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Article
Complete Plastid Genome Sequencing of Eight
Species from Hansenia,Haplosphaera and Sinodielsia
(Apiaceae): Comparative Analyses and
Phylogenetic Implications
Wei Gou 1,†, Sheng-Bin Jia 1,†, Megan Price 2, Xian-Lin Guo 1, Song-Dong Zhou 1
and Xing-Jin He 1, *
1Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences,
Sichuan University, Chengdu 610065, China; gouwei1@stu.scu.edu.cn (W.G.);
sdjiashengbin@gmail.com (S.-B.J.); xlguo@stu.scu.edu.cn (X.-L.G.); zsd@scu.edu.cn (S.-D.Z.)
2Sichuan Key Laboratory of Conservation Biology on Endangered Wildlife, College of Life Sciences,
Sichuan University, Chengdu 610065, China; meganprice@scu.edu.cn
*Correspondence: xjhe@scu.edu.cn
†Equal contributions to this work.
Received: 12 October 2020; Accepted: 6 November 2020; Published: 9 November 2020
Abstract:
Hansenia Turcz., Haplosphaera Hand.-Mazz. and Sinodielsia H.Wolffare three Apiaceae
genera endemic to the Hengduan Mountains and the Himalayas, which usually inhabit elevations
greater than 2000 m. The phylogenetic relationships between and within the genera were uncertain,
especially the placement of Hap. himalayensis and S. microloba. Therefore, we aimed to conduct
comparative (simple sequence repeat (SSR) structure, codon usage bias, nucleotide diversity (Pi)
and inverted repeat (IR) boundaries) and phylogenetic analyses of Hansenia,Haplosphaera and
Sinodielsia (also compared with Chamaesium and Bupleurum) to reduce uncertainties in intergeneric and
interspecific relationships. We newly assembled eight plastid genomes from Hansenia,Haplosphaera
and Sinodielsia species, and analyzed them with two plastid genomes from GenBank of Hap. phaea,
S. yunnanensis. Phylogenetic analyses used these ten genomes and another 22 plastid genome
sequences of Apiaceae. We found that the newly assembled eight genomes ranged from 155,435 bp to
157,797 bp in length and all had a typical quadripartite structure. Fifty-five to 75 SSRs were found
in Hansenia,Haplosphaera and Sinodielsia species, and the most abundant SSR was mononucleotide,
which accounted for 58.47% of Hansenia, 60.21% of Haplosphaera and 48.01% of Sinodielsia. There was
no evident divergence of codon usage frequency between the three genera, where codons ranged
from 21,134 to 21,254. The Pi analysis showed that trnE(UUC)-trnT(GGU),trnH(GUG)-psbA and
trnE(UUC)-trnT(GGU) spacer regions had the highest Pi values in the plastid genomes of Hansenia
(0.01889), Haplosphaera (0.04333) and Sinodielsia (0.01222), respectively. The ndhG-ndhI spacer regions
were found in all three genera to have higher diversity values (Pi values: 0.01028–0.2), and thus may
provide potential DNA barcodes in phylogenetic analysis. IR boundary analysis showed that the
length of rps19 and ycf1 genes entering IRs were usually stable in the same genus. Our phylogenetic
tree demonstrated that Hap. himalayensis is sister to Han. weberbaueriana; meanwhile, Haplosphaera
and Hansenia are nested together in the East Asia clade, and S. microloba is nested within individuals
of S. yunnanensis in the Acronema clade. This study will enrich the complete plastid genome dataset of
the Apiaceae genera and has provided a new insight into phylogeny reconstruction using complete
plastid genomes of Hansenia,Haplosphaera and Sinodielsia.
Keywords: Apiaceae; Hansenia;Haplosphaera; phylogeny; plastid genome; Sinodielsia
Plants 2020,9, 1523; doi:10.3390/plants9111523 www.mdpi.com/journal/plants
Plants 2020,9, 1523 2 of 17
1. Introduction
Hansenia Turcz., Haplosphaera Hand.-Mazz. and Sinodielsia H.Wolffare three endemic high-elevation
(typically >2000 m) genera of Apiaceae, mainly distributed in the Hengduan Mountains and the
Himalayas (Figure 1) [
1
]. According to the latest Apiaceae taxonomy, Hansenia,Haplosphaera and
Sinodielsia comprise five, two and four species, respectively [
2
–
5
]. Previous phylogenetic studies of
Hansenia,Haplosphaera and Sinodielsia based on two plastid genome regions (rpl16 and rps16 introns)
and nuclear internal transcribed spacers (nrITSs) found that Sinodielsia is within the Acronema clade,
while the closely related Hansenia and Haplosphaera are located in the East Asia clade, albeit from
limited sampling [
6
–
11
]. One study of 106 nrITS sequences representing 100 species from 52 genera
of Chinese Apiaceae found that the Acronema clade and the East Asia clade were well-supported
(posterior probability both valued 100% by Bayesian inference) [
8
]. Additionally, Hap. himalayensis and
S. microloba were unknown at the time of these previous studies and remain as two little-known species.
Plants 2020, 9, x FOR PEER REVIEW 2 of 19
1. Introduction
Hansenia Turcz., Haplosphaera Hand.-Mazz. and Sinodielsia H.Wolff are three endemic high-
elevation (typically >2000 m) genera of Apiaceae, mainly distributed in the Hengduan Mountains
and the Himalayas (Figure 1) [1]. According to the latest Apiaceae taxonomy, Hansenia, Haplosphaera
and Sinodielsia comprise five, two and four species, respectively [2–5]. Previous phylogenetic studies
of Hansenia, Haplosphaera and Sinodielsia based on two plastid genome regions (rpl16 and rps16
introns) and nuclear internal transcribed spacers (nrITSs) found that Sinodielsia is within the Acronema
clade, while the closely related Hansenia and Haplosphaera are located in the East Asia clade,
albeit fr
Figure 1. Plants of Hansenia, Haplosphaera and Sinodielsia. (A) Han. forbesii, (B) Han. forrestii, (C) Han.
oviformis, (D) Han. weberbaueriana, (E) Hap. himalayensis, (F) Hap. phaea, (G) S. microloba and (H) S.
yunnanensis.
With the development of second-generation sequencing technology, more plastid genomes have
been used in phylogeny and comparative studies, and fairly good results have been obtained [12].
Generally the circular genome consists of two inverted repeats (IRs) divided by two regions, the large
(LSC) and small single-copy (SSC) regions [13,14], and most angiosperm complete plastid genomes
are between 115 and 165 kb in length [15]. The gene content and order of plastid genomes are usually
highly conserved, and the substitution rate in plastid DNA is much lower than in plant nuclear DNA
[16]. The similarity of gene length and the low substitution rate of plant plastids make them valuable
sources of genetic markers for phylogenetic studies [17].
In our previous studies, we sequenced two plastid genomes of Hap. phaea [18] and S. yunnanensis
(HB: Zhongdian population) [19] and provided preliminary phylogenetic positions for the two
species. Following on from this, we aimed to conduct comparative (simple sequence repeat (SSR),
Figure 1.
Plants of Hansenia,Haplosphaera and Sinodielsia. (
A
)Han. forbesii, (
B
)Han. forrestii,
(
C
)Han. oviformis, (
D
)Han. weberbaueriana, (
E
)Hap. himalayensis, (
F
)Hap. phaea, (
G
)S. microloba and
(H)S. yunnanensis.
With the development of second-generation sequencing technology, more plastid genomes have
been used in phylogeny and comparative studies, and fairly good results have been obtained [
12
].
Generally the circular genome consists of two inverted repeats (IRs) divided by two regions, the large
(LSC) and small single-copy (SSC) regions [
13
,
14
], and most angiosperm complete plastid genomes are
between 115 and 165 kb in length [
15
]. The gene content and order of plastid genomes are usually
highly conserved, and the substitution rate in plastid DNA is much lower than in plant nuclear
DNA [
16
]. The similarity of gene length and the low substitution rate of plant plastids make them
valuable sources of genetic markers for phylogenetic studies [17].
In our previous studies, we sequenced two plastid genomes of Hap. phaea [
18
] and S. yunnanensis
(HB: Zhongdian population) [
19
] and provided preliminary phylogenetic positions for the two species.
Following on from this, we aimed to conduct comparative (simple sequence repeat (SSR), codon
Plants 2020,9, 1523 3 of 17
usage bias, nucleotide diversity (Pi) and IR) and phylogenetic analyses of Hansenia,Haplosphaera
and Sinodielsia to reduce uncertainties in intergeneric and interspecific relationships. Analyses were
conducted using a 32 complete plastid genome dataset, which was compiled from eight newly
assembled plastid genomes from Han. forbesii,Han. forrestii,Han. oviformis,Han. weberbaueriana,
Hap. himalayensis,S. microloba,S. yunnanensis (EY: Eryuan County pop.) and S. yunnanensis (KM:
Kunming pop.), as well as previously sequenced genomes. Our study provides newly complete plastid
genomes of Hansenia,Haplosphaera and Sinodielsia species, and since these three genera are endemic
to Pan-Himalayan regions, the information provided herein is indispensable for Apiaceae plastid
evolutionary and phylogenetic studies.
2. Results and Discussion
2.1. Phylogenetic Analysis
The phylogenetic tree (Figure 2) showed that Hansenia and Haplosphaera form a strongly supported
monophyly (Maximum Likelihood-Bootstrap Support (ML-BS) =100%) in the East Asia clade.
Hap. himalayensis is sister to Han. weberbaueriana (ML-BS =67%), and Hap. phaea, the type species of
Haplosphaera, is sister to Han. forrestii (ML-BS =100%), showing that Haplosphaera is nested within
Hansenia. This suggests that Hansenia and Haplosphaera should be combined to a single genus, as already
proposed in previous phylogenetic studies. [8,9,20].
Plants 2020, 9, x FOR PEER REVIEW 3 of 19
codon usage bias, nucleotide diversity (Pi) and IR) and phylogenetic analyses of Hansenia,
Haplosphaera and Sinodielsia to reduce uncertainties in intergeneric and interspecific relationships.
Analyses were conducted using a 32 complete plastid genome dataset, which was compiled from
eight newly assembled plastid genomes from Han. forbesii, Han. forrestii, Han. oviformis, Han.
weberbaueriana, Hap. himalayensis, S. microloba, S. yunnanensis (EY: Eryuan County pop.) and S.
yunnanensis (KM: Kunming pop.), as well as previously sequenced genomes. Our study provides
newly complete plastid genomes of Hansenia, Haplosphaera and Sinodielsia species, and since these
three genera are endemic to Pan-Himalayan regions, the information provided herein is
indispensable for Apiaceae plastid evolutionary and phylogenetic studies.
2. Results and Discussion
2.1. Phylogenetic Analysis
The phylogenetic tree (Figure 2) showed that Hansenia and Haplosphaera form a strongly
supported monophyly (Maximum Likelihood-Bootstrap Support (ML-BS) = 100%) in the East Asia
clade. Hap. himalayensis is sister to Han. weberbaueriana (ML-BS = 67%), and Hap. phaea, the type species
of Haplosphaera, is sister to Han. forrestii (ML-BS = 100%), showing that Haplosphaera is nested within
Hansenia. This suggests that Hansenia and Haplosphaera should be combined to a single genus, as
already proposed in previous phylogenetic studies. [8,9,20].
Figure 2. Maximum Likelihood (ML) phylogenetic tree of Apiaceae using the 32 plastid genomes
dataset. The studied taxa are bold. The numbers above the nodes are Maximum Likelihood-Bootstrap
Figure 2.
Maximum Likelihood (ML) phylogenetic tree of Apiaceae using the 32 plastid genomes
dataset. The studied taxa are bold. The numbers above the nodes are Maximum Likelihood-Bootstrap
Support (ML-BS) presented as percentages (>50%). The names of the clades follow the study of
Zhou et al. [8,9].
Plants 2020,9, 1523 4 of 17
The tree also showed that S. microloba is sister to S. yunnanensis (HB) (ML-BS =100%) and allied with
other S. yunnanensis (EY and KM) (ML-BS =100%) populations instead of other species in the Acronema
clade. The extremely close distance between S. microloba and S. yunnanensis is unusual compared
to other Apiaceae species, even though they are congeneric [
21
–
23
]. However, their morphological
characters are distinctive (Figure 1). We speculate there may have been a hybridization phenomenon
(i.e., recent or ongoing) between the two species or other intrageneric species, which have to also use
nuclear markers to disentangle the true phylogeny of the species. Otherwise, the observed closeness
may be caused by incomplete lineage sorting. More populations of S. microloba and other Sinodielsia
species are needed to explore their relationship and evolution.
2.2. The Plastid Genomes of Hansenia, Haplosphaera and Sinodielsia Species
The complete plastid genomes of Hansenia, Haplosphaera and Sinodielsia species exhibited a typical
quadripartite organization of a single circular DNA molecule (Figure 3). The lengths of the ten
genomes (four Hansenia, two Haplosphaera and four Sinodielsia species, including the additional two
populations of S. yunnanensis) ranged from 154,670 bp (S. yunnanensis: HB) to 157,797 bp (Han. forbesii)
(Table 1). Two identical IRs (including IRa and IRb, with lengths 26,404–26,542 bp) were found in the
plastid genomes, which were separated by LSC (85,233–86,968 bp) and SSC (17,370–17,891 bp) regions.
The quadripartite organization was found in most plastid genomes of higher plants [
13
,
14
], while IRs
were absent in Taxus chinensis var. mairei,Erodium species, Pisum sativum and Vicia faba [
21
,
24
,
25
],
which may lead to a reduction in the number of duplicated genes and the length of whole plastid
genomes. In Apiaceae, the IRa and IRb were both present in the studied plastid genomes from
GenBank, Bupleurum species [
26
] and Chamaesium species [
27
] ranging from 26,280–26,303 bp and
25,727–26,147 bp, respectively, and the difference of length is mainly caused by the loss or insertion of
the spacer regions. The Hansenia,Haplosphaera and Sinodielsia plastid genomes had almost identical GC
content (37.5–37.7%) to whole plastid genomes. Higher GC content (42.7–42.8%) was detected in the
IR regions compared to the average of a whole genome, which was possibly due to the presence of
rRNA sequences with high GC content (55.2–55.3%) in IR regions. Similarly, the high GC content of
rRNA sequences also occurs in other Apiaceae species [
26
,
27
]. The ten new plastid genomes contained
133 genes, including eight rRNA, 37 tRNA and 85 protein-coding genes (PCGs) (Table 2). Among these
133 genes, 95 genes only had one copy, while 19 genes were duplicated in the IRa and IRb regions,
including four rRNA genes (rrn4.5,rrn5,rrn16 and rrn23), seven tRNA genes (trnA-UGC,trnI-CAU,
trnI-GAU,trnL-CAA,trnN-GUU,trnR-ACG and trnV-GAC) and eight PCGs (ndhB,rpl2,rpl23,rps7,
rps12,rps19,ycf1 and ycf2). Four pseudogenes—
ψ
rps19,
ψ
ycf1 and two
ψ
ycf15—were found in all the
ten genomes. In comparison, Hansenia, Haplosphaera, Sinodielsia,Bupleurum [
26
] and Chamaesium [
27
]
plastid genes are consistent in their total number of predicted coding regions.
The PCGs in the Hansenia,Haplosphaera and Sinodielsia plastid genomes included five genes (psaA,
psaB,psaC,psaI and psaJ) encoding photosystem I subunits, while 15 genes (psbA,psbB,psbC,psbD,psbE,
psbF,psbH,psbI,psbJ,psbK,psbL,psbM,psbN,psbT and psbZ) were related to photosystem II subunits.
Nine (rpl2,rpl14,rpl16,rpl20,rpl22,rpl23,rpl32,rpl33 and rpl36) encoding large ribosomal protein
genes and 12 (rps2,rps3,rps4,rps7,rps8,rps11,rps12,rps14,rps15,rps16,rps18 and rps19) encoding
small ribosomal protein genes were detected. Additionally, six genes (atpA,atpB,atpE,atpF,atpH and
atpI) of ATP synthase subunits were detected. These PCGs were also detected in Bupleurum [
26
] and
Chamaesium species [27] and are generally involved in the important process of plant growth.
Plants 2020,9, 1523 5 of 17
Plants 2020, 9, x FOR PEER REVIEW 5 of 19
Figure 3. Plastid genome map of eight Hansenia, Haplosphaera and Sinodielsia species (for a better view,
these eight maps were combined into one derived from the map of H. forbesii because they have the
same order and composition of genes). The genes shown inside and outside of the circle indicate those
transcribed in the clockwise and counterclockwise direction, respectively. Genes of different
functional groups are colored differently. The GC contents are shown in the inner circle with darker
grey.
Figure 3.
Plastid genome map of eight Hansenia,Haplosphaera and Sinodielsia species (for a better view,
these eight maps were combined into one derived from the map of H. forbesii because they have the
same order and composition of genes). The genes shown inside and outside of the circle indicate those
transcribed in the clockwise and counterclockwise direction, respectively. Genes of different functional
groups are colored differently. The GC contents are shown in the inner circle with darker grey.
Plants 2020,9, 1523 6 of 17
Table 1.
The features of plastid genomes of eight Hansenia,Haplosphaera and Sinodielsia species (IRs: inverted repeats; LSC: large single-copy region; SSC: small
single-copy region).
Taxa Size (bp) LSC Length
(bp)
IR Length
(bp)
SSC Length
(bp)
Total
Genes
Protein Coding
Genes
tRNA
Genes
rRNA
Genes
Overall GC
Content (%)
Hansenia forbesii 157,797 86,968 26,517 17,795 131 85 37 8 37.6
Hansenia forrestii 156,911 86,245 26,435 17,796 131 85 37 8 37.6
Hansenia oviformis 157,175 86,412 26,494 17,775 131 85 37 8 37.6
Hansenia weberbaueriana 156,778 86,246 26,432 17,668 131 85 37 8 37.7
Haplosphaera himalayensis 157,286 86,479 26,542 17,723 131 85 37 8 37.6
Haplosphaera phaea 157,271 86,488 26,446 17,891 131 85 37 8 37.6
Sinodielsia microloba 155,709 85,320 26,479 17,431 131 85 37 8 37.5
Sinodielsia yunnanensis (EY) 155,552 85,276 26,441 17,394 131 85 37 8 37.6
Sinodielsia yunnanensis (HB) 154,670 84,446 26,427 17,370 131 85 37 8 37.6
Sinodielsia yunnanensis (KM) 155,435 85,233 26,404 17,394 131 85 37 8 37.5
Plants 2020,9, 1523 7 of 17
Table 2. List of genes encoded in the ten Hansenia,Haplosphaera and Sinodielsia plastid genomes.
Category Group of Genes Name of Genes
Self-replication
transfer RNAs (tRNAs)
trnA-UGC *, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU,
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
ribosomal RNAs
(rRNAs) rrn4.5 *, rrna5 *, rrn16 *, rrn23 *
RNA polymerase rpoA, rpoB, rpoC1, rpoC2
Small subunit of
ribosomal proteins
(SSU)
rps2, rps3, rps4, rps7 *, rps8, rps11, rps12, rps14, rps15, rps16, rps18, rps19 *
Large subunit of
ribosomal proteins
(LSU)
rpl2 *, rpl14, rpl16, rpl20, rpl22, rpl23 *, rpl32, rpl33, rpl36
Genes involved in
photosynthesis
Subunits of
NADH-dehydrogenase
ndhA, ndhB *, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
Subunits in
photosystem I psaA, psaB, psaC, psaI, psaJ
Subunits in
photosystem II
psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ,psbK, psbL, psbM, psbN,
psbT, psbZ
Subunits of cytochrome
b/f complex petA, petB, petD, petG, petL, petN
Subunits of ATP
synthase atpA, atpB, atpE, atpF, atpH, atpI
Large subunit of
rubisco rbcL
Other genes
Translational initiation
factor infA
Protease clpP
Maturase matK
Subunit of
Acetyl-CoA-carboxylase
accD
Envelope membrane
protein cemA
C-type cytochrome
synthesis gene ccsA
Conserved reading
frames
Conserved open
reading frames ψrps19, ycf1 * (ycf1, ψycf1), ycf2 *, ycf3, ycf4, ψycf15
* Duplicated genes.
2.3. IR Boundaries and Simple Sequence Repeats (SSRs) Structure Analysis
The IR boundaries of the ten Hansenia,Haplosphaera and Sinodielsia plastid genomes were compared
to analyze the fluctuations (expansion or contraction) in these regions (Figure 4). Although the ten
plastid genomes showed a similar structure and content, some variations were still identified. The rps19
gene entered the IRb region with 46 bp, 46 bp, 46 bp, 41 bp, 54 bp and 46 bp in the plastid genomes
of Han. forbesii,Han. forrestii,Han. oviformis,Han. weberbaueriana,Hap. himalayensis and Hap. phaea
(respectively), while 102 bp, 102 bp, 102 bp and 102 bp were entered in the plastid genomes of
S. microloba,S. yunnanensis (EY), S. yunnanensis (HB) and S. yunnanensis (KM), respectively. The ndhF
genes of Han. forrestii,Han. oviformis,Han. weberbaueriana,Hap. himalayensis,Hap. phaea,S. microloba
and S. yunnanensis (HB) are entirely within the SSC region, and a 6–48 bp intergenic region exists
between the ndhF gene and the JSB line (the border between IRb and SSC), while the ndhF genes of
Han. forbesii,S. yunnanensis (EY) and S. yunnanensis (KM) are partly included in IRb regions with 7 bp,
Plants 2020,9, 1523 8 of 17
5 bp and 5 bp, respectively. The ycf1 gene of the ten plastid genomes occupies the JSA line (the border
between SSC and IRa), with a length ranging from 1961 to 1997 bp in IRa regions, and ranging from 3475
to 3523 bp in close SSC regions. This also created a pseudogene ycf1 in the IRb regions. The trnH genes
of Han. forbesii,Han. forrestii,Han. oviformis,Han. weberbaueriana,Hap. himalayensis and Hap. phaea are
entirely within the LSC region. A 2–65 bp length of the intergenic region exists between the trnH genes
and JLA line (the border between IRa and SSC), while the trnH gene of S. microloba,S. yunnanensis (EY),
S. yunnanensis (HB) and S. yunnanensis (KM) is included in IRa regions with 1 bp.
Plants 2020, 9, x FOR PEER REVIEW 8 of 19
2.3. IR Boundaries and Simple Sequence Repeats (SSRs) Structure Analysis
The IR boundaries of the ten Hansenia, Haplosphaera and Sinodielsia plastid genomes were
compared to analyze the fluctuations (expansion or contraction) in these regions (Figure 4). Although
the ten plastid genomes showed a similar structure and content, some variations were still identified.
The rps19 gene entered the IRb region with 46 bp, 46 bp, 46 bp, 41 bp, 54 bp and 46 bp in the plastid
genomes of Han. forbesii, Han. forrestii, Han. oviformis, Han. weberbaueriana, Hap. himalayensis and Hap.
phaea (respectively), while 102 bp, 102 bp, 102 bp and 102 bp were entered in the plastid genomes of
S. microloba, S. yunnanensis (EY), S. yunnanensis (HB) and S. yunnanensis (KM), respectively. The ndhF
genes of Han. forrestii, Han. oviformis, Han. weberbaueriana, Hap. himalayensis, Hap. phaea, S. microloba
and S. yunnanensis (HB) are entirely within the SSC region, and a 6–48 bp intergenic region exists
between the ndhF gene and the JSB line (the border between IRb and SSC), while the ndhF genes of
Han. forbesii, S. yunnanensis (EY) and S. yunnanensis (KM) are partly included in IRb regions with 7
bp, 5 bp and 5 bp, respectively. The ycf1 gene of the ten plastid genomes occupies the JSA line (the
border between SSC and IRa), with a length ranging from 1961 to 1997 bp in IRa regions, and ranging
from 3475 to 3523 bp in close SSC regions. This also created a pseudogene ycf1 in the IRb regions. The
trnH genes of Han. forbesii, Han. forrestii, Han. oviformis, Han. weberbaueriana, Hap. himalayensis and
Hap. phaea are entirely within the LSC region. A 2–65 bp length of the intergenic region exists between
the trnH genes and JLA line (the border between IRa and SSC), while the trnH gene of S. microloba, S.
yunnanensis (EY), S. yunnanensis (HB) and S. yunnanensis (KM) is included in IRa regions with 1 bp.
Figure 4. Comparisons of inverted repeat (IR) boundaries between ten Hansenia, Haplosphaera and
Sinodielsia plastid genomes.
We found that the lengths of rps19 and trnH genes entering IRs are similar between Hansenia
(36–41 bp) and Haplosphaera (41–49 bp), which is different from these genes in Sinodielsia (102 bp). Not
Figure 4.
Comparisons of inverted repeat (IR) boundaries between ten Hansenia,Haplosphaera and
Sinodielsia plastid genomes.
We found that the lengths of rps19 and trnH genes entering IRs are similar between Hansenia
(36–41 bp) and Haplosphaera (41–49 bp), which is different from these genes in Sinodielsia (102 bp).
Not surprisingly, Hansenia is more closely related to Haplosphaera but distantly related to Sinodielsia,
which has also been confirmed by nrITS sequences and transcriptome data [
8
,
20
]. A collective analysis
of our results and previous studies [
26
–
29
] found that the lengths of rps19 and trnH genes entering IRs
are usually stable in the same genus or phylogenetically related groups. For example, the length of
rps19 gene entering IRs is 57–96 bp in Chamaesium species [
27
], and 49–84 bp in Bupleurum species [
26
],
which are the phylogenetically basal groups in the Chamaesium clade and Bupleureae. Whereas in
Apieae members, such as Anethum,Apium and Petroselium, the rps19 genes are not in the IRs. Instead,
the rpl2 genes are partly in the IRs [
26
,
30
]. Although the rpl2 genes are partly duplicated in the IRs of
some Apieae members, they are duplicated completely in the Hansenia,Haplosphaera and Sinodielsia
species. As for the two pseudogenes
ψ
rps19 and
ψ
ycf1 detected in the plastid genomes of Hansenia,
Haplosphaera and Sinodielsia, their incomplete duplication may be caused by the fluctuations in the
Plants 2020,9, 1523 9 of 17
IR boundary. The fluctuations may lead to gene loss (or pseudogene loss), which is a common
phenomenon in Apiaceae species [
26
,
27
]. Nevertheless, the length of land plants IRs can vary from
10 to 76 kbp, with most species having an IR of about 25 kbp, and often less than 15 kbp in lower
land plants and fern species [
31
]. Fluctuations in IR regions are the main reason for the differences in
plastid genome lengths in most species, which also leads to several genes entering the IR regions or the
single-copy sequences [32].
Sixty-six, 72, 65, 62, 65, 75, 61, 57, 55 and 56 SSRs were identified in the plastid genomes of
Han. forbesii,Han. forrestii,Han. oviformis,Han. weberbaueriana,Hap. himalayensis,Hap. phaea,S. microloba,
S. yunnanensis (EY), S. yunnanensis (HB) and S. yunnanensis (KM), respectively. The results hint that
plastids of related groups share similar numbers of SSRs, while that is not always the result [
26
].
Six repeat motifs (mono-, di-, tri-, tetra-, penta- and hexanucleotide repeats) of microsatellites were
detected in the plastid genome of the Hansenia,Haplosphaera and Sinodielsia species. Mononucleotide
repeats were the most abundant SSR, which accounted for 58.47% of Hansenia, 60.21% of Haplosphaera
and 48.01% of Sinodielsia. Dinucleotide SSRs were the second most abundant SSR, which accounted
for 18.02% of Hansenia, 17.69% of Haplosphaera and 29.29% of Sinodielsia. This was followed by
tetranucleotide repeats of 8.69%, 9.28% and 4.79%, and trinucleotide repeats of 10.65%, 11.38% and
14.41% in Hansenia, Haplosphaera and Sinodielsia, respectively. Pentanucleotide and hexanucleotide
were the least abundant SSR (average of the three genera: 2.73% and 0.62%, respectively) (Figure 5).
The repeat motifs with the highest content in SSRs were all mononucleotide, which is similar to
most species including Apiaceae plants [
26
,
27
], but different from Forthysia (dinucleotide) [
33
] and
Nitotiana (trinucleotide) species [
34
]. The similar contents of mononucleotide and dinucleotide repeats
of Hansenia and Haplosphaera indicates that both genera seem to belong together. In all Hansenia,
Haplosphaera and Sinodielsia species the repeats were composed almost entirely of A/T, except for
mononucleotide repeat motifs that also had G/C contents of 10.53%, 2.33%, 10.81%, 7.32% and 4.65 in
Han. forbesii,Han. forrestii,Han. weberbaueriana,Hap. himalayensis and Hap. phaea. The phylogenetic
closeness of these species suggests that the relatives may share similar mononucleotide repeated
contents. Most dinucleotide repeats were AT/TA, except for one TC repeat that was in the plastid
genome of S. yunnanensis (HB). Across all SSR loci, 94 SSRs (14.83%) were found in the IRs, 417 SSRs
(65.77%) in LSC regions and 123 SSRs (19.40%) in SSC regions of the plastid genomes (Figure 6).
Plants 2020, 9, x FOR PEER REVIEW 10 of 19
Figure 5. Frequency of detected simple sequence repeat (SSR) motifs in different repeat types in ten
Hansenia, Haplosphaera and Sinodielsia plastid genomes.
Figure 5.
Frequency of detected simple sequence repeat (SSR) motifs in different repeat types in ten
Hansenia,Haplosphaera and Sinodielsia plastid genomes.
Plants 2020,9, 1523 10 of 17
Plants 2020, 9, x FOR PEER REVIEW 11 of 19
Figure 6. Frequency of detected SSR motifs in LSC (blue), IR (orange) and SSC (green) regions of the
ten Hansenia, Haplosphaera and Sinodielsia plastid genomes.
2.4. Codon Usage Analysis
The codon usage bias and relative synonymous codon usage (RSCU) values were calculated
using 53 PCGs in the ten Hansenia, Haplosphaera and Sinodielsia plastid genomes. There was no evident
divergence of the codon usage frequency when we compared the three genera (Figure 7). The number
of codons of PCGs ranged from 21,134 (S. yunnanensis: KM) to 21,254 (Han. weberbaueriana)
(Supplementary Table S2). Among these codons, leucine was encoded by 2227–2236 and cysteine was
encoded by 214–224 codons, which presented the maximum and the minimum number of codons per
amino acids in our study species, respectively. AUU (850–872) involved in encoding isoleucine and
UAG (13–14) involved in encoding a terminator were the most and least used codons. Furthermore,
30 codons of Hansenia and Haplosphaera, and 31 codons of Sinodielsia plastid genomes had RSCU
values larger than 1, indicating that they were the preferred codons in those ten plastid genomes.
Among these 30/31 preferred codons, most codons terminated in A/T, except that UUG ended with
G, and C was not found at the third position. This demonstrated that the codon usages of the ten
plastid genomes were biased towards A/T at the third position of codons, which is generally
consistent with other reported genomes of angiosperm [35,36], including the Bupleurum [26] and
Chamaesium [27] species.
Figure 6.
Frequency of detected SSR motifs in LSC (blue), IR (orange) and SSC (green) regions of the
ten Hansenia,Haplosphaera and Sinodielsia plastid genomes.
2.4. Codon Usage Analysis
The codon usage bias and relative synonymous codon usage (RSCU) values were calculated
using 53 PCGs in the ten Hansenia,Haplosphaera and Sinodielsia plastid genomes. There was no
evident divergence of the codon usage frequency when we compared the three genera (Figure 7).
The number of codons of PCGs ranged from 21,134 (S. yunnanensis: KM) to 21,254 (Han. weberbaueriana)
(Supplementary Table S2). Among these codons, leucine was encoded by 2227–2236 and cysteine was
encoded by 214–224 codons, which presented the maximum and the minimum number of codons per
amino acids in our study species, respectively. AUU (850–872) involved in encoding isoleucine and
UAG (13–14) involved in encoding a terminator were the most and least used codons. Furthermore,
30 codons of Hansenia and Haplosphaera, and 31 codons of Sinodielsia plastid genomes had RSCU values
larger than 1, indicating that they were the preferred codons in those ten plastid genomes. Among these
30/31 preferred codons, most codons terminated in A/T, except that UUG ended with G, and C was not
found at the third position. This demonstrated that the codon usages of the ten plastid genomes were
biased towards A/T at the third position of codons, which is generally consistent with other reported
genomes of angiosperm [35,36], including the Bupleurum [26] and Chamaesium [27] species.
Codon usage bias is an important indicator for studying the evolutionary relationship of
genomes [
37
]. Studies have shown that many biological factors affect the preference of synonymous
codon usage, such as gene expression level [
38
], gene sequence length [
39
], tRNA abundance [
40
]
and GC distribution position [
41
]. Codon bias seems to be maintained by selection–mutation–drift
balance [
42
]. There is a general agreement that the strong bias towards highly expressed genes is due
to selection for speed or translational efficiency [
43
]. Our studied species of Hansenia,Haplosphaera and
Sinodielsia are from the Himalayan and Hengduan regions, usually inhabiting alpine areas over 3000 m,
and one of the reasons for their similar RSCU values may be the shared natural selection pressures,
even though they are not all closely related. This may provide another analytical technique to use
when studying nuclear genomes and can assist in understanding how species of separate lineages,
yet similar environments, have undergone parallel evolution. Increased numbers and improved
methods of analytical techniques are especially useful when studying many Pan-Himalayan region
Apiaceae species that are morphologically similar.
Plants 2020,9, 1523 11 of 17
Plants 2020, 9, x FOR PEER REVIEW 12 of 19
Figure 7. Codon content of 20 amino acids and the stop codon present in all 53 studied protein-coding
genes (PCGs) of the (A) Hansenia, (B) Haplosphaera and (C) Sinodielsia plastid genome. Different colors
of the histogram correspond to the different codons below.
Codon usage bias is an important indicator for studying the evolutionary relationship of
genomes [37]. Studies have shown that many biological factors affect the preference of synonymous
codon usage, such as gene expression level [38], gene sequence length [39], tRNA abundance [40] and
GC distribution position [41]. Codon bias seems to be maintained by selection–mutation–drift balance
[42]. There is a general agreement that the strong bias towards highly expressed genes is due to
selection for speed or translational efficiency [43]. Our studied species of Hansenia, Haplosphaera and
Sinodielsia are from the Himalayan and Hengduan regions, usually inhabiting alpine areas over 3000
m, and one of the reasons for their similar RSCU values may be the shared natural selection pressures,
even though they are not all closely related. This may provide another analytical technique to use
when studying nuclear genomes and can assist in understanding how species of separate lineages,
yet similar environments, have undergone parallel evolution. Increased numbers and improved
methods of analytical techniques are especially useful when studying many Pan-Himalayan region
Apiaceae species that are morphologically similar.
Figure 7.
Codon content of 20 amino acids and the stop codon present in all 53 studied protein-coding
genes (PCGs) of the (
A
)Hansenia, (
B
)Haplosphaera and (
C
)Sinodielsia plastid genome. Different colors
of the histogram correspond to the different codons below.
2.5. Nucleotide Diversity Analysis
Nucleotide diversity (Pi) values of the plastid genomes from the Hansenia,Haplosphaera and
Sinodielsia species were calculated to evaluate their sequence divergence level (Figure 8). In the four
Hansenia genomes, Pi values in the LSC regions ranged from 0 to 0.01889, with an average of 0.00371,
and ranged from 0 to 0.01472 in the SSC regions, with an average of 0.00263. The Pi values of IR regions
ranged from 0 to 0.00639 and had an average of 0.00089, the lowest Pi values of the three regions for
these four genomes. In the two Haplosphaera genomes, Pi values ranged from 0 to 0.04333 and averaged
0.00531 in the LSC regions, and from 0 to 0.02 in the SSC regions, with an average value of 0.0028.
The IR region values in the two Haplosphaera genomes were similarly low, with an average of 0.0015
and ranging from 0 to 0.01333. The two Haplosphaera species with high Pi values seem to be distant
species within the genus Hansenia (see further under phylogenetic studies). In the two Sinodielsia
genomes, Pi values ranged from 0 to 0.01222 with an average of 0.0021 in the LSC regions, and from
0 to 0.01028, averaging 0.00149 in the SSC regions. The Pi values of IR regions were again low for
the two Sinodielsia genomes and ranged from 0 to only 0.00833, with a value of 0.00112. Pi values
indicated mutations in the respective regions [
26
]. The Pi values of Sinodielsia were the lowest of the
three genera, indicating that S. microloba is more closely related to S. yunnanensis than the other species
within each genus.
Plants 2020,9, 1523 12 of 17
Plants 2020, 9, x FOR PEER REVIEW 14 of 19
Figure 8. The nucleotide diversity of (A) the four Hansenia, (B) the two Haplosphaera and (C) the four
Sinodielsia plastid genomes. The positions of 0.02 in all three graphs were marked by a line. Ten or
seven regions with the highest Pi values were named out.
We found that high Pi values of sequences were usually detected in spacer regions between
genes. Among these genomic spacer regions of the Hansenia species, trnK (UUU)-rps16, psbK-psbI,
trnE (UUC)-trnT (GGU), rps4-trnL (UAA), petL-psaJ, rpl22-rps19 and ndhG-ndhI had the highest Pi
values, ranging from 0.01083 to 0.01889. The highest Pi values of spacer regions of the Haplosphaera
species ranged between 0.015 and 0.04333 for trnH (GUG)-psbA, atpH-atpI, rps4-trnT (UGU), ycf4-
cemA, petB-petD and ndhG-ndhI. Within the spacer regions of Sinodielsia species, matK-rps16, trnE
(UUC)-trnT (GGU), ycf3-trnS (GGA), trnL (UAA)-ndhJ, rpl33-rps18, rps11-infA, rps3-rps19, ycf1-ndhF,
ndhF-rpl32 and ndhG-ndhI had the highest Pi values, ranging from 0.00722 to 0.01222. Although the
Figure 8.
The nucleotide diversity of (
A
) the four Hansenia, (
B
) the two Haplosphaera and (
C
) the four
Sinodielsia plastid genomes. The positions of 0.02 in all three graphs were marked by a line. Ten or
seven regions with the highest Pi values were named out.
We found that high Pi values of sequences were usually detected in spacer regions between
genes. Among these genomic spacer regions of the Hansenia species, trnK (UUU)-rps16,psbK-psbI,
trnE (UUC)-trnT (GGU),rps4-trnL (UAA),petL-psaJ,rpl22-rps19 and ndhG-ndhI had the highest Pi
values, ranging from 0.01083 to 0.01889. The highest Pi values of spacer regions of the Haplosphaera
species ranged between 0.015 and 0.04333 for trnH (GUG)-psbA,atpH-atpI,rps4-trnT (UGU),ycf4-cemA,
petB-petD and ndhG-ndhI. Within the spacer regions of Sinodielsia species, matK-rps16,trnE (UUC)-trnT
(GGU),ycf3-trnS (GGA),trnL (UAA)-ndhJ,rpl33-rps18,rps11-infA,rps3-rps19,ycf1-ndhF,ndhF-rpl32 and
Plants 2020,9, 1523 13 of 17
ndhG-ndhI had the highest Pi values, ranging from 0.00722 to 0.01222. Although the spacer regions
were diverse among the three genera, we found the spacer regions ndhG-ndhI occurred in all three
genera with relatively high values. Pending more specimen samples and plastid genome studies of
Chinese Apiaceae, the spacer regions (e.g., high Pi value ndhG-ndhI) may provide DNA barcodes for
molecular identification and phylogenetic studies in the large-scale clades such as East Asia clade and
Acronema clade, where there are many parallel branches whose support rate is less than 50% using ITS
or plastid DNA introns (rpl16 and rps16 genes) [8,9].
3. Materials and Methods
3.1. Material, DNA Extraction and Complete Genome Sequencing
The materials of Han. forbesii,Han. forrestii,Han. oviformis,Han. weberbaueriana,Hap. himalayensis,
S. microloba, S. yunnanensis (EY) and S. yunnanensis (KM) were newly obtained for this study, which were
collected from the type localities or their adjacent areas during 2018–2019 (Supplementary Table S1).
The different populations of S. yunnanensis are represented by EY: from Eryuan County, Yunnan; KM:
from Kunming, and Yunnan; HB: from Zhongdian, Yunnan (HB previously sequenced [
19
]). The total
genomic DNA was extracted from silica gel-dried leaves according to the protocols of the plant genomic
DNA kit (cwbio, Beijing, China), then sequenced using the Illumina Novaseq 6000 platform (Illumina,
San Diego, CA, USA) with Novaseq 150 sequencing strategy by Novogene (Beijing, China).
3.2. Genome Construction and Annotation
The clean data (removed connectors and low-quality reads) were assembled using NOVOPlasty
2.7.1 [
44
] with K-mer 39, where the rbcL gene of Han. oviformis and S. yunnanensis (amplificated and
sequenced beforehand) was used as a seed input and the reference sequence. The assembled complete
plastid genomes were checked then aligned with the reference plastid genome of Chuanminshen violaceum
(KU921430) using GENEIOUS R11 [
45
] to select the best option, which was then annotated using
PGA [
46
]. GENEIOUS R11 was then used to manually adjust the annotation for uncertain start and
stop codons based on the comparison with homologous genes from other annotated plastid genomes.
The eight annotated plastid genomes were submitted to GenBank, and their accession numbers are in
Supplementary Table S1. Their genome maps were drawn using OGDRAW version 1.3.1 [47].
3.3. Phylogenetic Analysis
To better infer phylogenetic relationships between Hansenia,Haplosphaera and Sinodielsia,
the 32 plastid genomes were applied for reconstructing the phylogenetic tree, of which 24 plastid
genomes were obtained from GenBank (Supplementary Table S1). All the plastid genomes were aligned
using MAFFT v7.308 [
48
,
49
]. Maximum Likelihood was then conducted for phylogenetic analyses
using RAxML version 8.2.4 [
50
] under the model GTR+G with 1000 rapid bootstraps. The scientific
names of plants followed the International Plant Names Index (https://www.ipni.org). Chamaesium and
Bupleurum species were selected as outgroups [8,9].
3.4. IRs Boundaries and SSR Analysis
The boundaries between the LSC, SSC and IR regions of the ten plastid genomes were compared
and drawn using the online program: IRscope (https://irscope.shinyapps.io/irapp/) [
51
]. The SSRs
were identified using MISA [
52
] with the repeat threshold settings: 10 repeats for mono-nucleotide,
5 for di-nucleotide, 4 for tri-nucleotide and 3 repeats for tetra-, penta-and hexanucleotide SSRs.
3.5. Codon Usage Bias Analysis
Codon usage bias analysis and calculation of RSCU values were conducted using the program
CodonW [
53
]. Fifty-three PCGs (more than 300 bp in length) of each Hansenia,Haplosphaera and
Sinodielsia plastid genomes were filtered. The codon adaptation index (CAI), the codon bias index (CBI),
Plants 2020,9, 1523 14 of 17
the effective number of codons (ENC), the frequency of optimal codons (Fop) and the GC content of the
synonymous third codons positions (GC3s) were calculated to assess the extent of the codon usage bias.
The RSCU values of the four Hansenia, two Haplosphaera and four Sinodielsia plastid genomes were also
calculated to assess their codon usages, including Hap. phaea and S. yunnanensis (HB) downloaded
from GenBank (Supplementary Table S1).
3.6. Nucleotide Diversity Analysis
The plastid genomes of Hansenia,Haplosphaera and Sinodielsia species were aligned using MAFFT
v7.308 [
48
,
49
]. DNA polymorphism analysis was then conducted to calculate the Pi values in DnaSP
v5 [
54
] in the sliding window. The setting parameters were as follows: (1) windows length: 600 sites;
(2) step size: 200 sites.
4. Conclusions
In this study, the plastid genomes of Han. forbesii,Han. forrestii,Han. oviformis,Han. weberbaueriana,
Hap. himalayensis,S. microloba,S. yunnanensis (KM) and S. yunnanensis (EY) were newly assembled
and annotated. All the eight plastid genomes exhibited a typical circular quadripartite organization
with similar whole length (155,435 bp to 157,797 bp) and gene contents. The IR boundary analysis
showed that length of rps19 and ycf1 genes entering IRs are usually stable in the same genus.
Hansenia shared relatively similar mononucleotide SSRs to Haplosphaera. Additionally, Hansenia,
Haplosphaera and Sinodielsia species had similar codon usage. Although Hansenia and Haplosphaera are
not phylogenetically close to Sinodielsia, there was no significant difference in their plastid genomes.
Furthermore, we found that the ndhG-ndhI spacer regions possessed higher nucleotide diversity in
the three genera and, therefore, may provide DNA barcodes for intra- and inter-genus identification
in Apiaceae. The phylogeny of the 32 plastid genomes, including the eight taxa mentioned above,
showed a close relationship between Hansenia and Haplosphaera, and S. microloba may be a species
of hybrid origin. This study will enrich the complete plastid genome dataset of the genera Hansenia,
Haplosphaera and Sinodielsia, and has provided a new insight into comparisons of distant taxa and
phylogeny reconstruction using complete plastid genomes of Apiaceae.
Supplementary Materials:
The following are available online at http://www.mdpi.com/2223-7747/9/11/1523/s1,
Table S1: Voucher details and GenBank accession numbers of taxa used in this study, Table S2: The indexes of the
codon usage bias in the Hansenia,Haplosphaera and Sinodielsia species.
Author Contributions:
Conceptualization, W.G., S.-B.J., X.-L.G., S.-D.Z. and X.-J.H.; Data curation, W.G. and
S.-B.J.; Formal analysis, W.G. and S.-B.J.; Funding acquisition, S.-D.Z. and X.-J.H.; Investigation, W.G. and S.-B.J.;
Methodology, W.G. and S.-B.J.; Resources, W.G. and S.-B.J.; Software, W.G. and S.-B.J.; Supervision, S.-D.Z. and
X.-J.H.; Writing-original draft, W.G. and S.-B.J.; Writing-review & editing, W.G., S.-B.J., M.P., X.-L.G., S.-D.Z. and
X.-J.H. All authors have read and agreed to the published version of the manuscript.
Funding:
This work was supported by the National Natural Science Foundation of China (Grant Nos. 31872647).
The Chinese Ministry of Science and Technology throng the National Science and Technology Infrastructure
Platform Project (Grant No. 2005DKA21403-JK). The fourth national survey of traditional Chinese medicine
resources (Grant No. 2019PC002).
Acknowledgments: We would like to thank Yan Yu, Dan-Mei Su and Fu-Min Xie for the help in software use.
Conflicts of Interest: The authors declare no conflict of interest.
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