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Citation: Wang, Y.; Zhao, X.; Chen, Q.;
Yang, J.; Hu, J.; Jia, D.; Ma, R.
Complete Chloroplast Genome of
Alternanthera sessilis and Comparative
Analysis with Its Congeneric Invasive
Weed Alternanthera philoxeroides.Genes
2024,15, 544. https://doi.org/
10.3390/genes15050544
Academic Editor: Qinghu Ma
Received: 3 April 2024
Revised: 22 April 2024
Accepted: 23 April 2024
Published: 25 April 2024
Copyright: © 2024 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 Chloroplast Genome of Alternanthera sessilis and
Comparative Analysis with Its Congeneric Invasive Weed
Alternanthera philoxeroides
Yuanxin Wang 1, Xueying Zhao 1, Qianhui Chen 1, Jun Yang 1, Jun Hu 1, Dong Jia 1 ,2 ,* and Ruiyan Ma 1,3, *
1College of Plant Protection, Shanxi Agricultural University, Taigu 030801, China;
wangyuanx1992@163.com (Y.W.); xyzhao1103@163.com (X.Z.); chenqianhui@163.com (Q.C.);
yangjuncau@163.com (J.Y.); hujun.yx@163.com (J.H.)
2Ministerial and Provincial Co-Innovation Centre for Endemic Crops Production with High-Quality and
Effciency in Loess Plateau, Taigu 030801, China
3State Key Laboratory of Sustainable Dryland Agriculture (in Preparation), Shanxi Agricultural University,
Taiyuan 030031, China
*Correspondence: biodong@hotmail.com (D.J.); mary@sxau.edu.com (R.M.)
Abstract: Alternanthera sessilis is considered the closest relative to the invasive weed Alternanthera
philoxeroides in China, making it an important native species for studying the invasive mechanisms
and adaptations of A. philoxeroides. Chloroplasts play a crucial role in a plant’s environmental
adaptation, with their genomes being pivotal in the evolution and adaptation of both invasive and
related species. However, the chloroplast genome of A. sessilis has remained unknown until now. In
this study, we sequenced and assembled the complete chloroplast genome of A. sessilis using high-
throughput sequencing. The A. sessilis chloroplast genome is 151,935 base pairs long, comprising two
inverted repeat regions, a large single copy region, and a small single copy region. This chloroplast
genome contains 128 genes, including 8 rRNA-coding genes, 37 tRNA-coding genes, 4 pseudogenes,
and 83 protein-coding genes. When compared to the chloroplast genome of the invasive weed A.
philoxeroides and other Amaranthaceae species, we observed significant variations in the ccsA,ycf1,
and ycf2 regions in the A. sessilis chloroplast genome. Moreover, two genes, ccsA and accD, were
found to be undergoing rapid evolution due to positive selection pressure. The phylogenetic trees
were constructed for the Amaranthaceae family, estimating the time of independent species formation
between A. philoxeroides and A. sessilis to be approximately 3.5186–8.8242 million years ago. These
findings provide a foundation for understanding the population variation within invasive species
among the Alternanthera genus.
Keywords: Alternanthera sessilis;Alternanthera philoxeroides; chloroplast genome; invasive plants;
phylogenetic analysis
1. Introduction
Alternanthera philoxeroides, commonly known as alligator weed, is a perennial herb
within the Alternanthera genus of the Amaranthaceae family. It originates from the Paraguay
and Parana River basins in South America. Over time, it has expanded its presence
across a vast geographical range, spanning from 32 degrees north to 38 degrees south
latitude, making it a globally pervasive invasive weed that poses significant threats to
both the environment and the economy [
1
–
3
]. Alternanthera sessilis, another perennial
herb belonging to the same Alternanthera genus as A. philoxeroides, is believed to be native
to tropical and subtropical regions of Asia, northeastern Australia, and the wetlands of
tropical
America [3–5]
. These two species share similarities in morphology, with upright or
prostrate growth habits, and the ability of all stem nodes to develop roots. Their flowers
are axillary in nature. While A. sessilis has sessile inflorescences, A. philoxeroides exhibits
Genes 2024,15, 544. https://doi.org/10.3390/genes15050544 https://www.mdpi.com/journal/genes
Genes 2024,15, 544 2 of 22
different characteristics. Their distribution areas overlap within China, with A. sessilis
primarily found along the wet edges of East China, South China, Central China, and
Southwest China. Conversely, A. philoxeroides is predominantly distributed in the broader
regions to the south of the Yangtze River and gradually extends into sporadic areas in
North China. It holds a larger territory and exhibits greater competitive strength compared
to A. sessilis.
Invasive weeds have a remarkable capacity for rapid adaptation to new environments,
making them excellent subjects for studying adaptive changes in plants [
6
–
8
]. One common
approach is to compare the adaptability and invasiveness of alien invasive species with
their local relatives. As a native species, A. sessilis has been frequently employed in studies
of the adaptation and invasive mechanisms of A. philoxeroides, yielding valuable insights. In
contrast to A. sessilis,A. philoxeroides demonstrates superior photosynthetic capacity, a faster
stem growth rate, a broader temperature tolerance range, enhanced competitive abilities,
and a greater capacity for invasion [
9
]. A. philoxeroides holds distinct advantages over A.
sessilis, whether facing biotic or abiotic stressors [
10
]. Following exposure to herbivores
and nematodes, A. philoxeroides displays increased branching, facilitating its expansion and
invasion [
11
]. Its defense responses surpass those of A. sessilis, making it more resistant to
the intrusion of pathogenic microorganisms [
12
]. Furthermore, A. philoxeroides exhibits a
wider environmental adaptability range and more robust phenotypic plasticity. It thrives in
various aquatic environments and demonstrates heightened tolerance to waterlogging [
13
].
Its osmotic adjustment capabilities exceed those of its native congener, A. sessilis [
14
,
15
].
Additionally, A. philoxeroides excels in clonal integration, enabling it to outcompete A.
sessilis within its ecological niche [
16
]. These findings shed light on the invasion mechanism
employed by A. philoxeroides to a considerable extent.
Chloroplasts are cellular organelles responsible for photosynthesis in plants and the
provision of energy for growth. They also serve as vital hubs for plant signal integration,
actively participating in adaptation to environmental stress [
17
]. Chloroplasts possess a
circular genome consisting of four main sections: a large single copy region (LSC), a small
single copy region (SSC), and two identical inverted regions (IRs) separated by two unique
single copy regions. Typically, chloroplast genomes (cp genome) have a size ranging from
107 kb to 218 kb, containing approximately 120 to 130 genes. The number and arrangement
of chloroplast genes (cp genes) exhibit a high degree of conservation, albeit with occasional
insertions, deletions, and rearrangements. These attributes of high conservation and slow
evolution of chloroplast genomes offer an effective means of distinguishing groups that
are challenging to classify based on morphology [
18
,
19
]. Cp genes also prove effective in
the identification of invasive plants, such as the combined utilization of matK and nucleic
ITS [20].
The cp genome plays a crucial role in elucidating the relationships and evolutionary
dynamics between invasive species and their congeners [
21
]. By comparing cp genomes
across different regions or among related species, researchers can analyze origins, evolu-
tionary pathways, and spread patterns. For example, this approach has been applied to
examine invasive and native individuals of Jacobaea vulgaris [
22
], invasive Mikania micrantha
and its native species M. cordata [
23
], and Sonchus asper and S. oleraceus [
24
]. This method
has increasingly become a potent tool for plant molecular systematics, phytogeography,
and the investigation of intraspecific polymorphism and interspecific divergence [
21
,
25
,
26
].
With advancements in next-generation sequencing technologies, there has been a growing
interest in studying the cp genomes of invasive species within specific regions. Despite
the sequencing and reporting of the cp genome of A. philoxeroides [
27
], research on the
cp genome sequences of its native congener, A. sessilis, and their comparative analysis
remains limited.
In this study, the complete cp genome of A. sessilis was sequenced and assembled.
The analysis of hotspot regions and repeat sequences was carried out in comparison to the
cp genome of the invasive weed, A. philoxeroides. Furthermore, highly divergent regions
between A. sessilis and A. philoxeroides were identified. Using protein-coding genes, a
Genes 2024,15, 544 3 of 22
phylogenetic tree of the Amaranthaceae family was constructed, and the divergence time
between A. sessilis and A. philoxeroides was estimated.
2. Materials and Methods
2.1. Plants Collection
Alternanthera sessilis were gathered from a natural population in Meizhou City, Guang-
dong, China, and subsequently cultivated within a greenhouse at Shanxi Agriculture
University (Taigu, China). Once they had bloomed, their seeds were harvested, dried,
and stored in a refrigerator for future cultivation. These seeds were then sown in small
black square pots measuring 7 cm
×
7 cm and cultured under controlled conditions at a
temperature of 25
±
1
◦
C with a photoperiod of 16 h of light and 8 h of darkness for six
weeks to obtain test plants. Fresh leaves were collected after exposure to more than 6 h of
light and were promptly frozen using liquid nitrogen for subsequent DNA extraction.
2.2. DNA Extraction and Sequencing
The frozen leaves of A. sessilis, as described earlier, were pulverized into a fine powder
using liquid nitrogen to facilitate total DNA extraction. Total DNA was extracted utilizing
the Plant DNA Isolation Kit (Tiangen, Beijing, China). Subsequently, the total DNA was
fragmented through ultrasound treatment. The resulting DNA fragments underwent pu-
rification, end repair, and adapter ligation. Following PCR enrichment, the fragments were
separated through agarose gel electrophoresis and subsequently employed for constructing
a DNA library. This library was subjected to sequencing on an Illumina NovaSeq platform
(San Diego, CA, USA), generating paired-end reads with a length of 150 bases (PE150).
2.3. Assembly and Annotation of A. sessilis Chloroplast Genome
Raw sequencing data were subjected to filtration using fastp 0.20.0 (https://github.
com/OpenGene/fastp, accessed on 4 December 2023), which entailed the removal of
adapter sequences and reads with an average quality score falling below Q5 or containing
more than five ambiguous bases (N) to obtain high-quality clean reads. To simplify the
assembly process, these clean reads were aligned against the chloroplast genome database
from Genepioneer Biotechnologies (Nanjing, China) using bowtie2 v2.2.4 (http://bowtiebio.
sourceforge.net/bowtie2/index.shtml, accessed on 4 December 2023) to specifically identify
sequencing reads corresponding to the chloroplast genome. These selected cp genome
sequencing reads were subsequently assembled into contigs using SPAdes v3.10.1 (http:
//cab.spbu.ru/software/spades/, accessed on 4 December 2023). These contigs were
further organized into scaffolds using SSPACE v2.0 (https://www.baseclear.com/services/
bioinformatics/basetools/sspace-standard/, accessed on 4 December 2023). To obtain a
complete, circular cp genome, any gaps between scaffolds were filled using Gapfiller v2.1.1
(https://sourceforge.net/projects/gapfiller/, accessed on 4 December 2023).
Annotation of the CDS within the A. sessilis cp genome was carried out using Prodi-
gal v2.6.3 (https://www.github.com/hyattpd/Prodigal, accessed on 4 December 2023).
Separately, the prediction of transfer RNA (tRNA) and ribosomal RNA (rRNA) was per-
formed using Aragorn [
28
] v1.2.38 (http://130.235.244.92/ARAGORN/, accessed on 4
December 2023), tRNAscan-SE [
29
] (http://trna.ucsc.edu/tRN-Ascan-SE/, accessed on 4
December 2023), and Hmmer v3.1b2 (http://www.hmmer.org/, accessed on 4 December
2023). Additionally, sequence alignment and annotation were conducted based on the
gene sequences of related species, and the assembled sequences were subjected to blast
v2.6 (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 4 December 2023) for further
annotation. The final annotation result was obtained after manually removing redundancy.
The complete cp genome sequence of A. sessilis was deposited in the NCBI GenBank with
the specific accession number PP239384.
Genes 2024,15, 544 4 of 22
2.4. Analysis Data Collection
The cp genome sequences of A. philoxeroides, which were used for comparative analysis,
were retrieved from the NCBI GenBank (accession number: NC_042798.1). These samples
were collected in Jinan, China.
2.5. Chloroplast Genome Structure
The analysis of inverted repeat regions (IRs) within the A. sessilis cp genome was
conducted using GeSeq [
30
] (https://chlorobox.mpimp-golm.mpg.de/geseq.html, ac-
cessed on 18 January 2024). Verification of the large single copy region (LSC), small
single copy region (SSC), and IRs was performed using Geneious 10.1 [
31
]. Visual-
ization of the structure of the A. sessilis cp genome was achieved using OGDraw [
32
]
(https://chlorobox.mpimpgolm.mpg.de/OGDraw.html, accessed on 18 January 2024).
2.6. Codon Usage Bias Analysis of A. sessilis and A. philoxeroides cp Genomes
Codon usage bias is a widespread phenomenon observed across various species
and stages of life. This bias is considered a result of long-term evolution and is influ-
enced by multiple factors, with directional mutation and neutral selection being primary
contributors [33,34]
. Relative synonymous codon usage (RSCU), a commonly employed
parameter for studying codon usage bias, represents the ratio between actual and expected
codon occurrences. It aids in the analysis of gene function and evolutionary patterns. An
RSCU value of 1 signifies no codon bias, while values greater than 1 indicate a higher
occurrence of a codon compared to other synonymous codons, and vice versa. The RSCU
values for the cp genomes of A. sessilis and A. philoxeroides were calculated using the protein-
coding genes from these cp genomes. This analysis was performed using codon usage
analysis in MEGA 11.0 [35].
2.7. Repeat Sequence Analysis
Repetitive sequences, distributed widely throughout the genome, are believed to play
a crucial role in gene recombination and rearrangement. The cp genome evolves at a
relatively slow pace, with repetitive sequences in non-coding regions exhibiting a higher
degree of variability. This characteristic facilitates the characterization of genetic variation at
lower taxonomic levels and aids in addressing population genetic inquiries [
25
,
36
,
37
]. The
identification of repeats in the cp genome holds great significance for the development of
novel molecular markers. REPuter [
38
] (https://bibiserv.cebitec.unibieleeld.de/reputer/,
accessed on 18 January 2024) was employed to detect various types of repeat sequences
within the cp genomes of both A. sessilis and A. philoxeroides. For the analysis of simple
tandem repeats, Tandem Repeats Finder [
39
] (http://tandem.bu.edu/trf/trf.html, accessed
on 18 January 2024) was utilized. Simple sequence repeats (SSRs) were identified using
MISA [
40
] (https://webblast.ipk-gatersleben.de/misa/, accessed on 18 January 2024). The
SSRs were searched for mononucleotide to hexanucleotide repeat motifs with a minimum
repeat number of 10, 5, 4, 3, 3, and 3 for mo, di, tri, tetra, penta, and hexanucleotide repeats,
respectively. The compound SSR was identified when the length of a sequence between
two SSRs to register was <100 bp.
2.8. Analysis of Hotspots and ka/ks and Identification of Highly Divergent Regions
The cp genome sequences of A. sessilis and A. philoxeroides were aligned using MAFFT
7.037 [
41
]. Nucleotide polymorphism (Pi) within the cp genomes of these species was ana-
lyzed using DnaSP 6.0 [
42
] to identify regions with high variability, employing a parameter
of a 200 bp step size and 600 bp window length. Seventy-one common protein-coding genes
were selected to assess the frequency of synonymous and non-synonymous substitution
events using DnaSP 6.0, providing insights into evolutionary selection pressures.
Genes 2024,15, 544 5 of 22
2.9. Contraction and Expansion Analysis of IRs Boundaries
The contraction and expansion of IRs have a substantial impact on the size of the cp
genome [
26
,
43
]. Irscope [
44
] (https://irscope.shinyapps.io/irapp/, accessed on 18 January
2024) was employed to analyze the contraction and expansion of IRs within the cp genomes
of A. sessilis and A. philoxeroides.
2.10. Genome Analysis and Comparison with Other Amaranthaceae Species cp Genomes
Using the annotation of the A. sessilis cp genome as a reference, a comprehensive
comparison was conducted with the cp genomes of other Amaranthaceae species using
mVISTA [
45
] (http://genome.lbl.gov/vista/mvista/submit.shtml, accessed on 18 January
2024). This analysis aimed to assess the distinctions between their respective cp genomes.
2.11. Phylogenetic Analysis and Divergence Time Estimate
To construct the phylogenetic tree, we utilized 59 common protein-coding genes
from a total of 28 species. This set included 25 species from the Amaranthaceae family
and 3 outgroups consisting of 2 species from the Achatocarpaceae family and Dianthus
caryophyllus. Sequence alignment was carried out using MAFFT 7.037 [
41
]. The maximum
likelihood (ML) tree was constructed with MEGA 11.0 [
35
], employing the best model
GTR + G + I and 1000 bootstrap replicates. ModelFinder [
46
] was utilized to determine the
best-fit model for constructing Bayesian inference phylogenies. The Bayesian phylogenetic
tree was generated using Mybayes 3.2.6 within Phyosuite 1.1.16 [
47
], employing the best-fit
model GTR + F + I + G4 with 2 parallel runs and 2,000,000 generations. The initial 25% of
the sampled data was discarded as burn-in.
Divergence times were estimated using the RelTime-ML method with the local molec-
ular clock in MEGA 11 [
35
]. Calibration points for divergence times were derived from
TimeTree [
48
] (http://timetree.org/, accessed on 23 January 2024), specifically, the diver-
gence time of the Amaranthus genus and Chenopodium genus (24.5–73.8 MYA) and the
Suaeda genus and Salicornia genus (12.1–39.7 MYA), based on data from 10 and 5 studies,
respectively. The time tree was constructed using the maximum likelihood method and the
GTR + G + I model.
3. Results
3.1. Sequencing, Assembly, and Annotation of A. sessilis cp Genome
High-quality clean reads, totaling 2.54 GB with a Q30 of 92.86%, were obtained and
employed for the assembly of the complete cp genome of A. sessilis. The A. sessilis cp
genome is 151,935 bp in length and follows the typical quadripartite structure, comprising
two inverted repeat regions (IRs), a large single copy region (LSC), and a small single copy
region (SSC). The LSC region spans 84,449 bp, while the SSC region is 17,298 bp long, with
a pair of Irs, each covering 25,095 bp (Figure 1). The overall GC content of the A. sessilis cp
genome is 36.3%, while the GC contents of LSC, SSC, and IRs are 33.3%, 29.8%, and 42.5%,
respectively (Table 1). It is noteworthy that IRs exhibit the highest GC content, primarily
due to the presence of high-GC-content rRNA genes. A comparison of the A. sessilis cp
genome with previously reported cp genomes from the Amaranthaceae species revealed
similarities with the A. philoxeroides cp genome (Table 1).
Genes 2024,15, 544 6 of 22
Genes2024,15,xFORPEERREVIEW6of23
Tab le1.ComparisonofbasiccharacteristicsofchloroplastgenomesinAmaranthaceaespecies.
SpeciesGenusGenBank
Number
Genome
Size(bp)
LSC
(bp)
SSC
(bp)
IR
(bp)
GC
(%)
tRNA
Gene
Number
rRNA
Gene
Number
Protein‐
Coding
Gene
Number
Total
Gene
Number
AlternantherasessilisAlternantheraPP239384151,93584,44917,29825,09536.337883128
Alternanthera
philoxeroidesAlternantheraNC_042798152,25584,67017,31925,13336.435883126
AmaranthustricolorAlternantheraKX094399150,02783,73517,60024,34636.633889130
Amaranthus
hypochondriacus
cultivarPlainsman
AmaranthusNC_030770150,51883,87317,94124,35236.631873112
A
maranthuscaudatusAmaranthusNC_040143150,52383,87817,94124,35236.637884129
A
maranthushybridus
subsp.PI566897AmaranthusMG836506150,75784,10117,95424,35136.637884129
CelosiaargenteaCelosiaNC_041294153,67385,14017,71125,41136.737885130
CelosiacristataCelosiaNC_045887153,47284,97517,68125,40836.736873117
CyathulacapitataCyathulaNC_041262151,55783,35017,12925,53936.437884129
Deeringia
amaranthoidesDeeringiaNC_041267155,10886,06518,30525,36936.835884127
Figure 1. Chloroplast genome map of Alternanthera sessilis. Genes coding forward are on the outer
circle, while genes coding backward are on the inner circle. The gray circle inside represents the
GC content.
Table 1. Comparison of basic characteristics of chloroplast genomes in Amaranthaceae species.
Species Genus GenBank
Number
Genome
Size (bp)
LSC
(bp)
SSC
(bp)
IR
(bp)
GC
(%)
tRNA Gene
Number
rRNA Gene
Number
Protein-
Coding Gene
Number
Total Gene
Number
Alternanthera sessilis Alternanthera PP239384 151,935 84,449 17,298 25,095 36.3 37 8 83 128
Alternanthera
philoxeroides Alternanthera
NC_042798
152,255 84,670 17,319 25,133 36.4 35 8 83 126
Amaranthus tricolor Alternanthera KX094399 150,027 83,735 17,600 24,346 36.6 33 8 89 130
Amaranthus
hypochondriacus
cultivar Plainsman
Amaranthus
NC_030770
150,518 83,873 17,941 24,352 36.6 31 8 73 112
Amaranthus caudatus Amaranthus
NC_040143
150,523 83,878 17,941 24,352 36.6 37 8 84 129
Amaranthus hybridus
subsp. PI566897 Amaranthus MG836506 150,757 84,101 17,954 24,351 36.6 37 8 84 129
Celosia argentea Celosia
NC_041294
153,673 85,140 17,711 25,411 36.7 37 8 85 130
Celosia cristata Celosia
NC_045887
153,472 84,975 17,681 25,408 36.7 36 8 73 117
Cyathula capitata Cyathula
NC_041262
151,557 83,350 17,129 25,539 36.4 37 8 84 129
Deeringia
amaranthoides Deeringia
NC_041267
155,108 86,065 18,305 25,369 36.8 35 8 84 127
Genes 2024,15, 544 7 of 22
The A. sessilis cp genome encodes a total of 128 genes, including 8 rRNA-coding
genes, 37 tRNA-coding genes, 4 pseudogenes, and 83 protein-coding genes. Among the
protein-coding genes, 44 are related to photosynthesis, 24 are involved in self-replication,
and the remaining 10 have diverse functions (Table 2). In comparison to its close relative,
the A. philoxeroides cp genome, A. sessilis possesses three additional tRNA-coding genes:
trnG-GCC,trnS-CGA, and trnfM-CAU. Furthermore, the gene trnM-CAU is present in a
single copy in the A. sessilis cp genome, whereas there are two copies in the A. philoxeroides
cp genomes. Additionally, the A. sessilis cp genome contains two unique protein-coding
genes, rpl22 and rps15, absent from the A. philoxeroides cp genome. Notably, the ndhA genes
have different structures in the two species, with ndhA in A. sessilis having one intron,
whereas that in A. philoxeroides is an all-exon structure. A. sessilis also harbors two specific
pseudogenes, ycf15 and ycf1, which contain introns. Within the A. sessilis cp genome, eight
tRNA-coding genes possess one intron. Among them, two copies of trnI-GAU and trnA-
UGC are located in IRs, while the remaining four tRNA-coding genes are situated in the
LSC region. Notably, trnK-UUU contains the largest intron, spanning 2538 bp, and encodes
the matK gene. Eleven protein-coding genes in the A. sessilis cp genome contain introns,
primarily associated with self-replication and photosynthesis. Nine of these genes possess
one intron, predominantly situated in the LSC region, except for ndhB in IRs and ndhA in
SSC. Two protein-coding genes, clpP, and ycf3, each exhibit two introns (Table 3).
Table 2. Genes in the chloroplast genome of Alternanthera sessilis and Alternanthera philoxeroides.
Function of
Genes Category of Genes Gene Name
Self-replication
Large subunit of ribosome
rpl2
a
, rpl14, rpl16
b
, rpl20, rpl22(ses), rpl23
a
(phi), rpl32, rpl33, rpl36
DNA-dependent RNA polymerase rpoA, rpoB, rpoC1 b, rpoC2
Ribosomal RNA genes rrn4.5 a, rrn5 a, rrn16 a, rrn23 a
Small subunit of ribosome rps2, rps3, rps4, rps7 a, rps8, rps11, rps12 abe, rps14, rps15(ses),
rps16 b, rps18, rps19 d(ses), rps19 a(phi)
Transfer RNA genes
trnA-UGC ab, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA,
trnG-GCC(ses), trnH-GUG, trnI-CAU a, trnI-GAU ab, trnK-UUU b,
trnL-CAA a, trnL-UAA b, trnL-UAG, trnM-CAU(ses), trnM-CAU
a
(phi), trnN-GUU
a
, trnP-UGG, trnQ-UUG, trnR-ACG
a
, trnR-UCU,
trnS-CGA b(ses), trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU,
trnT-UGU, trnV-GAC a, trnV-UAC b, trnW-CCA, trnY-GUA,
trnfM-CAU(ses)
Genes for
photosynthesis
Subunits of ATP synthase atpA, atpB, atpE, atpF b, atpH, atpI
Subunits of NADH dehydrogenase ndhA b(ses), ndhA(phi), ndhB ab, ndhC, ndhD, ndhE, ndhF, ndhG,
ndhH, ndhI, ndhJ, ndhK
Subunits of photosystem I psaA, psaB, psaC, psaI, psaJ
Subunits of photosystem II psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL,
psbM, psbN, psbT, psbZ
Subunits of cytochrome petA, petB b, petD b, petG, petL, petN
Large subunit of Rubisco rbcL
Other genes
Subunit of acetyl-CoA-carboxylase accD
C-type cytochrome synthesis gene ccsA
Envelop membrane protein cemA
ATP-dependent protease subunit p gene
clpP c
Maturase matK
Translation initiation factor infA
Genes of
unknown function
Conserved open reading frames ycf1 d(ses), ycf15 ad(ses), ycf1, ycf2 a, ycf3 c, ycf4
a
Two gene copies in IRs.
b
Gene containing a single intron.
c
Gene containing two introns.
d
Pseudogene.
e
Gene
divided into two independent transcription units. Ses—Genes that are particular to A. sessilis. phi—Genes that are
particular to A. philoxeroides.
Genes 2024,15, 544 8 of 22
Table 3. Genes containing introns in the A. sessilis chloroplast genome.
Gene Location Intron Number Exon I Intron I Exon II Intron II Exon III CDS Length
trnK-UUU LSC 1 37 2538 35 72
rps16 LSC 1 42 911 213 255
trnS-CGA LSC 1 31 713 60 91
atpF LSC 1 148 800 410 558
rpoC1 LSC 1 435 771 1602 2037
ycf3 LSC 2 124 764 230 774 153 507
trnL-UAA LSC 1 35 650 50 85
trnV-UAC LSC 1 38 595 35 73
rps12 IRa 114 - 232 538 26 372
clpP LSC 2 69 600 293 875 226 588
petB LSC 1 6 769 642 648
petD LSC 1 8 774 475 483
rpl16 LSC 1 9 1055 402 411
ndhB IRb 1 778 667 758 1536
rps12 IRb 232 - 26 538 114 372
trnI-GAU IRb 1 37 946 35 72
trnA-UGC IRb 1 38 825 36 74
ndhA SSC 1 556 959 539 1095
trnA-UGC IRa 1 38 825 36 74
trnI-GAU IRa 1 37 946 35 72
ndhB IRa 1 778 667 758 1536
3.2. Codon Usage Bias of A. sessilis and A. philoxeroides cp Genomes
The usage of synonymous codons in the cp genomes of A. sessilis was assessed using
relative synonymous codon usage (RSCU) and compared with that of A. philoxeroides. In
both genomes, Leu was found to have the highest amino acid frequency, accounting for
10.60% in A. sessilis and 10.37% in A. philoxeroides, while Cys exhibited the lowest frequency
at 1.17% in A. sessilis and 1.68% in A. philoxeroides (Figure 2). Regarding start codons, in the
A. sessilis cp genome, ACG was used as the start codon for psbL, while GTG was utilized
for rps19,ndhD, and ycf1. In the A. philoxeroides cp genome, psbL and ndhD employed ACG
as the start codon, while only rps19 used GTG. The RSCU values for stop codons UAA,
UAG, and UGA in the A. sessilis cp genome were 1.63, 0.72, and 0.65, respectively. UAA
was preferred as the primary stop codon in the A. sessilis cp genome. In contrast, a more
balanced preference for stop codon usage was observed in the A. philoxeroides chloroplast
genome, with RSCU values of 1.16 for UAA, 0.95 for UAG, and 0.89 for UGA (Table S1).
3.3. SSRs and Long Repeated Sequences
In the cp genomes of both A. sessilis and A. philoxeroides, a total of 96 and 113 SSRs
of four types were identified, respectively. Generally, A. philoxeroides exhibits a higher
abundance of SSRs compared to A. sessilis. Based on the length of the repeating motifs, the
A. sessilis cp genome contains 74 single-nucleotide repeat sequences, 10 dinucleotide repeats,
3 trinucleotide repeats, and 9 tetranucleotide repeats. In contrast, A. philoxeroides has an
equal number of dinucleotide repeats but shows a higher occurrence of single-nucleotide,
trinucleotide, and tetranucleotide repeats (Figure 3A). Regarding the type of repeating
motif, in the A. sessilis cp genome, the most abundant is the T single-nucleotide repeats,
followed by A single-nucleotide repeats. These two types of motif repeats account for
60.81% and 37.83%, respectively, out of all single-nucleotide repeats (Figure 3B). A similar
distribution pattern was observed in the A. philoxeroides cp genome (Figure 3B).
Genes 2024,15, 544 9 of 22
Genes2024,15,xFORPEERREVIEW9of23
Figure2.RelativesynonymouscodonusageintheA.sessilischloroplastgenomes.*:Terminator.
3.3.SSRsandLongRepeatedSequences
InthecpgenomesofbothA.sessilisandA.philoxeroides,atotalof96and113SSRsof
fourtypeswereidentified,respectively.Generally,A.philoxeroidesexhibitsahigherabun-
danceofSSRscomparedtoA.sessilis.Basedonthelengthoftherepeatingmotifs,theA.
sessiliscpgenomecontains74single-nucleotiderepeatsequences,10dinucleotiderepeats,
3trinucleotiderepeats,and9tetranucleotiderepeats.Incontrast,A.philoxeroideshasan
equalnumberofdinucleotiderepeatsbutshowsahigheroccurrenceofsingle-nucleotide,
trinucleotide,andtetranucleotiderepeats(Figure3A).Regardingthetypeofrepeating
motif,intheA.sessiliscpgenome,themostabundantistheTsingle-nucleotiderepeats,
followedbyAsingle-nucleotiderepeats.Thesetwotypesofmotifrepeatsaccountfor
60.81%and37.83%,respectively,outofallsingle-nucleotiderepeats(Figure3B).Asimilar
distributionpaernwasobservedintheA.philoxeroidescpgenome(Figure3B).
Figure 2. Relative synonymous codon usage in the A. sessilis chloroplast genomes. *: Terminator.
Genes2024,15,xFORPEERREVIEW10of23
Figure3.NumberofsimplesequencerepeatsinA.sessilisandA.philoxeroideschloroplastgenomes.
(A)NumberofsimplesequencerepeatsofdifferenttypesinA.sessilisandA.philoxeroideschloro-
plastgenomesbasedontherepeatingmotiflength.(B)Numberofsimplesequencerepeatswith
differentmotiftypesinA.sessilisandA.philoxeroideschloroplastgenomes.
Forty-nineandfiftyrepetitivesequenceslongerthan30basepairswereidentifiedin
thecpgenomesofA.sessilisandA.philoxeroides,respectively.Theseincluded21forward
repeatsand28palindromerepeatsintheA.sessiliscpgenomeand19forwardrepeats,29
palindromerepeats,and2reverserepeatsintheA.philoxeroidescpgenome(Figure4A).
ThemajorityoftheselargerepetitivesequencesarelocatedintheLSCandIRregions
(Figure4B).Repeatswithlengthsrangingfrom30to40basepairsaccountfor61.2%and
64%ofthetotalrepetitivesequencesinthecpgenomesofA.sessilisandA.philoxeroides,
respectively(Figure4C).
Figure 3. Number of simple sequence repeats in A. sessilis and A. philoxeroides chloroplast genomes.
(A) Number of simple sequence repeats of different types in A. sessilis and A. philoxeroides chloroplast
genomes based on the repeating motif length. (B) Number of simple sequence repeats with different
motif types in A. sessilis and A. philoxeroides chloroplast genomes.
Genes 2024,15, 544 10 of 22
Forty-nine and fifty repetitive sequences longer than 30 base pairs were identified in
the cp genomes of A. sessilis and A. philoxeroides, respectively. These included 21 forward
repeats and 28 palindrome repeats in the A. sessilis cp genome and 19 forward repeats,
29 palindrome
repeats, and 2 reverse repeats in the A. philoxeroides cp genome (Figure 4A).
The majority of these large repetitive sequences are located in the LSC and IR regions
(Figure 4B). Repeats with lengths ranging from 30 to 40 base pairs account for 61.2% and
64% of the total repetitive sequences in the cp genomes of A. sessilis and A. philoxeroides,
respectively (Figure 4C).
Genes2024,15,xFORPEERREVIEW11of23
Figure4.NumberofrepetitivesequencesinA.sessilisandA.philoxeroideschloroplastgenomes.(A)
NumberofrepetitivesequencesofdifferenttypesinA.sessilisandA.philoxeroideschloroplastge-
nomes.(B)NumberofrepetitivesequencesindifferentlocationsinA.sessilisandA.philoxeroides
chloroplastgenomes.(C)NumberofrepetitivesequencesofdifferentlengthsinA.sessilisandA.
philoxeroideschloroplastgenomes.
3.4.DivergenceHotspotsandKa/Ks
DespitethestructuralsimilaritybetweenthecpgenomesofA.sessilisandA.philoxe‐
roides,notablenucleotidedifferencesexist.Nucleotidepolymorphism(Pi)wasusedasan
indicatortomeasurenucleicaciddivergence,rangingfrom0to0.0433,withanaverage
valueof0.01159.Nineregionswithhighnucleotidepolymorphismswereidentified,in-
cludingtrnK‐rps16,trnC‐petN,petN‐trnD,trnT,petL‐petG,rps19‐rpl2,ndhF‐trnL,ccsA,and
ycf1(Pi>0.033).ThesehighlyvariableregionsareprimarilydistributedintheLSCand
SSCregions,whiletheIRregionremainsmoreconserved.Onlytworegionsexhibithigher
Pivalues:ycf2‐trnLintheIRaregionandycf2intheIRbregion,withPivaluesof0.02833
(Figure5).
Figure 4. Number of repetitive sequences in A. sessilis and A. philoxeroides chloroplast genomes.
(A) Number of repetitive sequences of different types in A. sessilis and A. philoxeroides chloroplast
genomes. (B) Number of repetitive sequences in different locations in A. sessilis and A. philoxeroides
chloroplast genomes. (C) Number of repetitive sequences of different lengths in A. sessilis and A.
philoxeroides chloroplast genomes.
3.4. Divergence Hotspots and Ka/Ks
Despite the structural similarity between the cp genomes of A. sessilis and A. philoxe-
roides, notable nucleotide differences exist. Nucleotide polymorphism (Pi) was used as an
indicator to measure nucleic acid divergence, ranging from 0 to 0.0433, with an average
value of 0.01159. Nine regions with high nucleotide polymorphisms were identified, in-
cluding trnK-rps16,trnC-petN,petN-trnD,trnT,petL-petG,rps19-rpl2,ndhF-trnL,ccsA, and
ycf1 (Pi > 0.033). These highly variable regions are primarily distributed in the LSC and
SSC regions, while the IR region remains more conserved. Only two regions exhibit higher
Pi values: ycf2-trnL in the IRa region and ycf2 in the IRb region, with Pi values of 0.02833
(Figure 5).
Genes 2024,15, 544 11 of 22
Genes2024,15,xFORPEERREVIEW12of23
Figure5.ThenucleotidepolymorphismforcpgenomesofA.sessilisandA.philoxeroidescalculated
usingDnaSP6.0employingparametersofa200bpstepsizeand600bpwindowlength.Eleven
mostdivergentregionsaresuggestedasmutationhotspots.Thenameofregionsinredindicate
theseregionsarelocatedinLSCregionorSSCregion,andthoseinblueindicatetheregionsare
locatedinIRs.
Synonymousandnon-synonymoussubstitutionrateswereanalyzedforthe73pro-
tein-codinggenessharedbythecpgenomesofA.sessilisandA.philoxeroides.Thesynon-
ymoussubstitutionraterangedfrom0to0.0789,withrps19exhibitingthehighestsynon-
ymoussubstitutionrate.Thenon-synonymoussubstitutionraterangedfrom0to0.0294,
andinfAdisplayedthehighestnon-synonymoussubstitutionrate(TableS2).
Theratioofsynonymoussubstitutionratetonon-synonymoussubstitutionrate
(Ka/Ks)wasfurthercalculatedtoassesstheselectionpressureon57protein-encoding
genes,withKa/Ksratiosrangingfrom0to3.475.Outofthese,55geneshadKa/Ksratios
below1,indicatingabiastowardpurificationselection.Notably,16genes,includingatpF
andpsbA,exhibitedaKa/Ksratioof0,suggestingthattheyareunderstrongpurification
selectionpressure.However,twogenes,ccsAandaccD,displayedKa/Ksratiosabove1,
specifically,1.237and3.475,respectively.Thissuggeststhatthesetwogenes,especially
accD,arerapidlyevolvingunderpositiveselectioninfluenceandmayplayacrucialrole
intheevolutionofthespecies.Fortheremaining16genes,theKa/Ksratiocouldnotbe
calculatedduetoKs=0(Figure6).
Figure6.TheKa/Ksratioof55protein-codinggenesinA.sessilisandA.philoxeroidescalculated
usingDnaSP6.0.Ka/KsvaluesofAccDandCcsAweremorethan1.
3.5.ComparisonofChloroplastGenomesintheAmaranthaceaeFamily
Figure 5. The nucleotide polymorphism for cp genomes of A. sessilis and A. philoxeroides calculated
using DnaSP 6.0 employing parameters of a 200 bp step size and 600 bp window length. Eleven most
divergent regions are suggested as mutation hotspots. The name of regions in red indicate these
regions are located in LSC region or SSC region, and those in blue indicate the regions are located
in IRs.
Synonymous and non-synonymous substitution rates were analyzed for the 73 protein-
coding genes shared by the cp genomes of A. sessilis and A. philoxeroides. The synonymous
substitution rate ranged from 0 to 0.0789, with rps19 exhibiting the highest synonymous
substitution rate. The non-synonymous substitution rate ranged from 0 to 0.0294, and infA
displayed the highest non-synonymous substitution rate (Table S2).
The ratio of synonymous substitution rate to non-synonymous substitution rate
(Ka/Ks) was further calculated to assess the selection pressure on 57 protein-encoding
genes, with Ka/Ks ratios ranging from 0 to 3.475. Out of these, 55 genes had Ka/Ks ratios
below 1, indicating a bias toward purification selection. Notably, 16 genes, including atpF
and psbA, exhibited a Ka/Ks ratio of 0, suggesting that they are under strong purification
selection pressure. However, two genes, ccsA and accD, displayed Ka/Ks ratios above 1,
specifically, 1.237 and 3.475, respectively. This suggests that these two genes, especially
accD, are rapidly evolving under positive selection influence and may play a crucial role
in the evolution of the species. For the remaining 16 genes, the Ka/Ks ratio could not be
calculated due to Ks = 0 (Figure 6).
Genes2024,15,xFORPEERREVIEW12of23
Figure5.ThenucleotidepolymorphismforcpgenomesofA.sessilisandA.philoxeroidescalculated
usingDnaSP6.0employingparametersofa200bpstepsizeand600bpwindowlength.Eleven
mostdivergentregionsaresuggestedasmutationhotspots.Thenameofregionsinredindicate
theseregionsarelocatedinLSCregionorSSCregion,andthoseinblueindicatetheregionsare
locatedinIRs.
Synonymousandnon-synonymoussubstitutionrateswereanalyzedforthe73pro-
tein-codinggenessharedbythecpgenomesofA.sessilisandA.philoxeroides.Thesynon-
ymoussubstitutionraterangedfrom0to0.0789,withrps19exhibitingthehighestsynon-
ymoussubstitutionrate.Thenon-synonymoussubstitutionraterangedfrom0to0.0294,
andinfAdisplayedthehighestnon-synonymoussubstitutionrate(TableS2).
Theratioofsynonymoussubstitutionratetonon-synonymoussubstitutionrate
(Ka/Ks)wasfurthercalculatedtoassesstheselectionpressureon57protein-encoding
genes,withKa/Ksratiosrangingfrom0to3.475.Outofthese,55geneshadKa/Ksratios
below1,indicatingabiastowardpurificationselection.Notably,16genes,includingatpF
andpsbA,exhibitedaKa/Ksratioof0,suggestingthattheyareunderstrongpurification
selectionpressure.However,twogenes,ccsAandaccD,displayedKa/Ksratiosabove1,
specifically,1.237and3.475,respectively.Thissuggeststhatthesetwogenes,especially
accD,arerapidlyevolvingunderpositiveselectioninfluenceandmayplayacrucialrole
intheevolutionofthespecies.Fortheremaining16genes,theKa/Ksratiocouldnotbe
calculatedduetoKs=0(Figure6).
Figure6.TheKa/Ksratioof55protein-codinggenesinA.sessilisandA.philoxeroidescalculated
usingDnaSP6.0.Ka/KsvaluesofAccDandCcsAweremorethan1.
3.5.ComparisonofChloroplastGenomesintheAmaranthaceaeFamily
Figure 6. The Ka/Ks ratio of 55 protein-coding genes in A. sessilis and A. philoxeroides calculated using
DnaSP 6.0. Ka/Ks values of AccD and CcsA were more than 1.
Genes 2024,15, 544 12 of 22
3.5. Comparison of Chloroplast Genomes in the Amaranthaceae Family
Among ten species in the Amaranthaceae sensu stricto, their cp genomes exhibit
remarkable structural conservation. However, noticeable variations in cp genome size are
attributed to the contraction and expansion of the IR boundaries (Figure 7). The IR regions
in these cp genomes vary in size, ranging from 24,346 to 25,539 base pairs (bp) (Table 1).
Regarding the LSC/IRb boundary, all Amaranthaceae species, except for A. philoxeroides,
harbor the rpl22 exclusively within the LSC region, devoid of any cross-boundary coding.
With the exception of Amaranthus hupochondriacus and Celosia cristata, the remaining eight
species feature the rps19 proximal to rpl22, extending into the IRb regions with segments
spanning 72 to 223 bp, overlapping the LSC/IRb boundary. In A. sessilis and A. philoxeroides,
the rps19 copy predominantly resides in the LSC region, with minor portions extending
into the IRb region (87 bp and 72 bp, respectively). Furthermore, an additional rps19 copy
is found solely within the IRa regions of A. sessilis,A. philoxeroides, and A. tricolor. At
the SSC/IRb boundary, a pseudogene of ycf1 is present in A. sessilis,Amaranthus tricolor,
Amaranthus caudatus, and Amaranthus hybridus, primarily located in IRbs, extending into the
SSC region by 10–15 bp. The premature termination of ORF was observed in the aforemen-
tioned pseudogene ycf1 as a result of the contraction and expansion of the IR boundaries.
Conversely, ndhF genes are primarily situated in the SSC regions, overlapping the SSC/IRb
boundary and containing segments approximately
1–34 bp
within the IRbs. Moving to the
SSC/IRa boundary, ycf1 genes in these Amaranthaceae species primarily inhabit the SSC
regions, extending 1387–1778 bp into the IRa regions. Notably, A. hypochondriacus and C.
cristata lack ycf1. Concerning the LSC/IRa boundary, the trnH genes in the cp genomes of
C. argentea and C. acpitata are predominantly found in the IRb region, while, in the other
species, they are mainly located in the LSC region, with a distance ranging from 1 to 25 bp
from the boundary (Figure 7).
Genes2024,15,xFORPEERREVIEW13of23
AmongtenspeciesintheAmaranthaceaesensustricto,theircpgenomesexhibitre-
markablestructuralconservation.However,noticeablevariationsincpgenomesizeare
aributedtothecontractionandexpansionoftheIRboundaries(Figure7).TheIRregions
inthesecpgenomesvaryinsize,rangingfrom24,346to25,539basepairs(bp)(Table1).
RegardingtheLSC/IRbboundary,allAmaranthaceaespecies,exceptforA.philoxeroides,
harbortherpl22exclusivelywithintheLSCregion,devoidofanycross-boundarycoding.
WiththeexceptionofAmaranthushupochondriacusandCelosiacristata,theremainingeight
speciesfeaturetherps19proximaltorpl22,extendingintotheIRbregionswithsegments
spanning72to223bp,overlappingtheLSC/IRbboundary.InA.sessilisandA.philoxe‐
roides,therps19copypredominantlyresidesintheLSCregion,withminorportionsex-
tendingintotheIRbregion(87bpand72bp,respectively).Furthermore,anadditional
rps19copyisfoundsolelywithintheIRaregionsofA.sessilis,A.philoxeroides,andA.tri‐
color.AttheSSC/IRbboundary,apseudogeneofycf1ispresentinA.sessilis,Amaranthus
tricolor,Amaranthuscaudatus,andAmaranthushybridus,primarilylocatedinIRbs,extend-
ingintotheSSCregionby10–15bp.TheprematureterminationofORFwasobservedin
theaforementionedpseudogeneycf1asaresultofthecontractionandexpansionofthe
IRboundaries.Conversely,ndhFgenesareprimarilysituatedintheSSCregions,overlap-
pingtheSSC/IRbboundaryandcontainingsegmentsapproximately1–34bpwithinthe
IRbs.MovingtotheSSC/IRaboundary,ycf1genesintheseAmaranthaceaespeciespri-
marilyinhabittheSSCregions,extending1387–1778bpintotheIRaregions.Notably,A.
hypochondriacusandC.cristatalackycf1.ConcerningtheLSC/IRaboundary,thetrnHgenes
inthecpgenomesofC.argenteaandC.acpitataarepredominantlyfoundintheIRbregion,
while,intheotherspecies,theyaremainlylocatedintheLSCregion,withadistance
rangingfrom1to25bpfromtheboundary(Figure7).
Figure 7. Comparison of the border positions of the LSC, IR, and SSC regions among ten Amaran-
thaceae species chloroplast genomes. Gene names are indicated in the boxes and their lengths in the
corresponding regions are displayed above the boxes.
Genes 2024,15, 544 13 of 22
Using the A. sessilis cp genome sequence as a reference, six known cp genome se-
quences from five related genera were aligned. The results revealed a high degree of
similarity among these sequences, with most regions displaying over 50 percent similarity.
Considering the chloroplast structure, relatively high similarity was noted in the IR regions,
while lower levels of similarity were observed in the LSC and SSC regions. From the
perspective of gene structure, extremely high sequence similarity was found in both exon
and UTR regions, except for ccsA,ycf1, and ycf2, which exhibited relatively high diversities.
The non-coding region displayed low similarity and significant variation, suggesting its
potential as a hotspot for the development of new molecular markers. The intergenic
regions, trnK-rps16,petN-trnD,petL-petG, and ndhF-trnL, showed large diversity, consistent
with the results of single nucleotide polymorphism analysis. Furthermore, significant
diversity was observed in the gene introns, such as those within petD,rpl16, and ndhA. In
our focused study on the cp genome of A. sessilis and A. philoxeroides, two regions of low
similarity were identified between ndhB and ycf2, adjacent to trnL-CAA (Figure 8).
Genes2024,15,xFORPEERREVIEW14of23
Figure7.ComparisonoftheborderpositionsoftheLSC,IR,andSSCregionsamongtenAmaran-
thaceaespecieschloroplastgenomes.Genenamesareindicatedintheboxesandtheirlengthsinthe
correspondingregionsaredisplayedabovetheboxes.
UsingtheA.sessiliscpgenomesequenceasareference,sixknowncpgenomese-
quencesfromfiverelatedgenerawerealigned.Theresultsrevealedahighdegreeofsim-
ilarityamongthesesequences,withmostregionsdisplayingover50percentsimilarity.
Consideringthechloroplaststructure,relativelyhighsimilaritywasnotedintheIRre-
gions,whilelowerlevelsofsimilaritywereobservedintheLSCandSSCregions.From
theperspectiveofgenestructure,extremelyhighsequencesimilaritywasfoundinboth
exonandUTRregions,exceptforccsA,ycf1,andycf2,whichexhibitedrelativelyhighdi-
versities.Thenon-codingregiondisplayedlowsimilarityandsignificantvariation,sug-
gestingitspotentialasahotspotforthedevelopmentofnewmolecularmarkers.Thein-
tergenicregions,trnK‐rps16,petN‐trnD,petL‐petG,andndhF‐trnL,showedlargediversity,
consistentwiththeresultsofsinglenucleotidepolymorphismanalysis.Furthermore,sig-
nificantdiversitywasobservedinthegeneintrons,suchasthosewithinpetD,rpl16,and
ndhA.InourfocusedstudyonthecpgenomeofA.sessilisandA.philoxeroides,tworegions
oflowsimilaritywereidentifiedbetweenndhBandycf2,adjacenttotrnL‐CAA(Figure8).
Figure8.SimilarityofchloroplastgenomesequencesamongsixAmaranthaceaespeciesfromdif-
ferentgenera.Sequenceidentityisportrayedwithacut-off of50%identity.TheY-scaleaxis
Figure 8. Similarity of chloroplast genome sequences among six Amaranthaceae species from different
genera. Sequence identity is portrayed with a cut-off of 50% identity. The Y-scale axis represents the
percent identity within 50–100%. Grey arrows indicate genes with their orientation and position.
Genome regions are color-coded as purple blocks for the conserved coding genes (exon), light red
blocks for the conserved non-coding sequences in intergenic regions (CNS), and aqua blue blocks for
UTR. The lines below the alignment indicate the chloroplast genomes. Black-bordered white peaks
that are shown in genome regions indicate the divergent regions with sequence variation among six
Amaranthaceae species.
Genes 2024,15, 544 14 of 22
3.6. Phylogenetic Analysis and Estimation of Divergence Time
For the construction of phylogenetic trees, we utilized fifty-nine chloroplast protein-
coding genes from 25 species within Amaranthaceae s.l as the inner group, while Acha-
tocarpus nigricans,Achatocarpus pubescens, and Dianthus caryophyllus were employed as
the outgroups. The phylogenetic trees were generated using both the maximum like-
lihood method and the Bayesian inference method independently. Remarkably, both
methods yielded similar tree topologies (Figures 9and 10). In these trees, A. sessilis and A.
philoxeroides clustered together with the Cyathula species, forming one branch, while the
genera Amaranthus,Celosia, and Deeringias grouped into another branch. These genera are
part of Amaranthaceae s.s and formed a single branch collectively. Species belonging to
Chenopodium,Beta,Haloxylon,Salicornia, and Suaeda genera constituted a distinct branch.
Notably, the Chenopodiaceae branch and the Amaranthaceae s.s branch together formed a
monophyletic group.
Genes2024,15,xFORPEERREVIEW15of23
representsthepercentidentitywithin50–100%.Greyarrowsindicategeneswiththeirorientation
andposition.Genomeregionsarecolor-codedaspurpleblocksfortheconservedcodinggenes
(exon),lightredblocksfortheconservednon-codingsequencesinintergenicregions(CNS),and
aquablueblocksforUTR.Thelinesbelowthealignmentindicatethechloroplastgenomes.Black-
borderedwhitepeaksthatareshowningenomeregionsindicatethedivergentregionswithse-
quencevariationamongsixAmaranthaceaespecies.
3.6.PhylogeneticAnalysisandEstimationofDivergenceTime
Fortheconstructionofphylogenetictrees,weutilizedfifty-ninechloroplastprotein-
codinggenesfrom25specieswithinAmaranthaceaes.lastheinnergroup,whileAchato‐
carpusnigricans,Achatocarpuspubescens,andDianthuscaryophylluswereemployedasthe
outgroups.Thephylogenetictreesweregeneratedusingboththemaximumlikelihood
methodandtheBayesianinferencemethodindependently.Remarkably,bothmethods
yieldedsimilartreetopologies(Figures9and10).Inthesetrees,A.sessilisandA.philoxe‐
roidesclusteredtogetherwiththeCyathulaspecies,formingonebranch,whilethegenera
Amaranthus,Celosia,andDeeringiasgroupedintoanotherbranch.Thesegeneraarepartof
Amaranthaceaes.sandformedasinglebranchcollectively.SpeciesbelongingtoChenopo‐
dium,Beta,Haloxylon,Salicornia,andSuaedageneraconstitutedadistinctbranch.Notably,
theChenopodiaceaebranchandtheAmaranthaceaes.sbranchtogetherformedamono-
phyleticgroup.
Figure9.ThephylogenetictreeconstructedusingthemaximumlikelihoodmethodinMEGA11.0
withtheGTR+G+Imodel,employing59chloroplastgenesequencesfrom28species.Thechloro-
plastgenomesequencesofAchatocarpusnigricans,Achatocarpuspubescens,andDianthuscaryophyllus
wereusedastheoutgroup.Thenumbersonthebranchesindicatethebootstrapvalues.
Figure 9. The phylogenetic tree constructed using the maximum likelihood method in MEGA 11.0
with the GTR + G + I model, employing 59 chloroplast gene sequences from 28 species. The chloroplast
genome sequences of Achatocarpus nigricans,Achatocarpus pubescens, and Dianthus caryophyllus were
used as the outgroup. The numbers on the branches indicate the bootstrap values.
Genes 2024,15, 544 15 of 22
Genes2024,15,xFORPEERREVIEW16of23
Figure10.ThephylogenetictreeconstructedusingtheBayesianinferencemethodinMybayes3.2.6
undertheGTR+I+G+Fmodel,incorporating59chloroplastgenesequencesfrom28species.The
chloroplastgenomesequencesofA.nigricans,A.pubescens,andD.caryophylluswereemployedas
theoutgroup.Thenumbersonthebranchesindicatetheposteriorprobabilities.
Additionally,atimetreewasconstructedusingtheaforementionedphylogenetic
treesandanalyzedtoestimatedivergencetimes.TheresultsindicatedthatAmaran-
thaceaes.s.andChenopodiaceaelikelyoriginatedduringthePaleogeneperiod,approxi-
mately47.24MYA.ThedivergencebetweentheAlternantheragenusandtheCyathulage-
nusoccurredaround20.04MYA.TheindependentspeciationeventsforA.philoxeroides
andA.sessilistookplaceroughlybetween3.5186and8.8242MYA(Figure11).
Figure 10. The phylogenetic tree constructed using the Bayesian inference method in Mybayes 3.2.6
under the GTR + I + G + F model, incorporating 59 chloroplast gene sequences from 28 species. The
chloroplast genome sequences of A. nigricans,A. pubescens, and D. caryophyllus were employed as the
outgroup. The numbers on the branches indicate the posterior probabilities.
Additionally, a time tree was constructed using the aforementioned phylogenetic trees
and analyzed to estimate divergence times. The results indicated that Amaranthaceae
s.s. and Chenopodiaceae likely originated during the Paleogene period, approximately
47.24 MYA
. The divergence between the Alternanthera genus and the Cyathula genus
occurred around 20.04 MYA. The independent speciation events for A. philoxeroides and A.
sessilis took place roughly between 3.5186 and 8.8242 MYA (Figure 11).
Genes 2024,15, 544 16 of 22
Genes2024,15,xFORPEERREVIEW17of23
Figure11.ThetimetreeestimatedunderthelocalmolecularclockusingtheRelTime-MLmethod
inMEGA11.0.ThecircularrepresentationwithfullredindicatesthedivergencetimesoftheAma‐
ranthusandChenopodiumgenus(estimatedat24.5–73.8MYA)aswellastheSuaedaandSalicornia
genus(estimatedat12.1–39.7MYA),derivedfrom10studiesand5studies,respectively,inTime-
Tree,usedforestimatingthedivergencetimebetweenA.sessilisandA.philoxeroides.
4.Discussion
Inthisstudy,weconductedsequencingandannotationofthechloroplastgenomeof
A.sessilis.Acomparativeanalysisofcpgenomeswasperformedbetweenrelativelocal
speciesA.sessilisandinvasiveweedA.philoxeroides.Thisstudyaimedtoidentifydiffer-
encesinthecpgenomesofthesetwoAlternantheraspecies,particularlyfocusingon
hotspotregionsandrepeatsequences.WealsoinvestigatedtheaccDandccsAgenes,
whichappeartobeevolvingrapidlyduetopositiveselection.Thedivergencetimebe-
tweenA.sessilisandA.philoxeroideswasestimated.
Overall,thecpgenomesofbothA.sessilisandA.philoxeroideshaveasizeofapproxi-
mately150kb,withGCpercentagesrangingfrom36.3%to36.8%(Figure1andTab l e1).
ThesecpgenomesexhibitthetypicaltetradstructureobservedinotherAmaranthaceae
species.Changesincpgenomesizesaretypicallyaributedtovariationsintheinverted
repeat(IR)region,geneloss,andalterationsingenespacerregions[49–51].Ourresults
revealthatvariousregionswithinthecpgenomesofAmaranthaceaes.s.specieshaveun-
dergonelengthchanges,rangingfromafewdozentothousandsofnucleotides.Notably,
thereisasizevariationofapproximately1kbwithintheIRregions.Tothebestofour
knowledge,A.sessilisandA.philoxeroidespossessmedium-sizedIRregions.AmongAm-
aranthaceaes.s.species,thesmallestIRsarefoundinA.tricolor,measuring24,346bp,
whilethelargestIRsareobservedinthecpgenomeofC.capitata,withalengthof25,539
bp.
Figure 11. The time tree estimated under the local molecular clock using the RelTime-ML method in
MEGA 11.0. The circular representation with full red indicates the divergence times of the Amaranthus
and Chenopodium genus (estimated at 24.5–73.8 MYA) as well as the Suaeda and Salicornia genus
(estimated at 12.1–39.7 MYA), derived from 10 studies and 5 studies, respectively, in TimeTree, used
for estimating the divergence time between A. sessilis and A. philoxeroides.
4. Discussion
In this study, we conducted sequencing and annotation of the chloroplast genome of A.
sessilis. A comparative analysis of cp genomes was performed between relative local species
A. sessilis and invasive weed A. philoxeroides. This study aimed to identify differences in the
cp genomes of these two Alternanthera species, particularly focusing on hotspot regions
and repeat sequences. We also investigated the accD and ccsA genes, which appear to be
evolving rapidly due to positive selection. The divergence time between A. sessilis and A.
philoxeroides was estimated.
Overall, the cp genomes of both A. sessilis and A. philoxeroides have a size of approxi-
mately 150 kb, with GC percentages ranging from 36.3% to 36.8% (Figure 1and Table 1).
These cp genomes exhibit the typical tetrad structure observed in other Amaranthaceae
species. Changes in cp genome sizes are typically attributed to variations in the inverted re-
peat (IR) region, gene loss, and alterations in gene spacer regions [
49
–
51
]. Our results reveal
that various regions within the cp genomes of Amaranthaceae s.s. species have undergone
length changes, ranging from a few dozen to thousands of nucleotides. Notably, there is a
size variation of approximately 1 kb within the IR regions. To the best of our knowledge, A.
sessilis and A. philoxeroides possess medium-sized IR regions. Among Amaranthaceae s.s.
species, the smallest IRs are found in A. tricolor, measuring 24,346 bp, while the largest IRs
are observed in the cp genome of C. capitata, with a length of 25,539 bp.
Genes 2024,15, 544 17 of 22
When comparing the genes and gene numbers in the cp genomes of A. sessilis and A.
philoxeroides with those of other Amaranthaceae species, it is evident that the protein-coding
genes in A. sessilis and A. philoxeroides are similar to those found in other Amaranthaceae
species. However, A. hypochondriacus and C. cristata exhibit a lower number of protein-
coding genes. Interestingly, although both A. sessilis and A. philoxeroides have the same
number of protein-coding genes, their genes are different. In the cp genome of A. sessilis,
genes such as rpl22 and rps15 are present, while rpl23 is absent. Conversely, the cp genome
of A. philoxeroides (NC_042798), collected from Jinan, exhibits the opposite situation (Table 2).
Another A. philoxeroides cp genome from Shiyan (MK450441) [
27
], which contains rpl22 and
rps15 but lacks rpl23, is similar to A. sessilis. In general, the loss of plastid genes can be
attributed to two main reasons. Firstly, non-essential gene loss results in permanent absence.
Secondly, the gene may transfer from the plastid genome to the nuclear genome [
52
,
53
].
The loss of rpl23 in the cp genomes of A. sessilis is a common occurrence in Amaranthaceae
s.l. Eleven species have demonstrated the loss of rpl23, while other species have shown
pseudogenization of rpl23 [
54
]. It is suggested that both rpl22 and rpl23 are essential for
leaf development, and their loss can lead to leaf deformities [
55
]. In Leguminosae, rpl22
transfers to the nucleus before being lost from the cp genome [
56
]. This suggests that there
may be nuclear transfer of rpl23/rpl22 in the cp genomes of A. sessilis and A. philoxeroides,
resulting in the loss of rpl23 or rpl22, which requires further investigation. Furthermore, the
absence of rps15, although not essential for plastid translation, can reduce the expression
efficiency of chloroplast genes, decrease accumulation levels in photosynthesis complexes,
affect ribosomal small subunit specificity, and impact leaf pigments, consequently retarding
plant growth and development. Cold stress can exacerbate these negative effects [
57
].
While the S15 protein encoded by the nucleus can be transported into plastids, it does
not fully compensate for the loss of plastid-encoded rps15 [
55
]. In the cp genomes of A.
philoxeroides, the sample from Jinan lacks rps15, whereas the sample from Shiyan possesses
it, suggesting that invasive weed A. philoxeroides in northern regions may have other cold
adaptation mechanisms to mitigate the negative effects caused by the loss of rps15. All
these differences between the two A. philoxeroides cp genomes may be attributed to their
different geographical locations, highlighting the potential impact of latitude on population
variations in alien species like A. philoxeroides, which needs more evidence.
The boundaries of the IR region in A. sessilis and the other nine Amaranthaceae cp
genomes were highly conserved, with minimal variation. This conservation aligns with the
typical pattern of IR region boundaries in most angiosperm species [
58
]. The expansion
and contraction of the IR region can lead to the pseudogenization of genes [
59
]. In our
study, we observed that pseudogenes
ψ
rps19 and
ψ
ycf1 were located at the IR boundary in
the chloroplast genome of A. sessilis (Figure 7), similar to findings for Mikania micrantha and
its native congener, Mikania cordata [
23
]. Specifically,
ψ
rps19 spanned the JLA (LSC/IRb)
boundary within Amaranthaceae s.s. Compared to the normal gene, we observed an
83 bp reduction in length at its 3
′
-end.
ψ
ycf1 crossed the JSB (SSC/IRb) boundary and
exhibited base deletions, causing a forward shift in its stop codon position and resulting
in a size reduction to 1483 bp. Generally, such genes may be transferred to the nuclear
genome [
60
,
61
]. However, it is worth noting that additional intact copies of rps19 and
ycf1 were also present in the chloroplast genome of A. sessilis, raising the possibility of
nuclear-transferred genes, which requires further investigation. Additionally, we found
two copies of pseudogene
ψ
ycf15 in the IR region, both with sizes of 519 bp and incomplete
ORFs due to base deletions. In chloroplast genomes, such as those observed in ANA-grade
species, monocots, most rosids, etc., ycf15 genes are fragmented or completely lost due to
nuclear gene transfers within the same plant lineages [
61
,
62
] or horizontal transfers from
different plant lineages [63].
The nucleotide polymorphism in the chloroplast genomes of A. sessilis and the re-
lated species A. philoxeroides was notably high, and significant nucleic acid divergence
regions were observed (Figure 5). These divergence regions were primarily situated in
single-copy regions and could serve as hotspots for designing DNA barcodes for species
Genes 2024,15, 544 18 of 22
identification. They offer potential molecular markers for investigating genetic variation
in invasive plants within the Amaranthaceae family. Notably, regions like trnK-rps16 and
trnC-petN, characterized by substantial nucleic acid divergence, have been successfully
used for identifying traditionally challenging classified species such as oaks [
64
] and the
Fragaria genus [
19
]. Furthermore, these regions can contribute to the phylogenetic analysis
of related species, as observed in wild grapes [
18
]. Additionally, large nucleic acid diver-
gences were also identified in the petL-petG,rps19-rpl2,ndhF-trnL,ccsA, and ycf1 regions.
Genes like petN,petL, and petG encode subunits of cytochrome b
6
f, which play a crucial
role in photosynthetic electron transport [
65
], suggesting that these divergences may be
related to adaptations to varying light conditions. Furthermore, our study revealed that
in the cp genomes of these two Alternanthera species, accD and ccsA are under positive
selection pressure (Figure 6). The accD encodes the beta subunit of the enzyme acetyl-CoA
carboxylase, which supplies malonyl-CoA primarily for fatty acid synthesis [
66
], playing an
important role in leaf development [
67
]. This positive selection of accD was also observed
in the chloroplast genome of Firmiana [
68
]. CcsA encodes cytochrome c biogenesis protein,
which mediates the attachment of heme to c-type cytochromes [
69
]. It has been shown that
ccsA was under positive selection in the epiphytic orchid [
70
] and Erigeron [
71
]. Both of
them have been found to contribute to adaption to environmental changes in other species,
for example, Lilium Ledebourii and the species in Malvaceae subfamilies [72,73].
Amaranthaceae s.l. is ranked as the second-largest family within the core Caryophyl-
lales, with a sister group relationship to Achatocarpaceae indicated by molecular phyloge-
netic evidence [
74
]. Within this family, Amaranthaceae s.s. forms a clearly monophyletic
group. In this study, phylogenetic trees were constructed using 59 chloroplast protein-
coding genes across 28 species, employing both the maximum likelihood and Bayesian
inference methods. Regardless of the methods and models used, the results were highly
consistent, displaying similar topological structures (Figures 9and 10), which aligned with
Yao’s plastid phylogenomic report of Caryophyllales [
54
]. The phylogenetic relationships
within the Alternanthera genus were supplemented in our study, a detail not covered in the
previous tree. Notably, a strong affinity was observed between the Alternanthera genus, com-
prising A. sessilis and A. philoxeroides, and the Cyathula genus. The incorporation of a broader
range of chloroplast protein-coding genes from various genera within Amaranthaceae s.l.
in our phylogenetic analysis provided a clearer understanding of the evolutionary and
ancestral relationships within this family compared to previous studies. Although previous
research had separately examined the phylogenetic relationships within the Alternanthera
genus in the Americas using combined sequences from the cp genome (trnL-F and rpl16)
and nuclear sequences (ITS sequences) [
75
], it did not elucidate the relationship between
A. philoxeroides and A. sessilis. The use of complete cp genome sequences to explain their
evolution was advantageous for closely related taxa and offered higher resolution [
26
]. It
is important to note, however, that complete cp genome sequences for Amaranthaceae
species remain limited. Further research is needed to explore the evolution and ancestral
relationships at the subfamily and genus levels within Amaranthaceae.
Subsequently, the divergence time between A. philoxeroides and A. sessilis was estimated
to have occurred approximately 3.5186–8.0242 million years ago, during the transition from
the Miocene to the Quaternary period (Figure 11). This timeframe coincided with the
wide proliferation of C4 plants and a shift in climate toward an ice age. The Alternanthera
genus comprises species with various photosynthetic pathways, including C3, C4, and
C3-C4 intermediate species [
75
], with A. sessilis being classified as a C3 species [
76
]. Our
results indicated that the rapidly evolving genes in these two Alternanthera species were
associated with photoadaptation and environmental adaptation, potentially contributing
to their invasive capabilities.
Genes 2024,15, 544 19 of 22
5. Conclusions
In this study, the complete chloroplast genome of A. sessilis was sequenced, assembled,
and compared with its relative species. This cp genome is 151,935 base pairs long with a
typical quadripartite structure and contains 128 genes, including 8 rRNA-coding genes,
37 tRNA-coding genes,
4 pseudogenes, and 83 protein-coding genes. This chloroplast
genome exhibited high conservation in structure and gene contents with other relative
species; however, some regions showed signification variations and rapidly evolving
genes involved in photosynthesis and environmental adaption were identified. The phy-
logenetic trees indicated that the Alternanthera genus is closely related to the Cyathula
genus, and A. philoxeroides and A. sessilis were estimated to have diverged approximately
3.5186–8.8242 million
years ago. Our findings lay the foundation for understanding the
population variation and environmental adaptability within invasive species among the
Alternanthera genus.
Supplementary Materials: The following supporting information can be downloaded at https://
www.mdpi.com/article/10.3390/genes15050544/s1, Table S1: Relative synonymous codon usage of
cp genomes in A. sessilis and A. philoxeroides; Table S2: Synonymous and non-synonymous substitution
rates and Ka/Ks ratio of 55 protein-coding genes in A. sessilis and A. philoxeroides.
Author Contributions: Conceptualization and methodology, R.M. and D.J.; performing the experi-
ments: Y.W. and Q.C.; software: Y.W., X.Z. and J.H.; validation, X.Z., Q.C. and D.J.; formal analysis,
Y.W., J.Y. and D.J.; writing—original draft preparation: Y.W.; writing—review and editing, D.J. and
R.M.; project administration and funding acquisition, R.M. and D.J. All authors have read and agreed
to the published version of the manuscript.
Funding: This research was supported by the special fund for Science and Technology Innovation
Teams of Shanxi Province (no. 202304051001006); Research Program Sponsored by State Key Lab-
oratory of Sustainable Dryland Agriculture (in preparation), Shanxi Agricultural University (no.
202003-4); Excellent Doctor Introduction Award Program of Shanxi Province (no. SXBYKY2022047);
Fundamental Research Program of Shanxi Province (no. 20210302123386); Research Program Spon-
sored by Ministerial and Provincial Co-Innovation Centre for Endemic Crops Production with
High-quality and Efficiency in Loess Plateau, Taigu 030801, China (no. SBGJXTZX-26); and Cultiva-
tion and Innovation Program for Scientific research, College of Plant Protection, Shanxi Agricultural
University (no. ZBXY23A-10).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The complete chloroplast genome sequence of Alternanthera sessilis is
publicly available online in the NCBI GenBank with the specific accession number PP239384.
Acknowledgments: We thank the assistance of everyone in the Biosafety and Biocontrol Laboratory,
Shanxi Agricultural University, Shanxi, China, for their generous help.
Conflicts of Interest: The authors declare no conflicts of interest.
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