ISSN 1346-7565 Acta Phytotax. Geobot. 71 (1): 13–21 (2020) doi: 10.18942/apg.201910
Asplenium serratipinnae (Aspleniaceae: Polypodiales), a New Allotetraploid Species in the A. normale Complex
1,†,* 1 2 1 Tao Fujiwara , Junki Ogiso , Sadamu Matsumoto and Yasuyuki Watano
1Department of Biology, Graduate School of Science, Chiba University, Yayoi-cho, Inage, Chiba 263-8522, Japan; 2Department of Botany, National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba 305-0005, Japan. †Present Address: Center for Integrative Conservation, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla 666303, Yunnan, China. *[email protected] (author for correspondence)
Asplenium serratipinnae (Aspleniaceae: Polypodiales), an allotetraploid species between the diploid race of A. normale and A. oligophlebium is described as new. It is endemic to Japan and morphologically most similar to A. normale, but differs in having narrower pinnae with an auriculate to hastate acroscopic base and deeply serrated margins.
Key words: Aspleniaceae, Asplenium normale, polyploidy, taxonomy, tetraploid
Asplenium L. (Aspleniaceae), one of the most Chang et al. (2013) collected samples of these species-rich genera of ferns, comprises approxi- species from regions throughout Japan, China, mately 700 species (PPG1 2016, Smith et al. Southeast Asia, Hawaii and Africa and conducted 2006). It occurs in all tropical and temperate re- phylogenetic analyses using chloroplast DNA gions. Asplenium is characterized by having the (cpDNA) and nuclear gene markers. Chang et al. highest proportion of polyploids among derived (2013) showed that recurrent reticulation events ferns (Schneider et al. 2017). In ferns, many spe- occurred among diploid members of this species cies complexes were formed through frequent re- complex and that the tetraploid race of A. nor- ticulate evolutionary events, e.g. the Appalachian male in Japan was an allotetraploid between a Asplenium complex (Werth et al. 1985), the New diploid race of A. normale in China and Southeast Zealand Asplenium complex (Shepherd et al. Asia and an unknown diploid race of A. boreale. 2008), and the A. monanthes complex (Dyer et al. In addition, Chang et al. (2013) suggested that A. 2012). The Asplenium normale complex belongs boreale was an autotetraploid and A. shimurae to the ‘black-stemmed’ spleenwort group (Sch- was an allotetraploid between an unknown dip- neider et al. 2005). In Japan, three tetraploid spe- loid race corresponding to a clade of A. shimurae cies, A. normale D. Don, A. boreale (Ohwi ex Sa. in their cpDNA phylogeny and the diploid race of Kurata) Nakaike and A. shimurae (H. Itô) Na- A. normale in China and Southeast Asia. Recent- kaike, and a diploid species, A. oligophlebium ly, Chang et al. (2018) recognized six diploid spe- Baker, have been recognized in the complex. cies within this complex, including two novel Their recognition is supported by sufficient dif- species, A. normaloides Y. Fen Chang & H. Sch- ferences in rbcL gene sequences (Murakami et neid. corresponding to the diploid race in the al. 1999), sterility of their hybrids (Matsumoto et clade of A. shimurae in the cpDNA phylogeny, al. 2003) and distinct flavonoid patterns (Iwashi- and A. guangdongense Y. Fen Chang & H. Sch- na & Matsumoto 1994, Matsumoto et al. 2003). neid. corresponding to the diploid race of A. boreale, 14 Acta Phytotax. Geobot. Vol. 71 by employing their ‘diploid first’ approach with 12 samples of cryptic species II, four samples of an integrated taxonomic analysis including cyto- cryptic species III, one sample of the tetraploid logical, morphological and phylogenetic analy- Asplenium normale from Taiwan (an allotetra- ses. Although diploid species or races have been ploid between the diploid race of A. normale and discovered in almost all major clades in the cpD- A. guangdongense) and four samples of the dip- NA phylogeny of this complex through their ef- loid A. normale from Taiwan (Appendix 1). All forts, the classification of the tetraploid lineages samples used in the present study were collected remains a major problem. by Fujiwara et al. (2017). We obtained a digital The Asplenium normale complex in Japan ex- image of a frond from a dried specimen of each hibits high variation in flavonoid content (Iwash- sample by using a scanner (GT-X980; Epson, Na- ina et al. 1990, 1993, Iwashina & Matsumoto gano, Japan). A middle pinna representative of 1994, Matsumoto et al. 2003, Fujiwara et al. each sample was selected and its morphological 2014). Tetraploid plants of A. normale can be di- characteristics were analyzed using ImageJ soft- vided into eight chemotypes, one of which has ware (Schneider et al. 2012). the same flavonoid composition as A. oligophle- To measure the shape and size of the pinnae, bium (Iwashina & Matsumoto 1994, Fujiwara et we marked the following points on each pinna al. 2014). By integrating our results from popula- (Fig. 1): the apex (A), the base (B), the intersect- tion genetics, chemotaxonomy and phylogenet- ing points of a perpendicular line at the midpoint ics, three cryptic species (I–III) with distinct fla- of line segment AB with the acroscopic and ba- vonoid compositions within Japanese tetraploid siscopic margins of the pinna (C and D), and the A. normale were noted (Fujiwara et al. 2017) The apex of the acroscopic basal lobe (E). Line seg- study revealed that cryptic species I and III origi- ments AB and CD were defined as pinna length nated from hybridization between the diploid and pinna width, respectively. Angles CAD and race of A. normale and A. guangdongense (treat- EBD were defined as the angle of the apex and the ed as an unknown diploid race of A. boreale in angle of the base, respectively. With respect to Fujiwara et al. 2017), whereas cryptic species II the extent of tooth development on the pinna mar- originated from hybridization between the dip- gin, we measured the relative area (S) and relative loid race of A. normale and A. oligophlebium. Ad- perimeter (L) of the pinna and defined the teeth ditionally, it was noted that cryptic species II index as L2S-1, which was expected to increase could be distinguished by morphological charac- with teeth development. Principal component teristics of the pinnae (Fujiwara et al. 2017). analysis (PCA) was conducted with R v.3.3 (R In this study, we measured morphological fea- Development Core Team 2013), using six vari- tures to discriminate cryptic species II (an allo- ables: 1) pinna length, 2) pinna width, 3) pinna tetraploid with A. oligophlebium as one of its dip- length to pinna width ratio (pinna length/pinna loid parents) from the diploid race of A. normale width), 4) apex angle, 5) base angle, and 6) the and the other allotetraploids with a different pa- tooth index. Differences in morphological char- rental species pair (the diploid race of A. normale acteristics between groups were tested for statis- and A. guangdongense) in the A. normale com- tical significance using Welch’s t test (Welch plex From our findings, we determined that cryp- 1947). tic species II represents a distinct species, which For the scanning electron microscope (SEM) we describe here as new. images, the spores were transferred from dried specimens to double-sided carbon tape on an alu- minum specimen holder. The samples were then coated with gold using a sputter coater (JEOL Materials and Methods JFC-1100; JOEL. Tokyo, Japan) and imaged us- ing a SEM (JEOL JSM-6510A; JOEL) at an accel- We analyzed 48 samples of cryptic species I, erating voltage of 15 kV. February 2020 Fujiwara & al. — A New Allotetraploid Species in Asplenium normale Complex 15
ently similar to A. normale × A. oligophlebium, which is a putative triploid hybrid (Ebihara 2016), but can be distinguished by the normally shaped spores 33–41.5 μm in diameter (Fujiwara et al. 2017, Fig. 1 and Supplemental Data). The present study revealed morphological differences between cryptic species II and the other samples of Asplenium normale. Cryptic species II is also unique among tetraploid taxa in Fig. 1. Diagram of pinna showing points used for morpho- having A. oligophlebium as one of its diploid par- logical measurements. ents. We therefore describe the allotetraploid spe- cies between the diploid race of A. normale and A. oligophlebium as a new species, A. serratipin- Results and Discussion nae T. Fujiw. & Watano. It should be noted that A. serratipinnae appears to be endemic to Japan, The six morphological characteristics were based on the specimens we examined, as is one of summarized using PCA; the first two axes, PC1 its diploid parents, A. oligophlebium (Nakaike and PC2, explained 62.3% and 26.1% of the vari- 1992). Genetically, A. serratipinnae is character- ation, respectively (Fig. 2). PC1 correlated posi- ized by sharing identical sequence alleles with A. tively with the pinna length to width ratio and the oligophlebium at all three nuclear loci examined tooth index, and negatively with the apex and (LFY, PgiC and NIA) (Fujiwara et al. 2017). Al- base angles. PC2 was negatively correlated with though Chang et al. (2013, 2018) examined the the two size variables, pinna length and width. PgiC gene in samples containing tetraploid A. The PCA result indicated that the samples could normale that were widely collected from China not be separated into the tetraploid cryptic spe- and Hawaii, samples that shared alleles with A. cies nor the diploid Asplenium normale, but rath- oligophlebium were not reported. This may be er into two groups: group 1, which contained all another indication that A. serratipinnae occurs samples of cryptic species I, cryptic species III only in Japan. and the Taiwanese A. normale samples including Chang et al. (2018) recognized ten species in diploids, and group 2, which contained only cryp- the A. normale complex; nine of them being ei- tic species II. This separation highlighted the ther diploid or tetraploid. Only A. normale con- morphological uniqueness of cryptic species II, tains two cytotypes. Asplenium normale in the which has A. oligophlebium as a diploid parent. A sense of Chang et al. (2018) was defined as a comparison of each of the six morphological members of the complex having buds at the distal characteristics showed that group 2 (cryptic spe- end of the rachis. Phylogenetically, members of cies II) was significantly different from other A. normale were included in the cpDNA clade I samples with respect to all characteristics except (Chang et al. 2013, 2018), which corresponded to pinna width (Fig. 3). In particular, the greater the NORMALE clade in the cpDNA tree of Fuji- pinna length to width ratio, the smaller apex an- wara et al. (2017). Asplenium normale is still tax- gle and greater tooth index in group 2 reflected onomically problematic, however, because it is a the narrower shape and serrated margins of the mixture of diploid A. normale and tetraploids of pinnae (Fig. 3) and the conspicuous characteris- distinct evolutionary units formed through allo- tics of one of its parental species, A. oligophlebi- polyploidization between diploid Asplenium nor- um. Therefore, the morphological characteristics male and other diploid species of the complex of group 2 (cryptic species II) appear to be inher- (Chang et al. 2013, Fujiwara et al. 2017). The rec- ited from A. oligophlebium. For this reason, the ognition of A. serratipinnae is the first attempt to plants of group 2 (cryptic species II) are appar- separate recognizable tetraploid taxa from A. 16 Acta Phytotax. Geobot. Vol. 71
Fig. 2. Biplot diagram of principal component analysis of samples of the Asplenium normale complex using six morphological characteristics of the pinna. Symbols represent cryptic species delimited by Fujiwara et al. (2017): solid square, cryptic species I; solid circle, cryptic species II; solid triangle, cryptic species III; solid diamond, tetraploid samples from Taiwan; open diamond, diploid samples from Taiwan. Arrows represent factor loadings of original morphological variables on PC1 and PC2.
Fig. 3. Box plots demonstrating differences between cryptic species II (Group 2 in Fig. 2) and other samples (Group 1 in Fig. 2) of Asplenium normale. In each box plot, thick line, edges of box, whiskers, and circles indicate median value, lower and upper quartiles, minimum and maximum values, and outliers, respectively. Significant difference, *: p < 0.05, **: p < 0.01. February 2020 Fujiwara & al. — A New Allotetraploid Species in Asplenium normale Complex 17 normale since Chang et al. (2018). To produce a pinnate, 2-forked or simple. Sori linear, 1–11 per new classifications for the remaining tetraploid pinna, borne along veinlets, indusiate; indusia taxa in the Asplenium normale complex, it is whitish or yellowish, semi-elliptic, membranous, clear that additional analyses are required. For entire, 1.5–2.5 mm long. Spores monolete, ellip- example, cryptic species I and III of Fujiwara et soid, perispore alate, crests dentate, surface al. (2017) were detected in sympatric populations slightly perforated, 33–41.5 µm in length. Spores in Japan through their genetic distinctness. The 64 per sporangium. Plants tetraploid: 2n = 144. allotetraploids between the diploid race of A. nor- Japanese name. Nankai-nuritoranoo male and A. guangdongense, including cryptic Distribution. JAPAN (Honshu, Shikoku, Ky- species I and III, are distributed in Japan, Taiwan ushu, Ryukyu islands) and China (Chang et al. 2018, Fujiwara et al. Notes. Asplenium serratipinnae corresponds 2017). However, reproductive isolation among the to cryptic species II and multi-locus genotype 2, Japanese, Taiwanese and Chinese samples cannot 6 and 8 in Fujiwara et al. (2017) and to flavonoid be assessed by population genetic approaches be- B- and C-chemotypes reported by Iwashina and cause they are not sympatric. Future taxonomic Matsumoto (1994). Asplenium serratipinnae has studies of A. normale should include artificial largely overlapping ranges of distribution with crossing tests or be based on quantitative analy- cryptic species I and III of A. normale and A. oli- ses of genetic differentiation. gophlebium in Japan (Fujiwara et al. 2017), but tends to grow in more southerly regions, includ- ing Okinawa, Yakushima and Hachijojima in the Taxonomic Treatment Pacific Ocean. The first part of the Japanese name, Nankai- (southern sea), is derived from the Asplenium serratipinnae T. Fujiw. & Watano, southerly distribution of A. serratipinnae. sp. nov. —Figs. 4 & 5 Specimens examined. JAPAN: Tokyo Pref. An allotetraploid species originating from hybridization Hachijojima, Miharayama, S. Matsumoto 130303-7 between the diploid race of Asprenium normale D. Don (TNS1310410); Shizuoka Pref., Kamo-gun, Nishiizu- and A. oligophlebium Baker; differing from A. normale in cho, T. Fujiwara & H. Sato FT120528-12 (TNS1303463); having narrower pinnae with an auriculate to hastate ac- Ibid., T. Fujiwara & H. Sato FT120528-13 (TNS1303464); roscopic base and deeply serrated margins and from A. Kamo-gun, Higashiizu-machi, A. Suzuki s.n. (TNS403555); oligophlebium in the serrated and broader pinnae. Aichi Pref., Chita-shi, Okusa, K. Inukai 5302 (TNS790114); Typus. JAPAN, Miyazaki Pref., Nobeoka-shi, Okato- Mie Pref., Kitamuro-gun, Kihoku-cho, T. Fujiwara & K. mi-cho, Jul. 16, 2013, T. Fujiwara, T. Minamitani & Y. Ohora FT121022-8 (TNS1303505); Minamimuro-gun, Akagi FT130716-9 (TNS). Kihou-cho, S. Iwanaka s.n. (TNS417762); Shiga Pref., Hikone-shi, Konki-cho, Hikonejo, T. Tatebe s.n. Herbs, 15–40 cm tall. Rhizome shortly erect, (TNS790076); Kyoto Pref., Soraku-gun, Waduka-cho, Sono, Y. Kubo 25 (TNS1226111); Wakayama Pref., Hi- apex scaly; scales dark brown, ovate-lanceolate gashimuro-gun, Nachikatsuura-cho, T. Fujiwara & K. or lanceolate with an opaque central band, ap- Ohora FT121023-14 (TNS1303519); Ibid., T. Fujiwara & proximately 2.5 mm × 0.5 mm, margin entire or K. Ohora FT121023-20 (TNS1303524); Nishimuro-gun, sparsely fimbriate. Fronds cespitose; stipe brown Shirahama-cho, Shagawa, T. Iwasaki s.n. (TNS833862); or purplish black, shiny, 0.5–2 mm in diameter, Tokushima Pref., Hiwasa-cho, Akamatsu, C. Abe 5544 (TNS397643); Saga Pref., Tosu-shi, Mt. Ishitaniyama, S. terete to tri- or tetragonous; lamina linear-lanceo- Tsutsui 3077 (TNS1228468); Nagasaki Pref., Sasebo- late, 10–20 cm × 2–4 cm, 1-pinnate; apex with a shi, Yoshii-cho, Naoya, S. Yamada s.n. (TNS1228476); reproductive bud. Rachis brown or purplish Miyazaki Pref., Nobeoka-shi, Kitagawa-cho, T. Fuji- black, shiny. Pinnae 20–35 pairs, alternate, ses- wara et al. FT130716-22 (TNS1303550); Ibid., T. Fuji- sile, trapeziform to falcate, 12–24 mm long, mar- wara et al. FT130716-25 (TNS1303546); Miyakonojo-shi, Yasuhisa-cho, Yunomoto, Y. Akagi s.n. (TNS1217102); gin on both sides deeply serrate, base asymmet- Kagoshima Pref., Izumi-shi, Nagareai, M. Shiroto s.n. ric, acroscopically auriculate to hastate, basiscop- (TNS403318); Tanegashima, Minamitane-cho, T. Kari- ically cuneate, apex subacute to obtuse. Veins yazaki s.n. (TNS514317); Yakushima, Funayuki, M. To- 18 Acta Phytotax. Geobot. Vol. 71
Fig. 4. Asplenium serratipinnae T. Fujiw. & Watano. A. Habit. (Location: Japan, Tokyo Pref. Hachijo Is. Mt. Mihara, Date: Dec. 7, 2017). B. Rhizome. C. Rhizome scales.
gashi s.n. (TNS148590); Tokunoshima, Tokunoshima- Pref., Isl. Okinawa, Kunigami-son, Mt. Yonaha, J. Hag- machi, R. Yoroi 4259 (TNS310348); Amamioshima, Yu- iniwa JH029626 (TNS403318). wandake, H. Tokashiki 4259 (TNS790095); Okinawa
Key to the species of the Asplenium normale complex in Japan
1a. Pinnae dissected ...... A. oligophlebium Baker 1b. Pinnae subentire to serrate ...... 2 2a. Fronds without buds on rachis ...... A. boreale (Ohwi ex Sa. Kurata) Nakaike 2b. Fronds with buds on rachis ...... 3 3a. Buds at apex and middle of rachis ...... A. shimurae (H.Itô) Nakaike 3b. Buds only at apex of rachis ...... 4 4a. Pinnae acroscopically auriculate to hastate, margin deeply serrate ...... A. serratipinnae T. Fujiw. & Watano 4b. Pinnae acroscopically truncate, margin subentire to crenate ...... A. normale D. Don February 2020 Fujiwara & al. — A New Allotetraploid Species in Asplenium normale Complex 19
Fig. 5. Asplenium serratipinnae T. Fujiw. & Watano. (T. Fujiwara et al. FT130716-9, holotype) A. Rhizome scales. B. Abaxial side of pinna (upper) and adaxial side of pinna (lower). C. Whole plant. D. Spore.
We thank A. Ebihara (National Museum of Nature and References Science, Tokyo) for permitting us to inspect herbarium specimens at TNS. We also thank two anonymous re- Chang, Y., A. Ebihara, S. Lu, L. Hongmei & H. Sch- viewers and the associate editor, whose comments greatly neider. 2018. Integrated taxonomy of the Asplenium improved the manuscript. This study was partly support- normale complex (Aspleniaceae) in China and adja- ed by a Grant-in-Aid for JSPS Fellows, Grant number cent areas. J. Plant Res. 131: 573–587. 15J03437, awarded to TF. Chang, Y., J. Li., S. Lu & H. Schneider. 2013. Species di- 20 Acta Phytotax. Geobot. Vol. 71
versity and reticulate evolution in the Asplenium nor- bundo, Tokyo (in Japanese). male complex (Aspleniaceae ) in China and adjacent PPG1. 2016. A community-derived classification for ex- areas. Taxon 62: 673–687. tant lycophytes and ferns. J. Syst. Evol. 54: 563-603. Dyer, R. J., V. Savolainen & H. Schneider. 2012. Apomix- R Development Core Team. 2013. R: A language and en- is and reticulate evolution in the Asplenium monan- vironment for statistical computing. R Foundation for thes fern complex. Ann. Bot. 110: 1515–1529. Statistical Computing, Vienna, Austria.
Received November 26, 2018; accepted May 14, 2019 February 2020 Fujiwara & al. — A New Allotetraploid Species in Asplenium normale Complex 21
Appendix 1. Voucher specimens of Asplenium normale and A. serratipinnae used for morphological analysis. All spec- imens were deposited in the National Museum of Nature and Science (TNS).
Asplenium normale, cryptic species I of Fujiwara et al. 26 (TNS1303585), -27 (TNS1303549), -29 (TNS1303587) (2017), (Allotetraploid between the diploid race of A. nor- & -31 (TNS1303589); Japan, Miyazaki Pref., Nobeoka- male and A. guangdongense—Japan, Kanagawa Pref., shi, Kitagawa-cho, T. Fujiwara et al. FT130716-17 Odawara-shi, Kuno, T. Fujiwara & T. Matsuoka (TNS1303545) & -20 (TNS1303551); Japan, Miyazaki FT120704-3 (TNS1303496) & -4 (TNS1303497); Shizuo- Pref., Nobeoka-shi, Kitakata-cho, T. Fujiwara et al. ka Pref., Kamo-gun, Nishiizu-cho, T. Fujiwara & H. Sato FT130716-15 (TNS1303544) & -16 (TNS1303545). FT120528-18 (TNS1303469) & -19 (TNS1303470); Mie Asplenium normale, cryptic species III of Fujiwara et Pref., Kumano-shi, Isato-cho, T. Fujiwara & K. Ohora al. (2017), (Allotetraploid plant between the diploid race FT121022-14 (TNS1303507) & -15 (TNS1303508); Mie of A. normale and A. guangdongense)— Japan, Wakaya- Pref., Kumano-shi, Kiwa-cho, T. Fujiwara & K. Ohora ma Pref., Higashimuro-gun, Nachikatsuura-cho, T. Fuji- FT121023-1 (TNS1303512) & -7 (TNS1303514); Wakaya- wara & K. Ohora FT121023-23 (TNS1303526) & -31 ma Pref., Higashimuro-gun, Nachikatsuura-cho, T. Fuji- (TNS1303533); Okayama Pref., Bizen-shi, Yagisan, T. wara & K. Ohora FT121023-12 (TNS1303518); Osaka Fujiwara et al. FT140512-11 (TNS1303603) & -13 Pref., Izumi-shi, Chichioni-cho, K. Tsuji FT120629-10 (TNS1303605). (TNS1303478) & -14 (TNS1303482); Osaka Pref., Izumi- Asplenium normale, tetraploid race from Taiwan, (Al- shi, Mt. Makio, K. Tsuji FT120629-6 (TNS1303475) & -7 lotetraploid plant between the diploid race of A. normale (TNS 1303476); Osaka Pref., Izumi-shi, Mt. Makio K. and A. guangdongense) —Taiwan, Chiayi County, Alis- Tsuji FT120629-1 (TNS1303471), -2 (TNS1303472) & -4 han, T. Fujiwara et al. 150701-3 (TNS1303662). (TNS1303473); Osaka Pref., Kawachinagano-shi, Mt. Asplenium normale, diploid race from Taiwan—Taiwan, Iwawaki, K. Tsuji FT120703-3 (TNS1303486) & -4 Taipei County, Mt. Chintan, T. Fujiwara et al. 150629-5 (TNS1303487); Hyogo Pref., Tanba-shi, Ichijima-cho, T. (TNS1303661); Chiayi County, Alishan, T. Fujiwara et al. Fujiwara et al. FT140512-94 (TNS1303648) & -95 150701-7 (TNS1303663); Pingtung County, Dahan Forest (TNS1303649); Hyogo Pref., Taka-gun, Taka-cho, T. Fuji- Road, T. Fujiwara et al. 150702-5 (TNS1303664), Ping- wara et al. FT140512-68 (TNS1303637) & -69 tung County, Jinshuiying, T. Fujiwara et al. 150702-14 (TNS1303638); Hyogo Pref., Kanzaki-gun, Ichikawa- (TNS1303665). cho, T. Fujiwara et al. FT140512-56 (TNS1303625) & -58 Asplenium serratipinnae sp. nov., cryptic species II of (TNS1303627); Hyogo Pref., Siso-shi, Yamazaki-cho, T. Fujiwara et al. (2017), (Allotetraploid plant between the Fujiwara et al. FT140512-50 (TNS1303619) & -51 diploid race of A. normale and A. oligophlebium) —Ja- (TNS1303620); Hyogo Pref., Kobe-shi, Kita-ku, Yamada- pan, Tokyo Pref., Hachijo Is., S. Matsumoto 130303-7 cho, T. Fujiwara et al. FT140512-5 (TNS1303597) & -7 (TNS1310410); Shizuoka Pref., Kamo-gun, Nishiizu-cho, (TNS1303599); Kochi Pref., Agawa-gun, Niyodogawa- T. Fujiwara & H. Sato FT120528-12 (TNS1303463) & -13 cho, M. Hyodo 20121231-1 (TNS1303666) & -2 (TNS1303464); Mie Pref., Kitamuro-gun, Kihoku-cho, T. (TNS1303667); Miyazaki Pref., Saito-shi, Kamiage, T. Fujiwara & K. Ohora FT121022-8 (TNS1303511); Fujiwara et al. FT130717-30 (TNS1303588) & -31 Wakayama Pref., Higashimuro-gun, Nachikatsuura-cho, (TNS1303589); Miyazaki Pref., Koyu-gun, Kijo-cho, T. T. Fujiwara & K. Ohora FT121023-14 (TNS1303519) & Fujiwara et al. FT130717-23 (TNS1303582) & -24 -20 (TNS1303524); Miyazaki Pref., Nobeoka-shi, Kitaga- (TNS1303583); Miyazaki Pref., Hyuga-shi, Togo-cho, wa-cho, T. Fujiwara et al. FT130716-22 (TNS1303550) & T. Fujiwara et al. FT130717-2 (TNS1303567), -5 -25 (TNS1303546); Miyazaki Pref., Nobeoka-shi, Okato- (TNS1303570), -9 (TNS1303573), -13 (TNS1303577), -16 mi-cho, T. Fujiwara et al. FT130716-8 (TNS1310411) & -9 (TNS1303552) & -18 (TNS1303578); Miyazaki Pref., (TNS1310242); Kagoshima Pref., Yaku Is., T. Fujiwara Nobeoka-shi, Kitaura-cho, T. Fujiwara et al. FT130716- FT130712-6 (TNS1303540) & -7 (TNS1303541).