A peer-reviewed open-access journal

PhytoKeys 214: 61—74 (2022)

&
doi: 10.3897/phytokeys.214.94294 46P h y toKe y S

https:/ / Pp hyto keys -pen soft.net Launched to accelerate biodiversity research

A new synonym of Enkianthus perulatus
(Ericaceae) in East Asia, based on
morphological and molecular evidence

Hua Liang', Lu Jiang', Dangi Li'’, Yi Yang'*, Dengmei Fan'?, Zhiyong Zhang!?

| Laboratory of Subtropical Biodiversity, Jiangxi Agricultural University, Nanchang, Jiangxi, China 2 Lushan
Botanical Garden, Chinese Academy of Sciences, Jiujiang, Jiangxi, China 3 Institute of Ecology, Jiangxi
Agricultural University, Nanchang, Jiangxi, China

Corresponding authors: Zhiyong Zhang (zhangzy@jxau.edu.cn), Dengmei Fan (dmf.625@163.com)

Academiceditor: Hugo de Boer | Received 2 September 2022 | Accepted 9 November 2022 | Published 25 November 2022

Citation: Liang H, Jiang L, Li D, Yang Y, Fan D, Zhang Z (2022) A new synonym of Enkianthus perulatus (Ericaceae)
in East Asia, based on morphological and molecular evidence. PhytoKeys 214: 61-74. https://doi.org/10.3897/
phytokeys.214.94294

Abstract

Enkianthus calophyllus was once treated as a synonym of E. serrulatus. However, field observations indicate
that E. calophyllus is distinct from E. serrulatus but resembles E. perulatus in flowers, leaves, fruits and
seeds. Hence, a taxonomic revision of these species was conducted based on morphological comparisons
of flowers, leaves, fruits and seeds, as well as molecular analyses of nuclear ribosomal internal transcribed
spacer (nrITS) and six plastid DNA markers (psbA-trnH, rp/32-trnL, trnL-trn¥, rps16-trnQ, psb)-petA
and matK). The morphological and molecular results reject the synonymization of E. calophyllus with
E. serrulatus, and instead show it to be placed in a clade with E. perulatus. Based on molecular evidence

and a reassessment of the morphology we synonymize E. calophyllus with the older name E. perulatus.

Keywords
Enkianthus calophyllus, Enkianthus perulatus, morphology, phylogeny, synonym

Copyright Hua Liang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

62 Hua Liang et al. / PhytoKeys 214: 61-74 (2022)

Introduction

Enkianthus Lout. is a small genus in Ericaceae with about 12—17 species (Anderberg
1994; Kron et al. 2002; Fang and Stevens 2005). It is only distributed in East Asia,
and most of its component species are in China and Japan. Enkianthus is ornamen-
tally important for its elegant flowers and ecologically valuable because it always
dominates in subtropical montane elfin forests (Hsu 1982). Accumulating molecu-
lar phylogenetic evidence suggests that Enkianthus is the first diverging lineage of
Ericaceae (Kron and Chase 1993; Kron 1996; Morton et al. 1996; Anderberg et al.
2002; Kron et al. 2002), indicating that this genus is key to understand the evolu-
tion of Ericaceae.

Species in Enkianthus are shrubs or small trees, leaves blade serrate or subentire,
inflorescences often umbels and racemes, corollas broadly campanulate to urceolate,
capsule loculicidal, seeds often lamellate-winged (Fang and Stevens 2005). The spe-
cies of Enkianthus vary in leaf texture, inflorescences structure, corolla shape, and
anther morphology, pollen and seed (Cheng and Lai 1988; Anderberg 1994; Kron et
al. 2002; Sarwar and Takahashi 2006). Infrageneric relationships of Enkianthus were
studied by Anderberg (1994), who proposed a classification comprising four sections
(sect. Enkianthus, sect. Andromedina, sect. Enkiantella and sect. Meisteria). Among
them, sect. Enkianthus is monophyletic according to phylogenetic analyses (Tsutsumi
and Hirayama 2012). However, due to the variable morphology in this genus (Hsu
1982), classification of some species, especially those with wide distribution range,
remains controversial.

During the past years, we have found several unique Enkianthus populations in
montane areas of Zhejiang and Jiangxi Province in China (Fig. 1). These plants are
1-3 m tall, white urceolate flowers with distinct basal gibbosities, rhombic-elliptic
leaves and erect capsule. After scrutinizing the protologue and type specimens,
we found that our collections matched the description of E. calophyllus T.Z. Hsu
exactly (Fig. 2, Suppl. material 1: fig. S1B; Hsu 1985). When Fang and Stevens
(2005) treated FE. calophyllus as a synonym of E. serrulatus (E.H. Wils.) C.K.
Schneid. (Fig. 3, Suppl. material 1: fig. S1C) in Flora of China (FOC), flowering
specimens of E. calophyllus were lacking (Fang and Stevens 2005). However, our
collections and £. calophyllus differ from E. serrulatus by urceolate corollas with
distinct basal gibbosities, margin with ciliate, smaller fruits (0.5—0.7 cm x 0.3-0.4
cm) and seeds without distinct wings (Figs 2, 3; Schneider 1911; Hsu 1985), and
such characters were described for E. perulatus C.K. Schneid. from Japan (Fig. 4,
Suppl. material 1: fig. SLA; Schneider 1911). These observations raise a question
about the taxonomic status of £. calophyllus and the identity of our collections.
To identify our new collections and clarify the taxonomic status of FE. calophyllus,
morphological comparisons and molecular phylogenetic analyses were performed to
study the taxonomic relationships amongst E. calophyllus, E. perulatus, E. serrulatus
and our new collections.

Enkianthus calophyllus is conspecific with E. perulatus 63

40°N

35°N

30°N

25°N

110°E 120°E 1305 140°E

Figure |. Distribution of Enkianthus perulatus based on specimen records and our field investigation.
Black triangles indicate EF. perulatus; red triangles indicate E. calophyllus (= E. perulatus).

on

Figure 2. Morphology of Enkianthus calophyllus (= E. perulatus) A flowering branch B fruiting branch
C leaves D flowers E seeds F leaf margin. A-F photographed by H. Liang.

64 Hua Liang et al. / PhytoKeys 214: 61-74 (2022)

Figure 3. Morphology of Enkianthus serrulatus A flowering branch B fruiting branch C leaves D flowers
E seeds F leaf margin. A-F photographed by H. Liang.

Materials and methods

Morphological studies

Our collections and digital images of Enkianthus perulatus, E. calophyllus and
E. serrulatus from the Chinese Virtual Herbarium (http://www.cvh.ac.cn/), the web
of Plants of Taiwan (http://tai2.ntu.edu.tw), the Kingdonia (http://kun.kingdonia.
org/) and the Global Biodiversity Information Facility (https://www.gbif.org/) were
examined to test whether there are significant differences in leaves and fruits of the
three species. The high-resolution images of those Enkianthus species were taken from
specimens deposited at CDBI, CSFI, CSH, GA, GXMI, GZTM, JIU, JXAU, K, KUN,
L, LGB, NAS, NTUE P, PE, SYS, TAI, USE and ZY. We randomly selected three well-
preserved leaves and/or fruits from each specimen (28 specimens of E. serrulatus, 18 of
E. calophyllus and 15 of E. perulatus). Among them, nine specimens of E. calophyllus
and seven of E. serrulatus were collected by ourselves. Eight morphological characters,
i.e., leaf length, leaf width, ratio of leaf length/width, pedicel length, fruit length, fruit
width, ratio of fruit length/width and carpopodium length, were measured in this
study. ImageJ (Rasband 1997) was applied to the examination of the focal characters.

ANOVA was performed to test the significance of pairwise difference of eight
characters using SPSS 26. Principal Component Analysis (PCA) was performed in

Enkianthus calophyllus is conspecific with E. perulatus 65

wal fit
(tis
1 eh

Figure 4. Morphology of Enkianthus perulatus A flowering branch (Hooker 1870) B fruiting branch
(USF 121400) C leaves (K-000780276) D flowers (Hooker 1870) E seeds F leaf margin (K-000780276).
E photographed by H. Liang.

ORIGIN 2021 to investigate the morphological variations among £. perulatus,
E. calophyllus and E. serrulatus. Morphological analysis was not carried out for flowers
and seeds, because there were only a few specimens available for analysis.

Sample collection, DNA extraction, PCR amplification, and sequencing

We collected 19 samples from 13 populations (1-3 individuals per population) of
five Enkianthus species (Suppl. material 1: table $1). Of these, 17 samples belonged
to species of sect. Enkianthus, i.e., six of E. calophyllus, four of E. serrulatus, four of
E. perulatus, and three of E. quinqueflorus Lour. (Suppl. material 1: table S1). Two
species of sect. Enkiantella, E. chinensis Franch. and E. deflexus (Griff.) Schneid. were
also collected. Based on previous studies (Tsutsumi and Hirayama 2012), we down-
loaded nuclear ribosomal internal transcribed spacer (nrITS) and plastid DNA mark-
ers of other Enkianthus species from the National Center for Biotechnology Informa-
tion’s (NCBI; http://www.ncbi.nlm.nih.gov/) nucleotide database (Suppl. material 1:
table S1). In addition, we selected species of genera Rhododendron, Vaccinium and
Clethra as outgroups to carry out the phylogenetic analysis of sect. Enkianthus ac-
cording to previous studies (Kron et al. 2002; Liu et al. 2014). Voucher specimens are
deposited in the Herbarium of Jiangxi Agricultural University (/XAU). Total genomic

66 Hua Liang et al. / PhytoKeys 214: 61-74 (2022)

DNA were extracted from the silica-dried leaves using a modified cetyltrimethylam-
monium bromide method (Doyle and Doyle 1987). Six chloroplast DNA (cpDNA)
regions (psbA-trnH, rpL32-traL, trnL-trn¥, rps16-trnQ, psbJ-petA and matK) (Taberlet
et al. 1991; Sang et al. 1997; Shaw et al. 2007) and nrITS (Sun et al. 1994) in 19 in-
dividuals from 13 populations of Enkianthus, were PCR amplified and sequenced. ‘The
PCR amplification protocols followed Cheng et al. (2021), and primers are listed in
supplementary (Suppl. material 1: table S2). Newly generated sequences in this study
are deposited in GenBank (Suppl. material 1: table S1).

Phylogenetic analysis with cpDNA and nrDNA sequence data

The matrices of DNA sequences were aligned using MAFFT v.7 (Katoh and Stand-
ley 2013), and improved manually using BioEdit 7.0.9 (Hall 1999). Bayesian infer-
ence (BI) and maximum likelihood (ML) were used for phylogenetic analysis on the
CIPRES Science Gateway 3.3 (www.phylo.org; Miller et al. 2015) with the best-fit
model of DNA substitution estimated by jModelTest v.2.1.4 (Darriba et al. 2012). The
alignments of nrITS, mazK and the concatenated plastid DNA (psbA-trnH + rpL32-
trnL + trnL-trnF + rps16-trnQ + psbJ-petA + matK) were analyzed with GTR + G,
GTR and GTR + G + I model, separately. We reconstructed a matK phylogeny of
Enkianthus because the other five chloroplast DNA regions were sequenced only in a
subset of species. Bayesian analysis was constructed using MrBayes v.3.2.7 (Ronquist
et al. 2012). We performed two independent BI runs with one cold and three heated
chains for 10,000,000 Markov chain Monte Carlo generations. We sampled trees every
1,000 generations and discarded the first 25% generations as burn-in. ML analysis was

conducted by RAxML-HPC (Stamatakis 2014) with 1000 bootstrap replications.

Results and discussion

Morphological analyses

Principal Component Analysis (PCA) showed that our collections of Enkianthus calophyllus
clustered with their type specimens and the 95% confidence ellipse of E. calophyllus
intersected marginally that of E. serrulatus, however, the ellipse of E. calophyllus almost
overlapped with that of E. perulatus (Fig. 5). In addition, pairwise comparisons of the
eight morphological traits among E. perulatus, E. calophyllus and E. serrulatus (ANOVA
analysis) showed there were significant differences between E. calophyllus and E. serrulatus
(Fig. 6), but no significant difference between EF. calophyllus and E. perulatus in all
compared traits except for leaf width and ratio of leaf length/width (Fig. 6). Last but
not least, morphological observation also found that £. calophyllus was almost the same
as E. perulatus, but differs from E. serrulatus in flowers and seeds (Table 1). ‘These results
suggest that E. calophyllus should be conspecific with E. perulatus rather than E. serrulatus.
The morphological description and comparison are elaborated in Table 1.

Enkianthus calophyllus is conspecific with E. perulatus 67

A. E. calophyllus
A FE. perulatus
AE serrulatus
O type specimens

PC2 (12.5%)

-4 -2 0 ”) 4 6
PCI (62.7%)

Figure 5. The Principal Component Analysis (PCA) plot for the morphological variations amongst
Enkianthus perulatus, E. calophyllus (= E. perulatus) and E. serrulatus. Red, gray and blue triangles repre-
sent E. perulatus, E. calophyllus and E. serrulatus, respectively. Yellow circles indicate type specimens. The

confidence ellipse level is 95%.

Phylogenetic relationships

Alignment length of nrITS sequences based on 10 species (E. calophyllus = E. perulatus)
of Enkianthus (approx. 83% species of Enkianthus, Fang and Stevens 2005) is 595 bp,
including 75 variable sites and 60 parsimony informative sites. Alignments of matK
consisting of the same 10 species contain 755 constant sites, 24 variable sites and 9 par-
simony informative sites. The concatenated length for six plastid DNA fragments based
on five species of Enkianthus is 4,768 bp, and the matrix contains 124 variable sites and
85 parsimony informative sites in total. Phylogenetic analyses based on nrITS or matk
supported that sect. Enkianthus was a monophyletic clade. Although the nrITS tree
showed that six accessions of E. calophyllus form a monophyletic clade (bootstrap value,
BS = 88, Bayesian posterior probability, PP = 0.86; Fig. 7A), this clade was nested
within E. perulatus (BS = 100, PP = 1; Fig. 7A). Notably, £. calophyllus intermingled
with £. perulatus, forming a highly supported clade in the matK tree (BS = 97, PP =
0.97; Suppl. material 1: fig. S2). Furthermore, the six plastid DNA tree supported that

68

Hua Liang et al. / PhytoKeys 214: 61-74 (2022)

12 5 3.0 3.0
A ; B ae Cc D
b b ;
s b
ca 10 on 4 3 ; ie 2.5 b
S| = = as &
2 & S25] 3 _
— Oo * —
= 8 =e b 0 S 2.0
a) s 5 Ey
= 6 & 3 2
o a oS q4 °
= vo @ 2.0; ‘B
a os, sis dh ear 2 a
=
‘= 2 /| i
au AF AL _ ra
1
EP EC. ES EP EC ES 15" =Pp EC ES
14
b b l¢é
E go) 4 a b
12 S26 te uk &
= E075 £ a €,
= 1.0 = a | 2
§ 2 0.60 ee: §
2 a z 2 ali. 3
E 0.8 ‘i 5 a a 2. 14 — &
= 0.45} 7 6 e
= = ec :
0.30, — ti a
EP EC ES EP EC ES “BP EC ES

Figure 6. Box plots of the character comparisons amongst Enkianthus perulatus (EP), E. calophyllus
(= E. perulatus; EC) and E. serrulatus (ES) A leaf length B leaf width C ratio of leaf length/width D pedi-
cel length E fruit length F fruit width G ratio of fruit length/width H carpopodium length. The different

lowercases on the top of the vertical line indicate significant differences (P < 0.05) between the three taxa.

Table |. Comparisons of morphological characters amongst Enkianthus perulatus, E. calophyllus and

E. serrulatus.

Characters E. perulatus E. calophyllus E. serrulatus
Habit shrubs deciduous, 1—2 m tall | shrubs deciduous, 1—3 m tall shrubs or small tree, deciduous,
3-6 m tall
Texture of leaf Papery Papery papery or thickly papery
Petiole 0.5-1.2 cm 0.5-1.4 cm 0.7—2.1 cm
Leaf blade oblong, obovate-oblong; 2—4 thombic-elliptic, elliptic; elliptic, oblong-elliptic or obovate-
(—5) cm x 0.8—2.0 cm 2.5-5.0 cm x 1.5-3.0 cm__| elliptic; 6-9 (-11) cm x 3-4 (-5) cm
Leaf margin margin with ciliate margin with ciliate margin without ciliate
Inflorescence umbellate, 1—5-flowered umbellate, 1—5-flowered umbellate, 2—6 (—9)-flowered
Corolla urceolate with distinct urceolate with distinct oblong-urceolate without distinct
basal gibbosities; white; basal gibbosities; white; basal gibbosities; greenish-white;
0.6-0.7 cm x 0.5—0.7 cm 0.6—-0.8 cm x 0.5—0.8 cm 1.3-1.6 cm x 0.8-1.0 cm
Length/width 1.0-1.3 1.0-1.4 1.5-2.0
of Corolla
Pedicel 1-2 cm 1.4—2.2 cm 2-3 cm
Fruit capsule erect, oblong, capsule erect, oblong, capsule erect, oblong, 0.8—
0.6-0.7 cm x 0.3—-0.4 cm 0.5—0.7 cm x 0.3-0.4 cm 1.2 cm x 0.5—0.8 cm
Seed without distinct wings without distinct wings with distinct wings
Distribution | Taiwan China, Japan (Honshu,| China (Zhejiang, Fujian, China (Jiangxi, Hubei, Hunan,
Shikoku and Kyushu) Jiangxi) Guangdong, Guangxi, Guizhou,
Chongqing, Sichuan, Yunnan)
Altitude 200-1600 m 600-1200 m 800-1800 m

Enkianthus calophyllus is conspecific with E. perulatus 69

the monophyly of £. perulatus and E. calophyllus was recovered again (BS = 100, PP = 1;
Fig. 7B), and E. calophyllus is paraphyletic with respect to E. perulatus (Fig. 7B). In all
the trees, E. serrulatus clustered with E. qguinqueflorus rather than with E. perulatus.

Taken together, we propose that E. calophyllus should be recognized as a new syno-
nym of £. perulatus rather than the synonym of E. serrulatus as suggested by Fang and
Stevens (2005). In addition, EF. serrulatus together with E. guinqueflorus may represent
a well differentiated lineage relative to E. perulatus.

Taxonomic treatments

Enkianthus perulatus (Miq.) C.K. Schneid

Andromeda perulata Miq., Ann. Mus. Bot. Lugduno-Batavi 1: 31. 1863. Basionym.
Type: Japan. W. Botanicus 57 (holotype: L-0007044!, Suppl. material 1: fig. SLA).

Enkianthus japonicus Hook. f., Bot. Mag. 96: 5822. 1870. Type: Japan. 1860, R. Alcock
sn. (holotype: K-000780276!). = Enkianthus perulatus var. japonicus (Hook. f.)
Nakai, J. Jap. Bot. 12(12): 896. 1936. = Enkianthus perulatus f. japonicus (Hook.
f.) Kitam., Acta Phytotax. Geobot. 25: 36. 1972.

—Enkianthus taiwanianus S.S. Ying, Quart. J. Chin. Forest. 9: 145. 1976. Type: Cura.
Taoyuan City, Peichiatienshan, 1976, Ying 5301 (holotype: NT UF-F00006903!;
isotype: NTUF-F00008372, NTUF-F00008372, NTUF-F00008372, NTUF-
F00008372!). = Enkianthus perulatus var. taiwanianus (S.S. Ying) Y.C. Liu, Tr.
Taiwan 514. 1988.

—Enkianthus calophyllus T.Z. Hsu, Acta Bot. Yunnan. 7(2): 151-152. 1985. syn.
nov. Type: Cuina. Zhejiang: Jingning County, 16 May 1959, S.Y Zhang 5286
(holotype: PE-00005629!, Suppl. material 1: fig. S1B; isotype: NAS-00063024!,
KUN-0001289!).

Description. Deciduous shrubs up to 3 m tall. Branchlets terete, glabrous. Leaves al-
ternate on young shoots, generally 4—6 crowded at apex of branchlets; petioles 0.5—1.4
cm long; blades papery, oblong, obovate-oblong, rhombic-elliptic or elliptic, 2-5 cm
long, 0.8—3.0 cm wide, apex acuminate, mucronate, base attenuate-cuneate or cuneate,
margin ciliate, upper surface glabrous except sparsely pubescent on midrib, lower sur-
face villous along lower part of midrib. Inflorescences terminal, umbellate, 1—5-flow-
ered, pendulous. Pedicels 1.0—2.2 cm long, glabrous, reflexed at flowering, straight at
fruiting. Bracts lanceolate, white or green, 0.8—1.8 cm long, caducous. Calyx lobes
triangular, 2-4 mm long, glabrous, deeply 5-lobed. Corollas urceolate, with distinct
basal gibbosities, white, 6-8 mm long, 5—8 mm wide, shallowly 5-lobcd; lobes widely
ovate, obtuse, much reflexed. Stamens 10, ca. 2/3 as long as corolla; filaments villous
at base, anthers with 2 awns on upper dorsal side. Ovaries glabrous. Capsules erect,
oblong, 5-ridged, 57 mm long, 3—4 mm wide. Seeds compressed, narrowly oblong,
ca. 4mm long, 1 mm wide, with marginal-like ridges, without distinct wings.
Phenology. Flowering from April to May; fruiting from May to November.

70 Hua Liang et al. / PhytoKeys 214: 61-74 (2022)

E. perulatus LC651259
E. perulatus Tok02

E. perulatus JE976288
E. perulatus AB697671
E. perulatus JF976287
E. perulatus Tok01

E. perulatus Toc02
E. perulatus Toc01

E. quinqueflorus WTS03
E. quinqueflorus NKS03
E. quinqueflorus WZS01
E. nudipes AB697669

E. subsessilis AB697670
E. deflexus EMS01

E. chinensis NGSO1

E. stkokianus AB697667
E. cernuus f. rubens LC667414
E. campanulatus KF963947
Vaccinium bracteatum KP092614
Rhododendron simsii KM605741
Clethra cubensis AY190560
Clethra barbinervis AY190573

. perulatus To
E. perulatus Toc01
E. perulatus Tok02
E. perulatus Toc02

0.01

E. quinqueflorus WZS01
E. deflexus EMS01

E. chinensis NGS01
Rhododendron simsii MW030509
Vaccinium bracteatum LC521967
Clethra barbinervis MW801007
Clethra fargesii MT742578

Figure 7. Phylogenetic relationships of Enkianthus A phylogenetic tree based on nuclear DNA loci (ITS)
B phylogenetic tree based on six plastid markers (psbA-trnH, rp/32-trnL, trnL-trn¥, rps16-trnQ, psb)-petA and
matk). Numbers and asterisksa above branches are Maximum Likelihood bootstrap values / Bayesian posterior
probability (> 50%). Asterisks indicate that the support values are 100%. The phylogenetic positions of E. peru-
latus, E. calophyllus (= E. perulatus) and E. serrulatus are highlighted in red, grey and blue, respectively.

Distribution and habitat. Enkianthus perulatus is distributed in China (Zhejiang
Fujian, Jiangxi and Taiwan) and Japan (Honshu, Shikoku and Kyushu) (Fig. 1). It
grows on open rocky slopes, mountain slopes, cliffs, serpentine area, by roadsides or at
forest margins at altitudes of 200-1600 m.

Additional specimens examined. Cuina. Zhejiang: Yueqing City, Yandangshan,
6 April 2015, XY. Ye 2015040609 (CSH); ibidem, 20 May 2019, H. Liang LSBZ-259

Enkianthus calophyllus is conspecific with E. perulatus al

(JXAU). Fujian: Taining County, Xingiao Town, 16 June 1978, G.L. Cai 445 (KUN). Ji-
angxi: Jinggangshan City, Jinggang Mountains, 15 July 1965, S.K. Lai et al. 4466 (LBG);
Lichuan County, Huixianfeng, 20 October 1985, S.K. Lai & D.FE Huang 473 (LBG);
ibidem, 16 November 2021, H. Liang 088 (JXAU). Tatwan: Taibei City, Tunlu, 14 April
1935, 7 Suzuki 19235 (TAI); Taibei City, Lupeishan, 16 April 1991, YB. Cheng &
LS. Hsieh 1202 (TAI); Taoyuan City, Peichatienshan, 28 September 1984, R. 7! Li 3532
(TAI); Chiayi City, Alishan, 5 April 1982, YE Chen 4604 (TAI), ibidem, 17 May 1982,
YE Chen 4902 (TAI). Javan. Honshu: Wakayama-Prefecture, Ohdaigahara Mountains,
23 May 1925, S. Saito (PE); Shizuoka-Prefecture, Tagata-gun, Sanagi Mountains, 24
April 1952, M. Furuse 24733 (PE); Nagano-Prefecture, lida-shi, 27 April 1962, M. Fu-
ruse 39658 (PE); Aichi-Prefecture, Minami-shitara-gun, 22 April 1978, M. Furuse 12532
(PE); Aichi-Prefecture, Shinshiro-shi, 6 August 1978, M/Z. Furuse 13039 (PE).

Additional specimens of Enkianthus serrulatus examined. Cina. Guangxi:
Debao County, 25 April 1977, D. Fang et al. 3-219 (GXMI); Longsheng County,
24 August 2018, H. Liang LSBZ-218 (JXAU); Longlin County, 17 May 1977, 7H.
Wei 3-0606 (GXMI); Xingan County, 26 July 1997, G.Z. Li 15137 (PE). Guizhou:
Chishui City, 24 May 2020, H. Liang LSBZ-297 (JXAU); Leishan County, 13 June
2020, H. Liang LSBZ-323 (JXAU); Songtao County, 22 July 1959, 7’P Zhu et al. 1592
(KUN); Suiyang County, 11 May 2010, YF Zhou KKS101197 (ZY); Zhengan Coun-
ty, 14 October 2014, H.W Zhang 520324141014031LY (GZTM). Hubei: Lichuan
County, 7 October 1980, B. Bartholomew et al. 2014 (PE); Tongshanx County, 14 May
2017, H.Y. Zhan et al. LXP5905 (LBG); Yichang City, 19 March 2017, D.G. Zhang et
al. ZCJ170319117 (JIU). Hunan: Sangzhi County, 4 August 2017, ZY. Zhang et al.
LSBZ-142 (JXAU); Xinning City, 5 July 2017, Z.K. Lin LSBZ-135 (JXAU); Yongshun
County, 3 August 2017, Z. ¥ Zhang et al. LSBZ-138 (JXAU); Zhangjiajie City, 11 Sep-
tember 2015, H. Zhou & D.S. Zhou 15091113 (CSF). Jiangxi: Jinggangshan City, 27
August 2020, YE Liu LSBZ-365 (JXAU); Luxi County, 23 June 1984, MX. Nie 113
(LBG); Suichuan County, 24 June 2016, Z.C. Liu et al. Lxp-13-18312 (SYS); Wuning
County, 22 May 2014, YH. Zhan et al. LXP0912 (LBG). Sichuan: Hechuan City,
1934, D.J. Yu 3112 (PE); Leibo County, 3 July 1983, Q.S. Zhao & Z.J. Zhao 121212
(PE); Xuyong County, 17 September 2013, WB. Ju e» HN. Deng HGX13668 (CDBI).
Yunnan: Maguan County, 31 July 1961, S.G. Wu 3597 (KUN); Suijiang County, 4
May 1973, B.X. Sun 0112 (KUN). Chongqing: Fengjie County, 28 June 1958, MY
Fang 24515 (NAS); Shizhu County, 2 June 1978, WH. Wang 1571 (CDBI).

Acknowledgements

We are grateful to the curators of the herbaria of CDBI, CSFI, CSH, GA, GXMI,
GZTM, JIU, JXAU, K, KUN, L, LGB, NAS, NTUE, P, PE, SYS, TAI, USF and ZY
for access to the specimens for our study. This study is supported by the National
Natural Science Foundation of China (No. 31660059, No. 31960049) and the Train-
ing Plan for Academic and Technical Leaders (leading talents) of major disciplines in

Jiangxi, China (No. 20213BCJ22006).

HD, Hua Liang et al. / PhytoKeys 214: 61-74 (2022)

References

Anderberg AA (1994) Cladistic analysis of Enkianthus with notes on the early
diversification of the Ericaceae. Nordic Journal of Botany 14(4): 385-401. https://doi.
org/10.1111/j.1756-1051.1994.tb00624.x

Anderberg AA, Rydin C, Kallersjo M (2002) Phylogenetic relationships in the order Ericales
s.1.: analyses of molecular data from five genes from the plastid and mitochondrial genomes.
American Journal of Botany 89(4): 677-687. https://doi.org/10.3732/ajb.89.4.677

Cheng ZB, Lai XR (1988) Studies on the pollen morphology and evolution of the
genus Enkianthus. Journal of Sichuan University 25(2): 221-229. https://doi.org/
CNKI:SUN:SCDX.0.1988-02-013 [Natural Science Edition]

Cheng SM, Zeng WD, Wang J, Liu L, Liang H, Kou YX, Wang HC, Fan DM, Zhang ZY
(2021) Species delimitation of Asteropyrum (Rannuculaceae) based on morphological,
molecular and ecological variation. Frontiers in Plant Science 12: 681864. https://doi.
org/10.3389/fpls.2021.681864

Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: More models, new heu-
ristics and parallel computing. Nature Methods 9(8): 772-772. https://doi.org/10.1038/
nmeth.2109

Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tis-
sue. Phytochemical Bulletin 19: 11-15. https://doi.org/10.1016/0031-9422(80)85004-7

Fang RZ, Stevens PF (2005) Enkianthus Lour. In: Wu ZY, Raven PH (Eds) Flora of China (Vol.
14). Science Press, Beijing & Missouri Botanical Garden Press, St. Louis, 243-245.

Hall TA (1999) BioEdit: A user-friendly biological sequence alignment editor and analysis
program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95—98. https://doi.
org/10.1021/bk-1999-0734.ch008

Hooker JD (1870) Japanese Enkianthus. Botanical Magazine 96: 5822.

Hsu TZ (1982) Classification, distribution and phylogeny of the genus Enkianthus. Acta
Botanica Yunnanica 4(4): 355-152.

Hsu TZ (1985) A new species of Enkianthus from China. Acta Botanica Yunnanica 7(2):
151-152.

Katoh K, Standley DM (2013) MAFFT Multiple sequence alignment software version 7:
Improvements in performance and usability. Molecular Biology and Evolution 30(4):
772-780. https://doi.org/10.1093/molbev/mst010

Kron KA (1996) Phylogenetic relationships of Empetraceae, Epacridaceae, Ericaceae, Mono-
tropaceae, and Pyrolaceae: Evidence from nuclear ribosomal 18S sequence data. Annals of
Botany 77(4): 293-303. https://doi.org/10.1006/anbo.1996.0035

Kron KA, Chase MW (1993) Systematics of the Ericaceae, Empetraceae, Epacridaceae and
related taxa based upon rbcL sequence data. Annals of the Missouri Botanical Garden
80(3): 735-741. https://doi.org/10.2307/2399857

Kron KA, Judd WS, Stevens PF, Crayn DM, Anderberg AA, Gadek PA, Quinn CJ, Lute-
yn JL (2002) Phylogenetic classification of Ericaceae: Molecular and morphological

Enkianthus calophyllus is conspecific with E. perulatus 73

evidence. Botanical Review 68(3): 335-423. — https://doi.org/10.1663/0006-
8101(2002)068[0335:PCOEMA]2.0.CO;2

Liu ZW, Jolles DD, Zhou J, Peng H, Milne RI (2014) Multiple origins of circumboreal taxa
in Pyrola (Ericaceae), a group with a Tertiary relict distribution. Annals of Botany 114(8):
1701-1709. https://doi.org/10.1093/aob/mcu198

Miller AM, Schwartz T, Pickett BE, He S, Klem EB, Scheuermann RH, Passarotti M, Kaufman
S, O’Leary MA (2015) A RESTful API for access to phylogenetic tools via the CIPRES
science gateway. Evolutionary Bioinformatics Online 11: 43—48. https://doi.org/10.4137/
EBO.S21501

Morton CM, Chase MW, Kron KA, Swensen SM (1996) A molecular evaluation of the mono-
phyly of the order Ebenales based upon rbcL sequence data. Systematic Botany 21(4):
567-586. https://doi.org/10.2307/2419616

Rasband W (1997) ImageJ. U. S. National Institutes of Health, Bethesda, Maryland, USA.
https://imagej.nih.gov/ ij

Ronquist F, Teslenko M, Mark PVD, Ayres DL, Darling A, Hohna S, Larget B, Liu L, Suchard
MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and
model choice across a large model space. Systematic Biology 61(3): 539-542. https://doi.
org/10.1093/sysbio/sys029

Sang T, Crawford DJ, Stuessy TF (1997) Chloroplast DNA phylogeny, reticulate evolution, and
biogeography of Paeonia (Paeoniaceae). American Journal of Botany 84(8): 1120-1136.
https://doi.org/10.2307/2446155

Sarwar AKMG, Takahashi H (2006) Pollen morphology of Enkianthus Lout.
(Ericaceae) and its taxonomic significance. Grana 45(3): 161-174. https://doi.
org/10.1080/00173130600813345

Schneider CK (1911) Ericaceae. In: Schneider CK (Ed.) Illustriertes Handbuch der Laubholz-
kunde (Vol. 2). Verlag von Gustav Fischer, Jena, 519-523.

Shaw J, Lickey EB, Schilling EE, Small RL (2007) Comparison of whole chloroplast genome se-
quences to choose noncoding regions for phylogenetic studies in angiosperms: ‘The tortoise
and the hare IN. American Journal of Botany 94(3): 275-288. https://doi.org/10.3732/
ajb.94.3.275

Stamatakis A (2014) RAxML version 8: A tool for phylogenetic analysis and post-analysis of
large phylogenies. Bioinformatics 30(9): 1312-1313. https://doi.org/10.1093/bioinfor-
matics/btu033

Sun Y, Skinner DZ, Liang GH, Hulbert SH (1994) Phylogenetic analysis of Sorghum and
related taxa using internal transcribed spacers of nuclear ribosomal DNA. Theoretical and
Applied Genetics 89(1): 26-32. https://doi.org/10.1007/BF00226978

Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal primers for amplification of three
non-coding regions of chloroplast DNA. Plant Molecular Biology 17(5): 1105-1109.
https://doi.org/10.1007/BF00037 152

Tsutsumi C, Hirayama Y (2012) The phylogeny of Japanese Enkianthus species (Ericaceae).
Bulletin of the National Museum of Nature and Science 38(1): 11-17.

74 Hua Liang et al. / PhytoKeys 214: 61-74 (2022)

Supplementary material |

Supplementary data

Authors: Hua Liang, Lu Jiang, Dangi Li, Yi Yang, Dengmei Fan, Zhiyong Zhang

Data type: figures and tables (word document)

Explanation note: Type specimen of Enkianthus perulatus, E. calophyllus and
E. serrulatus. Phylogenetic relationships of Enkianthus based on matK. Sample col-
lection information and GenBank accessions in this study. Sequences of primers
used for PCR amplification and sequencing.

Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODDbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.

Link: https://doi.org/10.3897/phytokeys.214.94294.suppl1