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Article

Ten Plastomes of Crassula (Crassulaceae) and Phylogenetic Implications

by
Hengwu Ding
1,†,
Shiyun Han
1,†,
Yuanxin Ye
1,
De Bi
2,
Sijia Zhang
1,
Ran Yi
1,
Jinming Gao
1,
Jianke Yang
1,
Longhua Wu
3 and
Xianzhao Kan
1,4,*
1
Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, College of Life Sciences, Anhui Normal University, Wuhu 241000, China
2
College of Landscape Engineering, Suzhou Polytechnic Institute of Agriculture, Suzhou 215000, China
3
CAS Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China
4
The Institute of Bioinformatics, College of Life Sciences, Anhui Normal University, Wuhu 241000, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Biology 2022, 11(12), 1779; https://doi.org/10.3390/biology11121779
Submission received: 3 November 2022 / Revised: 5 December 2022 / Accepted: 5 December 2022 / Published: 7 December 2022
(This article belongs to the Special Issue Advances in Plant Taxonomy and Systematics)
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Abstract

:

Simple Summary

Plastids are semi-autonomous plant organelles which play critical roles in photosynthesis, stress response, and storage. The plastid genomes (plastomes) in angiosperms are relatively conserved in quadripartite structure, but variable in size, gene content, and evolutionary rates of genes. The genus Crassula L. is the second-largest genus in the family Crassulaceae J.St.-Hil, that significantly contributes to the diversity of Crassulaceae. However, few studies have focused on the evolution of plastomes within Crassula. In the present study, we sequenced ten plastomes of Crassula: C. alstonii Marloth, C. columella Marloth & Schönland, C. dejecta Jacq., C. deltoidei Thunb., C. expansa subsp. fragilis (Baker) Toelken, C. mesembrianthemopsis Dinter, C. mesembryanthoides (Haw.) D.Dietr., C. socialis Schönland, C. tecta Thunb., and C. volkensii Engl. Through comparative studies, we found Crassula plastomes have unique codon usage and aversion patterns within Crassulaceae. In addition, genomic features, evolutionary rates, and phylogenetic implications were analyzed using plastome data. Our findings will not only reveal new insights into the plastome evolution of Crassulaceae, but also provide potential molecular markers for DNA barcoding.

Abstract

The genus Crassula is the second-largest genus in the family Crassulaceae, with about 200 species. As an acknowledged super-barcode, plastomes have been extensively utilized for plant evolutionary studies. Here, we first report 10 new plastomes of Crassula. We further focused on the structural characterizations, codon usage, aversion patterns, and evolutionary rates of plastomes. The IR junction patterns—IRb had 110 bp expansion to rps19—were conservative among Crassula species. Interestingly, we found the codon usage patterns of matK gene in Crassula species are unique among Crassulaceae species with elevated ENC values. Furthermore, subgenus Crassula species have specific GC-biases in the matK gene. In addition, the codon aversion motifs from matK, pafI, and rpl22 contained phylogenetic implications within Crassula. The evolutionary rates analyses indicated all plastid genes of Crassulaceae were under the purifying selection. Among plastid genes, ycf1 and ycf2 were the most rapidly evolving genes, whereas psaC was the most conserved gene. Additionally, our phylogenetic analyses strongly supported that Crassula is sister to all other Crassulaceae species. Our findings will be useful for further evolutionary studies within the Crassula and Crassulaceae.

1. Introduction

The family Crassulaceae comprises approximately 1400 species in 34 genera and three subfamilies (Crassuloideae Burnett, Kalanchoideae A. Berger, and Sempervivoideae Arn.) [1,2,3,4,5,6,7]. These subfamilies can be further subdivided into seven major clades: Crassula (Crassuloideae), Kalanchoe (Kalanchoideae), and the other five clades (Sempervivum, Leucosedum, Aeonium, Acre, and Telephium), which form the largest subfamily Sempervivoideae [3,4,5,6,8]. The genus Crassula, with about 200 accepted species, is the only unique genus in the clade Crassula, the second-largest genus of Crassulaceae, and significantly contributes to the diversity of Crassulaceae [3,9,10]. Previous taxonomic revision of Crassula recognized two subgenera: Crassula L. and Disporocarpa Fischer & C.A. Mey. [7,11,12]. The monophyly of the subgenus Crassula was well supported in two recent molecular phylogenetic studies [9,10]. Nevertheless, the monophyly of subgenus Disporocarpa is still controversial [9,10]. Thus, more evidence and further investigations are required to clarify the phylogenetic relationships of Crassula.
Plastids are semi-autonomous plant organelles which have many vital functions, such as photosynthesis, stress response, and storage [13]. In angiosperms, the plastid genome (plastome) generally exhibits a conserved quadripartite circular structure with a size of 120–170 kb, comprising two single copy regions (larger and small regions, namely LSC and SSC, respectively) and two inverted repeat regions (IRs) [14,15,16]. Owing to the low level of recombination, uniparental inheritance, and without interference from paralogs, plastome has been extensively utilized as a super-barcode for plant species identification and evolutionary studies [17,18,19,20,21,22,23,24]. Due to the rapid development and widespread application of high-throughput sequencing technologies (such as Illumina, PacBio, and Nanopore sequencing technologies), an increasing number of complete Crassulaceae plastomes (more than 70 sequences) have been deposited in public databases. However, within the Crassula, only one plastome has been reported to date [6]. The lack of plastome data has limited the progress in investigating the evolutionary history of Crassula. Therefore, more plastome data from Crassula are needed to address this issue.
Codon usage bias (CUB), indicating the preferential utilization of synonymous codons in protein-coding genes (PCGs), has evolved via combined effects of genetic drift, mutation, and natural selection [17,25,26,27,28]. Owing to different species having diverse codon usage patterns, investigations of CUB can reveal phylogenetic relationships between species [17,25,26,27,28]. Codon aversion is defined as the codon which is not used in a certain gene [29,30,31]. The codon aversion motif is phylogenetically conserved in some lineages [29,30,31]. Interestingly, our recent reports in Macaronesian species (Crassulaceae) and Bletilla Rchb.f. species (Orchidaceae Juss.) have suggested that plastid CUB and codon aversion patterns might harbor phylogenetic signals [17,26]. Therefore, the analyses of plastid genes in codon-usage aspects might broaden our understanding of the phylogeny of both Crassula and Crassulaceae.
Evolutionary rate, calculated by the ratio (dN/dS) of nonsynonymous rate (dN) and synonymous rate (dS), can quantify the intensity of the selective force acting on a PCG [32,33,34]. The evolutionary rate can also reflect the pattern of natural selection (dN/dS value >1, =1, and <1 indicate positive, neutral, and purifying selection, respectively) [33,34,35]. The dN/dS values in different genes are variable, which might be influenced by many factors, such as protein function, population size, generation time, and DNA-repair efficiency [36,37]. The dN/dS values of plastid genes have been measured in many plant lineages, and most values were lower than 1, indicating plastid genes were mainly under the purifying selection [13,22,38,39,40]. Currently, the detailed rates and patterns of plastid genes were largely unknow in Crassulaceae. Knowledge of the evolutionary rates and patterns will shed light on how the diversifying selection affected the plastome evolution in Crassulaceae.
To address these issues, we newly sequenced and assembled the plastomes of ten Crassula species (C. alstonii, C. columella, C. dejecta, C. deltoidea, C. expansa subsp. fragilis, C. mesembrianthemopsis, C. mesembryanthoides, C. socialis, C. tecta and C. volkensii) using Illumina sequencing technology. Together with the public data, we performed comprehensive analyses to investigate (1) structural characterizations of Crassula plastomes, (2) unique CUB and codon-aversion patterns for Crassula plastomes, (3) evolutionary rates and patterns of plastid genes of Crassulaceae, and (4) phylogenetic relationships among Crassulaceae species. Our findings will not only shed new insights into the plastome evolution of Crassulaceae, but also provide potential molecular markers for DNA barcoding.

2. Materials and Methods

2.1. Sample Collection, DNA Extraction, and Sequencing

The fresh leaf samples of ten Crassula species were collected from greenhouses of Anhui Normal University, with the voucher codes KL01739, KL01709, KL01449, KL01646, KL02048, KL01731, KL01444, KL01653, KL01657, and KL01688 for C. alstonii, C. columella, C. dejecta, C. deltoidea, C. expansa subsp. fragilis, C. mesembrianthemopsis, C. mesembryanthoides, C. socialis, C. tecta, and C. volkensii, respectively. The Plant Genomic DNA kit (Tiangen, Beijing, China) was used for Genomic DNA extraction. Furtherly, a TruSeq DNA PCR-Free Library Prep Kit (Illumina, San Diego, CA, USA) was employed for library construction. Then, these libraries were sequenced using the Illumina Hiseq X Ten (Illumina, San Diego, CA, USA) platform.

2.2. Plastome Assembly, Genome Annotation, and Comparative Genomic Analysis

All resulting high-quality clean reads were assembled by using GetOrganelle 1.7.5 [41] with the plastome of C. perforata Thunb. (NC_053949) [6] as reference. The plastomes were initially annotated with the online program GeSeq [42] and then checked manually. Bowtie 2.4.1 [43] and Chloroplot [44] were utilized for the sequencing depth estimation and the drawing of a gene map, respectively. Genome comparisons were visualized using mVISTA [45] in Shuffle-LAGAN mode. In order to detect highly variable regions (HVRs) among plastomes, the sliding-window nucleotide diversity (π) values were measured in DnaSP v6.12 (window length = 600 bp, and step size = 200 bp) [46]. The contiguous sliding windows with higher π values (π > πmean + 2 standard deviation) were merged as a HVR [47,48]. The contraction and expansion of IR regions at the junctions of plastomes were subsequently plotted using R package IRscope V0.1.R (Viikki Plant Science Centre, University of Helsinki, Helsinki, Finland) [49].

2.3. Codon Usage and Aversion Indices Analyses

To investigate the codon usage indices, we used CodonW v.1.4.2 (Peden, University of Nottingham, Nottingham, UK) to calculate the values of relative synonymous codon usage (RSCU), and the effective number of codons (ENC) of plastid genes (length ≥300 bp) among 87 Crassulaceae species (10 of which are new in this study, Table S1). The RSCU value for a codon represents the observed frequency divided by the expected frequency (RSCU >1 implies a codon use higher than expected, and vice versa) [50]. The RSCU heatmap was rendered using TBtools 1.098 [51]. In addition, the ENC values, ranging from 20 (extreme bias) to 61 (no bias), quantify the level of CUB of synonymous codons [52]. Furtherly, the parity rule 2 (PR2) plot was performed according to the two formulas: GC-bias = [G3/(G3 + C3)|4] and AT-bias = [A3/(A3 + T3)|4] (“|4” means 4-fold degenerate synonymous codons, and G3, C3, A3 and T3 denotes nucleotide composition at the 3rd codon sites, respectively) [53,54]. The points lying at the centre of plot (AT bias = 0.5 and GC bias = 0.5) indicate no bias, whereas the off-centred points reflect the direction and extent of bias [53,54]. Moreover, the codon aversion motifs harboring strong phylogenetic implications were identified by using CAM v.1.02 [31].

2.4. Nucleotide Substitution Rate Analyses

The 79 PCGs from 87 species of Crassulaceae were employed to evaluate the evolutionary rates (Table S1). The percentage of variable sites (PV) and average π values were measured with DnaSP v6.12 (Departament de Genètica, Universitat de Barcelona, Barcelona, Spain) [46]. The nucleotide substitution rates, including dN, dS, and dN/dS, were inferred with PAML v4.9 [55] under F3X4 and M0 model.

2.5. Phylogenetic Implications Analyses

Phylogenetic relationships among 87 Crassulaceae species were inferred by maximum-likelihood (ML) and Bayesian inference (BI) methods, based on 79 PCGs (Data S1). Recent studies of Lu et al. [9] and Bruyns et al. [10] revealed a sister relationship between Crassulaceae and Haloragaceae R.Br. Therefore, two species of Haloragaceae (Myriophyllum aquaticum (Vell.) Verdc., NC_048889 and Myriophyllum spicatum L., NC_037885) were selected as outgroups. Multiple sequence alignments were generated using MAFFT v7.505 in PhyloSuite v1.2.1 with codon model [56]. The best-fit nucleotide substitution models were evaluated with ModelTest-NG v0.1.7 [57]. Subsequently, we employed RAxML-NG 1.1 [58] and MrBayes v3.2.7a [59] for ML and BI analyses, respectively. For ML analyses, the reliabilities were assessed with 1000 bootstrap replicates, and the convergence was evaluated by using parameter “--bsconverge” in RAxML-NG package (Computational Molecular Evolution Group, Heidelberg Institute for Theoretical Studies, Heidelberg, Germany). For BI analyses, four independent Markov chains and two independent runs (running for 10,000,000 generations, and sampling every 1000 generations) were conducted, with Tracer 1.7.1 (Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, UK) [60] for the convergence. After discarding the first 25% trees as burn-in, the remaining 75% trees were used to estimate the consensus tree and Bayesian posterior probabilities.

3. Results

3.1. Plastome Organizations and Structural Features

Based on bowtie2 mapping, totally 3,246,461, 1,740,232, 3,915,950, 2,632,260, 2,801,895, 504,972, 5,440,628, 3,398,113, 1,877,288 and 1,530,319 paired reads were mapped to the plastomes of C. alstonii (coverage: 3344.02×), C. columella (coverage: 1762.18×), C. dejecta (coverage: 4020.78×), C. deltoidei (coverage: 5284.33×), C. expansa subsp. fragilis (coverage: 5796.17×), C. mesembrianthemopsis (coverage: 1010.16×), C. mesembryanthoides (coverage: 5557.15×), C. socialis (coverage: 3486.98×), C. tecta (coverage: 1918.07×), and C. volkensii (coverage: 3161.69×), respectively. The new complete plastomes of ten species of Crassula (accession numbers: OP729482–OP729487 and OP882297–OP882300) were typical circular and quadripartite biomolecules (Figure 1), with sizes ranging from 144,855 bp to 146,060 bp. These plastomes contains LSC (78,303–79,707 bp), SSC (16,568–16,871 bp), and IR (24,810–24,878 bp). The overall GC contents of Crassula plastomes were between 37.73% and 38.32%. Notably, the GC contents of IR regions (42.93–43.15%) were found to be higher than those of in the LSC (35.75–36.51%) and SSC regions (31.67–32.40%). In addition, these plastomes contain 134 genes, including 85 PCGs, 37 tRNA genes, 8 rRNA genes and 4 pseudogenes. Among these genes, 6 PCGs, 7 tRNA genes, 4 rRNA genes, and one pseudogene (ycf15), were completely duplicated in the IR regions (Table 1).
Furthermore, based on the results obtained with mVISTA, in all plastomes investigated it was found that the IR and coding regions (exons, tRNAs, and rRNAs) are more conserved than SC and conserved non-coding regions (CNS), respectively (Figure 2). Additionally, the results also revealed that 3 plastomes (labelled 8–10) of subgenus Disporocarpa exhibited higher divergences than 7 plastomes (labelled 1–7) of subgenus Crassula, when compared with the reference.
The sliding-window-based π values estimated for 11 plastomes of Crassula ranged from 0.00073 to 0.10315 (Table S2 and Data S2). The mean π value and its standard deviation were 0.02978 and 0.01954, respectively. Thus, a total of 11 HVRs were identified with relatively high variability (π > 0.06886) (Figure 3). These HVRs containing high π values (0.06912–0.08653) and abundant variable sites (111–559) might be used as potential DNA barcodes for species identification within Crassula (Table 2).
In our current study, all 11 plastomes of Crassula displayed similar IR junction patterns (Figure 4). The SSC/IRa borders are located in the coding regions of ycf1 gene, resulting in the fragmentations of ycf1 (ycf1-fragment) in IRb regions. Moreover, ndhF genes were discovered to occur mainly in SSC, and partly in IRb, regions. Notably, rps19 genes are located at the LSC/IRb junctions, with extension into the IRb regions for 110 bp. Similarly, trnH genes lie at the IRa/LSC junctions, with uniform 3 bp-sized expansions to the IRa regions.

3.2. Codon Usage and Aversion Patterns

To compare the patterns of codon usage and aversion between Crassula and other Crassulaceae species, four analyses (RSCU, ENC, PR2-plot, and codon aversion motif) of 53 plastid genes (length ≥300 bp) were performed.
The overall RSCU values ranged from 0.32 (CTC or AGC) to 2.07 (TTA) among Crassulaceae species (Table S3). Similar with other Crassulaceae species, seven taxa of Crassula exhibited significant preference for A/T-ending codons over G/C-ending codons in plastid genes (Figure 5). Importantly, the RSCU heatmap showed two subgenera within the Crassula: subgenus Disporocarpa included C. expansa subsp. fragilis, C. deltoidea and C. volkensii; subgenus Crassula consisted of the remaining eight taxa (Figure 5).
The ENC values ranged from 30.83 (ndhC in Sedum sarmentosum Bunge) to 57.74 (ndhJ in C. volkensii and C. expansa subsp. fragilis) among Crassulaceae species (Table S4). Generally, ENC values ≤35 indicate high codon preference [52,61,62]. The results show that most of the ENC values (99.48%) were higher than 35, indicating a weaker bias. Most surprisingly of all, we detected the ENC values of matK, from the Crassula clade, are significantly higher than those of all other clades (Table S4 and Figure 6). It might prove to be a unique feature for Crassula species. To further verify this finding, more sampling data and comprehensive analyses are need in future studies.
The PR2 plots of matK and 52 other PCGs are presented in Figure 7 and Figure S1, respectively. These results indicated the nucleotide usage at the 3rd codon site of 4-fold degenerate codons is uneven in different genes. For example, rps14, clpP, psbA, and pafII prefer to use A/G, A/C, T/C, and T/G in 4-fold degenerate sites, respectively (Figure S1). In addition, these unbalanced utilizations were also found in different species (Figure S1). Obviously divergent GC-biases were observed in matK genes between species of subgenus Crassula and others. Specifically, all GC-biases of clades from Kalanchoideae and Sempervivoideae, plus subgenus Disporocarpa, were less than 0.5. On the contrary, all these values for the subgenus Crassula were higher than 0.5, which might be unique characteristic for subgenus Crassula. Moreover, species with close relationships had identical nucleotide biases. For example, C. alstonii and C. columella had identical AT-biases (0.4074) and GC-biases (0.5455). Similar phenomena could also be observed in C. mesembryanthoides and C. tecta (AT-biases = 0.4286, and GC-biases = 0.5455).
Owing to the codon aversion motifs containing phylogenetic implication, we analyzed codon aversion patterns of genes among Crassulaceae species. Except for rpoB, rpoC2, ycf1 and ycf2, codon aversion motifs were found in the remaining 49 genes (Table S5). It is worth noting that 27 and 16 unique codon aversion motifs were detected for species of subgenus Crassula and subgenus Disporocarpa, respectively (Table 3), which might be used as potential biomarkers for species identification. Further to this, 8 consensus motifs might be considered as the feature of genus Crassula (Table 3). Moreover, the codon aversion motifs from 3 genes (matK, pafI and rpl22) could also divide 11 species into two subgenera (subgenus Crassula and subgenus Disporocarpa) (Figure 8), which is congruent with results from RSCU heatmap.

3.3. Evolutionary Rates and Patterns

The π (0.00447–0.0914) and PV (4.91–37.52%) values of 79 plastid PCGs of Crassulaceae species were plotted in Figure 9a. Two genes, referring to ycf1 (π = 0.0914, PV = 35.78%) and matK (π = 0.08239, PV = 37.52%), had obviously higher π and PV values than those of the other 77 genes, indicating they might accumulate more mutations than other plastid genes. The detailed data are listed in Table S6.
To further quantify the evolutionary rates of PCGs, the nucleotide substitution rates, including dN, dS and dN/dS, were calculated (Figure 9b, Table S6). The dN values ranged from 0 to 0.8671, with higher dN values for ycf1 (dN = 0.8671) and matK (dN = 0.7804) than for others. Compared with dN values, the dS values had relatively wide ranges (0.177–2.3917), resulting in corresponding dN/dS ratios (0–0.5891) of less than 1. This finding indicates the plastid genes from Crassulaceae appear to be evolving under a purifying selective constraint. Among 79 plastid PCGs, ycf2 is the most rapidly evolving gene, with the highest ratio (dN/dS = 0.5891), followed by ycf1, cemA, psaI, and matK. By contrast, psaC was the most conserved gene with the lowest ratio (dN/dS = 0).

3.4. Phylogenetic Implications

To investigate the evolutionary relationships among 87 species of Crassulaceae, phylogenetic analyses were performed. After a model test, GTR + G4 and GTR + I+G4 were inferred as the optimal substitution models for most genes (the detailed models can be seen in Table S7). As shown in Figure 10, the trees inferred from two methods displayed the same topology.
Ten species of Crassula that we sequenced, together with C. perforate, form the well-supported clade Crassula, which is sister to all other Crassulaceae species (maximum likelihood bootstrap [BS] = 100 and bayesian posterior probability [PP] = 1.00). In addition, our phylogenetic tree indicated that this monophyletic clade could be clustered into two subgenera: subgenus Disporocarpa harbored C. volkensii, C. expansa subsp. fragilis and C. deltoidea ([BS] = 100 and [PP] = 1.00). Subgenus Crassula included the remaining eight Crassula species (C. alstonii, C. columella, C. dejecta, C. mesembryanthoides, C. tecta, C. mesembrianthemopsis, C. socialis, and C. perforata) ([BS] = 100 and [PP] = 1.00). Within subgenus Crassula, two species (C. alstonii and C. columella) were sister to six other species (C. dejecta, C. mesembryanthoides, C. tecta, C. mesembrianthemopsis, C. socialis, and C. perforata) ([BS] = 100 and [PP] = 1.00). Further, C. tecta and C. mesembrianthemopsis formed the well-supported sister taxa ([BS] = 100 and [PP] = 1.00). Unfortunately, the sister group of C. dejecta and C. mesembryanthoides had relatively weak support ([BS] = 55 and [PP] =0.69). Due to the limited plastome data within Crassula, there are many unsolved phylogenetic problems in this clade. Therefore, more samples are needed to solve this issue.
As expected, the six species from genus Kalanchoe Adans. and genus Cotyledon L. formed the monophyletic clade Kalanchoe (or subfamily Kalanchoideae) ([BS] = 100 and [PP] = 1.00). The remaining 70 species, belonging to the subfamily Sempervivoideae, can be further grouped into 5 distinct clades: Acre, Aeonium, Leucosedum, Sempervivum, and Telephium. In detail, 7 Sedum L. species and 3 species from other genera respectively (Graptopetalum Rose, Echeveria DC., and Pachyphytum Link, Klotzsch & Otto) formed a well-supported clade Acre ([BS] = 100 and [PP] = 1.0). However, it is clear from our results that Sedum is not monophyletic, with some other taxa embedded within this genus.
In addition, 13 species from genus Aeonium Webb & Berthel. and genus Monanthes Haw. make up the clade Aeonium ([BS] = 100 and [PP] = 1.0). Furthermore, due to sampling in this study, only a single species form Leucosedum and Sempervivum clades, and full resolution of relationships within these clades requires sufficient molecular sequences. Notably, the clade Telephium, with 45 species, consists of clusters “Rhodiola” and “Hylotelephium” [63] ([BS] = 92 and [PP] = 1.0). Within cluster “Hylotelephium“, non-monophyly of Orostachys Fisch. was observed. Three Orostachys species, (O. japonica (Maxim.) A.Berger, O. minuta (Kom.) A.Berger, and O. fimbriata (Turcz.) A.Berger) belonging to the Subsection Orostachys [63] ([BS] = 100 and [PP] = 1.0), were sister to Meterostachys sikokianus (Makino) Nakai, while O. iwarenge f. magna Y.N.Lee (Subsection Appendiculata) and three Hylotelephium H.Ohba species formed a group with strong support ([BS] = 100 and [PP] = 1.0).

4. Discussion

Ten new plastomes of Crassula were reported in the present study. Combined with available data from public database, we conducted comprehensive analyses, including plastome organizations, codon usage and aversion patterns, evolutionary rates, and phylogenetic implications.
The expansion and contraction of IR regions are common evolutionary events and have been considered as the main mechanism for the length variation of angiosperm plastomes [64,65,66]. In our study, we performed comparative analyses among Crassula plastomes, and found that the IRb regions had uniform length (110 bp) expansions to the rps19 gene. This 110-bp expansion had been also observed in Aeonium, Monanthes, and most other taxa of Crassulaceae in our recent study [17]. This finding indicated that the conserved IR organization might act as a family-specific marker for Crassulaceae species.
Interestingly, it was reported that rps19 genes were completely located in the LSC regions in Forsythia suspensa (Thunb.) Vahl, Olea europaea Hoffmanns. & Link L., and Quercus litseoides Dunn [67,68], and were fully encoded by the IR regions in Polystachya adansoniae Rchb.f., Polystachya bennettiana Rchb.f., and Dracaena cinnabari Balf.f. [69,70]. There are several mechanisms that might explain the IR expansion and contraction [71,72,73]. For instance, Goulding et al. [71] proposed that short IR expansions may occur by gene conversion events, whereas large IR expansions involved in double-strand DNA breaks. In order to better reveal the mechanisms of IR expansion and contraction, more extensive investigations in Crassulaceae and Saxifragales are required.
Investigations of codon usage patterns could reveal phylogenetic relationships between organisms [25,74]. In particular, 11 species of Crassula can be divided into two subgenera from the RSCU heatmap, which agreed with the results of phylogenetic analyses. This finding further demonstrates that RSCU values contain phylogenetic implications [75,76,77,78,79,80]. Additionally, we observed codon usage patterns are gene-specific and/or species-specific, reflected in diversified ENC values and various distribution patterns in PR2 plots. Interestingly, we found the codon usage patterns of matK gene in Crassula species are unique among Crassulaceae species with elevated ENC values. Furthermore, the GC-biases of matK gene with specific preference (>0.5) might be the particular feature for subgenus Crassula. Due to rapid evolutionary rate, high universality, and significant interspecific divergence, the matK gene has been broadly used in plant evolutionary studies as one of the core DNA barcodes [9,10,81,82,83,84].
Codon aversion, a novel concept proposed by Miller et al. [29,30,31], is an informative character in phylogenetics. Specifically, the codon aversion motifs in orthologous genes are generally conserved in specific lineages [29,30,31]. To date, these analyses have only been performed in a few plant plastomes [17,26]. For example, the specific codon aversion motifs of rpoA gene could distinguish not only the two genera (Aeonium and Monanthes), but also the three subclades of Aeonium in our recent report [17]. In this work, genus-specific and subgenus-specific codon aversion motifs were identified for 11 Crassula species. These findings suggest codon aversion pattern could be used as a promising tool for phylogenetic study.
Generally, the dN/dS ratios of genes could reflect the extent of selection pressures during evolution [22]. Here, the dN/dS values of plastid PCGs ranged from 0 to 0.5891 within Crassulaceae, indicating all plastid genes were under purifying selection. Among these values, elevated dN/dS ratios were found for ycf1 (0.4349) and ycf2 (0.5891). Similarly, high dN/dS ratios of these two genes were also observed in other families, such as Asteraceae Bercht. & J.Presl [38], Mazaceae Reveal [22], and Musaceae Juss. [13]. The ycf1 gene was related to protein translocation [85]. The ycf2 gene is necessary for cell viability, but the detail function is still unknown [86]. Why ycf1 and ycf2 evolve relatively fast is interesting. The possible reason put forwarded by Barnard-Kubow et al. [87] considered that relaxed purifying selection or positive selection on ycf1, ycf2 and some other genes might result in the development of reproductive isolation and subsequent speciation in plants. Therefore, the results suggested that ycf1 and ycf2 might play important roles in the divergence of Crassulaceae.
Our phylogenetic tree divided 87 species into 3 subfamilies and 7 clades. The clade Crassula is sister to all other 6 clades, which agrees with the phylogeny reported by Gontcharova et al. [4], Chang et al. [6], and Han et al. [17]. Furtherly, 11 Crassula species could be furtherly divided into two subgenera, which generally accords with the morphological differences (floral shape) reported by Bruyns et al. [10] (Table S8). Nevertheless, there are still some unsolved phylogenetic problems within Crassulaceae. The first problem is that the plastid phylogeny of Crassula is not entirely clear due to the limited data. According to the classification proposed by Tölken [11,88], 11 and 9 sections were respectively identified in subgenus Crassula and subgenus Disporocarpa. However, Bruyns et al. [10] indicated that most sections were not monophyletic. Moreover, subgenus Disporocarpa recently has been regarded as a paraphyletic group [9,10]. The second is the genus Sedum, which is not monophyletic in our study, agreeing with the widely accepted viewpoint [3,4,5,89,90]. Finally, the genus Orostachys has been demonstrated to be non-monophyletic based on plastid data, which is consistent with previous analysis based on nuclear internal transcribed spacers (ITS) data [63]. In order to better understand the phylogeny of Crassula or Crassulaceae, more data are needed for the further detailed analyses.

5. Conclusions

In the present study, 10 new plastomes of Crassula species were reported. These plastomes exhibited identical gene content and order, and that they contained 134 genes (130 functional gene and 4 pseudogenes). The 11 identified HVRs with relatively high variability (π > 0.06886) might be used as potential DNA barcodes for species identification within Crassula. The unique expansion pattern, where the IRb regions had uniform length (110 bp) boundary expansions to rps19, might become a plesiomorphy of Crassulaceae. According to RSCU values, the A/T-ending codons were favored in plastid genes. Most importantly, we found the codon usage patterns of the matK gene in Crassula species are unique among Crassulaceae species with elevated ENC values. Furthermore, subgenus Crassula species have specific GC-biases in the matK gene. In addition, the codon aversion motifs from matK, pafI and rpl22 contained phylogenetic implications within Crassula. Compared with other Crassulaceae species, 27 and 16 unique codon aversion motifs were detected for subgenus Crassula and subgenus Disporocarpa, respectively. Additionally, the evolutionary rates analyses indicated all plastid genes of Crassulaceae were under purifying selection. Among these genes, ycf1 (dN/dS = 0.4349) and ycf2 (dN/dS = 0.5891) were the most rapidly evolving genes, whereas psaC (dN/dS = 0) was the most conserved gene. Finally, our phylogenetic analyses strongly supported Crassula is sister to all other Crassulaceae species. Our results will be benefit for further evolutionary studies within the Crassula and Crassulaceae.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biology11121779/s1, Figure S1: PR2 plots of 52 plastid genes; Table S1: Accession numbers of Crassulaceae plastomes sampled in this study; Table S2: The sliding window-based π values estimated by 11 plastomes of Crassula; Table S3: The RSCU values of codons among Crassulaceae species; Table S4: The ENC values of 53 plastid genes of Crassulaceae; Table S5: Plastid codon aversion motifs among Crassulaceae species; Table S6: Evolutionary rates of 79 plastid genes of Crassulaceae; Table S7: The best evolutionary models for 79 plastid genes; Table S8: The morphological characteristics of 11 Crassula species; Data S1: Phylogenetic datasets of 79 plastid genes; Data S2: Sequence matrix of 11 plastomes of Crassula for sliding-window analysis.

Author Contributions

Conceptualization, supervision, and project administration, X.K.; resources and validation, L.W. and D.B.; data curation, Y.Y. and J.G.; investigation and formal analysis, S.Z. and J.Y.; software, S.H. and R.Y.; methodology and writing—original draft, H.D.; funding acquisition, H.D., D.B. and X.K; writing—review & editing, X.K. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Opening Foundation of National Engineering Laboratory of Soil Pollution Control and Remediation Technologies, and Key Laboratory of Heavy Metal Pollution Prevention & Control, Ministry of Agriculture and Rural Affairs (Grant no. NEL&MARA-003), the Basic Research Program (Natural Science Foundation) of Jiangsu Province (Grant no. BK20211078), and the Scientific Research Project Foundation of Postgraduate of the Anhui Higher Education Institutions of China (Grant no. YJS20210136).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The sequence data generated in this study are available in GenBank of the National Center for Biotechnology Information (NCBI) under the access numbers: OP729482–OP729487 and OP882297–OP882300.

Acknowledgments

We kindly acknowledge three anonymous reviewers for the fruitful and critical comments.

Conflicts of Interest

The authors declare no conflict of interest.

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Biology 11 01779 g001 550
Figure 1. Annotation map of ten new plastomes from Crassula species. Directed with arrows, genes that are listed inside and outside of the circle are respectively transcribed clockwise and counterclockwise. Different colors represent different functional groups.
Figure 1. Annotation map of ten new plastomes from Crassula species. Directed with arrows, genes that are listed inside and outside of the circle are respectively transcribed clockwise and counterclockwise. Different colors represent different functional groups.
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Biology 11 01779 g002 550
Figure 2. Structure comparisons of ten new Crassula plastomes using the mVISTA program. Y-scale represents the percent identity between 50% and 100%. The labels 0 to 10 indicate C. perforata (reference), C. alstonii, C. columella, C. dejecta, C. mesembryanthoides, C. tecta, C. mesembrianthemopsis, C. socialis, C. volkensii, C. expansa subsp. fragilis, and C. deltoidei, respectively.
Figure 2. Structure comparisons of ten new Crassula plastomes using the mVISTA program. Y-scale represents the percent identity between 50% and 100%. The labels 0 to 10 indicate C. perforata (reference), C. alstonii, C. columella, C. dejecta, C. mesembryanthoides, C. tecta, C. mesembrianthemopsis, C. socialis, C. volkensii, C. expansa subsp. fragilis, and C. deltoidei, respectively.
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Biology 11 01779 g003 550
Figure 3. Sliding-window analysis of the plastomes of 11 Crassula species (window length: 600 bp; step size: 200 bp). x-axis: position of the midpoint of a window; y-axis: π value of each window. Regions with higher π values (π > 0.06886) were considered as HVRs.
Figure 3. Sliding-window analysis of the plastomes of 11 Crassula species (window length: 600 bp; step size: 200 bp). x-axis: position of the midpoint of a window; y-axis: π value of each window. Regions with higher π values (π > 0.06886) were considered as HVRs.
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Biology 11 01779 g004 550
Figure 4. Comparisons of LSC, SSC, and IR region borders among plastomes of 11 Crassula species. Blue, orange and green blocks represent the LSC, IR and SSC regions, respectively. Gene boxes represented above the block were transcribed clockwise and those represented below the block were transcribed clockwise. “fra.” is the abbreviation of “fragment”.
Figure 4. Comparisons of LSC, SSC, and IR region borders among plastomes of 11 Crassula species. Blue, orange and green blocks represent the LSC, IR and SSC regions, respectively. Gene boxes represented above the block were transcribed clockwise and those represented below the block were transcribed clockwise. “fra.” is the abbreviation of “fragment”.
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Biology 11 01779 g005 550
Figure 5. The heatmap of overall RSCU values among 7 clades of Crassulaceae species based on 53 concatenated plastid genes (length ≥300 bp). The x-axis: the cluster of different codons; y-axis: the clusters of species.
Figure 5. The heatmap of overall RSCU values among 7 clades of Crassulaceae species based on 53 concatenated plastid genes (length ≥300 bp). The x-axis: the cluster of different codons; y-axis: the clusters of species.
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Biology 11 01779 g006 550
Figure 6. The ENC value distributions of matK for 7 clades of Crassulaceae. The mean values with standard deviations are labeled.
Figure 6. The ENC value distributions of matK for 7 clades of Crassulaceae. The mean values with standard deviations are labeled.
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Biology 11 01779 g007 550
Figure 7. The PR2 plot of matK of Crassulaceae. Different colors represent different clades. Within the clade Crassula (or genus Crassula), red circles with black edges and cyan edges represent species of subgenus Crassula and Disporocarpa, respectively.
Figure 7. The PR2 plot of matK of Crassulaceae. Different colors represent different clades. Within the clade Crassula (or genus Crassula), red circles with black edges and cyan edges represent species of subgenus Crassula and Disporocarpa, respectively.
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Biology 11 01779 g008 550
Figure 8. The specific codon aversion motifs of matK, pafI and rpl22 gene for the 11 species of Crassula. The dots marked in different colors represent different species. Codons in red and green were specific for subgenus Crassula and subgenus Disporocarpa, respectively.
Figure 8. The specific codon aversion motifs of matK, pafI and rpl22 gene for the 11 species of Crassula. The dots marked in different colors represent different species. Codons in red and green were specific for subgenus Crassula and subgenus Disporocarpa, respectively.
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Biology 11 01779 g009 550
Figure 9. Sequence polymorphism among 79 PCGs of 87 Crassulaceae species. (a) Nucleotide diversity (π) and percentages of variable sites (PV). (b) Estimations of nonsynonymous (dN) and synonymous (dS) substitution rates and the dN/dS.
Figure 9. Sequence polymorphism among 79 PCGs of 87 Crassulaceae species. (a) Nucleotide diversity (π) and percentages of variable sites (PV). (b) Estimations of nonsynonymous (dN) and synonymous (dS) substitution rates and the dN/dS.
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Biology 11 01779 g010 550
Figure 10. The phylogenetic tree of 87 Crassulaceae species based on ML and BI methods. The maximum likelihood bootstrap (BS) and Bayesian posterior probability (PP) values for each node are indicated; * indicates 100% bootstrap or 1.00 PP.
Figure 10. The phylogenetic tree of 87 Crassulaceae species based on ML and BI methods. The maximum likelihood bootstrap (BS) and Bayesian posterior probability (PP) values for each node are indicated; * indicates 100% bootstrap or 1.00 PP.
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Table
Table 1. Plastome characteristics of Crassula species.
Table 1. Plastome characteristics of Crassula species.
SpeciesAccession NumberSize (bp)GC Content (%)Gene Number
GenomeLSCIRSSCGenomeLSCIRSSCTotalPCGstRNArRNAPseudo
C. alstonii†OP729482 *145,50779,30424,81316,57737.8235.8642.9631.85134 (19)85 (6)37 (7)8 (4)4 (2)
C. columella†OP729483 *145,55479,33224,82716,56837.8335.8642.9531.86134 (19)85 (6)37 (7)8 (4)4 (2)
C. dejecta†OP729484 *145,87079,49524,85916,65737.7335.7642.9331.67134 (19)85 (6)37 (7)8 (4)4 (2)
C. mesembrianthemopsis†OP882297 *146,04679,66424,85416,67437.7835.7842.9731.89134 (19)85 (6)37 (7)8 (4)4 (2)
C. mesembryanthoides†OP729485 *146,05779,70724,85816,63437.7835.7842.9431.9134 (19)85 (6)37 (7)8 (4)4 (2)
C. perforata†NC_053949145,73779,46524,81016,65237.7535.7542.9731.77134 (19)85 (6)37 (7)8 (4)4 (2)
C. socialis†OP729486 *145,84279,54924,82816,63737.7935.842.9531.86134 (19)85 (6)37 (7)8 (4)4 (2)
C. tecta†OP729487 *146,06079,66224,85416,69037.7835.7842.9731.88134 (19)85 (6)37 (7)8 (4)4 (2)
C. deltoidea#OP882298 *145,17578,58024,86216,87138.1336.2543.0832.32134 (19)85 (6)37 (7)8 (4)4 (2)
C. expansa subsp. Fragilis#OP882299 *144,96978,44924,85616,80838.3036.5143.1432.4134 (19)85 (6)37 (7)8 (4)4 (2)
C. volkensii#OP882300 *144,85578,30324,87816,79638.3236.5143.1532.4134 (19)85 (6)37 (7)8 (4)4 (2)
† These species belong to subgenus Crassula. #, These speciesbelong to subgenus Disporocarpa. *, The new plastomes were generated in this study. (n), The number of genes located on IRs.
Table
Table 2. The HVRs identified in the plastomes of 11 Crassula species.
Table 2. The HVRs identified in the plastomes of 11 Crassula species.
HVRCoordinatesRegion Size (bp)π ValueVariable SitesAnnotations
HVR16259–819719390.07521240rps16–trnQ-UUG
HVR228,684–29,6789950.07427119petN–psbM
HVR329,942–31,26213210.07064182psbM–trnD-GUC–trnY-GUA
HVR435,624–36,63710140.06912114psbZ–trnG-GCC
HVR547,003–48,28912870.08653223trnL-UAA–trnF-GAA–ndhJ
HVR655,345–56,2909460.07518158rbcL–accD
HVR763,273–64,1568840.07652164psbE–petL
HVR869,495–70,2127180.07133111clpP–psbB
HVR9109,945–111,09811540.06969192ndhF–rpl32–trnL-UAG
HVR10120,441–123,49030500.07259559rps15–ycf1
HVR11124,303–124,9086060.07227119ycf1
Table
Table 3. The specific codon aversion motifs of Crassula within Crassulaceae.
Table 3. The specific codon aversion motifs of Crassula within Crassulaceae.
GeneSpecific Codon Aversion Motifs
Subgenus CrassulaSubgenus DisporocarpaConsensus for Genus Crassula
accDTGC
atpBTGTCGG
atpITGT, CTG, CTA,
cemAGGG
ccsAGGC
ndhACAT, CCG, CAG, CGGCAT, CCGCAT, CCG
ndhECTC
ndhITCTTCTTCT
ndhJTGT, ACG
ndhKTGC, CAC, CTGTGC, CAC, CTGTGC, CAC, CTG
pafIIAAGAAGAAG
petBAAC, AGACAG
petDGGCGCC
psbB CGA
psbD ACG
rbcLGCG, CCC, AGG
rpl16GATGATGAT
rpl20 TGT
rps3 GCG, GTC
rps4TCTCGG
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Ding, H.; Han, S.; Ye, Y.; Bi, D.; Zhang, S.; Yi, R.; Gao, J.; Yang, J.; Wu, L.; Kan, X. Ten Plastomes of Crassula (Crassulaceae) and Phylogenetic Implications. Biology 2022, 11, 1779. https://doi.org/10.3390/biology11121779

AMA Style

Ding H, Han S, Ye Y, Bi D, Zhang S, Yi R, Gao J, Yang J, Wu L, Kan X. Ten Plastomes of Crassula (Crassulaceae) and Phylogenetic Implications. Biology. 2022; 11(12):1779. https://doi.org/10.3390/biology11121779

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Ding, Hengwu, Shiyun Han, Yuanxin Ye, De Bi, Sijia Zhang, Ran Yi, Jinming Gao, Jianke Yang, Longhua Wu, and Xianzhao Kan. 2022. "Ten Plastomes of Crassula (Crassulaceae) and Phylogenetic Implications" Biology 11, no. 12: 1779. https://doi.org/10.3390/biology11121779

APA Style

Ding, H., Han, S., Ye, Y., Bi, D., Zhang, S., Yi, R., Gao, J., Yang, J., Wu, L., & Kan, X. (2022). Ten Plastomes of Crassula (Crassulaceae) and Phylogenetic Implications. Biology, 11(12), 1779. https://doi.org/10.3390/biology11121779

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