DNA sequence variations
The three aligned cpDNA fragments, atp B-rbc L (delete the poly T), psb A-trn H and psb B-psb H, were 729, 617 and 644 bp in length for C. bifida ; 735, 581 and 644 bp in length for C. micholitzii , respectively. The 1990 bp combined cpDNA data set of C. bifida had 22 parsimony-informative polymorphic sites (S) and identified seven haplotypes (cpH1-cpH7). The nucleotide diversity per site (π) was 0.00191 and haplotype diversity (H d) was 0.7184. While, the 1960 bp combined cpDNA data set ofC. micholitzii had 24 parsimony-informative polymorphic sites (S) and identified 14 haplotypes (cpH8-cpH21). Its nucleotide diversity per site (π) was 0.00107 and haplotype diversity (H d) was 0.9078. The haplotype geographical distributions of the two species were presented in Figure 1a. For C. bifida , two populations in China, XS and SJ, had three haplotypes respectively and the remaining three populations (PX, TA and BT) had only one unique haplotype. For C. micholitzii , population ATT and KON had four haplotypes and the other five populations only had one unique haplotype.
The length of the five amplified nuclear genes ranged from 520 to 1005 bp (Table 1). All indels were excluded from subsequent analysis. The genetic diversity indices of C. bifida and C. micholitziiwere showed in Table 1. Recombination events occur frequently in the low copy nuclear genes. The number of segregating sites and number of haplotypes of C. micholitzii were generally higher than that ofC. bifida , while nucleotide diversity per site (π) and haplotype diversity (H d) of C. micholitzii were lower than that ofC. bifida . The haplotype geographic distributions of each gene for the two species were shown in Figure 1b-f. Results showed that there is one haplotype (A5) of the PHYP gene and two haplotypes (D6 and D10) of ATT that shared between C. bifida and C. micholitzii , and the haplotypes of the other three genes were all species-specific.