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PhytoKeys 252: 9-24 (2025) DOI: 10.3897/phytokeys.252.135155

Research Article

Lilium huanglongense (Liliaceae): a newly-discovered species in north-western Sichuan, China

Ting Wang'2®, Yumei Yuan'2®, Ting-Hong Zhou?®, Yundong Gao'2®

1 CAS Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization & Ecological Restoration and Biodiversity Conservation Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610213, China

2 University of Chinese Academy of Sciences, Beijing 100049, China

3 Administrative Bureau of the Huanglong Scenic and Historic Interest Area, Songpan 623300, China

Corresponding author: Yundong Gao (gaoyd@cib.ac.cn)

OPEN Qaceess

Academic editor: Lorenzo Peruzzi Received: 20 August 2024 Accepted: 30 December 2024 Published: 4 February 2025

Citation: Wang T, Yuan Y, Zhou T-H, Gao Y (2025) Lilium huanglongense (Liliaceae): a newly-discovered species in north-western Sichuan, China. PhytoKeys 252: 9-24. https://doi. org/10.3897/phytokeys.252.135155

Copyright: © Ting Wang et al.

This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0).

Abstract

In this study, we describe Lilium huanglongense, a newly-discovered lily species iden- tified following extensive surveys in an undeveloped area of the Huanglong National Nature Reserve in Sichuan, China. This region, located in the Hengduan Mountains of south-western China, is recognised as one of the world’s prominent biodiversity hotspots, providing diverse habitats for a wide range of plant species. Morphologically, L. huanglongense resembles Lilium fargesii Franch., which is distributed in central China, as well as other tepal-recurved members of the section Lophophora (Bureau & Franch.) F. T. Wang & Ts. Tang. This section comprises dwarf lilies predominantly found in the alpine scrub of the Hengduan Mountains, extending westwards into the Himalayas. Mo- lecular phylogenetic analyses using both nuclear ITS and chloroplast genomes confirm the independent status of the new species and its placement within the section Loph- ophora. The identification of this new species helps to fill the distribution gap between broad-leaved forest and alpine scrub species within the section, thereby enhancing our understanding of the diversity and distribution history of Lophophora.

Key words: Liliaceae, Lilium huanglongense, Lophophorum-clade, new species, section Lophophora

Introduction

Lilium, a genus in the tribe Lilieae of the family Liliaceae, comprises herbaceous, bulbous plants with scaled bulbs, dorsifixed anthers and loculicidal capsules (Peruzzi 2016). With approximately 123 recognised species (POWO 2024), the genus is widely distributed across the Northern Hemisphere in Asia, Europe and North America (Liang and Tamura 2000). China, in particular, hosts approx- imately 55 distinct species according to the latest flora records (Liang 1995; Liang and Tamura 2000), with key distribution areas including northeast, central and south-western China. Amongst these regions, south-western China stands out as a hotspot for the diversity of wild lilies due to its mountainous environ- ment (Gao et al. 2013b, 2015). These mountain ranges and deep valleys consti- tute an intricate topography, which provides unique habitats for various species.

Ting Wang et al.: Lilium huanglongense: a newly discovered species in northwestern Sichuan, China

The Daba Mountains and the Qinling Mountains of central China, as well as the Hengduan Mountains and the Himalayas, form a series of mountain rang- es from central to western China, together harbouring the greatest number of lilies in the world (Yundong Gao, unpublished data). Furthermore, the rugged terrain and sparse population in mountainous regions have constrained previ- ous explorations, indicating the potential presence of undiscovered species in these areas. Investigating plant groups within these continuous mountain rang- es would enhance our understanding of species diversification and dispersal history amongst the selected plant species, thereby contributing significantly to our overall knowledge of biodiversity.

Our prior investigation elucidated the taxonomic classification of the ge- nus Lilium (Gao et al. 2012, 2013b, 2015; Gao and Gao 2016; Yuan and Gao 2024). Specifically, the Lophophorum-clade sensu Gao et al. (2013b) was iden- tified, which comprises both the campanulate-flowered L. oxypetalum Baker, L. lophophorum (Bureau & Franch.) Franch., L. nanum Klotzsch, as well as the tepal-recurved L. fargesii Franch., L. stewartianum Balf. f. & W.W. Sm. and L. matangense J.M. Xu that span mountainous regions from central China (broad- leaved forests) to the Himalayas (alpine scrub and meadows). This clade is analogous to the subgeneric section Lophophora (Bureau & Franch.) F. T. Wang & Ts. Tang, as refined by Watanabe et al. (2021) in their recent work. Most mem- bers of this clade, which exhibit recurved perianths, have a limited and sporadic distribution. For example, L. fargesii exhibits a notable widespread distribu- tion at mid-elevation areas (second step) in China (mainly in Qinling and Daba Mountains), while L. matangense is found further westwards at higher altitudes of over 3000 m, with narrow distribution and very small population sizes and therefore is of conservation value. Additionally, L. stewartianum is distributed at an altitude of about 3500 m in the southern Hengduan Mountains (Fig. 1).

With advancements in molecular phylogenetics, the monophyly of the Lophophorum-clade has been confirmed (Gao et al. 2013b; Watanabe et al. 2021); however, our understanding of its composition and evolutionary his- tory remains incomplete. Moreover, the distribution of the entire Lophopho- rum-clade is disjunctive (Fig. 1), particularly between the northern Hengduan Mountains and the western Qinling Mountains, creating a “gap” where mem- bers of this clade have not been previously documented. This relic distribution pattern may suggest the presence of additional undiscovered taxa or those that are already extinct. In recent years, meticulous sampling and analysis of this group have yielded several novel findings. This paper highlights one such discovery: the putative new species L. huanglongense, identified by rangers in the Huanglong National Nature Reserve. This discovery can be partially at- tributed to the increased attention that the Chinese government has directed towards nature reserves and it represents a significant advancement in our understanding of this clade.

Currently, we aim to clarify the status and phylogenetic position of the puta- tive new species by comparing its morphology with that of the most morpho- logically similar species, in addition to conducting molecular phylogenetic anal- yses utilising both nuclear markers and chloroplast genomes. Furthermore, the analysis of the morphological and genetic distinctiveness of L. huanglongense is expected to offer additional insights into the Lophophorum-clade by address- ing the geographic distribution gap observed amongst its members.

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Ting Wang et al.: Lilium huanglongense: a newly discovered species in northwestern Sichuan, China

85°E 90°E 95°E 100°E 105°E 110°E

40°N

35°N

30°N

25°N

0 150 300 ent

Figure 1. Morphological characteristics and geographic distribution of Lilium huanglongense and related species. Lilium huanglongense fills the geographic gap west of the Qinling Mountains and at the confluence of the Hengduan Mountains.

Materials and methods

Field sampling

Leaf materials of the new species were collected from the Huanglong Nation- al Nature Reserve and temporarily preserved in silica gel for DNA extraction. During fieldwork, we captured many photographs of the individuals and collect- ed three complete specimens for conservation purposes. These images and specimens were used for subsequent measurements and descriptions. The voucher specimens have been deposited in the Herbarium of the Chengdu In- stitute of Biology (CDBI).

Morphological analysis

This study is grounded in an analysis of herbarium specimens, digital specimen images, field observations and relevant literature. We conducted a comprehen- sive literature review of pertinent taxa using online databases such as Tropicos (https://tropicos.org/) and the Biodiversity Heritage Library (BHL, https://www. biodiversitylibrary.org/), focusing on Lilium oxypetalum Baker (Baker 1874), L. lophophorum (Bureau & Franch.) Franch. (Franchet 1898), L. nanum Klotzsch (Klotzsch and Garcke 1862), L. fargesii Franch. (Franchet 1892), L. stewartia- num Balf. f. & W.W. Sm. (Smith 1923) and L. matangense J.M. Xu (Xu 1985).

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Ting Wang et al.: Lilium huanglongense: a newly discovered species in northwestern Sichuan, China

Specimens were meticulously examined through visits to the CDBI, IBSC, KUN, PE, SZ and WUK Herbaria (acronyms according to Thiers (2024), same below) and by accessing digital images from virtual herbarium platforms, in- cluding the China Virtual Herbarium (https://www.cvh.ac.cn/), the Kew Herbar- ium Catalogue (http://apps.kew.org/herbcat/gotoHomePage.do) and JSTOR Global Plants (https://plants.jstor.org/), as well as online images from herbaria B, E, GH, K and P. This approach aimed to facilitate a comparative analysis of morphological characters based on a substantial number of specimens. Morphological traits were selected, based on taxonomically significant fea- tures detailed in the “Flora of China” (Liang and Tamura 2000), including bulbs, stems, leaves and flowers. Specifically, the new species was morphologically compared to the tepal-recurved members of the Lophophorum-clade, namely L. fargesii, L. stewartianum and L. matangense. For comparative analysis, the dimensions of the bulbs, stems, leaves and floral organs were measured from both specimen images and photographs of fresh plants, utilising MATO (Liu et al. 2023) and PS software (Suppl. material 1: table S1). The Extent of Occur- rence (EOO) and Area of Occupancy (AOO) were calculated using the GeoCAT software (Bachman et al. 2011).

Molecular phylogeny inference

Genomic DNA was extracted from silica-gel dried leaves using a modified cet- yltrimethylammonium bromide (CTAB) method (Allen et al. 2006). Paired-end sequencing libraries were then constructed with insert sizes of approximately 350 bp, followed by sequencing on the DNBSEQ-T7 platform (Beijing Genomics Institute, BGI), with the depth of about 0.1 ~ 0.2 x (10G pair ending reads). About 13 Gb of raw data were filtered by fastp v.0.23.2 (Chen et al. 2018). The Inter- nal Transcribed Spacer (ITS1, 5.8S and ITS2) and chloroplast genome of new species were then assembled using GetOrganelle v.1.7.6.1 (Jin et al. 2020) with default parameters. Chloroplast genomes were annotated and manual correc- tions were made using Geneious Prime v.2023.1.2 (Biomatters Ltd. Auckland, New Zealand), based on the plastome of Lilium fargesii (NC_033908.1).

To deduce the phylogenetic position of the putative new species, we com- bined newly-generated DNA sequences and published sequences, including thirty-two ITS and twenty-eight cp genome from NCBI (https://www.ncbi.nIm. nih.gov/), to infer phylogenetic relationships, selecting the entire Lophopho- rum-clade species and 2-3 representative from closely-related clades (Sup- pl. material 1: table S2) based on previous studies (Gao et al. 2013a; Yuan and Gao 2024). Outgroups included four species of Fritillaria and Cardiocri- num (Suppl. material 1: table S2).

We utilised the online platform (https://ngphylogeny.fr/, Lemoine et al. (2019)) to construct Maximum Likelihood (ML) phylogenetic trees based on complete plastid sequences. The sequences were analysed through the Advanced Workflow, employing the PhyML + SMS/OneClick method. Detailed workflows for MAFFT, BMGE and PhyML + SMS (Maximum Like- lihood-Based Phylogenetic Tree Inference with Intelligent Model Selection) are provided in the Methods section of Lemoine et al. (2019). Bootstrap analysis (FBP + TBE) was conducted with 1000 replicates, while all other pa- rameters were kept at their default settings. ITS sequences were processed

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Ting Wang et al.: Lilium huanglongense: a newly discovered species in northwestern Sichuan, China

using PhyloSuite v.1.2.2 (Zhang et al. 2020). A total of 34 sequences were aligned in batches with MAFFT v.7.313 (Katoh and Standley 2013) using the ‘--auto’ strategy in normal alignment mode. The resultant files were sub- jected to additional manual corrections using MEGA v.11.0 (Tamura et al. 2021). Subsequent analyses were performed in PhyloSuite, where ambigu- ous sites and gaps were removed using Gblocks (Talavera and Castresana 2007). The sequences were then concatenated into a single alignment and converted into Nexus format files.

ModelFinder (Kalyaanamoorthy et al. 2017) was used for the selection of the most appropriate evolutionary model. Based on the Akaike Information Criterion, GTR + F + G4 was chosen as the optimal model of nucleotide evo- lution. Bayesian phylogenies were inferred using MrBayes 3.2.6 (Ronquist et al. 2012) under partition model (2 parallel runs, 10,000,000 generations), in which the initial 25% of sampled data were discarded as burn-in. The con- struction of the ITS Maximum Likelihood (ML) tree was performed using Phy- loSuite, with the sequence file generated through MAFFT and Gblocks. The file was then processed using the A La Carte option on the online tree-building platform to execute the PhyML analysis. Bootstrap analysis was conducted with 1000 replicates and all other parameters were set to their default values. The generated Maximum Likelihood (ML) and Bayesian Inference (BI) (Sup- pl. material 2) phylogenetic trees were visualised using iTOL v.6 (https://itol. embl.de, Letunic and Bork (2024)).

Results Morphology comparison (Figs 2-4, Table 1)

Lilium huanglongense shares with L. lophophorum a pair of marginal ridg- es along the central groove on the adaxial surface of tepals (Fig. 2B), which has been recognised as the most important character defining this section (Watanabe et al. 2021). This feature also provides morphological evidence supporting the new species’ placement within the Lophophorum-clade. Addi- tionally, the turk’s-cap perigone suggests that L. huanglongense is more closely related to tepal-recurved species such as L. fargesii.

While L. huanglongense shares reflexed perianth segments with L. fargesii, L. stewartianum and L. matangense, it differs notably in terms of floral organs.

Table 1. Morphological comparisons of Lilium huanglongense, L. fargesii, L. stewartianum, and L. matangense.

Characters

Bulb

Stem Leaves Flower

colour diam. length

basal colour tube length stigma

nectar glands

L. huanglongense L. fargesii L. stewartianum L. matangense yellow white yellow white T2-15-em approximately 1.5 cm approximately 2.0 cm 1.0-1.5:cm 15-40 cm 20-70 cm 20-50 cm. 23580.cM 5-12 x 0.3-0.7 cm 10=14*-0:2-0.5:cm 2,.5=7-*0.3-0:4 6m Pa2 AOS ACM yellow green, pink greenish to deep yellow white shorter shorter longer shorter three-lobed without three-lobed without three-lobed with inflation | three-lobed with inflation inflation inflation nectaries with cristate nectaries with cristate papillose nectaries that | inner ones with fimbriate projections on both projections on both form two ridges along the projections on both surfaces surfaces bases of the inner tepals surfaces of nectaries

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Ting Wang et al.: Lilium huanglongense: a newly discovered species in northwestern Sichuan, China

Figure 2. Comparison of floral structures of similar species A Lilium huanglongense flower B tepals of Lilium huanglon- gense with basal nectaries C Lilium matangense flower D Lilium fargesii flower E Lilium stewartianum flower F tepals of Lilium matangense with basal nectaries G tepals of Lilium fargesii with basal nectaries H tepals of Lilium stewartianum with basal nectaries. Photographed by Yundong Gao.

Firstly, the flower of the new species is about 3-4 cm in diameter and, when fully expanded, the perianth is nearly in the same plane as the androgynophore (Fig. 2A), whereas in the other species, the androgynophore is exposed to a greater extent (Fig. 2C—-E). Secondly, the stigma of L. huanglongense is three- lobed without inflation (Fig. 3F, G), whereas that of L. matangense is three-lobed with inflation (Fig. 2C).

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Ting Wang et al.: Lilium huanglongense: a newly discovered species in northwestern Sichuan, China

= F

Figure 3. Lilium huanglongense T.Wang & Y.D.Gao, sp. nov. A habit B dissected flower C outer perianth segment D inner perianth segment E stamen F pistil (frontal view) G pistil (lateral view). Drawn by T. Wang from the holotype.

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Ting Wang et al.: Lilium huanglongense: a newly discovered species in northwestern Sichuan, China

Figure 4. Habitat of Lilium huanglongense and morphological comparison with Lilium fargesii A habitat destroyed by mudslides B flowering plant C habit of Lilium huanglongense exhibiting a greater abundance of basal leaves, accom- panied by wider leaf blades compared to L. fargesii D habit of Lilium fargesii. Lilium huanglongense. Photographs were taken by multiple authors of present work.

Furthermore, L. huanglongense possesses a greater number of basal leaves com- pared to L. fargesii (Fig. 4C, D). The detailed differences between L. huanglongense and the most similar species are listed in Table 1. These morphological differenc- es effectively distinguish the new species from known congeners.

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Ting Wang et al.: Lilium huanglongense: a newly discovered species in northwestern Sichuan, China

Phylogenetic analyses (Fig. 5)

The analysis was based on molecular data, specifically ITS (ITS1, 5.8S and ITS2) sequences and the complete chloroplast genome. This study utilised two datasets, each including two individuals of the new species. The new species has an ITS sequences with 624 base pairs (bp) in length with a GC content of 61.5%, whereas the chloroplast genome was 152,597 bp long with a GC content of 37.0%. The chloroplast genome comprises double-stranded circular DNA and exhibits a characteristic quadripartite structure, including a large single-co- py (LSC) region spanning 81,965 bp, a small single-copy (SSC) region of 17,496 bp and two inverted repeat (IR) regions, each measuring 26,568 bp. We utilised 34 ITS sequences, with lengths ranging from 610 bp to 633 bp prior to align- ment and, after alignment correction, the sequence lengths were 641 bp with 223 variable sites and 411 conserved sites. In addition, we analysed 30 com- plete chloroplast genomes with sequence lengths ranging from 151,655 bp to 153,235 bp before alignment and 157,060 bp after alignment correction, con-

4100 Lilium stewartianum MN745202.1 Lilium 90/1 .00 a Lilium brownii var. viridulum HQ692117.1 100 Lilium matangense MN745201.1 93/1.00 r nt lancifolium HQ692093.1 100 Lilium lophophorum MK493298.1 Sinomartagon Il by are 76/1.00 ilium callosum E Za 100 Silat lied Seer h h Lilium bakerianumvar. bakerianum HM045428.1 5 ophnopnorum Sinomartacon | 6710.89} 97/4 09 Fees 400 Lilium huanglongense PP746761.1 g Lilium taliense HQ692109.1 j Lilium huanglongense PP746762.1 Lilium tsingtauense HQ687259.1 99/1.00 100 Lilium fargesii NC_033908.1 Martagon ium aistichum M045451.1 83/1.00 Lilium lankongense Mie e220d hartret | Liriot sare et acemeen YT Ri ium aucnartre! | Liriotypus a i Lilium duchartrei KY748300,1 SNe pean eee 88/1.00 Lilium pardanthinum HM045432.1 4100 Lilium bakerianum NC_035592.1 : P Sinomartagon | Nomocharis |; jum apertum sn785988.1 —J 84/099 100 Lilium taliense NC_034370.1 — 100 Lilium huanglongense PP468352.1 99/1.00 Lilium apertum NC_052786.1 7 . 92/1.00 _ 100 y Nomocharis Lilium huanglongense PP468353.1 Lilium pardanthinum NC_038193.1 Lilium stewartianum HQ692151.1 67/ Lilium japonicum NC_049018.1 als Lilium matangense HM045457.1 ia 97 400 a Archelirion 2 76/4.00 Lilium speciosum var. gloriosoides MN509267.1 Pesan Lilium lophophorum KC020204.1 tA Oopnophnorum ... #2 aa Lilium regale MW145135.1 Lilium fargesii HM045459.1 B Leucolirion | cium: h - lophophorum HQ692099.1 a Lilium sargentiae MK493303.1 ilium lophophorum var. lophophorum HQ692099. Lil dali mnosados4 Lilium oxypetalum AB020442.1 91/0.99 70/ Ly annie P iri Lili JN785979.1 5 seudolirium ilium nanum var.nanum | ie Lilium washingtonianum NC_037699.1 prea Sa AIS VOREEONIO A oe =, d 83/ 100 Lilium distichum MK493296.1 ar: Lilium pardalinum AB020439.1 100/1.00 olan Martagon | Pseudolirium vats Lilium tsingtauense KU230438.1 Lilium washingtonianum AB020438.1 96/1.00 s00f 7 Lilium lancifolium MW465411.1 Lili d hartr lankongense HQ692145.1 99/1.00 inom n | Lillum duchartrel 100 Lilium callosum ee 1 omartago Lilium duchartrei HQ692064.1 is 4007 Lilium brownii var. viridulum NC_050268.1 Archelirion "#7 @Panicum AB020451.1— 190/400 sc ili Lilium speciosum var. gloriosoides HM045461.1 ; Lilium longiflorum KC968977.1 Lilfunni| | tilt speciasum var aforigsos 60/0.59 Liriot Lilium sargentiae HQ692112.1 Lilium candidum NC_042399.1 Irlo us iri = yp Leucolirion Lilium regale HQ692090.1 100/1.00 100 Parties inuin-calneyanlm dali aad Cardiocrinum cathayanum HM045474.1 400/4.00

taining 6,417 variable sites and 148,565 conserved sites.

CP

Cardiocrinum cordatum KX575837.1

Fritillaria sinica NC_044631,1 | OUtgroup

Fritillaria cirrhosa NC_024728.1

Clade ITS

Lilium philippinense ABO20437.1

Cardiocrinum cordatum KP712019.1 Outgroup

Fritillaria cirrhosa HM045469.1 Fritillaria sinica KF906211.1

Figure 5. Maximum Likelihood (ML) phylogenetic analysis of selected species of Lilium based on A complete plas- tome DNA and B nuclear ITS sequence. Numbers at nodes indicate bootstrap percentages (BS) for ML. In B, the val- ues to the left of the “/” represent the bootstrap support (BS), while those to the right indicate the Bayesian posterior probability (PP).

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Ting Wang et al.: Lilium huanglongense: a newly discovered species in northwestern Sichuan, China

The phylogenetic analysis indicates that the Lophophorum-clade is mono- phyletic, supported by both chloroplast and ITS phylogenies, with support val- ues of 100% (Fig. 5A) and 91%/0.99 (Fig. 5B, Suppl. material 2), respectively. These results are consistent with previous works (Gao et al. 2013a; Yuan and Gao 2024). In the plastid phylogeny, L. fargesii is resolved as sister to all oth- er species within the Lophophorum-clade. Within this successively branching clade, both individuals of L. huanglongense form a monophyletic group, which is sister to L. nanum, L. lophophorum, L. matangense and L. stewartianum. In the ITS phylogeny, L. huanglongense is sister to L. stewartianum, whereas in the plastid phylogeny, L. stewartianum forms a clade with L. matangense (Fig. 5).

Taxonomic treatment

Lilium huanglongense T.Wang & Y.D.Gao, sp. nov. urn:lsid:ipni.org:names:77356314-1

Figs 1-4, 6, Table 1, Suppl. material 1: table S1 Common name: #2744 huang long bai he

Type. CHINA * Sichuan: Songpan County, Huanglong National Natural Reserve, 30 June 2023, Y.D. Gao GYD2023001 (holotype: CDBI 0285062) (Fig. 6).

Diagnosis. Lilium huanglongense is most similar to L. fargesii and L. stewar- tianum, but can be distinguished from L. fargesii by its yellow tepals and sta- mens that are longer than the pistil and, in contrast to L. stewartianum, L. huan- glongense lacks a deep, slender floral tubes (the height of the cone formed by the connivent tepals) and has a trilobed, non-inflated stigma. (Table 1, Suppl. material 1: table S1)

Description. Bulb ca. 1.2-1.5 cm in diam., ovoid; scales 1.5-3 x ca. 8 mm, lanceolate, yellow. Stem 15-40 cm long, smooth, basal part red, red colour gradually fading and becoming green with reddish-brown speckles towards the apex of stem. Leaves 5.0-12.0 x 0.3-0.7 cm, scattered, mostly in middle and distal parts of stem, linear, margin recurved, smooth. Flowers actinomorphic, solitary, ca. 4-5 cm in diameter, nodding, Tepals 3.0-3.5 x 0.7-1.0 cm, lance- olate, margin revolute, yellow, with scattered, purple or purplish-brown spots mainly concentrated in the basal part; inner tepal nectaries with cristate pro- jections on both surfaces, green; outer ones glabrous, with a green glistening nectarial channel at the base. Filaments 2—2.5 cm, glabrous; anthers 7-9 x ca. 2 mm, narrowly oblong, brown. Ovary 0.8-1 x ca. 0.3 cm, cylindrical. Style 0.8- 1.2 cm, shorter than filaments, three-lobed without inflation, curved upwards. Capsule ca. 2 x 1.5 cm, oblong.

Phenology. Flowering from June to July.

Habitat and distribution. Occurring in alpine meadows on limestone slopes near streams, at altitudes of 3000-3300 m. This species is only known from three locations (one destroyed) in Huanglong National Nature Reserve, Song- pan, NW Sichuan.

Etymology. The epithet adopted here is derived from Huanglong National Natural Reserve, the site of discovery of this species.

Conservation status and IUCN preliminary assessment. We conducted sur- veys in collaboration with staff from the Huanglong National Nature Reserve in Sichuan Province, China, covering approximately 150 km?. The species

PhytoKeys 252: 9-24 (2025), DOI: 10.3897/phytokeys.252.135155 18

Ting Wang et al.: Lilium huanglongense: a newly discovered species in northwestern Sichuan, China

ER Ste Ua a a Merariars ef Chengdu tnstitate of Bislogy, CAS [COB]

PSP RAB aT A PRRs Hse a aR He Hertariary of Chemie Inettu Chee ogy, CAS [CDE

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Figure 6. Type Specimens of Lilium huanglongense T.Wang & Y.D.Gao, sp. nov. A holotype CDBI0285062 B type CDBI0285063 C type CDBI0285064.

L. huanglongense was found at only three locations. The Extent of Occurrence (EOO) for this species was calculated to be approximately 5.361 km, while its Area of Occupancy (AOO) was estimated at around 0.509 km7?. During our field surveys, we observed that the species’ habitat is highly unstable due to annual summer floods and rockfalls. In the summer of 2023, one of the previously known sites was completely destroyed by a mudslide, resulting in the loss of all individuals at that location (Fig. 4A). At the remaining two locations, we re- corded approximately 30 mature individuals in total each year. Given the limited distribution, small population size and the instability of its habitat, we propose that L. huanglongense be classified as Critically Endangered (CR, B1itii, C1) according to IUCN Red List Criteria (IUCN 2024).

Discussion

Previously, we documented the presence of two distinct flower morphologies within the Lophophorum-clade, which may reflect parallel evolution as lilies rapidly adapt to diverse environments (Gao et al. 2015; Yuan and Gao 2024). This suggests that the former classification of Lilium into subgenera, based solely on floral morphological differences, may not be entirely valid (Watanabe et al. 2021). The Lophophorum-clade further supports the notion that parallel evolution is prevalent within the genus Lilium (Gao et al. 2015; Yuan and Gao 2024). Consequently, caution is warranted when assessing subgeneric affini- ties, based exclusively on morphological characteristics in this genus. Molecular phylogenetic analysis demonstrated that Lilium huanglongense occupies a distinct position within the Lophophorum-clade. The Maximum Like- lihood (ML) tree, based on chloroplast data, shows L. huanglongense as sister to L. nanum, L. lophophorum, L. matangense and L. stewartianum (Fig. 5A). In contrast, both the ML and Bayesian Inference (BI) trees, based on ITS data, place L. huanglongense and L. stewartianum in a monophyletic group (Fig. 5B), revealing a discordance between nuclear and plastid phylogenies (Fig. 5).

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Ting Wang et al.: Lilium huanglongense: a newly discovered species in northwestern Sichuan, China

This discrepancy may result from incomplete lineage sorting (ILS) and intro- gression. However, our previous studies suggest that plastid phylogeny better reflects the geographic relationships amongst species, with introgression be- ing a more plausible explanation (Gao et al. 2013b, 2015).

Within the plastid genome tree, Lilium fargesii occupies the earliest diverging position within the Lophophorum-clade (Fig. 5A). The emergence of L. huanglon- gense may have occurred during times of environmental fluctuation, such as the Quaternary Ice Age, followed by subsequent environmental isolation from its potential ancestral species during interglacial periods (Davis et al. 2005). This aligns with the observed distribution pattern, indicating that L. huanglongense is confined to a unique geographic position within the entire clade (Fig. 1), similar to other tepal-recurved members (e.g. L. matangense, L. stewartianum, Fig. 1) that are sporadically distributed in the Hengduan Mountains region. The limited pop- ulation size and few populations of these tepal-recurved members of the Lopho- phorum clade suggest that a widely-distributed common ancestor may have ex- isted previously, with the current relic pattern resulting from long-term isolation.

Morphologically, Lilium huanglongense is characterised by its compact stat- ure, pale yellow perianth (Fig. 2A), flattened and delicate floral structure and dense basal foliage, distinguishing it from morphologically similar species. The pale yellow perianth of L. huanglongense sets it apart from L. matangense (Fig. 2C) and L. fargesii (Fig. 2D), which typically exhibit flowers with perianth colours ranging from white to green. Additionally, its relatively small size and flattened floral structure (short tube length) differentiate it from L. stewartianum (Fig. 2E), which features a deep, slender floral funnel (the height of the cone formed by the connivent tepals). Our comparisons also revealed significant differences in the proportions of floral organs amongst these species. Lilium huanglongense has flattened floral parts, with the flowers and stamens nearly in the same plane (Fig. 2A). In contrast, the pistils and stamens of similar species, such as L. far- gesii, L. stewartianum and L. matangense, clearly protrude from the perianth.

The specialised perigone structure in L. huanglongense is likely the result of localised plant-environment interactions, particularly with its pollinators. These pollinators play a crucial role in driving morphological evolution (Van der Niet and Johnson 2012; Van der Niet et al. 2014) and have influenced the delicate floral features of L. huanglongense, such as its pale yellow perianth and flat- tened floral structure, which may have evolved to attract specific pollinators in its habitat. Lilium huanglongense exhibits a shorter style than L. fargesii and a shorter floral tube (the height of the cone formed by the connivent tepals) than L. stewartianum. These differences in floral morphology may have evolved in response to varying pollinators, indicating that pollination syndrome may play a key role in the speciation process of this new species.

Geographically, Lilium huanglongense bridges the distribution gap between L. fargesii, native to central China and other species inhabiting the south-western alpine mountains (Fig. 1). The entire Lophophorum-clade extends over a broad geographic range from Hunan (L. fargesii) in the east to the western Himalayas (L. nanum) in the west. This distribution encompasses mesic broadleaf forests in the central Daba Mountain system, alpine scrublands in the south-western Hengduan Mountains and extends to the alpine scrub meadows of the Himala- yas (Gao et al. 2013b). The divergence of the Lophophorum clade is likely due to historical geological and climatic changes in the Qinling-Dabashan-Heng-

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Ting Wang et al.: Lilium huanglongense: a newly discovered species in northwestern Sichuan, China

duan-Himalayan region (Gao et al. 2013b; Xing and Ree 2017). This region, characterised by a series of mountain ranges and diverse ecological niches, spans central and south-western China and supports substantial biodiversity. In conclusion, Lilium huanglongense is a morphologically and molecularly dis- tinct new species within the Lophophorum-clade. This discovery not only contrib- utes to the diversity of the genus, but also fills a geographical gap west of the Qinling Mountains, at the confluence of the Hengduan Mountains. However, our understanding of the Lophophorum-clade remains incomplete. To enhance our comprehension of its phylogenetic relationships and gain a comprehensive under- standing of the biogeographic processes involved, further literature reviews, field- work and additional collection of morphological and molecular data are necessary.

Acknowledgements

We gratefully acknowledge Professor Xin-Fen Gao and Ms. Qi Yu of Chengdu Institute of Biology, Professor Changgiu Liu of Guangxi Institute of Botany, Dr. Pan Li of Zhejiang University and Mr. Melvyn Herbert for their support with sam- ple collection. We appreciate Xiao-Juan Chen's assistance with the software and article submission.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This work was supported by the National Natural Science Foundation of China (NSFC Grant No. 32171605) to Yundong Gao, as well as the project "Research and Demonstra- tion of Key Technologies for Biodiversity Conservation in the Yellow River Basin of Sich- uan Province" (Grant No. 2023YFS0378) and the Natural Science Foundation of Sichuan Province (Grant No. 2023NSFSC0141).

Author contributions

All authors contributed equally to this study and made a significant contribution to the overall result.

Author ORCIDs

Ting Wang ® https://orcid.org/0009-0005-2338-7011 Yumei Yuan © https://orcid.org/0000-0002-4312-5167 Ting-Hong Zhou ® https://orcid.org/0000-0002-0647-478X Yundong Gao ® https://orcid.org/0000-0002-0534-2128

Data availability

All of the data that support the findings of this study are available in the main text or Supplementary Information.

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Ting Wang et al.: Lilium huanglongense: a newly discovered species in northwestern Sichuan, China

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Supplementary material 1

Supplementary information

Authors: Ting Wang, Yumei Yuan, Ting-Hong Zhou, Yundong Gao

Data type: xlsx

Explanation note: table S1. Comparison of measurements and morphology of specimens of Lilium huanglongense, Lilium stewartianum, Lilium fargesii and Lilium matangense. table S2. GenBank accession numbers for sequences utilised in phylogenetic analyses.

Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Link: https://doi.org/10.3897/phytokeys.252.135155.suppl1

Supplementary material 2 Phylogenetic tree constructed from 34 ITS sequences using Bayesian analysis

Authors: Ting Wang, Yumei Yuan, Ting-Hong Zhou, Yundong Gao

Data type: pdf

Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Link: https://doi.org/10.3897/phytokeys.252.135155.suppl2

PhytoKeys 252: 9-24 (2025), DOI: 10.3897/phytokeys.252.135155 DA