Imke Schmitt

Reconstructing the early evolution of Fungi using a six-gene phylogeny

The ancestors of fungi are believed to be simple aquatic forms with flagellated spores, similar to members of the extant phylum Chytridiomycota (chytrids). Current classifications assume that chytrids form an early-diverging clade within the kingdom Fungi and imply a single loss of the spore flagellum, leading to the diversification of terrestrial fungi. Here we develop phylogenetic hypotheses for Fungi using data from six gene regions and nearly 200 species. Our results indicate that there may have been at least four independent losses of the flagellum in the kingdom Fungi. These losses of swimming spores coincided with the evolution of new mechanisms of spore dispersal, such as aerial dispersal in mycelial groups and polar tube eversion in the microsporidia (unicellular forms that lack mitochondria). The enigmatic microsporidia seem to be derived from an endoparasitic chytrid ancestor similar to Rozella allomycis, on the earliest diverging branch of the fungal phylogenetic tree.

Ancient horizontal gene transfer from bacteria enhances biosynthetic capabilities of fungi.

Background Polyketides are natural products with a wide range of biological functions and pharmaceutical applications. Discovery and utilization of polyketides can be facilitated by understanding the evolutionary processes that gave rise to the biosynthetic machinery and the natural product potential of extant organisms. Gene duplication and subfunctionalization, as well as horizontal gene transfer are proposed mechanisms in the evolution of biosynthetic gene clusters. To explain the amount of homology in some polyketide synthases in unrelated organisms such as bacteria and fungi, interkingdom horizontal gene transfer has been evoked as the most likely evolutionary scenario. However, the origin of the genes and the direction of the transfer remained elusive. Methodology/Principal Findings We used comparative phylogenetics to infer the ancestor of a group of polyketide synthase genes involved in antibiotic and mycotoxin production. We aligned keto synthase domain sequences of all available fungal 6-methylsalicylic acid (6-MSA)-type PKSs and their closest bacterial relatives. To assess the role of symbiotic fungi in the evolution of this gene we generated 24 6-MSA synthase sequence tags from lichen-forming fungi. Our results support an ancient horizontal gene transfer event from an actinobacterial source into ascomycete fungi, followed by gene duplication. Conclusions/Significance Given that actinobacteria are unrivaled producers of biologically active compounds, such as antibiotics, it appears particularly promising to study biosynthetic genes of actinobacterial origin in fungi. The large number of 6-MSA-type PKS sequences found in lichen-forming fungi leads us hypothesize that the evolution of typical lichen compounds, such as orsellinic acid derivatives, was facilitated by the gain of this bacterial polyketide synthase.

Phylogenetic relationships of Lecanoromycetes (Ascomycota) as revealed by analyses of mtSSU and nLSU rDNA sequence data

The phylogeny of Lecanoromycetes (Ascomycota, Fungi) is investigated utilizing parsimony and Bayesian Markov Chain Monte Carlo analyses, of combined nLSU rDNA and mtSSU rDNA sequence datasets. The results suggest that Acarosporaceae, Candelariaceae, Phlyctis and Pycnora are not members of the monophyletic Lecanorales, and that Timdalia and Pleopsidium are members of a monophyletic Acarosporaceae. Pycnora, Candelariaceae and Acarosporaceae form a monophyletic group. Umbilicariaceae, Hypocenomyce scalaris, H. friesii, Ophioparmaceae, Boreoplaca, Elixia and Fuscidea form either a basal paraphyletic assemblage in Lecanoromycetes, or a monophyletic group which is the sister-group to Lecanorales and the rest of Lecanoromycetes (excluding Acarosporaceae). The Acarosporaceae forms a group with Pycnora and Candelariaceae, which may be outside the Lecanoromycetes. Chaetothyriales, Verrucariales, Eurotiales, Lichinales and Mycocaliciales form a monophyletic group, but with low support. We briefly discuss incongruence between datasets from different genetic markers, comparing the differences between the separate parsimony analyses, where the ILD test indicated a very significant incongruence. The phylogenetic significance of ascus-types that have influenced most recent Ascomycota classifications heavily is also discussed, and we finally point out risks with formalizing classifications too early.

Multiple evolutionary origins of legume traits leading to extreme rhizobial differentiation

SUMMARY *When rhizobia differentiate inside legume host nodules to become nitrogen-fixing bacteroids, they undergo a physiological as well as a morphological transformation. These transformations are more extreme in some legume species than others, leading to fundamental differences in rhizobial life history and evolution. Here, we analysed the distribution of different bacteroid morphologies over a legume phylogeny to understand the evolutionary history of this host-influenced differentiation. *Using existing electron micrographs and new flow cytometric analyses, bacteroid morphologies were categorized as swollen or nonswollen for 40 legume species in the subfamily Papilionoideae. Maximum likelihood and Bayesian frameworks were used to reconstruct ancestral states at the bases of all major subclades within the papilionoids. *Extreme bacteroid differentiation leading to swelling was found in five out of the six major papilionoid subclades. The inferred ancestral state for the Papilionoideae was hosting nonswollen bacteroids, indicating at least five independent origins of host traits leading to swollen bacteroids. *Repeated evolution of host traits causing bacteroid swelling indicates a possible fitness benefit to the plant. Furthermore, as bacteroid swelling is often correlated with loss of reproductive viability, the evolution of bacteroid cooperation or cheating strategies could be fundamentally different between the two bacteroid morphologies.

Meta‐analysis of deep‐sequenced fungal communities indicates limited taxon sharing between studies and the presence of biogeographic patterns

High-throughput amplicon sequencing gives new insights into fungal community ecology. Massively generated molecular data lead to the discovery of vast fungal diversity. However, it is unclear to what extent operational taxonomic units (OTUs) overlap among independent studies, because no comparative studies exist. We compared fungal diversity based on the internal transcribed spacer (ITS1) region among 10 published studies. Starting from the raw 454 pyrosequencing data, we used a uniform pipeline to prune the reads. We investigated fungal richness and taxonomic composition among phyllosphere and soil fungal communities, as well as biogeographic signals in the data. We did not find globally distributed OTUs, even when comparing fungal communities from similar habitats (phyllosphere or soil). This suggests that high local fungal diversity scales up to high global diversity. The most OTU-rich classes in the phyllosphere were Dothideomycetes (21%) and Sordariomycetes (14%), and in the soil were Sordariomycetes (13%) and Agaricomycetes (12%). The richness estimates suggest the presence of undiscovered fungal diversity even in deeply sequenced study systems. The small number of OTUs shared among studies indicates that globally distributed taxa and habitat generalists may be rare. Latitudinal diversity decline and distance decay relationships suggest the presence of biogeographic patterns similar to those in plants and animals.

An Illumina metabarcoding pipeline for fungi

High-throughput metabarcoding studies on fungi and other eukaryotic microorganisms are rapidly becoming more frequent and more complex, requiring researchers to handle ever increasing amounts of raw sequence data. Here, we provide a flexible pipeline for pruning and analyzing fungal barcode (ITS rDNA) data generated as paired-end reads on Illumina MiSeq sequencers. The pipeline presented includes specific steps fine-tuned for ITS, that are mostly missing from pipelines developed for prokaryotes. It (1) employs state of the art programs and follows best practices in fungal high-throughput metabarcoding; (2) consists of modules and scripts easily modifiable by the user to ensure maximum flexibility with regard to specific needs of a project or future methodological developments; and (3) is straightforward to use, also in classroom settings. We provide detailed descriptions and revision techniques for each step, thus giving the user maximum control over data treatment and avoiding a black-box approach. Employing this pipeline will improve and speed up the tedious and error-prone process of cleaning fungal Illumina metabarcoding data.

Relocation, high-latitude warming and host genetic identity shape the foliar fungal microbiome of poplars

Micro‐organisms associated with plants and animals affect host fitness, shape community structure and influence ecosystem properties. Climate change is expected to influence microbial communities, but their reactions are not well understood. Host‐associated micro‐organisms are influenced by the climate reactions of their hosts, which may undergo range shifts due to climatic niche tracking, or may be actively relocated to mitigate the effects of climate change. We used a common‐garden experiment and rDNA metabarcoding to examine the effect of host relocation and high‐latitude warming on the complex fungal endophytic microbiome associated with leaves of an ecologically dominant boreal forest tree (Populus balsamifera L.). We also considered the potential effects of poplar genetic identity in defining the reactions of the microbiome to the treatments. The relocation of hosts to the north increased the diversity of the microbiome and influenced its structure, with results indicating enemy release from plausible pathogens. High‐latitude warming decreased microbiome diversity in comparison with natural northern conditions. The warming also caused structural changes, which made the fungal communities distinct in comparison with both low‐latitude and high‐latitude natural communities, and increased the abundance of plausible pathogens. The reactions of the microbiome to relocation and warming were strongly dependent on host genetic identity. This suggests that climate change effects on host–microbiome systems may be mediated by the interaction of environmental factors and the population genetic processes of the hosts.

Evolution of complex symbiotic relationships in a morphologically derived family of lichen-forming fungi.

We studied the evolutionary history of the Parmeliaceae (Lecanoromycetes, Ascomycota), one of the largest families of lichen-forming fungi with complex and variable morphologies, also including several lichenicolous fungi. We assembled a six-locus data set including nuclear, mitochondrial and low-copy protein-coding genes from 293 operational taxonomic units (OTUs). The lichenicolous lifestyle originated independently three times in lichenized ancestors within Parmeliaceae, and a new generic name is introduced for one of these fungi. In all cases, the independent origins occurred c. 24 million yr ago. Further, we show that the Paleocene, Eocene and Oligocene were key periods when diversification of major lineages within Parmeliaceae occurred, with subsequent radiations occurring primarily during the Oligocene and Miocene. Our phylogenetic hypothesis supports the independent origin of lichenicolous fungi associated with climatic shifts at the Oligocene-Miocene boundary. Moreover, diversification bursts at different times may be crucial factors driving the diversification of Parmeliaceae. Additionally, our study provides novel insight into evolutionary relationships in this large and diverse family of lichen-forming ascomycetes.

Higher level phylogenetic relationships of euascomycetes (Pezizomycotina) inferred from a combined analysis of nuclear and mitochondrial sequence data

A combined data set of nuclear SSU rDNA, LSU rDNA, and mitochondrial SSU rDNA sequences was analyzed in order to examine the relationships of the major clades of euascomycetes. Partial sequences of 14 ascomycetes were determined and aligned with the corresponding sequences of 16 other ascomycetes retrieved from Genbank. The alignment was analyzed using maximum parsimony (MP) and a Bayesian analysis with Markov chain Monte Carlo (B/MCMC). The classification based on single-gene studies is supported, but the confidence is enhanced in the concatenated analysis. The monophyly of the superclass Leotiomyceta, which includes all euascomycetes with inoperculate asci, is strongly supported. The polyphyly of ascolocularous fungi is supported. The group is divided into two groups: the Dothideomycetes basal to all other Leotiomyceta and the Chaetothyriomycetes as sister-group to Eurotiomycetes. The Lecanoromycetes appear as a monophyletic group with strong support and form a sister-group to the Chaetothyriomycetes/Eurotiomycetes clade, but this lacks support. The Leotiomycetes and Sordariomycetes form a strongly supported sister-group. Alternative topologies are tested using parametric bootstrapping; a basal position of the Eurotiomycetes and Leotiomycetes in the Leotiomyceta cannot be rejected, while such a position can be rejected for Chaetothyriomycetes, Lecanoromycetes and Sordariomycetes. The character evolution with regard to ascoma type, ascus type and ascoma-ontogeny is examined using MP and maximum likelihood (ML). While it appears most likely that the ancestor of the inoperculate ascomycetes had apothecia and an ascohymenial ascoma-ontogeny using MP methods, the ML approach shows that there is some uncertainty at the current state of knowledge. The improvement of confidence of the combined data set in comparison with single-gene studies makes us confident that analyses with additional data sets will further improve the confidence and eventually uncover the branching order of euascomycetes.

Repeated evolution of closed fruiting bodies is linked to ascoma development in the largest group of lichenized fungi (Lecanoromycetes, Ascomycota).

Fruiting bodies are responsible for the effective dispersal of meiospores in ascomycetes. Different fruiting body types include open (apothecia) or closed (perithecia, cleistothecia) forms, which have traditionally been used as key paradigms for ascomycete classification. Molecular phylogenies show that most fruiting body types have multiple phylogenetic origins within the phylum, and are not suitable for the circumscription of classes. One exception are perithecia that are restricted in non-lichenized fungi to the monophyletic class Sordariomycetes. However, lichenized fungi with perithecioid fruiting bodies were found to belong to three other classes unrelated to Sordariomycetes. One of these is Lecanoromycetes, which includes the bulk of lichenized fungi. To understand the evolution of perithecioid fruiting bodies in the mostly apotheciate Lecanoromycetes, we assembled a combined data set of nuclear and mitochondrial ribosomal, and RPB1 DNA sequences, and traced the evolution of two morphological characters (fruiting body type and fruiting body development). We reconstructed ancestral character states using maximum likelihood and Bayesian methods. Additionally, we tested for correlation of character changes in a combined Bayesian/maximum likelihood framework. The results suggest that perithecia have evolved in unrelated groups of lichen-forming fungi. Within Lecanoromycetes they have evolved independently several times from apotheciate ancestors. Further, our analyses support a correlation between the type of fruiting body and the type of ascoma ontogeny. The evolution of angiocarpous ascoma development in Lecanoromycetes is a pre-adaptation for the repeated gain of perithecia. This finding is consistent with the hypothesis of a neotenic origin of perithecioid fruiting bodies in Lecanoromycetes.