2.1 Collections, rearing, imaging & digital sorting
About 11,000 y-larvae were collected with 75-µm-mesh plankton nets from
surface waters (0-5m) at three sites: the University of the Ryukyus
Sesoko Laboratory on Sesoko Island, Okinawa, Japan (JA) and Gongguan
Harbor on Green Island and WangHaiXiang Fishing Harbor in Keelung, both
in Taiwan (TA) (Olesen et al., 2022; Fig. 1; Table S1); 35 of these were
sequenced and included in this work. Supplementary material came from
Tioman Island (Malaysia; MAL), Piscinas do Pesquerio in Ponta Delgada,
Azores, Portugal (AZ, n=10), and waters off the White Sea Marine
Biological Station, Russia (RU; n=14) between 2017 and 2021. Larvae were
sorted from plankton samples with glass Pasteur pipettes under an
Olympus stereomicroscope and either (1) photographed and video-recorded
live with a Nikon ECLIPSE 80i compound microscope (JA) or either an
Olympus IX70 inverted compound microscope or a Zeiss AX10 light
microscope (TA), both of the latter being equipped with Nomarsky (DIC)
optics and a Canon EOS 5D Mark IV digital camera, or (2) bulk-fixed for
subsequent molecular work (TA, RU, AZ). All images were sorted digitally
to identify groups of morphologically similar specimens (i.e.,
morphotypes; Olesen et al., 2022), so that at least 2-3 specimens per
group were used for Sanger sequencing.