﻿Liparismacrosepala (Orchidaceae), a new species from southwest China with its phylogenetic position

﻿Abstract A new orchid species, Liparismacrosepala, is illustrated and described from Yunnan Province, China, based on morphological and molecular analyses. This plant is characterised by the ovoid-fusiform, slightly compressed pseudobulbs with 4 or 5 leaves with slightly crisped margins on their apical half, dorsal sepal heart-shaped, lip with a bituberculate basal callus and a thickened folded lateral lobe on each side, centrally with one cavity with slightly raised margins, the column with a single pair of broadly triangular, obtuse wings. Maximum Likelihood and Bayesian Inference analyses of combined nrITS and plastid matK DNA sequences place this species in section Cestichis.

from this genus are terrestrial, lithophytic, epiphytic and rarely mycoheterotrophic, with inflorescences laxly or densely many-flowered, lip often reflexed and usually with a basal callus, lacking a spur, column winged at apex and sometimes at base and four pollinia in two pairs (Chen et al. 2009).
During our field surveys in Xishuangbanna, Yunnan, China, an unknown species was found. In this paper, we analysed the morphological differences of the newlyfound species and its allied species and the phylogenetic position of the new entity is also discussed, based on molecular evidence from nrITS and plastid matK. After careful morphological comparison and phylogenetic analyses, we concluded that this species is new to science.

Morphological observations
Materials of the new species were collected from Xishuangbanna, Yunnan, China during a field expedition. Morphological characters were observed, measured and photographed based on five living individuals under a stereomicroscope (SZX16-6151, Olympus, Japan) and photographed with a digital camera (D750, Nikon, Japan). A voucher specimen, designated as the holotype, was deposited at Shanghai Chenshan Herbarium (CSH). Conservation assessment has been conducted following IUCN guidelines (IUCN 2019).
Taxonomic sampling DNA sequences of nrDNA ITS and plastid matK of the new species were sequenced and sequences of the same markers for 82 related species were downloaded from Gen-Bank, including five outgroup species from other subtribes (Table 1).

Phylogenetic analyses
DNA sequences were aligned using the MAFFT programme in Geneious v. 2020.2.4 (https://www.geneious.com, accessed on 10 March 2021). Phylogenetic analyses were conducted using Maximum Likelihood (ML) and Bayesian Inference (BI) in RAxML v.7.0.4 (Stamatakis 2006) andMrBayes v.3.2.6 (Huelsenbeck andRonquist 2001;Ronquist et al. 2012), respectively. The appropriate DNA substitution model under AIC criteria was estimated using jModelTest 2.1.10 (Posada 2008). ML analyses were conducted with bootstrap values calculated by running 1,000 replicates. For BI analysis, four chains were run with random initial trees, each for 1,000,000 generations, until the average standard deviation of the split frequency values was less than 0.01 to ensure convergence, sampling trees every 1,000 generations. After the first 20% of samples were discarded as burn-in, the remaining replicates were used to estimate the posterior probabilities.

Phylogenetic analyses
The length of nrITS matrix was 792 bp including 262 parsimony-informative sites and for matK, the length and parsimony-informative sites were 1443 bp and 120, respectively. Both analyses (MP and BI) recovered similar relationships. The ML tree with bootstrap percentages, on which the posterior probabilities from the BI analysis were also indicated, is shown in Fig. 1. The phylogenetic analyses indicate that Liparis is not monophyletic, being mingled with species of other genera of Malaxideae. This result agrees with what was found in previous studies (Cameron 2005;Margońska et al. 2012;Tang et al. 2015;Li et al. 2020;Kumar et al. 2022

Morphological comparisons
Liparis is defined as species with racemose inflorescences, resupinate lip lacking a spur, column without a conspicuous foot and four pollinia in two pairs with small viscidium, but no caudicle. The morphology of Liparis macrosepala is in  accordance with the characteristics of sect. Cestichis like the slightly flattened, narrowly winged rachis with alternating bracts. The morphological characters can distinguish Liparis macrosepala from its close relatives L. delicatula, L. fissipetala, L. assamica and L. resupinata. Diagnosis. Liparis macrosepala is characterised by the ovoid-fusiform, slightly compressed pseudobulbs with 4 or 5 alternate leaves on their apical half, these with slightly crispate margins, dorsal sepal ovate with cordate base, broadly elliptic, ca. 4 mm long, 2 callus-shaped and thickened folds, base with 2 oblong lobes on both sides, centrally with 1 thickened, concave callus, column with a single pair of arcuate wings.
Phenology: Flowering in November-December. Distribution and habitat. It is found on tree trunks on a limestone ridge-top evergreen broad-leaved forest at an elevation of 1500-1700 m in Mengna County, Xishuangbanna Autonomous Prefecture, Yunnan Province, People's Republic of China. The habitat presents a tropical monsoon climate.   Etymology. The species epithet refers to the large and conspicuous dorsal sepal of the flower.
Taxonomic notes. Liparis macrosepala differs from L. delicatula in its 4 to 5 leaves with slightly crispate margins on their apical half and single pair of wings on the column. Its entire, not Y-shaped petals and sessile lip (i.e. without a claw) easily distingush L. macrosepala from L. fissipetala. The dosal sepal of L. assamica is narrowly ovateoblong, in contrast with the heart-shaped dorsal sepal of Liparis macrosepala. Liparis resupinata is distinguished from L. macrosepala by its 10-50-flowered raceme and the column with a single pair of broad wings, each with a retrorse thread. The main differences between these closely-related species, according to our phylogenetic analyses, are summarised in Table 2.
Conservation assessment. The new species was found in a ridge-top evergreen broad-leaved forest on a limestone mountain. Despite numerous surveys in the areas, only six mature individuals were found without fruits or evidence of cross-pollination.
This extremely small effective population occurs in a touristic zone which is a serious threat to the survival of the species. Consequently, the species can be assessed as Critically Endangered (CR, D), based on current information and following IUCN guidelines (IUCN 2019).