Sedum lipingense (Crassulaceae) identifying a new stonecrop species in SE Guizhou, China, based on morphological and molecular evidence

Abstract We describe and illustrate Sedum lipingense (Crassulaceae), a new species of stonecrop found in the limestone areas of SE Guizhou, China. Based on the presence of adaxially gibbous carpels and follicles, this taxon belongs to sect. Sedum S.H. Fu. The new species superficially resembles S. subtile Miquel and S. bulbiferum Makino but differs from these two taxa in its development of a basal leaf rosette during florescence. The nrDNA internal transcribed spacer (ITS) sequences also support the claim that this plant is a new species in the Sedum genus.


Introduction
Sedum Linnaeus is the largest genus in the Crassulaceae family, containing about 430 species, with the greatest diversity centering in eastern Asia (Thiede andEggli 2007, Ito et al. 2017a). Approximately 121 Sedum species (91 endemics) occur in China, and 49 of these species (34 endemics) belong to sect. Sedum, a subclass which possess adaxially gibbous carpels and follicles (Wu et al. 2013). There are 23 species within five genera of Crassulaceae found in the Guizhou Province (Li et al. 1985). From 2005, a number of new species of Sedum were reported across mainland China, in areas includ-ing Zhejiang (Wang et al. 2005, Jin et al. 2010, Anhui (Xie et al. 2014, Chen et al. 2017) and the Guizhou Province (Yang et al. 2012). In China, only a few species in this genus retain rosette leaves during florescence, such as S. balfourii Hamet and S. drymarioides var. saxifragiforme X. F. Jin & H. W. Zhang. Sedum balfourii was formerly placed in sect. Aizoon, within the genus Sedum (Fu and Fu 1984), but was then moved to the genus Ohbaea (Raymond-Hamet) V. V. Byalt & I. V. Sokolova (Fu and Ohba 2001) based on its conspicuous lateral flowering stems that derive from rosettes during florescence.
During our fieldwork, a new species of Sedum was discovered in Liping County, Qiandongnan Prefecture, Guizhou Province, China. This particular species has conspicuous rosettes during florescence, an attribute similar to O. balfourii. However, the new species differs from O. balfourii as it possesses central flowering stems rather than lateral ones (Fig. 2D). It also differs from S. drymarioides var. saxifragiforme, a species which is glandular-pubescent throughout, despite its rosette leaves. Based on its adaxially gibbous carpels, we place the new species in Sect. Sedum. Macro-morphological character studies indicated that this species is also somewhat similar to S. subtile Miquel and S. bulbiferum Makino, sharing a number of traits with these species, including opposite leaves on proximal stems and alternate leaves mainly on distal stems. We conducted morphological comparisons and molecular phylogenetic analysis to elucidate the presumed new Sedum species.

Materials and methods
All morphological characters were measured using dissecting microscopes. Specimen checking was done at PE, IBK, ZY, with the additional use of some web database, including the Plant Photo Bank of China (http://ppbc.iplant.cn/) and Global Plants (http://plants.jstor.org/).
Leaf material from the presumed new species was collected in the field, and immediately dried in silica gel for DNA extraction. The nuclear ribosomal internal transcribed spacer (ITS) regions were used as molecular markers. ITS-F (TGAACCT-GCGGAAGGATCAT) and ITS-R (GGTAGTCCCGCCTGACCTG) primers (Wu et al. 2013) were selected to amplify the ITS sequences. DNA extraction and PCR amplification of the new species followed the procedure of Wu et al. (2013). Primer synthesis and PCR product sequencing were carried out at the Shanghai Sangon Biotech Institute, China.
The ITS sequence of the new species, as well as the ITS sequences of the congeners downloaded from GeneBank (Table 1), were aligned using MEGA7 and then manually adjusted. Bayesian inference was implemented using MrBayes v3.2.6. Prior to the Bayesian analysis, the Akaike information criterion (AIC) implemented in mrModel-Test v1.0 was used to select the best-fit model (GTR+I+G) of molecular evolution. For the BI analyses, four Markov Chain Monte Carlo (MCMC) chains were run, sampling one tree every 100 generations for 2,000,000 generations starting with a random tree (Xie et al. 2014). When the log-likelihood scores were found to have stabilized, a

Molecular analyses
In this study, the sequences of 40 species (44 samples) were treated as ingroups. Sequence length was 584 bp for the ITS region, of which 234 characters were constant, 45 characters were parsimony-uninformative and 305 characters were parsimony-informative. The sequence of the ITS region taken from S. lipingense aligned with the genus Sedum, confirming its generic identity (Fig. 1). The new species was resolved as sister to S. bulbiferum (Bayesian posterior probabilities (PP) was 97) but turned out to be genetically distant from S. subtile. There were 50 nucleotides differ between S. lipingense and S. bulbiferum, suggesting the high variation compared to the closest relatives was remarkable.
S. lipingense and S. bulbiferum were found to be nested with S. hangzhouense (PP = 41, suggesting a weak support), and then to be nested with S. baileyi and S. makinoi (PP = 100), all species with alternate or opposite stem leaves. Except for S. lipingense, the above four (or perhaps two-three) species were also clustered as a distinct clade (Wu et al. 2013, Xie et al. 2014, Ito et al. 2017a, suggesting that the four species are closely related. Sedum lipingense is a close member to this clade, but these species form a polytomy and it is hard to say for sure, which one is the closest relative of S. lipingense. Sedum subtile is not within the same clade as S. bulbiferum, S. hangzhouense, S. baileyi, and S. makinoi (Wu et al. 2013) or with S. hangzhouense, S. baileyi, and S. makinoi (Ito et al. 2017a), suggesting that the relationship between S. subtile and S. lipingense is relatively distant.  (Table 2).
May-Jun Jul-Aug Jun-Jul Distribution and habitat. At this time, based on our field observations, Sedum lipingense is only known to occur in Longxi village, Mengyan town, Liping County, Guizhou Province. It grows on moist limestone rocks, at ca. 800 m altitude, in groups of several hundred individuals.
Conservation status. This species is currently known to occur in a single valley and we suggest its placement in the Data Deficient category of IUCN (2017).
Phenology. This new species was observed flowering from April to May and fruiting from May to June.
Etymology. The specific epithet 'lipingense' is derived from the plant's locality, Liping County, Guizhou Province, China.