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\begin{document}

\title{Complete chloroplast genomes of hemiparasitic genus Cymbaria: Insights
into comparative analysis, development of molecular markers, and
phylogenetic relationships}



\author[1]{Yang Ma}%
\author[2]{Jordi López-Pujol}%
\author[1]{Dongqing Yan}%
\author[1]{Zhen Zhou}%
\author[1]{Zekun Deng}%
\author[1]{Jianming  Niu}%
\affil[1]{Inner Mongolia University}%
\affil[2]{Botanic Institute of Barcelona}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Cymbaria L. is a small genus nearly endemic to the Mongolian Plateau
with only two hemiparasitic species. Cymbaria daurica L. is an important
herb ``Xinba'' in traditional Mongolian medicine. However, C. daurica is
often confused with its sister species Cymbaria mongolica Maxim. because
they only differ in anther morphology. In addition, phylogenetic
placement and reductive evolution of this genus within the Orobanchaceae
remain poorly resolved. Here, we firstly sequenced two Cymbaria
chloroplast genomes and then conducted a comparative analysis among
those of 54 Orobanchaceae species. We found that the chloroplast genomes
of Cymbaria species were characterized by pseudogenization/loss events
of eight ndh genes and a unique rbcL-matK inversion. Unlike the
high-variability observed in holoparasites, Cymbaria and other
hemiparasites exhibited high similarity to autotrophs in genome size, GC
content, and intact genes. Notely, four pairs of specific DNA barcodes
(CymN1, CymN2, CymY, and CymR) were developed to distinguish the herb C.
daurica from its adulterant C. mongolica. Phylogenetic analyses revealed
that genus Cymbaria and the Schwalbea-Siphonostegia clade were grouped
into the tribe Cymbarieae. This tribe formed an independent sister clade
to the remaining parasitic lineages. Furthermore, the diversification of
monophyletic Cymbaria occurred during the late Miocene (6.72 Mya) in the
Mongol-Chinese steppe region. These findings provide valuable
information for regarding taxonomy, phylogeny and evolution of Cymbaria
and can be used to validate the authenticity of the herb ``Xinba''.%
\end{abstract}%



\sloppy


\textbf{RESEARCH ARTICLE}

Complete chloroplast genomes of hemiparasitic genus \emph{Cymbaria} :
Insights into comparative analysis, development of molecular markers,
and phylogenetic relationships

Yang Ma \textsuperscript{1}, Jordi L\selectlanguage{ngerman}ópez-Pujol \textsuperscript{2, 3},
Dongqing Yan \textsuperscript{1}, Zhen Zhou \textsuperscript{1}, Zekun
Deng \textsuperscript{1} and Jianming Niu \textsuperscript{1, 4, 5}

\textsuperscript{1} School of Ecology and Environment, Inner Mongolia
University, Hohhot 010020, China

\textsuperscript{2} Botanic Institute of Barcelona (IBB),
CSIC-Ajuntament de Barcelona, Barcelona 08038, Spain

\textsuperscript{3} Escuela de Ciencias Ambientales, Universidad
Espíritu Santo (UEES), Samborondón 091650, Ecuador

\textsuperscript{4} Ministry of Education Key Laboratory of Ecology and
Resource Use of the Mongolian Plateau, Hohhot 010020, China

\textsuperscript{5} Inner Mongolia Key Laboratory of Grassland Ecology
and the Candidate State Key Laboratory of Ministry of Science and
Technology, Hohhot 010020, China

Correspondence

Jianming Niu, School of Ecology and Environment, Inner Mongolia
University, Hohhot 010020, China; Ministry of Education Key Laboratory
of Ecology and Resource Use of the Mongolian Plateau, Hohhot 010020,
China; Inner Mongolia Key Laboratory of Grassland Ecology and the
Candidate State Key Laboratory of Ministry of Science and Technology,
Hohhot 010020, China

Email: \emph{111964401@imu.edu.cn}

Funding information

Major Science and Technology Projects of Inner Mongolia Autonomous
Region, Grant/ Award Number: 2019ZD008; National Natural Science
Foundation of China, Grant/ Award Number: 31860106 and 32260304

\textbf{Abstract}

\emph{Cymbaria} L. is a small genus nearly endemic to the Mongolian
Plateau with only two hemiparasitic species. \emph{Cymbaria daurica} L.
is an important herb ``Xinba'' in traditional Mongolian medicine.
However, \emph{C. daurica} is often confused with its sister
species\emph{Cymbaria mongolica} Maxim. because they only differ in
anther morphology. In addition, phylogenetic placement and reductive
evolution of this genus within the Orobanchaceae remain poorly resolved.
Here, we firstly sequenced two \emph{Cymbaria} chloroplast genomes and
then conducted a comparative analysis among those of 54 Orobanchaceae
species. We found that the chloroplast genomes of \emph{Cymbaria}species
were characterized by pseudogenization/loss events of eight\emph{ndh}
genes and a unique \emph{rbc} L-\emph{mat} K inversion. Unlike the
high-variability observed in holoparasites, \emph{Cymbaria} and other
hemiparasites exhibited high similarity to autotrophs in genome size, GC
content, and intact genes. Notely, four pairs of specific DNA barcodes
(CymN1, CymN2, CymY, and CymR) were developed to distinguish the herb
\emph{C. daurica} from its adulterant \emph{C. mongolica} . Phylogenetic
analyses revealed that genus \emph{Cymbaria} and the\emph{Schwalbea}
-\emph{Siphonostegia} clade were grouped into the tribe Cymbarieae. This
tribe formed an independent sister clade to the remaining parasitic
lineages. Furthermore, the diversification of monophyletic
\emph{Cymbaria} occurred during the late Miocene (6.72 Mya) in the
Mongol-Chinese steppe region. These findings provide valuable
information for regarding taxonomy, phylogeny and evolution
of\emph{Cymbaria} and can be used to validate the authenticity of the
herb ``Xinba''.

\textbf{Keywords}

Orobanchaceae, Hemiparasite, Plastome, Mongolian medicinal herb, DNA
barcoding, Reductive evolution

\textbf{TAXONOMY CLASSIFICATION}

Botany, Genomics, Phylogenetics, Taxonomy

1 \textbar{} \textbf{Introduction}

Plastids serve as crucial organelles that carry out photosynthesis in
green plants. Generally, autotrophic plants are highly conserved in the
chloroplast genomes (Wicke, Schneeweiss, dePamphilis, Müller, \& Quandt,
2011). By contrast, heterotrophic plants including mycoheterotrophs and
parasites exhibit varying degrees of degeneration in their chloroplast
genomes (Graham, Lam, \& Merckx, 2017). The family Orobanchaceae is
recognized as the largest single family of parasites, consisting of 101
genera and over 2100 species with autotrophic, hemiparasitic, and
holoparasitic lifestyles (Byng et al., 2016; Nickrent, 2020).
Orobanchaceae is thus considered as an excellent model to elaborate the
parasitic plants evolution (Westwood et al., 2012). Most previous
studies have focused on autotrophs and holoparasites (Xiaoqing Liu et
al., 2019; Wicke \& Naumann, 2018; Zeng et al., 2017). However, limited
investigations have been done on hemiparasites (Xin Li et al., 2021; R.
Zhang et al., 2020). The hemiparasitic tribe Cymbarieae is widely
distributed in Eurasia and comprises approximately 20 species in five
genera (Schneeweiss, 2013). Cymbarieae was originally thought to be
sister to various parasitic lineages in the family Orobanchaceae
(Bennett \& Mathews, 2006; McNeal, Bennett, Wolfe, \& Mathews, 2013),
while analysis of the tribe Orobancheae suggests that this original
classification merits reconsideration (Xi Li, Feng, Randle, \&
Schneeweiss, 2019). Furthermore, although chloroplast genomes of
\emph{Schwalbea americana}(Wicke et al., 2013) and \emph{Siphonostegia
chinensis} (Gao et al., 2019; Jiang et al., 2022) have been previously
published, no systematic comparative analysis of chloroplast genomes of
various groups within the tribe Cymbarieae has been conducted to date.

\emph{Cymbaria} L. \emph{sensu stricto} (i.e.,
excluding\emph{Cymbochasma} Endl.) is the type genus of the tribe
Cymbarieaea and usually parasitizes on roots of \emph{Stipa} (Poaceae)
and\emph{Caragana} (Fabaceae). It is now generally believed that this
genus includes only two facultative~hemiparasites (Zhao, 1999). \emph{C.
mongolica} Maxim. is endemic to the Loess Plateau of China,
while\emph{C. daurica} L. is a characteristic species of Mongolian
Plateau steppe. According to the Chinese Pharmacopoeia and the Mongolian
Medicinal Materials Standard, \emph{C. daurica} is the only original
plant species for elaborating the traditional Mongolian medicine, which
is referred to as ``Kanba-Arong'' in Mongolian and ``Xinba'' in Chinese.
It is widely used for treating several diseases, including pruritus,
psoriasis, fetotoxicity, impetigo, and diabetes (Zhang et al., 2013).
Its historical application dates back to the 19th-century Mongolian
pharmaceutical classic known as\emph{Mengyaozhengdian} (Fig. S1), which
provides a systematic approach to its usage. The dried whole plant is
included in several classical herbal formulations, such as
``Siweixinbasan'' and ``Baweixinbasan.'' Previous studies have shown
that \emph{C. daurica} contains 177 chemical components (Wu et al.,
2020), and the most common are flavonoids (Z.-H. Li et al., 2014) and
iridoid glycosides (Q. Wang, Bao, Hao, \& Han, 2018). This herb has
received much recent attention owing to its diverse bioactivities,
including anti-diabetic (Gong et al., 2020), anti-inflammatory (J.-J.
Guo, Liu, Zhu, Ren, \& Liang, 2017), and anti-bacterial (Shi et al.,
2020) activities. However, either accidental or intentional, the
adulteration and substitution of \emph{C. daurica}frequently occurs in
China and Mongolia, due to its minor morphological differences with
\emph{C. mongolica} (Hu, 2018; Liang, Liang, Yao, Liu, \& Li, 2016).
Specifically\emph{, C. daurica} has densely white sericeous anther
locules that are 4--4.5 mm and apically pilose, whereas\emph{C.
mongolica} has pilose anther locules that are 3--3.6 mm and glabrous
apically or occasionally with few hairs; thus, distinguishing between
these two species is a major challenge in the non-flowering stage. This
issue has an unforeseeable subsequent effect on the herb ``Xinba'',
jeopardizing its clinical use, potency, and safety.

In recent years, researchers have shifted their focus from morphological
and chemical identification to using DNA-based molecular markers as a
precise method to assess the authenticity of medicinal herbs. DNA
barcodes has become particularly popular for identifying species of
Chinese medicinal herbs owing to its accuracy and speed (Zhu, Liu, Qiu,
Dai, \& Gao, 2022). The universal barcodes include ITS, matK,\emph{rbc}
L, and \emph{trn} H--\emph{psb} A, either individually or in combination
(CBOL Plant Working Group et al., 2009; China Plant BOL Group et al.,
2011). However, these barcodes might be ineffective for complex
taxonomic groups, especially for radically evolved and closely related
taxa, because sufficient genetic variation is lacking (Z. F. Liu et al.,
2022). Therefore, more reliable specific DNA barcodes are needed to
distinguish the \emph{C. daurica} from its adulterant \emph{C.
mongolica,} and ensure the authenticity of the herb ``Xinba''.

Here, we provide the chloroplast genome sequences of these
two\emph{Cymbaria} species and then conducted a comparative analysis of
these two newly sequenced chloroplast genomes with those of 52 other
Orobanchaceae species. Our specific objectives were to (1)
characterize\emph{Cymbaria} chloroplast genomes, (2) develop the
specific molecular markers as DNA barcodes, and (3) investigate the
evolution and phylogeny of \emph{Cymbaria} . These findings will provide
key insights on the taxonomic identification, phylogenetic placement and
reductive evolution of hemiparasitic genus \emph{Cymbaria} , and
valuable genetic tools to validate the authenticity of the traditional
Mongolian medicine ``Xinba.''

2. \textbar{} \textbf{Materials and Methods}

2.1. \textbar{} Sampling, sequencing, assembly, and annotation

Wild \emph{C. mongolica} and \emph{C. daurica} plants were collected
from Binzhou, Shaanxi Province (35°14\selectlanguage{english}'38.63\selectlanguage{english}"N, 108deg13\selectlanguage{english}'06.11\selectlanguage{english}"E) and
Xilingol, Inner Mongolia Autonomous Region (43deg28\selectlanguage{english}'13.92\selectlanguage{english}"N,
116deg47\selectlanguage{english}'07.76\selectlanguage{english}"E) in China. One sample of young fresh leaves per species
was collected in a liquid nitrogen tank. The habitat and altitude were
also recorded. Voucher specimens were stored in the Herbarium of Inner
Mongolia University. Total genomic DNA was extracted using the modified
CTAB method (Allen, Flores-Vergara, Krasynanski, Kumar, \& Thompson,
2006). Illumina sequencing platform was used to generate raw reads. The
assembly was performed using clean data through SPAdes v. 3.14.0
(Bankevich et al., 2012) and GetOrganelle (Jin et al., 2020). The
graphic visualization and assembly quality was evaluated by Bandage
(Wick, Schultz, Zobel, \& Holt, 2015) and GetOrganelle (Jin et al.,
2020), respectively. Annotations were performed using GeSeq (Tillich et
al., 2017) and manually adjusted in Geneious (Kearse et al., 2012).
After identifying the boundaries by BLAST, the sequences were submitted
to GenBank and visualized as a single circular maps using the Organellar
Genome DRAW tool (Greiner, Lehwark, \& Bock, 2019).

2.2. \textbar{} Comparative analysis of chloroplast genomes

A comparative analysis of Orobanchaceae chloroplast genomes was
conducted including eight autotrophs, 21 hemiparasites, and 25
holoparasites (Table S1). The genome sizes, GC contents, and intact
genes were investigated using PhyloSuite (D. Zhang et al., 2020) and
drawn with RadarMap package in R software (Team, 2015). A heatmap of
genes in the chloroplast genome was generated using TBtools (Chen et
al., 2020). The PAML package v4.0 was used to calculate the
nonsynonymous (Ka) and synonymous (Ks) substitution rates and their
ratio (\selectlanguage{greek}ω \selectlanguage{english}= Ka/Ks) (Yang, 2007). Contractions and expansions of the
boundaries were detected using Irscope (Amiryousefi, Hyv\selectlanguage{ngerman}önen, \& Poczai,
2018). The Mauve program was used to align sequences against the
\emph{Schwalbea americana} reference sequence (Darling, Mau, \& Perna,
2010). CodonW (http://codonw.sourceforge.net/) was used to calculate
RSCU. REPuter (Kurtz et al., 2001) was employed to detect repeat
sequences and simple sequence repeats (SSRs) were identified by MISA
(Beier, Thiel, Münch, Scholz, \& Mascher, 2017).

2.3. \textbar{} Development and validation of DNA barcodes

Structural comparisons were conducted using mVISTA (Frazer, Pachter,
Poliakov, Rubin, \& Dubchak, 2004) with \emph{C. mongolica} as a
reference. Sliding window analysis was conducted to identify
hypervariable regions using DnaSP (Librado \& Rozas, 2009). Primer3web v
4.1.0 (\emph{https://primer3.ut.ee/} ) was used to design DNA barcoding
primers based on the hypervariable regions, and these were verified
using seven individuals from different regions of each species (Table
S4). PCR was conducted in 25 \selectlanguage{greek}µ\selectlanguage{english}L reactions with 12.5 \selectlanguage{greek}µ\selectlanguage{english}L of 2\selectlanguage{ngerman}×EasyTaq PCR
SuperMix, 1.0 \selectlanguage{greek}µ\selectlanguage{english}L of each primer (0.4 \selectlanguage{greek}µ\selectlanguage{english}M), 1 \selectlanguage{greek}µ\selectlanguage{english}L of template DNA, and 9.5
\selectlanguage{greek}µ\selectlanguage{english}L of ddH\textsubscript{2}O. A SimpliAmp Thermal Cycler (Applied
Biosystems, Carlsbad, CA, USA) was used to conduct all PCR reactions
with the following thermal cycling conditions: 94 @\selectlanguage{english}C for 5 min; 30
cycles of 94 @\selectlanguage{english}C for 30 s, a specific annealing temperature (Tm) for 30
s, and 72 @\selectlanguage{english}C for 30 s; and 72 @\selectlanguage{english}C for 10 min. Agarose gel electrophoresis
(1.5\%) was used to visualize PCR products. The DNA fragments were
purified and sequenced by Biomarker Technologies Co., Ltd.

2.4. \textbar{} Phylogenetic analyses and divergence time estimation

To identify early divergence events within Orobanchaceae, phylogeny was
inferred from 54 Orobanchaceae species with the two Paulowniaceae
species \emph{Tectona grandis} (GenBank accession number: NC\_020098)
and \emph{Paulownia tomentosa} (GenBank accession number: MK875778) as
outgroups. Phylogenetic trees were generated using three datasets: (1)
complete chloroplast genome sequences; (2) coding DNA sequences (CDSs);
and (3) shared CDSs (\emph{rps} 2,\emph{rps} 7, \emph{rps} 8, \emph{rps}
14, \emph{rpl} 2, \emph{rpl} 16,\emph{rpl} 36, and \emph{mat} K). The
standard phylogenetic workflow in Phylosuite v1.2.2 (D. Zhang et al.,
2020) was used as follows: alignment of multiple sequences with MAFFT
v7.313 (Katoh \& Standley, 2013), optimization of aligned regions with
Gblocks v0.91b (Castresana, 2000), concatenation of datasets with the
Concatenate Sequence function, selection of the optimal partitioning
scheme with PartitionFinder2, and phylogenetic inference with IQ-TREE
v1.6.8 (Nguyen, Schmidt, Von Haeseler, \& Minh, 2015) and MrBayes v3.2.6
(Drummond, Suchard, Xie, \& Rambaut, 2012). Phylogeny was inferred using
Maximum likelihood and Bayesian inference methods. Phylogenetic trees
visualized using iTOL v6 (Letunic \& Bork, 2021).

BEAST v1.7 (Drummond et al., 2012) was used to estimate divergence times
based on the shared concatenated CDSs. As no reliable fossil record is
available, the crown age for Orobanchaceae (C) was constrained as 56 \selectlanguage{ngerman}±
10 Mya using a normal model in TimeTree (http://timetree.org/). The
results of PartitionFinder were used to determine the nucleotide
substitution model of the unlinked subsets. The following parameters
were used in BEAUti software: ``Lognormal relaxed clock (Uncorrelated)''
for ``Clock model'' and ``Speciation: Yule Process'' for ``Tree model.''
Three independent MCMC chains were carried out under the same
parameters. Each MCMC was run for 50,000,000 generations, and sampling
was conducted every 5,000 generations. Convergence was evaluated using
Tracer v.1.6 (http://beast.bio.ed.ac.uk/Tracer). The LogCombiner program
was used to combine the three log files. The TreeAnnotator program was
used to generate the maximum clade credibility (MCC) tree. The effective
sample sizes in the combined three log outputs were greater than 200,
which indicated that the MCMC sampling was adequate. FigTree v1.4.3
(Rambaut, 2017) was used to visualize the MCC tree with 95\% highest
posterior density intervals.

\textbf{3.} \textbar{} \textbf{Results}

3.1. \textbar{} Characteristics of \emph{Cymbaria} chloroplast genomes

Illumina sequencing yielded more than 20 million bp clean reads. The
chloroplast genomes of \emph{C. mongolica} and \emph{C.
daurica}displayed a typical quadripartite structure with sizes of
149,431 bp (38.0\% GC content) and 151,545 bp (38.2\% GC content),
respectively. Each chloroplast genome comprised an LSC region (86,595 bp
and 87,376 bp), an SSC region (16,962 bp and 17,825 bp), and two IR
regions (22,937 bp and 23,172 bp). The GC content in the IR region
(44.1\%) was higher than that in the LSC (35.9--36.2\%) and SSC
(32.2--32.7\%) regions.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1.} Circular maps of \emph{Cymbaria} chloroplast genomes.

The chloroplast genome sequences of \emph{C. mongolica} and \emph{C.
daurica} contained 105 and 100 intact genes, respectively (Fig. 1; Table
S1). \emph{Cymbaria} contained 26 tRNA and four rRNA genes, while the
number of PCGs and pseudogenes varied between two species. In contrast
to the autotrophic \emph{Lindenbergia philippensis} , \emph{C.
mongolica}encoded 75 PCGs, and one pseudogene and lacked seven genes
while\emph{C. daurica} encoded 70 PCGs, and three pseudogenes and lacked
ten genes. Among the 15 intron-containing genes, 12 genes contained one
intron, and the remaining three genes harbored two introns (Table S1).
In addition, purifying or neutral selection was detected on all PCGs,
with the exception of \emph{ycf} 2, which was under positive selection.
No pronounced differences were detected in the boundary regions. An
inversion of large gene blocks (\emph{rbc} L-\emph{mat} K) was
identified in the LSC region according to the Mauve alignment (Fig. S2).
Four pairs of palindromic repeats (123, 67, 48, and 37 bp) and three
pairs of palindromic repeats (190, 152, and 121 bp) were detected at
both ends of the inverted region of \emph{C. mongolica} (54,654 bp size)
and \emph{C. daurica} (55,904 bp size), respectively.

We estimated the sequence divergence and gene content for 54 chloroplast
genomes within the family Orobanchaceae (Fig. 2; Table S2). The genome
sizes of the autotrophs (153,622--155,319 kb, mean: 154,213 kb) and
hemiparasites (142,733--160,910 kb, mean: 151,152 kb) were similar;
however, the genome sizes of the autotrophs were higher than those of
the holoparasites (45,673--150,504 kb, mean: 87,505 kb) (Fig. S3). The
GC content was lower in holoparasites (mean: 34.93\%) than in those of
hemiparasites (38.25\% on average) and autotrophs (mean: 37.90\%).
Autotrophs contained all intact genes; the number of intact genes was
lower in hemiparasites than in autotrophs, and this decrease primarily
stemmed from the pseudogenization/loss of \emph{ndh} genes (Fig. 2;
Table S2). Moreover, non-functionalization and gene loss were observed
in most photosynthetic genes (e.g., \emph{psa} /\emph{psb} ,\emph{ycf}
3/4, \emph{ndh} , and \emph{cem} A) in holoparasites.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2.} Heat map depicting the chloroplast gene content
across 54 Orobanchaceae species. Blocks in green, blue, and orange
indicate intact genes, pseudogenes, and lost genes, respectively.
Background colors in green, blue, and orange indicate autotrophs,
hemiparasites, and holoparasites, respectively.

3.2. \textbar{} Codon usage bias

The PCGs in the \emph{C. mongolica} and \emph{C. daurica} comprised
18,482 and 15,896 codons, respectively. Leucine (Leu, 10.50\% and
10.62\%) was the most commonamino acid, and cysteine (Cys, 1.09\% and
1.07\%) was the least common amino acid. A total of 30 codons (RSCU
\textgreater{} 1) were A/T-ending codons, with the exception of UUG
(Fig. 3A). The range of the ENC in \emph{C. mongolica} and \emph{C.
daurica} was 35.24--56.04 and 35.75--59.41, respectively, which
suggested weak codon usage bias. ENC was significantly negatively
correlated with GC2 in both species; a significant positive correlation
between ENC and GC3 was only detected in \emph{C. mongolica} (Fig. 3B).
There was no significant correlation between GC3 and GC12 according to
neutral plot analysis (Fig. 3C); the regression coefficient was 0.049
and 0.193 in \emph{C. mongolica} and \emph{C. daurica} , respectively.
Most genes were below and around the standard curve according to ENC
plot analysis (Fig. 3D), and the ENC ratio was from 0.05 to 0.15.
PR2-plot analysis (Fig. 3E) showed that T \textgreater{} A and G
\textgreater{} C in the base usage frequency. A total of 16 codons were
identified as preferred codons, of which 10 were shared by the two
species.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3.} Codon usage bias of \emph{Cymbaria} chloroplast
genomes. (A) RSCU. (B) Correlation heatmap. (C) Neutral plot analysis.
(D) PR2 plot analysis. (E) ENC plot analysis. Circle colors of green and
orange represent \emph{C. mongolica} (left) and \emph{C. daurica}
(right), respectively.

3.3. \textbar{} Repetitive sequence variation

A total of 134 repeats, consisting of 78 forward, four reverse, one
complementary, and 51 palindrome repeats, were detected from \emph{C.
mongolica} chloroplast genome. Meanwhile, a total of 225 repeats,
including 106 forward, 18 reverse, 13 complementary, and 88 palindrome
repeats, were identified from \emph{C. daurica} chloroplast genome (Fig.
4A; Fig. 4B). The size of approximately 90\% of the repeats ranged from
30 bp to 70 bp. \emph{C. mongolica} and \emph{C. daurica} contained 61
and 65 SSRs, and most were present in the LSC region (37 and 43 SSRs),
respectively (Fig. 4A; Fig. 4C). Hexa-nucleotide SSRs were only detected
in \emph{C. mongolica} ; the remaining SSRs were identified in both
species (Fig. 4D). Mono-nucleotides were the most plentiful, followed by
di- and tetra-nucleotides. The mononucleotide motifs A/T had the highest
proportion, accounting for 34.4\% in \emph{C. mongolica} and 32.3\%
in\emph{C. daurica} (Fig. 4E).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4.} Comparison of the Repeats and SSRs in \emph{Cymbaria}
chloroplast genomes. (A) Repeats and SSRs. (B) Repeat types. (C)
Occurrences of SSRs. (D) SSR types. (E) SSR motif types. Bar colors of
green and orange correspond to \emph{C. mongolica} (left) and \emph{C.
daurica} (right), respectively.

3.4. \textbar{} Development and validation of DNA barcodes

High conservation with some degree of divergence was observed in the two
chloroplast genomes (Figure 5). Most of the sequence differences were
observed in the non-coding regions. Nucleotide diversity (Pi) was
0.02099, and higher divergence was observed in the LSC and SSC regions
(Fig. S4). We also detected several divergence hotspot regions (Pi
\textgreater{} 0.05), including \emph{trn} M-CAU-\emph{ndh} C,\emph{psa}
A, \emph{mat} K,\emph{acc} D-\emph{psa} I,\emph{ycf} 4-\emph{cem} A,
\emph{rpl} 32-\emph{trn} L-UAG,\emph{ndh} D-\emph{ndh} G,\emph{rps}
15-\emph{ycf} 1, \emph{rrn} 23S, and\emph{trn} A-UGC-\emph{trn} E-UUC.
We designed four specific DNA markers (CymN1, CymN2, CymY, and CymR),
and they were validated using sequences from different regions from
seven individuals of each species (Table S3; Table S4). DNA fragments
varying in length were obtained from \emph{C. mongolica} and \emph{C.
daurica} using each primer, and both species could be distinguished via
agarose gel electrophoresis (Fig. 6). Likewise, the same results were
observed from the sanger sequencing alignment and chromatogram of the
amplification of DNA barcodes. Overall, these findings suggest that the
four pairs of DNA barcodes could be used to distinguish among
\emph{Cymbaria} species.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5.} Sequence alignment of \emph{Cymbaria} chloroplast
genomes.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 6.} Agarose gel electrophoresis, chromatogram, and
alignment of sequences obtained via Sanger sequencing of the amplified
DNA barcodes. The indel makers include (A) CymN1, (B) CymN2, (C) CymY,
and (D) CymR. The lanes correspond to the PCR products amplified from
seven individuals of \emph{C. mongolica} (left) and \emph{C.
daurica}(right). The red squares correspond to the Indel regions. The
original uncropped image is presented in Figure S5.

3.5. \textbar{} Phylogenetic relationships and divergence times

Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic
analyses yielded highly consistent topologies for the three datasets
(Fig. 7). The monophyly of Orobanchaceae was strongly supported. The
tribes Rehmannieae, Lindenbergieae, Cymbarieae, Pedicularideae,
Brandisieae, Rhinantheae, Orobancheae, and Buchnereae corresponded to
well-supported clades. The hemiparasitic tribe Cymbarieae was a clade
sister to the other parasitic lineages. \emph{C. mongolica} and \emph{C.
daurica} were grouped into the monophyletic genus \emph{Cymbaria} ,
which comprised a clade sister to the \emph{Schwalbea}
-\emph{Siphonostegia}clade. Divergence time analyses (Fig. 8) revealed
that the common ancestor of the Orobanchaceae originated in the early
Eocene (49.96 Mya), and parasites diverged from autotrophic plants in
the mid-Eocene (42.95 Mya). The emergence of Cymbarieae predates the
mid-Oligocene (31.44 Mya). Moreover, the diversification of
\emph{Cymbaria} was estimated to occur around the late Miocene (6.72
Mya).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

\textbf{Figure 7.} Phylogenetic relationship inferred from ML and BI
based on shared protein-coding genes of 54 Orobanchaceae species.
Numbers above the branches indicate bootstrap values (left) and
posterior probabilities (right). Background colors of gray, green, blue,
and orange indicate outgroups, autotrophs, hemiparasites, and
holoparasites, respectively. The stars () indicate the two newly
sequenced \emph{Cymbaria} species.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

\textbf{Figure 8.} Divergence time estimated from the MCC tree in BEAST.
Node numbers indicate posterior probabilities (below) and mean
divergence times (above). Node bars represent the 95\% HPD interval
(blue bar). Background colors of gray, green, blue, and orange indicate
outgroups, autotrophs, hemiparasites, and holoparasites, respectively.
The stars () indicate the two newly sequenced \emph{Cymbaria} species.

4. \textbar{} \textbf{Discussion}

4.1. \textbar{} Pseudogenization/loss events of \emph{ndh} genes and the
unique \emph{rbc} L-\emph{mat} K inversion

It is acknowledged that the lifestyle transition from autotrophy to
heterotrophy triggers the degradation of chloroplast genomes (Wicke \&
Naumann, 2018). Contrasting with the hypervariability of
holoparasites,\emph{Cymbaria} species and other Orobanchaceae
hemiparasites exhibit high similarity to autotrophs in length, GC
content, and intact genes. It has been confirmed that holoparasites have
the characteristic of chloroplast genome reduction (Wicke et al., 2013).
The high variability of holoparasites is explained by increases in
pseudogenization and gene loss. However, patterns of variation in
hemiparasites are diverse. The chloroplast genomes of hemiparasites in
the family Orobanchaceae were more similar to those of autotrophs, which
is in contrast to the reductions in the genome sizes of hemiparasites in
the order Santalales (X. Guo, Zhang, Fan, Liu, \& Ji, 2021; Y. Li et
al., 2017; Shin \& Lee, 2018). This might be attributed in part to
GC-biased gene conversion and mutational biases, which suggests that
sophisticated mechanisms contribute to the stability (Niu et al., 2017).

Angiosperms typically possess 113 plastid genes, consisting of 79
functional PCGs, 30 tRNA, and four rRNA genes (Wicke et al., 2011).
Within the Orobanchaceae family, pseudogenes and gene losses were
largely absent in autotrophs, occasionally observed in most
hemiparasites, and common in nearly all holoparasites. This can be
explained by the tendency for the chloroplast genomes of parasites to be
reduced in size (Naumann et al., 2016; Wicke \& Naumann, 2018). The
chloroplast NAD(P)H-dehydrogenase complex comprises 11 \emph{ndh} genes
(Ma, Liu, Bai, \& Yong, 2021), and the pseudogenization or loss of these
genes represents the initial stage of reductive evolution (Wicke \&
Naumann, 2018). The results of our study indicated that \emph{C.
mongolica} lost genes (\emph{ndh} I, \emph{ndh} J) and that \emph{C.
daurica} contained pseudogenes (\emph{ndh} F,\emph{ndh} H) and lost
genes (\emph{ndh} A, \emph{ndh} C, \emph{ndh} E,\emph{ndh} G, \emph{ndh}
I), indicating that these two species are in the initial phase of the
autotroph-to-heterotroph transition. A previous study has confirmed that
a hemiparasitic lifestyle can lead to an increase in the
pseudogenization/loss of \emph{ndh} genes (Xin Li et al., 2021). This
can be explained to some extent by the facultative root hemiparasitic
lifestyle of the two \emph{Cymbaria} species. The degradation of
\emph{ndh} genes affects several morphological and physiological traits
and enhances the adaptation of plants to environmental stress (Sabater,
2021).

The unique inversion including \emph{rbc} L-\emph{mat} K in the LSC
region, which most likely stems from a palindromic repeat-mediated
rearrangement. Inversions of the LSC fragments have also been observed
in \emph{Schwalbea americana} (Wicke et al., 2013)
and\emph{Siphonostegia chinensis}(Jiang et al., 2022); this distinct
evolutionary mechanism among Orobanchaceae members might explain the
unique phylogenetic position of the tribe Cymbarieae. This inversion has
also been observed in\emph{Codonopsis pilosula} subsp. \emph{tangshen}
(Yue et al., 2022) and\emph{Avena sativa} (Q. Liu et al., 2020).

The codon usage bias of the two \emph{Cymbaria} species might be
affected by natural selection and mutation, as has been observed in
several other angiosperms (Q.-F. Lu, Luo, \& Huang, 2020). The number of
repeats plays a key role in maintaining the stability of in several
angiosperms chloroplast genome (Jansen, Saski, Lee, Hansen, \& Daniell,
2010). The chloroplast genome of \emph{C. daurica} has more repetitive
sequences than that of \emph{C. mongolica} , suggesting that the
stability of the former might be higher than that of the latter. Our
findings suggest that A/T mononucleotide SSRs were dominant, and this is
consistent with the high prevalence of AT richness (Xia Liu, Li, Yang,
\& Zhou, 2018).

4.2. \textbar{} Specific DNA barcodes for distinguishing herb \emph{C.
daurica} from its adulterant \emph{C. mongolica} .

Traditional Mongolian medicine continues to receive much clinical
attention because of its distinctive properties and herb resources (Z.
F. Liu et al., 2022). \emph{C. daurica} ~has been used to treat
pruritus, psoriasis, fetotoxicity, impetigo, and diabetes. The incidence
of\emph{C. daurica} adulterated with its sister species \emph{C.
mongolica} is increasing, and this poses a threat to the clinical
efficacy of the herb. The high similarity in morphology between \emph{C.
daurica} and \emph{C. mongolica} is the root cause of this problem.
Distinguishing between these two \emph{Cymbaria} species is exceedingly
difficult because they only differ in anther morphology (Zhang et al.,
2013).

Both morphological and microscopic characteristics are currently used to
identify \emph{Cymbaria} species (Z.-W. Wang, Domg, Wu, \& Li, 2012).
However, both of these methods rely on the features of the anther
locule, which are only visible during the short flowering period, and
specialists are required to obtain accurate identifications. Several
divergence hotspot regions were first identified through sequence
divergence and nucleotide variability. We then developed and validated
four pairs of specific DNA barcodes to distinguish between these two
species. Each DNA barcode had at least one Indel locus and several SNP
loci. Some previous studies have suggested that specific barcodes are
superior to universal barcodes for identifying morphologically similar
species (Fang, Dai, Liao, Zhou, \& Liu, 2022; G. Y. Lu et al., 2022).
Overall, these four pairs of specific DNA barcodes could be used to
accurately and rapidly distinguish between \emph{C. daurica} and\emph{C.
mongolica} without the need to evaluate the morphological
characteristics of the anther during flowering nor specialized training.

4.3. \textbar{} Climate aridification and increasing host accelerate the
diversification of Cymbaria

Traditional Orobanchaceae has been merged with all hemiparasitic genera
as well as a few holoparasitic genera formerly placed in
Scrophulariaceae (Bennett \& Mathews, 2006; dePamphilis, Young, \&
Wolfe, 1997; Fischer, 2004; McNeal et al., 2013; Wolfe, Randle, Liu, \&
Steiner, 2005; Young, Steiner, \& dePamphilis, 1999) and the three
autotrophic genera previously considered as sister taxa to Orobanchaceae
(Schneeweiss, 2013; Xia, Li, Wen, \& Wang, 2021). Moreover, the two new
tribes Brandisieae and Pterygielleae have been proposed (Jiang et al.,
2022; Yu et al., 2018). To date, Orobanchaceae includes nine
well-supported clades corresponding to nine tribes, i.e., the two
autotrophic tribes Rehmannieae and Lindenbergieae, and the seven
parasitic tribes Cymbarieae, Buchnereae, Orobancheae, Brandisieae,
Pedicularideae, Rhinantheae, and Pterygielleae. The topology of the
major clades and autotroph--parasite sister relationships revealed by
our phylogenetic analyses (Fig. 5) were generally consistent with
previous findings.

The hemiparasitic tribe Cymbarieae is distinguished from other tribes in
the family Orobanchaceae by the presence of bracteoles, a tubular calyx
that is weakly dorsiventral, a highly two-lipped corolla, and anthers
with two mostly rounded and equal thecae (Fischer, 2004). Cymbarieae has
traditionally been considered sister to all other parasitic lineages
(Bennett \& Mathews, 2006; McNeal et al., 2013). However, a recent study
has challenged this classification after finding that the holoparasitic
tribe Orobancheae was sister to all other parasitic members (Xi Li et
al., 2019). Our findings are consistent with the traditional
classification of the Cymbarieae. The Cymbarieae was found to be sister
to the remaining parasitic lineages, and the hemiparasites evolved
earlier than the holoparasites; this is consistent with the progressive
nature of the evolution of increased host dependence (Xu et al., 2022).
This inconsistency in the placement of Cymbarieae might stem from
phylogenetic sources of error, such as incomplete lineage sorting or
deep coalescence. Resolving these early nodes will require a coalescent
approach that involves many genes with different histories.

Within parasitic plants, the hemiparasitic tribe Cymbarieae diverged
from the remaining lineages in the mid-Oligocene (31.44 Mya), which was
most likely induced by global climate cooling and the retreat of the
Tethys Sea during the Eocene--Oligocene Transition at 34 Mya (Abels,
Dupont-Nivet, Xiao, Bosboom, \& Krijgsman, 2011). The emergence and
divergence of hemiparasites might be attributed to the rapid expansion
of grasslands during the Oligocene (Torsvik \& Cocks, 2016), which would
have provided them with opportunities to exploit host plants.
\emph{Cymbaria} species diversified in the late Miocene (6.72 Mya),
which was driven by the final uplift of the Qinghai--Tibetan Plateau,
the onset of the East Asian monsoon, and the large accumulation of dust
in the Loess Plateau from 10 to 7 Mya {[}77--79{]}. Both climate
aridification and the increase in host steppe vegetation (Hurka et al.,
2019) likely accelerated the adaptive evolution of \emph{Cymbaria}
species in the Mongol--Chinese steppe region.

5. \textbar{} \textbf{Conclusions}

We characterized two \emph{Cymbaria} chloroplast genome and conducted a
comparative analysis using chloroplast genomes across 54 Orobanchaceae
species. The chloroplast genomes of \emph{C. mongolica} and \emph{C.
daurica} had a typical quadripartite structure, and their total lengths
were 149,431 bp and 151,545 bp, respectively. Although the chloroplast
genomes of holoparasites are hypervariable, the genome size, GC content,
and intact genes of \emph{Cymbaria} species, other hemiparasites, and
autotrophs are highly similar. The pseudogenization/loss of
\emph{ndh}genes might be associated with the facultative root
hemiparasitic habits of \emph{Cymbaria} . The \emph{rbc} L-\emph{mat} K
inversion in the LSC region most likely stemmed from a palindromic
repeat-mediated rearrangement. Specific DNA barcodes were developed
using four pairs of primers (CymN1, CymN2, CymY, and CymR) that
amplified sequences from the divergent hotspot regions to distinguish
the traditional Mongolian herb\emph{C. daurica} from its adulterant
\emph{C. mongolica} . The genus\emph{Cymbaria} and the \emph{Schwalbea}
-\emph{Siphonostegia} clade were clustered in the tribe Cymbarieae. This
tribe comprised an independent clade sister to the remaining parasitic
lineages, which is in contrast to the relationships hypothesized in a
recent study. The monophyletic genus \emph{Cymbaria} diversified during
the late Miocene period (6.72 Mya). The aridification of the climate and
increases in host steppe vegetation likely promoted the adaptive
evolution of \emph{Cymbaria}species in the Mongol-Chinese steppe region.
Our results provide key information for clarifying the taxonomic
identification, phylogenetic placement, and reductive evolution of
\emph{Cymbaria} ; our findings will also help assessments of the
authenticity of the traditional Mongolian medicine ``Xinba.''

\textbf{Author contributions}

Conceptualization, Yang Ma; Data curation, Yang Ma; Formal analysis,
Yang Ma; Funding acquisition, Jianming Niu; Investigation, Yang Ma, Zhen
Zhou and Zekun Deng; Methodology, Yang Ma; Project administration,
Jianming Niu; Resources, Yang Ma, Zhen Zhou and Zekun Deng; Software,
Yang Ma; Supervision, Jianming Niu; Validation, Yang Ma; Visualization,
Yang Ma; Writing -- original draft, Yang Ma; Writing -- review \&
editing, Yang Ma, Jordi L\selectlanguage{ngerman}ópez-Pujol, Dongqing Yan and Jianming Niu. All
authors have read and agreed to the published version of the manuscript.

\textbf{Acknowledgments}

This research was funded by the Chinese Scholarship Council, Major
Science and Technology Projects of Inner Mongolia Autonomous Region
(2019ZD008), and National Natural Science Foundation of China (31860106,
32260304). We also thank Qingcheng Light for providing computing
resources and TopEdit LLC for the linguistic editing and proofreading
during the preparation of this manuscript.

\textbf{CONFLICT OF INTERESTS}

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.

\textbf{Data Availability STATEMENT}

Data contained within the article are openly available in GeneBank
(NC\_064388 and NC\_064104).

\textbf{ORCID}

Yang Ma https://orcid.org/0000-0003-2566-4869

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\textbf{APPENDIX}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}

\textbf{Figure S1.} Mongolian pharmaceutical classic literature
describing~the herb \emph{C. daurica} (Huang et al., 2023)\emph{.}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image10/image10}

\end{center}
\end{figure}

\textbf{Figure S2.} Collinearity of two \emph{Cymbaria} chloroplast
genomes using Mauve program with\emph{Schwalbea americana} as the
reference.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image11/image11}

\end{center}
\end{figure}

\textbf{Figure S3.} Radar chart of complete chloroplast genome
comparison of 54 Orobanchaceae species. From outside to inside: genome
size, intact genes, and GC content. Background colors of green, blue,
and orange represent autotroph, hemiparasite, and holoparasite species,
respectively.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image12/image12}

\end{center}
\end{figure}

\textbf{Figure S4.} Sliding window analysis of two \emph{Cymbaria}
chloroplast genomes.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image13/image13}

\end{center}
\end{figure}

\textbf{Figure S5.} The original and uncropped agarose gel
electrophoresis of the amplification of DNA barcodes

\textbf{Table S1.} Gene contents in two \emph{Cymbaria} chloroplast
genomes.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Category & Gene group & Gene name & Gene name\tabularnewline
& & \emph{C. mongolica} & \emph{C. daurica}\tabularnewline
Photosynthesis & Subunits of photosystem I & \emph{psa}A, \emph{psa}B,
\emph{psa}C, \emph{psa}I, \emph{psa}J & \emph{psa}A, \emph{psa}B,
\emph{psa}C, \emph{psa}I, \emph{psa}J\tabularnewline
& Subunits of photosystem II & \emph{psb}A, \emph{psb}B, \emph{psb}C,
\emph{psb}D, \emph{psb}E, \emph{psb}F, \emph{psb}H, \emph{psb}I,
\emph{psb}J, \emph{psb}K, \emph{psb}L, \emph{psb}M, \emph{psb}N,
\emph{psb}T, \emph{psb}Z & \emph{psb}A, \emph{psb}B, \emph{psb}C,
\emph{psb}D, \emph{psb}E, \emph{psb}F, \emph{psb}H, \emph{psb}I,
\emph{psb}J, \emph{psb}K, \emph{psb}L, \emph{psb}M, \emph{psb}N,
\emph{psb}T, \emph{psb}Z\tabularnewline
& Subunits of NADH dehydrogenase & \emph{ndh}A,
\emph{ndh}B\textsuperscript{a}, \emph{ndh}C, \emph{ndh}D, \emph{ndh}E,
\emph{ndh}F, \emph{ndh}G, \emph{ndh}H, \emph{ndh}K &
\emph{ndh}F\selectlanguage{greek}\textsuperscript{Ψ}, \selectlanguage{english}\emph{ndh}H\selectlanguage{greek}\textsuperscript{Ψ},
\selectlanguage{english}\emph{ndh}B\textsuperscript{a}, \emph{ndh}D, \emph{ndh}F, \emph{ndh}H,
\emph{ndh}J, \emph{ndh}K\tabularnewline
& Subunits of cytochrome b/f complex & \emph{pet}A,
\emph{pet}B\textsuperscript{a}, \emph{pet}D, \emph{pet}G, \emph{pet}L,
\emph{pet}N & \emph{pet}A, \emph{pet}B\textsuperscript{a}, \emph{pet}D,
\emph{pet}G, \emph{pet}L, \emph{pet}N\tabularnewline
& Subunits of ATP synthase & \emph{atp}A, \emph{atp}B, \emph{atp}E,
\emph{atp}F\textsuperscript{a}, \emph{atp}H, \emph{atp}I & \emph{atp}A,
\emph{atp}B, \emph{atp}E, \emph{atp}F\textsuperscript{a}, \emph{atp}H,
\emph{atp}I\tabularnewline
& Large subunit of rubisco & \emph{rbc}L & \emph{rbc}L\tabularnewline
& Subunits photochlorophyllide reductase & - & -\tabularnewline
Self-replication & Proteins of large ribosomal subunit & \emph{rpl}14,
\emph{rpl}16, \emph{rpl}2\textsuperscript{a}, \emph{rpl}20,
\emph{rpl}22, \emph{rpl}23, \emph{rpl}32, \emph{rpl}33, \emph{rpl}36 &
\emph{rpl}14, \emph{rpl}16, \emph{rpl}2\textsuperscript{a},
\emph{rpl}20, \emph{rpl}22, \emph{rpl}23, \emph{rpl}32, \emph{rpl}33,
\emph{rpl}36\tabularnewline
& Proteins of small ribosomal subunit & \emph{rps}11,
\emph{rps}12\textsuperscript{b}, \emph{rps}14, \emph{rps}15,
\emph{rps}16\textsuperscript{a}, \emph{rps}18, \emph{rps}2,
\emph{rps}3\textsuperscript{a}, \emph{rps}4, \emph{rps}7(2), \emph{rps}8
& \emph{rps}11, \emph{rps}12\textsuperscript{b}, \emph{rps}14,
\emph{rps}15, \emph{rps}16\textsuperscript{a}, \emph{rps}18,
\emph{rps}2, \emph{rps}3\textsuperscript{a}, \emph{rps}4,
\emph{rps}7(2), \emph{rps}8\tabularnewline
& Subunits of RNA polymerase & \emph{rpo}A, \emph{rpo}B, \emph{rpo}C1,
\emph{rpo}C2 & \emph{rpo}A, \emph{rpo}B, \emph{rpo}C1,
\emph{rpo}C2\tabularnewline
& Ribosomal RNAs & \emph{rrn}16S, \emph{rrn}23S, \emph{rrn}4.5S,
\emph{rrn}5S & \emph{rrn}16S, \emph{rrn}23S, \emph{rrn}4.5S,
\emph{rrn}5S\tabularnewline
& Transfer RNAs & \emph{trn}A-UGC\textsuperscript{a}, \emph{trn}C-GCA,
\emph{trn}D-GUC, \emph{trn}E-UUC\textsuperscript{a}, \emph{trn}F-GAA,
\emph{trn}G-GCC, \emph{trn}H-GUG, \emph{trn}K-UUU\textsuperscript{a},
\emph{trn}L-CAA, \emph{trn}L-UAA\textsuperscript{a}, \emph{trn}L-UAG,
\emph{trn}M-CAU, \emph{trn}N-GUU, \emph{trn}P-UGG, \emph{trn}Q-UUG,
\emph{trn}R-ACG, \emph{trn}R-UCU, \emph{trn}S-CGA\textsuperscript{a},
\emph{trn}S-GCU, \emph{trn}S-GGA, \emph{trn}S-UGA, \emph{trn}T-GGU,
\emph{trn}T-UGU, \emph{trn}V-GAC, \emph{trn}W-CCA, \emph{trn}Y-GUA &
\emph{trn}A-UGC\textsuperscript{a}, \emph{trn}C-GCA, \emph{trn}D-GUC,
\emph{trn}E-UUC\textsuperscript{a}, \emph{trn}F-GAA, \emph{trn}G-GCC,
\emph{trn}H-GUG, \emph{trn}K-UUU\textsuperscript{a}, \emph{trn}L-CAA,
\emph{trn}L-UAA\textsuperscript{a}, \emph{trn}L-UAG, \emph{trn}M-CAU,
\emph{trn}N-GUU, \emph{trn}P-UGG, \emph{trn}Q-UUG, \emph{trn}R-ACG,
\emph{trn}R-UCU, \emph{trn}S-CGA\textsuperscript{a}, \emph{trn}S-GCU,
\emph{trn}S-GGA, \emph{trn}S-UGA, \emph{trn}T-GGU, \emph{trn}T-UGU,
\emph{trn}V-GAC, \emph{trn}W-CCA, \emph{trn}Y-GUA\tabularnewline
Other genes & Maturase & \emph{mat}K & \emph{mat}K\tabularnewline
& Protease & \emph{clp}P & \emph{clp}P\tabularnewline
& Envelope membrane protein & \emph{cem}A & \emph{cem}A\tabularnewline
& Acetyl-CoA carboxylase & \emph{acc}D\textsuperscript{a} &
\emph{acc}D\textsuperscript{a}\tabularnewline
& c-type cytochrome synthesis gene & \emph{ccs}A &
\emph{ccs}A\tabularnewline
& Translation initiation factor & \emph{inf}A &
\emph{inf}A\tabularnewline
& other & - & -\tabularnewline
Genes of unknown function & Conserved hypothetical chloroplast ORF &
\emph{ycf}1\selectlanguage{greek}\textsuperscript{Ψ}, \selectlanguage{english}\emph{ycf}2\textsuperscript{b},
\emph{ycf}3\textsuperscript{b}, \emph{ycf}4 &
\emph{ycf}1\selectlanguage{greek}\textsuperscript{Ψ}, \selectlanguage{english}\emph{ycf}2\textsuperscript{b},
\emph{ycf}3\textsuperscript{b}, \emph{ycf}4\tabularnewline
\bottomrule
\end{longtable}

Notes: Gene\textsuperscript{a}: containing one intron;
Gene\textsuperscript{b}: containing two introns;
Gene\selectlanguage{greek}\textsuperscript{Ψ}: \selectlanguage{english}pseudogenes

\textbf{Table S2.}Characteristics of 54 Orobanchaceae chloroplast
genomes.\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
Accession & Lifestlye & Species & Size (kbp) & GC content (\%) & Intact
genes & Protein-coding genes & tRNA genes & rRNA genes\tabularnewline
MW851293 & Holoparasites & \emph{Aeginetia\_indica} & 56.381 & 32.9 & 45
& 27 & 17 & 1\tabularnewline
MW262988 & Holoparasites & \emph{Centranthera\_grandiflora} & 147.655 &
38.2 & 109 & 75 & 30 & 4\tabularnewline
NC\_023131 & Holoparasites & \emph{Conopholis\_americana} & 45.673 &
33.9 & 43 & 24 & 19 & 0\tabularnewline
NC\_001568 & Holoparasites & \emph{Epifagus\_virginiana} & 70.028 & 36 &
44 & 22 & 22 & 0\tabularnewline
NC\_021111 & Holoparasites & \emph{Cistanche\_deserticola} & 102.657 &
36.8 & 58 & 26 & 28 & 4\tabularnewline
NC\_025642 & Holoparasites & \emph{Cistanche\_phelypaea} & 94.380 & 36.6
& 59 & 27 & 29 & 3\tabularnewline
MH499250 & Holoparasites & \emph{Aphyllon\_uniflorum\_var\_occidentale}
& 106.661 & 35.1 & 61 & 29 & 29 & 3\tabularnewline
MH580290 & Holoparasites & \emph{Aphyllon\_uniflorum\_var\_uniflorum} &
104.640 & 35.3 & 62 & 30 & 29 & 3\tabularnewline
NC\_039679 & Holoparasites & \emph{Aphyllon\_fasciculatum} & 106.796 &
35 & 60 & 28 & 29 & 3\tabularnewline
MH050785 & Holoparasites & \emph{Aphyllon\_epigalium\_subsp\_epigalium}
& 56.381 & 32.9 & 45 & 13 & 30 & 2\tabularnewline
MH050786 & Holoparasites &
\emph{Aphyllon\_epigalium\_subsp\_notocalifornicum} & 103.932 & 34.1 &
63 & 30 & 30 & 3\tabularnewline
NC\_025651 & Holoparasites & \emph{Aphyllon californicum} & 120.840 &
39.3 & 72 & 38 & 30 & 4\tabularnewline
KU212370 & Holoparasites & \emph{Phelipanche\_aegyptiaca} & 60.947 &
32.1 & 51 & 27 & 22 & 2\tabularnewline
NC\_023465 & Holoparasites & \emph{Phelipanche\_ramosa} & 62.304 & 32.1
& 54 & 28 & 24 & 2\tabularnewline
NC\_023132 & Holoparasites & \emph{Phelipanche\_purpurea} & 62.891 & 31
& 55 & 29 & 24 & 2\tabularnewline
NC\_031441 & Holoparasites & \emph{Orobanche\_austrohispanica} & 81.994
& 35.1 & 59 & 30 & 27 & 2\tabularnewline
NC\_023464 & Holoparasites & \emph{Orobanche\_gracilis} & 65.533 & 34.6
& 55 & 26 & 26 & 3\tabularnewline
NC\_031442 & Holoparasites & \emph{Orobanche\_densiflora} & 83.024 &
35.5 & 63 & 33 & 28 & 2\tabularnewline
KT387722 & Holoparasites & \emph{Orobanche\_cernua\_var\_cumana} &
89.851 & 35.5 & 58 & 28 & 27 & 3\tabularnewline
NC\_024845 & Holoparasites & \emph{Orobanche\_crenata} & 87.529 & 35.2 &
65 & 34 & 27 & 4\tabularnewline
NC\_031443 & Holoparasites & \emph{Orobanche\_pancicii} & 88.525 & 35.2
& 63 & 33 & 27 & 3\tabularnewline
NC\_031444 & Holoparasites & \emph{Orobanche\_rapum\_genistae} & 91.517
& 35.4 & 62 & 32 & 26 & 4\tabularnewline
NC\_025641 & Holoparasites & \emph{Orobanche\_latisquama} & 80.361 &
35.7 & 55 & 27 & 26 & 2\tabularnewline
NC\_043877 & Holoparasites & \emph{Diphelypaea\_coccinea} & 66.616 &
31.7 & 53 & 26 & 24 & 3\tabularnewline
NC\_027838 & Holoparasites & \emph{Lathraea\_squamaria} & 150.504 & 38.1
& 79 & 46 & 29 & 4\tabularnewline
NC\_045041 & Hemiparasites & \emph{Euphrasia\_regelii} & 153.026 & 38.2
& 103 & 69 & 30 & 4\tabularnewline
NC\_057523 & Hemiparasites & \emph{Melampyrum\_koreanum} & 143.865 &
38.2 & 101 & 67 & 30 & 4\tabularnewline
NC\_053791 & Hemiparasites & \emph{Melampyrum\_roseum} & 143.896 & 38.1
& 97 & 67 & 26 & 4\tabularnewline
NC\_042954 & Hemiparasites & \emph{Brandisia\_swinglei} & 155.344 & 38.1
& 113 & 79 & 30 & 4\tabularnewline
MT040753 & Hemiparasites & \emph{Pedicularis\_verticillata} & 142.733 &
38.6 & 103 & 69 & 30 & 4\tabularnewline
NC\_037433 & Hemiparasites & \emph{Pedicularis\_hallaisanensis} &
143.469 & 38.7 & 99 & 65 & 30 & 4\tabularnewline
NC\_046852 & Hemiparasites & \emph{Pedicularis\_longiflora} & 153.547 &
38.1 & 110 & 76 & 30 & 4\tabularnewline
NC\_046853 & Hemiparasites & \emph{Pedicularis muscicola} & 152.907 &
38.3 & 110 & 76 & 30 & 4\tabularnewline
NC\_046397 & Hemiparasites & \emph{Pedicularis\_resupinata} & 153.145 &
38.4 & 113 & 79 & 30 & 4\tabularnewline
NC\_056312 & Hemiparasites & \emph{Pedicularis\_dissecta} & 152.120 &
38.3 & 98 & 64 & 30 & 4\tabularnewline
NC\_046854 & Hemiparasites & \emph{Pedicularis\_oederi} & 153.139 & 38.4
& 111 & 77 & 30 & 4\tabularnewline
MW770457 & Hemiparasites & \emph{Pedicularis\_oederi\_var\_sinensis} &
152.770 & 38.3 & 110 & 76 & 30 & 4\tabularnewline
NC\_058762 & Hemiparasites & \emph{Pedicularis\_shansiensis} & 151.902 &
38.3 & 110 & 78 & 28 & 4\tabularnewline
NC\_029700 & Hemiparasites & \emph{Pedicularis\_ishidoyana} & 152.571 &
38.1 & 101 & 67 & 30 & 4\tabularnewline
NC\_053793 & Hemiparasites & \emph{Triphysaria\_versicolor} & 152.583 &
38.2 & 101 & 73 & 24 & 4\tabularnewline
NC\_031805 & Hemiparasites & \emph{Castilleja\_paramensis} & 152.926 &
38.2 & 106 & 72 & 30 & 4\tabularnewline
NC\_053792 & Hemiparasites & \emph{Phtheirospermum\_japonicum} & 153.397
& 38.1 & 107 & 79 & 24 & 4\tabularnewline
NC\_064388 & Hemiparasites & \emph{Cymbaria\_daurica} & 151.545 & 38.2 &
100 & 70 & 26 & 4\tabularnewline
NC\_064104 & Hemiparasites & \emph{Cymbaria mongolica} & 149.431 & 38 &
105 & 75 & 26 & 4\tabularnewline
NC\_023115 & Hemiparasites & \emph{Schwalbea\_americana} & 160.910 &
38.1 & 99 & 73 & 24 & 2\tabularnewline
NC\_046038 & Hemiparasites & \emph{Siphonostegia\_chinensis} & 148.961 &
38.4 & 112 & 78 & 30 & 4\tabularnewline
NC\_022859 & Autotrophs & \emph{Lindenbergia\_philippensis} & 155.103 &
37.8 & 113 & 79 & 30 & 4\tabularnewline
NC\_033534 & Autotrophs & \emph{Rehmannia\_chingii} & 154.055 & 38 & 113
& 79 & 30 & 4\tabularnewline
NC\_034312 & Autotrophs & \emph{Rehmannia\_elata} & 153.772 & 38 & 113 &
79 & 30 & 4\tabularnewline
NC\_034311 & Autotrophs & \emph{Rehmannia\_piasezkii} & 153.952 & 37.9 &
113 & 79 & 30 & 4\tabularnewline
NC\_034308 & Autotrophs & \emph{Rehmannia\_glutinosa} & 153.622 & 38 &
113 & 79 & 30 & 4\tabularnewline
NC\_034309 & Autotrophs & \emph{Rehmannia\_henryi} & 153.890 & 37.9 &
113 & 79 & 30 & 4\tabularnewline
NC\_034310 & Autotrophs & \emph{Rehmannia\_solanifolia} & 153.989 & 37.9
& 113 & 79 & 30 & 4\tabularnewline
NC\_039781 & Autotrophs & \emph{Triaenophora\_shennongjiaensis} &
155.319 & 37.7 & 113 & 79 & 30 & 4\tabularnewline
\bottomrule
\end{longtable}

Note: The statistics of gene numbers here refer to 113 unique plastid
genes in the typical angiosperms. The duplicated genes are not included.

\textbf{Table S3.} Four pairs of primers for amplifying DNA barcodes.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Name & Sequence (5' to 3') & Position\tabularnewline
CymN1\_F & agaccccattcctagaactaaca & \emph{ndhD-ndhG}\tabularnewline
CymN1\_R & agtcgctaaatctgcacaagt &\tabularnewline
CymN2\_F & gacccgacccgaaaattgat & \emph{ndhD-ndhG}\tabularnewline
CymN2\_R & tccccaccttgtaaattaggaaa &\tabularnewline
CymY\_F & atcatgcgcataccagtagc & \emph{rps15-ycf1}\tabularnewline
CymY\_R & agctactatgttcacagaagca &\tabularnewline
CymR\_F & ggcaattgtgaactctcggg & \emph{rpl32-trnL-UAG}\tabularnewline
CymR\_R & tcttcctcagtagctcagcg &\tabularnewline
\bottomrule
\end{longtable}

\par\null

\textbf{Table S4.} The list of sample numbers of the samples used in the
validation of DNA barcodes.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
Species & Sample No. & Location collected & Longitude & Latitude &
Altitude\tabularnewline
\emph{Cymbaria mongolica} & Cm1 & Binzhou, Shaanxi Province, China &
108\selectlanguage{ngerman}°13\selectlanguage{english}'06.11\selectlanguage{english}"E & 35\selectlanguage{ngerman}°14\selectlanguage{english}'38.63\selectlanguage{english}"N & 1035.3 m\tabularnewline
& Cm2 & Helan, Ningxia Hui Autonomous Region, China & 105\selectlanguage{ngerman}°59\selectlanguage{english}'35.67\selectlanguage{english}"E &
38\selectlanguage{ngerman}°42\selectlanguage{english}'05.28\selectlanguage{english}"N & 1357.6 m\tabularnewline
& Cm3 & Helan, Ningxia Hui Autonomous Region, China & 105\selectlanguage{ngerman}°59\selectlanguage{english}'35.67\selectlanguage{english}"E &
38\selectlanguage{ngerman}°42\selectlanguage{english}'05.28\selectlanguage{english}"N & 1357.6 m\tabularnewline
& Cm4 & Tianzhu, Gansu Province, China & 103\selectlanguage{ngerman}°04\selectlanguage{english}'44.68\selectlanguage{english}"E & 37\selectlanguage{ngerman}°01\selectlanguage{english}'12.47\selectlanguage{english}"N
& 2552.6 m\tabularnewline
& Cm5 & Tianzhu, Gansu Province, China & 103\selectlanguage{ngerman}°04\selectlanguage{english}'44.68\selectlanguage{english}"E & 37\selectlanguage{ngerman}°01\selectlanguage{english}'12.47\selectlanguage{english}"N
& 2552.6 m\tabularnewline
& Cm6 & Xining, Qinghai Province, China & 101\selectlanguage{ngerman}°49\selectlanguage{english}'33.59\selectlanguage{english}"E & 36\selectlanguage{ngerman}°34\selectlanguage{english}'13.16\selectlanguage{english}"N
& 2274 m\tabularnewline
& Cm7 & Xining, Qinghai Province, China & 101\selectlanguage{ngerman}°49\selectlanguage{english}'33.59\selectlanguage{english}"E & 36\selectlanguage{ngerman}°34\selectlanguage{english}'13.16\selectlanguage{english}"N
& 2274 m\tabularnewline
\emph{Cymbaria\_daurica} & Cd1 & Xilingol, Inner Mongolia Autonomous
Region, China & 116\selectlanguage{ngerman}°47\selectlanguage{english}'07.76\selectlanguage{english}"E & 43\selectlanguage{ngerman}°28\selectlanguage{english}'13.92\selectlanguage{english}"N & 1320.7 m\tabularnewline
& Cd2 & Yanqing, Hebei province, China & 115\selectlanguage{ngerman}°57\selectlanguage{english}'34.71\selectlanguage{english}"E & 40\selectlanguage{ngerman}°22\selectlanguage{english}'42.65\selectlanguage{english}"N
& 565 m\tabularnewline
& Cd3 & Yanqing, Hebei province, China & 115\selectlanguage{ngerman}°57\selectlanguage{english}'34.71\selectlanguage{english}"E & 40\selectlanguage{ngerman}°22\selectlanguage{english}'42.65\selectlanguage{english}"N
& 565 m\tabularnewline
& Cd4 & Yijinhuol Banner, Inner Mongolia Autonomous Region, China &
110\selectlanguage{ngerman}°11\selectlanguage{english}'42.41\selectlanguage{english}"E & 39\selectlanguage{ngerman}°33\selectlanguage{english}'08.35\selectlanguage{english}"N & 1372 m\tabularnewline
& Cd5 & Yijinhuol Banner, Inner Mongolia Autonomous Region, China &
110\selectlanguage{ngerman}°11\selectlanguage{english}'42.41\selectlanguage{english}"E & 39\selectlanguage{ngerman}°33\selectlanguage{english}'08.35\selectlanguage{english}"N & 1372 m\tabularnewline
& Cd6 & Daqing, Heilongjiang province, China & 124\selectlanguage{ngerman}°32\selectlanguage{english}'09.86\selectlanguage{english}"E &
46\selectlanguage{ngerman}°23\selectlanguage{english}'51.56\selectlanguage{english}"N & 159 m\tabularnewline
& Cd7 & Daqing, Heilongjiang province, China & 124\selectlanguage{ngerman}°32\selectlanguage{english}'09.86\selectlanguage{english}"E &
46\selectlanguage{ngerman}°23\selectlanguage{english}'51.56\selectlanguage{english}"N & 159 m\tabularnewline
\bottomrule
\end{longtable}

\selectlanguage{english}
\FloatBarrier
\end{document}

