﻿Goodyeramedogensis (Orchidaceae), a new species from Tibet, China

﻿Abstract A new species of Goodyera (Orchidaceae) from Tibet, China, G.medogensis, is described and illustrated here. Molecular phylogenetic results based on one nuclear (ITS) and two plastid markers (matK and trnL-F) support the recognition of G.medogensis as a new species in GoodyerasubsectionReticulum. Morphologically, the novelty is most similar to G.biflora, G.vittata and especially to G.hemsleyana, but differs by the thick grid lines of the reticulations with a diffused margin on the adaxial surface of the leaf blades, the inflorescence with more flowers, the obliquely obovate-rhombic petals, the yellow or yellowish labellum without a lamella on the blade, and the shorter trichomes on the floral bracts, sepals and ovary. Finally, a key to the species of Goodyerasubsect.Reticulum in China is also provided.

Different phylogenetic studies have revealed that Goodyera is polyphyletic (Juswara 2010;Hu et al. 2016;Chen et al. 2019), which has led Pace (2020) to subsequently propose a new generic arrangement for the Goodyera clade including 11 genera: Goodyera s.s., Cionisaccus Breda, Aspidogyne Garay, Microchilus C. Presl, Kreodanthus Garay, Lepidogyne Blume, Hylophila Lindl., Platylepis A. Rich., Eucosia Blume, Erythrodes Blume, Salacistis Rchb. f. and Paorchis M. C. Pace. Since the available molecular phylogenetic results are based on few markers, and the quality and representativeness of samples are limited, in this study, we still accept a broad concept of Goodyera. Goodyera s.l. consists of about 99 species distributed in Africa (Mozambique), Western Indian Ocean Islands, Asia, Southwest Pacific islands, Northeast Australia, Europe, Macaronesia (Madeira), North and Central America and the Caribbean (Chen et al. 2009;Zhou et al. 2020;Govaerts et al. 2021;Thiv et al. 2021). It is characterized by an elongate creeping rhizome, a cymbiform lip with a concave-saccate hypochile, sectile pollinia and one stigma.
There are 36 accepted species of Goodyera in China, of which 15 species are endemic (Chen et al. 2009;Guan et al. 2014;Jin and Yang 2015;Zhou et al. 2016;Liu et al. 2019;Wang et al. 2020;Zhou et al. 2020), and only G. repens (L.) R. Br. and G. brachystegia Hand.-Mazz. are distributed to the north of the Yangtze River. During a botanical survey in Medog County, Tibet Autonomous Region in January 2021, a few living plants with greenish reticulated leaves, resembling those of Macodes (Blume) Lindl., were collected and cultivated in Kunming Botanical Garden. Plants bloomed vigorously with many flowers in August. We also found flowering individuals in the field at the same time. The novelty is similar to G. hemsleyana King & Pantl. at first glance. However, it has greenish flowers with a yellow lip, while G. hemsleyana has pink flowers with a white lip. Besides, the reticulations on the leaf blades of the two species are also different.
Based on molecular phylogenetic evidence and a detailed examination of the morphological characters of our materials, the relevant types and a comprehensive array of additional herbarium specimens of similar species, we concluded that the plants discovered in Medog County in January 2021 represented a new species to science. It is described and illustrated here as G. medogensis H. Z. Tian, Y. H. Tong & B. M. Wang and included in a key to the species of Goodyera subsect. Reticulum in China.

Materials and methods
Based on our field observations of Goodyera in China in the past ten years, we identified three species that were closely related to the novelty, viz. G. hemleyana, G. biflora (Lindl.) Hook. f. and G. vittata Benth. ex Hook. f. (Fig. 1). Accordingly, we checked the types and additional material of these three taxa together with other pertinent specimens at HSNU, IBSC, K, KUN, LBG and PE. Photographs of specimens housed at AMES, AU, BR, CAL, CSFI, M and P were also examined. Among the target taxa, G. biflora is by far the most well-represented species in herbaria, and we did not find any additional specimens of the new species. Furthermore, we examined the distribution of multiple characters (e.g. leaf reticulation, flower number and indumentum, color and margin of the labellum) in all species of Goodyera subsect. Reticulum in China, and these observations were summarized in the form of an identification key. Voucher specimens of G. medogensis were collected in Medog County and preserved at the herbarium of South China Botanical Garden, Chinese Academy of Sciences (IBSC) and East China Normal University (HSNU). Fresh leaves used for molecular analyses were preserved in silica gel. The morphological description of the novelty is based on living material. Measurements were performed with a ruler (0.5 mm accuracy), and small plant parts were observed and measured under a stereo microscope (Mshot-MZ101).
To study the phylogenetic position of the new species within the genus Goodyera, three DNA fragments, viz. internal transcribed spacer (ITS) and two plastid DNA regions (matK and trnL-F), were selected for building the phylogenetic tree based on previous studies Zhou et al. 2020). In total, 33 species represented by 80 samples of Goodyera were analyzed with one sample of Zeuxine flava (Wall. ex Lindl.) Benth. ex Hook. f. as the outgroup. All sequences were downloaded from Gen-Bank except those of the new species. Species names and GenBank accession numbers are provided in the supplementary materials (Suppl. material 1: Table S1). Total genomic DNA of the new species was extracted from silica gel-dried leaves using a modified CTAB method (Doyle and Doyle 1987). Polymerase Chain Reaction (PCR) amplification was carried out on TAKARA TP600 thermocycler (TAKARA BIO INC, Japan) using 25 μl reactions containing 12.5 μl 2× Taq PCR Master Mix (HuaGene, China), 8.5 μl ddH 2 O, 1.5 μl of each primer (10 μM) and 1 μl target DNA template. Detailed information of primers of relevant DNA fragments used in PCR amplification and sequencing, as well as the procedures of PCR, can be found in Suppl. material 1: Table S2. The resulting amplicons were visualized by horizontal agarose gel electrophoresis (1%), colored with GoldView I (Solarbio, China). Clearly distinguishable bands were recorded, and then the corresponding PCR products were sequenced by Shanghai HuaGene Biotech Co., Ltd (Shanghai, China).
Sequences were firstly assembled and edited with Seqman (DNA STAR package, Madison, WI, F USA) and then adjusted manually. Phylogenetic analysis was conducted using PhyloSuite ver. 1.2.2 (Zhang et al. 2020). Sequences were aligned with MAFFT (Katoh and Standley 2013). Ambiguously aligned fragments were removed using Gblocks (Talavera and Castresana 2007) with all parameters at their default settings. Next, matK and trnL-F were concatenated as well as ITS, matK and trnL-F respectively to two datasets. Thus, three datasets were constructed in total: the cpDNA dataset (matK and trnL-F), the nrDNA dataset (ITS) and the nr+cpDNA dataset (ITS, matK and trnL-F). The three datasets were analyzed by using Bayesian inference (BI) and maximum likelihood (ML) methods respectively. Best-fit evolutionary models for Mrbayes and IQ-TREE were selected under the Bayesian Information Criterion (BIC) using ModelFinder (Kalyaanamoorthy et al. 2017). The best-fit models for the Maximum likelihood (ML) analysis are K80+R2 (ITS) and K3Pu+F+R2 (cpDNA, nr+cpDNA), and for Bayesian inference (BI) they are K2P+G4 (ITS) and GTR+F+G4 (cpDNA, nr+cpDNA).
Based on these models, the Maximum Likelihood (ML) analysis was performed with IQ-TREE (Nguyen et al. 2015) for 10000 ultrafast (Minh et al. 2013) bootstraps, and Bayesian Inference (BI) phylogenies were inferred using MrBayes 3.2.6 (Ronquist

Results
Our ML and BI phylogenetic trees constructed from the three datasets showed that our four samples of G. medogensis cluster into one separate subclade (Figs 2-4)  Diagnosis. Similar to G. hemsleyana, but distinguished by the greenish thick reticulations on the adaxial surface of leaf blades with diffused margin (vs. white thin reticulations with clear margin), inflorescence with more flowers ((6-)12-15 vs. 4-10), petals obliquely obovate-rhombic (vs. obliquely ovate-oblong), labellum yellow or yellowish (vs. white, with light greenish to pinkish tinge at apex) without lamella on blade (vs. with a low bi-lamellate callus), and floral bracts, sepals as well as ovary with shorter trichomes.
Etymology. The species epithet refers to the type locality, Medog County.

Discussion
The new species has the typical features of Goodyera sect. Reticulum, i.e., the leaves have reticulations on adaxial surface, and lateral sepals are not reflexed backwards. According to Hu et al. (2016), sect. Reticulum is one of the four sections of Goodyera, and can be further divided into two subsections, viz., G.