Langbiangia, a new genus of Gesneriaceae endemic to Langbiang Plateau, southern Vietnam and a taxonomic endeavor to achieve key targets of the post-2020 global biodiversity framework

Abstract

Situated in the southern end of the Annamite Mountain Range, Langbiang Plateau is a major biodiversity hotspot of southern Vietnam known for high species diversity and endemicity. To achieve effective conservation, parts of the plateau were designated as the Langbiang Biosphere Reserve, an UNESCO World Network aiming to improve relationships between inhabitants and their environments. Amongst the rich endemic flora of the plateau are three gesneriads ascribed to Primulina, a calciphilous genus with high species diversity in the vast limestone karsts stretching from southern China to northern Vietnam. However, a recent phylogenetic study questioned the generic placement of the Langbiang Primulina, corroborating with observations on the geographical distribution, habitat preference, and phyllotaxy of the three species. Based on phylogenetic analyses of nuclear ITS and plastid trnL-F DNA sequences of a comprehensive sampling covering nearly all genera of the Old World Gesneriaceae, we demonstrate that the three Langbiang Primulina species form a fully supported clade distantly related to other Primulina. As this clade is biogeographically, ecologically, morphologically, and phylogenetically distinct worthy of generic recognition, we propose to name it Langbiangia gen. nov. to highlight the rich and unique biodiversity of the Langbiang Plateau. By means of this taxonomic endeavor, we are hoping to raise the conservation awareness of this biodiversity heritage of southern Vietnam and promote the importance of Langbiang Biosphere Reserve that is crucial for achieving action-oriented global targets of the post-2020 global biodiversity framework (GBF) of the UN Convention on Biological Diversity (CBD)—effective conservation and management of at least 30% of biodiverse terrestrial, inland water, and costal and marine areas by 2030—that has been agreed at the COP15 in Montréal in December 2022.

Full text

RESEARCH ARTICLE
Langbiangia, a new genus of Gesneriaceae
endemic to Langbiang Plateau, southern
Vietnam and a taxonomic endeavor to
achieve key targets of the post-2020 global
biodiversity framework
Hong Truong Luu
1
, Chia-Lun HsiehID
2
, Chia-Rong Chuang
2
, Cheng-Wei Chen
2,3,4
, Ngoc
Toan Tran
1
, Ngoc Long Vu
1
, Kuo-Fang ChungID
2
*
1Southern Institute of Ecology, Institute of Applied Materials Science & Graduate University of Science and
Technology, Vietnam Academy of Science and Technology, Ho Chi Minh City, Vietnam, 2Research Museum
and Herbarium (HAST), Biodiversity Research Center, Academia Sinica, Taipei, Taiwan, 3Biodiversity
Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan, 4Department of Life
Science, National Taiwan Normal University, Taipei, Taiwan
*bochung@gate.sinica.edu.tw
Abstract
Situated in the southern end of the Annamite Mountain Range, Langbiang Plateau is a
major biodiversity hotspot of southern Vietnam known for high species diversity and
endemicity. To achieve effective conservation, parts of the plateau were designated as the
Langbiang Biosphere Reserve, an UNESCO World Network aiming to improve relationships
between inhabitants and their environments. Amongst the rich endemic flora of the plateau
are three gesneriads ascribed to Primulina, a calciphilous genus with high species diversity
in the vast limestone karsts stretching from southern China to northern Vietnam. However, a
recent phylogenetic study questioned the generic placement of the Langbiang Primulina,
corroborating with observations on the geographical distribution, habitat preference, and
phyllotaxy of the three species. Based on phylogenetic analyses of nuclear ITS and plastid
trnL-F DNA sequences of a comprehensive sampling covering nearly all genera of the Old
World Gesneriaceae, we demonstrate that the three Langbiang Primulina species form a
fully supported clade distantly related to other Primulina. As this clade is biogeographically,
ecologically, morphologically, and phylogenetically distinct worthy of generic recognition, we
propose to name it Langbiangia gen. nov. to highlight the rich and unique biodiversity of
the Langbiang Plateau. By means of this taxonomic endeavor, we are hoping to raise the
conservation awareness of this biodiversity heritage of southern Vietnam and promote the
importance of Langbiang Biosphere Reserve that is crucial for achieving action-oriented
global targets of the post-2020 global biodiversity framework (GBF) of the UN Convention
on Biological Diversity (CBD)—effective conservation and management of at least 30% of
biodiverse terrestrial, inland water, and costal and marine areas by 2030—that has been
agreed at the COP15 in Montre
´al in December 2022.
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OPEN ACCESS
Citation: Luu HT, Hsieh C-L, Chuang C-R, Chen C-
W, Tran NT, Vu NL, et al. (2023) Langbiangia, a
new genus of Gesneriaceae endemic to Langbiang
Plateau, southern Vietnam and a taxonomic
endeavor to achieve key targets of the post-2020
global biodiversity framework. PLoS ONE 18(5):
e0284650. https://doi.org/10.1371/journal.
pone.0284650
Editor: Abul Khayer Mohammad Golam Sarwar,
Bangladesh Agricultural University, BANGLADESH
Received: October 21, 2022
Accepted: March 17, 2023
Published: May 17, 2023
Copyright: ©2023 Luu et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in
any medium, provided the original author and
source are credited.
Data Availability Statement: All relevant data are
available within the paper and its Supporting
Information files.
Funding: Parts of this research were supported by
grants from Academia Sinica, Taiwan to K-FC. HTL,
NLV and part of field collection are funded by
Vietnam National Foundation for Science and
Technology Development (NAFOSTED) under grant
number 106.03-2020.38. The funders had no role

Introduction
The realization that collaboration with environmental and natural resource stakeholders
(Fig 1) is essential for effective conservation of biodiversity [1,2] has instigated the ideas of
“other effective area-based conservation measures (OECMs)” to complement traditional pro-
tected and preserved areas such as national parks and nature reserves [3]. An example of the
OECMs is UNESCO (United Nations Educational, Scientific and Cultural Organization) Bio-
sphere Reserves (BRs) [4]. First introduced in 1971, UNESCO Biosphere Reserves are a global
network of “learning places for sustainable development” using “interdisciplinary approaches to
understanding and managing changes and interactions between social and ecological systems,
including conflict prevention and management of biodiversity” [5]. As of now, the World Net-
work of Biosphere Reserves consists of 738 sites in 134 countries aiming to improve relation-
ships between inhabitants and their environments [5]. Given their integrated nature,
UNESCO BRs can facilitate greatly the achievement of the action-oriented global targets of the
post-2020 global biodiversity framework (GBF) agreed at the 15
th
meeting of the Conferences
of Parties (COP 15) to the UN Convention on Biological Diversity (CBD) in Montre
´al in
December 2022 [6]—effective conservation and management of at least 30% of biodiverse ter-
restrial, inland water, and costal and marine areas by 2030 [4]. Unfortunately, although current
Fig 1. Broad-leaved evergreen forest and expanding coffee plantation in Langbiang Plateau. As in much of the tropical countries, the biodiverse montane
forests of the Langbiang Plateau are being cleared for agriculture such as coffee plantation, threatening the native flora and fauna and the ecosystem functions
(photo: C.-W. Chen).
https://doi.org/10.1371/journal.pone.0284650.g001
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Langbiangia gen. nov.
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in study design, data collection and analysis,
decision to publish, or preparation of the
manuscript.
Competing interests: The authors have declared
that no competing interests exist.

UNESCO BRs cover more than 7 million km
2
and could contribute ca. 4.5% to the conven-
tional area-based protected areas [4], Biosphere Reserves are in general far less recognized
than traditional protected areas such as national parks [7]. The issue is particularly dire in
developing countries such as Vietnam, which boasts a remarkably high biodiversity [8,9] and
is home to 11 UNESCO Biosphere Reserves, the second-highest number in SE Asia [10–12].
To fully implement functions of UNESCO BRs, the importance of awareness-raising and out-
reach has constantly been emphasized [7,12,13].
Located within the Langbiang Plateau (Figs 1and 2), Langbiang Biosphere Reserve is the 9
th
UNESCO BRs in Vietnam [14]. Langbiang Plateau (also spelled as Lam Vien Plateau or known as
Da Lat Plateau) is situated in the southern end of the Annamite Mountain Range and bordered
by the Provinces of Dak Lak, Khanh Hoa, Lam Dong, and Ninh Thuan of Vietnam (Fig 2). As a
long-recognized biodiversity hotspot of southern Vietnam, a portion of the Langbiang Plateau
Fig 2. Distribution of Langbiangia and Primulina.The map was generated using QGIS 3.24.1 [60]. The basemap of
karst aquifers was downloaded from WHYMAP [61], which can be freely used and copied for educational and other
non-commercial purposes. The distributions were mapped based on occurrence records retrieved from Global
Biodiversity Facility [62]. The distribution of Langbiangia poilanei was plotted based on information taken from the
holotype. This work is licensed under a CC BY 4.0 license.
https://doi.org/10.1371/journal.pone.0284650.g002
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covering 275,439 ha has been protected as the Bidoup—Nui Ba National Park, which is also the
core zone of the Langbiang Biosphere Reserve designated by UNESCO in 2015 [14]. Langbiang
Biosphere Reserve is home to many rare and endangered mammals such as Indochinese tiger,
yellow-cheeked gibbon, Indian bison, etc. [14]. Additionally, a rich diversity in bats has also been
documented in the region [15]. In the recent decade, several new vertebrates including six fishes
[16,17], three snakes [18–20], and four amphibians [21–24] were described from the Langbiang
Plateau. Across the plateau, cryptic genetic diversity was also detected in the frog genus Leptolalax
[25]. The plateau is also known for its high fungal diversity [26,27] and the only habitat of the
rare holoparasitic plant Sapria himalayana Griff. (Rafflesiaceae) in Vietnam [28]. Langbiang Pla-
teau is also known as the prime habitats of numerous vascular plant species and in the recent
decade, a wide range of vascular plant taxa have also been described and/or recorded, including
16 ferns [29–32], one Schisandraceae [33], three Lauraceae [33,34], five orchids [35,36], one
bamboo [37], one Zingiberaceae [33], one legume [38], one aroid [39], one Menispermaceae [40],
one Daphniphyllaceae [33], four stone oaks [41,42], one Euphorbiaceae [43], two Rosaceae [33],
two begonias [44], one Clusiaceae [45], one Rutaceae [33], seven camellias [46–50], four Nyssa-
ceae [33,51], one ginseng [52], one Loranthaceae [53], two hollies [33], one Icacinaceae [33], one
Symplocaceae [33], one Rubiaceae [54], and five gesneriads [55–58]. However, the rich and
unique biological diversity of the Langbiang Plateau has continuously been threatened by grow-
ing land demands for agriculture (Fig 1) and residency [59]. These land-use changes thus under-
line the function of UNESCO World Network of Biosphere Reserves for sustainable development
of the biodiverse Langbiang Plateau.
The predominantly Old World subfamily Didymocarpoideae of the African Violet Family
Gesneriaceae is composed of ca. 2,500 species [63]. In recent decades, recorded diversity in the
subfamily has grown considerably as new taxa are being described continuously [e.g., 58, 64–66]
especially in Vietnam [55–58,66–71]. However, molecular phylogenetic studies in the past
decade also showed that traditional taxonomy of Gesneriaceae had utilized traits that are homo-
plasious [72–76]. Consequently, infrafamilial classification and generic delimitation of Gesneria-
ceae have undergone drastic changes in the past two decades [63,77]. One of the most extreme
examples of such changes has been Chirita Buch.-Ham. ex D.Don. Traditionally, Chirita was
known by the possession of a bifid stigma characterized by undeveloped upper lobe and bifidly
developed lower lobe known as the chiritoid stigma [75,78]. However, molecular phylogenetic
analyses showed that Chirita was highly polyphyletic [75,79,80], with the type species C.urticifo-
lia Buch.-Ham. ex D.Don intermingled with Henckelia Spreng. that was published earlier than
Chirita and thus has the taxonomic priority [75]. To establish a stable and phylogeny-based tax-
onomy of Gesneriaceae, the name Chirita was synonymized and its constituted species were
transferred to Codonoboea Ridl., Damrongia Kerr ex Craib, Henckelia,Microchirita (C.B.Clarke)
Yin Z.Wang (Chirita sect. Microchirita C.B.Clarke), Liebigia Endl. [Chirita sect. Liebigia
(Endl.) C.B.Clarke], and Primulina Hance (Chirita sect. Gibbosaccus C.B.Clarke) [75].
With the inclusion of former members of Chirita sect. Gibbosaccus as well as Chiritopsis W.
T.Wang and Wentsaiboea D.Fang & D.H.Qin [64,80], and the addition of more than 70 new
species, the recircumscribed Primulina has been expanded from a monotypic genus to its cur-
rent size of 227 species [64,81,82]. However, subsequent phylogenetic analyses revealed that
some former members placed in Chirita sect. Gibbosaccus by Wood 1974 [78] and transferred
to Primulina without DNA data by Weber et al. 2011 [75] should be placed in Deinostigma W.
T.Wang & Z.Y.Li [83,84]. More recently, Chirita umbrophila C.Y.Wu ex H.W.Li, a species
with uncertain generic placement [75], was shown to be conspecific with Loxostigma kurzii (C.
B.Clarke) B.L.Burtt [85]. On the other hand, phylogenetic studies of materials sampled from
further botanical exploration also revealed lineages that are distinct and worthy of generic rec-
ognition (e.g., Actinostephanus F.Wen, Y.G.Wei & L.F.Fu [65], Billolivia D.J.Middleton [86],
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Chayamaritia D.J.Middleton & Mich.Mo¨ller [87], Glabrella Mich.Mo¨ller & W.H.Chen [88],
Michaelmoelleria F.Wen, Y.G.Wei & T.V.Do [66], Middletonia C.Puglisi [89], Rachunia D.J.
Middleton & C.Puglisi [90], etc.). These recent taxonomic changes and novelties of the didy-
mocarpoid Gesneriaceae underscore the importance of incorporating molecular information
in achieving a stable classification of Asian Gesneriaceae.
Among the five recently described gesneriads from the Langbiang Plateau, Primulina scutel-
lifolia Luu, N.L.Vu & T.Q.T.Nguyen [58] is the latest described species. On the Langbiang Pla-
teau, P.scutellifolia is distributed in close proximity with P.poilanei (Pellegr.) Mich.Mo¨ller &
A.Weber (Fig 3) and often sympatric with P.annamensis (Pellegr.) Mich.Mo¨ller & A.Weber
[58] (Fig 2). Both P.annamensis and P.poilanei were initially described as Chirita Buch.-Ham.
ex D. Don (i.e., C.poilanei Pellegr. [91] and C.annamensis Pellegr. [92,93]) and placed under
Chirita sect. Gibbosaccus by Wood 1974 [78]. Along with other species of Chirita sect. Gibbo-
saccus,C.annamensis and C.poilanei were both transferred to Primulina in Weber et al. 2011
[75], though neither of these two species were sampled for phylogenetic analyses. Primulina
scutellifolia was described as a morphologically similar species of P.annamensis, differing from
the latter by several morphological characteristics including scutellate leaves, less densely
pilose petioles, and the stigma with enlarged lower lip but without upper lip [58]. Likewise, no
molecular data was generated for P.scutellifolia to assure its generic placement [58].
Biogeographically, Primulina annamensis,P.poilanei, and P.scutellifolia are all restricted to
the Langbiang Plateau, representing the southernmost and disjunctively distributed species of
Primulina (Fig 2). Ecologically, while a majority of Primulina are calciphilous plants on lime-
stone karsts (Fig 2) of lower elevations [64,94–96], P.annamensis,P.poilanei, and P.scutellifolia
grow on humid fertile soils of the evergreen broad-leaved forest at elevation of 1,450–2,000 m
[58,78,92,93,97]. Morphologically, P.annamensis,P.poilanei, and P.scutellifolia also differ
from the typical Primulina by their alternate and spirally arranged phyllotaxy [58,78], while a
majority of Primulina are characterized by decussate or whorled leaves (Fig 4). Additionally, the
peltate and scutellate leaf bases of P.scutellifolia are reminiscent to Deinostigma poilanei (Pel-
legr.) W.T.Wang & Z.Y.Li [98], D.serratum F.Wen, L.N.Tuan & D.Dien [99], and D.tamianum
(B.L.Burtt) D.J.Middleton & H.J.Atkins [58] not known in any other species of Primulina. Prior
to its current placement in Deinostigma W.T.Wang & Z.Y.Li, D.tamianum [83] was also first
described as Chirita (i.e., Chirita tamiana B.L.Burtt) and classified under Chirita sect. Gibbosac-
cus [78]. Along with C.annamensis and C.poilanei,C.tamiana was also transferred to Primu-
lina without molecular data [75], and was later transferred to the expanded Deinostigma [84].
Morphologically, however, in contrast to the caulescent habit and bilocular ovaries observed in
Deinostigma, all three Langbiang Primulina are acaulescent with unilocular ovaries. Phylogenet-
ically, Xu et al. 2021 [95] showed that their sampled Primulina annamensis (i.e., Primulina
annamensis ANNA) was not placed within the ‘true’ Primulina but sister to their sampled Dei-
nostigma poilanei (i.e., Deinostigma poilanei POIL), though no other Deinostigma species were
sampled and the taxonomic implications were not discussed. Therefore, it remains uncertain
whether the three Langbiang Primulina species belong to Primulina,Deinostigma, or even a
unique genus of itself. The biogeographical, ecological (Fig 2), morphological (Figs 3and 4),
and phylogenetic [95] uniqueness of the Langbiang Plateau Primulina thus prompted us to con-
duct phylogenetic analyses to confirm generic placements of the three species.
Materials and methods
Ethics statement
The samples used in this studied were collected under the permits granted by Van Huong Le,
the director of Bidoup—Nui Ba National Park.
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Inclusivity in global research
Additional information regarding the ethical, cultural, and scientific considerations specific to
inclusivity in global research is included in the S1 Checklist.
Fig 3. Langbiangia poilanei.(A) Habit. (B) Corolla, longitudinal dissection with stamens. (C) Disc, ovary and style.
Photos by Hong Truong Luu and Minh Tri Dang.
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Sampling and DNA extraction
To test generic placements of the three Langbiang Primulina species, we sampled two individ-
uals of P.annamensis, two P.poilanei, and one P.scutellifolia. The material of P.scutellifolia,
which was assessed as Critically Endangered [58], was sampled from the type collection. As P.
scutellifolia was considered morphologically similar to Deinostigma [58], we also collected D.
eberhardtii (Pellegr.) D.J.Middleton & H.J.Atkins (Bach 0512), which previously has never
been analyzed molecularly, to complement the sampling of the genus. Additionally, Bach
0513, which is an unknown gesneriad akin to Deinostigma, was also added to the analyses.
Voucher specimens (Table 1) were deposited in the Herbarium of Southern Institute of
Fig 4. Phyllotaxy of Langbiangia and Primulina.(A) Langbiangia annamensis, photo by Maxim S. Nuraliev / CC
BY-NC 4.0 (https://www.inaturalist.org/observations/38536524) (B) The acaulescent stem of Langbiangia scutellifolia.
(C) Primulina minutimaculata. (D) Primulina dryas. (E) Primulina lungzhouensis. (F) Primulina minor. B–E by C.-L.
Hsieh, and F by K.-F. Chung.
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Ecology (SGN), Ho Chi Minh City, Vietnam. The genomic DNAs were extracted from 0.2 g of
silica-dried leaf material using the CTAB method [100]. The quantity and quality of the
extracted DNAs were checked by electrophoresis on 1.5% agarose gels.
Based on the most updated classification of Gesneriaceae, Primulina and Deinostigma are
both placed in subtribe Didymocarpinae, tribe Trichosporeae, subfamily Didymocarpoideae
[63,77,96,101]. To maximize taxon sampling while exploiting DNA sequences available in
the GenBank, we sequenced the internal transcribed spacer (ITS) of the nuclear ribosomal
DNA (including the 5.8S gene) and the plastid trnL-trnF intergenic spacer (trnL-F), which
have been the most widely used molecular markers in reconstructing phylogenetic relation-
ships of Asian Gesneriaceae and delimitating new gesneriad genera [74–76,86–88,90,102,
103]. The ITS sequence was amplified by the universal primers, ITS4 and ITS5 [104]. The
trnL-F was amplified using three primer pairs: trnL-F_1F (5ʹ-GGCGAAATCGGTAGACGCTA
-3ʹ) and trnL-F_1R (5ʹ-CCCAGATACAGATTCGGGCC-3ʹ), trnL-F_2F (5ʹ-GGCCCGAATCTG
TATCTGGG-3ʹ) and trnL-F_2R (5ʹ-ACCGTTAACGAACAAAGCGG-3ʹ), and trnL-F_3F (5ʹ-C
CGCTTTGTTCGTTAACGGT-3ʹ) and trnL-F_3R (5ʹ-CCATGTGCCAGGAACCAGAT-3ʹ),
designed using Primer3 v.2.3.7 [105] implemented in the program Geneious Prime1
v.2022.1.1 [106]. The total volume for all polymerase chain reactions (PCRs) was 30 μl with
15 μl of Q-AmpTM 2x ScreeningFire Taq Master Mix (Bio-Genesis Technologies Inc., Tai-
wan), 9 μl of ddH2O, 4 μl of DNA, and 1 μl of each of the two primers. All PCRs were carried
out under the thermocycling program which starts from 5 minutes at 95˚ C for initial denatur-
ation, followed by 40 cycles of amplification of 1 minute at 95˚ C, 1 minute at 56˚ C for, and 1
minute at 72˚ C, and ends with the final extension step of 7 minutes at 72˚ C. The PCR prod-
ucts were Sanger sequenced by Genomics BioScience and Technology Co. (Taipei, Taiwan).
All newly generated DNA sequences (S1 Table) have been deposited in GenBank (https://
www.ncbi.nlm.nih.gov/genbank/).
Sequence alignment and phylogenetic analyses
Geneious was used to conduct sequencing quality check and sequence assembly. In addition to
the newly generated sequences, DNA sequences of 256 species, representing nearly all genera,
all subtribes, and all tribes (i.e., Epithemateae and Trichosporeae) of subfamily Didymocarpoi-
deae, were downloaded from GenBank (S1 Table). Our sampling of the subtribe Didymocarpi-
nae included 135 species representing all genera except for the doubtful genus Sepikea Schltr.
Table 1. Sampling information and GenBank accession numbers of newly sequenced individuals. All voucher specimens were deposited in the Herbarium of Southern
Institute of Ecology (SGN).
Code Species Voucher Locality GenBank accession
numbers (trnL-F/ITS)
Bach 0512 Deinostigma
eberhardtii
Tran Ngoc Toan Bach 0512 Da Nang City, Lien Chieu District, Nam Hai Van Special Use Forest OP617258/OP605498
Bach 0513 Deinostigma sp.Tran Ngoc Toan Bach 0513
(living collection, SGN)
Quang Nam Province, Nong Son District, Quang Nam Elephant
Sanctuary
OP617259/OP605499
BD77 Primulina
annamensis
Dang Minh Tri BD-TN3-077 Lam Dong Province, Lac Duong District, Da Chais Commune,
Bidoup—Nui Ba National Park, Bidoup Forest Dynamics Plot
OP508008/OP508034
CR024 Primulina
annamensis
C.-W.Chen s.n. Lam Dong Province, Lac Duong District, Giang Ly Ranger Station OP617260/OP605500
CR027 Primulina poilanei C.-W.Chen s.n. Khanh Hoa Province, Khanh Vinh District OP617261/OP605501
CR028 Primulina poilanei C.-W.Chen s.n. Khanh Hoa Province, Khanh Vinh District OP617262/OP605502
Luu
KH0945
Primulina
scutellifolia
Luu KH0945 Khanh Hoa Province, Khanh Vinh District, Son Thai Commune OP508007/OP508033
https://doi.org/10.1371/journal.pone.0284650.t001
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[63]. To ease the computational loading, we only sampled 16 species of Primulina that repre-
sent all major clades of the genus [64]. Titanotrichum oldhamii (Hemsl.) Soler. of tribe Titano-
tricheae, subfamily Gesnerioideae was chosen to root the tree based on recent studies [63,77].
After incorporating the newly generated sequences, each of the ITS and trnL-F matrix was
aligned separately by MAFFT v.7.490 [107] using Geneious with default settings. Each
sequence in the alignment was manually trimmed to the same length, and the sites with >98%
of gaps were excluded by applying the “mask alignment” function in Geneious.
Because of low phylogenetic resolutions in preliminary maximum likelihood (ML) analyses
of individual matrices (S1 and S2 Figs) using RAxML v.8.2.11 [108], the two matrices were
concatenated (S1 File) as executed in a majority of Gesneriaceae phylogenetic analyses [74–76,
86,87,90,102,103] using AMAS python scripts [109]. For the concatenated matrix, we con-
ducted phylogenetic analyses based on both (ML) and Bayesian inference (BI) using RAxML
v.8.2.11 and MrBayes v.3.2.7a [110], respectively. ML analyses were conducted with the set-
tings of GTR+GAMMA for nucleotide substitution model and rapid bootstrap (-f a option)
with 2000 replicates. Nodes in the ML tree with bootstrap support (BS) lower than 50% were
collapsed into polytomies by using TreeCollapserCL 4 [111]. Prior to the BI analyses, the best-
fit nucleotide model for each partition was determined by ModelFinder [112] in IQ-TREE
v.2.0.6 [113] with the setting of -mset mrbayes. For BI analyses, according to ModelFinder’s
results, the SYM+I+G and GTR+F+G models were applied to ITS and trnL-F partitions,
respectively, in MrBayes with all model parameters unlinked among partitions and allowing
the overall rate to vary across partitions. In MrBayes, two independent runs with four chains
each of the Markov Chain Monte Carlo (MCMC) simulations were set to perform 10,000,000
generations with sampling frequency of every 1000 generations and the first 25% of genera-
tions discarded as burn in. The resulting trees were summarized into a phylogram with clade
credibility (posterior probability) values. The final phylogenies of ML and BI analyses were
visualized in FigTree v.1.4.4 [114].
Nomenclature
The electronic version of this article in Portable Document Format (PDF) in a work with an
ISSN or ISSBN will represent a published work according to the International Code of
Nomenclature for algae, fungi, and plants, and hence the new names contained in the elec-
tronic publication of a PLOS ONE article are effectively published under that Code from the
electronic edition alone, so there is no longer any need to provide printed copies.
In addition, new names contained in this work have been submitted to IPNI, from where
they will be made available to the Global Names Index. The IPNI LSIDs can be resolved and
the associated information viewed through any standard web browser by appending the LSID
contained in this publication to the prefix http://ipni.org/. The online version of this work is
archived and available from the following digital repositories: PubMED Central and LOCKSS.
Results and discussion
The DNA alignment of 242 ITS sequences was 956 bp in length with 838 (87.7%) parsimony
informative sites, while the trnL-F alignment of 252 sequences was 931 bp in length with 604
(64.9%) parsimony informative sites. The final concatenated matrix of 256 accessions was
1,887 bp in length with 1,136 (60.2%) parsimony informative sites. Results of BI (Fig 5 and S3
Fig) and ML (S4 and S5 Figs) analyses are largely congruent, with BI analysis showing better
resolution and higher support values (S3 and S5 Figs). A simplified Bayesian inference phylog-
eny with ML bootstrap support values is shown in Fig 5. Rooted by Titanotrichum oldhamii of
the subfamily Gesnerioideae, the monophyly of both tribes Epithemateae and Trichosporeae
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Fig 5. Simplified Bayesian inference tree of Didymocarpoideae based on combined trnL-F and ITS sequences. Numbers at the node indicate Bayesian posterior
probability (0.5), with ML bootstrap value (50%) shown after slash (/). All genera except for Primulina,Deinostigma and Langbiangia gen. nov. are collapsed
into triangles, with gray triangles denoting genera/clades that are fully supported in both Bayesian and ML analyses. The infrafamilial classification of Weber et al.
2013 [77] and Weber et al. 2020 [63] is followed. This tree is simplified from the S3 Fig. ML bootstrap values are retrieved from S5 Fig.
https://doi.org/10.1371/journal.pone.0284650.g005
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of the subfamily Didymocarpoideae is recovered (Fig 5). Within the tribe Trichosporeae, sub-
tribes Jerdoniinae, Corallodiscinae, Tetraphyllinae, and Litostigminae formed successive sister
clades to the rest of the subtribes as revealed in previous studies [63,77]. Relationships among
the rest of the subtribes, i.e., Ramondinae, Leptoboeniae, Streptocarpinae, Didissandrinae,
Loxocarpinae, and Didymorcarpinae, remained unresolved or poorly resolved (Fig 5).
Within the subtribe Didymocarpinae, Rachunia,Codonoboea,Microchirita, and Henckelia
formed successive sister groups to an unresolved clade composed of all other genera of the sub-
tribe (i.e., ‘core’ Didymocarpinae), congruent with previous studies [66,90]. As shown in pre-
vious phylogenetic studies of Didymocarpinae using ITS and trnL-F sequences, relationships
within the ‘core’ Didymocarpinae remained poorly resolved [75,77,79,86,87,90]. The
employment of genomic data such as whole plastome sequences [96] and target capture [101]
will be essential to clarify and understand this apparent radiation of Asian gesneriad clade.
Together with Primulina annamensis ANNA and Deinostigma poilanei POIL that were
sequenced by Xu et al. 2021 [95], our newly sequenced Langbiang Primulina formed a fully sup-
ported clade within the ‘core’ Didymocarpinae (Fig 5), while other sampled species of ‘true’ Pri-
mulina formed a fully supported clade sister to Petrocodon Hance as revealed in previous studies
[64,96]. Except for D.poilanei POIL [95], all other sampled Deinostigma and its sister genus
Metapetrocosmea, including our newly sequenced D.eberhardtii Bach 0512 and Deinostigma sp.
Bach 0513, as well as D.poilanei KN201 that was used for recircumscribing Deinostigma by Mo¨l-
ler et al. 2016 [83], formed a fully support clade (Fig 5). Although no detailed voucher informa-
tion was provided for D.poilanei POIL [95], given that both ITS (S1 Fig) and trnL-F (S2 Fig)
sequences of D.poilanei POIL are almost identical to our sequenced Langbiang Primulina, our
phylogenetic results suggest that D.poilanei POIL could be a confusion with and/or misidentifica-
tion of Primulina poilanei, likely resulted from the identical species epithet ‘poilanei’.
Within the Langbiang Primulina clade, P.annamensis ANNA sequenced by Xu et al. 2021
[95] is sister to P.scutellifolia collected from the type collection in both the ITS tree (S1 Fig)
and the concatenated tree (Fig 5), rendering P.annamensis paraphyletic, though relationships
among the three Langbiang Primulina species are unresolved in the trnL-F tree (S2 Fig).
Because Primulina scutellifolia is morphologically similar to P.annamensis [58], P.annamensis
ANNA sampled by Xu et al. 2021 [95] might also be a misidentification of P.scutellifolia. Alter-
natively, given the geographical proximity between P.annamensis and P.scutellifolia [58], the
paraphyly of P.annamensis with P.scutellifolia nested within (Fig 5) also suggests the possibil-
ity of hybridization between the two species. Nevertheless, further study with increase sam-
pling and additional molecular markers will be needed to clarify the species boundaries
between these two species.
Given the biogeographical, ecological (Fig 2), morphological (Figs 3and 4), and phyloge-
netic distinctness (Fig 5) of the Langbiang Primulina, our study clearly shows that the clade
composed of these three species should be recognized as a distinct genus as those recently
described genera of the subtribe Didymocarpinae [65,66,86–90]. With the reduction of these
three species, the total number of Primulina known from Vietnam decreases from 24 [58] to
21 species. As members of this new genus are thus far only known from the Langbiang Plateau,
we propose to name the new genus Langbiangia to highlight the rich and unique biodiversity
of the region. In naming the new genus Langbiangia, we are also hoping to raise the conserva-
tion awareness and popularize the importance of Langbiang Biosphere Reserves that is crucial
for achieving action-oriented global targets of the post-2020 GBF of the UN CBD [6].
Taxonomic treatment
Langbiangia Luu, C.L.Hsieh & K.F.Chung, gen. nov. [urn:lsid:ipni.org:names:77306595–1]
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Type: Langbiangia poilanei (Pellegr.) Luu, C.L.Hsieh & K.F.Chung
Diagnosis. ngbiangia shares many similar floral traits with Primulina, but differs from the
latter by its spirally-arranged alternate phyllotaxy. Langbiangia is also reminiscent of Deinos-
tigma, but could be distinguished by the acaulescent habit and unilocular ovary in
Langbiangia.
Description. aulescent herbaceous perennials. Rhizomes terete, sometimes woody, vertical
or horizontal, 3–7 mm in diameter, up to 9 cm long. Leaves leathery, 3–13, spirally alternate,
rosulate; petioles rounded or cylindrical, 1.5–10 cm long, 3 mm in diameter, densely hairy
with reflexed hairs; leaf blades broadly ovate to narrowly elliptical, 2.7–14 ×0.6–5.2 cm, apexes
acute to obtuse, margins entire, slightly serrulate to sinuate, sometimes densely hairy to gla-
brous, bases cuneate, sub-cordate, or scutellate-foveate, variously hairy on both surfaces, lateral
veins 4–7 pairs, sunken adaxially, densely hairy abaxially. Inflorescences 1–5, axillary, cymose,
scapiform, flowers 1–3; peduncles 10–18 cm long, 2–3 mm in diameter, sparely hirsute; bracts
linear to narrowly triangular, 2–6 mm long, ca. 0.5 mm wide at base. Calyxes 5-lobed, divided
to the base, lobes equal, narrowly triangular to lanceolate-oblong, 8–14 ×0.8–3 mm, sparsely
hairy. Corollas infundibuliform, 3.5–5 cm long, 1–1.5 cm in diameter at mouth, ca. 0.5 cm in
diameter at base, not pouched, sparsely glandular hairy outside, white, violet or white at base
and gradually turning into violet to blue towards the lobes, hairiness inside with two yellow
strips on lower part of the corolla; corollas distinctly 2-lipped, upper-lips 2-lobed, lobes
broadly ovate, 3.5–4 ×7–8 mm, lower-lips 3-lobed, central lobe orbicular, 7–8 ×7–8 mm, lat-
eral lobes broadly ovate, 7–8 ×8–9 mm. Stamens 2, filaments 7–32 mm long, adnate to the
corolla tube 16–18 mm from the base, slightly curved or geniculate 2 mm from the point of
insertion, free part 13–14 mm long and slightly curved, glabrous or apically sparsely glandular
hairy; anthers fused by their entire adaxial surfaces, elliptic, ca. 3 mm long, yellowish, thecae
divergent. Staminodes 3, apex capitate, yellowish, glabrous, 15–21 mm long, adnate to 12–17
mm from the base of the corolla, free part 3.5–5 mm long. Disc a ring, ca. 1 mm high, slightly
5-lobed. Gynoecium 2.2–3.5 cm long, 1.4–2 mm in diameter at base, ca. 0.6 mm below the
stigma, glabrous or hairy; stigma obscurely 2-lobed, only the lower lip developed, trapeziform,
finely hairy. Capsules linear, slightly falcate, oblique in relation to the pedicel, reddish brown
or greenish and turn to light yellowish, 6–6.5 ×0.4–0.7 cm, glabrous or sparsely hairy on the
apical part, dehiscence along the dorsal side; calyx persistent. Seeds long ellipsoid, translucent
brownish.
Etymology. e new genus Langbiangia is named after the Langbiang Plateau. Situated in the
southern end of the Annamite Mountain Range and bordered by the Provinces Dak Lak,
Khanh Hoa, Lam Dong, and Ninh Thuan (Fig 1), Langbiang Plateau is a biodiversity hotspot
[26,28,30] and a portion of the plateau within Lam Dong Province was designated by
UNESCO as Langbiang Biosphere Reserve in 2015 for its high regional biodiversity that also
includes many endangered species [14]. By naming the clade Langbiangia, which is thus far
only known from the Langbiang Plateau, we are hoping to raise the conservation awareness of
this important heritage of biodiversity of Vietnam.
Distribution. Vietnam, known only from the Langbiang Plateau (Khanh Hoa and Lam
Dong Provinces).
Habitat. On fertile soils of forest floors of montane forests.
Key to the species of Langbiangia
1. Leaf blades narrowly elliptical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L.poilanei
1. Leaf blades broadly ovate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
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2. Petioles densely pilose; leaf blades flat, with obviously cordate base . . . . . . . . . .L.annamensis
2 Petioles appressed downward tomentose; leaf blades scutellate . . . . . . . . . . . . . . L.scutellifolia
1. Langbiangia annamensis (Pellegr.) Luu, C.L.Hsieh & K.F.Chung, comb. nov. (Fig 4A)
[urn:lsid:ipni.org:names:77306596–1] Chirita annamensis Pellegr. in Fl. Indo-Chine 4:
530 (1930) Primulina annamensis (Pellegr.) Mich.Mo¨ller & A.Weber in Taxon 60(3): 781
(2011).
Type: VIETNAM, Annam, Prov de Nha-trang, massif de Hon-ba, 1000 m, 3 July 1918, A.
Chevalier 38697 (lectotype, designated by Wood 1974 [78], p.140: P
[MNHN-P-P00602505]).
2. Langbiangia poilanei (Pellegr.) Luu, C.L.Hsieh & K.F.Chung, comb. nov. (Fig 3) [urn:lsid:ipni.
org:names:77306597–1] Chirita poilanei Pellegr., Bull. Soc. Bot. France. 73(3): 419. (1926) 
Primulina poilanei (Pellegr.) Mich.Mo¨ller & A.Weber in Taxon 60(3): 784 (2011). (Fig 5).
Type: VIETNAM, Annam, Giang Ly, Nhatrang dans le lit d’un sui (ruisseau), 2000 m alt. forêt
Ouest de Nhatrang, 22 May 1922, F.Poilane 3616 (holotype, P [MNHN-P-P00602520]).
3Langbiangia scutellifolia (Luu, N.L.Vu & T.Q.T.Nguyen) Luu, C.L.Hsieh, K.F.Chung,
comb. nov. (Fig 4B) [urn:lsid:ipni.org:names:77306598–1] Primulina scutellifolia Luu, N.
L.Vu & T.Q.T.Nguyen in PhytoKeys 187: 16, fig 1 (2021).
Type: VIETNAM, Khanh Hoa Province, Khanh Vinh District, Son Thai Commune, 12˚
11’39"N, 108˚43’30"E, at ca. 1485 m elevation, 01 November 2013, Luu Hong Truong
KH0945 (holotype, SGN!; isotypes SGN!, PHH!, VNMN!).
Supporting information
S1 Checklist. Checklist of inclusivity in global research.
(DOCX)
S1 Fig. Maximum likelihood phylogeny based on ITS sequences.
(PDF)
S2 Fig. Maximum likelihood phylogeny based on trnL-F sequences.
(PDF)
S3 Fig. Bayesian inference phylogeny of Didymocarpoideae based on combined ITS and
trnL-F sequences.
(PDF)
S4 Fig. A simplified maximum likelihood phylogeny based on combined ITS and trnL-F
sequences.
(PDF)
S5 Fig. Maximum likelihood phylogeny based on combined ITS and trnL-F sequences.
(PDF)
S1 Table. NCBI accession numbers of sampled species.
(XLSX)
S1 File. Nexus file for Bayesian inference. Concatenated alignment of ITS and trnL-F
sequences and parameters for MrBayes.
(NEX)
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Langbiangia gen. nov.
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Acknowledgments
We thank Van Huong Le, the director of Bidoup—Nui Ba National Park, for granting permis-
sion to sample the materials. Minh Tri Dang (SIE) is acknowledged for taking detailed photos
of Langbiangia poilanei.
Author Contributions
Conceptualization: Hong Truong Luu, Kuo-Fang Chung.
Data curation: Chia-Lun Hsieh, Chia-Rong Chuang.
Formal analysis: Chia-Lun Hsieh, Chia-Rong Chuang.
Funding acquisition: Hong Truong Luu, Kuo-Fang Chung.
Investigation: Hong Truong Luu, Chia-Lun Hsieh, Chia-Rong Chuang, Cheng-Wei Chen,
Ngoc Long Vu, Kuo-Fang Chung.
Methodology: Chia-Lun Hsieh, Chia-Rong Chuang.
Visualization: Hong Truong Luu, Chia-Lun Hsieh, Chia-Rong Chuang, Cheng-Wei Chen,
Ngoc Toan Tran, Ngoc Long Vu, Kuo-Fang Chung.
Writing – original draft: Chia-Lun Hsieh, Kuo-Fang Chung.
Writing – review & editing: Hong Truong Luu, Chia-Lun Hsieh, Chia-Rong Chuang,
Cheng-Wei Chen, Ngoc Toan Tran, Ngoc Long Vu, Kuo-Fang Chung.
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