Ann. Bot. Fennici 50: 187–194 ISSN 0003-3847 (print) ISSN 1797-2442 (online) Helsinki 28 May 2013 © Finnish Zoological and Botanical Publishing Board 2013 Lilium yapingense (Liliaceae), a new species from Yunnan, China, and its systematic significance relative to Nomocharis Yun-Dong Gao1,2, Song-Dong Zhou1 & Xing-Jin He1,* 1) Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Science, Sichuan University, Chengdu 610064, P. R. China (*corresponding author’s email: xjhe@ scu.edu.cn) 2) Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China Received 26 Nov. 2012, final version received 18 Feb. 2013, accepted 21 Mar. 2013 Gao, Y. D., Zhou, S. D. & He, X. J. 2013: Lilium yapingense (Liliaceae), a new species from Yunnan, China, and its systematic significance relative to Nomocharis. — Ann. Bot. Fennici 50: 187–194. We describe and illustrate Lilium yapingense sp. nova (Liliaceae) and show its position within the Lilium–Nomocharis complex (Liliaceae). It is similar in appearance to L. nanum but differs by (1) having no spots on the tepal bases, instead possessing sym- metric stripes; (2) nectaries lacking fimbriate projections on the surfaces, but having two dark grooves; and (3) an orange-colored instead of a white bulb. Phylogenetic analyses using nuclear ITS showed that L. yapingense merits specific rank and that it is more closely related to Nomocharis than to Lilium. However, the morphological synapomorphies thought to distinguish Nomocharis from Lilium are absent from the new species. The morphology and phylogeny of L. yapingense support previous stud- ies, which show that Nomocharis and Lilium have intergrading morphologies and that Lilium is paraphyletic with respect to Nomocharis. Introduction 1984, Liang & Tamura 2000, Gao et al. 2012). The relationship of Nomocharis to Lilium and The genus Lilium is comprised of approxi- Fritillaria has generated ongoing debate since mately 110 species that are widely distributed Nomocharis was established by Franchet in 1889 in the boreal and temperate northern hemisphere based on several specimens collected in south- (Liang 1995, Liang & Tamura 2000, Patterson & western China (Sealy 1950). In particular, the Givnish 2002). It is widely accepted that Lilium intermediate position of Nomocharis between is sister to Fritillaria, a genus roughly equal in Lilium and Fritillaria in several aspects of floral size and with a similar geographic distribution morphology has been the basis of numerous (Rønsted et al. 2005). Lilium is also consid- re-circumscriptions (Balfour 1918, Evans 1925, ered closely related to Nomocharis, a lesser Sealy 1950, 1983, Liang 1984). New delimita- known genus of eight species that are endemic to tions of Nomocharis have been proposed mainly the Hengduan Mountain region of southwestern by either integrating species into or removing China and adjacent areas (Sealy 1983, Liang them from Lilium and Fritillaria. 188 Gao et al. • ANN. BOT. FeNNICI Vol. 50 Molecular studies that focused on the Lilium– species, we performed an extensive literature Nomocharis complex showed that the species of survey and examined herbarium specimens. In Nomocharis are nested within Lilium and sup- the literature survey, we concentrated on Flora port uniting the two genera under the latter name Reipublicae Popularis Sinicae (Liang 1980), (Nishikawa et al. 1999, 2001, Hayashi & Kawano Flora of China (Liang & Tamura 2000), and 2000, Rønsted et al. 2005, Peruzzi et al. 2009, monographs on lilies of the world (Haw 1986, Gao et al. 2012). However, Lilium and Nomo- Jefferson-Brown 1995, McRae 1998). To exam- charis have been regarded as being morphologi- ine herbarium specimens, we visited PE, KUN, cally distinct (Sealy 1983, Liang 1984). Spe- CDBI, SZ in China and accessed images of addi- cifically,Nomocharis has been distinguished from tional specimens through the Chinese Virtual Lilium on the basis of (1) having a widely opened Herbarium (CVH, http://www.cvh.org.cn/), and perigone, (2) tepals having dark blotches at the digitization projects at P, K and E. Preliminary bases forming a showy “eye” in the center of the results showed strong resemblance between the flower, (3) outer tepals not channeled or nectarial, putative species and Lilium nanum on the basis (4) the dark “eye” being occupied by low ridges of alpine slope habitat and solitary nodding of flabellately arranged nectarial tissue on both flower with pink-purple tepals. Thus, to verify sides of a short median channel, and (5) having the independence of the new species, we com- fleshy, cylindrical filaments with an awn at the top pared its tepal morphology, bulb structure and (Sealy 1950). However, morphological intermedi- nectaries with those of L. nanum, because those acy between the two genera was recently demon- features have long been considered important strated by Gao et al. (2012) in N. gongshanensis. within Lilium (Comber 1949, Sealy 1950, Liang The affinity of N. gongshanensis to other species 1984, Liang & Tamura 2000). of Nomocharis is evidenced by its saucer-shaped flower and filaments with acute apices. However, its dark tepal bases, the absence of nectary proc- Taxon sampling for phylogenetic esses, and its yellow flowers resemble Lilium. analysis Gao et al. (2012) concluded that Lilium and Nomocharis may be morphologically intergrad- In order to infer the phylogenetic position of the ing, but thought that evidence from additional putative new species, we collected accessions species was needed to support that notion. for molecular analyses. Our taxonomic sampling In a recent expedition to Mt. Gaoligongshan was conducted broadly and thoroughly within in the central part of the Hengduan Mountains in the Lilium–Nomocharis complex and included China, we discovered a population of a liliaceous especially the morphologically similar species, plant, which appeared to represent an undescribed L. nanum and all species placed in Nomocharis. species. The primary goals of the present study Some accessions of Lilium–Nomocharis species were to (1) determine the status of the putative were obtained from GenBank. Those that were new species based on morphology and provide a newly sampled in this study were collected by description if necessary, (2) determine the phy- the authors in the field except three species: logenetic position of the putative new species Lilium longiflorum var. scabrum was collected within the Lilium–Nomocharis complex, and (3) from cultivated material in Hainan Province, and provide further evidence supporting a morpholog- samples of L. rosthonii and L. papiferum were ical continuity between Lilium and Nomocharis. obtained from herbarium specimens (SZ). Their leaves were placed in silica gel while in the field, allowed to dry, and stored at –80 ℃ until ana- Material and methods lysed. The voucher specimens were deposited in the Herbarium of Sichuan University (SZ). Taxonomic methods and morphological In total, we sampled 38 of the 55 species of analysis Lilium from China based on the classification presented in Flora of China (Liang & Tamura To infer the taxonomic status of the putative new 2000). They represent all four sections recog- ANN. BOT. FeNNICI Vol. 50 • Lilium yapingense (Liliaceae), a new species from China 189 nized in Flora Reipublicae Popularis Sinicae Data analysis (Liang 1980) as well as five of Comber’s (1949) seven sections native to China. We also sam- The ITS sequences for all samples were aligned pled six of the seven species of Nomocharis using ClustalX (Thompson et al. 1997) and accepted by Sealy (1950) and in Flora of China adjusted by eye in MEGA 4.0 (Tamura et al. (Liang & Tamura 2000), as well as the recently 2007) following the guidelines of Morrison described species N. gongshanensis (Gao et al. (2009). Gaps were positioned to minimize nucle- 2012). Thus, our sampling included all Nomo- otide mismatches, and were treated as missing charis native to China, and excluded only N. data in the phylogenetic analyses. The boundaries synaptica, which is endemic to India. For seven of ITS were determined by comparing the aligned Nomocharis species, we sampled 14 accessions, sequences with previously published Lilium of which three were new. Seven species of sequences (Nishikawa et al. 1999, Nishikawa et Notholirion, Cardiocrinum and Fritillaria were al. 2001) and sequences were trimmed accord- included as outgroups based on previous stud- ingly. Sequence lengths and G + C content were ies indicating a close relationship between these calculated using MEGA 4.0 (Tamura et al. 2007). groups and the Lilium–Nomocharis complex Bayesian phylogenetic analyses of the ITS (Patterson & Givnish 2002, Tamura et al. 2004, dataset were conducted using MrBayes ver. 3.1.2 Fay et al. 2006). (Ronquist & Huelsenbeck 2003). We applied the GTR + I + G model of nucleotide substitution, which was selected under the Akaike informa- DNA extraction, amplification and tion criterion (AIC, Akaike 1974) by MrMod- sequencing eltest ver. 2.2 (Nylander 2004). We performed two simultaneous Bayesian analyses using three For most samples, total DNA was isolated from hot chains and one cold chain starting from a silica-dried leaf tissue using a modification to random tree. Temperature increments between the cetyltrimethyl-ammonium bromide (CTAB) chains were adjusted to 0.2 based on mixing in protocol of Doyle and Doyle (1987). In several preliminary analyses. Analyses were run for 10 cases, the DNAQuick Plant System (TIANGEN million generations with sampling every 100 Biotech, Beijing, China) or Plant Genomic DNA generations. Variation in likelihood scores was Kit (TIANGEN Biotech, Beijing, China) was examined graphically for each independent run used following manufacturer protocols. using the program Tracer 1.4 (http://beast.bio. The ITS marker, including 5.8S, was ampli- ed.ac.uk/Tracer) to determine apparent stationar- fied using the ITS4 and ITS5 primers (5´-TCC- ity, and the program output from MrBayes was TC CGCTTATTGATATGC-3´ and 5´-GGAAG T- used to assess convergence between simultane- A A AAGTCGTAACAAGG-3´, respectively) of ous runs. The first 25% (or 25 000) trees were White et al.