Plastome Evolution and Comparative Analyses of a Recently Radiated Genus Vanda (Aeridinae, Orchidaceae)

Vanda R.Br. is an epiphytic orchid genus with significant horticultural and ornamental value. Previous molecular studies expanded Vanda including some members from five other genera. However, the interspecific relationships of this recently radiated genus have remained unclear based on several DNA markers until now. In this study, the complete plastome has been used to infer the phylogenetic relationships of Vanda s.l. The five newly obtained plastomes ranged from 146,340 bp to 149,273 bp in length, with a GC content ranging from 36.5% to 36.7%. The five plastomes contained 74 protein-coding genes (CDSs), 38 tRNAs, and 8 rRNAs, and their ndh genes underwent loss or pseudogenization. Comparative plastome analyses of 13 Vanda species revealed high conservation in terms of genome size, structure, and gene order, except for a large inversion from trnGGCC to ycf3 in V. coerulea. Moreover, six CDSs and five non-CDSs were selected as candidate DNA barcodes. Our phylogenetic analyses demonstrated that Vanda s.l. is a monophyletic group with high supporting values based on five different datasets (complete plastome with one IR, 68 CDSs, LSC, five hypervariable non-CDSs, and six hypervariable CDSs), while the phylogenetic relationships among species were fully resolved based on the complete plastome with one IR dataset. Our results confirmed that the complete plastome has a great power in resolving the phylogenetic relationships of recently radiated lineages.


Introduction
Vanda R.Br.(Aeridinae, Vandeae, Epidendroideae, and Orchidaceae) comprises approximately 73 species [1,2], and is found in subtropical and tropical Asia and Australia (Queensland).Over half of the species exhibit narrow endemism, with a center of species diversity in the South-East Asian archipelagos [3].Particularly, there is great diversity in floral characters, and thus, Vanda has been recognized as one of the five most horticulturally important orchid genera in the world [3][4][5].
Vanda is considered to be one of the most taxonomically complicated groups in Aeridinae, due to its morphological similarity to Ascocentrum, Euanthe, Trudelia, and Christensonia [4].The genus Vanda was established in 1820 by Robert Brown [6].Lindley [7] redefined this genus, and postulated five sections.Gardiner [8] transferred 17 species from Ascocentrum, Ascocentropsis, Christensonia, Eparmatostigma, and Neofinetia into Vanda, and proposed to establish Vanda s.l.Subsequently, Gardiner et al. [3] used three plastid regions (matK, psbA-trnH, and trnL-F) to partly resolve the phylogenetic relationships among Vanda s.l., and proposed 13 morphological sectional classifications.Chase et al. [2] adopted this broad taxonomic generic concept.Although the Vanda s.l. has been widely accepted, the infrageneric relationships remained controversial in phylogenetic research.For example, V. alpina and V. pumila were clustered together based on five DNA sequences (ITS, atpI-H, matK, psbA-trnH, and trnL-F) [9], but this relationship was not supported when using ITS and five plastid DNA regions (atpI-H, matK, psbA-trnH, trnL-F, and trnS-trnfM) [10].Moreover, many sections proposed by Gardiner et al. [3] were not supported as monophyletic groups based on ITS and three plastid DNA regions (matK, trnH-psbA, and trnL-F) [11].Zhang et al. [12] investigated the diversification of Orchidaceae and inferred that Vanda s.l.originated 8.19 million years ago (Ma), and then rapidly diversified following the Pliocene.Briefly, several DNA markers had limited power to reveal the interspecific relationships of the radiated Vanda s.l.Therefore, it is crucial to explore more genetically varied loci to elucidate the phylogenetic relationships within Vanda.
Until now, the complete plastome has successfully clarified the phylogenetic relationships of recently radiated lineages.For instance, Li et al. [13] used plastome data to explore the relationships within Holcoglossum, which had divergently radiated since the late Miocene [14].Moreover, the phylogenetic relationships of Eriocaulon, which diverged following the late Miocene and diversified in the Quaternary, were well resolved by the complete plastome [15].Wang et al. [16] successfully verified the species relationships within Lagerstroemia (Lythraceae) based on the plastome data, which rapidly radiated following the late Miocene.Thus, it is necessary to explore the plastome character of Vanda s.l. and employ the complete plastome to investigate these recently rapidly diverging lineages.
In this study, the plastomes of five Vanda species were newly obtained, and eight published plastomes were downloaded from GenBank.In total, 13 plastomes were combined to conduct plastome comparative analyses and phylogenetic reconstruction within Vanda s.l.The goals of this study are: (1) to reveal the gene content, gene order, and structures of Vanda plastomes; (2) to detect the hypervariable regions of these plastomes as prospective DNA markers for species identification; (3) to explore the phylogenetic relationships within this genus.Our study provides valuable genetic information on the phylogeny, species identification, and conservation of Vanda s.l.

The Plastome Characters of Vanda
The plastomes of Vanda species contained four regions: a large single copy (LSC) region, a small single copy (SSC) region, and two copies of inverted repeats (IRa/b) (Figure 1).The total length of the 13 plastomes in the genus Vanda ranged from 146,340 bp (V.ampullaceum) to 149,490 bp (V.subconcolor) (Table 1).The lengths of the LSC regions were 83,808-85,982 bp, those of the SSC regions were 11,424-12,002 bp, and a pair of IR regions ranged from 24,523 to 25,965 bp (Table 1).The total GC contents ranged from 36.5% to 36.7%.All of the 13 Vanda plastomes contained 120 genes, comprising 74 protein-coding genes (CDSs), 38 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes (Table 1).All the ndh genes of Vanda plastomes had been lost or pseudogenized (Figure 1; Table 1).The plastomes of V. cristata, V. falcata, V. richardsiana, and V. xichangensis possessed five pseudogenes (ndhB, ndhD, ndhE, ndhG, and ndhI) and lost six genes (ndhA, ndhC, ndhF, ndhH, ndhJ, and ndhK).The other nine species possessed eight pseudogenes (ndhB, ndhC, ndhD, ndhE, ndhG, ndhI, ndhJ, and ndhK), and lost three genes (ndhA, ndhF, and ndhH) (Figure 1).All the ndh genes of Vanda plastomes had been lost or pseudogenized (Figure 1; Table 1).The plastomes of V. cristata, V. falcata, V. richardsiana, and V. xichangensis possessed five pseudogenes (ndhB, ndhD, ndhE, ndhG, and ndhI) and lost six genes (ndhA, ndhC, ndhF, ndhH, ndhJ, and ndhK).The other nine species possessed eight pseudogenes (ndhB, ndhC, ndhD, ndhE, ndhG, ndhI, ndhJ, and ndhK), and lost three genes (ndhA, ndhF, and ndhH) (Figure 1).There was no expansion and contraction in the IR/SC boundary regions across the 13 species of Vanda (Figure S1).Rpl22 (318-329 bp) genes of the LSC crossed over into IRb.The trnN (72 bp) and rpl32 (174 bp) genes were near to the junction between SSC and IRb.Within the junction region between SSC and IRa, the ycf1 genes were across the SSC-IRa boundary, and primarily located in the SSC region, with lengths ranging from 5211 bp (V.coerulescens) to 5328 bp (V.concolor).The adjacent regions of LSC and IRb were located in the rps19 (279 bp) genes and psbA (1062 bp) genes.The collinearity analysis revealed that the SSC and IR regions of 13 Vanda plastomes were highly conserved.Additionally, conservation was observed in the LSC region of 12 Vanda plastomes, and a significant inversion from trnG GCC to ycf3 was identified in the LSC region of the V. coerulea plastome (Figure 2).There was no expansion and contraction in the IR/SC boundary regions across the 13 species of Vanda (Figure S1).Rpl22 (318-329 bp) genes of the LSC crossed over into IRb.The trnN (72 bp) and rpl32 (174 bp) genes were near to the junction between SSC and IRb.Within the junction region between SSC and IRa, the ycf1 genes were across the SSC-IRa boundary, and primarily located in the SSC region, with lengths ranging from 5211 bp (V.coerulescens) to 5328 bp (V.concolor).The adjacent regions of LSC and IRb were located in the rps19 (279 bp) genes and psbA (1062 bp) genes.The collinearity analysis revealed that the SSC and IR regions of 13 Vanda plastomes were highly conserved.Additionally, conservation was observed in the LSC region of 12 Vanda plastomes, and a significant inversion from trnG GCC to ycf3 was identified in the LSC region of the V. coerulea plastome (Figure 2).

Plastome Sequence Divergence and DNA Marker Investigation
The structural characteristics of 13 Vanda plastomes were investigated to evaluate the sequence of the plastomes (Figure S3).The coding regions exhibited higher conservation than the non-coding regions, and the LSC and SSC regions displayed greater variability than the IR regions (Figure S3).The nucleotide diversity (Pi) was calculated to identify the highly mutated hotspots of Vanda plastomes (Figure 4).The results showed that the Pi values of the LSC region, SSC region, and IR regions were 0.0075, 0.0064, and 0.0016, respectively.Based on the ranking of the Pi values, five hypervariable regions, including clpP-psbB, rps15-ycf1, trnN GUU -rpl32, trnS GCU -trnG GCC , and trnT UGU -trnL UAA were identified in the

Phylogenetic Analyses and Character Reconstruction
Maximum likelihood (ML) and Bayesian inference (BI) analyses yielded nearly identical topologies based on the complete plastome with one IR, 68 CDSs, LSC, five hypervariable non-CDSs and six hypervariable CDSs, respectively, except the uncertain systematic position of V. ampullaceum (Figures 5 and S4).The monophyly of Vanda s.l.have confirmed by the five datasets with high Bootstrap percentages (BS values) from the ML analyses and Posterior Probability (PP values) from the Bayesian analyses, respectively.The phylogenetic relationships among species were fully resolved based on the complete plastome (excluding one IR) matrix, and the phylogenetic tree was illustrated here.Vanda s.l. was well supported as a monophyletic group (PP-BI = 1.00,BS-ML = 100%) (Figure 5).Three species from sect.Neofinetia formed a clade and diverged first (PP-BI = 1.00,BS-ML = 100%).The other sections formed another clade with high to moderate supporting values (PP-BI = 1.00,BS-ML = 71%).The three species from sect.Cristatae and the two species from sect.Longicalcarata did not form a clade.
The reconstruction of ndh gene status showed that the most recent common ancestor (MRCA) of Vanda compared to its relatives involved the ndhI pseudogene.The ndhC, ndhJ, and ndhK were lost in sect.Neofinetia and V. cristata (Figure 5).

Phylogenetic Analyses and Character Reconstruction
Maximum likelihood (ML) and Bayesian inference (BI) analyses yielded nearly identical topologies based on the complete plastome with one IR, 68 CDSs, LSC, five hypervariable non-CDSs and six hypervariable CDSs, respectively, except the uncertain systematic position of V. ampullaceum (Figure 5 and Figure S4).The monophyly of Vanda s.l.have confirmed by the five datasets with high Bootstrap percentages (BS values) from the ML analyses and Posterior Probability (PP values) from the Bayesian analyses, respectively.The phylogenetic relationships among species were fully resolved based on the complete plastome (excluding one IR) matrix, and the phylogenetic tree was illustrated here.Vanda s.l. was well supported as a monophyletic group (PP-BI = 1.00,BS-ML = 100%) (Figure 5).Three species from sect.Neofinetia formed a clade and diverged first (PP-BI = 1.00,BS-ML = 100%).The other sections formed another clade with high to moderate supporting values (PP-BI = 1.00,BS-ML = 71%).The three species from sect.Cristatae and the two species from sect.Longicalcarata did not form a clade.
The reconstruction of ndh gene status showed that the most recent common ancestor (MRCA) of Vanda compared to its relatives involved the ndhI pseudogene.The ndhC, ndhJ, and ndhK were lost in sect.Neofinetia and V. cristata (Figure 5).

The Plastome Characteristics and Structural Evolution
In this study, the complete plastome sequences of five Vanda species were newly obtained.The Vanda plastomes contained one LSC, one SSC, and two IR regions (Figure 1), which is similar to most angiosperm [13,15,16].The genome sizes ranged from 146,340 bp to 149,490 bp and the GC contents of the plastomes range from 36.5% to 36.7% and fall within the range of other orchids, such as Aerides and Angraecum [17,18].A total of 120 genes were identified in the Vanda plastomes, consisting of 74 CDSs, 38 tRNA genes, and 8 rRNA genes (Table 1).Further, all ndh genes were pseudogenized or lost (Figure 5).The ndh genes have been involved in photosynthesis, the photosynthetic response and stress acclimation, and have been hypothesized to be closely related to plant transition to terrestrial habitats and are usually absent in epiphytic plants [19,20].Previous studies showed that all 11 ndh genes were lost or pseudogenized in nearly all photosynthetic and nonphotosynthetic orchids, which suggests that the ndh genes have been independently lost in various orchid lineages [21].As such, all of the ndh genes were truncated or pseudogenized in the epiphytic orchid genera Holcoglossum [13], Paraphalaenopsis [22], and Trichoglottis [23].
Our results reveal that the gene arrangement of the IR/SC boundary in Vanda plastomes was extremely conserved (Figure S1), and the gene content of this genus was identical.This finding implied that the length variation of Vanda plastomes did not attribute to the IR/SC boundary shift, although the IR/SC boundary shift has been considered to be the main factor for the variations in plastome length and gene content [24].
Previous studies have revealed that gene structure and gene order are highly conserved in orchid plastomes [25,26].Our collinearity analysis revealed that the structures of 13 Vanda plastomes were conserved, while there was a large inversion from trnG GCC to

The Plastome Characteristics and Structural Evolution
In this study, the complete plastome sequences of five Vanda species were newly obtained.The Vanda plastomes contained one LSC, one SSC, and two IR regions (Figure 1), which is similar to most angiosperm [13,15,16].The genome sizes ranged from 146,340 bp to 149,490 bp and the GC contents of the plastomes range from 36.5% to 36.7% and fall within the range of other orchids, such as Aerides and Angraecum [17,18].A total of 120 genes were identified in the Vanda plastomes, consisting of 74 CDSs, 38 tRNA genes, and 8 rRNA genes (Table 1).Further, all ndh genes were pseudogenized or lost (Figure 5).The ndh genes have been involved in photosynthesis, the photosynthetic response and stress acclimation, and have been hypothesized to be closely related to plant transition to terrestrial habitats and are usually absent in epiphytic plants [19,20].Previous studies showed that all 11 ndh genes were lost or pseudogenized in nearly all photosynthetic and non-photosynthetic orchids, which suggests that the ndh genes have been independently lost in various orchid lineages [21].As such, all of the ndh genes were truncated or pseudogenized in the epiphytic orchid genera Holcoglossum [13], Paraphalaenopsis [22], and Trichoglottis [23].
Our results reveal that the gene arrangement of the IR/SC boundary in Vanda plastomes was extremely conserved (Figure S1), and the gene content of this genus was identical.This finding implied that the length variation of Vanda plastomes did not attribute to the IR/SC boundary shift, although the IR/SC boundary shift has been considered to be the main factor for the variations in plastome length and gene content [24].
Previous studies have revealed that gene structure and gene order are highly conserved in orchid plastomes [25,26].Our collinearity analysis revealed that the structures of 13 Vanda plastomes were conserved, while there was a large inversion from trnG GCC to ycf3 existing in the V. coerulea plastome (Figure 2).Plastome rearrangement was also reported for the plastome of Orchidaceae.For example, Cypripedium formosanum has an inversion from atpA to petG [27], and there is a large inversion from trnS GCU to trnS GGA in the Apostasia wallichii plastome [28].
Our results showed that among 13 Vanda plastomes, the codon usage bias was extremely conserved (Figure S2).Leucine (Leu) had the highest amino acid frequency, while cysteine (Cys) had the lowest frequency.The high leucine frequency may be related to its critical function in photosynthesis-related metabolism [29].The measure of codon usage bias could be helpful to explore the evolutionary patterns of lineages [30].

The Barcoding Investigation and Phylogenetic Analyses
SSRs have been recognized as a crucial molecular marker in population genetics and conservation biology [31,32].A total of 775 SSRs were identified in the Vanda plastome, with 66.32% of them being mono-nucleotide repeats, which are mainly located in the intergeneric regions of the LSC (Figure 3).The majority of the nucleotides consisted of A/T motifs rather than C/G motifs, potentially attributed to the A/T-rich plastome structure, which was consistent with the results of previous studies [33,34].
Plastomes provide abundant genetic information to reveal the phylogenetic relationships within a genus [37].Previous research about the phylogenetic relationships of Vanda partly resolved the interspecific relationships based on several DNA markers [9][10][11]38].In this study, our phylogenetic results based on complete plastomes (excluding one IR) and four other datasets strongly supported that Vanda s.l. was a monophyletic group, which is consistent with previous studies [9][10][11].In addition, the complete plastome perfectly resolved the interspecific and infrageneric relationships of Vanda s.l.Among the seven sampled sections of 13 morphological sections proposed in Gardiner et al. [3], our phylogenetic results indicate that five sections were recognized, and two sections (sect.Longicalcarata and sect.Cristatae) were not monophyletic.The stem of Vanda s.l.diverged following the late Miocene, and rapidly diversified following the Pliocene [16].It is acknowledged that the phylogenetic relationships among the radiated groups were difficult to resolve with several markers.For instance, compared with the results of a few DNA markers, the complete plastome, except in the case of IR, was successfully used to clarify the interspecific relationships of Holcoglossum, which diverged and diversified following the latest Miocene [12,13].The plastomes also showed their power in reconstructing phylogenetic relationships of other recently diverged non-orchid lineages, such as Eriocaulon [14] and Lagerstroemia [15].Therefore, we posit that the plastome could be valuable for exploring the phylogenetic relationships of Vanda s.l., and may also be useful for other lineages that have recently undergone rapid radiation.

Sampling and Sequencing
In this study, five Vanda species, representing three sections (sect.Ascocentrum, sect.Cristatae and sect.Longicalcarata), were collected from Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences.Eight published plastomes of Vanda were downloaded from GenBank and the annotations were updated.In total, 13 species were sampled to represent seven sections of Vanda as accurately as possible.Additionally, two closely related species (Holcoglossum quasipinifolium and Luisia morsei) were selected as outgroups based on Zhang et al. [16].The detailed sampling information was listed in Table S2.
Using the modified CTAB method [39], we extracted the total DNA of five sampled Vanda species from silica gel-dried leaves, respectively.Then, following the manuals of NEB Next ® Ultra DNA Library Prep Kit (NEB, Ipswich, MA, USA), the libraries for paired-end 150 bp sequencing were constructed using an Illumina HiSeq 2000 platform.Finally, around 4 Gb of raw data was generated for each sampled species.

Sequence and Structure Divergence Analyses
The online program mVISTA [46] (https://genome.lbl.gov/vista/index.shtml,accessed on 15 June 2024) was used to visualize the sequence divergence of Vanda with the plastome of Holcoglossum quasipinifolium (NC_041516) as a reference.We analyzed the expansion and contraction of the IR boundary using IRscope v3.1 [47].Possible structure divergence (rearrangements and inversions) of plastomes were detected using the Mauve v1.1.3[48] plugin in Geneious v9.05 [45].Moreover, the IRa region was removed to avoid the overrepresentation of the inverted repeats.To further detect hypervariable regions within Vanda, the Pi values of complete plastomes and 68 CDSs of 13 Vanda species were estimated using DnaSP v6 [49].The window length was set to 100 sites with the step size of 25 sites.

Repetitive Sequence and Codon Usage Analyses
The SSRs were detected using the online program MISA [50] (https://webblast.ipkgatersleben.de/misa/,accessed on 17 June 2024), and the parameters for SSR motifs were set to 10, 5, 4, 3, 3, and 3 nucleotide repeats for mono-nucleotide, di-nucleotide, trinucleotide, tetra-nucleotide, penta-nucleotide, and hexa-nucleotide, respectively.Four long repeat types in 13 plastomes of Vanda were determined using the online program REPuter (https://bibiserv.cebitec.uni-bielefeld.de/reputer,accessed on 17 June 2024), including F (forward), P (palindrome), R (reverse), and C (complement) repeats.The maximum and minimum repeat sizes of long repeats in the REPuter program were set to 50 bp and 20 bp, respectively, with the Hamming distance as 3.In addition, RSCU values and amino acid frequencies were calculated using MEGA v11 [51].Protein-coding regions in the IRa region were also eliminated from the codon usage analyses to avoid overrepresentation.

Phylogenetic Analyses and Measurement of Divergence Variables
The phylogenetic relationships of Vanda were reconstructed based on the following five matrices: (1) the complete plastome sequences (excluding IRa), (2) concatenation of 68 CDSs, (3) LSC region, (4) hypervariable regions of the whole plastomes, and (5) hypervariable regions of 68 CDSs.We removed the IRa region from the complete plastome and CDSs to avoid the overrepresentation of the inverted repeats.Five matrices with 13 Vanda species and two outgroups were aligned using MAFFT v7 [52] and manually adjusted in BioEdit v7.0 [53].After the selection of the best nucleotide substitution model for each matrix using jModelTest2 [54], respectively, ML analyses were carried out in RAxML v8.2.12 [55] with the best-fit model.Moreover, BI analyses were conducted in MrBayes v3.2.6 [56] for 10,000,000 generations and sampled every 1000 generations.After the assessment of the convergence in Tracer v1.7 [57], we obtained the majority rule consensus tree with the first 25% samples as "burn-in".Finally, the phylogenetic trees were plotted using FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/Figtree/, accessed on 22 June 2024).

Character Reconstruction of Ndh Gene Status in Vanda
For all 13 Vanda species and two outgroups in our dataset, all 11 ndh genes were scored as lost and pseudogene.The character state reconstruction was conducted in Mesquite v3.51 (http://www.mesquiteproject.org,accessed on 12 August 2024) using the parsimony method.The best ML topology was selected as the input tree.

Conclusions
In the present study, we reported five Vanda plastomes (V.ampullaceum, V. alpina, V. coerulea, V. cristata, and V. pumila).The plastome characteristics and results of the comparative analyses indicate that the gene content and genomic structure of sampled Vanda plastomes were highly conserved, with the only difference was a large inversion from trnG GCC to ycf3 in the V. coerulea plastome.All ndh genes were found to be lost or pseudogenized.According to the ranking of Pi values, five non-CDSs (clpP-psbB, rps15-ycf1, trnN GUU -rpl32, trnS GCU -trnG GCC , and trnT UGU -trnL UAA ) and six CDSs (ccsA, psbI, rpl22, rpoA, rps15, and ycf1) were selected as candidate barcodes.The phylogenetic topologies based on complete plastome sequences (excluding one IR) showed the highest power in resolving the interspecific relationships of Vanda.Our results found that plastome can offer valuable insights into the phylogenetic relationships of recently diverged lineages.

Figure 1 .
Figure 1.The gene map of five Vanda plastomes.The darker gray in the inner circle corresponds to the GC content.The IRa/b, LSC, and SSC are shown inside the GC content.

Figure 1 .
Figure 1.The gene map of five Vanda plastomes.The darker gray in the inner circle corresponds to the GC content.The IRa/b, LSC, and SSC are shown inside the GC content.

Figure 2 .
Figure 2. Visualization of the collinearity.Structural alignment of 13 Vanda plastomes using progressive Mauve.The tree topology and species order were consistent with that in Figure 5.The lines linking the collinear blocks represent homology between different genomes.Numbers on the upper x-axis are genome map coordinates in kilobases (kb).

Figure 2 .
Figure 2. Visualization of the collinearity.Structural alignment of 13 Vanda plastomes using progressive Mauve.The tree topology and species order were consistent with that in Figure 5.The lines linking the collinear blocks represent homology between different genomes.Numbers on the upper x-axis are genome map coordinates in kilobases (kb).

Figure 3 .
Figure 3. Summary of the simple sequence repeats (SSR) and long repeat sequences of 13 Vanda plastomes.(A) SSR distributions in the LSC, SSC, and IR regions.(B) Number of different types of SSRs identified in plastomes.(C) Number of long repeats by sequence length (F: forward repeats; R: reverse repeats; P: palindromic repeats; C: complementary repeats).

Figure 4 .
Figure 4. Sliding window test of nucleotide diversity (Pi) in the Vanda plastomes.(A) The nucleotide diversity of the complete plastomes showing five mutation hotspot regions (dashed line marked the Pi > 0.54).The x-axis represented the position of the midpoint of a window, while the y-axis represents the Pi value of each window.(B) The nucleotide diversity of 68 CDSs showing six mutation hotspot regions (dashed line marked the Pi > 0.06).

Figure 4 .
Figure 4. Sliding window test of nucleotide diversity (Pi) in the Vanda plastomes.(A) The nucleotide diversity of the complete plastomes showing five mutation hotspot regions (dashed line marked the Pi > 0.54).The x-axis represented the position of the midpoint of a window, while the y-axis represents the Pi value of each window.(B) The nucleotide diversity of 68 CDSs showing six mutation hotspot regions (dashed line marked the Pi > 0.06).

Figure 5 .
Figure 5. Maximum likelihood phylogenetic tree of Vanda based on whole plastomes with one IR and reconstruction of ndh gene loss and pseudogenization.The numbers above represent the supporting values of Bayesian inference and maximum likelihood analyses, respectively, and the star reflects that the supporting value is equal to 100%.The black box represents the pseudogene of the ndh gene, and the blank box represents the absence of the ndh gene.

Figure 5 .
Figure 5. Maximum likelihood phylogenetic tree of Vanda based on whole plastomes with one IR and reconstruction of ndh gene loss and pseudogenization.The numbers above represent the supporting values of Bayesian inference and maximum likelihood analyses, respectively, and the star reflects that the supporting value is equal to 100%.The black box represents the pseudogene of the ndh gene, and the blank box represents the absence of the ndh gene.