﻿Gastrodiabawanglingensis (Orchidaceae, Epidendroideae), a new species from Hainan Island, China

﻿Abstract Gastrodiabawanglingensis, 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.

During our field investigation in April 2021, Gastrodia specimens with significantly different floral morphology from all the known species in China were collected in the forests of Bawangling, Hainan Tropical Rainforest National Park. Further studies, 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.

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, online annotation software Geseq (https://chlorobox.mpimp-golm.mpg.de/geseq.html)  (Tillich et al. 2017) and CpGAVAS (http://www.herbalgeno-mics.org/cpgavas)  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, phylogenies 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 Didymoplexis is sister to Gastrodia (Wen et al. 2022). The sequences of the species and related ones were aligned in MAFFT version 7 (https://mafft.cbrc.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 individuals) and dried herbarium specimens all belonging to the type specimen, which is 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. kept in the HUFB (Teaching Herbarium of the College of Forestry, Hainan University). All length and width of structures were measured by vernier calipers. Morphological characters of the new species were based on dried herbarium specimens. Furthermore, we examined the type specimens of Gastrodia albidoides Y.H.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.
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 region. This indicates that plastomes of Gastrodia are in the last stages of plastome degradation (see Barrett and Davis 2012;Liu et al. 2021;Jiang et al. 2022;Wen et al. 2022).

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. 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 lateral 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).
Phenology. Gastrodia bawanglingensis was observed flowering and fruiting in April and May.

Pollination implication. Flowers of
Gastrodia bawanglingensis barely open and pollen 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 selfpollinates. 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 Suetsugu 2013Suetsugu , 2014Suetsugu , 2016Suetsugu , 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 colonization 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 pollinators 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(Suetsugu , 2016. However, complete cleistogamy has arguably driven speciation Ogaki et al. 2019). We also found several other unpublished species that are different but very similar to G. bawanglingensis in 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 Yunnan, 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 literature 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