Bull. Natl. Mus. Nat. Sci., Ser. B, 47(2), pp. 71–77, May 21, 2021

Coryphopteris castanea (Thelypteridaceae),a Polymorphic Diploid Species Distributedin Taiwan and Japan
Atsushi Ebihara1, *, Narumi Nakato2, Yi-Hang Chang3 and Li-Yaung Kuo4
1

Department of Botany, National Museum of Nature and Science,
4–1–1 Amakubo, Tsukuba, Ibaraki 305–0005, Japan2
1–363 Narahashi, Higashiyamato 207–0031, Japan
3
Hengchun Research Center, Taiwan Forestry Research Institute, Hengchun,
Pingtung Co. 946005, Taiwan
4
Institute of Molecular & Cellular Biology, National Tsing Hua University, Hsinchu City, Taiwan*E-mail: ebihara@kahaku.go.jp(Received 9 February 2021; accepted 24 March 2021)

Abstract Diploid chromosome number 2n＝62 observed in the Taiwanese populations of Coryphopteris castanea supported the distinctness of this species from tetraploid Thelypteris (Coryphopteris) japonica. Although much variation in frond size was observed across its populations of
Taiwan and Iriomote Island in southern Ryukyu of Japan, their alleles of nuclear gapCp “short”
region formed a monophyletic clade, suggesting that those populations are highly likely to be populations of a single species. Spores of C. castnaea with membraneous ornamentation are apparently similar to those of T. japonica, but they are slightly smaller and more folds and small pits
were observed under the light and scanning electronic microscopes.
Keywords : chromosome number, fern, spore.

Thelypteris castanea (Tagawa) Ching [＝Parathelypteris castanea (Tagawa) Ching] is aterrestrial or epilithic fern species originallydescribed from northern Taiwan (Tagawa, 1935),
and later discovered in Iriomote Island in thesouthern Ryukyu, Japan (Hatusima, 1971; Serizawa, 1975) (Fig. 1). This species, morphologically closest to Thelypteris japonica (Baker)
Ching [＝Parathelypteris japonica (Baker)
Ching] distributed in temperate East Asia (China,
Japan and Korea), is usually distinguished fromT. japonica by its castaneous (vs. reddish purple
or stramineous) stipes of the fronds and denser(vs. sparse) hairs on the frond axis. There is a
conﬂicting opinion that T. castanea should not be
distinguished from T. japonica (e.g. DeVol andKuo, 1975; Kuo, 1985; Yang and Liu, 2002;© 2021 National Museum of Nature and Science

Knapp, 2011).
Nakato and Ebihara (2016) observed the chromosome number of T. castanea collected in Iriomote Island, and the result (2n＝62) suggested
that the Ryukyu population of the species is diploid—cytologically distinct from tetraploid T.
japonica (Nakato et al., 2004). The results ofmolecular phylogeny (Ebihara, 2011; Ebihara etal., 2020) also supported the identity of the
Ryukyu population of T. castanea as being independent from T. japonica. However, we cannot
draw a conclusion about the relationship of thetwo species until Taiwanese populations of T.
castanea are sampled for cytological and molecular phylogenetic studies. In fact, frond sizes of
the Ryukyu populations tend to be smaller thanthose of Taiwanese populations (Serizawa, 1975)(Figs. 1b, 1d). Thus, a careful study on infraspeciﬁc variation of T. castanea is necessary.

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Atsushi Ebihara et al.

Fig. 1. Coryphopteris castanea. a. Sihyuan Yakou, Taichung City, Taiwan; b. Iriomote Island, Japan; c. a specimen with normal-sized fronds collected from Iriomote Island (TNS VS-517507); d. a specimen with smaller
fronds collected from Iriomote Island (TNS VS-1027975).

Coryphopteris castanea in Taiwan and Japan

Materials and Methods
A total of ten living stocks from two locationsof Taiwan (the northern north-central parts) werecollected and used for chromosome count andmolecular analysis (Table 1). Chromosome number was counted in the metaphase of root tip cells
using the same methods as in Ebihara et al.
(2014). Plastid rbcL sequences and nuclear
gapCp “short” sequences (Schuettpelz et al.
2008) were obtained, following the methods usedin Ebihara et al. (2010, 2020). For the gapCp“short” sequences, molecular phylogenetic analysis was performed under the same methods as inEbihara et al. (2020) combining newly generated

73

sequences (GenBank accession numbers arelisted in Table 2) and the dataset used by thatstudy. Frond and spore sizes of T. castanea were
measured in a total of 21 specimens (8 fromJapan and 13 from Taiwan) and 18 specimens (8from Japan and 10 from Taiwan), respectively.
All of the specimens are deposited in the herbarium, National Museum of Nature and Science(TNS). For measuring frond sizes, one mature
frond was selected from each herbarium sheet.
For spore sizes, 20 spores derived from multiple
sporangia were selected and the length along thelong axis excluding perispore was measured.
Spores of a Taiwanese individual (Chang
20120707-011 [TAIF], Mt. Chitan, Dist. Xindian,

Table 1. The material of Coryphopteris castanea used in the present study
Locality & Coll. No.
Taiwan, Taipei city, Beitou district, Zhuzihu
Ebihara & Kuo 3136
Ebihara & Kuo 3137
Ebihara & Kuo 3138
Ebihara & Kuo 3139
Taiwan, Taichung city, Sihyuan Yakou
Ebihara & Kuo 3113
Ebihara & Kuo 3114
Ebihara & Kuo 3115
Ebihara & Kuo 3116
Ebihara & Kuo 3117
Ebihara & Kuo 3118
Japan, Okinawa Pref., Iriomote Island
Ebihara 060930-01

Voucher specimen[TNS VS-]

Chromosome
numbergapCp
genotype

Spore size
(μm)

1170755
11707541170753
1170752

2n＝62 (2x)2n＝62 (2x)2n＝62 (2x)
—

FK
II
IJ
IL

—
—
35.3±2.6—

1170785
1170784
1170783
1170782
11707811170778

2n＝62 (2x)2n＝62 (2x)2n＝62 (2x)2n＝62 (2x)2n＝62 (2x)
—

FF
HH
FH
FF*1
II
FF*1

36.4±2.1
36.1±2.1
36.6±2.038.5±1.935.8±2.036.9±2.3

759279—GG*2—

The following specimens were used only for morphological observation and measurements.
Taiwan, Datunshan
Sasaki s.n.
Taiwan, Yangmingshan
Masuda 8354
Taiwan, Chihsingshan
Liew 1190
Japan, Okinawa Pref., Iriomote Island
Shimabuku 999
Takara & Takara s.n.
Yasuda 3317
Kokubugata & al. 5441
Sugasawa s.n.
Ebihara 3177
Shimabuku 1787
Shimabuku 1790

387481——38.1±2.4

9539122——38.6±2.5

446647——38.6±1.7

1327691
533125102797595188966012551176780
517507533124—————2n＝62 (2x)*3
—
—

—
—
—
—
—
—
—
—

36.9±2.0
39.6±2.3
40.0±2.040.5±2.540.6±2.541.6±3.0
42.0±3.0
39.3±2.4*1 Alleles identiﬁed by a comparison of banding patterns of a SSCP gel.
*2 Ebihara et al. (2020).
*3 Nakato & Ebihara (2016).

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Atsushi Ebihara et al.
Table 2. GenBank accession numbers of gapCp“Short” sequences
Collection No.

AlleleGenBank accession
No.

Ebihara & Kuo 3136
Ebihara & Kuo 3136
Ebihara & Kuo 3137
Ebihara & Kuo 3138
Ebihara & Kuo 3138
Ebihara & Kuo 3139
Ebihara & Kuo 3139
Ebihara & Kuo 3113
Ebihara & Kuo 3114
Ebihara & Kuo 3115
Ebihara & Kuo 3115
Ebihara & Kuo 3117

F
K
I
I
J
I
L
F
H
F
H
I

LC605144
LC605145
LC605146
LC605147
LC605148
LC605149
LC605150
LC605151
LC605152
LC605153
LC605154
LC605155

New Taipei City, 7 July, 2012) were also
observed by Miniscope TM3000 scanning electronic microscope (SEM) (Hitachi High-Tech,
Tokyo).
Results
The diploid chromosome number, 2n＝62 was
successfully observed in eight of the ten living
samples (Fig. 2 shows one example; others weresimilar). Plastid rbcL sequences of the ten samples from Taiwan completely match that of Japan(GenBank accession AB575014). For nuclear
gapCp “short” sequences, a total of seven alleles(Tables 1, 2) were identiﬁed, and all the alleles ofthe samples from T. castanea of Taiwan andJapan formed a robustly supported monophyleticclade in the tree (Fig. 3). Frond sizes (lamina
length and width) were plotted in Fig. 5. Approximately 64 or more than 32 spores were observed
per sporangium. The spores are bilateral, and thesize (long axis) is 38.8±3.0 μm. Under a lightmicroscope, the perispore looks membraneous(Fig. 4a–c), and numerous folds and pits can beseen on the membraneous wings when observed
using an electron microscope (Fig. 4d).
Discussion
Our results of gapCp phylogeny and chromosome counts supported that both Taiwanese andJapanese populations of T. castanea are closely

Fig. 2. Microphotographs of mitotic metaphase
chromosomes of Coryphopteris castanea (Ebihara & Kuo 3117, Taiwan). 2n＝62. Scalebar＝10 μm.

related to each other and that they most likely
belong to a single species. The results also suggested that T. castanea is a diploid species, andthat it is not a progenitor of T. japonica, which isprobably allotetraploid. Therefore, T. japonica
should be excluded from the ﬂora of Taiwan as
was done by Taiwan Pteridophyte Group checklist (TPG) (2019, 2020).
It is interesting that relatively high allelic variation was detected in the gapCp “short” region of
T. castanea collected only in three locations incontrast to poor or almost no infraspeciﬁc variation in T. japonica, T. musashiensis, T. nipponica
and T. sylva-nipponica. (Ebihara et al., 2020).
The species is also polymorphic in frond size—
populations in Japan tend to have smaller fronds
than those in Taiwan (Fig. 5). It is still unclear
whether or not the frond size variation depends
on environmental conditions of the habitat. We

Coryphopteris castanea in Taiwan and Japan75

Fig. 3. A majority-rule consensus tree of nuclear gapCp “Short” region of Coryphopteris castanea and related
species inferred by the Bayesian method rooted with Parathelypteris cystopteroides. Support values of nodes:
posterior probabilities of Bayesian inference (＞0.80 are shown)/bootstrap values (%) in maximum likelihood
analysis (＞50% are shown).

noticed a specimen, Shimabuku 1787 TNS (VS517507) collected from Mt. Komidake, Iriomote
Island, which bears exceptionally large (＞20 cmlong) lamina among specimens collected inJapan. Although we have not yet succeeded
rediscovering any similar “large” individuals
there, it would be worth examining both morphological and genetic variation of the species in
Iriomote Island more carefully.
Spore morphology is often an important character for identifying species belonging to Coryphopteris sensu Fawcett et al. (in revision,see Taxonomic Treatment). In the case of T.
japonica and its closely related species, the regularity of spore shape is useful for recognizing
interspeciﬁc hybrids, which are frequently produced, and spore ornamentation pattern is themost reliable character for distinguishing diploid
T. musashinesis from tetraploid T. japonica(Nakato et al., 2004). According to our present
observation, the spore size of T. castanea(38.8±3.0 μm) tends to be slightly smaller thanT. japonica (44.7±3.2 μm, Nakato et al., 2004)
and T. musashiensis (44.4±3.8 μm, Nakato etal., 2004). The spore ornamentation of T. castanea is apparently similar to that of T. japonica inthat it has a membraneous or alate appearance
under a light microscope, though folds and small
pits are more frequently observed than in T.
japonica. The pits are more distinct in the spore
image by SEM (Fig. 4d). However, there is a certain degree of infraspeciﬁc variation in the spore
morphology of T. castanea. The spores on a
specimen collected in Taiwan (Liew 1190, TNS
446647, Fig. 4c) showed more or less echinate
perispore, an intermediate feature between T.
japonica and T. musashiensis.
In conclusion, T. castanea is a phylogeneticallydistinct diploid species distributed in Taiwan and
Iriomote Island in Japan. In spite of its restricteddistribution range, the species possesses abundantmorphological and genetic variation.

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Atsushi Ebihara et al.

Fig. 4. Spores of Coryphopteris castanea. a. Ebihara & Kuo 3116, Taiwan; b. Ebihara 3179, Japan; c. TNS
VS-446647, Taiwan; d. Chang 20120707-011, Taiwan. SEM image. Scale bar: 10 μm.

sion) proposed a generic revision based on an
updated global phylogeny of the family. They
subdivided Parathelypteris sensu PPG1 into twogenera Parathelypteris sensu stricto and Coryphopteris, classifying T. castanea, T. japonica, T.
nipponica and related species into the latter
genus. Taiwan Pteridophyte Group (TPG) (2019)
proposed a new generic combination “Coryphopteris castanea (Tagawa) Y.H.Chang”, but the
publication without citing the basionym did notmeet ICN Art. 41.1 (Turland, 2018), hence thisname has not been validly published.
Fig. 5. A plot diagram of lamina sizes of Coryphopteris castanea.

Taxonomic treatment
The generic classiﬁcation of Thelypteridaceae
accepted in PPG1 (Pteridophyte Phylogeny
Group, 2016) was premature, particularly forseveral polyphyletic genera including Parathelypteris, and more recently Fawcett et al. (in revi-

Coryphopteris castanea (Tagawa) Y.H.Chang,
A.Ebihara & L.Y.Kuo, comb. nov. —Dryopteris
castanea Tagawa, Acta Phytotax. Geobot. 4(3):132, 1935 —Thelypteris castanea (Tagawa)Ching, Bull. Fan Mem. Inst. Biol., Bot. 6(5): 315,1936 —Parathelypteris castanea (Tagawa) Ching,
Acta Phytotax. Sin. 8(4): 302, 1963.
Type: Taiwan, Taipei city, Beitou District, Zhuzihu ʻFormosa, prov. Taihoku, Sitiseigun,

Coryphopteris castanea in Taiwan and Japan

Tikusikoʼ, Tagawa 616 (holo KYO; iso BM [2sheets], MICH)
Acknowledgments
The authors thank K. Uno and C. Takaboshi
for their assistance in DNA sequencing. This
study was partly supported by JSPS KAKENHI(Grant Nos. 15K07204 and 19H03288 to AE).
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