Optimized DNA Extraction and Sequencing

Three cells of each species were isolated for DNA extraction. All cells were washed five times with filtered in situ water (0.22 µm, Millex-GP filter unit) to exclude contamination (Liu et al. 2022 (link)). Genomic DNA was extracted using the DNeasy Blood & Tissue kit (Qiagen, Germany) following the optimized manufacturer’s protocol, with 25% of the suggested volume used for each solution. The primers 82-F (5′-GAA ACT GCG AAT GGC TC-3′) and ITS-R (5′-TAC TGA TAT GCT TAA GTT CAG CGG-3′) were used for PCR amplifications of the 18S rRNA gene and ITS1-5.8S-ITS2 region (Gao et al. 2012 (link); Jerome et al. 1996 (link)). To minimize the possibility of PCR amplification errors, Q5^® Hot Start High-Fidelity 2 × Master Mix DNA Polymerase (New England BioLabs, USA) was used (Li et al. 2023 (link)). The thermal cycler program used was that described by Li et al. (2022 (link)). The quality of the amplified DNA was checked by 1% agarose gel electrophoresis. PCR products were purified using the EasyPure R Quick Gel Extraction Kit (TransGen Biotech Co., Ltd., Beijing, China), and then sequenced on an ABI-PRISM 3730 automatic sequencer (Applied Biosystems, Tsingke Biological Technology Company, Qingdao, China).