Jellyfish biodiversity and distribution patterns in the Xisha Islands revealed by environmental DNA metabarcoding

Samples were collected at 7 stations in Xisha Islands in the South China Sea in September 2023 (111°34′-112°19′ E, 16°26′-17°00′ N); four stations in the Lingyang reef (LYJ1-4), three stations in the Qilianyu Islands (QLY1-3) .  For seawater samples, six replicates of 1 L samples were collected from shallow seawater (depth<30 m) and mesophotic zones (30 m≤depth≤100 m) at 5 different sites (LYJ1-2 and QLY1-3) across the reef (n=120 samples total). Surface sediment samples from 7 stations were collected by diving. Some samples were lost, resulting in the final acquisition of 117 seawater and 10 sediment samples. eDNA of seawater samples (n=117) was extracted using a Qiagen DNeasy Blood & Tissue Kit according to the experimental protocols. eDNA of sediment samples was extracted using a DNeasy PowerSoil Pro Kit (Qiagen, Hilden, Germany).The eDNA extracted from seawater and sediment samples were dissolved in 80 µL of AE buffer and C6 Solution, respectively, and stored at -20℃. Final equimolar libraries were constructed using a TruSeqTM DNA Sample Preparation Kit (Illumina) and subjected to paired-end sequencing (2 × 300 bp) on an Illumina MiSeq platform (Shanghai Shuo Bei Biomedical Technology Co., Ltd.). The paired-end sequences were first spliced using FLASH v 1.2.11. The spliced sequences were quality controlled and filtered using QIIME v 1.9.1, and finally high quality clean reads were obtained. Non-repetitive sequences were detected, and the single sequences were removed (http://drive5.com/usearch/manual/dereplication.html). The high quality-filtered sequences were clustered into OTUs (operational taxonomic units), which were created by combining sequences over a 97% similarity threshold. Chimeras were removed while clustering. Representative sequences for each OTU were screened using Uparse software (version 7.0.1090) and annotated using the NT (Nucleotide Sequence Database) in the NCBI (National Center for Biotechnology Information) database based on BLAST (e-value=1e−5).