Saxifraga damingshanensis (S. sect. Irregulares, Saxifragaceae), a new species from Guangxi, China

Abstract Saxifraga damingshanensis (Saxifragaceae), a new species from Damingshan Nature Reserve in Guangxi Province, is described and illustrated. A morphological comparison between the new species and its putative relatives, S. mengtzeana and S. luoxiaoensis, is presented. The new species is morphologically similar to S. mengtzeana, but it can be easily distinguished by its non-peltate leaf, both surfaces of mature leaf blade covered with white glandular trichome, petals 3-veined and margin entire. Phylogenetic analysis, based on two chloroplast DNA regions (matK and psbA-trnH), confirmed that the new species belongs to S. sect. Irregulares. The new species is currently only known from Damingshan, Guangxi and we assign it an IUCN Red List preliminary status as Data Deficient.

During a botanical expedition to Damingshan National Nature Reserve, Wuming district, central Guangxi Province in September 2018, we discovered an unknown species of Saxifraga in Longtou Peak. Its mature leaves are densely covered with white trichomes and the abaxial surface is densely purple-spotted. After carefully checking specimens and literature, as well as morphological and molecular studies, we confirm that it is a new species of Saxifraga and it is described below.

Materials and methods
We collected more than 20 living individuals of the presumed new species for comparisons and taxonomical treatment. Specimens of Saxifraga sect. Irregulares, available at herbaria (PE, IBSC, SYS and IBK) and digital photos of all herbarium specimens of S. luoxiaoensis, S. mengtzeana Engl. & Irmsch., preserved in the Chinese Virtual Herbarium (http://www.cvh.org.cn/), have been checked. Five main characters (leaf shape, leaf margin, spots on the abaxial surface of leaf, petal shape and trichomes on plants) of these three species were compared both in the wild and in the herbarium.
To determine the systematic position of Saxifraga damingshanensis, we further sampled five individuals of the presumed new species for a phylogenetic study. The geographic sampling information of these individuals was recorded by a Garmin GPS unit (GPSMAP 62sc, Taiwan) and the voucher specimens were deposited at Sun Yatsen University Herbarium (SYS) ( Table 1). The final molecular dataset comprises 19 accessions representing eight species of S. sect. Irregulares, of which five accessions were newly generated and 14 accessions were downloaded from GenBank (Table 1).
The total DNA was extracted with the modified CTAB method (Doyle and Doyle 1987). The psbA-trnH2 and matK intergenic regions were amplified using previously reported primers (Tate and Simpson 2003;Zhang et al. 2019). PCR amplifications were performed following Chen et al. (2016). Sequences were aligned with MEGA version 6.0 and subsequently manually adjusted (Tamura et al. 2013). Phylogenetic reconstructions were carried out with Maximum Likelihood (ML) and Bayesian Inference (BI) analyses. ML was run by IQ-Tree 1.6.10 with 20,000 ultrafast bootstraps and SH-like approximate likelihood ratio test (aLRT) of 10,000 replicates (Nguyen et al. 2015). BI was executed in MrBayes version 3.2 (Ronquist et al. 2012), with four chains for at least 20,000,000 generations to make the average standard deviation of split frequencies (ASDFs) < 0.01, sampling every 1000 generations with the first 25% sampled trees discarded as burn-in. The 50% majority-rule consensus trees were finally generated. For both ML and BI analyses, F81+F+I was detected as the best-fitting nucleotide substitution model on the basis of Bayesian Information Criterion (BIC) detected by ModelFinder (Kalyaanamoorthy et al. 2017).

Morphological comparison
In morphology, the putative new species is closely related to Saxifraga mengtzeana and their morphology comparisons are presented in Table 2. These two species share such features as having stolons absent, inflorescences and pedicels covered with glandular hairs, white flowers without pink markings and base of three short petals with yellow plot. However, the new species differs from S. mengtzeana by having leaf base cordate to deep cordate (vs. usually peltate), leaves papery or nearly leathery (vs. leathery), adaxial surface of the mature leaf with glandular trichome (vs. nearly glabrous) and longest petal 3-veined, margin entire (vs. 8-veined, margin sparsely denticulate). Moreover, S. damingshanensis flowers from August to October, while S. mengtzeana flowers from March to August.  Phylogenetic placement of Saxifraga damingshanensis within S. sect. Irregulares The concatenated sequences of matK (740 bp) and psbA-trnH2 (297 bp) are 1037 bp in length and 81 parsimony-informative sites were detected. Our results showed that S. ser. Rufescentes J.T.Pan is monophyletic (SH-aLRT: 100; LP: 100; PP: 1.00, Fig. 1) which is coincident with the previous study (Zhang et al. 2018). The putative new species, S. damingshanensis, was nested into S. ser. Rufescentes J.T.Pan and was strongly supported as sister to a clade consisting of S. luoxiaoensis, S. daqiaoensis and S. shennongii (SH-aLRT: 97; LP: 98; PP: 1.0).

Discussion
Based on its basal leaves with long petiolate, flower zygomorphic and stamens with club-shaped filaments, the new species could be placed within S. sect. Irregulares. Our phylogeny also supports the inclusion of Saxifraga damingshanensis within S. sect. Irregulares (Fig. 1). All examined individuals of S. damingshanensis clustered into a single lineage, thus corroborating the evidence for the new species status, based on morphology.
Our study also recovered a sister relationship of the new species with a clade comprising Saxifraga luoxiaoensis, S. daqiaoensis and S. shennongii. The close relationship amongst these species was also supported by their similar morphological characteristics. All four species have white glandular trichomes on leaf and inflorescence and white and entire petals. However, S. damingshanensis differs from the latter three species by having mature leaf and petiole with glandular trichomes (vs. mature leaf sparsely hispid or glabrous) and the abaxial surface of the leaf blade with purple spots (vs. usually yellow-brown spots). Furthermore, their phenology and distribution are different. Saxifraga damingshanensis is flowering from August to October (vs. April to June) and endemic to Damingshan, Guangxi (vs. Guangdong, Jiangxi and Hunan).  (Fig. 2) Diagnosis. Saxifraga damingshanensis is morphologically most similar to S. mengtzeana, but differs by its leaf blade with glandular trichome and purple spots abaxially, short stamens and petal entire.
Phenology. Flowering from August to October, fruiting from September to November.
Etymology. The species epithet is based on the mountain name, Damingshan and the Latin suffix, -ensis, of origin, where the new species was collected.
Distribution, ecology and conservation status. Only three populations of Saxifraga damingshanensis were discovered from Damingshan National Nature Reserve, Guangxi Province. It was observed to grow on damp cliffs and rocks in broad-leaved forests at altitudes between 1300 and 1650 m. Its known localities are well protected and more field investigations are needed to determine its wild distribution. Therefore