G C A T

T A C G
G C A T
genes
Article
Comparative Analysis of Complete Chloroplast Genomes of
Nine Species of Litsea (Lauraceae): Hypervariable Regions,
Positive Selection, and Phylogenetic Relationships
Weicai Song 1 , Zimeng Chen 1 , Wenbo Shi 1 , Weiqi Han 1 , Qi Feng 1 , Chao Shi 1,2, *, Michael S. Engel 3
and Shuo Wang 1, *

1 College of Marine Science and Biological Engineering, Qingdao University of Science and Technology,
Qingdao 266042, China
2 Plant Germplasm and Genomics Center, Germplasm Bank of Wild Species in Southwest China,
Kunming Institute of Botany, the Chinese Academy of Sciences, Kunming 650204, China
3 Department of Ecology & Evolutionary Biology, University of Kansas, Lawrence, KS 66045, USA
* Correspondence: [email protected] (C.S.); [email protected] (S.W.)

Abstract: Litsea is a group of evergreen trees or shrubs in the laurel family, Lauraceae. Species of the
genus are widely used for a wide range of medicinal and industrial aspects. At present, most studies
related to the gene resources of Litsea are restricted to morphological analyses or features of individual
genomes, and currently available studies of select molecular markers are insufficient. In this study,
we assembled and annotated the complete chloroplast genomes of nine species in Litsea, carried out a
series of comparative analyses, and reconstructed phylogenetic relationships within the genus. The
genome length ranged from 152,051 to 152,747 bp and a total of 128 genes were identified. High
consistency patterns of codon bias, repeats, divergent analysis, single nucleotide polymorphisms
Citation: Song, W.; Chen, Z.; Shi, W.;
(SNP) and insertions and deletions (InDels) were discovered across the genus. Variations in gene
Han, W.; Feng, Q.; Shi, C.; Engel,
length and the presence of the pseudogene ycf1Ψ , resulting from IR contraction and expansion, are
M.S.; Wang, S. Comparative Analysis
reported. The hyper-variable gene rpl16 was identified for its exceptionally high Ka/Ks and Pi
of Complete Chloroplast Genomes of
Nine Species of Litsea (Lauraceae):
values, implying that those frequent mutations occurred as a result of positive selection. Phylogenetic
Hypervariable Regions, Positive relationships were recovered for the genus based on analyses of full chloroplast genomes and protein-
Selection, and Phylogenetic coding genes. Overall, both genome sequences and potential molecular markers provided in this
Relationships. Genes 2022, 13, 1550. study enrich the available genomic resources for species of Litsea. Valuable genomic resources and
https://doi.org/10.3390/ divergent analysis are also provided for further research of the evolutionary patterns, molecular
genes13091550 markers, and deeper phylogenetic relationships of Litsea.
Academic Editor: Zhiqiang Wu
Keywords: Litsea; chloroplast genome; structural variations; genetic relationship
Received: 29 July 2022
Accepted: 25 August 2022
Published: 28 August 2022

Publisher’s Note: MDPI stays neutral 1. Introduction

with regard to jurisdictional claims in Litsea is an evergreen tree or shrub and is one of the most diverse genera (about
published maps and institutional affil- 400 species) in the family Lauraceae (Mesangiospermae: Magnoliids: Laurales). It is widely
iations. distributed in tropical and subtropical Asia, North and South America [1,2], and 74 species
are located in China, at a maximum elevation of 2700 m above sea level [3] Species of
Litsea are utilized in a wide range of applications, covering medical, agricultural, industrial,
and many other fields. Litsea can be used to treat a variety of conditions such as diarrhea,
Copyright: © 2022 by the authors.
stomach pain, indigestion, the common cold, gastroenteritis, diabetes, edema, arthritis,
Licensee MDPI, Basel, Switzerland.
asthma, pain, and trauma [1]. In addition, Litsea is also known for the highly effective
This article is an open access article
distributed under the terms and
properties of its essential oil against food-borne pathogens [4]. Its essential oils can also
conditions of the Creative Commons
be resistant to several types of bacteria, have antioxidant, anti-parasitic, acute toxicity,
Attribution (CC BY) license (https://
genotoxic, and cytotoxic properties, and can even prevent several types of cancer [5–7].
creativecommons.org/licenses/by/ Despite the pharmaceutical applications of Litsea, it is also widely used as feed for silkworm
4.0/). pupae, especially for muga silkworms (Antheraea assama) [5]. In comparison with ordinary

Genes 2022, 13, 1550. https://doi.org/10.3390/genes13091550 https://www.mdpi.com/journal/genes

Genes 2022, 13, 1550 2 of 18

silk produced from other food sources, muga silk produced from Litsea possesses a higher
value and is considered to be of better quality, as reflected in its creamy and lustrous
appearance and texture. Some representative species of Litsea are industrially important
and have been utilized extensively [6]. For instance, Litsea cubebais is a spice shrub of
considerable economic importance. The essential oil prepared from the citric acid extracted
from the plant’s body is a natural spice, with a wide number of potential applications.
Moreover, it is also an important raw material for the synthesis of vital compounds, such
as vitamin A [7].
Chloroplasts are organelles that occur in green plants and algae, taking the responsi-
bility for photosynthesis and other housekeeping functions. Additionally, they are essential
for nitrate and sulfate assimilation as well as the synthesis of amino acids, fatty acids,
chlorophyll, and carotenoids [8]. In general, chloroplast (cp) genomes have a conservative
genome structure, gene content, and gene order in most monocotyledon plants [8,9]. The
complete cp genome of angiosperms is usually composed of four parts: a large single-copy
(LSC) region, a small single-copy (SSC) region, and two similar inverted repeat (IR) regions,
with a highly conservative structure [10]. The cp genome consists of 110 to 130 genes
primarily involved in photosynthesis, transcription, and translation. The contraction and
expansion of IR regions and gene and intron loss events have also occurred commonly
during evolution [11]. The sequences of cp genomes can provide information for genetic
relationships, gene transfer, cloning, and species domestication. The cp genome of ad-
vanced plants is inherited from a single parent [12], which can be used as an effective
barcode for species identification as well as the development of other potential identifica-
tion markers [13]. Identification of cp genomes promotes the sustainable development of
plant species, their utilization in a more rigorous scientific manner, as well as for species
conservation [14–16].
As the rapid development and iteration of methods for obtaining and analyzing whole
cp genome sequences, studies on the cp genome have shown an explosive growth [17,18].
However, in the genus Litsea, reports were mostly focus on chemical compositions or
species-specific genomic traits [19]. Genetic resources for Litsea still need to be supple-
mented. Moreover, studies of the selection pressure and high diversity sequences within
the genus Litsea are greatly in demand. Therefore, a detailed assembly and annotation of
the complete cp genomes of various species within Litsea will greatly enrich the existing
database, deepen the genetic recognition of the genus, and contribute to phylogenetic,
evolutionary, developmental, conservation, and taxonomic investigations. Advancing
our taxonomic knowledge for Litsea will enable us to refine conservation efforts and the
utilization of natural resources, providing sufficient genetic resources for artificial breeding
and drug development. In this study, we first sequenced and assembled the complete
cp genomes of nine species of Litsea. A comparative analysis was performed, including
gene features, GC content, codon usage, IR junction, repeats, Ka/Ks value, as well as nu-
cleotide diversity (Pi). Results of analysis provide informative and valid data regarding the
genotype and suitable DNA markers. Moreover, using 21 species from Litsea, evolutionary
relationships within the genus were analyzed using the complete cp genome as well as
protein-coding sequences. Ultimately, this study provides a reliable resource for further
utilization and conservation of genetic resources for Litsea.

2. Materials and Methods

2.1. Sample Collection, DNA Extraction, and Sequencing
In this study, samples of nine species of Litsea were collected from the Plant Germplasm
and Genomics Center, Kunming Institute of Botany, the Chinese Academy of Sciences.
The process of sample collection was approved by the Kunming Institute of Botany and
local policy and deposited in the Evolutionary Biology Laboratory of Qingdao University
of Science and Technology. Fresh leaf tissues were collected without apparent disease
symptoms and preserved in silica gel. Total genomic DNA was extracted from 150 mg
of silica-dried leaf tissues using modified CTAB [20]. The quantity and quality of the
Genes 2022, 13, 1550 3 of 18

extracted DNA was assessed by spectrophotometry while the integrity was evaluated using
a 1% (w/v) agarose gel electrophoresis [19] The Illumina TruSeq Library Preparation Kit
(Illumina, San Diego, CA, USA) was used to prepare approximately 500 bp of paired-end
libraries for DNA inserts, according to the manufacturer’s protocol. These libraries were
sequenced on the Illumina HiSeq 4000 platform in Novogene (Beijing, China).

2.2. Chloroplast Genome De Novo Assembly and Annotation

The raw data were preprocessed using Trimmomatic 0.39 software [21], including
the removal of adapter sequences and other sequences introduced during sequencing,
the removal of low-quality and over-N-base reads, etc. The quality of newly produced
clean short reads was assessed using FASTQC v0.11.9 [22] and MULTIQC software [23].
High-quality data with Phred scores averaging above 35 were screened out. According to
the reference sequence (Litsea glutinosa, KU382356), the chloroplast-like reads were isolated
from clean reads by BLAST [24]. Short reads were de novo assembled into long contigs
with SOAPdenovo 2.04 [25] by setting kmer values of 35, 44, 71, and 101. Finally, the long-
contigs complete sequence expansion and gap filling was done by Geneious ver 8.1 [26],
which forms the complete cp genome. The complete cp genome was further validated
and calibrated using de novo splicing software NOVOplsty 4.2 [27]. GeSeq [28] was used
to annotate the assembled genomes, and tRNAscanSE ver 1.21 [29] was applied to detect
tRNA genes with default settings. RNAmmer [30] was used to validate rRNA genes with
default settings. As a final check, we compared the results with the reference sequence and
corrected misannotated genes by GB2Sequin [31] by manual selection. The circular map of
the genomes was drawn using CHLOROPLOT [32]. The nine newly assembled Litsea cp
genomes were deposited in GenBank with the accession numbers NC_056809–NC_056817.

2.3. Analysis of Chloroplast Genome Characteristics

Information regarding the GC content, genome length, and number of each region in cp
genomes was obtained using Geneious ver 8.1 software [26]. Relative synonymous codon
usage (RSCU) was calculated by the Computer Codon Usage Bias function in MEGA X [33].
SSRs were identified using MISA [34], with a setting of ten repeats for mononucleotide SSRs,
four for dinucleotide and trinucleotide SSRs, and three for tetranucleotide, pentanucleotide,
and hexanucleotide SSRs. REPuter [35] was used to identify four types of repeats with the
minimum repetition unit set as 20 bp and the maximum as 300, and the remaining options
set to default parameters.

2.4. Comparative Analysis

To compare the gene differences among the nine species, Litsea garrettii (NC_050349)
was selected as the reference species and the online comparison tool mVISTA [36] was used
for sequence alignment. IRscope [37] was used to detect and visualize the contraction and
expansion of IRs boundaries. SNP and InDels were detected using Geneious ver 8.1. The
Ka/Ks value was batch evaluated by TBtools with the NG method [38]. DnaSP 6 was used
to analysis the nucleotide diversity (Pi) value [39].

2.5. Phylogenetic Analysis

We downloaded 12 further cp genomes of Litsea from NCBI (National Center for
Biotechnology Information). Two species from Lauraceae but in different genera, Actin-
odaphne obovate and Neolitsea sericea, were selected as outgroups to root our phylogenetic
networks. A total of 23 species were compared for phylogenetic evaluation using maximum
likelihood (ML) and Bayesian inference (BI) approaches. MAFFT v7 was used to perform
multiple genome alignment [40], and we used the complete cp genome sequence data as
well as a separate dataset of 64 protein-coding genes shared by all species to construct
individual maximum likelihood (ML) topologies. The GTR+G+I model was evaluated
as the best suit model for both CDS and all cp sequences by applying the Bayesian in-
formation criterion (BIC) using jmodeltest v2.1.7 [41]. The ML analyses were performed
Genes 2022, 13, 1550 4 of 18

using MEGA X [33], and bootstrap tests were performed with 1000 replicates with tree
bisection-reconnection branch swapping. MrBayes v3.2.7 [42] was used to the BI analysis,
for two million generations, sampled every 100 generations, with all other settings left at
their defaults and 25% of the trees discarded as burn-in.

3. Results and Discussion

3.1. Chloroplast Genome Features of Litsea
With reliable quality control, we filtered about 22.5 GB of high quality, 2 × 150 bp
pair-end reads generated by the Illumina HiSeq 4000 platform. The mean coverage of
sequencing was 1750 X. The cp genome features of nine species were analyzed and the
total length ranged from 152,051 to 152,747 bp (Figure 1). 128 genes were found in these
complete cp genomes, including 36 tRNA genes, eight rRNA genes, and 84 protein-coding
genes. These genes can be divided into three categories: self-replication related, photo-
synthesis related, and other genes. The large subunit of ribosomal proteins, small subunit
of ribosomal proteins, DNA-dependent RNA polymerase, rRNA genes, and tRNA genes
belong to the Self-replication category. Photosystem I, Photosystem II, NADH oxidoreduc-
Genes 2022, 13,tase,
x FORCytochrome
PEER REVIEW b6/f complex, ATP synthase, and Rubisco belong to the Photosynthesis
category. The remaining genes that have not been authorially classified yet were attributed
to the other genes category (Table 1) [43].

Figure 1. Complete genome

Figure 1.map of the chloroplast
Complete genome map genome
of the for representative
chloroplast genome species L. auriculata. species L
for representative
The inner gray ring isThe
divided
inner into
grayfour
ringareas, clockwise,
is divided andareas,
into four they are SSC, IRb,
clockwise, andLSC,
theyand
areIRa.
SSC,TheIRb, LSC, an
genes in the outer ring region
genes are outer
in the transcribed clockwise,
ring region while those
are transcribed in the inner
clockwise, ringthose
while are transcribed
in the inner ring are
counterclockwise. Incounterclockwise.
addition, this figureIn addition, this figure
also reflects the GC also reflectsthe
content; the inner
GC content; the inner
ring dark gray ring dar
catesthe
indicates the GC content, thelight
GC gray
content, the light
reaction gray reaction
AT content. AT content.
In the lower left is aIn the lower
legend left is a legend th
that classifies
cp genes according tocptheir
genes according to their functions.
functions.

Table 1. Gene content of the L. moupinensis chloroplast genome.

Group of Genes Gene Names

Pholosystem I psaA, psaB, psaC, psal, psaJ
Genes 2022, 13, 1550 5 of 18

Table 1. Gene content of the L. moupinensis chloroplast genome.

Group of Genes Gene Names Amount

Pholosystem I psaA, psaB, psaC, psal, psaJ 5
psbA, psbK, psbl, psbM, psbD, psbC, psbZ, psbG, psbL, psbF, psbE, psbB, psbT, psbN,
Photosystem II 15
psbH
Cytochrome petA, petG, petL, petN, petB, petD 6
ATP syntliase atpA, atpF, atpH, atpI, atpE, atpB 6
NADH dehydrogenase ndhJ, ndhB *, ndhK, ndhC, ndhD, ndhF, ndhE, ndhG, ndhl, ndhA, ndhH 12
RubisCO large subunit rbcL 1
RNA polymerase RpoCl, rpoC2, rpoB, rpoA 4
Ribosomal proteins (SSU) rps16, rpsl2 *, rps2, rps14, rps4, rps18, rps7 *, rps11, rps8, rps3, rps19, rps15 14
Ribosomal proteins (LSU) rpl33, rpl20, rpl36, rpll4, rpll6, rpl22, rpl2, rpl23, rpl32 9
trnH-GUG, trnK-UUU, trnQ-UUG, trnS-GCU, trnG-UCC, trnR-UCU, trnC-GCA,
trnD-GUC, trnY-GUA, trnE-UUC, trnT-GGU, trnS-UGA, trnG-UCC, trnT-GGU,
Transfer RNAs trnS-UGA, trnG-UCQ, trnM-CAU, trnS-GGA, trnT-UGU, trnL-UAA, trnF-GUU, 34
trnV-UAC, trnM-CAU, trnW-CCA, trnP-UGG, trnl-CAU, trnA-UGC, trnR-ACG,
trnL-UAG, trnN-GUU, trnR-GUG, trnA-UGC, trnl-GAU, trnL-CAA
Ribosomal RNAs rrn4.5 *, rrn5 *, rrn16 *, rrn23 * 8
Hypothetical chloroplast
ycfl, ycf2, ycf3, ycf4 4
reading frames (ycf)
Other genes accD, clpP, ccsA, cemA, infA, rpoA, matK 7
* Gene with two copies.

Typical quadripartite and circular structures were discovered. These cp genomes

contain a large single-copy (LSC) of 93,093–93,631 bp and a small single-copy region (SSC) of
18,813–18,902 bp, separated by two identical interspersed regions (IRs) of 20,014–20,117 bp.
Among the four types of regions, the LSC region contained the largest number of genes,
including 66 protein-coding genes and 23 tRNA genes. The SSC region contained only
11 protein-coding genes and one tRNA gene, but its average gene length was the longest
at 1100 bp. Two identical IR regions contained five protein-coding genes, six tRNA genes,
along with four rRNA genes (Table 1). The genome features of Litsea are consistent with
the basic structure of cps reported by other studies [44].
We also analyzed the GC content of the complete cp genome for the nine species of
Litsea, as well as the values of each region (Table 2). We discovered that the average GC
content of the full cp genome was 39.2% for all species except for L. sericea, which was
39.1%. In addition, the GC content of the IR region was firmly consistent at 44.4% and
significantly higher than the other two regions, which was assumed to be related to the
presence of many rRNA genes [45].

Table 2. Chloroplast genome features of nine species of Litsea.

L. auriculata L. chunii L. ichangensis L. moupinensis L. populifolia L. rubescens L. sericea L. tsinlingensis L. veitchiana

152,377 152,081 152,747 152,588 152,619 152,581 152,717 152,051 152,578
93,535 93,138 93,631 93,552 93,569 93,550 93,583 93,093 93,540
18,814 18,813 18,902 18,824 18,838 18,819 18,900 18,828 18,826
20,014 20,065 20,107 20,106 20,106 20,106 20,117 20,065 20,106
39.2 39.2 39.2 39.2 39.2 39.2 39.1 39.2 39.2
37.9 37.9 38.0 37.9 38.0 37.9 37.9 37.9 38.0
33.9 33.9 33.9 33.9 33.9 33.9 33.9 34.0 34.0
44.4 44.4 44.4 44.4 44.4 44.4 44.4 44.4 44.4
128 (113) 128 (113) 128 (113) 128 (113) 128 (113) 128 (113) 128 (113) 128 (113) 128 (113)
84 (79) 84 (79) 84 (79) 84 (79) 84 (79) 84 (79) 84 (79) 84 (79) 84 (79)
9 (4) 8 (4) 8 (4) 8 (4) 8 (4) 8 (4) 8 (4) 8 (4) 8 (4)
36 (30) 36 (30) 36 (30) 36 (30) 36 (30) 36 (30) 36 (30) 36 (30) 36 (30)
NC_056809 NC_056810 NC_056811 NC_056812 NC_056813 NC_056814 NC_056815 NC_056816 NC_056817
49.12 49.35 49.06 49.12 49.11 49.12 49.15 49.37 49.12
8.51 7.54 8.81 8.82 8.82 8.95 8.81 8.85 8.82
30.19 29.92 30.93 30.85 30.86 1.36 30.83 29.90 30.85
13 13 13 13 13 13 13 13 13
 36 (30) 36 (30) 36 (30) 36 (30) 36 (30) 36 (30) 36 (30) 36 (30) 36 (30)
NC_056809 NC_056810 NC_056811 NC_056812 NC_056813 NC_056814 NC_056815 NC_056816 NC_056817
49.12 49.35 49.06 49.12 49.11 49.12 49.15 49.37 49.12
8.51 7.54 8.81 8.82 8.82 8.95 8.81 8.85 8.82
30.19
Genes 2022, 13, 1550 29.92 30.93 30.85 30.86 1.36 30.83 29.90 30.856 of 18
13 13 13 13 13 13 13 13 13

3.2. Codon Usage Analysis

3.2. Codon Usage Analysis
All organisms share the same common codon table, reflecting the shared ancestry of
All organisms share the same common codon table, reflecting the shared ancestry of all
all life. But through the process of biological evolution, disproportionate biases have
life. But through the process of biological evolution, disproportionate biases have evolved.
evolved. Different species exhibit certain preferences for not only different synonymous
Different species exhibit certain preferences for not only different synonymous codons,
codons, but also different proteins within the same species may show a preference for the
but also different proteins within the same species may show a preference for the same
same amino acid, a phenomenon called codon bias. A measurement called RSCU is com-
amino acid, a phenomenon called codon bias. A measurement called RSCU is commonly
monly used to reflect the codon bias, which removes the effect of the amino acid compo-
used to reflect the codon bias, which removes the effect of the amino acid composition of a
sition of a codon [44]. Since L. moupinensis had the largest cp genome, we used it as an
codon [44]. Since L. moupinensis had the largest cp genome, we used it as an example to
example to calculate the codon usage bias and RSCU values of 84 CDS genes. The protein-
calculate the codon usage bias and RSCU values of 84 CDS genes. The protein-coding genes
coding genes in the complete cp genome of Litsea consist of 84 genes coded by 61 codons,
in the complete cp genome of Litsea consist of 84 genes coded by 61 codons, which encode
which encode 20 amino acids. The results showed that Leu (UUA), Ala (GCU), and Arg
20 amino acids. The results showed that Leu (UUA), Ala (GCU), and Arg (AGA) are the
(AGA) are the most
most frequently usedfrequently usedwhile
amino acids, amino Seracids,
(AGC) while
and Ser
Arg(AGC)
(CGC)andare Arg (CGC)
the least are the
abundant
least abundant amino acids (Figure 2). RSCU values greater than one mean
amino acids (Figure 2). RSCU values greater than one mean that there is significant codon that there is
significant codon bias. This results in a different use of amino acids, which correlates
bias. This results in a different use of amino acids, which correlates with protein-positive with
protein-positive
bias [45]. Analysis bias
of [45].
RSCU Analysis
values ofofthe
RSCU values
codons of the each
encoding codons encoding
amino each amino
acid revealed that
acid revealed that most codons with RSCU > 1 contained either an A-
most codons with RSCU > 1 contained either an A- or G-terminal. By contrast, RSCU or G-terminal. By
contrast, RSCU values for codons that ended with a C-terminal, such
values for codons that ended with a C-terminal, such as CGC (Arg), UGC (Cys), CACas CGC (Arg), UGC
(Cys), CAC
(His), and AGC(His), andwere
(Ser), AGC (Ser), were
relatively low. relatively
This resultlow.
wasThis result with
consistent was previous
consistent with
related
previous related
reports [46]. reports [46].

Figure 2. The frequency (Freq) and preference of codon usage (RSCU) in L. moupinensis protein-coding
region. Axis of abscissae indicate each amino acid and its abbreviation as well as the respective
codon, the blue bar in the figure is the frequency of codon usage, and the orange line represents
codon preference.

3.3. Long Repeat and SSR Analysis

Long repeat sequences and SSRs analysis commonly exist throughout the cp genome,
consisting of one to six nucleotide repeats [44]. Due to its variability at the intraspecific
level, SSRs are commonly used as markers in population genetic analyses [47,48]. In
the cp genome of nine species, the total number of repeats ranged from 109 (L. chuni) to
119 (L. auriculata) (Table S1). A total of 111 SSRs were detected from the cp genome of the
representative species L. moupinensis, including 62 mononucleotide, 36 dinucleotide, tree
trinucleotide, eight tetranucleotide, one pentanucleotide, and one hexanucleotide repeats.
In general, the SSR number decreased along with the increase in nucleotide number. The
percentage of tri-, tetra-, penta-, and hexa-nucleotide repeat sequences detected were
remarkably lower than that of mono- and di-nucleotide repeat. Mono-nucleotide repeats
were the largest class of SSRs that consisting of 56.97% of all repeats. These repeats were
notably rich in A/T bases, causing the differences in terms of base content, which was
in line with other angiosperm species [49]. We also analyzed the distribution of SSRs in
LSC/SSC/IR regions. The number of SSR markers in the LSC region of nine species of
Litsea ranged from 79 to 87, far exceeding that of SSC (19) and IR regions (12). In particular,
IR region contains the lowest number of SSRs, which further demonstrates the high degree
Genes 2022, 13, 1550 7 of 18

of conservatism of IR regions. This correlated phenomenon was previously reported in

other angiosperms studies [50].
Some repeats larger than 30 bp in length are called long repeat sequences, which
increase the rearrangement of the cp genome [51]. We investigated the interspersed repeated
sequences (IRs) including four types of long repeat sequences: complement repeats (C),
forward repeats (F), palindromic repeats (P), reverse repeats (R). In general, palindromic
repeatswere richest in most species, followed by forward repeats and reverse repeats.
Complement repeats (C) were notably rare among all species. However, in the cp genome of
L. ichangensis, the number of forward repeats were slightly higher than that of palindromic
repeats (16). What more, in the cp genome of L. auriculata, the ratio of reverse repeats (R)
was more than that of forward repeats (F), which is also different from the other eight
species (Figure 3A). Most repeats were found in LSC region, leaving SSC and R regions far
behind. This pattern is highly consistent in nine Litsea species analyzed (Figure 3B). We
also measured the number of long repeat sequences with different lengths (Figure 3C). It
was found that long repetitive sequences of length of 20 and 21 bp were most common,
while the remainder decreased in number with an increase in length in general. The repeats
with 29, 31, and 38 bp in length were almost absent. However, in 33 bp and 44 bp, the
repeats number presented to be tied for third place suddenly. This phenomenon varied
from different species, which may be affected by unknown molecular mechanisms [52,53].

3.4. IR Contraction Analysis and Sequence Identity Plot

The contraction and expansion of IR regions contribute greatly to variations of cp
genomes among different species, resulting in gene duplication, deletion, and the gener-
ation of pseudogenes. Studying the characteristic genes of the border region contributes
to species identification and phylogenetic analyses [54]. In this study, we analyzed and
visualized the genes located in the junction region of LSC and IRa (JSa) as well as the
junction of SSC and IRb (JSb) in the cp genome of the nine species of Litsea (Figure 4). JLa
represents the junction between LSC and IRa, and the same applies for JLb. In this study,
we observed that genes located in the junction of four regions were highly conserved,
with only a few variations. Most genes located at cp genome junctions in all nine species
differed only in the distance to their corresponding boundaries, such as ycf2, ndhF, trnH,
and psbA. To be more specific, the ycf2 gene spans LSC/IRb and is distributed in both
regions of similar length, with the LSC region being slightly longer. The ndhF gene exists
among nine species, completely located in SSC and a short distance from IRb except for
L. sericea, of which theirs was longer and closer to the JSb boundary. The trnH gene is
located in the LSC region, adjacent to the IRa/LSC border, and is 21–22 bp in length. PsbA
is located entirely in the LSC region. Yet, notable variations were found. The ycf1 gene was
absent in this junction, while the remaining eight species contain ycf1Ψ (pseudocopy, 50 end
missing) in JSb, which spans JSb with only 4–5 bp of length, located in SSC. Apart from
that, the contraction and expansion event located in the JSb was greater than that of the JLa
boundary. This pattern is consistent with previous IR region research [55].
The whole sequence identity plot of nine species within Litsea was analyzed using
mVISTA with L. garretti (NC_050349) set as the reference sequence for comparison (Figure 5).
Genome sequences of the nine Litsea exhibited a high degree of concordance. In this
study, we revealed that most of the variations in the cp genome of different species were
distributed in CNS (non-coding sequences) regions. Notable high-divergent regions in
CNS were atpF–atpH and ndhC–trnV-UAC, the divergent value of which exceeded 100%.
Other variant regions include: rps16–trnQ-UUG, ycf4–cemA, rps8–rpl14, and rps12–trnV-
GAC. Some of the coding genes, such as ndhK, ndhF, and ycf1, were found to were contain
variable regions. In general, the divergence in the IR region was significantly smaller
than that in the LSC and SSC regions, a result comparable to the previous divergence
analysis [50].
Genes 2022, 13, 1550 8 of 18
Genes 2022, 13, x FOR PEER REVIEW 9 of 20

Figure 3. Comparison of microsatellites and oligonucleotide repeats in the chloroplast genomes of

Figure 3. Comparison of microsatellites and oligonucleotide repeats in the chloroplast genomes of
Litsea species. (A) The number of SSR markers in the LSC/SSC/IR region for nine Litsea species. (B)
Litsea
Numberspecies. (A)long
of four Therepeat
number of SSR markers
sequences in the complement
in nine species: LSC/SSC/IRrepeats.regionFfor nine Litsea
represents species.
forward
(B)repeats,
Number of four long
P represents repeat sequences
palindromic repeats,inRnine species:reverse
represents complement
repeats,repeats. F represents
C represents forward
complement
repeats.P(C)
repeats, number of
represents long repeat repeats,
palindromic sequences R with different
represents lengths
reverse in nine C
repeats, species. Different
represents colors
complement
in the figure
repeats. represent
(C) number different
of long repeatlong repeat sequence
sequences types. Species
with different lengthsfrom leftspecies.
in nine to right are：L.
Differentauric-
colors
inulata, L. chunii,
the figure L. ichangensis,
represent differentL. moupinensis,
long L. populifolia,
repeat sequence types.L.Species
rubescens,
fromL.left
sericea, L. tsinlingensis,
to right L.
are: L. auriculata,
veitchiana.
L. chunii, L. ichangensis, L. moupinensis, L. populifolia, L. rubescens, L. sericea, L. tsinlingensis, L. veitchiana.
3.4.SNP
3.5. IR Contraction
and InDelsAnalysis and Sequence Identity Plot
ToThe contraction
further exploreandtheexpansion
divergence of of
IRnucleotides,
regions contribute greatly to
we compared andvariations
analyzedofsingle
cp
genomes among different species, resulting in gene duplication, deletion, and the gener-
nucleotide polymorphisms (SNPs) and insertions and deletions (InDels) using L. garrettii
ation of pseudogenes. Studying the characteristic genes of the border region contributes
(NC_050349) as a reference sequence. The polymorphism ratio of transition substitution
to species identification and phylogenetic analyses [54]. In this study, we analyzed and
(Ts) was higher than transversion substitutions (Tv) in the LSC region of nine cp genomes
visualized the genes located in the junction region of LSC and IRa (JSa) as well as the
(Table 3). The most substitutions were located in the LSC region, while IR regions contained
junction of SSC and IRb (JSb) in the cp genome of the nine species of Litsea (Figure 4). JLa
the lowest rate of polymorphisms. This result is consistent with previous studies [56]. In
represents the junction between LSC and IRa, and the same applies for JLb. In this study,
terms of transition substitutions, the polymorphism ratios of A/G and C/T were almost the
we observed that genes located in the junction of four regions were highly conserved, with
same, although the former took up a slightly larger proportion, with only three exceptions
only a few variations. Most genes located at cp genome junctions in all nine species dif-
auriculata,
(L.fered only inL.the
chunii, andtoL.their
distance tsinlingensis). As for
corresponding transversion
boundaries, suchsubstitutions,
as ycf2, ndhF,the polymor-
trnH, and
phism ratios of A/T and C/G were greatly lower than that of A/C and G/C
psbA. To be more specific, the ycf2 gene spans LSC/IRb and is distributed in both regionssubstitutions.
The same pattern
of similar length,applied
with thefor
LSCInDels (Table
region being4).slightly
LSC presented thendhF
longer. The largest
genenumber of InDels
exists among
in comparison with IR and SSC regions, while the average length of InDels in IR regions
was the longest, with the longest variation length being 678 bp (L. tsinlingensis).
 of which theirs was longer and closer to the JSb boundary. The trnH gene is located in the
LSC region, adjacent to the IRa/LSC border, and is 21–22 bp in length. PsbA is located
entirely in the LSC region. Yet, notable variations were found. The ycf1 gene was absent
in this junction, while the remaining eight species contain ycf1Ψ (pseudocopy, 5′ end miss-
ing) in JSb, which spans JSb with only 4–5 bp of length, located in SSC. Apart from that,
Genes 2022, 13, 1550 the contraction and expansion event located in the JSb was greater than that of the JLa9 of 18
boundary. This pattern is consistent with previous IR region research [55].

Figure 4. Comparison
Figure 4. Comparison of SSC, LSC,
of SSC, IRa,
LSC, and
IRa, IRb
and boundary
IRb boundaryregions
regionsininthe
thechloroplast
chloroplast genomes of nine
genomes of
nineofspecies
species Litsea.of Litsea.

The whole we
In particular, sequence
foundidentity
that inplot
the of
cpnine species
genome of within Litsea was
L. auriculata, theanalyzed
averageusing
length of
mVISTA with L. garretti (NC_050349) set as the reference sequence for comparison
InDels located in the IR regions contained a considerable number of small InDels rather (Figure
than5). Genome sequences of the nine Litsea exhibited a high degree of concordance. In this
only several long InDels, as was found in the other eight species, causing its average
study, we revealed that most of the variations in the cp genome of different species were
length to be three times shorter than others. This result indicated that L. auriculata may have
distributed in CNS (non-coding sequences) regions. Notable high-divergent regions in
experienced some degree of mutation during its evolution that differed from its related
CNS were atpF–atpH and ndhC–trnV-UAC, the divergent value of which exceeded 100%.
species (Vaccinium) [57]. include: rps16–trnQ-UUG, ycf4–cemA, rps8–rpl14, and rps12–trnV-
Other variant regions
GAC. Some of the coding genes, such as ndhK, ndhF, and ycf1, were found to were contain
3.6. Nucleotide Divergence
variable regions. and Selection
In general, Pressure
the divergence in the IR region was significantly smaller than
Despite
that in thegeneral
LSC andconsistency,
SSC regions, variations occurredtofrequently
a result comparable the previousduring the evolutionary
divergence analysis
[50]. forming different genotypes and phenotypes. These nucleotide variations (Pi)
process,
could be distinguished as high divergent regions [58]. Some may accumulate through
generations to better adapt to the environmental changes, which is called positive selection.
In bioinformatics, the Ka/Ks value is commonly used to evaluate selection pressure. Here,
we calculated and analyzed the Pi value of 79 unique protein-coding genes, 101 IGS
(intergenic spacer) sequences, and the Ka/Ks value of 79 unique protein-coding genes
(Table S2). Most of the protein-coding genes possessed relatively low diversity, while
the rpl16 gene presented with an extremely high Pi value (0.00892) among all samples
(Figure 6A). However, in IGS regions, the Pi values of 64 genes out of 101 exceeded
0.01 (Figure 6B). Moreover, 54 among them surpassed 0.1. As for selection pressure, after
filtering genes with no value, the Ka/Ks value of 23 of 25 genes were less than one using
L. Garrettii (NC_050349) as a reference sequence. In other words, these genes were under
negative selection pressure. Only two genes, rpl16 and ycf2, presented with a Ka/Ks value
of greater than one, undergoing positive selection. No gene presented with a suggested
neutral selection (Figure 6C).
Genes 2022,
Genes 2022,13,
13,1550
x FOR PEER REVIEW 11
10 of 20
of 18

Figure 5. Identification map of chloroplast genome of nine species of Litsea. From top to bottom:
Figure
L. 5. Identification
auriculata, L. chunii, L. map of chloroplast
ichangensis, genomeL.of
L. moupinensis, nine species
populifolia, of Litsea.L.From
L. rubescens, topL.to
sericea, bottom: L.
tsinlingensis,
auriculata, L. chunii, L. ichangensis, L. moupinensis, L. populifolia, L. rubescens, L. sericea, L.
L. veitchiana. The gray arrows above indicate the extension direction of the gene, and purple indicates tsinlingensis,
L. veitchiana. The gray arrows above indicate the extension direction of the gene, and purple indi-
the exon, blue indicates the untranslated region, pink indicates the non-coding sequences, and the
cates the exon, blue indicates the untranslated region, pink indicates the non-coding sequences, and
grayish part indicates mRNA.
the grayish part indicates mRNA.
Genes 2022, 13, 1550 11 of 18

Table 3. The number of SNP types in LSC, IR and SSC regions of nine Litsea chloroplast genomes.

Species Region Transition Substitutions Transversion Substitutions

A/G C/T A/T A/C C/G G/T
L. auriculata 109 106 25 46 6 63
L. chunii 137 129 20 52 11 80
Large
L. ichangensis 129 139 26 58 11 75
L. moupinensis single 134 139 21 58 10 78
L. populifolia 129 138 22 60 10 80
L. rubescens copy 134 140 22 58 10 78
L. sericea 123 129 23 55 10 78
L. tsinlingensis 136 127 19 56 11 73
L. veitchiana 127 128 21 59 10 75
L. auriculata 3 5 0 2 2 11
L. chunii 4 8 2 12 1 15
L. ichangensis 4 5 1 3 1 3
L. moupinensis 3 8 2 12 1 12
Inverted
L. populifolia 3 6 2 12 1 11
repeat
L. rubescens 3 8 2 12 1 11
L. sericea 3 6 3 12 1 12
L. tsinlingensis 2 9 2 12 1 14
L. veitchiana 3 7 2 12 1 12
L. auriculata 43 47 5 21 3 16
L. chunii 42 37 10 19 4 17
L. ichangensis Small 42 45 5 24 5 21
L. moupinensis 38 41 5 24 6 21
L. populifolia single 37 43 4 19 5 21
L. rubescens 38 41 5 10 6 21
L. sericea copy 38 39 6 19 5 24
L. tsinlingensis 42 37 5 20 4 18
L. veitchiana 37 43 4 18 5 22

Table 4. Comparative analyses of the number and average length of InDel sites in LSC, SSC, and IR
regions in the complete cp genomes of nine species of Litsea.

Comparative Analyses of InDel Sites

Species Large Single Copy Inverted Repeat Small Single Copy
No0 s
of InDels0 Average No0 s
of InDels0Average No0 s
of InDels0 Average
InDels Length (bp) InDels Length (bp) InDels Length (bp)
L. auriculata 86 4.40 16 103.3 18 1.3
L. chunii 89 8.4 3 458.7 20 1.4
L. ichangensis 99 3.7 5 276.0 19 1.6
L. moupinensis 88 3.7 4 339.0 16 1.9
L. populifolia 86 3.8 4 339.0 17 1.5
L. rubescens 88 3.9 5 272.4 16 1.9
L. sericea 86 3.7 4 339.0 19 2.0
L. tsinlingensis 87 9.1 2 678.0 19 1.4
L. veitchiana 88 3.7 4 339.0 18 1.8
Average 88.6 4.90 5.2 349.4 18.0 1.6
Genes 2022, 13, x FOR PEER REVIEW 14 of
Genes 2022, 13, 1550 12 of 18

Figure 6. 6.Nucleotide
Figure diversity
Nucleotide diversity in chloroplast
in chloroplast genomes
genomes of nineofspecies
of nine species ofabscissa
Litsea. The Litsea. represents
The abscissa rep
sents
thethe position,
position, and
and the redthe
linered line represents
represents the averagethe average
of the of the
nucleotide nucleotide
variations of the variations
nine species.of the ni
(A) Pi (A)
species. values for eachfor
Pi values gene region.
each gene(B). Pi values
region. (B). for each intergenic
Pi values for eachregion. (C) Ka/KS
intergenic values
region. for
(C) Ka/KS valu
for each
eachgene.
gene.
The hyper-variable regions detected in this study may provide a potential molecular
The hyper-variable
marker regions
for further studies. detected
In particular, theinrpl16
thisgene
study may provide
possesses a potential
both a high Pi valuemolecul
marker for further
and Ka/Ks value atstudies.
the sameIntime.
particular, thesuggest
This might rpl16 gene possesses
that the rpl16 geneboth
wentathrough
high Piavalue an
Ka/Ks
greatvalue at the
mutation thatsame time. to
was crucial This
the might suggest
evolution processthat the rpl16
of Litsea gene
species. went studies
Although through a gre
have reported
mutation that wasrpl16 to be one
crucial of the
to the highly divergent
evolution process genes [59]species.
of Litsea and a positive
Althoughselection
studies ha
site [60], as unique and significant as the present study is this is not a common
reported rpl16 to be one of the highly divergent genes [59] and a positive selection in studies of s
other angiosperms. The other positive selection site, the ycf2 gene, was more commonly
[60], as unique and significant as the present study is this is not a common in studies
described in previous studies [61,62].
other angiosperms. The other positive selection site, the ycf2 gene, was more common
3.7. Phylogenetic
described Analysis
in previous studies [61,62].
The expanding cp genome database provides an important basis for determining
3.7.evolutionary
Phylogeneticrelationships
Analysis [56,63–65] Phylogenetic trees based on different data had slightly
The expanding cp genome database provides an important basis for determinin
evolutionary relationships [56,63–65] Phylogenetic trees based on different data h
slightly varied topologies, with trees based on the whole cp genome and CDS data havin
Genes 2022, 13, 1550 13 of 18

varied topologies, with trees based on the whole cp genome and CDS data having the same
topology, and being more credible than trees based on the IR area and introns [61,66–69].
Genes 2022, 13, x FOR PEER REVIEW We found two similar topological structures with few changes based on the full cp genome
15 of 20
and the protein-coding sequences of 23 selected species, with N. sericea and A. obovate as
outgroup species (Table S3, Figure 7).

Figure 7.
Figure Phylogeneticanalysis.
7. Phylogenetic analysis.(A)
(A)Phylogenetic
Phylogenetictree
treebased
basedononthe
thecomplete
completechloroplast
chloroplastgenome.
genome.
(B)
(B) Phylogenetic
Phylogenetictree
tree based
based on
on 64
64 sets
sets of
of protein-coding
protein-coding genes.
genes. N.
N. sericea
sericea and
and Actinodaphne
Actinodaphneobovata
obovata
served
servedasas out
out groups.
groups. The
Thecolored
colored branches
branches show
show the
the difference
difference between
between twotwo trees.
trees. Numbers
Numbers at
at
branch
branchnodes
nodesare
arebootstrap
bootstrap values
values and
and posterior
posterior probability.
probability.

In
In general,
general,the theentire
entire phylogenetic
phylogenetictree treewas
was divided
dividedinto into three
three main
main branches,
branches, with with
the
the two
two outgroup
outgroup speciesspecies representing
representing two two distinct
distinct branches,
branches, eacheach withwith high
high bootstrap
bootstrap
values.
values.The Thefirst
firstsubclade
subcladeconsists
consistsof of11 species:L.L.moupinensis,
11species: moupinensis,L.L.rubescens,
rubescens,L.L.populifolia,
populifolia,
L.veitchiana,
L. veitchiana,L.L.pungens,
pungens,L.L. sericea,
sericea, L. ichangensis,
L. ichangensis, L. chunii,
L. chunii, L. tsinlingensis,
L. tsinlingensis, L. acutivena
L. acutivena and
and
L. L. glutivena.
glutivena. Among Amongthem,them, the clade
the clade L. chunii
of L.ofchunii and and L. tsinlingensis
L. tsinlingensis andand the the
clade clade of
of L.
L. acutivena
acutivena andand L. glutinosa
L. glutinosa formformsistersister
pairs,pairs, respectively.
respectively. Notably,
Notably, L. pungens
L. pungens switched
switched phy-
phylogenetic
logenetic positions
positions with with L. sericea,
L. sericea, with with relatively
relatively low bootstrap
low bootstrap values values
in bothin trees.
both trees.
An-
Another
other cladeclade included
included 10 10 species:
species: L. cubeba,
L. cubeba, L. L. mollis,
mollis, L. L. dilleniifolia,L.L.szemaois,
dilleniifolia, szemaois,L.L.auriculata,
auriculata,
L. coreana,
L. coreana, L. L. monpinensis,
monpinensis, L. L. garrettii,
garrettii, L.
L. elongata,
elongata, andand L. L. japonica.
japonica. Among
Among them, them, L. L. cubeba
cubeba
and L.
and L. mollis
mollis were
were grouped
grouped as as sisters
sisters and
and clustered
clustered withwith eight
eight other
other species.
species. ItIt isis worth
worth
noting that in topology based on the complete cp genome,
noting that in topology based on the complete cp genome, L. coreana and L. monopetala L. coreana and L. monopetala
were sisters
were sisters with
with lowlowsupport
support(only(only57).
57).However,
However, in in
thethe
CDS-based
CDS-based tree, L. dilleniifolia
tree, and
L. dilleniifolia
L. szemaois split into a clade that aggregated with the remaining
and L. szemaois split into a clade that aggregated with the remaining four species (L. four species (L. monpinensis,
L. garrettii, L.L.elongata,
monpinensis, garrettii,and L. japonica),
L. elongata, and L.and mergedand
japonica), with L. coreana
merged withto L.converge
coreana toas a single
converge
branch.
as a single Inbranch.
other words, in the
In other two in
words, different
the twoanalyses,
differentthe clade consisting
analyses, of L. dilleniifolia
the clade consisting of L.
and L. szemaois switched its position with L. coreana. The phylogenetic
dilleniifolia and L. szemaois switched its position with L. coreana. The phylogenetic trees trees resulting
from Bayesian
resulting inferenceinference
from Bayesian analyses analyses
(File S1) were generally
(File S1) consistentconsistent
were generally with the results
with the of
the maximum likelihood tree. However, in the Bayesian inference
results of the maximum likelihood tree. However, in the Bayesian inference tree, the po- tree, the positions of
L. pungens
sitions of L.and L. sericea
pungens andwere consistent
L. sericea with the results
were consistent with theof the maximum
results of the likelihood
maximumtree for
likeli-
the complete cp genome, while the relationships of L. coreana, L. dilleniifolia,
hood tree for the complete cp genome, while the relationships of L. coreana, L. dilleniifolia, and L. szemaois
wereL.consistent
and szemaois with were the results ofwith
consistent the maximum
the resultslikelihood
of the maximumtree constructed
likelihood by CDS.
tree con-
The development of low-copy nuclear DNA regions to investigate phylogenetic rela-
structed by CDS.
tionships among plant taxa has attracted growing interest [70]. Therefore, many studies
The development of low-copy nuclear DNA regions to investigate phylogenetic rela-
have tried to study the phylogenetic relationships of the genus Litsea using different meth-
tionships among plant taxa has attracted growing interest [70]. Therefore, many studies
ods, such as combined matK and ITS [71], rpb2 [72] gene fragments, and morphological
have tried to study the phylogenetic relationships of the genus Litsea using different meth-
ods, such as combined matK and ITS [71], rpb2 [72] gene fragments, and morphological
characters [73]. These studies focused on the analysis of the relationships between differ-
ent genera in Lauraceae. In comparative terms, the phylogenetic relationships constructed
from the complete chloroplast genome are more accurate than those constructed from a
Genes 2022, 13, 1550 14 of 18

characters [73]. These studies focused on the analysis of the relationships between different
genera in Lauraceae. In comparative terms, the phylogenetic relationships constructed
from the complete chloroplast genome are more accurate than those constructed from a
few fragments [74]. Zhang et al. (2021) [75] suggested that Litsea could be divided into four
sub-clades through the chloroplast genome. However, our study has suggested that Litsea is
more appropriately divided into two sub-clades (Figure 7). We discovered that both the ML
tree and the BI tree had greater support values for the phylogeny reconstructed from com-
plete cp genomes. Such different trees could originate from substitutions in the intergenic
spacer regions, which illustrates the importance of non-coding regions in phylogenetic
analyses [76]. Therefore, complete cp genomes can be used as a ‘super barcode’ [77], and
they have been demonstrated to be effective for preventing some identification errors and
the discovery of new species [78]. Despite minor differences, the phylogenetic relationships
of most species in the topologies were consistent, showing similar genetic affinities in the
topology, and which aligned nicely with the elevational distribution of the species [71,79].

4. Conclusions
In this study, we sequenced and reported the complete cp genome sequences of
nine species in Litsea, revealing typical quadripartite circular structures. We observed the
contraction and expansion of IR boundaries. This event caused gene loss, changes in gene
length, and the occurrence of pseudogenes, resulting in the differences between species.
In terms of alignment consistency, the LSC region had the largest number of nucleotide
variants, and IR regions showed a high degree of conservation. We found that the rpl16
and ycf2 genes underwent great positive selection pressure. Moreover, rpl16 gene also was
found to be the only hyper-variable protein-coding gene in the gene divergent analysis,
which was evaluated by the Pi value. This phenomenon is rare and further studies to
unfold the molecular mechanism behind is needed.
Phylogenetic relationships within the genus were explored using two sets of data
from the complete cp genome and another from 64 sets of protein-coding genes shared
by 21 Litsea and two outgroup species. Essentially the same conclusions were obtained: L.
moupinensis and Litsea rubescena, L. chunii, and L. tsinlingensis were sisters in the phylogenies
and showed similar genetic relationships consistent with their elevational distributions.
This study provides aid to taxonomic studies for Litsea, providing specific genetic markers
for taxon identification and for inferring evolutionary relationships among the species.
These data may also contribute to future conservation efforts as well as the practical use of
these species.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/genes13091550/s1, Table S1: Raw data of single sequence repeats
of nine Litsea species, grouped by mono-, di-, tri-, tetra, penta-, and hexanucleotide repeats, along with
the percentage of each group. Table S2: Raw data of Ka/Ks value and pi value of 79 protein-coding
genes. Raw data of pi value of 101 IGS sequences. Table S3: Details of taxonomy and accession
numbers of species mentioned in this study. File S1: The phylogenetic trees resulting from Bayesian
inference analyis.
Author Contributions: Conceptualization, Q.F.; Formal analysis, Z.C. and W.H.; Funding acquisition,
C.S. and S.W.; Investigation, W.S. (Weicai Song), W.S. (Wenbo Shi) and M.S.E.; Project administration,
W.S. (Weicai Song), C.S. and S.W.; Resources, Z.C. and W.S. (Wenbo Shi); Software, W.S. (Weicai Song);
Supervision, W.H.; Validation, Z.C.; Visualization, W.S. (Weicai Song), W.S. (Wenbo Shi) and Q.F.;
Writing—original draft, W.S. (Weicai Song) and Z.C.; Writing—review & editing, C.S., M.S.E. and
S.W. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the National Natural Science Foundation of China (NO.
31801022 and NO. 31701090) and Shandong Province Natural Science Foundation of China (NO.
ZR2019BC094).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Genes 2022, 13, 1550 15 of 18

Data Availability Statement: The data that support the findings of this study are openly available in
the Genbank database at https://www.ncbi.nlm.nih.gov/, under accession number [NC_056809 –
NC_056817] (accessed on 1 September 2021).
Acknowledgments: We are thankful to Beijing-based Novogene for their NGS service that was
instrumental to the execution of the project.
Conflicts of Interest: The authors declare no conflict of interest associated with the work described.

Abbreviations
AGE, agarose gel electrophoresis; BI, Bayesian inference; CDS, Coding sequences; Cp,
chloroplast; CTAB, Cetyltrimethylammonium bromid; DOGMA, Dual Organellar Genome
Annotator; IGS, Intergenic spacer; InDels, insertions/deletions; IR, inverted repeat; Ka,
Nonsynonymous; Ks, Synonymous; LSC, large single-copy; ML, Maximum likelihood;
NJ, neighbor joining; PCGs, Protein-coding genes; Pi, Nucleotide variance; RSCU, relative
synonymous codon usage; SBS, Sequencing By Synthesis; SNP, single nucleotide polymor-
phisms; SSC, small single-copy; SSR, simple sequence repeat.

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