Dna extraction

DNA extraction and sequencing

On average, eight Sinocalanus individuals (ranging from 1 to 24; based on preliminary morphological identification) per locality were randomly selected. DNA was extracted from each animal using the proteinase-K method ( Schwenk et al., 1998). Specifically, each animal was mixed with H3 buffer with proteinase K (20 μL), containing final concentrations of 10 mM Tris-HCl, 50 mM KCl, 0.005% Tween 20, 0.005% NP-40 and 10 mg /mL proteinase K (MERCK, Germany). Then, the mixed solution was incubated in a 55 °C water-bath with mild shaking for 16–20 h. Finally, proteinase K was denatured through a 12 min incubation at 95 °C. DNA samples were centrifuged briefly and stored at 4 °C for genetic analyses.

A total of 346 individuals (~eight individuals per location; table 1) were selected for the sequencing of a fragment of mtCOI

CONTRIBUTIONS TO ZOOLOGY 94 (2025) 142–179

(680 bp) gene. The amplification was carried out using the primer pair LCO1490 and HCO2918 ( Folmer et al., 1994). The PCR was conducted in a total volume of 20 μL, comprising 10 μL 2 × HieffTM PCR Master Mix (With Dye), 6 μL ddH2O, 1 μL 10 μM solution of each primer and 2 μL DNA sample. The PCR conditions were as follows: incubation at 94 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 45 s, annealing at 45 °C for 45 s and extension at 72 °C for 45 s; and a final extension at 72 °C for 7 min. Five individuals on average from each population (206 individuals in total; table 1) were also chosen for sequencing of a portion of the nuclear gene for 18S ribosomal RNA (18S; 1,805 bp). The PCR procedure was the same as for COI, except for the primers used: 18Sf (5′ -TAC CTG GTT GAT CCT GCC AG- 3′) and 18Sr (5′ -TAA TGA TCC TTC CGC AGG TTC AC- 3′). The PCR conditions were as follows: incubation at 94 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min and extension at 72 °C for 1 min 30 seconds; and a final extension at 72 °C for 7 min. Cloning was performed following the protocol established in our previous studies ( Ni et al., 2019; Wang et al., 2021) due to the potential for multiple heterozygous sites in 18S fragments. Ten clones were sequenced for each 18S PCR product, and only nucleotide sequences that were identical and obtained at least twice per PCR product were retained for further analysis. All COI PCR products were sequenced in the forward direction and all 18S PCR products were sequenced in both directions on an ABI PRISM 3730 DNA capillary sequencer using the PCR primers as sequencing primers (Sangon Biotech Co., Ltd, Shanghai, China). Subsequently, all the chromatograms of COI and 18S sequences underwent careful examination and manual correction for scoring errors in MEGA X ( Kumar et al., 2018). All new sequences have been submitted to GenBank under accession numbers: COI: PP750470–750551 and 18S: PP755206–755333.