A peer-reviewed open-access journal PhytoKeys 165: 69-84 (2020) & ie cee $¢PhytoKeys https:/ / Pp hyto keys -pen soft.net Launched to accelerate biodiversity research Deparia xnanakuraensis K.Hori (Athyriaceae), a new hybrid pteridophyte from Japan Kiyotaka Hori! | The Kochi prefectural Makino Botanical Garden, Kochi, Japan Corresponding author: Kiyotaka Hori (khori@makino.or.jp) Academic editor: Th. Almeida | Received 20 August 2020 | Accepted 6 October 2020 | Published 28 October 2020 Citation: Hori K (2020) Deparia xnanakuraensis K.Hori (Athyriaceae), a new hybrid pteridophyte from Japan. PhytoKeys 165: 69-84. https://doi.org/10.3897/phytokeys.165.57837 Abstract I describe Deparia xnanakuraensis hyb. nov. and discuss differences in morphological characteristics be- tween parental species D. pterorachis and D. viridifrons with chromosome counting, plastid, and nuclear DNA markers. The new hybrid is endemic to the eastern and northern parts of Japan. Based on the criteria of the International Union for Conservation of Nature and Natural Resources, this new species is here considered Data Deficient. The ploidy level is diploid sterile. Keywords Athyriaceae, Deparia, new hybrid, Japan Introduction The genus Deparia Hook. & Grev. is one of the largest groups in the Athyriaceae family. It contains 60-90 species mostly in East Asia, with some species distributed in Africa, western Indian Ocean, northeastern North America, the Hawaiian Islands, Australia, New Zealand, and South Pacific Islands (Kato 1984; Rothfels et al. 2012; He et al. 2013; Kuo et al. 2016, 2018; PPG I 2016; Moran et al. 2019). The genus is characterized by hair-like scales and disconnected grooves between rachises and costae (Kato 1973, 1977, 1984; Rothfels et al. 2012; Sundue and Roth- fels 2014; Kuo et al. 2018). These two features have not been observed in the genera Anisocampium C.Presl, Athyrium Roth., Diplazium Sw., Ephemeropteris R.C.Moran & Sundue, and Pseudathyrium Newman but in some species in the Athyriaceae family Copyright Kiyotaka Hori. 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. 70 Kiyotaka Hori / PhytoKeys 165: 69-84 (2020) (Kato 1973; Rothfels et al. 2012; Moran et al. 2019). In addition, narrowly U-shaped rachis grooves are also a unique character of the genus Deparia (Kuo et al. 2018). The basic chromosome number of Deparia is 40, contrary to Diplazium of 41 (Sano et al. 2000; Rothfels et al. 2012). In Japan, several hybrids of the genus Deparia have been described: D. x birii Fraser- Jenk. (Fraser-Jenkins 2008), D. xkiyozumiana (Sa.Kurata) Y.Shimura (Shimura 1980), pentaploid sterile D. dancea (Thunb.) Fraser-Jenk. (Nakato and Mitui 1979), D. xJo- batocrenata (Tagawa) M.Kato (Kato 1984; Ebihara 2017), D. xmusashiensis (H.Ohba) Seriz. (Serizawa 1981), pentaploid sterile D. petersenii (Kunze) M.Kato (Shinohara et al. 2003), D. xtogakushiensis Otsuka & Fujiw. (Otsuka and Fujiwara 1999), and D. xtomitaroana (Masam.) R.Sano (Sano et al. 2000). Furthermore, Ebihara (2017) mentioned several combinations of hybrids that are not still described. The Deparia okuboana complex (Athyriaceae) is recently defined by Ebihara (2017) as consisting of D. okuboana (Makino) M.Kato (apogamous triploid; Hira- bayashi 1970), D. coreana (Christ) M.Kato (sexual tetraploid, Nakato and Ebihara 2018), D. henryi (Baker) M.Kato (apogamous triploid, Nakato and Ebihara 2018), D. viridifrons (Makino) M.Kato (sexual diploid; Hirabayashi 1970), D. unifurcata (Bak- er) M.Kato (apogamous triploid; Hirabayashi 1970), D. pterorachis (Christ) M.Kato (sexual diploid; Hirabayashi 1970). There is continuous morphological variation be- tween D. coreana, D. henryi, D. okuboana, and D. unifurcata (Ebihara 2017). Kuo et al. (2018) identified that these members belong to sect. Dryoathyrium. Hori (2018) reported there were reticulate relationships in the D. okuboana complex with sect. Lu- nathyrium (Kuo et al. 2018) based on plastid and nuclear DNA marker. In addition, Ebihara (2017) mentioned undescribed diploid sterile hybrid between D. pterorachis and D. viridifrons based on morphology and ploidy level. This study described this new hybrid of D. pterorachis and D. viridifrons, Deparia xnanakuraensis K.Hori, based on morphological characteristics, chromosome number, plastid, and nuclear DNA marker. Materials and methods Plant materials, Chromosome count, and DNA extraction In this study, Deparia viridifrons, D. xnanakuraensis, and D. pterorachis were investigated in molecular DNA analysis. Other members of the D. okuboana complex (D. coreana, D. henryi, D. okuboana, D. unifurcata) and Japanese members of the sect. Lunathyrium (D. pycnosora var. albosquamata M.Kato, D. pycnosora (Christ) M.Kato var. pycnosora, D. pycnosora var. mucilagina M.Kato) were also used as materials. Voucher information for all samples is listed in Appendix I. All voucher specimens have been deposited in the Makino Herbarium of Tokyo Metropolitan University (MAK), and/or the Kochi Prefectural Makino Botanical Garden (MBK). The DNA sequences of Athyrium melanolepis Christ, A. crenulatoserrulatum Makino, A. opacum Depariaxnanakuraensis K.Hori (Athyriaceae) FA Copel., Diplazium chinense (Baker) C.Chr., Di. esculentum (Retz.) Sw., Di. wichurae (Mett.) Diels were used as outgroups, quoted from the Genbank database. Additionally, specimens from the Collection Database and Materials of TNS (http://db.kahaku.go.jp/webmuseum/), PE (http://pe.ibcas.ac.cn/en/), TAIF (http:// taif.tfri.gov.tw/search.php), and from the JSTOR Global Plants (https://plants.jstor. org/) as well as from the Global Biodiversity Information Facility (GBIF: https://www. gbif.org) database were checked. For the conservation assessment, the area of occupancy (AOO) and extent of oc- currence (EOO) were estimated using GeoCAT (Bachman et al. 2011), default set- tings for grid size were applied. In addition, mitotic chromosomes from D. xnanaku- raensis were counted. To observe mitotic chromosomes, root tips were collected in the field, and pre- treated with 0.004 M 8-hydroxyquinoline for 6 h at approximately 17—20 °C. After fixation in ethanol and acetic acid (3:1) for 15-30 min, the root tips were hydrolyzed in 1 N HCl at 60 °C for 1-3 min and then squashed in 2% aceto-orcein solution. The chromosomes were observed under a microscope (Leica DM2500) and then photo- graphed by using a digital camera (Leica MC170 HD). For the molecular analyses, total DNA was extracted from silica-dried leaves using cetyltrimethylammonium bromide solution, according to Doyle and Doyle (1990). Plastid and nuclear DNA sequencing trnL-F was used as the maternally-inherited (Gastony and Yatskievych 1992; Kuo et al. 2018) plastid DNA marker (F: 5'-ATTTGAACTGGTGACACGAG-3' and FernL 1 Irl: 5'-GGYAATCCTGAGCAAATC-3'; Taberlet et al. 1991; Li et al. 2009). AKI (AK4F: 5'-GATGAAGCCATCAAGAAACCA-3' and AKR2: 5'-ATGGATCCAGC- GACCAGTAA-3'; Hori and Murakami 2019) was used as a biparentally-inherited nu- clear marker for polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis, which was used to determine allelic variation in each individual (Hori and Murakami 2019). PCR amplification was performed using PrimeSTAR Max DNA Polymerase (Ta- kara, Kyoto, Japan). PCR entailed an initial denaturation step at 95 °C for 10 min, followed by 35 cycles of denaturation, annealing, and elongation steps at 98 °C for 10 s, 55 °C for 5 s, and 72 °C for 5 s, respectively, using a Model 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA). Gel electrophoresis of AKI PCR products was performed using gels of 5|0% MDE gel solution (Lonza) containing 2% glycerol at 15 °C for 16 h at 300 V, followed by silver staining. For sequencing of the bands separated on the gels, the polyacrylamide gel was dried after silver staining by sandwiching the gel between Kent paper and a cellophane sheet on an acrylic backplate at 55 °C for 4 h. To extract the DNA, a piece of the DNA band was peeled from the dried gel using a cutter knife and incubated in 50 uL of Tris-EDTA buffer (10-mM Tris-HCl and 1-mM EDTA, pH 8.0) at 4 °C 2 Kiyotaka Hori / PhytoKeys 165: 69-84 (2020) overnight. The supernatant solution was used as a template for further PCR amplifica- tion with the same primer set employed for initial PCR amplification. PCR products were purified using Illustra ExoStar 1-Step (GE Healthcare, Wis- consin, USA) and used as templates for direct sequencing. Reaction mixtures for se- quencing were prepared using the BigDye Terminator v.3.1 Cycle Sequencing Kit (Ap- plied Biosystems). The reaction mixtures were analyzed using an ABI 3130 Genetic Analyzer (Applied Biosystems). Molecular analysis The accession numbers of DNA sequences in the datasets were shown in Appendix I. The sequences were aligned using MUSCLE (Edgar 2004) and assessed with Bayesian inference (BI) analysis using MrBayes 3.2.6 (Ronquist et al. 2012), maximum par- simony (MP), and maximum likelihood (ML) analysis using the MEGA X software (Kumar et al. 2018). Indels were treated as missing characters in all analyses. In the BI analysis, the best-fit model (trnZ-F: HKY+I model; AK7: HKY model) of sequence evolution for each DNA region was selected using jModelTest 2.1.10 (Darriba et al. 2012). Four Markov chain Monte Carlo chains were run simultaneously and sampled every 100 generations for 1 million generations in total. Tracer 1.7.1 (Rambaut et al. 2018) was used to examine the posterior distribution of all parameters and their associ- ated statistics, including estimated sample sizes. ‘The first 2,500 sample trees from each run were discarded as burn-in periods. The MP tree was obtained using the Tree-Bi- section-Regrafting (TBR) algorithm (Nei and Kumar 2000) at search level 3, at which the initial trees were obtained by the random addition of sequences (100 replicates). The confidence level of the monophyletic groups was estimated with 1,000 MP boot- strap pseudo-replicates. In ML analysis, the best-fitting model of sequence evolution for each marker was selected using MEGA; Tamura 3-parameter + I model was used for trnL-F and HKY model for AK/. Initial trees for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood approach and then selecting the topology with superior log likelihood value. The bootstrap method with 1,000 replications was employed to estimate the confidence levels of monophyletic groups in MP and ML analysis. Results Chromosome count Mitotic metaphase chromosome number observed in an individual of D. xnanaku- raensis (Hori 3391) was 2n = 80 (Figure 1). This individual had shrunken sporan- gium with no spores. The basic chromosome numbers of the genus Deparia is x= 40 (Sano et al. 2000; Rothfels et al. 2012), and suitably, this sample was found to be a sterile diploid. Depariaxnanakuraensis K.Hori (Athyriaceae) 73 Figure |. Photograph and sketch of mitotic metaphase chromosomes (27 = 80) of D. x nanakuraensis (Hori 3391). Plastid and nuclear DNA phylogenetic trees We sequenced 653-746 bp of the zrnL-F intergenic spacer from different specimens. The aligned trnL-F matrix was 765 bp, of which 121 characters (15%) were parsi- mony-informative. For the AK/ intron, we sequenced 338-590 bp of the intron for each specimen, yielding a 604 bp aligned matrix, of which 74 characters (12%) were parsimony-informative. The ML trees according to the sequences of trnL-F (In L = -2309.05) and AKI (In Z = -1616.59) with bootstrap percentages (BPs), Bayesian posterior probabilities (PP) were shown in Figures 2, 3, respectively. In the trnL-F phylogeny, the haplo- type of D. pterorachis and D. viridifrons composed different clades with D. coreana, D. henryi, and D. okuboana which were supported by BP (>70) and PP (>0.90) values. In the AK/ phylogeny, the two clades containing D. pterorachis and D. vir- idifrons were supported by BP, but D. viridifrons was not supported by PP value. Deparia xnanakuraensis had the same haplotype of D. pterorachis and D. viridifrons in both trnL-F and AKI phylogenies. Other members of the D. okuboana complex (D. coreana, D. henryi, D. okuboana, D. unifurcata) shared the same alleles with D. viridifrons partly (Hori 2018), but the combination of alleles was different from D. xnanakuraensis. Japanese members of the sect. Lunathyrium (D. pycnosora var. albos- guamata, D. pycnosora vat. pycnosora, D. pycnosora var. mucilagina) did not share any alleles with D. xnanakuraensis. Therefore, D. xnanakuraensis can be of origin hybrid from D. pterorachis and D. viridifrons. Taxonomic treatment Deparia xnanakuraensis K.Hori, hyb. nov. urn:lsid:ipni.org:names:7721257 1-1 Figure 4 Type. Japan. Honshu: Akita prefecture, Noshiro city, Futatsui town, Nanakura-shrine, 40°12'9.48"N, 140°15'29.82"E, alt. 23 m, deciduous forest containing Acer miyabei 74 Kiyotaka Hori / PhytoKeys 165: 69-84 (2020) D. viridifrons Hori2971 D. viridifrons Hori3060 100/99/1 D. viridifrons Hori3061 D. henryi Hori3028 79/-/- D. okuboana Hori3033 D. coreana Hori3047 L_____-——. unifurcata Hori3029 91/83/- D. pterorachis Hori3053 D. pterorachis Hori3054 100/99/0.99 ae D. pterorachis Hori3055 D. x nanakuraensis Hori3391 100/100/1 D. x nanakuraensis Hori3392 D. x nanakuraensis Hori3393 __—— D. pycnosora var. albosquamata Hori3382 _— D. pycnosora var. mucilagina Hori3380 98/99/1 99/99/1 ‘— D. pycnosora var. pycnosora Hori3052 Diplazium wichurae LC468190* 98/97/1 Di. chinense LC468193* Athyrium melanolepis MG183541* 100/100/0.99 A. crenulatoserrulatum MG183578 100/99/1 ——_ A. opacum KX656057* 0.020 Figure 2. The ML tree based on the sequence variation of the gene tnL-F (In L = -2309.05) with PP (>0.90) and BP (+70) of ML/MP/BI analyses on each branch. The sequences with asterisks were quoted from Genbank. Maxim., Aesculus turbinata Blume, Cercidiphyllum japonicum Siebold & Zucc., Cryp- tomeria japonica (Thunb. ex L.f.) D.Don, Dryopteris monticola (Makino) C.Chr., and Pachysandra terminalis Siebold & Zucc., on soil, 7 Jul 2020, K. Hori 3391 (holotype: MAK467056; isotype: MBK). Description. Terrestrial, summer green fern. Rhizomes creeping, occasionally branched, with buds, stramineous, 15—25 x 4-7 cm, closely set with roots and persis- Depariaxnanakuraensis K.Hori (Athyriaceae) vay . x nanakuraensis Hori3391 . x nanakuraensis Hori3392 . x nanakuraensis Hori3393 . viridifrons Hori2971 D D D D 88/88)! 5 viridifrons Hori3060 D D D D . Viridifrons Hori3061 . coreana Hori3047 - /84 /- . okuboana Hori3033 . henryi Hori3028 r— D. unifurcata Hori3029-allele1 D. unifurcata Hori3029-allele2 a D. henryi Hori3028 D. okuboana Hori3033 ; _— D. pycnosora var. mucilagina Hori3380 77/82/0.90 D. pterorachis Hori3053 D. pterorachis Hori3054 99/99/1 | D. pterorachis Hori3055 82/87/0.97 91/96/0.92 D. x nanakuraensis Hori3391 D. x nanakuraensis Hori3392 99/99/1 -/74/0.99 D. x nanakuraensis Hori3393 ae D. pycnosora var. albosquamata Hori3382-allele1 | D. coreana Hori3047 = »— D. pycnosora var. pycnosora Hori3052-allele1 -/72/0.94 D. pycnosora var. pycnosora Hori3052-allele2 D. henryi Hori3028 97/93/1 '—— D. pycnosora var. albosquamata Hori3382-allele2 Athyrium melanolepis MN267422* ;_— A. crenulatoserrulatum LC421516* A. opacum MN267421* ———— A, decurrentialatum LC421512* Diplazium esculentum LC468186* 91/87/0.99 Di. wichurae LC468187* 100/100/1 ~— Di. wichurae LC468188* 98/99/1 99/99/1 } | 0.010 Figure 3. The ML tree based on the sequence variation of the gene AK/ (In L = -1616.59) with PP (>0.90) and BP (+70) of ML/MP/BI analyses on each branch. The sequences with asterisks were quoted from Genbank. tent, densely clothed by old stipe bases, glabrous; fronds 4-6 per rhizome; stipes whit- ish green, 30-40 x 0.8—1.5 cm, sparsely clothed with stramineous scales at the base (1-1.5 x 0.5-1 cm), ovate; blades yellowish green adaxially, 3-pinnate-pinnatifid at the 76 Kiyotaka Hori / PhytoKeys 165: 69-84 (2020) Cop el ef Figure 4. D. xnanakuraensis K.Hori A lower stipe scale B upper stipe scale C abaxial surface of frond and stipe D detail of adaxial pinnule E detail of abaxial pinnule, and F rhizome and base of stipes. A-F from the holotype (MAK467056) (illustration by K. Hori). base, in the middle to upper section, 2-pinnate at the apex, 40-70 x 30-40 cm, del- toid; rachises whitish green, glabrous, sparsely clothed with stramineous scales (2—5 x 1—2 mm) and black hairs adaxially; pinnae 10-15 pairs, ascending, lanceolate, shrunk- en at base, alternate, petiolated (2-5 mm), sessile near the apex, lowest pinnae slightly reduced, second lowest pair usually the largest, 25-30 x 4-8 cm; pinnules, alternate on the basal and middle sections of the blade, 20-30 pairs on the basal and middle sections of the blade, 15-20 pairs on the apex of the blade, reduced distally, lanceolate, deeply serrated, vein-free, close to or reaching to the margin, 10-15 pairs in the middle lobe; sori brown, tending to appear on the abaxial surface of the middle or upper part of blades, oblong- to J-shaped, 1.5—3 mm long, on the apex or middle of veinlets, 5—10 per ultimate segment, persistent; izdusium entire to serrated on margin, sporangium shrunken, spores absent. Depariaxnanakuraensis K.Hori (Athyriaceae) ra 190 195 140 (7) aa ae ane te CS 140 145 Figure 5. Map showing the known distribution of D. xnanakuraensis in Japan. Red star indicates type locality, other circles indicate examined specimens. Etymology. The name derives from Nanakura-shrine, Futasui town, Noshiro City, Akita prefecture, northeast Japan, where Deparia xnanakuraensis was first found. Specimens examined. Japan. Honshu: Akita pref., Noshiro city, Futatsui town, Nanakura-shrine, 40°12'9.48"N, 140°15'29.82"E, alt. 23 m, 7 Jul 2020, K. Hori 3392, loc. cit., K. Hori 3393, loc. cit., K. Hori 3394, loc. cit., 10 Jul 2012, Y. Horii 35548 (TNS 01167830), loc. cit., Y Horii 35549 (TNS 01167829); Aomori pref., Hachinohe city, Same town, Kamikoswa, alt. 100 m, 23 Aug 1975, coll. MZ. Neichi (TNS 1170337, image!); /oc. cit., Kitsunetai, alt. 30m, 9 Jul 2005, coll. M. Neichi (TNS 01183638, im- age!); Iwate pref., lwaizumi town, Atsuka, Matsugasawa, alt. 350 m, 18 Jul 1981, coll. M. Neichi (TNS 01161869, image!); loc. cit., Ichinoseki city, Higashiyama cho, Naga- saka, Nagahira, alt. 180 m, 22 Aug 1987, coll. MZ. Suzuki (TNS 932028, image!); loc. cit., Maikawa, Ohira, alt. 120 m, 22 Sep 1986, coll. M. Suzuki (TNS 9320284image!); Miyagi pref., Ishinomaki city, Mano, Uchihara, alt. 70 m, 25 May 1990, coll. K. Shogo (TNS01184195, image!); loc. cit., Sendai city, Akiu town, Baba, alt. 200 m, 15 Oct 1983, coll. K. Shogo (TNS01184194, image!); Yamagata pref., Kamiyama city, Takano, alt. 250 m, 5 Jun 1983, coll. N. Sakawa (TNS01161877, image!); Fukushima pref., Minamiaizu county, Shimosato town, Yunokami, alt. 500 m, 8 Sep 1972, coll. 77 Waku (TNS01161873, image!); Saitama pref., Hannnou city, Kasasugitouge, alt. 500 m, 21 78 Kiyotaka Hori / PhytoKeys 165: 69-84 (2020) Figure 7. Juvenile of D. xnanakuraensis. Depariaxnanakuraensis K.Hori (Athyriaceae) 7 5cm Figure 8. Indefinite growth through bud (red arrow) on rhizome of D. xnanakuraensis. Figure 9. Abaxial surface of pinnule and sori of A D. viridifrons B D. xnanakuraensis, and C D. ptero- rachis (illustration by K. Hori). Aug 1984, coll. 77 Iwata (TNS01140142, image!); loc. cit., 14 Sep 1980, coll. Y Kod- ayashi (MBK0233005); loc. cit., 14 June 1981, coll. Y Kobayashi (MBK0232983). Distribution and ecology. Deparia xnanakuraensis is known from the eastern and northern part of Honshu in Japan (Figure 5). It was observed to grow on soil under de- ciduous forest (Figure 6) or planted coniferous forest containing Cryptomeria japonica. This hybrid is endemic to Japan. In the type locality, this hybrid comprised a popula- tion of over 30 individuals with juveniles (Figure 7) although parents of D. viridifrons 80 Kiyotaka Hori / PhytoKeys 165: 69-84 (2020) and D. pterorachis were both absent, and sporangium had no spores. However, it is expected that Deparia xnanakuraensis can reproduce young individuals from buds on its rhizome (Figure 8). Conservation status. [UCN Red List Category. Based on estimates from Geo- CAT, the EOO of D. xnanakuraensis was 46,321 km?. The known AOO of D. xnana- kuraensis was 44 km’. The localities correspond to less than 20 points, but I could not check the population size on each locality. Therefore, available information is inad- equate to support the assessment of its extinction risk. According to the IUCN (2012) criteria, the category of Data Deficient (DD) is appropriate. Discussion Deparia xnanakuraensis presents almost intermediate morphologies between D. viridi- frons and D. pterorachis species. Deparia viridifrons is characterized by having deltoid- ovate or ovate-lanceolate fronds, reniform to U-shaped sori, pinnules with costal wing, rounded serration of pinnules, and acute apex of pinnules. In contrast, D. pterorachis has oblong fronds, oblong to J-shaped sori, pinnules truncated to costa; truncate serra- tion of pinnules, and obtuse apex of pinnules (He et al. 2013; Ebihara 2017). Deparia xnanakuraensis has deltoid fronds, oblong to J-shaped sori, pinnules with narrow costal wing, rather rounded serration of pinnules, and a rather acute apex of pinnules (Fig- ure 9, Table 1). Kuo et al. (2018) classified D. viridifrons and D. pterorachis as the members of sect. Dryoathyrium because lateral pinnules are not auricled, and these are closely related in plastid DNA phylogeny (Kuo et al. 2018). Therefore, Deparia xnanakuraensis is infra section hybrid in the sect. Dryoathyrium. The ploidy level of this hybrid is the same as its parents because D. viridifrons and D. pterorachis are both sexual diploid (Kurita 1963; Mitui 1966, 1968, 1970; Hira- bayashi 1970). In addition, this can be the first report of a diploid sterile hybrid of the genus Deparia from Japan although several hybrids have been described (Ebihara 2017). In conclusion, this study described Deparia xnanakuraensis based on morphology, cytology, and molecular DNA analysis. The morphological characteristics were inter- mediate between its parents D. viridifrons and D. pterorachis. This hybrid can produce young individuals from buds on its rhizome. Based on the criteria of the International Union for Conservation of Nature and Natural Resources, this new species is here Table |. Morphological comparison among D. xnanakuraensis and related species. Characteristics | Shape of frond Shape of sori Margin of Base of pinnule | Serration of Apex of D. viridifrons deltoid-ovate or | reniform to U-shaped serrated with costal wing rounded acute D. xnanakuraensis deltoid oblong to J-shaped | entire to serrated | with narrow costal} rather rounded | rather acute [ii jee i D. pterorachis oblong oblong to J-shaped truncated to costa obtuse Depariaxnanakuraensis K.Hori (Athyriaceae) 81 considered Data Deficient. This hybrid can be the first report of diploid sterile hybrid of the genus Deparia from Japan. 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D. pterorachis (Christ) M.Kato— JAPAN. Hokkaido Pref., Sapporo city, Minami- ku, Jouzannkei, 530m alt., 42°55'36.8"N, 141°10'6.1"E, July 30 2018, K. Hori 3053 (MBK); MT898441 (¢rnL-F); MT887299 (AK1). ibid., Ebetsu city, Nopporo nature park, July 30 2018, K. Hori 3054 (MBK); MT898442 (trnL-F); MT887300 (AK1). ibid., K. Hori 3055 (MBK); MT898443 (trnL-F); LC421964 (AKI, Hori 2018). D. viridifrons (Makino) M.Kato— JAPAN. Kochi pref., Takaoka county, Ochi town, Mt. Yokogura, May 30 2018, K. Hori 2971 (MBK); LC421960 (érnL-F, Hori and Murakami 2019); LC468191 (AKI, Hori 2018). ibid., Oct 17 2018, K. Hori 84 Kiyotaka Hori / PhytoKeys 165: 69-84 (2020) 3060 (MAK); MT898444 (trnL-F); MT887305 (AK). ibid., Oct 17 2018, K. Hori 3061 (MAK); MT898445 (¢rnL-F); MT887306 (AKZ). D. coreana (Christ) M.Kato— JAPAN. Aomori Pref., Kamikita county, Shichi- nohe town, Jul 26 2018, Hori 3047 (MBK); MW051518 (tmL-F); MW051522, MW051523 (AKI). D. henryi (Baker) M.Kato— JAPAN. Kyoto Pref., Kyoto City, Jul 14 2018, Hori 3028 (MBK); MW051514 (zrnL-F); MW051527, MW0515278, MW051529 (AK7). D. okuboana (Makino) M.Kato— JAPAN. Kyoto pref., Kyoto city, Jul 14 2018, Hori 3033 (MBK); MW051515 (¢rnZ-F); MW051530, MW051531 (AK). D. pycnosora (Christ) M.Kato var. albosquamata M.Kato— JAPAN. Nagano Pref., Nagano city, Togakushi shrine, Okusha, Jul 9 2020, K. Hori 3382 (MAK); MW051519 (trnL-F); MW051520, MW051521 (AKZ). D. pycnosora (Christ) M. Kato var. mucilagina M.Kato— JAPAN. Nagano Pref., Nagano city, Togakushi shrine, Okusha, Jul 9 2020, K. Hori 3380 (MAK); MW051516 (trnL-F); MW051526 (AK). D. pycnosora (Christ) M. Kato var. pycnosora M.Kato— JAPAN. Aomori Pref., Kamikita county, Touhoku town, Otsutomo, Jul 26 2018, K. Hori 3052 (MAK); MW051517 (trnL-F); MW051524, MW051525 (AKI). D. unifurcata (Baker) M.Kato— JAPAN. Kyoto Pref., Kyoto city, Jul 14 2018, K. Hori 3029 (MBK); LC468192 (trnL-F, Hori and Murakami 2019); LC421961, LC421962 (AKI, Hori 2018).