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Research Article
Hemipilia avisoides (Orchidaceae), a new species from Sichuan Province, China
expand article infoXue-Man Wang‡, Ying Tang‡, Pei-Hao Peng‡, Hua Peng§
‡ Chengdu University of Technology, Chengdu, China
§ Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China

Corresponding author: Ying Tang ( sanxiaqutang@sina.com )

Academic editor: Vincent Droissart
© 2022 Xue-Man Wang, Ying Tang, Pei-Hao Peng, Hua Peng.
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: Wang X-M, Tang Y, Peng P-H, Peng H (2022) Hemipilia avisoides (Orchidaceae), a new species from Sichuan Province, China. PhytoKeys 213: 1-18. https://doi.org/10.3897/phytokeys.213.90377
Open Access

Abstract

A new orchid species, Hemipilia avisoides, is described from Songpan County and Maoxian County, Sichuan Province, China. Morphologically, H. avisoides is most similar to H. hui, but can be distinguished by the combination of its involute middle lip lobe that is smaller than the lateral lobes, floral bracts that are 5 mm long and are always shorter than the ovary, a leaf that is appressed to the substrate and is adaxially green with white lines along 7–9 principal veins and the subterranean stem with a solitary sheath at its base. The floral morphology of H. avisoides is presented by utilising in vivo micro-CT scanning and 3D visualisation.

Keywords

Arid valley, Minjiang River Valley, Orchidinae, taxonomy

Introduction

The genus Hemipilia Lindl. sensu stricto (Orchideae, Orchidaceae) comprises ca 10 species that are characterised by a protruding, tongue-like rostellum (Luo and Chen 2000; Chen et al. 2009b). Nevertheless, molecular phylogenies revealed Hemipilia s.s. as monophyletic, but nested deeply within a strongly-supported clade that also included several species from closely-allied genera, i.e., Amitostigma hemipilioides (Finet) Tang & F.T.Wang, A. thailandicum Seidenf. & Thaithong, Ponerorchis brevicalcarata (Finet) Soó, P. limprichtii (Finet) Soó and Hemipiliopsis purpureopunctata (K.Y.Lang) Y.B.Luo & S.C.Chen (Luo 1999; Bateman et al. 2003; Jin et al. 2014, 2017; Tang et al. 2015; Lai et al. 2021). This clade was named “the Hemipilia Clade” by Tang et al. (2015). In addition, the recently published species Hemipilia galeata Y.Tang, X.X.Zhu & H.Peng and H. yajiangensis G.W.Hu, Jia X.Yang & Q.F.Wang, both of which do not possess a protruding rostellum, were also recovered as closely related to Hemipilia s.s. in the Hemipilia Clade (Tang et al. 2016; Yang et al. 2022).

Given the fact that Ponerorchis Rchb.f. and Amitostigma Schltr. are paraphyletic to several taxa, including Hemipilia s.s., Jin et al. (2014) formally combined the monotypic Hemipiliopsis with Hemipilia and expanded the circumscription of Ponerorchis. Tang et al. (2015) formally proposed Hemipilia sensu latissimo, lumping ca 65 species into a single broadly circumscribed and monophyletic genus. Under the latter treatment, seven sections that correspond to the seven major clades in nuclear trees were also established and Hemipilia s.s. and those basally divergent species were included in H. sect. Hemipilia (Tang et al. 2015, 2016; Yang et al. 2022).

When transferring Ponerorchis limprichtii to Hemipilia sensu latissimo, Tang et al. (2015) proposed a replacement name H. occidensichuanensis Y.Tang & H.Peng because of the existence of the name H. limprichtii Schltr. based on a different type. Unfortunately, the name H. occidensichuanensis turns out to be illegitimate. Schuiteman (2022) pointed out the problem and made a new combination and the correct name Hemipilia hui (Tang & F.T.Wang) Schuit. to replace P. limprichtii in Hemipilia. Coincidently, this particular species is the one most morphologically similar to the new taxon described in this study.

During the field trip in 2013 to collect Hemipilia physoceras (Schltr.) Y.Tang & H.Peng in Minjiang River Valley, Songpan County, Sichuan Province, China, one of the authors (Y. Tang) collected another orchid that morphologically fits into the category of Hemipilia sensu latissimo. It had been temporarily identified as Ponerorchis cf. limprichtii in the previous study by Tang et al. (2015). However, this taxon in Songpan not only differs in the morphology of the labellum and leaf but also diverges in DNA sequences, both of which suggest it is a potential new species (Tang et al. 2015). Here, we describe it in Hemipilia sect. Hemipilia sensu Tang et al. (2015) and present its floral morphology by using an in vivo micro-CT method.

Methods

Material collection

During our field investigation to Minjiang River Valley, Songpan, Sichuan, China in 7–9 June 2022, two populations of the new taxon with 12 flowering individuals were found. One population (ZJG) occurs at the same locality that was visited in 2013 by one of the authors (Y. Tang) and the other (JPY) is ca 11.2 km southwards in the Valley.

One living individual from the ZJG population and three from the JPY population with intact flowers were collected, each was packaged with soils and EPE pearl cotton in a plastic bottle and transported by air to the Key Laboratory of Stratigraphy and Paleontology, Ministry of Natural Resources for in vivo micro-CT scanning. After scanning, these individuals were pressed and conserved as dried specimens.

The leaf material of one individual from the JPY population was collected and dried with silica gel for DNA sequencing.

To compare the new taxon with morphologically similar species, one population of Hemipilia hui in Kangding, Sichuan, China was investigated in 18 June 2022. The population was found under shrubs at the elevation of ca 3470 m. Five blooming individuals with intact flowers were observed, which showed some variations in morphology but generally fit well with the description in Flora of China (Chen et al. 2009a). One individual of H. hui from this population was collected as a reference specimen. Digital images of herbarium specimens of H. hui at A, AMES, CDBI, IBSC, KUN, PE, SZ and WUK were examined.

All voucher specimens collected as part of this study were deposited at the Herbarium of Sichuan University (SZ).

Morphological observations

The morphological description of the new taxon was mainly based on living materials. The length and width of leaves and the height of the inflorescence were measured on seven living, flowering plants in the field. The morphology of subterranean parts was described based on the four plants collected (see Material collection). The morphology of a single flower was described mainly based on the 3D mesh model reconstructed by micro-CT data.

Micro-CT scanning and 3D Visualisation

X-ray Computed Tomography (CT) was completed at the Key Laboratory of Stratigraphy and Paleontology, Ministry of Natural Resources. The individual collected from the ZJG population (see Material collection) was finally selected for scanning and was then chosen as the holotype of the new taxon. Its inflorescence with the uppermost three flowers was scanned in vivo in a NIKON XTH 225ST CT scanner at a resolution of 18.6 μm and X-ray of 90 kV and 70 μA.

The 3D reconstructions were performed in the software VGSTUDIO MAX 3.0 with STL files being exported. For the 3D model of inflorescence, however, only the uppermost two flowers were reconstructed due to the trade-off between resolution and computing time. Acquired 3D mesh models were visualised and processed by the software GOM INSPECT PRO in GOM SUITE 3.1.1109.0.

Taxon sampling, DNA sequencing and phylogenetic analyses

Based on previous studies (Tang et al. 2015, 2016; Jin et al. 2017; Lin et al. 2021; Peng et al. 2022; Yang et al. 2022), a total of 66 accessions, representing 55 taxa and all seven sections of Hemipilia sensu latissimo, were selected to examine the phylogenetic position of the new taxon. Two species of the genus Brachycorythis Lindl. were chosen as outgroups. Voucher information and GenBank accession numbers are provided in Appendix 1.

Genomic DNA extraction, primer synthesis, PCR reactions and Sanger sequencing were completed by Tsingke Biotechnology Co., Ltd. (Chengdu, China). Four DNA markers, including one nuclear (nrITS) and three plastid markers (matK, trnL-F and trnS-trnG), were used in this study. The primer pairs for these regions were 17SE/26SE (Sun et al. 1994), 390F/1326R (Cuénoud et al. 2002), c/f (Taberlet et al. 1991) and trnS/trnG (Hamilton 1999), respectively. All regions were sequenced for both DNA strands. Contig sequences were assembled with SEQMAN 7.1.0.

Phylogenetic reconstruction was carried out using Bayesian inference (BI) and maximum likelihood (ML) analyses. Data for the plastid regions were combined, whereas the nrITS and combined plastid DNA datasets were analysed separately according to the results of Tang et al. (2015). Each region was individually aligned with MAFFT 7.313 (Katoh and Standley 2013) in PHYLOSUITE 1.2.2 (Zhang et al. 2020) using the “L-INS-I” strategy. Alignments were then manually adjusted in PHYDE 0.9971 (Müller et al. 2010) and ambiguously aligned characters in the trnL-F and trnS-trnG datasets were excluded prior to downstream analyses. ModelFinder (Kalyaanamoorthy et al. 2017) in PHYLOSUITE 1.2.2 (Zhang et al. 2020) was used to select the best-fit model for each dataset using the Bayesian information criterion (BIC) scores. Plastid regions were finally concatenated with PHYLOSUITE 1.2.2 (Zhang et al. 2020).

The best-fit models for BI are GTR+F+I+G4 (nrITS and matK) and GTR+F+G4 (trnL-F and trnS-trnG) and for ML analyses they are GTR+F+I+G4 (nrITS), K3Pu+F+R3 (matK), K3Pu+F+R2 (trnL-F) and K3Pu+F+G4 (trnS-trnG).

Partitioned BI analyses were conducted using MrBayes 3.2.7a (Ronquist et al. 2012) on XSEDE on the CIPRES Gateway (Miller et al. 2010). The Markov chain Monte Carlo (MCMC) analyses were run for 30,000,000 generations, sampling one tree every 1,000th generation. Convergence of runs was accepted when the average standard deviation of split frequencies (ASDSF) fell below 0.01. The initial 25% of sampled trees were discarded as burn-in. Partitioned ML analyses were conducted with IQ-TREE 2.1.2 (Nguyen et al. 2015) on XSEDE on the CIPRES Gateway (Miller et al. 2010) for 5,000 ultrafast (Minh et al. 2013) bootstraps. For the combined plastid dataset, each region was allowed to have its own evolution rate (“-spp”). TREEGRAPH 2.15.0-887 BETA (Stover and Muller 2010) was used to visualise the resulting trees with node support values. Nodes with a Bayesian posterior probability (BPP) ≥ 0.95 and/or a maximum likelihood bootstrap support (BSML) ≥ 80 were considered as strongly supported.

Data availability

The 3D mesh model of the uppermost two flowers on an inflorescence and photos of the corresponding micro-CT-scanned individual of Hemipilia avisoides are available on Zenodo via DOI: https://doi.org/10.5281/zenodo.6832154.

Results

Phylogenetic reconstruction

Trees reconstructed from the nrITS and combined plastid datasets in this study are similar to those of previous studies (e.g., Tang et al. 2015; Jin et al. 2017). Sequences of the accession “Hemipilia avisoides [Tang, Wang & Zhu 236]” generated in this study are nearly identical to those of the accession “Ponerorchis cf. limprichtii” identified and sequenced by Tang et al. (2015). The latter accession was labelled “Hemipilia avisoides [Tang 151]” in this study. The new species, represented by these two accessions, is revealed as a member of H. sect. Hemipilia sensu Tang et al. (2015). Both the nrITS and combined plastid trees recover the new species and H. hui as sister taxa with strong supports (Fig. 1: BPP = 1, BSML = 99; Fig. 2: BPP = 1, BSML = 95).

Figure 1. 

Phylogenetic placement of Hemipilia avisoides sp. nov. in the Bayesian analysis of the nrITS dataset. Bayesian posterior probabilities (BPP) and maximum likelihood bootstrap supports (BSML) are displayed above and below the branches, respectively. Only BPP ≥ 0.95 and BSML ≥ 80 are considered as strong supports and are shown. The scale bar denotes the estimated number of substitutions in Bayesian analysis.

Figure 2. 

Phylogenetic placement of Hemipilia avisoides sp. nov. in the Bayesian analysis of the combined plastid dataset. Bayesian posterior probabilities (BPP) and maximum likelihood bootstrap supports (BSML) are displayed above and below the branches, respectively. Only BPP ≥ 0.95 and BSML ≥ 80 are considered as strong supports and are shown. The scale bar denotes the estimated number of substitutions in Bayesian analysis.

Taxonomic treatment

Hemipilia avisoides Y.Tang, X.M.Wang & H.Peng, sp. nov.

urn:lsid:ipni.org:names:77307801-1
Figs 3A–D, 4, 5, 6A; see also Data availability

Type

China, Sichuan Province, Aba Tibetan and Qiang Autonomous Prefecture, Songpan County, 9 June 2022, Y. Tang, X.-M. Wang & Y.-T. Zhu 235 (holotype: SZ!); ibid., 9 June 2022, Y. Tang, X.-M. Wang & Y.-T. Zhu 236 (paratype: SZ!); Maoxian County, 1 June 1958, S.-Y. Chen, Z. He, M.-F. Zhong et al. 5078 (paratype: SZ!).

Diagnosis

Similar to Hemipilia hui (Tang & F.T.Wang) Schuit., from which H. avisoides can be distinguished by a set of characteristics: mid-lobe involute, suboblong in apical view, 2.2 × 1.2 mm, trapeziform when flattened, smaller than lateral lobes; floral bracts 5 mm long, always shorter than ovary; leaf appressed to the substrate, adaxially green with white lines along 7–9 principal veins; stem subterranean, with one sheath at the base (Table 1).

Table 1.
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Comparisons in morphology between Hemipilia avisoides sp. nov. and H. hui. Diagnostic characteristics of H. avisoides are in bold.

Species Hemipilia avisoides H. hui
Stem shape Subterranean, with 1 sheath at the base Partly subterranean, with 1 or 2 (or 3) sheaths at the base
Leaf position Appressed to the substrate Sub-basal
Leaf colour adaxially Green with white lines along 7–9 principal veins, sometimes also with purple spots Usually green with purple markings, sometimes green with white, reticulate venation or nearly uniformly green
Flora bract shape Elliptic, 5 mm long, always shorter than ovary Lanceolate or ovate-lanceolate, lower ones nearly as long as ovary, gradually smaller upwards to shorter than ovary
Dorsal sepal shape Oblong, apex rounded, sometimes concave at each side of central vein below middle Suboblong, apex subacute
Lateral lip lobe shape Pendulous, rhombic Usually horizontal, auricular or transversely suboblong
Middle lip lobe shape Involute, suboblong in apical view, 2.2 × 1.2 mm, trapeziform when flattened, smaller than lateral lobes, apex rounded or sometimes apiculate Usually open and flat, subsquare, 4–5 × 3–4 mm, larger than lateral lobes, apex obtuse-rounded, sometimes slightly emarginate or shortly apiculate

Description

Terrestrial, erect herbs, 8.5–31 cm tall. Tubers oblong, 2.5 cm long, 0.8 cm in diameter, neck with few roots. Stem subterranean, 2.7–5 cm long, 0.2 cm in diameter, with one sheath at the base. Sheath tubular, membranous, 1–2 cm long, pale yellow. Leaf appressed to the substrate, solitary, cordate, ovate or elliptic, 3–6.5 × 2–5.5 cm, apex acute, slightly fleshy, glabrous, abaxially purple, adaxially green with white lines along 7–9 principal veins, sometimes also with purple spots. Inflorescence terminal, erect, 3–14 cm long, 1–21-flowered, glabrous, dark purple. Flowers not secund, plum or purple plum, fragrant; floral bracts connivent to ovary, elliptic, 5 × 2.6 mm, shorter than ovary, apex acuminate, glabrous, dark purple; ovary curved, cylindrical, 10.5 mm long including pedicel, 1 mm in diameter, glabrous, dark purple. Dorsal sepal erect, oblong, cymbiform, 4.5 × 2.6 mm, apex rounded, sometimes concave at each side of central vein below middle, glabrous; lateral sepals spreading, obliquely ovate, cymbiform, 5.6 × 3.6 mm, apex obtuse, glabrous. Petals erect, connivent with dorsal sepal and forming a hood, apex bending similar to holding a fist in the other hand, obliquely ovate, 4 × 2.8 mm, apex obtuse, glabrous. Labellum spreading, broadly ovate when flattened, 7.1 × 5.4 mm, 3-lobed below middle, spurred, base collar-like raised on each side of spur entrance, glabrous, tinged white at base, disc dotted with purple; lateral lobes pendulous, rhombic, 3.4 × 2.5 mm, apex truncate, margin slightly undulate; mid-lobe horizontal, involute, suboblong in apical view, 2.2 × 1.2 mm, trapeziform when flattened, apex rounded or sometimes apiculate; spur horizontal, straight or curved upwards, cuneate, 9 mm long, ventrally carinate along central axis, entrance 2.5 mm wide, apex swollen, obtuse, 2.7 mm wide; anther reclined, 2.8 mm long, 2-locular, locules parallel and closely spaced, aubergine; pollinia 2, sectile, ovate, 1.2 × 0.7 mm; caudicles cuneate, 1.2 mm long; viscidia 2, closely spaced, oblong, transparent, each enclosed within a separate bursicle; bursicles formed by folding of rostellar arms, oblong, 0.6 × 0.3 mm; rostellum median lobe triangle, 0.7 mm long, lateral lobes grooved; stigma ventral, lobes 2, divergent, lamelliform, 1.2 × 0.5 mm, with hairs at base; auricles 2, each placed laterally at base of anther and behind collar of labellum base, 0.5 mm long.

Flowering

Peaking in early June.

Distribution and habitat

Hemipilia avisoides is currently known from two localities in Songpan County, which are ca 11.2 km apart along the Minjiang River Valley and one locality in Maoxian County according to the collection by S.-Y. Chen et al. in 1958. Individuals of the new taxon occur under arid-valley shrubs and on moss-covered rocks (see Discussion).

Etymology

Latin avis, bird, and suffix -oides, similar, alluding to appearance of flower arrangement simulating flying birds with flapped wings.

Chinese name

雁字舌喙兰 (Chinese Pinyin: yànzì shéhuìlán).

Additional specimens examined

Hemipilia avisoides: China, Sichuan Province, Aba Tibetan and Qiang Autonomous Prefecture, Songpan County, 30 June 2013, Y. Tang 151 (KUN!). Hemipilia hui: China, Sichuan Province, Ganzi Tibetan Autonomous Prefecture, Kangding City, 18 June 2022, Y. Tang, X.-M. Wang, W.-Q. Yuan & Y.-T. Zhu 237 (SZ!); ibid., 17 June 2017, Y.-L. Peng, Q. Yu & L.-L. Li THP-KD-1390 (CDBI!); ibid., 13 June 2014, Y. Tang 199 (KUN!); ibid., 28 May 1981, Z.-J. Zhao, J.-B. Shi & D.-G. Fan 114262 (SZ!); Luhuo County, 12 August 2005, D. E. Boufford, J.-H. Chen, K. Fujikawa, S. L. Kelley, R. H. Ree, H. Sun, J.-P. Yue, D.-C. Zhang & Y.-H. Zhang 34770 (A!); Xiangcheng County, 15 July 2004, D. E. Boufford, J.-H. Chen, S. L. Kelley, J. Li, R. H. Ree, H. Sun, J.-P. Yue & Y.-H. Zhang 30764 (A!); Daofu County, 10 June 1996, J.-S. Yang 91-270 (IBSC!; PE!); Xinlong County, 28 June 1974, Z.-S. Qin 06383 (CDBI!); ibid., 27 June 1974, Z.-S. Yu 06409 (CDBI!); Yajiang County, 15 June 1961, S. Jiang 05196 (KUN!). China, Sichuan Province, Aba Tibetan and Qiang Autonomous Prefecture, Xiaojin County, 2 July 2013, Y. Tang 156 (KUN!); ibid., 21 May 1959, Xiaojin Zu 0130 (SZ!); ibid., 21 May 1957, J. Zhou 34 (IBSC!); Maerkang City, 16 May 1957, X. Li 71047 (PE!; SZ!). China, Gansu Province, Longnan City, Wenxian County, 12 May 2007, Baishuijiang Caijidui 4839 (PE!); ibid., 9 May 2007, Baishuijiang Caijidui 4514 (PE!); Wudu District, 15 June 1959, Z.-Y. Zhang 4390 (WUK!); ibid., 5 June 1959, Z.-Y. Zhang 3379 (WUK!); ibid., 30 May 1959, Z.-Y. Zhang 3180 (PE!; WUK!). China, Gansu Province, Gannan Tibetan Autonomous Prefecture, Zhouqu County, 27 May 1999, Bailongjiang Exped. 1408 (PE!). China, sine loc., 1959, Chuan Jing A 0130 (KUN!); sine loc., July 1907, E. H. Wilson 1762 (the second individual from left on the sheet: AMES!).

Conservation status

Hemipilia avisoides seems narrowly distributed within the arid valley in the upper reaches of Minjiang River (see Discussion), with few populations and individuals being found. The habitat of H. avisoides could be easily disturbed by development as it is close to roads and villages. According to the IUCN Red List Categories and Criteria (IUCN Standards and Petitions Committee 2022), for H. avisoides, the area of occupancy (AOO) is 8 km2, the number of locations is one and the area, extent and/or quality of habitat are likely to decline due to disturbances. Moreover, the number of mature individuals is less than 50. Therefore, H. avisoides is here tentatively assigned to the IUCN category CR B2ab (Critically Endangered).

Figure 3. 

Comparisons between living plants of Hemipilia avisoides sp. nov. and H. hui in the wild A–D habit, flowers, leaf and habitat of H. avisoides E–H habit, flowers, leaf and habitat of H. hui. Photographs A–H by Y. Tang.

Figure 4. 

Floral morphology of Hemipilia avisoides sp. nov., based on 3D mesh model reconstructed by micro-CT data A inflorescence with uppermost two flowers B ventral and dorsal views of floral bract C lateral view of ovary, with floral bract at base D ventral and dorsal views of dorsal sepal E ventral and dorsal views of lateral sepal F ventral and dorsal views of petal G ventral view of gynostemium H ventral and dorsal views of labellum I lateral views of labellum. The 3D model in STL format is available on Zenodo (https://doi.org/10.5281/zenodo.6832154).

Figure 5. 

Specimen of S.-Y. Chen, Z. He, M.-F. Zhong et al. 5078 (SZ!) identified as Hemipilia avisoides in this study. Key features, which would facilitate the identification of this specimen, are highlighted and arrowed in yellow.

Figure 6. 

Leaves of Hemipilia avisoides sp. nov. and some related species in H. sect. Hemipilia sensu Tang et al. (2015) A H. avisoides B H. thailandica C H. hemipilioides D H. brevicalcarata E H. galeata F H. cordifolia. Photographs A–F by Y. Tang.

Discussion

The new species Hemipilia avisoides has oblong tubers, two erect anthers, two stigmas that are beneath the rostellum and two sectile pollinia with viscidium each enclosed within a bursicle. These characteristics fit well into the category of Hemipilia sensu latissimo (Tang et al. 2015). Hemipilia avisoides has also a solitary, slightly fleshy leaf that is appressed to the substrate, which is quite reminiscent of Hemipilia s.s. (Chen et al. 2009b). However, the median rostellum lobe of H. avisoides never protrudes between anther cells like that of Hemipilia s.s.

Molecular phylogenies did, however, reveal a close relationship between Hemipilia s.s. and H. avisoides in a clade (Figs 1, 2), namely, the Hemipilia Clade according to Tang et al. (2015). The sister relationship between H. avisoides and H. hui is strongly supported in both the nuclear and plastid trees, while they are most similar in morphology (see Taxonomic treatment and below). Moreover, H. avisoides and H. hui each occupies a relatively long branch in molecular trees (Figs 1, 2). It is also notable that one accession of H. hui (“Jin, Jin & Cui 14466”) shows obvious DNA sequence divergences from the other two accessions, though they still cluster into a monophyletic clade.

Amongst the specimens of Hemipilia hui, one collection by S.-Y. Chen et al. in 1958 caught our attention for it was gathered from Maoxian County at an elevation of 1780 m, this being close to the localities where we discovered H. avisoides. The environment of that region differs from the alpine habitat that H. hui usually favours. After careful examination, we believe that this specimen represents H. avisoides here described, although it was initially identified as H. hui. We highlighted in Fig. 5 the key features, which facilitated our identification of the specimen. Nevertheless, rather than on living plants, some subtle features could faintly be observed on pressed specimens of H. avisoides and its similar species. For example, the three-dimensional structure of flowers would collapse once pressed and the colours of leaves would fade away when drying. This might obscure the discrepancies between H. avisoides and H. hui or even other more distantly related species like H. chusua (D.Don) Y.Tang & H.Peng. We hope that the 3D mesh model reconstructed in this study (see Data availability) would become helpful for recognising H. avisoides in future research.

According to the spatial delimitation of the arid valley in the upper reaches of Minjiang River (Zheng et al. 2017), of the two Hemipilia avisoides populations we discovered, JPY is distributed within the range of the arid valley and ZJG is closely situated next to the arid valley. The locality of the collection by S.-Y. Chen et al. in 1958 was not precisely recorded, but the elevation of 1780 m implied that the specimen was collected from the range of the arid valley in that region. However, it is notable that, as climate changes, the spatial range of the arid valley varies (Zheng et al. 2017). Focusing on vegetation type, based on our field observations, the vegetation where our collections of H. avisoides occur could be classified into Form. Sophora davidii (Franch.) Skeels, Form. Prunus tangutica (Batal.) Korsh. and/or Form. Ostryopsis davidiana Decaisne, which are typical of arid-valley shrubs and span an elevation between ca 1700 and 2500 m (Yang 2007). To sum up, the habitats of Hemipilia avisoides are mostly within the arid valley in the upper reaches of Minjiang River. To our knowledge, other orchids in the same and sympatric habitats include Hemipilia physoceras, Habenaria acianthoides Schltr. and Cephalanthera erecta Blume, although each of these species is more widely distributed overall. We believe the ecological characteristics of these orchids, including Hemipilia avisoides, are worthy of future study.

The morphologically similar species Hemipilia hui is also distributed in Gansu Province, which is north of Sichuan Province. According to the vegetation regionalisation of China (Zhang 2007), the information of specimens (see Additional specimens examined) and the online photos (see below), the habitats of H. hui in Gansu probably range from arid-valley shrubs to deciduous broadleaved forests at an elevation between 1250 and 1850 m. Besides herbarium specimens, there are some photo records of H. hui in Gansu on the websites of Plant Photo Bank of China, PPBC and China Field Herbarium, CFH (all in Chinese; see http://ppbc.iplant.cn/tu/5920959 [by R.-B. Zhu in Zhouqu County in 21 May 2016], http://ppbc.iplant.cn/tu/5919232 [by R.-B. Zhu in Wenxian County in 16 May 2016], http://ppbc.iplant.cn/tu/5919279 [by R.-B. Zhu in Wenxian County in 16 May 2016], http://ppbc.iplant.cn/tu/7885080 [by X.-J. Liu in Chengxian County in 4 May 2021], http://ppbc.iplant.cn/tu/11011659 [by Z.-F. Bai in Wenxian County in 28 April 2021] and the remaining photos in each album and http://www.cfh.ac.cn/a5074ffa-60e3-4bf6-83a9-eb7cff45b0df.photo [by J.-H. Wang in Wenxian County in 23 April 2015]). The plants shown in those photos have a subsquare mid-lobe that is larger than the lateral lobes; therefore, we recognised them as H. hui. Surprisingly, their leaves are green with white, reticulate venation, which mainly resemble those of H. brevicalcarata Finet and H. yajiangensis in H. sect. Hemipilia.

A few individuals of Hemipilia avisoides were observed in the field to possess conspicuously purple spots, along with white lines along 7–9 principal veins, on their leaves (Fig. 6A). However, H. avisoides lacks reticulate venation that is distinct in H. brevicalcarata, H. yajiangensis and the Gansu populations of H. hui. Occasionally, H. hui has a nearly uniformly green leaf as shown in another online photo (see http://hengduan.huh.harvard.edu/fieldnotes/specimens/search/specimen_detail.zpt?specimen_id=21330&full_image=skelley04179 [by S. L. Kelley in Luhuo County, Sichuan in 12 August 2005]). Despite the variation of leaf-colour patterns within each species, H. avisoides could be distinguished from H. hui as their characteristics do not overlap.

Acknowledgements

Our deepest gratitude goes to Dr Shou-Ming Chen from the Key Laboratory of Stratigraphy and Paleontology, Ministry of Natural Resources for his work in completing the micro-CT scanning and 3D model reconstruction of the new species. We are grateful to Prof Wen-Lan Feng and Mr Jie Zheng from Chengdu University of Information Technology for their help in evaluating whether the new species occurs in the arid valley in the upper reaches of Minjiang River. We are indebted to Dr Jin-Bo Tan from the Herbarium of Sichuan University, Dr Fei Zhao from Kunming Institute of Botany, Chinese Academy of Sciences, Dr Yang-Jun Lai from Institute of Botany, Chinese Academy of Sciences and Prof Zhao-Yang Chang and Prof Zhen-Hai Wu from Northwest A&F University for their help in checking herbarium specimens. We thank Ms Wen-Qin Yuan and Mr Yu-Tong Zhu from Chengdu University of Technology for their assistance during our field trips. This study was supported by the Special Project of Orchid Survey of National Forestry and Grassland Administration (contract no. 2019073016).

References

  • Bateman RM, Hollingsworth PM, Preston J, Luo YB, Pridgeon AM, Chase MW (2003) Molecular phylogenetics and evolution of Orchidinae and selected Habenariinae (Orchidaceae). Botanical Journal of the Linnean Society 142(1): 1–40. https://doi.org/10.1046/j.1095-8339.2003.00157.x
  • Chen XQ, Cribb PJ, Gale SW (2009a) Ponerorchis limprichtii (Schlechter) Soó. In: Wu ZY, Raven PH, Hong DY (Eds) Flora of China Vol. 25. Science Press, Beijing & Missouri Botanical Garden Press, St. Louis, 94 pp.
  • Chen XQ, Gale SW, Cribb PJ (2009b) Hemipilia Lindley. In: Wu ZY, Raven PH, Hong DY (Eds) Flora of China Vol. 25. Science Press, Beijing & Missouri Botanical Garden Press, St. Louis, 98–100.
  • Cuénoud P, Savolainen V, Chatrou LW, Powell M, Grayer RJ, Chase MW (2002) Molecular phylogenetics of Caryophyllales based on nuclear 18S rDNA and plastid rbcL, atpB, and matK DNA sequences. American Journal of Botany 89(1): 132–144. https://doi.org/10.3732/ajb.89.1.132
  • Hamilton MB (1999) Four primer pairs for the amplification of chloroplast intergenic regions with intraspecific variation. Molecular Ecology 8(3): 521–523.
  • Jin WT, Jin XH, Schuiteman A, Li DZ, Xiang XG, Huang WC, Li JW, Huang LQ (2014) Molecular systematics of subtribe Orchidinae and Asian taxa of Habenariinae (Orchideae, Orchidaceae) based on plastid matK, rbcL and nuclear ITS. Molecular Phylogenetics and Evolution 77: 41–53. https://doi.org/10.1016/j.ympev.2014.04.004
  • Jin WT, Schuiteman A, Chase MW, Li JW, Chung SW, Hsu TC, Jin XH (2017) Phylogenetics of subtribe Orchidinae s.l. (Orchidaceae; Orchidoideae) based on seven markers (plastid matK, psaB, rbcL, trnL-F, trnH-psba, and nuclear nrITS, Xdh): Implications for generic delimitation. BMC Plant Biology 17(1): 222. https://doi.org/10.1186/s12870-017-1160-x
  • Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS (2017) ModelFinder: Fast model selection for accurate phylogenetic estimates. Nature Methods 14(6): 587–589. https://doi.org/10.1038/nmeth.4285
  • 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
  • Lai YJ, Han Y, Schuiteman A, Chase MW, Xu SZ, Li JW, Wu JY, Yang JB, Jin XH (2021) Diversification in Qinghai-Tibet Plateau: Orchidinae (Orchidaceae) clades exhibiting pre-adaptations play critical role. Molecular Phylogenetics and Evolution 157: 107062. https://doi.org/10.1016/j.ympev.2020.107062
  • Lin DL, Ya JD, Schuiteman A, Ma CB, Liu C, Guo XL, Chen SS, Wang XL, Zhang ZR, Yu WB, Jin XH (2021) Four new species and a new record of Orchidinae (Orchidaceae: Orchideae) from China. Plant Diversity 43(5): 390–400. https://doi.org/10.1016/j.pld.2021.05.003
  • Luo YB (1999) Studies on the orchid genus Hemipilia. PhD Thesis, Institute of Botany, Chinese Academy of Science, Beijing.
  • Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. Proceedings of the Gateway Computing Environments Workshop (GCE), 14 Nov. 2010, New Orleans, LA, 1–8. https://doi.org/10.1109/GCE.2010.5676129
  • Minh BQ, Nguyen MAT, von Haeseler A (2013) Ultrafast approximation for phylogenetic bootstrap. Molecular Biology and Evolution 30(5): 1188–1195. https://doi.org/10.1093/molbev/mst024
  • Müller K, Müller J, Quandt D (2010) PhyDE: Phylogenetic data editor, version 0.9971. http://www.phyde.de
  • Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution 32(1): 268–274. https://doi.org/10.1093/molbev/msu300
  • Peng S, Cheng YH, Mutie FM, Yang JX, Wang JJ, Lin HQ, He TM, Hu GW, Wang QF (2022) Ponerorchis wolongensis (Orchidaceae, Orchidinae), a new species with variable labellum from the Hengduan Mountains, western Sichuan, China. Nordic Journal of Botany 2022(2): e03295. https://doi.org/10.1111/njb.03295
  • Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539–542. https://doi.org/10.1093/sysbio/sys029
  • Schuiteman A (2022) Two new combinations in Paphiopedilum und[sic] Hemipilia. OrchideenJournal 29(2): 80–83.
  • Sun Y, Skinner DZ, Liang GH, Hulbert SH (1994) Phylogenetic analysis of Sorghum and related taxa using internal transcribed spacers of nuclear ribosomal DNA. Theoretical and Applied Genetics 89(1): 26–32. https://doi.org/10.1007/BF00226978
  • Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Molecular Biology 17(5): 1105–1109. https://doi.org/10.1007/BF00037152
  • Tang Y, Yukawa T, Bateman RM, Jiang H, Peng H (2015) Phylogeny and classification of the East Asian Amitostigma alliance (Orchidaceae: Orchideae) based on six DNA markers. BMC Evolutionary Biology 15(1): 96. https://doi.org/10.1186/s12862-015-0376-3
  • Tang Y, Zhu XX, Peng H, Ma JS (2016) Hemipilia galeata (Orchideae, Orchidaceae), a new species from Fujian Province, southeastern China. Phytotaxa 245(4): 271–280. https://doi.org/10.11646/phytotaxa.245.4.4
  • Yang QZ (2007) Study on the arid-valley scrubs in the upper reaches of Minjiang River. Journal of Mountain Science 25(1): 1–32.
  • Yang JX, Peng S, Wang JJ, Wang Y, Ding SX, Tian J, Hu GW, Wang QF (2022) Hemipilia yajiangensis (Orchidoideae, Orchidaceae), a new species from western Sichuan, China, based on molecular and morphological evidence. Kew Bulletin. https://doi.org/10.1007/s12225-022-10049-w
  • Zhang XS (2007) Vegetation Map of China and Its Geographic Pattern—Illustration of the Vegetation Map of The People’s Republic of China (1:1000000). Geological Publishing House, 625–628.
  • Zhang D, Gao FL, Jakovlić I, Zou H, Zhang J, Li WX, Wang GT (2020) PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Molecular Ecology Resources 20(1): 348–355. https://doi.org/10.1111/1755-0998.13096

Appendix 1

Table A1.
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Voucher information and GenBank accession numbers of taxa included in phylogenetic reconstruction. Sequences generated in this study are marked with asterisks (*). Missing data are indicated with “–”.

Accession Voucher/Reference ITS matK trnL-F trnS–trnG
Brachycorythis henryi Jin et al. 2017 MF944262 MF945438 MF945234 –
B. obcordata Jin et al. 2017 MF944263 MF945500 MF945301 –
Hemipilia alpestris Tang et al. 2015 KM651221 KM651385 KM651545 KM651627
H. amplexifolia Tang et al. 2015 KM651222 KM651386 KM651546 KM651628
H. avisoides [Tang 151] Tang et al. 2015 KM651296 KM651462 KM651621 KM651699
H. avisoides [Tang, Wang & Zhu 236] Tang, Wang & Zhu 236 OP597820* OP595696* OP595697* OP595698*
H. basifoliata [Jin 8361] Jin et al. 2017 MF944399 MF945455 MF945251 –
H. basifoliata [Tang & Su 166] Tang et al. 2015 KM651223 KM651387 KM651547 KM651629
H. brevicalcarata Tang et al. 2015 KM651285 KM651449 KM651611 KM651689
H. calcicola Tang et al. 2015 KM651279 KM651440 KM651605 KM651684
H. calophylla Tang et al. 2015 KM651269 KM651433 KM651595 KM651674
H. camptoceras Tang et al. 2015 KM651275 KM651439 KM651601 KM651680
H. capitata Tang et al. 2015 KM651224 KM651388 KM651548 KM651630
H. cf. amplexifolia Tang et al. 2015 KM651225 KM651415 KM651549 KM651631
H. cf. flabellata Jin et al. 2017 KJ460050 KJ452806 MF945327 –
H. chidori Tang et al. 2015 KM651287 KM651451 KM651612 KM651690
H. chusua [Jin 8272] Jin et al. 2017 MF944401 MF945460 MF945257 –
H. chusua [STET 0619] Jin et al. 2017 KJ460034 KJ452786 MF945189 –
H. chusua [Tang & Su 093] Tang et al. 2015 KM651288 KM651452 KM651616 KM651694
H. chusua [Tang 145] Tang et al. 2015 KM651290 KM651453 KM651615 KM651693
H. compacta Jin et al. 2017 JN696455 KJ452796 MF945321 –
H. cordifolia Jin et al. 2017 MF944329 MF945454 MF945250 –
H. crassicalcarata Tang et al. 2015 KM651270 KM651434 KM651596 KM651675
H. cruciata Jin et al. 2017 MF944330 MF945462 MF945259 –
H. cucullata Tang et al. 2015 KM651276 KM651442 KM651604 KM651683
H. faberi [Tang 158] Tang et al. 2015 KM651229 KM651391 KM651553 KM651635
H. faberi [Tang 161] Tang et al. 2015 KM651230 KM651389 KM651554 KM651636
H. farreri Tang et al. 2015 KM651231 KM651392 KM651555 KM651637
H. flabellata Tang et al. 2015 KM651271 KM651435 KM651597 KM651676
H. forrestii Jin et al. 2017 KJ460049 KJ452805 MF945326 –
H. fujisanensis Tang et al. 2015 KM651280 KM651444 KM651606 KM651685
H. galeata Tang et al. 2016 KT183499 KT183498 KT183500 –
H. gonggashanica Tang et al. 2015 KM651233 KM651394 KM651557 KM651639
H. gracilis Tang et al. 2015 KM651235 KM651397 KM651559 KM651641
H. graminifolia Tang et al. 2015 KM651294 KM651458 KM651619 KM651697
H. hemipilioides Tang et al. 2015 KM651238 KM651400 KM651562 KM651644
H. hui [Jin, Jin & Cui 14466] Jin et al. 2017 MF944398 MF945425 MF945220 –
H. hui [Tang 156] Tang et al. 2015 KM651297 KM651463 KM651622 KM651700
H. hui [Tang 199] Tang et al. 2015 KM651298 KM651461 KM651623 KM651701
H. keiskei Tang et al. 2015 KM651239 KM651401 KM651563 –
H. keiskeoides Tang et al. 2015 KM651240 KM651402 KM651564 KM651645
H. kinoshitai Tang et al. 2015 KM651241 KM651403 KM651565 KM651646
H. kiraishiensis Jin et al. 2017 MF944403 MF945445 MF945241 –
H. kwangsiensis Tang et al. 2015 KM651272 KM651436 KM651598 KM651677
H. lepida Tang et al. 2015 KM651242 KM651404 KM651566 KM651647
H. monantha [Tang & Jiang 171] Tang et al. 2015 KM651243 KM651405 KM651569 KM651650
H. monantha [Tang 192] Tang et al. 2015 KM651244 KM651407 KM651567 KM651648
H. monantha [Tang 193] Tang et al. 2015 KM651245 KM651406 KM651568 KM651649
H. oblonga Tang et al. 2015 KM651281 KM651445 KM651607 KM651686
H. omeishanica Tang et al. 2015 KM651299 KM651464 KM651624 KM651702
H. parceflora Jin et al. 2017 KJ460052 KJ452808 KM651571 –
H. physoceras Tang et al. 2015 KM651248 KM651410 KM651573 KM651654
H. pinguicula Tang et al. 2015 KM651250 KM651413 KM651576 KM651657
H. purpureopunctata Jin et al. 2017 KJ460051 KJ452807 MF945328 –
H. secundiflora Jin et al. 2017 MF944406 MF945458 MF945254 –
H. sichuanica Jin et al. 2017 KJ460059 KJ452815 MF945334 –
H. simplex Tang et al. 2015 KM651253 KM651416 KM651578 KM651659
H. tetraloba Tang et al. 2015 KM651255 KM651418 KM651580 KM651661
H. thailandica Tang et al. 2015 KM651256 KM651419 KM651581 KM651662
H. tibetica [Jin & Zhang 11075] Jin et al. 2017 MF944412 MF945449 MF945245 –
H. tibetica [Tang & Su 064] Tang et al. 2015 KM651257 KM651421 KM651582 KM651663
H. trifurcata Jin et al. 2017 KJ460055 KJ452811 KM651583 –
H. wenshanensis Tang et al. 2015 KM651258 KM651422 KM651584 KM651665
H. yajiangensis Yang et al. 2022 OM009240 OM009241 OM009241 OM009241
H. yueana Tang et al. 2015 KM651259 KM651423 KM651585 KM651666
Ponerorchis gongshanensis [Tang & Su 128] Tang et al. 2015 KM651226 KM651395 KM651550 KM651632
P. nana [Jin 9138] Jin et al. 2017 MF944404 MF945475 MF945273 –
P. wolongensis [PS-00309] Peng et al. 2022 MZ098270 – – –
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