Copyright by Brendan Paul Hodkinson 2011 Abstract Although common knowledge dictates that the lichen thallus is formed solely by a fungus (mycobiont) that develops a symbiotic relationship with an alga and/or cyanobacterium (photobiont), the non-photoautotrophic bacteria found in lichen microbiomes are increasingly regarded as integral components of lichen thalli and significant players in the ecology and physiology of lichens. Despite recent interest in this topic, the phylogeny, ecology, and function of these bacteria remain largely unknown. The data presented in this dissertation were generated using culture-free methods to examine the bacteria housed in these unique environments to ultimately inform an assessment of their status with regard to the lichen symbiosis. Microbiotic surveys of lichen thalli using new oligonucleotide-primers targeting the 16S SSU rRNA gene (developed as part of this study to target Bacteria, but exclude sequences derived from chloroplasts and Cyanobacteria) revealed the identity of diverse bacterial associates, including members of an undescribed lineage in the order Rhizobiales (Lichen-Associated Rhizobiales 1; ‘LAR1’). It is shown that the LAR1 bacterial lineage, uniquely associated with lichen thalli, is widespread among lichens formed by distantly related lichen-forming fungi and is found in lichens collected from the tropics to the arctic. Through extensive molecular cloning of the 16S rRNA gene and 454 16S amplicon sequencing, ecological trends were inferred based on mycobiont, photobiont, and iv geography. The implications for using lichens as microcosms to study larger principles of ecology and evolution are discussed. In addition to phylogenetic and ecological studies of lichen-associated bacterial communities, this dissertation provides a first assessment of the functions performed by these bacteria within the lichen microbiome in nature through 454 sequencing of two different lichen metatranscriptomes (one from a chlorolichen, Cladonia grayi , and one from a cyanolichen, Peltigera praetextata ). Non- photobiont bacterial genes for nitrogen fixation were not detected in the Cladonia thallus (even though transcripts of cyanobacterial nitrogen fixation genes from two different pathways were detected in the cyanolichen thallus), implying that the role of nitrogen fixation in the maintenance of chlorolichens may have previously been overstated. Additionally, bacterial polyol dehydrogenases were found to be expressed in chlorolichen thalli (along with fungal polyol dehydrogenases and kinases from the mycobiont), suggesting the potential for bacteria to begin the process of breaking down the fixed carbon compounds secreted by the photobiont for easier metabolism by the mycobiont. This first look at the group of functional genes expressed at the level of transcription provides initial insights into the symbiotic network of interacting genes within the lichen microbiome. v Contents Abstract .........................................................................................................................................iv List of Tables.................................................................................................................................xi List of Figures ..............................................................................................................................xii Acknowledgements .................................................................................................................. xiii Introduction ...................................................................................................................................1 1. A microbiotic survey of lichen-associated bacteria reveals a new lineage from the Rhizobiales.....................................................................................................................................8 1.1 Introduction.......................................................................................................................8 1.2 Materials and methods ..................................................................................................11 1.2.1 Primer design.............................................................................................................11 1.2.2 Specimen collection and storage .............................................................................12 1.2.3 Molecular methods....................................................................................................14 1.2.4 Phylogenetic analyses...............................................................................................16 1.2.4.1 Reference data set assembly and DNA sequence alignment.......................16 1.2.4.2 Chimera detection..............................................................................................18 1.2.4.3 Building a backbone constraint tree................................................................18 1.2.4.4 Topological and support inferences................................................................20 1.3 Results..............................................................................................................................21 1.3.1 PCR with new primers .............................................................................................21 1.3.2 Lichen-associated non-photobiont bacteria...........................................................22 vi 1.3.3 Phylogenetic relationships .......................................................................................22 1.4 Discussion........................................................................................................................24 1.4.1 A previously undescribed lineage ..........................................................................24 1.4.2 Symbiosis....................................................................................................................24 1.4.3 Diazotrophy ...............................................................................................................28 1.4.4 Potential effects of acidic secondary compounds .................................................29 1.4.5 The lichen thallus as a complex microbial community........................................30 2. Photoautotrophic symbiont and geography are major factors affecting highly- structured bacterial communities in the lichen microbiome ................................................32 2.1 Introduction.....................................................................................................................32 2.2 Materials and methods ..................................................................................................35 2.2.1 Sampling procedure..................................................................................................35 2.2.2 ‘SQERL’ DNA extraction..........................................................................................38 2.2.3 16S clone library construction..................................................................................38 2.2.4 Clone library analysis ...............................................................................................40 2.2.5 454 sequencing of 16S amplicons ............................................................................43 2.2.6 454 sequence processing and community analyses..............................................44 2.2.7 Sequence data archiving...........................................................................................46 2.3 Results..............................................................................................................................46 2.3.1 Clone library analyses...............................................................................................46 2.3.2 454 community analyses...........................................................................................51 2.4 Discussion........................................................................................................................57 vii 2.4.1 Additional factors to come to light .........................................................................57 2.4.2 Photobiont-bacterial interactions ............................................................................58 2.4.3 Mycobiont-bacterial interactions.............................................................................59 2.4.4 Effects of geography..................................................................................................59 2.4.5 Taxonomic profiles of lichen microbiomes............................................................60 2.4.6 The lichen as a microcosm .......................................................................................63 3. Metatranscriptomes provide insight into the role of non-photoautotrophic bacteria in the lichen symbiosis through carbon and nitrogen metabolism genes...............................65 3.1 Introduction.....................................................................................................................65 3.2 Materials and Methods..................................................................................................67 3.2.1 Specimen collection and storage .............................................................................67 3.2.2 DNA extraction..........................................................................................................67 3.2.3 16S amplicon prep, pyrosequencing,