LettersFine endophytes (Glomus tenue) are related to Mucoromycotina, not GlomeromycotaFine endophytes are arbuscule-producing fungi of unclear phylogenetic placement Fine endophytes (FE), Glomus tenue, are traditionally considered to be arbuscular mycorrhizal fungi (AMF) with distinctive microscopic morphology when stained. FE have fine hyphae (c. 1.5 lm diameter) which branch intra-cellularly in a distinctive fan-like pattern (Gianinazzi-Pearson et al., 1981;Abbott, 1982). The hyphae contain small swellings along their length, sometimes referred to as vesicle-like swellings (Hall, 1977). FE form arbuscules (or arbuscule-like structures) with fine elements in a tapered, conical shape (Greenall, 1963;Merryweather & Fitter, 1998). Spores of FE are very small (< 20 lm) compared to the majority of Glomeromycota, and colourless (Hall, 1977). Morphological variations indicate that FE may consist of multiple species (Thippayarugs et al., 1999), hence we use the term FE to indicate a species group.Within the kingdom Fungi, both morphological and genetic characteristics are used to determine taxonomic classification (St€ urmer, 2012). In 2001, all AMF were placed within the phylum Glomeromycota (Sch€ ußler et al., 2001). In the listing of glomeromycotan species by Sch€ ußler & Walker (2010), some members of the genus Glomus were not revised due to insufficient taxonomic knowledge, and this included FE. A key reason for classifying FE within the Glomeromycota was the presence of arbuscules, considered apomorphic for the phylum (Morton, 1990). However, the morphological features of root colonization by FE are distinct from other, coarse, AMF so their placement within the genus Glomus and the Glomeromycota was questioned (Hall, 1977;Sch€ ußler & Walker, 2010), and their status as mycorrhizal fungi is ambivalent.Accurate determination of FE usually requires magnification ≥ 9100, hence, where assessments of AMF colonization use lower magnifications they may not be identified. Furthermore, FE may be undetected if samples are not processed within 2 d of harvesting (Orchard et al., 2016a). Nevertheless, FE are globally distributed and prolific within many ecosystems, examples include: pastures and native bushland of New Zealand (Crush, 1973) and Australia (Abbott & Robson, 1982;McGee, 1989), Venezuelan cloud forests (Rabatin et al., 1993), riverine and alpine regions of Europe (Read & Haselwandter, 1981;Turnau et al., 1999;Binet et al., 2011) and an old-field in the United States (Hilbig & Allen, 2015). However, the difficulty of isolating and, hence, genetically characterizing FE has hindered the determination of their phylogenetic placement. A novel method to enrich colonization by fine endophytesTo clarify the identity of FE, we targeted the SSU (18S) ribosomal RNA gene using roots from two independent glasshouse experiments where individual pots contained multiple plants. For each pot we used one root system for DNA extraction and one root system to visually assess the percentage of total root length colonized (%TRL; see Sup...

A fungal colony is a syncytium composed of a branched and interconnected network of cells. Chimerism endows colonies with increased virulence and ability to exploit nutritionally complex substrates. Moreover, chimera formation may be a driver for diversification at the species level by allowing lateral gene transfer between strains that are too distantly related to hybridize sexually. However, the processes by which genomic diversity develops and is maintained within a single colony are little understood. In particular, both theory and experiments show that genetically diverse colonies may be unstable and spontaneously segregate into genetically homogenous sectors. By directly measuring patterns of nuclear movement in the model ascomycete fungus Neurospora crassa, we show that genetic diversity is maintained by complex mixing flows of nuclei at all length scales within the hyphal network. Mathematical modeling and experiments in a morphological mutant reveal some of the exquisite hydraulic engineering necessary to create the mixing flows. In addition to illuminating multinucleate and multigenomic lifestyles, the adaptation of a hyphal network for mixing nuclear material provides a previously unexamined organizing principle for understanding morphological diversity in the more-thana-million species of filamentous fungi.heterokaryon | hydrodynamics | biological networks G enetic diversity between individuals is important to the resilience of species (1) and ecosystems (2). However, physical and genetic barriers constrain internal genetic diversity within single organisms: Cell walls limit nuclear movement between cells, whereas separation of germ and somatic cell lines means that diversity created by somatic mutations is not transmitted intergenerationally. However, in syncytial organisms, including filamentous fungi and plasmodial slime molds (3), populations of genetically different and mobile nuclei may share a common cytoplasm ( Fig. 1A and Movie S1). Internal diversity may be acquired by accumulation of mutations as the organism grows or by somatic fusion followed by genetic transfer between individuals. For filamentous fungi, intraorganismic diversity is ubiquitous (4, 5). Shifting nuclear ratios to suit changing or heterogeneous environments enhances growth on complex substrates such as plant cell walls (6) and increases fungal virulence (7). Fusion between different fungal individuals is limited by somatic (heterokaryon) compatibility barriers (8), and most internal genetic diversity results from mutations within a single, initially homokaryotic individual (4). However, somatic compatibility barriers are not absolute (9), and exchange of nuclei between heterospecific individuals is now believed to be a motor for fungal diversification (10-12).A fungal chimera must maintain its genetic richness during growth. Maintenance of richness is challenging because fungal mycelia, which are made up of a network of filamentous cells (hyphae), grow by extension of hyphal tips. A continual tipward flow of vesicles and n...

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Hyphal fusion occurs at different stages in the vegetative and sexual life cycle of filamentous fungi. Similar to cell fusion in other organisms, the process of hyphal fusion requires cell recognition, adhesion, and membrane merger. Analysis of the hyphal fusion process in the model organism Neurospora crassa using fluorescence and live cell imaging as well as cell and molecular biological techniques has begun to reveal its complex cellular regulation. Several genes required for hyphal fusion have been identified in recent years. While some of these genes are conserved in other eukaryotic species, other genes encode fungal-specific proteins. Analysis of fusion mutants in N. crassa has revealed that genes previously identified as having nonfusion-related functions in other systems have novel hyphal fusion functions in N. crassa. Understanding the molecular basis of cell fusion in filamentous fungi provides a paradigm for cell communication and fusion in eukaryotic organisms. Furthermore, the physiological and developmental roles of hyphal fusion are not understood in these organisms; identifying these mechanisms will provide insight into environmental adaptation.

bThe evolution of multicellularity has occurred in diverse lineages and in multiple ways among eukaryotic species. For plants and fungi, multicellular forms are derived from ancestors that failed to separate following cell division, thus retaining cytoplasmic continuity between the daughter cells. In networked organisms, such as filamentous fungi, cytoplasmic continuity facilitates the long-distance transport of resources without the elaboration of a separate vascular system. Nutrient translocation in fungi is essential for nutrient cycling in ecosystems, mycorrhizal symbioses, virulence, and substrate utilization. It has been proposed that an interconnected mycelial network influences resource translocation, but the theory has not been empirically tested. Here we show, by using mutants that disrupt network formation in Neurospora crassa (⌬so mutant, no fusion; ⌬Prm-1 mutant, ϳ50% fusion), that the translocation of labeled nutrients is adversely affected in homogeneous environments and is even more severely impacted in heterogeneous environments. We also show that the ability to share resources and genetic exchange between colonies (via hyphal fusion) is very limited in mature colonies, in contrast to in young colonies and germlings that readily share nutrients and genetic resources. The differences in genetic/resource sharing between young and mature colonies were associated with variations in colony architecture (hyphal differentiation/diameters, branching patterns, and angles). Thus, the ability to share resources and genetic material between colonies is developmentally regulated and is a function of the age of a colony. This study highlights the necessity of hyphal fusion for efficient nutrient translocation within an N. crassa colony but also shows that established N. crassa colonies do not share resources in a significant manner.T he transition from unicellular to multicellular organisms has occurred on multiple occasions in diverse lineages over considerable evolutionary time (28,38,68). While an initial adaptive advantage may have accrued simply from being larger, multicellular organisms subsequently developed increased differentiation and specialization, leading to a more efficient division of labor (8). Multicellularity may have arisen by either the aggregation of individual cells to form a colony or by the failure of daughter cells to separate following division. Comparisons of unicellular animals and their multicellular relatives support the view that multicellularity is associated with expansion of the genetic families involved in cell adhesion, cell-cell signaling, and cell differentiation (63). In contrast, multicellular plants and fungi are derived from ancestors that failed to separate following cell division, providing an opportunity to retain cytoplasmic continuity between daughter cells (75). Thus, plant cells are linked by tissue-specific patterns of plasmodesmata (41, 47), while fungi are either coenocytic or have perforated septa that allow intercompartmental exchange (40).In ascomycete an...