﻿Oreocharisqianyuensis, a new species of Gesneriaceae from Southwest, China based on morphological and molecular evidence

﻿Abstract Oreocharisqianyuensis, a new species of Gesneriaceae from Southwest, China, is described and illustrated based on morphological comparisons and molecular phylogenetic analyses. Phylotranscriptomic analyses of the new species in the context of a comprehensive phylogeny with dense sampling of 88% (111/126) of all species of the genus indicated that the new species was most closely-related to O.fargesii. The new species is morphologically similar to O.fargesii and O.nanchuanica in the shape, color and structure of flowers and the number of stamens, but differs in the leaf blade shape, margin and the indumentum characters of the inflorescence. Its morphological relationship with similar species is discussed, the detailed descriptions, colour photographs, distribution, as well as the IUCN threatened status based on the IUCN Red List Categories and Criteria are also provided.

; Bournea sinensis Oliv. and B. leiophylla (W.T. Wang) W.T. Wang & K.Y. Pan were removed from the genus based on molecular and palynological evidence (Chen et al. 2020), together with the publication of some dozens of new species (e.g., Wei et al. 2016;Cai et al. 2017Cai et al. , 2020Guo et al. 2018;Pan et al. 2019;Yang and Shi 2021;Le et al. 2022), Oreocharis s.l. hitherto comprises ca. 160 species, mainly distributed in South and Southwest China (150 species), with several species occurring in North Vietnam (ten species with eight endemic), Myanmar (two species), Bhutan (one species), India (one species), Japan (one endemic species) and Thailand (one endemic species) (Wang et al. 1990(Wang et al. , 1998Li and Wang 2005;Cai et al. 2020;Wen et al. 2021). Oreocharis shows extremely diverse floral charateristics, particularly regarding the corolla shape ranging from narrowly or widely tubular, campanulate, urceolate, to flat; symmetry from zygomorphic to actinomorphic; color from white, red, yellow, pink to purple (Jin et al. 2021). A recent extensive study based on transcriptomic data of 88% (111/126) of all species of the genus revealed the spatiotemporal diversification and the possible driving forces (Kong et al. 2022). This study provided a robust phylogenetic hypothesis of the relationships of most species and acts as firm basis for further studies such as species identification and delimitation.
In 2019, during a field investigation in Kaili City, Guizhou, China, an anomalous plant of Gesneriaceae with few flowers caught the authors' attention, and in July 2020, it was recollected at flowering time. Coincidentally, similar specimens were collected by Si-Rong Yi from Pengshui County, Chongqing, China in 2021. We identified it as a member of the previously recognised genus of Isometrum Craib (Pan 1986;Wang et al. 1990;Li and Wang 2005), which now belongs to the genus Oreocharis s.l. based on the flower and fruit characteristics, such as: four stamens coherent in pairs, anther thecae not confluent, capsule dehiscent on one side (Wang et al. 1990(Wang et al. , 1998Li and Wang 2005). After examination of the specimens stored in the related herbaria (E, HITBC, IBK, HN, K, KUN, P, PE and VMN) including digital specimens online, such as Chinese Virtual Herbarium (http://www.cvh.ac.cn/) in China and Global Plants on JS-TOR (https://plants.jstor.org/), and consulting the related taxonomic publications of Oreocharis from the adjacent regions (Wang et al. 1990(Wang et al. , 1998Li and Wang 2005;Li and Li 2015;Guo et al. 2018;Cai et al. 2019;Fu et al. 2019a, b), we could not match the species to any previously published one of this genus. Then we carried out transcriptome sequencing using the leaves from two individuals of Guizhou Population and two individuals of Chongqing Population respectively, and added them to the recently published large data set of orthologous nuclear genes screened from the transcriptomic data of 111 Oreocharis species to reconstruct the phylogeny of the genus including the sus-pected new species (Kong et al. 2022). The results suggested that the plants were nested within Oreocharis and the analysed material was phylogenetically distinct from other species. Here, Oreocharis qianyuensis Lei Cai, J.W.Yang & Q.Zhang is described and illustrated based on the morphological comparisons and molecular phylogenetic analyses.

Materials and methods
We measured and recorded the morphological characters at least from more than ten mature individuals at flowering and fruiting from Guizhou and Chongqing populations. In addition, four relatively young leaves from each of the four individuals (two from Guizhou population and two from Chongqing population) were collected and sent to Novogene Technology Co., Ltd. for transcriptome sequencing. After filtering the low-quality reads, the remaining clean reads were used for denovo assembling with the package Trinity v2.11.0 (Grabherr et al. 2011). Referring to the published data consisting of 574 orthologous genes and including 111 Oreocharis species (Kong et al. 2022), we extracted the corresponding orthologous genes and added them to the data set for phylogenetic reconstruction. Phylogenetic tree was inferred based on the data set of the concatenated genes using maximum likelihood (ML) in RAxML v8.0.X (Stamatakis 2014) with parallel computation employing 100 threads on a server (ThinkSystem SR860). The parameters were set as GTR substitution model and a random starting tree with all others left as default. 100 bootstrap replicates were used to assess the robustness of the branches in the ML tree.

Data availability statement
The transcriptome data of four individuals in this study are openly available from NCBI: https://www.ncbi.nlm.nih.gov/sra/PRJNA813939 (the two individuals from Chongqing population) and https://www.ncbi.nlm.nih.gov/sra/PRJNA861104 (the two individuals from Guizhou population).
Phenology. Flowering from July to August in Guizhou and from September to October in Chongqing; time of fruiting unknown.
Etymology. The specific epithet 'qianyuensis' refers to the known distribution at the time of publication in Guizhou and Chongqing in China. Qian is an alternative name for Guizhou and Yu is an alternative name for Chongqing. Vernacular name. The Chinese name of the new species is "Qian Yu Ma Ling Ju Tai" (黔渝马铃苣苔). The first two characters mean this species is distributed in Guizhou and Chongqing, and the last four characters represent the Chinese name of the genus Oreocharis. Distribution and conservation status. Oreocharis qianyuensis was observed to grow on the surfaces of rocks under forest in karst region in Kaili City, Guizhou, and on limestone rock surface or crevices under deciduous forests in Pengshui County, Chongqing. The species is currently known from one population of ca. 2000 individuals within 5500 m 2 (AOO) in Guizhou and one population of ca. 300 individuals within 1000 m 2 (AOO) in Chongqing. Since no special surveys were carried out for its distribution, and the threat is that the population is close to roadside in Guizhou and possible continuous drought in Chongqing, so it is very likely to be damaged or excavated, so this species was provisionally considered to be Endangered [EN B2ab(iii)] in terms of IUCN Red List categories and criteria (IUCN 2022).

Taxonomic affinities
The molecular evidence (phylogenetic tree) clearly supports that this new species belongs to the genus Oreocharis s.l. (Fig. 1). Our phylogenetic results presented here are congruent with the results presented by Chen et al. (2020), and here, we choose to support the decision of removing the genus Bournea from the enlarged Oreocharis. Although the latest conclusions from the phylogenetic study of Oreocharis s.l. by Lv et al. (2022) are different, which may be related to the data these authors used providing a different view of early and rapid evolutionary radiation of the Oreocharis. Further work is needed to clarify these incongruent results. Another important aspect for us is to classify this species into this genus based on some characteristics of its flowers and capsules, such as: four stamens coherent in pairs, anther thecae not confluent, capsule dehiscent on one side (Wang et al. 1990(Wang et al. , 1998Li and Wang 2005). O. qianyuensis, O. fargesii and O. nanchuanica have characters shared with the previously recognized genus Isometrum Craib based on the anthers attached in pairs, corolla purple, tube campanulate and not swollen (Pan 1986;Wang et al. 1990Wang et al. , 1998Pan and Liu 1995;Li and Wang 2005    as members of Sect. Pachysiphon K.Y. Pan and can be distinguished from the other Isometrum species by their corolla tubes short and thick (e.g., 6-12 mm long, 5-9 mm in the diameter, the length is 1.2-1.5 times the width), outside glabrous (Pan 1986;Wang et al. 1990Wang et al. , 1998Li and Wang 2005). O. qianyuensis morphologically resembles O. fargesii and O. nanchuanica in the purple flowers, 5-parted to the base of calyx, campanulate corolla and coherent in pairs of anthers, however, O. qianyuensis can be easily differentiated from them by the shape, margin, apex and base of leaf blade shape, indumentum characters of the inflorescence. Detailed diagnostic characters of the new species are listed and compared with other morphologically similar species in Table 1.