Next Article in Journal
P2X4 Receptors Mediate Ca2+ Release from Lysosomes in Response to Stimulation of P2X7 and H1 Histamine Receptors
Previous Article in Journal
Establishment of Bovine-Induced Pluripotent Stem Cells
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 22
Issue 19
10.3390/ijms221910485
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

Lineage-Specific Variation in IR Boundary Shift Events, Inversions, and Substitution Rates among Caprifoliaceae s.l. (Dipsacales) Plastomes

by
Seongjun Park
1,
Minji Jun
2,
Sunmi Park
2 and
SeonJoo Park
2,*
1
Institute of Natural Science, Yeungnam University, Gyeongsan 38541, Gyeongbuk, Korea
2
Department of Life Sciences, Yeungnam University, Gyeongsan 38541, Gyeongbuk, Korea
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2021, 22(19), 10485; https://doi.org/10.3390/ijms221910485
Submission received: 30 August 2021 / Revised: 23 September 2021 / Accepted: 25 September 2021 / Published: 28 September 2021
(This article belongs to the Section Molecular Plant Sciences)
Browse Figures
Versions Notes

Abstract

:
Caprifoliaceae s.l. plastid genomes (plastomes) show that one inversion and two inverted repeat boundary shifts occurred in the common ancestor of this family, after which the plastomes are generally conserved. This study reports plastome sequences of five additional species, Fedia cornucopiae, Valeriana fauriei, and Valerianella locusta from the subfamily Valerianoideae, as well as Dipsacus japonicus and Scabiosa comosa from the subfamily Dipsacoideae. Combined with the published plastomes, these plastomes provide new insights into the structural evolution of plastomes within the family. Moreover, the three plastomes from the subfamily Valerianoideae exhibited accelerated nucleotide substitution rates, particularly at synonymous sites, across the family. The patterns of accD sequence divergence in the family are dynamic with structural changes, including interruption of the conserved domain and increases in nonsynonymous substitution rates. In particular, the Valeriana accD gene harbors a large insertion of amino acid repeat (AAR) motifs, and intraspecific polymorphism with a variable number of AARs in the Valeriana accD gene was detected. We found a correlation between intron losses and increased ratios of nonsynonymous to synonymous substitution rates in the clpP gene with intensified positive selection. In addition, two Dipsacoideae plastomes revealed the loss of the plastid-encoded rps15, and a potential functional gene transfer to the nucleus was confirmed.

1. Introduction

The plastid genome (plastome) of angiosperms is generally conserved and has a quadripartite structure with a pair of inverted repeats (IR) separated by large and small single copy (LSC and SSC) regions [1]. Plastomes generally range from 120 to 160 kb in length and contain 113 unique genes, 79 protein-coding genes, 30 tRNAs, and 4 rRNAs. However, accumulating data on complete plastome sequences exhibit variations in gene and intron content [2]. Plastid gene loss requires functional transfer to the nucleus before the loss in its plastome [3]. Extensive genome rearrangements, including IR boundary shifts, have also been reported for several lineages such as Campanulaceae [4], Caryophyllaceae [5], Fabaceae [6], Geraniaceae [7], Oleaceae [8], and Papaveraceae [9,10].
Lineage-specific variation in the rate of plastome sequence evolution has been documented in angiosperms [5,11,12,13]. Higher substitution rates are associated with structural rearrangements [5,14]. Accelerated rates of nucleotide substitution are affected by the whole genome as well as a subset of protein-coding genes. Compared with other plastid-encoded genes, acetyl-CoA carboxylase subunit β (accD), ATP-dependent Clp protease proteolytic subunit (clpP), DNA-directed RNA polymerase subunit α (rpoA), some subunits of ribosomal proteins, and the chloroplast factors ycf1, ycf2, and ycf4, show dynamic acceleration [5,9,11,15,16,17,18]. Multiple mechanisms, including dysfunction of DNA replication, repair, and recombination (DNA-RRR) machinery, localized hypermutation, mutagenic retroprocessing, and pseudogenization, have been hypothesized to explain this acceleration [11,14,16,19].
The most extreme accelerations are found in the plastid-encoded accD and clpP, which have undergone a history of insertions and deletions (indels) of amino acid sequences or intron losses in multiple independent lineages, respectively [9,15]. Several angiosperm lineages have experienced losses in the accD or clpP genes [10,20,21]. The plastid-encoded accD has been functionally replaced via gene transfer to the nucleus or gene substitution in eukaryotic ACCase [15,16,20,22,23,24,25,26]. However, clear evidence of plastid clpP transfer to the nucleus in angiosperms is lacking. Highly divergent accD and clpP genes are likely caused by compensatory mutations in the interactions between nuclear-encoded plastid-targeted subunits [27,28]. This is because the function of the two genes depends on nuclear-encoded proteins that assemble plastid-localized subunits. For example, the plastid-encoded accD plays an essential role in the fatty acid biosynthesis pathway [29], which is a subunit of the prokaryotic acetyl–CoA carboxylase (ACC) complex [30]. The prokaryotic ACC complex consists of four subunits, three of which are nuclear-encoded proteins: acetyl–CoA carboxylase subunit α (ACCA), biotin carboxyl carrier protein subunit (ACCB), and biotin carboxylase subunit (ACCC). Plastid-encoded clpP is a subunit of the caseinolytic protease (CLP) complex [31] and is involved in multiple processes of chloroplast development [32]. Multiple subunits (CLPP2 to CLPP6) of the CLP complex are encoded in the nucleus [31]. Thus, the nuclear-encoded subunits interact with plastid-encoded subunits.
The honeysuckle family (Caprifoliaceae sensu lato [s.l.]) comprises approximately 825 species in 28-42 genera with widespread cosmopolitan distribution [33]. Caprifoliaceae s.l. has been classified into seven major groups (six subfamilies and one genus): Diervilloideae, Caprifolioideae, Linnaeoideae, Morinoideae, Dipsacoideae, Valerianoideae, and Zabelia (Rehder) Makino [34]. A recent phylogenomic study based on nuclear loci and plastome sequences suggested that Zabelia is recognized as a new subfamily of Zabelioideae [35]. To date, complete plastomes of 22 genera have been sequenced (National Center for Biotechnology Information; NCBI, accessed on 18 August 2021). The sequenced Caprifoliaceae s.l. plastomes range in size from 151.3 to 161.6 kb with a quadripartite organization. Variations in inverted repeat (IR) boundary shifts and nucleotide substitution rates have been documented in the Caprifoliaceae s.l. plastomes [34,36]. Despite broader sampling and sequencing across the family, very little is known about the gene evolution, including structural changes and rate variation in the plastid-encoded accD and clpP genes.
In this study, we generated the complete plastome sequences of three species from the subfamily Valerianoideae and two species from the subfamily Dipsacoideae. Genome organization and nucleotide substitution rates were estimated and compared to the published Caprifoliaceae s.l. plastomes. In particular, the correlation between structural evolution and nucleotide substitution rates in the plastid-encoded accD and clpP genes across this family was examined. In addition, we examined intra- and infraspecific length variations in the accD coding region of Valeriana fauriei Briq. and V. sambucifolia f. dageletiana (Nakai ex F.Maek.) Hara.

2. Results

2.1. Plastome Organization

We sequenced and assembled the complete plastomes of three Valerianoideae (Fedia cornucopiae (L.) Gaertn., V. fauriei, and Valerianella locusta (L.) Laterr.) and two Dipsacoideae (Dipsacus japonicus Miq. and Scabiosa comosa Fisch. ex Roem. & Schult.) species (Figure S1, see Supplementary Materials). Among the five species, the plastome size ranged from 149,809 bp (V. locusta) to 160,243 bp (D. japonicus) (Table 1). Dipsacus japonicus had the largest LSC (87,066 bp), whereas F. cornucopiae had the smallest SSC (15,862 bp). The GC content of the D. japonicus (38.8%) was higher than that of the other species (Table 1). The three Valerianoideae plastomes encoded 79 protein-coding genes, 30 tRNA genes, and 4 rRNA genes (Table 1). However, the ribosomal protein subunit S15 (rps15) appears to be a pseudogene in two Dipsacoideae plastomes (Table 1). Functional replacement by gene transfer of rps15 from plastid to the nucleus was detected in Dipsacus transcriptome data (Figure S2). The plastome of D. japonicus was missing trnT-GGU and contained two trnE-UUC with 90.4% nucleotide identity (Figure S3). The duplicated gene content in the IR region varied as a result of IR expansion and contraction. The intron content also varied owing to the loss of intron in the clpP gene. The plastome of F. cornucopiae was missing the first intron of the clpP gene. The plastome of V. locusta was missing both introns in the clpP gene (Figure S1).
Among the analyzed Caprifoliaceae s.l. plastomes, the largest number of repeat pairs (95) was found in Morina, and the fewest repeats (11) were observed in Valerianella (Table S1). The average value of repeat pairs from Caprifoliaceae s.l. plastomes was ~10 times higher than that from the outgroups (Table S1). The number of repeat pairs from the subfamily Valerianoideae was 2–4 times higher than the average value of repeat pairs from the outgroups, but the number of the repeat pairs from Valerianella was similar to that from the outgroups.
To understand the evolutionary history of genome rearrangement in the family, we constructed a phylogenetic tree using 72 plastid genes (Figure 1). The inversions mainly occurred within the IR and SSC regions and some inversion is likely the result of a series of IR expansions and contractions (Figure 1). Based on the most parsimonious interpretation, the ancestral plastome of Caprifoliaceae s.l. had three structural changes: (1) an inversion associated with the ndhF gene, (2) a contraction at the IRB/SSC boundary to trnN resulting in the entire ycf1 gene into the SSC region, and (3) a contraction at the IRA/LSC boundary, from the rps19 to rpl23 gene (Figure 1). The plastome rearrangement model suggests that lineage- or species-specific events occurred independently after the ancestral structural changes (Figure 1). For example, an independent contraction to trnI at the IRA/LSC boundary and contraction to trnR at IRA/SSC occurred in early diverging Weigela. Mauve alignment among the subfamily Caprifolioideae identified seven locally collinear blocks (LCBs) with two inversions involving eight breakpoints (Figure S4). Within the subfamily Caprifolioideae, a contraction to ycf2 at IRA/LSC boundary in Leycesteria and a contraction to trnR at IRA/SSC occurred in Triosteum, respectively. The inversion in Leycesteria is the result of IR expansion at the IRB/SSC boundary to ndhH and IR contraction at the IRA/SSC boundary to trnN, resulting in the relocation of the ycf1-rps15 region (Figure 1). Mauve alignment among the subfamilies Valerianoideae and Dipsacoideae identified five LCBs with six inversions involving seven breakpoints (Figure S4). Contraction to trnI at the IRA/LSC boundary, followed by an expansion to trnH at the IRB/LSC boundary and an expansion back to ycf1, indicated synapomorphic events in the subfamily Valerianoideae (Figure 1). After that, four IR boundary shift events occurred in the common ancestor of the Fedia/Valerianella/Valeriana clade. The rpl32-ndhF region in Fedia, Valerianella, and Valeriana plastomes were relocated as a result of two expansions and two contractions. Expansion at the IRA/SSC boundary resulted in the duplication of ccsA and included a C-terminal portion of ndhD (Fedia: 1265 bp; Valerianella: 1286 bp; Valeriana: 1314 bp), generating a truncated ndhD fragment in IRB (Figure 1). Within the subfamily Dipsacoideae, an expansion from trnH to rps3 at the IRA/SSC boundary and an expansion from trnN to ycf1 occurred in the common ancestor of the Dipsacus/Scabiosa clade. The inversion event associated with ccsA-trnL-rpl32-ndhF is unique to Scabiosa (Figure 1). In Weigela, two IR contractions occurred at the IRA/LSC and IRA/SSC boundaries.

2.2. Elevated Substitution Rates in the Plastomes of the Subfamily Valerianoideae

The 24 Caprifoliaceae s.l. genera and five Adoxaceae genera shared 72 plastid-encoded genes. We excluded seven protein genes from the concatenated data set and substitution rate analysis because they appeared to be pseudogenes or losses (clpP, rps3, rps15, ycf1, and ycf2) and divergent (accD and ycf3) among some species within the family. To examine rate variation in the selected plastid genes among the 24 genera, nonsynonymous (dN) and synonymous (dS) substitution rates were estimated using the phylogenetic tree as a constraint tree (Figure 2). The dN and dS values in pairwise comparisons between Viburnum and Caprifoliaceae s.l. showed that Fedia, Valerianella, and Valeriana from the subfamily Valerianoideae had significantly higher dS rates than the other analyzed species (Wilcoxon rank-sum test, p < 0.001, after Bonferroni correction; Table S1), except for the comparison between Valeriana and Pterocephalus. Narodostachys and Patrinia had only significantly higher dS rates than Weigela (Wilcoxon rank-sum test, p < 0.05, after Bonferroni correction; Table S2). In the case of dN rates, only 11 comparisons between Fedia and six species (Kolkwitzia, Symphoricarpos, Triosteum, Triplostegia, Weigela, and Zabelia), between Valerianella and four species (Symphoricarpos, Triplostegia, Weigela, and Zabelia), and between Valeriana and Weigela showed that Fedia, Valerianella, and Valeriana had significantly higher rates than the other analyzed species (Wilcoxon rank-sum test, p < 0.05, after Bonferroni correction; Table S2).
In addition, multiple genes exhibit dN/dS ratios that are greater than one, but likelihood ratio tests (LRTs) with Bonferroni correction indicated that dN/dS for infA in Acanthocalyx, ndhD in Morinoideae/Linnaeoideae/Valerianoideae/Dipscacoideae/Zabelioideae, psbM in Vesalea, rbcL in Weigela, rpoC1 in Valeriana, rpl22 in Adoxa/Tetradoxa/Sinadoxa, rpl32 in Heptacodium, rps2 in Acanthocalyx, rps4 in Scabiosa, rps14 in Pterocephalus, rps16 in Dipsacus/Scabiosa/Pterocephalus, rps18 in Abelia, and rps19 in Acanthocalyx were significantly different (Table S3).

2.3. Structural Evolution of Plastid-Encoded accD Gene in Caprifoliaceae s.l. Plastomes

The length of the acetyl–CoA carboxylase beta subunit D (accD) open reading frame (ORF) varied remarkably among the examined Caprifoliaceae s.l. plastomes, ranging from 711 bp in Heptacodium to 2517 bp in Lonicera (Figure 3). The comparison revealed two histories of insertion events within the N- and C-terminal regions, resulting in the expansion or truncation of the accD ORF. The most parsimonious interpretation is that the accD genes were interrupted by the insertion of amino acids in the common ancestor of Caprifoliaceae s.l. (Figure 3). The second insertion of amino acids occurred in the common ancestor of Zabelia, Morinoideae, Linnaeoideae, Valerianoideae, and Dipscacoideae (except Pterocephalus and Triplostegia), which was split into two portions of the conserved domain (Figure 3 and Figure S4).
In particular, the accD ORFs of Morina, Linnaea, and Zabelia contain a part of the conserved domain, but the catalytic sites are included (Figure S2). Conserved domain (CD) searches identified a portion of Apolipoprotein, MSCRAMM_ClfB, MSCRAMM_SdrC, GAT1, PPK08581, PPK05901, SMC_N, pneumo_PspA, and rplD subfamilies surrounding the conserved domain of accD in the predicted ORF (Table S4). The presence of MSCRAMM_ClfB in Symphoricarpos, Linnaeoideae, and Adoxaceae indicated that this event occurred in the most recent common ancestor of each clade (Table S4). Protein sequence alignment of all inserted regions from the accD was highly divergent with low amino acid identities of 2.9–96.7% (Figure S4). However, two lineages—Dipsacus/Scabiosa and Linnaeoideae—had high amino acid identities of 94.3% and 83.1–96.7%, respectively.
The dN and dS were calculated for the selected Caprifoliaceae s.l. and outgroup using only the conserved domain sequences to test the effect of the insertion on nucleotide substitution rates of the accD gene. The dN values for accD in the selected Caprifoliaceae s.l. were significantly higher than in the outgroup (Wilcoxon rank-sum test, p < 0.05; Figure S3). Seven branches with dN/dS ratios > 1 were detected, but LTRs showed that three branches, Fedia/Valerianella, Triplostegia, and Zabelia, were significantly different (p < 0.00001 after Bonferroni correction, Figure 3). The RELAX analysis indicated that the accD experienced significantly intensified selection in the Zabelia, Morinoideae, Linnaeoideae, Valerianoideae, and Dipsacoideae clade (k = 1.96, p = 0.020, likelihood ratio [LR] = 5.38), and the Caprifoliaceae s.l. clade (k = 2.25, p = 0.032, LR = 4.60).

2.4. Length Variation in the Valeriana accD Gene

Valeriana fauriei plastome contains an expanded accD gene, which is interrupted by amino acid repeats (AARs) surrounding the conserved domains. Compared with the other available Valeriana plastomes, V. officinalis contains a truncated accD gene in its genome, whereas V. sambucifolia f. dageletiana contains an expanded accD like V. fauriei accD (Figure 4A). To evaluate the variability of the AAR motifs in the accD gene of V. fauriei and V. sambucifolia f. dageletiana, we designed a PCR primer that targets two hotspot regions of accD (Figure 4A). The amplicon sizes of the region ranged from 966 bp to 1239 bp (Table S5). Alignment of the two region sequences of the 50 individuals with two accD sequences from two plastomes revealed intra-and infraspecific variation of the accD in Valeriana (Figure 4). Two hotspot regions show length variation, consisting of (1) 3 to 10 repeats of “ESTTTESFAQR” and (2) 5 to 14 repeats of “SDSEEDLIKPD”, although there are one or three different amino acid sequences (Figure 4).

2.5. Correlation between Structural Change and Substitution Rates in the Plastid-Encoded clpP Gene

The phylogenetic distribution of clpP content in the selected Caprifoliaceae plastomes showed that this gene had been pseudogenized multiple times in the family (Figure 5A). BlastN searches using the plastid-encoded clpP from Viburnum identified partial exons or introns of clpP in Pterocephalus, Triplostegia, Heptacodium, Weigela, Linnaeoideae, Morinoideae, and Zabeliaoideae, which lacked a conserved domain (Figure 5). Only Dipsacus, Scabiosa, Fedia, Leycesteria, Nardostachys, Symphoricarpos, Valerianella, Valeriana, Lonicera, and Triosteum plastomes contain clpP genes that differ in intron content (Figure 5A,B). Similar to the Valerianella clpP gene, the clpP gene of Leycesteria, Lonicera, Symphoricarpos, and Triosterum are missing both introns. The phylogenetic distribution indicated that the loss of the second intron occurred in the Fedia/Valerianella clade (Figure 5).
To examine the correlation between structural changes and substitution rates, dN and dS were calculated using the 16 clpP gene sequences available (Figure 5C). Seven branches with dN/dS values >1 were detected. However, LRTs revealed that only four branches, Fedia/Valerianella/Valeriana/Nardostachys, Valerianoideae, Lonicera, and Caprifolioideae, were significantly under positive selection (p < 0.00001 after Bonferroni correction). The RELAX analysis indicated that clpP experienced significantly intensified selection in the analyzed Caprifoliaceae s.l. (k = 2.62, p = 0.001, LR = 10.96). Additional analyses indicated that the intronless clpP gene experienced significantly intensified selection in the subfamily Caprifolioideae (k = 3.18, p = 0.000, LR = 16.06). In Fedia and Valerianella, the clpP gene showed intensified selection, but this was not significant (k = 1.33, p = 0.443, LR = 0.59).

3. Discussion

Sequencing of the F. cornucopiae, V. fauriei, V. locusta, D. japonicus, and S. comosa plastomes revealed that they are distinct from the published plastomes of Caprifoliaceae s.l. These plastomes exhibit dynamic changes in structure, gene and intron content, and lineage-specific rate acceleration. Our results also showed a correlation between mutation rates and structural variation in the accD and clpP genes across Caprifoliaceae s.l. and interruption of the accD gene in the genus Valeriana. The loss of plastid-encoded rps15 was observed in D. japonicus and S. comosa plastomes. Comparative analysis of the gene content among Caprifoliaceae s.l. plastomes suggests the rps15 is lost in the common ancestor of Dipsacus, Scabiosa, and Pterocephalus. Functional replacement of the rps15 from plastid to the nucleus occurs in these lineages, although we found evidence for a plastid-to-nucleus gene transfer in the Dipsacus transcriptome. Additional nuclear transcriptome data for Scabiosa and Pterocephalus are needed for further investigation.
With the five plastomes, we selected the published plastomes of 19 additional genera from Caprifoliaceae s.l. and five genera from Adoxaceae to reconstruct the ancestral plastome in Caprifoliaceae s.l. and rearrangement events in each genus. Our results revealed three synapomorphic events (one inversion and two contractions of IR) in the Caprifoliaceae s.l. (Figure 1). A previous study showed that the inversion associated with the ndhF gene occurred in Adoxaceae [34]. However, compared with angiosperm plastomes, the ancestral Caprifoliaceae s.l. plastome has an inversion between ndhF. The comparison of the LCBs and IR boundary shift models indicates that independent events occurred in a lineage- or species-specific manner (Figure 1). The model suggests that the IR expansion and contraction were the main mechanisms for changes in gene order in F. cornucopiae, V. fauriei, and V. locusta plastomes. A double-strand break, followed by strand invasion, expansion, and recombination in IR [37], is a potential mechanism for IR expansion in the family. After ancestral inversion, one additional inversion event associated with ccsA-trnL-rpl32-ndhF was identified, unique to Scabiosa. Dispersed repeats can cause inversions and there is a correlation between the number of repeats and plastome rearrangements [6,38]. However, Scabiosa has a small number of repeats in the family, although it has a relatively large number of repeats compared to the outgroups (Table S1). Moreover, dispersed repeat sequences were not found surrounding the inversion block in the Scabiosa plastome, which suggests that a different mechanism of inversion may be involved. Several ebb-and-flow expansions and contractions were also observed in the analyzed Caprifoliaceae s.l. plastomes.
Lineage-specific variation occurred in the Caprifoliaceae s.l. plastomes, showing that Fedia, Valeriana, and Valerianella had significantly accelerated dS in comparison (Figure 2). Structural rearrangements have contributed to higher substitution rates [5,14]. Compared with the analyzed Caprifoliaceae s.l. plastomes, the three plastomes showed increased levels of structural divergence (Figure 1). However, IR boundary shifts are the main mechanisms responsible for the genomic changes in this lineage. This variation appears to result from genome-wide acceleration, arguing against localized hypermutation, mutagenic retroprocessing, and pseudogenization [16]. Mutated and changed DNA-RRR machinery could be a potential mechanism to explain these phenomena. In the subfamily Valerianoideae, expanded plastome sequencing and examination of organellar-targeted DNA-RRR genes would be needed to explain the causes and consequences of fast-evolving plastomes in this lineage.
Our analysis showed that interrupted accD evolution has occurred repeatedly across Caprifoliaceae s.l. (Figure 3). Many plastomes show that the accD is unrecognized and unannotated because of its extreme divergence [34,36]. In these cases, the accD appears to have been lost and possibly transferred to the nucleus, as intracellular gene transfer (IGT) is an ongoing process in angiosperms [3]. Evidence supports the functional replacement of the accD gene by gene transfer to the nucleus or gene substitution of nuclear homologs, including coexistence of the nuclear-encoded, plastid-targeted eukaryotic ACCase, prokaryotic ACCase, and the plastid-encoded accD [15,16,20,22,23,24,25,26]. However, previous studies have provided some evidence that highly divergent or truncated accD genes may be functional in plastids. For example, the divergent copy of the plastid-encoded accD in Lamprocapnos spectabilis (L.) Fukuhara (Papaveraceae) is transcribed [9]. A functional replacement of truncated accD to the nucleus was found in Trachelium caeruleum L. (Campanulaceae) [20], Hypseocharis bilobata Killip, and Monsonia emarginata (L.f.) L’Hér. (Geraniaceae) [15]. In the selected Caprifoliaceae s.l. plastomes, many accD reading frames remain intact, and several have truncated with the catalytic sites, which suggests that it probably encodes a functional protein. The intensity of both purifying (most branches are dN/dS < 1) and positive selection (three branches, Fedia/Valerianella, Triplostegia, and Zabelia with dN/dS > 1) also indicated that the accD is under selective constraint or adaptive changes. To fully understand the evolution of ACCase among Caprifoliaceae s.l. genomes, searching the assembled nuclear transcriptomes are required. In addition, we examined the variability of the AAR motifs in the accD gene and found evidence of intraspecific length variation in the Valeriana. One possible mechanism for length polymorphism is replication slippage and recombination [39]. Similar patterns of the accD gene have been previously shown to have repetitive amino acid sequence motifs in L. spectabilis [9] and Medicago truncatula Gaertn. [40]. Gurdon and Maliga [40] suggested that repetitive amino acid motifs within the accD could be recombinationally driven.
Previous studies have shown that multiple lineages experience independent loss [7,14,41,42,43]. Some cases of losses are because the clpP gene is unrecognized and unannotated owing to high divergence including structural changes. For example, the clpP gene was annotated as a loss in the five Actinidiaceae plastomes [42,43], but the plastomes contain clpP-like ORFs that are missing the two introns with a completely conserved domain. The lack of internal stop codons or frameshifts suggests the functionality of the plastid-encoded clpP. The Geranium and Monsonia clpP genes were annotated as pseudogenes or losses [7,14]; however, extremely divergent and intronless clpP ORFs are found in the lineages [15]. ClpP is involved in important chloroplast processes [32]. If the divergent ORFs are pseudogenes, functional replacement by gene transfer or gene substitution must occur. However, the evolutionary fate of the plastid-encoded clpP loss has not been reported in angiosperms. We identified at least six potential pseudogenizations of clpP and independent losses of one or both introns in the analyzed Caprifoliaceae s.l. (Figure 5). Using the conserved domain sequence of clpP as a query, we did not find any evidence of divergent clpP-like ORFs that were intact. Only two lineages contained the clpP gene, showing dynamics in intron content. The phylogenetic distribution showed the evolutionary history of intron loss events in the subfamily Valerianoideae, in which the loss of the second intron in the common ancestor of Fedia and Valerianella, followed by loss of the first intron independently in Valerianella (Figure 5). The subfamily Caprifolioideae plastome contains intronless clpP, but the history of loss events is unclear from the present data. Direct genomic deletion, exonization of introns, retroprocessing, and gene conversion with foreign copies are possible mechanisms of intron loss [44]. Intensified positive or negative selection in the clpP gene among Caprifoliaceae s.l. suggests that positive selection may act on the nuclear-encoded plastid-targeted genes. Cytonuclear coevolution between plastid- and nuclear-encoded subunits in Caprifoliaceae s.l. should be explored to test this hypothesis.

4. Materials and Methods

4.1. Genome Sequencing, Assembly, and Annotation

Total genomic DNA (gDNA) from V. fauriei and V. locusta from the subfamily Vaerianoideae, as well as D. japonicus, and S. comosa from the subfamily Dipsacoideae (Table S5), were isolated from fresh leaf tissues of a single individual using the Exgene Plant SV Mini Kit (GeneAll, Seoul, South Korea) following the manufacturer’s protocol. The gDNA of F. cornucopiae was provided by the Royal Botanic Gardens Kew DNA and Tissue Collection (Table S5). The gDNAs were sequenced using an Illumina Hiseq2500 sequencing platform (Illumina, San Diego, CA, USA), generating 6 Gb of 150 bp paired-end (PE) reads from a 550 bp insert library.
The PE reads were assembled de novo using Velvet v1.2.10 [45] using multiple k-mers (99 to 141). For each plastome, the longest contigs that reflected a complete plastome with only one copy of the IR were aligned manually, and the consensus was taken as the final genome sequence. Finished plastomes were annotated using a BLAST-like algorithm in Geneious Prime 2021.1.1 (www.geneious.com, accessed on 18 August 2021) with the genes of Nicotiana tabacum L. plastome (NC_001879) as the reference, and the open reading frames (ORFs) were confirmed using the “Find ORFs” option. Circular plastome maps were drawn using OrganellarGenomeDRAW (OGDRAW) v1.3.1 (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html, accessed on 18 August 2021) [46]. The plastomes were deposited in GenBank (accession numbers MZ934745-MZ934749).

4.2. Comparative Analyses

Repetitive DNA sequences in each plastome were identified by performing “blastn” searches using BLAST + v2.6.0 [47] against itself, with a word size of 11, an e-value of 1 × 10−6. The newly sequenced plastomes and the 19 published Caprifoliaceae s.l. plastomes were aligned with the outgroup Viburnum betulifolium Batalin from Adoxaceae using the “progressiveMauve” algorithm in Mauve v2.3.1 [48] in Geneious Prime. The National Center for Biotechnology Information (NCBI) Conserved Domain Database (CDD) v3.19 was used for functional domain annotation (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 18 August 2021) [49]. Transcriptome from Dipsacus asperoides C.Y.Cheng & T.M.Ai was assembled de novo with Trinity [50] using the Sequence Read Archive (SRA) (SRR2043985). The potential nuclear-encoded transcript was identified in the transcriptome by using “blastn” (e-value cutoff of 1 × 10−10) with the plastid-encoded rps15 gene sequence from Triplostegia glandulifera Wall. ex DC. as a query. Chloroplast transit peptide (cTP) was predicted by TargetP v1.1 [51].

4.3. Estimation of Substitution Rates

In total, 72 plastid protein-coding genes shared by all selected 29 taxa from newly sequenced plastomes and from the published plastomes were sampled (Table S1). Individual genes were aligned using the back-translation method with MAFFT [52] in Geneious Prime. The constraint tree was generated using the maximum likelihood method in IQ-TREE v2.1.2 [53] with concatenated sequence alignment. To estimate the rates of nucleotide substitution, all genes and the concatenated sequences were analyzed individually. The nonsynonymous (dN) and synonymous (dS) substitution rates for datasets were calculated in PAML v4.8 [54] with the constraint tree. Codon frequencies were estimated using the F3 × 4 model. LRTs were performed in Hyphy v2.5.23(MP) [55] to test dN/dS changes using the MG94xREV codon model. To test for potential relaxed selection, the RELAX [56] implemented in HyPhy was used on the Datamonkey Adaptive Evolution Server (https://www.datamonkey.org/, accessed on 18 August 2021) [57].

4.4. Survey of Variability in the Plastid-Encoded accD Gene

To examine length variation in the accD gene at the inter- and intraspecific levels, 38 V. fauriei individuals and 12 V. sambucifolia f. dageletiana individuals were sampled (Table S5). The gDNAs were extracted from the fresh leaves or herbarium specimens using the GeneAll Kit, or the methods described by Allen et al. [58]. Variable regions in the accD gene were amplified by PCR using specific primers designed with Primer3 in Geneious Prime (64F: 5′-AACTCTTATGATTCGGTTTCTCGT-3′ and 1328R: 5′-ATACCGGTTTGAATAGCCTCAGTT-3′). Each reaction was 50 μL in volume, including 38.75 μL of distilled water, 5 μL of 10 × Taq Reaction Buffer, 1 μL of dNTPs (10 mM), 0.25 μL of DiaStarTM Taq polymerase (5 units/μL, Solgent Co., Daejeon, Korea), 1 μL of each primer (10 pmole/μL), and 1 μL of total gDNA (20 ng). All reactions consisted of included an initial denaturation step (95 °C for 2 min), 35 cycles of denaturation (95  °C for 20 s), annealing (60  °C for 40 s), and extension (72  °C for 1 min 30 s), followed by a final extension (72  °C for 5 min). The PCR products were purified using a PCR purification kit (MGmed, Korea) according to the manufacturer’s protocol. Sequencing of PCR products was carried out using an ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA) at Solgent Co. The nucleotide sequences of the plastid accD copies were aligned using MUSCLE [59] in Geneious Prime.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/ijms221910485/s1, Figure S1: Maps for the newly sequenced plastomes, Figure S2: Nucleotide and amino acid sequences of the nuclear-encoded RPS15 gene from Dipsacus, Figure S3: Duplication of the trnE-UUC gene in Dipsacus japonicus plastome, Figure S4: Structural alignments of Caprifoliaceae s.l. plastomes using Mauve. Figure S5: Amino acid sequence alignments of the plastid-encoded accD of Caprifoliaceae s.l., Figure S6. Boxplots of the values of nonsynonymous and synonymous substitution rates of the plastid-encoded accD for Caprifoliaceae s.l. and outgroups, Table S1: GenBank accession numbers for taxa used in this study, Table S2: Pairwise Wilcoxon rank-sum tests of dN and dS values among plastid genes within Caprifoliaceae s.l., Table S3: Positive selection on Caprifoliaceae s.l. plastid genes, Table S4: CD-search results of the plastid-encoded accD gene from Caprifoliaceae s.l., Table S5: Material information and GenBank accession numbers for length variation in the plastid-encoded accD gene.

Author Contributions

Conceptualization, S.P. (Seongjun Park) and S.P. (SeonJoo Park); validation, S.P. (Seongjun Park); formal analysis, S.P. (Seongjun Park); investigation, M.J. and S.P. (Sunmi Park); data curation, S.P. (SeonJoo Park) and S.P. (Sunmi Park); writing—original draft preparation, S.P. (Seongjun Park); writing—review and editing, S.P. (Seongjun Park), M.J., S.P. (Sunmi Park), and S.P. (SeonJoo Park); visualization, S.P. (Seongjun Park); supervision, S.P. (SeonJoo Park); project administration, S.P. (SeonJoo Park); funding acquisition, S.P. (SeonJoo Park). All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the 2021 Yeungnam University Grant, Grant Number 221A061009.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available in a publicly accessible repository. GenBank accession numbers for the new sequences are MZ934745-MZ934749 and MZ954788-MZ954837.

Acknowledgments

We thank Eun Mi Lee for providing living materials of Valeriana sambucifolia f. dageletiana.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Ruhlman, T.A.; Jansen, R.K. The plastid genomes of flowering plants. Methods Mol. Biol. 2014, 1132, 3–38. [Google Scholar] [CrossRef] [PubMed]
  2. Jansen, R.K.; Cai, Z.; Raubeson, L.A.; Daniell, H.; dePamphilis, C.W.; Leebens-Mack, J.; Müller, K.F.; Guisinger-Bellian, M.; Haberle, R.C.; Hansen, A.K.; et al. Analysis of 81 genes from 64 plastid genomes resolves relationships in angiosperms and identifies genome-scale evolutionary patterns. Proc. Natl. Acad. Sci. USA 2007, 104, 19369–19374. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Timmis, J.N.; Ayliffe, M.A.; Huang, C.Y.; Martin, W. Endosymbiotic gene transfer: Organelle genomes forge eukaryotic chromosomes. Nat. Rev. Genet. 2004, 5, 123–135. [Google Scholar] [CrossRef] [PubMed]
  4. Cosner, M.E.; Raubeson, L.A.; Jansen, R.K. Chloroplast DNA rearrangements in Campanulaceae: Phylogenetic utility of highly rearranged genomes. BMC Evol. Biol. 2004, 4, 1–17. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Sloan, D.B.; Triant, D.A.; Forrester, N.J.; Bergner, L.M.; Wu, M.; Taylor, D.R. A recurring syndrome of accelerated plastid genome evolution in the angiosperm tribe Sileneae (Caryophyllaceae). Mol. Phylogenetics Evol. 2014, 72, 82–89. [Google Scholar] [CrossRef] [PubMed]
  6. Cai, Z.; Guisinger, M.; Kim, H.-G.; Ruck, E.; Blazier, J.C.; McMurtry, V.; Kuehl, J.V.; Boore, J.; Jansen, R.K. Extensive reorganization of the plastid genome of Trifolium subterraneum (Fabaceae) is associated with numerous repeated sequences and novel DNA insertions. J. Mol. Evol. 2008, 67, 696–704. [Google Scholar] [CrossRef] [PubMed]
  7. Guisinger, M.M.; Kuehl, J.V.; Boore, J.L.; Jansen, R.K. Extreme reconfiguration of plastid genomes in the angiosperm family Geraniaceae: Rearrangements, repeats, and codon usage. Mol. Biol. Evol. 2011, 28, 583–600. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  8. Lee, H.-L.; Jansen, R.K.; Chumley, T.W.; Kim, K.-J. Gene relocations within chloroplast genomes of Jasminum and Menodora (Oleaceae) are due to multiple, overlapping inversions. Mol. Biol. Evol. 2007, 24, 1161–1180. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  9. Park, S.; An, B.; Park, S. Reconfiguration of the plastid genome in Lamprocapnos spectabilis: IR boundary shifting, inversion, and intraspecific variation. Sci. Rep. 2018, 8, 13568. [Google Scholar] [CrossRef] [Green Version]
  10. Xu, X.; Wang, D. Comparative Chloroplast Genomics of Corydalis Species (Papaveraceae): Evolutionary Perspectives on Their Unusual Large Scale Rearrangements. Front. Plant Sci. 2021, 11. [Google Scholar] [CrossRef]
  11. Guisinger, M.M.; Kuehl, J.V.; Boore, J.L.; Jansen, R.K. Genome-wide analyses of Geraniaceae plastid DNA reveal unprecedented patterns of increased nucleotide substitutions. Proc. Natl. Acad. Sci. USA 2008, 105, 18424–18429. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  12. Schwarz, E.N.; Ruhlman, T.A.; Weng, M.-L.; Khiyami, M.A.; Sabir, J.S.M.; Hajarah, N.H.; Alharbi, N.S.; Rabah, S.O.; Jansen, R.K. Plastome-Wide Nucleotide Substitution Rates Reveal Accelerated Rates in Papilionoideae and Correlations with Genome Features Across Legume Subfamilies. J. Mol. Evol. 2017, 84, 187–203. [Google Scholar] [CrossRef] [PubMed]
  13. Shrestha, B.; Weng, M.-L.; Theriot, E.C.; Gilbert, L.E.; Ruhlman, T.A.; Krosnick, S.E.; Jansen, R.K. Highly accelerated rates of genomic rearrangements and nucleotide substitutions in plastid genomes of Passiflora subgenus Decaloba. Mol. Phylogenetics Evol. 2019, 138, 53–64. [Google Scholar] [CrossRef]
  14. Weng, M.-L.; Blazier, J.C.; Govindu, M.; Jansen, R.K. Reconstruction of the ancestral plastid genome in Geraniaceae reveals a correlation between genome rearrangements, repeats, and nucleotide substitution rates. Mol. Biol. Evol. 2014, 31, 645–659. [Google Scholar] [CrossRef] [Green Version]
  15. Park, S.; Ruhlman, T.A.; Weng, M.-L.; Hajrah, N.H.; Sabir, J.S.M.; Jansen, R.K. Contrasting Patterns of Nucleotide Substitution Rates Provide Insight into Dynamic Evolution of Plastid and Mitochondrial Genomes of Geranium. Genome Biol. Evol. 2017, 9, 1766–1780. [Google Scholar] [CrossRef]
  16. Magee, A.M.; Aspinall, S.; Rice, D.W.; Cusack, B.P.; Sémon, M.; Perry, A.S.; Stefanović, S.; Milbourne, D.; Barth, S.; Palmer, J.D.; et al. Localized hypermutation and associated gene losses in legume chloroplast genomes. Genome Res. 2010, 20, 1700–1710. [Google Scholar] [CrossRef] [Green Version]
  17. Blazier, J.C.; Ruhlman, T.A.; Weng, M.-L.; Rehman, S.K.; Sabir, J.S.M.; Jansen, R.K. Divergence of RNA polymerase α subunits in angiosperm plastid genomes is mediated by genomic rearrangement. Sci. Rep. 2016, 6, 24595. [Google Scholar] [CrossRef] [Green Version]
  18. Barnard-Kubow, K.B.; Sloan, D.B.; Galloway, L.F. Correlation between sequence divergence and polymorphism reveals similar evolutionary mechanisms acting across multiple timescales in a rapidly evolving plastid genome. BMC Evol. Biol. 2014, 14, 268. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  19. Parkinson, C.L.; Mower, J.P.; Qiu, Y.-L.; Shirk, A.J.; Song, K.; Young, N.D.; Claude, W.d.; Palmer, J.D. Multiple major increases and decreases in mitochondrial substitution rates in the plant family Geraniaceae. BMC Evol. Biol. 2005, 5, 1–12. [Google Scholar] [CrossRef] [Green Version]
  20. Rousseau-Gueutin, M.; Huang, X.; Higginson, E.; Ayliffe, M.; Day, A.; Timmis, J.N. Potential Functional Replacement of the Plastidic Acetyl-CoA Carboxylase Subunit (accD) Gene by Recent Transfers to the Nucleus in Some Angiosperm Lineages. Plant Physiol. 2013, 161, 1918–1929. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  21. Molina, J.; Hazzouri, K.M.; Nickrent, D.; Geisler, M.; Meyer, R.S.; Pentony, M.M.; Flowers, J.M.; Pelser, P.; Barcelona, J.; Inovejas, S.A.; et al. Possible Loss of the Chloroplast Genome in the Parasitic Flowering Plant Rafflesia lagascae (Rafflesiaceae). Mol. Biol. Evol. 2014, 31, 793–803. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Sabir, J.; Schwarz, E.; Ellison, N.; Zhang, J.; Baeshen, N.A.; Mutwakil, M.; Jansen, R.; Ruhlman, T. Evolutionary and biotechnology implications of plastid genome variation in the inverted-repeat-lacking clade of legumes. Plant Biotechnol. J. 2014, 12, 743–754. [Google Scholar] [CrossRef] [PubMed]
  23. Babiychuk, E.; Vandepoele, K.; Wissing, J.; Garcia-Diaz, M.; De Rycke, R.; Akbari, H.; Joubès, J.; Beeckman, T.; Jänsch, L.; Frentzen, M.; et al. Plastid gene expression and plant development require a plastidic protein of the mitochondrial transcription termination factor family. Proc. Natl. Acad. Sci. USA 2011, 108, 6674–6679. [Google Scholar] [CrossRef] [Green Version]
  24. Schulte, W.; Töpfer, R.; Stracke, R.; Schell, J.; Martini, N. Multi-functional acetyl-CoA carboxylase from Brassica napus is encoded by a multi-gene family: Indication for plastidic localization of at least one isoform. Proc. Natl. Acad. Sci. USA 1997, 94, 3465–3470. [Google Scholar] [CrossRef] [Green Version]
  25. Konishi, T.; Shinohara, K.; Yamada, K.; Sasaki, Y. Acetyl-CoA Carboxylase in Higher Plants: Most Plants Other Than Gramineae Have Both the Prokaryotic and the Eukaryotic Forms of This Enzyme. Plant Cell Physiol. 1996, 37, 117–122. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Gornicki, P.; Faris, J.; King, I.; Podkowinski, J.; Gill, B.; Haselkorn, R. Plastid-localized acetyl-CoA carboxylase of bread wheat is encoded by a single gene on each of the three ancestral chromosome sets. Proc. Natl. Acad. Sci. USA 1997, 94, 14179–14184. [Google Scholar] [CrossRef] [Green Version]
  27. Rockenbach, K.; Havird, J.C.; Monroe, J.G.; Triant, D.A.; Taylor, D.R.; Sloan, D.B. Positive Selection in Rapidly Evolving Plastid–Nuclear Enzyme Complexes. Genetics 2016, 204, 1507–1522. [Google Scholar] [CrossRef] [PubMed]
  28. Williams, A.M.; Friso, G.; van Wijk, K.J.; Sloan, D.B. Extreme variation in rates of evolution in the plastid Clp protease complex. Plant J. 2019, 98, 243–259. [Google Scholar] [CrossRef]
  29. White, S.W.; Zheng, J.; Zhang, Y.M.; Rock, C.O. The structural biology of type II fatty acid biosynthesis. Annu. Rev. Biochem. 2005, 74, 791–831. [Google Scholar] [CrossRef]
  30. Wakil, S.J.; Stoops, J.K.; Joshi, V.C. Fatty acid synthesis and its regulation. Annu. Rev. Biochem. 1983, 52, 537–579. [Google Scholar] [CrossRef]
  31. Adam, Z.; Adamska, I.; Nakabayashi, K.; Ostersetzer, O.; Haussuhl, K.; Manuell, A.; Zheng, B.; Vallon, O.; Rodermel, S.R.; Shinozaki, K.; et al. Chloroplast and Mitochondrial Proteases in Arabidopsis. A Proposed Nomenclature1. Plant Physiol. 2001, 125, 1912–1918. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  32. Shikanai, T.; Shimizu, K.; Ueda, K.; Nishimura, Y.; Kuroiwa, T.; Hashimoto, T. The Chloroplast clpP Gene, Encoding a Proteolytic Subunit of ATP-Dependent Protease, is Indispensable for Chloroplast Development in Tobacco. Plant Cell Physiol. 2001, 42, 264–273. [Google Scholar] [CrossRef] [PubMed]
  33. Christenhusz, M.J.; Byng, J.W. The number of known plants species in the world and its annual increase. Phytotaxa 2016, 261, 201–217. [Google Scholar] [CrossRef] [Green Version]
  34. Wang, H.-X.; Liu, H.; Moore, M.J.; Landrein, S.; Liu, B.; Zhu, Z.-X.; Wang, H.-F. Plastid phylogenomic insights into the evolution of the Caprifoliaceae s.l. (Dipsacales). Mol. Phylogenetics Evol. 2020, 142, 106641. [Google Scholar] [CrossRef] [PubMed]
  35. Wang, H.-X.; Morales-Briones, D.F.; Moore, M.J.; Wen, J.; Wang, H.-F. A phylogenomic perspective on gene tree conflict and character evolution in Caprifoliaceae using target enrichment data, with Zabelioideae recognized as a new subfamily. J. Syst. Evol. 2021, 59, 897–914. [Google Scholar] [CrossRef]
  36. Fan, W.-B.; Wu, Y.; Yang, J.; Shahzad, K.; Li, Z.-H. Comparative Chloroplast Genomics of Dipsacales Species: Insights Into Sequence Variation, Adaptive Evolution, and Phylogenetic Relationships. Front. Plant Sci. 2018, 9. [Google Scholar] [CrossRef] [Green Version]
  37. Goulding, S.E.; Wolfe, K.; Olmstead, R.; Morden, C. Ebb and flow of the chloroplast inverted repeat. Mol. Gen. Genet. MGG 1996, 252, 195–206. [Google Scholar] [CrossRef]
  38. Haberle, R.C.; Fourcade, H.M.; Boore, J.L.; Jansen, R.K. Extensive rearrangements in the chloroplast genome of Trachelium caeruleum are associated with repeats and tRNA genes. J. Mol. Evol. 2008, 66, 350–361. [Google Scholar] [CrossRef]
  39. Gemayel, R.; Vinces, M.D.; Legendre, M.; Verstrepen, K.J. Variable Tandem Repeats Accelerate Evolution of Coding and Regulatory Sequences. Annu. Rev. Genet. 2010, 44, 445–477. [Google Scholar] [CrossRef] [Green Version]
  40. Gurdon, C.; Maliga, P. Two Distinct Plastid Genome Configurations and Unprecedented Intraspecies Length Variation in the accD Coding Region in Medicago truncatula. DNA Res. 2014, 21, 417–427. [Google Scholar] [CrossRef] [Green Version]
  41. Erixon, P.; Oxelman, B. Whole-Gene Positive Selection, Elevated Synonymous Substitution Rates, Duplication, and Indel Evolution of the Chloroplast clpP1 Gene. PLoS ONE 2008, 3, e1386. [Google Scholar] [CrossRef] [Green Version]
  42. Wang, W.-C.; Chen, S.-Y.; Zhang, X.-Z. Chloroplast Genome Evolution in Actinidiaceae: clpP Loss, Heterogenous Divergence and Phylogenomic Practice. PLoS ONE 2016, 11, e0162324. [Google Scholar] [CrossRef]
  43. Yao, X.; Tang, P.; Li, Z.; Li, D.; Liu, Y.; Huang, H. The First Complete Chloroplast Genome Sequences in Actinidiaceae: Genome Structure and Comparative Analysis. PLoS ONE 2015, 10, e0129347. [Google Scholar] [CrossRef] [PubMed]
  44. Hepburn, N.J.; Schmidt, D.W.; Mower, J.P. Loss of two introns from the Magnolia tripetala mitochondrial cox2 gene implicates horizontal gene transfer and gene conversion as a novel mechanism of intron loss. Mol. Biol. Evol. 2012, 29, 3111–3120. [Google Scholar] [CrossRef] [Green Version]
  45. Zerbino, D.R.; Birney, E. Velvet: Algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008, 18, 821–829. [Google Scholar] [CrossRef] [Green Version]
  46. 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] [PubMed] [Green Version]
  47. Camacho, C.; Coulouris, G.; Avagyan, V.; Ma, N.; Papadopoulos, J.; Bealer, K.; Madden, T.L. BLAST+: Architecture and applications. BMC Bioinform. 2009, 10, 421. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  48. Darling, A.C.E.; Mau, B.; Blattner, F.R.; Perna, N.T. Mauve: Multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 2004, 14, 1394–1403. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  49. Marchler-Bauer, A.; Lu, S.; Anderson, J.B.; Chitsaz, F.; Derbyshire, M.K.; DeWeese-Scott, C.; Fong, J.H.; Geer, L.Y.; Geer, R.C.; Gonzales, N.R.; et al. CDD: A Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res. 2010, 39, D225–D229. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  50. Grabherr, M.G.; Haas, B.J.; Yassour, M.; Levin, J.Z.; Thompson, D.A.; Amit, I.; Adiconis, X.; Fan, L.; Raychowdhury, R.; Zeng, Q.; et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 2011, 29, 644–652. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  51. Emanuelsson, O.; Brunak, S.; von Heijne, G.; Nielsen, H. Locating proteins in the cell using TargetP, SignalP and related tools. Nat. Protoc. 2007, 2, 953–971. [Google Scholar] [CrossRef] [PubMed]
  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] [PubMed] [Green Version]
  53. Minh, B.Q.; Schmidt, H.A.; Chernomor, O.; Schrempf, D.; Woodhams, M.D.; von Haeseler, A.; Lanfear, R. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol. Biol. Evol. 2020, 37, 1530–1534. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  54. Yang, Z. PAML 4: Phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 2007, 24, 1586–1591. [Google Scholar] [CrossRef] [Green Version]
  55. Kosakovsky Pond, S.L.; Poon, A.F.Y.; Velazquez, R.; Weaver, S.; Hepler, N.L.; Murrell, B.; Shank, S.D.; Magalis, B.R.; Bouvier, D.; Nekrutenko, A.; et al. HyPhy 2.5—A Customizable Platform for Evolutionary Hypothesis Testing Using Phylogenies. Mol. Biol. Evol. 2019, 37, 295–299. [Google Scholar] [CrossRef]
  56. Wertheim, J.O.; Murrell, B.; Smith, M.D.; Kosakovsky Pond, S.L.; Scheffler, K. RELAX: Detecting Relaxed Selection in a Phylogenetic Framework. Mol. Biol. Evol. 2014, 32, 820–832. [Google Scholar] [CrossRef] [Green Version]
  57. Weaver, S.; Shank, S.D.; Spielman, S.J.; Li, M.; Muse, S.V.; Kosakovsky Pond, S.L. Datamonkey 2.0: A Modern Web Application for Characterizing Selective and Other Evolutionary Processes. Mol. Biol. Evol. 2018, 35, 773–777. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  58. Allen, G.; Flores-Vergara, M.; Krasynanski, S.; Kumar, S.; Thompson, W. A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nat. Protoc. 2006, 1, 2320–2325. [Google Scholar] [CrossRef]
  59. Edgar, R.C. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004, 32, 1792–1797. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Ijms 22 10485 g001 550
Figure 1. Plastome rearrangement in the analyzed Caprifoliaceae s.l. Schematic diagrams (gray open boxes) of the genomic regions surrounding the inverted repeat (IR) region. Genes drawn below the horizontal line indicate sequences found in an inverted orientation. Gray shadings indicate the IRA region. The hypothetical models for IR expansion and contraction in the common ancestor of Fedia, Valerianella, and Valeriana are illustrated (orange boxes).
Figure 1. Plastome rearrangement in the analyzed Caprifoliaceae s.l. Schematic diagrams (gray open boxes) of the genomic regions surrounding the inverted repeat (IR) region. Genes drawn below the horizontal line indicate sequences found in an inverted orientation. Gray shadings indicate the IRA region. The hypothetical models for IR expansion and contraction in the common ancestor of Fedia, Valerianella, and Valeriana are illustrated (orange boxes).
Ijms 22 10485 g001
Ijms 22 10485 g002 550
Figure 2. Plastid sequence divergence among the selected Caprifoliaceae s.l. Plastid phylograms of nonsynonymous (dN) and synonymous (dS) substitution rates based on 72 plastid genes. Boxplots of the values of dN and dS for individual genes. The box represents values between quartiles, solid lines extend to the minimum and maximum values, outliers are shown as circles and vertical lines in boxes show median values.
Figure 2. Plastid sequence divergence among the selected Caprifoliaceae s.l. Plastid phylograms of nonsynonymous (dN) and synonymous (dS) substitution rates based on 72 plastid genes. Boxplots of the values of dN and dS for individual genes. The box represents values between quartiles, solid lines extend to the minimum and maximum values, outliers are shown as circles and vertical lines in boxes show median values.
Ijms 22 10485 g002
Ijms 22 10485 g003 550
Figure 3. Rapid structural evolution of the accD gene. Phylograms show nonsynonymous (dN) and synonymous (dS) substitution rates for the accD among the analyzed Caprifoliaceae s.l. with outgroups. Scale bar indicates the number of substitutions per site. Gray boxes indicate the predicted open reading frames (ORFs). Red rectangles indicate the conserved domain in the ORFs. Asterisks indicate that the ORF has an alternative start codon. Branch lengths are drawn to the same scale based on dN and dS substitutions per site. Branches with significantly higher dN/dS ratios, as determined by likelihood ratio test are marked with asterisks (*, p < 0.05; ***, p < 0.001 after Bonferroni correction).
Figure 3. Rapid structural evolution of the accD gene. Phylograms show nonsynonymous (dN) and synonymous (dS) substitution rates for the accD among the analyzed Caprifoliaceae s.l. with outgroups. Scale bar indicates the number of substitutions per site. Gray boxes indicate the predicted open reading frames (ORFs). Red rectangles indicate the conserved domain in the ORFs. Asterisks indicate that the ORF has an alternative start codon. Branch lengths are drawn to the same scale based on dN and dS substitutions per site. Branches with significantly higher dN/dS ratios, as determined by likelihood ratio test are marked with asterisks (*, p < 0.05; ***, p < 0.001 after Bonferroni correction).
Ijms 22 10485 g003
Ijms 22 10485 g004 550
Figure 4. Length variation in the plastid accD of Valeriana. Schematic diagram of the genomic regions surrounding the accD from three Valeriana species. Gray boxes indicate the predicted open reading frames (ORFs). Red rectangles indicate the conserved domain in the ORFs. Dashed boxes indicate two hotspot regions in the accD gene. Each amino acid sequence of the two hotspot regions of the accD copies from 52 Valeriana individuals. Blue and green boxes indicate amino acid repeat (AAR) motifs. Asterisks indicate an amino acid sequence mismatch of the AARs (ESTTTESFAQR and SDSEEDLIKPD), respectively. V. fauriei: HB, MS, SBA, and SBI; V. sambucifolia f. dageletiana: UL.
Figure 4. Length variation in the plastid accD of Valeriana. Schematic diagram of the genomic regions surrounding the accD from three Valeriana species. Gray boxes indicate the predicted open reading frames (ORFs). Red rectangles indicate the conserved domain in the ORFs. Dashed boxes indicate two hotspot regions in the accD gene. Each amino acid sequence of the two hotspot regions of the accD copies from 52 Valeriana individuals. Blue and green boxes indicate amino acid repeat (AAR) motifs. Asterisks indicate an amino acid sequence mismatch of the AARs (ESTTTESFAQR and SDSEEDLIKPD), respectively. V. fauriei: HB, MS, SBA, and SBI; V. sambucifolia f. dageletiana: UL.
Ijms 22 10485 g004
Ijms 22 10485 g005 550
Figure 5. Structural evolution of the clpP gene: (A) phylogenetic distribution of pseudogene or intron loss among the selected Caprifoliaceae s.l. and five outgroups. Arrowheads indicate the positions of the first (green) and second (blue) intron; (B) schematic diagram of the structure of clpP. Pink boxes indicate the conserved domain of caseinolytic protease; (C) phylograms showing nonsynonymous (dN) and synonymous (dS) substitution rates for the clpP genes among 16 species that have intact gene sequences. Branch lengths are drawn to the same scale based on dN and dS substitutions per site. Branches with significantly higher dN/dS ratios determined by likelihood ratio test are marked with asterisks (*, p < 0.05; **, p < 0.01; ***, p < 0.001 after Bonferroni correction).
Figure 5. Structural evolution of the clpP gene: (A) phylogenetic distribution of pseudogene or intron loss among the selected Caprifoliaceae s.l. and five outgroups. Arrowheads indicate the positions of the first (green) and second (blue) intron; (B) schematic diagram of the structure of clpP. Pink boxes indicate the conserved domain of caseinolytic protease; (C) phylograms showing nonsynonymous (dN) and synonymous (dS) substitution rates for the clpP genes among 16 species that have intact gene sequences. Branch lengths are drawn to the same scale based on dN and dS substitutions per site. Branches with significantly higher dN/dS ratios determined by likelihood ratio test are marked with asterisks (*, p < 0.05; **, p < 0.01; ***, p < 0.001 after Bonferroni correction).
Ijms 22 10485 g005
Table
Table 1. Comparison of Caprifoliaceae plastomes sequenced in this study.
Table 1. Comparison of Caprifoliaceae plastomes sequenced in this study.
TaxonDipsacoideaeValerianoideae
Dipsacus
japonicus		Scabiosa comosaFedia
cornucopiae		Valeriana faurieiValerianella locusta
Size (bp)160,243159,651152,196155,302149,809
LSC length (bp)87,06687,47782,96085,54182,103
SSC length (bp)17,85018,71615,86215,15915,796
IR length (bp)27,66426,72926,68727,30125,955
Number of protein-coding genes78 (6)78 (7)79 (4)79 (4)79 (4)
Number of tRNA genes29 (8)30 (7)30 (7)30 (7)30 (7)
Number of rRNA genes4 (4)4 (4)4 (4)4 (4)4 (4)
Number of introns21 (5)20 (5)21 (5)21 (5)19 (5)
GC content (%)38.838.738.238.438.1
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Park, S.; Jun, M.; Park, S.; Park, S. Lineage-Specific Variation in IR Boundary Shift Events, Inversions, and Substitution Rates among Caprifoliaceae s.l. (Dipsacales) Plastomes. Int. J. Mol. Sci. 2021, 22, 10485. https://doi.org/10.3390/ijms221910485

AMA Style

Park S, Jun M, Park S, Park S. Lineage-Specific Variation in IR Boundary Shift Events, Inversions, and Substitution Rates among Caprifoliaceae s.l. (Dipsacales) Plastomes. International Journal of Molecular Sciences. 2021; 22(19):10485. https://doi.org/10.3390/ijms221910485

Chicago/Turabian Style

Park, Seongjun, Minji Jun, Sunmi Park, and SeonJoo Park. 2021. "Lineage-Specific Variation in IR Boundary Shift Events, Inversions, and Substitution Rates among Caprifoliaceae s.l. (Dipsacales) Plastomes" International Journal of Molecular Sciences 22, no. 19: 10485. https://doi.org/10.3390/ijms221910485

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