Lysimachia fanii, a new species of Primulaceae from limestone area of Guangxi, China

Abstract Lysimachia fanii, a new species of Lysimachia (Subgen. Idiophyton, Primulaceae), is described and illustrated from Guangxi, China based on morphological and molecular data. Lysimachia fanii differs from L. verbascifolia, L. rupestris and L. alpestris mainly by the habit being nearly rosulate, leaves congested at the apex of the rhizome, leaf blades spatulate to narrowly oblanceolate and flowers solitary. Phylogenetic analyses supported L. verbascifolia as sister to L. fanii. This new species is endemic to limestone areas in Liucheng county of Guangxi, China.

The south-western limestone karst area is one of China's biodiversity hotspots. These areas are fragile and sensitive to environmental change and, in the wake of the rapid economic development of China, they are facing serious threat. Documentation of the plant diversity in these regions is urgently needed. Thus, we are surveying traditional medicinal plants in the limestone areas of Guangxi and trying to increase our knowledge of these poorly studied areas. During fieldwork in May 2018, we discovered an unknown species in Lysimachia. This species is allied to subgen. Heterostylandra by having rosette leaves, but it differs in having heteromorphic flowers. It shows alliance to subgen. Idiophyton, subgen. Lysimachia and subgen. Palladia by having 5-merous flowers, but has unique filaments, anthers and glands. After morphological observation and consulting relevant literature (Chen and Hu 1979, Chen et al. 1989, Hu and Kelso 1996, Tong et al. 2017, we confirm that the rare plant is a new species and has been placed into subgen. Idiophyton, based on morphology and molecular analyses.

Taxon sampling
We followed the classification of Lysimachia of Chen et al. (1989) and Hu and Kelso (1996). Leaves were collected from the holotype (L.Y. Fan et al., FLY2018001 in GXMI) and paratypes (L.Y. Fan et al., FLY2018002 in IBK & GXMI) to represent the new species. Twenty related taxa within subgen. Idiophyton, one taxon within subgen. Heterostylandra and four taxa within subgen. Lysimachia were selected to ascertain the phylogenetic relationships within Lysimachia (Anderberg et al. 2002). Based on Yan et al. (2018), Pelletiera verna A. St.-Hil. and Anagallis monelli L. were selected as outgroups.

DNA sequencing
Total genomic DNA was extracted from silica-dried plant leaves by a modified CTAB protocol (Doyle and Doyle 1987). Four chloroplast DNA regions (atpF-atpH, rpl32-trnL, trnL-F and trnS-trnG) and one nuclear loci (ITS) were selected and amplified following Yan et al. (2018). Genebank Accession Numbers are listed in Table 1. Table 1. Species of Lysimachia and related taxa sampled and GenBank accession numbers of sequences used in this study.

Phylogenetic analysis
Sequences of each DNA region were aligned using MUSCLE 3.8.31 (Edgar 2004a(Edgar , 2004b and adjusted manually where necessary. Indels were treated as gaps and all regions were combined as a single region for further study. Maximum Parsimony (MP) analyses were conducted using PAUP v.4.0b10 (Swofford 2002). Heuristic searches were carried out with 1000 replicates and tree-bisectionreconnection (TBR) branch swapping. A strict consensus tree was summarised from all the most parsimonious trees. Node support was assessed by 500 bootstrap replicates using TBR branch swapping.
Bayesian Inference (BI) analyses were conducted using MrBayes version 3.1.2 (Ronquist and Huelsenbeck 2003). The Markov chain Monte Carlo (MCMC) chains were run for 100 000 generations while trees were sampled every 100 generations. The MCMC chains were stopped when the average standard deviation of the split frequencies was 0.008 after 100 000 generations, which meant that the chains were converged to a stationary distribution. A majority-rule consensus tree was constructed after removing a burn-in of 25% of the trees. Posterior Probability (PP) values were used to estimate branch support.

Molecular systematic relationship
In total, 29 atpF-atpH, rpl32-trnL, trnL-trnF and ITS sequences and 25 trnS-G sequences were included. The combined matrix has a length of 3649 aligned characters (ITS: 653bp, atpF-atpH: 512bp, rpl32-trnL: 728bp, trnL-trnF: 946bp, trnS-G: 810bp), of which 363 are parsimony informative. The inferred phylogenies using MP and BI analyses are congruent (Fig. 1). The two samples of the new species (L. fanii) are clustered into subgenus Idiophyton with strong support values in both MP and BI analyses (BS= 100%, PP = 0.99). L. verbascifolia is placed as the sister group to L. fanii with high support in the BI analysis (PP = 0.92). Diagnosis. Lysimachia fanii differs from congeneric species in subgen. Idiophyton mainly by the habit being nearly rosulate, leaves congested at the apex of the rhizome, leaf blades spatulate to narrowly oblanceolate and flowers being solitary.
Phenology. Flowering from May to June. Etymology. The new species is named after Mr. Li-Yong Fan, who first discovered and collected this rare species.
Distribution and habitat. Lysimachia fanii is known only from the type locality in Taiping Town, Liucheng County, Guangxi Zhuangzu Autonomous Region, China (Fig. 4). It grows on moist limestone rock surfaces at the entrance to caves.     (Tong et al. 2017), but it can be distinguished from the latter by its rhizome which is branched at the apex and without stolons from the base, leaf blade spatulate to narrowly oblanceolate and glabrous adaxially, lateral veins invisible on both sides. A comparison of the main characters of the four species is shown in Table 2.