Singleton-based species names and fungal rarity: Does the number really matter?

Fungi are among the least known organisms on earth, with an estimated number of species between 1.5 and 10 million. This number is expected to be refined, especially with increasing knowledge about microfungi in undersampled habitats and increasing amounts of data derived from environmental DNA sequencing. A significant proportion of newly generated sequences fail to match with already named species, and thus represent what has been referred to as fungal "dark taxa". Due to the challenges associated with observing, identifying, and preserving sporophores, many macro- and microfungal species are only known from a single collection, specimen, isolate, and/or sequence-a singleton. Mycologists are consequently used to working with "rare" sequences and specimens. However, rarity and singleton phenomena lack consideration and valorization in fungal studies. In particular, the practice of publishing new fungal species names based on a single specimen remains a cause of debate. Here, we provide some elements of reflection on this issue in the light of the specificities of the fungal kingdom and global change context. If multiple independent sources of data support the existence of a new taxon, we encourage mycologists to proceed with formal description, irrespective of the number of specimens at hand. Although the description of singleton-based species may not be considered best practice, it does represent responsible science in the light of closing the Linnean biodiversity shortfall.

Fungal Endophytes and Their Role in Agricultural Plant Protection against Pests and Pathogens.

Virtually all examined plant species harbour fungal endophytes which asymptomatically infect or colonize living plant tissues, including leaves, branches, stems and roots. Endophyte-host interactions are complex and span the mutualist-pathogen continuum. Notably, mutualist endophytes can confer increased fitness to their host plants compared with uncolonized plants, which has attracted interest in their potential application in integrated plant health management strategies. In this review, we report on the many benefits that fungal endophytes provide to agricultural plants against common non-insect pests such as fungi, bacteria, nematodes, viruses, and mites. We report endophytic modes of action against the aforementioned pests and describe why this broad group of fungi is vitally important to current and future agricultural practices. We also list an extensive number of plant-friendly endophytes and detail where they are most commonly found or applied in different studies. This review acts as a general resource for understanding endophytes as they relate to potential large-scale agricultural applications.

Foliar Endophytic Fungi from the Endangered Eastern Mountain Avens (Geum peckii, Rosaceae) in Canada.

Eastern Mountain Avens (Geum peckii Pursh, Rosaceae) is a globally rare and endangered perennial plant found only at two coastal bogs within Digby County (Nova Scotia, Canada) and at several alpine sites in the White Mountains of New Hampshire (USA). In Canada, the G. peckii population has declined over the past forty years due in part to habitat degradation. We investigated the culturable foliar fungi present in G. peckii leaves at five locations with varying degrees of human impact within this plant species' Canadian range. Fungal identifications were made using ITS rDNA barcoding of axenic fungal cultures isolated from leaf tissue. Differences in foliar fungal communities among sites were documented, with a predominance of Gnomoniaceae (Class: Sordariomycetes, Phylum: Ascomycota). Habitats with more human impact showed lower endophytic diversities (10-16 species) compared to the pristine habitat (27 species). Intriguingly, several fungi may represent previously unknown taxa. Our work represents a significant step towards understanding G. peckii's mycobiome and provides relevant data to inform conservation of this rare and endangered plant.

Fungal diversity and community structure from coastal and barrier island beaches in the United States Gulf of Mexico.

Fungi are an important and understudied component of coastal biomes including sand beaches. Basic biogeographic diversity data are lacking for marine fungi in most parts of the world, despite their important role in decomposition. We examined intertidal fungal communities at several United States (US) Gulf of Mexico sand beach sites using morphology and ITS rDNA terminal restriction fragment length polymorphism (T-RFLP) analyses. Fungal biogeographical patterns from sand beach detritus (wood, emergent plant [mangrove/ saltmarsh], or marine [algae, seagrass]) from Florida, Mississippi, and Texas were investigated using diversity indices and multivariate analyses. Fungal diversity increased with decreasing latitude at our study sites. Substrate type strongly influenced fungal community structure in this region, with different fungal communities on detrital marine versus emergent substrates, as well as detrital marine versus wood substrates. Thirty-five fungi were identified morphologically, including new regional and host records. Of these, 86% were unique to an individual collection (i.e., sampled once from one site). Rarefaction curves from pooled morphological data from all sites estimate the number of samples required to characterize the mycota of each substrate. As sampling occurred before the Deepwater Horizon oil spill (April-2010), our findings contribute pre-oil spill sand beach biodiversity data and marine fungal distribution trends within this economically important oceanographic region.

The Canadian Fungal Research Network: current challenges and future opportunities.

Fungi critically impact the health and function of global ecosystems and economies. In Canada, fungal researchers often work within silos defined by subdiscipline and institutional type, complicating the collaborations necessary to understand the impacts fungi have on the environment, economy, and plant and animal health. Here, we announce the establishment of the Canadian Fungal Research Network (CanFunNet, https://fungalresearch.ca), whose mission is to strengthen and promote fungal research in Canada by facilitating dialogue among scientists. We summarize the challenges and opportunities for Canadian fungal research that were discussed at CanFunNet's inaugural meeting in 2019, and identify 4 priorities for our community: (i) increasing collaboration among scientists, (ii) studying diversity in the context of ecological disturbance, (iii) preserving culture collections in the absence of sustained funding, and (iv) leveraging diverse expertise to attract trainees. We have gathered additional information to support our recommendations, including a survey identifying underrepresentation of fungal-related courses at Canadian universities, a list of Canadian fungaria and culture collections, and a case study of a human fungal pathogen outbreak. We anticipate that these discussions will help prioritize fungal research in Canada, and we welcome all researchers to join this nationwide effort to enhance knowledge dissemination and funding advocacy.

Saltmarsh rhizosphere fungal communities vary by sediment type and dominant plant species cover in Nova Scotia, Canada.

We surveyed Spartina saltmarsh sediment rhizosphere fungal communities at three saltmarshes and two timepoints in coastal Nova Scotia. Based on ITS2 Illumina miSeq rDNA data and multivariate analysis, neither sediment zone nor collection period correlated with fungal ASV richness, but collection site did. However, Shannon diversity indicated that sediment zone played a significant role in fungal diversity. For unweighted and weighted UniFrac distance, site was the major factor driving beta-diversity, with sediment zone and collection period having smaller roles. Sediment type and saltmarsh plant species may play important roles in structuring rhizosphere fungal assemblages, here dominated by ascomycetes. To our knowledge, our study is the first to assess fungal sediment communities in saltmarshes in Atlantic Canada using metabarcoding. It provides a biodiversity analysis of sediment fungi in a poorly studied but highly important ecosystem and points to their roles in nutrient cycling, blue carbon, coastal stability and coastal restoration. Our work will inform ongoing saltmarsh restoration in Atlantic Canada.

Shed Light in the DaRk LineagES of the Fungal Tree of Life-STRES.

The polyphyletic group of black fungi within the Ascomycota (Arthoniomycetes, Dothideomycetes, and Eurotiomycetes) is ubiquitous in natural and anthropogenic habitats. Partly because of their dark, melanin-based pigmentation, black fungi are resistant to stresses including UV- and ionizing-radiation, heat and desiccation, toxic metals, and organic pollutants. Consequently, they are amongst the most stunning extremophiles and poly-extreme-tolerant organisms on Earth. Even though ca. 60 black fungal genomes have been sequenced to date, [mostly in the family Herpotrichiellaceae (Eurotiomycetes)], the class Dothideomycetes that hosts the largest majority of extremophiles has only been sparsely sampled. By sequencing up to 92 species that will become reference genomes, the "Shed light in The daRk lineagES of the fungal tree of life" (STRES) project will cover a broad collection of black fungal diversity spread throughout the Fungal Tree of Life. Interestingly, the STRES project will focus on mostly unsampled genera that display different ecologies and life-styles (e.g., ant- and lichen-associated fungi, rock-inhabiting fungi, etc.). With a resequencing strategy of 10- to 15-fold depth coverage of up to ~550 strains, numerous new reference genomes will be established. To identify metabolites and functional processes, these new genomic resources will be enriched with metabolomics analyses coupled with transcriptomics experiments on selected species under various stress conditions (salinity, dryness, UV radiation, oligotrophy). The data acquired will serve as a reference and foundation for establishing an encyclopedic database for fungal metagenomics as well as the biology, evolution, and ecology of the fungi in extreme environments.

Spatial distribution of mercury and other potentially toxic elements using epiphytic lichens in Nova Scotia.

The use of naturally occurring epiphytic lichens can be an effective tool for regional monitoring of mercury (Hg) and other potentially toxic elements (PTEs). Nova Scotia, Canada is a hotspot for mercury and other trace metal accumulation in ecosystems; partially attributed to long-range transport of air pollution. The relative contribution of local and international sources of Hg to local air in Nova Scotia is unknown. This study assessed the potential of epiphytic lichens (Usnea spp.) as passive samplers for PTE air pollution in Nova Scotia. Lichens (n = 190) collected across mainland Nova Scotia were analyzed for PTEs. Results indicate that there are 3 distinct clusters of PTEs which suggest patterns and sources for each elemental cluster. Hg was correlated with longitude and prevailing wind direction, and Hg was not significantly different in site-specific hotspot sampling nor year of sampling. Our data support the hypothesis that Hg in lichens is from historical and ongoing long-range transport and diffuse emission patterns rather than localized pollution sources. PTE concentrations were shown to have median values that are similar to other remote regions (such as the Antarctic) however the maximum values were observed to be substantially higher for some elements (e.g. lead, cadmium). This research supports the use of lichens as biomonitors and provides a baseline for future monitoring efforts to identify changes in PTE distribution in Nova Scotia with ongoing industrial activity and a changing climate.

Fungi in the Marine Environment: Open Questions and Unsolved Problems.

Terrestrial fungi play critical roles in nutrient cycling and food webs and can shape macroorganism communities as parasites and mutualists. Although estimates for the number of fungal species on the planet range from 1.5 to over 5 million, likely fewer than 10% of fungi have been identified so far. To date, a relatively small percentage of described species are associated with marine environments, with ∼1,100 species retrieved exclusively from the marine environment. Nevertheless, fungi have been found in nearly every marine habitat explored, from the surface of the ocean to kilometers below ocean sediments. Fungi are hypothesized to contribute to phytoplankton population cycles and the biological carbon pump and are active in the chemistry of marine sediments. Many fungi have been identified as commensals or pathogens of marine animals (e.g., corals and sponges), plants, and algae. Despite their varied roles, remarkably little is known about the diversity of this major branch of eukaryotic life in marine ecosystems or their ecological functions. This perspective emerges from a Marine Fungi Workshop held in May 2018 at the Marine Biological Laboratory in Woods Hole, MA. We present the state of knowledge as well as the multitude of open questions regarding the diversity and function of fungi in the marine biosphere and geochemical cycles.

The Neglected Marine Fungi, Sensu stricto, and Their Isolation for Natural Products' Discovery.

Despite the rapid development of molecular techniques relevant for natural product research, culture isolates remain the primary source from which natural products chemists discover and obtain new molecules from microbial sources. Techniques for obtaining and identifying microbial isolates (such as filamentous fungi) are thus of crucial importance for a successful natural products' discovery program. This review is presented as a "best-practices guide" to the collection and isolation of marine fungi for natural products research. Many of these practices are proven techniques used by mycologists for the isolation of a broad diversity of fungi, while others, such as the construction of marine baiting stations and the collection and processing of sea foam using dilution to extinction plating techniques, are methodological adaptations for specialized use in marine/aquatic environments. To this day, marine fungi, Sensu stricto, remain one of the few underexplored resources of natural products. Cultivability is one of the main limitations hindering the discovery of natural products from marine fungi. Through encouraged collaboration with marine mycologists and the sharing of historically proven mycological practices for the isolation of marine fungi, our goal is to provide natural products chemists with the necessary tools to explore this resource in-depth and discover new and potentially novel natural products.

The homothallic mating-type locus of the conifer needle endophyte Phialocephala scopiformis DAOMC 229536 (order Helotiales).

We describe the complete mating-type (MAT) locus for Phialocephala scopiformis Canadian Collection of Fungal Cultures (DAOMC) 229536 - a basal lineage within Vibrisseaceae. This strain is of interest due to its ability to produce the important antiinsectan rugulosin. We also provide some of the first insights into the genome structure and gene inventory of nonclavicipitalean endophytes. Sequence was obtained through shotgun sequencing of the entire P. scopiformis genome, and the MAT locus was then determined by comparing this genomic sequence to known MAT loci within the Phialocephala fortinii s.l.-Acephala applanata species complex. We also tested the relative levels of sequence conservation for MAT genes within Vibrisseaceae (n = 10), as well as within the Helotiales (n = 27). Our results: (1) show a homothallic gene arrangement for P. scopiformis [MAT1-1-1, MAT1-2-1, and MAT1-1-3 genes are present], (2) increase the genomic survey of homothallism within Vibrisseaceae, (3) confirm that P. scopiformis contains a unique S-adenosyl-l-methionine-dependent methyltransferase (SAM-Mtase) gene proximal to its MAT locus, while also lacking a cytoskeleton assembly control protein (sla2) gene, and (4) indicate that MAT1-1-1 is the more useful molecular marker amongst the MAT genes for phylogenetic reconstructions aimed at tracking evolutionary shifts in reproductive strategy and/or MAT loci gene composition within the Helotiales.

The complete mitochondrial genome of the conifer needle endophyte, Phialocephala scopiformis DAOMC 229536 confirms evolutionary division within the fungal Phialocephala fortinii s.l. - Acephala appalanata species complex.

Despite the recent surge in mitochondrial (mt) genome sequencing, Kingdom Fungi remains underrepresented with respect to mtDNA. We describe the mt genome of the conifer needle endophyte, Phialocephala scopiformis DAOMC 229536 (Helotiales, Ascomycota). This strain is of interest to the Canadian forestry industry as it produces the anti-insectan compound rugulosin. Sequence was obtained from whole genome shotgun sequencing. Comparison to the only other published Phialocephala mt genome, Phialocephala subalpina, indicates that the suite of common mt genes - cox1-3, cob, nad1-6 and 4L, atp6, 8 and 9, as well as rrnL and rrnS - has retained an identical order. Nad4L remains one of the most conserved mitochondrial genes within Phialocephala. Members of the closely related Phialocephala fortinii s.l. - Acephala appalanata species complex (PAC) share too much sequence similarity to properly resolve lineages using ITS barcoding alone. Using P. scopiformis sequence as an outgroup, we determined ancestral gene states that help confirm clades within Phialocephala. Our results show: (1) the complete mt genome of P. scopiformis, representing the 10th complete mt genome for the order Helotiales (containing >3800 species), and (2) how large-scale genomic patterns, such as mitochondrial gene order, can be used to confirm lineages within fungal species complexes.

Full Genome of Phialocephala scopiformis DAOMC 229536, a Fungal Endophyte of Spruce Producing the Potent Anti-Insectan Compound Rugulosin.

We present the full genome of Phialocephala scopiformis DAOMC 229536 (Helotiales, Ascomycota), a foliar endophyte of white spruce from eastern Quebec. DAOMC 229536 produces the anti-insectan compound rugulosin, which inhibits a devastating forestry pest, the spruce budworm. This genome will enable fungal genotyping and host-endophyte evolutionary genomics in inoculated trees.

Antimicrobial dihydrobenzofurans and xanthenes from a foliar endophyte of Pinus strobus.

Foliar fungal endophytes of Pinus strobus (eastern white pine) were collected from different sites across south-eastern New Brunswick, Canada and screened for the production of bioactive metabolites. From one site, two fungal isolates representing a formerly unknown genus and species within the family Massarinaceae (Pleosporales, Dothideomycetes, Ascomycota) were resolved by phylogenetic analysis. These isolates produced crude organic extracts that were active against Microbotryum violaceum and Saccharomyces cerevisiae. From these strains, DAOM 242779 and 242780, four dihydrobenzofurans (1-4) and two xanthenes (5-6) were characterized. Structures were elucidated by HRMS, interpretation of NMR spectra and other spectroscopic techniques. All isolated metabolites displayed antimicrobial activity against the biotrophic fungal pathogen M. violaceum and Bacillus subtilis.

Marine fungal diversity: a comparison of natural and created salt marshes of the north-central Gulf of Mexico.

Marine fungal communities of created salt marshes of differing ages were compared with those of two reference natural salt marshes. Marine fungi occurring on the lower 30 cm of salt marsh plants Spartina alterniflora and Juncus roemerianus were inventoried with morphological and molecular methods (ITS T-RFLP analysis) to determine fungal species richness, relative frequency of occurrence and ascomata density. The resulting profiles revealed similar fungal communities in natural salt marshes and created salt marshes 3 y old and older with a 1.5 y old created marsh showing less fungal colonization. A 26 y old created salt marsh consistently exhibited the highest fungal species richness. Ascomata density of the dominant fungal species on each host was significantly higher in natural marshes than in created marshes at all three sampling dates. This study indicates marine fungal saprotroph communities are present in these manmade coastal salt marshes as early as 1 y after marsh creation. The lower regions of both plant hosts were dominated by a small number of marine ascomycete species consistent with those species previously reported from salt marshes of the East Coast of USA.