PHYLOGENETIC AND COPHYLOGENETIC ANALYSES OF THE LEAF-NODULE SYMBIOSIS IN ARDISIA SUBGENUS CRISPARDISIA (MYRSINACEAE): EVIDENCE FROM NUCLEAR AND CHLOROPLAST MARKERS AND BACTERIAL RRN OPERONS

摘要

Premise of research. Leaf-nodule symbiosis represents an intriguing relationship between angiosperms and bacteria. In leaf-nodulated Ardisia (subgenus Crispardisia), it has been hypothesized that the bacterial symbionts, which are also observed in reproductive organs, are transmitted to the next generation in a vertical and closed manner. We tested this hypothesis by using molecular approaches to locate the symbionts within hosts and to conduct host-symbiont cophylogenetic analyses. Methodology. Universal primers were used to amplify bacterial rrn operon from field-collected Crispardisia for identification of symbionts and design of symbiont-specific primers for PCR detection. The relationships of symbionts from 11 Crispardisia taxa with 60 validly named species in the same genus were analyzed. The monophyly of the hosts was also explored by constructing a phylogeny of Ardisia based on nuclear ribosomal internal transcribed spacer (nrITS) and three chloroplast markers from 36 taxa belonging to nine subgenera. Host-symbiont cophylogenetic analyses were conducted using TreeMap and ParaFit. Pivotal results. Ribosomal RNA operons amplified from the rod-shaped Gram-negative symbionts of Crispardisia suggested they form a clade within Burkholderia. The host taxa were also resolved as a mono-phyletic group in the phylogenetic analyses based on nrITS, trnL-trnF, atpB-rbcL, and psbA-trnH. PCR detection confirmed the speculation based on electron microscope observation that the same symbiotic bacteria occur in both vegetative and reproductive organs. The similarity of rrn operon sequences of symbionts from hosts collected from different localities and the overall Crispardisia-Burkholderia cophylogenetic pattern suggest that the symbionts have been confined to the host's life cycle since the formation of such an intimate association. Conclusions. Overall, this study supports the hypothesis that Ardisia leaf-nodule symbionts are vertically transmitted and should be recognized as an integral part of Crispardisia.