Chloroplast genome comparison and taxonomic reassessment of Polygonatum sensu Lato (Asparagaceae): implications for molecular marker development in traditional medicinal plants

Abstract

The Polygonati Rhizoma have generated significant market attention for their medicinal and culinary applications. However, morphological similarities and ambiguous species boundaries complicate the identification of genera and species, thereby impeding product development and utilization within Polygonatum sensu lato. Despite the widespread application of the chloroplast genome for taxonomic boundary revisions for Polygonatum s.l., a critical gap persist regarding their genomic applicability and the lack of standardized pipelines for developing species-specific molecular markers capable of rapid discrimination among species. This study aims to assess the effectiveness of chloroplast genomes in clarifying the current taxonomic status of the genera and species of Polygonatum s.l., and develop a reliable process for rapid identification of designated species from other species. A total of 21 chloroplast genomes were sequenced and assembled, and subsequent analyses included phylogenetic inference, multiple molecular species delimitation methods, and an automated screening framework were employed for subsequent analysis. Comparative analyses revealed relatively conserved chloroplast genomes, with notable variation limited primarily to the length of IR and LSC regions. By integrating multiple delimitation methods, the chloroplast genome validated 82.46% of the current classifications of Polygonatum s.l., demonstrating strong support (90.63%) for species represented by multiple sequences, yet only moderate support (70%) for those with single-sequence representation. Additionally, this study established and validated a scalable molecular marker development framework, spanning from identification of species-specific SNPs/InDels to the design of high-resolution molecular markers, illustrated through case studies involving Heteropolygonatum and three medicinally significant Polygonatum species.

Keywords: Heteropolygonatum; DNA barcoding; Molecular marker; Polygonati rhizoma.

Fig. 1

Phylogenetic relationships trees of Polygonatum and Heteropolygonatum based on 78 protein-coding genes. Phylogenetic tree with branch lengths based on Maximum Likelihood Method (A). A phylogenetic tree without branch lengths, generated using both Maximum Likelihood and Bayesian Inference (B); Asterisks represent BS = 100 / PP = 1; Branches with no support value; Samples highlighted in bold were newly generated in this study. Habitat (C) and rhizome (D) of P. cyrtonema. Rhizome (E) and habitat (F) of H. binatifolium

Fig. 2

Complete chloroplast genome map of Polygonatum (A) and Heteropolygonatum (B) and distribution of lengths and t-test results for total chloroplast genomes (C), large single-copy (D), inverted repeat (E), and small single-copy (F) regions of Polygonatum and Heteropolygonatum. Three asterisks indicate P < 0.001; Two asterisks indicate 0.01 > P > 0.001; one asterisk indicates P > 0.01

Fig. 3

Results of molecular species assessment. The different blocks in these columns represent the results of the Bayesian Poisson Tree Processes (bPTP), Generalized Mixed Yule Coalescent (GMYC), and Assemble Species by Automatic Partitioning (ASAP), respectively; Genetic distance relationships obtained by the ASAP method are shown on the right (A). Scatter plots of maximum intraspecific Kimura 2-parameter (K2P) distance vs. minimum interspecific K2P distance for complete chloroplast genomes for over two sequences per species (B)

Fig. 4

Identification and validation of species-specific sites for three medicinal Polygonatum species. Distribution (A) and categories (B) of species-specific sites; Validation of ASAP molecular species for region b (C)

Fig. 5

InDel primer sequences and electrophoretograms for Polygonatum and Heteropolygonatum. InDel primer sequences, with left and right arrows indicating forward and reverse primers, respectively (A). Electrophoretogram of trnH-psbA and InDel sequence fragments in 6 species: P. cyrtonema (1), P. sibiricum (2), P. kingianum (3), H. alternicirrhosum (4), H. ginfushanicum (5), and H. binatifolium (6), each species was tested in triplicate (B)

Fig. 6

Species-specific primers and validation results for three medicinal Polygonatum species. Species-specific primers sequence (A) and validation results (B)