title: s421
library_construction_protocol: The 2-step PCR was carried out for these representative samples. In the first PCR, fragments of the 96 single-copy genes were amplified using the forward and reverse primers listed in Table S2, which were designed containing an overlapping region of the forward and reverse Illumina sequencing primers (forward: 5?f-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNN [specific primer]-3?f, reverse: 5?f-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNN [specific primer]-3?f). The first PCR was performed in a 5 ??L reaction mixture containing 1?~ Ampdirect buffer with dNTPs, 2.5 pmol of both forward and reverse primers, 0.25 U of BIOTAQ Hot Start DNA Polymerase (Shimazu, Kyoto), and 5-30 ng of total DNA. Cycling parameters for the first PCR: initial denaturation at 95 ??C for 10 min, followed by 35-40 cycles stepdown PCR of denaturation at 94 ??C for 20 s, annealing at 60 - 54 ??C for 30 s (60??C: 5 cycles, 58??C: 5 cycles, 56??C: 5 cycles, 54??C: 20-25 cycles), and elongation at 72 ??C for 1 min, followed by final elongation for 72 ??C for 10 min. The Illumina sequencing adaptors plus the 8 bp identifier indices (Hamady et al. 2008) were added in the subsequent PCR process using a forward and reverse fusion primer (forward: AATGATACGGCGACCACCGAGATCTACAC-index-TCGTCGGCAGCGTC , reverse: CAAGCAGAAGACGGCATACGAGAT-index-GTCTCGTGGGCTCGG, respectively). The second PCR was conducted in a 50 ??L reaction mixture containing 1?~ PCR Buffer for KOD FX Neo, 20 nmol of each dNTP, 1 U KOD FX Neo polymerase (Toyobo, Osaka, Japan), 15 pmol of each primer and 1 ??L of the PCR products. Cycling parameters for the second PCR: initial denaturation at 94 ??C for 2 min, followed by 8 cycles of denaturation at 98 ??C for 10 s, annealing at 55 ??C for 30 s, and elongation at 68 ??C for 1 min, with a final elongation at 68 ??C for 7 min. The PCR products were quantified by Qubit fluorometer (Invitrogen) to adjust amplification efficiencies among samples. Because of poor results in PCR for two ingroup taxa, 28 taxa of the ingroup and one outgroup taxon (Table 1) were subject to the sequencing by the NGS Illumina MiSeq Platform. The adjusted PCR products were pooled then separated on an agarose gel and fragments between 450 and 600 bp in length were excised and extracted by QIAquick Gel Extraction Kit (QIAGEN). The amplicon libraries were sequenced by 2 ?~ 300 bp paired-end sequencing on the MiSeq platform using MiSeq v3 Reagent Kit according to the manufacturer?fs instructions.