3.2 Clonal diversity and structure of diploid and tetraploid
Both the diploid and tetraploid of C. udensis were clonal populations. There were 89 and 46 genets in tetraploid and diploid, respectively. For this species, it also presented relatively higher clonal diversity (Table 4).
The average clone size (N /G ) of the tetraploid was 1.146, the Simpson diversity index (D ) and Fager evenness index (E ) were 0.4695 and 0.6863, respectively. Correspondingly, these parameters for the diploid were 1.109, 0.3741 and 0.4612 respectively, indicating that the tetraploid had higher clonal diversity than the diploid.
No shared genotype was found between diploid and tetraploid (Table 4). The analysis of variance between Simpson diversity index (D ) and Fager evenness index (E ) showed that there were significant differences in clonal diversity between diploid and tetraploid (P <0.05).
There were no dominant clones in the C. udensis diploids and tetraploids of the Hualongshan Mountains. The diploids consisted of 46 genotypes (genets), of which three genets had two clonal ramets, and one genet had three clonal ramets, while the other 42 genets had only a single clonal ramet. For the tetraploid, there was 89 genotypes (genets), of which three genets had two clonal ramets, and two genets had three clonal ramets, while the other 78 genets had only a single clonal ramet (Fig. 3). Therefore, both the diploids and tetraploids were polyclonal. Further, no identical individual genotype was found between the different ploidies, which verified that differentiation occurred between the diploids and tetraploids.
It is worth noting that there was differentiation in the clonal reproduction characteristics between the diploids and tetraploids. In the diploids, the numbers of clones (number of genets) were relatively lower, and the distribution of clones or genets was scattered. While for the tetraploids, the distribution of genets was relatively close and dense, which indicated higher clonal diversity.
All of the sample C. udensis diploid and tetraploid individuals were amplified by PCR using SSR markers (Table 1). Finally, 51 clear and stable DNA fragments were obtained for eight pairs of SSR primers. The sizes of the DNA fragments were from 350-750 bp. No linkage disequilibrium loci were found. Among the 51 bands, 40 bands were polymorphic. The percentage of polymorphic loci (PPL ) was 78.4%, in addition, Nei’s gene diversity index H was 0.2358, and the Shannon information diversity index I was 0.6337. All these results confirmed a high level of genetic diversity in C. udensis (Table 4).
Three genetic diversity indices of the tetraploid were PPL = 68.5%, H = 0.2332, and I = 0.3416, respectively, which were slightly higher than that of the diploid (PPL = 63.5%, H = 0.2127, and I = 0.3012) (Table 2). It showed that the genetic diversity of the tetraploid was slightly higher than that of the diploid.
The gene differentiation coefficient G ST between the diploid and tetraploid was 0.6193, which indicated that 61.93% of the total genetic diversity existed between the different ploidies, and the genetic variation within different ploidies was 38.07%. This was consistent with the results of AMOVA analysis, namely, greatest genetic variation occurred between the diploid and the tetraploid (Table 5).