Evolutionary transitions across the tree of life : Phylogenomic case studies in environmental archaea, bacteria and eukaryotes

Microbes form the majority of life on Earth, but have been systematically underrepresented in historical research. The sequencing revolutions of the last two decades have completely changed the way we can study microbial genomic information, giving us the means to probe ever deeper into their evolution.  In a first study, we developed a new approach to screen large short-read datasets for the presence of lineages and genes of interest. By using gene-centric assembly and phylogenetic placement, we integrate the strengths of reference-based with phylogenetic screening methods to efficiently reconstruct gene sequences and label them taxonomically. This will enable researchers to more efficiently select datasets which are likely to contain desired lineages and genes. In the second study, we recover five metagenome-assembled genomes from the previously uncharacterized Hikarchaeia. We infer them to be the closest relatives of haloarchaea and study the evolution of genome content since the last common ancestor of these two aerobic groups with anaerobic methanogens. We detect patterns of gradual evolutionary transition, including the early loss of methanogenesis-related genes and the acquisition of the genetic basis for aerobic respiration. Furthermore, genomes of haloarchaea underwent significant expansion, mostly through transfers from diverse groups of bacteria, whereas Hikarchaeia display genome streamlining typical of marine prokaryotes. In a third study, we reconstructed two new bacterial families, Mitibacteraceae and Athabascaceae, that branch as sister clades to all previously known Rickettsiales. Their genomes lack the characteristic reduction of rickettsial bacteria but resemble free-living alphaproteobacteria. However, they contain several conserved features, such as the rvh type 4 secretion system. We reconstructed the last common ancestor of all Rickettsiales and inferred it as a free-living organism associated with marine biofilms. Further, we propose a model for the transition towards host-association found in all classical Rickettsiales. The fourth study explores the diversity and evolution of the eukaryotic orphan lineage Picozoa. With the help of 43 new single amplified genomes, Picozoa are placed within the supergroup Archaeplastida, in a clade together with red algae and the predatory rhodelphids. In contrast to other archaeplastids, picozoan genomes show no sign of a (cryptic) plastid or plastid-associated gene transfers. This suggests that Picozoa completely lost the primary plastid early in their evolution, or that in fact they never possessed it. These findings raise further questions about the acquisition of the plastid in the archaeplastidal ancestor and the frequency of plastid loss. In summary, this thesis explores several important evolutionary transitions across the tree of life using environmental genome reconstruction, phylogenomics and ancestral state reconstruction approaches.

Host generalists dominate fungal communities associated with alpine knotweed roots : a study of Sebacinales

Bistorta vivipara is a widespread herbaceous perennial plant with a discontinuous pattern of distribution in arctic, alpine, subalpine and boreal habitats across the northern Hemisphere. Studies of the fungi associated with the roots of B. vivipara have mainly been conducted in arctic and alpine ecosystems. This study examined the fungal diversity and specificity from root tips of B. vivipara in two local mountain ecosystems as well as on a global scale. Sequences were generated by Sanger sequencing of the internal transcribed spacer (ITS) region followed by an analysis of accurately annotated nuclear segments including ITS1-5.8S-ITS2 sequences available from public databases. In total, 181 different UNITE species hypotheses (SHs) were detected to be fungi associated with B. vivipara, 73 of which occurred in the Bavarian Alps and nine in the Swabian Alps-with one SH shared among both mountains. In both sites as well as in additional public data, individuals of B. vivipara were found to contain phylogenetically diverse fungi, with the Basidiomycota, represented by the Thelephorales and Sebacinales, being the most dominant. A comparative analysis of the diversity of the Sebacinales associated with B. vivipara and other co-occurring plant genera showed that the highest number of sebacinoid SHs were associated with Quercus and Pinus, followed by Bistorta. A comparison of B. vivipara with plant families such as Ericaceae, Fagaceae, Orchidaceae, and Pinaceae showed a clear trend: Only a few species were specific to B. vivipara and a large number of SHs were shared with other co-occurring non-B. vivipara plant species. In Sebacinales, the majority of SHs associated with B. vivipara belonged to the ectomycorrhiza (ECM)-forming Sebacinaceae, with fewer SHs belonging to the Serendipitaceae encompassing diverse ericoid-orchid-ECM-endophytic associations. The large proportion of non-hostspecific fungi able to form a symbiosis with other non-B. vivipara plants could suggest that the high fungal diversity in B. vivipara comes from an active recruitment of their associates from the co-occurring vegetation. The non-host-specificity suggests that this strategy may offer ecological advantages; specifically, linkages with generalist rather than specialist fungi. Proximity to co-occurring non-B. vivipara plants can maximise the fitness of B. vivipara, allowing more rapid and easy colonisation of the available habitats

Belowground fungal community diversity, composition and ecological functionality associated with winter wheat in conventional and organic agricultural systems

Understanding the impacts of agricultural practices on belowground fungal communities is crucial in order to preserve biological diversity in agricultural soils and enhance their role in agroecosystem functioning. Although fungal communities are widely distributed, relatively few studies have correlated agricultural production practices. We investigated the diversity, composition and ecological functionality of fungal communities in roots of winter wheat (Triticum aestivum) growing in conventional and organic farming systems. Direct and nested polymerase chain reaction (PCR) amplifications spanning the internal transcribed spacer (ITS) region of the rDNA from pooled fine root samples were performed with two different sets of fungal specific primers. Fungal identification was carried out through similarity searches against validated reference sequences (RefSeq). The R package 'picante' and FUNGuild were used to analyse fungal community composition and trophic mode, respectively. Either by direct or cloning sequencing, 130 complete ITS sequences were clustered into 39 operational taxonomic units (OTUs) (25 singletons), belonging to the Ascomycota (24), the Basidiomycota (14) and to the Glomeromycota (1). Fungal communities from conventional farming sites are phylogenetically more related than expected by chance. Constrained ordination analysis identified total N, total S and Pcal that had a significant effect on the OTU's abundance and distribution, and a further correlation with the diversity of the co-occurring vegetation could be hypothesised. The functional predictions based on FUNGuild suggested that conventional farming increased the presence of plant pathogenic fungi compared with organic farming. Based on diversity, OTU distribution, nutrition mode and the significant phylogenetic clustering of fungal communities, this study shows that fungal communities differ across sampling sites, depending on agricultural practices. Although it is not fully clear which factors determine the fungal communities, our findings suggest that organic farming systems have a positive effect on fungal communities in winter wheat crops

High diversity of fungal ecological groups in Andean–Patagonian Nothofagus forests

Native Andean–Patagonian Nothofagus forests harbour a unique diversity of microorganisms with diverse ecological roles. Although ectomycorrhizal associations constitute an important fragment of the biota associated with these forests, the factors affecting such communities are largely unknown. We investigated the biodiversity, relative abundance, and composition of ectomycorrhizal fungal communities in relation to two host tree species and ages and the soil properties in six monospecific and mixed evergreen–deciduous Nothofagus forests. We used the internal transcribed spacer (ITS2) region by sequencing 9,600 ectomycorrhizae (ECM) root tips for the identification of fungi. In total, 1,125 fungal taxa at the genus level distributed over 131 orders were identified. The phyla Ascomycota (34.5%) and Basidiomycota (62.1%) were the most abundant, whereas Mucoromycota (3.1%), Chytridiomycota, Cryptomycota, Olpidiomycota, and Zoopagomycota occurred less frequently. The highest taxon diversity was found in old trees, whereas young trees often exhibited a lower diversity of the associated fungi. The fungal taxa were grouped into seven broad ecological categories, of which saprotrophic associations were most common, followed by pathotrophic, pathotrophic–saprotrophic–symbiotrophic, pathotrophic–saprotrophic, and symbiotrophic associations. We did not detect significant differences in the number of taxa in each category between young and old N. dombeyi and N. obliqua. Overall, the scale of the Illumina sequencing approach allowed us to detect a fungal taxa diversity that would not be possible to find through surveys of fruiting bodies alone and that have never been observed in Nothofagus forests before. Our findings suggest the impact of the proximity between sites, the similarity of the soil conditions, and anthropogenic use of the forests on the belowground fungal community’s diversity and composition. Furthermore, there were differences between above- and belowground occurrences of the edible mushrooms B. loyo and Ramaria spp. However, future research, including on EMC tips found beneath fairy rings could provide significantly better correlations with the occurrence of aboveground fruiting body

The evolutionary origin of host association in the Rickettsiales

Phylogenomic analyses reveal novel environmental clades of Rickettsiales providing insights into their evolution from free-living to host-associated lifestyle. The evolution of obligate host-association of bacterial symbionts and pathogens remains poorly understood. The Rickettsiales are an alphaproteobacterial order of obligate endosymbionts and parasites that infect a wide variety of eukaryotic hosts, including humans, livestock, insects and protists. Induced by their host-associated lifestyle, Rickettsiales genomes have undergone reductive evolution, leading to small, AT-rich genomes with limited metabolic capacities. Here we uncover eleven deep-branching alphaproteobacterial metagenome assembled genomes from aquatic environments, including data from the Tara Oceans initiative and other publicly available datasets, distributed over three previously undescribed Rickettsiales-related clades. Phylogenomic analyses reveal that two of these clades, Mitibacteraceae and Athabascaceae, branch sister to all previously sampled Rickettsiales. The third clade, Gamibacteraceae, branch sister to the recently identified ectosymbiotic 'Candidatus Deianiraea vastatrix'. Comparative analyses indicate that the gene complement of Mitibacteraceae and Athabascaceae is reminiscent of that of free-living and biofilm-associated bacteria. Ancestral genome content reconstruction across the Rickettsiales species tree further suggests that the evolution of host association in Rickettsiales was a gradual process that may have involved the repurposing of a type IV secretion system

The origin and diversification of the Entorrhizales : deep evolutionary roots but recent speciation with a phylogenetic and phenotypic split between associates of the Cyperaceae and Juncaceae

Fungi belonging to the Entorrhizales (Entorrhizomycota) comprise biotrophic pathogens associated with roots of theCyperaceae and Juncaceae plant species. They are nearly globally distributed but rarely studied due to a hidden lifestyle without causing visible effects on host plants. Therefore, the evolutionary origin and phylogenetic relationships of the group are still poorly understood and it is not known whether species diversification was the result of co-evolution with their hosts or the result of host jumps. To infer hypotheses about the evolutionary history of the Entorrhizales, divergence times were estimated and plant-fungal tanglegrams calculated. Relaxed molecular clock analyses suggest that the Entorrhizomycota originated around the Neoproterozoic-Palaeozoic and diverged during the Late Cretaceous-Paleogene into the extant orders Entorrhizales and Talbotiomycetales. The split of the major lineages within the Entorrhizales took place in the Eocene, somewhat later than the divergence of the host families Cyperaceae and Juncaceae. Topology- and distance-based co-phylogenetic analyses of the fungi and their hosts revealed a large number of co-speciation and lineage sorting events in early fungal speciation, which resulted in a phylogenetic split corresponding to species infecting Cyperaceae or Juncaceae. Given that this split is congruent with spore differences, Entorrhiza s. str. is emended for species infecting hosts in the Cyperaceae, and a new genus Juncorrhiza is described for species restricted to hosts in the Juncaceae. Additionally, three new species are described: Entorrhiza fuirenae, Juncorrhiza maritima and J. oxycarpi

Gene gain facilitated endosymbiotic evolution of Chlamydiae

Additional data associated with the article "Gene gain facilitated endosymbiotic evolution of Chlamydiae".</p

The AEGEAN-169 clade of bacterioplankton is synonymous with SAR11 subclade V (HIMB59) and metabolically distinct

Bacterioplankton of the SAR11 clade are the most abundant marine microorganisms and consist of numerous subclades spanning order-level divergence (Pelagibacterales). The assignment of the earliest diverging subclade V (a.k.a. HIMB59) to the Pelagibacterales is highly controversial, with multiple recent phylogenetic studies placing them completely separate from SAR11. Other than through phylogenomics, subclade V has not received detailed examination due to limited genomes from this group. Here, we assessed the ecogenomic characteristics of subclade V to better understand the role of this group in comparison to the Pelagibacterales. We used a new isolate genome, recently released single-amplified genomes and metagenome-assembled genomes, and previously established SAR11 genomes to perform a comprehensive comparative genomics analysis. We paired this analysis with the recruitment of metagenomes spanning the open ocean, coastal, and brackish systems. Phylogenomics, average amino acid identity, and 16S rRNA gene phylogeny indicate that SAR11 subclade V is synonymous with the ubiquitous AEGEAN-169 clade and support the contention that this group represents a taxonomic family. AEGEAN-169 shared many bulk genome qualities with SAR11, such as streamlining and low GC content, but genomes were generally larger. AEGEAN-169 had overlapping distributions with SAR11 but was metabolically distinct from SAR11 in its potential to transport and utilize a broader range of sugars as well as in the transport of trace metals and thiamin. Thus, regardless of the ultimate phylogenetic placement of AEGEAN-169, these organisms have distinct metabolic capacities that likely allow them to differentiate their niche from canonical SAR11 taxa