Dryopteriswulingshanensis (Dryopteridaceae), a new species from Hunan, China

Abstract Dryopteriswulingshanensis, a new species growing on limestone in the Wulingshan Mountains, Hunan, China, is described and illustrated. This species is most similar to D.jishouensis and D.gymnophylla on general morphological traits, such as the form of scales, rhizome and sori, but differs by the number of vascular bundles at the base of the petiole, length to width ratio of lamina, stalk length of basal pinnae, division of the lamina, apex form of the pinnule and habitat. Moreover, molecular phylogenetic analysis using the chloroplast rbcL gene suggested that D.wulingshanensis, as the sister group of D.jishouensis, is a monophyletic clade. According to its restricted geographic range, small populations and few individuals, D.wulingshanensis should be considered endangered, according to the IUCN Red List criteria.


Introduction
Dryopteris Adans. (1763: 20, 551) is one of the largest fern genera with about 400 species, widely distributed all over the world (Wu et al. 2013). Based on molecular phylogenetic evidence, several genera are nested within Dryopteris, such as Acrophorus C. Presl, Acrorumohra (H. Itô) H. Itô, Diacalpe Blume, Dryopsis Holttum & P. J. Edwards, Nothoperanema (Tagawa) Ching and Peranema D. Don . Most species in Dryopteris share a short rhizome and catadromous arrangement of frond segments, compared to its sister genus, Arachniodes Blume, which has long-creeping rhizomes and anadromous laminae Wu et al. 2013). The species of this genus usually grow in forests, open vegetation and, occasionally, in the rocky area of temperate and tropical regions (Fraser-Jenkins 1986;Kramer et al. 1990;Wu et al. 2013). In China, the genus is widely distributed, especially in south-western regions, with about 167 species with 60 endemic species in four subgenera (D. subg. Pycnopteris, D. subg. Nothoperanema, D. subg. Dryopteris, and D. subg. Erythrovariae) (Wu et al. 2013).
During 2016-2021, we surveyed ferns in the Wulingshan Mountains, which occupy the border zone of four Provinces in China (Hubei, Chongqing, Guizhou and Hunan). This region, as one of the biodiversity hotspots, nurtures a large number of endemic plants and preserves many relict plants (Chen et al. 2004;Yan and Zhou 2021). When we arrived at the Pangu Peak of Dehang Scenic Area, Jishou City, Hunan, an epipetric species that grows in the limestone crevices caught our attention. It is most similar to Dryopteris gymnophylla (Baker) C. Chr. and Dryopteris jishouensis G.X. Chen & D.G. Zhang but differs by the length to width ratio of lamina, stalks of the basal pinnae, apex form of pinnules and habitat. Moreover, we found this unknown species was also distributed in Mt. Tianmenshan and Zhongli Grand Canyon of Zhangjiajie City, Hunan, China. In order to infer the phylogenetic position of this species, the chloroplast rbcL sequences of 32 individuals, representing 11 closely related species, were analyzed. Based on morphological and molecular phylogenetic evidence, we describe it as a new species, named Dryopteris wulingshanensis J.P. Shu, Y.H. Yan & R.J. Wang and illustrate it here.

DNA extraction and sequencing
A total of 32 samples, representing 11 species of the genus Dryopteris, were analyzed to infer the phylogenetic relationships amongst the unknown species and its closest relatives. Dryopteris aemula (Aiton) Kuntze was sampled as an outgroup based on the previous phylogenetic studies of the genus Dryopteris . The rbcL gene of 18 individuals were newly sequenced and submitted on the GenBank (Table 1), and the others were obtained from GenBank database. Total genomic DNA was extracted from silica gel-dried leaves by using a DNA secure Plant Kit (Tiangen Biotech, Beijing, China) according to the manufacturer's protocols. The primers and amplification reaction of rbcL gene followed the protocols of Shu et al. (2017). Sequencing reactions were set up to obtain both the forward and reverse sequences, and then sequenced on an ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, California, USA).

Molecular phylogenetic analysis
The consensus sequences were generated using SeqMan v7.1.0 (DNASTAR, USA) and then 32 sequences used for phylogenetic analysis were aligned with BioEdit v7.2.0 (Hall 1999). The Maximum Likelihood (ML) phylogenetic tree was constructed by IQ-TREE v2.1.3 (Minh et al. 2020), the best-fit model (K2P+I) was chosen according to Bayesian Information Criterion (BIC) with ModelFinder (Kalyaanamoorthy et al. 2017) and the branch support values of ultrafast bootstrap (UFBoot) approximation was performed with 1000 repetitions. Each bootstrap tree was optimized using a hill-climbing Nearest Neighbor Interchange (NNI) search, based directly on the corresponding bootstrap alignment to reduce the risk of overestimating branch supports with UFBoot (Hoang et al. 2017). The Bayesian Inference (BI) species tree was constructed by MrBayes v3.2.7 (Ronquist et al. 2012) with the GTR + I + G model. Four Markov Chain Monte Carlo (MCMC) chains were run simultaneously for two million generations, and sampled every 100 generations. The convergence was assessed with the average standard deviation of split frequencies lower than 0.01.
Additional specimens examined ( Etymology. The specific epithet "wulingshanensis" is derived from the name of type locality Wulingshan Mountains, where the new species is found. IUCN Conservation Assessment. EN(B1ab(iii)). Dryopteris wulingshanensis is only known from three locations of Wulingshan Mountains in Jishou and Zhangjiajie Cities, Hunan, China. Based on its restricted geographic range, small populations and few individuals, Dryopteris wulingshanensis should be considered endangered under the IUCN Red List criteria (IUCN 2019).