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.