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J
SE Journal of Systematics
and Evolution doi: 10.1111/jse.12650
Research Article
Molecular phylogeny and species delimitation of
Stachyuraceae: Advocating a herbarium specimen‐based
phylogenomic approach in resolving species boundaries
Jun‐Xia Su
1†
, Cong‐Cong Dong
1†
, Yan‐Ting Niu
2,3
,Li‐Min Lu
2
, Chao Xu
2
, Bing Liu
2
, Shi‐Liang Zhou
2
,
An‐Ming Lu
2
,Yu‐Ping Zhu
4
, Jun Wen
5
*, and Zhi‐Duan Chen
2
*
1
College of Life Sciences, Shanxi Normal University, Linfen 041000, China
2
State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
3
University of Chinese Academy of Sciences, Beijing 100049, China
4
School of Life Sciences, Nanjing University, Nanjing 210023, China
5
Department of Botany, National Museum of Natural History, Smithsonian Institution, Washington, DC 20013‐7012, USA
†
These authors contributed equally to this work.
*Author for correspondence. Jun Wen. E‐mail: wenj@si.edu; Zhi‐Duan Chen. E‐mail: zhiduan@ibcas.ac.cn
Received 12 March 2020; Accepted 9 May 2020; Article first published online 24 June 2020
Abstract Species concept and delimitation are fundamental to taxonomic and evolutionary studies. Both
inadequate informative sites in the molecular data and limited taxon sampling have often led to poor
phylogenetic resolution and incorrect species delineation. Recently, the whole chloroplast genome sequences
from extensive herbarium specimen samples have been shown to be effective to amend the problem.
Stachyuraceae are a small family consisting of only one genus Stachyurus of six to 16 species. However, species
delimitation in Stachyurus has been highly controversial because of few and generally unstable morphological
characters used for classification. In this study, we sampled 69 individuals of seven species (each with at least
three individuals) covering the entire taxonomic diversity, geographic range, and morphological variation of
Stachyurus from herbarium specimens for genome‐wide plastid gene sequencing to address species delineation in
the genus. We obtained high‐quality DNAs from specimens using a recently developed DNA reconstruction
technique. We first assembled four whole chloroplast genome sequences. Based on the chloroplast genome and
one nuclear ribosomal DNA sequence of Stachyurus, we designed primers for multiplex polymerase chain reaction
and high throughput sequencing of 44 plastid loci for species of Stachyurus. Data of these chloroplast DNA and
nuclear ribosomal DNA internal transcribed spacer sequences were used for phylogenetic analyses. The
phylogenetic results showed that the Japanese species Stachyurus praecox Siebold & Zucc. was sister to the rest
in mainland China, which indicated a typical Sino‐Japanese distribution pattern. Based on diagnostic
morphological characters, distinct distributional range, and monophyly of each clade, we redefined seven
species for Stachyurus following an integrative species concept, and revised the taxonomy of the family based on
previous reports and specimens, in particular the type specimens. Furthermore, our divergence time estimation
results suggested that Stachyuraceae split from its sister group Crossosomataceae from the New World at ca.
54.29 Mya, but extant species of Stachyuraceae started their diversification only recently at ca. 6.85 Mya.
Diversification time of Stachyurus in mainland China was estimated to be ca. 4.45 Mya. This research has provided
an example of using the herbarium specimen‐based phylogenomic approach in resolving species boundaries in a
taxonomically difficult genus.
Key words: chloroplast genome, DNA reconstruction, phylogeny, specimen‐based taxon sampling, Stachyuraceae.
1 Introduction
Species delimitation is central in systematics and evolu-
tionary biology (Simpson, 1951; Wiley, 1978; Agapow
et al., 2004; Baker & Bradley, 2006; De Queiroz, 2007;
Hausdorf, 2011). How species is delineated depends on the
species concept a taxonomist adopts. Numerous species
concepts (over 30) have been proposed so far, with the
“biological species concept”advocating reproductive iso-
lation being the most well known (Mayr, 1942, 1963;
Palumbi, 1994; Lu & Wang, 2016). Advances in molecular
phylogenetics in the 21st century have facilitated increased
applications of the “phylogenetic species concept,”which is
relatively easy to follow and uses monophyly to define
September 2020
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710–724© 2020 Institute of Botany, Chinese Academy of Sciences
species (De Queiroz, 2005; Zimmers et al., 2017). However,
the “integrative species concept”(Liu, 2016), which
harnesses population genetic evidence and harmonizes
different data sources, including morphological differences,
reproductive isolation, and niche differentiation, if available,
would be a better approach in delineating species
boundaries (De Queiroz, 1998, 2007; Stockman & Bond, 2007;
Bond & Stockman, 2008; Fujita et al., 2012; Hendrixson
et al., 2013; McKay et al., 2013). In plants, species delineation
has been challenging in many groups often due to the
complexity of morphological variation and the lack of
understanding of phylogenetic relationships and other
properties of species. Species recognition in floras of the
world or various regions has been largely morphology‐based
in most groups, which could be somewhat intuitive and has
led to inconsistent species boundaries among authors due to
differences in view of the importance of particular
morphological traits.
Recently, molecular techniques have been widely used to
resolve phylogenies and delimit species boundaries. How-
ever, both inadequate informative sites of DNA sequences
and a limited number of taxon sampling have sometimes led
to poor phylogenetic resolution and inappropriate taxonomic
treatment. The whole chloroplast genome, containing more
informative sites, has been regarded as a powerful data
source for resolving the phylogenetic relationships of
different taxonomic ranks, especially taxa with notorious
difficulty in species delimitation (Knox & Palmer, 1999;
Goremykin et al., 2004; Shaw et al., 2007; Tong et al., 2015;
Wen et al., 2018; Liu et al., 2019). With the development of
next‐generation sequencing technologies as well as the
decreasing cost, it has become practical to resolve difficult
phylogenies using the whole chloroplast genome (Wilkinson
et al., 2017; Valcárcel & Wen, 2019). In addition, sufficient
taxon sampling is crucial to resolve phylogenetic relation-
ships because many species exhibit abundant genetic
variation and different genotypes (Marshall, 1990). Incorrect
phylogenetic relationships might be retrieved if the sampling
cannot cover all geographic variations within a species. In
recent years, an increasing number of phylogenomic analyses
with denser taxon sampling have obviously improved the
resolution of deep phylogenetic relationships of taxonomi-
cally difficult genera (e.g., Niu et al., 2018, 2019; Wen
et al., 2018; Hammer et al., 2019; Zhang et al., 2019). But it
will be a big investment of time and money to acquire a large
amount of materials through fieldwork. Furthermore,
researchers often fail to obtain the species in concern due
to a series of factors, such as destroyed habitats, terrain, and
political issues. These problems can be overcome by utilizing
material from herbarium specimens. There are 3400 herbaria
storing approximately 350 million specimens over the past
400 years worldwide (http://sweetgum.nybg.org/science/ih/),
making it feasible for many plant taxonomists to obtain
species samples under their studies.
With increasing storage time, plastid DNA sequences could
degrade into very short fragments (<200 bp), and may be
easily contaminated during polymerase chain reaction (PCR)
amplification. However, Xu et al. (2015) explored the DNA
reconstruction technique (self‐primed PCR amplification,
Golenberg et al., 1996) to obtain 400–500 bp accurate
amplification of DNA fragments from specimens, which
proved effective for approximately one‐third of specimens
in the Herbarium of the Institute of Botany, Chinese
Academy of Sciences, Beijing, China (PE). Moreover, the
DNA reconstruction technique enables acquisition of high
quality whole plastome sequences from specimens.
Stachyuraceae, a family endemic to East Asia, consist of a
single genus Stachyurus Siebold & Zucc. with six to 16 species
according to different authors (Li, 1943; Chen, 1981;
Tang et al., 1983; Shan, 1999; Zhu et al., 2006; Yang &
Stevens, 2007). Stachyurus is confined to the temperate and
subtropical regions, ranging from the eastern Himalaya to
the Bonin Islands. The genus rarely stretches northward to
35°N on mainland Asia, but it can be found throughout the
Japanese archipelago, even as far north as Hokkaido
(Li, 1943; Fig. 1). Species of Stachyurus usually grow in
mountainous thickets or forests ranging from 30 to 2400 m
(up to 3300 m) a.s.l. (Zhu et al., 2006; Schneider, 2007).
The genus Stachyurus was first described by Siebold and
Zuccarini, and was placed in Pittosporaceae based on its
parietal placentation, superior ovary, numerous seeds, and the
lack of stipules (Siebold & Zuccarini, 1835). However, Bentham
(1861) and Bentham & Hooker (1862–1867) transferred
Stachyurus to Ternstroemiaceae, because they share some
morphological characters, such as the hermaphrodite flower,
superior ovary, and indehiscent berry. Gilg (1895) elevated
Stachyurus to the family rank, Stachyuraceae, which was
accepted by most classification systems (e.g., Hutchinson, 1967;
Cronquist, 1968; Takhtajan, 1969).
Stachyuraceae are well defined by some morphological
synapomorphies such as branches with large and distinct
pith, racemose or spicate inflorescences, 4‐merous flowers
(sepals 4, petals 4, and stamens 8), superior 4‐locular ovaries
with axile placentation, stigmas capitate, fruits as a berry,
and seeds with arils (Shan, 1999). Furthermore, their
monophyly is well supported by molecular studies based
on four chloroplast loci and one nuclear ribosomal (internal
transcribed spacer (ITS)) region (Zhu et al., 2006). Never-
theless, the infrafamilial classification of the monogeneric
Stachyuraceae has been highly controversial. Franchet (1898)
divided Stachyurus into two sections: sect. Callosurus and
sect. Stachyurus (=sect. Gymnosurus), mainly on the basis of
evergreen or deciduous habits. Section Callosurus is different
from sect. Stachyurus in having evergreen habits (vs.
deciduous habits) and short‐pedunculate inflorescences
with basal leaves (vs. sessile or subsessile inflorescences
without basal leaves). Although many authors showed that
intermediate conditions of the above characters also occur,
some workers (Li, 1943; Tang et al., 1983) still accepted
the sectional classification of Franchet (1898). However,
the subdivision in Stachyurus by Franchet (1898) was not
supported by other researchers (Chen, 1981; Shan, 1999;
Wei & Yang, 2001; Yang & Stevens, 2007). Molecular
systematic studies by Zhu et al. (2006) did not recover the
monophyly of sect. Callosurus and sect. Stachyurus.
The species circumscription of Stachyurus has been under
dispute. Stachyurus praecox Siebold&Zucc.,asthetypespecies,
is the first published species of the genus (Siebold, 1835).
Bentham (1861) described the second species, S. himalaicus
Hook. f. & Thomson ex Benth. Franchet (1898) recognized five
species and described three new species (S. salicifolius Franch., S.
chinensis Franch., and S. yunnanensis Franch.), and classified
711Integrative species delimitation of Stachyuraceae
J. Syst. Evol. 58(5): 710–724, 2020www.jse.ac.cn
them into two sections. Li (1943) identified 12 species and four
varieties of Stachyurus, mainly based on morphological
characters such as the leaf shape, apex, margin, and length of
inflorescence. Since then, the criterion for species delimitation of
Stachyurus mainly based on leaf morphology has been adopted
by most authors, and the number of species varies from 11 to 16
(Chen, 1981; Tang et al., 1983; Shan, 1999; Yang & Stevens, 2007).
However, Zhu et al. (2006) did not support leaf morphology as
the main taxonomic feature for Stachyurus due to its high level
of variation.
The most recent taxonomic revision of Stachyurus has
been carried out by using both morphological and molecular
data (Zhu, 2006), which recognized six species and eight
varieties. However, this study contained insufficient phylo-
genetic signal (four chloroplast DNA (cpDNA) markers, trnL‐
F,ndhF,rps16,trnS‐trnG; and one nuclear ribosomal DNA
(nrDNA), ITS) and a limited number of taxon samples (only
one individual sampled per species) not covering the
morphological and geographic diversity of the genus. To
date, the plastomes of Stachyurus have not been published.
In this study, we explore species delineation of Stachyur-
aceae using an integrative approach. We aim to: (i) investigate
the validity of an approach in plant taxonomic analysis, with all
samples from herbarium specimens and chloroplast genome
data obtained using next‐generation sequencing techniques; (ii)
reconstruct the phylogeny of Stachyuraceae; (iii) explore the
relationship between the species delimitation and geographic
distribution of Stachyurus; and (iv) revise the taxonomy of
Stachyurus based on molecular results, geographic range, and
morphological observations.
2 Material and Methods
2.1 Taxon sampling
For the convenience of discussion, we followed the species
delimitation of Stachyurus of Yang & Stevens (2007) in Flora
of China. A total of 69 individuals from seven species (each
with at least three individuals), spanning the taxonomic
diversity, geographic range, and morphological variation of
the genus, were sampled from the herbarium specimens
deposited in PE (Table S1). In this study, we sequenced 45
highly variable regions in both plastomes and ITS from the
above samples. Voucher information for each sample is
provided in Table S1. Two species of Sapindales, Boswellia
sacra Flueck. (Burseraceae) and Leitneria floridana Chapm.
(Simaroubaceae) were selected as outgroups for the
phylogenetic analyses, and their whole chloroplast genome
sequences were downloaded from GenBank (https://www.
ncbi.nlm.nih.gov/).
2.2 DNA extraction, purification, and reconstruction
In order to obtain high‐quality DNA templates, we improved
and combined the modified CTAB method (Li et al., 2013)
with the silicon‐based magnetic bead purification method
(Elkin et al., 2002) to process the samples. Most samples of
Fig. 1. Distribution of Stachyurus.
712 Su et al.
J. Syst. Evol. 58(5): 710–724, 2020 www.jse.ac.cn
this study were aged specimens, which were collected from
1933 to 2011 (Table S1), and the extracted DNAs were of
degraded quality. Most of them were short fragments with a
length of approximately 150 bp, which was not good for
direct PCR amplification. In order to solve this problem, we
adopted the DNA reconstruction technique to preprocess
the DNA samples, which could greatly improve the PCR
success rate for DNAs extracted from specimens (Xu
et al., 2015).
2.3 Chloroplast genome sequencing, assembly, annotation,
and primer design
In order to obtain the reference plastomes of Stachyurus,we
first undertook a phylogenetic analysis of 69 individuals in
Stachyurus using five DNA loci (four cpDNA: rps16 intron,
trnL‐F,ndhF,trnS‐trnG; and one nrDNA: ITS) according to Zhu
et al. (2006). Based on this result, four representative
individuals (S. chinensis JG1, S. chinensis WC1, S. retusus Yen C.
Yang EM1, and S. yunnanensis ML1) were finally selected to
obtain the whole chloroplast genome.
The DNAs of the four individuals were sent to Novogene
(Beijing, China) for library construction and sequencing. DNA
sequencing for these samples was carried out on an Illumina
(San Diego, CA, USA) HiSeq 2500 instrument with 2 ×150 bp
paired‐end reads. The raw Illumina data, including duplicate
reads, adapter contaminated reads, reads with more than five
Ns, and low‐quality reads with Q‐value ≤20 were subsequently
filtered out using Trimmomatic version 0.32 with the recom-
mended options (Bolger et al., 2014). Remaining high‐quality
reads were assembled de novo into contigs using SPAdes version
3.6.1 (http://cab.spbu.ru/software/spades/; Bankevich et al., 2012)
with a K‐mer length of 95. After filling the gaps and correcting
the ambiguities with Sanger sequencing, we obtained the whole
plastome sequences of the four samples. The assembled
chloroplast genome sequence was imported into MAFFT version
7.3 (Katoh & Standley, 2013), and was compared with clean reads
to check the reliability of the assembly.
Chloroplast genomes for the above four individuals were
annotated using Geseq (Tillich et al., 2017), and the final
annotated plastomes were deposited in GenBank (Table S2).
For plastid protein and rRNA‐coding genes, we referred to the
profile HMM database (http://hmmer.org/documentation.html)
and manually curated these misannotated exon–intron borders.
The plastome of B. sacra was referenced to make a correction
using the Geneious version 9.1.4 (http://www.geneious.com;
Kearse et al., 2012). Finally, we drew circular plastome maps
with OGDRAW (Lohse et al., 2013) (Fig. 2A).
The four chloroplast genomes were aligned using MAFFT
with default parameters. To evaluate sequence divergence
and find high mutation regions, DNA polymorphisms were
calculated in DnaSP version 5.0.0 (Librado & Rozas, 2009;
Fig. 2B). Furthermore, we designed 44 chloroplast primer
pairs (see Table S3 for details) in adjacent areas with the high
mutation regions in Geneious.
2.4 Polymerase chain reaction amplification with DNA‐
labeled primers and sequencing
For the remaining 65 samples, we selected 44 chloroplast
and one nrDNA (ITS) loci, and amplified the regions using the
newly designed primers (Table S3). Two short fragments of
each marker instead of one long fragment from the
degraded templates were amplified with internal primers in
combination with the DNA‐labeled primers. Polymerase chain
reactions were carried out in a 10 μL volume comprised of
1μL reconstructed DNA, 1 μL10×Taq buffer, 1 μL 2 mM dNTP,
0.5 μL5μM primers, 5.9 μL ddH
2
O, and 0.25 U Taq
polymerase, with the following program steps: 94 ℃for
3 min, 35 cycles of 94 ℃for 30 s, 50 ℃for 30 s, 72 ℃for 30 s,
and a final extension at 72 ℃for 10 min. The PCR products
were checked on 1% agarose gels. The mixture containing all
PCR products of different markers was used to construct a
DNA library and sequenced on an Illumina HiSeq 4000
sequencer (Novogene) with a mode of PE150.
2.5 Phylogenetic analyses
The sequencing reads of the Illumina sequencer were
classified into samples according to DNA labels, and further
divided into genes according to primer sequences. All reads
of the same gene in the same sample (>10×,Q‐value >20)
were used to produce consensus sequences under the
condition of >97% sequence similarity by Vsearch (Rognes
et al., 2016). The output sequences with the highest
sequencing depth were aligned in MAFFT.
In this study, we assembled two datasets: a cpDNA dataset
including 44 cpDNA loci (7266 bp) and one nrDNA (ITS)
dataset (161 bp). Before combining the ITS and cpDNA
datasets, we undertook rapid bootstrap (BS) analysis for
each dataset with 1000 replicates. Tree congruence was
assessed by visually comparing topologies and support
values between conflicting branches, and no remarkable
conflicts were observed between the two datasets (i.e.,
maximum likelihood (ML) BS >70%, Hillis & Bull, 1993;
posterior probability (PP) >0.99, Inácio et al., 2017). Un-
ambiguous aligned insertions/deletions (indels) were man-
ually coded and appended to the sequence matrix (Table S4).
In order to corroborate the veracity of the topology, we
used the ML and Bayesian inference (BI) approaches to
undertake phylogenetic analyses for the combined dataset of
44 cpDNA and ITS (Appendix 1, and please contact the author
for raw data). The ML analyses were implemented in RAxML
version 8.2.8 (Stamatakis, 2006, 2014) with 1000 bootstrap
replicates using the GTR +G model. Bayesian inference
analysis with the unpartitioned dataset was run using
MrBayes version 3.2.0 (Ronquist & Huelsenbeck, 2003) with
the GTR model. Two independent analyses were carried out
using four Markov chains Monte Carlo (one hot and three
cold chains), each running for 10 million generations and
sampling every 1000 generations. The stationarity was
considered to be reached when the average standard
deviation (SD) of split frequencies was below 0.01.
With the initial 25% of the trees of each run discarded as
burn‐in, the remaining trees were used to construct a 50%
majority‐rule consensus tree and to estimate the PP.
Phylogenetic trees were visualized using FigTree version 1.4.4
(Rambaut, 2018).
2.6 Divergence time estimation
To estimate the divergence times of Stachyuraceae, we
obtained sequences of three cpDNA regions (rbcL,matK, and
atpB) for 16 species that represent all major lineages of
Crossosomatales and four outgroup species (Wendtia gracilis
Meyen, Viviania marifolia Cav., Bersama lucens (Hochst.)
713Integrative species delimitation of Stachyuraceae
J. Syst. Evol. 58(5): 710–724, 2020www.jse.ac.cn
Fig. 2. Gene map and polymorphism analysis of chloroplast whole genome of Stachyuraceae. A, Genes shown outside and
inside the outer circle are transcribed clockwise and counter clockwise, respectively. Genes belonging to different functional
groups are color‐coded, and dashed area in the inner circle indicates the GC content of the chloroplast genome. B, The
abscissa is the genetic locus, and the ordinate “mutated value”represents the number of mutated sites per 100 bp of
the gene.
714 Su et al.
J. Syst. Evol. 58(5): 710–724, 2020 www.jse.ac.cn
Szyszył., and Francoa sonchifolia Cav.). Sequences of three
species (S. chinensis WC1, S. retusus EM1, and S. yunnanensis
ML1) were from our laboratory, those of the other 17 species
were downloaded from GenBank (Table S5).
The combined cpDNA dataset was conducted in BEAST version
1.7.3 (Drummond & Rambaut, 2007) with the GTR substitution
model after Bayesian searches for topologies. An uncorrelated
lognormal relaxed clock (Drummond et al., 2002) was applied,
and a Yule process was chosen as tree prior. The crown age of
Crossosomatales (54.42–115.31 Mya, Magallón et al., 2015)
was assumed as a normal distribution with a mean (±SD) of
84.63 ±30 Mya, which was used as a secondary calibration point
(Fig. S1, node F2). The oldest reliable fossil of Turpinia Vent. is
from the Lamar River Formation (late early to early middle
Eocene) in Yellowstone National Park (Wheeler et al., 1978), so
we followed Zhu (2006) and constrained the clade of
Turpinia–(Staphylea–Euscaphis)(Fig.S1,nodeF1)as49Mya
(mean =0, SD =1.0, offset =49 Mya) with a lognormal distribu-
tion. Markov chain Monte Carlo was run for 100 million
generations with sampling every 10 000 generations. Tracer
version 1.5.1 (Raymond & Rousset, 2009) was used to examine
theadequateeffective sample size values (>200). The maximum
clade credibility tree was constructed in TreeAnnotator version
1.7.3 (Drummond et al., 2012) and edited in FigTree.
2.7 Morphological examinations
Approximately 1500 specimens, including the types from A,
BM, E, IBSC, K, KUN, L, LBG, NAS, P, PE, US, and WUK, were
examined to check the morphological characters that were
previously used in the classification of Stachyuraceae. For
examination of morphological characters that need to use
experimental methods (such as the flower anatomy), we
made observations mainly on the specimens that were
sampled for the molecular analysis. Flowers were rehydrated
in hot water. Leaves and flowers were observed using a
dissecting microscope (SMZ1000; Nikon, Tokyo, Japan) in the
State Key Laboratory of Systematic and Evolutionary Botany,
Institute of Botany, Chinese Academy of Sciences. Morpho-
logical descriptions follow Li (1943), Chen (1981), Tang et al.
(1983), Shan (1999), Zhu (2006), and Yang & Stevens (2007).
3 Results
3.1 Evaluation of chloroplast genome polymorphism and
informative sites
The average length of the four plastomes of Stachyuraceae is
approximately 162 000 bp, ranging from 162 649 bp to
162 759 bp with 37.10% GC content and 98.6% aligned ratio.
DNA polymorphisms of the four plastomes are shown in
Fig. 2B. All the chloroplast genomes showed the typical
quadripartite structure: inverted repeat regions A and B, a
large single‐copy region, and a small single‐copy region
(Table S2). The plastomes of Stachyuraceae have 130 genes,
including 84 coding genes, 38 transfer RNA genes, and eight
ribosomal RNA genes (Tables S2, S6).
We generated three datasets of 71 individuals (including
two outgroups) containing the 44 aligned cpDNA regions
with a length of 7454 bp, the aligned ITS region with a length
of 162 bp, and the combined dataset with a length of 7616 bp
(Appendix 1), respectively. Eventually, 2244 DNA fragments
(72.27% of all DNA fragments) were obtained (Table S7;
Appendix 1). The combined dataset has 6955 constant sites
(93.67% of all sites), and 118 parsimony‐informative sites. In
the alignment, 42 unambiguous indels in total with 256 bp
were found, and 17 of them provided synapomorphic
information for phylogenetic analyses (Table S8).
3.2 Phylogenetic relationships and species delimitation
Due to the poorly resolved backbone relationships within
Stachyuraceae with the short ITS dataset, the phylogenetic
relationships were described below based on the results of
the combined cpDNA and ITS dataset. The tree topology of
the BI analysis was similar to that of the ML analysis (Fig. 3A).
Five clades of Stachyuraceae were identified in the ML
analysis. Clade A (BS =98%, PP =1.0) included eight
individuals of Stachyurus praecox from Japan and Taiwan,
China. The remaining taxa formed one big clade (BS =69%,
PP =0.73), which was divided into Clade B (BS =79%,
PP =0.95) and Clade C (BS =74%, PP =0.98). Clade B was
divided into two subclades (Clade B1 and Clade B2), with
Clade B1 including nine individuals of S. himalaicus distributed
in the high‐altitude areas of Tibet and adjacent areas
(BS =95%, PP =1.0), and Clade B2 (BS =99%, PP =1.0)
containing 26 individuals of S. chinensis collected from
central and southeastern China. Clade C included two small
clades (Clade C1 and Clade C2), with Clade C1 (BS =90%,
PP =1.0) consisting of 11 individuals of S. yunnanensis
distributed in the western and southern margin of the
Sichuan basin in China, and Clade C2 (BS =99%, PP =1.0)
composed of 15 individuals of S. retusus from the
northwestern Sichuan basin in China.
3.3 Divergence time estimation
The matrix of Crossosomatales with outgroups was 4415 bp
containing 338 parsimony‐informative sites. The stem and
crown ages of Stachyuraceae were estimated to be ca.
54.29 Mya (95% highest posterior density (HPD), 21.89–90.62
Mya; node A; Fig. S1) and 6.85 Mya (95% HPD, 0.43–20.38 Mya;
node B; Fig. S1), respectively. Divergence time of Stachyurus on
the Chinese mainland was estimated to be ca. 4.45 Mya (95%
HPD, 0.38–13.23Mya;nodeC;Fig.S1).
4 Discussion
4.1 Taxonomic approach of combining extensive herbarium
specimen sampling and plastid genome sequencing with DNA‐
labeled primers
Characters and taxon sampling are the most important
aspects for correct species delineation. In addition to
morphological characters, more and more molecular charac-
ters have been used for recognizing species. Extensive taxon
sampling can determine the variation range of morphological
and molecular characters, and help make optimal taxonomic
treatment of species. Both the “phylogenetic species
concept”and the “integrative species concept”are
emphasized on ample characters and extensive sampling
strategies in taxonomic studies. However, the species
recognition in many plant taxa is still disputed due to the
inadequate molecular characters and taxon sampling.
715Integrative species delimitation of Stachyuraceae
J. Syst. Evol. 58(5): 710–724, 2020www.jse.ac.cn
Fig. 3. Continued
716 Su et al.
J. Syst. Evol. 58(5): 710–724, 2020 www.jse.ac.cn
With the development of the next‐generation sequencing
technology, it has become common to use chloroplast
genome data in phylogenetic studies (Wen et al., 2015, 2017;
Zhang et al., 2019). However, for taxonomically difficult taxa
such as Stachyurus, adequate sampling and sufficient
phylogenetic signals are the key to advance the taxonomy
of these lineages (Funk, 2018). In this study, we used
extensive taxon sampling by drawing on the rich resources
of herbarium specimens. We also improved the phylogenetic
signals by obtaining enough informative sites through
sequencing the non‐conserved regions of the plastid genome
with DNA‐labeled primers for species of Stachyurus (Fig. 4).
Our sampling well represents the variation of each species in
its entire distribution range, and the abundant molecular
characters ensure the reconstruction of robust phylogenetic
trees with high resolution. The resolved clades showed
definite groupings for the infrageneric taxa of Stachyurus.
Our current approach of using extensive taxon sampling
and large datasets of molecular characters from next‐
generation sequencing provides better resolution than the
traditional molecular systematics approach with a few loci
sequenced using Sanger sequencing (Zhu et al., 2006; Fig. 4).
We can use the phylogenetic results from extensive taxon
sampling to help delimit species in Stachyurus. We adopted
the “phylogenetic species concept,”and accepted that the
monophyly was used as the criterion for species delimitation.
We also followed the “integrative species concept,”
delineating species in Stachyuaceae using multiple criteria
including phylogenetic monophyly, diagnostic morphological
characteristics, and geographic difference. In this study, we
yielded five clades that were supported by enough molecular
evidence with sampling that previously described Stachyurus
species throughout their distributional ranges. We checked if
each clade represented a species in the traditional systems of
classification, and then carefully examined the diagnostic
morphological characteristics of individuals in each clade. We
found that most of our strongly supported clades corre-
sponded to a species in previous systems of classification.
Some of them were treated differently in various systems,
and we finally provided a taxonomic treatment based on
specimens (including types) and previous reports, following
the rules of nomenclature.
4.2 Species identification with morphological and molecular
data
In our phylogenetic tree, five clades were recognized within
Stachyuraceae and grouped into three big clades (i.e., Clade
A, Clade B, and Clade C; Fig. 3A). Clade A was sister to the
rest of Stachyuraceae (Clade B +Clade C) in the ML and BI
trees. The position of Clade A was also found and strongly
supported in the ITS tree of Zhu et al. (2006). The remaining
species, except Clade C2, have been placed in sect.
Stachyurus based on their deciduous habit (Franchet, 1898;
Li, 1943; Tang et al., 1983). However, this study did not
recover the monophyly of sect. Stachyurus because the
evergreen Clade C1 was nested within the section of
deciduous taxa, which is consistent with Zhu et al. (2006).
Stachyurus praecox Clade A consists of only one species, S.
praecox. All eight individuals of S. praecox sampled by our
analyses in Clade A is allopatric to those of Clade B and Clade
C. Seven of them were collected from Japan and one
accession was from Taiwan, China (Fig. 3B). However, the
individuals of both Clade B and Clade C were from mainland
China. Morphologically, S. praecox has straight lateral veins
and larger petals (ca. 8–12 mm long), whereas other species of
Stachyurus have arcuate lateral veins and smaller petals
(ca. 4–7 mm long; Zhu, 2006). The taxonomic status of the
Stachyurus species from Taiwan, China has been controversial.
It has been treated as S. sigeyosii Masam. (Masamune, 1938),
S. himalaicus (Li, 1943; Chen, 1983; Tang et al., 1983;
Shan, 1999), or S. chinensis (Yang & Stevens, 2007). Zhu
(2006) treated S. sigeyosii as a variety of S. praecox,basedon
the phylogenetic analyses (Zhu et al., 2006) and morphological
evidence. The present study showed that the individual
collected from Taiwan, China was embedded within S. praecox
with high support in the ML and BI trees.
Stachyurus himalaicus and S. chinensis Clade B is a
monophyletic group with moderate support (BS =79%,
PP =0.95), which is supported by two deletions (4‐bp and
1‐bp) in the aligned matrix (Table S8). However, no obvious
morphological synapomorphies can be detected for Clade B
based on our morphological observations or search of
previous works. Most of the Clade B members belong to
the deciduous sect. Stachyurus, which has been considered
with a complicated taxonomic history (Li, 1943; Chen, 1981;
Tang et al., 1983; Shan, 1999; Zhu et al., 2006; Yang &
Stevens, 2007). Most researchers recognized the species
status of S. chinensis, but Zhu (2006) treated it as S.
himalaicus var. latus (H. L. Li) Y. P. Zhu. Furthermore, S.
himalaicus has been accepted as a separate species by all
authors; nevertheless, its species boundary was controver-
sial. For example, S. himalaicus of Zhu (2006) contained two
species, S. chinensis and S. salicifolius, and that of Li (1943)
included S. sigeyosii. Tang et al. (1983) argued that the two
varieties of S. himalaicus (var. dasyrachis C. Y. Wu ex S. K.
Chen and var. microphyllus C. Y. Wu ex S. K. Chen) recognized
by Chen (1981) should not be the members of S. himalaicus
(Fig. 4). Thus, the identifications of the Clade B samples have
been problematic. Most of them were identified as S.
himalaicus or its varieties, or S. chinensis, but there are also
other identifications such as S. yunnanensis,S. obovatus
(Rehder) Hand.‐Mazz., S. oblongifolius F. T. Wang & Tang, and
S. szechuanensis Fang in the PE.
Our result indicates that Clade B can be divided into two
well‐supported subclades, Clade B1 and Clade B2. All samples
of Clade B1 are limited to a small geographic area at
2000–2700 m a.s.l. in Linzhi and Bomi of Tibet, Baoxin,
Sichuan, and Lijiang, Yunnan, but those of Clade B2 occur in a
Fig. 3. Geographic distribution and phylogenetic trees of all samples of Stachyuraceae. A, Maximum likelihood phylogenetic tree of
Stachyuraceae based on the combined data of 44 chloroplast DNA loci and nrDNA ITS with support values above the branches
(maximum likelihood bootstrap percentages/Bayesian inference posterior probabilities). –, a node that does not appear in the
Bayesian inference trees. B, Solid or dotted lines represent the distribution range of each corresponding color clade.
717Integrative species delimitation of Stachyuraceae
J. Syst. Evol. 58(5): 710–724, 2020www.jse.ac.cn
Fig. 4. Development of taxonomic approaches and species delimitation of Stachyuraceae in different classification systems. A,
Three taxonomical approaches. B, Different systems of classification of Stachyurus.
718 Su et al.
J. Syst. Evol. 58(5): 710–724, 2020 www.jse.ac.cn
wide region covering central and southern China (Fig. 3B).
Furthermore, Clade B1 is distinguished from Clade B2 in
having three indels (1‐bp insert, 1‐bp deletion, and 7‐bp
deletion) in the aligned matrix (Table S8). Based on the
morphological investigation of our samples as well as the
type specimens, the samples of Clade B1 are different from
those of Clade B2 in having obovate petals (vs. ovate petals
in B2; Li, 1943; Shan, 1999; Yang & Stevens, 2007) and
stamens shorter than petals (vs. stamens with the same
length as petals in B2; Li, 1943; Shan, 1999). Combining the
molecular phylogenetic results, morphological observations,
and geographic distribution, we propose that each subclade
of Clade B merits the species status. According to our
observation on the specimens, we find that samples of Clade
B1 correspond to S. himalaicus in morphology (e.g., petals
obovate, stamens shorter than petals), which is largely
consistent with the identifications of both Y. P. Zhu and Q. E.
Yang in PE. As a result, we treated Clade B1 as S. himalaicus.
The type locality of S. chinensis is Yanjin, Yunnan, China,
which falls into the distribution region of Clade B2. Moreover,
the morphological characters of the samples in Clade B2 are
more similar to those of S. chinensis (e.g., petals ovate,
stamens with the same length as petals). We herein treated
Clade B2 as S. chinensis.
Stachyurus yunnanensis and S. retusus Clade C was
supported as a monophyletic group with moderate to strong
support (BS =74%, PP =0.98). Clade C contained two
subclades, Clade C1 (BS =90%, PP =1.0) and Clade C2
(BS =99%, PP =1.0). Samples of the two subclades in this
study share little geographic overlap (Fig. 3B), with Clade C2
mostly occurring in the northwest of the distribution region
of Clade C1. In addition, Clade C1 is different from Clade C2 in
having five insertions (one 3 bp, two 6‐bp, one 9‐bp, one 13‐
bp) in the alignment. Furthermore, specimens of Clade C1
differ markedly from those of Clade C2 in some morpho-
logical characters, including having short styles (vs. long
styles in Clade C2; Zhu, 2006), both leaf surfaces glabrous (vs.
leaf densely white tomentose to glabrous on the abaxial
surface in Clade C2; Li, 1943; Shan, 1999; Zhu, 2006; Yang &
Stevens, 2007), and lateral veins inconspicuous on both sides
(vs. lateral veins conspicuously raised on both sides in Clade
C2; Li, 1943; Shan, 1999; Yang & Stevens, 2007). Therefore,
each subclade of Clade C may be treated at the species level.
Samples of Clade C1 were collected from Muli and Dengcang
of Sichuan, Kunming and Malipo of Yunnan, Shanxia and
Xingshan of Hubei, and Daozheng, Guizhou, and Nanchuan of
Chongqing in China, corresponding to the distribution of S.
yunnanensis as defined by Tang et al. (1983). Thus, it is
feasible to recognize Clade C1 specimens as S. yunnanensis
morphologically. Most of the samples in Clade C2 were
collected from Daozheng, Guizhou, Emei, Dengcang, and
Tianquan of Sichuan, which largely match the distribution
region of S. retusus and S. obovatus. However, none of the
samples from Clade C2 shares similar morphological
characters with S. obovatus. Based on their monophyly and
geographic distribution as well as the nomenclatural priority
criterion, we herein treat Clade C2 as S. retusus.
Stachyurus salicifolius and S. cordatulus Merr. The species
status of S. salicifolius has been controversial. Zhu et al.
(2006) indicated that the two varieties of S. salicifolius (var.
salicifolius and lancifolius) did not group into the same clade
in the chloroplast tree. Additionally, the two clones of S.
salicifolius var. lancifolius were placed in two different clades
in the ITS tree. We found that the three samples of S.
salicifolius were assigned to two clades, one in Clade C1
collected from Yongshan, Yunnan and two in Clade C2
collected from Emei, Sichuan. Zhu et al. (2006) suggested
that the species S. salicifolius might be of hybrid origin. Based
on our results, we suggested that the taxonomic status of S.
salicifolius should be evaluated in future studies with more
data from the nuclear genome. Our current study did not
contain S. cordatulus, which is distributed in Gongshan,
Yunnan, China and northern Burma, but we tentatively
treated it at the species level based on the morphological
description from various workers (Merrill, 1941; Li, 1943;
Chen, 1981; Tang et al., 1983; Shan, 1999; Zhu et al., 2006;
Yang & Stevens, 2007).
Our research provided a valuable approach to resolve the
phylogeny of a taxonomically difficult genus, although some
unresolved problems of Stachyurus still remain. For example,
the interspecific hybridization origin in Stachyurus is not
clarified because of the lack of nuclear data. In addition,
statistical analyses of morphological variation and niche
differentiation are still not investigated. Furthermore, S.
cordatulus without available DNA was not included in our
phylogenetic analyses. To resolve these problems mentioned
above, further studies with denser sampling based on
nuclear and chloroplast genes, morphology, and ecological
niche analyses are needed to test the systematic framework
that we establish here.
4.3 Divergence times of Stachyuraceae
As an East Asian endemic family, Stachyuraceae seem to
have evolved more slowly compared with their sister group
Crossosomataceae from the New World (Zhu et al., 2006).
Stachyuraceae split from Crossosomataceae ca. 54.29 Mya
(95% HPD, 21.89–90.62 Mya; Fig. S1, node A), constituting an
ancient East Asian and New World disjunction (Wen
et al., 2010, 2016). But extant species of Stachyuraceae
were inferred to have not diverged until recently at ca. 6.85
Mya (95% HPD, 0.43–20.38 Mya; Fig. S1, node B). There might
also be potential extinction events in the early divergence
and evolution of Stachyuraceae. The modern distribution
pattern and fossil records of Stachyuraceae and Cross-
osomataceae indicate that their common ancestor was
widely distributed in the Northern Hemisphere before the
Tertiary period (Zhu et al., 2006). Zhu et al. (2006) suggested
that orogenies and the gradual climatic cooling might have
caused the early extinction of Stachyuraceae in Europe and
the discontinuous distribution of Crossosomatales in the
Northern Hemisphere (see also Harris et al., 2017).
One dispersal event in Stachyurus occurred between
southern East Asia and Japan during the Late Miocene to the
Early Pliocene (ca. 6.85 Mya; 95% HPD, 0.43–20.38Mya;Fig.S1,
node B), which is coincidentally similar to the biogeographic
pattern detected in many other groups, for example, Euptelea
Siebold & Zucc (Eupteleaceae) (Cao et al., 2016), Coptis Salisb.
(Ranunculaceae) (Xiangetal.,2018),andDiabelia Landrein
(Wang et al., 2020). One commonly accepted hypothesis is that
exchanges of plants between mainland Asia and the Japanese
Islands by way of the East China Sea land bridge occurred due
to the drop of sea level during the late Miocene to the early
719Integrative species delimitation of Stachyuraceae
J. Syst. Evol. 58(5): 710–724, 2020www.jse.ac.cn
Pliocene. Subsequently, the continuous distribution of some
extant ancestral species was interrupted due to the rise of the
sea level and global cooling (Herbert et al., 2016), as well as an
increasingly drier climate in East Asia (Li et al., 2014).
4.4 Taxonomic treatment
Stachyurus Siebold & Zucc., Fl. Jap. 1: 42, pl. 18. 1836.
Type Stachyurus praecox Siebold & Zucc.
Diagnostic characters Shrubs or small trees, sometimes
climbing. Evergreen, semi‐evergreen, or deciduous. Branch-
lets with obvious pith, most often glabrous. Leaves simple,
alternate; leaf blade papery to leathery. Racemes or spikes,
axillary. Flowers small; bract 1, bracteoles 2, connate at base.
Sepals 4 in 2 series, imbricate. Petals 4, imbricate. Stamens 8
in 2 series. Ovary superior, 4‐loculed, with basal nectary;
placentae axile at base, parietal at middle; style simple;
stigma capitate, shallowly 4‐lobed. Fruit a berry, pericarp
leathery. Seeds numerous, small, with soft aril. 2n=24.
Distribution In the temperate and subtropical regions of
East Asia, ranging from the eastern Himalaya to Japan.
Diversity Seven species.
Key to species of Stachyurus
1a. Evergreen or semi‐evergreen shrubs……………………2
1b. Deciduous shrubs or small trees…………………………3
2a. Leaf margin distinctly serrate; middle vein raised on
the abaxial surface; petiole long 1–2.5 cm; ovary glabrous
……………………………………………...S. yunnanensis
2b. Leaf margin inconspicuously serrate; middle veins raised
on both surfaces; petiole long ca. 4 mm; ovary pubescent
…………………………………………………S. salicifolius
3a. Lateral veins connected into a longitudinal vein parallel
to leaf margin………………………………..S. cordatulus
3b. Lateral veins irregularly anastomosing at margin………4
4a. Style ca. 10–15 mm long (distributed in southwest Sichuan,
China)……………………………………………..S. retusus
4b. Style ca. 1–4 mm long……………………………………5
5a. Leaf veins straight; petals 8–12 mm long (distributed in
Japan and Taiwan, China)……………………..S. praecox
5b. Leaf veins curving; petals 4–7 mm long…………………6
6a. Petals obovate; stamens shorter than petals (distributed
in eastern Himalaya)…………………………S. himalaicus
6b. Petals ovate; stamens as long as petals (distributed in
Chinese Mainland)…………………………….S. chinensis
(1) Stachyurus himalaicus Hook. f. & Thomson ex Benth., J.
Proc. Linn. Soc. Bot. 5: 55. 1861 [1860]. Type: INDIA,
Sikkim, Oct. 1849, J. D. Hooker s.n. (lectotype: designated
here, K, barcode K000190018!)
=S. himalaicus subsp. purpureus Y. P. Zhu & Zhi Y. Zhang,
Acta Phytotax. Sin. 42(5): 460. 2004 ≡S. himalaicus var.
purpureus (Y. P. Zhu & Zhi Y. Zhang) Y. P. Zhu in Ph.D.
thesis. 114. 2006, nom. inval. Type: NEPAL, Kabeli Khola,
Apr. 1967, J. D. A. Stainton 5822 (holotype: BM, barcode
BM000821600!); CHINA, Xizang, Mêdog, Apr. 1983,
B. S. Li & S. Z. Cheng 04255 (paratypes: PE, barcode
01432294!, 01432295!, 01432296!, 01432297!, 01432298!)
=S. himalaicus var. alatipes C. Y. Wu ex S. K. Chen, Acta
Bot. Yunnan. 3(2): 133. 1981. Type: CHINA, Yunnan,
Lijiang, May 1937, K. M. Feng 404 (holotype: KUN,
barcode 1208160!); isotype: A, barcode 00063256!)
(2) Stachyurus chinensis Franch., J. de Bot. 12: 254. 1898. Type:
CHINA, Yunnan, Yanjin, May 1894, J. M. Delavay s.n.
(lectotype: designated here, P, barcode P00067725!)
=S. chinensis var. brachystachyus C.Y.Wu&S.K.Chen,
Acta Bot. Yunnan. 3(2): 132. 1981. Type: CHINA, Yunnan,
Zhongdian, Jul. 1963, Zhongdian Exp. 63‐2512 (holotype:
KUN, barcode 1208139!); Jun. 1963, Zhongdian Exp. 63‐2711
(paratype: KUN, barcode 0478215!); CHINA, Yunnan, Lijiang,
Jul. 1959, K. M. Feng 22373 (paratypes: PE, barcode
01432275!, KUN, barcode 0478274!); Aug. 1962, Zhongdian
Exp. 1531 (paratype: KUN, barcode 0478282!); Jun. 1940, R.
C. Ching 30831 (paratype: KUN, barcode 0478226!); CHINA,
Yunnan,Weixi,Jun.1935,C. W. Wang 63694 (paratype:
KUN, barcode 0478539!); CHINA, Xizang, Chayu (Zayu),
Sept. 1935, C. W. Wang 66392 (paratypes: KUN, barcode
0478144!, PE, barcode 01432279!, 01432280!); Sept. 1937, T.
T. Yu 1025 9 (paratype: KUN, barcode 0478088!) ≡S.
brachystachyus (C. Y. Wu ex S. K. Chen) Y. C. Tang & Y. L.
Cao, Acta Bot. Yunnan. 10(3): 349. 1988 ≡S. chinensis
subsp. brachystachyus (C. Y. Wu ex S. K. Chen) Y. C. Tang &
Y.L.Cao,ActaPhytotax.Sin.21(3):246.1983
=S. caudatilimbus C. Y. Wu ex S. K. Chen, Acta Bot. Yunnan.
3(2): 130. 1981. Type: CHINA, Shaanxi, Ningqiang, Oct. 1958,
P. Y. Li 7 91 (holotype: KUN, barcode 1208137!; isotypes:
WUK, barcode 349498!, 105517!)
=S. chinensis var. cuspidatus H.L.Li,Bull.Torr.Bot.Club
70(6): 627, 1943. Type: CHINA, Sichuan, Wenchuan, May
1930, F. T. Wang 20 945 (lectotype: designated here, A,
barcode A00063254!; isolectotypes: PE, barcode
00025509!, IBSC, barcode 0193424!, KUN, barcode
0478403!) (note: H. L. Li incorrectly cited the collector F.
T. Wang as W. P. Fang)≡S. chinensis subsp. cuspidatus (H.
L.Li)Y.C.Tang&Y.L.Cao,ActaPhytotax.Sin.21(3):245.
1983 ≡S. himalaicus var. cuspidatus (H.L.Li)Y.P.Zhu,nom.
inval. in Ph.D. thesis. 112. 2006.
=S. chinensis var. latus H. L. Li, Bull. Torr. Bot. Club 70(6):
627, 1943. Type: CHINA, Henan, Songxian (Honan,
Sunghsien), Sept. 1919, J. Hers 1305 (holotype: A, barcode
A00063253!); CHINA, Hubei, Wan Tsao Shan, Aug. 1922, W.
Y. Chun 3901 (paratype: PE, barcode 01163737!); CHINA,
Hubei, Hsin Tien Tsze, Aug. 1922, W. Y. Chun 4041 (paratype:
PE,barcode01163739!);CHINA,Hubei,HuanTsao,Aug.
1922, W. Y. Chun 4138 (paratypes: N, barcode 117200029, PE,
barcode 01163740!); CHINA, Chongqing (Sichuan), Nan-
chuan, May 1928, W. P. Fang 830 (paratypes: PE, barcode
01163778, IBSC, barcode 0193428!); W. P. Fang 920
(paratypes: LBG, barcode 00071576!, IBSC, barcode
0193430!); Jun. 1928, W. P. Fang 1402 (paratypes: PE,
barcode 01163780!, IBSC, barcode 0193426!); CHINA,
Sichuan, Maoxian, Sept. 1928, W. P. Fang 5528 (paratypes:
LBG, barcode 00071580!, IBSC, barcode 0193427!, N,
barcode 117200004, PE, barcode 01281784!); CHINA,
Chongqing (Sichuan), Chengkou, May 1932, W. P. Fang
10309 (paratypes: IBSC, barcode 0193429!, PE, barcode
01281919!) ≡S. chinensis subsp. latus (H.L.Li)Y.C.Tang&
Y.L.Cao,ActaPhytotax.Sin.21(3):243.1983≡S. himalaicus
var. latus (H. L. Li) Y. P. Zhu in Ph.D. thesis. 104. 2006.
=S. duclouxii Pit. ex Chung, Mem. Sci. Soc. China 1:176.
1924. nom. nud.
720 Su et al.
J. Syst. Evol. 58(5): 710–724, 2020 www.jse.ac.cn
=S. himalaicus var. dasyrachis C. Y. Wu ex S. K. Chen, Acta
Bot. Yunnan. 3(2): 133. 1981. Type: CHINA, Yunnan, Funing,
Jun. 1964, S. C. Wang 64–1019 (holotype: KUN, barcode
1208135!); CHINA, Yunnan, Funing, Apr. 1940, C. W. Wang
88214 (paratypes:KUN,barcode0479026!,WUK,barcode
275341!);Oct.1965,WenShan Exp. 65–96 (paratype: KUN,
barcode 0479045!); CHINA, Yunnan, Xichou, May 1964, S. C.
Wang 153 (paratype: KUN, barcode 0478451!); Sept. 1947, K.
M. Feng 11644 (paratypes: WUK, barcode 195798!, KUN,
barcode 0479036!); Apr. 1959, C. A. Wu 7334 (paratypes:
WUK, barcode 273378!, KUN, barcode 0479046!); C. A. Wu
7812 (paratype: KUN, barcode 0478448!); May 1959, C. A. Wu
7893 (paratype:KUN,barcode0478447!);Oct.1962,C. A. Wu
62–254 (paratype: KUN, barcode 0479048!); Oct. 1939, C. W.
Wang 85321 (paratype: KUN, barcode 0478534!); CHINA,
Yunnan, MaGuan, Mar. 1933, H. T. Tsai 51851 (paratypes: IBSC,
barcode 0326939! PE, barcode 01288491!, KUN, barcode
0479035!); CHINA, Yunnan, Malipo, Feb. 1940, C. W. Wang
86768 (paratypes: WUK, barcode 273971! PE, barcode
01288542!, KUN, barcode 0479028!); CHINA, Yunnan,
Jinping, Apr. 1956, Yunnan Exp. 1010 (paratype: KUN,
barcode0478206!);CHINA,Yunnan,Pingbian,Sept.1939,
C. W. Wang 82123 (paratypes: PE, barcode 01288570!, KUN,
barcode 0479030!); C. W. Wang 82123 (paratypes: PE,
barcode 01288570!, KUN, barcode 0479030!); Oct. 1939, C.
W. Wang 82388 (paratypes: PE, barcode 01288569!, KUN,
barcode0479029!);CHINA,Guizhou,nopreciselocality,Apr.
1959, Qian‐nan Exp. 197 (paratype: KUN, barcode 0478731!)
=S. himalaicus var. microphyllus C. Y. Wu ex S. K. Chen, Acta
Bot. Yunnan. 3(2): 133. 1981. Type: CHINA, Yunnan, Jingdong,
Oct. 1956, B. Y. Qiu 52920 (holotype: KUN, barcode 1208164!;
isotype: LBG, barcode 00071549!); CHINA, Yunnan, Jingdong,
May 1959, S. G. Xu 4779 (paratypes: IBK, barcode
IBK00302072!, KUN, barcode 0478495! PE, barcode
01432293!); CHINA, Yunnan, Gongshan, Sept. 1940, K. M.
Feng 7986 (paratypes:KUN,barcode0478620!PE,barcode
01289130!);Oct.1935,C. W. Wang 66805 (paratypes: KUN,
barcode 0478535! PE, barcode 01289125!, 01289126!, WUK,
barcode 36484!)
(3) Stachyurus yunnanensis Franch., J. de Bot. 12: 253. 1898.
Type: CHINA, Yunnan, Mo‐So‐Yn, Apr. 1884, J. M. Delavay
822 (lectotype designated by Zhu & Zhang (2005): P,
barcode P00067730!)
=S. callosus C. Y. Wu ex S. K. Chen, Acta Bot. Yunnan.
3(2): 128. 1981. Type: CHINA, Yunnan, Malipo, Jan. 1940,
C. W. Wang 86275 (holotype: KUN, barcode 1208147!);
CHINA, Yunnan, Pingbian, Oct. 1954, K. M. Feng 5175
(paratype: KUN, barcode 0478059!)
=S. esquirolii H. Lév., Fl. Kouy‐Tcheou 416. 1915. Type:
CHINA, Guizhou, Than lo, Mar. 1912, J. Esquirol 3517
(lectotype: designated here, E, barcode E00030985!;
isolectotype: A, barcode 00135043!)
=S. oblongifolius F. T. Wang & Tang, Acta Phytotax.
Sin.1(3):325.1951.Type: CHINA, Guizhou, Suiyang,
Aug. 1928, P. C. Tsoong 434 (lectotype: designated
here, PE, barcode 00025515!; isolectotype: PE, barcode
00025514!)
=S. yunnanensis var. pedicellatus Rehder, Sarg. Pl.
Wils. 1(2): 288. 1912. Type: CHINA, Chongqing, Yunyang
(Szechwan, Yungyang), Jul. 1910, E. H. Wilson 4541
(lectotype: designated here, A, barcode 00063260!;
isolectotypes: US, barcode US00114919 photo!, K,
barcode K000651590 photo!)
(4) Stachyurus retusus Yen C. Yang, Contr. Biol. Lab. Sci.
China 12: 105, pl. 6. 1939. Type: CHINA, Sichuan, Mount
Emei, 1938, C. W. Yao 3365 (lectotype: designated here,
NAS, barcode NAS00071884!; isolectotypes: PE, barcode
00025517!, 00025518!)
=S. yunnanensis var. obovatus Rehder,J.Arn.Arb.11:165.
1930. Type: CHINA, Sichuan, Guanxian (Kuan‐hsien), Jul.
1928,W.P.Fang2000(lectotype:designatedhere,A,
barcode00063259photo!;isolectotypes:K,barcode
K000651591, PE, barcode 01432300!, E, barcode E00414141!,
E00414140!, IBSC, barcode 0000999!, NAS, barcode
NAS00071885!, NAS00071886!, NAS00082798!) syn. nov. ≡
S. obovatus (Rehder) Hand.‐Mazz., Oesterr. Bot. Z. 90: 118.
1941. ≡S. obovatus (Rehder) H. L. Li, Bull. Torr. Bot. Club
70(6): 620. 1943. isonym. ≡S. obovatus (Rehder) W. C.
ChengexW.P.Fang,Icon.Pl.Omei.1(2):pl.99.1944.
isonym.
=S. szechuanensis W.P.Fang,Icon.Pl.Omei.2(1):pl.103.
1945. Type: CHINA, Sichuan, Mount Emei, Sept. 1939, C. L.
Sun 1230 (holotype:SZ;isotype:KUN,barcode1208165!)
(5) Stachyurus cordatulus Merr., Brittonia 4: 122. 1941. Type:
MYANMAR, Adung River, Jan. 1931, Ward 9176 (lecto-
type: designated here, BM, barcode 000821622!; iso-
lectotype: A, barcode A00248621 photo!)
=S. coaetaneus Chatterjee, Kew Bull. 3(1): 60. 1948. Type:
MYANMAR, Kampti, Feb. 1912, S. M. Toppin 6273
(holotype: CAL)
(6) Stachyurus salicifolius Franch., J. de Bot. 12: 253. 1898.
Type: CHINA, Yunnan, Yanjin, Jul. 1894, J. M. Delavay s.n.
(lectotype: P, barcode P00067728!; isolectotype: P, barcode
P00067729!) ≡S. himalaicus var. salicifolius (Franch.) Y. P.
Zhu in Ph.D. thesis. 102. 2006, nom. inval.
=S. salicifolius var. lancifolius C. Y. Wu ex S. K. Chen, Acta
Bot. Yunnan. 3(2):127. 1981. Type: CHINA, Yunnan, Yongshan,
Aug. 1972, E. N‐Yunnan Exp. 418 (holotype: KUN, barcode
1208140!; isotype: PE, barcode 01432299!); CHINA,
Chongqing (Sichuan), Nanchuan, May 1957, K. F. Li 61126
(paratypes: PE, barcodes 01288952! IBSC, barcode 0327145!,
HIB, barcode 0070475); Jun. 1957, K. F. Li 62088 (paratypes:
PE, barcode 01288950! IBSC, barcode 0327146!); K. F. Li 62836
(paratypes: PE, barcode 01288949! IBSC, barcode 0327134!,
NAS, barcode NAS00082790!, KUN, barcode 0479149!) ≡S.
salicifolius subsp. lancifolius (C.Y.WuexS.K.Chen)Y.C.
Tang&Y.L.Cao,ActaPhytotax.Sin.21(3):247.1983
(7) Stachyurus praecox Siebold & Zucc., Fl. Jap. 1: 43, pl. 18.
1836. Type: JAPAN, Jan. 1829, von Siebold, PF, s.n.
(lectotype designated by Akiyama et al. (2016): M,
barcode M0172773!)
Acknowledgements
The authors thank the collectors and PE that provided access
to herbarium specimens for this project. We thank Zhiqiang
Wu for his assistance with chloroplast genome data analysis,
Qiu‐Yun (Jenny) Xiang, David Boufford, Qiner Yang, and
Xiangyun Zhu for their critical review and helpful sugges-
tions. This work was supported by the Strategic Priority
Research Program of CAS (XDB31000000 and XDA19050103),
National Natural Science Foundation of China (31590822 and
721Integrative species delimitation of Stachyuraceae
J. Syst. Evol. 58(5): 710–724, 2020www.jse.ac.cn
31300180), and Sino‐Africa Joint Research Center, Chinese
Academy of Sciences, CAS International Research and
Education Development Program (SAJC201613).
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Supplementary Material
The following supplementary material is available online for
this article at http://onlinelibrary.wiley.com/doi/10.1111/jse.
12650/suppinfo:
Fig. S1. Time‐tree of the stem and crown of Stachyuraceae
estimated using the uncorrelated lognormal method in
BEAST. Numbers next to nodes indicate the median age,
and blue bars correspond to the 95% highest posterior
density (HPD).
Table S1. Information of samples for phylogenetic analysis.
Table S2. Information on four chloroplast genomes.
Table S3. 44 chloroplast and one nuclear ribosomal
primers.
Table S4. Coded insertions/deletions (indels).
Table S5. Accessions of rbcL,atpB and matK sequences from
GenBank for estimating divergence time of the crown and
stem of Stachyuraceae.
Table S6. Information on chloroplast genes.
Table S7. Information on markers used in phylogenetic
analyse.
Table S8. Synapomorphy sites of each species of Stachyurus.
Appendix 1. The matrix of 44 cpDNA and one ITS dataset of
Stachyuraceae.
724 Su et al.
J. Syst. Evol. 58(5): 710–724, 2020 www.jse.ac.cn
