allopolyploid origin and distribution range of

Two species of Acystopteris have been record-ed in Japan (Kato 1995). Acystopteris japonica has been widely recorded from Honshu (exclud-ing the northernmost part), Shikoku and Kyushu (Kurata & Nakaike 1987), while A. tenuisecta is confined to the mountains of Yakushima. In 2008, an unknown plant of Acystopteris was col-lected in subalpine forests in Gifu Prefecture, the central part of Honshu, Japan. The plant differed from A. japonica, the sole species of Acystopteris on Honshu, in having a pale green stipe and ra-chis. We carefully compared it with other species of Acystopteris from various localities in Japan and Taiwan using morphology, cytology and DNA markers to try to identify it.

Materials and Methods

Plant materialsWe compared the unknown plant with all the

specimens of Acystopteris in TNS and TAIF. For

Acystopteris Nakai (Cystopteridaceae) is a genus of cystopteroid ferns comprising a few spe-cies in Asia (Kramer & Green 1990, Rothfels et al. 2012, PPG I 2016). According to the plastid phylogeny of the family (Rothfels et al. 2013), the members of the genus form a robust clade placed sister to Cystopteris. Acystopteris japonica (Lu-erss.) Nakai, the type species of the genus, occurs in China and Japan. A second species, A. tenui-secta (Blume) Tagawa, has a much wider distri-bution in eastern, southeastern and southern Asia (Wang et al. 2013). A third species, A. taiwaniana (Tagawa) Á. et D. Löve, which is only on Taiwan (Wang et al. 2013), was originally recognized as a variety of A. japonica (Tagawa 1935) and charac-terized by glandular hairs on the indusia. It is sometimes not distinguished from A. japonica (e.g. Shieh et al. 1994). A study by Rothfels et al. (2017) using PacBio single-molecule sequencing suggested an allopolyploid origin of A. taiwani-ana, but the results conflict with the report of a diploid cytotype by Tsai (1992).

Acta Phytotax. Geobot. 70 (1): 19–28 (2019)doi: 10.18942/apg.201812

ISSN 1346-7565

Allopolyploid Origin and Distribution Range of Acystopteris taiwaniana (Cystopteridaceae: Polypodiales)

aTsushi ebihara1,*, naruMi nakaTo2, li-Yaung kuo3, hiroki MiYazaki4

and shunsuke serizawa5

1Department of Botany, National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba, Ibaraki 305-0005, Japan. * [email protected] (author for correspondence); 2Narahashi 1–363, Higashiyamato, Tokyo 207-0031, Japan; 3Boyce Thompson Institute, Ithaca, New York 14853, USA; 4Ama, Aichi 490-1200, Japan; 5Aichi Green Association,

Nagamakiurahata 198-1, Oharu-cho, Ama-gun, Aichi 490-1131, Japan

Acystopteris taiwaniana, formerly known only from Taiwan, is newly recorded in Japan (northern and central Honshu and Shikoku). Chromosome counts and DNA sequences (plastid rbcL and nuclear PgiC) suggested that it is an allotetraploid species originating from hybridization between diploid A. japonica and an unknown diploid species. Acystopteris taiwaniana differs from A. japonica in having glandular hairs on the indusia of the sori, paler stipes and rachises, wider angles of divergence of pinnae from the rachis and larger spores, although there overlaps in the variation. Triploid sterile hybrids between the two species occur occasionally at the periphery of populations of one of the parental species in Japan.

Key words: Acystopteris, allotetraploid, hybrid, Japan, Taiwan

20 Vol. 70Acta Phytotax. Geobot.

Table 1. Specimens of Acystopteris used in this study, and results of chromosome counts and DNA sequences. PgiC alleles in parentheses are those estimated from SSCP banding patterns.

Collection No. (Voucher) Locality Chromosome number

rbcL haplotype

PgiC allele

PgiC genotype

Acystopteris japonica Ebihara 3609 (TNS VS-1286259) Japan. Yamagata Pref., Nishiokitama-gun,

Oguni-machi, Iwanasawa, alt. 400–500 m2n = ca. 84, 2x I A1 A1A1

Ebihara 3619 (TNS VS-1286270) Japan. Tochigi Pref., Kanuma-shi, Ozaku-san, alt. 470 m

2n = ca. 84, 2x I A1 A1A1

Ebihara 3620 (TNS VS-1286271) Ibid. 2n = 84, 2x - - -

Ebihara 060728-01 (TNS VS-763998)

Japan. Tokyo Pref., Nishitama-gun, Okutama-machi, Koizawa

unknown I (A1) A1A1?

Fujimoto 070612-1(TNS VS-765126)

Japan. Saitama Pref., Iruma-gun, Naguri-mura, Shirayasawa

unknown I A1 A1A1?

Nakato 3181 (TNS VS-1265723) Japan. Aichi Pref., Kitashitara-gun, Shitara-cho, Takouzu River

2n = 84, 2x - - -

Nakato 3182 (TNS VS-1265717) Ibid. 2n = 84, 2x (Figs. 1A, B)

I A1 A1A1

Y.C. Liu 9660 (TAIF 501123) China. Sichuan Prov., Yibin Co. unknown I A1/A2 A1A2

Kuo 783 (TAIF 502535) China. Hunan Prov., Tongdao Dong Autonomous Co.

unknown I A3 A3A3?

Acystopteris taiwanianaNakato & Ebihara 3008 (TNS VS-1222049)

Japan. Fukushima Pref., Aizuwakamatsu-shi, Kuwabara, alt. 420 m

2n = 168, 4x (Figs. 1G, H)

III A1/C A1A1CC

Ebihara 2614 (TNS VS-1112436) Japan. Fukushima Pref., Kitakata-shi, foot of Mt. Azuma, alt. 860 m

unknown III (A1C) A1A1CC?

Ebihara 2623 (TNS VS-1112430) Japan. Fukushima Pref., Minamiaizu-gun, Minamiaizu-machi, Aoyagi, alt. 850 m

unknown III A1/C A1A1CC?

Ebihara 2415 (TNS VS-1107846) Japan. Gunma Pref., Agatsuma-gun, Nakanojo-machi, Shima, alt. 850 m


Serizawa89669-2 (AICH; TNS) Japan. Nagano Pref., Shimoina-gun, Oshika-mura, Kashio, alt. 1,750 m


Ebihara 3329 (TNS VS-1200843) Japan. Gifu Pref., Takayama-shi, Hirayu, alt. 1,520 m

2n = 168, 4x III A1/C A1A1CC

Serizawa 90648-2(TNS VS-1265719)

Japan. Gifu Pref., Takayama-shi, Hirayu Pass, alt. 1,500 m

2n = ca. 168, 4x III A1/C A1A1CC

Ebihara 1985 (TNS VS-776951) Japan. Kochi Pref., Agawa-gun, Ino-ma-chi, Mt. Tsutsujo, alt. 1,750 m


Ebihara 1885 (TNS VS-776530) Taiwan. Ilan Co., Mt. Taiping-shan, alt. 1,960 m

unknown III (A1C) A1A1CC?

Kuo 175 (TAIF) Taiwan. Chiai Co., Mt. Alishan unknown III (A1C) A1A1CC?Ebihara 3390 (TNS VS-1222044) Taiwan. Nantou Co., Tefuye, alt. 2,160 m 2n = 168, 4x

(Figs. 1E, F)III A1/C A1A1CC

Ebihara 3392 (TNS VS-1222043) Ibid. 2n = ca. 168, 4x III (A1C) A1A1CCEbihara 3397 (TNS VS-1219682) Ibid. 2n = ca. 168, 4x III A1/C A1A1CCEbihara 3410 (TNS VS-1219673) Taiwan. Chiayi Co., Mt. Tashan, alt. 2,330 m 2n = ca. 168, 4x III (A1C) A1A1CCEbihara 3418 (TNS VS-1222038) Taiwan. Chiayi Co., Mt. Tashan, alt. 2,460 m 2n = 168, 4x - (A1C) A1A1CCEbihara 3420 (TNS VS-1222050) Ibid. 2n = ca. 168, 4x III A1/C A1A1CCLu 26851 (TNS VS-1222058) Taiwan. Hualien Co., Hehuan Valley 2n = ca. 168, 4x III (A1C) A1A1CCKuo 3945 (TNS VS-1222046) Taiwan. Nantou Co., Mt. Yushan, alt.

2,500 m2n = ca. 168, 4x III - -

Acystopteris japonica × A. taiwanianaEbihara 3621 (TNS VS-1286272) Japan. Tochigi Pref., Kanuma-shi,

Ozaku-san, alt. 400 m2n = 126, 3x (Figs. 1I, J)

III A1/C A1A1C

Serizawa 90649-3(TNS VS-1265721)

Japan. Gifu Pref., Takayama-shi, Hirayu Pass, alt. 1,500 m

2n = 126, 3x III A1/(C) A1A1C

Acystopteris tenuisectaTagane & Fuse 113 (TNS VS-763154)

Japan. Kagoshima Pref., Yakushima Isl. unknown II B BB?

F.-W. Li 887 (TAIF 370966) Taiwan. Taipei Co., Sunglo Lake unknown II (B) BB?Ebihara 3404 (TNS VS-1222036) Taiwan. Nantou Co., Tefuye, alt. 2,280 m 2n = 84, 2x II B BBEbihara 3412 (TNS VS-1222034) Taiwan. Chiayi Co., Mt. Tashan, alt. 2,330 m 2n = 84, 2x - B BBKuo 3947 (TNS VS-1222035) Taiwan. Nantou Co., Mt. Yushan, alt.

2,500 m2n = 84, 2x (Figs. 1C-D)

II B BB

February 2019 21ebihara & al. — Allopolyploids of Acystopteris taiwaniana

DNA analysis we collected 32 samples, including living stocks and silica-dried leaves, from Japan, Taiwan and China (Table 1). The living stocks were also used for chromosome counts. Voucher specimens were deposited in the herbaria TNS, TAIF or AICH (Table 1).

Chromosome countsThe chromosomes at metaphase of mitosis in

the root tips of living stocks were counted follow-ing the method of Ebihara et al. (2014b).

DNA sequencingAll leaf material for DNA sequencing was

dried in advance in plastic bags with silica gel. DNA was extracted using the DNeasy Plant Mini kit (Qiagen, Hilden, Germany). For the chloro-plast rbcL region, the methods of amplification and sequencing followed Ebihara et al. (2010). For the nuclear PgiC region, fragments including an exon and two introns were amplified using SapphireAmp Fast PCR Master Mix (Takara Bio, Shiga, Japan) with primers 14F and 16R (Ishika-wa et al. 2002) by 35 cycles of 98 ˚C (5 sec.)—53 ˚C (5 sec.)—72 ˚C (5 sec.). When possible, se-quence polymorphisms were detected by the SSCP method [general method following Ebihara et al. (2005); electrophoresis for 17 h at 17.0 ˚C in MDE gels containing 2% glycerol]. After silver staining, each band was cut out. The small gel blocks were kept at -80 ˚C for 2h, after which 20 μl of sterile water was added. After keeping them again at -80 ˚C for 2 h, they were brought back to room temperature for DNA elution. The second amplification was performed using the eluted DNA as a template under the same cycle condi-tions and with the same primers as in the first re-action, followed by sequencing as in the rbcL re-gion. In some samples, PgiC alleles were identi-fied by comparing band positions on the SSCP gels.

Spore size measurementsSpores were collected and mounted on glass

slides with Bioleit (Ohken Co., Tokyo, Japan). The long diameter of the spores, excluding the perispore, was measured under a light micro-

scope in both the voucher specimens for chromo-some observations and in specimens deposited in herbaria. Ten spores from each of the voucher specimens were measured.

Results

Chromosome countsChromosome counts were successful in 21

samples (Table 1, Fig. 1). From the assumed base number of x = 42 (Takamiya 1996, Rothfels et al. 2012), diploid (2n = 84), triploid (2n = 126) and tetraploid (2n = 168) were found. Individuals identified from their morphology as Acystopteris japonica (from Japan) and A. tenuisecta (from Taiwan) were principally diploid. In contrast, both A. taiwaniana from Taiwan and the uniden-tified plants from Japan were tetraploid. Addi-tionally, two triploid individuals were found in Japan—one growing with A. japonica and the other at the periphery of a population of the un-identified species.

DNA sequencesIn the 1,205 bp of chloroplast rbcL sequences,

only three haplotypes (named I, II and III) were found among the 30 samples examined (Table 1). Haplotypes I (corresponding to GenBank acces-sion No. AB574893) and II (corresponding to GenBank accession No. AB574894) were found in Acystopteris japonica and in A. tenuisecta, re-spectively. Haplotype III (corresponding to Gen-Bank accession No. JF832052) was not found in diploid plants, but was exclusively in the poly-ploids (triploid and tetraploid) in the plants we ex-amined. The sequence length varied from 670 to 672 bp in the nuclear PgiC region, except in in-complete sequences. Five different sequences [named A1 (GenBank accession No. LC390211/LC390215), A2 (LC390212), A3 (LC390213), B (LC390214) and C (LC390216)] were found. The A1, B and C sequences completely matched ac-cession numbers KX376729, KX376732 and KX376730 of Rothfels et al. (2017) deposited in GenBank. The alleles A2 and A3 did not match


Fig. 1. Mitotic metaphase chromosomes in species of Acystopteris. A, B. Acystopteris japonica (Nakato 3182, 2n = 84, dip-loid). C, D. A. tenuisecta (Kuo 3947, 2n = 84, diploid). E, F. A. taiwaniana from Taiwan (Ebihara 3390, tetraploid, 2n = 168). G, H. A. taiwaniana from Japan (Nakato & Ebihara 3008, tetraploid, 2n = 168). I, J. Hybrid between A. japonica and A. taiwaniana (Ebihara 3621, triploid, 2n = 126). A, C, E, G, I: microphotographs, B, D, F, H, J: explanatory illustrations. Scale bar = 10 μm.


any previously deposited sequences. Of the 31 samples, 11 exhibited a single allele each; the re-maining 20 exhibited two alleles each in a single individual. Multiple alleles in a single individual were isolated and sequenced by cutting out SSCP gels for 13 samples, then identifying them by comparison with the banding patterns on SSCP gels for the remaining ones. Diploid individuals confirmed by chromosome observations always had a single allele, while polyploids always had two different alleles. Acystopteris taiwaniana and the unidentified plants in Japan have the same two alleles, and one of which (A1) was the major allele in A. japonica.

Spore size measurementsSpore size was successfully measured in 17

voucher specimens used for chromosome counts. The longer diameter of the spore grains ranged from 25 to 35 μm in diploid and 30 to 40 μm in tetraploid individuals (Fig. 2). Additionally, ir-regularly shaped spores were observed in two triploid individuals as well as in several herbari-um specimens.

Discussion

Variations in the cytotypes (diploid, triploid and tetraploid) of Acystopteris corresponds well with morphological characters; i.e. diploid cyto-types were in A. tenuisecta and in typical indi-viduals of A. japonica; tetraploids were in A. tai-waniana and the unknown plants in Japan. Thus, A. taiwaniana from Taiwan was clearly distin-guishable from the diploid A. japonica. The treat-ment of A. taiwaniana as a distinct species was supported by our findings. Triploids, so far known only from Japan, are morphologically in-termediate between A. japonica and the unknown plant and produce irregularly shaped spores.

In both the chloroplast rbcL haplotypes and the nuclear PgiC genotypes, no differences were found between the unidentified tetraploid plant from Japan and Acystopteris taiwaniana of Tai-wan (Table 1). After confirming their morpholog-ical similarity, we concluded that the unidentified

plant in Japan represents a range extension of A. taiwaniana [although a preliminary report on the occurrence of A. taiwaniana in Japan was in Ebi-hara (2016). It is reported here for the first time accompanied by evidences]. The PgiC genotypes suggested that A. taiwaniana is probably an allo-tetraploid species derived from hybridization be-tween diploid A. japonica and an unknown dip-loid species and subsequent chromosome dou-bling. Because of the seven bp differences in the 1,205 bp sequence of the rbcL genes of A. japon-ica and A. taiwaniana, the unknown progenitor species is presumably the maternal progenitor, since the chloroplast genome has been found to be only maternally inherited in ferns (reviewed in Kuo et al. 2018). A comparison of our PgiC al-leles with those generated by Rothfels et al. (2017) suggested that the unknown diploid also served as one of the two progenitors of the tetra-ploid A. tenuisecta in Java. Both the molecular (Rothfels et al. 2017) and cytological evidence [e.g. diploids in Taiwan (Ebihara et al. 2014a, present study) and tetraploids and sterile triploids (Bir 1971)] suggest that A. tenuisecta is likely a species complex in need of geographically denser sampling. Considering the wide distribution of A. tenuisecta, unsampled infraspecific genetic variation and cryptic species might include the unknown maternal progenitor of A. taiwaniana.

We also found that several individuals in northern and central Honshu, Japan, formerly identified as Acystopteris japonica are the tetra-ploid A. taiwaniana. Additional occurrences in Honshu and Shikoku were suggested by the mor-phology of herbarium specimens (Fig. 3). Acystopteris taiwaniana in Japan generally oc-curs at higher elevations than A. japonica (rang-ing from 400 to 1,800 m), but with some overlap. Acystopteris japonica grows at ca. 400 m eleva-tion in Yamagata Prefecture and A. taiwaniana grows at nearly the same elevation in Fukushima Prefecture, both at almost the same latitude. Even though we have not discovered sympatric occur-rences of A. japonica and A. taiwaniana, triploid hybrids occasionally occur on the periphery of the range of one of the parental species in Hons-hu. We did not find evidence of A. japonica and


the hybrid in Taiwan based on specimens from Taiwan in TNS and TAIF.

Due to the triploid hybrid individuals, dis-crimination of Acystopteris taiwaniana from A. japonica based solely on gross morphology is not always easy (Figs. 4, 5). The recommended pro-cedures for separating the two species and their hybrid are: 1) observe mature spores; the triploid hybrid produces irregularly shaped spores; 2) ob-serve characters and characteristics in Table 2, but it should be noted that the stipe and rachis gradually darken with age and that the indusia are usually obscure after mid summer. Spore size (longer diameter) appears to be largely reflect the ploidy level, but because of the broad range over-lap, it cannot be used as the sole key characteris-tic.

Taxonomic treatment

Acystopteris japonica (Luerss.) Nakai, in Bot.

Mag. (Tokyo) 47: 180 (1933).—Cystopteris japonica Luerss., in Bot. Jahrb. Syst. 4: 363 (1883).

Japanese name. Usuhimewarabi Distribution. Japan [Honshu (north to

Yamagata and Miyagi prefectures), Shikoku, Ky-ushu and Yakushima] and S. China.

Acystopteris taiwaniana (Tagawa) Á. et D.Löve, in Taxon 26: 326 (1977).—Cystopteris japonica Luerss. var. taiwaniana Tagawa, in Acta Phytotax. Geobot. 4: 57 (1935).

Japanese name. Usuhimewarabi-modoki.Distribution. Japan (Honshu and Shikoku)

and Taiwan.

Representative specimens examined (excluding those from Taiwan). Japan, Akita Pref., Yuri-gun, Kisakata-machi, Daihango, Nov. 29, 1990, Y. Horii s.n. (TNS VS-795509); Fukushima Pref., Yama-gun, Atsushiokano-mu-ra, Nicchu, Sept. 8, 1981, Z. Kaneda s.n. (TNS VS-458023); Kitakata-shi, Iwatsukimachi, Jul. 6, 1980, Z. Kaneda 5915 (TNS VS-458024); Kitakata-shi, foot of Mt.

Ebihara 3609Ebihara 3619

Ebihara 3620Nakato 3181

Nakato 3182Nakato & Ebihara 3008

Serizawa 90648-2Ebihara 3390



Ebihara 3420Lu 26851

Kuo 3945Ebihara 3412

Kuo 3947

2530

3540

Long

er d

iam

eter

(μm

)

A. japonica(diploid)

A. taiwaniana(tetraploid)

A. tenuisecta(diploid)

Fig. 2. Box plots of spore size (longer diameter excluding perispore) of Acystopteris based on ten spores from each specimen drawn by R 3.3.0 with default settings. Boxes are located between higher and lower quartile values. Thick horizontal lines show median values; points are outliers.


Azuma, alt. 860 m, Jul. 26, 2010, (TNS VS-11124361); Ai-zuwakamatsu-shi, Kuwabara, alt. 420 m, Aug. 6, 2014, A. Ebihara & N. Nakato 3008 (TNS VS-1222049); Minami-aizu-gun, Shimogo-machi, Onodake, alt. 950 m, Jul. 29, 1978, T. Waku 8977 (TNS VS-366967); Minamiaizu-gun, Minamiaizu-machi, Aoyagi, alt. 850 m, July 26, 2010, A. Ebihara 2623 (TNS VS-1112430); Tateiwa-mura, Tashi-royama, Jul. 30, 1977, T. Waku 7010 (TNS VS-458026); Tochigi Pref., Shioya-gun, Kuriyama-mura, Yunishiga-wa, Sept. 27, 1981, K. Satoh s.n. (TNS VS-455054); Gun-ma Pref., Tone-gun, Niiharu-mura, Mt. Akazawa, Oct. 25, 1955, S. Namegata 6650 (TNS VS-128388), Akazawa, Nov. 11, 1987, M. Ono s.n. (TNS VS-1023740); Agatsu-ma-gun, Nakanojo-machi, Shima, alt. 850 m, Jul. 10, 2009, A. Ebihara 2415 (TNS VS-1107846); Jul. 18, 1954, T. Satow s.n. (TNS VS-106604); Kanra-gun, Shimonita-machi, Tsuchiyazawa, Jul. 28, 1955, T. Satomi s.n. (TNS VS-1046553); Agatsuma-gun, Kuni-mura, Iriyama, Aug. 29, 1975, T. Arai s.n. (TNS VS-587996); Saitama Pref.,

Chichibu-gun, Arakawa-mura, An-ya River, Sept. 5, 1993, T. Iwata 11284 (TNS VS-1026757); Shian-sawa, Sept. 20, 1987, T. Iwata 8149 (TNS VS-1026759); Nagano Pref., Komagane-shi, Akaho, Mt. Utsugi-dake, alt. 1,400–1,500 m, Jul. 29, 2016, J. Fujita TCCN16-0365 (AICH); Nishichikuma-gun, Kaida-mura, Nishino, Jun. 18, 1964, H. Okuhara s.n. (TNS VS-458967); Kiso-gun, Kisofuku-shima-machi, Shiroyama, Jul. 14, 1957, H. Okuhara s.n. (TNS VS-458968); Kamiina-gun, Miyada-mura, Miyada Highland, alt. 1,600 m, Aug. 10, 1981, H. Tsutsumi s.n. (TNS VS-420974); Shimoina-gun, Oshika-mura, Kashio, alt. 1,750m S. Serizawa 89669-2 (AICH; TNS); Omachi-shi, Tokiwa, Aug. 25, 1981, T. Yoshizawa s.n. (TNS VS-458974); Gifu Pref., Nakatsugawa-shi, Idenokojidani, alt. 989 m, Oct. 22, 2010, T. Ogiyama 9498 (TNS VS-1221159); Takayama-shi, R. branch of Gamata-gawa, alt. ca. 1,550 m, Sept. 17, 2012, S. Serizawa 87897 (AICH); Hirayu Pass, alt. 1,500 m, S. Serizawa 90648-2 (TNS VS-1265719); Hirayu, alt. 1,520 m, A. Ebihara 3329 (TNS

Fig. 3. Distribution map of Acystopteris japonica and A. taiwaniana in Japan and Taiwan. White rhomboids: localities of A. taiwaniana—those in Japan are based on specimens cited under taxonomic treatment in this study; localities in Taiwan are based on records from GBIF.org (15th May 2018, GBIF Occurrence Download https://doi.org/10.15468/dl.yixoij and https://doi.org/10.15468/dl.qtuidi, coordinates used without correction). Black triangles: localities of A. japonica based on specimens in TNS.


Fig.4. A. Specimen of Acystopteris japonica (TNS VS-1244737, Kanagawa Pref., Japan, collected on July 22, 2015). B. Spec-imen of A. taiwaniana (TNS VS-1107846, Gunma Pref., Japan, collected on July 10, 2009). Scale bars = 10 cm.

Fig.5. Acystopteris taiwaniana. A. Individual in Kitakata-shi, Fukushima Pref., Japan, on July 26, 2010. B. pale green stipe with whitish scales (photograph taken in Takayama-shi, Gifu Pref., Japan on June 29, 2009). C. glandular hairs on indu-sium of sorus. Scale bar = 0.1 mm.


VS-1200843); Ehime Pref., Uma-gun, Besshiyama-mura, Mt. Akaishi, Aug. 1950, H. Inoue 755 (TNS VS-1034097); Kochi Pref., Agawa-gun, Ino-machi, Mt. Tsutsujo, alt. 1,750 m, Sept. 27, 2008, A. Ebihara 1985 (TNS VS-776951).

Acystopteris tenuisecta (Blume) Tagawa, in Acta Phytotax. Geobot. 7: 73 (1938).—Aspidium tenuisectum Blume, Enum. Pl. Javae 2: 170 (1828) —Asplenium tenuisectum (Blume) Hook., Sp. Fil. 3: 222 (1859) —Athyrium tenui-sectum (Blume) T. Moore, Index Fil. 188 (1860) —Cornopteris tenuisecta (Blume) Tardieu, in Amer. Fern J. 48: 1 (1958).

Japanese name. Horai-usuhimewarabi Distribution. Japan (Yakushima), S. China,

Taiwan, Philippines, Myanmar, Thailand, Singa-pore, Vietnam, Peninsular Malaysia, Borneo, Su-lawesi, Java, Moluccas, Seram, New Guinea, In-dia, Nepal and Bhutan.

Acystopteris japonica (Luerss.) Nakai × A. taiwaniana (Tagawa) Á. et D. Löve

Japanese name. Ozaku-usuhimewarabiDistribution. Japan (Honshu: Tohoku, Kanto

and Chubu districts).

Representative specimens examined. Japan, Yamaga-ta Pref., Nishiokitama-gun, Iide-machi, Yachidaira, Jul. 30, 1968, S. Kato s.n. (TNS VS-222823, 460863); Mt. Iide, Budo-sawa, alt. ca. 1,020 m, Oct. 5, 1980, S. Seriza-wa 31911 (AICH); Fukushima Pref., Yama-gun, Atsush-iokano-mura, Mt. Akakuzure, Jul. 31, 1977, T. Waku 7094 (TNS VS-458027); Tochigi Pref., Kanuma-shi, Ozaku-san, alt. 400 m, Oct. 1, 2017, A. Ebihara 3621 (TNS VS-1286272); Saitama Pref., Iruma-gun, Naguri-mura, Ari-madani, Nov. 16, 1969, S. Serizawa 11308 (AICH); Gifu Pref., Takayama-shi, Hirayu Pass, alt. 1,500 m, S. Ser-

izawa 90649-3 (TNS VS-1265721); Ena-gun, Kashimo-mura. Doai, alt. 800 m, Aug. 19, 1987, M. Murase s.n. (AICH).

We wish to thank K. Hasunuma, F.-W. Li, Y.-C. Liu and N. Takahashi for their help in sampling the collections and C. Takaboshi and K. Uno for their assistance in the DNA analyses.

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Table 2. A comparison of morphological characters of Acystopteris.

A. japonica A. taiwaniana A. tenuisectaSpore longer diameter 25–35 μm 30–40 μm 27.5–35 μmColor of stipe brown or purplish brown pale green, pale brown at base pale greenColor of rachis brown or purplish brown pale green to brown pale greenAngle of pinnae against rachis 50–75° 60–75° 70–90°Glandular hairs on indusia absent present present


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Received June 5, 2018; accepted July 23, 2018

allopolyploid origin and distribution range of

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