Species identification, COII sequencing and microsatellite genotyping
Species were identified by combined analysis of the mitochondrial COII gene and morphology. Whole-genomic DNA was extracted from each termite sample using a TIANamp Genomic DNA Kit (Tian Gen Biotech Co., Ltd) according to the recommendations of the manufacturer. The extracted DNA was stored at 4°C until analysis. Subsequently, we obtained sequence data for a mitochondrial gene (COII) and nuclear markers (microsatellite loci) from each individual sample. For the mitochondrial COII gene, PCR was performed using the primers A-tLeu, 5’-CAGATAAGTGCATTGGATTT-3’, and B-tLys, 5’-GTTTAAGAGACCATTACTTA-3’ (Simon et al. 1994), with an annealing temperature of 56°C (Miura et al. 2000). For nuclear DNA, PCR was performed using ten pairs of microsatellite primers: Rs03, Rs78, Rs76, Ra50, Ra79, Ra95, Ra128, Ra141 and Ra144 (see supplementary materials, Table S1). The conditions for each PCR assay were based on those described by Wu et al. (2020). GeneMapper v4.0 (Applied Biosystems) and Peak Scanner v1.0 (Applied Biosystems) were used for the analysis of the results.
We only used sequences for which the two-direction chromatograms were well matched. Sequences were spliced using DNASTAR Lasergene v7.1 (http://www.dnastar.com/t-allproducts.aspx). After splicing was completed, all sequences were saved in Fasta format and imported into MEGA7 for comparison. The sequences that were not neat at both ends were deleted so that all sequences of the same length were retained. Haplotypes were estimated from the mitochondrial data using DnaSP v5 (Librado& Rozas 2009). The maximum likelihood phylogenetic tree, with 100 bootstrap replicates, and the estimated best substitution model (GTRGAMMA) were obtained using MEGA v. 7.
Genetic diversity is an important measure for evaluating the genetic structure and reproductive strategies of populations. We characterized the nuclear genetic diversity of R . flaviceps and R. chinensis using the following parameters: mean number of effective alleles per locus (Ne ), observed heterozygosity (Ho ) and expected heterozygosity (He ). These analyses were performed using GenAIEx v. 6.5 and GENEPOP v. 1.2. We also investigated genetic diversity based on mitochondrial data using DnaSP v5, including haplotype diversity and nucleotide diversity (h and π ). We tested the differences in genetic diversity (Ne ,He and Ho ) between the species via T-test analysis.