Viola shiweii Xiao C. Li & Z. W. Wang (Violaceae), a new species from Guizhou, China, is described, based on morphological and molecular evidence. The new species is morphologically most similar to V. kwangtungensis Melchior in its glabrous lateral petals and stoloniferous habit, but can be distinguished by its obtuse teeth along the leaf margin, its abaxially greyish-green leaf blade and its broader and entire sepals with a distinct basal appendage.

Morphology, phylogeny, sect. Plagiostigma, subg. Viola

Viola L. is the largest genus amongst the Violaceae, comprising approximately 580–620 species (Wahlert et al. 2014; Marcussen et al. 2015), which are widely distributed in temperate regions and tropical high mountain regions worldwide, with south-western China as one of its current centres of diversity (Wahlert et al. 2014). The diversity and high number of species has resulted in an extremely complex interspecific relationship in this genus due to hybridisation and horizontal evolution amongst sections and species (Marcussen et al. 2015). Since Becker (1925) provided the first infrageneric classification for Viola, several infrageneric classifications of the genus have been proposed (Clausen 1927, 1929, 1931, 1964; Gershoy 1934). In the latest taxonomical revision of Viola of China, 96 species were recognised as native (Chen et al. 2007). However, delimitation of the species with stolons distributed in southern and south-western China remains highly problematic and new species are still being discovered (Zhou and Xing 2007; Chen and Yang 2008, 2009; Zhou et al. 2008a; Dong et al. 2009; Ning et al. 2012; Huang et al. 2021).

During an expedition to Guizhou Province in November 2019, an unfamiliar violet whose habit was somewhat similar to that of Viola kwangtungensis Melchior caught the authors’ attention on the karst rock outcrops. Several specimens with cleistogamous flowers were collected from the field and living material was transplanted and cultivated in Chenshan Botanical Garden for further observation.

In this study, molecular phylogenetic analysis, based on the ITS dataset, was firstly conducted to resolve the phylogenetic position of the unfamiliar violet and its relationship with V. kwangtungensis Melchior. Subsequently, morphological characters of this unfamiliar violet and its related species were compared, based on living plants and herbarium specimens, including the digital resource of the Chinese Virtual Herbarium (https://www.cvh.ac.cn/) and the China Field Herbarium (https://www.cfh.ac.cn/). Herbarium specimens were examined in IBK and CSH. Original protologues and relevant literature were also investigated. Leaf material of the putative new species and its related species was collected and stored with silica. Six species, represented by eight individuals, were newly sampled. Voucher specimens were deposited in Chenshan Herbarium (CSH). Total genomic DNA was extracted with the modified CTAB method (Doyle and Doyle 1987) for library construction at Benagen (https://www.benagen.com). Paired-end sequencing of the whole sequences from both ends of 150 bp fragments was performed on the DNBSEQ T7, about 2 Gb clean data for every sample were produced. The nrDNA were de novo assembled using the GetOrganelle pipeline (Jin et al. 2020) and sequences of ITS1-5.8s-ITS2 were extracted with ITSx 1.1.3 (Bengtsson-Palme et al. 2013). Another 31 sample sequences were obtained from NCBI (Gong et al. 2010; Liang and Xing 2010). The sequences of the species and related ones were aligned in Geneious Prime 2021.2.2 (https://www.geneious.com) using MAFFT (Katoh and Standley 2013) by default setting. Phylogenetic construction was conducted by Maximum Likelihood with IQ-Tree 2 software (Minh et al. 2020), selecting the best-fit model of GTR+F+G4 with 2000 bootstraps. The tree file was visualised and annotated on iTOL (https://itol.embl.de/) (Ivica and Peer 2021). All the sequences accession numbers were listed in Table 1.

Although our phylogenetic analysis, based on ITS sequences, did not fully clarify the infrageneric relationships within Viola, it produced informative evidence for differentiation amongst lower taxa. V. shiweii can be placed in Viola ser. Australasiaticae (Okamoto et al. 1993), which is characterised by the stolons with scattered leaves, absent aerial stems, short spur of anterior petal and stigma beaked ventrally. The phylogenetic analysis in this study (Fig. 1) also confirmed this conjecture (Fig. 1); however, the monophyly of Viola ser. Australasiaticae was not supported, which was consistent with a previous study (Gong et al. 2010). Viola ser. Australasiaticae was proved to be nested in the subg. Viola sect. Plagiostigma Godr. (Marcussen et al. 2012).

Viola ser. Australasiaticae comprises ca. 27 species, widely distributed in the Himalayan Region, southern China, south-eastern Asia and Ryukyu Island of Japan, with 14 species occurring in China (Chen 2006), in which Viola davidii, V. schneideri W.Becker, V. kwangtungensis, V. mucronulifera and V. austrosinensis form a complex in this series and caused mass misidentification due to their high degree of morphological similarity.

Viola davidii Franchet was published by Adrien René Franchet (1885), based on the collection of David from Moupine (Baoxing County, Sichuan, China) (isotype: David#s.n., K000254222) in 1869 [1870]. It is a morphologically variable and widespread species characterised by its ovate or ovate-orbicular leaf blade with 6–8 rounded teeth along each side, bearded lateral petals and short spurred anterior petal. It was originally regarded as species similar to V. biflora L., but its beaked stigma (vs. bilobed), white and purple petals (vs. yellow) indicated a distinctly different affiliation amongst the genus. Later, Becker (1921) described a strikingly similar violet with ovate leaves, V. schneideri W.Becker, based on the collection of Schneider (isotype: Schneider C.K. #739, G00343327) from Te-chang (De-chang County, Sichuan, China), but the diagnostic leaf shape falls within the morphological variation of V. davidii Franchet and, for this reason, it was recently treated as its synonym, which is further supported by overlapping distributions (Chen 2006). The only collection of V. shiweii before this study was misidentified as V. davidii. Viola shiweii shares a similar leaf shape with V. davidii, but can be differed by its glabrous lateral petals and obtuse teeth along the leaf margin.

Viola kwangtungensis Melchior, which shows the highest resemblance to V. shiweii (Table 2, Figs 5, 6) is an overlooked species frequently being confounded with V. davidii or V. mucronulifera, but considered as a distinct taxon by Flora of China (Chen et al. 2007). Viola mucronulifera Hand.-Mazz. was published by Handel-Mazzetti (1931), based on the collection of R. C. Ching #7016 from Guangxi (holotype: PE00025463, isotypes: NY00097644 & A00067198) and is characterised by the distinctly stipitate tooth glands. Later, V. kwangtungensis Melchior (1933) was published, based on a collection of Woon-Young Chun and his assistants (isotype: P. Ko #50326, A00067196) from Guangdong, which can be easily recognised by its characteristic leaf crenation of the leaves, but it was subsequently reduced to a synonym of V. mucronulifera in Flora Reipublicae Popularis Sinicae (Wang 1991). Viola kwangtungensis has spinules at the apex of the teeth, as the horizontal extension of the teeth, while the spinules of V. mucronulifera are perpendicular to the leaf blade and placed between the teeth, which can be distinguished in field observation. Viola kwangtungensis also used to be considered conspecific with V. schneideri due to morphology transition in the spinose, based only on the specimen observation (Zhou et al. 2008b).

Figure 5. 

Flowers and leaves of Viola shiweii sp. nov. and Viola kwangtungensis A, B V. kwangtungensis, lower, front and side views C, D V. kwangtungensis, adaxial and abaxial leaf surfaces E, F Viola shiweii, flower, front and side views G, H V. kwangtungensis, adaxial and abaxial leaf surfaces.

More recently, as the latest supplement of this complex, a new species, V. austrosinensis, distinguished from V. kosanensis Hayata (ser. Rosulantes Borbas (Y.S.Chen)), was described, of which the leaves were coriaceous, glabrous, not glandular-dotted on the abaxial surface (Chen and Yang 2008). Viola austrosinensis is different from V. shiweii in its ovate leaf blade and acute anterior petal’s apex.

In China, V. mucronulifera was found to occur only in the Province of Yunnan and its type locality in Guangxi; its occurrence in Guizhou was a mistake caused by the misidentification of V. kwangtungensis in Flora of Guizhou (Yao 1989), as we personally observed in the locality cited in this work (Fanjingshan, Jiangkou). The morphology and distribution differences between V. shiweii, V. kwangtungensis and V. davidii are listed in Table 2. Comparision of V. shiweii and V. kwangtungensi was visualised in Figs 5, 6. Keys to V. shiweii and its allies were also presented.

Figure 6. 

Leaf margin of V. shiweii sp. nov. and V. kwangtungensis A V. shiweii, holotype B V. kwangtungensis, isotype: P. Ko #50326, A00067196.

Our deepest gratitude goes to three reviewers Thomas Marcussen, Juliana de Paula-Souza, and Chen You-sheng and Subject editor Yasen Mutafchiev, for their careful work and thoughtful suggestions that have helped improve this paper substantially. The authors are grateful to Mr. Yu Tian-yi for his excellent illustration in the manuscript, Dr. Jiang Kai from Chenshan Botanical Garden for his help in data analysis and Dr. Huang Yan-shuang from Sun Yat-Sen University, Dr. Zhu Xin-xin from Xinyang Normal University for their generous offer of samples and collection information. The first author is also indebted to Dr. Huang Yu-song and Huang Jin-quan from IBK for hosting our visit and Miss Pi for her company during the epidemic of COVID-19. This study was supported by the project of the National Wild Plant Germplasm Resource Center for Shanghai Chenshan Botanical Garden (ZWGX2102), the project of the Special Fund for Scientific Research of Shanghai Landscaping & City Appearance Administrative Bureau (G212416, G222404).