Details of Transcriptome sequencing.

Pagurus lanuginosus (De Haan, 1833–1850) and Pagurus maculosus (Komai and Imafuku, 1996) are two closely related species of the genus Pagurus, generally referred to as “right-handed hermit crabs”. Previously thought to be color morphs of the same species, recent studies have itemized their unique features. To investigate the molecular and gene expression evolution that have followed the divergence of these two species, we performed transcriptome sequencing and de novo transcriptome assembly using cephalothorax (head) and pereopod (leg) of the two species. Synonymous and nonsynonymous divergence between the two species was estimated to be 0.0246 and 0.0066, respectively. About 5% of the protein-coding transcripts had signatures of positive selection. The phylogenetic analyses showed that the two species are indeed closely related and diverged about 6.24 million years ago. Comparison of tissue expression showed that head expressions were more conserved, and that more genes were upregulated in the legs of P. maculosus leg; while more pigmentation genes were found to be upregulated in the legs of P. lanuginosus. Genes associated with the extracellular matrix and space, chitin binding and chitin-based larval cuticles were found to have higher expression in the legs of P. maculosus, suggesting the roles in morphological differences. This study sheds light on the evolutionary history of the two species at the molecular level.