Specimens and data collection

For this study, we sampled 32 different species of bobtail and bottletail squids. Samples were collected in different locations of the Japan Sea, Western North Pacific Ocean, Atlantic Ocean, Mediterranean Sea, Tasman Sea, Great Australian Bight, Banda Sea, and South Pacific Ocean. Specimens comprised members of the family Sepiadariidae and all the valid subfamilies of Sepiolidae (Sepiolinae, Heteroteuthinae, and Rossinae). For each individual, a small piece of tissue from the mantle was stored in 70% ethanol for DNA analyses and the remaining animals were stored in museums. Details of our specimens are found in Table S1.

Individuals collected off the coast of mainland Japan and the Ryukyu Archipelago were previously identified in Sanchez et al.¹¹. Mediterranean species were identified based on general morphology and/or the hectocotylus following Bello⁵¹. We also retrieved the full mitochondrial genome of Semirossia patagonica (E. A. Smith, 1881) ({"type":"entrez-nucleotide","attrs":{"text":"AP012226","term_id":"359270215","term_text":"AP012226"}}AP012226) and Sepiadarium austrinum Berry, 1921 ({"type":"entrez-nucleotide","attrs":{"text":"KX657686","term_id":"1177820278","term_text":"KX657686"}}KX657686), mitochondrial genes from the transcriptome of Euprymna tasmanica (Pfeffer, 1884) (SRR2984339), and the nuclear reads of Euprymna scolopes (PRJNA470951) from the GenBank database. For the outgroups, we retrieved the complete mitochondrial genome of Sepia esculenta Hoyle, 1885 ({"type":"entrez-nucleotide","attrs":{"text":"NC_009690","term_id":"153123921","term_text":"NC_009690"}}NC_009690), Sepia officinalis Linné, 1785 ({"type":"entrez-nucleotide","attrs":{"text":"NC_007895","term_id":"89255284","term_text":"NC_007895"}}NC_007895) and Idiosepius sp. ({"type":"entrez-nucleotide","attrs":{"text":"KF647895","term_id":"557954779","term_text":"KF647895"}}KF647895), the complete 18S rRNA gene of Idiosepius pygmaeus Steenstrup, 1881 ({"type":"entrez-nucleotide","attrs":{"text":"AY557477.1","term_id":"49482082","term_text":"AY557477.1"}}AY557477.1), and a partial region of the 28S rRNA gene of Idiosepius pygmaeus ({"type":"entrez-nucleotide","attrs":{"text":"AY293684.1","term_id":"34542093","term_text":"AY293684.1"}}AY293684.1). While the proximal outgroup of the Sepiolidae remains unclear (with different tree topologies found based on transcriptome sequences in Tanner et al¹⁵, Lindgren and Anderson⁵², and Anderson and Lindgren¹⁷), we believe the inclusion of Sepiadariidae should prevent any conflict relationships within Sepiolidae.