Oreocharis jasminina (Gesneriaceae), a new species from mountain tops of Hainan Island, South China

Abstract A new species of Gesneriaceae, Oreocharis jasminina S.J.Ling, F.Wen & M.X. Ren from Hainan Island, south China, is highlighted and described. The new species is distinguished by its actinomorphic corolla, narrow floral tube and ovate anthers hidden in the floral tube. The new species also showed clear geographic and altitudinal isolation from the three currently-recognised Oreocharis species on the Island. Molecular phylogenetic analysis, based on nuclear ITS1/2 and plastid trnL-trnF sequences, supported the delimitation of the new species, which forms a single lineage with all the other Oreocharis species from Hainan Island. The roles of geographic and floral isolation in the evolution of the new species and its affinities are discussed.


Introduction
The Oreocharis Bentham was recently re-circumscribed to a large genus by including ten more genera and over 135 species, based mainly on molecular phylogenetic studies (Möller et al. 2011(Möller et al. , 2016Xu et al. 2017;Möller 2019;Wen et al. 2019). The enlarged genus was predominantly distributed in China with some species in India, Myanmar, Thailand and Vietnam (e.g. Li and Wang 2005;Möller and Clark 2013;Möller et al. 2018). Regardless of the limited differences in habit and fruit structure, Oreocharis shows a strikingly-high diversity in floral syndromes (Li and Wang 2005;Wei 2010;Möller and Clark 2013).
As one of the globally-important biodiversity hotspots, Hainan Island harbours about 4000 seed-plant species, of which ca. 500 are endemics (Francisco-Ortega et al. 2010) and which are concentrated in the south-central mountains. Gesneriaceae, in Hainan Island especially, includes a high ratio of species endemism, eight out of the total of 24 species being endemic (Ling et al. 2017a). Currently, three taxa of Oreocharis are recorded on Hainan Island and all of them are Hainan-endemic and monophyletic, i.e. O. dasyantha Chun, O. dasyantha Chun var. ferruginosa Pan and O. flavida Merrill (Li and Wang 2005;Ling et al. 2020), while each of these species shows considerable variations in morphological traits (Wei 2010;Ling et al. 2020a).
During several fieldwork trips in the past three years, we found that some populations of Oreocharis on mountain tops in Hainan Island showed obvious differences in various morphological characters. After careful literature studies (Pan 1987;Li and Wang 2005;Wei 2010) and morphological and molecular examinations, we are convinced that populations from the mountain tops of Mt. Yingge and Mt. Limu represent a new species, which we report and describe here.

Morphological observations
The field study and conservation on Gesneriaceae were undertaken by two of the authors (SJL and MXR) over a long period of time, especially focusing on the Hainan-endemic species (Ling et al. 2017a, b;Xing et al. 2018;Li et al. 2019). Morphological observations and measurements were carried out, based on living plants during fieldwork. All available specimens of Oreocharis species, stored in the herbaria in China (PE, KUN, IBK and IBSC), were examined. We also downloaded all Oreocharis specimens from JSTOR Global Plants (http://plants.jstor.org), and Chinese Virtual Herbarium (http://www.cvh.ac.cn) to compare detailed morphological traits between the proposed new species with the currently-accepted species of Oreocharis. Specifically, we compared morphological traits of the possible new species with all the three currently-recognised Oreocharis species from Hainan Island, i.e. O. dasyantha, O. dasyantha var. ferruginosa and O. flavida. The specimens of new species were collected over the past two years and deposited in the herbarium of Hainan University (HUTB) and Kunming Institute of Botany, Chinese Academy of Sciences (KUN).

Taxonomic sampling, DNA extraction and molecular markers
The leaf samples of O. dasyantha, O. dasyantha var. ferruginosa, O. flavida and the putative new species were collected in the field and dried in a vascular bag with silica gel. Total genomic DNA extraction was conducted using CTAB methods (Doyle and Doyle 1987). One nuclear ribosomal DNA (nrDNA) sequence, the ITS region comprising spacer 1, the 5.8S gene and spacer 2 (White et al. 1990) and one chloroplast DNA (cpDNA) intron-spacer region trnL-trnF (Taberlet et al. 1991) were used in this study. Laboratory procedures followed Ling et al. (2020) and newly-acquired sequences were deposited in GenBank (Table 2).

Alignments and phylogenetic analyses
According to Möller et al. (2011), Chen et al. (2014 and Ling et al. (2020), Oreocharis sinohenryi (Chun) Mich.Möller & A.Weber which had the closest phylogenetic relationships with the Hainan Oreocharis taxa was used as outgroup with sequences (Genbank with accession numbers HQ632913 and HQ633009). The original chromatograms from both directions of the ITS1/2 and trnL-trnF sequences were evaluated using Bioedit (Hall 1999) for base confirmation and contiguous sequences editing, then we manually aligned sequences, where necessary, using MEGA v.6.5 (Kumar et al. 2008) and ambiguous positions were excluded from the alignments. The ITS1/2 and trnL-trnF were concatenated to a single matrix after a congruency test by PAUP* 4.0a164 (Swofford 2003). Bayesian Inference (BI) analysis was conducted using MrBayes version 3.1.2 (Huelsenbeck and Ronquist 2001) and Maximum Likelihood (ML) analysis was performed using MEGA v.6.5 (Kumar et al. 2008). Both procedures followed the Ling et al. (2020), based on the combined ITS1/2 and trnL-trnF sequences.

Phylogenetic reconstruction
The combined ITS1/2 and trnL-trnF datasets were 640 and 818 bp long, amongst which 64 and 17 were polymorphic sites and 27 and 6 were parsimony-informative sites, respectively. The aligned dataset was 1458 bp long and a total number of 81 polymorphic sites were measured, of which 33 were parsimony-informative sites. There was no significant incongruence, based on the incongruence length difference (ILD) test between the ITS1/2 and trnL-trnF (p > 0.05).
The new species also shows a clear geographic isolation from the three currently-recognised Oreocharis taxa on Hainan Island. The new species O. jasminina was only found in Mt. Limu and Mt. Yingge in the middle of the island, while O. dasyantha and O. dasyantha var. ferruginosa are restricted to the west side of the Island and O. flavida was only found in the east side (Fig. 5). They are isolated by a large river, the Changhua River (the second largest river on Hainan Island). Li et al. (2019) found that the geographic isolation by the Changhua River is a driving force for the great population differentiation in the two Hainan-endemic Gesneriaceae species, Primulina heterotricha (Merr.) Yan Liu and Metapetrocosmea peltata (Merr. et Chun) W. T. Wang. Thus, the geographic isolation by rivers or valleys may also play a key role in the evolution of O. jasminina and other Hainanendemic Oreocharis taxa. However, the relative contributions of such geographic isolation and altitudinal differentiation are still in need of further experimental examination.
Floral symmetry is widely recognised as a key trait in pollination and taxonomy. Normally, the zygomorphic corolla possesses higher pollen-transfer efficiency than the actinomorphic corolla (Sargent 2004). Oreocharis jasminina has yellow actinomorphic corolla with a long and narrow floral tube, differing from O. dasyantha and O. dasyantha var. ferruginosa (both have zygomorphic corolla). Although O. flavida has an actinomorphic flower, its campanulate corolla with four equivalent stamens and horseshoe-shaped anthers make it distinct from the new species O. jasminina (Table 1).
Floral shape was expected to be a vital factor in generating floral isolation and evolutionary shifts (Castellanos et al. 2004;Muchhala 2007). Generally, the floral shape has a strong connection with the expected pollinators in Gesneriaceae, for example, bees or hummingbirds for tubular flowers, bats for campanulate flowers and subcampanulate flowers having generalised pollination systems (Martén-Rodríguez et al. 2009). O. jasminina has thin-tubular corolla (Fig. 1), differing from O. dasyantha, O. dasyantha var. ferruginosa (both are conical corolla) and O. flavida (campanulatetubular corolla), indicating a possible pollination mechanism associated with the longtongued butterflies and moths. Such distinctive morphological differences indicate different pollination adaptation and clear reproductive isolation amongst these taxa, suggesting O. jasminina should be treated as a new species.