Fungal behaviour: a new frontier in behavioural ecology
Authors: Kristin Aleklett, Lynne Boddy
Journal: Trends in Ecology & Evolution (2021)

While there is increasing acceptance that non-neural organisms such as plants, slime moulds, and bacteria can perform behaviours, the vast kingdom of fungi is usually forgotten.We argue that fungi can also be studied through the theoretical framework of behavioural ecology. This would benefit both fungal biologists – yielding a better understanding of the lives of fungi – and behavioural ecologists, providing access to model organisms that can help to explain the evolution of primary senses and potentially discover behaviours new to science.Fungi have senses analogous to those of other organisms, they exhibit behaviour, and they have memory. This suggests a multitude of questions and new paths that could be taken to broaden our understanding of this forgotten and underestimated branch in the tree of life. As human beings, behaviours make up our everyday lives. What we do from the moment we wake up to the moment we go back to sleep at night can all be classified and studied through the concepts of behavioural ecology. The same applies to all vertebrates and, to some extent, invertebrates. Fungi are, in most people's eyes perhaps, the eukaryotic multicellular organisms with which we humans share the least commonalities. However, they still express behaviours, and we argue that we could obtain a better understanding of their lives – although they are very different from ours – through the lens of behavioural ecology. Moreover, insights from fungal behaviour may drive a better understanding of behavioural ecology in general. As human beings, behaviours make up our everyday lives. What we do from the moment we wake up to the moment we go back to sleep at night can all be classified and studied through the concepts of behavioural ecology. The same applies to all vertebrates and, to some extent, invertebrates. Fungi are, in most people's eyes perhaps, the eukaryotic multicellular organisms with which we humans share the least commonalities. However, they still express behaviours, and we argue that we could obtain a better understanding of their lives – although they are very different from ours – through the lens of behavioural ecology. Moreover, insights from fungal behaviour may drive a better understanding of behavioural ecology in general. All organisms, be they prokaryotic or eukaryotic, macroorganisms or microorganisms, have to solve a similar set of basic problems to survive: how to obtain energy and nutrients, avoid being eaten or killed, and spread their offspring and how to partition resources between these activities [1.Dusenbery David Life at small scale – the behavior of microbes. Scientific American Library, 1996Google Scholar]. To address these problems, they have all evolved different sets of solutions and behaviours (Figure 1 and Table 1).Table 1Comparison of key features and abilities important for the behaviour of major fungal phyla, and comparable features of vertebratesaThe classification of fungi is still in considerable flux as genome sequencing is increasing. The number of phyla currently ranges between five and 12 depending on the author. Here we consider five from the JGI Mycocosm Fungal Genomics Resource (https://mycocosm.jgi.doe.gov/mycocosm/home). Numbers are approximate and details often uncertain. Only vertebrates are used for comparison as behavioural ecology has largely been developed based on them.ChytridiomycotaZoopagomycota and MucoromycotaAscomycotaBasidiomycotaVertebratesProportion of species in Kingdom Fungi (approximate)<10<154525NAMain habitatAquatic/moisture filmsSoil or on plants and animalsAllTerrestrialTerrestrial/aquaticSpread of sporesSwimmingPassivePassivePassiveNAReproductionAsexual/sexualAsexual/sexualAsexualbSome are predominantly asexual./sexualAsexual/sexualSexualGrowth pattern: determinate, d; indeterminate, icBody form is hyphal, but some form yeasts.d/iiiidSpecialised hyphaedMorphology of some hyphae are specialised for specific function.nyyyNAYeast body formna fewyeSubphylum Saccharomycotina contains about 1000 species, which are predominantly yeasts.a fewNAMorphological switchingfMorphological switching includes the ability to change from hyphal to yeast form and vice versa or, in some, the production of different hyphal/mycelial forms with associated differing physiological activities.nyynCross walls in hyphaenngCross-walls are produced to delimit reproductive structures and to block hyphae following damage.yyNANetwork formationnn/yhSome, for example, Mortierella, have anastomoses.yyinternalComplex multicellulariSimple multicellular: all cells are in direct contact with the environment, while they are not in complex multicellular [62]., c; simple multicellular, sA few sss/cccMaximum size<1 cm<1 m?Usually <1 m? Occasionally several metresA few: many ha; >150 000 kg150 000 kgMaximum age1 week?<1 year<50 yearsA few >1000 years200 yearsMovement of water/nutrients over long distances within the bodynnjNutrient movement does occur within hyphae, but mycelia are relatively small.yyyAbility to use extremely complex molecules (e.g., lignin)nnA fewSomenTropic/taxic responsesyyyyya The classification of fungi is still in considerable flux as genome sequencing is increasing. The number of phyla currently ranges between five and 12 depending on the author. Here we consider five from the JGI Mycocosm Fungal Genomics Resource (https://mycocosm.jgi.doe.gov/mycocosm/home). Numbers are approximate and details often uncertain. Only vertebrates are used for comparison as behavioural ecology has largely been developed based on them.b Some are predominantly asexual.c Body form is hyphal, but some form yeasts.d Morphology of some hyphae are specialised for specific function.e Subphylum Saccharomycotina contains about 1000 species, which are predominantly yeasts.f Morphological switching includes the ability to change from hyphal to yeast form and vice versa or, in some, the production of different hyphal/mycelial forms with associated differing physiological activities.g Cross-walls are produced to delimit reproductive structures and to block hyphae following damage.h Some, for example, Mortierella, have anastomoses.i Simple multicellular: all cells are in direct contact with the environment, while they are not in complex multicellular [62.Nagy L.G. et al.Fungi took a unique evolutionary route to multicellularity: seven key challenges for fungal multicellular life.Fungal Biol. Rev. 2020; 34: 151-169Crossref Scopus (3) Google Scholar].j Nutrient movement does occur within hyphae, but mycelia are relatively small. Open table in a new tab Fungi constitute a vast kingdom of 2–6 million or more species [2.Hawksworth D.L. Lücking R. Fungal diversity revisited: 2.2 to 3.8 million species.in: The fungal kingdom. ASM Press, 2017: 79-95Crossref Scopus (92) Google Scholar,3.Baldrian P. et al.High-throughput sequencing view on the magnitude of global fungal diversity.Fungal Divers. 2021; (Published online February 19, 2021. https://doi.org/10.1007/s13225-021-00472-y)Crossref Scopus (18) Google Scholar] (Box 1 and Table 1), but despite our rapidly increasing understanding of fungal genetics, biochemistry, cell biology and physiology, there are a surprisingly large number of gaps in our basic understanding of their lives and behaviours. We believe that fungal ecology would greatly benefit from being studied under the framework of behavioural ecology and that behavioural ecology, in turn, will benefit from the challenges of including fungi.Box 1Fungal characteristics relevant to behaviourFungi are one of the five main multicellular lineages in the tree of life. Like animals and plants, they possess the key traits of complex multicellularity: cell–cell communication, cell–cell adhesion, long-range transport, programmed cell death, and a developmental program [40.Watkinson S.C. et al.The fungi.3rd edn. Academic Press, 2015Google Scholar]. However, they have fewer cell types (>12 compared with >100 in animals and ~30 in plants) with many different evolutionary origins [62.Nagy L.G. et al.Fungi took a unique evolutionary route to multicellularity: seven key challenges for fungal multicellular life.Fungal Biol. Rev. 2020; 34: 151-169Crossref Scopus (3) Google Scholar]. Although some fungi have unicellular body forms (e.g., yeasts), most are multicellular and mycelial, comprising fine filaments [hyphae (Figure I)]. Hyphae grow by apical extension to explore the environment (Figure I), feeding by extracellular digestion (secreting enzymes to break down large molecules to smaller ones that can be absorbed). Tip growth, including direction, is controlled by a multicomponent organizing centre – the Spitzenkörper – near the tip [40.Watkinson S.C. et al.The fungi.3rd edn. Academic Press, 2015Google Scholar]. Hyphae branch to form a fractal, tree-like system [63.Boddy L. Donnelly D.P. Fractal geometry and microorganisms in the environment.in: Biophysical chemistry of fractal structures and processes in environmental systems. John Wiley & Sons, 2008: 239-272Crossref Scopus (19) Google Scholar], and lateral branches often join adjacent hyphae resulting in indeterminate, adaptive networks [7.Boddy L. Saprotrophic cord-forming fungi: meeting the challenge of heterogeneous environments.Mycologia. 1999; 91: 13Crossref Scopus (208) Google Scholar,12.Fricker M.D. et al.The mycelium as a network.in: The fungal kingdom. American Society of Microbiology, 2017: 335-367Crossref Scopus (8) Google Scholar,64.Read N.D. et al.Hyphal fusion.in: Borkovich K.A. Ebbole D.J. Cellular and molecular biology of filamentous fungi. ASM Press, 2010: 260-273Crossref Google Scholar] (Figure I).In Dikarya (ascomycetes and basidiomycetes), hyphae are divided into compartments/cells by transverse partitions (septa), which extend from the wall inwards, leaving a small, central opening allowing cytoplasmic continuity and the passage of some organelles. Compartments commonly have two or more nuclei but the extent of co-ordination/competition is unclear [65.Mela A.P. et al.Syncytia in fungi.Cells. 2020; 9: 2255Crossref Scopus (7) Google Scholar]. Septal openings can be rapidly blocked off. This prevents loss of cytoplasm if a hypha is damaged and allows differentiation of morphology and activity, heterogeneous gene expression [65.Mela A.P. et al.Syncytia in fungi.Cells. 2020; 9: 2255Crossref Scopus (7) Google Scholar], the formation of large tissue-like structures, and dramatic behavioural responses.Fungi are heterotrophs that obtain their food from dead organic matter, by killing other organisms/tissues/cells, from living cells either as parasites or mutualists (lichen and mycorrhizal fungi), or by a combination of these methods. Fungi live in environments where food sources and the microclimatic environments are spatially heterogeneous and everchanging temporally [7.Boddy L. Saprotrophic cord-forming fungi: meeting the challenge of heterogeneous environments.Mycologia. 1999; 91: 13Crossref Scopus (208) Google Scholar]. They spread to new resources in space and time by spores, which are commonly single cells. Some (e.g., cord- and rhizomorph-forming basidiomycetes), can grow through soil in search of new resources, operating foraging strategies [21.Boddy L. Jones T.H. Mycelial responses in heterogeneous environments: parallels with macroorganisms.in: Gadd G. Fungi in the environment. Cambridge University Press, 2007: 112-140Crossref Scopus (21) Google Scholar], and some of these form the most extensive and persistent biological networks characterised to date [7.Boddy L. Saprotrophic cord-forming fungi: meeting the challenge of heterogeneous environments.Mycologia. 1999; 91: 13Crossref Scopus (208) Google Scholar,11.Heaton L. et al.Analysis of fungal networks.Fungal Biol. Rev. 2012; 26: 12-29Crossref Scopus (72) Google Scholar,20.Fricker M.D. et al.Mycelial networks: structure and dynamics.in: Boddy L. Ecology of saprotrophic basidiomycetes. Academic Press, 2008: 3-18Crossref Scopus (38) Google Scholar]. Fungi are one of the five main multicellular lineages in the tree of life. Like animals and plants, they possess the key traits of complex multicellularity: cell–cell communication, cell–cell adhesion, long-range transport, programmed cell death, and a developmental program [40.Watkinson S.C. et al.The fungi.3rd edn. Academic Press, 2015Google Scholar]. However, they have fewer cell types (>12 compared with >100 in animals and ~30 in plants) with many different evolutionary origins [62.Nagy L.G. et al.Fungi took a unique evolutionary route to multicellularity: seven key challenges for fungal multicellular life.Fungal Biol. Rev. 2020; 34: 151-169Crossref Scopus (3) Google Scholar]. Although some fungi have unicellular body forms (e.g., yeasts), most are multicellular and mycelial, comprising fine filaments [hyphae (Figure I)]. Hyphae grow by apical extension to explore the environment (Figure I), feeding by extracellular digestion (secreting enzymes to break down large molecules to smaller ones that can be absorbed). Tip growth, including direction, is controlled by a multicomponent organizing centre – the Spitzenkörper – near the tip [40.Watkinson S.C. et al.The fungi.3rd edn. Academic Press, 2015Google Scholar]. Hyphae branch to form a fractal, tree-like system [63.Boddy L. Donnelly D.P. Fractal geometry and microorganisms in the environment.in: Biophysical chemistry of fractal structures and processes in environmental systems. John Wiley & Sons, 2008: 239-272Crossref Scopus (19) Google Scholar], and lateral branches often join adjacent hyphae resulting in indeterminate, adaptive networks [7.Boddy L. Saprotrophic cord-forming fungi: meeting the challenge of heterogeneous environments.Mycologia. 1999; 91: 13Crossref Scopus (208) Google Scholar,12.Fricker M.D. et al.The mycelium as a network.in: The fungal kingdom. American Society of Microbiology, 2017: 335-367Crossref Scopus (8) Google Scholar,64.Read N.D. et al.Hyphal fusion.in: Borkovich K.A. Ebbole D.J. Cellular and molecular biology of filamentous fungi. ASM Press, 2010: 260-273Crossref Google Scholar] (Figure I). In Dikarya (ascomycetes and basidiomycetes), hyphae are divided into compartments/cells by transverse partitions (septa), which extend from the wall inwards, leaving a small, central opening allowing cytoplasmic continuity and the passage of some organelles. Compartments commonly have two or more nuclei but the extent of co-ordination/competition is unclear [65.Mela A.P. et al.Syncytia in fungi.Cells. 2020; 9: 2255Crossref Scopus (7) Google Scholar]. Septal openings can be rapidly blocked off. This prevents loss of cytoplasm if a hypha is damaged and allows differentiation of morphology and activity, heterogeneous gene expression [65.Mela A.P. et al.Syncytia in fungi.Cells. 2020; 9: 2255Crossref Scopus (7) Google Scholar], the formation of large tissue-like structures, and dramatic behavioural responses. Fungi are heterotrophs that obtain their food from dead organic matter, by killing other organisms/tissues/cells, from living cells either as parasites or mutualists (lichen and mycorrhizal fungi), or by a combination of these methods. Fungi live in environments where food sources and the microclimatic environments are spatially heterogeneous and everchanging temporally [7.Boddy L. Saprotrophic cord-forming fungi: meeting the challenge of heterogeneous environments.Mycologia. 1999; 91: 13Crossref Scopus (208) Google Scholar]. They spread to new resources in space and time by spores, which are commonly single cells. Some (e.g., cord- and rhizomorph-forming basidiomycetes), can grow through soil in search of new resources, operating foraging strategies [21.Boddy L. Jones T.H. Mycelial responses in heterogeneous environments: parallels with macroorganisms.in: Gadd G. Fungi in the environment. Cambridge University Press, 2007: 112-140Crossref Scopus (21) Google Scholar], and some of these form the most extensive and persistent biological networks characterised to date [7.Boddy L. Saprotrophic cord-forming fungi: meeting the challenge of heterogeneous environments.Mycologia. 1999; 91: 13Crossref Scopus (208) Google Scholar,11.Heaton L. et al.Analysis of fungal networks.Fungal Biol. Rev. 2012; 26: 12-29Crossref Scopus (72) Google Scholar,20.Fricker M.D. et al.Mycelial networks: structure and dynamics.in: Boddy L. Ecology of saprotrophic basidiomycetes. Academic Press, 2008: 3-18Crossref Scopus (38) Google Scholar]. Behaviour is not well defined in the literature, but broadly covers an organism's movements, interactions, cognition (see Glossary), and learning. Tinbergen introduced four classic ways of asking why an animal performs a certain behavioural act. How does the behaviour improve survival or reproduction? How has the behaviour changed over time? What factors lead to the behaviour seen in a specific instance? How does the behaviour in an individual change as it matures and which internal and external factors affect this? [4.Tinbergen N. On aims and methods of ethology.Z. Tierpsychol. 1963; 20: 410-433Crossref Scopus (2279) Google Scholar]. These questions are equally appropriate for fungi and through them we could gain a better understanding of the context in which fungi explore and forage for nutrients, interact with other organisms, and respond to their abiotic environment. There are several reasons why fungal behaviour is less well understood than the behaviour of animals. Fungal hyphae are microscopic and usually live in opaque environments, such as the soil matrix or plant and animal tissues, making it difficult to observe fungal behaviour in real time. Movement is often considered an important aspect of behaviour, and fungi are frequently considered sessile [5.Andrews J.H. Fungi and the evolution of growth form.Can. J. Bot. 1995; 73: 1206-1212Crossref Google Scholar]. However, movement can be defined as 'any translocation of biomass sustained by an organism's own energy resources, which is steered (navigated) in response to environmental cues and stimuli' [6.Bielčik M. et al.The role of active movement in fungal ecology and community assembly.Mov. Ecol. 2019; 7: 36Crossref PubMed Scopus (8) Google Scholar]; thus, as mycelia are actually very dynamic and responsive, changing locations by growth and the reallocation of mycelial biomass [7.Boddy L. Saprotrophic cord-forming fungi: meeting the challenge of heterogeneous environments.Mycologia. 1999; 91: 13Crossref Scopus (208) Google Scholar,8.Boddy L. et al.Fungal network responses to grazing.Fungal Genet. Biol. 2010; 47: 522-530Crossref PubMed Scopus (23) Google Scholar] (see Figure II in Box 2), they clearly exhibit movement. A further hindrance is that fungal ecology is understudied and the pivotal roles of fungi in ecosystems, as the main decomposers and recyclers of dead organic matter and as mutualistic mycorrhizas, are largely overlooked [9.Boddy L. Fungi: the unsung heroes of the planet.PAN Philos. Act. Nat. 2013; 10: 112-118Google Scholar]. However, despite this, major insights into mycelial behaviour (changes in its growth patterns, network architecture, spatial relationships, and function) and decision making have been gained, largely through soil microcosm studies of cord-forming fungi [7.Boddy L. Saprotrophic cord-forming fungi: meeting the challenge of heterogeneous environments.Mycologia. 1999; 91: 13Crossref Scopus (208) Google Scholar,8.Boddy L. et al.Fungal network responses to grazing.Fungal Genet. Biol. 2010; 47: 522-530Crossref PubMed Scopus (23) Google Scholar,10.Fukasawa Y. et al.Ecological memory and relocation decisions in fungal mycelial networks: responses to quantity and location of new resources.ISME J. 2020; 14: 380-388Crossref PubMed Scopus (11) Google Scholar, 11.Heaton L. et al.Analysis of fungal networks.Fungal Biol. Rev. 2012; 26: 12-29Crossref Scopus (72) Google Scholar, 12.Fricker M.D. et al.The mycelium as a network.in: The fungal kingdom. American Society of Microbiology, 2017: 335-367Crossref Scopus (8) Google Scholar], and we now have the tools to study fungal behaviour over a range of spatial scales (Box 2).Box 2Methods and examples of fungal behaviour at varying scalesThe behaviour of large mycelia operates, and must therefore be studied, at a range of scales from micrometres to many metres. Experimental setups can range from microfluidic chips at the hyphal scale, through laboratory microcosms of soil trays, to field systems. We present three case studies of directional memory at various scales (Figure II).Microfluidic chipsMicrofluidic chips, fabricated through a combination of computer design, soft lithography, and plasma bonding, contain microstructured environments of enclosed channels and chambers in a transparent and breathable material designed by the researcher (Figure IIA) [66.Aleklett K. et al.Build your own soil: exploring microfluidics to create microbial habitat structures.ISME J. 2018; 12: 312-319Crossref PubMed Scopus (64) Google Scholar]. These new techniques allow us to mimic the microscale structures of fungal environments (soil, plants, cells, etc.) [66.Aleklett K. et al.Build your own soil: exploring microfluidics to create microbial habitat structures.ISME J. 2018; 12: 312-319Crossref PubMed Scopus (64) Google Scholar] and to monitor fungal growth, behaviour, and decision making at the scale of individual hyphae, in real time with microscopic precision [32.Aleklett K. et al.Fungal foraging behaviour and hyphal space exploration in micro-structured Soil Chips.ISME J. 2021; 15: 1782-1793https://doi.org/10.1038/s41396-020-00886-7Crossref PubMed Scopus (12) Google Scholar,33.Held M. et al.Intracellular mechanisms of fungal space searching in microenvironments.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 13543-13552Crossref PubMed Scopus (20) Google Scholar]. For example, directional memory was shown in the hyphal tips of the basidiomycete Psilocybe cf. subviscida when they were growing through such labyrinths, but could sometimes be lost or confused when the hyphae were forced to navigate 'roundabouts' [32.Aleklett K. et al.Fungal foraging behaviour and hyphal space exploration in micro-structured Soil Chips.ISME J. 2021; 15: 1782-1793https://doi.org/10.1038/s41396-020-00886-7Crossref PubMed Scopus (12) Google Scholar] (Figure IIA).Soil microcosmsSoil microcosms, comprising compressed, non-sterile sieved soil, are more controlled than the field situation but provide some spatial heterogeneity and are appropriate for larger mycelia of, for example, cord-forming wood-decay fungi (Figure IIB). Studies using these systems have revealed behavioural responses involving the reallocation of mycelial biomass when new resources are encountered [7.Boddy L. Saprotrophic cord-forming fungi: meeting the challenge of heterogeneous environments.Mycologia. 1999; 91: 13Crossref Scopus (208) Google Scholar], 'decisions' on when to grow out from a resource in search of new ones [7.Boddy L. Saprotrophic cord-forming fungi: meeting the challenge of heterogeneous environments.Mycologia. 1999; 91: 13Crossref Scopus (208) Google Scholar], 'decisions' on when to abandon a resource in favour of a new one [10.Fukasawa Y. et al.Ecological memory and relocation decisions in fungal mycelial networks: responses to quantity and location of new resources.ISME J. 2020; 14: 380-388Crossref PubMed Scopus (11) Google Scholar], and 'memory' of the direction of a new resource relative to the original, when the original resource is severed from the network and placed on fresh soil [10.Fukasawa Y. et al.Ecological memory and relocation decisions in fungal mycelial networks: responses to quantity and location of new resources.ISME J. 2020; 14: 380-388Crossref PubMed Scopus (11) Google Scholar] (Figure IIB). This memory might simply be achieved by the development of more mycelium in one part of the wood block than elsewhere.Field systemsMycelial systems can be mapped and manipulated directly in the field by adding new resources or relocating large sections of the mycelium to study behaviour (Figure IIC). The fairy-ring-forming fungus Clitocybe nebularis provides an example of a field-scale study of directionality. It grows through the forest floor in an ever-increasing circle as a 30–40-cm-wide annulus of mycelium (Figure IIC). When turves containing the width of the annulus were cut and reoriented, the mycelium continued to grow only at approximately 90° to the annulus, not in any other direction [31.Dowson C.G. et al.Spatial dynamics and interactions of the woodland fairy ring fungus, Clitocybe nebularis.New Phytol. 1989; 111: 699-705Crossref PubMed Scopus (41) Google Scholar]. Mycelia of these fungi thus seem to be highly polar. The behaviour of large mycelia operates, and must therefore be studied, at a range of scales from micrometres to many metres. Experimental setups can range from microfluidic chips at the hyphal scale, through laboratory microcosms of soil trays, to field systems. We present three case studies of directional memory at various scales (Figure II). Microfluidic chips Microfluidic chips, fabricated through a combination of computer design, soft lithography, and plasma bonding, contain microstructured environments of enclosed channels and chambers in a transparent and breathable material designed by the researcher (Figure IIA) [66.Aleklett K. et al.Build your own soil: exploring microfluidics to create microbial habitat structures.ISME J. 2018; 12: 312-319Crossref PubMed Scopus (64) Google Scholar]. These new techniques allow us to mimic the microscale structures of fungal environments (soil, plants, cells, etc.) [66.Aleklett K. et al.Build your own soil: exploring microfluidics to create microbial habitat structures.ISME J. 2018; 12: 312-319Crossref PubMed Scopus (64) Google Scholar] and to monitor fungal growth, behaviour, and decision making at the scale of individual hyphae, in real time with microscopic precision [32.Aleklett K. et al.Fungal foraging behaviour and hyphal space exploration in micro-structured Soil Chips.ISME J. 2021; 15: 1782-1793https://doi.org/10.1038/s41396-020-00886-7Crossref PubMed Scopus (12) Google Scholar,33.Held M. et al.Intracellular mechanisms of fungal space searching in microenvironments.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 13543-13552Crossref PubMed Scopus (20) Google Scholar]. For example, directional memory was shown in the hyphal tips of the basidiomycete Psilocybe cf. subviscida when they were growing through such labyrinths, but could sometimes be lost or confused when the hyphae were forced to navigate 'roundabouts' [32.Aleklett K. et al.Fungal foraging behaviour and hyphal space exploration in micro-structured Soil Chips.ISME J. 2021; 15: 1782-1793https://doi.org/10.1038/s41396-020-00886-7Crossref PubMed Scopus (12) Google Scholar] (Figure IIA). Soil microcosms Soil microcosms, comprising compressed, non-sterile sieved soil, are more controlled than the field situation but provide some spatial heterogeneity and are appropriate for larger mycelia of, for example, cord-forming wood-decay fungi (Figure IIB). Studies using these systems have revealed behavioural responses involving the reallocation of mycelial biomass when new resources are encountered [7.Boddy L. Saprotrophic cord-forming fungi: meeting the challenge of heterogeneous environments.Mycologia. 1999; 91: 13Crossref Scopus (208) Google Scholar], 'decisions' on when to grow out from a resource in search of new ones [7.Boddy L. Saprotrophic cord-forming fungi: meeting the challenge of heterogeneous environments.Mycologia. 1999; 91: 13Crossref Scopus (208) Google Scholar], 'decisions' on when to abandon a resource in favour of a new one [10.Fukasawa Y. et al.Ecological memory and relocation decisions in fungal mycelial networks: responses to quantity and location of new resources.ISME J. 2020; 14: 380-388Crossref PubMed Scopus (11) Google Scholar], and 'memory' of the direction of a new resource relative to the original, when the original resource is severed from the network and placed on fresh soil [10.Fukasawa Y. et al.Ecological memory and relocation decisions in fungal mycelial networks: responses to quantity and location of new resources.ISME J. 2020; 14: 380-388Crossref PubMed Scopus (11) Google Scholar] (Figure IIB). This memory might simply be achieved by the development of more mycelium in one part of the wood block than elsewhere. Field systems Mycelial systems can be mapped and manipulated directly in the field by adding new resources or relocating large sections of the mycelium to study behaviour (Figure IIC). The fairy-ring-forming fungus Clitocybe nebularis provides an example of a field-scale study of directionality. It grows through the forest floor in an ever-increasing circle as a 30–40-cm-wide annulus of mycelium (Figure IIC). When turves containing the width of the annulus were cut and reoriented, the mycelium continued to grow only at approximately 90° to the annulus, not in any other direction [31.Dowson C.G. et al.Spatial dynamics and interactions of the woodland fairy ring fungus, Clitocybe nebularis.New Phytol. 1989; 111: 699-705Crossref PubMed Scopus (41) Google Scholar]. Mycelia of these fungi thus seem to be highly polar. One of the main obstacles to the discussion of fungal behaviour lies in the fact that fungi do not possess neurons or a brain in the classical sense. However, the concept of what constitutes a brain beyond the vertebrate paradigm is expanding [13.Pagán O.R. The brain: a concept in flux.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2019; 37420180383Crossref PubMed Scopus (8) Google Scholar,14.Solé R. et al.Liquid brains, solid brains.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2019; 37420190040Crossref PubMed Scopus (15) Google Scholar]. Solé et al.