Next Article in Journal
Influence of Different Evolutive Forces on GDF5 Gene Variability
Previous Article in Journal
Assessing the Expression of Long INterspersed Elements (LINEs) via Long-Read Sequencing in Diverse Human Tissues and Cell Lines
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Genes
Volume 14
Issue 10
10.3390/genes14101894
genes-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

Comprehensive Comparative Analyses of Aspidistra Chloroplast Genomes: Insights into Interspecific Plastid Diversity and Phylogeny

by
Jie Huang
1,2,
Zhaocen Lu
1,
Chunrui Lin
1,*,
Weibin Xu
2,* and
Yan Liu
1
1
Guangxi Key Laboratory of Plant Functional Phytochemicals and Sustainable Utilization, Guangxi Institute of Botany, Guangxi Zhuang Autonomous Region and Chinese Academy of Sciences, Guilin 541006, China
2
Guangxi Key Laboratory of Plant Conservation and Restoration Ecology in Karst Terrain, Guangxi Institute of Botany, Guangxi Zhuang Autonomous Region and Chinese Academy of Sciences, Guilin 541006, China
*
Authors to whom correspondence should be addressed.
Genes 2023, 14(10), 1894; https://doi.org/10.3390/genes14101894
Submission received: 26 August 2023 / Revised: 25 September 2023 / Accepted: 26 September 2023 / Published: 29 September 2023
(This article belongs to the Topic Plant Chloroplast Genome and Evolution)
Browse Figures
Review Reports Versions Notes

Abstract

:
Limestone karsts are renowned for extremely high species richness and endemism. Aspidistra (Asparagaceae) is among the highly diversified genera distributed in karst areas, making it an ideal group for studying the evolutionary mechanisms of karst plants. The taxonomy and identification of Aspidistra species are mainly based on their specialized and diverse floral structures. Aspidistra plants have inconspicuous flowers, and the similarity in vegetative morphology often leads to difficulties in species discrimination. Chloroplast genomes possess variable genetic information and offer the potential for interspecies identification. However, as yet there is little information about the interspecific diversity and evolution of the plastid genomes of Aspidistra. In this study, we reported chloroplast (cp) genomes of seven Aspidistra species (A. crassifila, A. dolichanthera, A. erecta, A. longgangensis, A. minutiflora, A. nankunshanensis, and A. retusa). These seven highly-conserved plastid genomes all have a typical quartile structure and include a total of 113 unique genes, comprising 79 protein-coding genes, 4 rRNA genes and 30 tRNA genes. Additionally, we conducted a comprehensive comparative analysis of Aspidistra cp genomes. We identified eight divergent hotspot regions (trnC-GCA-petN, trnE-UUC-psbD, accD-psaI, petA-psbJ, rpl20-rps12, rpl36-rps8, ccsA-ndhD and rps15-ycf1) that serve as potential molecular markers. Our newly generated Aspidistra plastomes enrich the resources of plastid genomes of karst plants, and an investigation into the plastome diversity offers novel perspectives on the taxonomy, phylogeny and evolution of Aspidistra species.

1. Introduction

Limestone karsts, with their unique physical and chemical substrates, shape a range of microhabitats and unique island habitats, such as karst caves, and terrestrial islands [1]. These special habitats have ecological conditions that significantly differ from the surroundings and thus provide relatively isolated environments for species isolation and differentiation [2,3]. Karst areas have harboured a high level of plant richness and endemism through a long period of evolution [2]. For example, the limestone ecosystem has reported plant groups with rich and marvellous flora, such as Gesneriaceae and Begonia, that exhibit prominent morphological variation [4,5,6]. Many karst plant species often have very narrow distribution ranges. However, damage to the fragile ecological environments threatens the karst plants partly because of growing human activities (tourism industry, etc.). Thus, accurate species identification is particularly important for the utilization and protection of these plants.
Aspidistra (Asparagaceae) is one of the represented plant groups of evergreen perennials in limestone karsts, with a center of diversity in southwest China and northern Vietnam [7]. Over the last forty years, the number of recognized Aspidistra species has experienced a remarkable increase [8], rapidly rising from 11 (until the year 1978) [9] to more than 200 [10,11,12,13]. However, the diversity of Aspidistra is still far from being revealed, with more new species being discovered during field investigations every year [8,11,12,14]. Despite its richness in species, Aspidistra plants are morphologically similar in vegetative characters (such as habit and leaf, etc.). Most Aspidistra species are characterized by creeping rhizomes, except several members which have an erect stem (e.g., A. erecta Yan Liu & C.I Peng and A. brachypetala C.R. Lin & B. Pan) [15]. The species of Aspidistra can be classified into two main groups based on their leaf growth patterns. One group has solitary and elliptical (oblong) or oval-shaped leaves with petioles (e.g., A. elatior Bl., A. austroyunnanensis G.W. Hu, Lei Cai & Q.F. Wang and A. chunxiusis C.R. Lin & Yan Liu) [16,17,18]. In contrast, the other group possesses linear leaves lacking a petiole or indistinctly differentiated from leaf blade (e.g., A. longifolia Hook.f. and A. hainanensis W.Y. Chun & F.C. How) [10,19]. In most cases, accurate identification of Aspidistra is almost impossible in non-flowering conditions [10]. Interspecific identification of the genus relies mainly on remarkably varied floral structures. However, most Aspidistra species have cryptic flowering and fruiting. The inconspicuous Aspidistra flowers, commonly found near the ground and concealed beneath leaf litter [20,21], can be easily overlooked or difficult to collect during field investigations, which might have hindered identifying species within the genus [22]. In addition, even supplemented with evidence of cytophylogenies (e.g., chromosome number, size, and morphology of chromosome), micromorphological features and palynological characters (pollen), these are far from sufficient for species identification of the genus [10,23].
DNA barcoding is considered a reliable and cost-efficient method for authenticating plants. Molecular markers in plants hitherto mainly come from chloroplast DNA (cpDNA). The cpDNA is a DNA fragment derived from the chloroplast (cp) genome, which encodes proteins involved in photosynthesis and generally has unique characteristics. The cp genome is usually maternal inherited and exhibits a high level of conservation in its genome structure and coding sequences. With a size range of 120–160 kb, the cp genome is composed of four distinct regions, namely a pair of inverted repeat sequences (IRs), a large single-copy (LSC) and a small single-copy (SSC) region. The plastid genome offers valuable information for examining evolution, DNA barcoding, taxonomy, and phylogeny [24]. However, some frequently used plastid regions (such as psbA-trnH, matK, psbK-psbI and rbcL, etc.) are insufficient to discriminate Aspidistra at the species level [25]. Therefore, few DNA molecular markers have been suitable for identifying Aspidistra species until now.
With the development of high-throughput sequencing technologies, plastid genomes (plastomes) and genome-wide nuclear sequence data of numerous plant species have been sequenced and applied in phylogenetic studies. As for the Asparagaceae, cp genomes of over 200 species have been reported (data from CGIR (Chloroplast Genome Information Resource)) [26,27,28,29]. Although four plastomes of Aspidistra species are available (accessed on 1 August 2023) in the National Center for Biotechnology Information (NCBI), there is still a lack of information regarding the plastid phylogenomics of the genus. To address this gap, we conducted a study in which we newly sequenced seven Aspidistra chloroplast genome sequences. Additionally, we conducted a comparative analysis of these cp genomes with the previously available four Aspidistra plastomes. We investigated the general plastome features, structural characteristics and sequence divergence of the Aspidistra plastomes. We identified highly variable regions within the cp genomes that could potentially serve as barcodes for species identification. Moreover, we utilized the complete plastome sequence of the genus to infer the phylogenetic relationships. Our comprehensive examination of chloroplast genomes provides a valuable theoretical foundation for future taxonomic and evolutionary studies of Aspidistra plants and helps fill the knowledge gap regarding the plastid phylogenomics of the genus.

2. Results

2.1. Characteristics of Chloroplast Genome Sequences

We sequenced and assembled seven chloroplast genomes of Aspidistra species (Table S1). All the Aspidistra complete cp sequences were typical circular DNA molecules, ranging from 156,335 to 156,475 bp (Figure 1 and Table 1). They were conserved in length and had a typical plastid genome structure, consisting of a pair of IR regions (26,498 to 26,540 bp), and one LSC (85,102 to 85,181 bp) and SSC region (18,199 to 18,232 bp). The average GC contents of the genomes ranged from 37.61% to 37.65%, with a significantly higher value in the IR regions (42.95–43%) than in the LSC (35.54–35.59%) and SSC (31.61–31.74%) regions. The chloroplast genomes are available in the NCBI database with accession numbers listed in Table 1.

2.2. Genome Annotation

The genomic structures of the seven cp genomes exhibited a high level of conservation, with the same gene numbers, composition, order and orientation. Table 1 presents the fundamental genomic characteristics of the Aspidistra species. The seven plastid genomes all contain 113 unique genes, including 79 protein-coding genes, 4 rRNA genes and 30 tRNA genes (Table S2).
Seven protein-coding genes (rpl2, rpl23, rps12, rps19, rps7, ndhB and ycf2), four rRNA (rrn16S, rrn23S, rrn5S and rrn4.5S) and eight tRNA genes (trnA-UGC, trnH-GUG, trnI-CAU, trnI-GAU, trnL-CAA, trnN-GUU, trnR-ACG and trnV-GAC) were located in the IR regions, resulting in two copies of these genes. The chloroplast genome contained a total of 17 genes that had introns. Out of the identified genes, there were 11 protein-coding genes (rps16, atpF, rpoC1, ycf3, clpP, petB, petD, rpl16, rpl2, ndhB, and ndhA) and six tRNA genes (trnK-UUU, trnG-UCC, trnL-UAA, trnV-UAC, trnI-GAU and trnA-UGC). The trans-spliced gene rps12 has two copies, with the 5′ end located in the LSC region and the 3′ ends in the IRa and IRb regions, respectively.

2.3. Repeat Sequences

The total numbers of simple sequence repeats (SSRs) we identified for the sampled Aspidistra species were as follows: 62 (A. minutiflora), 57 (A. retusa), 60 (A. dolichanthera), 64 (A. crassifila), 66 (A. erecta), 66 (A. longgangensis), and 67 (A. nankunshanensis) (Figure 2A and Table S3). Most SSRs in the seven Aspidistra plastid genomes were mononucleotide repeats, with numbers of repeats ranging from 29 (A. retusa) to 37 (A. longgangensis and A. nankunshanensis), accounting for 50.88–56.06% of the total SSRs (Figure 2A). Other types of SSRs included dinucleotide repeats (range in number of repeats and proportion of all SSRs of these Aspidistra plastomes: 12–14, 18.46–21.67%), trinucleotide repeats (5, 7.46–8.77%), tetranucleotide repeats (9–10, 13.64–15.79%), and pentanucleotide repeats (2–3, 2.99–4.84%) (Figure 2A and Table S3). No hexanucleotide repeats were found in any Aspidistra plastomes.
In addition to SSRs, we also detected four other interspersed repeats (forward, reverse, palindromic, and complement repeats) (Figure 2B). Among them (37–47 repeat sequences for seven chloroplast genomes), most were forward repeats (17–21; 44.19–47.37%) and palindromic repeats (19–24; 47.73–52.63%). There were two reverse repeats in A. erecta, while only one was found in the other six Aspidistra species. Complement repeats were found in four Aspidistra species (two repeats in A. longgangensis; one in A. retusa, A. crassifila and A. nankunshanensis).

2.4. Genomic Divergence

Genomic variation in the seven Aspidistra species was examined in mVISTA [30,31], with the cp genome of Aspidistra minutiflora as a reference sequence (Figure 3). The plastomes were highly conserved, but some different levels of sequence variation were still observed, particularly in the LSC and SSC regions (Figure 3 and Figure 4). Most high-variable sites belonged to the conserved noncoding sequences (CNS) regions.
We detected DNA polymorphisms using DnaSP v6.0 [32] based on the 11 aligned Aspidistra chloroplast genomes (including the other four Aspidistra plastomes available in NCBI) (Figure 4). The Aspidistra cp genomes were highly conserved. The nucleotide diversity (Pi) values of Aspidistra species (500 bp sliding widows) ranged from 0 to 0.013 (average value of 0.002394), with relatively high variation rates in the LSC and SSC regions and low rates in the two IR regions. There were eight positions with Pi values ≥ 0.007. Among them, six regions were located in the LSC region (trnC-GCA-petN (0.01033), trnE-UUC-psbD (0.01171), accD-psaI (0.00796), petA-psbJ (0.00898), rpl20-rps12 (including two adjacent sliding windows with Pi values of 0.00771 and 0.00764, respectively) and rpl36-rps8 (0.00727)), and the other two were in the SSC region (ccsA-ndhD (0.01207) and rps15-ycf1 (0.00749)).

2.5. Contraction and Expansion of IR Regions

Plastid genomes of the Aspidistra species were highly conserved, showing very limited variations of SC/IR boundaries (Figure 5). Several genes (such as genes rpl22, rps19, ndhF, ycf1, rpl2, trnH and psbA) were found spanning across or near the junctions of the IR and SC regions. rps19 and trnH both had two copies and completely accessed the two different IR (IRa and IRb) regions. The rps19 gene was located at the boundary between the LSC and IR regions, with a distance of 45 to 47 bp from the border. Similarly, the rpl22 was found within 8 bp to 10 bp of the JLB line. In all 11 Aspidistra plastomes, ndhF crossed the IRb/SSC boundary, with 29 bp in the IRb region and the remaining 2182 bp in the SSC region. The ycf1 gene was located in the boundary between the SSC and IRa regions, with 4486–4510 bp of its length in the SSC region and 899 bp in the IRa region. In all 11 chloroplast genomes, a ycf1 pseudogene is present in the IRa region, with a corresponding fragment ranging from 898 bp to 901 bp in the IRb region.

2.6. Phylogenetic Relationships

Phylogenetic analyses were performed in IQ-TREE version 2.2.0.3 using maximum likelihood (ML) methods. We sampled a total of 40 representative species from three subfamilies of Asparagaceae, namely, Nolinoideae (36 species), Asparagoideae (two species), and Lomandroideae (two species). The phylogenomic analysis (Figure 6) recovered the six tribes in subfamily Nolinoideae as major clades with strong bootstrap (BS): Convallarieae (19 species, BS = 100%), Ophiopogoneae (five species, BS = 100%), Nolineae (two species, BS = 100%), Polygonateae (seven species, BS = 100%), Draceaneae (two species, BS = 100%) and Ruscineae (with only one representative species). The monophyly of the genus Aspidistra was strongly supported (BS = 100%), with A. erecta as the earliest branching species. Four species were successively diverging, namely, A. nankunshanensis, A. retusa, A. elatior and A. crassifila. The remaining six species form a clade (BS = 81%) containing three subclades (all subclades BS = 100%), with two species of each subclade, namely, A. yingjiangensis and A. minutiflora, A. cavicola and A. dolichanthera, and A. obliquipeltata and A. longgangensis.

3. Discussion

3.1. Chloroplast Genome Evolution with Conserved Genome Structure

We reported and analyzed the seven cp genomes of the genus Aspidistra, which have lengths ranging from 156,335 to 156,475 bp and a typical tetrad structure. These Aspidistra plastomes showed similarities to those of other members in the Asparagaceae family. We observed no gene gain and loss in the seven plastomes, suggesting that cp genomes were conserved in Aspidistra plants. Previous research has highlighted unique evolutionary events in the chloroplast genomes of certain Asparagaceae species, such as the incorporation of mitochondrial DNA sequences into the plastid genome in the Convallaria genus [33,34]. Expansion and contraction of the IR regions are commonly observed in angiosperms, as documented in previous studies [35,36]. In some cases, certain species have even experienced the complete loss of an IR region [37,38]. Our comparative analysis revealed that the IR regions exhibited no significant variation among the seven Aspidistra cp genomes, with lengths ranging from 26,498 bp to 25,540 bp.

3.2. Repeat Sequence Analysis

In the study, we discovered a range of 57 (A. retusa) to 67 (A. nankunshanensis) SSRs in seven different species of Aspidistra. Most of these SSRs were mononucleotide repeats, making up 50.88% to 56.06% of all the SSRs, similar to that in other angiosperm chloroplast genomes [39,40]. Most mononucleotide repeats were A/T repeats, generally polyadenine (Poly-A) or polythymine (Poly-T) repeats, and thus significantly impacted the overall G/C content of chloroplast genomes [41]. The lengths of SSRs were shown to be polymorphic in different species and are thought to be potential markers in species identification. Huang et al. [42] developed 48 SSR loci based on the transcriptome data from the flowers of Aspidistra saxicola. They discovered that approximately a third of the loci were polymorphic in four distinct populations of A. elatior. Our research also identified a significant number of SSRs within the Aspidistra cp genomes (Table S3), which might also be valuable sources for developing markers to identify species within the genus.

3.3. Phylogenetic Relationship Based on the Plastid Genome

The complete chloroplast genome has been frequently employed in resolving the phylogenetic relationships of plants across various classification levels [3,29,43,44,45,46]. This study used 40 Asparagaceae species (including 36 species for the subfamily Nolinoideae; see Section 4.4) to construct the ML tree based on plastid genomes. Our results provide a well-resolved phylogenetic framework for the subfamily Nolinoideae (Figure 6). The first diverging clade (BS = 100%) consists of two tribes (Draceaneae and Ruscineae). The remaining Nolinoideae species are divided into two clades: one (BS = 100%) containing the tribe Convallarieae; and the other (BS = 100%) encompassing the tribes Ophiopogoneae, Nolineae, and Polygonateae. The phylogenetic relationships among these six tribes were consistent with the Nolinoideae phylogeny from plastid phylogenomics [29]. The monophyly of Aspidistra was well supported with 100% BS (Figure 6). In our results, the sister genus of Aspidistra was Tupistra, a finding similar to those of previous studies based on the plastid genome (containing four Aspidistra species) [29] and transcriptome data (with one Aspidistra species) [47]. Within the Aspidistra, the first branching clade was A. erecta, representing a group with distinguishable vegetative morphology (erect stem) [10,48]. After the divergence of A. nankunshanensis and A. retusa, the branching order of A. elatior, A. crassifila and a clade with the remaining six Aspidistra species was not well defined (BS = 48% and 43%); the six Aspidistra species group into three well-supported subclades (each containing two species), and the result can also be supported by evidence from morphology. The subclade of A. yingjiangensis and A. minutiflora represents a group of Aspidistra species with tufted (clustered) linear leaves [49,50]. A. cavicola and A. dolichanthera have a white, wide campanulate (broad bell-shaped) to rotate perianth [51,52]; while the perianth tube was urceolate in the subclade including A. obliquipeltata and A. longgangensis [53]. In conclusion, the chloroplast genome sequences offered valuable genetic information regarding the phylogenetic relationships of Aspidistra. However, the results are somewhat limited due to the plastid genome’s highly conserved nature and maternal inheritance property. Furthermore, more species needed to be sequenced to determine the relationships among Aspidistra.

3.4. Eight Highly Variable Cp Genome Regions Show Great Potential as DNA Barcodes

The chloroplast genomes of plants exhibit a high degree of conservation, making DNA fragments within these genomes effective tools in phylogenetic and evolutionary analyses. The complete chloroplast sequence has recently been utilized as a super-barcode to accurately identify and distinguish plant species [54,55].
As for the Aspidistra, one previous study [25] tested the efficiency of four universal DNA barcodes (matK, psbA-trnH, psbK-psbI and rbcL) in 19 species of the genus, and the individual successful identification rates of the barcode sequences ranged from 43.6% (psbA-trnH) to 88.7% (rbcL). Using a combination of markers had a higher identification efficiency than that of single sequences. The identification success rate using matK + (psbK-psbI) reached 100% in the 19 Aspidistra species, while the identification rates of these two sequences individually were 78% (matK) and 88.7% (psbK-psbI) [25]. Using this sequence combination (i.e., matK + (psbK-psbI)) also resulted in a 100% success identification rate in another study with 11 Aspidistra species [56].
With the growing number of identified Aspidistra species (currently exceeding 200), it became necessary to test the discriminatory power of these DNA markers in a broader range of species within the genus. In addition, new and more efficient DNA barcodes are necessary for the Aspidistra species. We evaluated the nucleotide diversity of the cp sequence using a sliding window analysis and detected eight DNA loci having Pi values greater than 0.007. The hypervariable regions were trnC-GCA-petN (Pi = 0.01033), trnE-UUC-psbD (Pi = 0.01171), accD-psaI (Pi = 0.00796), petA-psbJ (Pi = 0.00898), rpl20-rps12 (Pi = 0.00771 and 0.00764 for the two adjacent sliding windows, respectively), rpl36-rps8 (Pi = 0.00727), ccsA-ndhD (Pi = 0.01207) and rps15-ycf1 (Pi = 0.00749), indicating their potential to be developed as molecular markers. The previously used four barcodes (matK, psbA-trnH, psbK-psbI and rbcL) were not among the eight highly variable regions we detected, suggesting a possible higher identification efficiency of the potential markers. These findings are significant for identifying, protecting and rationally utilizing Aspidistra plants in karst areas.

4. Materials and Methods

4.1. Plant Sampling and Data Collection

We procured fresh leaves from seven Aspidistra species cultivated at the Guangxi Institute of Botany, Chinese Academy of Sciences, Guilin, China. Professor Chunrui Lin (Guangxi Institute of Botany) conducted the formal identifications of all samples. The selected Aspidistra species included members with different types of forms or morphologies and geological distribution regions. For example, we sampled species with erect stems (A. erecta) or creeping rhizomes (A. nankunshanensis, A. retusa, etc.), as well as species with clustered linear leaves (A. minutiflora) or solitary oblong leaves with petioles (A. obliquipeltata and A. longgangensis). The sampled taxa also included members with high ornamental value (having unique flowers), such as A. dolichanthera, A. longgangensis and A. crassifila. All sampled vouchers were deposited in the Herbarium, Guangxi Institute of Botany (IBK; Guilin, China), with detailed information and deposition numbers shown in Supplementary Infomation (Table S1). The total genomic DNA of each sample was extracted following a modified CTAB method [57]. We then constructed a DNA library for each sample with 350 bp inserted fragments following the Illumina NovaSeq protocol. Raw reads were sequenced in the Illumina Nova6000 platform. After eliminating low-quality reads, clean data were obtained. The rest of the 33 plastomes (including four Aspidistra species and 29 representatives in the family Asparagaceae) were obtained from the NCBI.

4.2. Genome Annotation

Clean data were de novo assembled into the cp genome using GetOrganelle [58]. The average coverage depth of Aspidistra plastomes ranged from 63 to 512× (Table S1). Initially, the gene annotation of each plastome was implemented in CpGAVAS2 [59] with Reineckea carnea as a reference (GenBank: MK801116), then manually corrected in Apollo genome editor v1.11.8 [60]. Necessary corrections included the identification of missing genes, checking and adjusting the start and stop positions of genes, annotating small exons for some genes (such as petB, petD, rpl16, etc.), and curating gene structures (such as rps12). We utilized the OGDRAW program to create genome maps [61]. The seven Aspidistra chloroplast genomes have been submitted and are now available in the GenBank database (Table 1).

4.3. Repeats Sequences Analysis and Genome Comparison

The GC content of the plastid genomes was estimated using EMBOSS [62]. SSRs of each plastid genome were detected on the website MISA “https://webblast.ipk-gatersleben.de/misa/ (accessed on 9 June 2023)”, including mononucleotides (with a minimum number of repetitions ≥10), dinucleotides (≥5), trinucleotides (≥4), tetranucleotides (≥3), pentanucleotides (≥3) and hexanucleotides (≥3). Other repetitive sequences (i.e., palindromic repeats, forward repeats, reverse repeats, and complementary repeats) were identified by REPuter “https://bibiserv.cebitec.uni-bielefeld.de/reputer/ (accessed on 10 July 2023)” with parameters being 3 (hamming distance) and of 30 bp (minimum repeat size).
The seven Aspidistra (A. minutiflora, A. retusa, A. dolichanthera, A. crassifila, A. erecta, A. longgangensis, and A. nankunshanensis) plastomes were compared in mVISTA [30,31] to explore interspecific variations and identify hotspot regions “http://genome.lbl.gov/vista/mvista/submit.shtml (accessed on 7 August 2023). The junction boundaries of these chloroplast genomes were visualized in IRscope [63] “https://irscope.shinyapps.io/irapp/ (accessed on 10 August 2023). Four other publicly available Aspidistra plastomes (A. yingjiangensis, A. cavicola, A. obliquipeltata and A. elatior) and three outgroup plants (Tupistra grandistigma, Rohdea chinensis and Reineckea carnea) were also included in the analysis to compare junction boundaries. The nucleotide polymorphism (Pi) among the 11 Aspidistra species was calculated by DnaSP [32] using the aligned chloroplast sequences matrix, with the sliding window length being 500 bp.

4.4. Phylogenetic Analysis

In addition to 11 Aspidistra species, including four other publicly available Aspidistra plastomes (A. yingjiangensis, A. cavicola, A. obliquipeltata and A. elatior), a total of 40 Asparagaceae species were included in the phylogenetic analyses. These species belong to three subfamilies, i.e., Lomandroideae (two species), Asparagoideae (two species), and Nolinoideae (36 species), with Nolinoideae species from tribes of Convallarieae (19 species), Ophiopogoneae (five species), Polygonateae (seven species), Nolineae (two species), Draceaneae (two species) and Ruscineae (one species). The 40 complete plastome sequences were aligned in MAFFT [64] with default parameters and then used to infer the phylogenetic tree implemented in IQ-TREE multicore version 2.2.0.3 [65] with the ML method (1000 bootstrap replicates).

5. Conclusions

In this study, we reported the chloroplast genomes of seven Aspidistra species and conducted comprehensive analyses of these plastid genomes with the other available plastomes within the genus. Each Aspidistra plastome has a typical quartile structure similar to that in most angiosperms, and is highly conserved in regard to GC content, gene number, order and structural characteristics. A comprehensive analysis of these plastomes shows limited interspecific diversity within the genus. In addition, we identified eight relatively hypervariable regions of the Aspidistra chloroplast genome as candidate markers. These analyses of chloroplast genomes present a new perspective into the taxonomy, identification, phylogeny and evolution of Aspidistra species, providing a basis for the utilization and conservation strategy of this representative karst plant group.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/genes14101894/s1. Table S1: Information of the seven newly sequenced Aspidistra taxa in this study; Table S2: Gene composiApption in the Aspidistra plastid genomes; Table S3: Simple sequence repeats (SSRs) in the seven Aspidistra plastid genomes.

Author Contributions

Conceptualization and project administration, J.H. and C.L.; data curation, investigation, conceptualization and resources, J.H., C.L., Z.L., W.X. and Y.L.; writing—original draft preparation, J.H.; writing—review and editing, C.L., Z.L., W.X. and Y.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Biological Resources Programme of the Chinese Academy of Sciences (Grant No. KFJ-BRP-007-012) and the National Natural Science Foundation of China (Grant No. 31760061).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data generated in this study are available at Genbank (accession numbers: OP598581-OP598584 and OP612671-OP612673).

Acknowledgments

We are sincerely grateful to Jingjian Li for help with the analyses and to Lin Zhang and Jing Guo for assistance in improving our manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Tao, J.; Feng, C.; Ai, B.; Kang, M. Adaptive molecular evolution of the two-pore channel 1 gene TPC1 in the karst-adapted genus Primulina (Gesneriaceae). Ann. Bot. 2016, 118, 1257–1268. [Google Scholar] [CrossRef] [PubMed]
  2. Wang, J.; Ai, B.; Kong, H.; Kang, M. Speciation history of a species complex of Primulina eburnea (Gesneriaceae) from limestone karsts of southern China, a biodiversity hot spot. Evol. Appl. 2017, 10, 919–934. [Google Scholar] [CrossRef] [PubMed]
  3. Wang, Y.; Wen, F.; Hong, X.; Li, Z.; Mi, Y.; Zhao, B. Comparative chloroplast genome analyses of Paraboea (Gesneriaceae): Insights into adaptive evolution and phylogenetic analysis. Front. Plant Sci. 2022, 13, 3895. [Google Scholar] [CrossRef]
  4. Chung, K.F.; Leong, W.C.; Rubite, R.R.; Repin, R.; Kiew, R.; Liu, Y.; Peng, C.I. Phylogenetic analyses of Begonia sect. Coelocentrum and allied limestone species of China shed light on the evolution of Sino-Vietnamese karst flora. Bot. Stud. 2014, 55, 1–15. [Google Scholar] [PubMed]
  5. Qin, H.; Liu, Y.; Yu, S.; Xu, W.; Hou, M. A Checklist of Vascular Plants of Guangxi; Science Press: Beijing, China, 2010. [Google Scholar]
  6. Li, Z.; Wang, W. Plants of Gesneriaceae in China; Henan Science and Technology Publication: Zhengzhou, China, 2005. (In Chinese) [Google Scholar]
  7. Chase, M.W.; Reveal, J.L.; Fay, M.F. A subfamilial classification for the expanded asparagalean families Amaryllidaceae, Asparagaceae and Xanthorrhoeaceae. Bot. J. Linn. Soc. 2010, 161, 132–136. [Google Scholar] [CrossRef]
  8. Averyanov, L.V.; Nguyen, K.S.; Son, H.T.; Tillich, H.J.; Wynn-Jones, B.; Maisak, T.V. New taxa and new records in Aspidistra (Convallariaceae s.s.) of Laos and Vietnam. Nord. J. Bot. 2020, 38, e02877. [Google Scholar] [CrossRef]
  9. Lang, K.Y. Aspidistra. In Flora of China; Wang, F.Z., Tang, J., Eds.; Science Press: Beijing, China, 1978; Volume 15, pp. 18–24. (In Chinese) [Google Scholar]
  10. Vislobokov, N.A.; Fu, L.F.; Wei, Y.G.; Nuraliev, M.S. Leaf epidermal micromorphology in Aspidistra (Asparagaceae): Diversity and taxonomic significance. PhytoKeys 2021, 185, 65–86. [Google Scholar] [CrossRef]
  11. Lin, C.R.; Xu, W.B.; Huang, Y.S.; Wang, B.M.; Liu, Y. Four new species of Aspidistra (Asparagaceae) from southern China. Phytotaxa 2023, 587, 121–135. [Google Scholar] [CrossRef]
  12. Tillich, H.J.; Averyanov, L.V. A critical survey of infraspecific taxa in the genus Aspidistra (Asparagaceae). Feddes Repert. 2018, 129, 185–188. [Google Scholar] [CrossRef]
  13. Tillich, H.J. 200 years Aspidistra (Asparagaceae), and now more than 200 species: A new comprehensive determination key, and an annotated bibliography of the genus. Nord. J. Bot. 2023, 2023, e03818. [Google Scholar] [CrossRef]
  14. Averyanov, L.V.; Truong, B.V.; Trinh, B.N.; Dang, S.V.; Bui, V.H.; Le, T.A.; Nguyen, T.H.A.; Ha Do, T.T.; Nguyen, C.V.; Maisak, T.V.; et al. Aspidistra peltata, Convallariaceae (Asparagaceae s.l.) a new species from northern Vietnam. Nord. J. Bot. 2021, 39, e03090. [Google Scholar] [CrossRef]
  15. Pan, B.; Yuan, Q.; Lin, C.R. Aspidistra brachypetala (Asparagaceae), a new species from Guangxi, China. Taiwania 2020, 66, 195. [Google Scholar]
  16. Cai, L.; Peng, S.; Tian, J.; Dao, Z.L.; Wei, N.; Hu, G.W.; Wang, Q.F. Aspidistra austroyunnanensis (Asparagaceae), a new species from southern Yunnan, China. Phytotaxa 2018, 356, 233–237. [Google Scholar] [CrossRef]
  17. Lin, C.R.; Huang, X.Y.; Pan, B.; Xu, W.B.; Liu, Y. Two new species of Aspidistra (Asparagaceae) from Guangxi, China: A. chunxiuensis and A. longshengensis. Phytotaxa 2015, 208, 163–169. [Google Scholar] [CrossRef]
  18. Blume, C.L. Aspidistra elatior  . Tijdschr. Voor Nat. Geschied. Physiol. 1834, 1, 76–79. [Google Scholar]
  19. Tillich, H.J.; Averyanov, L.V. Four new species of Aspidistra Ker Gawl. (Asparagaceae) from China and Vietnam with a comment on A. longifolia Hook. f. and A. hainanensis WY Chun & FC How. Gard. Bull. Singap. 2012, 64, 201–209. [Google Scholar]
  20. Phonsena, P.; De Wilde, W.J. The genus Aspidistra Ker Gawl. (Asparagaceae/Ruscaceae) in Thailand. Thai For. Bull. 2010, 38, 48–58. [Google Scholar]
  21. Lin, W.; Xu, Y.; Jiang, Y.; Ma, Z. To cheat or to treat? Fungus gnat pollination in Aspidistra. Ecology 2022, 103, e3729. [Google Scholar] [CrossRef]
  22. Tillich, H.J. The genus Aspidistra Ker-Gawl. (Asparagaceae) In Vietnam. Taiwania 2014, 59, 1–8. [Google Scholar] [CrossRef]
  23. Chen, J.S.; Gao, Q.; Zhou, H.; Huang, Y.S.; Ogisu, M.; Cao, M. Distribution, karyomorphology, and morphology of Aspidistra subrotata (Asparagaceae) at different ploidy levels in limestone areas of Asia. Comp. Cytogenet. 2017, 11, 1–13. [Google Scholar] [CrossRef]
  24. Liu, X.; Zhou, B.; Yang, H.; Li, Y.; Yang, Q.; Lu, Y.; Gao, Y. Sequencing and analysis of Chrysanthemum carinatum Schousb and Kalimeris indica. The complete chloroplast genomes reveal two inversions and rbcL as barcoding of the vegetable. Molecules 2018, 23, 1358. [Google Scholar] [CrossRef] [PubMed]
  25. Lv, D.; Liu, A.; Xu, W.; He, S. Study on the genus Aspidistra based on DNA barcoding. Plant Sci. J. 2018, 36, 784–789. [Google Scholar]
  26. Seberg, O.; Petersen, G.; Davis, J.I.; Pires, J.C.; Stevenson, D.W.; Chase, M.W.; Fay, M.F.; Devey, D.S.; Jørgensen, T.; Sytsma, K.J. Phylogeny of the Asparagales based on three plastid and two mitochondrial genes. Am. J. Bot. 2012, 99, 875–889. [Google Scholar] [CrossRef] [PubMed]
  27. Kim, H.T.; Kim, J.S.; Kim, J.-H. The complete plastid genome sequence of Eustrephus latifolius (Asparagaceae: Lomandroideae). Mitochondrial DNA Part A 2016, 27, 1549–1551. [Google Scholar] [CrossRef] [PubMed]
  28. Sheng, W.; Chai, X.; Rao, Y.; Tu, X.; Du, S. Complete chloroplast genome sequence of Asparagus (Asparagus officinalis L.) and its phylogenetic position within asparagales. J. Plant Breed. Genet. 2017, 5, 121–128. [Google Scholar]
  29. Ji, Y.; Landis, J.B.; Yang, J.; Wang, S.; Zhou, N.; Luo, Y.; Liu, H. Phylogeny and evolution of Asparagaceae subfamily Nolinoideae: New insights from plastid phylogenomics. Ann. Bot. 2023, 131, 301–312. [Google Scholar] [CrossRef]
  30. Frazer, K.A.; Pachter, L.; Poliakov, A.; Rubin, E.M.; Dubchak, I. VISTA: Computational tools for comparative genomics. Nucleic Acids Res. 2004, 32 (Suppl. S2), W273–W279. [Google Scholar] [CrossRef]
  31. Thiel, T.; Michalek, W.; Varshney, R.; Graner, A. Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). Theor. Appl. Genet. 2003, 106, 411–422. [Google Scholar] [CrossRef]
  32. Rozas, J.; Ferrer-Mata, A.; Sánchez-DelBarrio, J.C.; Guirao-Rico, S.; Librado, P.; Ramos-Onsins, S.E.; Sánchez-Gracia, A. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol. Biol. Evol. 2017, 34, 3299–3302. [Google Scholar] [CrossRef]
  33. Raman, G.; Lee, E.M.; Park, S. Intracellular DNA transfer events restricted to the genus Convallaria within the Asparagaceae family: Possible mechanisms and potential as genetic markers for biographical studies. Genomics 2021, 113, 2906–2918. [Google Scholar] [CrossRef]
  34. Raman, G.; Park, S.; Lee, E.M.; Park, S. Evidence of mitochondrial DNA in the chloroplast genome of Convallaria keiskei and its subsequent evolution in the Asparagales. Sci. Rep. 2019, 9, 5028. [Google Scholar] [CrossRef] [PubMed]
  35. He, J.; Yao, M.; Lyu, R.D.; Lin, L.L.; Liu, H.J.; Pei, L.Y.; Yan, S.X.; Xie, L.; Cheng, J. Structural variation of the complete chloroplast genome and plastid phylogenomics of the genus Asteropyrum (Ranunculaceae). Sci. Rep. 2019, 9, 15285. [Google Scholar] [CrossRef] [PubMed]
  36. Zhu, A.; Guo, W.; Gupta, S.; Fan, W.; Mower, J.P. Evolutionary dynamics of the plastid inverted repeat: The effects of expansion, contraction, and loss on substitution rates. New Phytol. 2016, 209, 1747–1756. [Google Scholar] [CrossRef] [PubMed]
  37. Rousseau-Gueutin, M.; Bellot, S.; Martin, G.E.; Boutte, J.; Chelaifa, H.; Lima, O.; Michon-Coudouel, S.; Naquin, D.; Salmon, A.; Ainouche, K.; et al. The chloroplast genome of the hexaploid Spartina maritima (Poaceae, Chloridoideae): Comparative analyses and molecular dating. Mol. Phylogenetics Evol. 2015, 93, 5–16. [Google Scholar] [CrossRef] [PubMed]
  38. Hao, Z.; Cheng, T.; Zheng, R.; Xu, H.; Zhou, Y.; Li, M.; Lu, F.; Dong, Y.; Liu, X.; Chen, J.; et al. The complete chloroplast genome sequence of a relict conifer Glyptostrobus pensilis: Comparative analysis and insights into dynamics of chloroplast genome rearrangement in Cupressophytes and Pinaceae. PLoS ONE 2016, 11, e0161809. [Google Scholar] [CrossRef] [PubMed]
  39. Bessega, C.F.; Pometti, C.L.; Miller, J.T.; Watts, R.; Saidman, B.O.; Vilardi, J.C. New microsatellite loci for Prosopis alba and P. chilensis (Fabaceae). Appl. Plant. Sci. 2013, 1, 1200324. [Google Scholar] [CrossRef]
  40. Li, J.; Tang, J.; Zeng, S.; Han, F.; Yuan, J.; Yu, J. Comparative plastid genomics of four Pilea (Urticaceae) species: Insight into interspecific plastid genome diversity in Pilea. BMC Plant Biol. 2021, 21, 25. [Google Scholar] [CrossRef]
  41. Gichira, A.W.; Avoga, S.; Li, Z.; Hu, G.; Wang, Q.; Chen, J. Comparative genomics of 11 complete chloroplast genomes of Senecioneae (Asteraceae) species: DNA barcodes and phylogenetics. Bot. Stud. 2019, 60, 17. [Google Scholar] [CrossRef]
  42. Huang, D.; Zhang, Y.; Jin, M.; Li, H.; Song, Z.; Wang, Y.; Chen, J. Characterization and high cross-species transferability of microsatellite markers from the floral transcriptome of Aspidistra saxicola (Asparagaceae). Mol. Ecol. Resour. 2014, 14, 569–577. [Google Scholar] [CrossRef]
  43. Gitzendanner, M.A.; Soltis, P.S.; Wong, G.K.; Ruhfel, B.R.; Soltis, D.E. Plastid phylogenomic analysis of green plants: A billion years of evolutionary history. Am. J. Bot. 2018, 105, 291–301. [Google Scholar] [CrossRef]
  44. Fu, C.N.; Mo, Z.Q.; Yang, J.B.; Ge, X.J.; Li, D.Z.; Xiang, Q.Y.J.; Gao, L.M. Plastid phylogenomics and biogeographic analysis support a trans-Tethyan origin and rapid early radiation of Cornales in the Mid-Cretaceous. Mol. Phylogenetics Evol. 2019, 140, 106601. [Google Scholar] [CrossRef] [PubMed]
  45. Li, H.T.; Luo, Y.; Gan, L.; Ma, P.F.; Gao, L.M.; Yang, J.B.; Cai, J.; Gitzendanner, M.A.; Fritsch, P.W.; Zhang, T.; et al. Plastid phylogenomic insights into relationships of all flowering plant families. BMC Biol. 2021, 19, 232. [Google Scholar] [CrossRef] [PubMed]
  46. Yan, L.J.; Zhu, Z.G.; Wang, P.; Fu, C.N.; Guan, X.J.; Kear, P.; Zhang, C.Z.; Zhu, G.T. Comparative analysis of 343 plastid genomes of Solanum section Petota: Insights into potato diversity, phylogeny, and species discrimination. J. Syst. Evol. 2023, 61, 599–612. [Google Scholar] [CrossRef]
  47. Meng, R.; Luo, L.Y.; Zhang, J.Y.; Zhang, D.G.; Nie, Z.L.; Meng, Y. The deep evolutionary relationships of the morphologically heterogeneous Nolinoideae (Asparagaceae) revealed by transcriptome data. Front. Plant Sci. 2020, 11, 584981. [Google Scholar] [CrossRef] [PubMed]
  48. Liu, Y.; Kono, Y.; Lin, C.R.; Xu, W.B.; Peng, C.I. Aspidistra erecta (Asparagaceae), a new species from limestone areas in Guangxi, China. Bot. Stud. 2011, 52, 367–373. [Google Scholar]
  49. Cai, L.; Pan, B.; Lin, C.R.; Dao, Z.L.; Hu, G.W. Aspidistra xichouensis (Asparagaceae), a new species from the karst region in southeastern Yunnan, China. Phytotaxa 2020, 439, 143–149. [Google Scholar] [CrossRef]
  50. Dilip, K.R.; Lytan, R.; Odyuo, N. Aspidistra yingjiangensis (Asparagaceae: Nolinoidae), a new species record for the flora of India. Turczaninowia 2022, 25, 67–74. [Google Scholar]
  51. Ding, F.; Ke-chien, Y.; Ling, Z. Two new species of Aspidistra (Liliaceae) from Guangxi, China. J. Syst. Evol. 1993, 31, 180. [Google Scholar]
  52. Chen, X.X.; Fang, D. Two new species of the Aspidistra (Liliaceae) from Guangxi (In Chinese). Guihaia 1982, 2, 77–79. [Google Scholar]
  53. Lin, C.R.; Nong, Z.Q.; Huang, Y.S.; Meng, T.; Liu, Y. Aspidistra longgangensis sp. nov. (Asparagaceae) from limestone areas in Guangxi, China. Nord. J. Bot. 2015, 33, 377–380. [Google Scholar] [CrossRef]
  54. Ji, Y.; Yang, J.; Landis, J.B.; Wang, S.; Yang, Z.; Zhang, Y. Deciphering the taxonomic delimitation of Ottelia acuminata (Hydrocharitaceae) using complete plastomes as Super-Barcodes. Front. Plant Sci. 2021, 12, 681270. [Google Scholar] [CrossRef]
  55. Fu, C.N.; Wu, C.S.; Ye, L.J.; Mo, Z.Q.; Liu, J.; Chang, Y.W.; Li, D.Z.; Chaw, S.M.; Gao, L.M. Prevalence of isomeric plastomes and effectiveness of plastome super-barcodes in yews (Taxus) worldwide. Sci. Rep. 2019, 9, 2773. [Google Scholar] [CrossRef] [PubMed]
  56. Liu, A.l.; He, S.Z.; Yao, H.; Chen, S.L.; Song, J.Y. Molecular identification of medicinal plants in Aspidistra (in Chinese). World Sci. Technol./Mod. Tradit. Chin. Med. Mater. Med. 2012, 14, 1166–1171. [Google Scholar] [CrossRef]
  57. Doyle, J.J.; Doyle, J.L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 1987, 19, 11–15. [Google Scholar]
  58. 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]
  59. Shi, L.; Chen, H.; Jiang, M.; Wang, L.; Wu, X.; Huang, L.; Liu, C. CPGAVAS2, an integrated plastome sequence annotator and analyzer. Nucleic Acids Res. 2019, 47, W65–W73. [Google Scholar] [CrossRef]
  60. Misra, S.; Harris, N. Using Apollo to browse and edit genome annotations. Curr. Protoc. Bioinform. 2005, 12, 9.5.1–9.5.28. [Google Scholar] [CrossRef]
  61. 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]
  62. Rice, P.; Longden, I.; Bleasby, A. EMBOSS: The European molecular biology open software suite. Trends Genet. 2000, 16, 276–277. [Google Scholar] [CrossRef]
  63. Amiryousefi, A.; Hyvönen, J.; Poczai, P. IRscope: An online program to visualize the junction sites of chloroplast genomes. Bioinformatics 2018, 34, 3030–3031. [Google Scholar] [CrossRef]
  64. Katoh, K.; Standley, D.M. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol. Biol. Evol. 2013, 30, 772–780. [Google Scholar] [CrossRef] [PubMed]
  65. Nguyen, L.T.; Schmidt, H.A.; von Haeseler, A.; Minh, B.Q. IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 2014, 32, 268–274. [Google Scholar] [CrossRef] [PubMed]
Genes 14 01894 g001
Figure 1. Structural map and images of seven Aspidistra species. All seven plastid genomes possess the characteristic quartile structure, comprising a pair of IR regions, an LSC region, and an SSC region. The genes inside the circle are transcribed in a clockwise direction, while the genes outside the circle are transcribed in a counterclockwise direction. Genes are color-coded to represent distinct functions, with the dark gray of the inner circle indicating the GC content in the genome and the light gray indicating the AC content. The template map is based on the structure of A. erecta.
Figure 1. Structural map and images of seven Aspidistra species. All seven plastid genomes possess the characteristic quartile structure, comprising a pair of IR regions, an LSC region, and an SSC region. The genes inside the circle are transcribed in a clockwise direction, while the genes outside the circle are transcribed in a counterclockwise direction. Genes are color-coded to represent distinct functions, with the dark gray of the inner circle indicating the GC content in the genome and the light gray indicating the AC content. The template map is based on the structure of A. erecta.
Genes 14 01894 g001
Genes 14 01894 g002
Figure 2. Sequence repeats in chloroplast genomes of the seven Aspidistra species. (A) Types and numbers of SSRs in the seven plastid genomes. (B) Comparison of interspersed repeats in the seven plastid genomes.
Figure 2. Sequence repeats in chloroplast genomes of the seven Aspidistra species. (A) Types and numbers of SSRs in the seven plastid genomes. (B) Comparison of interspersed repeats in the seven plastid genomes.
Genes 14 01894 g002
Genes 14 01894 g003
Figure 3. Comparative analysis with mVISTA of the plastomes of seven Aspidistra species. The chloroplast genome of A. minutiflora was used as a reference. The annotated genes and their direction are indicated by gray arrows. The different regions on the genome are marked with different colors. The vertical scale represents the similarity among sequences.
Figure 3. Comparative analysis with mVISTA of the plastomes of seven Aspidistra species. The chloroplast genome of A. minutiflora was used as a reference. The annotated genes and their direction are indicated by gray arrows. The different regions on the genome are marked with different colors. The vertical scale represents the similarity among sequences.
Genes 14 01894 g003
Genes 14 01894 g004
Figure 4. Nucleotide diversity (Pi) of the 11 Aspidistra plastomes. Each black diamond represents the Pi value per 500 bp window. A total of eight positions had Pi values greater than 0.007, and all were intergenic regions (trnC-GCA-petN (0.01033), trnE-UUC-psbD (0.01171), accD-psaI (0.00796), petA-psbJ (0.00898), rpl20-rps12 (0.00771 and 0.00764 for the two adjacent sliding windows, respectively), rpl36-rps8 (0.00727), ccsA-ndhD (0.01207) and rps15-ycf1 (0.00749)).
Figure 4. Nucleotide diversity (Pi) of the 11 Aspidistra plastomes. Each black diamond represents the Pi value per 500 bp window. A total of eight positions had Pi values greater than 0.007, and all were intergenic regions (trnC-GCA-petN (0.01033), trnE-UUC-psbD (0.01171), accD-psaI (0.00796), petA-psbJ (0.00898), rpl20-rps12 (0.00771 and 0.00764 for the two adjacent sliding windows, respectively), rpl36-rps8 (0.00727), ccsA-ndhD (0.01207) and rps15-ycf1 (0.00749)).
Genes 14 01894 g004
Genes 14 01894 g005
Figure 5. Comparison of the borders of the LSC, SSC, and IR regions of 11 Aspidistra species and three outgroups. The tree on the left is simplified from our phylogenetic results (see Section 2.6, Phylogenetic relationships in the Results). The genes near the boundary are shown above and below the mainlines. JLB, JSB, JSA and JLA represent the border loci of LSC/IRb, IRb/SSC, SSC/IRa and IRa/LSC, respectively.
Figure 5. Comparison of the borders of the LSC, SSC, and IR regions of 11 Aspidistra species and three outgroups. The tree on the left is simplified from our phylogenetic results (see Section 2.6, Phylogenetic relationships in the Results). The genes near the boundary are shown above and below the mainlines. JLB, JSB, JSA and JLA represent the border loci of LSC/IRb, IRb/SSC, SSC/IRa and IRa/LSC, respectively.
Genes 14 01894 g005
Genes 14 01894 g006
Figure 6. The phylogenetic tree of 40 Asparagaceae species. Species names are shown as tip labels on the phylogenetic tree, with NCBI accessions in brackets. Newly acquired Aspidistra plastomes in this study are in bold. Species in different tribes are highlighted with distinct colored backgrounds, with the name of each tribe to the right. Subfamilies are indicated by gray bars. Numbers at nodes are BS supports.
Figure 6. The phylogenetic tree of 40 Asparagaceae species. Species names are shown as tip labels on the phylogenetic tree, with NCBI accessions in brackets. Newly acquired Aspidistra plastomes in this study are in bold. Species in different tribes are highlighted with distinct colored backgrounds, with the name of each tribe to the right. Subfamilies are indicated by gray bars. Numbers at nodes are BS supports.
Genes 14 01894 g006
Table 1. Basic features of the seven Aspidistra plastid genomes.
Table 1. Basic features of the seven Aspidistra plastid genomes.
SpeciesAccession Number in NCBILength (bp)GC Content (%)Gene Numbers
TotalLSCSSCIRTotalLSCSSCIRTotalProtein-coding GenetRNA GenerRNA Gene
A. minutifloraOP598584156,38985,13118,23226,51337.6235.5931.6142.9513086388
A. retusaOP598583156,34885,10818,20626,51737.6135.5431.6942.9713086388
A. dolichantheraOP598582156,36785,11918,21626,51637.6435.5831.6742.9813086388
A. crassifilaOP598581156,37385,15918,21826,49837.6235.5731.6142.9713086388
A. erectaOP612671156,38885,10918,22326,52837.6535.5931.664313086388
A. longgangensisOP612673156,33585,10218,19926,51737.6435.5831.7242.9813086388
A. nankunshanensisOP612672156,47585,18118,21426,54037.6535.5831.7442.9913086388
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

Huang, J.; Lu, Z.; Lin, C.; Xu, W.; Liu, Y. Comprehensive Comparative Analyses of Aspidistra Chloroplast Genomes: Insights into Interspecific Plastid Diversity and Phylogeny. Genes 2023, 14, 1894. https://doi.org/10.3390/genes14101894

AMA Style

Huang J, Lu Z, Lin C, Xu W, Liu Y. Comprehensive Comparative Analyses of Aspidistra Chloroplast Genomes: Insights into Interspecific Plastid Diversity and Phylogeny. Genes. 2023; 14(10):1894. https://doi.org/10.3390/genes14101894

Chicago/Turabian Style

Huang, Jie, Zhaocen Lu, Chunrui Lin, Weibin Xu, and Yan Liu. 2023. "Comprehensive Comparative Analyses of Aspidistra Chloroplast Genomes: Insights into Interspecific Plastid Diversity and Phylogeny" Genes 14, no. 10: 1894. https://doi.org/10.3390/genes14101894

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