Cryptic species identification

We used the methods described by Shen and his research group^15,16 to identify cryptic species among the observed samples. In general, we extracted DNA from fish tissue using the DNeasy Blood & Tissue Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s protocol and conducted a multiplex COI haplotype-specific PCR (MHS-PCR). Cryptic species were detected based on the length of PCR products run on gel agarose (2%). Six PCR product samples were sequenced using Sanger sequencing after being cloned into plasmid vectors. Nucleotide BLAST on Genbank was used to identify sequences. The cloned genes showed more than 97% identity with the COI gene of grey mullet. In addition, a phylogeny tree (not shown) was constructed, and cryptic species samples identified in our study were clustered with corresponding cryptic species from previous studies. As we used the method from previous studies^15,16, we decided to keep the same abbreviations for the three grey mullet cryptic species(i.e. NWP1, NWP2, and NWP3). The list of fish samples is presented in Supplementary Table ¹ and is made up of 16 adult fish samples—eight NWP1, four NWP2, and four NWP3—and 12 juvenile fish samples—six each of NWP1 and NWP2. We did not observe any juvenile NWP3 samples, probably because NWP3 juveniles are scarce in Taiwanese waters (unpublished data). However, comprehensive data on juvenile and adult samples of NWP1 and NWP2 suggested that more information is needed on the differences in gut microbial diversity among the different life stages, as well as the potential influence of migratory tracts on the intestinal microbiota.