Lespedezadanxiaensis (Fabaceae), a new species from Guangdong, China, based on molecular and morphological data

Abstract Lespedezadanxiaensis (Fabaceae), a new species from Danxiashan National Nature Reserve in Guangdong Province, is described and illustrated. The new species is morphologically similar to Lespedezapilosa, but it can be easily distinguished by its thin leathery leaflets and long peduncles. Phylogenetic analysis based on ITS confirmed that the new species belongs to Lespedezasubg.Macrolespedeza. The new species is the first known species of Lespedeza endemic to Danxia landform and is currently only known from Mount Danxia, Guangdong.

During a botanical expedition to Danxiashan National Nature Reserve, Renhua County, Guangdong Province from May to October, 2020, we discovered an unknown species of Lespedeza. It is similar to L. pilosa (Thunb.) Siebold & Zucc. in indumentum (densely villous throughout), procumbent stems and ovate to obovate leaflets, but differs from the latter by its leathery leaflets, pinkish corolla and longer peduncles of chasmogamous flowers. After carefully checking specimens and literature, together with a molecular phylogenetic analysis based on Internal Transcribed Spacers (ITS), we demonstrated it is indeed a new species; thus here, we describe and illustrate it.

Morphological study
The morphological characters were examined, based on the living plants and specimens kept in the herbaria IBSC, NPH, SWFC and SYS, herbarium acronyms as in Thiers (2021).

Taxon sampling and molecular analyses
Three individuals of L. danxiaensis were collected from Danxiashan National Park, Guangdong province, China from July to September in 2020 (Fig. 1). Voucher specimens were deposited in the Herbarium of Sun Yat-sen University (SYS). The nuclear DNA Internal Transcribed Spacers (ITS) was used for reconstructing the phylogeny of the new species and its related taxa (Xu et al. 2012). A total of 45 ac-cessions, representing 33 species of Lespedeza [including two nominal species viz. L. nipponica Nakai and L. japonica L. H. Bailey, which had been synonymised with L. formosa (Vogel) Koehne (Hatusima 1967) or L. thunbergii (DC.) Nakai (Ohashi et al. 2009)] and one species of a related genus, Campylotropis macrocarpa (Bunge) Rehder was sampled for outgroup comparison. The GenBank accession numbers are listed in Appendix I. Most sequences were downloaded from GenBank, except for the new species, which was newly sequenced in the present study. Three samples of the new species were sequenced and were identical, of which only one sequence (MZ468553) was selected for the phylogenetic analysis. Genomic DNA was extracted from silica-gel-dried leaves using the modified 2 × CTAB procedure of Doyle and Doyle (1987). The ITS sequences were amplified with primer pairs ITS4/ ITSA, with PCR amplification and sequencing following Xu et al. (2012). The phylogenetic relationships were assessed using the Maximum Likelihood (ML) method, which was constructed using the programme IQ-TREE (Nguyen et al. 2015).

Molecular phylogenetics
The aligned sequences of ITS for phylogenetic analyses are 702 bp in length. Lespedeza was recovered as monophyletic in the resulting phylogenetic tree in this study (LP: 100, Fig. 2). The North American Lespedeza taxa were clustered into a clade

Morphological comparison
A detailed morphological comparisons of the new species with the five closely related species within subclade C-1 are summarized in Table 1. In morphology, the putative new species is most similar to L. pilosa, sharing such features as procumbent stem, ovate to obovate leaf blades, and plant covered densely villous indumentum. However, the new species differs from the latter by leathery leaflets, longer peduncles of chasmogamous flowers, and pink to pale purple corolla (Table 1, Fig.  3). The other four species included in subclade C-1 could be easily distinguishable from the new species by their habits (stem erect vs. stem procumbent), narrow leaf shape (oblong-linear to narrowly obovate leaf vs. ovate, obovate to subrounded), and shorter peduncles (0.5-1.0 mm vs. 11-28 mm) (Table 1). Diagnosis. L. danxiaensis is most similar to L. pilosa morphologically both being densely villous throughout, and having procumbent stems with ovate to obovate leaflets, but differs from the latter by its leathery leaflets with obviously concave veins (vs. leaflets papery, veins slightly concave), pink to pale purple corolla (vs. corolla yellowish-white to white, with purple spots at base of the standard) and longer peduncles of chasmogamous flowers (1.1-2.8 cm vs. peduncles of chasmogamous flowers rather short, 0.5-1.0 mm in L. pilosa).
Distribution, ecology and habitat. Lespedeza danxiaensis is currently known only from a few populations on Mount Danxia in Renhua County, Guangdong Province of China. It was observed to occur in bushwood on the mountaintop of Danxia landform at elevations between 270 and 310 m; plants in association included Osteomeles subrotunda K. Koch, Abelia chinensis R. Br., Lagerstroemia indica L., Selaginella tamariscina (P. Beauv.) Spring etc.
Conservation status. The known localities of Lespedeza danxiaensis are in Danxiashan National Nature Reserve where they are well protected. However, its population size is quite small. There are fewer than 100 individuals surviving in an area of about 200 m 2 in the currently known localities. We carried out several field surveys in 2020

Discussion
It is obvious that the new species belongs to Lespedeza due to its persistent bracts with two flowers inside, non-articulate pedicels, and 1-seeded pods (Fig. 3). Our molecular phylogenetic results further support the inclusion of the new species within Lespedeza subg. Macrolespedeza re-circumscribed by Ohashi and Nemoto (2014) (Fig. 2). The most conspicuous character of L. danxiaensis is its procumbent stems. There are only three procumbent Lespedeza species formerly recorded in China, i.e., L. fasciculiflora, L. hengduanshanensis, and L. pilosa. However, the former two species, occurring in western China (northwestern Yunnan, western Sichuan and Tibet) (Huang et al. 2010;Xu et al. 2014), are distantly related to the new species in the phylogenetic tree (Fig. 2). The third species L. pilosa is close to the new species, but they differ in the leaf texture, flower color, and the peduncle length of the chasmogamous flowers as described above. In addition, the ITS sequences of the three individuals of the new species are identical and no heterozygous sites were detected in these sequences, indicating that L. danxiaensis is not of hybrid origin, but a distinct species.
Lespedeza danxiaensis is current only known from the type locality, i.e. Mount Danxia, and only one population with fewer than 100 individuals was found by the authors. They grow in the special habitat of the Danxia landform, confined to the sub-top area of a peak. The special habitat may lead the phenomenon in which the number of this species is extremely small, thus the conservation of the species, including ex situ and in situ conservation, is urgently needed. Lespedeza danxiaensis has a procumbent habit, usually growing in patches on the ground, and is drought-tolerant. Our observations found that the above-ground part of the species survives drought by dropping many leaves during the dry season. Thus, this species may be suitable as a slope protection or soil-and-water conservation plant, which has potential development and application value.