A new synonym of Polygonatum in China, based on morphological and molecular evidence

Abstract PolygonatumkingianumCollett et Hemsl.var.grandifolium D.M. Liu & W.Z. Zeng (1981), which sprouts twice a year, once in spring and once in autumn, differs from Polygonatumkingianum in leaves, bracts, perianth and filaments. Morphological comparison and molecular phylogeny indicate that it is identical to the newly-published Polygonatumhunanense H.H. Liu & B.Z. Wang (2021). Hence, we propose that P.kingianumvar.grandifolium should be recognised as a new synonym of P.hunanense. In addition, phylogenetic analyses confirmed that P.hunanense is sister to Polygonatumsect.Polygonatum, rather than P.kingianum of Polygonatumsect.Verticillata.

During fieldwork in the last few years, we found several populations of a unique Polygonatum species in Sichuan Province, Chongqing Municipality and Hubei Province of China ( Figure 1). The plants are 1-3 m high with 3-5 whorled leaves per round, yellowish-white or greenish-white flowers and yellow or orange berries ( Figure 2). It sprouts twice a year, once in spring (March to April) and once in autumn (September), whereas other species sprout only once in spring. It is likely belonging to the section Verticillata, according to the phenotype. After carefully checking the protologue and type specimens (Figure 3), we found that our collections matched the description of Polygonatum kingianum Collett et Hemsl. var. grandifolium D.M. Liu et W.Z. Zeng, which was published in Flora Sichuanica and is differing from other P. kingianum varieties by having broader leaves ((1.5-) 2.4-5 cm wide) with green leaf base (vs. 0.2-1.0 (-1.5) cm wide, leaf base red), 2-5 mm long bracts at base of pedicel (vs. 1-2 mm long, on pedicel), yellowish-or greenish-white perianth (vs. pink, red or white) (Figures 2, 4;Xu 1981 (Liu et al. 2021). In this study, molecular phylogenetic analyses were performed to reveal the phylogenetic relationships amongst P. kingianum var. grandifolium, P. hunanense and P. kingianum.

Morphologic observation
Morphological characters of the living individuals from Mt. Emei, Sichuan Province, China were observed. In addition, 16 herbarium specimens of Polygonatum kingianum var. grandifolium in IMC, CDCM and CDBI were examined. Subsequently, mor- phological comparisons were conducted with the living individuals, specimens and descriptions of Polygonatum hunanense and Polygonatum kingianum from flora and previous research (Jeffrey 1980;Xu 1981;Chen and Tamura 2000;Liu et al. 2021).

Sequencing, plastome assembly and annotation
In order to determine the phylogenetic status of the taxon, we sequenced three samples from Nanchuan, Chongqing Municipality, Enshi, Hubei Province and Mt. Emei, Sichuan Province, respectively (Figure 1), as well as two Polygonatum kingianum, one Polygonatum sibiricum and one Polygonatum zanlanscianense (Table 1). Representative voucher specimens are currently deposited at the Herbarium of Zhejiang University (HZU). Genomic DNAs were extracted from silica-gel dried leaves using DNA Plantzol Reagent (Invitrogen), following the manufacturer's instructions. The libraries were prepared and sequenced using paired-end 150 bp at Beijing Genomics Institute (BGI, Shenzhen, China) on a BGISEQ-500 sequencing platform. Approximately 3G raw data were generated for each sample. Raw data were trimmed by removing adapters and low-quality reads and then a de novo approach was applied to assemble plastomes using the NOVOPlasty v.3.8.3 (Dierckxsens et al. 2017) with K-mer = 39. The plastome and rbcL gene sequences of Polygonatum stenophyllum Maxim. (KX822773) were adopted as reference and seed sequence, respectively. DOGMA (Wyman et al. 2004) was used for plastome annotation with manually checking the start/stop codons in Geneious 10.2.3 (http://www.geneious.com). In addition, plastome data of Polygonatum and outgroups (Heteropolygonatum and Disporopsis Hance) from Floden & Schilling (2018) were used for phylogenetic analyses (Table 1). To study the phylogenetic relationship between P. kingianum var. grandifolium and P. hunanense, rbcL, trnK, psbA-trnH and trnC-petN, sequences from Liu et al. (2021) were downloaded for further phylogenetic analyses.

Phylogeny of Polygonatum
The sequence of 78 protein coding genes (CDS) shared by all plastomes were aligned using MAFFT v.7 (Katoh and Standley 2013) in Geneious 10.2.3. The rbcL, trnK, psbA-trnH and trnC-petN sequences from Liu et al. (2021) and those from the seven newlyreported plastomes were aligned using MUSCLE in Geneious 10.2.3. DNASP6 was used to do statistics of site information (Rozas et al. 2017). The phylogenetic trees were constructed using both Maximum Likelihood (ML) and Bayesian Inference (BI) methods, implemented on CIPRES Science Gateway website (https://www.phylo.org, Miller et  (Ronquist and Huelsenbeck 2003). The first 25% of calculated trees were discarded as burn-in and the remaining trees were used to construct a consensus tree to estimate the posterior probability (PP).

Results and discussion
Morphological comparisons showed that P. kingianum var. grandifolium is almost the same as P. hunanense, except that the latter has narrower leaves (Table 2). However, they both differ from other P. kingianum varieties in leaves, bracts, perianth and fila-  ments (Table 2). In addition, we have observed stout and no thickening filaments in P. kingianum var. grandifolium ( Figure 2G and Suppl. material 1: Figure S1) and slender filaments in P. kingianum ( Figure 4G and Suppl. material 1: Figure S1). Tamura has reported that species of sect. Verticillata has slender filaments, whereas sect. Polygonatum has stout filaments and filaments of the ser. Bracteata, ser. Polygonatum and ser. Inflata are thickening in the upper part, thickening in the middle or without thickening and thickening in the lower part, respectively (Tamura 1991(Tamura , 1993Tamura et al. 1997b). The length of seven plastomes ranged from 155,549 bp to 155,911 bp, the accession numbers being MW373516−MW373522 (Table 1). They displayed the typical quadripartite structure with 132 genes in the same order, of which 112 were unique genes including 78 protein-coding genes, 30 tRNA genes and 4 rRNA genes. The alignment CDS matrix has 66,589 characters in length, in which 1827 are variable (polymorphic) sites and 512 are parsimony-informative sites. In addition, the alignment matrix of four plastid fragments has 4,652 characters in length, of which 85 are variable (polymorphic) sites and 58 are parsimony-informative sites. The phylogenetic tree of 78 CDS supports a robust monophyletic clade of three samples of P. kingianum var. grandifolium (BS = 100, PP = 1; Figure 5). However, it was not closely related to P. kingianum of section Verticillata, but was sister to the section Polygonatum (BS = 100, PP = 1; Figure 5). Additionally, the phylogeny, based on four plastid fragments, supported the monophyly of P. hunanense and       Figure 6), which suggested they should be conspecific.
Therefore, we propose that P. kingianum var. grandifolium should be recognised as a new synonym of P. hunanense. In addition, both morphology and phylogeny showed that P. hunanense is different from P. kingianum and has a close relationship with sect. Polygonatum.  Figure 3; isolectotype: CDCM [barcode 00044022]!).
Distribution and habitat. Polygonatum hunanense is relatively common in Chongqing Municipality, Sichuan, Hubei and Hunan Provinces, China (Figure 1). It grows in evergreen broad-leaved forests, thickets or on moist grassy slopes, at an elevation of 200 m to 1200 m. In addition, it is also widely cultivated in those areas for harvesting the rhizomes.
Conservation status. To our knowledge, this species is widely distributed in low elevations of southwest China. Therefore, we propose to treat it as Least Concern (LC) according to the IUCN Red List Categories and Criteria version 14 (August 2019). However, due to the medicinal value of the genus, many of its populations are destroyed by unmanaged exploitation.