A new species of Liparis (Orchidaceae: Epidendroideae) based on molecular and morphological dat

Liparis lihengiana is described and illustrated here as a new species discovered in the Dulongjiang of Yunnan province (China). It morphologically resembles L. bootanensis in having one leaf, and dense pseudobulbs, but can be clearly distinguished from the latter by having a labellum with 2 very small calli united basally (versus 2 variously shaped calli, rounded, triangular in L. bootanensis), a column with a triangular wings (versus with a falcate wings), and a heart-shaped and obtuse anther cap (versus oblong and apex with two long horns). The results of molecular phylogenetic analysis based on nrITS and one chloroplast matK fragment of 50 Liparis species supported that L. lihengiana is a novel species, forming an independent lineage sister to L. balansae and L. bautingensis (95% bootstrap support, 1.00 posterior probability). A new species of Liparis (Orchidaceae: Epidendroideae) based on molecular and morphological data

Liparis lihengiana is described and illustrated here as a new species discovered in the Dulongjiang of Yunnan province (China). It morphologically resembles L. bootanensis in having one leaf, and dense pseudobulbs, but can be clearly distinguished from the latter by having a labellum with 2 very small calli united basally (versus 2 variously shaped calli, rounded, triangular in L. bootanensis ), a column with a triangular wings (versus with a falcate wings), and a heart-shaped and obtuse anther cap (versus oblong and apex with two long horns). The results of molecular phylogenetic analysis based on nrITS and one chloroplast mat K fragment of 50 Liparis species supported that L. lihengiana is a novel species, forming an independent lineage sister to L. balansae and L. bautingensis (95% bootstrap support, 1.00 posterior probability).

Liparis lihengiana, morphological, Orchidaceae, phylogeny, taxonomy

Liparis is an orchid genus encompassing 444 species (Xiao et al. 2024, Long et al. 2025, POWO 2025). These species are mainly epiphytic, growing on trees or rocks within mountain forests. The native range is cosmopolitan. In China, 92 Liparis species have been recorded (Chen et al. 2009, Feng and Jin 2010, Tian et al. 2012, Hsu 2013, Huang, M.Z. et al. 2016, Wu, S.S. et al. 2017, Fan et al. 2017, Li et al. 2020, Tu et al. 2020, Nong, S.Y. et al. 2021, Xiao et al. 2024, Wen et al. 2025, Long et al. 2025). In the current study, a new species, Liparis lihengiana XX & XXX, is described and illustrated. During a routine survey in 2019, a lithophytic orchid of Liparis was first discovered in Dulongjiang (Gongshan County, Yunnan Province, China). It was tentatively identified as a species within Liparis sect. Cestichis (Li et al. 2020). Nevertheless, a morphological examination found that this species exhibits an unusual combination of characteristics that does not correspond to any known species of Liparis sect. Cestichis . Molecular data confirmed that this Liparis species was a new species via the phylogenetic position analysis using nrITS (ITS1-5.8S-ITS2) and plastid mat K. The aims of this study are (1) to describe this new species and (2) to examine both the molecular and morphological affinities of L. lihengiana XX & XXX sp. nov.

Morphology The measurements and description of L. lihengiana were based on the living plant individuals, respectively. Voucher specimens were deposited at the Herbarium of Yunnan University (holotype YUKU! [barcode YUKU02074881], isotypes YUKU! [barcode YUKU02074882]) . DNA extraction, amplification and sequencing The sequences of the four individuals of the new species newly generated in this study, and the sequences of the remaining 53 species used in the molecular phylogenetic analysis, were retrieved from GenBank. Detailed information concerning the DNA markers, sampled taxa, and GenBank accession numbers are listed in Table 1. Extraction of Total DNA employed a modified 2×CTAB protocol on fresh leaf tissue ground in liquid nitrogen. The nrITS region (ITS1-5.8S-ITS2) was amplified via polymerase chain reaction (PCR) with the primers 17SE and 26SE (Sun et al. 1994). Furthermore, the mat K gene region was amplified and sequenced, following primer sets described by Whitten et al. (2000) and Cameron (2005). All DNA samples were sent to TSINGKE Biotech Co. Ltd (Kunming, China) for sequencing. The obtained DNA sequences of the nrITS and plastid mat K regions were submitted to GenBank (https://www.ncbi.nlm.nih.gov/genbank, accession numbers: PV866665–PV866668; PV871814–PV871817). Detailed information regarding the nrITS and mat K sequences is provided in Table 1. Phylogenetic methods We selected three species from the tribe Epidendreae as outgroups for phylogenetic tree construction (Table 1 and Figure 3), employing both Maximum Likelihood (ML) and Bayesian Inference (BI) approaches. 1. Data Processing Workflow: (1) Sequence Alignment: nrITS and mat K sequences were batch-aligned using MAFFT v7 (Katoh and Standley 2013) in PhyloSuite v1.2.3 (Zhang et al. 2020) under the –auto strategy with default parameters. 2. Region Filtering: Ambiguously aligned regions were removed from both alignments using Gblocks (Talavera and Castresana 2007) with parameters: (1)Minimum conserved/flanking sequences: 43/43; (2) Maximum contiguous non-conserved positions: 8; (3)Minimum block length: 10; (4) Allowed gap positions: with half. 3. Sequence Concatenation: Aligned nrITS and mat K sequences were merged into a supermatrix. 4. Evolutionary Model Selection: ModelFinder (Kalyaanamoorthy et al. 2017) identified K3Pu+F+R3 as the optimal substitution model under the Bayesian Information Criterion (BIC). 5. Phylogenetic Tree Reconstruction: (1) ML Analysis: IQ-TREE (Nguyen et al. 2015) constructed phylogenies under the K81u+R3+F model with 5,000 ultrafast bootstrap replicates (Minh et al. 2013); SH-aLRT branch support test (Shimodaira-Hasegawa approximate likelihood ratio test (Guindon et al. 2010). (2) BI Analysis: MrBayes 3.2.6 (Ronquist et al. 2012) generated trees under the GTR+I+G+F model through: Two independent Markov chain Monte Carlo (MCMC) runs (2 million generations each); 25% burn-in discard of initial samples.

and discussion Morphological comparison L. lihengiana morphologically resembles L. bootanensis Griff. (1851: 278) in having one leaf, and dense pseudobulbs, but can be clearly distinguished from the latter by having a labellum with 2 very small calli united basally (versus 2 variously shaped calli, rounded, triangular to digitate), a column with a triangular wings (versus with a falcate wings), a heart-shaped and obtuse anther cap (versus oblong and apex with two long horns) ; L. lihengiana is also closely related to L. balansae Gagnep. (1932: 165), but differs by the smaller inforescence 15-26 flowered (versus 3-5 flowered), the labellum’s base with 2 very small calli united basally (versus with 1 callus divided into 2 narrowly conic lobes), and the column with triangular wings (versus with 2 straight to falcate, acute wings); The new species also similar to L. bautingensis Tang & Wang (1974: 39), but differs by the inforescence 15-26 flowered (versus 3-4 flowered), pseudobulbs dense without rhizomes (versus rhizomes elongate, pseudobulbs borne 1.5-4 cm apart from each other) and the column with triangular wings (versus decurved, hooked wings) (Table 3 and Figures 2). Phylogenetic analyses The concatenated nrITS and mat K matrix spanned 2,223 bp, containing 559 parsimony-informative sites. Maximum Likelihood (ML) and Bayesian Inference (BI) generated highly congruent trees. The bootstrap percentages of BS and BPP were shown on the Bayesian trees (Figure 3). The result is in accordance with prior research on Liparis . Phylogenetic analyses provide strong evidence that Liparis is not monophyletic genus, as it is divided into two lineages: the epiphytic lineage A (PP=1.00, BS=99.6) and the terrestrial lineage B (PP=1.00, BS=98.8) (Li et al. 2020; Wang et al. 2022; Xiao et al. 2024; Long et al. 2025). The cladograms indicated that L. lihengiana is the sister species of L. balansae, L. bautingensis based on phylogenetic trees and forms a lineage with L. bootanensis, and L. stricklandiana (PP= 0.85, BS= 74) (Figure 3). Taxonomic treatment Liparis lihengiana XX & XXX sp. nov. ( Fig. 1, 2 A.E.I.M ) Diagnosis Liparis lihengiana is most similar to L. bootanensis in having a leaf and dense pseudobulbs, but it can be clearly distinguished by having a labellum with 2 very small calli united basally, a column with triangular wings, and a heart-shaped anther cap (Table 3 and Fig. 3) Type: China, Yunnan, Nujiang city, Gongshan county, Dulongjiang countryside, evergreen broadleaved forests alt. 1500 m, 16 Nov., 2019, XX & XXX 101 (holotype YUKU! [barcode YUKU02074881], isotypes YUKU! [barcode YUKU02074882]). Epiphytic herbs. Pseudobulbs clustered, usually ovoid-oblong, 2.7-5 cm × 0.5-0.7 cm, the new one enveloped by 3 clasping basal foliaceous sheaths. Leaves 1, blade narrowly elliptic-oblong, 13.5-17.5 cm× 2-3 cm, papery, apex acuminate, attenuate from apex to base, contracted into a short petiole at base, petiole 1-2 cm, articulate. Inflorescence 11-15 cm long; peduncle somewhat compressed; rachis 0.3-0.5 cm long, loosely 15-26 flowered, 0.5–0.6 cm apart. Floral bracts nearly subulate, 3-5 mm long. Flowers yellow–greenish or orange; pedicel and ovary 8-11 mm. Dorsal sepal broadly linear or broadly linear-lanceolate, 5 × 0.5 mm, with obtuse apex and revolute margin; lateral sepals slightly wider than dorsal sepal, broadly linear-lanceolate, 5 × 0.6-0.7 mm, oblique from middle occasionally. Petals nearly filiform, 4-5 × 0.2-0.3 mm, obtuse at apex; Lip waxy, broadly oblong, 4-5 × 2.5-3.5 mm, margin somewhat irregularly serrulate, round and apex mucronate, lip recurved, middle part with a slightly thick green longitudinal lamella extending from callus base to margin, base with 2 very small calli united basally, in profile appearing very low. Column arcuate at the front, 2.5-3 mm, slightly dilated at base, upper part triangular winged 0.3 mm, rostellum obtuse. Anther cap heart-shaped or broadly ovoid, yellow-green, obtuse, ca. 0.6 × 0.4 mm; pollinia 4 in two pairs, oblong, yellow, waxy, ca. 0.3 mm long. Phenology Flowers and fruits have been recorded from September to February. Etymology Named in memory of Prof. Li Heng, a Chinese botanist who has made significant contributions to the knowledge of the flora in the Dulongjiang region. “ Chinese pinyin: lǐ héng yáng ěr suàn ” (李恒羊耳蒜), is suggested here. Distribution and ecology Dulongjiang Countryside, Gongshan County, Nujiang City, Yunnan province, China. It is a predominantly lithophytic species that grows in the evergreen broadleaved forest at an elevation of 1500 m. Conservation status In 2019, approximately 200 individuals of L. lihengiana were discovered at one location around Dulongjiang Countryside, Gongshan County, Nujiang City, Y unnan province, China. Consequently, in accordance with the IUCN Red List Categories and Criteria (IUCN 2024), we classified the conservation status of this new species as Data Deficient (DD).

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Taxon sampling in this study. | Species | nrlTS | mat k | | Dendrobium catenatum | KP265010 | AB847713 | | Eria ferruginea | AF521071 | AF263660 | | Eulophia schweinfurthii | KF318924 | KF358133 | | Liparis anopheles | AY907075 | AY907139 | | Liparis assamica | KJ459276 | KJ459306 | | Liparis balansae 1 | KF589874 | KF589880 | | Liparis bistriata | KJ459279 | KJ459309 | | Liparis bootanensis | KJ459280 | KJ459310 | | Liparis bracteata | AY907076 | AY907140 | | Liparis brunnescens | AY907098 | AY907165 | | liparis bautingensis | KJ459278 | KJ459308 | | Liparis cespitosa | AY907077 | AY907141 | | Liparis chalandei | AY907078 | AY907142 | | Liparis condylobulbon | AY907080 | AY907144 | | Liparis cordicallosa 2 | PQ842535 | PQ850568 | | Liparis cordifolia | KJ459282 | KJ459312 | | Liparis delicatula | KJ459283 | KJ459313 | | Liparis distans | KJ459284 | KJ459314 | | Liparis disticha | AY907081 | AY907145 | | Liparis elliptica | KJ459285 | KJ459315 | | Liparis fissilabris | KJ459286 | KJ459316 | | Liparis fissipetala | KJ459287 | KJ459317 | | Liparis formosana | AY907082 | AY907147 | | Liparis fujisanensis | EU024936 | EU024937 | | Liparis gibbosa 1 | AY907083 | AY907148 | | Liparis gibbosa 2 | AY907084 | AY907149 | | Liparis glossula | KJ459289 | KJ459319 | | Liparis guangxiensis | KF589875 | KF589881 | | Liparis latifolia | AY907088 | AY907153 | | Liparis latilabris | KJ459291 | KJ459321 | | Liparis lilifolia | AF521067 | AF263667 | | Liparis lihengiana 1 | PV866665 | PV871814 | | Liparis lihengiana 2 | PV866666 | PV871815 | | Liparis lihengiana 3 | PV866667 | PV871816 | | Liparis lihengiana 4 | PV866668 | PV871817 | | Liparis macrosepala | ON642332 | ON642331 | | Liparis maguanensis 1 | PP969756 | PP990742 | | Liparis maguanensis 2 | PP969757 | PP990743 | | Liparis maguanensis 3 | PP969753 | PP990741 | | Liparis maguanensis 4 | PP969758 | PP990744 | | Liparis maguanensis 5 | PP969755 | PP990740 | | Liparis maguanensis 6 | PP969754 | PP990745 | | Liparis mannii | Kj459293 | KJ459323 | | Liparis napoensis 1 | MT012899 | MT019986 | | Liparis napoensis 2 | MT012900 | MT019987 | | Liparis nervosa | AY907092 | AY907158 | | Liparis nugentiae | AY907093 | AY907159 | | Liparis pandurata 1 | AY907094 | AY907160 | | Liparis pandurata 2 | AY907095 | AY907161 | | Liparis pingxiangensis | KF589870 | KF589876 | | Liparis resupinata | KJ459297 | KJ459327 | | Liparis rheedei | AY907097 | AY907164 | | Liparis somae 1 | MT012897 | MT019984 | | Liparis somae 2 | MT012898 | MT019985 | | Liparis stricklandiana 1 | KF589873 | KF589879 | | Liparis stricklandiana 2 | MT012903 | MT019990 | | Liparis sula | AY907104 | AY907171 | | Liparis truncicola | AY907106 | AY907173 | | Liparis viridiflora 1 | MT012901 | MT019988 | | Liparis viridiflora 2 | MT012902 | MT019989 | | Liparis wenshanensis | KM391980 | KM391981 | | Oberonia ensiformis | KJ459300 | KJ459331 | | Oberonia equitans | AY907130 | AY907198 | | Oberonia falconeri | JN114632 | JN004526 | | Oberonia heliophila | AY907131 | AY907199 | | Oberonia iridifolia | AY907132 | AY907200 | | Oberonia mucronata | JN114641 | JN004530 | | Oberonia padangensis | AY907135 | AY907203 | NOTES: The ITS and mat K sequence of Liparis lihengiana . All sequences in this article are from NCBI, except L. lihengiana . The novel species is shown in bold. Table 3. Morphological comparison of Liparis lihengiana and related species. FIGURE 1. Morphological characters of Liparis lihengiana . A. Habit; B. Plant; C. Inflorescence; D–E. Flower; F. Column and ovary, G. Anther cap and Pollinarium. Photographed by XX. FIGURE 2. Morphological comparison of Liparis lihengiana and other three Liparis species. L. lihengiana (A.E.I.M); L. balansae (B.F.J.N); L. bautingensis (C.G.K.O); L. bootanensis . (D.H.L.P) Photographed by XX. FIGURE 3. Phylogram based on nrITS and plastid mat K DNA sequence data. ML bootstrap values /Bayesian posterior probabilities are indicated above the branches, respectively. Liparis lihengiana is shown in red. Information & Authors Information Version history Copyright This work is licensed under a Non Exclusive No Reuse License.

Authors Metrics & Citations Metrics Article Usage 349views 230downloads Citations Download citation Bo Long, Jie BI, Ze Zhang, et al. A new species of Liparis (Orchidaceae: Epidendroideae) based on molecular and morphological dat. Authorea. 13 July 2025. DOI: https://doi.org/10.22541/au.175240994.49240411/v1 DOI: https://doi.org/10.22541/au.175240994.49240411/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.