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.