Morphological analysis
Of the three leaf characters (leaf length, leaf width and ratio of leaf length to width), putative hybrid individuals consistently had two morphological characters, leaf length and leaf width, intermediate in value between B. alternifolia and B. crispa (Table 1). Ratios of leaf length to leaf width were significantly larger in B. alternifolia than the other two taxa (Table 1). Of the seven floral characters, corolla tube width and anther height of B. × wardii were intermediate between the values of the two assumed parental species, whereas herkogamy did not differ significantly between the three taxa (Table 1). The remaining four floral characters, corolla tube length (TL), corolla lobe length(CLL), corolla lobe width (CLW) and style length (SL) all showed a similar pattern, with the characters in B. alternifolia having significantly lower values than those measured in B. crispa or B. × wardii (Table 1).
Table 1
Morphological traits used to distinguish between B. alternifolia, B. crispa and B. × wardii.
Characters
|
B. alternifolia
|
B. × wardii
|
B. crispa
|
F
|
Welch
|
P value
|
L (mm)
|
16.00 ± 3.15a
|
27.31 ± 10.02b
|
64.65 ± 25.47c
|
|
62.971
|
< 0.001
|
W (mm)
|
3.62 ± 0.66a
|
14.00 ± 3.26b
|
36.61 ± 11.72c
|
|
235.112
|
< 0.001
|
L/W
|
4.52 ± 0.99a
|
1.94 ± 0.47b
|
1.76 ± 0.31b
|
|
97.410
|
< 0.001
|
TL (mm)
|
7.10 ± 0.80a
|
9.77 ± 1.04b
|
10.25 ± 1.51b
|
|
79.584
|
0.017
|
TW (mm)
|
0.93 ± 0.17a
|
1.09 ± 0.15b
|
1.20 ± 0.18c
|
17.584
|
|
0.641
|
CLL (mm)
|
1.43 ± 0.27a
|
2.29 ± 0.37b
|
2.17 ± 0.40b
|
49.695
|
|
0.073
|
CLW (mm)
|
1.60 ± 0.20a
|
2.41 ± 0.35b
|
2.45 ± 0.46b
|
|
80.282
|
0.004
|
AH (mm)
|
5.22 ± 0.76a
|
5.97 ± 0.93b
|
6.66 ± 0.96c
|
18.266
|
|
0.539
|
SL (mm)
|
2.89 ± 0.47a
|
4.14 ± 0.91b
|
4.41 ± 1.05b
|
|
46.621
|
0.001
|
HE (mm)
|
1.08 ± 0.67
|
0.75 ± 0.72
|
0.95 ± 0.87
|
1.261
|
|
0.588
|
1 mean ± standard deviation are shown for the three traits. |
2 L: leaf length, W: leaf width, L/W: ratio of leaf length to leaf width, TL: corolla tube length, TW: corolla tube width, CLL: corolla lobe length, CLW: corolla lobe width, AH: anther height, SL: style length, HE: herkogamy. |
3 a, b, c: the means with different superscripts are significantly different from each other at the 0.05 level and based on Tamhane’s T2 test |
The two parental species are morphologically clearly distinct. In the PCA of 10 morphological characteristics, the first and second principal components explained 52.17% and 12.98% of the total variation, respectively. The two-dimensional scatter diagram based on PC1 and PC2 showed clearly the separation of B. alternifolia and B. crispa. Individuals of B. × wardii fell between the two parent species, with a slight overlap with B. alternifolia and a large overlap with B. crispa. Apart from the character HE, there is little difference in the correlation coefficients between the other nine traits (0.29–0.38) (Fig. 2a).
Petal color reflectance
The reflectance spectrum of the petals showed some variation between the different species. In B. crispa and B. × wardii there was a clearly marked peak in the reflectance spectrum at 485 nm, with extremely low variation. However, there was no obvious peak in the reflectance spectrum in B. alternifolia (Fig. 2 b).
Sequence analyses of the four nuclear genes in the BH population
NrETS: The total length of the nrETS region alignment was 380 bp in all individuals, including 20 nucleotide substitutions (for variation sites, see Additional file 1: Table S1). A total of 19 haplotypes were observed from these loci, among them five, six and thirteen haplotypes from B. alternifolia, B. crispa and B. × wardii, respectively. Haplotype network analysis identified two major clusters separated by eight nucleotide substitutions. One cluster comprised five haplotypes of B. alternifolia, one haplotype of B. crispa and seven haplotypes of B. × wardii. The other cluster comprised five haplotypes of B. crispa and six haplotypes of B. × wardii (Fig. 3 a1).
Only one haplotype from B. crispa nested with the B. alternifolia cluster, and was found in BHCR2 (H10/H11) and BHCR7 (H10/H12), which show different haplotypes from both clusters. Of the putative hybrid individuals, all but one individual (BHWI9) had two divergent haplotypes nested with each of the two divergent clusters. The individual BHWI9 was homozygous for a B. crispa haplotype at this locus (Fig. 3 a1).
GapC2: The total length of the gapC2 region alignment was 606 bp for all individuals, including 21 nucleotide substitutions, one 2-bp insertion/deletion and one 1-bp insertion/deletion (for variation sites, see Additional file 2: Table S2). A total of 13 haplotypes were observed for these loci, among them one, nine and nine haplotypes belonging to B. alternifolia, B. crispa and B. × wardii, respectively. In the haplotype network analysis, this region was divided into two clusters by twelve nucleotide substitutions. One cluster contained the only one haplotype of B. alternifolia, one haplotype of B. crispa and two haplotypes of B. × wardii. The other cluster contained eight haplotypes of B. crispa and seven haplotypes of B. × wardii. Two individuals of B. crispa, BHCR2 and BHCR7, had a haplotype found in the B. alternifolia cluster but all their other haplotypes were found in the B. crispa cluster. All B. × wardii individuals but one (BHWI9) showed two divergent haplotypes originating from both clusters, and the individual BHWI9 was homozygous for a haplotype from B. crispa cluster (H5/H5) (Fig. 3 b1).
PPR24: The total length of the PPR24 region alignment was 647 bp for all individuals, including 43 nucleotide substitutions (for variation sites, see Additional file 3: Table S3). A total of 20 haplotypes were observed for these loci, among them four, ten and fourteen haplotypes from B. alternifolia, B. crispa and B. × wardii, respectively. In the haplotypes network analysis, this region was divided into two clusters by twenty-one nucleotide substitutions. One cluster contained four haplotypes of B. alternifolia, one haplotype of B. crispa and seven haplotypes of B. × wardii. The other cluster contained nine haplotypes of B. crispa and seven haplotypes of B. × wardii. One B. crispa individual (BHCR7) showed different haplotypes from both clusters (H1/H14). All B. × wardii individuals showed two divergent haplotypes originating from both B. alternifolia and B. crispa clusters (Fig. 3 c1).
PPR123: After sequence alignment, the PPR123 region was 809 bp in length, and included 23 nucleotide substitutions (for variation sites, see Additional file 4: Table S4). A total of nine haplotypes were observed for these loci, among them two, four and eight haplotypes from B. alternifolia, B. crispa and B. × wardii, respectively. Haplotype network analysis identified two major clusters separated by eight nucleotide substitutions. One cluster comprised two haplotypes of B. alternifolia, one haplotype of B. crispa and five haplotypes of B. × wardii. The other cluster comprised three haplotypes of B. crispa and three haplotypes of B. × wardii (Fig. 3 d1).
The only one haplotype from B. crispa nested within the B. alternifolia cluster derived from BHCR7 (H1/H7). Of the putative hybrid individuals, all but one individual (BHWI9) had two divergent haplotypes nested with each of the two divergent clusters. The individual BHWI9 had two haplotypes found in the B. crispa cluster (H7/H8) (Fig. 3 d1).
Sequence analyses for the combined chloroplast regions
The combined length of the cpDNA fragment alignment (including rpl16, trnD-trnT, trnS-trnfM) from the three taxa was 2054 bp, and contained 10 nucleotide substitutions (for variation sites, see Additional file 5: Table S5). Seven haplotypes were inferred in total, and each taxon had three haplotypes. Haplotype network analysis indicated that most putative hybrid individuals (75%) shared two haplotypes with B. crispa, while they did not share any haplotypes with B. alternifolia. The remaining four B. × wardii individuals had a unique haplotype (H7), which was one mutational step away from the predominant haplotype of B. crispa (H4). Even though B. alternifolia and B. crispa did not share any haplotypes, the differences between their haplotypes were only one or two mutational steps. The haplotype network of cpDNA could not be divided into two clades. The haplotype H3 from B. alternifolia (BHAL13) was closer to the haplotype H6 from B. crispa and B. × wardii (two mutation sites away) than the haplotype H2 from the closest B. alternifolia (six mutation sites away) (Fig. 3 e1).
NewHybrids analysis
Analysis of the four studied nuclear genes using NewHybrids among the three taxa showed that all individuals with B. alternifolia morphology were assigned to a pure parental species with high posterior probabilities (>0.999). Of the 16 individuals morphologically identified as B. crispa, all but two (BHCR2 and BHCR7) could be identified as B. crispa with high posterior probabilities (>0.996). The individuals BHCR2 and BHCR7 were assigned to B. crispa and F1 with the lower probabilities of 0.775 and 0.785, respectively. Of the 15 individuals morphologically identified as B. × wardii, 13 individuals were assigned to the F1 class with high posterior probabilities (>0.974). The remaining individual BHWI2 was assigned to the F1 class with probability of 0.778 whereas BHWI9 was assigned to be B. crispa with a high posterior probability (0.997) (Fig. 4 a1).
Population structure analysis
The Bayesian clustering-based structure analysis of all 48 individuals based on all variation loci showed that the highest △K value was found with K=2, indicating that the individuals sampled in this study were divided into two genetic clusters. When K=2, the genetic component of the individuals morphologically identified as B. alternifolia in the BH population mostly formed one cluster and 14 of the 16 individuals identified as B. crispa formed another cluster. One of the remaining two individuals of B. crispa (BHCR7) as well as 15 B. × wardii individuals showed equal proportions from both clusters. The remaining B. × wardii (BHWI9) and B. crispa (BHCR2) individuals had large genetic component belonging to B. crispa (BHWI9: 0.840; BHCR2: 0.755), indicating a backcrossed generation (Fig. 4 b1).
Sequence analyses of the four nuclear genes in the TJ population
NrETS: The total length of the nrETS region alignment was 380 bp in all individuals, including 17 nucleotide substitutions (for variation sites, see Additional file 1: Table S1). A total of 12 haplotypes were observed from these loci, among them six, three and seven haplotypes from B. alternifolia, B. crispa and B. × wardii, respectively. Haplotype network analysis identified two major clusters separated by eight nucleotide substitutions. One cluster comprised six haplotypes from B. alternifolia, one haplotype from B. crispa and three haplotypes from B. × wardii. The other cluster comprised two haplotypes from B. crispa and four haplotypes from B. × wardii (Fig. 3 a2).
Only one haplotype from B. crispa, derived from TJCR13 (H4/H11), fell into the B. alternifolia cluster, and shows different haplotypes from both clusters. Of the putative hybrid individuals, all but one (TJWI1) had two divergent haplotypes from each of the two divergent clusters. The remaining one individual (TJWI1) was homozygous for a B. crispa haplotype at this locus (H12/H12) (Fig. 3 a2).
GapC2: The total length of the gapC2 region alignment was 606 bp for all individuals, including 22 nucleotide substitutions and one 1-bp insertion/deletion (for variation sites, see Additional file 2: Table S2). A total of 11 haplotypes were observed for these loci, among them three, six and ten haplotypes from to B. alternifolia, B. crispa and B. × wardii, respectively. In the haplotype network analysis, this region was divided into two clusters by twelve nucleotide substitutions. One cluster contained two haplotypes from B. alternifolia and three haplotypes from B. × wardii. The other cluster contained six haplotypes from B. crispa and seven haplotypes from B. × wardii. In B. alternifolia, there was an exception of two individuals, TJAL6 and TJAL12, which had a haplotype (H9) found in the B. crispa cluster, but all the other haplotypes were found in B. alternifolia cluster. All B. × wardii individuals showed two divergent haplotypes originating from both clusters (Fig. 3 b2).
PPR24: The total length of the PPR24 region alignment was 647 bp for all individuals, including 46 nucleotide substitutions (for variation sites, see Additional file 3: Table S3). A total of 22 haplotypes were observed for these loci, among them one, eleven and fifteen haplotypes belong to B. alternifolia, B. crispa and B. × wardii, respectively. In the haplotypes network analysis, this region was divided into two clusters by twenty-one nucleotide substitutions. One cluster contained the only haplotype from B. alternifolia and eight haplotypes from B. × wardii. The other cluster contained all ten haplotypes from B. crispa and seven haplotypes from B. × wardii. All B. × wardii individuals showed two divergent haplotypes originating from both the B. alternifolia and B. crispa clusters (Fig. 3 c2).
PPR123: After sequence alignment, the total length of the PPR123 region was 735 bp, including 45 nucleotide substitutions (for variation sites, see Additional file 4: Table S4). A total of 17 haplotypes were observed for these loci, among them two, ten and ten haplotypes from B. alternifolia, B. crispa and B. × wardii, respectively. The haplotype network analysis identified three major clusters separated by seven or eight nucleotide substitutions. One cluster comprised two haplotypes from B. alternifolia and one haplotype from B. × wardii. The second cluster comprised five haplotypes from B. crispa and two haplotypes from B. × wardii. The third cluster comprised five haplotypes rom B. crispa and seven haplotypes from B. × wardii (Fig. 3 d2).
All haplotypes from B. alternifolia individuals fell into the first cluster. For the B. crispa individuals, ten individuals (TJCR3/5/6/7/9/10/11/12/13/15) had two divergent haplotypes, falling into the second and third clusters, two individuals (TJCR2/14) had haplotypes nested in the second cluster and four individuals (TJCR1/4/8/16) had haplotypes nested in the third cluster. Of the putative hybrid individuals, all had two divergent haplotypes, one in the first cluster and the other in either the second or the third cluster (Fig. 3 d2).
Sequence analyses for the combined chloroplast regions
The combined length of the aligned cpDNA fragments (rpl16, trnD-trnT, trnS-trnfM) from the three taxa was 2065 bp, containing 5 nucleotide substitutions (for variation sites, see Additional file 5: Table S5). All individuals had only two haplotypes, of them one was derived from six individuals (TJAL2/3/4/5/8/11), and the remaining 42 individuals shared another haplotype consistent with the haplotype only in individual BHAL13 (H3) from the BH population (Fig. 3 e2).
NewHybrids analysis
Analysis of four nuclear genes using NewHybrids on the three taxa showed that all individuals with B. alternifolia morphology were assigned to a pure parental species with high posterior probabilities (>0.988). Of the 16 individuals morphologically identified as B. crispa, all but one (TJCR13) were identified as B. crispa with high posterior probabilities (>0.997). The individual TJCR13 was assigned to B. crispa with lower probability (0.840). Of the 17 individuals morphologically identified as B. × wardii, all were assigned to the F1 class with high posterior probabilities (>0.974) (Fig. 4 a2).
Population structure analysis
The Bayesian clustering-based structure analysis of all 48 individuals sampled from the TJ population based on all variable loci showed that the highest △K value was obtained for K=2, indicating that the individuals sampled in this study could be divided into two genetic clusters. When K=2, the genetic component of 13 of the 15 individuals morphologically identified as B. alternifolia from the TJ population mostly formed one cluster, whereas 15 of 16 individuals identified as B. crispa formed another cluster. The remaining two individuals of B. alternifolia (TJAL6 and TJAL12) showed low genetic admixture from B. crispa, moreover, the remaining single B. crispa (TJCR13) contained a large genetic proportion similar to that of B. crispa (0.830), indicating a backcross. All individuals morphologically identified as B. × wardii displayed an approximately equal proportion of genetic material from both clusters (Fig. 4 b2).