Characterization of the complete chloroplast genome of Wolffia arrhiza and comparative genomic analysis with relative Wolffia species

Abstract

Lemnoideae, commonly referred to as the duckweed, are aquatic plants found worldwide. Wolffia species are known for their extreme reduction in size and complexity, lacking both roots and leaves, and they hold the distinction of being the smallest plants among angiosperms. Interestingly, it belongs to the Araceae family, despite its apparent morphological differences from land plants in the same family. Traditional morphological methods have limitations in classifying these plants, making molecular-level information essential. The chloroplast genome of Wolffia arrhiza is revealed that a total length of 169,602 bp and a total GC content of 35.78%. It follows the typical quadripartite structure, which includes a large single copy (LSC, 92,172 bp) region, a small single copy (SSC, 13,686 bp) region, and a pair of inverted repeat (IR, 31,872 bp each) regions. There are 131 genes characterized, comprising 86 Protein-Coding Genes, 37 Transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. Moreover, 48 simple sequence repeats and 32 long repeat sequences were detected. Comparative analysis between W. arrhiza and six other Lemnoideae species identified 12 hotspots of high nucleotide diversity. In addition, a phylogenetic analysis was performed using 14 species belonging to the Araceae family and one external species as an outgroup. This analysis unveiled W. arrhiza and Wolffia globosa as closely related sister species. Therefore, this research has revealed the complete chloroplast genome data of W. arrhiza, offering a more detailed understanding of its evolutionary position and phylogenetic categorization within the Lemnoideae subfamily.

Full text

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Characterization of the complete
chloroplast genome of Wola
arrhiza and comparative genomic
analysis with relative Wola
species
Halim Park
1, Jin Hwa Park
2 & Yang Jae Kang
1,3*
Lemnoideae, commonly referred to as the duckweed, are aquatic plants found worldwide. Wola
species are known for their extreme reduction in size and complexity, lacking both roots and leaves,
and they hold the distinction of being the smallest plants among angiosperms. Interestingly, it
belongs to the Araceae family, despite its apparent morphological dierences from land plants in the
same family. Traditional morphological methods have limitations in classifying these plants, making
molecular-level information essential. The chloroplast genome of Wola arrhiza is revealed that
a total length of 169,602 bp and a total GC content of 35.78%. It follows the typical quadripartite
structure, which includes a large single copy (LSC, 92,172 bp) region, a small single copy (SSC,
13,686 bp) region, and a pair of inverted repeat (IR, 31,872 bp each) regions. There are 131 genes
characterized, comprising 86 Protein-Coding Genes, 37 Transfer RNA (tRNA) genes, and 8 ribosomal
RNA (rRNA) genes. Moreover, 48 simple sequence repeats and 32 long repeat sequences were
detected. Comparative analysis between W. arrhiza and six other Lemnoideae species identied
12 hotspots of high nucleotide diversity. In addition, a phylogenetic analysis was performed using
14 species belonging to the Araceae family and one external species as an outgroup. This analysis
unveiled W. arrhiza and Wola globosa as closely related sister species. Therefore, this research has
revealed the complete chloroplast genome data of W. arrhiza, oering a more detailed understanding
of its evolutionary position and phylogenetic categorization within the Lemnoideae subfamily.
Lemnoideae, commonly known as duckweed, is a monocotyledonous aquatic plant belonging to the Araceae
family1,2. It exhibits a growth pattern of oating freely or being submerged3,4. It is extensively distributed world-
wide, with a particular prevalence in tropical and subtropical regions1,5–7. It thrives in freshwater ponds, rivers,
and various other aquatic environments. ese were classied into a total of ve genera and 38 species: Spirodela
SCHLEID (containing 2 species), Landoltia LES & D. J. CRAWFORD (comprising 1 species), Lemna L. (with 14
species), Wol ella HEGELM (including 10 species), and Wola SCHLEID (encompassing 11 species). ese
ndings were initially documented by Landolt in 19861,8,9. Of these, the Wola genus is notable for being the
smallest angiosperm plant in the world, measuring merely 1mm in diameter, and it does not possess stems or
roots. Instead, it has spherical fronds10. It features a level upper surface that hovers above the water’s top layer,
with parallelly arranged stomata (Fig.1). Additionally, this plant generates infrequently small owers with only
one stamen and pistil, which emerge from a hole on the upper side of the frond. However, the main method of
propagation is vegetative reproduction11,12. is is the way in which a new leaf bud emerges from the reproductive
pouch of the parent leaf, undergoing gradual growth and eventual separation13. Consequently, Wola has the
capacity to rapidly double its population in as little as 2–3 days14,15 in the optimal conditions such as temperatures
within the range of 20–30°C and a pH level spanning from 5.0 to 7.016,17. is exceptional ability establishes
Wola as one of the fastest-growing plants globally.
Because of its rapid growth, high reproductive rate, and straightforward cultivation and harvesting, it was
regarded as an ideal candidate for an experimental model plant in numerous elds. Wola arrhiza, specically,
OPEN
1Division of Bio and Medical Bigdata Department (BK4 Program), Gyeongsang National University, Jinju 52828,
Republic of Korea. 2DEEVO Inc., Jinju 52828, Republic of Korea. 3Division of Life Science Department at
Gyeongsang National University, Jinju, Republic of Korea. *email: kangyangjae@gnu.ac.kr
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has the capacity to absorb and cleanse excessive nitrogen and phosphorus that may result in eutrophication in
aquatic environments18. Furthermore, its nutritional potential is noteworthy, as 40% of its dry weight is con-
stituted by protein, and it contains notable levels of amino acids, calcium, magnesium, and vitamins that hold
signicance in the human diet19. ese characteristics have captured interest, leading to research into the appli-
cation of W. arrhiza in elds such as wastewater management20, components of human food21 or animal feed22.
e morphological classication of these duckweed species is exceptionally challenging due to their sig-
nicantly diminished size and simplied intricacy. Accordingly, molecular taxonomy has been vital for species
classication, and to establish the phylogenetic relationships within the Lemnoideae subfamily, the chloroplast
barcode has been adopted and utilized23–25. In contrast to the nuclear genome, the chloroplast genome presents
advantages in species classication owing to its smaller genome size, haploid inheritance, conserved structure,
and slower mutation rate26,27. is enabled to the classication of Lemnoideae within the Araceae family, along-
side land plants that have considerable morphological dissimilarities28. Moreover, it has been indicated that
Landoltia, previously grouped within the Spirodela genus, is now a novel and separate genus, distinct from both
Spirodela and Lemna29. us, in order to establish a robust basis for gaining insight into the genetic variation
and its placement in the phylogenetic tree of Lemnoideae subfamily, it is imperative to gather more chloroplast
information from a diverse of duckweed species9,30.
In this study, the full chloroplast genome of W. arrhiza was assembled from whole genome sequencing data
from MGI DNBSEQ-G50 second-generation sequencing platform. e overview for conducting research is
as follows: (i) characterization of the complete chloroplast genome of W. arrhiza; (ii) a comparative analysis
of chloroplast genome data using six species available from NCBI; (iii) Analyzing evolution progression and
phylogenetic relationship. e goal is to improve the clarify of relationships within Lemnoideae and establish a
fundamental basis for a coherent classication system.
Results
Chloroplast genome characteristics of W. arrhiza
e chloroplast genome of W. arrhiza is 169,602bp in the quadripartite structure with one large single copy (LSC)
region of 92,172bp, one small single copy (SSC) region of 13,686bp, and a pair of inverted repeat (IR) regions of
31,872bp each (Fig.2). e total guanine and cytosine (GC) content of the chloroplast genome is 35.78%. e
relative occupation ratio of the LSC, SSC, and IR regions in the chloroplast genome were 33.63%, 30.79%, and
39.97%, respectively. It contains a total of 131 predicted genes, which are divided into three groups: 86 protein-
coding genes (PCGs), 37 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. e entire set of
genes exhibited a GC content of 38.52%. Within this, PCGs demonstrated a GC content of 37.08%, tRNA genes
had a GC content of 52.50%, and rRNA genes exhibited a GC content of 54.69%. e LSC region contained a
total of 83 genes, comprising 61 PCGs and 22 tRNA genes. In the SSC region, there were 11 genes, including 10
PCGs and one tRNA gene. e IR regions consisted of 36 genes, with seven PCGs, seven tRNA genes, and four
rRNA genes duplicated (Table1). Additionally, the rps12 gene is a trans-spliced gene that exons found in both
the LSC and IRs, while the rps19 gene extended across two regions between the LSC and IRb.
According to the chloroplast genome annotation of W. arrhiza, 112 unique genes were categorized into four
functional groups. ere were 59 transcription and translation-related genes, 46 photosynthesis-related genes,
ve biosynthesis-related genes, and two genes whose functions were unidentied (Table2).
Concurrently, a total of 17 unique intron genes were detected, and they were distributed across the LSC (11),
IR (5), and SSC (1, ndhA) regions. It was comprised 11 PCGs and 6 tRNA genes. Among these, 15 genes (atpF,
ndhA, ndhB, petB, rpl16, rpl2, rpoC1, rps12, rps16, trnA-UGC , trnG-UCC , trnI-GAU , trnK-UUU , trnL-UAA , trnV-
UAC ) had a single intron each, while the remaining 2 genes (clpP1, pafI) contained two introns each (Table3).
Figure1. Microscopic photos of Wola arrhiza taken at (A) 40×, (B) 100×, and (C) 400× magnication. e
majority is composed of chloroplast, and stomata can be seen in the lower part of the specimen. e scale bar in
the lower right of each gure is 100μm for each magnication.
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Figure2. e gene map of the chloroplast genome of Wola arrhiza. e map identies three distinct regions:
the large single copy region (LSC), the small single copy region (SSC), and the inverted repeat A/B regions
(IRA/B). Additionally, the innermost dark gray track represents the GC contents. Genes on the inner side of the
map are transcribed counterclockwise, while those on the outer side are transcribed clockwise.
Table 1. Chloroplast genome structure and feature of Wola arrhiza.
Genome feature Length (bp)/Numbers GC content (%)
Structure length
Tot al 169,602 35.78
LSC region 92,172 33.63
SSC region 13,686 30.79
IR (a/b) region 31,872 39.97
Counts of genes in dierent categories
Genes 131 38.52
PCGs 86 37.08
tRNA 37 52.50
rRNA 8 54.69
Detailed counts of genes in dierent regions
LSC region 61 PCGs, 22 tRNA –
SSC region 10 PCGs, 1 tRNA –
IR (a/b) region 7 PCGs, 7 tRNA, 4 rRNA –
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Repeat sequences analysis
e web application Misa successfully identied a total of 48 Simple Sequence Repeats, SSRs, with lengths
ranging from 10 to 16bp. ere was a total of 42 mononucleotide and 6 dinucleotide repeat types observed,
all composed of A or T bases. ere were 26 mononucleotides consisting solely of A and 16 mononucleotides
consisting solely of T. Likewise, four dinucleotides comprised of AT repeats and two dinucleotides comprised of
TA repeats. Among the identied SSRs, the LSC region contains the highest number, accounting for the majority
(72.92%) with a total of 35 SSRs. e SSC region hosts seven SSRs (14.58%), while the IR region holds six SSRs
(12.5%). At the same time, the Intergenic spacer (IGS) region presents the largest number of SSRs, totaling 40
(83.3%) of the total SSRs. Five SSRs (10.42%) were found in introns, and the remaining three SSRs (6.25%) were
in PCG regions. Notably, each of the introns within the petB, rps16, trnK-UUU , pafI, and clpP1 genes contained
Table 2. Genetic classication of the chloroplast genome of Wola arrhiza. Duplicated genes are denoted by
an asterisk (*).
Category (number) Group (number) Gene name
Transcription and translation (59)
Ribosomal RNAs (4) rrn16*, rrn23*, rrn4.5*, rrn5*
Transfer RNAs (30)
trnA-UGC *, trnC-GCA , trnD-GUC , trnE-UUC , trnF-GAA , trnG-GCC , trnG-UCC , trnH-
GUG , trnI-CAU *, trnI-GAU *, trnK-UUU , trnL-CAA *, trnL-UAA , trnL-UAG , trnM-CAU
, trnN-GUU *, trnP-UGG , trnQ-UUG , trnR-ACG *, trnR-UCU , trnS-GCU , trnS-GGA , trnS-
UGA , trnT-GGU , trnT-UGU , trnV-GAC *, trnV-UAC , trnW-CCA , trnY-GUA , trnfM-CAU
Small subunit of ribosome (SSU) (12) rps2, rps3, rps4, rps7*, rps8, rps11, rps12*, rps14, rps15*, rps16, rps18, rps19
Large subunit of ribosome (LSU) (9) rpl2*, rpl14, rpl16, rpl20, rpl22, rpl23*, rpl32, rpl33, rpl36
DNA-dependent RNA polymerase (4) rpoA, rpoB, rpoC1, rpoC2
Photosynthesis (46)
Photosystem I (7) psaA, psaB, psaC, psaI, psaJ, pafI, pafII
Photosystem II (15) psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbT, psbZ, pbf1
Subunit of cytochrome (6) petA, petB, petD, petG, petL, petN
ATP synthase (6) atpA, atpB, atpE, atpF, atpH, atpI
RubisCO (1) rbcL
NADH dehydrogenase (11) ndhA, ndhB*, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
Biosynthesis (5)
Maturase (1) matK
ATP-dependent Protease (1) clpP1
Acetyl-CoA-carboxylase (1) accD
Envelop membrane protein (1) cemA
C-Type cytochrome synthesis (1) ccsA
Unknown (2) Hypothetical chloroplast reading frames(ycf) (2) ycf1*, ycf2*
Table 3. Introns and exons length information of the Wola arrhiza. Duplicated genes are denoted by an
asterisk (*). e duplicated 3’ ends of the rps12 gene, which is trans-spliced, are found in the IR regions with
the 5’ end of the gene located in the LSC region.
Gene Location Exon I (bp) Intron I (bp) Exon II (bp) Intron II (bp) Exon III (bp)
atpF LSC 144 839 402
clpP1* LSC 71 811 295 656 243
ndhA SSC 551 916 532
ndhB* IR 777 703 756
pafI* LSC 126 750 226 789 155
petB LSC 6 752 642
rpl16 LSC 9 1575 399
rpl2* IR 391 660 431
rpoC1 LSC 453 744 1620
rps12* LSC, IR 114 540 232 – 26
rps16 LSC 40 1025 200
trnA-UGC * IR 37 801 36
trnG-UCC LSC 24 619 48
trnI-GAU * IR 42 804 35
trnK-UUU LSC 36 2545 43
trnL-UAA LSC 37 507 50
trnV-UAC LSC 38 615 37
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one SSR. Moreover, SSRs within PCG were observed in one instance within the rpoB gene and one each in the
ycf1 genes of IRa and IRb (Table4).
A total of 32 long repeat sequences were detected using the REPuter web application. Among these repeats,
there were 16 forward repeats (F), 1 reverse repeat (R), and 15 palindromic repeats (P). e lengths exhibited a
distribution ranging from 30 to 69bp, and among them, a unique palindromic repeat measuring 31,872bp in
length was identied. Out of these, 13 were exclusively located within the LSC region (40.63%), while 6 were
uniquely situated in the IR region (18.75%). Additionally, 7 were suspended across both IRa and IRb (21.87%),
with the remaining 6 spanning across the LSC and IR (18.75%), covering two structural regions. Furthermore,
there were a total of 12 repeats solely present within a single PCG (37.5%), and all these repeats were located in
the ycf2 gene. ere was also one repeat that distributed across both the intron and the PCG (3.12%), and it was
the longest repeat in terms of length. is was present across a total of 20 genes. One repeat was identied in
both the IGS and the PCG (3.12%), and PCG corresponded to the pbf1 gene. ere were three repeats spanning
two PCGs (9.38%), and all these PCGs were identied as tRNA genes. In addition, six repeats were observed,
spanning across introns and the IGS (18.75%), with four of them positioned in introns within the pafI gene, and
the other two in the petB gene. e remaining nine repeats, containing the only reverse repeat that was detected,
were exclusively located in the IGS (28.13%) (Table5).
Codon usage
A total of 86 PCGs and their CDS were extracted from the chloroplast genome of W. arrhiza. ese sequences
have a combined length of 84,507bp and consist of 28,169 codons. Leucine (Leu) was the most commonly
encoded amino acid, comprising 10.50% of the total with 2959 codons. Conversely, Cysteine (Cys) was the
least frequently encoded amino acid, making up only 1.10% of the total with 310 codons. e RSCU values for
each codon fell within the range of 0.3 (CGG, Arg) to 2.01 (AGA, Arg). Out of a total of 30 codons with a high
frequency of usage (RSCU > 1), except for UUG (Leu), 29 of these preferred synonymous codons ended with A
or U(T) nucleotides. For the 32 codons with RSCU < 1, the 29 codons ended with C or G nucleotide, excluding
CUA(Leu), AUA(Ile), and UGA(TER). Additionally, the terminator most preferred was UAA, showing an RSCU
value of 1.60. In contrast, the codons AUG (Met) and UGG (Trp) demonstrated an RSCU value of 1, suggesting
there is no bias as they each encode only one amino acid (TableS1).
Comparison of chloroplast genomes within Lemnoideae
e lengths of genes and IGS regions were compared among the chloroplast genomes of seven species of duck-
weed within the Lemnoideae subfamily (Fig.3). e gene regions exhibited a range in length from 109,650bp to
Table 4. e types of SSRs in Wola arrhiza and their corresponding regions and locations.
Repeat type Repeat unit Number of repeats Repeat length Number of SSRs Region Location
Mono-nucleotide
A
10 10 18 LSC(15)
IGS(12)
Intron(3, petB, rps16,
trnK-UUU )
SSC(3) IGS(3)
11 11 5
LSC(2) IGS(2)
SSC(1) IGS(1)
IRb(2) IGS(1)
PCG(1, ycf1)
13 13 3 LSC(2) IGS(2)
IRb(1) IGS(1)
T
10 10 9 LSC(7)
IGS(5)
Intron(1, pafI)
PCG(1, rpoB)
SSC(2) IGS(2)
11 11 5
LSC(3) IGS(2)
Intron(1, clpP1)
IRa(2) IGS(1)
PCG(1, ycf1)
12 12 1 LSC(1) IGS(1)
13 13 1 IRa(1) IGS(1)
Di-nucleotide
AT 6 12 3 LSC(3) IGS(3)
7 14 1 LSC(1) IGS(1)
TA 6 12 1 SSC(1) IGS(1)
8 16 1 LSC(1) IGS(1)
Tot al 48 LSC(35, 72.92%)
SSC(7, 14.58%)
IR(6, 12.5%)
IGS(40, 83.3%)
Intron(5, 10.42%)
PCG(3, 6.25%)
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114,821bp. Among the seven Lemnoideae species, W. arrhiza possessed the longest gene region with 114,821bp
(Fig.3A). On the other hand, IGS regions had lengths that ranged from 51,306bp to 59,471bp, with W. arrhiza
possessing the second shortest IGS region at 54,781bp, following Lemna minor (Fig.3B). e gene regions
were further categorized into coding sequences (CDS) and intron regions. It was observed that CDS regions
had a length range from 94,398bp to 96,193bp. Notably, W. arrhiza possessed the second-longest CDS region,
measuring 96,189bp, which was 4bp shorter than W. globosa (Fig.3C). Conversely, the lengths of intron regions
ranged from 16,173bp to 20,159bp, with W. arrhiza having the longest intron region at 20,159bp (Fig.3D).
To gain further insight into these changes, an analysis was conducted on events such as insertions, deletions,
duplications, and intron changes in genes across Lemnoideae species (TableS2). Compared to other Lemnoideae
species, W. arrhiza was represented by the genes pafI, pafII, clpP1, and pbfI, which are synonymous with the ycf3,
ycf4, clpP, and psbN genes in other species. When comparing other genes, the most signicant alterations were
the deletion events of pseudogenes ycf68 and ycf15 in the IR region (Fig.4). Upon a more detailed examination,
it was observed that the gene ycf68, which had perfect overlap with trnI-GAU in other species, was deleted in
W. arrhiza, leaving only trnI-GAU . However, another deleted gene, ycf15, which has been alone in other species,
underwent deletion in W. arrhiza and sequences remained at an IGS region. e length between ycf2 and trnL-
CAA anking ycf15 in W. australiana, Wolella lingulata, Lemna minor, and Spirodela polyrhiza were 1005bp,
1019bp, 1027bp, and 1027bp, respectively. In W. arrhiza, W. globosa, and W. brasiliensis, where ycf15 was deleted,
the IGS length between ycf2 and trnL-CAA was 988bp, 993bp, and 1023bp, respectively.
To identify variations in introns, the gap between the longest and shortest length values for each species
within the same gene was calculated (TableS2). As a result, it was determined that the lengths of the petB and
rpl16 genes in W. arrhiza are 1400bp and 1983bp, respectively. ese lengths exceed twice the sizes observed in
other species, where they typically range from 642 to 701bp for petB and 411bp (the exception of Lemna minor,
which has a length of 1714bp) for rpl16. When analyzing the exons and introns of these genes in each species,
the exons exhibit consistent lengths across all species (ranging from 642 to 654bp for petB and 408–411bp for
Table 5. e types of long repeat in Wola arrhiza and their corresponding regions and locations. In the case
of diering regions or locations between the repeats, they are connected using ’-’ and indicated as a repeat set.
e genes follow the same pattern. F for forward repeat, R for reverse repeat, and P for palindromic repeat.
Repeats match type Repeats length range Repeats length Number of repeats set Region of repeats set Location of repeats set
F
30–39
30 4 LSC(3) IGS(2)
PCG(1, trnS(GCU )-trnS(UGA ))
LSC-IRb(1) Intron-IGS(1, pafI)
31 3
LSC(1) IGS(1)
IRa(1) PCG(1, ycf2)
IRb(1) PCG(1, ycf2)
32 1 LSC(1) IGS(1)
34 1 LSC(1) IGS(1)
36 1 LSC-IRa(1) Intron-IGS(1, petB)
37 2 IRa(1) PCG(1, ycf2)
IRb(1) PCG(1, ycf2)
39 1 LSC-IRb(1) Intron-IGS(1, pafI)
40–49 41 2 IRa(1) PCG(1, ycf2)
IRb(1) PCG(1, ycf2)
60–69 63 1 LSC(1) IGS(1)
P
30–39
30 2 LSC(1) IGS(1)
LSC-IRa(1) Intron-IGS(1, pafI)
31 4 LSC(2) IGS(1)
IGS-PCG(1, pbf1)
IRa-IRb(2) PCG(2, ycf2)
32 2 LSC(2) PCG(2,
trnS(GCU )-trnS(GGA ) /
trnS(UGA )-trnS(GGA ))
36 1 LSC-IRb(1) Intron-IGS(1, petB)
37 2 IRa-IRb(2) PCG(2, ycf2)
39 1 LSC-IRa(1) Intron- IGS(1, pafI)
40–49 41 2 IRa-IRb(2) PCG(2, ycf2)
70~ 31,872 1 IRa-IRb(1)
Intron-PCG(1,
rps7-ycf2-ndhB-ycf1-trnA(UGC )-rpl2-rpl23-trnI(CAU )-rrn5-
rrn23-rrn16-trnN(GUU )-trnV(GAC )-trnR(ACG )-rps12-
rrn4.5-rps15-trnI(GAU )-trnL(CAA )- trnM(CAU )
R 30–39 32 1 LSC(1) IGS(1)
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rpl16). However, in W. arrhiza, the introns are notably longer, with the petB gene containing a 752bp intron and
the rpl16 gene containing a 1575bp intron (TableS3).
Sequence homology among the chloroplast genomes of seven species within the Lemnoideae subfamily was
assessed and visualized via the shue-LAGAN mode in mVista. e annotation data relied upon the reference
strain, W. australiana (strain 8730). As a result, it was established that the chloroplast genome sequence of
duckweed maintains a high degree of sequence conservation, with very few regions exhibiting sequence identity
below 90% (Fig.5). In detail, the IR region showed a higher level of preservation when contrasted with the LSC
and SSC regions. In addition, the mutation rate was greater in the IGS region in contrast to the PCG region.
e majority of PCGs were generally well-preserved, but signicant variations were observed in some PCGs,
including matK, rpoC2, ndhF, cssA, ndhD, and ndhH. In contrast to the PCG regions, the non-coding regions
showed a relatively higher mutation rate in numerous locations. Within non-coding regions, intergenic regions
displayed the highest variability rate. Upon visual examination of the gure, the most notable segments appeared
to be trnC(GCA )-petN, petN-psbM, and trnE(UUC )-trnT(GGU ).
To clearly identify the variable regions within the mVista results, a sliding window analysis was executed using
DnaSP v.6.10 soware, followed by the calculation of nucleotide diversity values (π, Pi). ere were 769 nucleotide
diversity point observed, with values ranging from 0.00000 to 0.21294, and an average value of 0.04589 (Fig.6).
e nucleotide diversity value was highest (0.21294) in the LSC region, while the IR region had the lowest value
(0.00048), excluding zero. In this regard, the IR region exhibited signicantly lower variability compared to the
LSC and SSC regions. Among them, 12 locations demonstrated high Pi values greater than 0.15. Eight of them
were found in the LSC region, while four were located in the SSC region. Within the LSC region, 5 locations
were detected in intergenic regions including rps16-trnQ(UUG ), trnS(GCU )-trnG(UCC ), atpH-atpI, petA-psbJ,
psbE-petL, while 3 locations were found in the coding regions of trnC(GCA ), trnT(GGU ), and trnT(UGU ). In
the SSC region, one of the four positions was located in the intergenic region of ndhF-rpl32, whereas the other
three were found in the coding regions of ndhF, rpl32, and ndhE. e coding region and non-coding region
with the highest nucleotide diversity values were trnT(GGU ) (0.18841) and trnS(GCU )-trnG(UCC ) (0.21294),
respectively, located in the LSC.
Figure3. Length for each region of the Lemnoideae, including Wola arrhiza. (A) Gene region length (B) IGS
region length (C) CDS region length (D) Intron region length. e X-axis represents the species names, while
the Y-axis depicts the lengths of the regions. e arrangement of each graph is based on the ascending order of
region lengths.
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To delve deeper into the nucleotide diversity results, SNPs and InDels were analyzed in seven Lemnoideae spe-
cies, utilizing Wola australiana as a reference. is revealed 17,269 SNPs and 2030 InDels. e majority of SNPs
appeared in the IGS regions (51.91%), followed by exon regions (35.20%) and intron regions (12.89%) (TableS6).
ese ndings aligned with earlier results (Fig.6), particularly noting that the IGS in trnS(GCU )—trnG(UCC )
represented 5.30% of the total IGS region, and the IGS in ndhF—rpl32 comprised 4.03% of the total IGS region.
Additionally, InDels were predominantly distributed in IGS regions (76.35%), with lesser occurrences in intron
(17.10%) and exon regions (6.55%) (TableS7). Most were short InDels of 10 base pairs or fewer, accounting for
80.59% of the total, and one long InDel of 1000 bp was detected. Similarly with SNPs aspect, the IGS regions in
trnS(GCU )—trnG(UCC ), and ndhF—rpl32, constituted 4.54% and 1.86% of the total IGS region, respectively.
e gene distribution at the boundaries of the LSC/SSC and IR regions in the chloroplast genomes of the
seven species was compared using IRscope. Overall, the distribution of genes at each boundary region appeared
to be similar, with rpl22, rps19, rpl2, rps15, ndhF, ndhH, trnH, and psbA. However, it was observed that the rpl2
gene is found solely in the IRb region and is absent in the IRa region of W. australiana and Wolella lingulata
(Fig.7, TableS5). Although not shown in the gure due to their location at the boundaries and greater distance,
other genes did not undergo any loss. Nevertheless, variations were noted in the association between genes and
the boundary lines. e JLB (LSC/IRB) boundary displayed three dierent congurations: positioned within
the rps19 gene, within the rpl2 gene, or within IGS between the rps19 and rpl2 genes. For W. arrhiza, the JLB
boundary can be found within the rps19 gene. e rps19 gene spans 240bp in the LSC region, and the remaining
39bp extend into the IRB region. Similar cases are apparent in the W. australiana, W. brasiliensis, and Wol ell a
lingulata. ese species have respectively occupied 277bp, 249bp, and 250bp within the LSC region, along with
extensions of 2bp, 30bp, and 29bp into the IRB. In the case of Lemna minor, it was observed that the bound-
ary of the JLB was located within the rpl2 gene. e rpl2 gene spanned 1100bp within the IRB region, with the
Figure4. Comparative analysis of alterations resulting from the ycf68, and ycf15 deletion in Wola
arrhiza. is represents one of the IR regions, and the other IR region exhibits a same aspect with reverse
complementarity. e squares represent genes, where those transcribed on the forward strand are positioned
at the top of the line, and those transcribed on the reverse strand are located at the bottom of the line. e
numbers adjacent to the squares represent the lengths of individual genes, while the numbers above the lines
are the lengths of IGS between each gene. ycf2 is depicted by the color red, ycf15 by orange, trnL-CAA by yellow,
trnI-GAU by green, and ycf68 by blue.
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Figure5. Analyzed the chloroplast genome sequences of seven Lemnoideae species, including Wola arrhiza,
using mVista, with Wola australiana as the reference. e X-axis represents the coordinates of the chloroplast
genome sequence position, while the Y-axis indicates the range of sequence identity from 50 to 100%. e
direction and position of the genes are depicted by the gray arrows on the graph. e graph’s shaded colors have
the following meanings: the dark blue regions correspond to protein coding sequences (CDS), the pink regions
represent Conserved Non-Coding Sequences (CNS), and the light-blue regions indicate UTRs.
Figure6. Nucleotide diversity of chloroplast genome sequences in Lemnoideae, including Wola arrhiza. e
X-axis represents the alignment sequence’s position, while the Y-axis indicates the values for nucleotide diversity.
e use of a hyphen to connect two genes signies a non-coding region, whereas the representation of a single
gene indicates a coding region.
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remaining 386bp extending towards the LSC region. W. globosa and Spirodela polyrhiza were both found to
have the JLB boundary positioned within the IGS between the rps19 and rpl2 genes. Additionally, the rps19 and
rpl2 genes were identied within the LSC and IRB regions, respectively. In the instance of the JSA (SSC/IRA)
boundary, it presented in two dierent cases, with one found within the ndhH gene and the other within the IGS
region lying between the ndhH and rps15 genes. For W. arrhiza, the ndhH and rps15 genes were contained within
their respective SSC and IRA regions, rather than extending beyond them. is same pattern was also observed
in W. globosa, W. australiana, W. brasiliensis, and Spirodela polyrhiza. Nevertheless, for Wolella lingulata and
Lemna minor, the boundary of the JSA was positioned within the ndhH gene, with each extension 1183bp and
1144bp into the SSC region, while the remaining 5bp and 44bp entered the IRA region.
Phylogenetic analysis
To explore the phylogenetic relationship of W. arrhiza, a phylogenetic tree was created that included a total of
15 species. is species set comprised 7 species from the Lemnoideae subfamily, which includes W. arrhiza, and
7 species within the Araceae family to which Lemnoideae subfamily belongs, along with one outgroup species,
Zea mays. Excluding genes in the IR, there are 44 PCGs shared between them (TableS4). In addition, based on
the results from Prottest, the CpREV + G + I model was determined to be the best t model for explaining protein
evolution across the 15 species. e Bayesian analysis was performed using BEAST v1.10.4 soware, employ-
ing 50,000,000 Markov Chain Monte Carlo (MCMC) chains with the previously identied shared PCG and the
most suitable model. Consequently, the phylogenetic tree was constructed, with Bayesian posterior probability
values that ranged from 0.5643 to 1 (Fig.8). e tree can be divided into three parts: W. arrhiza—Spirodela pol-
yrhiza, Colocasia esculenta—Symplocarpus renifolius, and the outgroup. W. arrhiza is classied as part of the W.
arrhiza—Spirodela polyrhiza section, belonging to the Lemnoideae subfamily. It exhibits the nearest evolutionary
relationship with W. globosa.
Discussion
e complete chloroplast genome of W. arrhiza was assembled using the short reads from MGI DNBSEQ-G50
next-generation sequencing platform, revealing a common circular quadripartite genomic structure. Despite W.
arrhiza is an aquatic plant, it exhibited a similar pattern to land plants, having a length of 169,602bp falling within
the range of 130–170 kbp and a GC content of around 35.78%, which is in proximity to the average of 36.3% in
land plants31. Additionally, the analysis of SSRs indicated that in W. arrhiza, SSRs are composed exclusively of A
or T nucleotides, predominantly distributed in the LSC region out of the four regions (LSC, SSC, IRs) and in the
IGS among the three locations (IGS, PCG, intron) (Table4). A strong A/T bias and high concentration within
Figure7. Comparing the boundaries of chloroplast genome regions in seven species, including Wola arrhiza,
from the Lemnoideae subfamily: LSC, IRs, and SSC. e junctions between each pair of genomic regions are
indicated as JLB (LSC/IRB), JSB (SSC/IRB), JSA (SSC/IRA), and JLA (LSC/IRA). Genes transcribed on the
forward strand are depicted above the line, whereas genes transcribed on the reverse strand are exhibited below
the line. Furthermore, the numbers above the genes signify the gap between the gene’s start or end and the
region’s boundary.
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the LSC region and IGS were similar to patterns observed in other angiosperms chloroplast genome32–34. e
analysis of codon usage revealed that all but one of the codons with RSCU values exceeding 1 ended in either A
or T (TableS1). is corresponds with research results indicating a preference for A/T-ending codons among
synonymous codons, as it oers advantages in codon optimization during gene expression compared to codons
ending in C/G35. Codon usage patterns are fundamentally dependent on the gene sequences, thereby mirror-
ing nucleotide substitutions that lead to variations of gene, protein structure, and function. Consequently, by
understanding codon usage, it was possible to gain a foundational framework for insights into the history of W.
arrhiza in the context of genome evolution36.
e analysis of genome information for the Lemnoideae subfamily, including W. arrhiza, was conducted to
identify the various changes that occurred during evolution. First, diversity within the Lemnoideae subfamily,
including W. arrhiza, was analyzed at the nucleotide level, and a total of 12 hotspots were detected. ere were
several peaks in the data with signicantly high Pi values, such as trnS(GCU )-trnG(GCC ) (0.21294)37, rps16-
trnQ(UUG ) (0.18500)38, and ndhF-rpl32 (0.19456)39, indicating a substantial nucleotide variability in other
angiosperm plants. One can surmise that these genes are inclined to undergo rapid nucleotide substitutions,
suggesting at their applicability as molecular markers for phylogenetic analysis and species identication. Sub-
sequently, within the Lemnoideae, it was conrmed that W. arrhiza had the longest chloroplast genome length
among its fellow species (TableS5). Various elements inuencing chloroplast length have been reported as the
contraction and expansion of the IR region, gene insertions, deletions, duplications, inversions, and alterations
in introns40–43. In the case of W. arrhiza, the JLB and JSA boundaries were positioned similarly to most other
Lemnoideae species. ere was no noticeable expansion or contraction in the IR region, which was 31,872bp in
size. is falls within the average IR range of 31,223 to 31,930bp observed in other species (Fig.7 and TableS5).
To explore whether there were additional alterations, such as gene rearrangements or inversions, a synteny
analysis of the Lemnoideae chloroplast genome was performed utilizing the MAUVE program44. e seven
Lemnoideae species visually demonstrated a highly similar gene position pattern, suggesting a signicant level
of collinearity (Fig.S1). e events such as the deletion of ycf68 and ycf15, resulted in changes to the types and
number of genes (Fig.4). However, they could not be considered to have had a signicant impact on the length
of the whole chloroplast genome. On the other hand, when comparing the lengths of genes, CDS, introns, and
intergenic regions, there was a noticeable increase in the total intron length (Fig.3). is suggests that intron
evolution may have occurred in the W. arrhiza chloroplast genome. As a result, signicant alterations were
identied in the intron analysis of petB and rpl16 showing from 642–701bp to 1400bp, and from 411 to 1983bp,
respectively (TablesS2, S3). While the exact reasons for the insertion of these introns require additional explo-
ration, it implied the signicance of both the petB and rpl16 genes. From this, it can be inferred that changes
Figure8. Bayesian phylogenetic tree of Araceae species based on the chloroplast genome data. e colors of
the branches in the tree represent the Bayesian posterior probability, as indicated by the color bar. e numerical
values displayed on the branches represent precise posterior values. At the bottom of each species name is the
genbank accession number.
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in introns impacted the chloroplast genome length of W. arrhiza, resulting in it possessing the longest genome
length among Lemnoideae species. Additionally, the alterations in introns were commonly detected in various
angiosperms, indicating their potential utility as molecular markers for conducting phylogenetic studies and
identifying dierent species45–49.
e chloroplast, which contains all of this information, was used to explore the phylogenetic history of the
species. e phylogenetic tree was partitioned into three sections: W. arrhiza—Spirodela polyrhiza, Colocasia
esculenta—Symplocarpus renifolius, and the outgroup (Fig.8, TableS4). In the W. arrhiza—Spirodela polyrhiza
section, the majority of posterior values were 1.0, providing a strong representation of the Lemnoideae subfamily
lineage. Specically, the relationship between W. arrhiza and W. globosa is armed with a posterior value of 1,
signifying that they are sister species. W. brasiliensis showed unclear taxonomic results that did not align with
the same genus, Wola, as observed in previous research8,9,23. Nevertheless, through conducting a phylogenetic
classication of the Lemnoideae subfamily, which incorporated W. arrhiza, valuable insights into the evolution-
ary dynamics aecting populations and species were gained.
Conclusion
In this study, an analysis of the complete chloroplast genome of W. arrhiza is provided. e chloroplast genome
of W. arrhiza exhibits resemblances in terms of size, structure, gene composition, GC content, and codon prefer-
ences when compared to the typical characteristics observed in land plants and angiosperms. e comparison
of Lemnoideae species with W. arrhiza also demonstrates a signicant level of conservation in the chloroplast
genome. It also oers details about genes containing nucleotide or intron variations that can be used as molecular
markers in the species classication and evolutionary research. e phylogenetic analysis veried that W. arrhiza
is closely related as a sister species to W. globosa. In summary, the characterization of chloroplast genomic data
for W. arrhiza has provided insights and enriched understanding of the phylogeny of the challenging-to-classify
Lemnoideae subfamily using traditional methods.
Materials and methods
Plant materials, DNA extraction, and sequencing
e samples of W. arrhiza were obtained from the Rutgers Duckweed Stock Cooperative (RDSC) located at
Rutgers University in New Jersey (http:// www. ruduc kweed. org/, ruduckweed@gmail.com). e plant collection
and use were in accordance with all the relevant guidelines. Among several strains of W. arrhiza, voucher number
of 7193 was used, which was collected in Masaka, Uganda, located at 0°19′36.3″S 31°45′13.5″E. Total genomic
DNA was isolated from the whole plant by the modied cetyltrimethylammonium bromide (CTAB) method
and quantied by Nanodrop spectrophotometer (ermo Fisher Scientic, ND-1000) and Qubit Fluorometer
(Invitrogen, ermo Fisher Scientic, Qubit 4). e paired-end libraries were constructed using the MGI Eazy
FS DNA Library Prep Kit, with an insert length of 350bp. Sequencing was performed using reads of 150bp on
the MGI DNBSEQ-G50 second-generation sequencing platform, resulting in a total of approximately 37.82GB
of raw reads being generated.
Chloroplast genome assembly and annotations
GetOrganelle v1.7.5.350 was utilized to assemble the chloroplast genome using following command (get_orga-
nelle_from_reads.py -1 le.fq -2 right.fq -k 21,45,65,85,105 -t 3 -o result_folder -F embplant_pt), and annotation
was done with GeSeq51. e circular maps for newly sequenced plastomes were generated using the OGDRAW
v1.3.152.
Repeat sequences analysis
e web application MISA53 was utilized to detect microsatellites, known as simple sequence repeats (SSRs). e
minimum number of repetitions were set as follows: 10 repeat units for mononucleotide SSRs, 6 repeat units
for dinucleotide SSRs, and 5 repeat units for tri-, tetra-, penta-, and hexanucleotide SSRs. Furthermore, the web
application REPuter54 was used to identify long repeats. is process encompassed detecting forward, reverse,
complement, and palindromic repeats with a minimum repeat size set at 30bp and a Hamming distance of three.
Codon usage
e chloroplast genome’s Protein-Coding Genes (PCGs) and their protein coding sequences (CDS) were
extracted from the genbank le using Python’s SeqIO object. Following that, the Relative Synonymous Codon
Usage (RSCU) was calculated using codonW v1.4.455. Synonymous codons refer to codons encoding the same
amino acid, and RSCU assesses the relative frequency of such synonymous codons. An RSCU value greater than
1.00 signies a relatively higher frequency of codon usage, while values less than 1.00 indicate the opposite.
Chloroplast genome comparison
e tool mVISTA56, with the Shue-LAGAN mode, was used to identify variations of whole chloroplast
genome sequences using Wola australiana (MN912638.1, strain 8730) as the reference. It visually represents
the similarities and dierences among seven species, namely Wola arrhiza (OQ079152.1), Wola globosa
(MN881100.1), Wola australiana (NC_015899.1, strain 7733), Wola brasiliensis (MN850406.1), Wol ell a
lingulata (NC_015894.1), Lemna minor (DQ400350.1), and Spirodela polyrhiza (NC_015891.1). is entailed
utilizing reference information as annotation notes to gain a deeper comprehension of the observed patterns.
Additionally, the genome sequences of seven chloroplasts were aligned using the multiple sequence alignment
program, MAFFT v7.52057. In this alignment, nucleotide diversity (π, Pi) was calculated using DnaSP (DNA
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Sequences Polymorphism) v6.12.0358, employing a window length of 600bp and a step size of 200bp. It was also
utilized for the calling of SNPs and InDels. e visualization and comparison of the genes located at the junctions
of chloroplast genomes in the seven species were carried out using the web application IRscope59.
Phylogenetic analysis
e phylogenetic analysis encompassed a total of 14 species from the Araceae family, which included W. arrhiza.
To facilitate the comparative analysis, Zea mays was chosen as the outgroup. e chloroplast genome information
was acquired from GenBank, and Python’s SeqIO object was employed for parsing and extracting the protein-
coding genes and their corresponding protein sequences, which were shared among 15 species. Aerward, the
alignment process was conducted using PRANK60, and to select the most suitable amino acid substitution model
for the alignment data, ProtTest61 was employed. e Beast v1.10.4 soware62 performed Bayesian-based evo-
lutionary analysis, with the CpREV + G + I substitution model, Yule model for prior tree, and the uncorrelated
relaxed clock, widely regarded as the most suitable model for datasets at the species level63. e Markov Chain
Monte Carlo (MCMC) was also set for 50,000,000 generations. Following that, a maximum credibility tree was
constructed using TreeAnnotator v1.10.4, discarding the initial 10% of trees as burn-in using Tracer v1.7.1. e
phylogenetic tree was then created using FigTree v1.4.4.
Data availability
e genome sequence data that support the ndings of this study are openly available in GenBank of NCBI at
[https:// www. ncbi. nlm. nih. gov/] under the Accession No. OQ079152.
Received: 23 November 2023; Accepted: 6 March 2024
References
1. Landolt, E. Biosystematic investigations in the family of duckweeds (Lemnaceae). II: e family of Lemnaceae: a monographic
study. 1. Veröentlichungen des Geobotanischen Institutes der ETH, Stiung Rübel, Zürich (1986).
2. Landolt, E. Taxonomy and ecology of the section Wola of the genus Wola (Lemnaceae). Ber. Geobot. Inst. ETH, Stiung Rübel,
Zürich 60, 137–151 (1994).
3. Xu, Y. et al. Species distribution, genetic diversity and barcoding in the duckweed family (Lemnaceae). Hydrobiologia 743, 75–87
(2015).
4. Cheng, J. J. & Stomp, A. M. Growing duckweed to recover nutrients from wastewaters and for production of fuel ethanol and
animal feed. Clean-Soil Air Water 37, 17–26 (2009).
5. Bog, M. et al. A taxonomic revision of Lemna sect: Uninerves (Lemnaceae). Taxo n 69, 56–66 (2020).
6. Bog, M. et al. Key to the determination of taxa of Lemnaceae: An update. Nordic J. Botany https:// doi. org/ 10. 1111/ njb . 02658 (2020).
7. Les, D. H. Aquatic Monocotyledons of North America: Ecology, Life History, and Systematics (CRC Press, 2020).
8. Les, D. H. et al. Phylogeny and systematics of Lemnaceae, the duckweed family. Syst. Botany 27, 221–240 (2002).
9. Tippery, N. et al. Evaluation of phylogenetic relationships in Lemnaceae using nuclear ribosomal data. Plant Biol . 17, 50–58 (2015).
10. Les, D. H. et al. Systematics of the Lemnaceae (duckweeds): Inferences from micromolecular and morphological data. Plant Syst.
Evol. 204, 161–177 (1997).
11. Pan, S. & Chen, S. Morphology of Wola arrhiza: a scanning electron microscopic study. Botanical bulletin of Academia Sinica.
New series (1979).
12. Appenroth, K. J. et al. Telling duckweed apart: Genotyping technologies for the Lemnaceae. Chin. J. Appl. Environ. Biol 19, 1–10
(2013).
13. Yang, J. et al. Frond architecture of the rootless duckweed Wola globosa. BMC Plant Biol. 21, 1–10 (2021).
14. Iqbal, S. Duckweed aquaculture. Potentials, possibilities and limitations for combined wastewater treatment and animal feed produc-
tion in developing countries. SAn-DEC Report (1999).
15. Skillicorn, P. et al. Duckweed Aquaculture: A New Aquatic Farming System for Developing Countries (World Bank, 1993).
16. Ansal, M. et al. Duckweed based bio-remediation of village ponds: An ecologically and economically viable integrated approach
for rural development through aquaculture. Livestock Res. Rural Dev. 22, 129 (2010).
17. Jorgensen, S. E. & Fath, B. D. Encyclopedia of Ecology (Elsevier, 2008).
18. Fujita, M. et al. Nutrient removal and starch production through cultivation of Wola arrhiza. J. Biosci. Bioeng. 87, 194–198 (1999).
19. Czerpak, R. & Szamrej, I. e eect of β-estradiol and corticosteroids on chlorophylls and carotenoids content in Wola arrhiza
(L.) Wimm.(Lemnaceae growing in municipal Bialystok tap Tap T water. Pol. J. Environ. Stud. 12, 677 (2003).
20. Körner, S. et al. e capacity of duckweed to treat wastewater: ecological considerations for a sound design. J. Environ. Qual. 32,
1583–1590 (2003).
21. Bhanthumnavin, K. & Mcgarry, M. G. Wola arrhiza as a possible source of inexpensive protein. Nature 232, 495–495 (1971).
22. Chareontesprasit, N. & Jiwyam, W. An evaluation of Wola meal (Wola arrhiza) in replacing soybean meal in some formulated
rations of Nile tilapia (Oreochromis niloticus L.). (2001).
23. Wang, W. et al. DNA barcoding of the Lemnaceae, a family of aquatic monocots. BMC Plant Biol. 10, 1–11 (2010).
24. Borisjuk, N. et al. Assessment, validation and deployment strategy of a two-barcode protocol for facile genotyping of duckweed
species. Plant Biol. 17, 42–49 (2015).
25. Bog, M. et al. Duckweed (Lemnaceae): Its molecular taxonomy. Front. Sustain. Food Syst. 3, 117 (2019).
26. Palmer, J. D. et al. Chloroplast DNA variation and plant phylogeny. Ann. Missouri Botanical Garden, 1180–1206 (1988).
27. Li, X. et al. Plant DNA barcoding: From gene to genome. Biol. Rev. 90, 157–166 (2015).
28. Nauheimer, L. et al. Global history of the ancient monocot family Araceae inferred with models accounting for past continental
positions and previous ranges based on fossils. New Phytol. 195, 938–950 (2012).
29. Les, D. H. & Crawford, D. J. Landoltia (Lemnaceae), a new genus of duckweeds. Novon, 530–533 (1999).
30. Mardanov, A. V. et al. Complete sequence of the duckweed (Lemna minor) chloroplast genome: Structural organization and
phylogenetic relationships to other angiosperms. J. Mol. Evol. 66, 555–564 (2008).
31. Guo, Y.-Y. et al. Chloroplast genomes of two species of Cypripedium: Expanded genome size and proliferation of AT-biased repeat
sequences. Front. Plant Sci. 12, 609729 (2021).
32. Morton, B. R. & Clegg, M. T. Neighboring base composition is strongly correlated with base substitution bias in a region of the
chloroplast genome. J. Mol. Evol. 41, 597–603 (1995).
33. Morton, B. R. e inuence of neighboring base composition on substitutions in plant chloroplast coding sequences. (1997).
Content courtesy of Springer Nature, terms of use apply. Rights reserved

14
Vol:.(1234567890)
Scientic Reports | (2024) 14:5873 | https://doi.org/10.1038/s41598-024-56394-7
www.nature.com/scientificreports/
34. Zhang, R.-S. et al. A high level of chloroplast genome sequence variability in the Sawtooth Oak Quercus acutissima. Int. J. Biol.
Macromol. 152, 340–348 (2020).
35. Wang, Z. et al. Comparative analysis of codon usage patterns in chloroplast genomes of six Euphorbiaceae species. PeerJ 8, e8251
(2020).
36. Wicke, S. et al. Disproportional plastome-wide increase of substitution rates and relaxed purifying selection in genes of carnivorous
Lentibulariaceae. Mol. Biol. Evol. 31, 529–545 (2014).
37. Jiang, H. et al. Comparative and phylogenetic analyses of six Kenya Polystachya (Orchidaceae) species based on the complete
chloroplast genome sequences. BMC Plant Biol. 22, 1–21 (2022).
38. Song, Y. et al. Development of chloroplast genomic resources for Oryza species discrimination. Front. Plant Sci. 8, 1854 (2017).
39. Shaw, J. et al. Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angio-
sperms: e tortoise and the hare III. Am. J. Botany 94, 275–288 (2007).
40. Tsudzuki, J. et al. Chloroplast DNA of black pine retains a residual inverted repeat lacking rRNA genes: Nucleotide sequences of
trnQ, trnK, psbA, trnI and trnH and the absence of rps16. Mol. Gener. Genet. MGG 232, 206–214 (1992).
41. Lin, C.-P. et al. e complete chloroplast genome of Ginkgo biloba reveals the mechanism of inverted repeat contraction. Genome
Biol. Evol. 4, 374–381 (2012).
42. White, E. Chloroplast DNA in Pinus monticola: 1 Physical map. eor. Appl. Genet. 79, 119–124 (1990).
43. Li, C. et al. Initial characterization of the chloroplast genome of Vicia sepium, an important wild resource plant, and related infer-
ences about its evolution. Front. Genet. 11, 73 (2020).
44. Darling, A. C. et al. Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 14, 1394–1403
(2004).
45. Butterworth, C. A. et al. Molecular systematics of tribe Cacteae (Cactaceae: Cactoideae): A phylogeny based on rpl16 intron
sequence variation. Syst. Botany 27, 257–270 (2002).
46. Kelchner, S. A. & Clark, L. G. Molecular evolution and phylogenetic utility of the chloroplast rpl16 intron in Chusquea and the
Bambusoideae (poaceae). Mol. Phylogen. Evol. 8, 385–397 (1997).
47. Downie, S. R. et al. A phylogeny of the owering plant family Apiaceae based on chloroplast DNA rpl16 and rpoC1 intron
sequences: Towards a suprageneric classication of subfamily Apioideae. Am. J. Botany 87, 273–292 (2000).
48. Zhang, W. Phylogeny of the grass family (Poaceae) from rpl16 intron sequence data. Mol. Phylogen. Evol. 15, 135–146 (2000).
49. Fukuda, T. et al. Molecular phylogeny of the genus Asparagus (Asparagaceae) inferred from plastid petB intron and petD–rpoA
intergenic spacer sequences. Plant Species Biol. 20, 121–132 (2005).
50. Jin, J.-J. et al. GetOrganelle: a fast and versatile toolkit for accurate de novo assembly of organelle genomes. Genome Biol. 21, 1–31
(2020).
51. Tillich, M. et al. GeSeq–versatile and accurate annotation of organelle genomes. Nucleic Acids Res. 45, W6–W11 (2017).
52. Greiner, S. et al. OrganellarGenomeDRAW (OGDRAW) version 1.3.1: Expanded toolkit for the graphical visualization of organellar
genomes. Nucleic Acids Res. 47, W59–W64 (2019).
53. Beier, S. et al. MISA-web: A web server for microsatellite prediction. Bioinformatics 33, 2583–2585. https:// doi. org/ 10. 1093/ bioin
forma tics/ btx198 (2017).
54. Kurtz, S. et al. REPuter: e manifold applications of repeat analysis on a genomic scale. Nucleic Acids Res. 29, 4633–4642 (2001).
55. Peden, J. F. Analysis of codon usage. (2000).
56. Frazer, K. A. et al. VISTA: Computational tools for comparative genomics. Nucleic Acids Res. 32, W273–W279 (2004).
57. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment soware version 7: improvements in performance and usability.
Mol. Biol. Evol. 30, 772–780 (2013).
58. Rozas, J. et al. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol. Biol. Evol. 34, 3299–3302 (2017).
59. Amiryouse, A. et al. IRscope: An online program to visualize the junction sites of chloroplast genomes. Bioinformatics 34,
3030–3031. https:// doi. org/ 10. 1093/ bioin forma tics/ bty220 (2018).
60. Löytynoja, A. Phylogeny-aware alignment with PRANK. Multiple sequence alignment methods, 155–170 (2014).
61. Darriba, D. et al. ProtTest 3: fast selection of best-t models of protein evolution. Bioinformatics 27, 1164–1165. https:// doi. org/
10. 1093/ bioin forma tics/ btr088 (2011).
62. Drummond, A. J. et al. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol. Biol. Evol. 29, 1969–1973 (2012).
63. Bouckaert, R. et al. BEAST 2: A soware platform for Bayesian evolutionary analysis. PLoS Comput. Biol. 10, e1003537 (2014).
Acknowledgements
We would like to express our thanks to the professors in the Division of Bio & Medical Big Data Department at
Gyeongsang National University for their priceless advice and assistance during this research project.
Author contributions
H.P. and Y.J.K.conceptualized and designed this research project. H.P. and J.H.P. collected samples and conducted
DNA extraction and sequencing. H.P. assembled, annotated, and analyzed the sequencing data, and wrote the
manuscript. All authors read and approved the manuscript and agreed to be accountable for all aspects of the
work.
Funding
is work was supported by a grant from the "Cooperative Research Program for Agriculture Science and
Technology Development (Project No. PJ01627602)" from the Rural Development Administration, Republic of
Koreaand was supported byLearning & Academic research institution for Master’s·PhD students, and Postdocs
(LAMP) Program of the National Research Foundation of Korea (NRF) grant funded by the Ministry of Educa-
tion (RS-2023-00301974).
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 024- 56394-7.
Correspondence and requests for materials should be addressed to Y.J.K.
Reprints and permissions information is available at www.nature.com/reprints.
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