Fungi are phylogenetically and functionally diverse ubiquitous components of almost all ecosystems on Earth, including aquatic environments stretching from high montane lakes down to the deep ocean. Aquatic ecosystems, however, remain frequently overlooked as fungal habitats, although fungi potentially hold important roles for organic matter cycling and food web dynamics. Within a broad ecological framework, we conceptualize the spatio-temporal dimensions, diversity, functions and organismic interactions of fungi in structuring aquatic foodwebs. We focus on currently unexplored fungal diversity, highlighting poorly understood ecosystems, including emerging artificial aquatic habitats. Recent methodological improvements have facilitated a greater appreciation of the importance of fungi in many aquatic systems, yet a conceptual framework is still missing. To date, aquatic fungi and their interactions have largely remained "hidden" and require interdisciplinary efforts to be explored in an ecosystem context. There remain obvious methodological and knowledge gaps to explore potential functions of aquatic fungi, moving from the microscale to the global scale. This knowledge is urgently needed since we humans strongly interfere with structure and function of natural ecosystems by permanently reshaping most of the Earth's surface and creating vast areas of novel urban habitats. Introduction: Recent advances in DNA sequencing technology have revealed that fungi are abundant in many, if not all aquatic ecosystems, however their diversity, quantitative abundance, ecological function and, in particular, their interactions with other microorganisms, remain largely speculative, unexplored and missing from current general concepts in aquatic ecology and biogeochemistry 1-4. This is surprising since terrestrial-focused research has understood the outstanding ecological role of fungi for >100 years, and therefore fungi constitute a major component of general concepts in terrestrial science 5,6. In aquatic ecosystems, the systematic analysis of fungal diversity and their ecological roles has faced several setbacks due to methodological limitations and a too small scientific community, in particular in the marine environment 7-11. This review focusses on aquatic fungi, which form a morphologically, phylogenetically, and ecologically diverse group 7. We here broadly define "aquatic fungi" as fungi that rely for the whole or part of their life cycle on aquatic habitats (FIG. 1). Three groups (indwellers, periodic immigrants and versatile immigrants) based on their degree of adaptation and dependence on aquatic habitats have been previously defined 12. We highlight the numerous knowledge gaps in their diversity, interactions and functional roles, as well as methodological limitations. In this review we propose new research avenues to set aquatic fungi in a broad ecological framework. Here, we do not explore the many existing gaps in the fungal phylogenetic tree. In aquatic systems, fungi constitute a significant proportion of eukaryo...

SummaryChytridiomycota, often referred to as chytrids, can be virulent parasites with the potential to inflict mass mortalities on hosts, causing e.g. changes in phytoplankton size distributions and succession, and the delay or suppression of bloom events. Molecular environmental surveys have revealed an unexpectedly large diversity of chytrids across a wide range of aquatic ecosystems worldwide. As a result, scientific interest towards fungal parasites of phytoplankton has been gaining momentum in the past few years. Yet, we still know little about the ecology of chytrids, their life cycles, phylogeny, host specificity and range. Information on the contribution of chytrids to trophic interactions, as well as coevolutionary feedbacks of fungal parasitism on host populations is also limited. This paper synthesizes ideas stressing the multifaceted biological relevance of phytoplankton chytridiomycosis, resulting from discussions among an international team of chytrid researchers. It presents our view on the most pressing research needs for promoting the integration of chytrid fungi into aquatic ecology.

1. We describe the dynamics of host-parasite interactions over a period of more than 30 years between the freshwater diatom Asterionella formosa and two highly virulent chytrid parasites (Rhizophydium planktonicum and Zygorhizidium planktonicum) in Lake Maarsseveen, The Netherlands. This period is characterised by a significant warming trend which is strongest in spring. 2. The key spring event in lakes, the diatom bloom, was in many years dominated by Asterionella. We examine whether and how climate warming has affected the prevalence of infection in Asterionella by chytrids. 3. In years with cold winters/early springs, a dense Asterionella bloom is followed by epidemic development of disease as high Asterionella densities greatly facilitate transmission of chytrid zoospores. This sequence of events is absent in milder winters. 4. Earlier experimental studies have shown that the parasite is almost non-infective at water temperatures below 3 degrees C, offering a disease-free window of opportunity for growth of Asterionella. Climate warming has reduced periods in which water temperature remains < 3 degrees C, narrowing the window of opportunity for uninfected growth. Consequently, Asterionella continuously suffers from infection, albeit at low levels. 5. Population reduction as a result of low level infection allows other diatoms to take over as dominant species, possibly through priority effects. 6. In mild winters, chytrid infections no longer reach epidemic levels, but remain at low prevalence since transmission is impaired at low host densities. Climate warming thus affects both host and parasite in intricate ways, with the host denied a bloom and consequently the parasite denied an epidemic. 7. A shift from Asterionella to a mixed diatom community in years with mild winters may benefit the food web, because of the poor edibility of Asterionella, unless the numerous chytrid zoospores produced during epidemics significantly contribute to zooplankton nutrition. 8. Our study demonstrates the potential complexity of climate change impacts on disease. A reduction in the likelihood of epidemic development of a virulent parasite would seem to be of great benefit to the host, but this was not the case. Unexpected, sometimes paradoxical consequences of climate change can be expected and suggest that the view of a 'warmer hence sicker world' may not always apply

Sequence comparison and analysis of the various ribosomal genetic markers are the dominant molecular methods for identification and description of fungi. However, new environmental fungal lineages known only from DNA data reveal significant gaps in our sampling of the fungal kingdom in terms of both taxonomy and marker coverage in the reference sequence databases. To facilitate the integration of reference data from all of the ribosomal markers, we present three sets of general primers that allow for amplification of the complete ribosomal operon from the ribosomal tandem repeats. The primers cover all ribosomal markers: ETS, SSU, ITS1, 5.8S, ITS2, LSU and IGS. We coupled these primers successfully with third-generation sequencing (PacBio and Nanopore sequencing) to showcase our approach on authentic fungal herbarium specimens (Basidiomycota), aquatic chytrids (Chytridiomycota) and a poorly understood lineage of early diverging fungi (Nephridiophagidae). In particular, we were able to generate high-quality reference data with Nanopore sequencing in a high-throughput manner, showing that the generation of reference data can be achieved on a regular desktop computer without the involvement of any large-scale sequencing facility. The quality of the Nanopore generated sequences was 99.85%, which is comparable with the 99.78% accuracy described for Sanger sequencing. With this work, we hope to stimulate the generation of a new comprehensive standard of ribosomal reference data with the ultimate aim to close the huge gaps in our reference datasets. K E Y W O R D S discussions on the implementation of long-read sequencing. The authors would like to acknowledge support from Science for Life Laboratory, the National Genomics Infrastructure, NGI and Uppmax for providing assistance in massive parallel sequencing and computational infrastructure. CW and RHN gratefully acknowledge financial support from Stiftelsen Olle Engkvist Byggmästare, Stiftelsen Lars Hiertas Minne, Kapten Carl Stenholms Donationsfond and Birgit och Birger Wålhströms Minnesfond.

The spatiotemporal changes in abundance and biomass of heterotrophic bacteria, of three major bacterial phylogenetic groups, and of picocyanobacteria in the upper 20 m of a deep prealpine lake (Lake Zurich, Switzerland) were monitored during a seasonally persistent bloom of the toxigenic filamentous cyanobacterium Planktothrix rubescens. In addition, bacterial 16S ribosomal deoxyribonucleic acid (rDNA) sequences were collected at one instance from the bloom layer and from waters above and below this zone. P. rubescens comprised up to 70% of particulate organic carbon during summer stratification and autumnal mixis and thus by far exceeded the total biomass both of other phytoplankton and of prokaryotes. A strong negative correlation was found between the estimated basin-wide biomass of P. rubescens and of heterotrophic bacteria, and there was different spatial niche preference of filamentous vs. picocyanobacteria. Only members of the CytophagaFlavobacterium lineage of Bacteroidetes showed an increasing tendency of association with the P. rubescens population, in particular at the onset of autumnal mixing. Although the filamentous cyanobacterium was the dominant primary producer throughout the year, it did not seem to be a carbon source for heterotrophic bacteria at all. We conclude that P. rubescens represents a powerful competitor of autotrophic and heterotrophic prokaryotes, likely due to both its specific physiological (photoheterotrophic) properties and its protection against zooplankton grazing. This competitiveness might be regarded as another reason for its mass occurrence in numerous lakes of the Northern hemisphere.

Microbial interactions in aquatic environments profoundly affect global biogeochemical cycles, but the role of microparasites has been largely overlooked. Using a model pathosystem, we studied hitherto cryptic interactions between microparasitic fungi (chytrid Rhizophydiales), their diatom host Asterionella, and cell-associated and free-living bacteria. We analyzed the effect of fungal infections on microbial abundances, bacterial taxonomy, cell-to-cell carbon transfer, and cell-specific nitrate-based growth using microscopy (e.g., fluorescence in situ hybridization), 16S rRNA gene amplicon sequencing, and secondary ion mass spectrometry. Bacterial abundances were 2 to 4 times higher on individual fungal-infected diatoms compared to healthy diatoms, particularly involving Burkholderiales. Furthermore, taxonomic compositions of both diatom-associated and free-living bacteria were significantly different between noninfected and fungal-infected cocultures. The fungal microparasite, including diatom-associated sporangia and free-swimming zoospores, derived ∼100% of their carbon content from the diatom. By comparison, transfer efficiencies of photosynthetic carbon were lower to diatom-associated bacteria (67 to 98%), with a high cell-to-cell variability, and even lower to free-living bacteria (32%). Likewise, nitrate-based growth for the diatom and fungi was synchronized and faster than for diatom-associated and free-living bacteria. In a natural lacustrine system, where infection prevalence reached 54%, we calculated that 20% of the total diatom-derived photosynthetic carbon was shunted to the parasitic fungi, which can be grazed by zooplankton, thereby accelerating carbon transfer to higher trophic levels and bypassing the microbial loop. The herein termed “fungal shunt” can thus significantly modify the fate of photosynthetic carbon and the nature of phytoplankton–bacteria interactions, with implications for diverse pelagic food webs and global biogeochemical cycles.

Chytrids are zoosporic fungi that play an important, but yet understudied, ecological role in aquatic ecosystems. Many chytrid species have been morphologically described as parasites on phytoplankton. However, the majority of them have rarely been isolated and lack DNA sequence data. In this study we isolated and cultivated three parasitic chytrids, infecting a common volvocacean host species, Yamagishiella unicocca. To identify the chytrids, we characterized morphology and life cycle, and analyzed phylogenetic relationships based on 18S and 28S rDNA genes. Host range and specificity of the chytrids was determined by cross-infection assays with host strains, characterized by rbcL and ITS markers. We were able to confirm the identity of two chytrid strains as Endocoenobium eudorinae Ingold and Dangeardia mamillata Schröder and described the third chytrid strain as Algomyces stechlinensis gen. et sp. nov. The three chytrids were assigned to novel and phylogenetically distant clades within the phylum Chytridiomycota, each exhibiting different host specificities. By integrating morphological and molecular data of both the parasitic chytrids and their respective host species, we unveiled cryptic host-parasite associations. This study highlights that a high prevalence of (pseudo)cryptic diversity requires molecular characterization of both phytoplankton host and parasitic chytrid to accurately identify and compare host range and specificity, and to study phytoplankton-chytrid interactions in general.

During the last decade, the classification system of chytrids has dramatically changed based on zoospore ultrastructure and molecular phylogeny. In contrast to well-studied saprotrophic chytrids, most parasitic chytrids have thus far been only morphologically described by light microscopy, hence they hold great potential for filling some of the existing gaps in the current classification of chytrids. The genus Zygorhizidium is characterized by an operculate zoosporangium and a resting spore formed as a result of sexual reproduction in which a male thallus and female thallus fuse via a conjugation tube. All described species of Zygorhizidium are parasites of algae and their taxonomic positions remain to be resolved. Here, we examined morphology, zoospore ultrastructure, host specificity, and molecular phylogeny of seven cultures of Zygorhizidium spp. Based on thallus morphology and host specificity, one culture was identified as Z. willei parasitic on zygnematophycean green algae, whereas the others were identified as parasites of diatoms, Z. asterionellae on Asterionella, Z. melosirae on Aulacoseira, and Z. planktonicum on Ulnaria (formerly Synedra). According to phylogenetic analysis, Zygorhizidium was separated into two distinct order-level novel lineages; one lineage was composed singly of Z. willei, which is the type species of the genus, and the other included the three species of diatom parasites. Zoospore ultrastructural observation revealed that the two lineages can be distinguished from each other and both possess unique characters among the known orders within the Chytridiomycetes. Based on these results, we accommodate the three diatom parasites, Z. asterionellae, Z. melosirae, and Z. planktonicum in the distinct genus Zygophlyctis, and propose two new orders: Zygorhizidiales and Zygophlyctidales.