Mitochondrial and nuclear DNA analysis revealed a cryptic species and genetic introgression in Littorina sitkana (Mollusca, Gastropoda) Noriko Azuma • Tomoyasu Yamazaki • Susumu Chiba Received: 24 November 2011 / Accepted: 16 February 2012 / Published online: 29 February 2012 Ó Springer Science+Business Media B.V. 2012 Abstract We investigated mitochondrial and nuclear DNA genotypes in nominal Littorina sitkana samples from 2 localities in Eastern Hokkaido, northern Japan. Our results indicated the existence of cryptic species. In the analysis of partial mitochondrial Cytchrome b gene sequences, haplotypes of L. sitkana samples were mono- phyletic in a phylogenetic tree with orthologous sequences from other Littorina species, but were apparently separated in 2 clades. One included typical L. sitkana (CBa clade) samples, which formed a clade with an allopatric species, L. horikawai. The other, CBb, was independent from CBa and L. horikawai. Haplotypes of the mitochondrial 16S rRNA gene also separated into 2 clades. We additionally examined intron sequence of the heat shock cognate 70 (HSC70) nuclear gene and identified 17 haplotypes. These were also separated into 2 clades, HSCa and HSCb. Among the examined Hokkaido samples, 60% of individuals were heterozygotes. However, each heterozygote consisted of haplotypes from the same clade, HSCa or HSCb, and no admixture of HSCa and HSCb haplotypes was observed. These results indicate reproductive isolation between the 2 clades. Among the genotyped Hokkaido samples, 93% of individuals had CBa ? HSCa or CBb ? HSCb genotypes, and 7% had CBb ? HSCa genotypes. The discrepancy between the mtDNA and nuclear DNA haplotypes in a few individuals may have been caused by genetic introgression due to past hybridization. Keywords Periwinkle Á Speciation Á Reproductive isolation Á Cytb Á 16S Á HSC70 Introduction Recent applications of molecular genetic approaches for reconstructing phylogenies have successfully uncovered reproductive isolation and speciation among closely related species within the same locality (e.g., Avise 2000; Barraclough and Vogler 2000; Templeton 2001). In shell- bearing mollusks, shell characters have contributed to taxonomy and are regarded as useful tools for species identification. However, in some cases, molecular evidence has revealed the existence of cryptic species that cannot be distinguished by shell morphologies (e.g., Liu et al. 2011; Puillandre et al. 2009). There are several examples of gastropod phenotypes, which can become genetically adapted or plastically changed in response to environ- mental factors (Dewitt and Scheiner 2004; Piersma and Gils 2010). Therefore, species identification using shell characters may be misleading if within species variation is high and/or a high level of similarity in shell morphology can be observed among species. High levels of intraspecific shell variation are common in the genus Littorina (Mollusca, Gastropoda), and con- vergent evolution and/or ancestral effect can lead to shell character similarities in different Littorina species (Reid 1996; Johannesson 2003). Such variable shell characters have attracted many researchers, and Littorina species have Present Address: N. Azuma (&) Center for Environmental Biology and Ecosystem Studies, National Institute for Environmental Studies, Tsukuba, Ibaraki, Japan e-mail: norikoazuma@gmail.com T. Yamazaki Shellfish Museum of Rankoshi, Rankoshi, Hokkaido, Japan S. Chiba Faculty of Bioindustry, Tokyo University of Agriculture, Abashiri, Hokkaido, Japan 123 Genetica (2011) 139:1399–1408 DOI 10.1007/s10709-012-9638-9