Geum sunhangii (Rosaceae), a new species from Hubei Province, China

Abstract Geum sunhangii – first discovered in Shennongjia National Nature Reserve, Hubei Province, China – is described as a new species of Rosaceae. Compared to all known Chinese Geum species, the new species differs by possessing jointed styles, imbricate petals and a reniform radical leaf terminal leaflet. Most significantly, the jointed style is curved at an obtuse or a right angle. In addition, the inclusion of this species within the genus Geum was supported by phylogenetic analysis using the sequence data of a nuclear ribosomal internal transcribed spacer (nrITS) and a chloroplast trnL–trnF intergenic spacer. The new species was found to be closely related to G. rivale and G. aleppicum.


introduction
The genus Geum L. (1753: 500) (Rosaceae), also known as Avens, contains ca. 56 species distributed throughout the temperate, tropical and arctic regions of the world and is found mainly in the Northern Hemisphere (Gajewski 1959). The morphology of Geum is very complex. Most species are herbaceous perennials that form rosettes consisting of imparipinnate leaves and a thick caudex, but a few species are small shrubs. In addition, most species in this genus have fish-hook shaped fruits (Rafinesque 1833). Only three species G. aleppicum Jacq., G. rivale L. and G. japonicum var. chinense F.Bolle are present in China (Li et al. 2003). Bolle (1933) divided Geum into several genera, including Novosieversia F.Bolle and Oncostylus F.Bolle (Bolle, 1933), amongst others. However, based on cytological data, Gajewski (1957) concluded that Geum is a polyploid complex and did not support any previous circumscriptions of Geum; instead, Gajewski recognised most of the previously segregate genera as subgenera within the genus Geum (Gajewski 1957(Gajewski , 1968. Finally, his point of view was further supported by later morphological and molecular studies (Schulze-Menz 1964;Hutchinson 1967;Robertson 1974;Kalkman 1988;Smedmark and Eriksson 2002).
The Shennongjia National Nature Reserve is located in the northwest of China's Hubei Province. It is a world diversity hotspot defined by its unique geographical location and complex topography (Hu et al. 2004). The Nature Reserve is also characterised by its high species diversity (including many rare and endangered animals and plants) and relict plant species . Correspondingly, it has recently attracted considerable attention from many researchers (Ma 2016;Xie et al. 2017 (Deng et al. 2016) and Impatiens baokangensis Q.L. Gan & X.W. Li (Gan and Li 2016).
During a biodiversity survey of the Shennongjia National Nature Reserve, we discovered an undescribed species belonging to the Rosaceae. This species was distinguished by the presence of a rosette of basal leaves, petals and jointed styles. Our morphological and molecular studies revealed that the newly-collected material belongs to an unknown Geum species which is described here.

Materials and methods
Plant materials. Specimens of the new species were collected from a site in Shennongjia National Nature Reserve, Hubei Province. Leaves of the new species were obtained for molecular studies. All herbaria used in this study were obtained from KUN (Herbarium, Kunming Institute of Botany, CAS).
Anatomical observation. Specimens of G. sunhangii were dissected and directly observed, before being placed under an anatomical lens for further observation under magnification. The cauline leaf, radical leaves, seeds, petal, style joint and stamen were inspected. DNA sequencing and molecular analyses. Total DNA was extracted using a DP305 Plant Genomic DNA Kit (Tiangen, Beijing, China) and we selectively amplified the nuclear ribosomal internal transcribed spacer (nrITS) and chloroplast trnL-trnF intergenic spacer (trnL-trnF) regions by Polymerase Chain Reaction (PCR). Successfully amplified DNA fragments were then sequenced commercially. Molecular analysis was performed using two outgroups (i.e. Fallugia paradoxa Endl. and Sanguisorba officinalis L.; Potter et al. 2007), one piece of material from the putative new species and fourteen samples with similar morphology. Partial sequences were obtained from GenBank (https://www. ncbi.nlm.nih.gov/genbank); GenBank accession numbers for all species are listed in Table 2. Multiple sequence alignments were initially performed using CLUSTAL W ver. 1.4 (Thompson et al. 1994) and were manually adjusted using BioEdit ver. 7.0.5.3 (Hall 1999). We used SequenceMatrix ver. 1.7.8 to combine the sequences (Vaidya et al. 2011).
Phylogenetic trees were constructed using Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI) algorithms. All characters were weighted equally, with gaps treated as missing data. MP analysis was conducted using PAUP ver. 4.0a (Swofford 2002). MP trees were obtained from a heuristic search of 1000 random addition replicates using tree bisection-reconnection (TBR). Finally, we obtained 1000 bootstrap values to evaluate the support for each branch.

Taxonomic treatment
Conservation status. Based on the results of field investigations, Geum sunhangii was only discovered in Nantianmen, Shennongjia National Nature Reserve, where there is almost no human pressure on the distribution location of this species. About 60,000 individuals were present and the extent of occurrence is ca. 30,000 m 2 . It is possible that additional populations may be discovered during future excursions. We initially define the category of G. sunhangii as Near Threatened (NT) by the Guidelines for Using the IUCN Red List Categories and Criteria (IUCN 2017).
Vernacular name. 神农花, shén nóng huā in Chinese Pinyin. Etymology. The new species is named after the famous Chinese botanist Hang Sun, who made significant contributions to plant taxonomy, floral identification and biogeography in China.

Phylogenetic analyses
We conducted a phylogenetic analysis of 15 species, based on nrITS and trnL-trnF sequence data. The aligned combined data matrix included 1649 characters and 397 variables. The three topologies inferred by the Bayesian Inference analyses, MP analyses and ML analyses were similar. The Bayesian tree, including MP bootstrap (BP), ML bootstrap (LP) and PP values, is presented in Figure 5. Bayesian analysis of the combined dataset showed that samples were divided into three clades. Clade III included most species of the genus Geum, while Coluria geoides (Pall.) Ledeb., Waldsteinia geoides Willd. and Taihangia rupestris T.T. Yu & C.L. Li were grouped in Clade II. Clade I was a sister clade to Clades II and III and consisted of only one species, Geum andicola Reiche. Finally, G. sunhangii was nested into a monophyletic group (PP = 1, LP = 100%, BP = 100%; Fig. 5) with G. rivale, G. aleppicum and G. montanum Gouan ex Steud. The new species was clustered with G. rivale, but with a weak support (PP = 0.82, LP = 73%, MP = 67%; Fig. 5).

Discussion
Geum aleppicum and G. japonicum var. chinense are similar in morphology; they both have similar yellow petals and green sepals, but these species differ in receptacle hair (Fig. 4Aa, Ba; Table 1). In contrast, G. rivale is easily recognisable due to its purplish sepals and purple-brown striped petals (Table 1). The nodding flower of the new spe-   cies is similar to that of G. rivale. Therefore, the combination of green sepals, nodding flower, yellow and imbricate petals is distinctly different from those found in the other three species present in China. In addition, the radical leaf and style joint of the new species are distinct in this genus. The terminal largest leaflet of the radical leaf is reniform (Fig. 3G) and different from the other species, which possess rhombic-ovate or compressed orbicular leaflets (Table 1). Curved styles are also important and recognisable features of the new species. Obtuse or right-angle curves of the style joint can be used to distinguish between the new species and its close relatives (Figs 3F, 4a). All distinguishing features are shown in Fig. 4 and Table 1.
We obtained samples of fifteen species for phylogenetic analysis. The topologies of the Bayesian, ML and MP trees were identical and were consistent with previous studies (Smedmark and Eriksson 2002). According to our results, Geum is probably a poly-phyletic group and the boundary between Geum and species in Clade II is not clear (Fig. 5). At the same time, species in the genera Taihangia T.T. Yu & C.L. Li, Coluria R.Br. and Waldsteinia Willd. are likely congeneric to the Geum species. Therefore, further studies of the taxonomic and phylogenic relationships of Geum, Taihangia, Coluria and Waldsteinia species are needed. At present, the new species was confirmed as a member of Geum, since G. sunhangii was nested within a group of Geum species (PP = 1, LP = 100%, MP = 100%; Fig. 5) that form a monophyletic group (Fig. 5). In addition, the new species can be easily identified by its morphological features.