Molecular cytogenetic characterization of 9 populations of four species in the genus Polygonatum (Asparagaceae)

For centuries, the knobby rhizomes of Polygonatum species have been ground into powders and brewed into teas as part of traditional Chinese medicine, where they are prized as Yin-nourishing herbs believed to delay senescence and treat ailments ranging from osteoporosis and fatigue to diabetes and lung disorders. Now, a team of Chinese cytogeneticists has taken the first deep molecular look at the chromosomes of four of these medicinal species, and what they found is a genome-level story of dramatic chromosome shuffling, hidden number changes, and evolutionary relationships that finally line up with what DNA sequence studies have been saying for a decade. The study, published in the journal Comparative Cytogenetics, marks the first time fluorescence in situ hybridization, or FISH, has been systematically applied to the genus, opening a new window onto one of Asia’s most important medicinal plant lineages.

The genus Polygonatum, commonly known as Solomon’s seal, is the largest genus in the tribe Polygonateae within the asparagus family, Asparagaceae, comprising roughly 70 species distributed across the temperate Northern Hemisphere. About 50 of those species occur in east Asia, from the Himalayas through China to Japan, with the main diversification centered in southwest China and northeast Asia. At least 37 species and one variety have been used as traditional medicine and functional food, with the rhizome being the most commonly used plant part. In the Chinese Pharmacopoeia, the dry rhizome of P. odoratum is known as Yuzhu, while the dry rhizomes of P. sibiricum, P. kingianum, and P. cyrtonema are known collectively as Huangjing. Despite this commercial and cultural importance, the classification of the genus has long been controversial, complicated by high morphological variation and wide overlaps in geographic distribution that make species identification notoriously difficult.

Conventional cytogenetic work stretching back to the middle of the last century had already revealed something unusual about Polygonatum chromosomes: significant variation in basic chromosome number among species, dysploid variation within species, and a peculiar bimodality in the karyotypes of most populations studied, meaning the chromosome sets consist of a few pairs of conspicuously large chromosomes alongside several pairs of conspicuously small ones. More than 50 species have been conventionally karyotyped, but these analyses could provide only limited information on species identification and karyotype evolution because they lacked effective chromosomal markers. The only previous molecular cytogenetic report in the genus was a 2001 study that detected 45S ribosomal DNA in just two species. Until now, no comprehensive FISH-based chromosome characterization of Polygonatum had ever been attempted.

The research team, led by cytogeneticists at the Agricultural Environment and Ecology Institute of the Hunan Academy of Agricultural Sciences, analyzed nine populations representing four species: P. cyrtonema, P. kingianum, P. odoratum, and P. sibiricum. Plants were collected from different regions of China and cultivated in Huangjing germplasm gardens. The researchers prepared chromosome spreads from young root tips using an enzymatic digestion protocol with cellulase and pectolyase, then applied dual-color FISH with synthetic oligonucleotide probes targeting the 5S and 45S ribosomal DNA genes. The 5S probes were labeled with 6-carboxyl fluorescein, producing green signals, while the 45S probes were labeled with 6-carboxyl-tetramethyl rhodamine, producing red signals. After overnight hybridization at 37 degrees Celsius and careful washing, the chromosomes were counterstained with DAPI and visualized under a fluorescence microscope equipped with a high-resolution CCD camera.

The results were striking. The four populations of P. cyrtonema displayed three different chromosome numbers: 2n = 18, 2n = 22, and 2n = 26, with the last of these reported for the first time in the species. Both populations of P. kingianum shared the same number, 2n = 30, while the two populations of P. odoratum differed, with one at 2n = 20 and the other at 2n = 22. The single population of P. sibiricum showed 2n = 24. In terms of basic chromosome numbers, the team detected x = 9, 11, and 13 for P. cyrtonema, x = 15 for P. kingianum, x = 10 and 11 for P. odoratum, and x = 12 for P. sibiricum. The total length of the haploid chromosome complement ranged from 72.23 micrometers in one P. cyrtonema population to 93.40 micrometers in one P. kingianum population, showing both inter- and intra-specific variation. All chromosomes were classified as metacentric, submetacentric, or subtelocentric, and all nine karyotypes fell into moderate asymmetry categories on the Stebbins classification system.

The FISH mapping of ribosomal DNA sites revealed a remarkably consistent pattern across most populations. Seven of the nine populations carried a single locus of 5S rDNA and two loci of 45S rDNA, suggesting that this configuration represents the ancestral state for the genus. The two exceptions added complexity: one population of P. cyrtonema carried an additional 5S locus on a large chromosome pair, and one population of P. odoratum showed what the researchers described as one and a half 5S loci, with only one member of a chromosome pair displaying the signal. Similarly, two populations, one of P. cyrtonema and one of P. kingianem, carried three 45S loci instead of the typical two. The team suggested that these extra loci probably originated from chromosomal rearrangements or from the action of transposable elements that accumulate in the proximity of rDNA loci. Almost all 45S loci generated visible secondary constrictions, the pinched regions on chromosomes where the nucleolus organizer activity is concentrated.

Perhaps the most intriguing finding concerns the mechanism of dysploidy, the change in chromosome number without a change in ploidy level. The genus displays two levels of dysploid variation: interspecific variation in basic chromosome number ranging from x = 8 to x = 16, and intraspecific dysploid variation within more than fourteen species. By analyzing the frequency of each basic chromosome number across all reported populations, the team inferred that the most frequently occurring number in each species should represent the original character. For P. cyrtonema, x = 11 occurs most frequently, accounting for about 61 percent of reports, while for P. odoratum, x = 10 accounts for 75 percent. For P. sibiricum, x = 12 accounts for nearly 86 percent. The new detection of x = 13 in P. cyrtonema further demonstrates what the researchers call continuous dysploidy within that species. Crucially, the team found that the intraspecific dysploid changes were not classic Robertsonian transformations, the simple chromosomal fissions or fusions that produce one-to-one changes in chromosome number. Instead, they observed a one-to-two or two-to-one relationship between changes in the numbers of large and small chromosomes, accompanied by changes in relative lengths and arm ratios, and even the loss of karyotype bimodality in some populations. The researchers concluded that complex chromosomal rearrangements, probably including centromere fission or fusion, unequal translocations, and pericentric inversions, have driven the continuous dysploid variation within these species.

The karyological relationships among the four species, revealed by comparing rDNA patterns and by a principal coordinate analysis based on six quantitative karyological parameters, turned out to be basically consistent with the phylogenetic relationships established by molecular sequence studies. The PCoA scatter plot divided the nine populations into two groups along the first coordinate axis, which captured nearly 58 percent of the original data variation. When only populations carrying the inferred original basic chromosome numbers were considered, P. cyrtonema and P. odoratum clustered closely together, P. sibiricum was placed nearby in the same major group, and P. kingianum occupied a distinctly isolated position in the other group. This arrangement mirrors the three-section classification of the genus proposed in 2014, which placed P. cyrtonema and P. odoratum in section Polygonatum, P. sibiricum as the sole species of section Sibirica, and P. kingianum in section Verticillata. The rDNA pattern comparison told the same story: the conserved 5S locus in P. cyrtonema, P. odoratum, and P. sibiricum sits in the interstitial or distal regions of the long arms of a pair of small chromosomes, while in P. kingianum it sits in the proximal regions of the short arms of a pair of large subtelocentric chromosomes, marking that species as the most distantly related of the four.

The study also raises a fundamental evolutionary question that remains unresolved: whether the interspecific dysploidy in Polygonatum proceeds primarily in an ascending or descending direction. Morphological evidence has generally been interpreted to suggest that species with alternate leaf arrangement are relatively primitive and those with whorled leaves are relatively evolved, which would imply ascending dysploidy as the main evolutionary mode. Bayesian analyses of chloroplast genome sequences supported alternate leaf arrangement as the ancestral state. However, a recent comparative analysis of chloroplast genomes suggested that the whorled leaf might actually be the ancestral condition, which would flip the direction of chromosome number evolution. The perennial and clonal nature of Polygonatum species, the researchers note, makes geographically diverse populations prone to preserving large-scale chromosomal rearrangements, and it is precisely this abundant rearrangement and the resulting dysploid variation that produces the high morphological variation within widely distributed species such as P. cyrtonema and P. odoratum. For a genus whose rhizomes have been harvested for millennia as medicine, the chromosomes are now telling a story of restless genomic change that may ultimately explain why these plants have diversified so successfully across Asia, Europe, and North America.

Subject of Research: Molecular cytogenetic characterization of 9 populations of four species in the genus Polygonatum (Asparagaceae)

Article Title: Molecular cytogenetic characterization of 9 populations of four species in the genus Polygonatum ( Asparagaceae )

Article References: Wei, Y.-F., Jiang, X.-H., Song, R., & She, C.-W. (2024). Molecular cytogenetic characterization of 9 populations of four species in the genus Polygonatum (Asparagaceae). Comparative Cytogenetics, 18, 73-95. https://doi.org/10.3897/compcytogen.18.122399

Image Credits: AI Generated

DOI: 10.3897/compcytogen.18.122399

Keywords: Molecular, cytogenetic, characterization, populations, four, species, genus, Polygonatum, Asparagaceae, scientific research