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Ecological understanding of root-infecting fungi using trait-based
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approaches
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Carlos A. Aguilar-Trigueros1,2, Jeff R. Powell3, Ian C. Anderson3, Janis Antonovics4,
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Matthias C. Rillig1,2
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Germany
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Germany
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Freie Universität Berlin, Institut für Biologie, Plant Ecology, D-14195 Berlin,
Berlin-Brandenburg Institute of Advanced Biodiversity Research, D-14195 Berlin,
University of Western Sydney, Hawkesbury Institute for the Environment, Penrith
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NSW 2751, Australia
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Corresponding author: Rillig M.C. (rillig@zedat.fu-berlin.de)
University of Virginia, Department of Biology, Charlottesville, VA 22904, USA
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Word (main text): 2187
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Abstract: 115
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Additional elements: 1 Glossary box; 1 Text box (315 words); 1 Table; 1 Figure
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Classification schemes have been popular to tame the diversity of root infecting
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fungi. However, the usefulness of these schemes is limited to descriptive purposes.
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We propose that a shift to a multidimensional trait-based approach to disentangle
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the saprotrophic-symbiotic continuum will provide a better framework to
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understand fungal evolutionary ecology. Trait information reflecting the
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separation of root infecting fungi from free living soil relatives will help to
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understand the evolutionary process of symbiosis; the role that species interactions
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play in maintaining their large diversity in soil and in planta; and their
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contributions at the ecosystem level. Methodological advances in several areas such
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as microscopy, plant immunology and metatranscriptomics represent emerging
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opportunities to populate trait data bases.
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Limitations of categorical approaches to study plant–soil fungal interactions
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Understanding the effects of plant–soil fungal interactions in natural communities
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has become a major research area in plant science [1]. Interest in these interactions
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stem from increasing awareness that soil biota play an important role in plant
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performance, plant community assembly and ecosystem functioning [2].
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However the complexity of soil fungal communities challenges our ability to
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understand the effects of such interactions on plant performance and on ecosystems
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processes. Recent surveys show that roots interact with phylogenetically diverse
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groups of fungi [3]. Moreover, the effects of particular plant-fungal combinations
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depend on environmental conditions and on the host and fungal genotypes [4]. In
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diverse communities and variable environments, net responses may be due to
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complex indirect interactions among co-occurring fungi and plants [5].
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Given these complex associations, researchers regularly classify fungal taxa
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into broad categories according to particular criteria. These criteria may be based on
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nutritional mode (e.g., “biotroph”, “necrotroph” or “saprotroph” [6] (see Glossary)),
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presence of hyphal melanization and formation of septa (e.g."dark septate
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endophytes" [7]), or on a mix of taxonomic, morphological and physiological
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characteristics (e.g. “arbuscular mycorrhizal”; “ectomycorrhizal” [8]). These
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classificatory approaches have been important for distilling broad generalities from
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the rich brew of fungal–plant interactions such as the recognition of contrasting
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plant defense mechanisms against infecting fungi with different nutritional modes
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[9].
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However, assignment of root infecting fungi into fixed categories is
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problematic for species labeled as “endophytes”. For example, some of the criteria
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used in delineating endophyte classes [10] are quantitative (number of potential
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hosts; number of co-infections within a host or degree of tissue colonization) but
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their delineation is imprecise (narrow vs. broad host range; low vs. high in planta
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diversity; extensive vs. limited in planta colonization). Similarly, a suggestion that
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“endophytic functional groups” should be based on their effects on host fitness
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resulted in the rather unsatisfying conclusion that "some endophytes may be latent
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pathogens, some may be derived from pathogens, and others may be latent
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saprotrophs, but many are neither"[11]. Such classification schemes can provide a
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useful initial framework to understand poorly studied plant–fungal interactions, but
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the resulting generalizations often include the listing of so many exceptions to the
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proposed scheme that the framework is not useful operationally.
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We argue that a shift in focus from classification schemes to a
multidimensional trait-based approach reflecting the biology of the fungi is necessary
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for a better understanding of the ecology and evolution of root infecting fungi. These
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approaches consider species as a conglomerate of unique combinations of multiple
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traits, which could be depicted as species being points defined by multiple traits
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represented as dimensions (Fig 1a). This view directly link particular ecological and
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evolutionary questions with trait information. The proposed multidimensional trait-
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based program presented here is focused on the traits that allocate “endophytic” or
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“pathogenic” root fungi to a symbiotic lifestyle and separate them from free-living
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saprotrophic relatives. We explain how trait information can be used to address three
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essential questions: What are the mechanisms behind the evolution of root
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endophytic or pathogenic lifestyles from free-living fungi and vice versa? What is
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the importance of trait similarity in explaining the co-occurrence patterns of fungal
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genotypes or species in planta and in the soil? And, which traits might be used to
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understand the functional diversity of soil fungi? This is illustrated conceptually in
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Figure 1.
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A fungal-trait approach: understanding the saprotrophic-symbiotic continuum.
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Trait-based approaches rely on measurements of phenotypic characters or traits to
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guide inferences about particular ecological or evolutionary processes (Box 1). For
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example, plant scientists have successfully used such approaches to understand how
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fire has influenced the evolution within the Pinacea by combining trait information
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with phylogenetic reconstructions [12]; measure the relative importance of abiotic
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factors and biotic interactions in shaping community assembly of tropical trees by
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measuring trait overdispersion in local communities [13]; or understanding how plant
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diversity influences the variability of decomposition rates within climate regions by
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combining decomposition data with leaf traits from databases [14].
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We explain how application of such trait-based approaches may be valuable at a
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conceptual level in understanding the saprotrophic-symbiotic continuum in root
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infecting fungi. This continuum is pertinent to this set of fungi as most root
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endophytes constitute a gray area along the saprotrophic–symbiotic spectrum [10]
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and facultative saprotrophy is a characteristic of most studied root pathogens [15].
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Indeed, this issue has been debated among plant pathologists ever since the seminal
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work of Garrett [16]. More recently it has been a major focus of study among
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researchers on ectomycorrhizal fungi [17, 18].
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Understanding the saprotrophic-symbiotic continuum is of particular
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relevance to plant–fungal associations that have been labeled "endophytic" or "root
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pathogenic". Despite being considered major components of natural communities
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[10, 19], "endophytes" are not well defined in terms of their nutritional mode, there
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is little empirical understanding of their life history strategies, which in turns makes
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predicting their community and ecosystem impacts difficult.
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Specifically we aim at explaining how characterizing this continuum using a
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trait-based approach will make inroads on: first, explaining the evolutionary
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trajectories of symbiotic fungi from soil-inhabiting relatives (and vice versa).
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Second, understanding and predicting the functional role of fungi along this
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continuum in nutrient and carbon dynamics within ecosystems. Third, providing a
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mechanistic understanding of fungal community assembly and diversity maintenance
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both in soil and in planta. In the following section, we lay out the basis for such an
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approach, defining classes of traits relevant to this continuum, and suggest examples
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of relevant and measureable traits.
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Symbiotic and saprotrophic traits: a starting point
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In Table 1 we provide some examples of traits that we consider to be critical in
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assessing the ecology and evolution of “endophytic/pathogenic” root infecting fungi.
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As will be evident from the brief discussion of the rationale for each of them, these
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broad traits will themselves consist of many component traits that are operationally
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measurable. We expand on some of these points below.
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Symbiotic traits are often related to the ability of fungi to avoid or overcome
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resistance responses driven by the plant immune system as host resistance is the main
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filter preventing the use of the root niche [20]. Other traits may be related to the
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metabolic interactions that occur in such symbioses; for example, systems for
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translocation and bidirectional transport of nutrient and carbon resources [21].
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Identification of relevant component traits for “root endophytic” associations
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depends on understanding the molecular and physiological activities in fungal
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colonization structures [22]. Saprotrophic traits are related to the enzymatic
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machinery required to degrade the diversity of recalcitrant carbon sources [23],
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production of antimicrobial compounds during competition with other fungi [24] and
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with bacteria [25], and preventing or responding to fungivory by soil animals [26].
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The traits in this continuum may help us to understand the evolutionary
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process behind the transitions from saprotrophism to symbiosis and vice versa [27].
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This could be achieved using comparative phylogenetic approaches quantifying the
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variation in saprotrophic and symbiotic traits and identifying potential life history
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trade-offs, instead of using categories such as endophyte, saprotroph or pathogen as
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if they were characters [28]. It should also be possible to compare the evolutionary
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processes underlying the colonization and exploitation of roots vs. leaves by fungal
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endophytes or pathogens. The contrast between these two organs regarding their
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structure and their local environments [29] may help explain differences in
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colonization patterns between root and leaf “pathogens" or "endophytes” [4].
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Trait-based approaches could also be used to unravel compositional
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differences in fungal communities inhabiting roots, the rhizosphere, and bulk soils.
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As roots are living organs, fungal species with “symbiotic" traits should dominate in
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the root compartment while species with “saprotrophic” ones should be more
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abundant in the rhizosphere and bulk soil. Trait information, if mapped onto fungal
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communities found in various soil compartments could be used to understand the
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ecological basis and predictability of these assemblages. For instance, canonical
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examples in agricultural research have led to the common view that root necrotrophic
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pathogens, despite having saprotrophic abilities, are outcompeted by “strict” soil
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saprotrophs [30]. Trait information could be used to test whether such putative trade-
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offs apply to root “endophytes” or “pathogens” in natural systems.
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Trait information also allows the formulation of novel hypotheses. For
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example, does high trait diversity at the community level increase resistance to
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invasion by other fungi? Successful invasion depends on the number of open niches
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[31], and established fungal communities exhibiting high trait diversity may reduce
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resources (space, nutrients, organic matter) or limit access to resources (by priming
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host defenses or producing antibiotics). For fungi at the symbiotic end of the
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spectrum, is growth in the soil environment limited by the diversity of resident
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saprotrophic traits in the rhizosphere? Relationships between trait diversity and
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invasion resistance may help predict the likelihood of disease outbreaks by highly
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virulent root-infecting fungi in agricultural systems.
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Assessing trait similarity provides a better means to assess the effect of
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competition among co-infecting fungi in the evolution, maintenance and frequency
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of mutualistic or pathogenic interactions [32]. Another application might be in
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coupling trait information with the comparisons of root infecting fungal communities
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among invasive plants in their native and introduced ranges: such information may
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help explain the alleged reduction of pathogen attack of invasive plants in the
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introduced ranges if infecting fungi do not possess the traits to effectively exploit the
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new host [33].
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A trait based approach for the saprotrophic-symbiotic continuum may better
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capture the importance of root infecting fungi in carbon and nutrient cycling at the
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ecosystem level, in a manner completely analogous to the use of "functional trait
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analysis" in plants [34, 35]. Little is currently known about the effects of fungal trait
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diversity on ecosystem processes although the recent frameworks proposed for
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mycorrhizal associations [36, 37] suggest that other root-inhabiting fungi may be
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important contributors to ecosystem function. Trait-based approaches would allow
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the recognition of factors that correlate with the main ecosystem processes, to
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identify the taxa driving such effects, and lead to a better understanding of ecosystem
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drivers.
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Trait measurement and storage of trait information in high quality databases
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The full application of any trait-based approach depends on the accessibility of
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databases that are well curated, well funded, and linked to genomic and phylogenetic
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information. Phylogenetic databases have already been created for taxonomic
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purposes (e.g. UNITE [38]; http://www.deemy.de for ectomycorrhizal fungi). Thus,
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there is an excellent window of opportunity to integrate trait information generated
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from morphological and physiological characterization of newly isolated fungi with
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such platforms. Traits are measured on particular individuals, and meaningful
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extrapolations to species (or higher ranks) also depend on having estimates of trait
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variability among individuals and populations, as well as on the adequacy of the
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species concept for fungi [36].
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Additionally, to be meaningful, trait information should be presented together
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with data on the conditions and circumstances under which the traits were measured
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(“metadata”). Trait information collected from culturable fungi under controlled
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conditions is useful to quantify a particular trait (e.g. phytotoxin production).
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Analogously, plant ecologists have populated trait-databases with measurements
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from controlled experimental conditions with standardized methods [39]. This level
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of control enables the use of meta-analytical tools to filter out the effect of variable
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conditions to better address broad scale functional diversity-ecosystem functioning
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questions [14].
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Coupling this approach with in situ trait measurements under natural
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conditions (in mesocosms or the field) and with species abundance data will allow
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estimates of the contribution of a species in the context of environmental variability.
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Furthermore, in situ measurement of intraspecific trait variability could be used to
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refine models of community assembly [40]. However, caution must be taken when
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such trait information is intended to be used outside the particular system from which
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the traits were measured. It has been shown that traits with high levels of plasticity
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and measured from extreme habitats do not match well average values from
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databases [41].
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In situ measurements are the only option to incorporate trait information for
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non-culturable fungi and are analogous to measurements for long-lived plant species
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(e.g., wood traits for tree species in the TRY database [42]). Hyphal length in soil or
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nutrient concentration in the hyphae can be measured following extraction of hyphae
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directly from the environment or from substrate-filled compartments [43, 44].
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Enzyme activity assays performed on ectomycorrhizal root tips [45] or fungal carbon
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substrate usage by fungi [46] are usually measured on excised material, but they
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may not accurately reflect process rates due to the effects of the manipulations.
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Metatranscriptomic and other gene expression profiling approaches are promising
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and have the potential to lead to major insights at the community level [47, 48] and
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would be especially valuable if these could be targeted at fungal gene expression in
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particular. More sophisticated approaches at the individual level using "omics" tools
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(e.g., single cell genomics [49]; laser microdissection [50]) that can simultaneously
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examine fungal characters and identify the species being examined, hold exciting
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promise for future studies of soil fungal communities.
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Concluding remarks
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Rigorous comparative studies linked with phylogenetic information that is focused
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on traits, rather than qualitative categories, are necessary to determine what
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constitutes an adaptation to a particular life-style. In turn, traits can be used to make
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predictions about the impact and causes of community structure, and traits that
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strongly influence ecosystem function can be highlighted and measured for
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predicting outcomes. In this paper, as illustrated in Figure 1, we advocate a more
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objective trait-based approach to characterizing the processes involved in the
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interactions of root and soil inhabiting fungi, and hence provide a way to assess their
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importance at the evolutionary, community, and ecosystem levels. Others have
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strongly supported the use of trait-based approaches to move forward research on
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plant–fungal interactions [51, 52] and on microbial ecology [53]. In particular, there
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has been a recent call to explicitly use conceptual frameworks from functional
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ecology in ectomycorrhiza research, which promote integration of this important
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fungal group into research on diversity-ecosystem functioning relations .
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Although broad categories have their place in the initial development of a discipline,
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they provide a rather abstract view of multi component processes at such a level of
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simplicity that they may actually inhibit understanding. In studies of root-inhabiting
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fungi, there has been a tendency to use qualitative categories to describe the
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saprotrophic-symbiotic continuum, rather than search for generalizations that come
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from more quantitative studies. Here we advocate a trait-based approach to
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understand the evolutionary ecology of root-inhabiting fungi and illustrate how this
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approach promises a clear way forward in this complex and technically difficult
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field.
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Acknowledgements
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We thank the Alumni Network Program of Freie Universität Berlin for funding. CA
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was also supported by the German Academic Exchange Service (DAAD), ICA by a
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fellowship from the German Federal Ministry of Education and Research, and JA by
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an Alexander von Humboldt Research Award. We thank Sarah Hortal, Anna
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Simonin, Jen Walker, and anonymous reviewers for constructive comments.
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72 Agerer, R. (2001) Exploration types of ectomycorrhizae. Mycorrhiza 11, 107-114
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Figure legend:
411
Figure 1. From categorization to trait analysis for root-infecting fungi. Schematic
412
representation of a trait-based approach to understanding the ecology and evolution of
413
root infecting fungi. Instead of placing species into fixed categories such as pathogen
414
endophyte or saprotroph trait-based multivariate approaches represent species as
415
particular combinations of traits in various dimensions. Such information can be
416
coupled with: (A) phylogenic data () to understand evolutionary change; (B) their effect
417
on species performance under different ecological factors () to understand mechanisms
418
of community assembly and species co-existence and; (C) with their ecosystem function
419
to explore their role on ecosystem processes. Note: The graphic in (B) represent the
420
lower resource boundaries above which species 1 and 2 can still grow (zero net growth
421
isoclines as in [54]). In this figure, the position of lines depends on the traits the species
422
posses to exploit two resources: carbon from the host or from decaying matter.
423
424
Glossary box
425
Biotroph: nutritional mode in which a fungal symbiont relies exclusively on living host
426
cells as a source of nutrients.
427
Functional trait: Species traits directly linked with a particular ecosystem process.
428
Different species sharing similar functional traits are pooled into functional groups.
15
429
Life history trait: Traits reflecting allocation of resources of an individual into different
430
fitness components.
431
Necrotroph: nutritional mode in which a fungal symbiont causes host cell death in
432
order to acquire nutrients.
433
Saprotroph: nutritional mode in which a free-living fungus obtains nutrients from
434
decaying organic matter, without inducing the death of the tissue.
435
Symbiosis: a physiological or structurally intimate interaction between phylogenetically
436
unrelated organisms, without implying a specific effect on either organisms’ fitness.
437
Trait: any morphological, physiological, or phenological character of an organism.
438
439
Box 1. Rationales for trait-based approaches
440
The rationale for collecting data for particular traits may differ. Traits may be chosen
441
because they are likely to contribute to answer particular questions: a "top-down"
442
approach. Alternatively, traits may be chosen because they can be integrated with the
443
ever-increasing body of genomic and gene expression data allowing linkage of
444
particular genes with tangible phenotypes, or traits, of ecological importance. This
445
would be a "bottom-up" approach. Additionally, there is tremendous value in
446
including traits simply because they are "easy to measure": databases are as valuable
447
in generating hypotheses as they are in testing them, and unusual and unpredicted
448
trait associations are a stimulus for deeper investigation.
449
Traits can be used to test hypotheses of evolutionary processes in conjunction
450
with independently derived, usually sequence-based, phylogenetic information. Thus,
16
451
they provide objective ways to identify and test potential evolutionary trade-offs in
452
life history traits and relate them to environmental conditions using phylogenetic
453
comparative methods. For example, this approach has been used to evaluate
454
allocation of carbon by arbuscular mycorrhiza fungal species to hyphal structures in
455
roots and soil where percentage of root colonization and length of extra-radical
456
mycelia were used as traits [55].
457
Trait-based approaches can also be used to understand community assembly.
458
First, they allow the identification of potentially important physiological and
459
ecological mechanisms by correlating traits with species performance along
460
environmental gradients [56]. Second, trait similarity among species can be used to
461
infer niche similarity and determine its effects on species sorting [57]. For example,
462
the existence of trait trade-offs in important ecological factors or niche axes is
463
fundamental for a mechanistic understanding of species co-existence [54].
464
At the ecosystem level, species sharing traits that influence particular
465
ecosystem properties or species’ responses to environmental conditions can be
466
pooled into particular functional groups [58]. For example, Tilman [34] showed that
467
functional trait diversity is a better predictor of community productivity than
468
taxonomic diversity. This functional ecology perspective has focused on determining
469
which traits allow species to face environmental changes –response traits- as well as
470
which traits determine how species influence the environment –effect traits; and
471
establishing the link between these set of traits to make better trait-based models of
472
community assembly and ecosystem functioning [59]. Recently there has been a call
473
to use this conceptual framework for ectomycorrhizal fungi [37].
474
17
475
18
476
Table 1. Examples of traits that are potentially important for characterizing fungi that fall along the symbiotic and saprotrophic
continuum.
Fungal traits
Description and rationale
Refs
Production of cell-wall degrading
enzymes
They broaden the spectrum of carbon sources for saprotrophic growth, but at the
same time they trigger host defenses as a result of disruption of cell walls in living
cells.
The ability to decompose lignin (e.g. by white rot saprotrophs) expands availability
of carbon substrates in woody systems. This trait seems rare in ectomycorrhizal
fungi.
Key traits to cope with interference competition by fungi and bacteria are the
production of antibiotics as well as the enzymes that degrade them.
Soil fungi may produce toxic metabolites, melanin or crystalline cell inclusions to
avoid fungivory by soil animals.
Resting structures permit survival under adverse conditions: a lack of appropriate
hosts or inadequate carbon sources. Whether this is more or less likely in
endophytic fungi is not known, and may depend on root or plant longevity.
Root symbiotic fungi have both vertical and horizontal transmission, but how these
transmission modes differ from corresponding dispersal modes of more
saprotrophic fungi is not understood.
Root exudates (either inducible or constitutive) constrain growth patterns of fungal
species and are a component of host resistance. Whether and how they are
degraded, is likely to be important in colonization.
Many symbiotic fungi have specialized structures for initial penetration of host
tissue. These structures also produce physical, enzymatic and chemical signals
that enable the early infection process.
Extensive intercellular growth (apolastic growth) may reflect the ability of fungi to
avoid the disruption of host cell walls, and so avoid the activation of resistance
responses such as by reactive oxygen species, phenols, pathogenesis-related
proteins, and phytoalexins.
Some symbionts have structures that include the invagination of plant membranes
(e.g. haustoria and arbuscules), but such invaginations may also occur in fungi
with saprotrophic abilities (e.g. Piriformospora indica).
Cell wall thickening is one of the first general responses against pathogen
invasion. The ability to avoid or prevent such responses during hyphal penetration
may be essential for a symbiotic lifestyle.
Some fungi depend on the production of phytotoxins and the ability to degrade
plant derived antimicrobial compounds for successful entry.
Symbiotic fungi can obtain sugars from the host by either secreting fungal
invertases or by being reliant on plant invertases. Fungal invertases are
seemingly absent in ectomycorrhizal symbionts.
In some fungi, establishment of symbiosis requires effective exchange of
resources with the host plant. Gene expression is regulated in fungi based on
their location in the host cells, the compounds being transported, and the direction
of transport. While such processes are well established in mycorrhizal
associations, there is little evidence whether other root symbionts exhibit similar
mechanisms of resource exchange.
Root fungal symbionts often maintain dual growth in the root and the soil.
Allocation of biomass to the extra-radical mycelium and the spatial distribution of
hyphae in soil affect the transfer of nutrients. Several morphological “foraging
types” have been identified in ectomycorrhizae and are likely in other root
symbionts.
[60]
Lignolytic ability
Antibiotic production
Palatability
Resting structures
Dispersal/Transmission mode
Degradation of antimicrobial root
exudates
Penetration structures
Ratio of inter- to intracellular
colonization
Perifungal membrane
Induction of host cell wall
reinforcements
Phytotoxin production and
detoxification enzymes
Fungal invertases
Transporter traits and their expression
Extra-radical mycelia and "foraging"
pattern.
477
19
[61]
[62]
[26]
[63]
[10]
[64]
[65]
[9]
[66, 67]
[68]
[60, 69]
[70]
[21, 50]
[55, 71, 72]