Next Article in Journal
Optimizing the Comet Assay-Based In Vitro DNA Repair Assay for Placental Tissue: A Pilot Study with Pre-Eclamptic Patients
Next Article in Special Issue
Complete Chloroplast Genomes and the Phylogenetic Analysis of Three Native Species of Paeoniaceae from the Sino-Himalayan Flora Subkingdom
Previous Article in Journal
The Ralstonia solanacearum Type III Effector RipAW Targets the Immune Receptor Complex to Suppress PAMP-Triggered Immunity
Previous Article in Special Issue
Complete Chloroplast Genome of Hypericum perforatum and Dynamic Evolution in Hypericum (Hypericaceae)
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 25
Issue 1
10.3390/ijms25010184
ijms-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 thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Characterization of Angraecum (Angraecinae, Orchidaceae) Plastomes and Utility of Sequence Variability Hotspots

by
Cheng-Yuan Zhou
1,†,
Wen-Jun Lin
2,†,
Ruyi Li
1,
Yuhan Wu
1,
Zhong-Jian Liu
1,2,* and
Ming-He Li
1,2,*
1
Key Laboratory of National Forestry and Grassland Administration for Orchid Conservation and Utilization at Landscape Architecture and Arts, Fujian Agriculture and Forestry University, Fuzhou 350002, China
2
Fujian Colleges and Universities Engineering Research Institute of Conservation and Utilization of Natural Bioresources, Fujian Agriculture and Forestry University, Fuzhou 350002, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2024, 25(1), 184; https://doi.org/10.3390/ijms25010184
Submission received: 27 November 2023 / Revised: 15 December 2023 / Accepted: 16 December 2023 / Published: 22 December 2023
(This article belongs to the Special Issue Plant Phylogenomics and Genetic Diversity 2.0)
Browse Figures
Versions Notes

Abstract

:
Angraecum, commonly known as Darwin’s orchid, is the largest genus of Angraecinae (Orchidaceae). This genus exhibits a high morphological diversity, making it as a good candidate for macroevolutionary studies. In this study, four complete plastomes of Angraecum were firstly reported and the potential variability hotspots were explored. The plastomes possessed the typical quadripartite structure and ranged from 150,743 to 151,818 base pair (bp), with a guanine–cytosine (GC) content of 36.6–36.9%. The plastomes all contained 120 genes, consisting of 74 protein-coding genes (CDS), 38 transfer RNA (tRNA) genes and 8 ribosomal RNA (rRNA) genes; all ndh genes were pseudogenized or lost. A total of 30 to 46 long repeats and 55 to 63 SSRs were identified. Relative synonymous codon usage (RSCU) analysis indicated a high degree of conservation in codon usage bias. The Ka/Ks ratios of most genes were lower than 1, indicating that they have undergone purifying selection. Based on the ranking of Pi (nucleotide diversity) values, five regions (trnSGCU-trnGGCC, ycf1-trnNGGU, trnNGUU-rpl32, psaC-ndhE and trnSGCU-trnGGCC) and five protein-coding genes (rpl32, rps16, psbK, rps8, and ycf1) were identified. The consistent and robust phylogenetic relationships of Angraecum were established based on a total of 40 plastomes from the Epidendroideae subfamily. The genus Angraecum was strongly supported as a monophyletic group and sister to Aeridinae. Our study provides an ideal system for investigating molecular identification, plastome evolution and DNA barcoding for Angraecum.

1. Introduction

Darwin’s most famous hypothesis was that a hawkmoth with an 11-inch (approximately 28 cm) proboscis would pollinate the Madagascan orchid [1]. This orchid belongs to Angraecum, commonly known as Darwin’s orchid, and is the largest genus of Angraecinae (Orchidaceae), comprising over 220 species [2,3]. Members of the Angraecum genus are mainly distributed from Madagascar to Africa and the Mascarene Islands [4]. Madagascar is the diversity center of this genus with approximately 142 species, 90% of which are endemic [5]. This genus is primarily characterized by its white to green flowers, labellum with a clavate or filiform spur, and two pollinia [6]. The high morphological variation among Angraecum species makes it one of the most valuable ornamental orchids. Approximately 190 artificial interspecific hybrids of Angraecum have been produced and registered with the Royal Horticultural Society (http://apps.rhs.org.uk/horticulturaldatabase/orchidregister/orchidregister.asp, accessed on 2 November 2023). Additionally, this genus also plays a critical role in pollination biology, evolution and ecological research [7,8,9].
Due to its extensive morphological diversity, this genus has generally been considered one of the most complicated groups within Angraecinae since its establishment. Morphologically, Garay proposed 19 sections to categorize the Angraecum species based on their floral characters [10]. However, recent molecular phylogenetic studies have revealed that Angraecum was non-monophyletic, and most of the sections defined by Garay were also non-monophyletic [8,11,12,13,14]. Additionally, all studies consistently showed unstable topologies with weak to moderate support. It seems that a limited number of traditional molecular markers have hindered our understanding of Angraecum phylogeny, making it challenging to distinguish between different Angraecum species. Exploring the molecular phylogenetic markers with more loci suitable for Angraecum is needed.
Advancements in next-generation sequencing (NGS) technology have greatly facilitated the acquisition of complete plastomes, which can provide more loci to clarify the phylogenetic relationships of complex taxa [15,16,17]. Plastomes are suitable for phylogenetic analysis due to their uniparental inheritance, abundance of informative loci and moderate mutation rate [18]. In recent years, whole plastome data have significantly advanced our understanding of the relationships within Orchidaceae [19,20,21]. Liu et al. used plastome sequences to elucidate the phylogenetic relationships within the Cleisostoma–Gastrochilus clades, revealing strong support and a stable topological structure [22]. Based on 79 protein-coding sequences of 46 species from 16 genera, Tu et al. showed a robust phylogenetic framework of the Cheirostylis and Goodyera clades of Goodyerinae [23]. Moreover, the comparison of plastome structures has proven valuable in understanding the molecular evolutionary patterns involving gene duplication, loss, rearrangement, and transfer within Orchidaceae [24,25,26,27,28]. However, no studies of Angraecum plastomes have been reported, hindering our understanding of the plastome evolution and phylogenetic relationships of this genus.
To enhance our understanding of Angraecum plastome characteristics, structural diversity and evolution, we firstly present four Angraecum plastomes. This study aims to evaluate variations in high-variability sites and simple sequence repeats, characterizing and contrasting Angraecum plastomes in order to understand the evolutionary pattern of the plastome and resolving phylogenetic relationships in Angraecum for accurate authentication of Angraecum species.

2. Results

2.1. Characteristics of the Plastome

A total of four newly sequenced Angraecum plastomes comprised an LSC region (87,889–88,904 bp), an SSC region (11,599–11,922 bp) and a pair of IRs (25,387–25,982 bp) (Figure 1). Plastome sizes ranged from 150,743 bp (A. borbonicum) to 151,818 bp (A. sesquipedale). Each Angraecum plastome possessed the quadripartite structure with similar percentages in each region (LSC 58.1–58.6%, IR 16.8–17.1%, and SSC 7.7–7.9%) (Table 1). The GC content of the whole plastome exhibited minimal variation, ranging from 36.6% to 36.9%.
All four plastomes of Angraecum encoded 120 genes, encompassing 74 CDSs, 38 tRNA genes and 8 rRNA genes (Table 1). Among them, 18 genes were replicated in the IR regions, comprising 5 protein-coding genes (rpl2, rpl23, rps7, rps19 and ycf2), 4 rRNA genes (rrn4.5, rrn5, rrn16, and rrn23), and 8 tRNA genes (trnAUGC, trnHGUG, trnICAU, trnIGAU, trnLCAA, trnNGUU, trnRACG, and trnVGAC) (Figure 1). The loss or pseudogenization of the ndh genes was widespread among all Angraecum plastomes (Figure 1, Table 1). All four plastomes shared the same pseudogenes (ndhB/C/D/E/J/K). No significant rearrangements among these plastomes were detected by a Mauve analysis (Figure 2).
The comparative analysis of plastome boundary genes for four Angraecum plastomes revealed a highly conserved distribution pattern (Figure 3). The rpl22 gene in all species spanned from LSC to IRb, with a length of 31 bp to 33 bp. The ycf1 gene was entirely located in the SSC region, and there were no ycf1 fragments near the junction between the IRb and the SSC (JSB). For the junction between the IRa and the LSC (JLA), the trnH and psbA genes were detected.

2.2. Repeated Analysis

Six types of simple sequence repeats (SSRs) (mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide) were examined to explore potential genetic markers suitable for clarifying intragenus variations in Angraecum. All categories were detected, with a total of 55 (A. borbonicum) to 63 (A. lecomtei) SSRs (Figure 4, Supplementary Table S2). Mononucleotide repeats were the most frequent type, followed by dinucleotide repeats, with a range of 7 (A. lecomtei) to 11 (A. borbonicum). Among these classified repeat types, the A/T mononucleotide repeats were the most frequently observed, with a range of 30 (A. borbonicum) to 44 (A. lecomtei).
A total of 155 long repeats were detected in Angraecum plastomes, comprising 4 types of long repeats (palindrome, forward, reverse, and complement) (Figure 4, Supplementary Table S3). Among them, all types were detected within two species (A. lecomtei and A. sesquipedale), three types were detected within A. borbonicum, and only two types were detected within A. sororium (Figure 4). The number of long repeats ranges from 30 (A. sororium) to 46 (A. lecomtei) (Figure 4, Supplementary Table S3). Palindrome repeats were the dominant type of long repeats, followed by forward. The length of long repeats in all species mostly ranged from 30 bp to 40 bp. There were two extremely long repeat sequences found within A. lecomtei, 104 bp and 80 bp, respectively.

2.3. Codon Usage Analyses

The concatenated sequences of 68 CDSs (ndh genes were widespread pseudogenized or lost) were used to calculate the RSCU values and codon usage frequency of Angraecum plastomes. Visualization of the RSCU values for Angraecum plastomes revealed a highly conserved codon usage bias (Figure 5, Supplementary Table S4). All species possessed 64 different types of codons and encoded a total of 19,377–19,389 codons. Among these codons, leucine (Leu) was the most frequent amino acid, while cysteine (Cys) had the lowest frequency (Supplementary Table S4). The codon GCU exhibited the highest RSCU value, while the codon CGC had the lowest RSCU value. The most frequently used stop codon was UAA, and then UAG and UGA.

2.4. Selective Pressure Analysis

The nonsynonymous (Ka), synonymous substitution rates (Ks), and the ratio Ka/Ks were calculated to explore whether the protein-coding genes of four Angraecum plastomes have undergone selection (Figure 6, Supplementary Table S5). A. borbonicum and A. lecomtei exhibited relatively high Ka and Ks values among the four Angraecum plastomes. A. sesquipedale had the highest average Ka/Ks value (0.2815). The genes atpH, infA, psaC, psaJ, psbC, psbE and psbI were found to undergo neutral evolution (Ka/Ks = 1). The majority of the protein-coding genes were found to have undergone purifying selection. (Ka/Ks < 1).

2.5. Plastome Sequence Divergence and Barcoding Investigation

The plastome divergence among Angraecum species was calculated using an mVISTA program with the annotated plastome of Thrixspermum centipeda as a reference genome (Figure 7). The results showed that the greatest variation was localized within the LSC and SSC regions, whereas the IR regions exhibited higher conservation. The coding regions were highly conserved in comparison to the non-coding regions. These results indicate that several regions may be suitable for DNA barcodes that can distinguish different Angraecum species easily.
To further explore the mutation hotspots of Angraecum plastomes to develop specific DNA barcodes, Pi values was calculated using DnaSP6 (Figure 8). The average Pi value among the four plastomes was 0.00777, with the IR region averaging 0.00251, the LSC region averaging 0.00919, and the SSC region averaging 0.02019, respectively (Supplementary Table S6). According to the ranking of Pi values, five hypervariable regions were identified: trnSGCU-trnGGCC, ycf1-trnNGGU, trnNGUU-rpl32, psaC-ndhE and trnSGCU-trnGGCC. In terms of protein-coding genes, rpl32, rps16, psbK, rps8, and ycf1 showed high Pi values and may be used as DNA barcodes for further phylogenetic analyses and species identification.

2.6. Phylogenetic Analysis

In the present study, we obtained a robust phylogenetic framework of the Epidendroideae using three methods (ML, MP and BI), including 40 species from 34 genera (Figure 9). The species of Angraecum formed a well-supported monophyletic group (BS = 100, PP = 1.00), which was revealed as a sister to Aeridinae. The intrageneric relationships of Angraecum showed that Angraecum could be divided into two diverging clades with strong support (BS = 100, PP = 1.00). The taxa A. lecomtei, together with A. borbonicum, formed the first distinct clade. A. sororium was grouped together with A. sesquipedale, supported as the second clade.

3. Discussion

3.1. The Plastome Characteristics and Structural Evolution

In the present study, we firstly reported four Angraecum plastomes and provided genetic resources for understanding the evolution of plastomes in this group. All Angraecum plastomes had the typical quadripartite structure (Figure 1), consisting of one LSC region, one SSC region, and two IR regions, similar to most common angiosperms. Limited variation in overall plastome size was detected among Angraecum species: A. borbonicum possessed the smallest plastome at 150,743 bp, and A. sesquipedale had the largest at 151,818 bp. The plastome size falls within the previously reported range of Orchidaceae plastomes, which ranged from 19,047 bp (Epipogium roseum) [29] to 212,688 bp (Cypripedium tibeticum) [24]. No significant variation in GC content was found among Angraecum plastomes (36.7–36.9%) in this study. In addition, no unusual structural features were detected among Angraecum plastomes according to the result of Mauve (Figure 2).
The loss and pseudogenization of the ndh genes were commonly observed in Orchidaceae (Figure 1, Table 1). The phenomenon has been observed in several orchid lineages, including Vanilla [30], Dendrobium [25], Bulbophyllum [31], Goodyerinae [23], Neottieae [32], Polystachya [26], and Aeridinae [20,21]. Our study showed that all Angraecum plastomes were ndh-deleted: ndhA/F/G/H/I genes were completely lost and the other ndh genes were pseudogenes. The previous study suggests that the loss of ndh genes may be associated with the epiphytic lifestyle of plants [33]. Angraecum species are usually epiphytic or lithophytic [6], supporting the pseudogenization or loss of ndh genes in epiphytic habitats.
Previous studies showed that the IR/SC boundary shift is one of the main factors contributing to differences in plastome length and gene content [34,35]. However, the gene arrangement of the IR/SC boundary in Angraecum plastomes was extremely conserved (Figure 3), indicating that the variations in plastome length and gene content in Angraecum were not caused by the IR/SC boundary shift.
Simple sequence repeats (SSRs) are commonly found in plastomes, serving as a crucial molecular marker in phylogenetics, population genetics, and evolutionary studies [36,37]. The present study investigated the dispersion of repeat sequences in four Angraecum plastomes, which showed a similar SSR motif distribution (Figure 4). Numerous long repeat sequences were identified, with the majority falling within the range of 30 to 40 bp, consistent with ranges previously recorded in other Orchidaceae lineages [24,25,26,27,28]. However, we detected two extremely long repeat sequences within the plastome of A. lecomtei with lengths of 104 bp and 80 bp. This result indicated that these long repeat sequences could potentially be DNA barcodes in future studies of this species. Our results significantly contribute to understanding the development of specific DNA barcodes in Angraecum.
Relative synonymous codon usage (RSCU) values were used to measure codon usage bias in coding sequences, which could provide evidence for exploring the evolutionary patterns of species [38]. Our results indicated that codon usage bias was highly conserved among four Angraecum plastomes (Figure 5). Previous studies indicated that similar codon selection strategies may contribute to the close phylogenetic relationships between closely related species. Our results were consistent with previous studies of codon preference in Orchidaceae [26,27].
We also conducted a selective pressure analysis to compare the protein-coding genes evolution in the four Angraecum plastomes (Figure 6, Supplementary Table S6). The Ka/Ks ratios were crucial for understanding the adaptive evolution among species [39]. Our results showed that no genes were identified with positive selection (Ka/Ks > 1) and most genes were found to have undergone purifying selection (Ka/Ks < 1). These phenomena might reflect that most cp-genes in these Angraecum species were likely to undergo deleterious nonsynonymous substitutions [40].

3.2. Plastid Genomic Evolutionary Hotspots

To identify mutational hotspots for phylogenetic reconstruction of taxonomically problematic groups, numerous plastomes comparative analyses within Orchidaceae had been reported [24,25,26,27,28]. In this study, a total of five hotspots regions (trnSGCU-trnGGCC, ycf1-trnNGGU, trnNGUU-rpl32, psaC-ndhE and trnSGCU-trnGGCC) and five CDSs (rpl32, rps16, psbK, rps8, and ycf1) were selected for candidate barcodes, respectively (Figure 8). These findings may contribute to the development of specific DNA barcoding markers and the resolution of phylogenetic relationships in Angraecum.

3.3. Phylogenetic Analysis

Angraecum presented a considerable challenge to its phylogenetic reconstruction and classification because of the numerous disparities between morphology and molecular analyses. Based on matK, trnL-F and ITS, Carlsward et al. [11] revealed that Angraecum was non-monophyletic and exhibited collapsed relationships with low to moderate support [11]. However, based on a broader sampling and molecular markers, the phylogenetic relationships of Angraecum, as revealed by Micheneau et al. [12], Andriananjamanantsoa et al. [5], and Simo-Droissart et al. [13], are still unresolved due to incongruent topology and weak support. In addition, the non-monophyletic status of most sections defined by Garay [10] was commonly found in these studies [5,12,13]. Therefore, the most recent molecular phylogenetic study suggested that using genomic data to resolve the phylogeny of Angraecum is needed [14]. Our phylogenomic analyses revealed that the phylogenetic resolution within Angraecum has been greatly improved with strong support (Figure 9). Four Angraecum species were clustered into a monophyletic group (BS = 100, PP = 1.00) and could be further divided into two diverging clades. This result indicated that the plastome sequences were ideal molecular markers for resolving the intrageneric relationships of Angraecum.
According to the new Orchidaceae classification system [41], Angraecum was placed in the subtribe Angraecinae, sister to the subtribe Aeridinae of the subfamily Epidendroideae. Previous studies commonly indicated that Epidendroideae was the most taxonomically problematic subfamily due to its significant diversification (comprising approximately 76% of Orchidaceae species) [42]. To explore the phylogenetic position of Angraecinae, we reconstructed the phylogeny relationships within Epidendroideae, including a total of 40 species from 34 genera. Our results showed that Angraecinae was sister to Aeridinae with strong support in all analyses (BS = 100, PP = 1.00) (Figure 9), consistent with previous studies [19]. Additionally, we identified several extremely short branches within Epidendroideae (Figure 9). Short branch lengths in phylogenetic trees could be attributed to the rapid radiation events, resulting in few opportunities for molecular changes [43]. The widespread rapid radiation events among Epidendroideae may explain why the numerous species and genera within this subfamily.

4. Materials and Methods

4.1. Taxon Sampling and Sequencing

Four Angraecum species were selected for the study: A. borbonicum, A. lecomtei, A. sesquipedale, and A. sororium. Fresh and healthy leaf tissues of Angraecum were obtained from Fujian Agriculture and Forestry University (Fuzhou, Fujian, China) and Shanghai Chenshan Botanical Garden (Shanghai, China). Four Angraecum species were selected. Based on the previous study [41], a total of 40 plastomes from 34 genera were selected, including six species from five genera from Lower Epidendroideae as the outgroups. Voucher information and GenBank accession numbers are provided in Supplementary Table S1.
Total DNA was extracted from fresh leaves with a Plant Mini Kit (Qiagen, Redwood City, CA, USA) based on the manufacturer’s protocol, which included prewashing with STE buffer to remove inhibitory chemicals. DNA degradation and contamination were evaluated on 1% agarose gels. Next-generation sequencing (NGS) was performed on an Illumina Hiseq 4000 sequencing platform (Illumina, San Diego, CA, USA), generating 150-bp paired-end reads. Scripts were used to filter the Illumina data in the cluster with the default parameters. Paired reads were excluded from the analysis if they contained more than 50% low-quality (Q ≤ 5) bases or if the N content exceeded 10% of the reads’ base number. More than 10 Gb clean data were obtained for each species.

4.2. Plastome Assembly and Annotation

To obtain complete plastomes, we used a GetOrganelle pipe-line (https://github.com/Kinggerm/GetOrganelle, accessed on 1 November 2023) [44] to filter the paired-end reads with default parameters. Then, the SPAdes 3.10 [45] were employed to assemble the filtered reads. To obtain pure contigs, we further filtered the “fastg” files by the GetOrganelle script. The filtered De Bruijn graphs were then examined and corrected by Bandage [46]. Finally, four high-quality and complete plastomes were obtained.
PGA software [47] was used to annotate the newly assembled Angraecum plastome, and the published sequence of Thrixspermum centipeda (MW057769) was used as a reference. The start and stop codons in protein-coding genes were manually visualized and corrected by aligning them with the reference plastome in Geneious R11.1.5 [48]. The annotation maps were drawn using OGDRAW [49].

4.3. Plastome Comparative and Codon Usage Analysis

The rearrangements of Angraecum plastomes were identified and plotted by Mauve [50]. The genes on the boundary regions of LSC/IRb/SSC/IRa were visualized by the IRscope online program [51]. The online software MISA (http://misaweb.ipk-gatersleben.de/, accessed on 1 November 2023) [52] was employed to detect simple sequence repeats (SSRs). Parameters for SSR motifs were 10, 5, 4, 3, 3, and 3 nucleotide repeats set for mono-, di-, tri-, tetra-, penta- and hexa-motif microsatellites (mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide) set as the minimum threshold, respectively. The REPuter software [53] was used to detect four types of long repeat sequences, including forward (F), palindrome (P), reverse (R), and complement (C). The minimum repeat size of oligonucleotide repeats was set at 30 bp, and the Hamming distance was set at 3. Results were visualized with the R package ggplot2 [54].
A total of 68 CDSs of each Angraecum plastome were extracted and concatenated using PhyloSuite v1.2.2 [55]. Relative synonymous codon usage (RSCU) values for each Angraecum species were calculated by DAMBE [56]. Finally, a heatmap was generated using TBtools [57].

4.4. Selective Pressure Estimation

A total of 68 CDSs were retrieved and used to investigate substitution rates, respectively. The non-synonymous (Ka) and synonymous (Ks) rates, as well as the Ka/Ks ratio, were calculated using Ka/Ks calculator ver. 2.0 [58]. When Ka/Ks > 1 indicates positive (adaptive) selection, Ka/Ks = 1 indicates neutral evolution, while Ka/Ks < 1 signifies negative (purifying) selection.

4.5. Sequence Divergence, Barcoding Investigation and Phylogeny

The online program mVISTA was used to analyze the diversity of Angraecum plastomes using the Shuffle-LAGAN [59] alignment program and Thrixspermum centipeda (MW057769) was used as a reference. The nucleotide variability (Pi) of whole plastomes and 68 CDSs of Angraecum were calculated by DnaSP 6 [60] with the default parameters.
A total of 40 plastomes were aligned by MAFFT [61] and we employed TrimAL v1.4 [62] to trim the poorly aligned positions with a default parameter. Then, the matrix was used to reconstruct the phylogenetic tree. The phylogenetic trees were inferred by maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI) on the website CIPRES Science Gateway web server (RAxML-HPC2 on XSEDE 8.2.12, PAUP on XSEDE 4.a 168 and MrBayes on XSEDE 3.2.7a) [63]. For ML analysis, the GTRGAMMA model was specified for all datasets [64] and calculated bootstrap values from 1000 bootstrap replicates using heuristic searches [65]. For BI analysis, we used MrBayes v. 3.2.7a under the GTR + I + Γ substitution model. The Markov chain Monte Carlo (MCMC) algorithm was run for 10,000,000 generations, with one tree sampled every 100 generations. The first 25% of trees were discarded as burn-in to construct majority-rule consensus trees and estimate posterior probabilities (PP).

5. Conclusions

In the present study, we firstly reported four Angraecum plastomes (A. borbonicum, A. lecomtei, A. sesquipedale, and A. sororium). The characteristics and comparative analysis results indicate that the genomic structure and gene content of Angraecum plastomes are highly conserved. All ndh genes were found to be lost or pseudogenized. According to the ranking of Pi values, a total of five hotspots regions (trnSGCU-trnGGCC, ycf1-trnNGGU, trnNGUU-rpl32, psaC-ndhE and trnSGCU-trnGGCC) and five protein-coding genes (rpl32, rps16, psbK, rps8, and ycf1) were identified for DNA barcodes. Based on whole plastome sequences, we explored the intrageneric and intergeneric relationships of Angraecum and found that plastome data offer valuable insights into the phylogenetic relationships of Angraecum. These findings shed new light on plastome evolution and the phylogenetic relationships of Angraecum and its related lineages.

Supplementary Materials

The supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms25010184/s1.

Author Contributions

Z.-J.L. and M.-H.L.: Conceptualization. C.-Y.Z. and W.-J.L.: Methodology, Software; C.-Y.Z., W.-J.L., R.L. and M.-H.L.: Data curation, Writing—Original draft preparation, Writing—Reviewing and editing. C.-Y.Z., R.L. and Y.W.: Validation; Resources. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Outstanding Youth Scientific Fund of Fujian Agriculture and Forestry University (XJQ202005), the Nature Science Foundation of Fujian Province, China (2021J01134), and the Forestry Peak Discipline Construction Project of Fujian Agriculture and Forestry University (72202200205).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All the data are provided within this manuscript and supplementary materials.

Acknowledgments

We acknowledge the technical support of laboratory staff during the conduction of laboratory experiments, Ding-Kun Liu, Xiong-De Tu, and Jin-Liao Chen.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Harder, L.D.; Johnson, S.D. Darwin’s beautiful contrivances: Evolutionary and functional evidence for floral adaptation. New Phytol. 2009, 183, 530–545. [Google Scholar] [CrossRef] [PubMed]
  2. Hermans, J.; Verlynde, S.; Rajaovelona, L.; Cribb, P.J.; Hervouet, J.M. New species and nomenclatural changes in Angraecum (Orchidaceae) from Madagascar. Kew Bull. 2020, 75, 49. [Google Scholar] [CrossRef]
  3. Hermans, J.; Rajaovelona, L.; Cribb, P. Angraecum inflatum, a new species in Angraecinae (Orchidaceae) from Madagascar. Kew Bull. 2021, 76, 513–517. [Google Scholar] [CrossRef]
  4. Simo-Droissart, M.; Micheneau, C.; Sonké, B.; Droissart, V.; Plunkett, G.M.; Lowry, P.P., II; Hardy, O.J.; Stévart, T. Morphometrics and molecular phylogenetics of the continental African species of Angraecum section Pectinaria (Orchidaceae). Plant Ecol. Evol. 2013, 146, 295–309. [Google Scholar] [CrossRef]
  5. Andriananjamanantsoa, H.N.; Engberg, S.; Louis, E.E., Jr.; Brouillet, L. Diversification of Angraecum (Orchidaceae, Vandeae) in Madagascar: Revised phylogeny reveals species accumulation through time rather than rapid radiation. PLoS ONE 2016, 11, e0163194. [Google Scholar] [CrossRef] [PubMed]
  6. Pridgeon, A.M.; Cribb, P.J.; Chase, M.W.; Rasmussen, F.N. Genera Orchidacearum Volume 6: Epidendroideae (Part 3); OUP Oxford Press: Oxford, UK, 2014. [Google Scholar]
  7. Nilsson, L.A.; Jonsson, L.; Rason, L.; Randrianjohany, E. Monophily and pollination mechanisms in Angraecum arachnites Schltr. (Orchidaceae) in a guild of long-tongued hawk-moths (Sphingidae) in Madagascar. Biol. J. Linn. Soc. 1985, 26, 1–19. [Google Scholar] [CrossRef]
  8. Micheneau, C.; Fournel, J.; Pailler, T. Bird Pollination in an Angraecoid Orchid on Reunion Island (Mascarene Archipelago, Indian Ocean). Ann. Bot. 2006, 97, 965–974. [Google Scholar] [CrossRef]
  9. Buyun, L.I.; Cherevchenko, T.M.; Kovalska, L.A.; Ivannikov, R.V. Reproductive biology of Angraecum eburneum subsp. superbum (Orchidaceae) under glasshouse conditions. Environ. Exp. Biol. 2015, 13, 33–39. [Google Scholar]
  10. Garay, L.A. Systematics of the genus Angraecum (Orchidaceae). Kew Bull. 1973, 28, 495–516. [Google Scholar] [CrossRef]
  11. Carlsward, B.S.; Whitten, W.M.; Williams, N.H.; Bytebier, B. Molecular phylogeny of Vandeae (Orchidaceae) and the evolution of leaflessness. Am. J. Bot. 2006, 93, 770–786. [Google Scholar] [CrossRef]
  12. Micheneau, C.; Carlsward, B.S.; Fay, M.F.; Bytebier, B.; Pailler, T.; Chase, M.W. Phylogenetics and biogeography of Mascarene angraecoid orchids (Vandeae, Orchidaceae). Mol. Phylogenet. Evol. 2008, 46, 908–922. [Google Scholar] [CrossRef] [PubMed]
  13. Simo-Droissart, M.; Plunkett, G.M.; Droissart, V.; Edwards, M.B.; Farminhão, J.N.M.; Ječmenica, V.; D’haijère, T.; Lowry, P.P.; Sonké, B.; Micheneau, C.; et al. New phylogenetic insights toward developing a natural generic classification of African angraecoid orchids (Vandeae, Orchidaceae). Mol. Phylogenet. Evol. 2018, 126, 241–249. [Google Scholar] [CrossRef] [PubMed]
  14. Farminhão, J.N.M.; Verlynde, S.; Kaymak, E.; Droissart, V.; Simo-Droissart, M.; Collobert, G.; Martos, F.; Stévart, T. Rapid radiation of angraecoids (Orchidaceae, Angraecinae) in tropical Africa characterized by multiple karyotypic shifts under major environmental instability. Mol. Phylogenet. Evol. 2021, 159, 107105. [Google Scholar] [CrossRef]
  15. Niehuis, O.; Hartig, G.; Grath, S.; Pohl, H.; Lehmann, J.; Tafer, H.; Donath, A.; Krauss, V.; Eisenhardt, C.; Hertel, J.; et al. Genomic and morphological evidence converge to resolve the enigma of Strepsiptera. Curr. Biol. 2012, 22, 1309–1313. [Google Scholar] [CrossRef] [PubMed]
  16. Lyu, R.; Xiao, J.; Li, M.; Luo, Y.; He, J.; Cheng, J.; Xie, L. Phylogeny and Historical Biogeography of the East Asian Clematis Group, Sect. Tubulosae, Inferred from Phylogenomic Data. Int. J. Mol. Sci. 2023, 24, 3056. [Google Scholar] [CrossRef] [PubMed]
  17. 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] [PubMed]
  18. Ohyama, K. Chloroplast and mitochondrial genomes from a liverwort, Marchantia polymorpha—Gene organization and molecular evolution. Biosci. Biotechnol. Biochem. 1996, 60, 16–24. [Google Scholar] [CrossRef] [PubMed]
  19. Givnish, T.J.; Spalink, D.; Ames, M.; Lyon, S.P.; Hunter, S.J.; Zuluaga, A.; Iles, W.J.; Clements, M.A.; Arroyo, M.T.; Leebens-Mack, J.; et al. Orchid phylogenomics and multiple drivers of their extraordinary diversification. Proc. Biol. Sci. B 2015, 282, 2108–2111. [Google Scholar] [CrossRef]
  20. 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]
  21. 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]
  22. Liu, D.K.; Tu, X.D.; Zhao, Z.; Zeng, M.Y.; Zhang, S.; Ma, L.; Zhang, G.Q.; Wang, M.M.; Liu, Z.J.; Lan, S.R.; et al. Plastid phylogenomic data yield new and robust insights into the phylogeny of Cleisostoma-Gastrochilus clades (Orchidaceae, Aeridinae). Mol. Phylogenet. Evol. 2020, 145, 106729. [Google Scholar] [CrossRef] [PubMed]
  23. Tu, X.D.; Liu, D.K.; Xu, S.W.; Zhou, C.Y.; Gao, X.Y.; Zeng, M.Y.; Zhang, S.; Chen, J.L.; Ma, L.; Zhou, Z.; et al. Plastid phylogenomics improves resolution of phylogenetic relationship in the Cheirostylis and Goodyera clades of Goodyerinae (Orchidoideae, Orchidaceae). Mol. Phylogenet. Evol. 2021, 164, 107269. [Google Scholar] [CrossRef] [PubMed]
  24. Guo, Y.Y.; Yang, J.X.; Bai, M.Z.; Zhang, G.Q.; Liu, Z.J. The chloroplast genome evolution of Venus slipper (Paphiopedilum): IR expansion, SSC contraction, and highly rearranged SSC regions. BMC Plant Biol. 2021, 21, 248. [Google Scholar] [CrossRef] [PubMed]
  25. Niu, Z.; Zhu, S.; Pan, J.; Li, L.; Sun, J.; Ding, X. Comparative analysis of Dendrobium plastomes and utility of plastomic mutational hotspots. Sci. Rep. 2017, 7, 2073. [Google Scholar]
  26. Jiang, H.; Tian, J.; Yang, J.; Dong, X.; Zhong, Z.; Mwachala, G.; Zhang, C.; Hu, G.; Wang, Q. Comparative and phylogenetic analyses of six Kenya Polystachya (Orchidaceae) species based on the complete chloroplast genome sequences. BMC Plant Biol. 2022, 22, 177. [Google Scholar] [CrossRef]
  27. Chen, Y.; Zhong, H.; Zhu, Y.; Huang, Y.; Wu, S.; Liu, Z.; Lan, S.; Zhai, J. Plastome structure and adaptive evolution of Calanthe s.l. species. PeerJ 2020, 8, e10051. [Google Scholar] [CrossRef]
  28. Li, L.; Wu, Q.; Fang, L.; Wu, K.; Li, M.; Zeng, S. Comparative Chloroplast Genomics and Phylogenetic Analysis of Thuniopsis and Closely Related Genera within Coelogyninae (Orchidaceae). Front. Genet. 2022, 13, 850201. [Google Scholar] [CrossRef]
  29. Schelkunov, M.I.; Shtratnikova, V.Y.; Nuraliev, M.S.; Selosse, M.A.; Penin, A.A.; Logacheva, M.D. Exploring the limits for reduction of plastid genomes: A case study of the mycoheterotrophic orchids Epipogium aphyllum and Epipogium roseum. Genome Biol. Evol. 2015, 7, 1179–1191. [Google Scholar] [CrossRef]
  30. Kim, Y.K.; Jo, S.; Cheon, S.H.; Joo, M.J.; Hong, J.R.; Kwak, M.; Kim, K.J. Plastome Evolution and Phylogeny of Orchidaceae, with 24 New Sequences. Front. Plant Sci. 2020, 11, 22. [Google Scholar] [CrossRef]
  31. Zavala-Páez, M.; Vieira, L.D.N.; Baura, V.A.D.; Balsanelli, E.; Souza, E.M.D.; Cevallos, M.C.; Smidt, E.D.C. Comparative plastid genomics of neotropical Bulbophyllum (Orchidaceae; Epidendroideae). Front. Plant Sci. 2020, 11, 799. [Google Scholar] [CrossRef]
  32. Feng, Y.L.; Wicke, S.; Li, J.W.; Han, Y.; Lin, C.S.; Li, D.Z.; Zhou, T.T.; Huang, W.C.; Huang, L.Q.; Jin, X.H. Lineage-specific reductions of plastid genomes in an orchid tribe with partially and fully mycoheterotrophic species. Genome Biol. Evol. 2016, 8, 2164–2175. [Google Scholar] [CrossRef] [PubMed]
  33. Sanderson, M.J.; Copetti, D.; Burquez, A.; Bustamante, E.; Charboneau, J.L.M.; Eguiarte, L.E.; Kumar, S.; Lee, H.O.; Lee, J.; McMahon, M.; et al. Exceptional reduction of the plastid genome of saguaro cactus (Carnegiea gigantea): Loss of the ndh gene suite and inverted repeat. Am. J. Bot. 2015, 102, 1115–1127. [Google Scholar] [CrossRef] [PubMed]
  34. 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]
  35. Dugas, D.V.; Hernandez, D.; Koenen, E.J.; Schwarz, E.; Straub, S.; Hughes, C.E.; Jansen, R.K.; Nageswara-Rao, M.; Staats, M.; Trujillo, J.T.; et al. Mimosoid legume plastome evolution: IR expansion, tandem repeat expansions, and accelerated rate of evolution in clpP. Sci. Rep. 2015, 5, 16958. [Google Scholar] [CrossRef] [PubMed]
  36. Cavalier-Smith, T. Chloroplast evolution: Secondary symbiogenesis and multiple losses. Curr. Biol. 2002, 12, R62–R64. [Google Scholar] [CrossRef] [PubMed]
  37. Gu, C.; Ma, L.; Wu, Z.; Chen, K.; Wang, Y. Comparative analyses of chloroplast genomes from 22 Lythraceae species: Inferences for phylogenetic relationships and genome evolution within Myrtales. BMC Plant Biol. 2019, 19, 281. [Google Scholar] [CrossRef] [PubMed]
  38. Iriarte, A.; Lamolle, G.; Musto, H. Codon Usage Bias: An endless tale. J. Mol. Evol. 2021, 89, 589–593. [Google Scholar] [CrossRef]
  39. Fay, J.C.; Wu, C.-I. Sequence Divergence, Functional Constraint, and Selection in Protein Evolution. Annu. Rev. Genom. Hum. Genet. 2003, 4, 213–235. [Google Scholar] [CrossRef]
  40. Zhang, J. Rates of conservative and radical nonsynonymous nucleotide substitutions in mammalian nuclear genes. J. Mol. Evol. 2000, 50, 56–68. [Google Scholar] [CrossRef]
  41. Chase, M.W.; Cameron, K.M.; Freudestein, J.V.; Pridgeon, A.M.; Salazar, G.; Van den Berg, C.; Schuiteman, A. An update classification of Orchidaceae. Bot. J. Linn. Soc. 2015, 177, 151–174. [Google Scholar] [CrossRef]
  42. 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] [PubMed]
  43. Mitchell, N.; Lewis, P.O.; Lemmon, E.M.; Lemmon, A.R.; Holsinger, K.E. Anchored phylogenomics improves the resolution of evolutionary relationships in the rapid radiation of Protea L. Am. J. Bot. 2017, 104, 102–115. [Google Scholar] [CrossRef] [PubMed]
  44. Jin, J.-J.; Yu, W.-B.; Yang, J.-B.; Song, Y.; DePamphilis, C.W.; Yi, T.S.; 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]
  45. Bankevich, A.; Nurk, S.; Antipov, D.; Gurevich, A.A.; Dvorkin, M.; Kulikov, A.S.; Lesin, V.M.; Nikolenko, S.I.; Pham, S.; Prjibelski, A.D.; et al. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 2012, 19, 455–477. [Google Scholar] [CrossRef] [PubMed]
  46. 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]
  47. Qu, X.J.; Moore, M.J.; Li, D.Z.; Yi, T.S. PGA: A software package for rapid, accurate, and flexible batch annotation of plastomes. Plant Methods. 2019, 15, 50. [Google Scholar] [CrossRef]
  48. 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]
  49. Greiner, S.; Lehwark, P.; Bock, R. OrganellarGenomeDRAW (OGDRAW) version 1.3.1: Expanded toolkit for the graphical visualization of organellar genomes. Nucleic Acids Res. 2019, 47, W59–W64. [Google Scholar] [CrossRef]
  50. Rissman, A.I.; Mau, B.; Biehl, B.S.; Darling, A.E.; Glasner, J.D.; Perna, N.T. Reordering contigs of draft genomes using the Mauve aligner. Bioinformatics 2009, 25, 2071–2073. [Google Scholar] [CrossRef]
  51. Amiryousefi, A.; Hyvonen, J.; Poczai, P. Irscope: An online program to visualize the junction sites of chloroplast genomes. Bioinformatics 2018, 34, 3030–3031. [Google Scholar] [CrossRef]
  52. Beier, S.; Thiel, T.; Münch, T.; Scholz, U.; Mascher, M. MISA-web: A web server for microsatellite prediction. Bioinformatics 2017, 33, 2583–2585. [Google Scholar] [CrossRef] [PubMed]
  53. Kurtz, S.; Choudhuri, J.V.; Ohlebusch, E.; Schleiermacher, C.; Stoye, J.; Giegerich, R. REPuter: The manifold applications of repeat analysis on a genomic scale. Nucleic Acids Res. 2001, 29, 4633–4642. [Google Scholar] [CrossRef] [PubMed]
  54. Villanueva, R.A.M.; Chen, Z.J. ggplot2: Elegant Graphics for Data Analysis (2nd Ed.). Meas. Interdiscip. Res. Perspect. 2019, 17, 160–167. [Google Scholar] [CrossRef]
  55. Zhang, D.; Gao, F.; Jakovlić, I.; Zou, H.; Zhang, J.; Li, W.X.; Wang, G.T. PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Mol. Ecol. Resour. 2020, 20, 348–355. [Google Scholar] [CrossRef] [PubMed]
  56. Xia, X. Dambe7: New and improved tools for data analysis in molecular biology and evolution. Mol. Biol. Evol. 2018, 35, 1550–1552. [Google Scholar] [CrossRef] [PubMed]
  57. Chen, C.J.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.H.; Xia, R. Tbtools: An integrative toolkit developed for interactive analyses of big biological data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef] [PubMed]
  58. Li, J.; Zhang, Z.; Vang, S.; Yu, J.; Wong, G.K.; Wang, J. Correlation between Ka/Ks and Ks is related to substitution model and evolutionary lineage. J. Mol. Evol. 2009, 68, 414–423. [Google Scholar] [CrossRef] [PubMed]
  59. Brudno, M.; Malde, S.; Poliakov, A.; Do, C.B.; Couronne, O.; Dubchak, I.; Batzoglou, S. Glocal alignment: Finding rearrangements during alignment. Bioinformatics 2003, 19, i54–i62. [Google Scholar] [CrossRef]
  60. Rozas, J.; Sánchez-DelBarrio, J.C.; Messeguer, X.; Rozas, R. Dnasp, dna polymorphism analyses by the coalescent and other methods. Bioinformatics 2003, 19, 2496–2497. [Google Scholar] [CrossRef]
  61. 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]
  62. Capella-Gutierrez, S.; Silla-Martinez, J.M.; Gabaldon, T. Trimal: A tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 2009, 25, 1972–1973. [Google Scholar] [CrossRef] [PubMed]
  63. Miller, M.A.; Pfeiffer, W.; Schwartz, T. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In Proceedings of the 2010 Gateway Computing Environments Workshop (GCE), New Orleans, LA, USA, 14 November 2010; pp. 1–8. [Google Scholar]
  64. Stamatakis, A.; Hoover, P.; Rougemont, J. A rapid bootstrap algorithm for the RAxML Web servers. Syst. Biol. 2008, 57, 758–771. [Google Scholar] [CrossRef] [PubMed]
  65. Felsenstein, J. Confidence limits on phylogenies: An approach using the bootstrap. Evol. Int. J. Org. Evol. 1985, 39, 783–791. [Google Scholar] [CrossRef]
Ijms 25 00184 g001
Figure 1. The annotation map of Angraecum plastome. The darker gray in the inner circle corresponds to GC content. The IRA and IRB (two inverted repeating regions), LSC (large single-copy region), and SSC (small single-copy region) are indicated outside the GC content.
Figure 1. The annotation map of Angraecum plastome. The darker gray in the inner circle corresponds to GC content. The IRA and IRB (two inverted repeating regions), LSC (large single-copy region), and SSC (small single-copy region) are indicated outside the GC content.
Ijms 25 00184 g001
Ijms 25 00184 g002
Figure 2. Plastome comparison of four species of Angraecum using a progressive MAUVE algorithm. The locally collinear blocks are represented by blocks of the same color connected by lines. Genome regions are color-coded as CDS, tRNA, rRNA, and non-coding region.
Figure 2. Plastome comparison of four species of Angraecum using a progressive MAUVE algorithm. The locally collinear blocks are represented by blocks of the same color connected by lines. Genome regions are color-coded as CDS, tRNA, rRNA, and non-coding region.
Ijms 25 00184 g002
Ijms 25 00184 g003
Figure 3. Comparison of junctions between the LSC (large single-copy region), SSC (small single-copy region), and IR (inverted repeat regions) regions among four Angraecum plastomes.
Figure 3. Comparison of junctions between the LSC (large single-copy region), SSC (small single-copy region), and IR (inverted repeat regions) regions among four Angraecum plastomes.
Ijms 25 00184 g003
Ijms 25 00184 g004
Figure 4. Summary of sequence repeats across the Angraecum plastomes. (A) Frequency of identified SSR motifs (mono-, di-, tri-, tetra-, penta- and hexa-); (B) Frequency of classified repeat types (considering sequence complementary); (C) Variation in repeat abundance and type (P, palindromic; F, forward; R, reverse; C, complement); (D) The number of long repeats sequences by length.
Figure 4. Summary of sequence repeats across the Angraecum plastomes. (A) Frequency of identified SSR motifs (mono-, di-, tri-, tetra-, penta- and hexa-); (B) Frequency of classified repeat types (considering sequence complementary); (C) Variation in repeat abundance and type (P, palindromic; F, forward; R, reverse; C, complement); (D) The number of long repeats sequences by length.
Ijms 25 00184 g004
Ijms 25 00184 g005
Figure 5. The RSCU (relative synonymous codon usage) values of concatenated 68 CDSs for four plastomes. Color key: the red values mean higher RSCU values and the blue values mean lower RSCU values.
Figure 5. The RSCU (relative synonymous codon usage) values of concatenated 68 CDSs for four plastomes. Color key: the red values mean higher RSCU values and the blue values mean lower RSCU values.
Ijms 25 00184 g005
Ijms 25 00184 g006
Figure 6. Selective pressure of shared protein-coding genes in four Angraecum species. (A–C) Ka, Ks, and Ka/Ks values of four Angraecum plastomes. (D–F) Ka, Ks, and Ka/Ks of different genes or gene groups in four Angraecum plastomes.
Figure 6. Selective pressure of shared protein-coding genes in four Angraecum species. (A–C) Ka, Ks, and Ka/Ks values of four Angraecum plastomes. (D–F) Ka, Ks, and Ka/Ks of different genes or gene groups in four Angraecum plastomes.
Ijms 25 00184 g006
Ijms 25 00184 g007
Figure 7. Global alignment of four Angraecum plastomes by mVISTA with Thrixspermum centipeda as a reference. The y-axis shows the coordinates between the plastomes.
Figure 7. Global alignment of four Angraecum plastomes by mVISTA with Thrixspermum centipeda as a reference. The y-axis shows the coordinates between the plastomes.
Ijms 25 00184 g007
Ijms 25 00184 g008
Figure 8. Sliding window test of nucleotide diversity for Angraecum plastomes. (A) The nucleotide diversity of the whole plastome; five mutation hotspot regions were annotated. (B) The nucleotide diversity of 68 CDSs. The window size was set to 100 bp and the sliding windows size was 25 bp. x-axis, position of the midpoint of a window; y-axis: nucleotide diversity of each window or genes.
Figure 8. Sliding window test of nucleotide diversity for Angraecum plastomes. (A) The nucleotide diversity of the whole plastome; five mutation hotspot regions were annotated. (B) The nucleotide diversity of 68 CDSs. The window size was set to 100 bp and the sliding windows size was 25 bp. x-axis, position of the midpoint of a window; y-axis: nucleotide diversity of each window or genes.
Ijms 25 00184 g008
Ijms 25 00184 g009
Figure 9. Phylogenetic tree obtained by maximum-likelihood analysis based on the complete plastome. The numbers near the nodes are bootstrap percentages and Bayesian posterior probabilities (BPML, BPMP, PP). * node is the 100 bootstrap percentage or 1.00 posterior probability.
Figure 9. Phylogenetic tree obtained by maximum-likelihood analysis based on the complete plastome. The numbers near the nodes are bootstrap percentages and Bayesian posterior probabilities (BPML, BPMP, PP). * node is the 100 bootstrap percentage or 1.00 posterior probability.
Ijms 25 00184 g009
Table 1. Characteristics of the complete plastomes of Angraecum.
Table 1. Characteristics of the complete plastomes of Angraecum.
TaxaSize (bp)GC Content (%)LSC Size in bp (%)IR Size in bp (%)SSC Size in bp (%)Total Number of GeneCDStRNA GenerRNA GeneNumber of ndh Fragment
A. borbonicum150,74336.788,206 (58.5)25,387 (33.7)11,763 (7.8)120743887
A. lecomtei151,27736.887,889 (58.1)25,733 (34.0)11,922 (7.9)120743887
A. sesquipedale151,81836.888,904 (58.6)25,982 (34.2)11,760 (7.7)120743887
A. sororium151,34136.987,992 (58.1)25,875 (34.2)11,599 (7.7)120743887
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

Zhou, C.-Y.; Lin, W.-J.; Li, R.; Wu, Y.; Liu, Z.-J.; Li, M.-H. Characterization of Angraecum (Angraecinae, Orchidaceae) Plastomes and Utility of Sequence Variability Hotspots. Int. J. Mol. Sci. 2024, 25, 184. https://doi.org/10.3390/ijms25010184

AMA Style

Zhou C-Y, Lin W-J, Li R, Wu Y, Liu Z-J, Li M-H. Characterization of Angraecum (Angraecinae, Orchidaceae) Plastomes and Utility of Sequence Variability Hotspots. International Journal of Molecular Sciences. 2024; 25(1):184. https://doi.org/10.3390/ijms25010184

Chicago/Turabian Style

Zhou, Cheng-Yuan, Wen-Jun Lin, Ruyi Li, Yuhan Wu, Zhong-Jian Liu, and Ming-He Li. 2024. "Characterization of Angraecum (Angraecinae, Orchidaceae) Plastomes and Utility of Sequence Variability Hotspots" International Journal of Molecular Sciences 25, no. 1: 184. https://doi.org/10.3390/ijms25010184

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