Oxytropis shennongjiaensis (Fabaceae), a new species from Hubei, Central China

Abstract Here we describe Oxytropis shennongjiaensis, a new species of Fabaceae from Central China (Hubei Province). Morphologically, O. shennongjiaensis is closely similar to O. sitaipaiensis, O. melanocalyx and O. kansuensis, but differs in stem characters, with less conspicuous internodes; persistent herbaceous stipules; pale yellow to white corolla; and stipitate legumes, 3–5 mm with a long beak. Phylogenetic analysis, based on the internal transcribed spacers (ITS) and two chloroplast markers (trnL–F and psbA–trnH), also identified O. shennongjiaensis as a new species, which is consistent with our morphological analyses. Considering the morphological data and phylogenetic data presented here, we believe that this evidence satisfies the required diagnostic criteria to identify O. shennongjiaensis as a new species.


Introduction
About 310 species of Oxytropis DC. have been described, mainly distributed in East and Central Asia, as well as Europe, Africa and North America (Zhu et al. 2010). Zhang (1998) recorded 146 species of Oxytropis (incorporating 12 varieties) as native to China. However, in the Flora of China, Zhu et al. (2010) only recognised 133 species of Oxytropis after having eliminated taxa of uncertain taxonomic status and those based on specimen misidentifications. Oxytropis in China is mainly distributed in Xinjiang, Tibet, Qinghai, northwest Yunnan, western Sichuan, Gansu, Inner Mongolia, Shaanxi, Shanxi, Henan, Hebei, Liaoning, Jilin and Heilongjiang Provinces.
China has a vast territory with a wide range of complex and diverse topographies and soils and covering several climate zones, which contribute to the wealth of Chinese botanical diversity (Chen et al. 2018a). The Shennongjia National Park in Hubei Province is a world-famous natural heritage site for biodiversity richness and, in recent years, many new species have been described from the region (Chen et al. 2018b;Deng et al. 2018). In 2016, during a comprehensive collecting expedition within this Park, we discovered a species of Oxytropis that was very unusual in its morphological characters. After consulting local floras (Fu 1979;Qian 1990;Yang et al. 2009;Li and Liu 2010) and newly published species Zhu et al. 2002;Zhu 2003), especially from the vicinity of the Park (Hubei, Anhui, Jiangxi, Hunan, Guizhou and Chongqing), we were unable to find any record of Oxytropis in these regions. However, there are eight species of Oxytropis recorded in the neighbouring Henan Province (Ding and Wang 1988). Additionally, in the neighbouring Shaanxi Province, which has the closest geographical connection, nine species and two varieties of Oxytropis are recorded in Flora Tsinlingensis (Northwest Institute of Botany, Chinese Academy of Sciences 1981).
After three years of observations of wild living plants, herbarium specimens and laboratory studies, we determined that the morphological characters of this entity were stable and did not match with any other species of Oxytropis known to us. Accordingly, combined with a molecular phylogenetic analysis, based on the internal transcribed spacers (ITS) and two chloroplast markers trnL-F and psbA-trnH, we determined that this entity was indeed a species new to science and, therefore, we describe it below as O. shennongjiaensis D.G. Zhang, J.T. Chen, T. Deng & H. Sun, sp. nov. As Oxytropis was first discovered in the mountains of Central China (Hubei Province), this new species is particularly valuable for further study of the origins, dispersal and current geographical distribution of the genus.

Morphology
The specimens of Oxytropis shennongjiaensis were collected from Shennongjia National Park in Hubei Province. Morphological characters, recorded for the new species, were  Table 1.

Molecular analyses
Molecular analysis was performed, based on 35 samples from 34 species (incorporating one variety) belonging to 11 sections of Oxytropis and, as such, represents the most comprehensive phylogeny of Chinese Oxytropis undertaken to date. Astragalus daenensis daenensis Boissier and A. penetratus Maassoumi were chosen as outgroups, following Shahi-Shavvon et al. (2017). Sequences for 34 related Oxytropis taxa and the two outgroup taxa were obtained from the NCBI GenBank. The GenBank accession numbers are listed in Appendix I. DNA of O. shennongjiaensis was isolated Stems with 2 or more conspicuous internodes. Stems with (0 or)1-4 conspicuous internodes.

Leaves
Leaves with sparsely subappressed white trichomes.
Leaves with sparsely white trichomes.
Leaves with sparse yellow, white and black long trichomes.
Leaves with glabrescent or sparsely spreading white villous.

Flowers
Corolla pale yellow to white; standard 16-18 mm long, lamina broadly ovate, 12-13 × 10-11 mm, apex emarginate to 2-lobed, margin lightly undulately entire or with irregular repand teeth; wings 12-15 mm long, lamina obovate; keel 15 mm long, beak 3 mm long.  Lu et al. 2010;Li et al. 2011). Sequences were assembled and a multiple alignment was initially performed using MAFTT in Geneious version 9.0.2 (Kearse et al. 2012), followed by minor manual corrections. Gaps were treated as missing data. Phylogenetic relationships were assessed using Bayesian Inference (BI) analyses, maximum parsimony (MP) and maximum likelihood (ML). A MP phylogenetic tree was constructed using PAUP* version 4.0a (Swofford 2002). The heuristic search was selected using 1000 replicates of random addition sequence and tree bisection-reconnection (TBR). Branch support was evaluated by 1000 bootstrap values. The ML phylogenetic tree was conducted in the IQ-TREE webserver (Trifinopoulos et al. 2016, http://iqtree.cibiv.univie.ac.at). Substitution model options were set to Auto and analysis, followed by 1,000 replicates. BI analyses were calculated in MrBayes version 3.2.7 (Ronquist and Huelsenbeck 2003). Models of sequence evolution for each partition were determined following the Akaike Information Criterion (AIC), as implemented in jModelTest, version 2.1.6 (Posada 2008). The results showed that the TIM3ef+I model was identified as the best-fit for ITS, the TIM1+I model for psbA-trnH and the TIM2+I model for trnL-F. These models cannot be found in MrBayes and GTR+I was thus selected as a replacement. Bayesian analyses were done using the settings: Bayesian trees were started from random trees; four Markov Chain Monte Carlo (MCMC) simulations were run simultaneously and sampled every 1,000 generations for a total of 10 million generations; and the first 20% of trees were discarded as burn-in. shennongjiaensis appears to be closely similar to O. sitaipaiensis, from which it can be distinguished by its stems with less conspicuous internodes and 5-15 mm internodes (stems with two or more conspicuous internodes in O. sitaipaiensis); stipules ovate,   Description. Perennial herbs, 10-15 cm tall. Yellowish-brown, cylindrical roots, up to 25 cm long, with lateral roots. Caulescent from a multi-headed caudex, slightly subterranean sometimes rhizomatous. Stems sprawling, 3-15 cm long, basally with persistent stipules; nodes of stems slightly swollen; internodes 5-15 mm long, invested with sparse, white trichomes. Leaves (4-) 6-9 (-11) cm long, 13-17 (-19)-foliolate; leaflets ovate, 5-11 × 2-4 mm, apex acuminate, with sparse, subappressed white trichomes, abaxially mid-vein slightly raised (obvious after drying), with denser trichomes along vein; dark purplish-red or green rachis, with sparse white trichomes; stipules ovate, 7-10 × 3-4 mm, herbaceous, basally connate, apex acuminate, abaxially sparsely hairy with white trichomes, adaxially glabrous, margins scarious, ciliate with black and white trichomes. Racemes rather lax, 3-6-flowered; peduncles 2.5-4.5 cm long, erect, villous, with white trichomes, sparsely intermixed with black trichomes below, with densely black trichomes above. Bracts ovate, 6-8 × 2-3 mm, membranous, with sparse, dark brown trichomes intermixed with white trichomes abaxially. Calyx campanulate, 9-11 × 2-4 mm, with dark brown trichomes sparsely intermixed with white trichomes outside; lobes subulate, 4-5 mm long, as long as or sometimes slightly shorter than tube. Pale yellow to white corolla; standard 16-18 mm long, lamina broadly ovate, 12-13 × 10-11 mm, apex emarginate to 2-lobed, margins slightly undulated entire or with irregular repand teeth; wings 12-15 mm, lamina obovate, 7 × 4 mm long, apex obtuse, claw 4-5 mm long; keel 15 mm long, beak 3 mm long. Ovary linear, with dense white trichomes. Legumes stipitate (stipe 5-7 mm long), oblong-ellipsoid, 20-25 × 5-7 mm, erect, inflated and slightly flattened, with sparsely white trichomes, beak 3-5 mm long. Conservation status. The new species was only discovered in Jinsiyanya, Shennongjia National Park, from our expeditions during the past few years. About 300 individuals were observed and the extent of occurrence is ca. 50,000 m 2 . The precise conservation status of the population(s) has not been determined, so further explorations are needed to assess its conservation status. Based on available data, the new species is assigned to the category 'Data Deficient' (DD) of International Union for Conservation of Nature (IUCN 2019).

Oxytropis shennongjiaensis
Molecular phylogenetic analysis. Based on the combined datasets (ITS, trnL-F and psbA-trnH), BI, MP and ML trees were reconstructed and their topologies are quite similar. The ML tree is presented in Figure 5 and shows the posterior probability (PP), ML bootstrap support (ML BS) and MP bootstrap support (MP BS) values. Our This new species is shown to be separated from other species and, to some extent, it can be identified as a new species.

Discussion
These above-detailed characters indicate that, according to Zhang (1998), this new species belongs to Oxytropis sect. Oxytropis with 19 other species (incorporating one variety) and, according to Zhu et al. (2010), it belongs to the Oxytropis sect. Mesogaea Bunge with 32 other species (incorporating three varieties). It can be distinguished from all other species of these two sections in branches, leaves, racemes, flowers and legumes characters, as described above. Morphologically, the new species shows some similarities with Oxytropis sitaipaiensis, O. melanocalyx and O. kansuensis and we also examined the specimens of these species , but they are also easily distinguished (Table 1). Additionally, there is no previous record of this genus in Hubei Province.
Phylogenetic analyses, based on 35 samples from 34 species (incorporating one variety), show that their topologies of the BI, MP and ML trees were quite similar and were consistent with previous studies (Shahi-Shavvon et al. 2017). Oxytropis is a monophyletic group. However, partial PP and BS of the tree were relatively low, which might be caused by the rapid radiation of Oxytropis (Shahi-Shavvon et al. 2017) and phylogenetic relationships of the new species and Oxytropis require further study. O. melanocalyx and O. latibracteata were shown to be sister to O. shennongjiaensis. These species share some morphological similarities. However, morphologically, O. latibracteata also shows the greatest differences in the following characters: acaulescent; racemes rather dense, 5-13-flowered or more; bluish-purple to pale purple corolla; standard 21-27 mm, lamina narrowly elliptic; wings 17-19 mm; keel 16-17 mm, beak 1-1.5 mm; legume sessile.
Considering the morphological data and phylogenetic results, we believe that this evidence satisfies the required diagnostic criteria to identify O. shennongjiaensis as a new species.