A peer-reviewed open-access journal PhytoKeys 220: 39-50 (2023) & Re nee $¢PhytoKeys https:/ / Pp hyto keys -pen soft.net Launched to accelerate biodiversity research Gastrodia bawanglingensis (Orchidaceae, Epidendroideae), a new species from Hainan Island, China Zhi-Heng Chen'”, Zhong-Yang Zhang'’, Xi-Qiang Song'??, Zhe Zhang!? | Key Laboratory of Genetics and Germplasm Innovation of Tropical Special Forest Trees and Ornamental Plants, Ministry of Education, Hainan University, Haikou 570228, China 2 College of Forestry, Hainan University, Haikou 570228, China 3 Key Laboratory of Biology of Tropical Flower Resources of Hainan Province, Haikou 570228, China Corresponding author: Xi-Qiang Song (songstrong@hainanu.edu.cn) Academiceditor:M.Simo-Droissart | Received2 1 September2022 | Accepted3 1 January2023 | Published23 February2023 Citation: Chen Z-H, Zhang Z-Y, Song X-Q, Zhang Z (2023) Gastrodia bawanglingensis (Orchidaceae, Epidendroideae), a new species from Hainan Island, China. PhytoKeys 220: 39-50. https://doi.org/10.3897/phytokeys.220.95 137 Abstract Gastrodia bawanglingensis, a new species of Orchidaceae from Hainan Island, China, is described and illustrated. It is morphologically similar to G. theana, G. albidoides and G. albida with dwarf habits, scarcely opening flowers, elongated fruit stems, curved and fleshy perianth tubes and similar columns and lips, but can be easily distinguished from them by having a pair of lateral wings bent outwards at the apex of the column and lateral wings with acuminate tips lower than the anther. According to the IUCN Red List Categories and Criteria, the new species is assessed as Endangered (EN). The plastome of G. bawanglingensis is greatly reduced and reconfigured with approximately 30876 bp in size and 25.36% in GC content. Morphological characteristics and molecular phylogenetic results based on chloroplast gene sequences support the recognition of G. bawanglingensis as a new species within Gastrodia. Keywords Gastrodieae, Hainan Tropical Rainforest National Park, holomycotrophic orchids, taxonomy, tropical rainforest Introduction Gastrodia Brown (1810: 330) (Epidendroideae, Gastrodieae) comprises approximately 100 species and is widespread from northeast India through the eastern Himalayas and southern China to Japan and eastern Siberia, southwards to Malaysia and Aus- Copyright Zhi-Heng Chen et al. 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. 40 Zhi-Heng Chen et al. / PhytoKeys 220: 39-50 (2023) 08°30'0"E 109°0'0" E 109°30'0"E 110°0'0" E 110°30'0"E 111°O'0"E 111°30'0"E 20°30'0" N 20°30'0"N North Bay Qiongzhou Strait 20°0'0"N 20°0'0"N South China Sea Hainan Island 19°30'0"N 19°30'0"N Legend G. sp. nov. @ 19°0'0"N 19°0'0"N G, punctata & G. longitubularis @ G. menghaiensis @ Hainan Island [__] Hainan Tropical Rainforest National Park 18°30'0"N 18°30'0"N 0 20 40 80 es ki 08°30'0" E 109°0'0" E 109°30'0"E 110°0'0"E 110°30'0"E 111°0'0"E 111°30'0"E Figure |. Pictures and distribution of Gastrodia species in Hainan Island based on our field investigation in the past three years. G., Gastrodia A G. sp. nov. B G. punctata C G. longitubularis D G. menghaiensis. tralia, eastwards to the Pacific Islands as far as Samoa and westwards to Madagascar, Mascarene Islands and tropical Africa (Pridgeon et al. 2005; Chen et al. 2009; Cribb et al. 2010; Chase et al. 2015; Jin and Kyaw 2017; Suetsugu 2019, 2021; Bandara et al. 2020; Liu et al. 2021). There are 33 known species (16 endemic) of Gastrodia in China, mainly distributed in southern China, including Tibet, Fujian, Hainan, Yun- nan, Sichuan and Taiwan (Liu et al. 2021; Zhou et al. 2021). In Hainan Island, three species, namely Gastrodia longitubularis Q.W.Meng, X.Q.Song & Y.B.Luo (Meng et al. 2007), G. punctata Aver. (Lu et al. 2017) and G. menghaiensis Z.H.Tsi & S.C.Chen (Huang et al. 2021), have been reported from the tropical rainforest (Fig. 1). During our field investigation in April 2021, Gastrodia specimens with signifi- cantly different floral morphology from all the known species in China were collected in the forests of Bawangling, Hainan Tropical Rainforest National Park. Further stud- ies, based on examination of specimens and literature of Gastrodia (Averyanov 2005; Hsu and Kuo 2011; Tan et al. 2012) and comparison with type specimens, showed that those specimens represent a new species that is morphologically distinct from previously-known taxa of the genus Gastrodia and is described below. Materials and methods DNA extraction and sequencing The next generation sequencing technology (high-throughput sequencing) was applied to extract the total genomic DNA of plant materials and chloroplast splicing software GetOrganelle was used to assemble the plant genome (Jin et al. 2020). Moreover, on- line annotation software Geseq (https://chlorobox.mpimp-golm.mpg.de/geseq. html) A new species of Gastrodia (Orchidaceae) is described 41 (Tillich et al. 2017) and CpGAVAS (http://www.herbalgeno-mics.org/cpgavas) (Liu et al. 2012) were used to determine the chloroplast genome start position and IR region and annotate the genes on the chloroplast genome. Finally, we used manual proofreading to verify the correctness of the annotations, according to the reference of NC_024662.1. Phylogenetic analysis To estimate the phylogenetic position of the Gastrodia sp. nov. within Gastrodia, phy- logenies were reconstructed by Maximum likelihood (ML) and Bayesian Inference (BI) analyses using the coding sequences (CDSs). All plastomes were downloaded from the NCBI database except Gastrodia sp. nov. (Wen et al. 2022). In the phylogenetic tree, Epipogium roseum (D.Don) Lindl. and Didymoplexis pallens Griff. were selected as outgroup; Epipogium belongs to Nervilieae, a sister tribe to Gastrodieae while Didymo- plexis is sister to Gastrodia (Wen et al. 2022). The sequences of the species and related ones were aligned in MAFFT version 7 (https://mafft.cbre.jp/alignment/server/) using MAFFT (Katoh and Standley 2013) by default setting. Phylogenetic construction was conducted by Maximum Likelihood with MEGA11 software (Tamura et al. 2021), selecting the best-fit model of GTR+G with 1000 bootstraps (Nei and Kumar 2000), and Bayesian Inference (BI) tree in MrBayes 3.2.7 using the GTR+G model (Ronquist et al. 2012), runs for 20 million generations. Phylogenetic trees were sampled every one thousand generations, the first 25% of trees generated were discarded as burn-in and the remaining trees were used to construct majority-rule consensus tree. Finally, the tree file was visualised and annotated on iTOL (https://itol.embl.de/) (Ivica and Peer 2021). All the sequences’ accession numbers were listed in Fig. 2. Morphological description Morphological observations of Gastrodia sp. nov. were based on living plants (four in- dividuals) and dried herbarium specimens all belonging to the type specimen, which is Tree scale: 0.1: -- E. roseum NC026448.1 D. pallens ON515488.1 Outgroup --------G, javanica ON515482.1 ; cit peer cee, G, elata MF 163256, 1 aus Woes G. angustaON515479.1 vnooer G. Alexistyla ON515480.1 . | sa G. crispaON515481.1 roopouG, Uraiensis ON515487.1 Gastrodia ot aC. sp. nov. OP219766.1 aneo favor CG. sp. ON515486.1 lor G. shimizuana ON515485.1 vam FG. longistyla ON515483.1 61 a'G, peichatieniana ON515484.1 “eG. menghaiensis ON515489.1 Figure 2. Phylogenetic tree reconstruction of Gastrodia using the maximum likelihood (ML) method based on chloroplast gene sequences of Gastrodia sp. nov. and 11 other species. Only the ML tree is shown, because its topology is nearly identical to that of the obtained BI tree. Numbers associated with the branches are BI posterior probabilities (PP) and ML bootstrap value (BS). The species name is followed by the accession number of the GenBank accession. D, Didymoplexis; E, Epipogium; G, Gastrodia. 42 Zhi-Heng Chen et al. / PhytoKeys 220: 39-50 (2023) kept in the HUFB (Teaching Herbarium of the College of Forestry, Hainan Universi- ty). All length and width of structures were measured by vernier calipers. Morphologi- cal characters of the new species were based on dried herbarium specimens. Further- more, we examined the type specimens of Gastrodia albidoides YH.Tan & T.C.Hsu, which is the most morphologically similar species to Gastrodia sp. nov. and housed in HFTC. High resolution photographs of living plants were provided by Zhong-Yang Zhang and Zhi-Heng Chen. Results Plastome of Gastrodia sp. nov. The plastome of the novelty is 30876 bp in length with its GC content approximately 25.36% (GenBank accession number: OP219766) (Fig. 3), which is similar to the 11 other species of Gastrodia (29,696-36,812 bp, Table 1). The plastome contains 19 protein-coding genes, five transfer RNA and three ribosomal RNA genes. Several genes and typical plastome regions appear to have been either lost or pseudogenised in G. sp. nov. The G. sp. nov. plastome does not contain housekeeping genes and lacks an IR re- gion. This indicates that plastomes of Gastrodia are in the last stages of plastome degra- dation (see Barrett and Davis 2012; Liu et al. 2021; Jiang et al. 2022; Wen et al. 2022). Gastrodia sp. nov chloroplast genome 30,876 bp ribosomal proteins (SSU) ff ribosomal proteins (LSU) i transfer RNAs i ribosomal RNAs i clpP, matK i other genes CJ hypothetical chloroplast reading frames (ycf) Figure 3. Plastome of Gastrodia sp. nov. A new species of Gastrodia (Orchidaceae) is described 43 Table |. Information on the chloroplast genomes of Gastrodia sp. nov. and other 11 species of Gastrodia. Species Length of chloroplast genome (bp) GC content (%) Number of genes Gastrodia angusta 36.812 25.4 19 5 4 Gastrodia crispa 30.582 25.7 19 5 4 Gastrodia elata 35.304 26.8 20 5 3 Gastrodia flexistyla 30.797 25.4 19 5 4 Gastrodia javanica 31.896 24.8 18 4 4 Gastrodia longistyla 30.464 24.8 18 5 3 Gastrodia menghaiensis 30.118 24.9 19 4 3 Gastrodia peichatieniana 29.696 25.9 18 5 4 Gastrodia shimizuana 30.019 25.5 18 5 4 Gastrodia sp. 29.944 25.8 18 5 4 Gastrodia sp. nov. 30.876 25.4 19 5 3 Gastrodia uraiensis 30.746 24.9 19 5 4 Phylogenetic analysis Our ML and BI phylogenetic trees constructed from the chloroplast gene sequences showed that the novelty belongs to the genus Gastrodia, and is related to G. uraiensis, G. flexistyla and G. crispa. Taxonomic treatment Gastrodia bawanglingensis Z.H.Chen, Z.Y.Zhang & X.Q.Song, sp. nov. urn:lsid:ipni.org:names:77314677-1 Fig. 4 Type. Cuina. Hainan Province: Bawangling, Hainan Tropical Rainforest National Park, in tropical rainforest, 850-950 m elevation, 25 April 2022, Z.Y. Zhang 006 (Holotype, HUFB!). Diagnosis. Gastrodia bawanglingensis is similar to G. albidoides with dwarf habits, scarcely opening flowers, elongated fruit stems, curved and fleshy perianth tubes and similar columns and lips, but can be easily distinguished from the latter by having lat- eral sepals adnate to 4/5 of total length (vs. lateral sepals adnate to 1/2 of total length), lip with four ridges (vs. lip with two ridges), the absence of a column foot (vs. the presence of a column foot) and a pair of lateral wings bent outwards (vs. lateral wings upright) at the column apex (Table 2). Description. Terrestrial, leafless, achlorophyllous herbs. Roots few, slender, 1-7 cm long, ca. 0.5-0.7 mm in diameter. Rhizome fleshy, tuberous, fusiform, 3-4 cm long, 5-7 mm in diameter, dark brown, covered with numerous scales. Scales verticillate, lanceolate, dark brown,1—2 mm long. Inflorescence erect, termi- nal, 2.0—6.5 cm long, ca. 2.2 mm in diameter, white to orange-brown, peduncle 3—4 noded, ovate to broadly ovate, sheath membranous, 3—5 x 2—3 mm; rachis often 44 Zhi-Heng Chen et al. / PhytoKeys 220: 39-50 (2023) less than 5 mm long. Bracts membranous, ovate to ovate-oblong, apex pointed, pale yellowish-brown, 4-6 mm long, 1.5-3 mm wide. Ovary 3-6 mm long, 2-3 mm in diameter. Flowers (1—) 2—4 (—6), erect, bell-shaped, slightly curved, not opening widely, 8-10 mm long, 4—5 mm in diameter. Flowers whitish on both surfaces, apex brownish, lip red at the base, light green at the middle, reddish-brown apically and marginally; column white. Sepals and petals united, forming a 5—lobed perianth tube, 8-10 mm long, slightly verrucous in the middle and upper part, distinctly verrucose apically. Sepals fleshy, thickened, similar. Lateral sepals fused to 4/5 of their length, whitish on both surfaces, apex is brownish; free lobe of dorsal sepal triangle, ca. 2.5 x 2.0 mm; free lobes of lateral sepals ovate, ca. 2.0 x 2.0 mm. Petals connate with sepals, free portions brownish, whitish on both sides, triangular-ovate, ca. 1.5 mm long, 1 mm wide, connate portions distinctly thickened and the inside is obviously reddish-brown, forming a pair of ridge-like structures inside the perianth tube and the other side of the ridge-like structures is flesh-coloured. Lip rhombic-ovate, base adnate to perianth tube, 3.5—4.5 x 2.0—2.2 mm; hypochile with two whitish, globose, subsessile, nectarless calli, ca. 0.5 mm in diameter; epichile 5—7 nerved, truncate at base, entire, disc thickened with four ridges, a pair of low ridges outside the two main ridges; the two main ridges fused into one before reaching the tip, main ridges much raised and tinged orange near apex. Column 4.2—4.5 x 1.6—1.8 mm, apex with a pair of lateral wings bent outwards; lateral wings with acuminate tips lower than anther; column foot absent; rostellum 0.2 x 1 mm; stigma located near base. Anther hemi- spherical, 0.6-0.7 mm in diameter; pollinia 2. Capsule ellipsoid, 1.2—1.8 cm long, 0.5—0.8 cm in diameter; pedicel elongating to 10-25 cm in fruit. Seeds fusiform, 1.6—2.2 mm long. Etymology. The new species is named after Bawangling, the mid-west State of Hainan Island where it was discovered in a vast area of primitive montane rainforest. Vernacular name. #3) EIQ (Chinese pinyin: ba wang ling tian m4). Distribution and habitat. Gastrodia bawanglingensis is a terrestrial mycohetero- trophic species that grows in montane rainforests which are dominated by Dysoxylum gotadhora (Buch.-Ham.) Mabb., Livistona saribus (Lour.) Merr. and A.Chev., Hancea hookeriana Seem. and Engelhardia roxburghiana Lindl. at elevations from 850 m to 950 m and associated with other orchids, such as Anoectochilus hainanensis H.Z. Tian, EW.Xing & L.Li, A. roxburghii (Wall.) Lindl., Oxystophyllum changjiangense (S.J. Cheng & C.Z.Tang) M.A.Clem., Dendrobium hainanense Rolfe, Cymbidium kanran Makino and Micropera poilanei (Guillaumin) Garay. So far, only the type subpopulation has been found in the tropical rainforest of Bawangling, in Hainan. Conservation status. Endangered [EN D1]. Gastrodia bawanglingensis was dis- covered in the mountain rainforest of Bawangling in Hainan Tropical Rainforest Na- tional Park. Until now, only the type subpopulation, consisting of ca. 100 individuals, has been discovered in Bawangling. Since its number of mature individuals is fewer than 250, we assess it as Endangered (EN) using criterion D1 (IUCN Standards and Petitions Subcommittee 2022). Phenology. Gastrodia bawanglingensis was observed flowering and fruiting in April and May. A new species of Gastrodia (Orchidaceae) is described 45 Figure 4. Gastrodia bawanglingensis Z.H.Chen, Z.Y.Zhang & X.Q.Song, sp. nov. A plant B flowers C flattened perianth tube D lip, column and ovary E column F, G lip H fruiting specimen. Illustration by Ling-Yi Cao, based on the holotype of Z.Y. Zhang 006 (HUFB). 46 Zhi-Heng Chen et al. / PhytoKeys 220: 39-50 (2023) Table 2. Differences between Gastrodia bawanglingensis, G. albidoides, G. theana and G. albida. Character G. bawanglingensis G. albidoides G. theana G. albida Perianth tube] slightly verrucous in the middle slightly verrucose distinctly striate and verrucose | distinctly verrucose and upper part, distinct verrucose | towards apex, otherwise throughout throughout apically smooth Lateral sepals adnate, to 4/5 of their length adnate, to 1/2 adnate, 1/3—1/4 their length adnate, 1/5—1/6 their length their length Petals brownish, fleshy, petals whitish on | whitish, thin in texture, salmon-pink, thin in whitish outside, both sides, triangular-ovate, ca. triangular-ovate, texture, narrowly triangular, | orange inside, fleshy, 1.5 x 1.0 mm 0.8—1.0 x 0.6-0.8 mm 0.4-0.8 x 0.2-0.3 mm oblong-ovate, ca. 1.5 x 1.0mm Lip red at the base, light green at the pale green, epichile green, epichile ovate, white, epichile middle, reddish-brown apically and rhombic-ovate, 5-nerved, disc slightly elevate triangular, disc marginally, epichile rhombic-ovate, 6—7-nerved, disc longitudinally at middle, thickened with two 5-7 nerved, disc thickened with four} thickened with two with four ridges four ridges, ridges, truncate ridges, a pair of low ridges outside ridges, rounded at arranged one behind the other. | at base; hypochile the two main ridges. truncate at base, hypochile with cordate at base, hypochile with two whitish, base, hypochile with two whitish, two whitish, globose, with two whitish, globose, | globose, subsessile, globose, subsessile, nectarless calli, | subsessile, nectarless calli,| subsessile, nectarless calli, nectarless calli, ca. 0.5 mm in diameter ca.1 mm in diameter ca.0.8 mm in diameter ca.1 mm in diameter Column apex with a pair of lateral wings | apex with a pair of lateral) apex with a pair of lateral | with a pair of lateral bent outwards; lateral wings with | wings; lateral wings with | wings bent inwards; lateral | wings distally; edges acuminate tips lower than anther | acuminate tips superior | wings with acuminate tips of lateral wings to anther superior to anther parallel to column Column foot Absent 1.5-1.8 mm 1.5-1.8 mm column foot very short Rostellum 0.2 x 1.0 mm 0.2 x 1.5 mm 0.2 x 1.5 mm Absent Pollination implication. Flowers of Gastrodia bawanglingensis barely open and pol- len massulae were observed on the stigma when flowers were dissected. Through field observation, it was found that the fruiting rate is very high. We bagged buds on 3 plants with 10 flowers in total prior to the anthesis, and found that each of them has evolved into fruit after 15 days. These observations indicate that the new species probably self- pollinates. Gastrodia is probably the only genus that contains species with completely cleistogamous flowers as confirmed by intensive monitoring. Self-pollination might be an adaptation to ensure reproduction, compensating for the defi- ciency of pollinators in the habitat (Suetsugu 2022; Suetsugu et al. 2022). Currently, complete cleistogamy has been reported in five Gastrodia species: G. clausa, G. takeshimensis, G. flexistyloides, G. kuroshimensis and G. amamiana (Hsu et al. 2012; Suetsugu 2013, 2014, 2016, 2019), G. bawanglingensis is likely to be the sixth species reported. Similar to other five species, G. bawanglingensis is also distributed on the island, further confirming island coloniza- tion may be one of the factors of evolution of complete cleistogamy. And compared with the mainland, there are more frequent geological and climate changes on the island, which may cause the rapid change of its living environment and lead to the loss of pol- linators in its distribution area. Unreliable pollinator services and the cost of maintaining open flowers probably drove the completely cleistogamous Gastrodia species to abandon insect-mediated pollination (Suetsugu 2014, 2016). However, complete cleistogamy has arguably driven speciation (Kishikawa et al. 2019; Ogaki et al. 2019). We also found sev- eral other unpublished species that are different but very similar to G. bawanglingensis in A new species of Gastrodia (Orchidaceae) is described 47 our field survey in Hainan Island, which also confirms the above point of view. It is also notable that although lack of rostellum often facilitates selfing in the genus (Suetsugu 2022; Suetsugu et al. 2022), the new species has somewhat well-developed rostellum. Further observations are needed on how the species accomplishes autogamy. Discussion Gastrodia bawanglingensis is most similar to G. albidoides (Tan et al. 2012) from Yun- nan, G. theana (Averyanov 2005) from Vietnam and G. albida (Hsu and Kuo 2011) from Taiwan. They share dwarf habits, scarcely opening flowers, fleshy curved perianth tubes with verruca and similar columns and lips. After comparison of available litera- ture and specimens, we conclude that G. bawanglingensis could be clearly differentiated from G. albidoides, G. theana and G. albida by several floral characters (Table 2). Key to the species of Gastrodia found in Hainan Island, China 1 Sepals adnate to 4/5 of their length; lip light green at the middle, reddish- brown apically and marginally; lip disc with two ridges ranging from base to NOL Gh rrr Wea tree a Myr nN TE RAN RAS Me aoe ES Bree G. bawanglingensis - Sepals adnate up to 1/2 of their length; lip green or white at the middle, uni- form coloured or orange-red towards apex; lip disc without distinct ridges, Butewithe ave lacor keels cette Ws tiecvnt slacks obacet pener tec ohesdvensction vets ha cioetdeeet 2 2 Flowers white, sub-erect; petals margin wrinkled; column foot very short, pedicelclongated in: frit 1s. ctv. a. Juctpnese sate cabpateobeaetes G. menghaiensis = Flowers grey-brownish, horizontal or slightly bending; petals margin entire, column foot distinct; pedicel not elongated in fruit 0.0... ccc eeeereeeeeeee 3 3 Tepal tube without white spots; column cylindrical and thick; lip disc with a pair of longitudinal lamellae near apex... eee eeeeeeeeee G. longitubularis = Tepal tube with white spots; column flat and thin; lip disc with four keels... Labi dinette ncetsth lida kas bi thestlimh Relat atti cmon enmecntbactin wide G. punctata Acknowledgements We acknowledge the support from the Bawangling Region of the Hainan Tropical Rainforest National Park. We are very grateful to Prof. Yi-Bo Luo and Prof. Xiao-Hua Jin for their guidance. Mrs. Ling-Yi Cao is thanked for preparing the illustrations. We also thank Mrs. Min-Ting Jin and Mrs. Meng-Xue Wang for facilitating the check- ing of specimens and Mr. Jin-Qiang Wang from Hainan Tropical Rainforest National Park for his kind help with fieldwork. This study was supported by the Project for Orchidaceae Plant Resources Special Investigation of National Forestry and Grassland Administration (Grant No.2020070708). 48 Zhi-Heng Chen et al. / PhytoKeys 220: 39-50 (2023) References Averyanov LV (2005) New orchids from Vietnam. Rheedea 15: 1-19. Bandara C, Priyankara T, Atthanagoda AG, Lakkana T, Ediriweera S, Kumar P (2020) Gastrodia gunatillekeorum (Gastrodieae, Epidendroideae, Orchidaceae), a new species from a lowland rainforest of Sri Lanka. Phytotaxa 436: 55-62. https://doi.org/10.11646/phy- totaxa.436.1.5 Barrett CF, Davis JI (2012) The Plastid Genome of the Mycoheterotrophic Corallorhiza striata (orchidaceae) Is in the Relatively Early Stages of Degradation. American Journal of Botany 99(9): 1513-1523. https://doi.org/10.3732/ajb. 1200256 Brown R (1810) Prodromus Florae Novae Hollandiae, et Insulae van Diemen. Johnson, Lon- don, 446 pp. Chase MW, Cameron KM, Freudenstein JV, Pridgeon AM, Salazar G, Van den Berg C, Schu- iteman A (2015) An updated classification of Orchidaceae. Botanical Journal of the Lin- nean Society 177(2): 151-174. https://doi.org/10.1111/boj.12234 Chen XQ, Gale SW, Cribb PJ (2009) Gastrodia. In: Wu ZY, Raven PH, Hong DY (Eds) Flora of China (Vol. 25). Science Press and Missouri Botanical Garden Press, Beijing and St. Louis, 201-205. http://www. iplant.cn/info/Gastrodia?t=foc Cribb P, Fischer E, Killmann D (2010) A revision of Gastrodia (Orchidaceae: Epidendroideae, Gastrodieae) in tropical Africa. Kew Bulletin 65: 315-321. https://link.springer.com/arti- cle/10.1007/s12225-010-9193-4 Hsu T-C, Kuo C-M (2011) Gastrodia albida (Orchidaceae), a New Species from Taiwan. An- nales Botanici Fennici 48(3): 272-275. https://doi.org/10.5735/085.048.0308 Hsu T-C, Chung S-W, Kuo C-M (2012) Supplements to the Orchid Flora of Taiwan 57(VI): 1-7. Huang M-Z, Wang Y, Wang Q-L, Li S-N, Chen Z, Yang G-S (2021) Miscellaneous Notes on Orchidaceae from Hainan (III). Redai Zuowu Xuebao 42(3): 703-706. IUCN Standards and Petitions Subcommittee (2022) Guidelines for Using the IUCN Red List Categories and Criteria. Version 13. Prepared by the Standards and Petitions Subcommit- tee. http://www.iucnredlist.org/documents/RedListGuide-lines.pdf Ivica L, Peer B (2021) Interactive Tree Of Life ((TOL) v5: An online tool for phylogenetic tree display and annotation. Nucleic Acids Research 49(W 1): W293-W296. https://doi. org/10.1093/nar/gkab301 Jiang Y, Hu X-D, Yuan Y, Guo X-L, Chase MW, Ge S, Li J-W, Fu J-L, Li K, Hao M, Wang Y-M, Jiao Y-N, Jiang W-K, Jin X-H (2022) The Gastrodia menghaiensis (Orchidaceae) ge- nome provides new insights of orchid mycorrhizal interactions. BMC Plant Biology 22(1): 179. https://doi.org/10.1186/s12870-022-03573-1 Jin X-H, Kyaw M (2017) Gastrodia putaoensis sp. nov. (Orchidaceae, Epidendroideae) from North Myanmar. Nordic Journal of Botany 35(6): 730-732. https://doi.org/10.1111/ njb.01581 Jin J-J, Yu W-B, Yang J-B, Song Y, dePamphilis CW, Yi T-S, Li D-Z (2020) GetOrganelle: A fast and versatile toolkit for accurate de novo assembly of organelle genomes. Genome Biology 21(1): 241. https://doi.org/10.1186/s13059-020-02154-5 A new species of Gastrodia (Orchidaceae) is described 49 Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution 30(4): 772-780. https://doi.org/10.1093/molbev/mst010 Kishikawa K, Suetsugu K, Kyogoku D, Ogaki K, Iga D, Shutoh K, Isagi Y, Kaneko S (2019) Development of microsatellite markers for the completely cleistogamous species Gastrodia takeshimensis (Orchidaceae) that are transferable to its chasmogamous sister G. nipponica. Genes & Genetic Systems 94(2): 95-98. https://doi.org/10.1266/ggs.18-00057 Liu C, Shi L, Zhu Y, Chen H, Zhang J, Lin X, Guan X (2012) CpGAVAS, an integrated web server for the annotation, visualization, analysis, and GenBank submission of com- pletely sequenced chloroplast genome sequences. BMC Genomics 13(1): 715. https://doi. org/10.1186/1471-2164-13-715 Liu Q, Ya J-D, Wu X-E, Shao B-Y, Chi K-B, Zheng H-L, Li J-W, Jin X-H (2021) New taxa of tribe Gastrodieae (Epidendroideae, Orchidaceae) from Yunnan, China and its conservation implication. Plant Diversity 43(5): 420-425. https://doi.org/10.1016/j.pld.2021.06.001 Lu G, Hu A-Q, Xiao Y, Huang J-Q, Zhen X-L (2017) Gastrodia punctata Aver., a newly tre- corded species of Orchidaceae from China. Guihaia 34(2): 228-230. Meng Q-W, Song X-Q, Luo Y-B (2007) A new species of Gastrodia (Orchidaceae) from Hainan Island, China and its conservation status. Nordic Journal of Botany 25(1—2): 23-26. https://doi.org/10.1111/j.0107-055X.2007.00067_17.x Nei M, Kumar S (2000) Molecular Evolution and Phylogenetics. Oxford University Press, Oxford, 333 pp. https://www.nature.com/articles/688923 1 Ogaki K, Suetsugu K, Kishikawa K, Kyogoku D, Shutoh K, Isagi Y, Kaneko S (2019) New microsatellite markers recognize differences in tandem repeats among four related Gas- trodia species (Orchidaceae). Genes & Genetic Systems 94(5): 225-229. https://doi. org/10.1266/ges.19-00025 Pridgeon AM, Cribb PJ, Chase MW, Rasmussen FN (2005) Genera Orchidacearum (Vol. 4). Epidendroideae (Part one). Oxford University Press, Oxford, 444 pp. Ronquist E, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: efficient bayesian phylogenetic infer- ence and model choice across a large model space. Systematic Biology 61(3): 539e542. https://doi.org/10.1093/sysbio/sys029 Suetsugu K (2013) Gastrodia takeshimensis (Orchidaceae), a New Mycoheterotrophic Species from Japan. Annales Botanici Fennici 50(6): 375-378. https://doi.org/10.5735/085.050.0613 Suetsugu K (2014) Gastrodia flexistyloides (Orchidaceae), a new mycoheterotrophic plant with complete cleistogamy from Japan. Phytotaxa 175(5): 270. https://doi.org/10.11646/phy- totaxa; 1'75.5.5 Suetsugu K (2016) Gastrodia kuroshimensis (Orchidaceae), a new mycoheterotrophic and com- plete cleistogamous plant from Japan. Phytotaxa 278(3): 265. https://doi.org/10.11646/ phytotaxa.278.3.6 Suetsugu K (2019) Gastrodia amamiana (Orchidaceae; Epidendroideae; Gastrodieae), a new completely cleistogamous species from Japan. Phytotaxa 413(3): 225-230. https://doi. org/10.11646/phytotaxa.413.3.3 50 Zhi-Heng Chen et al. / PhytoKeys 220: 39-50 (2023) Suetsugu K (2021) Gastrodia longiflora (Orchidaceae: Epidendroideae: Gastrodieae), a new mycoheterotrophic species from Ishigaki Island, Ryukyu Islands, Japan. Phytotaxa 502: 107-110. https://doi.org/10.11646/phytotaxa.502.1.9 Suetsugu K (2022) Living in the shadows: Gastrodia orchids lack functional leaves and open flowers. Plants, People, Planet 4(5): 418-422. https://doi.org/10.1002/ppp3.10281 Suetsugu K, Fukushima K, Makino T, Ikematsu S, Sakamoto T, Kimura S (2022) Transcrip- tomic heterochrony and completely cleistogamous flower development in the mycohet- erotrophic orchid Gastrodia. New Phytologist 237(1): 323-338. https://doi.org/10.1111/ nph.18495 Tamura K, Stecher G, Kumar S (2021) MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Molecular Biology and Evolution 38: 3022-3027. https://doi.org/10.1093/ molbev/msab120 Tan Y-H, Hsu T-C, Pan B, Li J-W, Liu Q (2012) Gastrodia albidoides (Orchidaceae: Epi- dendroideae), a new species from Yunnan, China. Phytotaxa 66(1): 1-38. https://doi. org/10.11646/phytotaxa.66.1.6 Tillich M, Lehwark P, Pellizzer T, Ulbricht-Jones ES, Fischer A, Bock R, Greiner S (2017) GeSeq — versatile and accurate annotation of organelle genomes. Nucleic Acids Research 45(W1): W6-W11. https://doi.org/10.1093/nar/gkx39 1 Wen Y, Qin Y, Shao B, LiJ, Ma C, Liu Y, Yang B, Jin X (2022) The extremely reduced, diverged and reconfigured plastomes of the largest mycoheterotrophic orchid lineage. BMC Plant Biology 22(1): 448. https://doi.org/10.1186/s12870-022-03836-x Zhou Z, Shi R, Zhang Y, Xing X, Jin X (2021) Orchid conservation in China from 2000 to 2020: Achievements and perspectives. Plant Diversity 43(5): 343-349. https://doi. org/10.1016/j.pld.2021.06.003