Pleione jinhuana (Arethuseae; Epidendroideae; Orchidaceae), a new species from China based on morphological and DNA evidence

Abstract

Pleione jinhuana, a new orchid from China, is illustrated and described based on morphological and phylogenetic evidence. Detailed morphological comparisons between the new species and other members of Pleione indicate that P. jinhuana is similar to P. bulbocodioides and P. formosana but differs in having a lip with an erose apical margin and lateral lobes not enfolding the column. To explore the phylogenetic position of this species, nuclear ribosomal ITS and plastid DNA (matK exon, trnT-trnL spacer, trnL exon and trnL-trnF spacer) were sequenced for P. jinhuana and individuals of P. formosana collected in Taiwan, Fujian and Zhejiang Provinces. The morphological and molecular evidence support the hypothesis that P. jinhuana is a new species.

Full text

Phytotaxa 345 (1): 043–050
http://www.mapress.com/j/pt/
Copyright © 2018 Magnolia Press Article PHYTOTAXA
ISSN 1179-3155 (print edition)
ISSN 1179-3163 (online edition)
Accepted by Mark Chase: 24 Feb. 2018; published: 20 Mar. 2018
https://doi.org/10.11646/phytotaxa.345.1.5
43
Pleione jinhuana (Arethuseae; Epidendroideae; Orchidaceae), a new species from
China based on morphological and DNA evidence
MING-TAO JIANG1,2,3, SHA-SHA WU1,3, ZHONG-JIAN LIU1,2 & SI-REN LAN1,3
1College of Landscape Architecture, Fujian Agriculture and Forestry University, Fuzhou 350002, China;
E-mail (S.R. Lan): lkzx@fafu.edu.cn
2Shenzhen Key Laboratory for Orchid Conservation and Utilization, The National Orchid Conservation Centre of China and The Orchid
Conservation and Research Centre of Shenzhen, Shenzhen 518114, China
3Fujian Agriculture and Forestry University Cross-Straits Orchids Conservation and Research Center, Fuzhou 350002, Fujian, China
Abstract
Pleione jinhuana, a new orchid from China, is illustrated and described based on morphological and phylogenetic evidence.
Detailed morphological comparisons between the new species and other members of Pleione indicate that P. jinhuana is
similar to P. bulbocodioides and P. formosana but differs in having a lip with an erose apical margin and lateral lobes not
enfolding the column. To explore the phylogenetic position of this species, nuclear ribosomal ITS and plastid DNA (matK
exon, trnT-trnL spacer, trnL exon and trnL-trnF spacer) were sequenced for P. jinhuana and individuals of P. formosana
collected in Taiwan, Fujian and Zhejiang Provinces. The morphological and molecular evidence support the hypothesis that
P. jinhuana is a new species.
Keywords: Arethuseae, Asian orchids, Pleione phylogenetics, window-sill orchid
Introduction
Pleione Don (1825: 36; Orchidaceae) comprises 22 species and five natural hybrids (Govaerts et al. 2016) distributed
in China, India, Nepal, Bhutan, Myanmar, Thailand, Laos and Vietnam (Cribb & Butterfield 1999, Chen et al. 2009).
Recently, a new species, Pleione arunachalensis, was discovered by Vadakkoot et al. (2017). Due to different views
on key morphological characters of the genus, four infrageneric classifications of Pleione have been proposed.
Phylogenetic relationships within Pleione were first evaluated by Gravendeel et al. (2004) using plastid and nuclear
DNA, and three groups were suggested for the genus. As new species are discovered and new data added from DNA
studies, the number of species and classification of Pleione species continues to change.
During fieldwork in 2013 in Jinhua, Zhejiang Province, China, we discovered plants that morphologically were
similar to P. formosana. We cultivated some individuals in the nursery and then observed them again in 2016. Upon
careful examination of their morphological characters, we confirmed the stability of their morphology and identified
this material as representing a new species. It is generally similar to P. bulbocodioides (Franchet) Rolfe (1903: 291)
and P. formosana Hayata (1911: 64) but differs significantly in the structure of the lip. In this study, we describe the
features of this new species and use DNA sequences to explore its phylogenetic position.
Materials and methods
Morphological observations:—Morphological data were obtained during flowering, and specimens were deposited
in the National Orchid Conservation Centre Herbarium (NOCC). Measurements, shapes, colours and other details
given in the description are based on the examination of living material. The floral structure of the fully opened flowers
was observed using a Leica M205a microscope (Leica Microsystems Ltd., Heerbrugg, Germany).
Taxonomic sampling:—To explore the phylogenetic position of the new species, we used a total of 26 taxa as
ingroups, and two taxa, Dendrochilum longifolium and Thunia alba, were selected as outgroups following Gravendeel
et al. (2004). GenBank accession numbers and origin of specimens are provided in Table 1.

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44 • Phytotaxa 345 (1) © 2018 Magnolia Press
TABLE 1. GenBank accession numbers for sequence data generated in this study.
Species Origin ITS matK trnT-trnL trnL trnL-trnF
Pleione jinhuana Liu, Jiang & Lan Jinhua,
Zhejiang, China MG195686 MG195698 MG195734 MG195710 MG195722
P. formosana Hayata Pingtung,
Taiwan, China MG195676 MG195688 MG195724 MG195700 MG195712
P. formosana Hualien,
Taiwan, China MG195679 MG195691 MG195727 MG195703 MG195715
P. formosana Nantou, Taiwan,
China MG195682 MG195694 MG195730 MG195706 MG195718
P. formosana Zhenghe,
Fujian, China MG195678 MG195690 MG195726 MG195702 MG195714
P. formosana Dehua, Fujian,
China MG195683 MG195695 MG195731 MG195707 MG195719
P. formosana Wuyishan,
Fujian, China MG195685 MG195697 MG195733 MG195709 MG195721
P. formosana Taishun,
Zhejiang, China MG195684 MG195696 MG195732 MG195708 MG195720
P. formosana Lin’an,
Zhejiang, China MG195677 MG195689 MG195725 MG195701 MG195713
P. albiflora Cribb & Tang China AY101967 AF503730 AF503655 AF503705 AF503680
P. aurita Cribb & Pfennig China AF461467 AF503732 AF503658 AF503708 AF503683
P. bulbocodioides (Franchet) Rolfe China AF461482 AF302704 AF503657 AF503682 AF503707
P. coronaria Cribb & Tang Nepal AF461470 AF503734 AF503660 AF503710 AF503685
P. formosana Hayata unknown AF461485 AF302705 AF503661 AF503711 AF503686
P. forrestii Schlechter China AF461478 AF503735 AF503662 AF503712 AF503687
P. grandifora (Rolfe) Rolfe China AF461477 AF503736 AF503663 AF503713 AF503688
P. hookeriana (Lindley) Rollisson China AF461468 AF503737 AF503664 AF503714 AF503689
P. hookeriana India AF461469 AF503738 AF503665 AF503715 AF503690
P. humilis (Smith) Don Nepal AF461495 AF503739 AF503666 AF503716 AF503691
P. limprichtii Schlechter China AF461490 AF503740 AF503667 AF503717 AF503692
P. maculata (Lindley) Lindley &
Paxton unknown AF461493 AF503741 AF503668 AF503718 AF503693
P. pleionoides (Kraenzlin) Braem &
Mohr China AF461480 AF503743 AF503670 AF503720 AF503695
P. praecox (Smith) Don unknown AF461491 AF503742 AF503669 AF503719 AF503694
P. saxicola Tang & Wang ex Chen China AF461492 AF503745 AF503672 AF503722 AF503697
P. scopulorum Smith unknown AF461471 AF503746 AF503673 AF503723 AF503698
P. yunnanensis (Rolfe) Rolfe China AF461487 AF503748 AF503675 AF503725 AF503700
Dendrochilum longifolium
Reichenbach New Guinea AF281121 AY003874 AF503678 AF503728 AF503703
Thunia alba (Lindley) Reichenbach Nepal AY008466 AF263684 AF503679 AF503729 AF503704
Molecular markers:—One nuclear region (rITS) and four plastid DNA regions (matK exon, trnT-trnL spacer, trnL
intron and trnL-trnF spacer) were used in the phylogenetic analyses. DNA extraction, PCR amplification, sequencing,
sequence editing and sequence assembly were performed according to Zhang et al. (2013). The primers used for the
PCR amplification were specified in Li et al. (2016) and Taberlet et al. (1991).
Phylogenetic analyses:—The matrices include nrITS alone, plastid DNA alone and all data combined. Data
analyses including Bayesian inference (BI), maximum-likelihood (ML), maximum-parsimony (MP) methods were
performed as previously described by Zhang et al. (2013).
Results
Morphological analysis:—A detailed comparison of the new species and other similar species in Pleione is presented
in Table 2. Pleione jinhuana is similar to P. bulbocodioides and P. formosana but differs from them in having a lip
with apically erose margin and lateral lobes not enfolding the column (Fig. 1). In addition, the new species can be
distinguished from P. bulbocodioides by having two calluses and from P. formosana by having a conical pseudobulbs
and narrower sepals and petals.

PLEIONE JINHUANA Phytotaxa 345 (1) © 2018 Magnolia Press • 45
FIGURE 1. Pleione jinhuana and morphologically similar species. A. Pleione jinhuana. B. Pleione bulbocodioides. C. Pleione
formosana.

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46 • Phytotaxa 345 (1) © 2018 Magnolia Press
TABLE 2. Morphological comparisons of P. jinhuana, P. bulbocodioides, P. formosana, P. limprichtii and P. pleionoides
Character P. jinhuana P. bulbocodioides P. formosana P. limprichtii P. pleionoides
Pseudobulb 2.3–2.8 × 1.4–1.7 cm 2.0–2.6 × 1.2–2.0 cm 1.3–3.0 × 1.7–3.7 cm 3.0–4.0 × 2.0–2.5 cm 2.5–3.0 × ca. 1.5 cm
Dorsal sepal 4.1–4.5 × 0.5–0.6 cm 3.4–4.5 × 0.5–0.8 cm 4.2–5.7 × 0.9–1.5 cm 3.0–3.5 × 0.5–0.9 cm 4.0–6.5 × 0.6–1.3 cm
Lateral sepal 4.0–4.5 × 0.7–0.9 cm 3.3–4.4 × 0.6–1.0 cm 4.0–5.5 × 1.0–1.5 cm 2.8–3.5 × 0.6–1.0 cm 4.0–6.5 × 0.7–1.4 cm
Petals 4.0–4.8 × 0.4–0.5 cm 3.7–4.6 × 0.4–0.7 cm 4.2–6.0 × 1.0–1.5 cm 3.0–3.5 × 0.4–0.5 cm 4.2–6.4 × 0.5–1.0 cm
Lip 4.1–5.1 × 2.5–3.3 cm 3.2–4.5× 2.5–3.5 cm 4.0–5.5 × 3.2–4.6 cm 2.5–4.0 × 2.5–3.5 cm 4.2–5.5 × 3.5–4.2 cm
Lip shape apical margin erose,
lateral lobes not
enfolding the column
apical margin
lacerate, lateral lobes
enfolding the column
apical margin
lacerate, lateral lobes
enfolding the column
apical margin
lacerate, lateral lobes
enfolding the column
strongly deflexed in
apical 1/3, apical margin
denticulate, lateral lobes
enfolding the column
Callus 2 erose lamellae 4–5 erose lamellae 2–5 entire or erose
lamellae
4 regularly denticulate
or erose lamellae.
2 or 4 denticulate
lamellae
FIGURE 2. Bayesian tree produced by combined matrix of nrITS and plastid DNA. Numbers at nodes are Bayesian posterior probabilities
and bootstrap percentages (ML/MP). Analyses of the portion of the tree containing the new species based on separate nrITS (a) and plastid
DNA (b) data are shown in the top left corner. “-” indicates a node that is inconsistent between the topology of the MP/ML and Bayesian
trees.
Phylogenetic analysis:—The length of the aligned nrITS matrix was 649 bp, of which 98 were variable and 54
were potentially parsimony-informative. For the plastid regions combined, the aligned length was 2,999 positions, of
which 151 were variable and 65 were potentially parsimony-informative. The combined matrix of nrITS and plastid
regions consisted of 3648 characters, of which 249 were variable and 119 were potentially parsimony-informative.
Detailed information for each dataset is listed in Table 3. The phylogenetic relationships determined with the methods
described above are shown in Figure 2. In the combined analysis, P. jinhuana is sister to P. bulbocodioides with strong
support (PP = 1.00, bootstrap percentage, BPML = 96, BPMP = 94).
TABLE 3. Statistics from the phylogenetic analyses.
DNA region No. of taxa Aligned length No. Variable
characters
No. Informative
characters (%)
Tree length Consistency
index
Retention
index
ITS 31 649 98 54 (8.32%) 205 0.86 0.81
matK 31 1667 84 27 (1.62%) 120 0.93 0.89
trnT-trnL 31 564 38 21 (3.72%) 72 0.86 0.87
trnL 31 484 19 12 (2.48%) 34 0.91 0.91
trnL-trnF 31 284 10 5 (1.76%) 16 0.94 0.90
Plastid 31 2999 151 65 (2.17%) 248 0.89 0.86
Combined 31 3648 249 119 (3.26%) 470 0.84 0.79

PLEIONE JINHUANA Phytotaxa 345 (1) © 2018 Magnolia Press • 47
FIGURE 3. Pleione jinhuana. A. Plant with flower. B. Flower, front view. C. Sepal and petal. D. Lip. E. Pollinia. Drawn by P. W. Zhang
from Liu 7084 (the holotype).
Discussion
The phylogenetic analyses show that individuals of P. formosana from Taiwan, Fujian, and Zhejiang cluster together
and are distinct from P. jinhuana, which is found in central Zhejiang Province. A close relationship was found between
P. jinhuana and P. bulbocodioides based on the plastid DNA and the combined DNA matrices. However, the new
species differs from P. bulbocodioides in having two erose calluses (vs. 4–5 calluses), a lip with an erose apical margin
(vs. lacerate apical margin) and lateral lobes of the lip not enfolding the column (vs. lateral lobes of the lip enfolding
the column).
Several authors have treated P. bulbocodioides, P. formosana, P. limprichtii Schlechter (1922: 346) and P.
pleionoides (Kraenzlin) Braem & Mohr (1989: 124) as one variable species due to the minor variation in chromosome
number and morphology (Hunt & Vosa 1971). Gravendeel et al. (2004) supported this treatment because DNA sequence
diversity is low within the P. bulbocodioides complex. However, this treatment has not been widely followed; all these

JIANG ET AL.
48 • Phytotaxa 345 (1) © 2018 Magnolia Press
names are accepted in the World Checklist (Govaerts et al. 2016), and the Royal Horticultural Society continues to
register hybrids between these species, such as P. Fuego (P. formosana × P. bulbocodioides), P. Jorullo (P. limprichtii
× P. bulbocodioides), and P. Versailles (P. formosana × P. limprichtii) (Cribb & Butterfield 1999). The new species
and the four previously described species can be distinguished from each other by morphology, and phylogenetic
analysis of the combined nuclear plus plastid DNA supports the hypothesis that P. jinhuana is a new species.
Relationships among species in this genus have not been well resolved in previous studies. Incomplete lineage
sorting and historical episodes of hybridisation may be invoked to explain this phenomenon. In addition, analysis of
the combined data indicated that P. formosana is divided into Taiwan and Fujian/Zhejiang groups, suggesting that
genetic diversity does exist in these groups. Therefore, more variable molecular markers should be used and population
genetic studies need to be undertaken to clarify the relationships between different species of this genus.
Taxonomy
Pleione jinhuana Z.J.Liu, M.T.Jiang & S.R.Lan, sp. nov. (金华独蒜兰, Figs. 3, 4)
Type:—CHINA. Zhejiang (浙江): Jinhua (金华), on the slopes of evergreen broad-leaved forests, 1100 m, 24 April 2013, Liu 7084
(holotype NOCC!; isotype NOCC!).
FIGURE 4. Pleione jinhuana. A. Plant with flower. B. Flower, front view. C. Flower, side view. D. Flower, back view. E. Anther cap. F.
Pollinia.
This new species is similar to P. bulbocodioides and P. formosana but differs from them by having a lip with an
erose apical margin and lateral lobes of the lip not enfolding the column. In addition, it can be distinguished from P.
bulbocodioides by having two calluses and from P. formosana by having conical pseudobulbs and narrower sepals and
petals.

PLEIONE JINHUANA Phytotaxa 345 (1) © 2018 Magnolia Press • 49
Lithophytic herbs with dark green, ovoid pseudobulbs, 2.3–2.8 × 1.4–1.7 cm, each bearing a single leaf. Leaf
developing at anthesis, elliptic-lanceolate, base attenuate into a petiole-like stalk 1.0–4.0 cm, apex acute. Inflorescence
erect; peduncle ca. 6.0 cm, with 2 sheaths at base; flowers pale purple, with purple marks on the middle and apical
regions of lip; ovary ca. 1.0 cm. Floral bracts narrowly elliptic, ca. 2.0 × 0.5 cm, exceeding ovary, apex acute. Dorsal
sepal spatulate-oblanceolate, 4.1–4.5 × 0.5–0.6 cm, apex acute; lateral sepals narrowly elliptic-oblanceolate, slightly
oblique, 4.0–4.5 × 0.7–0.9 cm, apex subacute. Petals linear-oblanceolate, 4.0–4.8 × 0.4–0.5 mm, apex subacute; lip
broadly elliptic when flattened, 4.0–5.1 × 2.5–3.3 cm, apical margin erose, lateral lobes not enfolding the column,
callus consisting of 2 interrupted lamellae with yellowish, erose margins and purple marks on the middle and apical
regions of lip. Column ca. 3.3 cm, apex obscurely toothed.
Phenology:—Flowering April to May.
Distribution and Habitat:—Pleione jinhuana is lithophytic on the slopes of evergreen broad-leaved forests at
1100 m in Jinhua, Zhejiang Province, China.
Etymology:—The specific epithet refers to the locality where this new species was first found.
Acknowledgements
We would like to thank Hai-jun Yang, Li-Jun Chen, Wen-Hui Rao, Pei-Wen Zhang, Xin-Yi Wu, Jun-wen Zhai and
Qing Xu for their kind help during the preparation of this article. This work was financially supported by the National
Natural Science Foundation of China (31500270), Fujian Agriculture and Forestry University Fund for Distinguished
Youth Scholars (XJQ201620), and Innovation of Characteristic Flower Varieties and Industrialization Project of
Seedling Breeding Facilities in Fujian Province (2014S1477-7).
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