The Evolutionary Significance of Leaf Nodulation: Evidence from Ardisia and Its Relatives (Primulaceae: Myrsinoideae)

Interactions between plants and microorganisms have long been a central topic in biological research. Bacterial symbiosis on leaf surfaces represents a distinctive and mutually beneficial system within the phyllosphere microbiome. Leaf nodules are the visible manifestation of the symbiosis and confer ecological advantages to host plants by enhancing host resistance against pathogens and herbivores. It has been hypothesized that these advantages promote higher diversification rates in host lineages, but this remains uncertain. Ardisia subg. Crispardisia and its close relatives (Amblyanthopsis and Amblyanthus) within Primulaceae are typical plant groups with leaf nodule symbiosis, making them an ideal system for testing this hypothesis. In this study, we conducted extensive sampling of “Ardisioids” (Ardisia and its allies) and reconstructed their phylogenetic relationships and evolutionary history using plastid genomes and nuclear datasets (i.e., nuclear ribosomal DNA (nrDNA) and genome-wide single nucleotide polymorphisms (SNPs)). We clarified the phylogenetic positions of several “Ardisioids” genera (e.g., Sadiria, Tapeinosperma, Amblyanthus, and Amblyanthopsis) and multiple subgenera within Ardisia. We further detected a rapid radiation during the middle Miocene in Ardisia and its allies. Notably, we found that the leaf-nodulated clade appears to have originated during this period, approximately 11–8 Ma. BAMM (Bayesian Analysis of Macroevolutionary Mixtures) analyses revealed elevated diversification rates in leaf-nodulated lineages, while HiSSE (Hidden State Speciation and Extinction) analyses indicated that leaf nodule symbiosis might have increased speciation rates without significantly affecting extinction rates. These results provide strong evidence that leaf nodule symbiosis, together with other abiotic and biotic factors, represents a key evolutionary innovation that has promoted diversification in Ardisia and its close relatives.