ISSN 1346-7565 Acta Phytotax. Geobot. 71 (2): 177–184 (2020) doi: 10.18942/apg.201922

Interspecific Hybridization between australis and S. sinensis () in Southern Taiwan

1,†,* 2 3 4,† Kenji Suetsugu , Jing-Zhi Lin , Tian-Chuan Hsu and Hiroshi Hayakawa

1Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe, Hyogo 657- 8501, Japan. *[email protected] (author for correspondence); 2Department of Earth and Life Sciences, University of Taipei, Taipei 10048, Taiwan; 3Botanical Garden Division, Taiwan Forestry Research Institute, No. 53, Nanhai Rd., Taipei 100, Taiwan; 4Museum of Natural and Environmental History, Shizuoka, 5762 Oya, Suruga-ku, Shizuoka 422-8017, Japan. †These authors contributed equally to this work

Two closely related , and S. australis (Orchidaceae), have often been distin- guished based on the presence or absence of hairs in their and on the stems and ovaries: the stems and ovaries of S. australis are generally pubescent, whereas those of S. sinensis are reported to be glabrous. Although S. australis has not been documented to occur in Taiwan, we found a population of Spiranthes with slightly pubescent stems and ovaries at Tahanshan, southern Taiwan. Because the slight- ly pubescent may represent an undocumented extension of the distribution of S. australis, we at- tempted to identity the slightly pubescent plants by sequencing the ITS and trnL–F intergenic spacer regions. We also compared the lip and column morphology, which have diagnostic characteristics in Spi- ranthes, and the molecular and morphological data from S. australis, S. sinensis, their close relative, S. hongkongensis, and the unknown plants. The molecular analysis revealed the slightly pubescent plants to be heterozygous for the ITS sequences from S. australis and S. sinensis or from S. hongkongensis and S. sinensis, whereas the trnL–F intergenic spacer region was identical to that of S. sinensis. While we could not completely exclude the possibility that the unknown plants were hybrids between S. hongkon- gensis and S. sinensis based on the molecular data, we concluded that they were hybrids between the pubescent S. australis and the glabrous S. sinensis (Taiwan type) based on the lip and column morphol- ogy.

Keywords: Flowering phenology, hybrid, , Spiranthes sinensis species complex

Spiranthes L. C. Rich. (Orchidaceae) contains [syn. S. sinensis var. australis (R.Br.) H. Hara & ca. 50 species widely distributed across tropical S. Kitam. and S. sinensis var. amoena (M. Bieb.) and temperate regions of the Americas, Eurasia, H. Hara] and S. sinensis, have similar morpho- and Australia (Dueck et al. 2014, Pace & Camer- logical features with some overlap in both foliar on 2017, Surveswaran et al. 2017, 2018, Frericks and floral characteristics e.g( . Surveswaran et al. et al. 2018, Pace et al. 2019). The classification of 2017, Pace et al. 2019). Spiranthes australis has species within the genus is often complicated by often been treated as a synonym of S. sinensis morphological polymorphism. For example, S. si- (e.g. Surveswaran et al. 2017). However, Pace et nensis (Pers.) Ames s.l., has been intensively al. (2019) clearly showed that despite the morpho- studied taxonomically, morphologically, geo- logical resemblance, S. australis is genetically graphically, cytologically and ecologically (Vij & fully differentiated from S. sinensis and should Vohra 1974, Tsukaya 1994, 2005, Surveswaran et be treated as a distinct species. Spiranthes aus- al. 2017, 2018, Tao et al. 2018, Pace et al. 2019). tralis is therefore a cryptic species harboring ge- Within the S. sinensis species complex, two netic uniqueness, yet high morphological similar- closely related species, S. australis (R.Br.) Lindl. ity with S. sinensis. The presence or absence of 178 Acta Phytotax. Geobot. Vol. 71

Fig. 1. Flowering plants of hybrid between Spiranthes australis and S. sinensis from Tahanshan, Pingtung, Taiwan; hybrid with slightly pubescent inflorescence, stems and ovaries. hairs in the inflorescence and on the stems and for distinguishing Spiranthes australis and S. si- ovaries are the only reliable morphological trait nensis (Tsukaya 1994, 2005, Pace et al. 2019). June 2020 Suetsugu & al. — Hybridization between Two Spiranthes species 179

Table 1. The sequences obtained from Spiranthes spp. samples.

a a trnL–F ITS NCBI Accession Taxon and collection site 1 1 1 2 2 2 3 4 1 1 1 1 2 2 4 4 5 5 5 No. Reference Voucher ID 7 2 4 7 4 5 8 9 1 1 8 2 5 9 9 0 6 5 6 3 8 9 7 0 9 4 4 1 1 1 2 6 8 3 3 4 6 6 6 5 0 2 3 7 trnL-F ITS S. australis J. Yokoyama s.n. Japan: Hokkaido, Hidaka C G C A A C C C A G T C C A G C A ? ? ? ? ? AB187135 AB187151 Tsukaya 2005 (TUS) J. Yokoyama s.n. Japan: Hokkaido, Samani C G C A A C C C A G T C C A G C R ? ? ? ? ? AB187136 AB187152 Tsukaya 2005 (TUS) H. Tsukaya s.n. Japan: Shizuoka, Sessokyo C G C A A C C C A G T C C A G C A ? ? ? ? ? AB187137 AB187153 Tsukaya 2005 (TI) H. Tsukaya s.n. Japan: Aichi, Okazaki C G C A A C C C A G T C C A G C A ? ? ? ? ? AB187138 AB187154 Tsukaya 2005 (TI) H. Tsukaya et al. Japan: Hiroshima, Miyajima Isl. C G C A A C C C A G T C C A G C A ? ? ? ? ? AB187139 AB187155 Tsukaya 2005 s.n. (TI) H. Tsukaya s.n. Japan: Mie, Mt. Asama C G C A A C C C A G T C C A G C A ? ? ? ? ? AB187140 AB187156 Tsukaya 2005 (TI) F. Miyamoto Japan: Miyazaki, Takanabe C G C A A C C C A G T C C A G G A ? ? ? ? ? AB187141 AB187157 Tsukaya 2005 010101 (TI) Hayakawa et al. Japan: Ibaraki, Tsukuba (A) C G C A A C C C A G T C C A G C A G T G G T AB823668 AB740173 2013 Hayakawa et al. Japan: Ibaraki, Tsukuba (B) C G C A A C C C A G T C C A G C A R T G G T AB823669 AB740174 2013 Japan: Kochi, Nankoku Hayakawa et al. C G C A A C C C A G T C C A G C A G T G G T AB823667 AB740176 (puberulous) 2013 Japan: Kochi, Nankoku Hayakawa et al. MBK0235286 C G C A A C C C A G T C C A G C A G T G G T AB823666 AB740175 (glabrous) 2013 (MBK) Japan: Kumamoto, Suetsugu & H.Hayakawa108 C G C A A C C C A G T C C A G C A G T G G T LC155425 LC155423 Kamiamakusa (glabrous) Hayakawa 2016 (TNS) Japan: Kumamoto, Suetsugu & H.Hayakawa109 C G C A A C C C A G T C C A G C A G T G G T LC155425 LC155423 Kamiamakusa (glabrous) Hayakawa 2016 (TNS)

S. hongkongensis T.C. Hsu 7674 Taiwan: New Taipei G T C C A G G A G T G G C LC475429 Currect study (TAIF) Taiwan: New Taipei A C G C A T A G C LC475430 Currect study J.Z. Lin 1 (TAIF)

b Surveswaran Hong Kong G T C C A G G A G T G G C MH002639 et al. 2018

b Surveswaran Hong Kong G T C C A G G A G T G G T MH002630 et al. 2018

S. sinensis J. Yokoyama s.n. Japan: Okinawa, Iriomote Isl. A C C C C C A C C T Y Y C A T G A ? ? ? ? ? AB187148 AB187165 Tsukaya 2005 (TUS) Taiwan: New Taipei A C C C A C A C C G T T A - G G A G C G C T LC455736 LC455733 Currect study J.Z. Lin 2 (TAIF) T.C. Hsu 8998 Taiwan: Taipei G T T A - G G A G C G C T LC455732 Currect study (TAIF) T.C. Hsu 9045 Taiwan: Taichung A C C C A T A C C G T T A - G G A G C G C T LC455739 LC455735 Currect study (TAIF) Taiwan (Commercial cultivar) A C C C A T A C C G T T A - G G A G C G C T LC455737 LC455731 Currect study

Spiranthes sp. (hybrid between S. australis and S. sinensis) Taiwan: Pingtung Country A C C C A T A C C G T Y M A/- G G A G Y R S T LC455738 LC455734 Currect study J.Z. Lin 3 (TAIF) a Underlining indicates nucleotides that differ in identity from those of Japanese S. australis. b Only representative haplotype of cloned ITS sequences is shown. ? indicates nucleotides that unknown. - indicates nucleotides that is insertion or deletion. 180 Acta Phytotax. Geobot. Vol. 71

The two species also vary greatly in their geo- cided to sequence the ITS and trnL–F intergenic graphical distribution, at least in Japan and Tai- spacer regions of the slightly pubescent plants wan; Spiranthes australis is mainly on the Japa- and compare them to known sequences of S. aus- nese mainland (Northern Ryukyus and north- tralis, S. hongkongensis and S. sinensis. ward), whereas S. sinensis is restricted to the cen- tral and southern Ryukyus and Taiwan (Maeka- wa 1971, Satomi 1982). Materials and Methods While glabrous plants of Spiranthes have rarely been documented on the Japanese main- All the flowering individuals (~20) of Spiran- land, molecular analyses of the internal tran- thes that we observed in Tahanshan, Chunri scribed spacer (ITS) and trnL–F intergenic spac- Township, Pingtung Country, Taiwan had inflo- er regions suggest that glabrous plants on the Jap- rescence stems and ovaries with fewer hairs than anese mainland should be considered to be vari- in typical individuals of S. australis. To compare ants of S. australis rather than a range extension their morphology with typical S. australis and S. for S. sinensis (Hayakawa et al. 2013, Suetsugu & sinensis, we collected two plants from the Taiwan Hayakawa 2016). Although S. sinensis and S. population on 25 April 2017. We also examined australis co-occur on some islands in the central herbarium specimens of the S. sinensis species Ryukyus (e.g., Amami and Okinawa islands; Su- complex in several herbaria (TI, TNS, KYO, etsugu & Hayakawa, unpublished data), S. sinen- KPM, and OSA) and digital images in JSTOR sis on those islands blooms at least two months Global Plants (http://plants.jstor.org/). earlier (mostly in March) than S. australis (June For molecular analysis, we used the DNeasy to July), which suggests that there is little oppor- Mini Kit (Qiagen) according to the manu- tunity for the two species to hybridize. facturer’s protocol to isolate total DNA from the In 2017, we discovered a population of slightly leaves of one slightly pubescent plant of Spiran- pubescent plants of Spiranthes in Tahanshan, thes, two plants of S. hongkongensis and five Chunri Township, Pingtung Country, Taiwan plants of S. sinensis collected in Taiwan. We then (Figs. 1 & 2). We speculated that the pubescent amplified the ITS and trnL–F intergenic spacer plants represented a range extension for S. aus- regions, using the primers ITS4 and ITS5 (White tralis, since puberulous plants of Spiranthes had et al. 1990) and c and f (Taberlet et al. 1991), re- not been reported from Taiwan (Chung 2015, Hsu spectively. The 16-µL PCR solutions each con- & Chung 2016). However, because glabrous tained 0.5 µM of each forward and reverse prim- plants from the Japanese mainland are apparently er, 1.5 mM MgCl2, 0.2 mM of each dNTP, and glabrous variants of S. australis (Hayakawa et al. 0.05 units TaKaRa Ex Taq DNA polymerase in a 2013, Suetsugu & Hayakawa 2016), the pubescent 1× concentration buffer (Takara Bio Inc., Shiga, plants from Tahanshan might be pubescent vari- Japan) and were subject to the following thermal ants of S. sinensis, rather than a range extension cycle profile: 30 s at 95 °C, 30 s at 55 °C, and 1 for S. australis. Because the plants were only min at 72 °C for 35 cycles, followed by a 5-min slightly pubescent, we speculated that they could final extension at 72 °C. After amplification, 3 µL also be hybrids between S. australis and S. sinen- of each product was visualized on 1.0% agarose sis. Since another pubescent species within the S. gels. The PCR products were purified using Exo- sinensis species complex, S. hongkongensis Hu & SAP-IT (USB Corporation, Cleveland, OH, USA) Barretto, has been reported from Taiwan (Hsu & and were directly sequenced in both directions Chung 2014), they could also be hybrids between using a BigDye Terminator kit v.3.1 (Applied S. sinensis and S. hongkongensis. Biosystems, Foster City, CA, USA) and an ABI Since plastid trnL–F and nuclear ITS se- Prism 3130 Genetic Analyzer (Applied Biosys- quences can be used to distinguish species in the tems), following the manufacturer’s instructions, S. sinensis complex (e.g. Tsukaya 2005), we de- and the same primers used for amplification. The June 2020 Suetsugu & al. — Hybridization between Two Spiranthes species 181

Fig. 2. Closeup of ovaries and bracts. (A) Glabrous in Spiranthes sinensis collected in Taipei, Taiwan (KPM-NA010545). (B) Slightly pubescent in hybrid collected at Tahanshan, Pingtung, Taiwan (Lin 3; TAIF). Arrowheads point to hairs. (C) Densely pubescent in S. australis collected at Kanagawa, Japan (KPM-NA1072974). All bars = 1 mm.

Fig. 3. Closeup of column. (A–D) Side view. (E–H) Ventral view. (A, E) Spiranthes hongkongensis collected in Taipei, Taiwan (TAIF-446306). (B, F) S. sinensis collected in New Taipei, Taiwan (TAIF-469828). (C, G) Hybrid collected at Tahanshan, Pingtung, Taiwan (in present study). (D, H) Spiranthes australis collected in Hyogo, Japan (TNS-1302470). 182 Acta Phytotax. Geobot. Vol. 71

Table 2. Morphological comparison among hybrid between S. australis and S. sinensis and its morphologically similar spe- cies.

hybrid between Characters S. hongkongensis S. australis S. sinensis S. australis and S. sinensis

inflorescence stem pubescent pubescent slightly pubescent glabrous

lip status weakly recurved strongly recurved strongly recurved strongly recurved

stigma shape distinctly 3-lobed suborbiculate suborbiculate suborbiculate

ovate-truncate rostel- ovate-truncate rostellum, ovate-truncate rostel- membranous, narrow rostellum shape lum, acuminate and acuminate and bilobed at lum, acuminate and and truncate bilobed at the apex the apex bilobed at the apex

viscidium status absent well-developed well-developed well-developed resulting sequences were deposited in the DDBJ, sinensis. The trnL–F sequences from the slightly EMBL, and GenBank International DNA data- pubescent plants were identical with those from bases (Table 1). After adding the sequences of the individuals of S. sinensis from Taichung and three species determined by previous studies from a commercial cultivar in Taiwan (Table 1). (Tsukaya 2005, Hayakawa et al. 2013, Suetsugu While we could not completely exclude the & Hayakawa et al 2016, Surveswaran et al. 2018), possibility that the unknown plants were hybrids the obtained sequences were aligned by using between S. hongkongensis and Taiwanese S. si- ClustalW in the MEGA v.7 (Kumar et al. 2016). nensis based on the molecular data, we concluded that they might be hybrids between pubescent S. australis and glabrous S. sinensis (Taiwan type) Results and Discussion based on both molecular and morphological evi- dence. The slightly pubescent plants of Spiran- The ITS sequences that we obtained from the thes are morphologically similar, other than in Taiwanese plants of Spiranthes sinensis differed degree of pubescence, with S. australis and S. si- from the ITS sequences of S. australis at five nensis (Figs. 1–3, Table 2). In contrast, our plants sites: 123, 153, 194, 460, and 583 bp and of those differed greatly from S. hongkongensis in having from common type of S. hongkongensis at six an ovoid, truncate rostellum with a well-devel- sites: 123, 153, 194, 460, 583, and 597bp (Table 1). oped viscidium (vs. membranous, narrow, trun- Therefore, the sequences from the slightly pubes- cate rostellum attached to the middle of the pol- cent plants of Spiranthes were probably a combi- linia, without a viscidium) and suborbicular stig- nation of sequences between those of S. australis ma (vs. distinctly three-lobed stigma; Fig. 3) (Hu and Taiwanese S. sinensis (Table 1). However, we & Barretto 1976, Hsu & Chung 2014, Pace et al. note that seventy-two cloned ITS sequences from 2019, Suetsugu & Hayakawa 2019). The morpho- 11 individuals of S. hongkongensis in the DDBJ logical evidence indicated that our plants were tended to differ (66/72 sequences), but were not not hybrids between S. hongkongensis and S. si- fully distinguishable (6/72 sequences) from those nensis. of Taiwanese S. australis at the 597 bp site It is interesting to note that the ITS and trnL– (Surveswaran et al. 2018, Table 1). The results in- F sequences of plants of S. sinensis collected dicated that the slightly pubescent plants of Spi- from Iriomote Island and Taiwan were not identi- ranthes were more likely to be hybrids between cal (Table 1). Future research should attempt to S. australis and Taiwanese S. sinensis, but might determine whether S. sinensis in the Ryukyu Is- possibly be S. hongkongensis and Taiwanese S. lands and Taiwan contains multiple taxonomic June 2020 Suetsugu & al. — Hybridization between Two Spiranthes species 183 identities. It is also noteworthry that the ITS se- We thank Mr. Chih-Kai Fan for his assistance in the field study. We thank Mr. Toshiharu Mitsuhashi for providing quences of the slightly pubescent Spiranthes had a commercial cultivar of the Taiwanese Spiranthes sinen- R (A/G) at the 532 bp site, be we were unable to sis. We also thank the curators of TAIF, TI, TNS, KYO, find the sequences of the A type at the site in in- KPM, and OSA for access to their specimens. This work dividuals of S. australis, S. hongkongensis and S. was financially supported by the JSPS KAKENHI (Grant sinensis we used (Table 1), suggesting that addi- Number 17H05016). tional sequence diversity among them. 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Received October 28, 2019; accepted December 12, 2019