Discovery of novel intermediate forms redefines the fungal tree of life.

Fungi are the principal degraders of biomass in terrestrial ecosystems and establish important interactions with plants and animals. However, our current understanding of fungal evolutionary diversity is incomplete and is based upon species amenable to growth in culture. These culturable fungi are typically yeast or filamentous forms, bound by a rigid cell wall rich in chitin. Evolution of this body plan was thought critical for the success of the Fungi, enabling them to adapt to heterogeneous habitats and live by osmotrophy: extracellular digestion followed by nutrient uptake. Here we investigate the ecology and cell biology of a previously undescribed and highly diverse form of eukaryotic life that branches with the Fungi, using environmental DNA analyses combined with fluorescent detection via DNA probes. This clade is present in numerous ecosystems including soil, freshwater and aquatic sediments. Phylogenetic analyses using multiple ribosomal RNA genes place this clade with Rozella, the putative primary branch of the fungal kingdom. Tyramide signal amplification coupled with group-specific fluorescence in situ hybridization reveals that the target cells are small eukaryotes of 3-5 µm in length, capable of forming a microtubule-based flagellum. Co-staining with cell wall markers demonstrates that representatives from the clade do not produce a chitin-rich cell wall during any of the life cycle stages observed and therefore do not conform to the standard fungal body plan. We name this highly diverse clade the cryptomycota in anticipation of formal classification.

Molecular diversity and distribution of marine fungi across 130 European environmental samples.

Environmental DNA and culture-based analyses have suggested that fungi are present in low diversity and in low abundance in many marine environments, especially in the upper water column. Here, we use a dual approach involving high-throughput diversity tag sequencing from both DNA and RNA templates and fluorescent cell counts to evaluate the diversity and relative abundance of fungi across marine samples taken from six European near-shore sites. We removed very rare fungal operational taxonomic units (OTUs) selecting only OTUs recovered from multiple samples for a detailed analysis. This approach identified a set of 71 fungal 'OTU clusters' that account for 66% of all the sequences assigned to the Fungi. Phylogenetic analyses demonstrated that this diversity includes a significant number of chytrid-like lineages that had not been previously described, indicating that the marine environment encompasses a number of zoosporic fungi that are new to taxonomic inventories. Using the sequence datasets, we identified cases where fungal OTUs were sampled across multiple geographical sites and between different sampling depths. This was especially clear in one relatively abundant and diverse phylogroup tentatively named Novel Chytrid-Like-Clade 1 (NCLC1). For comparison, a subset of the water column samples was also investigated using fluorescent microscopy to examine the abundance of eukaryotes with chitin cell walls. Comparisons of relative abundance of RNA-derived fungal tag sequences and chitin cell-wall counts demonstrate that fungi constitute a low fraction of the eukaryotic community in these water column samples. Taken together, these results demonstrate the phylogenetic position and environmental distribution of 71 lineages, improving our understanding of the diversity and abundance of fungi in marine environments.

Horizontal gene transfer facilitated the evolution of plant parasitic mechanisms in the oomycetes.

Horizontal gene transfer (HGT) can radically alter the genomes of microorganisms, providing the capacity to adapt to new lifestyles, environments, and hosts. However, the extent of HGT between eukaryotes is unclear. Using whole-genome, gene-by-gene phylogenetic analysis we demonstrate an extensive pattern of cross-kingdom HGT between fungi and oomycetes. Comparative genomics, including the de novo genome sequence of Hyphochytrium catenoides, a free-living sister of the oomycetes, shows that these transfers largely converge within the radiation of oomycetes that colonize plant tissues. The repertoire of HGTs includes a large number of putatively secreted proteins; for example, 7.6% of the secreted proteome of the sudden oak death parasite Phytophthora ramorum has been acquired from fungi by HGT. Transfers include gene products with the capacity to break down plant cell walls and acquire sugars, nucleic acids, nitrogen, and phosphate sources from the environment. Predicted HGTs also include proteins implicated in resisting plant defense mechanisms and effector proteins for attacking plant cells. These data are consistent with the hypothesis that some oomycetes became successful plant parasites by multiple acquisitions of genes from fungi.

Newly identified and diverse plastid-bearing branch on the eukaryotic tree of life.

The use of molecular methods is altering our understanding of the microbial biosphere and the complexity of the tree of life. Here, we report a newly discovered uncultured plastid-bearing eukaryotic lineage named the rappemonads. Phylogenies using near-complete plastid ribosomal DNA (rDNA) operons demonstrate that this group represents an evolutionarily distinct lineage branching with haptophyte and cryptophyte algae. Environmental DNA sequencing revealed extensive diversity at North Atlantic, North Pacific, and European freshwater sites, suggesting a broad ecophysiology and wide habitat distribution. Quantitative PCR analyses demonstrate that the rappemonads are often rare but can form transient blooms in the Sargasso Sea, where high 16S rRNA gene copies mL(-1) were detected in late winter. This pattern is consistent with these microbes being a member of the rare biosphere, whose constituents have been proposed to play important roles under ecosystem change. Fluorescence in situ hybridization revealed that cells from this unique lineage were 6.6 ± 1.2 × 5.7 ± 1.0 µm, larger than numerically dominant open-ocean phytoplankton, and appear to contain two to four plastids. The rappemonads are unique, widespread, putatively photosynthetic algae that are absent from present-day ecosystem models and current versions of the tree of life.

Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water.

Sequencing of ribosomal DNA clone libraries amplified from environmental DNA has revolutionized our understanding of microbial eukaryote diversity and ecology. The results of these analyses have shown that protist groups are far more genetically heterogeneous than their morphological diversity suggests. However, the clone library approach is labour-intensive, relatively expensive, and methodologically biased. Therefore, even the most intensive rDNA library analyses have recovered only small samples of much larger assemblages, indicating that global environments harbour a vast array of unexplored biodiversity. High-throughput parallel tag 454 sequencing offers an unprecedented scale of sampling for molecular detection of microbial diversity. Here, we report a 454 protocol for sampling and characterizing assemblages of eukaryote microbes. We use this approach to sequence two SSU rDNA diversity markers-the variable V4 and V9 regions-from 10 L of anoxic Norwegian fjord water. We identified 38 116 V4 and 15 156 V9 unique sequences. Both markers detect a wide range of taxonomic groups but in both cases the diversity detected was dominated by dinoflagellates and close relatives. Long-tailed rank abundance curves suggest that the 454 sequencing approach provides improved access to rare genotypes. Most tags detected represent genotypes not currently in GenBank, although many are similar to database sequences. We suggest that current understanding of the ecological complexity of protist communities, genetic diversity, and global species richness are severely limited by the sequence data hitherto available, and we discuss the biological significance of this high amplicon diversity.

Marine fungi: their ecology and molecular diversity.

Fungi appear to be rare in marine environments. There are relatively few marine isolates in culture, and fungal small subunit ribosomal DNA (SSU rDNA) sequences are rarely recovered in marine clone library experiments (i.e., culture-independent sequence surveys of eukaryotic microbial diversity from environmental DNA samples). To explore the diversity of marine fungi, we took a broad selection of SSU rDNA data sets and calculated a summary phylogeny. Bringing these data together identified a diverse collection of marine fungi, including sequences branching close to chytrids (flagellated fungi), filamentous hypha-forming fungi, and multicellular fungi. However, the majority of the sequences branched with ascomycete and basidiomycete yeasts. We discuss evidence for 36 novel marine lineages, the majority and most divergent of which branch with the chytrids. We then investigate what these data mean for the evolutionary history of the Fungi and specifically marine-terrestrial transitions. Finally, we discuss the roles of fungi in marine ecosystems.

Validation and justification of the phylum name Cryptomycota phyl. nov.

The recently proposed new phylum name Cryptomycota phyl. nov. is validly published in order to facilitate its use in future discussions of the ecology, biology, and phylogenetic relationships of the constituent organisms. This name is preferred over the previously tentatively proposed "Rozellida" as new data suggest that the life-style and morphology of Rozella is not representative of the large radiation to which it and other Cryptomycota belong. Furthermore, taxa at higher ranks such as phylum are considered better not based on individual names of included genera, but rather on some special characteristics - in this case the cryptic nature of this group and that they were initially revealed by molecular methods rather than morphological discovery. If the group were later viewed as a member of a different kingdom, the name should be retained to indicate its fungal affinities, as is the practice for other fungal-like protist groups.