﻿ Coptishuanjiangensis, a new species of Ranunculaceae from Guangxi, China

﻿Abstract Coptishuanjiangensis, a new species of Ranunculaceae distributed in the valleys of Jiuwanshan National Natural Reserve in Huanjiang county (Guangxi, China), is described and illustrated for the first time based on morphological and plastome sequences data. It differs from C.chinensis, C.deltoidei and C.omeiensis mainly by having notably longer petiole, scape, bigger leaf blade with lobes obviously remote and robust rhizomes without stolons. Phylogenetic analyses support that C.huanjiangensis is sister to C.omeiensis and C.deltoidei.


Introduction
The genus Coptis Salisb. (Ranunculaceae), containing 15 recognized species, is one of the most medicinally important genera in China and demonstrates a classical eastern Asian and North American disjunct distributional pattern. According to the morphology, especially flower and leaf, genus representatives distributed in China had been classified into six species and one variant, i. e., C. chinensis Franch. (endemic to SW China), C. chinensis var. brevisepala W. T. Wang & P. K. Hsiao (endemic to SE China), C. deltoidei C. Y. Cheng & P. K. Hsiao (endemic to Sichuan, China), C. omeiensis (Chen) C. Y. Cheng (endemic to Sichuan, China), C. quinquefolia Miq. (distributed in Taiwan province and Japan), C. quinquesecta W. T. Wang (endemic to Yunnan, China) and C. teeta Wall. (endemic to SW China) (Tamura 1995;Dezhi and Robinson 2001).
All these species have branched rhizomes, basal and long petioled leaves splitting into three-five segments, small and actinomorphic flowers. Most of these species are less than 30 cm in height and grow in shady places in forest valleys at an altitude of 600-2500 meters. Due to the richness in benzylisoquinoline alkaloids, various Coptis species have been used in China ethnomedicine, and three of them, C. chinensis, C. deltoidea, and C. teeta, are used as official Huanglian 'Weilian', 'Yalian' and 'Yunlian' in the Chinese Pharmacopeia respectively Wang et al. 2022).
The south-western limestone area is one of the biodiversity centers in China, especially in Guangxi (López-Pujol et al. 2011;Huang et al. 2019). During the Fourth National Survey of Chinese Materia Medica Resources in Guangxi (August, 2018), we found an unusual species of Coptis with an outstanding plant size and robust rhizomes that are distinctive from other species in Jiuwanshan National Natural Reserve, Huanjiang county. Subsequently, an in-depth field investigation, detailed morphological observations and phylogenetic reconstruction by plastomes were carried out. The comprehensive morphology and molecular results suggested that it is a new species, which is described as follows.

Taxon sampling and DNA extraction
Samples of the new species were collected in the field and 12 related species of Coptis (a total of nineteen accessions) were obtained from the herbarium of PE (Herbarium, Institute of Botany, CAS, Beijing, China) and CMMI (Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China). Asteropyrum peltatum and A. cavaleriei were taken as outgroups, and the plastome sequences were downloaded from GenBank (http://www.ncbi.nlm.nih.gov) with accession numbers MG734862.1 and MG734861.1, respectively. Sample information is listed in Suppl. material 1.
Total genomic DNA was extracted from specimens using a modified cetyl trimethyl ammonium bromide (CTAB) method and purified with the Genebetter DNA clean-up kit (GeneBetter Biotech Corporation, Beijing, China) (Li et al. 2013). All the DNA and molecular material were deposited in the herbarium of the Institute of Chinese Materia Medica (CMMI).
Plastome sequencing and assembly PE150 sequencing was conducted on an Illumina HiSeq XTen platform at Novogene (Tianjin, China). The raw data of the PE150 sequencing were filtered using the Trim-momatic 0.39 software to obtain high-quality reads (Bolger et al. 2014). The de novo assembly of the high-quality reads was performed by GetOrganelle v1.7.5 with the following settings: -F embplant_pt, -R 15 and -K 105 (Jin et al. 2020). Ambiguous regions and four junctions between IRs and SCs in the plastid were confirmed manually in Geneious v8.1 Dong et al. 2022).

Phylogenetic reconstruction
A total of 23 plastid sequences were aligned using the MAFFT online service and manually adjusted using MEGA X (Kumar et al. 2018;Katoh et al. 2019). And ambiguous regions were trimmed by the Gblocks 0.91b program (Castresana 2000). Phylogenetic reconstruction was carried out using the maximum likelihood (ML) and Bayesian inference (BI) methods in PhyloSuite (Zhang et al. 2020). The program ModelFinder was used to select the best-fit model according to the Bayesian information criterion (Kalyaanamoorthy et al. 2017). The ML tree was inferred using IQ-TREE with the TVM+F+R2 model and 5,000 ultrafast bootstraps (Nguyen et al. 2015). The BI tree was implemented with the GTR+F+I+G4 model and the Markov chain Monte Carlo (MCMC) chains were run for 1,000,000 generations. The trees were sampled every 1000 generations and the initial 25% were discarded as burn-in. Trees were visualized in FigTree v1.3.1.

Taxonomic treatment
Distribution and habitat. This species has only been found in the valleys of Jiuwanshan National Natural Reserve, Huanjiang County up until now. It grows in shaded places in valleys at 800-1200 m. a. s. l.
Etymology. The specific epithet is derived from the type locality, Huanjiang County, Guangxi.
Phenology. The species was observed flowering in February -March and fruiting in April-June.
Note. There are seven species and one variant of Coptis distributed in China. An identification key is presented below.

Discussion
Plastoms have been extensively used in phylogeny reconstruction and species delimitation studies because of their moderate evolution rate and abundant phylogenetic information (Dong et al. 2021;Wang et al. 2021;Wang et al. 2022). The relationships of Coptis species were clearly resolved by phylogenetic studies. Coptis huanjiangensis possesses an independent phylogenetic position and is located in the clade formed by C. chinensis, C. deltoidei, and C. omeiensis. And the phylogenetic relationship of these four species is also supported by the morphological characters of these species in having a similar leaf blade shape (leaves three-sects), leaf blade texture (papery to subleathery), and a long and erect scape with five to ten small actinomorphic flowers. However, C. huanjiangensis can be distinguished from the latter three species by having notably longer petioles (15-40 cm) (vs other species having petioles shorter than 18 cm), spatulate petals (vs lanceolate or linear in other species), bigger leaf blades with lobes obviously remote and robust rhizomes without stolons. The detailed comparison between C. huanjiangensis and close species is represented in Table 1. Herein, both morphological and molecular studies indicated that C. huanjiangensis is an independent species.