RAD sequencing and population structure analysis
We performed RAD-sequencing for 69 S. schlegeli individuals, obtaining a total of 120.08 Gb raw sequence data, of which 118.13 Gb clean data remained after filtering (Table S3). The sequencing reads were aligned to the reference genome (gulf pipefish Syngnathus scovelli ) to evaluate the mapping rate, sequencing depth, and coverage. The results shows that the average mapping rate of the sequencing reads was 85.28%, the average sequencing depth was 10 fold, and the average coverage of the data was 20.70% (coverage at least 1×) and 10.82% (coverage at least 4×) (Table S4). We identified a total of 1,607,529 SNPs from the S. schlegeli populations, and retained 93,845 high-quality SNP markers after filtering (Table S5).
We reconstructed phylogenetic relationships among the 69 sequencedS. schlegeli individuals based on the SNPs data using the neighbor-joining method. The result showed three distinct clusters that represent three geographical lineages (Figure 2a). Principal components analysis (PCA) recovered the same clusters revealed by the phylogenetic analysis (Figure 2b), revealed that the YS population is closely related to the ECS population, whereas the SCS population formed a separate cluster. Multi-level (K = 2, 3, 4, and 5) population structures showed that SCS pipefish comprise a relatively stable and independent group, with a change in K value from 2 to 5, indicating that the SCS population has low genetic exchange with other populations. In contrast, the YS and ECS populations were well connected with appreciable gene exchange (Figure 2c).