A new species of Viola (Violaceae) from Guangdong Province, China

Abstract Viola huizhouensis (Violaceae), a new species from Xiangtoushan National Nature Reserve of Guangdong Province in China, is described and illustrated. The new species is most similar to V. guangzhouensis, but it can be easily distinguished by its much stouter rhizome, lack of aerial stem, dense pubescence of the basal pedicel and the whole plant. Our phylogenetic analysis, based on ITS sequences, confirms that the new species belongs to V. sect. Diffusae.


Introduction
Mount Xiangtoushan in Guangdong Province, China, is located in a subtropical zone with abundant rainfall, mainly a low-lying hilly landform, but has an extremely high altitude compared with the surrounding environment. A large area of granite in this region is exposed on the surface due to intense erosion and denudation and there are climax lithophytic vegetation communities with a high level of biodiversity. During fieldwork in March 2018 for the investigation of the biodiversity patterns in this mountainous region, a distinct new species, Viola huizhouensis, was collected on Mount Xiangtoushan. Viola L. is the largest genus of family Violaceae, with approximately 525-600 species around the world (Ballard et al. 1998;Clausen 1929). This genus has a high level of morphological differentiation and there are hybridisation and horizontal evolution amongst sections and species (Marcussen et al. 2015). There are about 93-108 native Viola species in China which belong to four subgenera according to Yuzepchuk and Klokov's (1949) classification, i.e. Melanium, Chamaemelanium, Dischidium and Viola. Amongst them, Viola is the largest subgenus, which includes nine sections and 78-95 species in China (Wang 1991;Chen et al. 2007).

Material and methods
Leaf material of the putative new species and its related species V. guangzhouensis, was collected and stored with silica gel in zip-lock plastic bags until use for comparisons and taxonomical treatment. Specimens of V. huizhouensis and V. guangzhouensis were collected respectively from Darenyan, Xiangtoushan National Nature Reserve and Shaoshangling, Liuxi River State Forest Park in March 2018. Voucher specimens were deposited in the Herbarium of Sun Yat-sen University (SYS).
Total DNA was extracted with the modified CTAB method (Doyle and Doyle 1987). The regions of partial internal transcribed spacer 1, 5.8S ribosomal RNA gene and partial internal transcribed spacer 2 were amplified using previously-reported primers ITS1, ITS4 (White et al. 1990). PCR amplifications were performed following Fan et al. (2015). The sequences of the species and related ones, downloaded from NCBI, were aligned using MEGA 6.0 (Tamura et al. 2013) with ClusterW and subsequently manually adjusted. Phylogenetic constructions were carried out with Maximum Likelihood (ML). ML was run by Iqtree 2.0.3 (Minh 2020), selecting best-fit model TIM+F+G4 with 2000 bootstraps. Phylogenetic analyses, based on Bayesian Inference (BI) were carried out using MrBayes version 3.1.2 (Huelsenbeck and Ronquist 2001). Bayesian analysis was run with four chains for 200,000 generations with the first 25% of sampled trees discarded as burn-in. Main quantitative characteristics of the putative new species and V. guangzhouensis were statistically analysed using IBM SPSS version 22.0.

Result
The aligned length of ITS sequences was 638 bps in total. ML and BI analyses produced similar topology ( Fig. 1  tuted a monophyletic clade (BS = 99%). Quantitative characteristics and a statistical analysis showed that there was a significant difference between V. huizhouensis and V. guangzhouensis in the diameter of rhizome and the leaf shape (Table 2). Diagnosis. Viola huizhouensis is most similar to V. guangzhouensis, but differs by its much stouter rhizome, lack of aerial stem, different leaf shape and dense pubescence of the basal pedicel and the whole plant.
Note. Based on its slightly 2-lobed stigma and stolons topped by rosettes of leaves, Viola huizhouensis should be a member of section Diffusae (W. Beck.) C.J. Wang, which was formerly treated as subsection Diffusae under section Viola by Becker (1925). The closest relative of V. huizhouensis on morphological grounds could be V. guangzhouensis. They shared several characteristics, for example, the well-developed rhizome and the bearded lateral petals. The new species can be distinguished from V. guangzhouensis, however, by its much stouter rhizome; lack of aerial stem; different leaf shape (apex obtuse, never acute vs. apex acute); and dense pubescence of the basal pedicel and the whole plant (vs. the basal pedicel sparsely puberulous or subglabrous and the stem glabrous) (Tables 1, 2; Fig. 2).
The ITS tree shows that V. huizhouensis is sister to V. guangzhouensis (BS = 100%), then they form a well-supported clade with V. yunnanensis, V. diffusa, V. nanlingensis and V. lucens (BS = 99%) (Fig. 1). Viola guangzhouensis and the other four species in this clade all belong to section Diffusae (Dong et al. 2009). Thus, the phylogenetic analysis supports V. huizhouensis as being close to V. guangzhouensis and belongs to section Diffusae.
In conclusion, the morphological differences and the molecular phylogenetic results provide sufficient evidence for treating V. huizhouensis as a distinct new species and it is a member of section Diffusae (W. Beck.) C.J. Wang (Wang 1991).

Appendix 1
GenBank accessions for phylogenetic analysis.