Analysis of genetic variation with RAPD
28 RAPD primers used to analyse the genomic DNAs of total 60 individuals from five different populations of A. spinulosa generated 118 clear and reproducible bands, varying in size from 300 bp to 3000 bp. The number of bands per primer ranged from 1 to 8 with an average of 4.21 bands/primer. Seven out of 28 (25%) random primers detected 26 polymorphic loci, giving the percentage of polymorphic loci as 22.03% (Table 2). In TLS, only 2 out of the 28 primers, S64 and S97, detected 4 and 2 polymorphic sites, respectively. For DXS, 2 primers, S97 and S512, amplified 2 and 1 polymorphic fragments separately. In DHS, 1, 1, 2, and 2 polymorphic bands were detected by primers S64, S97, S145 and S12, respectively. As for JFL, 1, 1, 6 and 2 polymorphic band(s) were yielded using primers S64, S97, S145 and S506, respectively. Finally, in DLS 2 polymorphic bands were detected by primer S145. The percentage of polymorphic loci varied among populations with the highest value in JFL (9.09%) and the lowest in DLS (1.96%) (Table 2).
Table 2. Percentage of polymorphic loci revealed by 28 random primers in five A. spinulosa populations.
|Populations||No. of samples||No. of loci||No. of polymorphic loci||Percentage of polymorphic sites|
|Tang Lang Shan||14||101||6||0.0594|
|Da Xi Shan||11||104||3||0.0288|
|Ding Hu Shan||9||106||6||0.0566|
|Diao Luo Shan||10||102||2||0.0196|
|Jian Feng Ling||16||110||10||0.0909|
A total of 17 different RAPD phenotypes were distinguished by the 28 random primers in the five sampled populations. 2, 2, 3, 3 and 7 phenotypes were found in DLS, DHS, TLS, DXS and JFL populations, respectively. No RAPD phenotype was shared among the populations, suggesting a high degree of population differentiation.
Shannon diversity indices based on RAPD banding patterns, ranging from 0 to 0.283, revealed the highest diversity in JFL (0.0276), intermediate in DHS (0.0207), TLS (0.0089) and DLS (0.0066) and the lowest in DXS (0.0044). The diversity within populations on average was 0.0136, whereas the average diversity within species was 0.0560. This study detected that most of the variation from the total diversity occurred among populations (1−Hpop/Hsp=75.71%), compared with the diversity within populations (24.29%) (Table 3).
Table 3. Phenotypic diversity estimated by Shannon's index and its partition within and among populations of A. spinulosa.
|Tang Lang Shan||Da Xi Shan||Ding Hu Shan||Diao LuoShan||Jian Feng Ling||Diversity within populations (Hpop)||Diversity within species (Hsp)||Hpop/Hsp||(Hsp−Hpop)/Hsp|
Results of Nei's gene diversity demonstrated that JFL possessed the highest diversity (0.0220), followed in order by the TLS (0.0153), DHS (0.0152), DXS (0.0127) and DLS (0.0069). The GST value was 0.7763 (Table 4), suggesting significant gene differentiation among populations, which was in agreement with the result of Shannon diversity.
Table 4. Nei's gene diversity and its partition within and among populations of A. spinulosa.
|Populations||Number of alleles||Number of effective alleles||Gene diversity (H)||Gene diversity within populations (HS)||Gene diversity among populations (DST)||Total gene diversity (HT)||Gene differentiation coefficient (GST)|
|Tang Lang Shan||1.0594||1.0263||0.0153||0.0132||0.0458||0.0590||0.7763|
|Da Xi Shan||1.0288||1.0288||0.0127|| || || || |
|Ding Hu Shan||1.0508||1.0218||0.0152|| || || || |
|Diao Luo Shan||1.0169||1.0119||0.0069|| || || || |
|Jian Feng Ling||1.0909||1.0325||0.0220|| || || || |
Hierachical analysis of phenotypic diversity was performed to investigate the partitioning of the RAPD variation within and among populations using AMOVA procedure with the modification proposed for RAPD data by Stewart and Excoffier (1996). Significant ϕst value among populations (ϕst=0.8145, p<0.001) was uncovered, suggesting highly phenotypic differentiation. Then, five sampled populations were defined as two groups (TLS/DHS/DXS vs JFL/DLS) according to their geographical locations (Guangdong vs Hainan) for further dissection. The results demonstrated that most of the genetic diversity (47.44%) was attributable to the variance among regions, 34.01% partitioned among populations within regions, whereas only 18.55% genetic diversity occurring within populations (Table 5).
Table 5. AMOVA analysis for the partition of RAPD variation of A. spinulosa populations.
|Source of variation||Df||Sum of squares||Variance components||% Total variance||p-value|
|Among populations within regions||3||87.939||1.83554||0.3401||<0.001|
|Total||59||234.583||5.39670|| || |
Cluster analysis of the 17 different RAPD phenotypes based on Jaccard's similarity coefficient using UPGMA method led to the identification of two major clades: TLS, DXS and DHS populations distributing in Guangdong form a group; whereas JFL and DLS populations from Hainan constitute the other. (Fig. 1). The result was supported by PCA (principle components analysis) based on RAPD phenotypic data. The three-dimensional representation established from the first three principle components differentiated two groups: one consisting of Hainan populations (JFL/DLS) and the other consisting of Guangdong populations (TLS/DXS/DHS) (Fig. 2).
Figure 1. UPGMA tree showing the relationships among the 17 different RAPD phenotypes obtained in Alsophila spinulosa. The Jaccard similarity coefficient was used.
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Figure 2. Plot of principal components analysis based on RAPD phenotypic characters obtained in Alsophila spinulosa.
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Gene genealogies of cpDNA haplotypes
Sequences of atpB-rbcL intergeic spacer of cpDNA were also determined for conducting a phylogenetic analysis of populations of A. spinulosa. Sequence length varied from 724 bp to 730 bp, with a consensus length of 731 bp; 46 sites (6.3%) were variable. Nucleotides A and T are common in the chloroplast sequence, with contents between 63.17% and 63.70%, which is consistent with the nucleotide composition of most noncoding regions. In total, 13 haplotypes of cpDNA atpB-rbcL spacers were identified in A. spinulosa. Both species, A. podophylla and A. denticulata, were utilized as outgroups since molecular phylogeny of family Cyatheaceae indicates that subgenus Gymnosphaera to which Alsophila podophylla and Alsophila denticulata belong is dichotomized as sister group of subgenus Alsophila which contains A. spinulosa (Wang et al. 2003). Figure 3 shows a minimum spanning network reconstructed on mutational changes between cpDNA haplotypes. Haplotypes of JFL and DLS firstly separated from those identified in TLS, DHS, and DLS, suggesting that A. spinulosa may be subdivided into two geographical groups: Hainan populations and Guangdong populations. Within Hainan region, 2 haplotypes, JFL02 and JFL03, coalesced to JFL01 (=JFL04=JFL05=DLS01) with 3 mutations. As for Guangdong region, 4 haplotypes (DHS01, DHS03, TLS04 and DXS03) coalesced to TLS01 (=TLS02=DXS04) with 1 to 3 mutations, this branch and haplotype DXS01 again coalesced to TLS05 (=DHS02) with 1 mutation each; on another branch haplotypes TLS03 and TLS06 coalesced to DXS02 (=DXS05) with 1 and 4 mutation(s), respectively.
Figure 3. Minimum spanning network relating haplotypes of atpB-rbcL spacer of cpDNA found in populations of Alsophila spinulosa. A. podophylla (AP) and A. denticulate (AD) were used to root the tree. Major links between haplotypes are represented as thick lines. Other possible link is given as thin line. Numbers in circles on links between haplotypes indicate the number of mutational differences between haplotypes.
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A neighbour-joining (NJ) tree was also constructed based on sequences of haplotypes of atpB-rbcL intergenic spacer of cpDNA (Fig. 4). Haplotypes of A. spinulosa formed a monophyletic group within which two major clades were identified. One consisted of populations from Hainan, and the other corresponded to populations from Guangdong (bootstrap values=100%). Two clades formed sister groups to each other. Of the Guangdong clade, except for TLS03, TLS06, DXS02 and DXS05, most of the rest haplotypes coalesced near the tip of the tree; as for Hainan clade, JFL02, JFL03 and DLS01 coalesced exactly at the tip of the tree, suggesting the relatively recent occurrences of the coalescence events. In addition, sequences of the same populations were never grouped into a monophyletic clade. Consequently, the branches from different locations were highly mixed, indicating great amount of gene flow between them.
Figure 4. Neighbour-joining tree of Alsophila spinulosa, rooted using Alsophila podophylla and Alsophila denticulata as outgroups, based on sequences of haplotypes of the atpB-rbcL intergenic spacer of cpDNA. Numbers above branches indicate the bootstrap values of 1000 replicates.
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