Genetic Diversity And Structure Of Camellia Chrysanthoides (Theaceae) And Its Closely Related Species

Camellia chrysanthoides,Camellia micrantha and Camellia parvipetala are three Camellia species distributed within southern Guangxi,China,with a very limited and parapatric distribution.On account of their ornamental value,the three Camellia species have been transplanted excessively from their native habitats by local farmers and in addition the habitats have been damaged.The natural populations of the three Camellia species have been declining rapidly and their distributions have become obviously fragmented.C.chrysanthoides and C.micrantha have been listed as endangered species in the Threatened Species List of China's Higher Plants.In the present study,we evaluated genetic diversity and population structure,analyzed the composition and distribution of haplotypes of C.chrysanthoides,C.micrantha and C.parvipetala using two single-copy nuclear genes?PAL and waxy?and chloroplast DNA?cpDNA?sequences,as well as simple sequence repeats?SSR?.The main results were as follows:1)The alignment of the PAL gene sequences had a length of 653 bp,including four parsimony-informative sites,eight single-nucleotide polymorphisms,and no indels?insertions and deletions?,resulting in 11 distinct haplotypes.The alignment of the waxy gene sequences had a length of 684 bp,including 15 parsimony-informative sites,2 single-nucleotide polymorphisms,and two indels?insertions or deletions?,resulting in 8 distinct haplotypes.For the cpDNA sequences,alignment produced a consensus sequence of 5,192 bp,with five nucleotide substitutions,resulting in 4 distinct haplotypes.For PAL sequences,the haplotype H11 was the most frequent and widely shared haplotype,being found in 6 of the total of 7 populations across the three species,and was situated in the middle of the network.For waxy sequences,the haplotype W6 was the most frequent and widely shared haplotype,being found in 6 populations across the three species,and was situated in the middle of the network.And second,the W4 was frequent and widely haplotype shared by 7 populations.In the cpDNA network diagram,haplotype C3 was located at the central position,shared by 4 populations?BH2,NB1,NB2,NB3?.C4?BH1?was separated from C3 by only one mutational step.C1?PT2?and C2?PT1?were separated from C3 by four and three mutational steps,respectively.2)The three Camellia species studied here all showed a low level of genetic diversity?for both haplotype diversity and nucleotide diversity?based on PAL,waxy and cpDNA than the congeners.Based on the SSR dataset,in the species level,the mean number of alleles?3.9?,the effective number of alleles?2.328?,observed heterozygosity?0.520?and expected heterozygosity?0.501?of C.parvipetala were higher than C.chrysanthoides and C.micrantha.In the population level,the mean number of alleles varied from 2.7?NB1?to 5.1?NB3?,with an average of 3.8,the effective number of alleles ranged from 1.788?NB1?to 2.466?PT1?,with an average of 2.161,the observed heterozygosity ranged from 0.409?NB1?to 0.543?NB3?,with an average of 0.487 and the expected heterozygosity ranged from 0.379?NB1?to 0.543?NB3?,with an average of 0.471.3)High levels of genetic differentiation were detected in these three Camellia species.The F_STT value is less than 0.15 only between the populations NB1 and NB2,indicated a moderate level of genetic differentiation,and other F_STT values between any pair of populations were higher than 0.15,indicated a high or very high level of genetic differentiation.The gene flow?Nm?between each pair of populations are low,with only three of them are larger than 1.Population fragmentation,limited dispersal,small population and genetic drift may lead to increase the degree of inter-population differentiation.4)The AMOVA analysis revealed that,for PAL,13.25%of the genetic variation was among three Camellia species,20.14%of the genetic variation was among population within species and 66.61%was within populations.For waxy,35.18%of the genetic variation was among three Camellia species,0.86%of the genetic variation was among population within species and 63.95%was within populations.In species,most of the genetic variation was found within populations based on PAL and waxy.We failed to observe sequence variation within individual populations based on cpDNA.For SSR,9.66%of the genetic variation was among three Camellia species,24.62%of the genetic variation was among population within species and 65.72%was within populations.In species,the result is similar to the result of PAL and waxy.The STRUCTURE and PCoA analysis showed that 7 populations were clustered into two groups.One group comprised 5 populations from C.chrysanthoides and C.micrantha,and the other consisted of 2populations from C.parvipetala.Among all populations,neither two single-copy nuclear genes?PAL and waxy?nor cpDNA showed significant phylogeographic structure.Correlation between genetic and geographic distances was nonsignificant?R²=0.0847,p=0.23?,which was supported by the result of the Mantel test.Neutrality test and mismatch analysis showed that these three Camellia species did not experience a bottleneck effect or population expansion.5)Analysis of SAMOVA and NETWORK based on PAL,waxy and cpDNA,STRUCTURE analysis based on SSR dataset showed that relative lower genetic differentiation was observed between C.chrysanthoides and C.micrantha than between C.parvipetala and C.chrysanthoides or C.micrantha.Based on the reports before,we think that 2 populations of C.chrysanthoides and 3 populations of C.micrantha are likely to be the same species.6)In the field surveys,we found that the habitats of these three Camellia species had damaged.STRUCTURE analysis show that the most likely number of groups was two?K=2?,and These two groups should be regarded as two distinct units for conservation purposes.In situ and ex situ actions should be urgently implemented as soon as possible.