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Rhododendron kuomeianum (Ericaceae), a new species from northeastern Yunnan (China), based on morphological and genomic data


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Rhododendron kuomeianum Y. H. Chang, J. Nielsen & Y. P. Ma, a new species of Rhododendron (Ericaceae) within subsect. Maddenia in sect. Rhododendron from Yiliang County, NE Yunnan, China, is described and illustrated. The new species is similar to R. valentinianum, but it can be easily distinguished by its sparse scales on the abaxial surface of the leaf blade, fewer flowers per inflorescence and white corolla with pale red margins. There are also differences in the widths of calyx lobes, leaf blade shape and indumentum characteristics of the petiole between the new species and R. linearilobum. We confirmed that Rhododendron kuomeianum is a new species closely related to R. valentinianum and R. changii with phylogenomic studies of 10 species within this subsection based on restriction site-associated DNA sequencing (RAD-seq) data. These phylogenomic analyses also clarified additional taxonomic problems in this subsection previously raised by morphological analysis. Our findings make a strong case for using next-generation sequencing to explore phylogenetic relationships and identify new species, especially in plants groups with complicated taxonomic problems.
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Research paper
Rhododendron kuomeianum (Ericaceae), a new species from
northeastern Yunnan (China), based on morphological and genomic
Yu-Hang Chang
, Gang Yao
, Jens Neilsen
, De-Tuan Liu
, Lu Zhang
Yong-Peng Ma
Key Laboratory for Integrative Conservation of Plant Species with Extremely Small Populations, Kunming Institute of Botany, Chinese Academy of Sciences,
Kunming, Yunnan 650201, China
Key Laboratory for Plant Diversity and Biogeography of East Asia, Chinese Academy of Sciences, Kunming, Yunnan 650201, China
Dobbies Garden Centre, UK-DD2 Perth, Scotland, UK
Flower Research Institute, Yunnan Academy of Agricultural Sciences, Kunming, Yunnan 650051, China
article info
Article history:
Received 28 September 2020
Received in revised form
7 April 2021
Accepted 13 April 2021
Available online xxx
New species
Phylogenetic position
Rhododendron kuomeianum Y.H. Chang, J. Nielsen &Y.P. Ma, a new species of Rhododendron (Ericaceae)
within subsect. Maddenia in sect. Rhododendron from Yiliang County, NE Yunnan, China, is described and
illustrated. The new species is similar to R. valentinianum, but it can be easily distinguished by its sparse
scales on the abaxial surface of the leaf blade, fewer owers per inorescence and white corolla with pale
red margins. There are also differences in the widths of calyx lobes, leaf blade shape and indumentum
characteristics of the petiole between the new species and Rhododendron linearilobum. We conrmed
that R. kuomeianum is a new species closely related to R. valentinianum and R. changii with phylogenomic
studies of 10 species within this subsection based on restriction site-associated DNA sequencing (RAD-
seq) data. These phylogenomic analyses also claried additional taxonomic problems in this subsection
previously raised by morphological analysis. Our ndings make a strong case for using next-generation
sequencing to explore phylogenetic relationships and identify new species, especially in plants groups
with complicated taxonomic problems.
Copyright ©2021 Kunming Institute of Botany, Chinese Academy of Sciences. Publishing services by
Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC BY-
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1. Introduction
Rhododendron L. is the largest genus in Ericaceae. It contains
about 1025 species and can be classied into eight subgenera (Fang
and Min, 1995;Chamberlain et al.,1996). Members of this genus are
distributed widely in the northern hemisphere, with the Sino-
Himalayan as the main center of abundance and diversity (Fang
and Min, 1995). Because of its high ornamental value and long
history of cultivation, rhododendrons have become popular horti-
cultural plants worldwide. Rhododendron species richness is high-
est in China, which also contains numerous endemic species (Fang
et al., 2005). In fact, an estimated 600 Rhododendron species have
been described in China, and large numbers of new taxa are still
being discovered since the publication of Flora of China (Gao and
Zhang, 2008;Gao and Li, 2009;Chen et al., 2010,2012;Ma et al.,
2013,2015;Liao et al., 2015;Cai et al., 2016;Liu et al., 2018;Tian
et al., 2019).
In recent years, eld investigations in Yiliang County (Zhaotong
prefecture), within the Wumeng Mountain system in northeastern
Yunnan, China, have discovered several new species of Rhododen-
dron (Gao and Zhang, 2008;Gao and Li, 2009;Tian et al., 2019),
suggesting that Rhododendron diversity in this region has been
*Corresponding author. Key Laboratory for Integrative Conservation of Plant Species with Extremely Small Populations, Kunming Institute of Botany, Chinese Academy of
Sciences, Kunming, Yunnan 650201, China.
E-mail address: (Y.-P. Ma).
Peer review under responsibility of Editorial Ofce of Plant Diversity.
These authors contributed equally to this work.
Contents lists available at ScienceDirect
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2468-2659/Copyright ©2021 Kunming Institute of Botany, Chinese Academy of Sciences. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This
is an open access article under the CC BY-NC-ND license (
Plant Diversity xxx (xxxx) xxx
Please cite this article as: Y.-H. Chang, G. Yao, J. Neilsen et al.,Rhododendron kuomeianum (Ericaceae), a new species from northeastern Yunnan
(China), based on morphological and genomic data, Plant Diversity,
underestimated. During eld investigations in this region in June
2018, we collected specimens from a Rhododendron population
with white owers that were growing on steep cliffs and rocks.
After a detailed examination of relevant specimens and litera-
ture, we concluded that these specimens represent a species
new to science in subsect. Maddenia (Hutch.) Sleumer in subgen.
Morphologically, this species is similar to Rhododendron valen-
tinianum Forrest ex Hutch. and Rhododendron changii (W. P. Fang)
W. P. Fang: it has a broadly elliptic to obovate leaf blade, rusty-
yellow setae on the petiole and funnelform-campanulate corollas.
However, the new species can be distinguished from these species
by having sparse scales on the abaxial surface of the leaf blade, 1e2
owers per inorescence, and a larger, white corolla. Although both
the new species and Rhododendron linearilobum R. C. Fang &A. L.
Chang have similar corollas, the new species can be distinguished
by having a broadly elliptic to obovate leaf blade, dense rusty-
yellow setae on the petioles, and wider calyx lobes.
In recent years, as many new species have been described,
molecular marker technology has greatly advanced our under-
standing of Rhododendron phylogeny (Gao et al., 2003;Ma et al.,
2013;Yan et al., 2014;Liu et al., 2018;Du et al., 2020). However,
these studies have mainly relied on a few common markers, which
have limited their resolution. Next Generation Sequencing (NGS)
offers a solution to these issues (Hohenlohe et al., 2010). For
example, restriction site-associated DNA sequencing (RAD-seq) has
been widely used in non-model species (e.g., temperate bamboos)
to resolve evolutionary relationships (Heckenhauer et al., 2018;
Zhang et al., 2018;Liu et al., 2020;Guo et al., 2020).
Here we conrmed that our newly collected specimen is a
hitherto undescribed species using RAD-seq data to reconstruct the
phylogenetic relationships of 11 Rhododendron species.
2. Materials and methods
2.1. Morphological analysis
During eld investigations in June 2018 in Xiaocaoba Nature
Reserve, Zhaotong prefecture, northeastern Yunnan, China, speci-
mens of the new species were collected. Some living plants were
introduced and cultivated at the Kunming Botanical Garden,
Kunming Institute of Botany, CAS. The microscopic morphology of
leaves was observed through a KEYENCE VHX-6000 stereoscopic
microscope (KEYENCE Corp., Osaka, Japan). In addition, three
similar species were sampled for morphological comparison. All
voucher specimens in this study were deposited in the herbarium
of the Kunming Institute of Botany (KUN), CAS.
2.2. Restriction site-associated DNA sequencing and SNP
A total of 11 individuals were sampled to construct the phylo-
gram (Appendix A). Because the morphological characters of the
new species are very similar to species within subsect. Maddenia in
Rhododendron, we sampled nine additional species within the
subsection and selected Rhododendron simsii Planch. as the out-
group for subsequent phylogenetic analysis. High quality genomic
DNA was extracted from silica gel dried young leaves using a
modied CTAB protocol (Doyle, 1991).
The RAD-seq library was prepared using EcoRI to digest DNA,
following the adapted protocol of Miller et al. (2007). The de novo
assembly of the RAD-seq library and SNP genotyping from short-
read sequences was performed using the STACKS 2.5.2 pipeline
(Rochette, 2017,2019). The raw data was demultiplexed and ltered
using the process_radtags program (key parameters included -c -q
-E -t 135). For each sample, short-reads were merged into loci
based on a maximum likelihood framework using the program
ustacks, and were then aligned into exactly-matching stacks. The
stackedepth parameter was set to three (m ¼3) and the within-
individual distance parameter was ve (M ¼5). The catalog of
loci from all samples was built using cstacks with a between-
individual distance parameter of three (n ¼3). Lastly, the loci of
each sample were matched against the catalog to conrm alleles
via the program sstacks. To reconstruct a phylogenetic tree, we
randomly selected one SNP from each locus via the program pop-
ulations. The SNP obtained from the previous STACKS pipeline was
rst applied by VCFtools (Danecek, 2011) with the following key
parameters: min-alleles 2 max-alleles 2 remove-indels maf
0.05 max-missing 0.2, minGQ 30 minDP 3.
After ltering with VCFtools, 42,083 SNPs were obtained and
then tested for neutrality using the key parameter Tajima's D with
the sliding windows of 2500 and a 95% condence interval (Tajima,
1989). The VCF le of neutral site was converted to phy format
using Tassel 5.0 (Bradbury et al., 2007). Phylogenetic analysis of SNP
matrices in phy format was performed using the maximum likeli-
hood method of concatenation using IQTREE (Minh et al., 2020;
Kalyaanamoorthy et al., 2017;Hoang et al., 2018) on partition A of
the Beijing Supercloud Computing Center, and the best base sub-
stitution model selected from 242 DNA base substitution models
was TVMþFþASC, and the bootstrap was set as 1000. Phylogenetic
trees were graphically visualized using FigTree (Price et al., 2010).
To capitalize on phylogenetic information from the sequencing
data and obtain a robust taxonomic status for the new species, SNP
loci were genotyped based on the de novo assembly of the RAD-seq
data. After non-linkage ltering and neutral tests, we genotyped 12,
380 loci. By calculating and sorting the BIC scores for these geno-
typed sites, the TVMeþASC model was selected as the best-t
model for the maximum likelihood method of the phylogenetic
3. Results
3.1. Morphological characters
The morphological characters of the new species differ from all
species previously described in Rhododendron. Several traits of the
new species indicate that it belongs to the subsect. Maddenia;
specically, it has a large corolla, the abaxial surface of the leaf is
scaly, and a quarter of the base of the style has scales (Figs. 1 and 2).
The new species can be distinguished from the two most similar
species (i.e., R. valentinianum and R. changii) by having sparse scales
(ca. 1e2their own diameter apart) on the abaxial surface of leaf
blade (Fig. 3), fewer owers (1e2) per inorescence, and a larger,
white corolla (3.5e4.5 cm) (Table 1). R. linearilobum has a corolla
with similar morphology and color as the new species, but can be
easily distinguished from the new species by having a narrowly
obovate leaf blade, densely rusty-red-villous petiole, and calyx
lobes ca. 2 mm in width (Table 1).
3.2. Data generation and molecular phylogeny
We constructed a RAD-seq libraries and genotyped SNPs from
11 species of Rhododendron. A total of 4.6 gigabase pairs (gb) of
data was obtained, with an average of 428.22 megabase pairs (Mb)
of data per sample (see Appendix B for details).
Phylogenetic trees based on the de novo assembly of our RAD-
seq library reveal that all 10 species within subsect. Maddenia are
clustered into one clade, with Rhododendron maddendii subsp.
crassum (Franchet) Cullen as the basal species (Fig. 4). One subclade
includes R. valentinianum,R. changii,Rhododendron pachypodum
Y.-H. Chang, G. Yao, J. Neilsen et al. Plant Diversity xxx (xxxx) xxx
Balf. f. &W. W. Smith and Rhododendron kuomeianum Y.H. Chang, J.
Nielsen &Y.P. Ma sp. nov., which veries that R. kuomeianum is
genetically distinct and supports its status as a new species.
Notably, the topological structure of the ML tree indicates that the
phylogenetic position of R. valentinianum is very close to that of
R. changii. The remaining ve species (Rhododendron liliiorum
Levl., Rhododendron chunienii Chun &Fang, Rhododendron maddenii
subsp. maddenii (Batalin) H. Hara, Rhododendron excellens Hemsl. &
Wils. and Rhododendron ciliatum Hook. f.) cluster into a separate
subclade, although R. liliiorum and R. chunienii cannot be distin-
guished from each other. In addition, R. maddenii subsp. maddenii is
genetically distant from the subspecies R. maddenii subsp. crissum
(Fig. 4).
4. Discussion
In the present study, morphological comparisons and phyloge-
netic analysis based on de novo assembly of a RAD-seq library
conrmed that R. kuomeianum is a new species. R. kuomeianum has
similar oral traits as R. linearilobum; however, the new species has
an oblong-elliptic leaf blade similar to R. valentinianum and R. changii.
Due to unavailability of sequencing materials of R. linearilobum,
which was estimated to be <50 plants and hence evaluated to be
critically endangered by IUCN criteria (Gibbs et al., 2011), the most
genetically close relative remains to be unclear.
Rhododendron is the largest genus of seed plants in China, and
recognized as one of the most taxonomically challenging plants due
to recent adaptive radiations and natural hybridization (Yan et al.,
2014). One major taxonomic problem for this genus is that many
hybrids have been incorrectly identied as new taxa. Previous
Fig. 1. Illustrations of Rhododendron kuomeianum Y.H. Chang, J. Nielsen &Y.P. Ma sp.
nov. Drawn by R.M. Zhang. (A) habit; (B) corolla; (C) dissected corolla; (D) inorescence
bud; (E) ower bud; (F) stamen; (G) pistil and calyx; (H) leaf adaxial surface; (I) leaf
abaxial surface; (J) distribution of scales on the adaxial surface of the leaf blade; (K)
single scale on the abaxial surface of leaf blade; (L) capsule.
Fig. 2. Rhododendron kuomeianum Y.H. Chang, J. Nielsen &Y.P. M a sp. nov. (AeB) plant and habitat; (CeD) leaf; (E) inorescence bud; (F) corolla anatomy; (G) fruit. Scale bar ¼1 cm.
Y.-H. Chang, G. Yao, J. Neilsen et al. Plant Diversity xxx (xxxx) xxx
studies at the Baili Rhododendron Nature Reserve in Guizhou have
conrmed that many of the new taxa described in this area are
actually different genotypes produced by natural hybridization
between Rhododendron delavayi Franch. and Rhododendron irror-
atum Franch (Marczewski et al., 2016;Zhang et al., 2017).
Morphological evidence has been used to describe taxa for over a
century; however, this approach often leads to uncertainty and
debate. For instance, R. changii was initially described as a variety of
R. valentinianum; subsequently, it was upgraded to species status
because of its larger, scaleless corolla, glabrous style, and the
absence of hairs on the pedicel or calyx (Fang, 1983). After
comparing holotypes and native plants, Geng (2014) restored
R. changii as a variety of R. valentinianum based on morphological
similarities and habitats. Our phylogenetic tree clusters these
two species together, indicating that R. changii can reasonably
be treated as a variety of R. valentinianum. Similarly, Li (1995) and
Geng (2004) proposed that the holotype of R. chunienii, which
had previously been described as having ve stamens (the most
unique character in this subsection), had been misdiagnosed
because of damage to the corolla. Both living plants and specimens
of R. chunienii collected at the type locality have 10 stamens;
furthermore, other characteristics of R. chunienii are the same as in
R. liliiorum. Thus, R. chunienii has been treated as a synonym for
R. liliiorum. Our phylogenetic analysis supports this classication.
Rhododendron maddenii contains two subspecies, R. maddenii
subsp. maddenii and R. maddenii subsp. crassum. Although these
subspecies are morphologically similar, they are geographically
isolated from one another. R. maddenii subsp. maddenii is mainly
distributed in Cuona County and Naidong District in southern Tibet,
China, as well as in Bhutan and northeastern India; in contrast,
R. maddenii subsp. crassum is mainly distributed in western
Yunnan. In this study, the high-condence phylogenetic tree
Fig. 3. The micrograph of the leaf surface of Rhododendron kuomeianum Y.H. Chang, J. Nielsen &Y.P. M a sp. nov. (A) distribution of scales on the adaxial surface of the leaf blade; (B)
distribution of scales on the abaxial surface of leaf blade; (C) single scale on the on the adaxial surface of the leaf blade; (D) single scale on the on the abaxial surface of leaf blade; (E)
indumentum characteristics of the petiole.
Table 1
Main morphological comparisons of Rhododendron kuomeianum with similar Rhododendron species.
Characters R. kuomeianum R. valentinianum R. changii R.linearilobum
Geographic distribution NE Yunnan SE W Yunnan
NW NE Guizhou
Chongqing SE Yunnan
Elevation 1800e2000 m 2400e3000 m 1600e2000 m 2200 m
Petiole setose setose setose villous
Shape of the leaf blade broadly elliptic to obovate obovate to oblong-elliptic obovate to oblong-elliptic elliptic
Size of leaf blade 3.5e5.5 2.5e3.5 cm 3e41.5e2cm 3e4.5 2e2.5 cm 4e7.5 1.5e2.5 cm
Scale density (as own diameter
1e2 0.5 or contiguous nearly contiguous 1e2
Inorescence 1 or 2-owered 2e4-owered 3 or 4-owered 2e4-owered
Pedicel glabrous setose glabrous pubescent
Calyx lobes 4e73e5 mm, ovate or
broadly elliptic
84e5 mm, ovoid or oblong-
10 4e5 mm, ovoid or oblong-
6e12 2 mm, linear
Color of the corolla white with pale red margins light to bright yellow light to bright yellow white with pale red margins
Corolla size 3.5e4.5 cm long, tube 2e3cm
2e3.5 cm long, tube 1.8e2cm
3.5e4 cm long, tube 1.8e2cm
ca. 4 cm long, tube 1.8e2cm
Proportion of scales to style 1/4 1/8 1/8 2/5
Indumentum characteristics of
the style
glabrous glabrous glabrous base pubescent
Y.-H. Chang, G. Yao, J. Neilsen et al. Plant Diversity xxx (xxxx) xxx
reveals that the genetic difference between these two subspecies is
very large, which has also been conrmed by DNA barcoding (Ya n
et al., 2014). One explanation for the genetic differences between
these two subspecies is that long-term geographic isolation has
allowed mutations to accumulate in their respective populations.
4.1. Taxonomic treatment
4.1.1. Rhododendron kuomeianum Y.H. Chang, J.Nielsen &Y.P. Ma
sp. nov (Figs. 1 and 2)
. Type. CHINA. Yunnan: Yiliang County Xiaocaoba Nature
Reserve, 25
N, 104
E, alt. 2000 m, 15 Apr. 2019, Y.H.
Chang,Y.P. Ma &D.T. Liu, Cyh20190402 (holotype KUN! 1498888;
isotype KUN! 1498889, 1498890). Diagnosis. Rhododendron kuomeianum resembles
R. valentinianum in having a broadly elliptic to obovate leaf
blade, dense rusty-yellow setae on the petiole, and a funnelform-
campanulate corolla. It differs from R. valentinianum in having a
white corolla with a pale red margin (versus light to bright yellow),
fewer owers (1e2) per inorescence (versus 2e4), and sparser
distribution of scales on the abaxial surface of the leaf blade
(1e2scales diameter apart) (versus 0.5 scales diameter apart)
(Table 1). Description. Multi-branched shrubs, evergreen, 40e10 0 cm
tall; branches short, old branches deep red to brown, young shoots
scaly, hispid. Petiole 5e9 mm, scaly, hispid; leaf blade leathery,
broadly elliptic to obovate, 3.5e5.5 2.5e3.5 cm; base broadly
cuneate or rounded; margin entire and slightly revolute, sparsely
setose; apex obtuse or rounded, apiculate; abaxial surface pale
green, scales 1e2their own diameter apart, about equal in size,
golden, seldom contiguous, adaxial surface deep green and shiny,
densely scaly, brown hispid along midrib; midveins raised adax-
ially and lateral veins hardly raised; midrib concave adaxially.
Inorescence terminal, cymose, 1 or 2-owered. Pedicel stout,
0.6e0.9 cm, scaly, without hairs; calyx 4e5-lobed to the half, lobes
pale green, 4e73e5 mm, ovate or broadly elliptic, persistent in
fruit, scaly abaxially and on margin, margin sparsely white-ciliate;
corolla funnelform-campanulate, white with pale red margin and
without blotch, 3.5e4.5 cm, tube 2e3 cm, outer surface glabrous,
lobes orbicular or broadly elliptic, ca. 1.8e2.5 2.0e2.5 cm; sta-
mens 10, 1.5e3.7 cm, unequal, shorter than corolla; laments
densely pubescent in lower 1/5; anthers oblong-elliptic, red
brown, ca. 2.5e3 mm; ovary 5-locular, ca. 4.5 mm, densely scaly;
style slightly arched, as long as corolla or exserted from corolla,
3.5e4.2 cm base densely sca ly in lower 1/4. Capsule ovoid, c a. 1 cm,
dehiscing from top, densely scaly. Flowering: AprileMay, Fruiting:
after June. Paratype. China. Yunnan: Yiliang county Xiaocaoba Nature
Reserve, 25
N, 104
E, alt. 1950 m, 5 Apr. 2019, YH.
Chang &F.M. Yang, Cyh20190401 (KUN! 1498891). Same locality,
alt. 2030 m, 6 June 2018, Y.P. Ma,Y.H. Chang &J. Neilsen,
Ma20180603 (KUN! 1498892, 1498893).
Fig. 4. Maximum likelihood phylogenetic topology based on 12380 unlinked high-quality and neutral SNP sites without a reference genome. The phylogenetic tree bootstrap
probabilities 75 are shown at branches.
Y.-H. Chang, G. Yao, J. Neilsen et al. Plant Diversity xxx (xxxx) xxx
5 Distribution and habitat. To date, Rhododendron kuomeia-
num has only been found in the type locality at Xiaocaoba Nature
Reserve, Zhaotong City, NE Yunnan, China, where it is represented
by one population at elevations of 1800e2000 m (Fig. 5). In addi-
tion, we found all plants growing on rocks and cliffs, which are
steep and barren (Fig. 2A). Based on the known occurrences, we
conclude that R. kuomeianum is well-adapted to cold and wet
environments. Conservation status. During many eld investigations since
2018, we have only found this species in the type locality, and there
are about 200 mature individuals in this population. Detailed ex-
amination of relevant specimens and literature available for
Rhododendron species collected from Xiaocaoba Nature Reserve
and adjacent regions revealed no additional information about the
distribution of this species; therefore, this species is likely to be
endemic to this region. Based on the limited information currently
available, we thus tentativelyassign this species to an IUCN Red List
status of Data Decient (DD) (IUCN, 2019). Further eld investiga-
tion is urgently needed. Etymology. The new species Rhododendron kuomeianum
was named after Professor Kuo Mei Feng, a botanist and horti-
culturalist from the Kunming Institute of Botany, Chinese Academy
of Sciences, to honor his great contribution to research on the genus
Rhododendron in China. In pinyin, the Chinese name is Gu
ei dù
Author contributions
Y.P. Ma conceived and designed experiments. Y.H. Chang, Y.P.
Ma, D.T. Liu, and J. Neilsen performed eld investigations and
collected the specimens. G. Yao and L. Zhang analyzed the molec-
ular data. Y.H. Chang wrote the manuscript. Y.H. Chang, G. Yao and
Y.P. Ma revised the manuscript.
Declaration of competing interest
We declare that the named authors have no conicts of interest,
nancial or otherwise.
We are grateful to Mrs. Rongmei Zhang for the botanical line
drawing, Mr. Lianyi Li for the microscopic scanning of the leaf
surface, Ms. Songting Du, Mrs. Wei Wei, and Mr. Yechun Xu for
providing photos and Mr. Fengmao Yang for help during eldwork.
We also thank Mr. Shiwei Guo for his constructive comments on the
manuscript. This study was nancially supported by the National
Natural Science Foundation of China (Grant No. 31770418 and No.
31760231) the Youth Innovation Promotion Association, Chinese
Academy of Sciences (Grant No. 2018428), and the Reserve Talents
for Academic and Technical Leaders of Middle-aged and Young
People in Yunnan Province (Grant No. 2018HB066).
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... Ss. Maddenia is the largest among all the subsections in subgenus Rhododendron (if the vireyas are treated as subgenus Vireya rather than the broad section Schistanthe under subgenus Rhododendron), and several new species (Mao and Bhaumik 2015, Chang et al. 2021, Rushforth et al. 2022) have been published since Chamberlain et al. (1996). With 'lumping' species as synonyms and/or changes in placement of species over time, the number of accepted species in ss. ...
... According to conservation assessments for 51 ss. Maddenia taxa, 33 were placed in either a threatened category or Data Deficient (Gibbs et al. 2011, Chang et al. 2021. Due to the variable taxonomy and species definitions derived from traditional morphology, conservation assessments and subsequent conservation action are subject to debate (Cubey 2003, Gibbs et al. 2011, Donald 2012, Jamieson 2021 In this study, we collected leaf samples from living accessions to examine the ploidy variation in ss. ...
... (Gibbs et al. 2011, Donald 2012. Three new species published post-1996 were examined: R. pseudomaddenii (Mao and Bhaumik 2015), R. leptocladon (Rushforth and Nguyen 2019), R. kuomeianum (Chang et al. 2021). R. ...
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Polyploidy, which is common in plants, can confound taxon recognition and hence conservation assessments. In the taxonomically complex genus Rhododendron, 25% of the over 1,300 taxa are considered under threat and 27% Near Threatened or Data Deficient, with their taxonomy needing to be resolved urgently. Although ploidy levels of Rhododendron taxa range from diploid (2x) to dodecaploid (12x) according to previous reports, the extent of polyploidy across the genus has not been examined. We first summarised the taxonomic distribution of polyploids in the genus based on the literature. Then as a case study, we estimated ploidy levels of 47 taxa in subsection Maddenia (subgenus Rhododendron, section Rhododendron) using flow cytometry, together with verification of meiotic chromosome counts for representative taxa. The summary of reported ploidy in Rhododendron indicates that polyploidy is most common in subgenera Pentanthera and Rhododendron. In subsection Maddenia, all examined taxa are diploids except for the R. maddenii complex that shows a high ploidy variation (2–8x, 12x). We investigated ploidy level of 12 taxa in subsection Maddenia for the first time, and estimated genome sizes of two Rhododendron species. Knowledge of ploidy levels will inform phylogenetic analysis of unresolved species complexes. Overall, our study of subsection Maddenia provides a model for examining multiple issues including taxonomic complexity, ploidy variation and geographic distribution in relation to biodiversity conservation.
... They were obtained from the Flora of China ( and previous taxonomic works (Chen et al., 2010a(Chen et al., , 2010b(Chen et al., , 2010c(Chen et al., , 2010d(Chen et al., , 2012Geng, 2014;Tian et al., 2019;Chang et al., 2021), and the recoded character matrices are available in Table S5 and S6. These traits were selected based on the following criteria: 1) they are extremely vital for diagnosing taxa in Rhododendron, especially at the subgenus level, or they are important ornamental characters; 2) they have stable and discrete state within same species; 3) they are available and easily categorized. ...
... Rhododendron is the largest genus belonging to the family Ericaceae, which is known for colorful flowers [1]. In recent years, varieties of Rhododendron have been grown as ornamental plants, making them become the most popular evergreen shrubs all over the world [2]. ...
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Rhododendron pulchrum Sweet is a traditional ornamental plant cultivated in China and presents a great variation in petal coloration. However, few studies have been performed to reveal the genes involved and the regulatory mechanism of flower color formation in this plant. In this study, to explore the underlying genetic basis of flower color formation, transcriptome analysis was performed by high-throughput sequencing techniques on four petal samples of different colors: purple, pink, light pink, and white. Results show that a total of 35.55 to 40.56 million high-quality clean reads were obtained, of which 28.56 to 32.65 million reads were mapped to the reference genome. For their annotation, 28,273, 18,054, 24,301, 19,099, and 11,507 genes were allocated to Nr, Swiss-Prot, Pfam, GO, and KEGG databases, correspondingly. There were differentially expressed genes among the four different petal samples, including signal-transduction-related genes, anthocyanin biosynthesis genes, and transcription factors. We found that the higher expressed levels of genes associated with flavonol synthase (FLS) might be the key to white formation, and the formation of red color may be related to the higher expression of flavanone 4-reductase (DFR) families. Overall, our study provides some valuable information for exploring and understanding the flower color intensity variation in R. pulchrum.
... There are about 125 genera of Ericaceae worldwide, widely distributed in temperate regions and tropical mountains (Yoichi et al. 2016;Khan et al. 2021). Rhododendron is the largest genus, with about 967 species, mainly located in Asia, Europe and North America, about 700 species growing in China especially in Southwest China (Chang et al. 2021;Yan et al. 2015). ...
This study was designed to investigate the insecticidal activity of the essential oils (EOs) and extracts from Rhododendron rufum and Rhododendron przewalskii. The EOs were extracted from the leaves of R. Rufum and R. przewalskii by hydro-distillation and their chemical components were analyzed by gas chromatography–mass spectrometry (GC–MS). The repellency, contact toxicity and antifeedant activity of the EOs and extracts were evaluated against Sitophilus oryzae and Tribolium castaneum along with those of their main components. A total of nine compounds were identified from the EO of R. Rufum, and the most abundant component was myristicin (79.72%). The EO of R. Rufum exhibited repellent activities at different levels and its main compound myristicin showed contact toxicity and repellent effects against S. oryzae and T. castaneum. Meanwhile, by bioassay-guided fractionation, four compounds with strong antifeedant activities against T. castaneum, 24-methylenecycloartanyl-2'E, 4'Z-tetradecadienoate (1), methyl thyrsiflorin B acetate (2), friedelin (3) and Excoecarin R1 methyl ester (4) were separated and identified from the ethanol extract of R. przewalskii for the first time. Considering the significant anti-insect activities, the EOs and extracts of R. Rufum and R. przewalskii might be used in integrated pest strategies, establishing a good perspective for the comprehensive use of natural plant resources of Rhododendron genus.
... It is likely that more than half of these are in the Himalaya-Hengduan Mountains (HHM). These two global biodiversity hotspots harbor a remarkable diversity of endemic species (Wu, 1988;Xing and Ree, 2017;Ding et al., 2020) and new species of other genera from these and adjacent areas are being reported at a rapid pace (Sun et al., 2017;Du et al., 2020;Chang et al., 2021;Li et al., 2021;Olonova et al., 2021;Ya et al., 2021;Ye et al., 2021;Zheng et al., 2021). It has been noted, however, that more than half of the species diversity is unevenly concentrated in particular taxa, Berberis being one of them (others include Aconitum L., Gentiana L., Pedicularis L., Primula L., Rhododendron L. and Saxifraga L.; Ding et al., 2020). ...
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Though Berberis (Berberidaceae) is widely distributed across the Eurasian landmass it is most diverse in the Himalaya–Hengduan Mountain (HHM) region. There are more than 200 species in China where it is one of the most common mountain shrubs. The study on the taxonomy and evolution of Berberis in this region can thus provide an important insight into the origin and diversification of its flora. A prerequisite to this is mapping and describing the various species of Berberis in the region – a task that despite recent progress is by no means complete. It is clear that in China there may be a significant number of species still to be described and that even with published species much about their distribution remains to be discovered. As a contribution to the first of these tasks seven new species from the northern Hengduan Mountain of N. Sichuan and S. Qinghai: B. chinduensis, B. degexianensis, B. jiajinshanensis, B. jinwu, B. litangensis, B. longquensis and B. riparia, are described here. Differences in overall morphology and especially in floral structures with each other and with similar species of Berberis in the same region are presented. The report is the result of phylogenetic analyses based on plastome and partial nrDNA sequences of both the seven proposed new species and a significant number of similar species already published. Provisional conclusions as to the insights provides on the history of the genetic divergence are discussed.
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Rhododendron vialii (subgen. Azaleastrum) is an evergreen shrub with high ornamental value. This species has been listed as a plant species with extremely small populations (PSESP) for urgent protection by China’s Yunnan provincial government in 2021, due to anthropogenic habitat fragmentation. However, limited genomic resources hinder scientifically understanding of genetic threats that the species is currently facing. In this study, we assembled a high-quality haplotype-resolved genome of R. vialii based on PacBio HiFi long reads and Hi-C reads. The assembly contains two haploid genomes with sizes 532.73 Mb and 521.98 Mb, with contig N50 length of 35.67 Mb and 34.70 Mb, respectively. About 99.92% of the assembled sequences could be anchored to 26 pseudochromosomes, and 14 gapless assembled chromosomes were included in this assembly. Additionally, 60,926 protein-coding genes were identified, of which 93.82% were functionally annotated. This is the first reported genome of R. vialii, and hopefully it will lay the foundations for further research into the conservation genomics and horticultural domestication of this ornamentally important species.
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From 2000 to 2019, 11,895 new names or new additions to the Chinese vascular flora were proposed by 4226 individuals (4086 articles and 140 books), as documented in the Chinese Plant Names Index (CPNI). During those 20 years, 4407 new taxa of vascular plants were described from China, including 7 new families, 132 new genera, 3543 new species, 68 new subspecies, 497 new varieties and 160 new forms. Additionally, 3562 new combinations and names at new rank and 306 new replacement names were also proposed. Among these various new names were 150 invalid names and 108 illegitimate names, including some that have not been resolved. Six hundred and forty three vascular plants were reported as new to China, while 2349 names were reduced to synonyms of 1406 taxa. The data show that the Chinese flora increased in size at the rate of about 200 taxa annually during those years. Despite the increased attention given to biodiversity in recent years, the evidence indicates that a large number of species in China have yet to be discovered. Further basic investigation of the Chinese flora is needed. Additionally, in the past two decades only 8.5% of the newly published species have been based on molecular evidence, but in the past five years such data have increased significantly, reaching about 20%. Molecular data will undoubtedly become increasingly significant in the discovery of new species in the coming years. Yunnan, Guangxi, Sichuan, Xizang and Taiwan were important sources of new discoveries, with more than 3300 new taxa and records from these five provinces. By area, Taiwan and Hainan, two islands in southern China, have the highest density of newly discovered species. Regional plant surveys are still needed, especially in areas in the southwest and on the southern islands.
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Rapid evolutionary radiations are among the most challenging phylogenetic problems, wherein different types of data (e.g., morphology, molecular) or genetic markers (e.g., nuclear, organelle) often yield inconsistent results. The tribe Arundinarieae i.e., the temperate bamboos, is a clade of tetraploid originated 22 million years ago and subsequently radiated in East Asia. Previous studies of Arundinarieae have found conflicting relationships and/or low support. Here, we obtain nuclear markers from ddRAD data for 213 Arundinarieae taxa and parallel sampling of chloroplast genomes from genome-skimming for 147 taxa. We first assess the feasibility of using ddRAD-seq data for phylogenetic estimates of paleopolyploid and rapidly radiated lineages, optimize clustering thresholds and analysis workflow for orthology identification. Reference-based ddRAD data assembly approaches perform well and yield strongly supported relationships that are generally concordant with morphology-based taxonomy. We recover five major lineages, two of which are notable (the pachymorph and leptomorph lineages), in that they correspond with distinct rhizome morphologies. By contrast, the phylogeny from chloroplast genomes differed significantly. Based on multiple lines of evidence, the ddRAD tree is favored as the best species tree estimation for temperate bamboos. Using a time-calibrated ddRAD tree we find that Arundinarieae diversified rapidly around the mid-Miocene corresponding with intensification of the East Asian monsoon and the evolution of key innovations including the leptomorph rhizomes. Our results provide a highly resolved phylogeny of Arundinarieae, shed new light on the radiation and reticulate evolutionary history of this tribe, and provide an empirical example for the study of recalcitrant plant radiations.
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Though it is well-acknowledged that next generation sequencing (NGS) technologies can provide further insights into plant conservation management than traditional molecular markers, studies employing NGS to address conservation genomics and subsequent conservation strategies for threatened plants are still rare. Rhododendron is the largest genus of woody plants in China, and many species are threatened, however, to date there has been no conservation genetic research using NGS in this genus. In the present study, we investigated the conservation genetics of R. cyanocarpum, a threatened species endemic to the Cangshan Mountains in Yunnan, China, using a double digest restriction-site-associated DNA-sequencing (ddRAD-seq) approach. Due to the availability of sufficient SNPs, we were able to distinguish between neutral and putatively selected SNPs and were able to further investigate the genetic diversity, population structure, and differentiation in R. cyanocarpum, as well as make an estimation of its demographic history. A total of 6,584 SNPs were obtained, of which 5,729 were neutral (detected using Tajima's D). In terms of the 5,729 neutral SNPs, R. cyanocarpum had a higher genetic diversity (π = 0.0702 ± 0.0017, H e = 0.0675 ± 0.0016) than other plant species assessed using Rad-seq methods, while population differentiation (F st from 0.0314 to 0.0452) was weak. Interestingly, contrasting patterns of population structure were revealed from all neutral and selected SNPs, with distinct genetic clusters forming for all SNPs and neutral SNPs, but no distinct subgroups for selected ones. Moreover, we were able to detect changes in effective population size (N e ) of R. cyanocarpum from 150,000 years ago, including a bottleneck event ca. 60,000 years ago, followed by recovery of N e over a short period, and a subsequent gradual decline in N e to date. Implications for conserving R. cyanocarpum based on these main results are then discussed.
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IQ-TREE (, last accessed February 6, 2020) is a user-friendly and widely used software package for phylogenetic inference using maximum likelihood. Since the release of version 1 in 2014, we have continuously expanded IQ-TREE to integrate a plethora of new models of sequence evolution and efficient computational approaches of phylogenetic inference to deal with genomic data. Here, we describe notable features of IQ-TREE version 2 and highlight the key advantages over other software.
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The conservation and management of endangered species requires information on their genetic diversity, relatedness and population structure. The main genetic markers applied for these questions are microsatellites and single nucleotide polymorphisms (SNPs), the latter of which remain the more resource demanding approach in most cases. Here, we compare the performance of two approaches, SNPs obtained by restriction‐site‐associated DNA sequencing (RADseq) and 16 DNA microsatellite loci, for estimating genetic diversity, relatedness and genetic differentiation of three, small, geographically close wild brown trout (Salmo trutta) populations and a regionally used hatchery strain. The genetic differentiation, quantified as FST, was similar when measured using 16 microsatellites and 4,876 SNPs. Based on both marker types, each brown trout population represented a distinct gene pool with a low level of interbreeding. Analysis of SNPs identified half‐ and full‐siblings with a higher probability than the analysis based on microsatellites, and SNPs outperformed microsatellites in estimating individual‐level multilocus heterozygosity. Overall, the results indicated that moderately polymorphic microsatellites and SNPs from RADseq agreed on estimates of population genetic structure in moderately diverged, small populations, but RADseq outperformed microsatellites for applications that required individual‐level genotype information, such as quantifying relatedness and individual‐level heterozygosity. The results can be applied to other small populations with low or moderate levels of genetic diversity.
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The key process in speciation concerns the formation and maintenance of reproductive isolating barriers between diverging lineages. Although species boundaries are frequently investigated between two species across many taxa, reproductive isolating barriers among multiple species (>2) that would represent the most common phenomenon in the nature, remain to be clarified. Here, we use double digest restriction‐site associated DNA (ddRAD) sequencing to examine patterns of hybridization at a sympatric site where three Ligularia species grow together and verify whether those patterns contribute to the maintenance of boundaries among species. The results based on the RAD SNP datasets indicated hybridization L. cyathiceps × L. duciformis and L. duciformis × L. yunnanensis were both restricted to F1s plus a few first‐generation backcrosses and no gene introgression were identified, giving rise to strong reproductive isolation among hybridizing species. Moreover, hybrid swarm simulation, using HYBRIDLAB, indicated the RAD SNP datasets had sufficient discriminatory power for accurate hybrid detection. We conclude that parental species show strong reproductive isolation and they still maintain species boundaries, which may be the key mechanism to maintain species diversity of Ligualria in the eastern Qinghai‐Tibetan Plateau and adjacent areas. Moreover, this study highlights the effectiveness of RAD sequencing in hybridization studies.
For half a century population genetics studies have put type II restriction endonucleases to work. Now, coupled with massively‐parallel, short‐read sequencing, the family of RAD protocols that wields these enzymes has generated vast genetic knowledge from the natural world. Here we describe the first software natively capable of using paired‐end sequencing to derive short contigs from de novo RAD data. Stacks version 2 employs a de Bruijn graph assembler to build and connect contigs from forward and reverse reads for each de novo RAD locus, which it then uses as a reference for read alignments. The new architecture allows all the individuals in a meta population to be considered at the same time as each RAD locus is processed. This enables a Bayesian genotype caller to provide precise SNPs, and a robust algorithm to phase those SNPs into long haplotypes – generating RAD loci that are 400‐800bp in length. To prove its recall and precision, we test the software with simulated data and compare reference‐aligned and de novo analyses of three empirical datasets. We show that the latest version of Stacks is highly accurate and outperforms other software in assembling and genotyping paired‐end de novo datasets.
The genus Rhododendron Linnaeus (1753: 392) has more than 1,000 species that have been recognized in the temperate areas of the world (Chamberlain et al. 1996). In China, ca. 600 species of Rhododendron have been confirmed, including numerous species described after the publishing of Flora of China (Chen et al. 2010, Chen et al. 2012, Ma et al. 2013, Liao et al. 2015, Ma et al. 2015, Cai et al. 2016). They occur in most of China’s provinces except Xinjiang and Ningxia (Ma et al. 2014). Among these provinces, Yunnan, Sichuan and Tibet have the greatest diversity.
Rhododendron microcarpum R.L. Liu & L.M. Gao sp. nov. is described as a new species which is confined to Jiangxi, China. It has been discovered only in Yingpan Mountain located in the junction area of Nanling Mountains and Luoxiao Mountains. Morphologically, R. microcarpum is significantly distinguishable from closely related species R. ovatum by elliptic leaves with small size of 2.56 (± 0.11) × 1.25 (± 0.04) cm, pink-white corolla, short calyx with 0.23 (±0.003) cm in length, and small flower and fruit. ITS Neighbor-joining (NJ) tree indicates that R. microcarpum represents a distinct species and is genetically closest to R. ovatum. Evidences from morphological, ecological and molecular data supported R. microcarpum as a new species of R. subgenus Azaleastrum Planch..
A supra-annual, community-level synchronous flowering prevails in several parts of the tropical forests of Southeast Asia and its evolution has been hypothesized to be linked to pollinator shifts. The aseasonal Southeast Asian lowland rainforests are dominated by Dipterocarpaceae, which exhibit great floral diversity, a range of pollination syndromes and include species with annual and supra-annual gregarious flowering. Phylogenetic relationships within this family are still unclear, especially in the tribe Shoreeae. Here, we develop a pipeline to maximize recovery of genome-wide SNPs from restriction-site associated DNA sequencing (RADseq) in non-model organisms across wide phylogenetic scales. We then infer phylogenomic relationships in the tribe Shoreeae using both traditional and coalescent analyses. The phylogenetic trees obtained with these methods are congruent to each other and highly resolved. They allow reconstructing the evolutionary patterns of floral traits (number of stamens, anther structure and anther/appendage size) in the group. Our inferences indicate that species with many stamens, but smaller, globose anthers and longer appendages and have evolved multiple times from species with fewer stamens, but larger, oblong anthers and shorter appendages. This could have happened in parallel to iterative shifts in pollinators across the uncovered phylogeny from larger, longer generation to smaller, shorter-generation insects that can quickly build up the necessary population sizes during mass flowering episodes.