Lichens are biological associations comprised of a fungus (mycobiont) and an alga and/or cyanobacterium (photobiont). Certain lichens have been shown to host diverse communities of bacteria that are not photobionts, though these communities are very poorly known. Many lichens are able to grow to large sizes in very nutrient-poor habitats such as rock outcrops. This observation supports the notion that members of the lichen-associated bacterial community may provide the lichen with crucial nutrients (e.g., fixed nitrogen) that otherwise would be unavailable. The main objectives of this study are to conduct the first large-scale survey of bacteria associated with lichens and to determine what role nitrogen requirements play in determining the composition of these bacterial communities.
The data generated as part of this study will reveal additional new lineages of microorganisms and contribute to the general understanding of bacterial diversity. The examination of these microbial communities may also shed light on the evolution of interactions among bacteria and the organisms that comprise lichens. In conjunction with this project, the funded graduate student will lead local 'lichen walks' to educate the public. Additionally, websites will be generated and maintained in order to make research findings accessible to all.
INTRODUCTION The main goals of this project were to investigate the diversity of bacteria associated with lichens and to understand the factors that affect which bacteria live in these environments. A lichen is a specific structure comprised of a fungus (mycobiont) and a single-celled photosynthetic partner (photobiont); lichens are typically long-lived and the major partners in the association have been quite successful in colonizing nearly every type of terrestrial habitat on Earth (Fig. 1). Recent work has shown that lichens host communities of micro-organisms that are highly-structured and diverse. The work funded under this grant (NSF DEB-1011504) illuminated precisely which types of bacteria are present and provided insight into what the bacteria are doing and why they are there. INTELLECTUAL MERIT One major aspect of this work was the production of detailed taxonomic profiles of the bacteria found in association with multiple diverse individual lichens (Fig. 2). As previous work had shown, the most abundant class of bacteria in lichens is Alphaproteobacteria. However, we further demonstrated the predominance of Rhodospirillales and Rhizobiales, orders of bacteria that are within Alphaproteobacteria. We also showed how diverse the lichen-associated members of Rhizobiales are, with multiple families of that order being present in large numbers (unlike in any of the other bacterial orders detected in lichens). The majority of the Rhizobiales bacteria associated with lichens are closely related to symbiotic bacteria found in other systems (e.g., plant root nodules), suggesting that lineages of bacteria specifically adapted for a symbiotic lifestyle may be present in lichens. This work also further illuminated the significance of the 'LAR1' ('Lichen-Associated Rhizobiales 1') sequence group and its unique association with lichens. Besides being nearly absent from all other metagenomic sequence libraries generated by researchers studying other types of environmental samples, this sequence group was found to represent one of the four most abundant family-level groupings found in lichens overall. Additionally, ecological analyses of the communities of bacteria associated with lichens revealed several factors that influence the composition of these diverse communities (Fig. 3). Samples were collected from across the North American continent from Alaska to Costa Rica. Analyses of these samples revealed that geography and spatial scale are significant factors influencing which bacteria are associated with lichens. The lichen samples collected also had various types of primary fungal and algal partners, and the types of partners also turned out to influence the types of bacteria living in association with lichens. The most striking difference was in the bacterial communities associated with lichens that had cyanobacterial photobionts and those with green-algal photobionts. These differences most likely reflect the major differences in the ways that lichens with these types of partners process nitrogen and carbon. The work conducted as a result of this funding has demonstrated that major bacterial community trends are significantly correlated with differences in large-scale geography, mycobiont-type and photobiont-type, indicating that the factors determining community composition in the lichen microbiome are more complex than previously demonstrated. Additionally, the presence of geographic structure implies that dispersal of lichen-associated bacteria is more limited than in other environmental microbial communities (e.g., soil), indicating that lichens may act as excellent microcosms for studying the specific effects of various types of experimental manipulation and global change on microbial communities. BROADER IMPACTS The graduate student trained as part of this grant has provided exposure to science and technology for pre-college teachers, young people, and other non-scientist members of the public by leading local 'Lichen Walks' (Durham, NC), participating in the WOODSmont Children's Festival (Durham, NC), attending a booth at the North Carolina Science Festival (Chapel Hill, NC), volunteering at the USA Science and Engineering Festival (Washington, DC), and maintaining a science-oriented weblog (http://squamules.blogspot.com/). During the period of funding, the student also presented research findings at five national/international conferences, posted multiple science-oriented videos on YouTube, and released a lichen-themed hip-hop song through the 'Squamules' weblog. A number of specific tools and methodologies developed as part of this project are significant for the advancement of the biological sciences. A new DNA extraction protocol known as SQERL (Simultaneous QuickExtract/ReadyLyse) has been developed for the rapid extraction of microbial DNA from many individual samples, opening up greater opportunities to study microbes on a large scale. New molecular primers were developed to facilitate the study of bacterial communities closely associated with algae and plants. Various computer scripts have been developed for the processing and statistical analysis of large sequence data sets. The student developed a bioinformatics pipeline for processing data generated through next-generation sequencing, which could prove useful for various applications, including clinical diagnostic work.
