Deep Sequencing of Subseafloor Eukaryotic rRNA Reveals Active Fungi across Marine Subsurface Provinces

The deep marine subsurface is a vast habitat for microbial life where cells may live on geologic timescales. Because DNA in sediments may be preserved on long timescales, ribosomal RNA (rRNA) is suggested to be a proxy for the active fraction of a microbial community in the subsurface. During an investigation of eukaryotic 18S rRNA by amplicon pyrosequencing, unique profiles of Fungi were found across a range of marine subsurface provinces including ridge flanks, continental margins, and abyssal plains. Subseafloor fungal populations exhibit statistically significant correlations with total organic carbon (TOC), nitrate, sulfide, and dissolved inorganic carbon (DIC). These correlations are supported by terminal restriction length polymorphism (TRFLP) analyses of fungal rRNA. Geochemical correlations with fungal pyrosequencing and TRFLP data from this geographically broad sample set suggests environmental selection of active Fungi in the marine subsurface. Within the same dataset, ancient rRNA signatures were recovered from plants and diatoms in marine sediments ranging from 0.03 to 2.7 million years old, suggesting that rRNA from some eukaryotic taxa may be much more stable than previously considered in the marine subsurface.     

Tracing carbon flow in an arctic marine food web using fatty acid-stable isotope analysis

Global warming and the loss of sea ice threaten to alter patterns of productivity in arctic marine ecosystems because of a likely decline in primary productivity by sea ice algae. Estimates of the contribution of ice algae to total primary production range widely, from just 3 to >50%, and the importance of ice algae to higher trophic levels remains unknown. To help answer this question, we investigated a novel approach to food web studies by combining the two established methods of stable isotope analysis and fatty acid (FA) analysis--we determined the C isotopic composition of individual diatom FA and traced these biomarkers in consumers. Samples were collected near Barrow, Alaska and included ice algae, pelagic phytoplankton, zooplankton, fish, seabirds, pinnipeds and cetaceans. Ice algae and pelagic phytoplankton had distinctive overall FA signatures and clear differences in delta(13)C for two specific diatom FA biomarkers: 16:4n-1 (-24.0+/-2.4 and -30.7+/-0.8 per thousand, respectively) and 20:5n-3 (-18.3+/-2.0 and -26.9+/-0.7 per thousand, respectively). Nearly all delta(13)C values of these two FA in consumers fell between the two stable isotopic end members. A mass balance equation indicated that FA material derived from ice algae, compared to pelagic diatoms, averaged 71% (44-107%) in consumers based on delta(13)C values of 16:4n-1, but only 24% (0-61%) based on 20:5n-3. Our estimates derived from 16:4n-1, which is produced only by diatoms, probably best represented the contribution of ice algae relative to pelagic diatoms. However, many types of algae produce 20:5n-3, so the lower value derived from it likely represented a more realistic estimate of the proportion of ice algae material relative to all other types of phytoplankton. These preliminary results demonstrate the potential value of compound-specific isotope analysis of marine lipids to trace C flow through marine food webs and provide a foundation for future work.     

Seasonality of parasitic and saprotrophic zoosporic fungi: linking sequence data to ecological traits

Zoosporic fungi of the phylum Chytridiomycota (chytrids) regularly dominate pelagic fungal communities in freshwater and marine environments. Their lifestyles range from obligate parasites to saprophytes. Yet, linking the scarce available sequence data to specific ecological traits or their host ranges constitutes currently a major challenge. We combined 28 S rRNA gene amplicon sequencing with targeted isolation and sequencing approaches, along with cross-infection assays and analysis of chytrid infection prevalence to obtain new insights into chytrid diversity, ecology, and seasonal dynamics in a temperate lake. Parasitic phytoplankton-chytrid and saprotrophic pollen-chytrid interactions made up the majority of zoosporic fungal reads. We explicitly demonstrate the recurrent dominance of parasitic chytrids during frequent diatom blooms and saprotrophic chytrids during pollen rains. Distinct temporal dynamics of diatom-specific parasitic clades suggest mechanisms of coexistence based on niche differentiation and competitive strategies. The molecular and ecological information on chytrids generated in this study will aid further exploration of their spatial and temporal distribution patterns worldwide. To fully exploit the power of environmental sequencing for studies on chytrid ecology and evolution, we emphasize the need to intensify current isolation efforts of chytrids and integrate taxonomic and autecological data into long-term studies and experiments.Subject terms: Fungal ecology, Limnology     

Pathogenic fungi of marine animals: A taxonomic perspective

Fungi cause diseases in a variety of marine animal hosts. After a thorough review of published literature, we identified 225 fungal species causing infections of 193 animal species, for a total of 357 combinations of pathogenic fungi and marine animal hosts. Among the 193 animal host species, Chordata (100 species, 51.8 %) and Arthropoda (68 species, 35.2 %) were discovered to be the most frequently reported hosts of fungal pathogens. Microsporidia (111 species, 49.3 %) constitutes over half of the described pathogenic fungal species of marine animals, followed by Ascomycota (85 species, 37.8 %), Mucoromycota (22 species, 9.8 %), Basidiomycota (6 species, 2.7 %) and Chytridiomycota (1 species, 0.4 %). Microsporidia primarily parasitize marine arthropods and Teleostei fish, while Basidiomycota are primarily known to cause respiratory diseases of marine mammals. Ascomycota has a diverse host range, from mammals, fish, crustaceans, soft corals and sea turtle. Few Mucoromycota and Chytridiomycota were reported to infect marine animals. Fungal diseases documented in this review likely represent a fraction of fungal diseases in the ocean, where it was estimated to be inhabited by 2.15 million animal species. Intensification of aquaculture practices, global warming and marine pollution may increase fungal disease outbreak of marine animals. All the topics mentioned above will be discussed in greater details in this review.     

Phylogenetic detection of novel Cryptomycota in an Iowa (United States) aquifer and from previously collected marine and freshwater targeted high‐throughput sequencing sets

Fungi are everywhere and interact with humans in countless ways, but a large group of fungi called ‘Cryptomycota’ has escaped detection until very recently. Still, the extent of diversity and ecological habits of this group remain largely unknown. We interrogated publically available 18S rRNA gene datasets, obtained via high‐throughput sequencing from marine and freshwater samples, for Cryptomycota sequences. Contrary to previous work, we found evidence of substantial Cryptomycota diversity in the marine upper water column. Additionally, we produced a sequencing set from a groundwater aquifer, an environment unrepresented among 18S rRNA gene pyrosequencing sets. The Cryptomycota community in this aquifer sample appears distinct from the community in both freshwater and marine environments with evidence of a unique aquifer clade. This study significantly expands the boundary of known Cryptomycota sequence diversity and characterizes the phylogenetic distribution of this diversity in aquatic environments. Furthermore, the approach utilized is generalizable to discovery of novel micro‐eukaryotic diversity from any lineage.     

Unveiling fungal zooflagellates as members of freshwater picoeukaryotes: evidence from a molecular diversity study in a deep meromictic lake

This study presents an original 18S rRNA PCR survey of the freshwater picoeukaryote community, and was designed to detect unidentified heterotrophic picoflagellates (size range 0.6-5 microm) which are prevalent throughout the year within the heterotrophic flagellate assemblage in Lake Pavin. Four clone libraries were constructed from samples collected in two contrasting zones in the lake. Computerized statistic tools have suggested that sequence retrieval was representative of the in situ picoplankton diversity. The two sampling zones exhibited similar diversity patterns but shared only about 5% of the operational taxonomic units (OTUs). Phylogenetic analysis clustered our sequences into three taxonomic groups: Alveolates (30% of OTUs), Fungi (23%) and Cercozoa (19%). Fungi thus substantially contributed to the detected diversity, as was additionally supported by direct microscopic observations of fungal zoospores and sporangia. A large fraction of the sequences belonged to parasites, including Alveolate sequences affiliated to the genus Perkinsus known as zooparasites, and chytrids that include host-specific parasitic fungi of various freshwater phytoplankton species, primarily diatoms. Phylogenetic analysis revealed five novel clades that probably include typical freshwater environmental sequences. Overall, from the unsuspected fungal diversity unveiled, we think that fungal zooflagellates have been misidentified as phagotrophic nanoflagellates in previous studies. This is in agreement with a recent experimental demonstration that zoospore-producing fungi and parasitic activity may play an important role in aquatic food webs.     

Carbon assimilating fungi from surface ocean to subseafloor revealed by coupled phylogenetic and stable isotope analysis

Fungi are ubiquitous in the ocean and hypothesized to be important members of marine ecosystems, but their roles in the marine carbon cycle are poorly understood. Here, we use ¹³C DNA stable isotope probing coupled with phylogenetic analyses to investigate carbon assimilation within diverse communities of planktonic and benthic fungi in the Benguela Upwelling System (Namibia). Across the redox stratified water column and in the underlying sediments, assimilation of ¹³C-labeled carbon from diatom extracellular polymeric substances (¹³C-dEPS) by fungi correlated with the expression of fungal genes encoding carbohydrate-active enzymes. Phylogenetic analysis of genes from ¹³C-labeled metagenomes revealed saprotrophic lineages related to the facultative yeast Malassezia were the main fungal foragers of pelagic dEPS. In contrast, fungi living in the underlying sulfidic sediments assimilated more ¹³C-labeled carbon from chemosynthetic bacteria compared to dEPS. This coincided with a unique seafloor fungal community and dissolved organic matter composition compared to the water column, and a 100-fold increased fungal abundance within the subseafloor sulfide-nitrate transition zone. The subseafloor fungi feeding on ¹³C-labeled chemolithoautotrophs under anoxic conditions were affiliated with Chytridiomycota and Mucoromycota that encode cellulolytic and proteolytic enzymes, revealing polysaccharide and protein-degrading fungi that can anaerobically decompose chemosynthetic necromass. These subseafloor fungi, therefore, appear to be specialized in organic matter that is produced in the sediments. Our findings reveal that the phylogenetic diversity of fungi across redox stratified marine ecosystems translates into functionally relevant mechanisms helping to structure carbon flow from primary producers in marine microbiomes from the surface ocean to the subseafloor.Subject terms: Microbial ecology, Fungal ecology, Microbiome, Biogeochemistry     

DNA分子标记技术在真菌系统学研究中的应用及影响

DNA分子标记技术为真菌系统进化研究提供了许多新的方法,真菌分子系统学已成为一门成熟的学科。简述了真菌分子系统学的发展简史和代表性的研究方法以及对真菌系统学的主要贡献,包括将广义的真菌划分为3个类群,粘菌和卵菌不再属于真菌界成员。真菌生命之树项目的研究结果对真菌界高阶分类系统作出重大调整,将先前的4个门(壶菌门、接合菌门、子囊菌门和担子菌门)变为7个门(微孢子虫门、壶菌门、新丽鞭毛菌门、芽枝霉门、球囊菌门、子囊菌门和担子菌门)和4个亚门,并对真菌各类群概念作出修订。此外,DNA分子标记技术对真菌种概念的认识、有性型-无性型关联及分子生态学等研究领域产生了重要影响。     

High‐throughput sequencing reveals neustonic and planktonic microbial eukaryote diversity in coastal waters

Neustonic organisms inhabit the sea surface microlayer (SML) and have important roles in marine ecosystem functioning. Here, we use high‐throughput 18S rRNA gene sequencing to characterize protist and fungal diversity in the SML at a coastal time‐series station and compare with underlying plankton assemblages. Protist diversity was higher in February (pre‐bloom) compared to April (spring bloom), and was lower in the neuston than in the plankton. Major protist groups, including Stramenopiles and Alveolata, dominated both neuston and plankton assemblages. Chrysophytes and diatoms were enriched in the neuston in April, with diatoms showing distinct changes in community composition between the sampling periods. Pezizomycetes dominated planktonic fungi assemblages, whereas fungal diversity in the neuston was more varied. This is the first study to utilize a molecular‐based approach to characterize neustonic protist and fungal assemblages, and provides the most comprehensive diversity assessment to date of this ecosystem. Variability in the SML microeukaryote assemblage structure has potential implications for biogeochemical and food web processes at the air‐sea interface.     

On the structure and development of Arctic pack ice communities in Fram Strait: a multivariate approach

Summary The distribution of ice organisms was investigated in Fram Strait in May 1988 during the ARK V/1 expedition on RV Polarstern. Over a 3 week period the abundances of bacteria, diatoms, auto- and heterotrophic flagellates as well as various groups of meiofauna organisms were observed in the lowermost 30 cm of an ice floe. Data were obtained from three experimental fields under three different light regimes as a result of manipulations of the snow cover. The application of multivariate factor analysis on this time series data set resulted in the characterization of four succession stages of an Arctic sea ice community: 1) the diatom bottom assemblage, 2) the mixed autotrophic assemblage, 3) the mixed auto- and heterotrophic supra-bottom assemblage, and 4) the heterotrophic supra-bottom assemblage. The two most abundant meiofauna groups (Turbellaria, Ciliata) showed different preferences according to algal distribution. While turbellarians were most abundant in samples with mixed populations of diatoms and flagellates, ciliates reached their abundance maxima in samples dominated by diatoms, suggesting different prey selections. We have developed a model for the explanation of the spatial separation of auto- and heterotrophic organisms, highlighting the possible role of DOC production by ice algae and DOC transport with brine flow.     

Protists in Arctic drift and land‐fast sea ice

Global climate change is having profound impacts on polar ice with changes in the duration and extent of both land‐fast ice and drift ice, which is part of the polar ice pack. Sea ice is a distinct habitat and the morphologically identifiable sympagic community living within sea ice can be readily distinguished from pelagic species. Sympagic metazoa and diatoms have been studied extensively since they can be identified using microscopy techniques. However, non‐diatom eukaryotic cells living in ice have received much less attention despite taxa such as the dinoflagellate Polarella and the cercozoan Cryothecomonas being isolated from sea ice. Other small flagellates have also been reported, suggesting complex microbial food webs. Since smaller flagellates are fragile, often poorly preserved, and are difficult for non‐experts to identify, we applied high throughput tag sequencing of the V4 region of the 18S rRNA gene to investigate the eukaryotic microbiome within the ice. The sea ice communities were diverse (190 taxa) and included many heterotrophic and mixotrophic species. Dinoflagellates (43 taxa), diatoms (29 taxa) and cercozoans (12 taxa) accounted for ~80% of the sequences. The sympagic communities living within drift ice and land‐fast ice harbored taxonomically distinct communities and we highlight specific taxa of dinoflagellates and diatoms that may be indicators of land‐fast and drift ice.     

Timing of blooms,algal food quality and Calanus glacialis reproduction and growth in a changing Arctic

The Arctic bloom consists of two distinct categories of primary producers, ice algae growing within and on the underside of the sea ice, and phytoplankton growing in open waters. Long chain omega‐3 fatty acids, a subgroup of polyunsaturated fatty acids (PUFAs) produced exclusively by these algae, are essential to all marine organisms for successful reproduction, growth, and development. During an extensive field study in the Arctic shelf seas, we followed the seasonal biomass development of ice algae and phytoplankton and their food quality in terms of their relative PUFA content. The first PUFA‐peak occurred in late April during solid ice cover at the onset of the ice algal bloom, and the second PUFA‐peak occurred in early July just after the ice break‐up at the onset of the phytoplankton bloom. The reproduction and growth of the key Arctic grazer Calanus glacialis perfectly coincided with these two bloom events. Females of C. glacialis utilized the high‐quality ice algal bloom to fuel early maturation and reproduction, whereas the resulting offspring had access to ample high‐quality food during the phytoplankton bloom 2 months later. Reduction in sea ice thickness and coverage area will alter the current primary production regime due to earlier ice break‐up and onset of the phytoplankton bloom. A potential mismatch between the two primary production peaks of high‐quality food and the reproductive cycle of key Arctic grazers may have negative consequences for the entire lipid‐driven Arctic marine ecosystem.     

Molecular diversity of fungal phylotypes co-amplified alongside nematodes from coastal and deep-sea marine environments

Nematodes and fungi are both ubiquitous in marine environments, yet few studies have investigated relationships between these two groups. Microbial species share many well-documented interactions with both free-living and parasitic nematode species, and limited data from previous studies have suggested ecological associations between fungi and nematodes in benthic marine habitats. This study aimed to further document the taxonomy and distribution of fungal taxa often co-amplified from nematode specimens. A total of 15 fungal 18S rRNA phylotypes were isolated from nematode specimens representing both deep-sea and shallow water habitats; all fungal isolates displayed high pairwise sequence identities with published data in Genbank (99-100%) and unpublished high-throughput 454 environmental datasets (>95%). BLAST matches indicate marine fungal sequences amplified in this study broadly represent taxa within the phyla Ascomycota and Basidiomycota, and several phylotypes showed robust groupings with known taxa in phylogenetic topologies. In addition, some fungal phylotypes appeared to be present in disparate geographic habitats, suggesting cosmopolitan distributions or closely related species complexes in at least some marine fungi. The present study was only able to isolate fungal DNA from a restricted set of nematode taxa; further work is needed to fully investigate the taxonomic scope and function of nematode-fungal interactions.     

Microplastics alter composition of fungal communities in aquatic ecosystems

Despite increasing concerns about microplastic (MP) pollution in aquatic ecosystems, there is insufficient knowledge on how MP affect fungal communities. In this study, we explored the diversity and community composition of fungi attached to polyethylene (PE) and polystyrene (PS) particles incubated in different aquatic systems in north‐east Germany: the Baltic Sea, the River Warnow and a wastewater treatment plant. Based on next generation 18S rRNA gene sequencing, 347 different operational taxonomic units assigned to 81 fungal taxa were identified on PE and PS. The MP‐associated communities were distinct from fungal communities in the surrounding water and on the natural substrate wood. They also differed significantly among sampling locations, pointing towards a substrate and location specific fungal colonization. Members of Chytridiomycota, Cryptomycota and Ascomycota dominated the fungal assemblages, suggesting that both parasitic and saprophytic fungi thrive in MP biofilms. Thus, considering the worldwide increasing accumulation of plastic particles as well as the substantial vector potential of MP, especially these fungal taxa might benefit from MP pollution in the aquatic environment with yet unknown impacts on their worldwide distribution, as well as biodiversity and food web dynamics at large.     

Molecular detection of fungal communities in the Hawaiian marine sponges Suberites zeteki and Mycale armata

Symbiotic microbes play a variety of fundamental roles in the health and habitat ranges of their hosts. While prokaryotes in marine sponges have been broadly characterized, the diversity of sponge-inhabiting fungi has barely been explored using molecular approaches. Fungi are an important component of many marine and terrestrial ecosystems, and they may be an ecologically significant group in sponge-microbe interactions. This study tested the feasibility of using existing fungal primers for molecular analysis of sponge-associated fungal communities. None of the eight selected primer pairs yielded satisfactory results in fungal rRNA gene or internal transcribed spacer (ITS) clone library constructions. However, 3 of 10 denaturing gradient gel electrophoresis (DGGE) primer sets, which were designed to preferentially amplify fungal rRNA gene or ITS regions from terrestrial environmental samples, were successfully amplified from fungal targets in marine sponges. DGGE analysis indicated that fungal communities differ among different sponge species (Suberites zeteki and Mycale armata) and also vary between sponges and seawater. Sequence analysis of DGGE bands identified 23 and 21 fungal species from each of the two sponge species S. zeteki and M. armata, respectively. These species were representatives of 11 taxonomic orders and belonged to the phyla of Ascomycota (seven orders) and Basidiomycota (four orders). Five of these taxonomic orders (Malasseziales, Corticiales, Polyporales, Agaricales, and Dothideomycetes et Chaetothyriomcetes incertae sedis) have now been identified for the first time in marine sponges. Seven and six fungal species from S. zeteki and M. armata, respectively, are potentially new species because of their low sequence identity (< or =98%) with their references in GenBank. Phylogenetic analysis indicated sponge-derived sequences were clustered into "marine fungus clades" with those from other marine habitats. This is the first report of molecular analysis of fungal communities in marine sponges, adding depth and dimension to our understanding of sponge-associated microbial communities.     

Identification of the sea ice diatom biomarker IP25 in Arctic benthic macrofauna: direct evidence for a sea ice diatom diet in Arctic heterotrophs

Currently, the impact of declining seasonal sea ice extent in the Arctic on polar food webs remains uncertain. Previously, a range of proxy techniques has been employed to determine links between sea ice or phytoplankton primary production and the Arctic marine food web, although it is accepted that such approaches have their limitations. Here, we propose a novel approach to tracing sea ice primary production through Arctic food webs using the sea ice diatom biomarker, IP₂₅. Various benthic macrofaunal specimens were collected between March and May 2008 from Franklin Bay in the Amundsen Gulf, Arctic Canada, as part of the International Polar Year–Circumpolar Flaw Lead system study. Each specimen was analysed for the presence of the sea ice diatom biomarker IP₂₅ in order to provide evidence for feeding by benthic organisms on sea ice algae. IP₂₅ was found in nineteen out of the twenty-one specimens analysed, often as the most abundant of the highly branched isoprenoid biomarkers detected. The stable isotope composition of IP₂₅ (δ¹³C = −17.1 ± 0.5‰) in the sea urchin (Strongylocentrotus sp.) specimens was similar to that reported previously for this biomarker in Arctic sea ice, sedimenting particles and sediments. It is concluded that detection of IP₂₅ in Arctic benthic macrofauna represents a novel approach to providing convincing evidence for feeding on sea ice algae. It is also proposed that analysis of IP₂₅ may be used to trace trophic transfer of sea ice algal-derived organic matter through Arctic food webs in the future.     

CHARACTERIZATION OF A cDNA ENCODING GLUTAMINE SYNTHETASE FROM THE MARINE DIATOM SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)

A cDNA-encoding glutamine synthetase (GS) was isolated from the marine diatom Skeletonema costatum (Greville) Cleve by PCR amplification. Nucleic acid and deduced amino acid sequences of the diatom GS were greater than 50% identical to GS from green algae and vascular plants, and phylogenetic analysis established the diatom GS as a member of the GSII gene family. The presence of an N-terminus signal sequence, identified on the basis of sequence similarity with other chloroplast-localized proteins from diatoms, suggests that the encoded GS isoenzyme is localized to the chloroplast. The GS mRNA was present in log-phase cells grown with either nitrate or ammonium as the sole added nitrogen source. Results from Southern blot analysis of genomic DNA suggested that the cDNA isolated in this study was either a member of a small, highly conserved gene family or that there was allelic variation within the region examined. Phylogenetic analyses further indicated that genes encoding GS from the diatom and two species of green algae diverged prior to the gene duplication, to the isoenzymes in vascular plants, supporting the hypothesis that GS isoenzymes in diatoms, green algae, and vascular plants arose through independent evolutionary events.