DNA extraction, amplification and sequencing
The total genomic DNA was extracted from the muscles of one hind leg by AxyPrep Genomic DNA Miniprep Kit (AXYGEN), according to the manufacturer’s instructions. We used previously published sequences from the family Rhaphidophoroidea to jointly complete this sampling, including six species of the genus Diestrammena Brunner von Wattenwyl, 1888, four species of the genus RhaphidophoraServille, 1838, twenty species of the subfamily Dolichopodainae Brunner von Wattenwyl, 1888, nineteen species of the subfamily Troglophilinae Krauss, 1879 (Allegrucci & Sbordoni, 2019). The samples and sequence information were listed in Table S1. The following six gene regions were amplified: Mitochondrial ribosomal 16S, nuclear ribosomal 28S, mitochondrial cytochrome oxidase I (COI), ATPase catalytic subunit A-like (ACSL), cytoplasmic-like (CPL) and water dikinase-like (WDL). PCR was performed using 2×Es Taq Master Mix (Dye) manufactured by Cowin Bio (China), with a final volume of 50μL, composed of: 25μL of the 2×Es Taq Master Mix (Dye), 19μL of the ddH2O, 2μL of forward and reverse primer each and 2μL of the sample DNA extract. The PCR annealing temperature for each gene was modified (Table S2). PCR products were directly sequenced.