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.

Outcrossing plant species are more likely to exhibit autonomous selfing in marginal habitats to ensure reproduction under conditions of limited pollinator and/or mate availability. Distyly is a classical paradigm that promotes outcrossing; however, little is known about the variation in floral traits associated with distylous syndrome in marginal populations. In this study, we compared the variation in floral traits including stigma and anther height, corolla tube length, herkogamy, and corolla diameter between the central and peripheral populations of the distylousPrimula wannanensis, and assessed the variation of floral traits at early and late florescence stages for each population. To evaluate the potential consequences of the variation in floral traits on the mating system, we investigated seed set in each population under both open-pollinated and pollinator-excluded conditions. The flower size of both short- and long-styled morphs was significantly reduced in late-opening flowers compared with early opening flowers in both central and peripheral populations. Sex-organ reciprocity was perfect in early opening flowers; however, it was largely weakened in the late-opening flowers of peripheral populations compared with central populations. Of these flowers, disproportionate change in stigma height (elongated in S-morph and shortened in L-morph) was the main cause of reduced herkogamy, and seed set was fairly high under pollinator-excluded condition. Our results provide empirical support for the hypothesis on the evolution of delayed autonomous selfing in marginal populations of distylous species. Unsatisfactory pollinator service is likely to have promoted reproductive assurance of distylous plants with largely reduced herkogamy mimicking “homostyles.”