Marco Polo: How Do Lichen-Forming Fungi Find Their Cyanobacterial Partners?

This summer, I will compare lichenized cyanobacteria communities to environmental cyanobacteria communities (cyanobacteria living outside the lichens) to inform how lichen-forming fungi associate with their cyanobacterial partners. For context, lichens are organisms made of symbiotic associations between algae/cyanobacteria and fungi. Fungi in lichen can reproduce sexually via fungal spores, which are essentially sterile, free of cyanobacteria, and it is largely unknown how the fungi find and associate with their symbiotic partners. In nature, fungal species have been observed associating with different cyanobacterial species, sometimes “switching” between cyanobacterial partners. The mechanism behind symbiosis formation is unclear; it is unknown where and how often the fungi find their cyanobacterial partners, and we want to know if the same cyanobacteria are found in the surrounding environment. 

My project focuses on lichens made of Peltigera, a genus of lichen-forming fungi, and Nostoc, a genus of blue-green algae or cyanobacteria. Using lichen and environmental samples, we want to ask: are lichenized cyanobacteria similar to, or different from, the environmental cyanobacteria found outside the lichens? The Lutzoni Lab collected lichen and environmental samples across Alberta, Canada, to help answer this question. The lab collected roughly 2500 lichen samples and 1800 environmental samples across 15 sites spread over five natural regions of Alberta (3 sites per natural region). The environmental samples consist of substrate sampled next to and between lichens and are split into 900 “top” and 900 “bottom” samples. The top samples were collected closer to the earth’s surface, and the bottom samples were collected deeper underground. 

This summer, I aim to answer two primary questions to determine the best method for extracting and sequencing the Nostoc DNA from lichen and environmental samples!

First, we want to know if the 16S rRNA or rbcLX gene is a more accurate marker for detecting Nostoc in the samples. The rbcLX gene is more specific to cyanobacteria and has more variation than the 16S rRNA gene, so it could more accurately classify between species of Nostoc. However, Nostoc contain one copy of the rbcLX gene compared to multiple copies of the 16S rRNA gene. The environmental samples may have low abundances of Nostoc, so sequencing the 16S rRNA gene may be more suitable for detecting the low cyanobacteria levels. To answer this question, we are testing the 16S rRNA and rbcLX approaches on 12 environmental samples which have already undergone metagenomic and metatranscriptomic analyses. By comparing the resulting 16S rRNA and rbcLX sequences to pre-existing data, we will select the marker with which we will sequence the remaining environmental samples. We hypothesize that 16S rRNA will more accurately detect the Nostoc because we are trying to detect microbes presumably in trace amounts, and because the 16S rRNA gene is the standard marker in microbiology. 

Next, we want to determine how the cyanobacterial communities in the top environmental samples compare to those of the bottom. As mentioned above, the 900 top and 900 bottom samples are from the same areas but different depths into the substrate. We want to see if the bottom cyanobacterial communities are a subset of those of the top samples, and if so, only extract DNA from the top samples to save time and money. We hypothesize that the bottom samples will be a subset of the top, and more Nostoc will be detected in the top samples because Nostoc require sunlight to photosynthesize ☀️