Next Article in Journal
Harnessing the Genetic Basis of Sorghum Biomass-Related Traits to Facilitate Bioenergy Applications
Next Article in Special Issue
Comparative Analysis of Plastomes in Elsholtzieae: Phylogenetic Relationships and Potential Molecular Markers
Previous Article in Journal
Immune Activation and Inflammatory Response Mediated by the NOD/Toll-like Receptor Signaling Pathway—The Potential Mechanism of Bullfrog (Lithobates catesbeiana) Meningitis Caused by Elizabethkingia miricola
Previous Article in Special Issue
Characterization and Comparative Analysis of the Complete Plastomes of Five Epidendrum (Epidendreae, Orchidaceae) Species
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 24
Issue 19
10.3390/ijms241914544
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

Characteristics and Comparative Analysis of Seven Complete Plastomes of Trichoglottis s.l. (Aeridinae, Orchidaceae)

by
Cheng-Yuan Zhou
1,†,
Meng-Yao Zeng
1,†,
Xuyong Gao
1,
Zhuang Zhao
1,
Ruyi Li
1,
Yuhan Wu
1,
Zhong-Jian Liu
1,2,
Diyang Zhang
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. 2023, 24(19), 14544; https://doi.org/10.3390/ijms241914544
Submission received: 17 August 2023 / Revised: 20 September 2023 / Accepted: 22 September 2023 / Published: 26 September 2023
(This article belongs to the Special Issue New Trends in Genomics and Metabolomics of Horticultural Plants at Fujian Agriculture and Forestry University)
Browse Figures
Review Reports Versions Notes

Abstract

:
Trichoglottis exhibits a range of rich variations in colors and shapes of flower and is a valuable ornamental orchid genus. The genus Trichoglottis has been expanded by the inclusion of Staurochilus, but this Trichoglottis sensu lato (s.l.) was recovered as a non-monophyletic genus based on molecular sequences from one or a few DNA regions. Here, we present phylogenomic data sets, incorporating complete plastome sequences from seven species (including five species sequenced in this study) of Trichoglottis s.l. (including two species formerly treated as Staurochilus), to compare plastome structure and to reconstruct the phylogenetic relationships of this genus. The seven plastomes possessed the typical quadripartite structure of angiosperms and ranged from 149,402 bp to 149,841 bp with a GC content of 36.6–36.7%. These plastomes contain 120 genes, which comprise 74 protein-coding genes, 38 tRNA genes, and 8 rRNA genes, all ndh genes were pseudogenized or lost. A total of 98 (T. philippinensis) to 134 (T. ionosma) SSRs and 33 (T. subviolacea) to 46 (T. ionosma) long repeats were detected. The consistent and robust phylogenetic relationships of Trichoglottis were established using a total of 25 plastid genomes from the Aeridinae subtribe. The genus Trichoglottis s.l. was strongly supported as a monophyletic group, and two species formerly treated as Staurochilus were revealed as successively basal lineages. In addition, five mutational hotspots (trnNGUU-rpl32, trnLUAA, trnSGCU-trnGUCC, rbcL-accD, and trnTGGU-psbD) were identified based on the ranking of PI values. Our research indicates that plastome data is a valuable source for molecular identification and evolutionary studies of Trichoglottis and its related genera.

1. Introduction

Trichoglottis Blume is a genus of Aeridinae within the family Orchidaceae, which was established by Carl Blume in 1825 with the type species Trichoglottis retusa Blume [1], comprising approximately 85 species [2]. Members of Trichoglottis are mainly distributed in tropical Asia and the northwest Pacific [3]. Many species are narrow endemics, and there is a major center of diversity in Indonesia and the Philippines [3,4]. Species of Trichoglottis are mainly characterized by monopodial growth, an axillary inflorescence with one or several brightly colored flowers, and a hairy strap-shaped lip firmly fused to a column [4,5]. Trichoglottis exhibits a range of rich variations in colors and shapes of flower and is a valuable ornamental orchid. To date, approximately 75 artificial interspecific hybrids have been produced and registered with the Royal Horticultural Society (http://apps.rhs.org.uk/horticulturaldatabase/orchidregister/orchidregister.asp, accessed on 10 July 2023).
Since the establishment of Trichoglottis, the intergeneric phylogenetic relationships of this genus have remained unclear and are generally considered to be one of the most complicated groups in Aeridinae. Molecular phylogenetics, based on two markers (matK and ITS), indicated that Trichoglottis is sister to Staurochilus, and both together are sisters to Ceratochilus and Ventricularia [6]. Trichoglottis has been expanded by the inclusion of Ceratochilus, Staurochilus, and Ventricularia [3]. However, the subject of Staurochilus versus Trichoglottis is still debated. Based on three combined markers (ITS, matK, trnL-F), Staurochilus was revealed as a sister to Acampe [7]. Additionally, based on five combined markers (ITS, atpI-H, matK, psbA-trnH, and trnL-F), the results showed that Trichoglottis was polyphyletic, Staurochilus (including S. luchuensis and S. ionosma) was paraphyletic, and T. latisepala was embedded in Staurochilus [8]. Nevertheless, many of the relationships among these clades/genera remain uncertain due to weak support. Briefly, traditional plant classification methods based on morphological features are not always adequate for the delimitation of Trichoglottis. Furthermore, the use of DNA barcoding for analysis has limitations in terms of resolution, making it difficult to differentiate between Trichoglottis and Staurochilus.
One traditional approach for increasing the resolution in molecular phylogenetic data sets is to sample more loci [9,10]. Plastids are usually uniparentally inherited and have their own genomes with dense gene content and a moderate mutation rate [11]. The complete plastid genome (plastome) data have proved to be effective tools for orchid species delimitation and molecular marker development [12,13,14,15]. Here, we report new complete plastomes for five Trichoglottis species. These new sequences are compared with the two previously described Trichoglottis plastomes [15], which (we refer to Trichoglottis s.l.) include two species (T. ionosma and T. philippinensis) formerly placed in Staurochilus. Simple sequence repeats (SSRs) were used to investigate the genetic diversity of these plastomes. Codon usage analysis was conducted to find the codon bias. The sequence divergence was calculated for barcoding investigation. Further, a total of 25 previously sequenced plastomes representing all previously recognized major lineages of Aeridinae were integrated for phylogenetic analysis with the aim of understanding the generic delimitation of Trichoglottis s.l. The present results provide a useful genetic resource for molecular identification and evolutionary studies of Trichoglottis and its related genera.

2. Results

2.1. Characteristics of the Plastome

The plastomes of Trichoglottis displayed a typical quadripartite structure (Figure 1), consisting of a pair of IRs (25,775–25,812 bp), a LSC region (85,681–86,210 bp), and a SSC region (12,004–12,171 bp) (Table 1). The size of the plastomes varied from 149,402 bp (T. latisepala) to 149,841 bp (T. ionosma), which fell within the typical range observed for common angiosperm plastome sizes. The GC content of whole plastomes exhibited minimal variation. The T. ionosma had a content of 36.6%, and the remaining six species exhibited a content of 36.7%.
The plastomes of Trichoglottis encoded 120 genes, comprising 74 protein-coding genes, 38 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes (Table 1). All the plastomes had undergone loss or pseudogenization of the ndh genes (Figure 1, Table 1). The plastome of T. ionosma possessed seven pseudogenes (ndhB/C/D/E/G/J/K), the other six species possessed eight pseudogenes (ndhB/C/D/E/G/I/J/K). The result of Mauve analysis revealed no significant rearrangements among these plastomes (Supplementary Figure S1).
Visualization of boundary genes in the plastomes revealed a highly conserved distribution pattern among Trichoglottis (Supplementary Figure S2). The rpl22 gene in all species spanned from LSC to IRb, with a consistent length of 31 bp. At the junction between SSC and IRb (JSB), the ycf1 gene of T. latisepala and T. philippinensis was entirely located within IRb, while, for the other five species, the ycf1 gene spanned from IRb to SSC, with a range of 11 to 41 bp. In all species, the ycf1 gene spanned the junction between the SSC and IRa (JSA), and the trnH and psbA genes were found adjacent to the junction between the IRa and LSC (JLA).

2.2. Repeated Analysis

The different types of SSR (mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide) were analyzed in the seven plastomes of Trichoglottis. A total of 98 (T. philippinensis) to 134 (T. ionosma) SSRs were detected (Figure 2, Supplementary Table S2). Mononucleotide repeats were the most frequent type of SSRs (50–90), followed by dinucleotide repeats (14–20). Hexanucleotide occurred with the lowest frequency, and it was exclusively detected in T. subviolacea (2). Additionally, it was observed that A/T (48–90) dominated over C/G (0–2) in mononucleotide repeats. In dinucleotide repeats, the number of AT/AT (10–16) exceeded that of AG/CT (4). Furthermore, trinucleotide repeats exclusively contained A/T.
Four types of long repeats (palindrome, forward, reverse, and completement) were also detected in Trichoglottis plastomes. The number of long repeats ranged from 33 (T. subviolacea) to 46 (T. ionosma) (Figure 2, Supplementary Table S3). Except for T. subviolacea, which only possessed three types of long repeats (palindrome, forward, and reverse), the other six species possessed all four types of long repeats. The length of long repeats in all species mostly ranged from 30 bp to 40 bp. For long repeats above 50 bp, only T. ionosma had one, while the other species possessed two. Complement repeats were only detected below 40 bp in length.

2.3. Codon Usage Analyses

The concatenated sequences of 68 protein-coding genes were used to calculate the codon usage frequency of Trichoglottis plastomes. These protein-coding genes encoded 19,285–19,351 codons. The heatmap showed that the codon usage bias was highly conserved (Supplementary Figure S3, Supplementary Table S4). The range of relative synonymous codon usage (RSCU) values was observed to be 0.346–1.894, with 30 codons having RSCU values greater than 1. Among these codons, leucine (Leu) had the highest number of amino acids, and cysteine (Cys) had the lowest frequency (Supplementary Table S4). The GCU codon had the highest RSCU value (1.881–1.894), while GAC exhibited the lowest RSCU value (0.348–0.353). Among the three types of termination codons (UAA, UAG, UGA), the UAA codon had the highest RSCU values, ranging from 1.412 to 1.456.

2.4. Plastome Sequence Divergence and Barcoding Investigation

To assess the variations of Trichoglottis plastomes, the mVISTA was employed to investigate the structural characteristics of seven plastomes with the reference Thrixspermum centipeda. Supplementary Figure S4 shows that the coding regions were highly conserved in comparison to the non-coding regions. The IR regions were more conserved than the LSC and SSC regions.
In order to explore the highly mutated hotspots of Trichoglottis plastomes, the DnaSP6 was employed to calculate nucleotide diversity (Pi). The results showed Pi values ranging from 0 to 0.12857 (trnNGUU-rpl32) (Figure 3A, Supplementary Table S5). The IR regions were relatively conserved (Pi = 0.0031) and high levels of nucleotide diversity were detected in the SSC region (Pi = 0.01058), followed by the LSC region (Pi = 0.0052). According to the ranking of Pi values, five hypervariable regions, including trnNGUU-rpl32, trnLUAA, trnSGCU-trnGUCC, rbcL-accD, and trnTGGU-psbD, were identified. Protein-coding genes were also employed for the analysis of nucleotide diversity and showed high conservation (Figure 3B, Supplementary Table S5). Among these genes, psbK had the highest Pi, but with only 0.0109.

2.5. Phylogenetic Analysis

The phylogenetic trees constructed based on complete plastomes and 68 protein-coding genes revealed similar topological structures, and the main clades were recovered with high-level support (Figure 4, Supplementary Figure S5). The species of Trichoglottis formed a well-supported monophyletic group (BS = 100, PP = 1.00) and was revealed as a sister to Acampe. The phylogenetic relationship inferred by complete plastomes showed strong support within Trichoglottis (BS ≥ 98, PP = 1.00), but relatively moderate support was shown in trees inferred by 68 protein-coding genes (BSML ≥ 72, BSMp ≥ 43, PP ≥ 0.71). The phylogenetic trees showed that Trichoglottis could be divided into four diverging lineages. T. ionosma (formerly treated as Staurochilus ionosmus) was supported as the first lineage. T. philippinensis (formerly treated as S. philippinensis) was supported as the second lineage. T. subviolacea was supported as the third lineage. Four species, T. lanceolaria, together with T. latisepala, T. cirrhifera, and T. orchidea, were supported as the fourth lineage. Extremely short branch lengths were observed within the fourth lineage.

3. Discussion

3.1. The Plastome Characteristics and Structural Evolution

In this study, we expanded plastome sampling in Trichoglottis and provided a valuable opportunity to further understand plastome evolution in this complex taxon. All plastomes of Trichoglottis (Figure 1) displayed the typical quadripartite structure, consisting of one LSC region, one SSC region, and two IR regions, consistent with that of most angiosperm plastomes. The length of Trichoglottis plastomes (Table 1) ranged from 149,402 bp (T. latisepala) to 149,841 bp (T. ionosma), falling entirely within the reported range of plastomes in Orchidaceae, which ranged from 19,047 bp (Epipogium roseum) to 212,688 bp (Cypripedium tibeticum) [16,17]. The GC content (36.6–36.7%) also fell within the range reported in previous studies, ranging from 23.1% (Gastrodia flexistyla) to 37.8% (Cypripedium macranthos) [16,18].
In terms of gene content, Trichoglottis encoded a total of 120 genes, including 74 protein-coding genes, 38 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes, with the pseudogenization or loss of all ndh genes (Figure 1, Table 1). The pseudogenization and loss of ndh genes were commonly observed in Orchidaceae [19,20,21]. It has been hypothesized that this might be associated with epiphytic habitats [22]. Epiphytic orchids usually undergo pseudogenization or loss of the ndh genes, such as Dendrobium [23], Polystachya [24], and Aeridinae [13,15]. For Trichoglottis, the results showed that four genes (ndhA/F/H/I) were completely lost and the other seven genes (ndhB/C/D/E/G/J/K) were pseudogenes; in T. ionosma, three genes (ndhA/F/H) were completely lost and the other eight genes (ndhB/C/D/E/G/I/J/K) were pseudogenes in the other six species. Species of Trichoglottis are usually epiphytic on tree trunks or lithophytic on rocks. Therefore, the results provide support for a close association between pseudogenization or loss of ndh genes and epiphytic habitats.
IR/SC boundary shift is a common phenomenon in the evolutionary process of angiosperms and serves as the main factor for the difference in plastome length and gene content [25,26]. In this study, the gene arrangement of the IR/SC boundary in seven Trichoglottis plastomes was extremely conserved; except for a slight difference at JSB, the ycf1 in T. latisepala and T. philippinensis was entirely located within the IRb region, while in the remaining species, the ycf1 crossed over JSB. These results indicated that no significant expansion or contraction occurred in the IR regions of Trichoglottis, which may be one of the primary factors contributing to the high conservation of plastome structure.
Simple sequence repeats (SSRs), widely distributed in plastomes, are effective molecular markers used for detecting genome rearrangement, species identification, and analyses of genetic differences among individuals [27,28]. A total of 98 (T. philippinensis) to 134 (T. ionosma) SSRs were detected, which is more than previous results [24,29,30,31]. This might be associated regions of increased nucleotide diversity [32]. Most SSRs were composed of A/T repeats rather than G/C repeats; this may be due to A/T having a more stable framework compared to G/C [33]. Additionally, hexanucleotide (AAGAAT/ATTCTT) was exclusively detected in the plastomes of T. subviolacea, making it a specific molecular marker for identifying T. subviolacea. The number of long repeats ranged from 33 (T. subviolacea) to 46 (T. ionosma), which is similar to previous studies [24,29,30]. The results contributed valuable molecular resources for the development of DNA markers in Trichoglottis.
Relative synonymous codon usage (RSCU) is an important measure to calculate the preference for synonymous codon usage. When the RSCU value is greater than 1, it indicates a preferred selection of that codon, whereas when it is less than 1, there is no preference [34]. The codon usage bias is highly conserved among plastomes of Trichoglottis. Leucine (Leu) had the highest amino acid frequency, while cysteine (Cys) had the lowest frequency. Knill et al. [35] proposed that the high leucine frequency may be attributed to the substantial demand for leucine in photosynthesis-related metabolism. When cysteine is accumulated above a specific threshold tolerated by the host, it can be considered toxic, resulting in the lowest abundance among amino acids [36]. Our results were consistent with previous results regarding codon preference in Orchidaceae [24,29,30,31].

3.2. The Barcoding Investigation and Phylogenetic Analysis

The phylogenetic relationship of Trichoglottis s.l. has remained unresolved for a long time. Based on nuclear ribosomal ITS and a number of plastid sequences, a conflicting relationship between Staurochilus and Trichoglottis s.s. was revealed, but weak support and unstable topology were found [6,7,8]. The genus Trichoglottis s.l. was strongly supported as a monophyletic group, and two species, T. ionosma and T. philippinensis, formerly treated as Staurochilus, were nested as successively basal lineages (Figure 4, Supplementary Figure S5). The relationship was not consistent with previous studies based on traditional molecular markers [6,7,8], which could be due to the expanded sampling and markers with more parsimony-informative sites. However, our study also revealed extremely short branch lengths within Trichoglottis s.s. (Figure 4, Supplementary Figure S5), indicating that our expanded taxonomic sample did not permit us to clarify the intrageneric relationships of Trichoglottis s.l. This phenomenon could be attributed to Trichoglottis s.l. having undergone rapid radiation, resulting in few opportunities for molecular changes due to the short time of speciation [37]. Therefore, future studies based on broader sampling and markers with different genetic backgrounds is needed.
Nucleotide diversity (Pi) was used to measure the degree of polymorphism. Similar to other Orchidaceae taxa, the non-coding regions of Trichoglottis plastomes had higher Pi values than the coding regions [24,29,31]. A total of five regions (trnNGUU-rpl32 > trnLUAA > trnSGCU-trnGUCC > rbcL-accD > trnTGGU-psbD) were identified as mutation hotspots and could be used in the phylogenetic and identification of Trichoglottis and its related genera.

4. Materials and Methods

4.1. Taxon Sampling and Sequencing

Seven species of Trichoglottis were selected, among which five plastomes were newly sequenced in this study. Plant materials were collected from Fujian Agriculture and Forestry University (Fuzhou, Fujian Province, China). Based on previous molecular systematic studies [15], a total of 31 plastid genomes from 31 species were selected in this study, including six species from five genera (Calanthe, Calypso, Cattleya, Masdevallia, and Tridactyle) as the outgroups. The taxa with voucher information and GenBank accession numbers are provided in Supplementary Table S1.
Total DNA was extracted from fresh leaves with the Plant Mini Kit (Qiagen, CA. USA) based on the manufacturer’s protocol, and DNA degradation and contamination were evaluated on 1% agarose gels. Next-generation sequencing (NGS) was conducted using the Illumina Hiseq 4000 sequencing platform (Illumina, CA, United States), and 150-bp paired-end reads were produced. Scripts were used to filter the Illumina data in the cluster with the default parameter. When the low-quality (Q ≤ 5) base number in sequencing reads surpassed 50% of the reads base number and the N content in reads exceeded 10% of the reads base number, paired reads were eliminated from the analysis. More than 10 Gb of clean data were obtained for each species.

4.2. Plastome Assembly and Annotation

The paired-end sequencing reads were filtered and assembled into complete plastid genomes by using the GetOrganelle pipe-line [38] with default parameters. The obtained complete plastid genomes were annotated using PGA software (https://github.com/quxiaojian/PGA, accessed on 10 July 2023) [39], and the published plastome of Trichoglottis philippinensis (MN124404) was used as a reference. The annotated plastomes were manually examined using Geneious 11.1.5 [40]. Genes containing one or more internal stop codons, when compared to homologous genes, were classified as pseudogenes or partial copies. Genes with a ≥ 50% loss of the complete protein-coding genes or a similarity ≤ 50% were considered the lost genes. Finally, five high-quality complete plastid genomes were obtained. The annotation circle maps were drawn using OGDRAW v 1.3.1 [41].

4.3. Plastome Comparative Analysis

The Perl script MISA [42] was employed to detect simple sequence repeats (SSRs), with 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. Four long repeat types in seven plastid genomes, F (forward), P (palindrome), R (reverse), and C (complement), were detected using REPuter [43] with default parameters. Results were visualized with the R package ggplot2 [44]. The rearrangements between different Trichoglottis plastomes were identified by Mauve [45]. The IRscope online program [46] was used to compare the genes in the boundary regions of LSC/IRb/SSC/IRa.

4.4. Codon Usage Analysis

A total of 68 protein-coding genes from Trichoglottis plastomes were extracted using PhyloSuite v1.2.2 [47]. These protein-coding genes were concatenated using Phylosuite v1.2.2 and analyzed for the relative synonymous codon usage (RSCU) for each species of Trichoglottis using DAMBE [48]. Finally, a heatmap was generated using Tbtools v 1.120 [49].

4.5. Sequence Divergence and Barcoding Investigation

The online program mVISTA was used to analyze the diversity of plastome sequences of Trichoglottis using the Shuffle-LAGAN [50] alignment program. The Thrixspermum centipeda (MW057769) plastome was used as a reference. The nucleotide variability (Pi) of complete plastomes and 68 protein-coding genes of seven Trichoglottis species was calculated using DnaSP 6 [51] with a window length of 100 sites and a step size of 25 sites.

4.6. Phylogenetic Reconstruction

In this study, phylogenetic trees were constructed using a concatenated matrix of 68 protein-coding genes and a matrix of complete plastomes. The 68 protein-coding genes (ndh genes were widespread, pseudogenized, or lost in Aeridinae species) were extracted using PhyloSuite v1.2.2 [47] and aligned using MAFFT [52]. The aligned 68 protein-coding genes were concatenated using PhyloSuite v1.2.2 [47]. The complete plastid genomes were aligned by MAFFT, and TrimAL v1.4 was employed [53] to trim the poorly aligned positions with the default parameter.
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) [54]. For ML analysis, the GTRGAMMA model was specified for all datasets [55] and bootstrap values were calculated from 1000 bootstrap replicates using heuristic searches [56]. 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 (PPs).

5. Conclusions

In this study, five plastomes of Trichoglottis (T. cirrhifera, T. ionosma, T. lanceolaria, T. orchidea, and T. subviolacea) were newly assembled. Our results showed that the structure and gene content of Trichoglottis plastomes are highly conserved. All ndh genes were lost or pseudogenized. The Trichoglottis s.l. (including Staurochilus) was strongly supported as a monophyletic group, and two species formerly treated as Staurochilus were revealed as successively basal lineages. Five non-coding regions (trnNGUU-rpl32 > trnLUAA > trnSGCU-trnGUCC > rbcL-accD > trnTGGU-psbD) were identified to further elucidate the phylogeny of Trichoglottis. These findings provide valuable insights into plastome evolutionary patterns and phylogenetic relationships for Trichoglottis and its related genera and even extend to the orchid family.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms241914544/s1.

Author Contributions

Z.-J.L., D.Z. and M.-H.L., conceptualization; C.-Y.Z. and M.-Y.Z.: methodology, software; C.-Y.Z., X.G., Z.-J.L. and M.-H.L., data curation, writing—original draft preparation, writing—reviewing and editing; C.-Y.Z., Z.Z., R.L. and Y.W., validation, resources; D.Z., supervision. 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 is provided within this manuscript.

Acknowledgments

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

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Blume, C.L. Bijdragen Tot de Flora van Nederlandsch Indië (Part 7); Ter Lands Drukkerij: Batavia, The Netherlands, 1825. [Google Scholar]
  2. Bandara, C.; Atthanagoda, A.G.; Bandara, N.L.; Ranasinghe, B.; Kumar, P. Trichoglottis longifolia (Orchidaceae: Epidendroideae: Vandeae: Aeridinae), a new species from Sri Lanka. Phytotaxa 2022, 567, 71–78. [Google Scholar] [CrossRef]
  3. 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]
  4. Cootes, J. Philippine Native Orchid Species; Katha Publishing: Quezon, Philippines, 2011. [Google Scholar]
  5. Wood, J.J.; Lamb, A.; Chan, C.L. A new species of Trichoglottis (Orchidaceae: Vandeae: Aeridinae) from Borneo. Sandaicania 1998, 11, 43–47. [Google Scholar]
  6. Topik, H.; Yukawa, T.; Ito, M. Molecular phylogenetics of subtribe Aeridinae (Orchidaceae): Insights from plastid matK and nuclear ribosomal ITS sequences. J. Plant Res. 2005, 118, 271–284. [Google Scholar] [CrossRef] [PubMed]
  7. 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]
  8. Zou, L.H.; Huang, J.X.; Zhang, G.Q.; Liu, Z.J.; Zhuang, X.Y. A molecular phylogeny of Aeridinae (Orchidaceae: Epidendroideae) inferred from multiple nuclear and chloroplast regions. Mol. Phylogenet. Evol. 2015, 85, 247–254. [Google Scholar] [CrossRef]
  9. 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]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. 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]
  16. 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]
  17. 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]
  18. Wen, Y.; Qin, Y.; Shao, B.; Li, J.; Ma, C.; Liu, Y.; Yang, B.; Jin, X. The extremely reduced, diverged and reconfigured plastomes of the largest mycoheterotrophic orchid lineage. BMC Plant Biol. 2022, 22, 448. [Google Scholar] [CrossRef] [PubMed]
  19. 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]
  20. Lin, C.S.; Chen, J.J.W.; Huang, Y.T.; Chan, M.T.; Daniell, H.; Chang, W.J.; Hsu, C.T.; Liao, D.C.; Wu, F.H.; Lin, S.Y.; et al. The location and translocation of ndh genes of chloroplast origin in the Orchidaceae family. Sci. Rep. 2015, 5, 9040. [Google Scholar] [CrossRef]
  21. 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]
  22. 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]
  23. 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]
  24. 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]
  25. 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]
  26. Xi, J.; Lv, S.; Zhang, W.; Zhang, J.; Wang, K.; Guo, H.; Hu, J.; Yang, Y.; Wang, J.; Xia, G.; et al. Comparative plastomes of Carya species provide new insights into the plastomes evolution and maternal phylogeny of the genus. Front. Plant Sci. 2022, 13, 990064. [Google Scholar] [CrossRef]
  27. He, S.L.; Wang, Y.S.; Volis, S.; Li, D.Z.; Yi, T.S. Genetic diversity and population structure: Implications for conservation of wild soybean (Glycine soja Sieb. et Zucc) based on nuclear and chloroplast microsatellite variation. Int. J. Mol. Sci. 2012, 13, 12608–12628. [Google Scholar] [CrossRef]
  28. Qi, W.; Lin, F.; Liu, Y.; Huang, B.; Cheng, J.; Zhang, W.; Zhao, H. High-throughput development of simple sequence repeat markers for genetic diversity research in Crambe abyssinica. BMC Plant Biol. 2016, 16, 139. [Google Scholar] [CrossRef]
  29. Xiao, T.; He, L.; Yue, L.; Zhang, Y.; Lee, S.Y. Comparative phylogenetic analysis of complete plastid genomes of Renanthera (Orchidaceae). Front. Genet. 2022, 13, 998575. [Google Scholar] [CrossRef]
  30. 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]
  31. 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]
  32. McDonald, M.J.; Wang, W.C.; Huang, H.D.; Leu, J.Y. Clusters of nucleotide substitutions and insertion/deletion mutations are associated with repeat sequences. PLoS Biol. 2011, 9, e1000622. [Google Scholar] [CrossRef]
  33. Gragg, H.; Harfe, B.D.; Jinks-Robertson, S. Base composition of mononucleotide runs affects DNA polymerase slippage and removal of frameshift intermediates by mismatch repair in Saccharomyces cerevisiae. Mol. Cell. Biol. 2002, 22, 8756–8762. [Google Scholar] [CrossRef]
  34. Yang, X.; Luo, X.; Cai, X. Analysis of codon usage pattern in Taenia saginata based on a transcriptome dataset. Parasites Vectors 2014, 7, 527. [Google Scholar] [CrossRef]
  35. Knill, T.; Reichelt, M.; Paetz, C.; Gershenzon, J.; Binder, S. Arabidopsis Thaliana Encodes a Bacterial-Type Heterodimeric Isopropylmalate Isomerase Involved in Both Leu Biosynthesis and the Met Chain Elongation Pathway of Glucosinolate Formation. Plant Mol. Biol. 2009, 71, 227–239. [Google Scholar] [CrossRef]
  36. Hildebrandt, T.M.; Nesi, A.N.; Araújo, W.L.; Braun, H.P. Amino Acid Catabolism in Plants. Mol. Plant 2015, 8, 1563–1579. [Google Scholar] [CrossRef]
  37. Leaché, A.D.; Banbury, B.L.; Linkem, C.W.; De Oca, A.N.-M. Phylogenomics of a rapid radiation: Is chromosomal evolution linked to increased diversification in north american spiny lizards (Genus Sceloporus)? BMC Evol. Biol. 2016, 16, 63. [Google Scholar] [CrossRef]
  38. 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]
  39. 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]
  40. 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]
  41. 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]
  42. 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]
  43. 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]
  44. 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]
  45. 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]
  46. 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]
  47. 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]
  48. 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]
  49. 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]
  50. 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]
  51. 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]
  52. 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]
  53. 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]
  54. 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]
  55. 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]
  56. Felsenstein, J. Confidence limits on phylogenies: An approach using the bootstrap. Evol. Int. J. Org. Evol. 1985, 39, 783–791. [Google Scholar] [CrossRef]
Ijms 24 14544 g001
Figure 1. The plastome annotation map of Trichoglottis cirrhifera, Trichoglottis ionosma, Trichoglottis lanceolaria, Trichoglottis orchidea, and Trichoglottis subviolacea. 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 of GC content.
Figure 1. The plastome annotation map of Trichoglottis cirrhifera, Trichoglottis ionosma, Trichoglottis lanceolaria, Trichoglottis orchidea, and Trichoglottis subviolacea. 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 of GC content.
Ijms 24 14544 g001
Ijms 24 14544 g002
Figure 2. Summary of simple sequence repeats (SSR) across the Trichoglottis 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. (D) Number of long repeats by sequence length.
Figure 2. Summary of simple sequence repeats (SSR) across the Trichoglottis 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. (D) Number of long repeats by sequence length.
Ijms 24 14544 g002
Ijms 24 14544 g003
Figure 3. Sliding window test of nucleotide diversity for Trichoglottis plastomes. (A) The nucleotide diversity of the complete plastome; five mutation hotspot regions were annotated. (B) The nucleotide diversity of 68 protein-coding sequences. The window size was set to 100 bp, and the sliding window size was 25 bp. X-axis: position of the midpoint of a window; Y-axis: nucleotide diversity of each window or gene.
Figure 3. Sliding window test of nucleotide diversity for Trichoglottis plastomes. (A) The nucleotide diversity of the complete plastome; five mutation hotspot regions were annotated. (B) The nucleotide diversity of 68 protein-coding sequences. The window size was set to 100 bp, and the sliding window size was 25 bp. X-axis: position of the midpoint of a window; Y-axis: nucleotide diversity of each window or gene.
Ijms 24 14544 g003
Ijms 24 14544 g004
Figure 4. Phylogenetic tree obtained by maximum-likelihood analysis based on complete plastome. The numbers near the nodes are bootstrap percentages and Bayesian posterior probabilities (BPML, BPMP, PP). A dash (-) indicates that a node is inconsistent between the topology of the MP/ML trees and the Bayesian tree; * node is 100 bootstrap percentage or 1.00 posterior probability.
Figure 4. Phylogenetic tree obtained by maximum-likelihood analysis based on complete plastome. The numbers near the nodes are bootstrap percentages and Bayesian posterior probabilities (BPML, BPMP, PP). A dash (-) indicates that a node is inconsistent between the topology of the MP/ML trees and the Bayesian tree; * node is 100 bootstrap percentage or 1.00 posterior probability.
Ijms 24 14544 g004
Table 1. Characteristics of the complete plastomes of Trichoglottis. An asterisk (*) indicates those newly sequenced in this study.
Table 1. Characteristics of the complete plastomes of Trichoglottis. An asterisk (*) indicates those newly sequenced in this study.
Species NameSize (bp)GC Content (%)LSC Size in bp (%)IR Size in bp (%)SSC Size in bp (%)Total Number of GeneProtein-Encoding GenetRNArRNANumber of ndh Fragment
T. cirrhifera *149,44236.785,721 (57.36)25,775 (17.25)12,171 (8.14)120743888
T. ionosma *149,84136.686,210 (57.53)25,812 (17.23)12,007 (8.01)120743887
T. lanceolaria *149,46736.785,728 (57.36)25,784 (17.25)12,171 (8.14)120743888
T. latisepala149,40236.785,681 (57.35)25,784 (17.26)12,153 (8.13)120743888
T. orchidea *149,44936.785,715 (57.35)25,782 (17.25)12,170 (8.14)120743888
T. philippinensis149,66336.786,069 (57.51)25,784 (17.23)12,026 (8.04)120743888
T. subviolacea *149,43936.785,853 (57.45)25,791 (17.26)12,004 (8.03)120743888
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.; Zeng, M.-Y.; Gao, X.; Zhao, Z.; Li, R.; Wu, Y.; Liu, Z.-J.; Zhang, D.; Li, M.-H. Characteristics and Comparative Analysis of Seven Complete Plastomes of Trichoglottis s.l. (Aeridinae, Orchidaceae). Int. J. Mol. Sci. 2023, 24, 14544. https://doi.org/10.3390/ijms241914544

AMA Style

Zhou C-Y, Zeng M-Y, Gao X, Zhao Z, Li R, Wu Y, Liu Z-J, Zhang D, Li M-H. Characteristics and Comparative Analysis of Seven Complete Plastomes of Trichoglottis s.l. (Aeridinae, Orchidaceae). International Journal of Molecular Sciences. 2023; 24(19):14544. https://doi.org/10.3390/ijms241914544

Chicago/Turabian Style

Zhou, Cheng-Yuan, Meng-Yao Zeng, Xuyong Gao, Zhuang Zhao, Ruyi Li, Yuhan Wu, Zhong-Jian Liu, Diyang Zhang, and Ming-He Li. 2023. "Characteristics and Comparative Analysis of Seven Complete Plastomes of Trichoglottis s.l. (Aeridinae, Orchidaceae)" International Journal of Molecular Sciences 24, no. 19: 14544. https://doi.org/10.3390/ijms241914544

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