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Citation: Ji, J.; Luo, Y.; Pei, L.; Li, M.;
Xiao, J.; Li, W.; Wu, H.; Luo, Y.; He, J.;
Cheng, J.; et al. Complete Plastid
Genomes of Nine Species of
Ranunculeae (Ranunculaceae) and
Their Phylogenetic Inferences. Genes
2023,14, 2140. https://doi.org/
10.3390/genes14122140
Academic Editor: Zhiqiang Wu
Received: 20 October 2023
Revised: 22 November 2023
Accepted: 24 November 2023
Published: 27 November 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
genes
G C A T
T A C G
G C A T
Article
Complete Plastid Genomes of Nine Species of Ranunculeae
(Ranunculaceae) and Their Phylogenetic Inferences
Jiaxin Ji 1, Yike Luo 1, Linying Pei 2, Mingyang Li 3, Jiamin Xiao 1, Wenhe Li 1, Huanyu Wu 1, Yuexin Luo 1,
Jian He 1, Jin Cheng 3,4 and Lei Xie 1, *
1
State Key Laboratory of Efficient Production of Forest Resources, School of Ecology and Nature Conservation,
Beijing Forestry University, Beijing 100083, China; jx9024@bjfu.edu.cn (J.J.); luoyk@bjfu.edu.cn (Y.L.);
xiaojiamin0916@bjfu.edu.cn (J.X.); liwenhe@bjfu.edu.cn (W.L.); wuhuanyu22@bjfu.edu.cn (H.W.);
yxluo01@bjfu.edu.cn (Y.L.); hejian@bjfu.edu.cn (J.H.)
2
College of Agriculture and Forestry, Longdong University, Qingyang 745000, China; peilinying0724@163.com
3State Key Laboratory of Efficient Production of Forest Resources, College of Biological Sciences
and Technology, Beijing Forestry University, Beijing 100083, China; lmylmy2220103@bjfu.edu.cn (M.L.);
chengjin@bjfu.edu.cn (J.C.)
4National Engineering Research Center of Tree Breeding and Ecological Restoration, Beijing Key Laboratory
of Ornamental Plants Germplasm Innovation and Molecular Breeding, College of Biological Sciences and
Technology, Beijing Forestry University, Beijing 100083, China
*Correspondence: xielei@bjfu.edu.cn
Abstract:
The tribe Ranunculeae, Ranunculaceae, comprising 19 genera widely distributed all over
the world. Although a large number of Sanger sequencing-based molecular phylogenetic studies have
been published, very few studies have been performed on using genomic data to infer phylogenetic
relationships within Ranunculeae. In this study, the complete plastid genomes of nine species
(eleven samples) from Ceratocephala,Halerpestes, and Ranunculus were de novo assembled using
a next-generation sequencing method. Previously published plastomes of Oxygraphis and other
related genera of the family were downloaded from GenBank for comparative analysis. The complete
plastome of each Ranunculeae species has 112 genes in total, including 78 protein-coding genes,
30 transfer RNA genes, and four ribosomal RNA genes. The plastome structure of Ranunculeae
samples is conserved in gene order and arrangement. There are no inverted repeat (IR) region
expansions and only one IR contraction was found in the tested samples. This study also compared
plastome sequences across all the samples in gene collinearity, codon usage, RNA editing sites,
nucleotide variability, simple sequence repeats, and positive selection sites. Phylogeny of the available
Ranunculeae species was inferred by the plastome data using maximum-likelihood and Bayesian
inference methods, and data partitioning strategies were tested. The phylogenetic relationships were
better resolved compared to previous studies based on Sanger sequencing methods, showing the
potential value of the plastome data in inferring the phylogeny of the tribe.
Keywords:
complete plastid genome; next-generation sequencing; phylogeny; positive selection;
Ranunculeae; IR contraction
1. Introduction
The complete plastid genome (plastome) has become an increasingly popular tool for
phylogenetic studies in recent years [
1
–
4
]. Plastid is a common organelle found in plant
cells that contains its own genome which is typically circular and relatively conserved
across plant species [
5
]. The plastomes are often uniparentally inherited [
6
] and typically
include about 80 protein-coding genes and more than 30 RNA genes [
7
,
8
]. The high degree
of evolutionary conservation, large amount of data, uniparental inheritance, ability to
identify polymorphisms, and easy availability make the plastome an ideal marker for
studying phylogenetic relationships among plant taxa at different taxonomic levels [9].
Genes 2023,14, 2140. https://doi.org/10.3390/genes14122140 https://www.mdpi.com/journal/genes
Genes 2023,14, 2140 2 of 17
The tribe Ranunculeae comprises 16 to 19 genera and about 650 species distributed
worldwide, making it the most representative and diverse group within the buttercup
family (Ranunculaceae) [
10
–
13
]. Among all the genera in this tribe, Ranunculus stands out
as the species-rich genus of the family, with about 650 wild species in the world, whereas
all the other genera are small or even monotypic [
12
]. There are four genera: Ranunculus L.
(with the inclusion of Batrachium (DC.) Gray), Oxygraphis Bunge, Helerpestes E. L. Greene,
and Ceratocephala Moench distributed in China, and Ranunculus is also the largest one of
the tribes with more than 120 wild species in China [
10
,
14
]. Plants of Ranunculeae include
numerous ornamental and medicinal species, with a particularly rich species diversity in
temperate and alpine regions [10,15].
In recent years, numerous molecular phylogenetic studies on the tribe Ranunculeae
have been published [
11
–
13
]. However, all of these studies used a small number of DNA
fragments for phylogenetic inference, and their results had inevitable limitations such as
low resolution and statistical support due to insufficient phylogenetic signal. The complete
plastid genomes of the family Ranunculaceae gained more and more attention in the last few
years [
16
–
19
]. Both sequence and structural variations (such as IR expansion/contraction,
gene inversion, and gene transposition) in the plastomes of Ranunculaceae showed the
potential to yield phylogenetic significance when comprehensive data are available [
17
].
There is an urgent need to incorporate genomic data to deepen our insights into the
phylogeny of Ranunculeae. However, a very small number of the plastid genomes of this
tribe have been published up to now.
In this study, the complete plastomes of nine species (eleven samples), represent-
ing three genera of Ranunculeae, were assembled using the next-generation sequencing
method and reference-guided assembly. We described the bioinformatic characteristics of
the plastomes, such as gene content, codon usage, RNA editing sites, repeat sequences,
and positive selection. We also compared the synteny of the plastid genome sequences
across the family to investigate their plastid genome structural variation and gene order.
Finally, combining all the currently available plastome sequences of Ranunculeae species
in GenBank, we reconstructed the phylogenetic framework to assess the potential value
of the plastome sequences across the tribe. The aims of this study are to: understand the
variation of the plastomes across Ranunculus and its close allies, to compare the plastome
structures (gene order and arrangement) of Ranunculeae with those of the other genera
of Ranunculaceae, and to advance the phylogenetic and evolutionary understanding of
Ranunculeae.
2. Materials and Methods
2.1. Plant Sampling and Next-Generation Sequencing
Leaf samples of eleven new accessions representing three genera (Ceratocephala,Haler-
pestes, and Ranunculus) and nine species of tribe Ranunculeae were collected from field
(Table 1). The identification of the specimens was conducted by LX (Lei Xie) and all the
vouchers were deposited in the herbarium of Beijing Forestry University (BJFC). In addition,
we retrieved all the available complete plastome sequences of tribe Ranunculeae as well as
plastomes of its allies in Ranunculaceae from GenBank for comparative and phylogenetic
analyses. In total, 11 genera and 33 species (36 samples) of Ranunculaceae (Table 1) were
included for different analyses (see below in detail).
For each new sample, about 50 mg of dried leaf tissue was ground for DNA extraction.
We used DNA extraction kits (Tiangen Biotech Co., Ltd., Beijing, China) to obtain total
genomic DNA. Extracted DNAs were checked by 1.0% agarose gel electrophoresis and then
were sent to BerryGenomics (Beijing, China) for library construction and next-generation
sequencing (NGS). NGS was run on the Illumina NovaSeq 6000 platform (Illumina Inc.,
San Diego, CA, USA) to generate paired-end reads of 2 ×150 bp.
Genes 2023,14, 2140 3 of 17
Table 1. Sample information pertaining to the present study.
Tribe Species Collecting Site Voucher Number GenBank No.
Ranunculeae Ceratocephala testiculata * Altay, Xinjiang, China L. Xie 2016S3 (BJFC) OR625574
Ranunculeae Ce. testiculata * Altay, Xinjiang, China L. Xie 2016S46 (BJFC) OR625575
Ranunculeae Ranunculus monophyllus * Altay, Xinjiang, China L. Xie 2016S2 (BJFC) OR625578
Ranunculeae R. polyrhizos * Altay, Xinjiang, China L. Xie 2016S47 (BJFC) OR625579
Ranunculeae R. tanguticus * Daocheng, Sichuan, China W.H. Li WH072 (BJFC) OR625580
Ranunculeae R. mongolicus * Qinghe, Xinjiang, China C. Shang et al. I-4186 (BJFC) OR625576
Ranunculeae R. trichophyllus * Tingri, Xizang, China W.H. Li DR008 (BJFC) OR625577
Ranunculeae R. bungei * Xinglong, Hebei, China L. Xie et al. PL002 (BJFC) OR625572
Ranunculeae R. trichophyllus * Xiangrila, Yunnan, China L. Xie et al. T-20220808
016 (BJFC) OR625582
Ranunculeae R. pekinense * Yanqing, Beijing, China L. Xie and Y.K. Luo
20200916001 (BJFC) OR625573
Ranunculeae Halerpestes tricuspis * Zhongba, Xizang, China W.H. Li ZB006 (BJFC) OR625581
Anemoneae Anemoclema glaucifolium MH205609
Anemoneae A. tomentosa NC_039451
Anemoneae Pulsatilla chinensis NC_039452
Anemoneae A. trullifolia MH205608
Anemoneae Clematis brevicaudata MT796620
Anemoneae Cl. terniflora KJ956785
Adonideae Adonis coerulea MK253469
Delphinieae Aconitum barbatum MK253470
Ranunculeae Ce. falcata MK253464
Ranunculeae H. sarmentosa MK253457
Ranunculeae Oxygraphis glacialis MK253453
Ranunculeae R. austro-oreganus KX639503
Ranunculeae R. cantoniensis NC_045920
Ranunculeae R. cassubicifolius OP250948
Ranunculeae R. chinensis ON500677
Ranunculeae R. japonicus MZ169045
Ranunculeae R. macranthus NC_008796
Ranunculeae R. macranthus DQ359689
Ranunculeae R. membranaceus NC_065303
Ranunculeae R. occidentalis NC_031651
Ranunculeae R. reptans NC_036977
Ranunculeae R. sceleratus MK253452
Ranunculeae R. silerifolius ON462450
Ranunculeae R. ternatus OQ943173
Ranunculeae R. yunnanensis MZ703201
* newly sequenced in this study.
2.2. Plastid Genome Assembling and Annotating
After obtaining raw reads, we used the FASTX Toolkit (http://hannonlab.cshl.edu/
fastx_toolkit, accessed on 18 June 2022) to remove the adaptors and low-quality reads.
The plastid genome sequences were then de novo assembled according to our previous
study [
17
]. GetOrganelle (https://github.com/Kinggerm/GetOrganelle, accessed on 15
July 2022) was used with SPAdes 3.10.1 as the assembler [
20
]. Contigs were connected into
larger ones using RepeatFinder option in Geneious v. Prime [
21
], and when necessary, the
gaps were bridged using 100 replicates of Fine Tuning in Geneious Prime [
21
] to generate
complete plastome sequences. The gaps and junctions between IRs and LSC/SSC regions
were further verified by Sanger sequencing PCR amplifications. The assembled plastome
sequences were then annotated using the Plastid Genome Annotator [
22
]. Plastid genome
circles were drawn using the Organellar Genome DRAW v. 1.3.1 [23].
2.3. Comparative Analyses of the Plastomes
The newly sequenced plastomes were aligned and compared with those of the previ-
ously published Ranunculaceae species. Geneious Prime [
21
] and CodonW v. 1.4.2 [
24
] were
used to calculate amino acid frequency and codon usage for the new samples. We checked
putative RNA editing sites in protein-coding genes by the PREP-cp suite [
25
] for the new
samples. The plastome sequences across Ranunculaceae were aligned using mVISTA [
26
]
for the synteny analysis. We used LAGAN and Shuffle-LAGAN modes with default param-
eters to detect possible plastome structural variation. IR expansion/contraction of all the
Genes 2023,14, 2140 4 of 17
available Ranunculeae samples were checked using IRscope [
27
]. The nucleotide variability
(Pi) of the plastomes of both Ranunculeae and Ranunculus (which have the most species)
were calculated using a sliding window analysis implemented in DnaSP v. 5 [28].
We searched plastid microsatellites by using software MIcroSAtellite (MISA) [
29
] with
a minimum threshold of ten nucleotides for mononucleotide repeats, five for di-, four for
tri-, and three for tetra-, penta-, and hexanucleotide repeats according to our previous
study [
17
]. We also searched forward (F), reverse (R), complement (C), and palindromic
(P) oligonucleotide repeats using the REPuter program [
30
] with a minimum repeat size of
30 bp and similarity value of 90%.
2.4. Positive Selection Analysis
All the available Ranunculeae samples (25 species, 28 samples) and outgroups (four
species from Trib. Anemoneae) were used for CDS extraction using Geneious Prime [
21
].
The program CODEML implemented in PAML v. 4.10.6 package [
31
,
32
] was applied for
the positive selection site analysis. We estimated a single dN:dS ratio (
ω
) of the entire
alignment for the null model. Then, the branch model (model = 2; NSsites = 0) was used
to estimate a single
ω
of all the lineages of tribe Ranunculeae as the foreground, and a
different
ω
of the lineages from the outgroups tribe Anemoneae as the background. Finally,
a chi-square distribution was applied to assess the significance of the results. On the other
hand, the Bayes Empirical Bayes (BEB) method was also applied to identify specific amino
acid sites in genes to calculate posterior probability values (PP). High PP values (P > 0.9) of
the codon sites were considered to be positive selection sites [
33
,
34
]. According to previous
studies, we take the genes with a p-value < 0.05 and at least one positively selected site
with high PP values as a positive selection gene [35].
2.5. Phylogenetic Analysis
The phylogenetic framework was reconstructed for all the available species (28 sam-
ples representing 25 species) of tribe Ranunculeae. Previous studies showed that tribe
Anemoneae is sister to Ranunculeae in the family [
16
,
17
,
19
], so we chose four samples
from Anemoneae as the outgroups. For phylogenetic tree reconstruction, the IRa region
was excluded from the analysis. Inversion and translocation regions in tribe Anemoneae
were manually adjusted. To investigate potential differences in phylogenetic reconstruc-
tion using different partitions, we divided the complete plastome sequences under the
following partition strategies. The complete dataset was first separated into coding regions
(CDS), intergenic spacer regions (IGS), and introns. Each dataset was further separated
by their positions: LSC, SSC, and IR, respectively. We ultimately obtained 13 datasets for
phylogenetic analysis. They are the complete plastome, the complete CDS sequence, the
complete IGS, the complete intron, the LSC-CDS, the LSC-IGS, the LSC intron, the SSC-CDS,
the SSC-IGS, the SSC-intron, the IR-CDS, the IR-IGS, and the IR-intron datasets. Multiple
alignments for all the datasets were conducted by MAFFT v. 6.833 [
36
]. We removed
ambiguous alignments using a Python script written in our previous study [17].
For each dataset, the maximum likelihood (ML) and the Bayesian inference (BI) meth-
ods were applied for phylogenetic reconstruction. Substitution models and data partitions
of the complete plastome dataset were tested by PartitionFinder v2.1.1 [
37
]. We tested six
partitioning schemes for the complete plastome dataset according to previous studies [
38
].
They are (1) no partitions, (2) by coding and non-coding regions, (3) by positions of LSC,
SSC, and IRs, (4) by genes for the CDS and non-coding region as a separate partition, (5) by
genes and codon positions for the CDS and non-coding region as separate partition, (6) by
the third codon position for the coding region. The Bayesian information criterion (BIC)
was applied to assess the best partitioning scheme. The ML analysis was carried out using
RAxML v.8.1.17 [
39
] with the GTR + G model recommended in the user’s manual. We run
500 replicates of resampling analysis to obtain the ML bootstrap support values. The BI
analysis was conducted using MrBayes v3.2.3 [
40
], with the default priors for tree search.
Two Markov chain Monte Carlo (MCMC) chains, each with three heated and one cold chain,
Genes 2023,14, 2140 5 of 17
were independently run for 2,000,000 generations with tree sampling every 100 generations.
The first 25% of the trees were discarded as burn-in, and the remaining 75% of trees were
then summarized to yield the Bayesian consensus phylogram.
3. Results
3.1. Plastome Characterization of Ranunculeae Genera and Species
We obtained up to 12 Gb raw NGS data to assemble the plastid genome sequences.
By using reference sequences, we filtered out 412,658–651,511 plastid reads from the raw
reads for plastome assembly, which was 391 to 626
×
coverage of the plastid genome of
Ranunculeae. When assembling, we successfully bridged gaps by our previous method [
17
],
and those gaps and IR/SC boundaries were confirmed by PCR amplification. All the newly
assembled plastome sequences were deposited in the public online database GenBank
under accession numbers from OR625572 to OR625582 (Table 1).
The length of all the newly assembled plastome sequences of Ranunculeae ranged
from 150,820 bp (C. testiculata) to 158,344 bp (H. tricuspis) with the overall GC content of 36.7
to 37.4% (Figure 1; Supplementary Table S1). Within the genus Ranunculus, the length of
plastome sequences ranged from 155,973 bp (R. monophyllus) to 158,314 bp (R. trichophyllus),
with the overall GC content of 36.7 to 36.8%. In Ranunculeae samples, all the plastome
sequences contained a LSC (83,575–86,441 bp), an SSC region (17,619–21,735 bp), and a
pair of IRs (24,168–27,868 bp) regions and showed a typical structure in Angiosperms. A
set of 112 genes were present in the plastomes of Ranunculeae samples, among which 78
are protein-coding genes, 30 are transfer RNAs, and 4 are ribosomal RNA genes (Table 2).
A total of 16 (Ceratocephala samples) and 17 (other newly sequenced samples) genes were
located in a single IR region. A total of 18 (in Ranunculus and Halerpestes samples) and 17
(in Ceratocephala samples) genes have introns (Supplementary Table S2). In Ranunculus and
Halerpestes samples, the longest intron is in the clpP gene (1497 bp in R. polyrhizos
−1562 bp
in H. tricuspis), whereas in Ceratocephala samples, the longest intron is in the ycf 3 gene
(1442 bp).
Table 2. Genes present in the plastid genomes of the 11 newly sequenced Ranunculeae samples.
Gene Type Gene Name
Ribosomal RNA genes 16S rRNA 23S rRNA 4.5S rRNA 5S rRNA
Transfer RNA genes trnA-UGC gene trnC-GCA gene trnD-GUC gene trnE-UUC gene trnF-GAA gene
trnfM-CAU gene trnG-GCC gene trnG-UCC gene trnH-GUG gene trnI-CAU gene
trnI-GAU gene trnK-UUU gene trnL-CAA gene trnL-UAA gene trnL-UAG gene
trnM-CAU gene trnN-GUU gene trnP-UGG gene trnQ-UUG gene trnR-ACG gene
trnR-UCU gene trnS-GCU gene trnS-GGA gene trnS-UGA gene trnT-GGU gene
trnT-UGU gene trnV-UAC gene trnV-GAC gene trnW-CCA gene trnY-GUA gene
Small subunit of the ribosome
rps2 gene rps3 gene rps4 gene rps7 gene rps8 gene
rps11 gene rps12 gene rps14 gene rps15 gene rps16 gene
rps18 gene rps19 gene
The large subunit of the
ribosome rpl2 gene rpl14 gene rpl16 gene rpl20 gene rpl22 gene
rpl23 gene rpl32 gene rpl33 gene rpl36 gene
RNA polymerase subunits rpoA gene rpoB gene rpoC1 gene rpoC2 gene
NADH dehydrogenase ndhA gene ndhB gene ndhC gene ndhD gene ndhE gene
ndhF gene ndhG gene ndhH gene ndhI gene ndhJ gene
ndhK gene
Photosystem I psaA gene psaB gene psaC gene psaI gene psaJ gene
Cytochrome b/f complex petA gene petB gene petD gene petG gene petL gene
petN gene
ATP synthase atpA gene atpB gene atpE gene atpF gene atpH gene
atpI gene
Large subunit of rubisco rbcL gene
Maturase matK gene
Protease clpP gene
Genes 2023,14, 2140 6 of 17
Table 2. Cont.
Gene Type Gene Name
Envelope membrane protein cemA gene
Subunit of
acetyl-CoA-carboxylase accD gene
Photosystem II psbA gene psbB gene psbC gene psbD gene psbE gene
psbF gene psbH gene psbI gene psbJ gene psbK gene
psbL gene psbM gene psbN gene psbT gene psbZ gene
Copper chaperone for
superoxide dismutase ccsA gene
Conserved open
reading frames Ycf 1,2,3,4
Genes 2023, 14, x FOR PEER REVIEW 6 of 18
Figure 1. Gene maps of the newly sequenced plastome sequences of Ranunculus using Organellar
Genome DRAW (A,B), Ceratocephala (C), and Halerpestes (D). For each circle, bold lines on the outer
circle show the IR regions, while unbold lines indicate LSC and SSC regions. The inner track shows
the G + C content. Genes transcribed in a clockwise direction are located on the outside of circle,
while genes transcribed in a counterclockwise direction are on the inside of the map. LSC: large
single copy region; SSC: small single copy region; IR: inverted repeat region. Arrows point the dif-
ferent IR-SC boundaries. Yellow and blue arrows indicate different changes at the same location in
each of the four gene maps.
Table 2. Genes present in the plastid genomes of the 11 newly sequenced Ranunculeae samples.
Gene Type Gene Name
Ribosomal RNA genes 16S rRNA 23S rRNA 4.5S rRNA 5S rRNA
Transfer RNA genes trnA-UGC gene trnC-GCA gene trnD-GUC gene trnE-UUC gene trnF-GAA
gene
trnfM-CAU gene trnG-GCC gene trnG-UCC gene trnH-GUG gene trnI-CAU
gene
trnI-GAU gene trnK-UUU gene trnL-CAA gene trnL-UAA gene trnL-UAG
gene
trnM-CAU gene trnN-GUU gene trnP-UGG gene trnQ-UUG gene trnR-ACG
gene
trnR-UCU gene trnS-GCU gene trnS-GGA gene trnS-UGA gene trnT-GGU
gene
trnT-UGU gene trnV-UAC gene trnV-GAC gene trnW-CCA gene trnY-GUA
gene
Figure 1.
Gene maps of the newly sequenced plastome sequences of Ranunculus using Organellar
Genome DRAW (
A
,
B
), Ceratocephala (
C
), and Halerpestes (
D
). For each circle, bold lines on the outer
circle show the IR regions, while unbold lines indicate LSC and SSC regions. The inner track shows
the G + C content. Genes transcribed in a clockwise direction are located on the outside of circle,
while genes transcribed in a counterclockwise direction are on the inside of the map. LSC: large single
copy region; SSC: small single copy region; IR: inverted repeat region. Arrows point the different
IR-SC boundaries. Yellow and blue arrows indicate different changes at the same location in each of
the four gene maps.
3.2. Comparative Results of the Plastomes
Multiple alignments using mVISTA were carried out for Ranunculeae samples to
investigate plastid genome structural variations. Species with both normal and specific
Genes 2023,14, 2140 7 of 17
(in Adonis and tribe Anemoneae) plastome structures were also included. Two methods,
LAGAN and Shuffle-LAGAN, were conducted and shown in Figure 2. When using the
LAGAN method, Ranunculeae plastomes showed the same gene order as that of the
Aconitum samples, but large empty (mismatch) regions were found in the LSC regions of
Adonis and tribe Anemoneae samples due to gene inversion or gene translocation events.
Genes 2023, 14, x FOR PEER REVIEW 8 of 18
Figure 2. Multiple sequence alignments of Ranunculeae samples and its allies by mVISTA program.
(A): alignment with LAGAN method, the white (empty) regions in the Anemoneae and Adonideae
samples are the inverted and transposed regions; (B): alignment with shuffle LAGAN method. Blue
regions show the coding regions, while green shows the rRNA regions, and pink shows the non-
coding regions.
Figure 2.
Multiple sequence alignments of Ranunculeae samples and its allies by mVISTA program.
(
A
): alignment with LAGAN method, the white (empty) regions in the Anemoneae and Adonideae
samples are the inverted and transposed regions; (
B
): alignment with shuffle LAGAN method.
Blue regions show the coding regions, while green shows the rRNA regions, and pink shows the
non-coding regions.
Genes 2023,14, 2140 8 of 17
Because the IR expansion/contraction may carry important phylogenetic information
in Ranunculaceae [
17
], the IR/SC boundaries of the newly sequenced plastomes were com-
pared with other outgroups in the family. The newly sequenced Ranunculus and Halerpestes
samples as well as the published Oxygraphis sample in Ranunculeae have 17 genes in their
IR region, which is the same as many other genera in Ranunculaceae (such as Aconitum
L., Caltha, L., Coptis Salisb, Delphinium L., and Thalictrum L.) and other angiosperm taxa
such as Amborella Baill. and Arabidopsis Heynh. [
17
,
34
]. Therefore, this 17-gene IR region
of Ranunculus and Halerpestes can be taken as the primitive type in Ranunculaceae [
17
].
Whereas the IR regions of Ceratocephala samples showed slight contraction with incomplete
rpl2 genes on the LSC/IR borders compared to the Ranunculus and Halerpestes samples
(Figure 3).
Genes 2023, 14, x FOR PEER REVIEW 9 of 18
Figure 3. Detailed IR-SC boundaries of the newly sequenced samples. SC: single copy region; IR:
inverted repeats.
Nucleotide variability was assessed by sliding window analysis, and the results (Fig-
ure 4) showed that the IR region has a lower variability than the SC regions in Ranunculus
samples. When taking all the Ranunculeae samples into consideration, the trend of lower
nucleotide variability in the IR region is also obvious. In Ranunculus samples, our result
discovered extremely high variations at the border of the IR/SSC regions.
Figure 3.
Detailed IR-SC boundaries of the newly sequenced samples. SC: single copy region;
IR: inverted repeats.
Genes 2023,14, 2140 9 of 17
Nucleotide variability was assessed by sliding window analysis, and the results
(
Figure 4
) showed that the IR region has a lower variability than the SC regions in Ranun-
culus samples. When taking all the Ranunculeae samples into consideration, the trend of
lower nucleotide variability in the IR region is also obvious. In Ranunculus samples, our
result discovered extremely high variations at the border of the IR/SSC regions.
Genes 2023, 14, x FOR PEER REVIEW 10 of 18
Figure 4. Graph of sliding window analysis showing plastome nucleotide variability (Pi) of Ranun-
culus (A) and Ranunculeae (B).
3.3. Synonymous Codon Usage
This study calculated the relative synonymous codon usage (RSCU) for the newly
assembled plastome sequences using all the protein-coding genes. We presented results
of amino acid frequency and putative RNA editing sites in Figure 5 and Supplementary
Tables S2 and S3. We detected 95 putative RNA editing sites in the 24 protein-coding genes
of Ceratocephala, 92 sites in 27 protein-coding genes of Halerpestes, 93 sites in 27 protein-
coding genes of R. bungei and R. pekinensis, and 91 sites in 27 protein-coding genes of the
other five Ranunculus species. In the Ranunculus samples, ndhF has the most RNA editing
sites (10 and 11 sites), and the second was matK (9 sites). In the Ceratocephala samples,
rpoC2 gene has the most RNA editing sites (12 sites), and the second was ndhF (11 sites).
For the Halerpestes samples, both rpoC2 and ndhF genes have the most RNA editing sites
(10 sites), and then was ndhB gene (8 sites).
The substitution from serine to leucine was tested to be the most common type
(30.1%) in R. bungei and R. pekinensis, followed by serine to phenylalanine (15.1%),
whereas in the other Ranunculus species, serine to leucine was the most common one
(29.7%), followed by threonine to isoleucine (14.3%). In Ceratocephala, substitution from
serine to leucine accounted for 26.3% of the editing sites, and from serine to phenylalanine
was 13.7%. In Halerpestes, 31.5% of editing sites substituted from serine to leucine, and
Figure 4.
Graph of sliding window analysis showing plastome nucleotide variability (Pi) of Ranuncu-
lus (A) and Ranunculeae (B).
3.3. Synonymous Codon Usage
This study calculated the relative synonymous codon usage (RSCU) for the newly assem-
bled plastome sequences using all the protein-coding genes. We presented results of amino
acid frequency and putative RNA editing sites in Figure 5and
Supplementary Tables S2 and S3
.
We detected 95 putative RNA editing sites in the 24 protein-coding genes of Ceratocephala,
92 sites in 27 protein-coding genes of Halerpestes, 93 sites in 27 protein-coding genes of R.
bungei and R. pekinensis, and 91 sites in 27 protein-coding genes of the other five Ranunculus
species. In the Ranunculus samples, ndhF has the most RNA editing sites (10 and 11 sites),
and the second was matK (9 sites). In the Ceratocephala samples, rpoC2 gene has the most
RNA editing sites (12 sites), and the second was ndhF (11 sites). For the Halerpestes samples,
both rpoC2 and ndhF genes have the most RNA editing sites (10 sites), and then was ndhB
gene (8 sites).
Genes 2023,14, 2140 10 of 17
Genes 2023, 14, x FOR PEER REVIEW 11 of 18
15.2% from serine to phenylalanine, and among all its RNA editing sites, 23 substitutions
appeared at the first nucleotide positions while 71 substitutions occurred at the second
nucleotide position. Plastomes of the other two genera showed similar results in the sub-
stitution site on the codon positions (Supplementary Table S2).
Figure 5. The values of relative synonymous codon usage for the 20 amino acids and stop codons in
the plastomes of the newly sequenced samples.
3.4. SSR, Repetitive Sequences and Positive Selection Analysis
Rich SSRs including mononucleotide to hexonucleotide repeats were detected rang-
ing from 47 to 70 in the newly sequenced plastomes (Supplementary Table S4). Among all
the tested species, C. testiculata has the fewest SSRs, whereas H. tricuspis has the most. The
most common SSR is mononucleotide repeat (A/T) among the nine species. For the tested
species, the least proportion (53.2%) of the mononucleotide repeats was in C. testiculata,
whereas the highest proportion (70.0%) was in H. tricuspis. The rare mononucleotide re-
peat (G/C) was only found in R. mongolicus, R. monophyllus, and R. trichophyllus. The sec-
ond most common SSR is dinucleotide repeat (AT/TA) with six, eight, and nine replicates,
respectively. The third most common SSR is tetranucleotide repeat (AATG/TGAA) with
six, seven, nine, and ten replicates, respectively, and its total number was slightly smaller
than the dinucleotide repeats. The fourth most common SSR is trinucleotide repeat
(AAT/TTA), whereas pentanucleotide repeats were present in all the tested samples but R.
mongolicus, and hexanucleotide repeats were only present in the plastomes of H. tricuspis,
R. bungei, and R. pekinense. Within the newly sequenced plastomes, the largest proportion
of SSR loci were found in IGS, followed by CDS and Intron. Within the plastid genome
circle, SSRs are most common in the LSC region, followed by SSC, and the least in IR
regions (Supplementary Table S4).
The eleven newly sequenced plastomes had a total of 281 direct, reverse, palindromic,
and complement repeats (Figure 6), which may serve as potential molecular markers for
further population genetic studies. Direct repeat was tested to be the most common repeat
type, which accounted for 54.8% of the total repeats. It was followed by palindromic re-
peat (38.8%), reverse repeat (5.3%), and complement repeat (1.1%). The only three com-
plement repeats were found in R. pekinense, R. polyrhizos, R. tanguticus, respectively. Re-
peats were usually short with 30–49 bp in length. We also found several longer direct and
reverse repeats up to 82 bp in some Ranunculus samples. The largest proportion of repeats
was found in the IGS region (73%), followed by CDS (21%) and Intron (6%) (Figure 6).
Figure 5.
The values of relative synonymous codon usage for the 20 amino acids and stop codons in
the plastomes of the newly sequenced samples.
The substitution from serine to leucine was tested to be the most common type (30.1%)
in R. bungei and R. pekinensis, followed by serine to phenylalanine (15.1%), whereas in the
other Ranunculus species, serine to leucine was the most common one (29.7%), followed
by threonine to isoleucine (14.3%). In Ceratocephala, substitution from serine to leucine
accounted for 26.3% of the editing sites, and from serine to phenylalanine was 13.7%. In
Halerpestes, 31.5% of editing sites substituted from serine to leucine, and 15.2% from serine
to phenylalanine, and among all its RNA editing sites, 23 substitutions appeared at the
first nucleotide positions while 71 substitutions occurred at the second nucleotide position.
Plastomes of the other two genera showed similar results in the substitution site on the
codon positions (Supplementary Table S2).
3.4. SSR, Repetitive Sequences and Positive Selection Analysis
Rich SSRs including mononucleotide to hexonucleotide repeats were detected ranging
from 47 to 70 in the newly sequenced plastomes (Supplementary Table S4). Among all the
tested species, C. testiculata has the fewest SSRs, whereas H. tricuspis has the most. The
most common SSR is mononucleotide repeat (A/T) among the nine species. For the tested
species, the least proportion (53.2%) of the mononucleotide repeats was in C. testiculata,
whereas the highest proportion (70.0%) was in H. tricuspis. The rare mononucleotide repeat
(G/C) was only found in R. mongolicus,R. monophyllus, and R. trichophyllus. The second
most common SSR is dinucleotide repeat (AT/TA) with six, eight, and nine replicates,
respectively. The third most common SSR is tetranucleotide repeat (AATG/TGAA) with six,
seven, nine, and ten replicates, respectively, and its total number was slightly smaller than
the dinucleotide repeats. The fourth most common SSR is trinucleotide repeat (AAT/TTA),
whereas pentanucleotide repeats were present in all the tested samples but R. mongolicus,
and hexanucleotide repeats were only present in the plastomes of H. tricuspis,R. bungei,
and R. pekinense. Within the newly sequenced plastomes, the largest proportion of SSR
loci were found in IGS, followed by CDS and Intron. Within the plastid genome circle,
SSRs are most common in the LSC region, followed by SSC, and the least in IR regions
(Supplementary Table S4).
The eleven newly sequenced plastomes had a total of 281 direct, reverse, palindromic,
and complement repeats (Figure 6), which may serve as potential molecular markers for
further population genetic studies. Direct repeat was tested to be the most common repeat
type, which accounted for 54.8% of the total repeats. It was followed by palindromic repeat
(38.8%), reverse repeat (5.3%), and complement repeat (1.1%). The only three complement
Genes 2023,14, 2140 11 of 17
repeats were found in R. pekinense,R. polyrhizos,R. tanguticus, respectively. Repeats were
usually short with 30–49 bp in length. We also found several longer direct and reverse
repeats up to 82 bp in some Ranunculus samples. The largest proportion of repeats was
found in the IGS region (73%), followed by CDS (21%) and Intron (6%) (Figure 6).
Genes 2023, 14, x FOR PEER REVIEW 12 of 18
Figure 6. Graphs of repeated sequence analyses for the newly assembled plastomes. (A) Histogram
of four repeat type numbers; (B) Histogram of palindromic repeats by length; (C) Pie chart showing
proportion of repeats in different locations; (D) Histogram of forward repeats by length.
Positive selection of 67 CDS was tested for all the available Ranunculeae samples and
its close allies. The likelihood ratio analysis showed that p-values of most genes were >0.05
(insignificant), except that atpB, ndhC, ndhG, ndhJ, psaC, rps2, rps15, ycf2 (p < 0.05). Further-
more, the nonsynonymous/synonymous rate ratio (ω = dN/dS) of only one gene, accD, is
>1, but its p-value is >0.05. However, the BEB test showed that accD, atpF, ccsA, ndhF, petD,
rbcL, rpoA, rpoC2 and ycf2 have high posterior probability values (≥0.9) (Supplementary
Table S5). Previous studies considered that a coding region with a high posterior proba-
bility value of the BEB analysis can be taken as a positive selection gene [35]. Under this
measure, nine genes, accD, atpF, ccsA, ndhF, petD, rbcL, rpoA, rpoC2, and ycf2 can be con-
sidered as positive selection genes.
3.5. Partitioning and Phylogenetic Reconstruction Results
We tested the complete plastome dataset by using six data partitioning strategies.
The results showed that those six partitioning treatments obtained quite different results,
indicating that different partitioning methods may greatly affect phylogenetic reconstruc-
tion. It showed that the partitioning strategy by LSC, SSC, and IRs obtained the best results
(Table 3). According to this result, this partitioning strategy was applied for CDS, IGS,
introns, and complete datasets, and no partitioning strategy was applied for the rest of the
datasets. The GTR model for each partition of CDS, IGS, introns, and complete datasets
was applied for BI analysis, while the GTR + I + G model was used for the rest of the
datasets for both ML and BI analyses according to our partition results.
Table 3. Partitioning strategy tests for the complete plastid genome dataset using PartitionFinder.
Dataset Partitioning Strategy Parameters Subsets ln L BIC
Complete
Plastome
Dataset
No partition 63 1 −492,025.19 984,804.90
Coding and non-coding 74 2 −600
,
890.34 1
,
202
,
691.59
LSC, SSC, IRs 85 3 −489,387.16 979,792.32
By gene 162 11 −710,387.28 1,422,765.77
By gene and codon position 219 17 −708,937.30 1,420,566.42
Figure 6. Graphs of repeated sequence analyses for the newly assembled plastomes. (A) Histogram
of four repeat type numbers; (
B
) Histogram of palindromic repeats by length; (
C
) Pie chart showing
proportion of repeats in different locations; (D) Histogram of forward repeats by length.
Positive selection of 67 CDS was tested for all the available Ranunculeae samples and
its close allies. The likelihood ratio analysis showed that p-values of most genes were >0.05
(insignificant), except that atpB, ndhC, ndhG, ndhJ, psaC, rps2, rps15, ycf 2 (
p< 0.05
). Further-
more, the nonsynonymous/synonymous rate ratio (
ω
= dN/dS) of only one gene, accD, is
>1, but its p-value is >0.05. However, the BEB test showed that accD, atpF, ccsA, ndhF, petD,
rbcL, rpoA, rpoC2 and ycf 2 have high posterior probability values (
≥
0.9) (Supplementary
Table S5). Previous studies considered that a coding region with a high posterior probability
value of the BEB analysis can be taken as a positive selection gene [
35
]. Under this measure,
nine genes, accD, atpF, ccsA, ndhF, petD, rbcL, rpoA, rpoC2, and ycf 2 can be considered as
positive selection genes.
3.5. Partitioning and Phylogenetic Reconstruction Results
We tested the complete plastome dataset by using six data partitioning strategies. The
results showed that those six partitioning treatments obtained quite different results, indi-
cating that different partitioning methods may greatly affect phylogenetic reconstruction.
It showed that the partitioning strategy by LSC, SSC, and IRs obtained the best results
(Table 3). According to this result, this partitioning strategy was applied for CDS, IGS,
introns, and complete datasets, and no partitioning strategy was applied for the rest of the
datasets. The GTR model for each partition of CDS, IGS, introns, and complete datasets was
applied for BI analysis, while the GTR + I + G model was used for the rest of the datasets
for both ML and BI analyses according to our partition results.
Genes 2023,14, 2140 12 of 17
Table 3. Partitioning strategy tests for the complete plastid genome dataset using PartitionFinder.
Dataset Partitioning Strategy Parameters Subsets ln L BIC
Complete
Plastome
Dataset
No partition 63 1 −492,025.19 984,804.90
Coding and non-coding 74 2 −600,890.34 1,202,691.59
LSC, SSC, IRs 85 3 −489,387.16 979,792.32
By gene 162 11 −710,387.28 1,422,765.77
By gene and codon position 219 17 −708,937.30 1,420,566.42
By the third codon position 84 3 −802,253.60 1,605,550.34
Phylogenies reconstructed by both complete and separate datasets and both methods
(ML and BI) were generally the same, especially in strongly supported clades (Figure 7,
Supplementary Figure S2). The complete plastome dataset generated the most robustly
resolved phylogeny of Ranunculeae. For this reason, our discussion was based on the phy-
logenetic framework inferred from the complete plastome dataset. All four tested genera
of the tribe Ranunculeae were strongly supported. Two genera Halerpestes and Oxygraphis
were sister groups and formed a clade sister to another clade including Ranunculus and
Ceratocephala. Three major clades were resolved in the largest genera Ranunculus. Clade
1 includes species mainly from sect. Auricomus (Spach) Schur [
15
]. Clade 2 comprises
aquatic species from sect. Batrachium DC. and a hydrophilus species R. sceleratus L from
sect. Hecatonia. Clade 3 includes species mainly from sect. Flammula and sect. Acris
Schur [10].
Genes 2023, 14, x FOR PEER REVIEW 13 of 18
By the third codon position 84 3 −802,253.60 1,605,550.34
Phylogenies reconstructed by both complete and separate datasets and both methods
(ML and BI) were generally the same, especially in strongly supported clades (Figure 7,
Supplementary Figure S2). The complete plastome dataset generated the most robustly
resolved phylogeny of Ranunculeae. For this reason, our discussion was based on the phy-
logenetic framework inferred from the complete plastome dataset. All four tested genera
of the tribe Ranunculeae were strongly supported. Two genera Halerpestes and Oxygraphis
were sister groups and formed a clade sister to another clade including Ranunculus and
Ceratocephala. Three major clades were resolved in the largest genera Ranunculus. Clade 1
includes species mainly from sect. Aurico mus (Spach) Schur [15]. Clade 2 comprises
aquatic species from sect. Batrachium DC. and a hydrophilus species R. sceleratus L from
sect. Hecatonia. Clade 3 includes species mainly from sect. Flammula and sect. Acris Schur
[10].
Figure 7. The Bayesian phylogenetic tree of all the currently available Ranunculaceae samples in-
ferred from the complete plastome data. Numbers on nodes indicate maximum likelihood (ML)
bootstrap values/posterior probability (PP) values. Bold branches show the fully supported clades
with the ML bootstrap values =100 and PP values = 1.
4. Discussion
Many previous studies have reported that most of the plastid genomes of Ranuncu-
laceae have a set of 112 genes, and this is also the case in Ranunculeae species [17,19,35].
All our sequenced Ranunculeae samples also showed the same results as previous reports.
Gene inversions and gene loss in Ranunculaceae plastomes were revealed more than 20
years ago. Johansson [41] studied Adonis species using the restriction site mapping
method and found large inversions and gene loss presented in their plastid genomes. In
Figure 7.
The Bayesian phylogenetic tree of all the currently available Ranunculaceae samples
inferred from the complete plastome data. Numbers on nodes indicate maximum likelihood (ML)
bootstrap values/posterior probability (PP) values. Bold branches show the fully supported clades
with the ML bootstrap values =100 and PP values = 1.
Genes 2023,14, 2140 13 of 17
4. Discussion
Many previous studies have reported that most of the plastid genomes of Ranuncu-
laceae have a set of 112 genes, and this is also the case in Ranunculeae species [
17
,
19
,
35
].
All our sequenced Ranunculeae samples also showed the same results as previous re-
ports. Gene inversions and gene loss in Ranunculaceae plastomes were revealed more
than 20 years ago. Johansson [
41
] studied Adonis species using the restriction site mapping
method and found large inversions and gene loss presented in their plastid genomes. In
recent years, structural variations including gene inversions, gene translocations, and IR
variations have been explicitly reported in Ranunculaceae [
17
,
19
]. The plastome structural
variations of tribe Anemoneae also have structural variations, but in comparison with Ado-
nis, their variations have differed in many aspects. In genera Anemone s. l. and Anemoclema,
there are three large inversions in the LSC region of their plastomes, while in Clematis there
are two large inversions and one large transposition in the LSC region of the plastome [
17
].
In Ranunculeae species, gene orders of the plastome are the same as those of most other
genera (such as Aconitum,Thalictrum, and so on), and no gene inversions and translocations
are found.
The IR region normally has 17 genes, and this type of IR was considered to be primi-
tive in Ranunculaceae [
17
]. However, IR expansion/contractions are also very common in
Ranunculaceae. He et al. [
17
] reported that many genera (such as Asteropyrum,Anemone,
Anemoclema,Clematis,Dichocarpum,Hepatica,Hydrastis,Naravelia, and Pulsatilla) in Ranun-
culaceae have expanded IR regions, whereas only two genera, Helleborus and Ceratocephala,
were found to have slightly contracted IR regions. Up to 27 genes in Asteropyrum peltatum
were found in the plastome of the family, and IR expansion/contractions may carry impor-
tant phylogenetic information [
17
]. In tribe Ranunculeae, the majority of tested species have
17 genes in their IR regions except for Ceratocephala, which showed a little contraction in
the IR regions (Supplementary Table S1). IR contraction is rare in Ranunculaceae, and only
found in Helleborus and Ceratocephala [
17
]. In these two genera, rpl2 is not completely located
in the IR region (Figure 3), and these two generic cases seemed to have no phylogenetic
relationship in Ranunculaceae [
17
]. However, both species of Ceratocephala tested to have
the same contracted IR regions, indicating that this IR contraction may be a synapomorphy
in the genus Ceratocephala within the Ranunculeae clade. Our results showed that plastome
structural variation is not characteristic of Ranunculeae, but IR expansion/contraction may
have phylogenetic information.
Simple sequence repeats (SSRs) for microsatellites have been widely applied for
population genetics and evolutionary studies of Angiosperm species [
42
]. However, the
use of the plastid SSRs has not been fully developed in Ranunculaceae. Our results showed
that 47 to 70 plastid SSRs are found in the 11 new samples (Supplementary Table S4), and
pentanucleotide repeats are very common in the plastomes of Ranunculeae species. The
rich plastid SSR diversity can provide opportunities for future population genetic studies
on Ranunculeae species.
In Ranunculaceae, the tribe Ranunculeae is characterized by its ascending unitegmic
ovules (except Myosurus which has pendent ovules), often smaller sepals and larger petals,
and petals with one or more nectary glands near the base [
10
]. Some taxonomists in-
cluded Callianthemum and Adonis into Ranunculeae [
43
,
44
]. However, this treatment was
not supported by molecular phylogenetic analysis [
45
,
46
]. A large number of molecular
phylogenetic studies of Ranunculeae have been published [
11
–
13
,
47
–
49
] which helped us
understand the delimitation and generic relationship of this tribe. Based on molecular
phylogeny and comprehensive sampling, 19 genera were recognized within the tribe Ra-
nunculeae [
11
–
13
]. However, most of them were based on small numbers of DNA regions
(nrITS and plastid DNA fragments), and the phylogenetic relationship within the tribe
was still not robustly resolved. In this study, the generic relationship of the tribe inferred
from the complete plastome data was congruent with previous studies and more robustly
resolved (Figure 7and Supplementary Figure S2), therefore demonstrating that plastome
data may provide the opportunity for the reconstruction of generic phylogeny of Ranun-
Genes 2023,14, 2140 14 of 17
culeae in the future with comprehensive sampling. Our current sampling covered all the
generic representatives in China. The results showed the aquatic sect. Batrachium should be
included in Ranunculus but as a distinct genus. Generic statuses of Ceratocephala,Halerpestes,
and Oxygraphis can be kept.
Ranunculus is the largest genus in both Ranunculeae and Ranunculaceae with about
650 species around the world [
10
]. Taxonomy of Ranunculus has been considered extremely
difficult and there are considerable differences among different classifications [
9
,
15
,
50
–
53
].
For this reason, this genus also attracted great attention in its phylogeny using molecular
markers [
47
,
54
–
59
]. Based on a comprehensive sampling and nrITS and three plastid DNA
regions, Emadzade et al. [
57
] resolved nine major clades in Ranunculus. Although we
combined all the available complete plastome sequences of Ranunculus from GenBank,
the sampling is still limited. Three major clades were robustly resolved by our plastome
data. Clade 1 (Figure 6) corresponded to clade IV of Emadzade et al. [
57
] which included
species of sect. Auricomus. Clade 1 also included R. ternatus from the sect. Tuberifer whose
phylogenetic position has never been tested. Sect. Tuberifer is characterized by its tuberous
roots. Wang [
60
] considered that R. ternatus may be closely related to sect. Auricomus
and this prediction was supported by our phylogenetic analysis. Clade 2 included an
aquatic sect. Batrachium and sect. Hectonia in wetland, and well corresponded to cluster
III of Emadzade et al. [
57
]. The monophyly of cluster III in Emadzade et al. [
57
] was not
supported in their study. However, clade 2 is fully supported showing the advantage of
using the plastome data for phylogenetic reconstruction over the small number of DNA
regions by Sanger sequencing. Clade 3 was also fully supported. In this clade, the first
diverged R. reptans was in clade V of Emadzade et al. [
57
], and the other two subclades
(R. japonicus–R. occidentalis) and (R. cantoniensis–R. chinensis) correspond with clade VI and
Clade VIII of Emadzade et al. [
57
], respectively. Phylogenetic position of R. macranthus has
never been inferred, and this species is also nested in clade 3. In general, the phylogenetic
relationship within Ranunculus inferred by the complete plastome sequences was fully
congruent with previous molecular studies and showed advantages of high resolution.
Plastid phylogenomic analysis is needed for future studies with a comprehensive sampling.
5. Conclusions
The complete plastomes of eleven samples representing nine species of tribe Ranun-
culeae were de novo assembled using a next-generation sequencing method. The plastome
sequences from all the Ranunculaceae samples and their allies were compared in various
aspects including gene content, nucleotide variability, codon usage, RNA editing sites,
simple sequence repeats, and positive selection sites through bioinformatic analyses. The
phylogeny of Ranunculeae was reconstructed for the complete and separated datasets
using both ML and BI methods to infer generic and specific relationships within the tribe.
Our results showed that the majority of the Ranunculeae genera and species have the most
common plastid genome type, which is widely shared in the family [
17
], and there are
potential values of the plastome sequences for reconstructing the phylogeny of both the
tribe and the genus Ranunculus in future studies.
Supplementary Materials:
The following supporting information can be downloaded at:
https://www.mdpi.com/article/10.3390/genes14122140/s1, Figure S1: Detailed IR-SC boundaries
of all the tested samples. SC: single copy region; IR: inverted repeats. Figure S2: The Bayesian
phylogenetic tree of all the currently available Ranunculaceae samples inferred from the separated
data. Numbers on nodes indicate maximum likelihood (ML) bootstrap values/posterior probability
(PP) values. Bold branches show the fully supported clades with the ML bootstrap values = 100
and
PP values = 1
. Table S1: Major plastid genome features of the 11 newly sequenced samples
of Ranunculeae. Table S2: Prediction of RNA editing by the PREP-cp program. Table S3: Base
composition in the plastid genome of each sample. Table S4: Information of simple sequence repeats
(SSRs) for the 11 newly sequenced Ranunculeae samples. Table S5: The potential positive selection
test based on the branch-site model.
Genes 2023,14, 2140 15 of 17
Author Contributions:
Conceptualization, L.X., J.C., J.H. and L.P.; Data curation, J.J., J.H., Y.L.
(Yike Luo) and J.X.; Formal analysis, J.J. and Y.L. (Yike Luo); Funding acquisition, L.X. and J.C.;
Investigation, M.L., W.L., H.W., Y.L. (Yvexin Luo) and L.X.; Project administration, J.H.; Super-
vision, L.X.; Validation, L.X. and J.C.; Visualization, L.P.; Writing—original draft, J.J. and L.X.;
Writing—review and editing, L.X. and J.C. All authors have read and agreed to the published version
of the manuscript.
Funding:
This research was funded by the National Natural Science Foundation of China (Nos. 32270223
and 31670207).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The genome sequence data that support the findings of this study are
openly available from NCBI GenBank (https://www.ncbi.nlm.nih.gov, accessed on 30 September 2023)
under accession numbers from OR625572 to OR625582. The aligned datasets are available on Zenodo,
with the identifier https://doi.org/10.5281/zenodo.10012320, accessed on 20 September 2023.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
Abbreviations
SC Single copy region
LSC Large single copy region
SSC Small single copy region
IR Inverted repeat region
CDS Coding regions
IGS Intergenic spacer regions
dN Nonsynonymous substitution
dS Synonymous substitution
Pi Nucleotide variability
BEB Bayes Empirical Bayes
PP Posterior probability values
ML Maximum likelihood
BI Bayesian inference
BIC Bayesian information criterion
SSRs Simple sequence repeats
RSCU Relative synonymous codon usage
NGS Next-generation sequencing
MISA MIcroSAtellite
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