. Yet it seems likely that much might be learned from birds about the evolution, organization, and function of this intricate complex of polymorphic genes. In this study a close relative of the chicken, the ring-necked pheasant (Phasianus colchicus), was examined for the presence and organization of Mhc B-G genes. The patterns of restriction fragments revealed by chicken B-G probes in Southern hybridizations and the patterns of pheasant erythrocyte polypeptides revealed in immunoblots by antisera raised against chicken B-G polypeptides provide genetic, molecular, and biochemical data confirming earlier serological evidence for the presence of B-G genes in the pheasant, and hence, the presence of a family of B-G genes in at least a second species of birds. The high polymorphism exhibited by the pheasant B-G gene family allowed genetic differences among individuals within the small experimental population in this study to be detected easily by restriction fragment patterns. Further evidence was found for the organization of the pheasant Mhc class I and class II genes into genetically independent clusters. Whether these gene clusters are fully comparable to the B and Rfp-Y systems in the chicken or whether yet another organization of Mhc genes has been encountered in the pheasant remains to be determined.     

Diversity of<Emphasis Type="Italic"> Mhc</Emphasis> class I and IIB genes in house sparrows (<Emphasis Type="Italic">Passer domesticus</Emphasis>)

In order to understand the expression and evolution of host resistance to pathogens, we need to examine the links between genetic variability at the major histocompatibility complex (Mhc), phenotypic expression of the immune response and parasite resistance in natural populations. To do so, we characterized the Mhc class I and IIB genes of house sparrows with the goal of designing a PCR-based genotyping method for the Mhc genes using denaturing gradient gel electrophoresis (DGGE). The incredible success of house sparrows in colonizing habitats worldwide allows us to assess the importance of the variability of Mhc genes in the face of various pathogenic pressures. Isolation and sequencing of Mhc class I and IIB alleles revealed that house sparrows have fewer loci and fewer alleles than great reed warblers. In addition, the Mhc class I genes divided in two distinct lineages with different levels of polymorphism, possibly indicating different functional roles for each gene family. This organization is reminiscent of the chicken B complex and Rfp-Y system. The house sparrow Mhc hence appears to be intermediate between the great reed warbler and the chicken Mhc, both in terms of numbers of alleles and existence of within-class lineages. We specifically amplified one Mhc class I gene family and ran the PCR products on DGGE gels. The individuals screened displayed between one and ten DGGE bands, indicating that this method can be used in future studies to explore the ecological impacts of Mhc diversity.     

Mhc‐linked survival and lifetime reproductive success in a wild population of great tits

Major histocompatibility complex (Mhc) genes are frequently used as a model for adaptive genetic diversity. Although associations between Mhc and disease resistance are frequently documented, little is known about the fitness consequences of Mhc variation in wild populations. Further, most work to date has involved testing associations between Mhc genotypes and fitness components. However, the functional diversity of the Mhc, and hence the mechanism by which selection on Mhc acts, depends on how genotypes map to the functional properties of Mhc molecules. Here, we test three hypotheses that relate Mhc diversity to fitness: (i) the maximal diversity hypothesis, (ii) the optimal diversity hypothesis and (iii) effect of specific Mhc types. We combine mark–recapture methods with analysis of long‐term breeding data to investigate the effects of Mhc class I functional diversity (Mhc supertypes) on individual fitness in a wild great tit (Parus major) population. We found that the presence of three different Mhc supertypes was associated with three different components of individual fitness: survival, annual recruitment and lifetime reproductive success (LRS). Great tits possessing Mhc supertype 3 experienced higher survival rates than those that did not, whereas individuals with Mhc supertype 6 experienced higher LRS and were more likely to recruit offspring each year. Conversely, great tits that possessed Mhc supertype 5 had reduced LRS. We found no evidence for a selective advantage of Mhc diversity, in terms of either maximal or optimal supertype diversity. Our results support the suggestion that specific Mhc types are an important determinant of individual fitness.     

Evolution of Mhc–DRB Introns: Implications for the Origin of Primates

Introns are generally believed to evolve too rapidly and too erratically to be of much use in phylogenetic reconstructions. Few phylogenetically informative intron sequences are available, however, to ascertain the validity of this supposition. In the present study the supposition was tested on the example of the mammalian class II major histocompatibility complex (Mhc) genes of the DRB family. Since the Mhc genes evolve under balancing selection and are believed to recombine or rearrange frequently, the evolution of their introns could be expected to be particularly rapid and subject to scrambling. Sequences of intron 4 and 5 DRB genes were obtained from polymerase chain reaction-amplified fragments of genomic DNA from representatives of six eutherian orders—Primates, Scandentia, Chiroptera, Dermoptera, Lagomorpha, and Insectivora. Although short stretches of the introns have indeed proved to be unalignable, the bulk of the intron sequences from all six orders, spanning >85 million years (my) of evolution, could be aligned and used in a study of the tempo and mode of intron evolution. The analysis has revealed the Mhc introns to evolve at a rate similar to that of other genes and of synonymous sites of non-Mhc genes. No evidence of homogenization or large-scale scrambling of the intron sequences could be found. The Mhc introns apparently evolve largely by point mutations and insertions/deletions. The phylogenetic signals contained in the intron sequences could be used to identify Scandentia as the sister group of Primates, to support the existence of the Archonta superorder, and to confirm the monophyly of the Chiroptera. Received: 26 October 1998 / Accepted: 21 December 1998     

Mhc-DRB Genes and the Origin of New World Monkeys

The major histocompatibility complex (Mhc) is a family of loci characterized by its relatively rapid evolutionary turnover, large genetic distances between genes, and long persistence of allelic lineages effected by balancing selection. These features render the Mhc highly suitable for answering questions concerning speciation and adaptive radiation. The aim of the present study was to use Mhc-DRB genes to make inferences about the founding population of the Platyrrhini. Three segments, each approximately 300 base pairs in length, of the platyrrhine DRB genes were amplified by the polymerase chain reaction and sequenced. The segments were derived from intron 2, exon 3, and exon 6 of DRB genes from different species of New World monkeys. The results of the study have revealed that on a phylogenetic tree, all of the tested platyrrhine genes appear to form a single cluster, while all catarrhine DRB genes form a distinct cluster, although the bootstrap values fail to provide statistically significant support for the separation of these two clades. This observation suggests that the multiple platyrrhine genes originated from a single ancestral gene after the divergence of the Platyrrhini and Catarrhini and thus contradicts the results of an earlier study in which some exon 2 DRB sequences appeared to predate the split of the two primate groups. The inconsistency in the DRB gene phylogeny can be explained by postulating convergent evolution for the peptide-binding region of the DRB exon 2 sequences. The phylogeny of the platyrrhine DRB genes (except for exon 2) is relatively "shallow"; the distances between genes are relatively short (in comparison to the catarrhine DRB genes), and there is a tendency for sequences of individual species to cluster together. The phylogeny of the platyrrhine DRB genes is consistent with the postulate that a small population founded the group and that there is an ongoing adaptive radiation from small, relatively isolated founding populations.     

Major histocompatibility complex class II A gene polymorphism in the striped bass

Adaptions of the polymerase chain reaction were used to isolate cDNA sequences encoding the Major histocompatibility complex(Mhc) class II A gene(s) of the striped bass (Morone saxatilis). Four complete Mhc class II A genes were cloned and sequenced from a specimen originating in the Roanoke River, North Carolina, and another three A genes from a specimen originating from the Santee-Cooper Reservoir, South Carolina, identifying a total of seven unique sequences. The sequence suggests the presence of at least two Mhc class II A loci. The extensive sequence variability observed between the seven different Mhc class II clones was concentrated in the 1 encoding domain. The encoded 2, transmembrane, and cytoplasmic regions of all seven striped genes correlated well with those of known vertebrate Mhc class II proteins. Overall, the striped bass sequences showed greatest similarity to the Mhc class II A genes of the zebrafish. Southern blot analysis demonstrated extensive polymorphism in the Mhc class II A genes in members of a Roanoke river-caught population of striped bass versus a lesser degree of polymorphism in an aquacultured Santee-Cooper population of striped bass.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers (Mosa-A-S5) L35062, (Mosa-A-S8) L35066, (Mosa-A-R7) L35067, and (Mosa-A-S7) L35072 L35066, (Mossa-A-R7) L35067, and (Mosa-A-S7) L35072     

The function of mate choice in sticklebacks: optimizing Mhc genetics*

Sexual reproduction is an evolutionary ‘puzzle’. A sexual female ‘throws away’ half of her genes (during meiosis), and ‘fills up’ what she lost with genes from a male. Thus, sexual reproduction can only be successful if the offspring with the new mixture of genes should be more than twice as fit as if she had just made a copy of herself. A challenging hypothesis assumes that infectious diseases select for females that reshuffle the immune genes for their offspring in each generation. The required increase in quality could be achieved by females selectively ‘smelling out’ suitable immune‐genes (i.e. Mhc alleles) in potential partners, which, in combination with the female's genes, offer optimal resistance against quickly changing infectious diseases. It was found that most three spined sticklebacks Gasterosteus aculeatus in natural populations around Plön, Germany, had intermediate instead of maximal numbers of different Mhc class IIB alleles. Furthermore, fish with an intermediate number of different Mhc alleles were infected with the lowest number of both parasite species and parasites per species. This suggests that Mhc heterozygosity was optimized instead of maximized. Can this immunogenetic optimum be achieved through female choice? In a flow channel design that allowed the detection of olfactory signals only, it was found that female three‐spined sticklebacks that were ready to spawn preferred males as mates that in combination with their Mhc alleles would allow the production of offspring with the optimal number of Mhc alleles. Thus, mate choice in three‐spined sticklebacks could have the two‐fold advantage over asexual reproduction that is required to maintain sexual reproduction. The interaction of olfactory with visual signals in three‐spined stickleback mate choice is discussed. The three‐spined stickleback is a suitable model organism for studying the evolution of sexual reproduction in relation to optimizing offspring immune genetics although other fishes may be as suitable.     

Songbird Genomics: Analysis of 45 kb Upstream of a Polymorphic Mhc Class II Gene in Red-Winged Blackbirds (Agelaius phoeniceus)

Here we present the sequence of a 45 kb cosmid containing a previously characterized poly-morphic Mhc class II B gene (Agph-DAB1) from the red-winged blackbird (Agelaius phoeniceus). We compared it with a previously sequenced cosmid from this species, revealing two regions of 7.5 kb and 13.0 kb that averaged greater than 97% similarity to each another, indicating a very recent shared duplication. We found 12 retroelements, including two chicken repeat 1 (CR1) elements, constituting 6.4% of the sequence and indicating a lower frequency of retroelements than that found in mammalian genomic DNA. Agph-DAB3, a new class II B gene discovered in the cosmid, showed a low rate of polymorphism and may be functional. In addition, we found a Mhc class II B gene fragment and three genes likely to be functional (encoding activin receptor type II, a zinc finger, and a putative γ-filamin). Phylogenetic analysis of exon 2 alleles of all three known blackbird Mhc genes indicated strong clustering of alleles by locus, implying that large amounts of interlocus gene conversion have not occurred since these genes have been diverging. Despite this, interspecific comparisons indicate that all three blackbird Mhc genes diverged from one another less than 35 million years ago and are subject to concerted evolution in the long term. Comparison of blackbird and chicken Mhc promoter regions revealed songbird promoter elements for the first time. The high gene density of this cosmid confirms similar findings for the chicken Mhc, but the segment duplications and diversity of retroelements resembles mammalian sequences.     

Trans-specific Mhc polymorphism and the origin of species in primates

The major histocompatibility complex (Mhc) is a cluster of loci controlling the specific immune response in vertebrates. Mhc alleles often differ by a large number of nucleotide substitutions, some of which began to accumulate before the emergence of extant species. We have applied the theory of allelic genealogy to the primate Mhc genes with the aim of estimating the size of the founding populations. The calculations indicate that the long-term effective population size of the studied species was between 10⁴ and 10⁵ individuals and that it most likely never dropped below 10³ individuals.     

An Mhc class I allele associated to the expression of T-dependent immune response in the house sparrow

The major histocompatibility complex (Mhc) encodes for highly variable molecules, responsible for foreign antigen recognition and subsequent activation of immune responses in hosts. Mhc polymorphism should hence be related to pathogen resistance and immune activity, with individuals that carry either a higher diversity of Mhc alleles or one specific Mhc allele exhibiting a stronger immune response to a given antigen. Links between Mhc alleles and immune activity have never been explored in natural populations of vertebrates. To fill this gap, we challenged house sparrows (Passer domesticus) with two T-dependent antigens (phytohemagglutinin and sheep red blood cells) and examined both primary and secondary immune responses in relation to their Mhc class I genotypes. The total number of Mhc alleles had no influence on either primary or secondary response to the two antigens. One particular Mhc allele, however, was associated with an increased response to both antigens. Our results point toward a contribution of the Mhc, or of other genes in linkage disequilibrium with the Mhc, in the regulation of immune responses in a wild animal species.     

A polymorphic system related to but genetically independent of the chicken major histocompatibility complex

Analyses of the major histocompatibility complex (Mhc) in chickens have shown inconsistencies between serologically defined haplotypes and haplotypes defined by the restriction fragment patterns of Mhc class I and class II genes in Southern hybridizations. Often more than one pattern of restriction fragments for Mhc class I and/or class II genes has been found among DNA samples collected from birds homozygous for a single serologically defined B haplotype. Such findings have been interpreted as evidence for variability within the Mhc haplotypes of chickens not detected previously with serological methods. In this study of a fully pedigreed family over three generations, the heterogeneity observed in restriction fragment patterns was found to be the result of the presence of a second, independently segregating polymorphic Mhc-like locus, designated Rfp-Y. Three alleles (haplotypes) are identified in this new system.     

Mhc supertypes confer both qualitative and quantitative resistance to avian malaria infections in a wild bird population

Major histocompatibility complex (Mhc) genes are believed to play a key role in the genetic basis of disease control. Although numerous studies have sought links between Mhc and disease prevalence, many have ignored the ecological and epidemiological aspects of the host–parasite interaction. Consequently, interpreting associations between prevalence and Mhc has been difficult, whereas discriminating alleles for qualitative resistance, quantitative resistance and susceptibility remains challenging. Moreover, most studies to date have quantified associations between genotypes and disease status, overlooking the complex relationship between genotype and the properties of the Mhc molecule that interacts with parasites. Here, we address these problems and demonstrate avian malaria (Plasmodium) parasite species-specific associations with functional properties of Mhc molecules (Mhc supertypes) in a wild great tit (Parus major) population. We further show that correctly interpreting these associations depends crucially on understanding the spatial variation in risk of infection and the fitness effects of infection. We report that a single Mhc supertype confers qualitative resistance to Plasmodium relictum, whereas a different Mhc supertype confers quantitative resistance to Plasmodium circumflexum infections. Furthermore, we demonstrate common functional properties of Plasmodium-resistance alleles in passerine birds, suggesting this is a model system for parasite–Mhc associations in the wild.     

Identification of major histocompatibility complex genes in the guppy,Poecilia reticulata

The guppy, Poecilia reticulata, a teleostean fish of the order Cyprinodontiformes, has been used extensively in studies of host-parasite interactions, courtship behavior, and mating preference, as well as in ecological and evolutionary genetics. A related species was among the first poikilotherm vertebrates to be used in the study of histocompatibility genes. All these studies could benefit from the identification and characterization of the guppy major histocompatibility complex (Mhc) genes. Here, both class I and class II genes of the guppy are described. The number of expressed loci, as determined by representation of clones in a cDNA library, sequencing, and Southern blot analysis, may be low in both Mhc classes: combined evidence suggests that there may be one expressed class II locus only and one or two expressed class I loci. The variability of aquaristic guppy stocks is very low: only three and two genes have been detected at the class I and class II loci, respectively, in the stocks examined. This genetic paucity is most likely the consequence of breeding practices employed by aquarists and commercial establishments. Limited sampling of wild guppy populations revealed extensive Mhc polymorphism at loci of both classes in nature. Comparison of guppy Mhc sequences with those of other vertebrates has revealed the existence of a set of insertions/deletions which can be used as characters in cladistic analysis to infer phylogenetic relationships among vertebrate taxa and the Mhc genes themselves. These indels are particularly frequent in the regions coding for the loops of 1 and 2 domains of class I proteins.The nucleotide sequence data reported in this publication have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers Z54076-Z54095     

<Emphasis Type="Italic">Mhc</Emphasis> class II diversity and balancing selection in greater prairie-chickens

The major histocompatibility complex (Mhc) of domestic chickens has been characterized as small and relatively simple compared with that of mammals. However, there is growing evidence that the Mhc of many bird lineages may be more complex, even within the Order Galliformes. In this study, we measured genetic variation and balancing selection at Mhc loci in another galliform, the greater prairie-chicken. We cloned and sequenced a 239 bp fragment of Mhc Class II β-chain (BLB) exon 2 in 14 individuals. There was a total of 10 unique sequences and a minimum of four BLB loci. The d _N/d _S ratio at peptide-binding codons was significantly greater than one, suggesting balancing selection is acting on the BLB. We also recovered two YLB sequences, which clustered tightly with YLB sequences from three other species: domestic chicken, black grouse and common quail. The relatively large number of loci revealed in our study suggests that even closely related galliforms differ in the level of Mhc variation and structure.     

Polymorphism and transcription of Mhc class I genes in a passerine bird, the great reed warbler

 The class I genes of the major histocompatibility complex (Mhc) are here investigated for the first time in a passerine bird. The great reed warbler is a rare species in Sweden with a few semi-isolated populations. Yet, we found extensive Mhc class I variation in the study population. The variable exon 3, corresponding to the α₂ domain, was amplified from genomic DNA with degenerated primers. Seven different genomic class I sequences were detected in a single individual. One of the sequences had a deletion leading to a shift in the reading frame, indicating that it was not a functional gene. A randomly selected clone was used as a probe for restriction fragment length polymorphism (RFLP) studies in combination with the restriction enzyme Pvu II. The RFLP pattern was complex with 21–25 RFLP fragments per individual and extensive variation. Forty-nine RFLP genotypes were detected in 55 tested individuals. To study the number of transcribed genes, we isolated 14 Mhc class I clones from a cDNA library from a single individual. We found eight different sequences of four different lengths (1.3–2.2 kilobases), suggesting there are at least four transcribed loci. The number of nonsynonymous substitutions (d _N ) in the peptide binding region of exon 3 were higher than the number of synonymous substitutions (d _S ), indicating balancing selection in this region. The number of transcribed genes and the numerous RFLP fragments found so far suggest that the great reed warbler does not have a "minimal essential Mhc" as has been suggested for the chicken. Received: 13 May 1998 / Revised: 18 August 1998     

Genotyping and segregation analyses indicate the presence of only two functional MIC genes in rhesus macaques

MIC molecules are stress-inducible ligands of the activating receptor NKG2D, which is expressed on natural killer cells and subsets of T lymphocytes. In rhesus macaques (Macaca mulatta), three different MIC sequences (MIC1, MIC2, MIC3) have been described that are closely related to but, according to phylogenetic analysis, do not represent orthologues of the human MICA and MICB genes. Although a single haplotype of the rhesus macaque Mhc (Mamu) has been completely sequenced, it remained unknown so far whether these three sequences are derived from two or three Mamu-MIC genes. We genotyped a cohort of 115 rhesus macaque individuals for the presence of MIC1, MIC2, and MIC3 sequences and analysed the segregation in families. All individuals were positive for MIC2, whereas only 66.1 and 80.9 % were positive for MIC1 and MIC3, respectively. MIC1 and MIC3 sequences segregated in offspring, indicating that they behave as alleles. Thus, we conclude that two MIC genes are present in the rhesus macaque Mhc, which we propose to designate as Mamu-MICA (MIC1 and MIC3) and Mamu-MICB (MIC2). “MIC1” and “MIC3” are regarded as divergent allelic lineages of the Mamu-MICA gene. Mamu-MIC genotyping of DNA of a cohort of 68 experimentally simian immunodeficiency virus (SIV)-infected rhesus macaques revealed no significant association of either of the two Mamu-MICA allelic lineages with differences in progression to AIDS-like symptoms. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorize users. Anne Averdam and Sandra Seelke contributed equally.     

Analysis of the sequence variations in the Mhc DRB1-like gene of the endangered Humboldt penguin (Spheniscus humboldti)

The Major Histocompatibility Complex (Mhc) genomic region of many vertebrates is known to contain at least one highly polymorphic class II gene that is homologous in sequence to one or other of the human Mhc DRB1 class II genes. The diversity of the avian Mhc class II gene sequences have been extensively studied in chickens, quails, and some songbirds, but have been largely ignored in the oceanic birds, including the flightless penguins. We have previously reported that several penguin species have a high degree of polymorphism on exon 2 of the Mhc class II DRB1-like gene. In this study, we present for the first time the complete nucleotide sequences of exon 2, intron 2, and exon 3 of the DRB1-like gene of 20 Humboldt penguins, a species that is presently vulnerable to the dangers of extinction. The Humboldt DRB1-like nucleotide and amino acid sequences reveal at least eight unique alleles. Phylogenetic analysis of all the available avian DRB-like sequences showed that, of five penguin species and nine other bird species, the sequences of the Humboldt penguins grouped most closely to the Little penguin and the mallard, respectively. The present analysis confirms that the sequence variations of the Mhc class II gene, DRB1, are useful for discriminating among individuals within the same penguin population as well those within different penguin population groups and species.The nucleotide sequence and amino acid sequence data reported in this paper have been submitted to the DDBJ database and have been assigned the accession numbers AB088371–AB088374, AB089199, AB154393–AB154399, and AB162144.     

Dynamics of Mhc evolution in birds and crocodilians: amplification of class II genes with degenerate primers

Genes of the major histocompatibility complex (Mhc) are the most polymorphic functional loci in mammalian populations, but little is known of Mhc variability in natural populations of nonmammalian vertebrates. To help extend such studies to birds and relatives, we present a pair of degenerate primers that amplify polymorphic segments of one chain (the β chain) of the class II genes from the major histocompatibility complex (Mhc) of archosaurs (birds + crocodilians). The primers target two conserved regions lying within portions of the antigen-binding site (ABS) encoded by the second exon and amplify multiple genes from both genomic DNA and cDNA. The pattern of nucleotide substitution in ABS codons of 51 sequences amplified and cloned from five species of passerine birds and an alligator (Alligator mississippiensis) indicates that archosaurian class II β genes are subject to selective forces similar to those operating in mammalian populations. Hybridization of a genomic clone generated by the primers revealed highly polymorphic bands in a sample of Florida scrub jays (Aphelocoma coerulescens coerulescens). Because the primers amplify only part of the ABS from multiple class II genes, they will be useful primarily for generating species specific clones, thereby providing a critical inroad to more detailed structural and evolutionary studies.     

Partitioning of genetic variation between regulatory and coding gene segments: the predominance of software variation in genes encoding introvert proteins

Summary  In considering genetic variation in eukaryotes, a fundamental distinction can be made between variation in regulatory (software) and coding (hardware) gene segments. For quantitative traits the bulk of variation, particularly that near the population mean, appears to reside in regulatory segments. The main exceptions to this rule concern proteins which handle extrinsic substances, here termed extrovert proteins. The immune system includes an unsually large proportion of this exceptional category, but even so its chief source of variation may well be polymorphism in regulatory gene segments. The main evidence for this view emerges from genome scanning for quantitative trait loci (QTL), which in the case of the immune system points to a major contribution of pro-inflammatory cytokine genes. Further support comes from sequencing of major histocompatibility complex (Mhc) class II promoters, where a high level of polymorphism has been detected. These Mhc promoters appear to act, in part at least, by gating the back-signal from T cells into antigen-presenting cells. Both these forms of polymorphism are likely to be sustained by the need for flexibility in the immune response. Future work on promoter polymorphism is likely to benefit from the input from genome informatics.     

Evaluating the occurrence of cryptic invasions of a rocky shore barnacle,Semibalanus cariosus,between the north-eastern Pacific and Japan

Many coastal barnacles are introduced to non-native regions. However, data are lacking on cryptic invasion, which is defined as an invasion that remains unrecognised because the invader is mistaken for a native or previously introduced species or clade. In this work, cryptic invasions of an intertidal barnacle, Semibalanus cariosus, between Japan and the north-eastern Pacific were evaluated based on population genetic analyses. A significant genetic differentiation was found between the Japanese and north-eastern Pacific populations, suggesting a limited introduction of non-native genotypes between these regions. Haplotype frequencies did not differ significantly between the past (museum samples collected in 1971 from Hokkaido, Japan) and present Japanese populations, implying the rare occurrence of human-mediated migration from the north-eastern Pacific to Japan. Migrate-n analysis revealed a low level of directional gene flow in S. cariosus from the north-eastern Pacific to Japan, possibly by natural stepping-stone dispersal via directional water currents or human-mediated transport.