Phytotaxa 392 (1): 045–053 ISSN 1179-3155 (print edition) https://www.mapress.com/j/pt/ PHYTOTAXA Copyright © 2019 Magnolia Press Article ISSN 1179-3163 (online edition) https://doi.org/10.11646/phytotaxa.392.1.4

Paris lihengiana (Melanthiaceae: Parideae), a new species from Yunnan, China

ZHUN XU1,2,3§, NENG WEI1,2,3§, YING TAN1, 4§, SHUAI PENG1, 5, VERONICAH MUTELE NGUMBAU1,2,3, GUANG- WAN HU1,2 & QING-FENG WANG1,2 1Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, China: E-mail: [email protected], [email protected] 2Sino-Africa Joint Research Center, Chinese Academy of Sciences, Wuhan, China 3University of Chinese Academy of Sciences, Beijing, China 4Institute of Molecular Plant Sciences, University of Edinburgh, Edinburgh, UK 5Colleage of Life Sciences, Hunan Normal University, Changsha, China §These authors contributed equally to this work

Abstract

Paris lihengiana (Melanthiaceae), a new species from Yunnan Province, China, is described and illustrated based on evi- dence from morphological characters and molecular phylogeny. It differs from other species of Paris in its pubescent stem, pedicel and abaxial leaf surface, as well as other characters. Molecular phylogenetic analysis of 33 taxa in Paris was con- ducted based on nuclear ribosomal ITS and six plastid markers. Paris lihengiana is supported as a new species by both morphological characters and molecular data.

Keywords: Chinese flora, Chinese traditional medicine, Liliales, new species, Parideae

Introduction

Paris Linnaeus (1753: 367), tribe Parideae Bartling (1830: 53; Melanthiaceae) includes 27 species names accepted in The Plant List (2013). These perennial herbs are widespread in Europe and Asia, with most distributed in China. As a Chinese traditional medicine, the dried rhizomes of most species of Paris have been used for treatment of sores, injury, stings and bites by poisonous insects and snakes and are important ingredients in some Chinese traditional patent medicines, such as Yunnan baiyao (ChPC 2015). Most other species of Paris are also regarded as effective medicinal herbs in China, mainly because of saponins in their rhizomes (Man et al. 2010). Parideae are a distinctive tribe of Melanthiaceae, and their inclusion in this family has been controversial (Li 1984, Farmer et al. 2002, 2006, Ji et al. 2006, Huang et al. 2016, Kim et al. 2016). Paris and Trillium Linnaeus (1753: 329) are two pivotal genera, and merosity is the most important morphological trait to distinguish them. In most studies, Paris is considered monophyletic (Li 1984, Farmer et al. 2002, 2006, Ji et al. 2006, Kim et al. 2013). However recent phylogenetic research based on plastid genome sequences showed Paris to be paraphyletic, twelve Paris taxa were divided into two segregate genera: Paris s.s. and Daiswa Raf. (1838: 18; Huang et al. 2016). A sister relationship between Daiswa and Trillium was supported (Huang et al. 2016). Although the plastid genome provides more characters, the number and choice of taxa also matters. Abundant data with inadequate taxon sampling might lead to strong support but incorrect evolutionary reconstructions (Soltis et al. 2004). In that case, more taxa should be included in phylogenomic analyses to support Daiswa as a segregate genus (Huang et al. 2016). So far, the subgeneric circumscription of Paris has not reached an agreement. During the fieldwork in northeastern Yunnan Province in April 2011, a unique population of Paris caught our attention. When compared with cultivated plants and herbarium specimens, it is easily distinguished from all of other species of Paris. Combined with the evidence from molecular phylogenetics, it seems clear that it should be considered a new species.

Accepted by Mark Chase: 11 Jan. 2019; published: 12 Feb. 2019 45 Materials and Methods

Materials:—This new species was collected from Daxueshan Forest in Weixin County, Yunnan Province, China. Morphologically, the new species was described based on the examination of fresh plants and herbarium specimens. For morphological comparison with similar species, relevant specimens deposited at KUN, PE and HIB were examined, as well as taxonomic descriptions from recently published literature (Ji et al. 2017, Li et al. 2017, Liu et al. 2017, Wang et al. 2017, Yang et al. 2017). Phylogenetic analysis:—Total genomic DNA of the new species was extracted from the specimen (Hu, Wang & Zhao HGW-00655 at HIB) with Mag-MK Plant Genomic DNA extraction kits (Sangon Biotech, Shanghai). In total, 34 taxa were included in phylogenetic analysis, 32 of them were downloaded from GenBank (Table 1).

TABLE 1. GenBank accession numbers for samples in the phylogenetic analysis. Species ITS trnL-trnF rbcL atpB ndhF matK psbA-trnH Paris axialis DQ404210 DQ404278 JN417469 KM242704 KM242852 JN417379 DQ404244 Paris bashanensis DQ404205 DQ404273 DQ404239 Paris caobangensis JF977269 JF942760 JF954856 JN045699 Paris cronquistii DQ404214 DQ404281 JF954859 DQ404248 Paris cronquistii var. DQ404221 DQ404289 DQ404255 xichouensis Paris daliensis DQ404226 DQ404294 JF954861 DQ404260 Paris delavayi DQ404215 DQ404283 KM242936 KM242705 KM242853 KM242778 DQ404249 Paris dulongensis DQ404207 DQ404275 KM242937 KM242706 KM242854 KM242779 DQ404241 Paris dunniana DQ404225 DQ404293 KM242938 KM242707 KM242855 KM242780 DQ404259 Paris fargesii DQ404217 DQ404285 KM242939 KM242708 KM242856 JF954875 DQ404251 Paris fargesii var. petiolata DQ404220 DQ404288 DQ404254 Paris forrestii DQ404208 DQ404276 KM242940 KM242709 KM242857 KM242781 DQ404242 Paris incompleta DQ404203 DQ404271 JF942774 KM242710 HG475404 DQ404237 Paris japonica DQ404202 DQ404270 KM242941 KM242711 KM242858 KM242782 DQ404236 Paris luquanensis DQ404219 DQ404287 KM242942 KM242712 KM242859 KM242783 Paris mairei DQ404213 DQ404282 KM242943 KM242713 KM242860 KM242784 DQ404247 Paris marmorata DQ404222 DQ404290 JF942784 JF954899 DQ404256 Paris polyphylla DQ404224 DQ404292 KM242945 KM242715 KM242862 KM242786 DQ404258 Paris polyphylla var. alba DQ663680 Paris polyphylla var. chinensis DQ404218 DQ404286 KM242944 KM242714 KM242861 KM242785 DQ404252 Paris polyphylla var. JF977326 KM242946 KM242716 KM242863 KM242787 JN045743 stenophylla Paris polyphylla var. DQ404223 DQ404291 KM242947 KM242717 KM242864 KM242788 DQ404257 yunnanensis Paris quadrifolia DQ404204 DQ404272 KM360917 KM242718 KM242865 JF954931 DQ404238 Paris rugosa DQ404211 DQ404279 KM242948 KM242719 KM242866 KM242789 DQ404245 Paris tetraphylla AB018806 D28159 AJ417584 AB018833 Paris thibetica DQ404216 DQ404284 KM242949 KM242720 KM242867 KM242790 DQ404250 Paris thibetica var. apetala DQ486016 Paris undulata AY192533 Paris vaniotii DQ404209 DQ404277 KM242950 KM242721 KM242868 KM242791 DQ404243 Paris verticillata DQ404206 DQ404274 JN417471 KM242722 KM242869 JN417381 DQ404240 Paris vietnamensis DQ404212 DQ404280 KM242951 KM242723 KM242870 KM242792 DQ404246 Pseudotrillium rivale AB018822 HG475380 KM242952 KM242724 KM242871 KM242793 AY727185 Paris nitida MK483720 MK492271 MK492269 MK488087 MK492265 MK488085 MK492267 Paris ligengiana MK483719 MK492270 MK492268 MK488086 MK492264 MK488084 MK492266

46 • Phytotaxa 392 (1) © 2019 Magnolia Press XU et al. For phylogenetic analyses, we used one nuclear region, ribosomal ITS (White et al. 1990), and six plastid markers, trnL-trnF, psbA-trnH, rbcL, atpB, ndhF, matK (Taberlet et al. 1991, Olmstead et al. 1994, Hoot et al. 1995, Terry et al. 1997, Osaloo et al. 1999, Hayashi et al. 2000, Molvray et al. 2000). PCR products were sequenced by Sangon Biotech using 3730xl DNA Analyzer. Sequence assembly and editing was conducted in Geneious v.5.6.4 (Kearse et al. 2012). MAFFT v.7.222 (Katoh et al. 2013), and Mesquite v.3.2 (Maddison et al. 2017) was used for alignments and dataset editing. For Bayesian inference (BI), suitable DNA substitution models were selected by MrModeltest v.2.3 (Nylander 2004); GTR+I+G was chosen under the Akaike information criterion as the best-fitting model for nrITS and plastid DNA. MrBayes v.3.2.6 (Ronquist et al. 2012) was used to perform Bayesian phylogenetic inference. BI was run with four Markov chains, starting with a random tree, for 10 million generations and sampled every 100th generations. The first 25% of sampled trees were discarded as burn-in. RAxML v.8.2.10 (Stamatakis 2014) was used in maximum likelihood (ML) analysis with GTRGAMMA model, with 1000 ML bootstrap replicates.

Taxonomy

Paris lihengiana G.W.Hu & Q.F.Wang, sp. nov. (Figs. 1, 2). Type:—CHINA. Yunnan Province: Zhaotong City, Weixin County, Daxueshan Forest, 27°53′N, 104° 46′E, elev. 1440 m, 24 April, 2011, Hu, Wang, Zhao HGW-00655 (holotype: HIB; isotype: HIB).

Paris lihengiana is distinguished from all other Paris species by its pubescent stem, abaxial veins and pedicel and other characters (Table 2). Perennial herbs, erect. Rhizome 5.0–8.0 cm long, 0.8–1.0 cm in diam., cylindric, brown externally, white internally, with numerous nodes. Stem 16–40 cm long, green or brownish, covered with silver-grey pubescence. Leaves 5–6 in a whorl at stem apex, 10.0–15.5 × 1.6–2.5 cm, linear-oblong to narrowly elliptic-lanceolate, dark green; apex acuminate, base cuneate or subrotund; petiole ca. 0.5 cm long; adaxial surface glabrous, abaxial surface covered with scattered hairs on veins; leaf margin entire, slightly sinuous, ciliolate; trinerved, veins sunken adaxially and protrudent abaxially. Flower solitary, pedicel 13–16 cm long, green or brownish, with silver-grey hairs. Flower tetramerous or pentamerous; sepals 4 or 5, 2.8–4.0 × 0.8–1.1 cm, lanceolate to narrowly ovate, light green, apex acuminate, base gradually narrowed into a claw, margin ciliolate. Petals 4 or 5, 6.5–7.0 cm, length ca. twice that of sepals, less than 1 mm broad, filiform, green, stamens 12, 16 or 20 in 3 or 4 whorls; filament ca. 5 mm long, green; anthers ca. 8 mm long, basifixed, with convex connective apically, yellow; free portion of connective ca. 0.8 mm long, apex acute. Ovary ca. 8 mm in diam., 3–5-locules distally, uniloculate basally, ribbed, glabrous, bluish violet, incomplete axile placentation, 3–5; stylar base glabrous, sometimes angular; style reddish violet, stigmas 4–5, 4–5 mm long, revolute. Fruits unknown. Phenology:—Flowering April. Distribution and habitat:—Known from one location in Daxueshan Forest, Weixin County, Yunnan Province, in subtropical evergreen forest at 1440 m. Etymology:—Named in honor of Prof. Heng Li, who has made significant contributions to our knowledge of the genus Paris. Conservation Status:—Because of over-harvesting and lacking of experience in cultivation, most members of Paris are endangered, with numbers of populations sharply declining in the last decade. Increasing demand for medicinal herbs aggravates risk of their extinction. In this case, no additional populations of the new species have been found, although several surveys were done in the original and adjacent areas. According to current information and IUCN Red List criteria (2010), the status of Paris lihengiana would be tentatively classified as critically endangered (CR B2a; B2b(ii)). More extensive fieldwork in surrounding similar habitats is needed to accurately assess its conservation status. Notes:—Paris lihengiana is morphologically unique although its linear-oblong to narrowly elliptic-lanceolate leaf shape is similar to that of Paris polyphylla var. stenophylla Franchet (1888: 97). However, the latter has more leaves, (3–)4–7(–8)-merous flower, two whorls of stamens and a loculate ovary. The pubescence on the stem, abaxial leaf surface and pedicel match the basic description of P. mairei Léveillé (1912: 302). Nonetheless, the latter has a thicker rhizome, obovate to oblanceolate leaves, (4–)5–8(–9)-merous flowers, two whorls of stamens and axile placentation. Paris vaniotii Léveillé (1906: 355) and Paris undulata Li & Soukup (1992: 16) are also similar to P. lihengiana. These three species share features such as thick rhizome, long leaves and angulate ovary. Characters distinguishing P. lihengiana from the other two species are provided (Table 2).

Paris lihengiana Phytotaxa 392 (1) © 2019 Magnolia Press • 47 FIGURE 1. Paris lihengiana. (A) Rhizome and stem. (B) Leaves. (C) Flower. (D) Stamen. (E) Pistil. (F) Ovary (cross section). Drawing by Ling Wang.

48 • Phytotaxa 392 (1) © 2019 Magnolia Press XU et al. FIGURE 2. Paris lihengiana. (A) Habit. (B) Stem, pedicel and abaxial leaf surface (showing pubescence). (C) Leaf adaxial surface. (D) Rhizome. (E–H) Flower. (I) Ovary (cross section). Photographs taken by Guangwan Hu.

Molecular phylogenetic analyses:—The topology of BI trees and ML trees are highly congruent, except for the placement of Paris axialis Li (1978: 273) and Paris thibetica var. apetala Handel-Mazzetti (1925: 149) in the ITS analysis. Paris subgen. Daiswa is monophyletic in the plastid trees. Paris lihengiana is placed in subgen. Daiswa and is well supported by the phylogenetic analyses of nrITS (Fig. 3) and plastic DNA data (Fig. 4). Although P. lihengiana is well resolved as a new species, several phylogenetic problems have been identified in Paris. (1) There is strong discordance between the topology of nrITS and plastid trees. (2) A long branch is generated in plastid DNA analysis. (3) Varieties of Paris polyphylla Smith (in Rees, 1819: 2) are not monophyletic.

Paris lihengiana Phytotaxa 392 (1) © 2019 Magnolia Press • 49 TABLE 2. Morphological comparison of Paris lihengiana, P. vaniotii and P. undulata. (Dash indicates data not available.) Characters P. lihengiana P. vaniotii P. undulata Rhizome diam. 0.8–1.0 cm in diam. 1.0–1.2 cm in diam. 1.0–1.5 cm in diam. Stem 16.0–40.0 cm long, pubescent 20.0–30.5 cm long, glabrous 70.0–80.0 cm long, glabrous Leaves arrangement 5–6 in a whorl 5–6 in a whorl 7–9 in a whorl Leaf blade and petiole 10.0–15.5 × 1.6–2.5 cm, linear- 7.5–14 × 2.5–5.5 cm, oblanceolate- 10.0–12.0 × 4.0–4.5 cm, oblong to narrowly elliptic- elliptic, petiole ca. 1.0 cm suboblong, petiole ca. 3.5 cm lanceolate, petiole ca. 0.5 cm Leaf base Cuneate or subrotund Subcuneate Rotund Leaf margin Slightly sinuous, ciliolate Entire, non-ciliolate Entire, non-ciliolate Pedicel 13–16 cm long, pubescent 10–15 cm long, glabrous 20–30 cm long, glabrous Flower merous 4–5-merous 5–6-merous 4–5-merous Sepal 4–5, lanceolate to narrowly ovoid, 5–6, ovoid-lanceolate, 2.5–3.5 × 4–5, lanceolate to oblanceolate, 2.8–4.0 × 0.8–1.1 cm, margin 0.7–1.2 cm, margin glabrous 5.0–6.0 × ca. 2.0 cm, margin ciliolate glabrous Petal Filiform-linear, about twice length of Filiform-linear, longer than sepals, Linear, equal length with sepals, sepals, 6.5–7.0 cm × 1 mm, margin 3.5–5.0 cm × 1.0–2.0 mm, margin 5.0–6.0 cm × 2.0–3.0 mm, entire entire margin undulate Stamen 3–4 whorls, filaments ca. 5.0 mm 2 whorls, filaments 3.5–4.5 mm 2 whorls, filaments 5.0–6.0 long, anther ca. 8.0 mm long, free long, anther 5.5–9.0 mm long, free mm long, anther 11.0–12.0 mm portion of connective ca. 0.8 mm portion of connective 0.5–1.5 mm long, free portion of connective long long 11.0–15.0 mm long Ovary 3–5-loculate at most upper part, 5-loculate, axile placentation 1-loculate, parietal placentation locules fused into one at base, incomplete axile placentation Style 4–5, reddish violet, 4–5 mm 5–6, orange, - 4–5, purple, 6–7 mm

FIGURE 3. Bayesian tree based on nuclear ribosomal ITS. Bootstrap value (BS) for maximum likelihood (ML) analyses and posterior probability (PP) for Bayesian analysis are shown (BSML/PPBI). Dashes indicate BSML<50 or PPBI<0.50.

50 • Phytotaxa 392 (1) © 2019 Magnolia Press XU et al. FIGURE 4. Bayesian tree based on plastid DNA data. Bootstrap value (BS) for maximum likelihood (ML) analyses and posterior probability (PP) from Bayesian analysis are shown (BSML/PPBI). Dashes indicate BSML<50 or PPBI<0.50.

Discussion

Paris lihengiana is distinct from others members of Paris in both nrITS and plastid trees. Morphologically, it differs from other species by pubescent stem, abaxial veins and pedicel, linear-oblong to narrowly elliptic-lanceolate leaves, 3–5-loculate, bluish violet ovary and incomplete axile placentation. The nrITS tree here is consistent with the tree based on plastid data (Huang et al. 2016). Our results also support the eastern and western clades in P. subgen. Daiswa (Huang et al. 2016). Since the varieties of P. polyphylla are not supported here as a monophyletic and in most of the phylogenetic studies (Ji et al. 2006, Huang et al. 2016, Kim et al. 2016), these subspecific taxa should be reconsidered. Due to the high divergence of trnL-trnF in P. lihengiana, a long branch has been generated in combined plastid analysis. As first mentioned for Paris in Ji et al. (2006), there is strong discordance between nrITS and plastid DNA trees, which we also found in this study. This might be caused by natural hybridization between sympatric species (Ji et al. 2006). In this case, nuclear genes with biparental genetic information might permit a more accurate assessment of phylogenetic relationships than plastid markers.

Acknowledgments

We are grateful to Heng Li from Kunming Institute of Botany (CAS) for encouraging us to publish this new species, and we also thank Ling Wang for the illustration. This research was supported by the National Natural Science Foundation of China (grant number: 31270244). We also thank an anonymous reviewer for useful comments.

Paris lihengiana Phytotaxa 392 (1) © 2019 Magnolia Press • 51 References

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