Comparative chloroplast genomes and phylogenetic analyses of Pinellia

Abstract

Background
Pinellia Tenore (Araceae) is a genus of perennial herbaceous plants, all of which have medicinal value. The chloroplast (cp) genome data of Pinellia are scarce, and the phylogenetic relationship and gene evolution remain unclear.

Methods and results
We sequenced and annotated the Pinellia pedatisecta cp genome and combined it with previously published genomes for other Pinellia species. We used bioinformatics methods to analyse the genomic structure, repetitive sequences, interspecific variation, divergence hotspots, phylogenetic relationships, divergence time estimation and selective pressure of four Pinellia plastomes. Results showed that the cp genomes of Pinellia varied in length between 168,178 (P. pedatisecta MN046890) and 164,013 bp (P. ternata KR270823). A total of 68–111 SSR loci were identified as candidate molecular markers for further genetic diversity study. Eight mutational hotspot regions were determined, including psbI-trnG-UCC, psbM-rpoB, ndhJ-trnT-UGU, trnP-UGG-trnW-CCA, ndhF-trnN-GUU, ndhG-ndhE, ycf1-rps15 and trnR-ycf1. Gene selection pressure suggested that four genes were subjected to positive selection. Phylogenetic inferences based on the complete cp genomes revealed a sister relationship between Pinellia and Arisaema plants whose divergence was estimated to occur around 22.48 million years ago. All Pinellia species formed a monophyletic evolutionary clade in which P. peltata, rather than P. pedatisecta, earlier diverged, indicating that P. pedatisecta is not the basal taxon of Pinellia but P. peltata may be.

Conclusions
The cp genomes of Pinellia will provide valuable information for species classification, identification, molecular breeding and evolutionary exploration of the genus Pinellia.

Full text

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Molecular Biology Reports (2022) 49:7873–7885
https://doi.org/10.1007/s11033-022-07617-5
ORIGINAL ARTICLE
Comparative chloroplast genomes andphylogenetic analyses
ofPinellia
NingCui1 · WeixuChen2· XiwenLi3· PingWang1
Received: 19 October 2021 / Accepted: 18 May 2022 / Published online: 11 June 2022
© The Author(s) 2022
Abstract
Background Pinellia Tenore (Araceae) is a genus of perennial herbaceous plants, all of which have medicinal value. The
chloroplast (cp) genome data of Pinellia are scarce, and the phylogenetic relationship and gene evolution remain unclear.
Methods and results We sequenced and annotated the Pinellia pedatisecta cp genome and combined it with previously
published genomes for other Pinellia species. We used bioinformatics methods to analyse the genomic structure, repetitive
sequences, interspecific variation, divergence hotspots, phylogenetic relationships, divergence time estimation and selective
pressure of four Pinellia plastomes. Results showed that the cp genomes of Pinellia varied in length between 168,178 (P.
pedatisecta MN046890) and 164,013bp (P. ternata KR270823). A total of 68–111 SSR loci were identified as candidate
molecular markers for further genetic diversity study. Eight mutational hotspot regions were determined, including psbI-
trnG-UCC, psbM-rpoB, ndhJ-trnT-UGU, trnP-UGG-trnW-CCA, ndhF-trnN-GUU, ndhG-ndhE, ycf1-rps15 and trnR-ycf1.
Gene selection pressure suggested that four genes were subjected to positive selection. Phylogenetic inferences based on the
complete cp genomes revealed a sister relationship between Pinellia and Arisaema plants whose divergence was estimated
to occur around 22.48 million years ago. All Pinellia species formed a monophyletic evolutionary clade in which P. peltata,
rather than P. pedatisecta, earlier diverged, indicating that P. pedatisecta is not the basal taxon of Pinellia but P. peltata
may be.
Conclusions The cp genomes of Pinellia will provide valuable information for species classification, identification, molecular
breeding and evolutionary exploration of the genus Pinellia.
Keywords Pinellia· Phylogeny· Evolution· Divergence time estimation· Chloroplast genome
Abbreviations
cp Chloroplast
SSRs Simple sequence repeats
WGS Whole-genome sequencing
PE Paired-end
BLAST Basic Local Alignment Search Tool
LSC Large single copy
SSC Small single copy
IR Inverted repeat
indel Insertion-deletion
Pi Nucleotide diversity
CDS Protein-coding sequence
ML Maximum likelihood
Mya Million years ago
ESS Effective sample size
LRT Likelihood ratio test
BEB Bayes Empirical Bayes approach
HPD Highest posterior density
MP Maximum parsimony
LCBs Locally collinear blocks
Introduction
Pinellia Tenore is a small eastern Asian genus in the Araceae
family. Although there are only seven perennial herbaceous
species in Pinellia genus [1], every member it contains
* Xiwen Li
XWLi@icmm.ac.cn
* Ping Wang
wangpingjinan@163.com
1 Central Laboratory, Shandong Academy ofChinese
Medicine, Ji’nan, China
2 Shang Yao Hua Yu (LinYi) Traditional Chinese Medicine
Resources Co., Ltd, Linyi, China
3 Institute ofChinese Materia Medica, China Academy
ofChinese Medical Sciences, Beijing, China
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7874 Molecular Biology Reports (2022) 49:7873–7885
1 3
is important traditional Chinese medicinal plant and was
recorded in Chinese herb classics more than 2000years ago;
the most famous among them is P. ternata, with an annual
demand of 5500–6000 tons [2]. They have been used for
the treatment of viper bites, lumbago, allergic reaction and
externally to treat traumatic injury, abscesses, neck lympho-
sarcoma, breast mastitis and uterine cancer [3, 4].
In recent years, the phylogeny and evolution of monocots
have come under intense scrutiny with the rapid develop-
ment of molecular phylogenetic systematics, and multiple
studies have highlighted the phylogeny of Pinellia as prob-
lematic [5, 6]. The phylogenetic position of the Pinellia
genus in Araceae has been controversial, and sister groups
of Pinellia show discrepancy under different classification
systems and studies [7, 8]. The six major taxonomic systems
of Araceae, namely, Schott system [9], Engler system [10],
Hutchinson system [11], Grayum system [12], Bogner and
Nicolson system [13] and Mayo etal. system [14], have dif-
ferent views on the sister genus of Pinellia, with four genera
(Crytocoryne, Langenandra, Ambrosina and Arisaema) as
candidates. With the development of molecular systematics,
analyses of the plastome gene and restriction-site sequences
suggested that the Arisaema genus is strongly related to
Pinellia and is the sister genus of Pinellia [7, 15, 16]. Con-
trary to the above classification, Keating [17] and Bogner
and Petersen [18] based on morpho-anatomical data argued
that Arisaema and Pinellia cannot gather into a unique clade
for their morphological discrepancy of stamens.
Members of Pinellia exhibit wide variations in flower,
leaf, bulblet, spathe and ovule characteristics [19, 20]. The
low-level taxonomy and interspecific phylogeny of Pinellia
are difficult to address based on morphological traits. The
most comprehensive below-genus phylogenetic analysis to
date has been provided by Yin, a study using a matrix of all
seven Pinellia species and ITS and trnL-F DNA barcode
sequences [21]. Combined with the morphological traits,
Yin concluded that P. pedatisecta is the basal taxon of Pinel-
lia and suggested that its taxonomic rank should be elevated
to a section. However, our group previously conducted inter-
specific phylogenetic analyses of Pinellia by using four DNA
barcode sequences (ITS, matK, rbcL and trnL-F) with the
same method as Yin. The topologies of four barcodes were
not consistent, with P. pedatisecta in the outer layer of ITS
and matK trees and in the inner layer of rcbL and trnL-F
trees, receiving considerably lower bootstrap values (Sup-
plementary Fig. S1) probably due to insufficient sequence
length and interspecific variations.
The chloroplast (cp) is an important self-replicating
organelle that plays a crucial role in photosynthesis and in
the synthesis of pigment, protein and starch [22]. The cp
contains its own circular double-stranded genome, which is
inherited maternally in most angiosperms or paternally in
some gymnosperms [23]. Unlike the nuclear genome, the cp
lacks meiotic recombination. These properties, along with
adequate levels of polymorphism, make it a suitable mol-
ecule for studies on phylogeny and evolution [24]. Scientists,
especially Henriquez CL and Abdullah research group, have
long been devoted to the plastome phylogeny of the Araceae
family [25–28], and sequenced the cp genome of P. pedati-
secta (MN046890) in 2020 [22]. Nevertheless, their studies
focused more on the backbone phylogeny at the taxonomic
level of the entire or subfamilies of Araceae, and local phy-
logenetic relationships of Pinellia genus were rarely paid
attention to. Moreover, we previously performed a compara-
tive genomics analysis within Pinellia genus, and found that
the published P. pedatisecta cp genome was relatively differ-
ent from those of two other species, P. peltata (NC052862)
[29] and P. ternata (KR270823) [30] in sequence length,
gene content, GC content, etc. We determined to identify,
sample and sequence the cp genome of P. pedatisecta inde-
pendently for a reliable phylogeny of Pinellia species. The
plastome evolution of Pinellia was also discussed, including
the estimations of gene selection pressure and divergence
time which have not been studied before.
To reveal the interspecific diversification pattern of Pinel-
lia, we determined the complete cp genome of P. pedati-
secta and compared its sequence features with three other
Pinellia plastomes. The main goals of this study were to (1)
characterize and compare the cp genomes of Pinellia and
detect the sequence differences between Pinellia species and
between published and newly assembled cp genomes of P.
pedatisecta; (2) identify simple sequence repeats (SSRs),
long repeats and genetically variable regions and select
divergence hotspots as candidate DNA barcodes; (3) recon-
struct phylogenetic relationships of Pinellia species based
on the cp genome alignments and verify their phylogenetic
position within Araceae; and (4) estimate genes selection
pressure and divergence time for determining the relative
order and spacing of speciation events of Pinellia. Compara-
tive cp genomic analysis could provide theoretical basis to
further understand the evolution of the Araceae family and
additional insights into the long-standing controversial inter-
genus and intragenus phylogeny of Pinellia.
Materials andmethods
Plant material, DNA extraction, sequencing,
assembly andannotation
In our study, the cp genome of P. pedatisecta was sequenced
to explore the phylogeny and evolution of Pinellia. Fresh
leaves of P. pedatisecta from Linyi City, Shandong Prov-
ince were sampled. Voucher specimens were deposited in
Shandong Academy of Chinese Medicine. Total genomic
DNA was extracted from 100mg of silica-dried leaf by
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7875Molecular Biology Reports (2022) 49:7873–7885
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using a DNeasy Plant MiniKit (Qiagen, CA, USA) accord-
ing to the manufacturer’s instructions [31]. The quantity and
quality of genomic DNA were examined by using ND-2000
spectrometer (ThermoFisher Scientific, Wilmington, DE,
USA) and 0.8% agarose gel electrophoresis. The chloroplast
genome of Salvia plebeia (NC050929) was assembled in
our previous study [31]. Taking this work as a guidance,
the DNA sample pre-treatment (Covaris M220 [Covaris,
US; 250bp] and VAHTS™ Universal DNA Library Prep
Kit [Vazyme, China]), whole genome sequencing (Illumina
Hiseq 1500 platform [Illumina Inc., USA]), cp genome
assembly (Skewer, Basic Local Alignment Search Tool
[BLAST], SOAPdenovo v.2.04, GapCloser and MUMmer),
junction validation, and annotation (Plann v. 1.1.2, BLAST
and Apollo) were performed in turn with the cp genome of
P. peltata (NC052862) as a reference sequence. The primers
for junction validation are listed in Supplementary TableS1.
The cp genome obtained in this study has been submitted to
the NCBI database (www. ncbi. nlm. nih. gov). The physical
map of P. pedatisecta cp genome was produced with Chlo-
roplot (https:// irsco pe. shiny apps. io/ Chlor oplot/).
Repeat structure identification
SSRs were identified by MISA [32], and the minimum
thresholds for mono-, di-, tri-, tetra-, penta- and hexa-nucle-
otides were set to 10, 6, 5, 5, 5 and 5, respectively. REPuter
[33] was used to detect two kinds of long repeats: forward
and palindromic repeats which have been reported relatively
more prevalent in the cp genomes of Araceae family [22,
26]. Detection parameter settings were used as follows: rep-
find -d -p -h 3 -l 30 -best 50. Tandem Repeats Finder (http://
tandem. bu. edu/ trf/ trf. html) was used to find tandem repeats
with the default settings.
Genome comparative analysis
In addition to the newly sequenced cp genome, 26 avail-
able cp genome sequences of Pinellia and related spe-
cies were downloaded from the NCBI database: P. peda-
tisecta (MN046890), P. ternata (KR270823), P. peltata
(NC052862), Arisaema franchetianum (MN046885),
Arisaema ringens (MK111107), Arisaema erubescens
(MT676834), Arisaema nepenthoides (MW338731),
Typhonium blumei (NC051872), Sauromatum giganteum
(NC050648), Alocasia navicularis (MN046882), Coloca-
sia esculenta (JN105689), Pistia stratiotes (MN885890),
Amorphophallus konjac (MK611803), Calla palus-
tris (MN046887), Epipremnum aureum (KR872391),
Epipremnum amplissimum (MN477424), Aglaonema cos-
tatum (MN046881), Monstera adansonii (MN046888),
Zantedeschia aethiopica (KY792991), Pothos scandens
(MN046891), Symplocarpus renifolius (KY039276),
Symplocarpus nipponicus (MK341566), Lemna minor
(DQ400350), Acorus calamus (AJ879453), Acorus ameri-
canus (EU273602) and Acorus gramineus (KP099646). The
multiple sequence alignment of the 27 cp genome sequences
was performed using MAFFT v.7 with the default settings
and adjusted manually where necessary with BioEdit v.7.2.5
software. On the basis of the aligned sequence matrix of the
cp genomes of Pinellia, interspecific nucleotide diversity (K)
was evaluated by sliding window analysis with a step size
of 1000bp and window length of 2000bp in DnaSP v.5.10
[34]. The evolutionary divergences of the four Pinellia cp
genomes were evaluated using nucleotide differences and
p-distance by MEGA v.10.0.4. The protein-coding sequences
(CDSs) were extracted using Geneious v.2019.1.3 [35].
Phylogenetic analyses
Two datasets (cp genome and coding sequence) were used
to construct the phylogenetic topology of 27 Pinellia and
related species with maximum likelihood (ML) methods,
respectively. ML analyses were performed using RAxML
8.2.9 under the GTRGAMMA model with 1000 rapid boot-
strap replicates. Three species of Acorus (A. calamus, A.
americanus and A. gramineus) were used as outgroups.
Divergence time estimation
Accurate estimation of the divergence time in a taxon is
important to understand its evolutionary history. Divergence
times were estimated using PAML mcmctree (PAML v.4.9j)
[36] with the approximate likelihood calculation method.
The analysis was performed on 27 complete cp genome
sequences used in the phylogenetic analysis with three
known calibration times: (1) divergence between P. peda-
tisecta and T. blumei was 36–32 million years ago (Mya);
(2) divergence between A. navicularis and P. stratiotes was
85–46 Mya; and (3) all species except the genus Acorus from
the Araceae family arose 122–117 Mya inferred from the
published knowledge-based TimeTree (timetree.org). Pos-
terior distributions of parameters were approximated using
two independent mcmctree analyses of 10,000,000 genera-
tions with 20% burn-in. Tracer v.1.4.1 was used for checking
the convergence of the chains through adequate effective
sample sizes (ESSs).
Gene selection site analysis
A total of 74 single-copy protein-coding genes shared by
four Pinellia plastid genomes (Table1) were extracted
and aligned by Geneious v.9.0.5 [35] and MAFFT v.7.
The ML tree was constructed using RAxML V.8.2.9 with
the GTRGAMMA model based on complete cp genomes.
Protein-coding exon and each value of dN, dS and ω were
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7876 Molecular Biology Reports (2022) 49:7873–7885
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calculated using the site-specific model in the Codeml
program (seqtype = 1, model = 0, Nsites = 0, 1, 2, 3, 7, 8)
of PAML v.4.9j [36]. To determine the selected sites, we
compared the model M0 (one ratio) versus M3 (discrete),
M1 (neutral) versus M2 (positive selection) and M7 (beta)
versus M8 (beta and ω) and carried out the three likelihood
ratio tests (LRTs). Only consistent sites of positive selec-
tion with significant support from posterior probability (p
of (ω > 1) ≥ 0.99; Bayes Empirical Bayes approach [BEB])
were identified. BEB recognized by Models M2 and M8
were further considered.
Results
Chloroplast genome features inPinellia
In our study, the cp genome of P. pedatisecta was sequenced,
assembled and validated (Fig. S2). A total of 49,904,104
PE raw reads were generated using the Illumina Sequencing
System. The novel cp genome sequence has been preserved
in GenBank (MZ702636, Table1). The cp genome of P.
pedatisecta was circular double-stranded DNA and displayed
a quadripartite structure (Fig.1). The cp genome assembled
in this study was 164,682bp in length, which was 3,496bp
shorter than the published one (MN046890, 168,178bp)
mainly due to the contraction of the LSC region. Moreo-
ver, the GC content in the SSC region of the cp genome
MZ702636 was 2.58% higher than that of MN046890. The
cp genome MN046890 had the same rRNA gene content
as that of MZ702636 but contained one more tRNA gene
(tRNA-His) (Supplementary TableS2).
After downloading from the NCBI database, protein-
coding gene number variations of 24 cp genomes of the
Araceae family, including four Pinellia cp genomes, were
analysed (Fig. S3). Compared with the cp genome of the
model plant Arabidopsis thaliana, two genes, infA and ycf68,
were inserted among most Araceae plants, while one-copy
ycf1 gene was missing. In the Araceae family, there were
significant differences in the number of ycf68 genes among
species. The ycf68 genes in 60% of Araceae cp genomes
were completely missing, while the remaining 40% of the
cp genomes had two copies. Even within the Pinellia genus,
the number of ycf68 genes was also inconsistent. Never-
theless, there was no discrepancy in the type and number
of protein-coding genes between our newly assembled cp
genome (MZ702636) and the published one (MN046890).
SSR andrepeat sequence analyses
The number of SSRs in the four Pinellia cp genomes ranged
from 68 (P. peltata) to 111 (P. ternata, Fig.2A and Supple-
mentary TableS3). Three kinds of SSRs were discovered,
namely, mononucleotide, dinucleotide and trinucleotide.
Among each Pinellia species, mononucleotide repeats were
the most common, whereas trinucleotide repeats accounted
for the lowest proportion of SSRs. The number of A/T mon-
onucleotide repeats exceeded that of the other three types
combined (Fig.2A). Interspersed repeated sequences were
identified by using REPuter for four plastomes (Fig.2B and
Supplementary TableS4). Except for IR regions, the repeats’
length ranged from 31 to 328bp with forward (F) repeats as
the relative prevalent type. Most interspersed repeats of two
P. pedatisecta cp genomes were located in the LSC regions,
while the long repeats of P. peltata and P. ternata were pre-
dominantly in the IRb/SSC and SSC regions, respectively.
The total number of tandem repeats for four plastid genomes
was in the range of 34–274. P. peltata had the least repeats,
and P. pedatisecta (MN046890) had the most (Supplemen-
tary TableS5).
Comparative genomic analyses
The differences and evolutionary divergences among four
Pinellia cp genomes were compared using nucleotide sub-
stitutions and sequence distance (Supplementary TableS6).
Across all four Pinellia cp genomes, the p-distance was
0.000402–0.013668, and the value of nucleotide differences
was 66–2459. The comparison results of nucleotide substi-
tution and genetic distance were consistent. The sequence
divergence level of two cp genomes of P. pedatisecta was
the lowest. The differentiation between P. pedatisecta and
P. ternata was less than that between P. pedatisecta and P.
peltata. Among three Pinellia species, P. ternata and P. pel-
tata were the most genetically distant. Moreover, the K value
(sequence divergence between species) was calculated, and
Table 1 Summary of features of four Pinellia chloroplast genomes
Taxon Accession number Length (bp) Number of genes GC content (%)
Genome LSC SSC IR Total Protein coding tRNA rRNA Genome LSC SSC IR
P. pedatisecta MZ702636 164,682 90,947 22,523 25,606 129 85 36 8 35.71 33.92 29.43 42.09
P. pedatisecta MN046890 168,178 92,963 23,981 25,617 130 85 37 8 35.08 33.35 26.85 42.09
P. peltata NC052862 164,923 90,089 24,871 24,981 130 86 36 8 36.51 34.53 31.77 42.44
P. ternata KR270823 164,013 89,783 22,980 25,625 131 86 37 8 36.66 34.6 31.66 42.53
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7877Molecular Biology Reports (2022) 49:7873–7885
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the sliding windows of the K values were constructed by
DnaSP (Fig.2C and Supplementary TableS7). Figure2C
showed that the sequence divergence between P. pedatisecta
and P. ternata was much lower than the two other K val-
ues. The sequence divergence between P. pedatisecta and
P. peltata was not so different from that between P. ternata
and P. peltata. Eight highly variable regions with great K
values were detected, namely, psbI-trnG-UCC, psbM-rpoB,
ndhJ-trnT-UGU, trnP-UGG-trnW-CCA, ndhF-trnN-GUU,
ndhG-ndhE, ycf1-rps15 and trnR-ycf1. Four of these regions
were located in the LSC region, and the remaining four were
located in the SSC region, all of which were present in the
non-coding regions.
Phylogenetic analyses
To analyze the phylogenetic relationship of Pinellia species,
we constructed phylogenetic trees using whole cp genome
Fig. 1 Chloroplast genome map of P. pedatisecta assembled in this
study. The centre of the figure provides length information of the cp
genome. In the first inner circle, the proportion of the shaded parts
represents the GC content of each part. The gene names are labelled
on the outermost layer. The transcription directions for the inner and
outer genes are listed clockwise and anticlockwise, respectively
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7878 Molecular Biology Reports (2022) 49:7873–7885
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sequences and their CDSs by ML methods. For Pinellia spe-
cies, the topological structure obtained by either complete cp
genome or CDSs was roughly identical (Fig.3). The topol-
ogy based on entire cp genomes showed that Pinellia species
were monophyletic and clustered into three clades: one for
two P. pedatisecta plastomes, one for P. ternata and one
for P. peltata with strong support (bootstrap value 100%).
P. pedatisecta and P. ternata clustered together, exhibiting
the highest genetic similarity among the studied representa-
tives of Pinellia genus. In contrast to the CDSs phylogeny
(Fig.3B), in which Pinellia species were placed most closely
to Arisaema, Sauromatum and Typhonium clade, the whole-
length plastome phylogeny placed some Arisaema species
as sister to the genus Pinellia with 100% bootstrap support
(Fig.3A).
Divergence time estimation
In this study, we used full-length sequences of cp genomes
of 23 Araceae family plants (including four Pinellia cp
Fig. 2 The SSRs (A), interspersed repeated sequences (B), and inter-
specific nucleotide diversity (C) among the chloroplast genomes of
Pinellia. X-axis in Fig.3C: position of a window. Y-axis in Fig.3C:
sequence divergence (K values) between species of each window.
K(a): K values between P. pedatisecta and P. peltata; K(b): K values
between P. pedatisecta and P. ternata; K(c): K values between P. ter-
nata and P. peltata
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7879Molecular Biology Reports (2022) 49:7873–7885
1 3
genomes) and three outgroups to estimate the divergence
times of major clades in the Araceae family. Our dat-
ing analysis resulted in estimates for the crown node of
the Araceae family of 91.99 Mya (95% highest posterior
density [HPD] = 46.69–125.17 Mya; node 1 in Fig. S4;
Supplementary TableS8) in the early Cretaceous. The
divergence time of the four genera Pinellia, Arisaema,
Sauromatum and Typhonium was estimated at 25.65 Mya
(95% HPD = 12.93–36.35 Mya; node 2) in the Oligocene.
The age estimation for the crown node of Pinellia and
most species of Arisaema was dated to 22.48 Mya (95%
HPD = 11.39–33.32 Mya; node 3) in the late Oligocene
Fig. 3 Phylogenetic trees constructed with the whole cp genomes (A) and their protein-coding sequences (B) by using ML method. Numbers
near each branch are bootstrap values
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7880 Molecular Biology Reports (2022) 49:7873–7885
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and early Miocene. Pinellia diverged from its sister clade
at 13.09 Mya (95% HPD = 4.91–21.96 Mya; node 4 in Fig.
S4; Supplementary TableS8) in the Miocene.
Gene selection pressure analysis ofprotein
sequences
The site-specific selective pressure on four Pinellia plasto-
mes was assumed using the site model in PAML program.
Three pairs of site model comparisons (M0 vs M3, M1 vs
M2a and M7 vs M8) showed four protein-coding genes sub-
jected to positive selection (LRT of the three comparisons all
p < 0.05; Supplementary Tables S9 and S10), namely, petB,
rpoC1, rps12 and ycf1. Of these genes, ycf1 had the highest
number of sites (21 sites; TableS9), followed by rpoC1 (4
sites), petB (1 site) and rps12 (1 site).
Discussion
Phylogenetic position ofthePinellia genus
inAraceae
In the previous classification systems, the Pinellia genus
belonged to the Araceae family, but its sister genus remains
controversial. In the Schott system [9], which is the first clas-
sification system of the Araceae family, the genus Pinellia
was placed in the tribus Alleluehieae, related to two genera
of Crytocoryne and Langenandra. In the Engler system [10],
Pinellia belonged to the subtrib. Pinelliinae, trib. Areae, sub-
fam. Aroideae, in the same tribus as the Arisaema genus. In
the Hutchinson system [11], this genus was placed in trib.
Areae, related to two genera of Crytocoryne and Ambro-
sina. In the classification system of Grayum [12], Pinellia
belonged to trib. Pinellia, subfam. Aroideae. After modify-
ing the Engler system by Bogner and Nicolson [13], the
Pinellia genus changed to the subtrib. Atherurinae, trib.
Pinellia, subfam. Aroideae. Mayo etal. [14] conducted
taxonomic analysis of 106 taxa in the Araceae family using
63 morphological and anatomical traits and proposed the
latest classification system of the Araceae family. In this
system, Pinellia was placed in trib. Arisaemateae, subfam.
Aroideae with the Arisaema genus as sister group. Since the
21st century, with the development of molecular systemat-
ics, many studies have investigated the phylogenetic position
of the Pinellia genus in Araceae [7, 15–18], yet controversy
regarding molecular systematics and morphological clas-
sification persists. French etal. [16], Cabrera etal. [7] and
Cusimano etal. [15] performed molecular phylogenetic
analyses by applying different types and numbers of DNA
marker sequence data, and they discovered that the Arisaema
genus is strongly related to Pinellia among Araceae plants,
and is the sister genus of Pinellia. Contrary to the above
classification, Keating [17] and Bogner and Petersen [18]
based on morpho-anatomical data argued that Arisaema
and Pinellia cannot be grouped into a unique clade. One
significant morphological discrepancy between them was
that almost all Arisaema species have at least partially fused
stamens, whereas Pinellia and other related genera (e.g. Sau-
romatum and Typhonium) have free stamens.
The cp genome is one of three subcellular compartments
in the plant genome, and it is mainly inherited from the
maternal parent [37]. Given its high conservation and abun-
dant interspecific variation, the cp genome has the potential
to provide distinguishing differences that can help molecu-
larly classify closely related species [38]. With advances
in high-throughput sequencing, achieving the cp genome is
easily acquirable at a large scale with low costs. Research-
ers have proposed the entire cp genome as a super barcode
to discriminate and classify closely related species [39]. To
date, phylogenies of several genus-level taxa have been fur-
ther clarified by using cp genome sequences, such as Epi-
medium [40], Paris [41] and Sanguisorba [42]. Hence, we
compared 24 cp genomes of the Araceae family, including
four plastomes of Pinellia, to explore the cp genome molec-
ular phylogeny of Pinellia. The resulting phylogeny based
on entire cp genomes in this study showed that Pinellia and
Arisaema plants were gathered into one branch and sister
groups to each other with a well-supported bootstrap value
(100%; Fig.3A). Although the Araceae family is a major
group of monocotyledons, there is still a limited number
of cp genomes available from Araceae species, which may
result in some congeneric species, such as Arisaema plants,
not clustering together in one phylogenetic branch (Fig.3).
The absence of plastid genomes from three other candidates
of the Pinellia sister groups, Crytocoryne, Langenandra and
Ambrosina [21], could also hinder our discovery of the inter-
genus phylogeny of Pinellia.
Phylogeny withinthePinellia genus
Despite recent advances in molecular phylogenetic stud-
ies, deep evolutionary relationships and below-genus taxo-
nomic classification of Pinellia remain unresolved. Yin
[21] made the first comprehensive phylogenetic analyses
of all seven Pinellia species by using the sequences of
ITS and trnL-F DNA barcodes. The resulting phylogeny
of both ITS and trnL-F sequences supported that P. peda-
tisecta and the six remaining Pinellia species were sis-
ter species, and P. pedatisecta served as a basal taxon in
Pinellia. Moon etal. [43] reconstructed the phylogenetic
trees of three species, P. ternata, P. tripartita and P. peda-
tisecta, with matK and rbcL sequences; they found that
the topology of two trees is inconsistent. The basal taxon
in the matK tree was P. pedatisecta, congruent with that
by Yin [21], while the basal taxon in the rbcL tree was P.
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7881Molecular Biology Reports (2022) 49:7873–7885
1 3
ternata. Furthermore, after morphological investigation,
Yin found that P. pedatisecta has many characteristics of
the Pinellia genus. These traits include perennial herb with
a small tuber at the top of the main tuber, persistent spathe,
female inflorescence adnate to the spathe, unisexual flow-
ers, no perianth, one straight ovule and green fruit. Hence,
combined with the molecular phylogeny, Yin concluded
that the basal taxon of the Pinellia genus is P. pedatisecta,
the species native to shady woodland areas, forested slopes
and valleys in northern and western China; he suggested
that the taxonomic rank of P. pedatisecta should be ele-
vated to a section of Pinellia [21], which was the first and
only below-genus taxonomic recommendation of Pinellia.
In our opinion, P. pedatisecta indeed has some of the
characteristic traits of Pinellia, but it also possesses so
many characteristics significantly different from other con-
generic species. For example, lanceolate spathe, no con-
striction in the tube and eaves of spathe, no diaphragm
between the female inflorescence and male inflorescence
and no bulblet on the petiole [15, 19, 20]. Our group pre-
viously conducted phylogenetic analyses of Pinellia by
using four different barcode sequences with maximum par-
simony (MP) method. The topologies of the four barcodes
were not consistent, with poor bootstrap values (Fig. S1)
probably due to insufficient sequence length and interspe-
cific variation. Hence, plastid genomes, an ideal model for
evolutionary and comparative genomic studies of related
species, were necessary here for a molecular phylogeny of
Pinellia with a significantly higher resolution.
After comparing the structural organization of three
previously published cp genomes of Pinellia, we found
that the sequence similarity between the published P.
pedatisecta plastome (MN046890) and the cp genomes
of P. peltata (NC052862) and P. ternata (KR270823) was
clearly lower than expected in the following five aspects.
(1) The length of the P. pedatisecta plastome was sig-
nificantly longer than that of the two other species
(Table1), specifically 3.2kb longer than that of P. pel-
tata and even 4.1kb longer than that of P. ternata. The
length of the LSC region in P. pedatisecta was 3.18kb
longer than that of P. ternata.
(2) With regard to protein-coding gene content, all two
ycf68 genes were missing in the published cp genome
of P. pedatisecta (Fig. S3), compared with P. peltata
and P. ternata, which could have the potential to affect
the physiological functions of plants, although some
authors suggested that the ycf68 gene likely does not
encode a protein [44].
(3) The GC content of published P. pedatisecta was lower
than that in P. peltata and P. ternata (Table1), espe-
cially in the SSC region, which was 4.92% lower than
that in P. peltata and 4.81% lower than that in P. ter-
nata.
(4) After the alignment of three published cp genomes
and further estimation of locally collinear blocks
(LCBs) with MAUVE 2.4.0, the gene order compari-
son revealed one rearrangement (~ 128bp) between the
published plastomes of P. pedatisecta and P. ternata
(Fig. S5A and S5B).
(5) A significant insertion/deletion variation was noted
across three cp genomes in this group, located between
tRNA-Ser (GCU) and tRNA-Ser (CGA) genes in
the LSC region with the length of ~ 1.2kb (10,714–
11,950bp in the published P. pedatisecta cp genome
and 10,192–10,234bp in the self-assembled one; Fig.
S5C and S5D).
These five sequence discrepancies do not follow the rule
of “chloroplast genomes of related species, especially those
within the same genus, are generally highly conserved”
[45, 46]. Therefore, we determined to identify, sample and
sequence the plastome of P. pedatisecta independently for
further phylogenetic analyses of Pinellia.
In this study, the phylogenetic tree based on whole cp
genomes showed that the species of Pinellia were clustered
into three clades: one for two P. pedatisecta plastomes, one
for P. ternata and one for P. peltata with high support values
(Fig.3). P. pedatisecta and P. ternata, which possess pedate
and three full-lobed leaves, respectively, grouped together
in topology, exhibiting the highest genetic similarity among
the studied representatives of Pinellia genus. In contrast,
P. peltata, characterized by undivided and peltata leaf and
development of stem tuber, was placed in the outer layer
of Pinellia topology. P. pedatisecta is buried inside the cp
genome phylogeny, incongruent with the phylogeny from
ITS and trnL-F sequences by Yin [21]. Furthermore, com-
parative genomic analyses showed that the nucleotide sub-
stitutions and sequence distance between P. pedatisecta and
P. ternata were the smallest (1275 and 0.007206; TableS6),
while that between P. ternata and P. peltata was the largest
(2459 and 0.013668). The interspecific nucleotide diver-
sity (K value) between P. pedatisecta and P. ternata was
considerably lower than the two other K values (Fig.2C),
both of which confirmed that P. peltata shared less sequence
similarity with P. pedatisecta and P. ternata. Limited by the
number of Pinellia cp genomes published so far, we cannot
determine whether P. peltata is the ancestor of Pinellia spe-
cies, but it is possible. P. peltata has the same floral morpho-
logical characteristics as other Pinellia species, except for P.
pedatisecta [29]. For example, solitary inflorescence, persis-
tent spathe, with constriction in the tube and eaves of spathe,
female inflorescence adnate to the spathe, with diaphragm
between the female inflorescence and male inflorescence,
unisexual flowers, no perianth, one locular ovary and one
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7882 Molecular Biology Reports (2022) 49:7873–7885
1 3
straight ovule [21]. However, the peltata leaf characteristics
of P. peltata are considerably different. Thus, the below-
genus taxonomic classification of Pinellia is suggested to be
based on differences in leaves or other tissue morphological
characteristics between species, rather than flowers.
Variations andevolution ofPinellia cp genomes
In this study, the cp genome of P. pedatisecta was sequenced
(MZ702636) with the length of 164,682bp, which fell within
the cp genome size range for angiosperms but much smaller
than its published one (MN046890, 168,178bp; Table1).
Among four Pinellia plastid genomes, LSC regions showed
the most difference in size, with the shortest 89,783bp of P.
ternata KR270823 and the longest 92,963bp of P. pedati-
secta MN046890. Additionally, the inferred structures and
protein-coding gene contents were in accordance except
for the infA and ycf68 genes (Fig.1 and S3). Analysis with
DnaSP inferred that some of the most divergent regions of
psbI-trnG-UCC, psbM-rpoB, ndhJ-trnT-UGU, trnP-UGG-
trnW-CCA, ndhF-trnN-GUU, ndhG-ndhE, ycf1-rps15 and
trnR-ycf1, as shown in Fig.2C, were found for further
related species identification of Pinellia.
SSRs in the cp genome are an efficient marker tool for
population genetic structure and phylogeography [47]. In
this study, 68–111 SSR loci were identified between Pinel-
lia species (Supplementary TableS3). These SSR loci could
provide candidate molecular markers for the genetic diver-
sity study of Pinellia. The composition of SSR loci in the cp
genomes of three Pinellia species was similar to that of most
angiosperms, with A/T mononucleotide repeats dominating
all the repeat units. This phenomenon may be one of the rea-
sons for the abundance of A/T bases in cp genomes. Repeat
units in the cp genome can cause sequence polymorphism,
providing information for further genetic diversity study of
Pinellia.
At present, specific studies on the divergence time esti-
mation of Pinellia by using molecular data, particularly
plastid genomes, are lacking. Li etal. [48] first supposed
that species with simple leaves (e.g. P. peltata) are less
evolved than those with pedate or lobed leaves (e.g. P. peda-
tisecta and P. ternata) on the basis of the palynology and
isozyme characteristics of Pinellia species. Yin [21] specu-
lated that the Pinellia genus first appeared in the Paleogene
according to geographic distribution and the origin time
of its related genus (Arisaema). Here, based on whole cp
genome sequences, our divergence time estimation indi-
cated that Pinellia species originated at ~ 22.48 Mya (95%
HPD = 11.39–33.32 Mya; node 3 in Fig. S4; Supplementary
TableS8) in the late Oligocene and early Miocene, con-
gruent with that estimated by Yin and further refined, and
diverged diversely at ~ 13.09 Mya (95% HPD = 4.91–21.96
Mya; node 4 in Fig. S4) in the Miocene. Furthermore, within
the Pinellia genus, P. peltata diverged first at ~ 5.34 Mya
earlier than two other species, P. pedatisecta and P. ternata;
these findings were consistent with the results of Li etal.
[48]. The present estimation of divergence times based on
plastid genomes and fossil data provides new insights and a
hypothetical foundation for future studies on the origin and
earlier evolution of Pinellia.
Genes subjected to positive selection have a significant
impact on the creative effects of populations, changes under
selection stress, and genetic drift led to the rapid transfor-
mation of genes into new common adaptive combinations
[49]. In this study, we calculated the non-synonymous/syn-
onymous substitution rate ratio (ω = dN/dS) for each of the
74 single copy protein-coding genes shared by the analyzed
plastid genomes of Pinellia. Four genes with high posterior
probability of codon sites in the BEB test were acquired
and considered as genes under positive selection (Fig.4
and Tables S9 and S10). These genes included one gene
for cytochrome b/f complex subunit protein in the photo-
system II reaction (petB) [50], one DNA-dependent RNA
Fig. 4 Positive selection sites logo of four Pinellia plastomes
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7883Molecular Biology Reports (2022) 49:7873–7885
1 3
polymerase gene (rpoC1), one gene for ribosome small
subunit protein (rps12) and ycf1 gene. All these genes were
also detected in other plants [51–54], and these genes may
have played a significant role in the adaptive evolution of
Pinellia. The specific role needs to be further studied.
The genus Pinellia includes seven species in total. Only
three Pinellia species were studied here, and as a conse-
quence, it might not fully show the real effect of the phylog-
eny of Pinellia. However, our study was the first compre-
hensive phylogenetic analysis of Pinellia at the cp genome
level, and the resulting topologies based on either whole
cp genome or CDS datasets both definitively show that P.
pedatisecta cannot be the ancestor of Pinellia species. The
main habitat of all three Pinellia species in our study is in
the southeastern portion of Asia, where hybridization and
introgression have been reported [55, 56], which may cause
interspecific gene flow in response to ecological selection
and lead to difficulties in molecular phylogenetic analysis.
Given that the cp and nuclear genomes evolve independently,
the phylogeny of cp genomes alone is insufficient in making
taxonomic decisions about Pinellia. Therefore, more meth-
ods and more species are needed in further study to improve
our ability to better understand the phylogeny and evolution
of Pinellia.
Conclusions
The genus Pinellia Tenore is comprised of seven species,
all of which are important medicinal plant resources. Spe-
cies phylogenetic classification is vital for protecting species
diversity and selecting high quality germplasm resources.
But due to members of Pinellia exhibit rather wide varia-
tions in morphological structures, the low-level taxonomy
and interspecific phylogeny of Pinellia are difficult to
address based on morphology. With the development of
next-generation sequencing technology, complete chloro-
plast genomes have been widely employed to explore phy-
logenetic relationships of intra-or inter-genus. However, the
variation and evolution of the whole chloroplast genomes in
the genus Pinellia have been ignored. Here, we sequenced
the chloroplast genome of P. pedatisecta, compared it with
previously published plastid genomes, and reconstructed
phylogenetic relationships of Pinellia based on chloroplast
genomes and their CDSs. The divergence time estimation
and selective pressure of Pinellia plastomes were also inves-
tigated. The results showed some variations and adaptive
evolution between Pinellia complete chloroplast genomes
including size, structure and nucleotide diversity, which
provided valuable information for species classification and
evolution. In addition, our results revealed a sister relation-
ship between Pinellia and Arisaema plants whose diver-
gence was estimated to occur around 22.48 million years
ago. All Pinellia species formed a monophyletic evolution-
ary clade in which P. peltata, rather than P. pedatisecta,
earlier diverged, indicating that P. pedatisecta is not the
basal taxon of Pinellia but P. peltata may be. In conclu-
sion, our results could provide insight into the chloroplast
genome evolution and phylogeny of Pinellia genus and even
of Araceae family, which would be useful for selecting high
quality Pinellia germplasm resources in the future.
Supplementary Information The online version of this article (https://
doi. org/ 10. 1007/ s11033- 022- 07617-5) contains supplementary mate-
rial, which is available to authorized users.
Acknowledgements The authors thank Baosheng Liao for his support
in data analysis.
Author contributions NC and XL conceived and designed the study;
NC and WC collected and analyzed the data; NC drafted the initial ver-
sion of the manuscript; NC, XL and PW contributed to later versions
of the manuscript.
Funding This study was supported by the Major Special Project of
Scientific and Technological Cooperation of Bijie City (Grant No.
2021-02), Research Incubation Fund Project of Shandong Academy
of Chinese Medicine (Grant No. 2021SACM-3), Medical and Health
Science and Technology Development Project of Shandong Province
(Grant No. 202102041135) and Key Research and Development Pro-
gram of Shandong Province (Grant No. 2020CXGC010505-04).
Data availability The chloroplast genome sequence of Pinellia peda-
tisecta assembled here is accessible via GenBank with the accession
number of MZ702636 and Global Pharmacopoeia Genome Database
(GPGD,
http:// www. gpgen ome. com/ speci es/ 40314). Raw sequencing data
is available at NCBI SRA database with the accession number of
SRR15328795.
Declarations
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with human
participants performed by any of the authors.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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7884 Molecular Biology Reports (2022) 49:7873–7885
1 3
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