Symbiotic germination and development of fully mycoheterotrophic plants convergently targeting similar Glomeraceae taxa.

Plants producing dust seeds often meet their carbon demands by exploiting fungi at the seedling stage. This germination strategy (i.e. mycoheterotrophic germination) has been investigated among orchidaceous and ericaceous plants exploiting Ascomycota or Basidiomycota. Although several other angiosperm lineages have evolved fully mycoheterotrophic relationships with Glomeromycota, the fungal identities involved in mycoheterotrophic germination remain largely unknown. Here, we conducted in situ seed baiting and high-throughput DNA barcoding to identify mycobionts associated with seedlings of Burmannia championii (Burmanniaceae: Dioscoreales) and Sciaphila megastyla (Triuridaceae: Pandanales), which have independently evolved full mycoheterotrophy. Subsequently, we revealed that both seedlings and adults in B. championii and S. megastyla predominantly associate with Glomeraceae. However, mycorrhizal communities are somewhat distinct between seedling and adult stages, particularly in S. megastyla. Notably, the dissimilarity of mycorrhizal communities between S. megastyla adult samples and S. megastyla seedling samples is significantly higher than that between B. championi adult samples and S. megastyla adult samples, based on some indices. This pattern is possibly due to both mycorrhizal shifts during ontogenetic development and convergent recruitment of cheating-susceptible fungi. The extensive fungal overlap in two unrelated mycoheterotrophic plants indicates that both species convergently exploit specific AM fungal phylotypes.

Mycorrhizal communities of two closely related species, Pyrola subaphylla and P. japonica, with contrasting degrees of mycoheterotrophy in a sympatric habitat.

Mycoheterotrophic plants typically form associations with a narrow range of mycorrhizal fungi. Consequently, mycorrhizal specialization is often considered to be an important step in mycoheterotrophic evolution. However, it remains unclear whether such specialization is likely to occur in plants of the genus Pyrola, which are generally associated with fungi in multiple ectomycorrhizal families. Here, we investigated the mycorrhizal communities of a nearly fully mycoheterotrophic Pyrola species (Pyrola subaphylla), a closely related partially mycoheterotrophic Pyrola species (Pyrola japonica), and a co-occurring autotrophic ectomycorrhizal tree, Quercus crispula, which is their potential carbon source, in a cool-temperate Japanese forest. High-throughput DNA sequencing revealed that numerous common ectomycorrhizal OTUs interact with the two Pyrola species and Q. crispula, thereby providing an opportunity to exploit a certain amount of carbon from common mycorrhizal networks. In addition, not only P. japonica but also P. subaphylla exhibited exceptionally high alpha mycobiont diversity, with 52 ectomycorrhizal OTUs belonging to 12 families being identified as P. subaphylla mycobionts and 69 ectomycorrhizal OTUs in 18 families being detected as P. japonica mycobionts. Nonetheless, the beta mycobiont diversity of P. subaphylla and P. japonica individuals was significantly lower than that of Q. crispula. Moreover, the beta mycobiont diversity of P. subaphylla was found to be significantly lower than that of P. japonica. Therefore, despite their seemingly broad mycorrhizal interactions, the two Pyrola species (particularly P. subaphylla) showed consistent fungal associations, suggesting that mycorrhizal specialization may have developed during the course of mycoheterotrophic evolution within the genus Pyrola.