Six populations of the putative novel species (Polygonatum praecox) were found in Anhui, Zhejiang and Shaanxi Provinces (Fig. 1, Table 1) and some individuals from three populations (JZ, LY and QY) were transplanted to the Botanical Garden in Anhui Normal University for further observation of their morphology and flowering phenology. Some populations of P. cyrtonema (TTZ, QLF and JH, Table 1), P. odoratum (QS and CZ, Table 1) and P. caulialatum (KZ and TB, Table 1) were also collected and transplanted to the Garden for further morphological assessment. In the field, more than five living plants in each population were randomly selected for rhizomes, leaves, flowers morphological observations. The stability and variation patterns of these morphological traits (especially the filaments characteristics) and the flowering period were further observed in transplanted populations. All voucher specimens were deposited at the Herbarium of Anhui Normal University (ANUB). The specimens of Polygonatum in Herbaria PE, CSH, K, KUN, WU, JSPC, XBGH and NAS were also examined through digital platforms (CVH, GBIF, NSII), with special attention on the type specimens of P. cyrtonema and its synonyms and morphological similar species.

Figure 1. 

Distribution map of Polygonatum praecox (red dots showing locations found in this study; black dots showing locations identified by specimen examination), P. caulialatum (black triangle) and P. cyrtonema (black square).

Fresh leaves of one individual per population in five populations (SY, JZ, LY, LA of P. praecox and KZ of P. caulialatum, Table 1) were collected and dried in silica for molecular analysis. Genomic DNA was extracted using Tiangen DNAsecure Plant Kit (DP320). All libraries were built through optimisation of the construction process and sent to the Germplasm Bank of Wild Species in Southwest China (GBOWS, Kunming, China) for Illumina sequencing. Approximately 3 GB of raw data were generated for each sample. Raw data were trimmed by removing adapters and low-quality reads by FastQC/Trimmomatic. The complete chloroplast genome was assembled using Getorganelle v.1.7.5.2, through the original data reads (fastq / FQ file) with K-mer = 21, 65 and 105 (Jin et al. 2020). The plastome gene sequences of P. odoratum (MZ150858) were adopted as reference and seed sequences. PGA (Qu et al. 2019) was used for plastome annotation with manually checking the start/stop codons in Geneious 10.2.3 (http://www.geneious.com). The plastid genome map was drawn using OGDRAW (Greiner et al. 2019). Basic characteristics of chloroplast genomes were read in Geneious (Table 2).

In order to reveal the phylogenetic relationship of the putative novel species and its related species, plastome sequence data of Polygonatum and outgroup (Heteropolygonatum ogisui) from GenBank were downloaded (Floden and Schilling 2018; Xia et al. 2021, 2022; Wang et al. 2022). All sequences were aligned using MACSE v.2 and one of the inverted repeats (IRa) was deleted using Geneious (e.g. Ranwez et al. 2018) before further analysis. The phylogenetic tree was constructed using Maximum Likelihood (ML) methods and Bayesian Inference (BI) methods. The best substitution model was determined by ModelFinder in PhyloSuite (Kalyaanamoorthy et al. 2017; Zhang et al. 2020). The ML analysis was performed using IQ-TREE v.1.6.12 with 1000 bootstrap replicates and (GTR) + G +I (Nguyen et al. 2015). The BI analysis was conducted in MrBayes v.3.2 (Ronquist et al. 2012). The Markov Chain Monte Carlo (MCMC) algorithm was run for 20 million generations and the trees were sampled every 1000 generations. Convergence was determined by examining the average standard deviation of the split frequencies (< 0.01). 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).

The length of chloroplast complete genome of Polygonatum praecox samples was 155,115–155,256 bp (Fig. 2) and P. caulialatum was 155,318 bp; both possessed typical quadripartite structure (IRa, IRb, LSC and SSC). The characteristics and statistics of their plastid genomes are summarised in Table 2.

Figure 2. 

Plastid genome map of Polygonatum praecox Y.F.Hu & J.W.Shao, sp. nov.

As in previous phylogenetic analyses of Polygonatum (Meng et al. 2014; Xia et al. 2022), three well-supported clades corresponding to monophyletic sections were also resolved, i.e. Verticillata, Sibirica and Polygonatum (Fig. 3). The four individuals of the putative novel species from different geographic populations grouped into a monophyletic clade (Bootstrap Support (BS) = 100%, Bayesian Inference (BI) = 1), which were not sister to the three morphologically similar species (P. cyrtonema, P. odoratum and P. caulialatum, Fig. 3), although they are all in section Polygonatum. Unexpectedly, the putative novel species is closely related to the two European species (P. multiforum Kunth and P. latifolium (Jacq.) Desf.) (Fig. 3).

Figure 3. 

Phylogenetic relationships of the new species and its related species in Polygonatum, inferred from Maximum Likelihood (ML) and Bayesian Inference (BI) methods, based on the plastid genomes without one of the inverted repeats (IRa). Numbers on the branches indicate the bootstrap support of the ML and the posterior probability of BI analyses. The phylogenetic position of P. praecox is highlighted in red and P. caulialatum in brown. GenBank accession numbers are displayed after the species name.

The new species is morphologically similar to Polygonatum cyrtonema and P. caulialatum in its alternate oblong leaves, thick moniliform rhizome and large flowers (corolla about 1.7–2.2 cm long) (Figs 4, 5, Table 3), but it differs from P. cyrtonema in racemose inflorescence (vs. umbel-like), cylindrical and hairless filaments and its apex without saccate-convex (vs. papillose or shortly cottony, apex slightly dilated or with saccate-convex) and flowering from mid-March to early April (vs. from late April to late May); it differs from P. caulialatum in its terete stem (vs. obviously angled in upper part), yellowish-green corolla and lobes excurved (vs. green-white and lobes not excurved) and earlier flowering period (mid-March to early April vs. May to June). As to inflorescence type and flowering phenology, Polygonatum praecox is also similar to P. odoratum (raceme inflorescence and flowering from mid-March to early April), but they are very different in rhizome morphology (moniliform vs. terete) and stem (terete vs. angled). In morphology, this new species is also easily distinguished from its genetically related species P. multiforum and P. latifolium by its moniliform rhizome (vs. terete) and campanulate-cylindrical yellowish-green floral tube (vs. cylindrical, but somewhat contracted in the middle and whitish). In summary, there are obvious differences between the new species and its related species in morphology, especially in filament traits. However, because of the short flowering period, most previously collected specimens of Polygonatum were without blooming flowers and the stamen morphology is not easy to observe on dry specimens, which may be the reason for this new species being neglected for a long term.

Figure 4. 

Polygonatum praecox Y.F.Hu & J.W.Shao, sp. nov. A habitat B general habit C longitudinal section of floral tube, showing stamens and pistil D seeds and fruits, soaked in alcohol E rhizome with roots. All Photos by Yingfeng Hu.

Figure 5. 

Illustration of Polygonatum praecox Y.F.Hu & J.W.Shao, sp. nov. A morphology of aboveground part B longitudinal section of floral tube, showing stamens and pistil C moniliform rhizome. Drawn according to the holotype by Wei Wu.