﻿ Polygonatumpraecox (Asparagaceae), a new species from mid-eastern China revealed by morphological and molecular evidence

﻿Abstract A new species, Polygonatumpraecox Y.F.Hu & J.W.Shao (Asparagaceae), is described and illustrated. This species is similar to P.cyrtonema, P.odoratum and P.caulialatum, but can be distinguished from P.cyrtonema by its racemose inflorescence, cylindrical hairless filaments and apex without a retrorse spur; from P.odoratum by its stout moniliform rhizome, straight stem and longer (1.7–2.2 cm long) floral tube; and from P.caulialatum by its upper part straight stem, yellowish-green corolla, lobes excurved and earlier flowering. The complete chloroplast genome of this new species is 155,115–155,256 bp in length. Phylogenetic analysis revealed that P.praecox is not genetically related to the above three morphological similar species, but is closely related to the two European species (P.multiforum and P.latifolium). This species is relatively common in mid-eastern China and has previously been confused with P.cyrtonema. As its wild resources have decreased in recent years due to over-exploitation for medicinal or edible purposes, we classify it as Near Threatened (NT) according to the IUCN Red List Criteria.


Introduction
Polygonatum Mill., the largest genus of Tribe Polygonateae in Asparagaceae, contains more than 70 species that are widely distributed in the warm temperate, subtropical and boreal zones of the Northern Hemisphere (Tang 1978;Chen and Tamura 2000;Meng et al. 2014;Wang et al. 2016;Xia et al. 2022). The eastern Himalaya and Hengduan Mountains and also North East Asia are the centres of diversity where ca. 50 of the species occur (Floden 2017;Zhao et al. 2019;Xia et al. 2022). Polygonatum is one of the most important medicinal taxa in Asia, with some species being widely used in traditional Chinese medicine, such as P. cyrtonema Hua, P. sibiricum Redouté and P. kingianum Coll. & Hemsl. (Zhao et al. 2018;Chinese Pharmacopoeia Commission 2020;Fan et al. 2020;Li et al. 2021). Most species in this genus are edible and can be cultivated in forests without occupying farmland; thus, some of them are emerging as a new generation crop that offers high yield and nutrition, but do not require fertile land for growth (Si and Zhu 2021).
Accurate species delimitation has become of practical importance in conservation and utilisation of plant resources (Isaac et al. 2004). Flower features, especially filament shape and vestiture and its position in the perianth tube, are vital in Polygonatum species identification (Tamura 1991(Tamura , 1993Tamura et al. 1997;Floden 2012). However, the observation of these flower features is relatively difficult due to the short flowering period of most species, while the easily observed vegetative organ features show high plasticity in different habitats. The systematics and species classification of Polygonatum still requires study to understand the diversity as shown by the synonym lists for some species according to the Flora of China, such as P. cyrtonema, P. odoratum (Mill.) Druce and P. kingianum (Chen and Tamura 2000) and by the recent description of distinctive new species which have been published in recent years (e.g. Cai et al. 2015;Floden 2015;Yang et al. 2020;Chen et al. 2021).
During an investigation of wild germplasm resources of Polygonatum in eastern China, we made several collections of a possibly unknown plant with alternate leaves, thick moniliform rhizome and large yellow-green flowers (1.7-2.2 cm long). This plant is in appearance similar to and has usually been identified as P. cyrtonema. However, we found that this plant differs from P. cyrtonema in its filaments (inserted near the distal 1/3 of the perianth tube, hairless and apical part without saccateconvex), flowering phenology (mid-March to early April) and inflorescence type (racemose). After further observation of its morphology and flowering phenology, chloroplast sequencing and phylogenetic analysis, we identified it as a new species which has been overlooked. Thus, we report the results of our investigation and the new species, named as Polygonatum praecox Y.F.Hu & J.W.Shao, sp. nov., is described and illustrated here.

Morphological assessment
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.

Genome sequencing, assembly and annotation
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  (Greiner et al. 2019). Basic characteristics of chloroplast genomes were read in Geneious (Table 2).

Phylogenetic analyses
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. 2021Xia et al. , 2022Wang 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).

Results and discussion
Characteristics of the complete plastid genome 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.

Phylogenetic relationship
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).

Morphological assessment
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 saccateconvex) 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 campanulatecylindrical 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. Diagnosis. Most similar to P. cyrtonema, but differs in racemose inflorescence, cylindrical and glabrous filaments and apex not saccate convex.
Phenology. Flowering from mid-March to early April and fruiting from May to September.
Etymology. The specific epithet praecox alludes to early flowering of the new species as compared to Polygonatum cyrtonema, a morphologically similar species. The Chinese name of the new species is here given as 早花黄精 (Zǎo huā huáng jīng).
Distribution and habitat. Currently, Polygonatum praecox is known from more than 10 populations and it is fairly widely distributed in middle-eastern China (Fig. 1).    Conservation status. Near Threatened. Polygonatum praecox is relatively common in middle-eastern China. As it is similar to P. cyrtonema in morphology and these two species occasionally co-exist in the wild, this new species is usually recognised as P. cyrtonema and has been exploited for medicinal or edible purposes. Its wild resources have clearly decreased in recent years. Therefore, we classify it as Near Threatened (NT) according to the IUCN Red List Criteria (IUCN 2019).