Research Article |
Corresponding author: Cheng-Jian Zheng ( cjzheng1984@126.com ) Academic editor: Timothée Le Péchon
© 2023 Yan-Bin Wu, Yu Han, Xu-Hui He, Hui-Ling Chen, Jin-Zhong Wu, Qi Ye, Cheng-Jian Zheng.
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.
Citation:
Wu Y-B, Han Y, He X-H, Chen H-L, Wu J-Z, Ye Q, Zheng C-J (2023) Anoectochilus zhongshanensis (Orchidaceae), a new species from Guangxi, China. PhytoKeys 234: 203-218. https://doi.org/10.3897/phytokeys.234.111106
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A new species of Anoectochilus (Orchidaceae) from Guangxi, China, A. zhongshanensis, is described here, which was identified based on phylogenetic studies adopting combined plastid markers (rbcL-matK-trnL-F), morphological observation and chemical analysis. Molecular phylogenetic results support the systematic status of A. zhongshanensis as a new species in Anoectochilus genus. Morphologically, this new species is similar to A. zhejiangensis and A. malipoensis, but differs by its characteristic labellum and column, including the hastate or scalpel-shaped lobes of epichile, forward curved and pinnately divided cristate lobes at both sides of the mesochile and inverted triangle column wings. Furthermore, HPLC-ELSD analysis of these three species revealed the interesting chemotaxonomic difference that the principle and characteristic lactone glycoside in this new species was kinsenoside, rather than its diastereoisomer, goodyeroside A, a major glycoside in A. zhejiangensis and A. malipoensis.
Anoectochilus, new species, phylogeny, taxonomy
The genus Anoectochilus Blume (Goodyerinae, Cranichideae, Orchidaceae) consists of about 40 reported species in the world, distributed mainly from south and southeast Asia to Australia and the southwest Pacific islands (
In the course of our comprehensive resource survey of Anoectochilus species in China from 2015 to 2021, we have reported three new record species: A. elatus (
During our plant resource investigation in Zhongshan, Guangxi Province, China in Aug 2020, an Anoectochilus species was found to be difficult to identify, which was finally clarified as a new species on the basis of detailed morphological, molecular and chemical studies, and described below as A. zhongshanensis C.J. Zheng & Y.B. Wu. In this study, we deciphered the morphological differences between the new species and its nearest congener, A. zhejiangensis Z. Wei & Y. B. Chang. We also used three combined cpDNA sequences (matK, trnL-F and rbcL) to infer the phylogenetic relationships and substantiate the systematic status of A. zhongshanensis as a new species in Anoectochilus. In addition, potential chemotaxonomic markers, kinsenoside and goodyeroside A, were also employed to disclose the chemical difference between the new species and its nearest congeners, A. zhejiangensis and A. malipoensis.
Voucher specimens of A. zhongshanensis were collected in Zhongshan, Guangxi Province, and preserved at the herbarium of Fujian Agriculture and Forestry University (FAFU!). Fresh leaves were washed and dried with filter paper in the field, and then stored in a plastic bag with silica gel for molecular experiments. The living plants of A. zhongshanensis were carefully observed for detailed morphological description and local observation of the plant′s small parts was performed using a stereo microscope (SZ61). Tissue cultures of all collected Anoectochilus species in our lab have been successfully established for resource protection and further chemical and biological studies.
A total of 42 samples representing 18 Anoectochilus species were included for molecular analysis, and all sequences used for constructing the phylogenetic tree were downloaded from GenBank (Suppl. material
Ezup column plant tissue genomic DNA extraction kit (Sangon B518261) was used to extract the total genomic DNA from silica gel-dried leaves of the new species. The concentration of DNA samples used in this study were ≥ 20 ng/µL, and the working DNA was stocked in refrigerator at 4 °C for use. Polymerase Chain Reaction (PCR) amplification was performed on a Veriti 96-well thermal cycler (Verity, ABI, USA) using a 25 μL reaction system containing 2.5 μL 10× Taq Buffer (with MgCl2), 0.2 μL Taq enzyme, 1.0 μL Dntp (mix), 1.0 μL forward and reverse primers, 1.0 μL target DNA template and 18.3 μL ddH2O. Information on primers and amplification protocols for each DNA region is listed in Suppl. material
DNA quality was detected by electrophoresis using 1% agarose and 1× TAE buffer solution (voltage 120–180V). The concentration and purity were detected by spectrophotometer, and gel imager FR-980A (Shanghai Furi Technology Co., LTD.) was used to record and take photos. The qualified PCR products were sequenced bi-directionally on a 3730XL sequencer (ABI, USA) after purified by a SanPrep column DNA gel extraction kit (Sangon B518131). Sequences were first assembled and edited with SEQMAN (DNA STAR package, USA), followed by sequence alignment with MEGA11 to trim the irregular bases at both ends of the aligned sequences. Bayesian inference (BI), maximum likelihood (ML) and maximum parsimony (MP) methods were used to construct the dataset of multi-gene tandem (rbcL-matK-trnL-F). All characters are considered as unordered and equally weighted, while the indels were processed as missing data after sequence alignment.
MP analysis was performed using MEGA11 (
nr/cpDNA data partitions and best-fit models estimated by IQ-TREE model selection for BI analysis.
Partition | Model |
---|---|
ITS | K80 |
rbcL | HKY |
matK | GTR+I+G |
trnL-F | HKY |
HPLC-ELSD was performed using EasySep®-3030 HPLC system (Shanghai Tongwei Analytical Technology Co., LTD., China) equipped with an AQ-C18 chromatographic column (3 µm, 4.6 × 250 mm) and an ELSD detector. The mobile phase was ultrapure water (100%) and the flow rate was set at 0.5 mL/ min. The column temperature and ELSD spray chamber temperature were 30 °C and 70 °C, respectively, while the nitrogen flow rate was 2.5 mL/min (
Since ITS2 sequence alone could not confirm the systematic status of the new species with convincing infrageneric relationships in the tested Anoectochilus species (Suppl. materials
Phylogenetic relationships based on concatenated rbcL, matK and trnL-F sequences in Anoectochilus species inferred by Bayesian inference. PPBI is shown above the branches, while BSML and BSMP are displayed below the brancher (left, BSMP; right, BSML). “*” indicates that the value is not supported or is smaller than 50.
In addition, HPLC-ELSD analysis displayed interesting chemotaxonomic difference that the principle and characteristic lactone glycoside in A. zhongshanensis was kinsenoside rather than its diastereoisomer, goodyeroside A, a major glycoside in A. zhejiangensis and A. malipoensis (Fig.
China. Guangxi province: Zhongshan County, Hezhou City, under evergreen broad-leaved forest or shady and humid valleys, cultivated at the Medicinal Botanical Garden of the Second Military Medical University, 12 August 2020, Wu20200812003 (holotype: FAFU!).
A. zhongshanensis is similar to A. zhejiangensis, but can be distinguished by the hastate or scalpel-shaped lobes of epichile (vs. semiovoid), forward curved and pinnately divided cristate lobes at both sides of the mesochile (vs. backward curved, the same orientation as the spur), unbowed conical spur (vs. bowed) and inverted triangle column wings (vs. squarish) (Fig.
Terrestrial herb, 8~22 cm tall, with an erect stem and 2~6 leaves. Leaf ovate or orbicular, 1.2–4.0 × 1.0–2.8 cm, adaxially black with fine golden red net veins with silk luster, abaxially purplish red, apex acute, base subtruncate or rounded, abruptly narrowed into a stalk; petiole 4–12 mm long, base enlarged into a cauline sheath. Racemose inflorescence, 1–6 flowered, inflorescence rachis pubescent; peduncle long and slender, mauve red, pubescent, with 2–4 mauve sheath-like bracts; floral bracts reddish, ovate-lanceolate, ca. 6 × 3 mm, apex acuminate, abaxially pubescent, subequal length as the ovary or slightly longer; ovary cylindrical, not twisted, reddish brown, white pubescent, connected with pedicel ca. 13 mm long; flowers not resupinate (labellum held uppermost); sepals reddish, subequal, ca. 5 mm long, abaxially puberulent; dorsal sepal ovate, sunken navicular, apex acute, joined with petals to form a hood; lateral sepals oblong, slightly oblique; petals white greenish, obliquely falcate, ca.5 mm long, middle part ca. 1.5 mm wide, base narrow, apex acute; lip white, upstretched, Y-shaped, 13 mm long, epichile longitudinally dilated, 2-lobed, lobes hastate or scalpel-shaped, 5 mm long, ca. 2 mm wide, apex blunt, diverging at an acute angle; mesochile ca. 4 mm long, flange bearing forward curved and pinnately divided cristate lobes at both sides; conical spur, ca. 10 mm long, obliquely upward, subvertical to ovary, apex shallowly bilobed, containing 2 subcuneate calli. Column ca. 4 mm long, ventrally with an inverted triangular column wing on both sides. Anther cap ovate, ca. 3 mm. Pollinia 2, yellow, obliquely clavate, ca. 3 mm long. Rostellum erect, apical concave and bifurcated. Stigma lobes 2, distinct, located to the sides of the rostellum. Fruit not seen.
Referring to the locality (Zhongshan County) where this new species was found.
钟山金线兰 (Chinese pinyin: zhong shan jin xian lan).
A. zhongshanensis is currently known only from Zhongshan County, Hezhou City, Guangxi Province, China. This species grows in evergreen broad-leaved forests or shady and humid valleys, elev. 500–1200 m.
During our three surveys in April, August and September 2020, Anoectochilus zhongshanensis was found in the forests or shady and humid valleys of Zhongshan County only at two separate locations, where we counted fewer than 100 individuals at each site. Due to the highly medicinal and edible value of Anoectochilus plants (
Flowering in August-October.
Anoectochilus zhongshanensis (Fig.
The single use of nrDNA (ITS) (Suppl. materials
The authors thanked Li-Xiang Zheng for drawing Fig.
The authors have declared that no competing interests exist.
No ethical statement was reported.
This study was funded by the National Natural Science Foundation of China (82174081), Shanghai Pujiang Program (21PJD082), Key project at central government level: The ability establishment of sustainable use for valuable Chinese medicine resources (2060302) and Industry-University Cooperative Project from Fujian Provincial Department of Science and Technology (2020Y4015).
Conceptualization: YBW, CJZ. Data curation: HLC, YBW, YH, JZW, QY, XHH. Funding acquisition: CJZ. Investigation: XHH. Methodology: YH. Resources: YBW. Supervision: CJZ. Writing – original draft: YH. Writing – review and editing: YBW, CJZ.
Yan-Bin Wu https://orcid.org/0000-0001-9401-366X
Cheng-Jian Zheng https://orcid.org/0000-0002-7867-3438
All of the data that support the findings of this study are available in the main text or Supplementary Information.
Phylogenetic relationships based on nrDNA (ITS) in Anoectochilus species inferred by maximum likelihood (ML)
Data type: jpg
Phylogenetic relationships based on nrDNA (ITS) in Anoectochilus species inferred by Bayesian inference (BI)
Data type: jpg
Phylogenetic relationships based on nrDNA (ITS) in Anoectochilus species inferred by maximum parsimony (MP)
Data type: jpg
Phylogenetic relationships based on concatenated rbcL, matK and trnL-F sequences in Anoectochilus species inferred by maximum parsimony (MP)
Data type: png
Explanation note: BSMP is shown below the brancher.
Phylogenetic relationships based on concatenated ITS, rbcL, matK and trnL-F sequences in Anoectochilus species inferred by Bayesian inference (BI)
Data type: jpg
Information of samples for phylogenetic analysis in this study
Data type: docx
Primers and amplification protocols used in this study
Data type: docx