Interrelationships of the 11 gasterosteiform families (sticklebacks, pipefishes, and their relatives): a new perspective based on whole mitogenome sequences from 75 higher teleosts.

The fishes currently recognized as members of the order Gasterosteiformes (sticklebacks, pipefishes, and their relatives) number 278 species, classified into two suborders (Gasterosteoidei and Syngnathoidei), 11 families and 71 genera. Members of this group exhibit unique appearances, many of which are derived from armored bodies with bony plates in various forms. Although recent molecular phylogenetic studies have repeatedly questioned the monophyly of this order, none of the studies examined all of the representative families and the phylogenetic reality of the group has remained unclear. In this study, we examined whole mitochondrial genome (mitogenome) sequences from 13 gasterosteiform species representing all 11 families in the order, and subjected them to partitioned maximum likelihood and Bayesian analyses, with additional data from other percomorphs and outgroups (75 mitogenome sequences considered overall, including 10 newly determined). The resultant phylogenies indicated explicitly that previously recognized members of Gasterosteiformes had diverged basally within the Percomorpha into three different clades with the following subgroups: Syngnathoidei, Gasterosteoidei (minus Indostomidae), and Indostomidae. Monophyly of the order Gasterosteiformes and any combinations of the three subgroups were confidently rejected by statistical tests. Syngnathoidei (together with Dactylopteroidei) formed a monophyletic group, a sister-group relationship between Gasterosteoidei (minus Indostomidae) and Zoarcoidei was reconfirmed and Indostomidae was nested within the Synbranchiformes, rendering the latter group paraphyletic. Our study demonstrates a new perspective of gasterosteiform phylogeny, which will provide fundamental information for future studies of phylogeny, systematics, and evolution.

Extensive lineage-specific gene duplication and evolution of the spiggin multi-gene family in stickleback.

BACKGROUND: The threespine stickleback (Gasterosteus aculeatus) has a characteristic reproductive mode; mature males build nests using a secreted glue-like protein called spiggin. Although recent studies reported multiple occurrences of genes that encode this glue-like protein spiggin in threespine and ninespine sticklebacks, it is still unclear how many genes compose the spiggin multi-gene family. RESULTS: Genome sequence analysis of threespine stickleback showed that there are at least five spiggin genes and two pseudogenes, whereas a single spiggin homolog occurs in the genomes of other fishes. Comparative genome sequence analysis demonstrated that Muc19, a single-copy mucous gene in human and mouse, is an ortholog of spiggin. Phylogenetic and molecular evolutionary analyses of these sequences suggested that an ancestral spiggin gene originated from a member of the mucin gene family as a single gene in the common ancestor of teleosts, and gene duplications of spiggin have occurred in the stickleback lineage. There was inter-population variation in the copy number of spiggin genes and positive selection on some codons, indicating that additional gene duplication/deletion events and adaptive evolution at some amino acid sites may have occurred in each stickleback population. CONCLUSION: A number of spiggin genes exist in the threespine stickleback genome. Our results provide insight into the origin and dynamic evolutionary process of the spiggin multi-gene family in the threespine stickleback lineage. The dramatic evolution of genes for mucous substrates may have contributed to the generation of distinct characteristics such as "bio-glue" in vertebrates.

Diversification and adaptive evolution of putative sweet taste receptors in threespine stickleback.

The threespine stickleback Gasterosteus aculeatus is known to include several morphologically and ecologically divergent forms. Its phenotypic traits related to feeding vary among forms, and are considered to be a result of adaptations to various environments to find foods effectively. To examine whether the diversification of feeding modes in the stickleback involves genetic changes of the sense of taste, taste receptor family 1 (T1R) genes in stickleback were analyzed and compared with those in other model fishes. Ten T1R genes and 2 pseudogenes were identified from the stickleback genomic sequences. In particular, putative sweet taste receptors (T1R2s) highly increased in number in stickleback (8 genes and 2 pseudogenes) compared to other fishes (2-3 genes). Maximum likelihood estimations of nonsynonymous-synonymous nucleotide substitution rate have indicated that stickleback T1R2 are under positive selection. Expression analysis by RT-PCR revealed that most stickleback T1R genes were expressed in the taste organs; however, at least two T1R2 genes were not expressed in the taste organs, suggesting that the expression levels of these T1R2 genes may be fluctuated through the life history. In addition, sequencing analysis showed that several T1R2 genes in an anadromous form stickleback individual collected from the western Pacific (Japan) were substantially different from those in genomic data derived from a freshwater form individual collected in North America. This suggested that intra-specific variations of stickleback T1R2 genes were considerably large. Our results imply that, in stickleback, T1R2s have diversified through adaptation to various environments, probably to perceive substances important for its survival and reproduction.

Multiple occurrences of spiggin genes in sticklebacks.

The threespine stickleback (Gasterosteus aculeatus) is known to have a unique reproductive mode including male nest-building with a secreted glue-like protein named spiggin. Spiggin is a key character for studying the molecular mechanism in the evolution of this intriguing nest-building behavior, since the glue-like protein is essential for nest-building. However, it is unclear whether spiggin is encoded by a single gene or multiple genes; there are conflicting reports on this point. To resolve this discrepancy, we cloned this gene in a threespine stickleback collected in Japan. We found seven types of cDNAs including at least four genes, indicating that spiggin is encoded by a multi-gene family. On close inspection of the cDNAs, we found that one of these spiggin genes had four splicing variants. Although the cDNA sequences conserved functional domain structures, considerable diversity was observed in the sequence of each domain. Altogether, the current results imply that the structure and function of the spiggins are more complex than ever previously thought. Phylogenetic analysis of the cDNAs with related sequences including ninespine stickleback (Pungitius pungitius) spiggin genes and homologues of zebrafish (Danio rerio), torafugu (Takifugu rubripes), and spotted green pufferfish (Tetraodon nigroviridis) implied the existence of an ancestral "spiggin" gene and multiple duplication events of the gene in the stickleback lineage before and after the divergence of the threespine and ninespine sticklebacks.

Increase in the number of integrinbeta1-immunoreactive monocyte-lineage cells in experimentally-induced adenomyosis in mice.

Uterine adenomyosis is a disease in which hyperplastic endometrial stroma and glands invade the myometrium. We have previously demonstrated that hyperprolactinemia leads to the development of adenomyosis in mice. In the present study, a subtracted cDNA library was made by suppression subtractive hybridization to find specific genes that are abundantly expressed in the adenomyotic but not normal tissue in mice. A cDNA fragment of integrinbeta1 (ibeta1) was found in the library, and the expression of the gene product was increased in the adenomyotic uteri at mRNA and protein levels. Intense ibeta1-immunoreactivity was localized on a group of cells dispersing throughout the endometrial stroma. The number of ibeta1-immunoreactive (ibeta1-ir) cells was significantly greater in the uteri of mice with adenomyosis than normal mice. The majority of the ibeta1-ir cells expressed CD14-ir signal, a marker for monocyte-lineage cells, whereas an increase in the number of CD14-ir cells was also evident in the adenomyotic uteri, especially in the ectopic endometrial tissue. Thus, the adenomyotic stromal tissue contained numerous monocyte-lineage cells with higher expression levels of ibeta1, one of their products. The relationship between the increased number of monocyte-lineage cells and the hyperplastic proliferation of endometrial tissues was discussed with a view to understanding the progressive mechanism of adenomyosis.