Next Article in Journal
Collections for the Public Good: A Case Study from Ohio
Previous Article in Journal
Phylogeography and Past Distribution of Peripheral Individuals of Large Hairy Armadillo Chaetophractus villosus
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Diversity
Volume 17
Issue 6
10.3390/d17060391
diversity-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Comparative Plastomics of Tropidia (Orchidaceae): Unraveling Structural Evolution and Phylogenetic Implications in Epidendroideae

by
Deng-Li Yu
1,
Zi-Qing Wei
1,
Rong-Rong Yan
2,
Shi-Peng Fei
1,
Wei Wu
2 and
Guo-Xiong Hu
2,*
1
Management Department of Maolan National Nature Reserve, Libo 558400, China
2
College of Life Sciences, Guizhou University, Guiyang 550025, China
*
Author to whom correspondence should be addressed.
Diversity 2025, 17(6), 391; https://doi.org/10.3390/d17060391
Submission received: 8 April 2025 / Revised: 28 May 2025 / Accepted: 29 May 2025 / Published: 31 May 2025
(This article belongs to the Section Phylogeny and Evolution)
Browse Figures
Versions Notes

Abstract

:
Tropidia, a type genus of Tropidieae (Orchidaceae, Epidendroideae), represents an important lineage for investigating plastome evolution and phylogenetic relationships within Epidendroideae. Despite its importance, the lack of available plastid genomic data has hindered comprehensive analyses of its genome structure and phylogenetic relationships. In this study, we assembled and characterized the complete plastid genomes of Tropidia angulosa and T. nipponica, providing valuable insights into plastome evolution and phylogenetic placement of Tropidieae. The plastomes of T. angulosa and T. nipponica exhibited a highly conserved quadripartite structure, sharing similar genomic size (161,395 bp and 160,801 bp) and GC content (36.87% and 36.90%). Both plastomes contained identical gene content and gene order, with 79 protein-coding genes (PCGs), 30 tRNA genes, and four rRNA genes. A total of 169 simple-sequence repeats (SSRs) and 92 long-sequence repeats (LSRs) were identified, most of which were distributed in large single-copy (63.91% and 66.30%) and non-coding regions (83.43% and 65.22%). Comparative plastomes analyses revealed the overall structural stability among photosynthetic lineages, whereas structural variation was primarily detected in mycoheterotrophic lineages. Phylogenomic reconstruction based on plastid-coding sequences revealed that Tropidieae occupies a relatively isolated phylogenetic position within Epidendroideae. These findings contribute to a more comprehensive understanding of plastome evolution and the phylogenetic framework of Epidendroideae.

1. Introduction

With more than 28,000 species, Orchidaceae is one of the most species-rich groups of flowering plants and is divided into five subfamilies, namely Apostasioideae, Vanilloideae, Cypripedioideae, Orchidoideae, and Epidendroideae [1,2,3,4,5]. Epidendroideae, the largest subfamily of Orchidaceae, includes approximately 21,000 species across 16 tribes and exhibits significant diversification in adaptations [3,6,7]. Within the Epidendroideae, the basal lineages comprise nine tribes, namely, Gastrodieae, Neottieae, Nervilieae, Sobralieae, Thaieae, Triphoreae, Tropidieae, Wullschlaegelieae, and Xerorchideae [8]. Previous molecular phylogenetic studies have indicated that resolving the relationships among tribes or subtribes within Epidendroideae is challenging, predominantly due to the low phylogenetic resolution and recent rapid radiations [1,2,3,8,9,10,11,12]. Especially in these basal tribes, some lineages have historically been taxonomically complicated [13,14]. A type case is the tribe Tropidieae, comprising Tropidia Lindl. and Corymborkis Thouars [15]. Historically, Tropidia and Corymborkis were placed in the subtribe Corymborkidinae within the tribe Neottieae of the subfamily Orchidoideae [16,17]. Dressler [18] reclassified these two genera into the tribe Tropidieae and placed them within Spiranthoideae based on seed, pollinia, and columnar structure. Nevertheless, their tall, reed-like, and semi-woody stems, plicate leaves, and robust floral morphology differ markedly from the typical characteristics of spiranthoids [8]. Subsequent molecular phylogenetic analyses indicated that Tropidia and Corymborkis clustered within Epidendroideae [8]. Consequently, the tribe Tropidieae was reassigned to this subfamily, a taxonomic treatment that has been widely accepted [3,6,12,19]. As the type genus of Tropidieae, Tropidia currently includes previously recognized Kalimantanorchis Tsukaya, M.Nakajima & H. Okada and approximately 30 species, mainly distributed in tropical Asia and Australasia [15,20]. Interestingly, most members of this genus are terrestrial autotrophic plants, except three mycoheterotrophic species [T. saprophytica J.J.Sm., T. connata J.J.Wood & A.Lamb, and T. nagamasui (Tsukaya, M.Nakaj. & H.Okada) Ormerod & Juswara], which is an ideal system for exploring the evolution of photosynthetic ability in orchids [15,21].
Chloroplast, originating from ancestral photosynthetic bacteria, is essential for plant survival, adaptation, and evolutionary diversification [22,23,24]. As the primary site of photosynthesis, chloroplast possesses its own genetic system, known as the plastome, characterized by maternal inheritance, a moderate nucleotide substitution rate, and a relatively small genome size [22,25]. Recently, plastid phylogenomic studies have been widely applied to resolve phylogenetic relationships in most angiosperms, such as Asteraceae [26], Lamiaceae [27,28], Linderniaceae [29], Liliaceae [30], and Orchidaceae [31,32,33]. Although most plastid genomes are relatively conserved in terms of genomic structure, gene content, and sequence composition, they exhibit varying degrees of variation among families or genera, including gene loss/duplication, expansion/contraction of inverted repeat (IR) regions, and rearrangement [34,35,36,37]. Therefore, comparative plastid genomic analyses provide valuable insight into plant phylogeny, genome evolution, and adaptive diversification [38,39]. However, previous plastid genome studies in orchids have mainly focused on economically important genera (e.g., Dendrobium Sw. and Liparis Rich.) or on a limited number of non-photosynthetic lineages (e.g., Dipodium R. Br., Epipogium J.F. Gmel. ex Borkh., and Wullschlaegelia Rchb. f.) [40,41,42]. Although plastid data from a few Tropidia species have been included in phylogenetic studies, their structural features remain uncharacterized, and the genomes have not been made publicly available [43,44]. To date, no available plastomes related to Tropidia or even the tribe Tropidieae have been published (https://www.ncbi.nlm.nih.gov/, accessed on 20 February 2025), which limits our understanding of the plastome evolution within this group. In this study, the complete plastomes of two Tropidia species, T. angulosa (Lindl.) Blume and T. nipponica Masam., were assembled and annotated. Also, we characterized their plastome features and performed comparative analyses with previously published plastomes from other basal tribes of Epidendroideae. Specifically, our aims were to (1) analyze the structure and variation in Tropidia plastomes, (2) investigate the dynamic evolution of plastomes within the basal tribes of Epidendroideae, and (3) provide further insight into the phylogenetic placement of Tropidieae using plastid data.

2. Materials and Methods

2.1. Sampling, DNA Extraction, and Sequencing

Neither Tropidia angulosa nor T. nipponica are considered endangered species. The plant materials of the two Tropidia species used in this study were sampled from Orchid Conservation Center at the Management Department of Maolan National Nature Reserve, Libo, Guizhou, China. These two Tropidia species were originally introduced by orchid expert Shi-Peng Fei in 2021 from the Maolan National Nature Reserve in Libo County, Guizhou, China, at coordinates 108°05′20.8764” E, 25°12′15.1549” N for T. angulosa and 108°04′46.4057” E, 25°14′55.7382” N for T. nipponica. The voucher specimens were deposited in the herbarium of the Natural Museum of Guizhou University (GACP), with voucher number 2024Tang001 for T. angulosa and 2024Tnip001 for T. nipponica. Fresh leaves from healthy plants were dried using silica, and genomic DNA was extracted using a modified CTAB procedure [45]. The DNA purity and concentration were quantified using agarose gel electrophoresis (Liuyi Biotechnology, Beijing, China) and a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively. Qualified DNA libraries were sequenced using the DNBseq platform (PE 150bp) at BGI Technology Service Co., Ltd. (Wuhan, China). Low-quality sequences of raw reads were filtered using the software Trimmomatic v.0.32 [46], setting the parameters to SLIDINGWINDOW:4:20 and MINLEN:50. Clean reads totaling 6.73 GB (T. angulosa) and 6.74 GB (T. nipponica) were retained for plastome assembly.

2.2. Plastome Assembly and Annotation

De novo assembly was performed using GetOrganelle v1.7.5, with extension rounds (-R) set to 15 and k-mer sizes (-k) set to 21, 45, 65, 85, and 105 [47]. The assembly results were subsequently checked using Bandage v0.8.1 [48], with average base coverages of 186.8× and 196.8× for T. angulosa and T. nipponica, respectively. The well-assembled plastomes were annotated using CPGAVAS2 [49] and Geseq [50], with Dendrobium cariniferum Rchb. F. (NC_080348) and D. comatum (Blume) Lindl. (NC_063511) as references. Subsequently, the results were checked and adjusted to ensure annotation accuracy by the software Geneious v9.0.2 [51]. The gene maps of plastomes were drawn using OrganellarGenomeDRAW (OGDRAW) [52]. The final annotated plastomes were submitted to GenBank, with accession numbers PV424134 for T. angulosa and PV424135 for T. nipponica.

2.3. Plastome Structure and Features Analyses

Basic structural features of plastomes, including length, GC content, and gene numbers, were summarized in Geneious. Simple-sequence repeats (SSRs), including mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats, were detected using MISA [53], with the minimum repeat thresholds set to 10, 5, 4, 3, 3, and 3, respectively. Long-sequence repeats (LSRs), comprising forward, reverse, complement, and palindromic repeats, were identified using REPuter [54] with the following parameters: a Hamming distance of 3 (three mismatches), maximum computed repeats of 5000, and a minimal repeat size of 30 bp. Amino-acid frequency and relative synonymous codon usage (RSCU) of protein-coding genes (PCGs) were calculated in CodonW v1.4.4 [55]. To statistically validate codon usage patterns, chi-square tests were performed for each amino acid using R v4.2.

2.4. Plastome Comparative Analyses

To further explore the plastome characteristics of basal epidendroids, eight species representing five basal tribes were selected for comparative analyses after excluding unavailable (Gastrodieae, Thaieae, and Xerorchideae) or full mycoheterotrophic (Wullschlaegelieae) data, with accession numbers in Supplementary Table S1. Genomic size and gene content were compared using CPStools v2.5 [56]. Whole genome alignments for these plastomes were performed in progressiveMauve v2.3.1 [57] to detect rearrangement or inversion events. The expansion and contraction of inverted repeat (IR) regions were visualized using online program CPJSdraw v1.0 [58].

2.5. Phylogenetic Analyses

Phylogeny reconstruction was carried out using the shared coding sequences (CDSs), with 22 species representing 12 tribes of Epidendroideae included as the ingroup, and four species from the remaining four subfamilies of Orchidaceae serving as outgroups (Supplementary Table S1). These shared CDSs were extracted using CPStools v2.5 [56] and subsequently aligned using MAFFT v7.407 under the auto strategy [59]. Poorly aligned positions were trimmed using trimAL v1.4.1 following default settings [60]. The maximum likelihood (ML) and Bayesian inference (BI) methods were used to reconstruct the phylogenetic relationships. An ML tree was constructed in IQTREE v1.6.12 [61] with 5000 bootstraps, in which GTR+F+R3 was identified as the best-fit model according to the Akaike information criterion (AIC) [62]. The jModelTest2 [63] was used to select the BI-best substitution model based on the AIC method. A BI tree was conducted using MrBayes v3.2.7a [64] by running 5,000,000 generations and sampling every 1000 generations under the GTR+F+I+G4 model. Two independent runs were performed, each consisting of four Markov chain Monte Carlo (MCMC) chains. The initial 25% of trees were discarded as burn-in, and the remaining trees were used to construct a consensus tree. Convergence of the MCMC chains was confirmed when the average standard deviation of the split frequencies (ASDSF) reached ≤0.01.

3. Results

3.1. Plastome Structure and Characteristics

The plastid genomes of two Tropidia species exhibited a typical quadripartite structure (Figure 1). The complete plastome of T. angulosa was 161,395 bp in length, comprising a large single-copy (LSC) region of 88,572 bp, a small single-copy (SSC) region of 18,615 bp, and two inverted repeats (IR) of 27,104 bp each, with an overall GC content of 36.87%. The GC contents of the LSC, SSC, and IR regions were 34.52%, 30.18%, and 43.01%, respectively. Similarly, T. nipponica plastome was 160,801 bp in length, with the LSC, SSC, and IR regions of 88,014, 18,623, and 27,082 bp, respectively. Its overall GC content was 36.90%, with 34.58%, 30.11%, and 43% in the LSC, SSC, and IR regions, respectively.
Both Tropidia species were annotated with 113 unique genes, including 79 protein coding genes (PCGs), 30 tRNA genes, and four rRNA genes (Table 1). Among these, eight PCGs, eight tRNA genes, and four rRNA genes located in the IR region, with two copies. In terms of gene structure, 18 genes were identified with introns, of which the rps12, clpP, and ycf3 genes contained two introns and the others possessed a single intron. Functionally, these genes were categorized into 44 involved in photosynthesis, 59 associated with self-replication, six related to other functions, and four with unknown functions (Table 1).

3.2. Codon Usage Bias

After excluding duplicated genes, genes containing non-canonical start codons and genes shorter than 300 bp, a total of 50 PCGs from each species were retained for codon usage bias analyses (Supplementary Table S2). The total number of codons encoded by PCGs exhibited a minor difference between T. angulosa (20,457) and T. nipponica (20425), with a discrepancy of 32 codons. These codons encode 20 amino acids and three stop codons, among which leucine (Leu) and cysteine (Cys) were the most and least abundant, respectively (Figure 2A). The relative synonymous codon usage (RSCU) values were highly similar, ranging from 0.33 (CGC) to 1.91 (AGA) for T. angulosa and T. nipponica (Supplementary Table S3). Of these, the AGA codon encoding arginine (Arg) exhibited the highest RSCU values, whereas CGC (Arg) showed the lowest (Figure 2B). Both species had 30 codons with RSCU values greater than 1, nearly all of which ended with A/U, except for UUG. Methionine (Met) and tryptophan (Trp) each displayed a single preferred codon (AUG and UGG, respectively), with RSCU = 1. The chi-square tests revealed that synonymous codon usage significantly deviated from uniform expectations in nearly all amino acids (p < 0.05), confirming the presence of strong codon usage bias (Supplementary Table S4). Furthermore, the total GC contents of codons were 38.24% and 38.21% for T. angulosa and T. nipponica, respectively, with a higher GC content at the first codon position compared to the second and third positions (Supplementary Table S2).

3.3. Abundance and Distribution of Repeat Sequences

A total of 169 simple-sequence repeats (SSRs) were identified, with 88 and 81 in T. angulosa and T. nipponica, respectively (Figure 3). These SSRs were classified into six types based on repeat unit length (mono- to hexanucleotide), with mononucleotide repeats being the most abundant (63.6% and 66.7%), while hexanucleotide repeats were the least frequent (1.1% and 2.5%) (Figure 3A). Furthermore, the majority of SSRs located in the LSC region (65.9% and 61.7%), while the IR regions contained the fewest SSRs (11.4% and 9.9%) (Figure 3B). The majority of SSRs were found in intergenic spacers (IGS) (67.0% and 59.3%), with exons containing the lowest proportion (14.8% and 18.5%) (Figure 3C). Moreover, 92 long-sequence repeats (LSRs) were detected, with 55 and 37 repeats in T. angulosa and T. nipponica, respectively (Figure 3). Palindromic repeats were most abundant (45.4% and 37.8%), while complement repeats were least frequent (5.4% and 2.7%) (Figure 3D). Similar to the distribution pattern of SSRs, LSRs were predominantly located in the LSC region (65.4% and 67.6%) and IGS regions (45.4% and 48.6%), while the SSC region (7.3% and 10.8%) and introns (16.4% and 21.6%) contained the lowest proportions (Figure 3E,F). Overall, no statistically significant differences were found in either SSR or LSR type composition (p > 0.4) or regional distribution (p > 0.8) between T. angulosa and T. nipponica (Supplementary Table S5).

3.4. Plastome Structural Variation

Among these eight species from five basal tribes of Epidendroideae, plastome sizes varied from 151,898 bp in Triphora trianthophoros to 163,909 bp in Palmorchis pabstii (Supplementary Table S6). The plastid genomes of T. angulosa and T. nipponica were similar to those of other photosynthetic lineages in genome length, whereas the partially mycoheterotrophic T. trianthophoros exhibited notable variation (Figure 4A). The length of the LSC region ranged from 82,518 to 90,710 bp, the SSC region from 860 to 18,854 bp, and the IR regions from 26,610 to 34,260 bp. Notably, T. trianthophoros (Triphoreae) exhibited a significantly expanded IR region (34,260 bp) accompanied by a markedly reduced SSC region (860 bp) (Figure 4A). These selected species contained 113 unique genes, with the exception of T. trianthophoros, which lacked 11 ndh genes and exhibited duplications of 5 additional genes (ccsA, psaC, rpl32, ycf1, and trnL-UAG) (Figure 4B). On the other hand, no gene rearrangement or inversion events were detected (Supplementary Figure S1), although slight variations were observed in the gene composition at the IR boundary (Figure 5). At the LSC/IRb junction (JLB), the rpl22 gene spanned the junction, including 110–329 bp in the LSC and 37–256 bp in the IRb. The SSC/IRb junction (JSB) was crossed by the ndhF gene in all species, except T. trianthophoros, where the junction was located near the psaC and rps15 genes instead. Similarly, the ycf1 gene was located in the SSC/IRa junction (JSA), while the junction was placed between the psaC and rps15 genes for T. trianthophoros. The LSC/IRa junction (JLA) was typically located in the IGS of the rps19 and psbA genes.

3.5. Phylogenetic Relationships

After removing ambiguously aligned regions, the coding sequences consisted of 66,608 bp, with 6150 parsimony-informative sites. The inferred phylogenetic topology revealed strong support for the monophyly of the subfamily Epidendroideae [bootstrap (BS) = 100, posterior probability (PP) = 1], with 12 tribes recognized (Figure 6). In the early divergent tribes of Epidendroideae, five strongly supported clades were recovered, with Neottieae resolved as sister to the remaining Epidendroideae (BS = 100, PP = 1), followed by Triphoreae and Nervilieae (BS = 99.6, PP = 1). The tribe Sobralieae was sister to the clade comprising Tropidieae and seven other tribes, with full posterior probability support (PP = 1), despite a relatively low ML bootstrap value (BS = 42.9). The next supported clade was the monophyletic Tropidieae, with T. angulosa and T. nipponica, which was a sister group to the well-supported clade comprising Arethuseae, Malaxideae, Podochileae, Collabieae, Vandeae, Cymbidieae, and Epidendreae (BS = 98.9, PP = 1). Excluding the five basal tribes of Epidendroideae, the next well-supported clade comprising the remaining seven tribes showed strong support for most nodes, except for the relationship between Cymbidieae and Epidendreae, which presented moderate support (BS = 70.8, PP = 0.89).

4. Discussion

4.1. Conserved Plastome Structure of Two Tropidia Species

As in most angiosperms [65,66,67,68,69], the plastid genomes of Tropidia angulosa and T. nipponica exhibited the typical quadripartite structure (Figure 1). The plastome sizes of T. angulosa and T. nipponica were 161,395 bp and 160,801 bp, respectively, both slightly exceeding the typical size range of 120–160 kb reported for most photosynthetic land plants [22]. The inverted repeat (IR) regions in both species measured approximately 27 kb, marginally larger than the average IR length of ~25 kb in angiosperms [70], which may contribute to their slight expansion of plastome size. These two plastid genomes encoded 113 unique genes, including 79 protein-coding genes (PCGs), 30 tRNA genes, and four rRNA genes (Table 1), which is a gene composition comparable to that of other orchid species [71,72,73]. In terms of gene expression patterns, codon usage bias not only influences gene function and expression efficiency but also serves as an important indicator of gene origin and evolutionary processes [74]. In T. angulosa and T. nipponica, the relative synonymous codon usage (RSCU) patterns were highly similar, with most codons showing RSCU values greater than 1 ending in A or U (Supplementary Table S3). The A/U-ending preference is consistent with patterns reported in other orchids, such as Herminium monorchis (L.) R. Br. [75], Platanthera ussuriensis (Regel & Maack) Maxim. [76], and Taeniophyllum complanatum Fukuy. [77]. Additionally, notable variation in the GC content was observed across codon positions, with the third codon position predominantly composed of A or U (Supplementary Table S2). This codon usage pattern is a conserved feature widely documented across plastid genomes of land plants [78,79,80]. Although codon usage bias in plastid genomes is largely shaped by nucleotide composition, previous studies have shown that it may also be influenced by multiple interacting factors, including mutational pressure, translational selection, and tRNA gene copy number and availability [81,82]. Further investigation of these factors could provide a deeper understanding of codon usage pattern in plastid genomes.
Generally speaking, simple-sequence repeats (SSRs) are considered efficient molecular markers due to their high polymorphism, reproducibility, and low cost, which have been widely employed in genetic analyses [83,84]. Consistent with previous studies [85,86,87], SSRs are primarily concentrated in the large single-copy (LSC) and non-coding regions of the plastome, with relatively fewer occurrences in the IR regions and coding sequences (Figure 3B,C). Similarly, long-sequence repeats (LSRs), which play an important role in genome recombination and structural rearrangement [88,89], were also predominantly distributed in the LSC and non-coding regions here (Figure 3E,F). These findings further revealed the notion that IR regions are more conserved than the single-copy regions, which is related to their relatively higher GC content. The GC content across different regions of the plastome is uneven, with the notably higher GC content in the IR regions likely associated with their structural stability and the presence of rRNA genes [79,80]. Previous studies have shown that the nucleotide substitution rate in the IR regions of plastid genomes is significantly lower than that in the single-copy regions [90], further supporting the notion that IR regions are more conserved. Additionally, mononucleotide SSRs, particularly A/T-rich motifs, were the most abundant type identified, consistent with previous observations in plastid genomes [29,78,79,80]. This pattern is likely attributable to the AT-rich nature of plastid genomes and the high slippage potential of homopolymeric A/T tracts during DNA replication [22].

4.2. Plastome Evolution in Basal Lineages of Epidendroideae

Seven photosynthetic species and one partial mycoheterotrophic species from five basal tribes within Epidendroideae were comparatively analyzed to investigate the dynamic evolution of plastome structure. The results demonstrated that the most plastid genomes were relatively conservative in structure, genomic size, gene content, and gene order (Supplementary Table S6), suggesting a stable evolutionary trajectory. For the partial mycoheterotrophic T. trianthophoros, however, the plastome exhibits a markedly contracted small single-copy (SSC) region and an expanded IR region (Figure 4A). The expansion or contraction of IR regions can result in variations in plastome size, as well as associated gene loss, duplication, or pseudogenization [91]. This structural variation in T. trianthophoros is accompanied by the loss of 11 ndh genes originally located in the SSC region and the expansion of 5 genes (ccsA, psaC, rpl32, ycf1, and trnL-UAG) into the IR regions, resulting in their duplication (Figure 4B). The loss of ndh genes of T. trianthophoros plastome likely represents an evolutionary response to its partially mycoheterotrophic lifestyle. In plant lineages with reduced dependence on photosynthesis, genes associated with photosynthetic function frequently become functionally redundant and are subsequently lost or pseudogenized, a pattern commonly observed among non-photosynthetic species [92,93,94]. Such reduction is commonly attributed to relaxed selective pressure on photosynthesis-related genes, which allows for the accumulation of deleterious mutations and leads to eventual gene loss [22,95,96]. A representative example is Wullschlaegelia, a fully mycoheterotrophic genus within Epidendroideae, whose plastome has been reported to be approximately 37 kb in length and to exhibit extensive genomic reduction, including the complete loss of IR regions [94]. All genes associated with photosynthesis and ATP synthesis are absent, with only 22 protein-coding genes (PCGs), seven tRNA genes, and all four rRNA genes retained. The retention of rRNA genes in Wullschlaegelia may reflect the minimal plastome hypothesis, which posits that plastomes of heterotrophs are maintained primarily to support organelle translation machinery [97,98]. Overall, these findings suggest a potential continuum of plastome reduction in Epidendroideae, from relatively conserved plastome in photosynthetic species to varying degrees of gene loss and structural modification in partially and fully mycoheterotrophic lineages, possibly reflecting gradual adaptations to reduced photosynthetic reliance. The genus Tropidia represents a promising genus for exploring genome evolution, as it contains both photosynthetic and mycoheterotrophic species [15]. This dual presence allows us to hypothesize that plastomes in mycoheterotrophic Tropidia species may also exhibit a trend of plastome reduction. To gain a more comprehensive understanding of plastome evolution and gene loss in orchid species, further studies should integrate comparative analyses across photosynthetic, partially, and fully mycoheterotrophic lineages, alongside functional investigations to elucidate the physiological consequences of gene loss.

4.3. Phylogenetic Placement of Tropidieae

Despite significant progress in resolving orchid phylogeny, the relationships among basal tribes of Epidendroideae remain problematic, particularly in studies based on a limited number of DNA markers, which often exhibit low phylogenetic resolution [2,11,12,99,100]. Recent phylogenetic reconstructions of Orchidaceae using high-throughput sequencing data have substantially improved resolution, providing valuable insights into tribal relationships [43,44,101,102]. The phylogram inferred from mitochondrial data revealed that Tropidieae formed a distinct clade and was closely related with the clade including Arethuseae, Thaieae, Malaxideae, Epidendreae, Collabieae, Podochileae, Cymbidieae, and Vandeae (BS = 96, PP = 0.99) [43]. Based on plastid data, however, Serna-Sánchez et al. [44] suggested that Tropidieae is sister to Nervilieae with a low bootstrap value (BS = 40). Here, our results reveal that Tropidieae is sister to a well-supported clade comprising Malaxideae, Epidendreae, Collabieae, Podochileae, Cymbidieae, and Vandeae, with strongly supported values (BS = 98.9, PP = 1), demonstrating that Tropidieae occupies a relatively isolated phylogenetic position within Epidendroideae. This result is similar to the findings based on mitochondrial data [43], although our sampling lacked representatives of Thaieae, as the corresponding plastome sequences were not made publicly available. Furthermore, current phylogenetic inference is largely congruent with recent phylogenomic evidence based on low-copy nuclear genes [103]. Despite this, our study is based on limited taxon sampling, particularly with respect to the early-divergent tribes of Epidendroideae, which may have influenced the inferred topologies and support values. Therefore, future studies should consider expanding the taxon sampling of Tropidieae and incorporating additional representatives from other tribes to construct a more comprehensive phylogenetic tree, which may provide stronger support for the precise phylogenetic placement of Tropidieae.

5. Conclusions

In this study, we assembled and characterized the complete plastid genomes of Tropidia angulosa and T. nipponica, both displaying the typical quadripartite structure and a highly conserved gene content. The two plastomes showed a high degree of similarity in GC content, repeat sequence distribution, and codon usage patterns. Comparative plastome analyses across five basal tribes of Epidendroideae revealed overall structural stability among photosynthetic lineages, while mycoheterotrophic taxa showed early signs of plastome reduction, indicating lineage-specific evolutionary dynamics. Plastid phylogenomics provided strong support for the phylogenetic relationship of Tropidieae, with a relatively isolated phylogenetic position. Future studies incorporating additional plastid genomes of Tropidia, particularly from mycoheterotrophic taxa, will be essential for further understanding the plastome characteristics and evolution of this genus. Altogether, our findings provide evidence supporting the phylogenetic position of Tropidieae and contribute valuable plastome data for further investigations of genome evolution in orchids.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/d17060391/s1, Figure S1: Mauve alignment result within the basal Epidendroideae; Table S1: Genbank accession numbers of plastomes used in this study; Table S2: Codon number and GC content of plastomes of two Tropidia; Table S3: Relative synonymous codon usage of plastomes of two Tropidia; Table S4: Chi-square test results of codon usage bias by amino acid in T. angulosa and T. nipponica; Table S5: Statistical comparison of repeat types and distribution between two Tropidia plastomes; Table S6: Comparisons of plastome size among different species.

Author Contributions

Conceptualization, D.-L.Y. and G.-X.H.; investigation, D.-L.Y., Z.-Q.W., S.-P.F. and W.W.; resources, D.-L.Y., Z.-Q.W. and S.-P.F.; formal analysis, W.W. and R.-R.Y.; writing—original draft preparation, D.-L.Y. and R.-R.Y.; writing—review and editing, Z.-Q.W. and G.-X.H.; funding acquisition, G.-X.H. and D.-L.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Management Department of Maolan National Nature Reserve (MLHT2024-17, MLHT2025-155, 2023-07) and the Natural Science Foundation of Guizhou Province (Qiankehezhongyindi [2023] 029).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The Tropidia plastomes generated in this study are available in the NCBI repository (https://www.ncbi.nlm.nih.gov), with the accession numbers PV424134 and PV424135.

Acknowledgments

We are grateful to Min Zhan from the Central South University of Forestry and Technology for the help in data analyses.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Cameron, K.M. Utility of plastid psaB gene sequences for investigating intrafamilial relationships within Orchidaceae. Mol. Phylogenet. Evol. 2004, 31, 1157–1180. [Google Scholar] [CrossRef] [PubMed]
  2. Górniak, M.; Paun, O.; Chase, M.W. Phylogenetic relationships within Orchidaceae based on a low-copy nuclear coding gene, Xdh: Congruence with organellar and nuclear ribosomal DNA results. Mol. Phylogenet. Evol. 2010, 56, 784–795. [Google Scholar] [CrossRef]
  3. Chase, M.W.; Cameron, K.M.; Freudenstein, J.V.; Pridgeon, A.M.; Salazar, G.; van den Berg, C.; Schuiteman, A. An updated classification of Orchidaceae. Bot. J. Linn. Soc. 2015, 177, 151–174. [Google Scholar] [CrossRef]
  4. Christenhusz, M.J.; Byng, J.W. The number of known plant species in the world and its annual increase. Phytotaxa 2016, 261, 201–217. [Google Scholar] [CrossRef]
  5. Govaerts, R.; Lughadha, E.N.; Black, N.; Turner, R.; Paton, A. The World Checklist of Vascular Plants, a continuously updated resource for exploring global plant diversity. Sci. Data 2021, 8, 215. [Google Scholar] [CrossRef]
  6. Freudenstein, J.V. Orchid phylogenetics and evolution: History, current status and prospects. Ann. Bot. 2024, 15, mcae202. [Google Scholar] [CrossRef]
  7. Pérez-Escobar, O.A.; Bogarín, D.; Przelomska, N.A.S.; Ackerman, J.D.; Balbuena, J.A.; Bellot, S.; Bühlmann, R.P.; Cabrera, B.; Cano, J.A.; Charitonidou, M.; et al. The origin and speciation of orchids. New Phytol. 2024, 242, 700–716. [Google Scholar] [CrossRef]
  8. Cameron, K.M.; Chase, M.W.; Whitten, W.M.; Kores, P.J.; Jarrell, D.C.; Albert, V.A.; Yukawa, T.; Hills, H.G.; Goldman, D.H. A phylogenetic analysis of the Orchidaceae: Evidence from rbcL nucleotide sequences. Am. J. Bot. 1999, 86, 208–224. [Google Scholar] [CrossRef]
  9. Chase, M.W.; Cameron, K.M.; Barrett, R.L.; Freudenstein, J.V. DNA Data and Orchidaceae Systematics: A New Phylogenetic Classification. In Orchid Conservation; Dixon, K.W., Kell, S.P., Barrett, R.L., Cribb, P.J., Eds.; Natural History Publications (Borneo): Kota Kinabalu, Malaysia, 2003; pp. 69–89. [Google Scholar]
  10. Freudenstein, J.V.; van den Berg, C.; Goldman, D.H.; Kores, P.J.; Molvray, M.; Chase, M.W. An expanded plastid DNA phylogenetic analysis of Orchidaceae and analysis of jackknife clade support strategy. Am. J. Bot. 2004, 91, 149–157. [Google Scholar] [CrossRef]
  11. Freudenstein, J.V.; Chase, M.W. Phylogenetic relationships in Epidendroideae (Orchidaceae), one of the great flowering plant radiations: Progressive specialization and diversification. Ann. Bot. 2015, 115, 665–681. [Google Scholar] [CrossRef]
  12. Li, M.; Zhang, G.; Lan, S.; Jin, X.; Guo, S.X. A molecular phylogeny of Chinese orchids. J. Syst. Evol. 2016, 54, 349–362. [Google Scholar] [CrossRef]
  13. Dressler, R.L. The major clades of the Orchidaceae–Epidendroideae. Lindleyana 1990, 5, 117–125. [Google Scholar]
  14. Dressler, R.L. The Neottieae in orchid classification. Lindleyana 1990, 5, 102–109. [Google Scholar]
  15. Kikuchi, I.A.B.S.; Keßler, P.J.A.; Schuiteman, A.; Tsukaya, H.; Yukawa, T. Molecular phylogenetic study of the tribe Tropidieae (Orchidaceae, Epidendroideae) with taxonomic and evolutionary implications. PhytoKeys 2020, 140, 11. [Google Scholar] [CrossRef] [PubMed]
  16. Seidenfaden, G. The Orchids of Indochina. Opera Bot. 1992, 114, 1–502. [Google Scholar]
  17. Chen, S.C.; Tsi, Z.H.; Lang, K.Y.; Zhu, G.H. Orchidaceae. In Flora Reipublicae Popularis Sinicae; Wu, C.Y., Ed.; Science Press: Beijing, China, 1999; Volume 18, pp. 1–412. [Google Scholar]
  18. Dressler, R.L. Phylogeny and Classification of the Orchid Family; Harvard University Press: Cambridge, MA, USA, 1993; pp. 1–212. [Google Scholar]
  19. Chen, X.Q.; Liu, Z.J.; Zhu, G.H.; Lang, K.Y.; Ji, Z.H.; Luo, Y.B.; Jin, X.H.; Cribb, P.J.; Wood, J.J.; Gale, S.W. Orchidaceae. In Flora of China; Wu, Z.Y., Raven, P.H., Hong, D.Y., Eds.; Science Press: Beijing, China, 2009; Volume 25, pp. 1–506. [Google Scholar]
  20. Dressler, R.L. Classification of the Orchidaceae and their probable Origin. Telopea 1983, 2, 413–424. [Google Scholar] [CrossRef]
  21. Kikuchi, I.A.B.S.; Tsukaya, H. Epitypification with an emended description of Tropidia connata (Orchidaceae, Epidendroideae, Tropidieae). PhytoKeys 2017, 80, 77. [Google Scholar] [CrossRef]
  22. Wicke, S.; Schneeweiss, G.M.; DePamphilis, C.W.; Müller, K.F.; Quandt, D. The evolution of the plastid chromosome in land plants: Gene content, gene order, gene function. Plant Mol. Biol. 2011, 76, 273–297. [Google Scholar] [CrossRef]
  23. Daniell, H.; Lin, C.S.; Yu, M.; Chang, W.J. Chloroplast genomes: Diversity, evolution, and applications in genetic engineering. Genome Biol. 2016, 17, 134. [Google Scholar] [CrossRef]
  24. Dopp, I.J.; Yang, X.; Mackenzie, S.A. A new take on organelle mediated stress sensing in plants. New Phytol. 2021, 230, 2148–2153. [Google Scholar] [CrossRef]
  25. Twyford, A.D.; Ness, R.W. Strategies for complete plastid genome sequencing. Mol. Ecol. Resour. 2017, 17, 858–868. [Google Scholar] [CrossRef] [PubMed]
  26. Chu, R.; Xu, X.; Lu, Z.; Ma, Y.; Cheng, H.; Zhu, S.; Bakker, F.T.; Schranz, M.E.; Wei, Z. Plastome-based phylogeny and biogeography of Lactuca L. (Asteraceae) support revised lettuce gene pool categories. Front. Plant Sci. 2022, 13, 978417. [Google Scholar] [CrossRef] [PubMed]
  27. Wu, H.; Ma, P.F.; Li, H.T.; Hu, G.X.; Li, D.Z. Comparative plastomic analysis and insights into the phylogeny of Salvia (Lamiaceae). Plant Divers. 2021, 43, 15–26. [Google Scholar] [CrossRef]
  28. Qian, M.L.; Chen, Y.P.; Dirmenci, T.; Zhao, Y.; Hu, G.X.; Zhao, F.; Celep, F.; Li, B.; Xiang, C.L. Phylogenomics of the tribe Leonureae reveals a new genus: Paraleonurus (Lamiaceae, Lamioideae). Turk. J. Bot. 2024, 48, 427–439. [Google Scholar] [CrossRef]
  29. Yan, R.; Geng, Y.; Jia, Y.; Xiang, C.; Zhou, X.; Hu, G. Comparative analyses of Linderniaceae plastomes, with implications for its phylogeny and evolution. Front. Plant Sci. 2023, 14, 1265641. [Google Scholar] [CrossRef] [PubMed]
  30. Chen, Q.; Hu, H.; Zhang, D. DNA barcoding and phylogenomic analysis of the genus Fritillaria in China based on complete chloroplast genomes. Front. Plant Sci. 2022, 13, 764255. [Google Scholar] [CrossRef]
  31. Barrett, C.F.; Freudenstein, J.V.; Skibicki, S.V.; Sinn, B.T.; Chung, S.-W.; Hsu, T.-C.; Liao, W.; Lee, S.Y.; Luo, Y.-B.; Yukawa, T.; et al. Phylogenomics and intergenomic conflict in a challenging orchid clade (Calypsoinae): Monophyly of Corallorhiza, paraphyly of Oreorchis, and resurrection of Kitigorchis. Bot. J. Linn. Soc. 2024, boae092. [Google Scholar] [CrossRef]
  32. Chen, H.Y.; Zhang, Z.R.; Yao, X.; Ya, J.D.; Jin, X.H.; Wang, L.; Lu, L.; Li, D.Z.; Yang, J.B.; Yu, W.B. Plastid phylogenomics provides new insights into the systematics, diversification, and biogeography of Cymbidium (Orchidaceae). Plant Divers. 2024, 46, 448–461. [Google Scholar] [CrossRef]
  33. Simpson, L.; Clements, M.A.; Orel, H.K.; Crayn, D.M.; Nargar, K. Plastid phylogenomics clarifies broad-level relationships in Bulbophyllum (Orchidaceae) and provides insights into range evolution of Australasian section Adelopetalum. Front. Plant Sci. 2024, 14, 1219354. [Google Scholar] [CrossRef]
  34. Cosner, M.E.; Raubeson, L.A.; Jansen, R.K. Chloroplast DNA rearrangements in Campanulaceae: Phylogenetic utility of highly rearranged genomes. BMC Evol. Biol. 2004, 4, 27. [Google Scholar] [CrossRef]
  35. Cai, Z.; Guisinger, M.; Kim, H.G.; Ruck, E.; Blazier, J.C.; McMurtry, V.; Jansen, R.K. Extensive reorganization of the plastid genome of Trifolium subterraneum (Fabaceae) is associated with numerous repeated sequences and novel DNA insertions. J. Mol. Evol. 2008, 67, 696–704. [Google Scholar] [CrossRef] [PubMed]
  36. Lee, H.L.; Jansen, R.K.; Chumley, T.W.; Kim, K.J. Gene relocations within chloroplast genomes of Jasminum and Menodora (Oleaceae) are due to multiple, overlapping inversions. Mol. Biol. Evol. 2007, 24, 1161–1180. [Google Scholar] [CrossRef] [PubMed]
  37. Wu, H.; Li, D.Z.; Ma, P.F. Unprecedented variation pattern of plastid genomes and the potential role in adaptive evolution in Poales. BMC Biol. 2024, 22, 97. [Google Scholar] [CrossRef] [PubMed]
  38. Zhong, Q.W.; Yang, S.P.; Sun, X.M.; Wang, L.H.; Li, Y. The complete chloroplast genome of the Jerusalem artichoke (Helianthus tuberosus L.) and an adaptive evolutionary analysis of the ycf2 gene. PeerJ 2019, 7, e7596. [Google Scholar] [CrossRef]
  39. Gu, J.; Li, M.; He, S.; Li, Z.; Wen, F.; Tan, K.; Bai, X.; Hu, G. Comparative chloroplast genomes analysis of nine Primulina (Gesneriaceae) rare species from the karst region of southwest China. Sci. Rep. 2024, 14, 30256. [Google Scholar] [CrossRef]
  40. Goedderz, S.; Clements, M.A.; Bent, S.J.; Nicholls, J.A.; Patel, V.S.; Crayn, D.M.; Schlüter, P.M.; Nargar, K. Plastid phylogenomics reveals evolutionary relationships in the mycoheterotrophic orchid genus Dipodium and provides insights into plastid gene degeneration. Front. Plant Sci. 2024, 15, 1388537. [Google Scholar] [CrossRef]
  41. Zhao, Z.; Li, Y.; Zhai, J.W.; Liu, Z.J.; Li, M.H. Organelle genomes of Epipogium roseum provide insight into the evolution of mycoheterotrophic orchids. Int. J. Mol. Sci. 2024, 25, 1578. [Google Scholar] [CrossRef]
  42. Zhang, F.P.; Deng, J.J.; Guo, Y.; Han, L.J.; Yin, Z.L. Comparative chloroplast genomic analysis of an important horticultural plant, Dendrobium sulcatum (Orchidaceae), and phylogenetic position in Dendrobium. Hortic. Environ. Biotechnol. 2025, 66, 13–24. [Google Scholar] [CrossRef]
  43. Li, Y.X.; Li, Z.H.; Schuiteman, A.; Chase, M.W.; Li, J.W.; Huang, W.C.; Hidayat, A.; Wu, S.S.; Jin, X.H. Phylogenomics of Orchidaceae based on plastid and mitochondrial genomes. Mol. Phylogenet. Evol. 2019, 139, 106540. [Google Scholar] [CrossRef]
  44. Serna-Sánchez, M.A.; Pérez-Escobar, O.A.; Bogarín, D.; Torres-Jimenez, M.F.; Alvarez-Yela, A.C.; Arcila-Galvis, J.E.; Hall, C.F.; de Barros, F.; Pinheiro, F.; Dodsworth, S.; et al. Plastid phylogenomics resolves ambiguous relationships within the orchid family and provides a solid timeframe for biogeography and macroevolution. Sci. Rep. 2021, 11, 6858. [Google Scholar] [CrossRef]
  45. Doyle, J.J.; Doyle, J.L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 1987, 19, 11–15. [Google Scholar]
  46. Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30, 2114–2120. [Google Scholar] [CrossRef] [PubMed]
  47. Jin, J.J.; Yu, W.B.; Yang, J.B.; Song, Y.; Li, D.Z. GetOrganelle: A fast and versatile toolkit for accurate de novo assembly of organelle genomes. Genome Biol. 2020, 21, 241. [Google Scholar] [CrossRef] [PubMed]
  48. Wick, R.R.; Schultz, M.B.; Zobel, J.; Holt, K.E. Bandage: Interactive visualization of de novo genome assemblies. Bioinformatics 2015, 31, 3350–3352. [Google Scholar] [CrossRef] [PubMed]
  49. Shi, L.; Chen, H.; Jiang, M.; Wang, L.; Wu, X.; Huang, L.; Zhang, W.; Liu, C. CPGAVAS2, an integrated plastome sequence annotator and analyzer. Nucleic Acids Res. 2019, 47, W65–W73. [Google Scholar] [CrossRef]
  50. Tillich, M.; Lehwark, P.; Pellizzer, T.; Ulbricht-Jones, E.S.; Fischer, A.; Bock, R.; Greiner, S. GeSeq—Versatile and accurate annotation of organelle genomes. Nucleic Acids Res. 2017, 45, W6–W11. [Google Scholar] [CrossRef]
  51. Kearse, M.; Moir, R.; Wilson, A.; Stones-Havas, S.; Cheung, M.; Sturrock, S.; Buxton, S.; Cooper, A.; Markowitz, S.; Duran, C.; et al. Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 2012, 28, 1647–1649. [Google Scholar] [CrossRef]
  52. Lohse, M.; Drechsel, O.; Kahlau, S.; Bock, R. OrganellarGenomeDRAW—A suite of tools for generating physical maps of plastid and mitochondrial genomes and visualizing expression data sets. Nucleic Acids Res. 2013, 41, W575–W581. [Google Scholar] [CrossRef]
  53. Beier, S.; Thiel, T.; Munch, T.; Scholz, U.; Mascher, M. MISA-web: A web server for microsatellite prediction. Bioinformatics 2017, 33, 2583–2585. [Google Scholar] [CrossRef]
  54. Kurtz, S.; Choudhuri, J.V.; Ohlebusch, E.; Schleiermacher, C.; Stoye, J.; Giegerich, R. REPuter: The manifold applications of repeat analysis on genomic scale. Nucleic Acids Res. 2001, 29, 4633–4642. [Google Scholar] [CrossRef]
  55. Peden, J.F. Analysis of Codon Usage; University of Nottingham: Nottingham, UK, 2000; pp. 73–74. [Google Scholar]
  56. Huang, L.; Yu, H.; Wang, Z.; Xu, W. CPStools: A package for analyzing chloroplast genome sequences. iMetaOmics 2024, 1, e25. [Google Scholar] [CrossRef]
  57. Darling, A.C.E.; Mau, B.; Blattner, F.R.; Perna, N.T. Mauve: Multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 2004, 14, 1394–1403. [Google Scholar] [CrossRef]
  58. Li, H.; Guo, Q.; Xu, L.; Gao, H.; Liu, L.; Zhou, X. CPJSdraw: Analysis and visualization of junction sites of chloroplast genomes. PeerJ 2023, 11, e15326. [Google Scholar] [CrossRef] [PubMed]
  59. Katoh, K.; Standley, D.M. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol. Biol. Evol. 2013, 30, 772–780. [Google Scholar] [CrossRef] [PubMed]
  60. Capella-Gutiérrez, S.; Silla-Martínez, J.M.; Gabaldón, T. trimAl: A tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 2009, 25, 1972–1973. [Google Scholar] [CrossRef]
  61. Nguyen, L.T.; Schmidt, H.A.; von Haeseler, A.; Minh, B.Q. IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 2015, 32, 268–274. [Google Scholar] [CrossRef]
  62. David, P.; Buckley, T.R. Model selection and model averaging in phylogenetics: Advantages of Akaike information criterion and Bayesian approaches over likelihood ratio tests. Syst. Biol. 2004, 53, 793–808. [Google Scholar] [CrossRef]
  63. Darriba, D.; Taboada, G.L.; Doallo, R.; Posada, D. jModelTest 2: More models, new heuristics and parallel computing. Nat. Methods 2012, 9, 772. [Google Scholar] [CrossRef]
  64. Ronquist, F.; Teslenko, M.; van der Mark, P.; Ayres, D.L.; Darling, A.; Höhna, S.; Larget, B.; Liu, L.; Suchard, M.A.; Huelsenbeck, J.P. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 2012, 61, 539–542. [Google Scholar] [CrossRef]
  65. Jansen, R.K.; Ruhlman, T.A. Plastid genomes of seed plants. In Genomics of Chloroplasts and Mitochondria; Bock, R., Knoop, V., Eds.; Springer: Dordrecht, The Netherlands, 2012; pp. 103–126. [Google Scholar]
  66. Gu, L.; Su, T.; An, M.T.; Zhang, Q.; Kang, M. The complete chloroplast genome of the vulnerable Oreocharis esquirolii (Gesneriaceae): Structural features, comparative and phylogenetic analysis. Plants 2020, 9, 1692. [Google Scholar] [CrossRef]
  67. Zhao, F.; Drew, B.T.; Chen, Y.P.; Hu, G.X.; Li, B.; Xiang, C.L. The chloroplast genome of Salvia: Genomic characterization and phylogenetic analysis. Int. J. Plant Sci. 2020, 181, 812–830. [Google Scholar] [CrossRef]
  68. Du, Y.P.; Bi, Y.; Yang, F.P.; Zhang, M.F.; Chen, X.Q.; Xue, J.; Zhang, X.H. Complete chloroplast genome sequences of Lilium: Insights into evolutionary dynamics and phylogenetic analyses. Sci. Rep. 2017, 7, 5751. [Google Scholar] [CrossRef]
  69. Wu, B.; Jia, J.; Luo, D.; Zhang, Y.; Zhao, N. The conservative chloroplast genomic features and comparative analysis provide new evidence for the unification of Pennisetum and Cenchrus. Ind. Crops Prod. 2025, 226, 120747. [Google Scholar] [CrossRef]
  70. Kolodner, R.; Tewari, K.K. Inverted repeats in chloroplast DNA from higher plants. Proc. Natl. Acad. Sci. USA 1979, 76, 41–45. [Google Scholar] [CrossRef]
  71. Luo, J.; Hou, B.W.; Niu, Z.T.; Liu, W.; Xue, Q.Y.; Ding, X.Y. Comparative chloroplast genomes of photosynthetic orchids: Insights into evolution of the Orchidaceae and development of molecular markers for phylogenetic applications. PLoS ONE 2014, 9, e99016. [Google Scholar] [CrossRef]
  72. Kim, Y.K.; Jo, S.; Cheon, S.H.; Kwak, M.; Kim, K.J. Plastome evolution and phylogeny of Orchidaceae, with 24 new sequences. Front. Plant Sci. 2020, 11, 22. [Google Scholar] [CrossRef] [PubMed]
  73. Chen, X.H.; Ding, L.N.; Zong, X.Y.; Xu, H.; Wang, W.B.; Ding, R.; Qu, B. The complete chloroplast genome sequences of four Liparis species (Orchidaceae) and phylogenetic implications. Gene 2023, 888, 147760. [Google Scholar] [CrossRef] [PubMed]
  74. Botzman, M.; Margalit, H. Variation in global codon usage bias among prokaryotic organisms is associated with their lifestyles. Genome Biol. 2011, 12, R109. [Google Scholar] [CrossRef]
  75. Liu, L.; Du, J.; Liu, Z.; Zuo, W.; Wang, Z.; Li, J.; Zeng, Y. Comparative and phylogenetic analyses of nine complete chloroplast genomes of Orchidaceae. Sci. Rep. 2023, 13, 21403. [Google Scholar] [CrossRef]
  76. Han, C.; Ding, R.; Zong, X.; Zhang, L.; Chen, X.; Qu, B. Structural characterization of the Platanthera ussuriensis chloroplast genome and comparative analyses with other species of Orchidaceae. BMC Genom. 2022, 23, 84. [Google Scholar] [CrossRef]
  77. Zhou, Z.; Chen, J.L.; Wang, F.; Wu, X.P.; Liu, Z.J.; Peng, D.H.; Lan, S.R. The complete chloroplast genome of an epiphytic leafless orchid, Taeniophyllum complanatum: Comparative analysis and phylogenetic relationships. Horticulturae 2024, 10, 660. [Google Scholar] [CrossRef]
  78. Cui, Y.; Chen, X.; Nie, L.; Sun, W.; Hu, H.; Lin, Y.; Cheng, J.; Song, J. Comparison and phylogenetic analysis of chloroplast genomes of three medicinal and edible Amomum species. Int. J. Mol. Sci. 2019, 20, 4040. [Google Scholar] [CrossRef] [PubMed]
  79. Yang, J.X.; Hu, G.X.; Hu, G.W. Comparative genomics and phylogenetic relationships of two endemic and endangered species (Handeliodendron bodinieri and Eurycorymbus cavaleriei) of two monotypic genera within Sapindales. BMC Genom. 2022, 23, 27. [Google Scholar] [CrossRef]
  80. Yan, R.; Gu, L.; Qu, L.; Wang, X.; Hu, G. New insights into phylogenetic relationship of Hydrocotyle (Araliaceae) based on plastid genomes. Int. J. Mol. Sci. 2023, 24, 16629. [Google Scholar] [CrossRef]
  81. Xiao, M.; Hu, X.; Li, Y.; Liu, Q.; Shen, S.; Jiang, T.; Zhang, L.; Zhou, Y.; Li, Y.; Luo, X.; et al. Comparative analysis of codon usage patterns in the chloroplast genomes of nine forage legumes. Physiol. Mol. Biol. Plants 2024, 30, 153–166. [Google Scholar] [CrossRef]
  82. Chen, X.; Zhao, Y.; Xu, S.; Zhou, Y.; Zhang, L.; Qu, B.; Xu, Y. Analysis of codon usage bias in the plastid genome of Diplandrorchis sinica (Orchidaceae). Curr. Issues Mol. Biol. 2024, 46, 9807–9820. [Google Scholar] [CrossRef] [PubMed]
  83. Provan, J.; Powell, W.; Hollingsworth, P.M. Chloroplast microsatellites: New tools for studies in plant ecology and evolution. Trends Ecol. Evol. 2001, 16, 142–147. [Google Scholar] [CrossRef]
  84. Flajoulot, S.; Ronfort, J.; Baudouin, P.; Barre, P.; Huguet, T.; Huyghe, C.; Julier, B. Genetic diversity among alfalfa (Medicago sativa) cultivars coming from a breeding program, using SSR markers. Theor. Appl. Genet. 2005, 111, 1420–1429. [Google Scholar] [CrossRef]
  85. Feng, L.; Zhao, G.; An, M.; Wang, C.; Yin, Y. Complete chloroplast genome sequences of the ornamental plant Prunus cistena and comparative and phylogenetic analyses with its closely related species. BMC Genom. 2023, 24, 739. [Google Scholar] [CrossRef]
  86. Qin, J.; Ma, Y.; Liu, Y.; Wang, Y. Phylogenomic analysis and dynamic evolution of chloroplast genomes of Clematis nannophylla. Sci. Rep. 2024, 14, 15109. [Google Scholar] [CrossRef]
  87. Tamboli, A.S.; Youn, J.S.; Kadam, S.K.; Pak, J.H.; Choo, Y.S. Chloroplast genome of Arisaema takesimense: Comparative genomics and phylogenetic insights into the Arisaema. Biochem. Genet. 2025, 63, 1–10. [Google Scholar] [CrossRef] [PubMed]
  88. Tang, S.J. New evidence for the theory of chromosome organization by repetitive elements (CORE). Genes 2017, 8, 81. [Google Scholar] [CrossRef]
  89. Thode, V.A.; Lohmann, L.G. Comparative chloroplast genomics at low taxonomic levels: A case study using Amphilophium (Bignonieae, Bignoniaceae). Front. Plant Sci. 2019, 10, 796. [Google Scholar] [CrossRef] [PubMed]
  90. Perry, A.S.; Wolfe, K.H. Nucleotide substitution rates in legume chloroplast DNA depend on the presence of the inverted repeat. J. Mol. Evol. 2002, 55, 501–508. [Google Scholar] [CrossRef] [PubMed]
  91. Blazier, J.C.; Jansen, R.K.; Mower, J.P.; Govindu, M.; Zhang, J.; Weng, M.L.; Ruhlman, T.A. Variable presence of the inverted repeat and plastome stability in Erodium. Ann. Bot. 2016, 117, 1209–1220. [Google Scholar] [CrossRef]
  92. Carlsward, B.S.; Stern, W.L. Vegetative anatomy and systematics of Triphorinae (Orchidaceae). Bot. J. Linn. Soc. 2009, 159, 203–210. [Google Scholar] [CrossRef]
  93. Thangavelu, M.; Muthu, S. Vegetative anatomical adaptations of Epidendrum radicans (Epidendroideae, Orchidaceae) to epiphytic conditions of growth. Mod. Phytomorphol. 2017, 11, 117–130. [Google Scholar] [CrossRef]
  94. Barrett, C.F.; Pace, M.C.; Corbett, C.W.; Kennedy, A.H.; Thixton-Nolan, H.L.; Freudenstein, J.V. Organellar phylogenomics at the epidendroid orchid base, with a focus on the mycoheterotrophic Wullschlaegelia. Ann. Bot. 2024, 134, 1207–1228. [Google Scholar] [CrossRef]
  95. Barrett, C.F.; Davis, J.I. The plastid genome of the mycoheterotrophic Corallorhiza striata (Orchidaceae) is in the relatively early stages of degradation. Am. J. Bot. 2012, 99, 1513–1523. [Google Scholar] [CrossRef]
  96. Wicke, S.; Naumann, J. Molecular evolution of plastid genomes in parasitic flowering plants. Adv. Bot. Res. 2018, 85, 315–347. [Google Scholar] [CrossRef]
  97. Wolfe, K.H.; Morden, C.W.; Palmer, J.D. Function and evolution of a minimal plastid genome from a nonphotosynthetic parasitic plant. Proc. Natl. Acad. Sci. USA 1992, 89, 10648–10652. [Google Scholar] [CrossRef] [PubMed]
  98. Barrett, C.F.; Sinn, B.T.; Kennedy, A.H. Unprecedented parallel photosynthetic losses in a heterotrophic orchid genus. Mol. Biol. Evol. 2019, 36, 1884–1901. [Google Scholar] [CrossRef] [PubMed]
  99. Whitten, W.M.; Williams, N.H.; Chase, M.W. Subtribal and generic relationships of Maxillarieae (Orchidaceae) with emphasis on Stanhopeinae: Combined molecular evidence. Am. J. Bot. 2000, 87, 1842–1856. [Google Scholar] [CrossRef] [PubMed]
  100. Pridgeon, A.M.; Solano, R.; Chase, M.W. Phylogenetic relationships in Pleurothallidinae (Orchidaceae): Combined evidence from nuclear and plastid DNA sequences. Am. J. Bot. 2001, 88, 2286–2308. [Google Scholar] [CrossRef]
  101. Kim, Y.K.; Jo, S.; Cheon, S.H.; Kwak, M.; Kim, Y.D.; Kim, K.J. Plastome evolution and phylogeny of subtribe Aeridinae (Vandeae, Orchidaceae). Mol. Phylogenet. Evol. 2020, 144, 106721. [Google Scholar] [CrossRef]
  102. Tang, H.; Tang, L.; Shao, S.; Peng, Y.; Li, L.; Luo, Y. Chloroplast genomic diversity in Bulbophyllum section Macrocaulia (Orchidaceae, Epidendroideae, Malaxideae): Insights into species divergence and adaptive evolution. Plant Divers. 2021, 43, 350–361. [Google Scholar] [CrossRef]
  103. Zhang, G.; Hu, Y.; Huang, M.Z.; Huang, W.C.; Liu, D.K.; Zhang, D.; Hu, H.; Downing, J.L.; Liu, Z.J.; Ma, H. Comprehensive phylogenetic analyses of Orchidaceae using nuclear genes and evolutionary insights into epiphytism. J. Integr. Plant Biol. 2023, 65, 1204–1225. [Google Scholar] [CrossRef]
Diversity 17 00391 g001
Figure 1. Gene maps of the plastid genomes of Tropidia angulosa and T. nipponica. Genes shown on the inside of the circle are transcribed in a clockwise direction, while those on the outside are transcribed counterclockwise. Genes are color-coded based on functional categories. The darker gray and lighter gray in the inner circle represent the GC content and AT content, respectively. LSC, large single copy; SSC, small single copy; IR, inverted repeat.
Figure 1. Gene maps of the plastid genomes of Tropidia angulosa and T. nipponica. Genes shown on the inside of the circle are transcribed in a clockwise direction, while those on the outside are transcribed counterclockwise. Genes are color-coded based on functional categories. The darker gray and lighter gray in the inner circle represent the GC content and AT content, respectively. LSC, large single copy; SSC, small single copy; IR, inverted repeat.
Diversity 17 00391 g001
Diversity 17 00391 g002
Figure 2. Amino-acid frequency (A) and relative synonymous codon usage (B) of T. angulosa and T. nipponica.
Figure 2. Amino-acid frequency (A) and relative synonymous codon usage (B) of T. angulosa and T. nipponica.
Diversity 17 00391 g002
Diversity 17 00391 g003
Figure 3. Simple-sequence repeats (SSRs) and long-sequence repeats (LSRs) in the two Tropidia plastomes: (A) number of six types of SSRs; (B) number of SSRs in the LSC, SSC, and IR regions; (C) number of SSRs in the intergenic spacers (IGS), introns, and exons; (D) number of four types of LSRs; (E) number of LSRs in the LSC, SSC, and IR regions; (F) number of LSRs in the IGS, introns, and exons.
Figure 3. Simple-sequence repeats (SSRs) and long-sequence repeats (LSRs) in the two Tropidia plastomes: (A) number of six types of SSRs; (B) number of SSRs in the LSC, SSC, and IR regions; (C) number of SSRs in the intergenic spacers (IGS), introns, and exons; (D) number of four types of LSRs; (E) number of LSRs in the LSC, SSC, and IR regions; (F) number of LSRs in the IGS, introns, and exons.
Diversity 17 00391 g003
Diversity 17 00391 g004
Figure 4. Comparative analyses of plastome size and gene content: (A) length of the complete plastome, LSC, SSC, and IR regions; (B) gene loss and duplication.
Figure 4. Comparative analyses of plastome size and gene content: (A) length of the complete plastome, LSC, SSC, and IR regions; (B) gene loss and duplication.
Diversity 17 00391 g004
Diversity 17 00391 g005
Figure 5. Comparison of the four junctions between the IR and single-copy regions within the basal taxon of Epidendroideae.
Figure 5. Comparison of the four junctions between the IR and single-copy regions within the basal taxon of Epidendroideae.
Diversity 17 00391 g005
Diversity 17 00391 g006
Figure 6. Phylogenetic tree of Epidendroideae and related taxa inferred from plastid-coding sequences. As the topologies generated from the maximum likelihood (ML) and Bayesian inference (BI) methods are identical, only the ML tree is presented with the bootstrap support (BS) and posterior probability (PP) values shown above the branches. Blue, pink, purple, orange, and green represent Epidendroideae, Orchidoideae, Vanilloideae, Cypripedioideae, and Apostasioideae, respectively.
Figure 6. Phylogenetic tree of Epidendroideae and related taxa inferred from plastid-coding sequences. As the topologies generated from the maximum likelihood (ML) and Bayesian inference (BI) methods are identical, only the ML tree is presented with the bootstrap support (BS) and posterior probability (PP) values shown above the branches. Blue, pink, purple, orange, and green represent Epidendroideae, Orchidoideae, Vanilloideae, Cypripedioideae, and Apostasioideae, respectively.
Diversity 17 00391 g006
Table 1. List of gene content in the plastomes of T. angulosa and T. nipponica.
Table 1. List of gene content in the plastomes of T. angulosa and T. nipponica.
CategoryGene FunctionsName of Genes
Self-replicationLarge subunits of ribosomerpl2 *+, rpl14, rpl16 *, rpl20, rpl22, rpl23+, rpl32, rpl33, rpl36
Small subunits of ribosomerps2, rps3, rps4, rps7+, rps8+, rps11, rps12 **+, rps14, rps15, rps16 *, rps18, rps19+
DNA-dependent RNA polymeraserpoA, rpoB, rpoC1 *, rpoC2
Ribosomal RNAsrrn16+, rrn23+, rrn4.5+, rrn5+
Transfer RNAstrnA-UGC *+, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnG-GCC, trnG-UCC *, trnH-GUG+, trnI-CAU+, trnI-GAU *+, trnK-UUU *, trnL-CAA+, trnL-UAA *, trnL-UAG, trnM-CAU, trnN-GUU+, trnP-UGG, trnQ-UUG, trnR-ACG+, trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC+, trnV-UAC *, trnW-CCA, trnY-GUA
PhotosynthesisSubunits of photosystem IpsaA, psaB, psaC, psaI, psaJ
Subunits of photosystem IIpsbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbL, psbK, psbM, psbN, psbT, psbZ
Subunits of NADH dehydrogenasendhA *, ndhB *+, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
Subunits of cytochrome b/f complexpetA, petB *, petD *, petG, petL, petN
Subunits of ATP synthaseatpA, atpB, atpE, atpF *, atpH, atpI
Large subunit of rubiscorbcL
Other genesMaturasematK
ProteaseclpP **
Envelope membrane proteincemA
Acetyl-CoA carboxylaseaccD
C-type cytochrome synthesis geneccsA
Translation initiation factorinfA
Genes of unknownProteins of unknown functionycf1, ycf2+, ycf3 **, ycf4
* Genes with one intron. ** Genes with two introns. + Genes with two copies from IR regions.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Yu, D.-L.; Wei, Z.-Q.; Yan, R.-R.; Fei, S.-P.; Wu, W.; Hu, G.-X. Comparative Plastomics of Tropidia (Orchidaceae): Unraveling Structural Evolution and Phylogenetic Implications in Epidendroideae. Diversity 2025, 17, 391. https://doi.org/10.3390/d17060391

AMA Style

Yu D-L, Wei Z-Q, Yan R-R, Fei S-P, Wu W, Hu G-X. Comparative Plastomics of Tropidia (Orchidaceae): Unraveling Structural Evolution and Phylogenetic Implications in Epidendroideae. Diversity. 2025; 17(6):391. https://doi.org/10.3390/d17060391

Chicago/Turabian Style

Yu, Deng-Li, Zi-Qing Wei, Rong-Rong Yan, Shi-Peng Fei, Wei Wu, and Guo-Xiong Hu. 2025. "Comparative Plastomics of Tropidia (Orchidaceae): Unraveling Structural Evolution and Phylogenetic Implications in Epidendroideae" Diversity 17, no. 6: 391. https://doi.org/10.3390/d17060391

APA Style

Yu, D.-L., Wei, Z.-Q., Yan, R.-R., Fei, S.-P., Wu, W., & Hu, G.-X. (2025). Comparative Plastomics of Tropidia (Orchidaceae): Unraveling Structural Evolution and Phylogenetic Implications in Epidendroideae. Diversity, 17(6), 391. https://doi.org/10.3390/d17060391

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop