Metagenomic analyses of worldwide Thamnolia lichens reveal new photo- and mycobiont lineages that correlate with temperature

Sammanfattning

Lichen are a symbiosis between a mycobiont and a photobiont. Sanger sequencing is a popular method to identify the photobiont species, though it has recently been suggested that it tends to only amplify the most common genotype. Using next generation sequencing (NGS) has revealed that the lichen thalli often include more than one photobiont. The majority of discoveries in lichen biology, including both the photo- and mycobiont, are made using the internal transcribed spacer (ITS). It is a good tool for distinguishing between species but makes sequence alignment difficult. This study proposes that using the whole rDNA operon, including both the conserved 18- and 28S as well as the ITS, will resolve this issue. Therefore, this study examines whether metagenomic data sequenced at 30X can be used to retrieve the rDNA operon of photo- and mycobionts in Thamnolia lichen, as well as if it is able to identify more than one photobiont. It also examines which photobionts associate with Thamnolia lichens and if there is a correlation between them as well as between them and the temperature in which they grew. Further, if the identified photobionts are found in other mycobiont species beyond Thamnolia. To establish this, 150 Thamnolia samples from all continents of the world except Africa were collected between 1994-2023, their DNA was extracted and sequenced. The rDNA operon of both the photo- and mycobiont of 39 of the samples, including the 18S, ITS and 28S, were retrieved through metagenomic assembly using metaSPAdes. With the obtained data, phylogenies, maps, one-way ANOVAs and Fisher’s Exact test were made. With this method, six Thamnolia mycobiont lineages were discovered: A, B, C1, C2, C3 and C4, through their distribution it could be established that they had different regions of origin and were impacted by the average temperature in which they grew. Five Trebouxia photobiont lineages were discovered: ‘pink’, ‘red’, ‘yellow’, ‘green’ and ‘blue’, from their distribution the same conclusions regarding origins and temperature could be established, as for the mycobionts. Fisher’s Exact test also showed that the lineages have specific associations with each other. The results indicate that the Thamnolia and Trebouxia lineages have spatial and thermal patterns, meaning that their distribution differs depending on origin and temperature, as well as certain associations with each other and that this is worth investigating further to establish the true depiction of reality.