Sample collection and DNA extraction
A total of 280 adult individuals of seaweed pipefish were collected from 9 sites along the coastal waters of China ranging from the Yellow Sea (YS) to the South China Sea (SCS) (Table 1, Figure S1). We derived morphological data from 163 seaweed pipefish specimens: 63 from the YS, 34 from the East China Sea (ECS), and 66 from the SCS. The body of the remaining 117 specimens was damaged, preventing to obtain reliable morphological measures. The total length (TL) of sampled specimens ofS. schlegeli ranged 18.1–21.6 cm, and no significant differences in fish sizes existed among sampled localities (ANOVA, Duncan’s multiple range tests, p > 0.05). Genomic DNA was extracted from muscle samples and then treated with RNase A to digest any RNAs present in the samples. The quality and integrity of the DNA samples were checked by agarose gel electrophoresis, and DNA concentrations were quantified using a Qubit 2.0 fluorometer.
We performed RAD-sequencing for 69 individuals (31 from the Yellow Sea, 22 from the East China Sea, and 16 from the South China Sea) (Table 1, Figure S1). The mitochondrial Cytochrome b (cytb ) and nuclear Sorting nexin 33 (snx33 ) amplificated from 270 pipefish individuals, and we obtained 248 and 265 valid sh3 andcytb sequences after data correction (Table 1). All experimental procedures performed in this study were in accordance with relevant animal ethics guidelines and approved by the Ethical Committee of South China Sea Institute of Oceanology, Chinese Academy of Sciences.