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.