ISSN 1346-7565 Acta Phytotax. Geobot. 71 (3): 243–248 (2020) doi: 10.18942/apg.201924
A New Form of the Mycoheterotrophic Plant Lecanorchis japonica (Orchidaceae) from Japan
1,* 2 3 2 Kenji Suetsugu , Shingo Kaida , Hirokazu Fukunaga and Shinichiro Sawa
1Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe, Hyogo 657-8501, Japan. *[email protected] (author for correspondence); 2Graduate School of Science and Technology, Kumamoto University, Kumamoto 860-8555, Japan; 3Tokushima-cho, Tokushima city, Tokushima 770-0852, Japan
A new color variant of Lecanorchis japonica Blume, L. japonica f. lutea (Orchidaceae), is described from Kagoshima and Chiba prefectures, Japan. Lecanorchis japonica f. lutea differs from L. japonica f. japonica only by its brilliant yellow coloration. Additionally, L. japonica f. lutea is superficially similar to L. hokurikuensis f. kiiensis but differs in having widely open flowers vs( . barely open flowers) and a nearly entire column wing (vs. apparent projections below column wing).
Keywords: color variation, Lecanorchis hokurikuensis f. kiiensis, Lecanorchis japonica f. lutea, taxonomy
The mycoheterotrophic genus Lecanorchis etsugu et al. 2016, 2017a,b, 2018a,c,d). Owing to Blume (Orchidaceae) comprises ca. 30 widely the difficulty in identifying Lecanorchis, species distributed species with endemic taxa reported diversity in the genus is likely to be underesti- from many regions of Southeast and Eastern Asia mated (Suetsugu et al. 2016, 2018c). In fact, dur- (Seidenfaden 1978, Lin 1987, Hashimoto 1990, ing recent botanical surveys in Japan, we discov- Pearce & Cribb 1999, Szlachetko & Mytnik 2000, ered flowering plants of an unknown Lecanor- Pridgeon et al. 2003, Averyanov 2005, 2011, chis. Although the samples were superficially 2013, Suddee & Pedersen 2011, Suetsugu & Fu- similar to L. hokurikuensis Masam. f. kiiensis kunaga 2016, Suetsugu et al. 2018a,b). Lecanor- (Murata) Seriz. in having a brilliant yellow-col- chis can be distinguished from other genera of or- ored plants, we found them to be genetically dif- chids by the numerous long, thick, horizontal ferentiated from L. hokurikuensis f. kiiensis. Af- roots that extend from a short rhizome, the long- ter careful consideration, we concluded that they lived thin stems, a cup-like structure (i.e., calycu- are more similar to L. japonica Blume than to L. lus) located between the base of the perianth and hokurikuensis (including L. hokurikuensis f. ki- apex of the ovary, and an elongate column that iensis). Therefore, we here propose them to be a bears a small wing on each side of the anther (Se- new forma of L. japonica. idenfaden 1978, Hashimoto 1990). Precise identification of the species of Lecanorchis is often hindered by their overall Materials and Methods morphological similarity and brief flowering pe- riods and herbarium specimens often lack impor- Morphological observation tant diagnostic characteristics. Flowers on her- To compare the recently discovered plants of barium material of Lecanorchis are easily lost or Lecanorchis, we undertook a thorough literature damaged during preservation (Reviewed by Su- review, conducted field sampling throughout Ja- 244 Acta Phytotax. Geobot. Vol. 71 pan and consulted specimens from multiple her- Results and Discussion baria (KAG, KANA, KPM, KYO, MBK, OSA, TI and TNS; abbreviations follow Index Herbari- Morphological analysis revealed that the un- orum; Thiers 2019, http://sweetgum.nybg.org/ known specimens of Lecanorchis were similar to science/ih/) as well as online digital images of plants of the L. japonica complex, including L. plant specimens on JSTOR Global Plants (http:// japonica, L. hokurikuensis, L. thalassica and plants.jstor.org/). their intraspecific taxon, and to L. purpurea Ma- sam., L. malaccensis Ridl. and L. sarawakensis Phylogenetic analysis Suetsugu & Naiki, in having relatively large Flowers of the unknown Lecanorchis and flowers (tepals ca. 20 mm long) with a tri-lobed those of its putative close relatives L. japonica, L. lip. The unknown specimen could be easily hokurikuensis, L. thalassica T. P. Lin and L. distinguished from L. purpurea, L. malaccensis, thalassica var. laoensis Suetsugu & T. C. Hsu and L. sarawakensis by its much longer rachis were collected and desiccated in the field using [>5 cm vs. <1.5 cm; (Hashimoto 1990, Serizawa silica gel (Hashimoto 1990, Serizawa 2005, Lin 2005, Lin et al. 2016, Suetsugu et al. 2018e]. In et al. 2016; Table 1). DNA was then isolated from this respect, the unidentified specimens of the silica-dried materials using the CTAB meth- Lecanorchis should belong to the L. japonica od (Doyle & Doyle 1987), and the rDNA internal complex (Hashimoto 1990, Serizawa 2005, Lin transcribed spacer (ITS) region was PCR-ampli- et al. 2016). fied in a 2720 Thermal Cycler (Applied Biosys- In particular, the unknown Lecanorchis spec- tems, USA) using 20-μL reaction mixtures that imens were superficially similar to L. hokurikue- contained 2-μL extracted DNA, 10-μL Sapphire- nsis f. kiiensis in having brilliant yellow-colored Amp Fast PCR Master Mix (Takara Bio, Japan), plants (Fig. 1). Due to the distinct color variation 0.2 μM of each primer (AB101 and AB102; Dou- (bright yellow in both floral and vegetative parts), zery et al. 1999), under the following conditions: L. hokurikuensis f. kiiensis was originally de- initial denaturation at 94 °C for 1 min; 30 cycles scribed as the distinct species, L. kiiensis (Mura- of 98 °C for 5 s, 55 °C for 5 s, and 72 °C for 15 s; ta 1973). However, color polymorphism within final elongation at 72 °C for 7 min. Finally, the the same species occurs in many mycoheterotro- amplified PCR products were purified using phic species (Fukunaga et al. 2008, Tsukaya & EconoSpin columns (Gene Design, Inc., Japan) Okada 2012, Suetsugu & Yagame 2014, Suetsugu and sent to Eurofins Genomics (Ebersberg, Ger- 2016a,b), likely due to selective pressure for pho- many) to be sequenced using the same primers tosynthesis and photoprotection pigments being used for PCR amplification. The obtained se- relaxed in mycoheterotrophs. In fact, similar col- quences were aligned by using ClustalW in or variants have been the basis for the description MEGA X (Kumar et al. 2018). The aligned se- of infraspecific taxa in other species of Lecanor- quences were used to construct phylogenetic rela- chis [i.e., L. suginoana (Tuyama) Seriz. f. flava tionships with MEGA X (Kumar et al. 2018) us- Seriz. and L. kiusiana Tuyama f. lutea Sawa, H. ing the Neighbor-joining (NJ) method. The reli- Fukunaga & S. Sawa; Serizawa 2005, Fukunaga ability of each node was assessed using bootstrap et al. 2008, Suetsugu 2012]. Hence, recent studies analysis (1,000 replicates). Lecanorchis kiusiana considered that it is preferable to treat L. kiiensis and L. suginoana were included as outgroups as an intraspecific taxon rather than a distinct (Suetsugu et al. 2019), since they are superficially species (Hashimoto 1990, Serizawa 2005). similar due to the existence of brilliant yellow- In spite of superficial similarity between the colored intraspecific variants although both spe- unknown plants and L. hokurikuensis f. kiiensis, cies differ greatly in morphology from the un- we found the unknown plants to differ morpho- known plants (Serizawa 2005, Fukunaga et al. logically from L. hokurikuensis and L. hokuri- 2008, Suetsugu 2012). kuensis f. kiiensis in having a nearly entire col- October 2020 Suetsugu & al. A New Form of Lecanorchis japonica 245
Table 1. Lecanorchis taxa included in the molecular analysis for the present paper. Collection GeneBank Taxon Location date Collection number numbers L. hokurikuensis JAPAN. Niigata Pref., Niigata City 20180614 R. Fujita KS292-1 (TNS) LC504443 L. hokurikuensis JAPAN. Niigata Pref., Niigata City 20180614 R. Fujita KS292-2 (TNS) LC504444 L. hokurikuensis JAPAN. Niigata Pref., Niigata City 20180614 R. Fujita KS292-3 (TNS) LC504445 L. hokurikuensis JAPAN. Osaka Pref., Kawachinagano City 20180613 I. Yamazumi Teramoto- LC504446 No.108-KS294 (TNS) L. hokurikuensis JAPAN. Kagawa Pref., Takamatsu City 20160601 H. Fukunaga 290 (MBK) LC504447 L. hokurikuensis f. kiiensis JAPAN. Niigata Pref., Niigata City 20180614 R. Fujita KS293 (TNS) LC504448 L. japonica JAPAN. Kagoshima Pref., Amami Island 20180421 H. Morita M24-003-KS258 LC457488 (KYO) L. japonica JAPAN. Kagoshima Pref., Amami Island 20180423 H. Morita Y13-16-KS259 LC457489 (KYO) L. japonica JAPAN. Kumamoto Pref., Chuo-ku, Kurokami-cho 20180521 A. Kinoshita KS263 (TNS) LC457490 L. japonica JAPAN. Osaka Pref., Minamikawachi-gun, 20180613 I. Yamazumi Azumazaka- LC457491 Chihayaakasaka Village No.12-KS297 (TNS) L. japonica f. lutea JAPAN. Kagoshima Pref., Amami Island 20190407 Y. Tashiro et al. AN016 (TNS) LC504449 L. japonica f. lutea JAPAN. Kagoshima Pref., Amami Island 20190414 Y. Tashiro et al. AN019 (TNS) LC504450 L. kiusiana JAPAN. Kyoto Pref., Kyoutanabe City 20160523 S. Mitsuta s.n. (KYO) LC457495 L. suginoana JAPAN. Shiga Pref., Higashiomi City 20160602 S. Mori s.n. (KYO) LC457492 L. thalassica TAIWAN. Nantou County 20090514 T. C. Hsu 2201 (TAIF) LC421225 L. thalassica var. laoensis LAOS. Xiangkhoang Province, Phonsavan 20180504 T. Nishioka KS257 (FOF) LC421224
umn wing (vs. apparent projections below the col- Taxonomy umn wing). The difference in column wing mor- phology is known diagnostic for distinguishing Lecanorchis japonica Blume f. lutea Suetsugu Lecanorchis hokurikuensis and L. japonica (Mu- & Fukunaga, forma nov.— Fig. 1. rata 1973, Hashimoto 1990, Serizawa 2005). It is Lecanorchis japonica f. lutea is the same as L. japonica f. also distinguishable from L. hokurikuensis f. ki- japonica in morphology and nrITS sequence, but differs iensis by having widely open vs. barely open only in the brilliant yellow coloration. flowers. The unknown plants are also distin- Typus. JAPAN. Kagoshima Pref., Amami-Oshima Is- guished from L. thalassica (including L. thalas- land, Amami City, Naze, 7 April 2019, Y. Tashiro, K. Ya- mamuro, C. Hara AN016 (holo- TNS, one flower in spirit sica var. laoensis) by the tepal apex (acute vs. ob- collection). tuse or slightly retuse), lip and column adnation (ca. one third of lip length vs. ca. half of lip length) Japanese name. Kibana-no-muyou-ran, nom. and floral condition (widely openvs . barely open). nov. The ITS sequences of the unidentified specimen and L. japonica were also identical, whereas Distribution. Lecanorchis japonica f. lutea is there were sequence divergences among the un- currently known only from two populations (in identified specimens,L. hokurikuensis (including Amami-Ohshima and Chiba). About 40 flower- L. hokurikuensis f. kiiensis) and L. thalassica ing individuals were at the type locality, which is (Fig. 2). The evidence indicates that the unidenti- dominated by Castanopsis sieboldii (Makino) fied specimens are closer toL. japonica than to L. Hatus. ex T. Yamaz. et Mashiba. The population hokurikuensis and L. thalassica. Both molecular is not sympatric with L. japonica f. japonica. and morphological data support describing the There were three flowering plants of L. japonica unknown plants as a intraspecific taxon of L. ja- f. lutea in dense forest dominated by Quercus ponica. glauca Thunb. and C. sieboldii in the Chiba pop- 246 Acta Phytotax. Geobot. Vol. 71
Fig. 1. Lecanorchis japonica f. lutea at Amami-Oshima Island, Kagoshima Prefecture, the type locality (A–C; photographed by Hidekazu Morita) and Chiba Prefecture (D–E; photographed by Emiko Kato), Japan. (A) Flowering plant. (B, D) Side view of flower. (C, E) Front view of flower.
Fig. 2. NJ tree of Lecanorchis japonica f. lutea and its close relatives based on nrITS sequences. Numbers above branches are bootstrap probabilities with 1,000 replicates (≥ 50 %). October 2020 Suetsugu & al. A New Form of Lecanorchis japonica 247 ulation. They were sympatric with a few plants of Murata, G. 1973. Taxonomical notes 11. Acta Phytotaxon. Lecanorchis japonica f. japonica. Geobot. 26: 144–148. Pearce, N. & P. Cribb. 1999. Notes relating to the flora of Additional specimens examined. JAPAN. Kagoshima Bhutan: XXXVII. New species and records of Orchi- Pref., Amami-Oshima Island, Amami City, Naze, 7 April daceae from Bhutan and India (Sikkim). Edinburgh J. 2019, Y. Tashiro, K. Yamamuro, C. Hara AN017 (TNS, Bot. 56: 273–284. spirit collection), Kagoshima Pref., Amami-Oshima Is- Pridgeon, A. M., P. J. Cribb, M. W. Chase & F. N. Ras- land, Amami City, Naze, 14 April 2019, Y. Tashiro, C. mussen. 2003. Genera Orchidacearum 3. Orchidoide- Hara, H. 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Received October 18, 2019; accepted December 30, 2019