Entomopathogenic Nematodes-Killed Insect Cadavers in the Rhizosphere Activate Plant Direct and Indirect Defences Aboveground.

Plants can perceive and respond to external stimuli by activating both direct and indirect defences against herbivores. Soil-dwelling entomopathogenic nematodes (EPNs), natural enemies of root-feeding herbivores, carry symbiotic bacteria that grow and reproduce once inside arthropod hosts. We hypothesized that the metabolites produced by EPN-infected insect cadavers could be perceived by plants, thereby activating plant defences systemically. We tested this hypothesis by adding three EPN-infected Galleria mellonella cadavers to maize plants and testing plant responses against a major maize pest (Spodoptera frugiperda) and one of its parasitoids (Trichogramma dendrolimi). We found that S. frugiperda females deposited fewer, and caterpillars fed less on maize plants growing near EPN-infected cadavers than on control plants. Accordingly, EPN-infected cadavers triggered the systemic accumulation of defence hormones (SA), genes (PR1), and enzymes (SOD, POD, and CAT) in maize leaves. Furthermore, four volatile organic compounds produced by plants exposed to EPN-infected cadavers deterred S. frugiperda caterpillars and female adults. However, these compounds were more attractive to T. dendrolimi parasitoids. Our study enhances the understanding of the intricate relationships within the above- and belowground ecosystems and provides crucial insights for advancing sustainable pest management strategies.

Beneath the blooms: Unearthing the effect of rhizospheric bacteria on floral signals and pollinator preferences.

The relationship between plants and pollinators is known to be influenced by ecological interactions with other community members. While most research has focused on aboveground communities affecting plant-pollinator interactions, it is increasingly recognized that soil-dwelling organisms can directly or indirectly impact these interactions. Although studies have examined the effects of arbuscular mycorrhizal fungi on floral traits, there is a gap in research regarding similar effects associated with plant growth-promoting rhizobacteria (PGPR), particularly concerning floral scent. Our study aimed to investigate the influence of the PGPR Bacillus amyloliquefaciens on the floral traits of wild (Solanum habrochaites, Solanum pimpinellifolium and Solanum peruvianum) and cultivated tomato (Solanum lycopersicum), as well as the impact of microbially-driven changes in floral scent on the foraging behaviour of the stingless bee Melipona quadrifasciata. Our findings revealed that inoculating tomatoes with PGPR led to an increased number of flowers and enhanced overall floral volatile emission. Additionally, we observed higher flower biomass and pollen levels in all species, except S. peruvianum. Importantly, these changes in volatile emissions influenced the foraging behaviour of M. quadrifasciata significantly. Our results highlight the impact of beneficial soil microbes on plant-pollinator interactions, shedding light on the multiple effects that plant-microbial interactions can have on aboveground organisms.

Soil legacy effects of plants and drought on aboveground insects in native and range-expanding plant communities.

Soils contain biotic and abiotic legacies of previous conditions that may influence plant community biomass and associated aboveground biodiversity. However, little is known about the relative strengths and interactions of the various belowground legacies on aboveground plant-insect interactions. We used an outdoor mesocosm experiment to investigate the belowground legacy effects of range-expanding versus native plants, extreme drought and their interactions on plants, aphids and pollinators. We show that plant biomass was influenced more strongly by the previous plant community than by the previous summer drought. Plant communities consisted of four congeneric pairs of natives and range expanders, and their responses were not unanimous. Legacy effects affected the abundance of aphids more strongly than pollinators. We conclude that legacies can be contained as soil 'memories' that influence aboveground plant community interactions in the next growing season. These soil-borne 'memories' can be altered by climate warming-induced plant range shifts and extreme drought.

Agricultural soil legacy influences multitrophic interactions between crops, their pathogens and pollinators.

Soil legacy influences plant interactions with antagonists and below-ground mutualists. Plant-antagonist interactions can jeopardize plant-pollinator interactions, while soil mutualists can enhance plant-pollinator interactions. This suggests that soil legacy, either directly or mediated through plant symbionts, affects pollinators. Despite the importance of pollinators to natural and managed ecosystems, information on how soil legacy affects plant-pollinator interactions is limited. We assessed effects of soil management legacy (organic versus conventional) on floral rewards and plant interactions with wild pollinators, herbivores, beneficial fungi and pathogens. We used an observational dataset and structural equation models to evaluate hypothesized relationships between soil and pollinators, then tested observed correlations in a manipulative experiment. Organic legacy increased mycorrhizal fungal colonization and improved resistance to powdery mildew, which promoted pollinator visitation. Further, soil legacy and powdery mildew independently and interactively impacted floral traits and floral reward nutrients, which are important to pollinators. Our results indicate that pollination could be an overlooked consequence of soil legacy and suggests opportunity to develop long-term soil management plans that benefit pollinators and pollination.

Aboveground herbivory can promote exotic plant invasion through intra- and interspecific aboveground-belowground interactions.

Aboveground herbivores and soil biota profoundly affect plant invasions. However, how they interactively affect plant invasions through plant-soil feedbacks (PSFs) remains unclear. To explore how herbivory by the introduced beetle Agasicles hygrophila affects Alternanthera philoxeroides invasions in China, we integrated multiyear field surveys and a 2-yr PSF experiment, in which we examined how herbivory affects PSFs on the performance of native and invasive plants and the introduced beetles. Despite increased herbivory from A. hygrophila, A. philoxeroides dominance over co-occurring congeneric native Alternanthera sessilis remained constant from 2014 to 2019. While occurring at lower abundances, A. sessilis experienced similar herbivore damage, suggesting apparent competitive effects. Our experiments revealed that herbivory on A. philoxeroides altered soil microbial communities, prolonged its negative PSF on A. sessilis, and decreased A. hygrophila larvae performance on the next-generation invasive plants. Consequently, A. hygrophila larvae performed better on leaves of natives than those of invasives when grown in soils conditioned by invasive plants defoliated by the introduced beetles. Our findings suggest that aboveground herbivory might promote rather than suppress A. philoxeroides invasion by enhancing its soil-mediated self-reinforcement, providing a novel mechanistic understanding of plant invasions. These findings highlight the need to incorporate an aboveground-belowground perspective during the assessment of potential biocontrol agents.

Microbiome resilience of Amazonian forests: Agroforest divergence to bacteria and secondary forest succession convergence to fungi.

An alarming and increasing deforestation rate threatens Amazon tropical ecosystems and subsequent degradation due to frequent fires. Agroforestry systems (AFS) may offer a sustainable alternative, reportedly mimicking the plant-soil interactions of the natural mature forest (MF). However, the role of microbial community in tropical AFS remains largely unknown. This knowledge is crucial for evaluating the sustainability of AFS and practices given the key role of microbes in the aboveground-belowground interactions. The current study, by comparing different AFS and successions of secondary and MFs, showed that AFS fostered distinct groups of bacterial community, diverging from the MFs, likely a result of management practices while secondary forests converged to the same soil microbiome found in the MF, by favoring the same groups of fungi. Model simulations reveal that AFS would require profound changes in aboveground biomass and in soil factors to reach the same microbiome found in MFs. In summary, AFS practices did not result in ecosystems mimicking natural forest plant-soil interactions but rather reshaped the ecosystem to a completely different relation between aboveground biomass, soil abiotic properties, and the soil microbiome.

Water stress and disruption of mycorrhizas induce parallel shifts in phyllosphere microbiome composition.

Water and nutrient acquisition are key drivers of plant health and ecosystem function. These factors impact plant physiology directly as well as indirectly through soil- and root-associated microbial responses, but how they in turn affect aboveground plant-microbe interactions are not known. Through experimental manipulations in the field and growth chamber, we examine the interacting effects of water stress, soil fertility, and arbuscular mycorrhizal fungi on bacterial and fungal communities of the tomato (Solanum lycopersicum) phyllosphere. Both water stress and mycorrhizal disruption reduced leaf bacterial richness, homogenized bacterial community composition among plants, and reduced the relative abundance of dominant fungal taxa. We observed striking parallelism in the individual microbial taxa in the phyllosphere affected by irrigation and mycorrhizal associations. Our results show that soil conditions and belowground interactions can shape aboveground microbial communities, with important potential implications for plant health and sustainable agriculture.

Microbial drivers of plant richness and productivity in a grassland restoration experiment along a gradient of land-use intensity.

Plant-soil feedbacks (PSFs) underlying grassland plant richness and productivity are typically coupled with nutrient availability; however, we lack understanding of how restoration measures to increase plant diversity might affect PSFs. We examined the roles of sward disturbance, seed addition and land-use intensity (LUI) on PSFs. We conducted a disturbance and seed addition experiment in 10 grasslands along a LUI gradient and characterized plant biomass and richness, soil microbial biomass, community composition and enzyme activities. Greater plant biomass at high LUI was related to a decrease in the fungal to bacterial ratios, indicating highly productive grasslands to be dominated by bacteria. Lower enzyme activity per microbial biomass at high plant species richness indicated a slower carbon (C) cycling. The relative abundance of fungal saprotrophs decreased, while pathogens increased with LUI and disturbance. Both fungal guilds were negatively associated with plant richness, indicating the mechanisms underlying PSFs depended on LUI. We show that LUI and disturbance affect fungal functional composition, which may feedback on plant species richness by impeding the establishment of pathogen-sensitive species. Therefore, we highlight the need to integrate LUI including its effects on PSFs when planning for practices that aim to optimize plant diversity and productivity.

Drought legacy effects on root morphological traits and plant biomass via soil biota feedback.

Drought causes soil feedback effects on plant performance. However, how the linkages between conditioned soil biota and root traits contribute to explain plant-soil feedback (PSF) as a function of drought is unknown. We utilized soil inoculum from a conditioning experiment where grassland species grew under well-watered and drought conditions, and their soil fungi were analyzed. Under well-watered conditions, we grew 21 grassland species with those inocula from either conspecific or heterospecific soils. At harvest, plant biomass and root traits were measured. Negative PSF (higher biomass in heterospecific than in conspecific soils) was predominant, and favored in drought-conditioned soils. Previous drought affected the relationship between root traits and fungal groups. Specific root surface area (SRSA) was higher in heterospecific than in conspecific droughted soils and was linked to an increase in saprotroph richness. Overall, root diameter was higher in conspecific soils and was linked to mutualist and pathogen composition, whereas the decrease of root : shoot in heterospecific soils was linked to pathogenic fungi. Drought legacy affects biomass and root morphological traits via conditioned soil biota, even after the drought conditions have disappeared. This provides new insights into the role that soil biota have modulating PSF responses to drought.

Root mass carbon costs to acquire nitrogen are determined by nitrogen and light availability in two species with different nitrogen acquisition strategies.

Plant nitrogen acquisition requires carbon to be allocated belowground to build roots and sustain microbial associations. This carbon cost to acquire nitrogen varies by nitrogen acquisition strategy; however, the degree to which these costs vary due to nitrogen availability or demand has not been well tested under controlled conditions. We grew a species capable of forming associations with nitrogen-fixing bacteria (Glycine max) and a species not capable of forming such associations (Gossypium hirsutum) under four soil nitrogen levels to manipulate nitrogen availability and four light levels to manipulate nitrogen demand in a full-factorial greenhouse experiment. We quantified carbon costs to acquire nitrogen as the ratio of total root carbon to whole-plant nitrogen within each treatment combination. In both species, light availability increased carbon costs due to a larger increase in root carbon than whole-plant nitrogen, while nitrogen fertilization generally decreased carbon costs due to a larger increase in whole-plant nitrogen than root carbon. Nodulation data indicated that G. max shifted relative carbon allocation from nitrogen fixation to direct uptake with increased nitrogen fertilization. These findings suggest that carbon costs to acquire nitrogen are modified by changes in light and nitrogen availability in species with and without associations with nitrogen-fixing bacteria.

Root infection by the nematode Meloidogyne incognita modulates leaf antiherbivore defenses and plant resistance to Spodoptera exigua.

Studies on plant-mediated interactions between root parasitic nematodes and aboveground herbivores are rapidly increasing. However, outcomes for the interacting organisms vary, and the mechanisms involved remain ambiguous. We hypothesized that the impact of root infection by the root-knot nematode Meloidogyne incognita on the performance of the aboveground caterpillar Spodoptera exigua is modulated by the nematode's infection cycle. We challenged root-knot nematode-infected tomato plants with caterpillars when the nematode's infection cycle was at the invasion, galling, and reproduction stages. We found that M. incognita root infection enhanced S. exigua performance during the galling stage, while it did not affect the caterpillar's performance at the invasion and reproduction stages. Molecular and chemical analyses performed at the different stages of the nematode infection cycle revealed that M. incognita root infection systemically affected the jasmonic acid-, salicylic acid-, and abscisic acid-related responses, as well as the changes in the leaf metabolome triggered during S. exigua feeding. The M. incognita-induced leaf responses varied over the nematode's root infection cycle. These findings suggest that specific leaf responses triggered systemically by the nematode at its different life-cycle stages underlie the differential impact of M. incognita on plant resistance against the caterpillar S. exigua.

Land-use drives the temporal stability and magnitude of soil microbial functions and modulates climate effects.

Soil microbial community functions are essential indicators of ecosystem multifunctionality in managed land-use systems. Going forward, the development of adaptation strategies and predictive models under future climate scenarios will require a better understanding of how both land-use and climate disturbances influence soil microbial functions over time. Between March and November 2018, we assessed the effects of climate change on the magnitude and temporal stability of soil basal respiration, soil microbial biomass and soil functional diversity across a range of land-use types and intensities in a large-scale field experiment. Soils were sampled from five common land-use types including conventional and organic croplands, intensive and extensive meadows, and extensive pastures, under ambient and projected future climate conditions (reduced summer precipitation and increased temperature) at the Global Change Experimental Facility (GCEF) in Bad Lauchstädt, Germany. Land-use and climate treatment interaction effects were significant in September, a month when precipitation levels slightly rebounded following a period of drought in central Germany: compared to ambient climate, in future climate treatments, basal respiration declined in pastures and increased in intensive meadows, functional diversity declined in pastures and croplands, and respiration-to-biomass ratio increased in intensive and extensive meadows. Low rainfall between May and August likely strengthened soil microbial responses toward the future climate treatment in September. Although microbial biomass showed declining levels in extensive meadows and pastures under future climate treatments, overall, microbial function magnitudes were higher in these land-use types compared to croplands, indicating that improved management practices could sustain high microbial ecosystem functioning in future climates. In contrast to our hypothesis that more disturbed land-use systems would have destabilized microbial functions, intensive meadows and organic croplands showed stabilized soil microbial biomass compared to all other land-use types, suggesting that temporal stability, in addition to magnitude-based measurements, may be useful for revealing context-dependent effects on soil ecosystem functioning.

Effects of plant species diversity on nematode community composition and diversity in a long-term biodiversity experiment.

Diversity loss has been shown to change the soil community; however, little is known about long-term consequences and underlying mechanisms. Here, we investigated how nematode communities are affected by plant species richness and whether this is driven by resource quantity or quality in 15-year-old plant communities of a long-term grassland biodiversity experiment. We extracted nematodes from 93 experimental plots differing in plant species richness, and measured above- and belowground plant biomass production and soil organic carbon concentrations (Corg) as proxies for resource quantity, as well as C/Nleaf ratio and specific root length (SRL) as proxies for resource quality. We found that nematode community composition and diversity significantly differed among plant species richness levels. This was mostly due to positive plant diversity effects on the abundance and genus richness of bacterial-feeding, omnivorous, and predatory nematodes, which benefited from higher shoot mass and soil Corg in species-rich plant communities, suggesting control via resource quantity. In contrast, plant-feeding nematodes were negatively influenced by shoot mass, probably due to higher top-down control by predators, and were positively related to SRL and C/Nleaf, indicating control via resource quality. The decrease of the grazing pressure ratio (plant feeders per root mass) with plant species richness indicated a higher accumulation of plant-feeding nematodes in species-poor plant communities. Our results, therefore, support the hypothesis that soil-borne pathogens accumulate in low-diversity communities over time, while soil mutualists (bacterial-feeding, omnivorous, predatory nematodes) increase in abundance and richness in high-diversity plant communities, which may contribute to the widely-observed positive plant diversity-productivity relationship.

Insights into the Intraspecific Variability of the above and Belowground Emissions of Volatile Organic Compounds in Tomato.

The in-vivo monitoring of volatile organic compound (VOC) emissions is a potential non-invasive tool in plant protection, especially in greenhouse cultivation. We studied VOC production from above and belowground organs of the eight parents of the Multi-Parent Advanced Generation Intercross population (MAGIC) tomato population, which exhibits a high genetic variability, in order to obtain more insight into the variability of constitutive VOC emissions from tomato plants under stress-free conditions. Foliage emissions were composed of terpenes, the majority of which were also stored in the leaves. Foliage emissions were very low, partly light-dependent, and differed significantly among genotypes, both in quantity and quality. Soil with roots emitted VOCs at similar, though more variable, rates than foliage. Soil emissions were characterized by terpenes, oxygenated alkanes, and alkenes and phenolic compounds, only a few of which were found in root extracts at low concentrations. Correlation analyses revealed that several VOCs emitted from foliage or soil are jointly regulated and that above and belowground sources are partially interconnected. With respect to VOC monitoring in tomato crops, our results underline that genetic variability, light-dependent de-novo synthesis, and belowground sources are factors to be considered for successful use in crop monitoring.

Putting soil invertebrate diversity on the map.

Ecologists have had a very good foundational knowledge of the global distribution of plants and aboveground animals for many decades. But despite the immense diversity of soil organisms, our knowledge of the global distribution, drivers and threats to soil biodiversity is very limited. In this issue of Molecular Ecology, Bastida et al. (2020) produce the first global maps of soil invertebrate diversity that have been sampled at 83 locations, across six continents, using standardised methods and DNA sequencing. Using data from nematodes, arachnids and rotifers, and structural equation models, they find that diversity of these taxa is primarily driven by vegetation and climate. Given the anthropogenic changes that are occurring, and are projected to continue, this study provides important baseline information for future soil biodiversity and function monitoring, as well as exciting working hypotheses for targeted experiments.

Soil Inoculation Alters Leaf Metabolic Profiles in Genetically Identical Plants.

Abiotic and biotic properties of soil can influence growth and chemical composition of plants. Although it is well-known that soil microbial composition can vary greatly spatially, how this variation affects plant chemical composition is poorly understood. We grew genetically identical Jacobaea vulgaris in sterilized soil inoculated with live soil collected from four natural grasslands and in 100% sterilized soil. Within each grassland we sampled eight plots, totalling 32 different inocula. Two samples per plot were collected, leading to three levels of spatial variation: within plot, between and within grasslands. The leaf metabolome was analysed with 1H Nuclear magnetic resonance spectroscopy (NMR) to investigate if inoculation altered the metabolome of plants and how this varied between and within grasslands. Inoculation led to changes in metabolomics profiles of J. vulgaris in two out of four sites. Plants grown in sterilized and inoculated soils differed in concentrations of malic acid, tyrosine, trehalose and two pyrrolizidine alkaloids (PA). Metabolomes of plants grown in inoculated soils from different sites varied in glucose, malic acid, trehalose, tyrosine and in one PA. The metabolome of plants grown in soils with inocula from the same site was more similar than with inocula from distant sites. We show that soil influences leaf metabolomes. Performance of aboveground insects often depends on chemical composition of plants. Hence our results imply that soil microbial communities, via affecting aboveground plant metabolomes, can impact aboveground plant-insect food chains but that it is difficult to make general predictions due to spatial variation in soil microbiomes.

Soil multifunctionality and drought resistance are determined by plant structural traits in restoring grassland.

It is increasingly recognized that belowground responses to vegetation change are closely linked to plant functional traits. However, our understanding is limited concerning the relative importance of different plant traits for soil functions and of the mechanisms by which traits influence soil properties in the real world. Here we test the hypothesis that taller species, or those with complex rooting structures, are associated with high rates of nutrient and carbon (C) cycling in grassland. We further hypothesized that communities dominated by species with deeper roots may be more resilient to drought. These hypotheses were tested in a 3-yr grassland restoration experiment on degraded ex-arable land in southern England. We sowed three trait-based plant functional groups, assembled using database derived values of plant traits, and their combinations into bare soil. This formed a range of plant trait syndromes onto which we superimposed a simulated drought 2 yr after initial establishment. We found strong evidence that community weighted mean (CWM) of plant height is negatively associated with soil nitrogen cycling and availability and soil multifunctionality. We propose that this was due to an exploitative resource capture strategy that was inappropriate in shallow chalk soils. Further, complexity of root architecture was positively related to soil multifunctionality throughout the season, with fine fibrous roots being associated with greater rates of nutrient cycling. Drought resistance of soil functions including ecosystem respiration, mineralization, and nitrification were positively related to functional divergence of rooting depth, indicating that, in shallow chalk soils, a range of water capture strategies is necessary to maintain functions. Finally, after 3 yr of the experiment, we did not detect any links between the plant traits and microbial communities, supporting the finding that traits based on plant structure and resource foraging capacity are the main variables driving soil function in the early years of grassland conversion. We suggest that screening recently restored grassland communities for potential soil multifunctionality and drought resilience may be possible based on rooting architecture and plant height. These results indicate that informed assembly of plant communities based on plant traits could aid in the restoration of functioning in degraded soil.

Legacy effects of drought on plant-soil feedbacks and plant-plant interactions.

Interactions between aboveground and belowground biota have the potential to modify ecosystem responses to climate change, yet little is known about how drought influences plant-soil feedbacks with respect to microbial mediation of plant community dynamics. We tested the hypothesis that drought modifies plant-soil feedback with consequences for plant competition. We measured net pairwise plant-soil feedbacks for two grassland plant species grown in monoculture and competition in soils that had or had not been subjected to a previous drought; these were then exposed to a subsequent drought. To investigate the mechanisms involved, we assessed treatment responses of soil microbial communities and nutrient availability. We found that previous drought had a legacy effect on bacterial and fungal community composition that decreased plant growth in conspecific soils and had knock-on effects for plant competitive interactions. Moreover, plant and microbial responses to subsequent drought were dependent on a legacy effect of the previous drought on plant-soil interactions. We show that drought has lasting effects on belowground communities with consequences for plant-soil feedbacks and plant-plant interactions. This suggests that drought, which is predicted to increase in frequency with climate change, may change soil functioning and plant community composition via the modification of plant-soil feedbacks.

A whole-plant perspective reveals unexpected impacts of above- and belowground herbivores on plant growth and defense.

Trade-offs between plant growth and defense are central to theoretical frameworks used to study the ecology and evolution of plant defense against herbivores. However, these frameworks, as well as the experiments designed to test them, rarely include belowground herbivores. We experimentally challenged seedlings of the tropical shrub Solanum lycocarpum (Solanaceae) with either aboveground foliar herbivores (Spodoptera caterpillars) or belowground root herbivores (the nematode Meloidogyne incognita) and measured the resulting changes in plant growth rates, biomass allocation, and the concentration of defensive terpenoids in roots and leaves. We found that plants that suffered aboveground herbivory responded with aboveground growth but belowground defense. Similarly, belowground herbivory resulted in root growth but elevated defenses of leaves. These results underscore the importance of belowground plant-herbivore interactions, and suggest that, in contrast to theoretical predictions, plants can simultaneously invest in both growth and defense. Finally, they emphasize the need for a "whole-plant" perspective in theoretical and empirical evaluations of plant-herbivore interactions.

Responses of belowground communities to large aboveground herbivores: Meta-analysis reveals biome-dependent patterns and critical research gaps.

The importance of herbivore-plant and soil biota-plant interactions in terrestrial ecosystems is amply recognized, but the effects of aboveground herbivores on soil biota remain challenging to predict. To find global patterns in belowground responses to vertebrate herbivores, we performed a meta-analysis of studies that had measured abundance or activity of soil organisms inside and outside field exclosures (areas that excluded herbivores). Responses were often controlled by climate, ecosystem type, and dominant herbivore identity. Soil microfauna and especially root-feeding nematodes were negatively affected by herbivores in subarctic sites. In arid ecosystems, herbivore presence tended to reduce microbial biomass and nitrogen mineralization. Herbivores decreased soil respiration in subarctic ecosystems and increased it in temperate ecosystems, but had no net effect on microbial biomass or nitrogen mineralization in those ecosystems. Responses of soil fauna, microbial biomass, and nitrogen mineralization shifted from neutral to negative with increasing herbivore body size. Responses of animal decomposers tended to switch from negative to positive with increasing precipitation, but also differed among taxa, for instance Oribatida responded negatively to herbivores, whereas Collembola did not. Our findings imply that losses and gains of aboveground herbivores will interact with climate and land use changes, inducing functional shifts in soil communities. To conceptualize the mechanisms behind our findings and link them with previous theoretical frameworks, we propose two complementary approaches to predict soil biological responses to vertebrate herbivores, one focused on an herbivore body size gradient, and the other on a climate severity gradient. Major research gaps were revealed, with tropical biomes, protists, and soil macrofauna being especially overlooked.