Exploring and applying symbioses between a Hawaiian endemic orchid and mycorrhizal partner for conservation

Abstract

Orchids rely on associated orchid mycorrhizal fungi (OMF) from the earliest stages of development to provide vital nutrients and even carbon. This carbon dependence, termed mycoheterotrophy, can be variable over time and environmental conditions, with many orchids remaining partially mycoheterotrophic even once they produce leaves capable of photosynthesis. Due to the marked difference between photosynthetic C3 plant and fungal pathways of carbon acquisition, previous studies have used stable isotope signatures to estimate the percent of fungal-derived carbon, and thus the degree of mycoheterotrophy, of orchids. However, orchids associating with so-called rhizoctonia fungi (orders Cantharellales and Sebacinales) have shown carbon signatures similar to autotrophic plants, despite nitrogen isotopic signatures characteristic of mycoheterotrophy, spurring a hypothesized condition coined “cryptic mycoheterotrophy”. C and N isotopic analyses were performed on fungal hyphal pelotons (Ceratobasidium, order Cantharellales) from roots of the rhizoctonia-associated Hawaiian endemic orchid Anoectochilus sandvicensis from three bog sites at Ka‘ala Natural Area Reserve (KNAR), Oʻahu, Hawaiʻi, alongside leaves of the orchid and surrounding autotrophic reference plants. Previous research shows that the adult Hawaiian orchid Anoectochilus sandvicensis engages in unexpected symbiotic specificity, associating with only three operational taxonomic units of the genus Ceratobasidium throughout the Hawaiian archipelago. Cultures of one Ceratobasidium sp. from these Oʻahu bog sites were produced and employed to support the germination of Anoectochilus sandvicensis seeds collected from Volcanoes National Park, Hawaiʻi Island. Upon isotope analysis, fungal samples were found to be significantly enriched in 13C, though considerably less so than ectomycorrhizal or other saprotrophic fungi. Orchids had significantly higher 15N enrichment and nitrogen concentration values consistent with expectations for a food chain, due to accumulations of 15N-heavy compounds and digestion of N-rich fungal biomass. Additionally, the presence of the fungal partner significantly increased seed germination rates, particularly on oatmeal agar media and under 14hr light/10hr dark conditions. Additionally, data shows the compatibility of A. sandvicensis seeds with a fungal operational taxonomic unit previously undetected on the originating island. However, as previously observed in other studies, failure to swiftly advance into higher stages of seed development suggests that this close mycorrhizal relative initiates germination but does not support later seedling growth. These data support prior findings of cryptic mycoheterotrophy of this species, provide a clearer picture of rhizoctonia isotopic signatures, and offer insight for future OMF culturing and symbiotic germination of these and other orchid species.