Enhancing consistency in arbuscular mycorrhizal trait-based research to improve predictions of function

1Pedro M Antunes*, 2Sidney L. Stürmer*, 3James D. Bever, 4Pierre-Luc Chagnon, 5V. Bala Chaudhary, 10Coline Deveautour, 1Catherine Fahey, 6Vasilis Kokkoris, 7Ylva Lekberg, 8Jeff R. Powell, 9Carlos A. Aguilar-Trigueros, 8Haiyang Zhang1Biology Department, Algoma University, Sault Ste. Marie, Ontario, Canada, P6A 2G42Departamento de Ciências Naturais, Universidade Regional de Blumenau, Blumenau, SC, 89030-903, Brazil3Kansas Biological Survey and Center for Ecological Research and Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, Kansas, USA4Institut de Recherche en Biologie Vegetale, Universite de Montreal, 4101 Sherbrooke Est, Montreal, QC, H1X2B2, Canada5Department of Environmental Studies, Dartmouth College, Hanover, New Hampshire, USA6Amsterdam Institute for Life and Environment (A-LIFE), Faculty of Science, Section Systems Ecology, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, the Netherlands7MPG Ranch & Department of Ecosystem and Conservation Sciences, W.A. Franke College of Forestry and Conservation, University of Montana, Missoula, Montana, USA8Hawkesbury Institute for the Environment, Western Sydney University, Penrith, New South Wales, Australia9Institute of Biology, Freie Universität Berlin, Altensteinstrasse 6, 14195 Berlin, Germany.10 Institut Polytechnique UniLaSalle, Unité AGHYLE, Campus Rouen, 76130, Mont-Saint-Aignan, Normandie, France *Equal contributionCorresponding: Pedro M. Antunes [pedro.antunes [at] algomau.ca] ORCID:Pedro M. Antunes: 0000-0003-3596-6983Sidney L. Stürmer: 0000-0003-3213-1841James D. Bever: 0000-0003-4068-3582Pierre-Luc Chagnon: 0000-0002-5054-5813Bala Chaudhary: 0000-0002-7232-1757Coline Deveautour: 0000-0001-6887-0414Catherine Fahey: 0000-0002-6987-0456Vasilis Kokkoris: 0000-0002-1667-0493Ylva Lekberg: 0000-0003-1033-8032 Jeff Powell: 0000-0003-1091-2452Carlos A. Aguilar-Trigueros: 0000-0003-0512-9500Haiyang Zhang 0000-0001-7951-0502AbstractArbuscular mycorrhizal (AM) fungi (phylum Glomeromycota) are obligate symbionts with plants influencing plant health, soil a(biotic) processes, and ecosystem functioning. Despite advancements in molecular techniques, understanding the role of AM fungal communities on a(biotic) processes based on AM fungal taxonomy remains challenging. This review advocates for a standardized trait-based framework to elucidate the life-history traits of AM fungi, focusing on their roles in three dimensions: host plants, soil, and AM fungal ecology. We define morphological, physiological, and genetic key traits, and propose methodologies for their consistent measurement, enabling cross-study comparisons towards improved predictability of ecological function. We aim for this review to lay the groundwork for establishing a baseline of AM fungal trait responses under varying environmental conditions. Furthermore, we emphasize the need to include underrepresented taxa in research and utilize advances in machine learning and microphotography for data standardization. Keywords: symbiosis; trait-based ecology; ecosystem processes; standardization; functional diversity; environmental adaptation. The problem: identifying and associating AM fungal traits with functional outcomes A trait is an attribute influencing an organism’s performance within its environment, encompassing morphological, genetic and physiological characteristics measured at the individual or population levels (Salguero-Gómez et al. 2018; Zhang et al. 2023b). Understanding the ecology of species using a trait-based approach can contribute to a mechanistic explanation of processes mediated by microbes, including those that affect ecosystem functioning (Romero-Olivares et al. 2021). This approach holds particular significance for arbuscular mycorrhizal (AM) fungi – Phylum Glomeromycota. As obligate symbionts of plants, where multiple fungi colonize both roots and soils in a network, predicting the functional outcomes (e.g., host growth, plant community diversity, changes in soil processes) of individual AM fungal genotypes and communities within ecosystems remains challenging, despite major developments in molecular methods in the last two decades (Tisserant et al. 2013; Montoliu-Nerin et al. 2021). Indeed, establishing relationships between AM fungal taxa and/or genotypes (i.e., accounting for within species variability) and their functional roles is a laborious process, which is expected to continue in the foreseeable future (Serghi et al. 2021; Manley et al. 2023; Corradi et al. 2024). However, it is important to go beyond taxonomy and morphological characteristics, incorporating physiological and genetic traits. This work is needed due to the complex associations that exist between AM fungi and various functional outcomes for hosts (e.g., plant growth and fitness, nutrient uptake and stress tolerance) and soil processes (e.g., carbon storage, aggregate stability, biotic diversity), all of which currently appear highly context dependent and relatively poorly predicted by taxonomy alone (Munkvold et al. 2004; Koch et al. 2017; Yang et al. 2017; Qiu et al. 2021). However, this effort is also required because AM fungal traits have not been systematically assessed alongside with hypotheses of adaptation or with specific mechanisms in mind. For example, small-spored AM fungi may be dispersed longer distances by wind than large-spored AM fungi, which leads to the reasonable hypothesis that small spore size is an adaptation for wind dispersal. One could empirically observe that small-spored AM fungi are geographically more widespread than large-spored fungi and this potential result could be viewed as evidence in support of this hypothesis. However, this finding would not necessarily prove that such dispersal difference has “functional” or “adaptive” value. Alternatively, producing small spores is a correlated response to producing many spores (arguably more quickly if they are structurally simpler), which itself could be an adaptive response to the likelihood of unpredictable soil disturbance (e.g., caused by soil animals or from tillage). In this scenario, the adaptation and/or function is the production of many spores quickly to confer resistance to disturbance and then, after soil disturbance, with wind erosion, small spores may also be blown farther (which may or may not improve fitness). Measuring traits like spore size, spore production rates, and their ability to resist or respond to environmental disturbances can help to disentangle different hypotheses about how these traits contribute to AM fungal success and distribution. Another example to illustrate the complexity of associating traits with function is the variation in rooting depth among plants in a community, which may contribute to resource partitioning, but the mechanism (differential resource depletion with depth) still needs to be demonstrated separately from the trait evidence. AM fungi could contribute to equalize resource partitioning if plants with short roots associate with AM fungi that form more extensive extra-radical mycelium and vice-versa. As such, plants and fungal traits cannot be considered in isolation. Given these complexities, we consider the development of a robust, universally applicable trait-based framework towards predicting key AM fungal functional outcomes a priority. To achieve this objective, first we must identify AM fungal traits that can be measured not only at morphological levels of organization but also at physiological, and genetic levels. Second, considering the important roles of AM fungi in ecosystems, affecting host plants, soil processes, and the AM fungi themselves, we need to discern/hypothesize how measuring AM fungal traits impacts each of these components. For the host plant, it is crucial to consider nutrition, biomass, fitness, and survival in face of nutrient limitations, pathogens, heavy metals, salinity, drought, etc. (Delavaux et al. 2017; Wehner et al. 2010). Within the soil environment, AM fungal effects on soil structure (Rillig and Mummey 2006), nutrient cycling, carbon storage, and other members of the soil food-web are relevant (Antunes and Koyama 2016; Frew et al. 2021; Horsch et al. 2023a). Regarding the fungal organism itself, we should focus on key aspects of their life-history strategies: reproduction and fitness, survival, dispersal, competitive ability, infectivity and abundance both within the host and soil environments (Aguilar-Trigueros et al. 2019; Chaudhary et al. 2020; Deveautour et al. 2020). Connecting traits to functional outcomes requires identifying relevant proxies (sometimes termed “soft traits” in the plant ecophysiology literature) to provide easy-to-measure quantitative metrics for such complex facets of fungal life-history that can be measured across several species. For example, small spore size and high spore production rates can serve as proxies for functional traits such as effective dispersal in face of disturbance. Hyphal growth rate and branching pattern can serve as proxies for resource foraging efficiency. Third, we need to evaluate existing standardized methods and experimental designs, or develop new ones, to measure such relevant (soft) traits, as it has been done in plant ecophysiology (Pérez-Harguindeguy et al. 2013). Measurement standardization and relevant metadata for hypothesis-driven analysis and interpretation is essential if we are to eventually aggregate trait information from different studies into a public database, facilitating their incorporation into earth system models (e.g., Fry et al. 2019) and enhancing the predictability of functional processes and/or adaptations associated with AM fungi. Analogous libraries on plant traits (Kattge et al. 2020) have proved useful to better understand trait variation along global climatic gradients (Butler et al. 2017). Here, we aim to: To comprehensively catalog and define AM fungal functional traits (morphological, physiological/phenological, and genetic) while avoiding redundancy. To elucidate the relationships between these traits and their functional outcomes for host plants, soil environments, and the AM fungi themselves. To critically review the historical methods and experimental designs employed in measuring AM fungal traits, highlighting their strengths and limitations. To propose standardized methodologies and protocols for measuring AM fungal traits. To explore the integration of AM fungal trait information into ecological models to potentially enhance ecosystem processes’ predictability. Historical perspective of trait-based approaches The scientific literature on the life-history traits of AM fungi (i.e., the biological characteristics and features that influence their growth, reproduction, and survival) has predominantly centered on aspects related to plant growth and nutrition, largely through an agronomic lens. Although not explicitly reported as such, early studies employing experimental approaches to assess, for example, AM fungal root colonization, abundance of external hyphae, and spore counts for specific species under certain experimental conditions have yielded insights into AM fungal trait variation (Abbott 1982; Reich 1988; Jakobsen et al. 1992a; Gazey et al. 1992; Bever et al. 1996). Given the wide variation observed, these and other seminal studies provided a foundation for further inquiry into the complex dynamics of AM fungal life-history traits and their broader implications to the AM symbiosis.Studies of distinct traits within a taxonomic framework started with the comparison of mycelium form and function, and root colonization strategies among major families of the Glomeromycota. For example, Dodd et al. (2000) compared the morphology and mycelial architecture of different AM fungal genera, discussing form and function. In a comparative study of 21 AM fungal isolates (i.e., defined as an AM fungus isolated in the laboratory into pure culture but without genetic characterization, at which point it becomes a certified strain with a collection number) spanning 16 species from North America, Hart and Reader (2002a) showed that the isolates of the Glomeraceae family, on average, colonized roots before those of Acaulosporaceae and Gigasporaceae families. Additionally, the proportion of fungal biomass in roots versus soil also diverged, on average, among isolates of those families. Those in the Glomeraceae exhibited high root colonization but low soil colonization, Gigasporaceae tended to have low root colonization but high soil colonization, and Acaulosporaceae displayed low colonization in both roots and soil. These findings revealed a strong association between AM fungal morphological characteristics and taxonomy for these fungi, as isolates from the main families could be differentiated based on colonization rate, biomass allocation, and the onset of sporulation. These observations were corroborated by subsequent studies, albeit using AM fungi from the same community and, possibly, the same isolates (Hart and Reader 2002a, 2005; Maherali and Klironomos 2007; Powell et al. 2009; Sikes et al. 2009). In fact, using the same data, Aguilar-Trigueros et al. (2019) showed that large-spore species produced, on average, fewer spores than small-spore species, suggesting that AM fungi experience similar resource allocation constraints during reproduction as plants seeds (Moles et al. 2005). However, to what extent plant trait-frameworks may be applicable to AM fungi is unknown. At present, evidence suggest differences between Glomeraceae and Gigasporaceae concerning life-history traits and their relationship with host benefits. However, new comparative studies that include more fungal species isolated from other ecological contexts are necessary to confirm these differences. More recently, a distinction between ‘edaphophilic’ and ‘rhizophilic’ life-history strategies has been introduced to categorize AM fungi that allocate more biomass to growth within roots versus soil (Weber et al. 2019), and data show that long-term P enrichment in subtropical forests shifts AM fungal communities toward edaphophilic guilds (Wang et al. 2023). The patterns described above demonstrate the utility of employing a comparative framework to test hypotheses concerning AM fungal function by examining trait expression. For instance, based on soil mycelium production, Gigasporaceae would be expected to outperform Glomeraceae in nutrient uptake (Maherali and Klironomos, 2007). Alternatively, if early or extensive root colonization (with abundant coils/arbuscules) is more important for nutrient delivery to the host, then Glomeraceae could be more beneficial partners under nutrient limiting conditions (e.g., (Horsch et al. 2023b). Despite inconsistencies among studies, which may to some extent be explained by variability in mycorrhizal dependency among hosts (Pringle and Bever 2008; Sikes et al. 2009), a meta-analysis (Yang et al. 2017) suggested that, on average, fungi of the familiy Glomeraceae were better at acquiring P and reducing pathogen growth compared to other AM fungal families. It is also of interest, that this family also appears to be the most abundant in many locations (Öpik et al. 2010). Despite these advances towards better consistency in predicting functional outcomes from morphological and taxonomy data, we argue that only a robust database integrating morphological, physiological, and genetic trait variation under different environmental conditions can establish a basis for more accurately predicting the functions of these fungi.Previous studies lacked a comprehensive environmental perspective. For instance, considering diverse environmental conditions, such as varying soil types or climatic factors, could unveil how AM fungal traits respond and adapt. Currently, most data reporting the impact of different AM fungi on their host originate from short-term experiments, using fungal taxa that readily sporulate and are easily amenable to pure cultures (Ohsowski et al. 2014). This may not reflect the reality in natural environments. Both the study by Sikes et al. (2009) investigating differences in plant pathogen protection between AM fungal taxa, as well as that of (Lerat et al. 2003) on C-sink strength among different AM fungal families suggest that certain functional outcomes resulting from the symbiosis depend on the combination of plant and fungal traits et al. As such, considering fungal traits alone (i.e., in of plant and soil may of functional outcomes of the symbiosis Chaudhary et al. This an of complexity to the study of AM fungal ecophysiology and trait-based as relationships between fungal and plant traits are to be expected et al. 2013). trait-based for AM fungi and the first comprehensive review of AM fungal traits to plant growth and ecosystem functioning. The provided a of AM fungal functional traits, which they into morphological traits (e.g., rate and extent of root colonization, spore and physiological traits (e.g., fungal carbon host nutrient uptake of into the and trait variation among AM fungal species Koch et al. the of traits and their incorporation into functional trait To this they a framework for trait variation of AM fungal species within the context of nutrient cycling, based on experimental and trait to and AM fungal genetic should be to diverse environmental conditions (e.g., host plants, soil nutrient would the of trait relationships among traits, the adaptive of and the potential for variation to these could traits an and into functional models for predicting nutrient Chaudhary et al. the in traits for such as fungi that establish mycorrhizal a trait by a standardized with the of establishing a between trait-based mycorrhizal AM fungal and community The traits into three main morphological, physiological, and Within each of these they mycorrhizal traits specific to both the host plant (e.g., growth and the fungal (e.g., spore size, and these traits by plant or fungal traits, Chaudhary et al. introduced the of mycorrhizal traits. These are that during symbiosis and are on both aspects such as root plant mycorrhizal and resource This framework an understanding of mycorrhizal ecology and as a basis for the framework et al. an AM fungal trait-based framework on framework which disturbance and as the major trait and in plant natural to be potential between fungal traits (e.g., carbon growth and environmental (e.g., soil from host, low soil this framework could identify traits for measurement, and of host and fungal traits that may to the benefits. on the of many traits or responses to environmental were between AM fungal major families and and However, as by et al. this association is and to AM fungal responses in complex et al. 2024). In it to consider several AM fungal families (e.g., or more like and It also to consider the of different AM fungal families in certain or at certain For example, is a in the where it can be both in natural forests and under where traits are crucial (e.g., et al. The significance of the framework in AM fungal trait-based ecology should not be considered as a framework for associating families with it should be as a for life-history in plant ecology to fungal traits that should be into research We two to a more understanding of AM fungal ecology. among studies due to experimental Second, the of a comprehensive database on AM fungal traits further in this the and of the isolates and species employed in these studies are on the taxa in culture or from a natural A of taxa, or other taxa fungal in with AM fungi, such as as suggested by et al. remains an important Given the existing data variability in plant and soil responses to the AM symbiosis both among and within AM fungal species, we must these to and to what AM fungal traits plant growth responses or effects on and and physiological traits mycorrhizal fungal traits, including for example or the of and spore can key with not only to the of the fungus itself but also the associated plant and the soil and for of key traits, their function, and methods for trait Here, we define AM fungal traits as which serve as of mycorrhizal function and depend on the morphological, physiological, or characteristics of the fungal et al. In this AM fungal traits are most in ecosystem and to environmental as certain fungal isolates with specific traits may demonstrate or under the form and function of AM fungal traits becomes within the of the fungal We can categorize the of an AM fungus into two the in which the dispersed spores other are and explore the soil for a host, and the which of root of within the root of mycelium into the soil and other and spore production and dispersal. and colonized root as the three types of the conditions et al. 2017; et al. from these identify a host to its and root A from which a the root to the A of and molecular processes into at these enabling the plant to the of the AM fungus by and et al. 2007; and and 2017). the root the fungus the mycelium This mycelium then into such as or and, in some taxa, and a certain of root colonization, beyond the root system into the soil the mycelium which of hyphae, spore associated and The network, and the traits that several processes to AM fungi (e.g., nutrient cycling, soil carbon soil pathogen As we explore these traits impact not the host plants and soil environment, but also the fungal organism

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