The Complete Chloroplast Genomes of Bulbophyllum (Orchidaceae) Species: Insight into Genome Structure Divergence and Phylogenetic Analysis

Bulbophyllum is one of the largest genera and presents some of the most intricate taxonomic problems in the family Orchidaceae, including species of ornamental and medical importance. The lack of knowledge regarding the characterization of Bulbophyllum chloroplast (cp) genomes has imposed current limitations on our study. Here, we report the complete cp genomes of seven Bulbophyllum species, including B. ambrosia, B. crassipes, B. farreri, B. hamatum, B. shanicum, B. triste, and B. violaceolabellum, and compared with related taxa to provide a better understanding of their genomic information on taxonomy and phylogeny. A total of 28 Bulbophyllum cp genomes exhibit typical quadripartite structures with lengths ranging from 145,092 bp to 165,812 bp and a GC content of 36.60% to 38.04%. Each genome contained 125–132 genes, encompassing 74–86 protein-coding genes, 38 tRNA genes, and eight rRNA genes. The genome arrangements, gene contents, and length were similar, with differences observed in ndh gene composition. It is worth noting that there were exogenous fragment insertions in the IR regions of B. crassipes. A total of 18–49 long repeats and 38–80 simple sequence repeats (SSRs) were detected and the single nucleotide (A/T) was dominant in Bulbophyllum cp genomes, with an obvious A/T preference. An analysis of relative synonymous codon usage (RSCU) revealed that leucine (Leu) was the most frequently used codon, while cysteine (Cys) was the least used. Six highly variable regions (rpl32-trnLUAG > trnTUGU-trnLUAA > trnFGAA-ndhJ > rps15-ycf1 > rbcL-accD > psbI-trnSGCU) and five coding sequences (ycf1 > rps12 > matK > psbK > rps15) were identified as potential DNA markers based on nucleotide diversity. Additionally, 31,641 molecular diagnostic characters (MDCs) were identified in complete cp genomes. A phylogenetic analysis based on the complete cp genome sequences and 68 protein-coding genes strongly supported that 28 Bulbophyllum species can be divided into four branches, sects. Brachyantha, Cirrhopetalum, and Leopardinae, defined by morphology, were non-monophyly. Our results enriched the genetic resources of Bulbophyllum, providing valuable information to illustrate the complicated taxonomy, phylogeny, and evolution process of the genus.


Introduction
Bulbophyllum, comprising approximately 2200 species, is one of the largest genera of Orchidaceae and serves as an excellent model system for investigating orchid biodiversity [1][2][3].Its distribution spans pantropical regions, including Africa, Madagascar, the Americas, and the Asia-Pacific region [4].Members of this genus exhibit epiphytic or lithophytic habits and typically possess one-or two-leaved pseudobulbs with a labellum attached to the base of the floral column via an elastic hinge [4,5].Bulbophyllum demonstrates remarkable adaptability, flourishing in a variety of environments including subtropical dry forests and wet montane cloud forests [4,6].Owing to its morphologically diverse lateral sepals that vary in size, shape, color, and surface ornamentation, Bulbophyllum has economic importance that is attributable to its ornamental uses [5].Additionally, the aromatic compounds found in these orchids are significant for their medical benefits [7,8].
Phylogenetic analyses of Bulbophyllum using DNA sequence data have made significant progress recently.Most phylogenetic results supported the monophyly of a broadly defined Bulbophyllum and its continental taxa, such as Asian, African, and Neotropical clades [1,4,17].Hu et al. reconstructed the phylogenetic relationship in the Asian Cirrhopetalum alliance of Bulbophyllum based on combining four DNA sequence data (nrDNA: ITS, Xdh; cp DNA: matK and psbA-trnH, 117 taxa), supporting an amended Cirrhopetalum alliance was monophyly [5].Based on eight DNA sequence data (nrDNA: ITS, Xdh, OrcPI; cp DNA: atpI-atpH, ycf1, matK, trnD-trnE, psbA-trnH, 179 taxa), Gamisch et al. divided the Malagasy taxa into four clades [18].These studies have clarified the phylogenetic relationships of different regions in this group, but nodal support values of the main clades or lineages were moderate to low or lacking for some relationships.The number of accepted species continues to grow as new discoveries are reported [19][20][21]; taxonomic work on Bulbophyllum became a major challenge, and further investigation into the relationships within the genus necessitates more detailed study.
With the continuous reduction in sequencing costs, the chloroplast (cp) genome has become a pivotal tool for investigating phylogenetic relationships within complex taxa.The cp genome offers several advantages, including a unique mode of inheritance, highly conserved genome structure, and a moderate evolutionary rate [22,23].Owing to these unique characteristics, cp genomes are widely used to explore the phylogenetic relationship among orchid clades.Liu et al. reconstructed the phylogenetic relationships of the Cleisostoma-Gastrochilus clades in Aeridinae based on the cp genomes, robustly supporting that this clade can be divided into six subclades with higher support rates and more stable topological structures than before [24].Additionally, many studies have comprehensively compared differences in orchid cp genomes to understand the structural characteristics and evolution patterns, such as Pholidota (13 species) and Paraphalaenopsis (three species) [25,26].Yang et al. compared and analyzed cp genomes of 18 species from Asian and Neotropical Bulbophyllum.The results show that the cp genome structure of Asian and Neotropical clades was different due to selection pressures under the condition of geographical isolation [27].Furthermore, integrative analyses of multiple cp genomes can help to develop applicable molecular markers for species identification [28].Five highly variable regions (ycf1, ndhA, ndhF, trnQ, and trnK), the potential DNA markers, were found in four Liparis cp genomes [29].Tang et al. analyzed the cp genomes of sect.Macrocaulia in Bulbophyllum and proposed 20 intergenic regions and three coding genes of the most variable hotspot regions as candidate effective molecular markers [30].Although the species identification rate of multiple DNA molecular markers has been improved to different degrees, the species identification rate of closely related groups is still not optimistic [31].Complete cp genome sequences harbor many more sequence variations, making them far more sensitive and effective than standard DNA barcodes, referred to as ultrabarcodes, can effectively improve the identification rate [32].Sawicki et al. analyzed the cp genomes of Apopellia endiviifolia using the ultrabarcode technique.They suggested that the genomes could be clearly separated into two evolutionary lineages, and the number of detected molecular diagnostic characters (MDCs) indicated a level of genetic divergence in the dataset of cp genomes [33].
To date, only a few cp genomes of Bulbophyllum have been sequenced.Detailed cp genomic comparisons and phylogenetic analyses are lacking, which hinders our ability to further elucidate its interspecific relationships.In order to further clarify the phylogenetic relationships among species of the genus and to obtain useful genetic resources, we sequenced and assembled the cp genomes of seven Bulbophyllum species (B.ambrosia, B. crassipes, B. farreri, B. hamatum, B. shanicum, B. triste, B. violaceolabellum) and compared them with other Bulbophyllum species published to investigate their relationships.Our results will provide valuable information for cp genome evolution, phylogenetic relationships, and species identification of Orchidaceae.

General Characteristics of the Chloroplast Genomes
The seven newly sequenced Bulbophyllum cp genomes were circular with the typical quadripartite structure, including a large single copy (LSC), a small single copy (SSC), and a pair of inverted repeats (IRs) (Figure 1).We combined the published cp genomes of 21 Bulbophyllum orchids with this study's seven species to compare the basic cp genome features within the genus.The number of genes, GC content, etc., of the 28 cp genomes are summarized in Table 1.As shown in Table 1, the 28 Bulbophyllum cp genome sizes ranged from 145,092 bp (B.kwangtungense) to 165,812 bp (B.crassipes).The cp genomes were variable in LSC and SSC regions, with 77,088 to 87,177 bp and 11,089 to 18,632 bp, while being conserved in IR regions, with sizes ranging from 25,465 to 30,927 bp.The GC content was relatively consistent, ranging from 36.60% to 38.04%, and the distribution of the GC content across different regions was uneven, with about 43.18%, 34.93%, and 29.67% for the IR, LSC, and SSC regions, respectively (   optimistic [31].Complete cp genome sequences harbor many more sequence variations, making them far more sensitive and effective than standard DNA barcodes, referred to as ultrabarcodes, can effectively improve the identification rate [32].Sawicki et al. analyzed the cp genomes of Apopellia endiviifolia using the ultrabarcode technique.They suggested that the genomes could be clearly separated into two evolutionary lineages, and the number of detected molecular diagnostic characters (MDCs) indicated a level of genetic divergence in the dataset of cp genomes [33].
To date, only a few cp genomes of Bulbophyllum have been sequenced.Detailed cp genomic comparisons and phylogenetic analyses are lacking, which hinders our ability to further elucidate its interspecific relationships.In order to further clarify the phylogenetic relationships among species of the genus and to obtain useful genetic resources, we sequenced and assembled the cp genomes of seven Bulbophyllum species (B.ambrosia, B. crassipes, B. farreri, B. hamatum, B. shanicum, B. triste, B. violaceolabellum) and compared them with other Bulbophyllum species published to investigate their relationships.Our results will provide valuable information for cp genome evolution, phylogenetic relationships, and species identification of Orchidaceae.

General Characteristics of the Chloroplast Genomes
The seven newly sequenced Bulbophyllum cp genomes were circular with the typical quadripartite structure, including a large single copy (LSC), a small single copy (SSC), and a pair of inverted repeats (IRs) (Figure 1).We combined the published cp genomes of 21 Bulbophyllum orchids with this study's seven species to compare the basic cp genome features within the genus.The number of genes, GC content, etc., of the 28 cp genomes are summarized in Table 1.As shown in Table 1, the 28 Bulbophyllum cp genome sizes ranged from 145,092 bp (B.kwangtungense) to 165,812 bp (B.crassipes).The cp genomes were variable in LSC and SSC regions, with 77,088 to 87,177 bp and 11,089 to 18,632 bp, while being conserved in IR regions, with sizes ranging from 25,465 to 30,927 bp.The GC content was relatively consistent, ranging from 36.60% to 38.04%, and the distribution of the GC content across different regions was uneven, with about 43.18%, 34.93%, and 29.67% for the IR, LSC, and SSC regions, respectively (Table 1).Note: An asterisk (*) indicates the sequences are newly sequenced in this study, "-" indicated the missing data.
Each cp genome was annotated with a total of 106-113 unique genes, which included 68-79 protein-coding genes, 30 transfer RNAs (tRNAs), and four ribosomal RNAs (rRNAs) (Table 1).Most genes existed as single copies in either LSC or SSC regions.However, 19 genes were duplicated in IRs, encompassing seven protein-coding genes (ycf1, rpl2, rps7, ndhB, ycf2, rpl23, and rps12), eight tRNAs (trnN GUU , trnR ACG , trnA UGC , trnL GAU , trnV GAC , trnL CAA , trnI CAU , and trnH GUG ), and four rRNAs (rrn23, rrn16, rrn5, and rrn4.5).Nine protein-coding genes and six tRNA genes contained one intron each, whereas genes ycf3 and clpP possessed two introns.The lengths of introns varied among different genes, with the longest intron found in the trnK UUU gene.Notably, the ndh genes were truncated or completely lost in more than half of the species (Tables 1 and S10).The highest degree of loss was the ndhF gene, which was observed in 11 species.The highest degree of pseudogenization was the ndhD gene, which was pseudogenized in nine species.S1).

Repeat Sequence Characterization
We identified four types of long repeats-palindromic (P), forward (F), complementary (C), and reverse (R) elements (Figure 2A, Supplementary Table S2) in 28 Bulbophyllum cp genomes.Among these, all four categories were observed in 14 species, while 12 species contained three categories of repeats (C/R, F, and P); two species (B.crassipes and B. farreri) exhibited two categories (P and F).The number of long-repeat sequences ranged from 17 (B.S3).
A total of 38 (B.leopardinum) to 80 (B.mentosum) SSRs were detected in the cp genome of the 28 Bulbophyllum species, and six categories of SSRs (mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats) were identified (Figure 2C,D and Supplementary Tables S4 and S5).And SSRs were mostly located in the intergenic region of LSC (Supplementary Figure S1).Mono-nucleotide repeats (SSR loci A/T) were the most abundant, accounting for 58.3% (B.lingii) to 81.7% (B.ambrosia), with counts varying from 47 to 58.This was followed by di-nucleotide repeats (6 to 13 occurrences, 8.8.% to 22.0%), tri-nucleotide repeats (zero to four occurrences, 5.9%), tetra-nucleotide repeats (2 to 13 occurrences, 2.6% to 17.6%), penta-nucleotide repeats (zero to five occurrences, 6.4%), and hexa-nucleotide repeats, with the least number of SSRs (zero to two occurrences, 4.1%).All mononucleotide SSRs belonged to the A or T type, and the majority of di-, tri-, tetra-, penta-, and hexa-nucleotide SSRs were particularly rich in A or T (Figure 2D, Supplementary Table S5).In general, the distribution pattern of SSRs was uneven across the 28 species.The mono-, di-, and tetranucleotide repeat categories were observed in all species, while tri-and penta-nucleotide repeats were absent in 10 different species.Hexa-nucleotide repeats were only present in B.   S3).

Relative Synonymous Codon Usage Analysis
We analyzed a total of 68 protein-coding genes among the 28 Bulbophyllum cp genomes, with the exception of the ndh genes due to incomplete gene loss and pseudogenization.These genes were encoded by a range of 17,226 codons in B. plumosum to 22,758 codons in B. shanicum (Figure 3, Supplementary Table S6).The codon usage patterns revealed a highly conserved codon usage bias (CUB).Leucine (Leu) was one of the most frequently occurring amino acids, appearing a total of 57,130 times across all 28 cp genomes.In contrast, cysteine (Cys) was the least frequent, occurring only 6515 times.An analysis of the relative synonymous codon usage (RSCU) indicated that UUA and AGA had the highest CUB, with average values of 1.934 and 1.894, respectively, while CGC and CUC had the lowest CUB, with average values of 0.374 and 0.397, respectively.Among the three stop codons, the frequency of UAA was the highest, accounting for 39.9%.The results also showed that 30 codons exhibited RSCU values greater than one, and 32 codons exhibited values less than one (Figure 3, Supplementary Table S6).The RSCU values of AUG encoding for methionine (Met) and UGG encoding for tryptophan (Trp) were determined to be one in all seven species.A total of 38 (B.leopardinum) to 80 (B.mentosum) SSRs were detected in the cp genome of the 28 Bulbophyllum species, and six categories of SSRs (mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats) were identified (Figure 2C,D and Supplementary Tables S4  and S5).And SSRs were mostly located in the intergenic region of LSC (Supplementary Figure S1).Mono-nucleotide repeats (SSR loci A/T) were the most abundant, accounting for 58.3% (B.lingii) to 81.7% (B.ambrosia), with counts varying from 47 to 58.This was followed by di-nucleotide repeats (6 to 13 occurrences, 8.8.% to 22.0%), tri-nucleotide repeats (zero to four occurrences, 5.9%), tetra-nucleotide repeats (2 to 13 occurrences, 2.6% to 17.6%), penta-nucleotide repeats (zero to five occurrences, 6.4%), and hexa-nucleotide repeats, with the least number of SSRs (zero to two occurrences, 4.1%).All mononucleotide SSRs belonged to the A or T type, and the majority of di-, tri-, tetra-, penta-, and hexa-nucleotide SSRs were particularly rich in A or T (Figure 2D, Supplementary Table S5).In general, the distribution pattern of SSRs was uneven across the 28 species.The mono-, di-, and tetra-nucleotide repeat categories were observed in all species, while tri-and penta-nucleotide repeats were absent in 10 different species.

Relative Synonymous Codon Usage Analysis
We analyzed a total of 68 protein-coding genes among the 28 Bulbophyllum cp genomes, with the exception of the ndh genes due to incomplete gene loss and pseudogenization.These genes were encoded by a range of 17,226 codons in B. plumosum to 22,758 codons in B. shanicum (Figure 3, Supplementary Table S6).The codon usage patterns revealed a highly conserved codon usage bias (CUB).Leucine (Leu) was one of the most frequently occurring amino acids, appearing a total of 57,130 times across all 28 cp genomes.In contrast, cysteine (Cys) was the least frequent, occurring only 6515 times.An analysis of the relative synonymous codon usage (RSCU) indicated that UUA and AGA had the highest CUB, with average values of 1.934 and 1.894, respectively, while CGC and CUC had the lowest CUB, with average values of 0.374 and 0.397, respectively.Among the three stop codons, the frequency of UAA was the highest, accounting for 39.9%.The results also showed that 30 codons exhibited RSCU values greater than one, and 32 codons exhibited values less than one (Figure 3, Supplementary Table S6).The RSCU values of AUG encoding for methionine (Met) and UGG encoding for tryptophan (Trp) were determined to be one in all seven species.

Expansion and Contraction of IRs, Sequence Divergence, and Nucleotide Diversity
A comprehensive comparison of the boundaries between the LSC, IRs, and SSC regions was conducted across the 28 Bulbophyllum species (Figures 4 and S2).The junctions between the IRs and SC regions exhibited a high degree of conservation.In the cp genomes of these 28 Bulbophyllum species, several key genes, namely rpl22, ndhF, ycf1, rps19, and psbA, were found at the junction of the LSC/IRb, IRb/SSC, SSC/IRa, and IRa/LSC borders.The rpl22 gene, spanning from LSC to IRb, was primarily located in the LSC region and ranged from 279 to 423 bp in length.

Expansion and Contraction of IRs, Sequence Divergence, and Nucleotide Diversity
A comprehensive comparison of the boundaries between the LSC, IRs, and SSC regions was conducted across the 28 Bulbophyllum species (Figure 4, Supplementary Figure S2).The junctions between the IRs and SC regions exhibited a high degree of conservation.In the cp genomes of these 28 Bulbophyllum species, several key genes, namely rpl22, ndhF, ycf1, rps19, and psbA, were found at the junction of the LSC/IRb, IRb/SSC, SSC/IRa, and IRa/LSC borders.The rpl22 gene, spanning from LSC to IRb, was primarily located in the LSC region and ranged from 279 to 423 bp in length.The divergence of sequences in the cp genomes of 28 Bulbophyllum species was plotted using the mVISTA program with the annotated B. affine (LC556091) sequence as a reference (Supplementary Figure S3).The results revealed sequences with significant conservation in Bulbophyllum cp genomes, particularly in the coding region.The highest variation was observed in the SSC region, followed by the LSC region and IR regions.Mauve visualization graphs also indicated that no significant gene rearrangement was detected among these cp genomes (Supplementary Figure S4).Exogenous fragment insertions were detected at rps12~trnV GAG in the IR regions of B. crassipes, with a length of 3975 bp (Figure 1).
The nucleotide diversity value (Pi) for the coding regions and intergenic regions was calculated using DnaSP to further analyze the mutation hotspots in 28 Bulbophyllum species.The results showed that the Pi values ranged from 0 to 0.21413 (rpl32-trnL UAG ) (Figure 5A, Supplementary Table S7).The IR regions exhibited the highest conservation, with a value of 0.0035.The SSC region displayed the greatest nucleotide diversity (Pi = 0.0307), followed by the LSC region (0.0141).According to the ranking of the Pi values, six hypervariable regions were identified for candidate barcodes, including rpl32-trnL UAG (0.21413), trnT UGU -trnL UAA (0.09500), trnF GAA -ndhJ (0.09138), rps15-ycf1 (0.09315), rbcL-accD (0.07534), and psbI-trnS GCU (0.06529).Additionally, the protein-coding genes displayed higher conservation (Figure 5B, Supplementary Table S8).Among these genes, ycf1 (0.02956), rps12 (0.02643), matK (0.02178), psbK (0.01599), and rps15 (0.01511) exhibited the highest Pi values appropriate for phylogeny.The divergence of sequences in the cp genomes of 28 Bulbophyllum species was plotted using the mVISTA program with the annotated B. affine (LC556091) sequence as a reference (Supplementary Figure S3).The results revealed sequences with significant conservation in Bulbophyllum cp genomes, particularly in the coding region.The highest variation was observed in the SSC region, followed by the LSC region and IR regions.Mauve visualization graphs also indicated that no significant gene rearrangement was detected among these cp genomes (Supplementary Figure S4).Exogenous fragment insertions were detected at rps12~trnV GAG in the IR regions of B. crassipes, with a length of 3975 bp (Figure 1).

Molecular Diagnostic Characters (MDCs)
The ABGD analysis revealed a consistent count of species division; there were 20 groups in the 28 Bulbophyllum species.The prior intraspecific distance ranged between 0.0021 and 0.0010.The results correctly recognized 14 species, and the other  S11).
A complete cp genome analysis revealed 31,641 MDCs in the 28 Bulbophyllum species, which comprised 17.73% of the total length (Figure 6, Supplementary Table S12).The number of MDCs that distinguished species from the others of the genus was extremely variable, ranging from 149 to 8603 (B.crassipes).The MDC analysis was also carried out using the four datasets (five coding genes, six noncoding regions, ycf1, and rpl32-trnL UAG ) obtained by Pi values.The higher number of MDCs identified was with the dataset of five coding genes; the sum of MDCs was 2203 (26.32% of the total length), ranging from 3 to 750 (B.exaltatum).The sums of MDCs of the latter three datasets were 1534, 1435, and 249, accounting for 33.48%, 24.08%, and 29.05%, respectively.The datasets of ycf1 and six noncoding regions ranged from 2 to 372 (B.plumosum) and 3 to 232 (B.kwangtungense), respectively.The lowest number of MDCs was identified in the dataset of rpl32-trnL UAG ; the number of MDCs was 45, and three species did not have MDCs.

Molecular Diagnostic Characters (MDCs)
The ABGD analysis revealed a consistent count of species division; there were 20 groups in the 28 Bulbophyllum species.The prior intraspecific distance ranged between 0.0021 and 0.0010.The results correctly recognized 14 S11).
A complete cp genome analysis revealed 31,641 MDCs in the 28 Bulbophyllum species, which comprised 17.73% of the total length (Figure 6, Supplementary Table S12).The number of MDCs that distinguished species from the others of the genus was extremely variable, ranging from 149 to 8603 (B.crassipes).The MDC analysis was also carried out using the four datasets (five coding genes, six noncoding regions, ycf1, and rpl32-trnL UAG ) obtained by Pi values.The higher number of MDCs identified was with the dataset of five coding genes; the sum of MDCs was 2203 (26.32% of the total length), ranging from 3 to 750 (B.exaltatum).The sums of MDCs of the latter three datasets were 1534, 1435, and 249, accounting for 33.48%, 24.08%, and 29.05%, respectively.The datasets of ycf1 and six noncoding regions ranged from 2 to 372 (B.plumosum) and 3 to 232 (B.kwangtungense), respectively.The lowest number of MDCs was identified in the dataset of rpl32-trnL UAG ; the number of MDCs was 45, and three species did not have MDCs.

Phylogenetic Analysis
The phylogenetic analysis of 28 Bulbophyllum species, based on two datasets comprising complete cp genomes and 68 protein-coding genes, revealed that the species formed four major clades (Figure 7, Supplementary Figure S5).The alignment matrix of complete cp genomes was 131,138 bp, with 12,031 variable sites and 6201 parsimony informative sites.The matrix of 68 protein-coding genes was 59,417 bp and included 4869 variable sites, along with 2404 parsimony informative sites.The topologies remained largely consistent within the two datasets, demonstrating strong support based on complete cp genomes (Bootstrap Support, BS ≥ 98; posterior probability, PP = 1.00), while the support was relatively moderate, inferred by 68 protein-coding genes (BS ≥ 75, PP ≥ 0.70) (Figure 7, Supplementary Figure S5).

Phylogenetic Analysis
The phylogenetic analysis of 28 Bulbophyllum species, based on two datasets comprising complete cp genomes and 68 protein-coding genes, revealed that the species formed four major clades (Figures 7 and S5).The alignment matrix of complete cp genomes was 131,138 bp, with 12,031 variable sites and 6201 parsimony informative sites.The matrix of 68 protein-coding genes was 59,417 bp and included 4869 variable sites, along with 2404 parsimony informative sites.The topologies remained largely consistent within the two datasets, demonstrating strong support based on complete cp genomes (Bootstrap Support, BS ≥ 98; posterior probability, PP = 1.00), while the support was relatively moderate, inferred by 68 protein-coding genes (BS ≥ 75, PP ≥ 0.70) (Figures 7 and S5

The Characteristics of Chloroplast Genomes
Owing to the highly conserved structure, uniparental inheritance, and mutation rates between those shown in the mitochondrial and nuclear genomes, cp genomes have been widely employed for investigating phylogenetic relationships [22,23].Recently, orchids have become a focal point in phylogenetic studies due to their rich diversity, wide distribution, and epiphytic habits.With the decreasing costs of sequencing, an increasing number of cp genome evolutions in Orchidaceae have been studied [34,35].The genus Bulbophyllum serves as one of the representative groups of orchid biodiversity [1,2,4]; the cp genomes of their diversity patterns and evolutionary adaptations attract much attention [24,30,36].
This study sequenced the complete cp genomes of seven orchid species in the genus Bulbophyllum and compared them with other 21 Bulbophyllum species in order to broaden the knowledge about the genome organization and molecular evolution of the Orchidaceae species.The obtained seven cp genomes of the Bulbophyllum species in this

The Characteristics of Chloroplast Genomes
Owing to the highly conserved structure, uniparental inheritance, and mutation rates between those shown in the mitochondrial and nuclear genomes, cp genomes have been widely employed for investigating phylogenetic relationships [22,23].Recently, orchids have become a focal point in phylogenetic studies due to their rich diversity, wide distribution, and epiphytic habits.With the decreasing costs of sequencing, an increasing number of cp genome evolutions in Orchidaceae have been studied [34,35].The genus Bulbophyllum serves as one of the representative groups of orchid biodiversity [1,2,4]; the cp genomes of their diversity patterns and evolutionary adaptations attract much attention [24,30,36].
This study sequenced the complete cp genomes of seven orchid species in the genus Bulbophyllum and compared them with other 21 Bulbophyllum species in order to broaden the knowledge about the genome organization and molecular evolution of the Orchidaceae species.The obtained seven cp genomes of the Bulbophyllum species in this study possessed a typical quadripartite structure, with the genome sizes of these cp genomes varying from 145,092 bp (B.kwangtungense) to 165,812 bp (B.crassipes), and the GC content ranging from 36.60% (B.plumosum) to 38.04% (B.leopardinum), all of which fell within the normal range of cp genomes reported in previous studies [37,38].The gene order and content were not different from those of other closely related Bulbophyllum species [27,30,36,39,40].Notably, an exogenous fragment insertion appeared at rps12~trnV GAG in the IR regions of B. crassipes, leading to the length of the IRs being obviously larger than other Bulbophyllum species (Figures 1, 4 and S2).The source remains to be further explored.
Although the general structure of Bulbophyllum cp genomes is conserved, differences in ndh gene composition were detected.The ndh genes encode the thylakoid NADH complex [41], which is frequently pseudogenized or lost in Orchidaceae [42,43].Recently, studies of the orchid cp genomes have revealed that rampant independent loss of the ndh genes occurred in different orchid clades.The cp genome of E. pusilla contains truncated versions of ndhJ, C, D, B, G, and H, and lacks sequences for ndhK, F, E, A, and I [44], and the pseudogenization of ndh genes in the Cleisostoma-Gastrochilus clades is widespread [24].In this study, all of the cp genomes showed evidence of gene pseudogenization or loss  1 and S10).Some studies have suggested that the inactivation of ndh genes may be associated with epiphytic habitats [45] and connected to the extreme water availability and use of CAM (Crassulacean acid metabolism) photosynthesis [24,46], such that the ndh genes were extensively pseudogenized in Cymbidium mannii, an epiphyte with constitutive CAM [47].Although Bulbophyllum is primarily an epiphytic group and utilizes the CAM pathway [18,48], more research is needed to understand the relationship between the evolution of the CAM pathway or growth form and the cp genomes.

Repeat Sequence Analysis
As an inherent variation, long-repeat sequences with lengths greater than 30 bp are universal in angiosperms and considered to play an important role in genome stability and structural variation [49].There were abundant long-repeat sequences in the cp genomes of Bulbophyllum species in previous studies [36], and a total of 18-49 long repeats were detected in our study (Figure 2B).The palindromic (P) and forward (F) repeats were the most common long-repeat sequences in our study (Figure 2A).Slight variation in the number of repeat units and their proportions occurred in different species.Additionally, the GC content of IR regions was much higher than that of the LSC and SSC regions (Table 1), and these characteristics were also revealed in other plant species, primarily because of the presence of rRNA (rrn4.5, rrn5, rrn16, and rrn23) genes in this region [50].
Simple sequence repeats (SSRs) are highly abundant and randomly distributed throughout the genome, making them valuable genetic molecular markers for population genetic relationships and phylogenetic studies [51].A number of SSR markers were discovered in several orchid genera such as Vanda [52] and Dendrobium [53].The most abundant SSR type was the mononucleotide repeat, and the majority of SSRs in the Bulbophyllum species were composed of A/T SSRs [27,30,36].In this study, a total of 38-80 SSRs and six types of SSRs (mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats) were detected (Figure 2C,D).A/T SSRs were found to be more abundant than G/C SSRs (G/C was only detected in B. triste) and may be due to a bias towards A/T in cp genomes [54].Of dito hexa-nucleotide SSRs among Bulbophyllum species, most SSRs were specific to each species (Figure 2D).These SSRs were distributed widely and randomly throughout the chloroplast genomes, and were usually located in the intergenic spacer (IGS) region, which is consistent with angiosperm cp genomes [30].Most of the previous studies revealed that the richness of SSR types is various in different species, which may contribute to the genetic variations among species [55].Notably, some of the SSRs repeats were highly specific, such as AC/GT and AAATCC/ATTTGG SSRs only being detected in the cp genomes of B. farreri, AGATAT/ATATCT SSRs only being detected in B. gedangense, and ATCCCC/ATGGGG only being detected in B. hamatum.Furthermore, B. crassipes and B. orientale (the members of sect Racemosae) possessed ACT/AGT SSRs; B. hirtum, B. shanicum, and B. triste (the members of sect Lemniscata) possessed AATCT/AGATT SSRs, which is consistent with some results of the phylogenetic analysis (Figure 2C,D).Thus, these SSRs have the potential to be specific molecular markers for establishing the molecular evolutionary history and demographic diversity of Bulbophyllum species.These results are significant for identifying and analyzing genetic diversity in Bulbophyllum.

Codon Usage Analysis
Codons are involved in protein translation, vital for the genetic information transfer process of an organism.Codon usage bias is a significant factor in cp genome evolution, influencing gene function expression.Organisms with close genetic relationships exhibit similar codon usage bias [56].These studies can help to clarify evolutionary relationships and improve the efficiency of gene expression in research utilizing genetic transformation [57].More recently, a variety of orchid cp genomes have been sequenced, allowing for the comprehensive analysis of codon preferences [58,59].The codon usage bias in Bulbophyllum cp genomes showed similar patterns, as indicated by the comparative analysis of RSCU values (Figure 3, Supplementary Table S6).According to the RSCU analysis, it was found that most of the frequently used codons (RSCU > 1) ended in A or U, while the less frequently used codons (RSCU < 1) ended in C or G.Among all codons, leucine (Leu) had the highest occurrence, while cysteine (Cys) had the lowest frequency.This trend is consistent with observations in most angiosperm cp genomes [60] and further demonstrates the high conservation in these 28 Bulbophyllum species.

Expansion and Contraction of IRs, Sequence Divergence, and Nucleotide Diversity
Boundary shifts between the IR and SC regions are a common occurrence in the evolution of angiosperms and are the main factors contributing to the differences in the length and gene content of cp genomes [58].For instance, the IR region of the cp genome of Pelargonium × hortorum was expanded extensively; its length was increased to 76 kb [61].In general, the gene arrangement of the IR/SC boundary was highly conserved (Figures 4 and S2), with some differences in the IR/SSC junction detected.In B. hamatum and B. tianguii, the ycf1 gene was completely located within the SSC region, while in the other species, the ycf1 gene crossed over JSA.At the junction between JSB, some species lost the ndhF gene.This result indicated that there was no significant expansion or contraction in the IR regions of Bulbophyllum.This may be one of the primary factors contributing to the high conservation of the cp genome structure.

Molecular Diagnostic Characters (MDCs)
Within uncharacterized groups, DNA barcodes, short DNA sequences that are present in a wide range of species, can be used to assign organisms into species.Automatic Barcode Gap Discovery (ABGD) can assign these sequences into potential species based on the barcode gap [66].A previous ABGD analysis showed that clear taxa assignments mostly corresponded to the diverged lineages in the phylogenetic trees [67].In this study, half of the species were identified correctly using this method, while other species were clustered together with their relatives.This corresponded with sections based on morphology to some extent (Supplementary Table S11).
Recently, the development of methods based on molecular diagnostic characters (MDCs) comes into view; the number of observed MDCs has a particularly large impact on the efficiency of species identification [68].We conducted an MDC analysis of five datasets (complete cp genomes, five coding genes, six noncoding regions, ycf1, and rpl32-trnL UAG ); the results revealed that B. crassipes possessed the most abundant MDCs of the Bulbophyllum species, possibly related to the insertion observed in the genome structure (Figures 1 and 6).The number of MDCs of complete cp genomes was remarkable, while three species could not be identified on the basis of differences in the datasets of rpl32-trnL UAG (Figure 6 and Supplementary Table S12).

Phylogenetic Analysis
Complete cp genomes are valuable resources for analyzing phylogenetic relationships; they have been extensively used for phylogenetic analysis across different plant groups [24,32].Our phylogenetic analysis of Bulbophyllum, based on complete cp genome and 68 CDSs (Figures 7 and S5), provided strong support for the monophyly of the Neotropical clade, sects Lemniscata, Racemosae, and Macrostylida (BS ≥ 98, PP = 1.00), in agreement with previous studies [5,27,30,36].The branch topology and node support rates compared to the phylogenetic relationships constructed using traditional molecular markers also improved [4,5] (Figures 7 and S5).In addition, B. ambrosia, previously assigned to sect Leopardinae, was distantly related to other two species (B.leopardimum and B. pectinatum) [5], a result corroborated here.It was noteworthy that B. hamatum, being a member of sect Cirrhopetalum and closely related to B. omerandrum based on a morphological comparison [19], was close to two species from sect Brachyantha (B.tianguii and B. violaceolabellum) with high support (Figures 7 and S5).Two species, i.e., B. ningboense and B. gedangense, were identified as unplaced along the spine of Bulbophyllum by Lin et al. and Luo et al. [69,70].B. ningboense, a species similar to B. chrondriophorum morphologically, was a sister to B. pingnanense and B. inconspicuum (Figure 7), with lateral sepals connected partly and subumbellate raceme [19], basically in accordance with the characteristics of sect Cirrhopetalum.The phylogenetic analysis further strongly supported that B. ningboense is closely related to B. pingnanense within the sect Cirrhopetalum.B. gedangense, morphologically similar to B. psychoon and B. scabractum, was close to the single species B. ambrosia.It appears that more sampling and more evidence are required to understand the evolutionary history of B. gedangense.Our results generally indicated that there was an overlap of species from different sections, especially sects Brachyantha, Cirrhopetalum, and Leopardinae.The conclusions of previous studies found that the boundaries between these sections should be re-evaluated [5,27,71].However, our phylogenetic analysis showed that species from sect Ephippium and sect Desmosanthes, as well as sect Stenochilus and sect Reptantia, respectively, were sister groups, which might due to limited sampling.Therefore, additional cp genomes from Bulbophyllum individuals are necessary to further investigate phylogeny, especially at lower taxonomic levels.

Taxon Sampling and DNA Sequencing
In this study, we sequenced seven Bulbophyllum species (B.ambrosia, B. crassipes, B. farreri, B. hamatum, B. shanicum, B. triste, and B. violaceolabellum), and their voucher specimens were stored at the herbarium of the College of Forestry, Fujian Agriculture and Forestry University (FJFC).Total genome DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) method [72].Sequencing was carried out at Berry Genomics (Beijing, China) using the Illumina HiSeq 2500 platform, with a read length of 150 bp.Approximately 10 Gb of raw data was obtained for each species.In addition to our newly sequenced data, we downloaded the available chloroplast genomes of 21 other Bulbophyllum species from GenBank (Table 1).

Relative Synonymous Codon Usage Analysis
Codon usage and relative synonymous codon usage (RSCU) values were estimated using Codon W, accessible at http://codonw.sourceforge.net/(accessed on 1 November 2023) [82].To minimize sampling errors, we excluded repeat sequences and protein-coding regions (CDSs) shorter than 300 bp from the codon usage calculations.This step was necessary since short CDSs can lead to estimation errors in codon usage.TBtools v1.1047 was employed to create the heat map for the RSCU analysis [83].

Sequence Divergence and Nucleotide Diversity
To investigate variations in the boundaries of the LSC/IR/SSC regions in 28 Bulbophyllum chloroplast genomes, we conducted the SC/IR boundary analyses using the Perl script CPJSdraw.pl(https://github.com/xul962464/CPJSdraw,accessed on 1 November 2023).For visualizing identity across the 28 cp genomes, we employed the shuffle-LAGAN mode of the mVISTA program, with B. affine (LC556091) as the reference genome (http://genome.lbl.gov/vista/mvista/submit.shtml, accessed on 1 November 2023) [84].Mauve was utilized to perform analyses of cp genome rearrangement using default "seed families" and default values.In all sequences, one of the IR regions was consistently removed [85].The nucleotide variability (Pi) for the 28 Bulbophyllum cp genomes and the 68 protein-coding genes was calculated using DnaSP v6.0, with a window length of 100 bp and a step size of 25 bp [86].

Molecular Diagnostic Characters (MDCs)
The Automatic Barcode Gap Discovery (ABGD) analysis was conducted online (https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html,accessed on 9 February 2024) using the Kimura [K80] TS/TV 2.0 model and specific settings (Pmin = 0.001, Pmax = 0.01, Steps = 10, X = 0.1, Nb bins = 20) for primary species delimitation [66].Five protein-coding genes and six noncoding sequences were selected for species discrimination analysis according to the Pi values.The MDC analysis was performed using the FastaChar v. 0.2.4 software [87].The analysis was performed by comparing each species with the other members of Bulbophyllum included in this study.

Phylogenetic Analysis
In accordance with previous molecular systematic studies [27,36,40], we selected a total of 33 chloroplast genomes from 33 species for this study.The selection includes 28 species from Bulbophyllum and five species from Dendrobium (D. chrysanthum, D. findlayanum, D. hercoglossum, D. longicornu, and D. moschatum), which serve as the outgroups (Supplementary Table S9).A total of 68 protein-coding genes (excluding ndh genes due to their widespread loss or truncation in Bulbophyllum) were extracted using PhyloSuite v1.2.2 [88], and we aligned them using MAFFT v.7 [89].The complete chloroplast genomes were aligned using MAFFT and trimmed using TrimAl v1.2 to remove poorly aligned positions [90].For the phylogenetic analysis, we utilized the CIPRES Science Gateway, specifically RaxML-HPC2 on XSEDE 8.2.12, PAUP on XSEDE 4.a168, and MrBayes on XSEDE 3.2.7,applying three methods, including maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI) [91].In the ML analysis, we specified the GTRGAMMA model for all datasets and calculated bootstrap values on 1000 bootstrap replicates using heuristic searches [92,93].In the MP analysis, we conducted a heuristic search involving 1000 random addition sequence repeats, employing TBR branch switching.All characters were treated as equally weighted and unordered.In the BI analysis, we utilized the GTR + I + Γ substitution model with MrBayes v. 3.2.7 [94].The Markov chain Monte Carlo (MCMC) algorithm was run for 10,000,000 generations, with one tree sampled every 100 generations.We discarded the first 25% of trees as burn-ins to construct majority-rule consensus trees and estimate posterior probabilities (PPs).

Conclusions
In this study, we obtained the cp genomes of seven Bulbophyllum species (B.ambrosia, B. crassipes, B. farreri, B. hamatum, B. shanicum, B. triste, and B. violaceolabellum) and compared them with 21 related species to investigate cp genome differences.We found that most cp genomes exhibited high similarity in terms of the genome size, gene content, and gene order, and differences were observed in their ndh gene composition.Additionally, long-repeat sequences in the cp genomes of Bulbophyllum species were abundant, with an obvious A/T preference.A number of exclusive SSRs, present in some species, are useful molecular markers for species identification and detecting genetic diversity.The RSCU analysis revealed that the codon usage bias in Bulbophyllum cp genomes showed similar patterns.Six highly variable regions (rpl32-trnL UAG , trnT UGU -trnL UAA , trnF GAA -ndhJ, rps15-ycf1, rbcL-accD, and psbI-trnS GCU ) and five coding sequences (ycf1, rps12, matK, psbK, and rps15) were identified as potential DNA markers based on nucleotide diversity.Additionally, the number of MDCs that distinguish a species from the others varied from 149 to 8603 in complete cp genomes.Based on the cp genome sequences, 28 Bulbophyllum species can be divided into four clades, sects.Brachyantha, Cirrhopetalum, and Leopardinae, defined by morphology, were non-monophyly.This study further supports the significance of cp genomes in elucidating the phylogeny of Bulbophyllum.

Figure 1 .
Figure 1.Chloroplast genome maps for seven Bulbophyllum species.Genes on the inside of the large circle are transcribed clockwise, and those on the outside are transcribed counter-clockwise.The color coding of the genes is determined according to their annotation functions.The GC content of the chloroplast genomes is represented by the dashed area.Pseudogenes are marked by Ψ.

Figure 1 .
Figure 1.Chloroplast genome maps for seven Bulbophyllum species.Genes on the inside of the large circle are transcribed clockwise, and those on the outside are transcribed counter-clockwise.The color coding of the genes is determined according to their annotation functions.The GC content of the chloroplast genomes is represented by the dashed area.Pseudogenes are marked by Ψ.
kwangtungense) to 49 (B.disciflorum, B. gedangense, B. reptans, and B. violaceolabellum).Across these 28 cp genomes, P was the most prevalent, ranging from five occurrences in B. hirtum to 25 occurrences in B. inconspicuum and B. pingnanense.The Bulbophyllum cp genomes had fewer R and C repeats, and the highest counts of the two types were 25 Rs in B. reptans and 10 Cs in B. hirtum, respectively.Long-repeat sequences in the range of 30-40 bp were the most frequently observed and ranged from 15 occurrences in B. shanicum to 47 occurrences in B. disciflorum.B. inconspicuum displayed the highest count of 40-50 bp repeats.The 50-60 bp repeat sequences were detected in 18 Bulbophyllum species, ranging from one to six occurrences.The 60-70 bp repeat sequences were only present in B. crassipes, B. lingii, B. menghaiense, B. pentaneurum, B. pingnanense, B. shanicum, and B. triste, ranging from one to four occurrences.The longest repeat sequences were 77 bp in B. ningboense (Figure 2B, Supplementary Table affine, B. farreri, B. gedangense, B. hamatum, B. ningboense, B. pentaneurum, B. pingnanense, and B. plumosum.
15 occurrences in B. shanicum to 47 occurrences in B. disciflorum.B. inconspicuum displayed the highest count of 40-50 bp repeats.The 50-60 bp repeat sequences were detected in 18 Bulbophyllum species, ranging from one to six occurrences.The 60-70 bp repeat sequences were only present in B. crassipes, B. lingii, B. menghaiense, B. pentaneurum, B. pingnanense, B. shanicum, and B. triste, ranging from one to four occurrences.The longest repeat sequences were 77 bp in B. ningboense (Figure 2B, Supplementary Table

Figure 2 .
Figure 2. Summary of sequence repeats across the 28 Bulbophyllum cp genomes.(A) Variation in repeat abundance and type; (B) number of long repeats by sequence length; (C) frequency of identified SSR motifs (mono-, di-, tri-, tetra-, penta-, and hexa-); (D) frequency of classified repeat types (considering sequence complement).

Figure 2 .
Figure 2. Summary of sequence repeats across the 28 Bulbophyllum cp genomes.(A) Variation in repeat abundance and type; (B) number of long repeats by sequence length; (C) frequency of identified SSR motifs (mono-, di-, tri-, tetra-, penta-, and hexa-); (D) frequency of classified repeat types (considering sequence complement).

Figure 3 .
Figure 3. RSCU values of the codons in the 28 Bulbophyllum cp genomes.Figure 3. RSCU values of the codons in the 28 Bulbophyllum cp genomes.

Figure 3 .
Figure 3. RSCU values of the codons in the 28 Bulbophyllum cp genomes.Figure 3. RSCU values of the codons in the 28 Bulbophyllum cp genomes.
B. hamatum and B. tianguii, comprising 279 bp in the LSC region and 87 bp in the IRb region, were the shortest.Furthermore, the IRb/SSC border of B. ambrosia, B. crassipes, B. farreri, B. gedangense, and B. mentosum was located in the ndhF pseudogene, with just 70 bp located in the IRb region.The ndhF pseudogene was close, too, but did not overlap with the IRb/SSC junction in B. affine, B. andersonii, B. disciflorum, B. hirtum, B. kwangtungense, B. leopardimum, B. orientale, B. pectinatum, or B. reptans.Within the SSC/IRa (JSA) region, the ycf1 gene spanned the SSC/IRa boundary, primarily residing in the SSC region, with lengths ranging from 4308 bp (B.crassipes) to 5469 bp (B.hamatum).In the case of B. hamatum and B. tianguii, the ycf1 gene was positioned to the left side of the JSA line, with a distance of 6 bp and 9 bp, respectively.In the IRa/LSC (JLA) region, the rps19 gene was situated on the left side of the JLA line, and the distance from rps19 to the JLA line ranged from 229 bp (B.crassipes) to 290 bp (B.hamatum).The psbA gene was located on the right side of the JLA line, with distances ranging from 10 bp (B.kwangtungense, B. leopardinum, and B. pectinatum) to 132 bp (B.ambrosia) (Figures 4 and S2).
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 7 of 19 B. hamatum and B. tianguii, comprising 279 bp in the LSC region and 87 bp in the IRb region, were the shortest.Furthermore, the IRb/SSC border of B. ambrosia, B. crassipes, B. farreri, B. gedangense, and B. mentosum was located in the ndhF pseudogene, with just 70 bp located in the IRb region.The ndhF pseudogene was close, too, but did not overlap with the IRb/SSC junction in B. affine, B. andersonii, B. disciflorum, B. hirtum, B. kwangtungense, B. leopardimum, B. orientale, B. pectinatum, or B. reptans.Within the SSC/IRa (JSA) region, the ycf1 gene spanned the SSC/IRa boundary, primarily residing in the SSC region, with lengths ranging from 4308 bp (B.crassipes) to 5469 bp (B.hamatum).In the case of B. hamatum and B. tianguii, the ycf1 gene was positioned to the left side of the JSA line, with a distance of 6 bp and 9 bp, respectively.In the IRa/LSC (JLA) region, the rps19 gene was situated on the left side of the JLA line, and the distance from rps19 to the JLA line ranged from 229 bp (B.crassipes) to 290 bp (B.hamatum).The psbA gene was located on the right side of the JLA line, with distances ranging from 10 bp (B.kwangtungense, B. leopardinum, and B. pectinatum) to 132 bp (B.ambrosia) (Figure 4, Supplementary Figure S2).

Figure 5 .
Figure 5.The nucleotide diversity (Pi) of 28 Bulbophyllum cp genomes and 68 protein-coding sequences.(A) For the nucleotide diversity of the complete cp genomes using a sliding window test, four mutation hotspot regions were annotated.The window size was set to 100 bp and the sliding window size was 25 bp.X-axis, the position of the midpoint of a window; Y-axis, Pi values of each window.(B) The nucleotide diversity of 68 protein-coding sequences.X-axis, gene; Y-axis, Pi values of each gene.

Figure 5 .
Figure 5.The nucleotide diversity (Pi) of 28 Bulbophyllum cp genomes and 68 protein-coding sequences.(A) For the nucleotide diversity of the complete cp genomes using a sliding window test, four mutation hotspot regions were annotated.The window size was set to 100 bp and the sliding window size was 25 bp.X-axis, the position of the midpoint of a window; Y-axis, Pi values of each window.(B) The nucleotide diversity of 68 protein-coding sequences.X-axis, gene; Y-axis, Pi values of each gene.

Figure 6 .
Figure 6.Molecular diagnostic characteristics (MDCs) in whole cp genomes and four datasets of 28 Bulbophyllum species.

Clade 1 (
Neotropical clade) consisted of four species from different sections and Clade 2 primarily consisted of species from sect Macrocaulia with robust support.In Clade 3, sects Lemniscata (B.shanicum, B. triste, and B. hirtum) and Racemosae (including B. crassipes and B. orientale) were sister groups with generally high support values in one subclade, while another subclade consisted of

Figure 6 .
Figure 6.Molecular diagnostic characteristics (MDCs) in whole cp genomes and four datasets of 28 Bulbophyllum species.
). Clade 1 (Neotropical clade) consisted of four species from different sections and Clade 2 primarily consisted of species from sect Macrocaulia with robust support.In Clade 3, sects Lemniscata (B.shanicum, B. triste, and B. hirtum) and Racemosae (including B. crassipes and B. orientale) were sister groups with generally high support values in one subclade, while another subclade consisted of species from sects Leopardinae, Trias, Stenochilus, and Repantia.Clade 4 contained species assigned to sects Cirrhopetalum, Brachyantha, Leopardinae, Ephippium, and Desmosanthes.A single species of sect Brachyantha (B.farreri) appeared as a sister to two species from sect Cirrhopetalum (including B. pingnanense and B. inconspicuum) with strong support (BP = 100, PP = 1.00).Additionally, B. ambrosia and B. gedangense formed a separate and strongly supported clade.Notably, B. hamatum, a newly described species belonging to sect Cirrhopetalum and recently published by Yan et al. [19], appeared as a sister to B. tianguii (sect Brachyantha).Subsequently, B. violaceolabellum (sect Brachyantha) was also a sister to these two species with strong support (BP = 100, PP = 1.00).species from sects Leopardinae, Trias, Stenochilus, and Repantia.Clade 4 contained species assigned to sects Cirrhopetalum, Brachyantha, Leopardinae, Ephippium, and Desmosanthes.A single species of sect Brachyantha (B.farreri) appeared as a sister to two species from sect Cirrhopetalum (including B. pingnanense and B. inconspicuum) with strong support (BP = 100, PP = 1.00).Additionally, B. ambrosia and B. gedangense formed a separate and strongly supported clade.Notably, B. hamatum, a newly described species belonging to sect Cirrhopetalum and recently published by Yan et al.[19], appeared as a sister to B. tianguii (sect Brachyantha).Subsequently, B. violaceolabellum (sect Brachyantha) was also a sister to these two species with strong support (BP = 100, PP = 1.00).

Figure 7 .
Figure 7. Phylogenetic tree of 28 Bulbophyllum species and five outgroups based on complete cp genomes.The numbers near the nodes are bootstrap percentages and Bayesian posterior probabilities (BPML, BPMP, PP).* Node is 100 bootstrap percentage or 1.00 posterior probability.The annotations of sections referred to Pridgeon et al. [4] and Hu et al. [5] are highlighted by the color of branches.

Figure 7 .
Figure 7. Phylogenetic tree of 28 Bulbophyllum species and five outgroups based on complete cp genomes.The numbers near the nodes are bootstrap percentages and Bayesian posterior probabilities (BP ML , BP MP , PP). * Node is 100 bootstrap percentage or 1.00 posterior probability.The annotations of sections referred to Pridgeon et al. [4] and Hu et al. [5] are highlighted by the color of branches.

Table 1 .
Features of the complete chloroplast genomes of 28 Bulbophyllum species.