Lessons from Ethiopian coffee landscapes for global conservation in a post-wild world.

The reality for conservation of biodiversity across our planet is that all ecosystems are modified by humans in some way or another. Thus, biodiversity conservation needs to be implemented in multifunctional landscapes. In this paper we use a fascinating coffee-dominated landscape in southwest Ethiopia as our lens to derive general lessons for biodiversity conservation in a post-wild world. Considering a hierarchy of scales from genes to multi-species interactions and social-ecological system contexts, we focus on (i) threats to the genetic diversity of crop wild relatives, (ii) the mechanisms behind trade-offs between biodiversity and agricultural yields, (iii) underexplored species interactions suppressing pest and disease levels, (iv) how the interactions of climate change and land-use change sometimes provide opportunities for restoration, and finally, (v) how to work closely with stakeholders to identify scenarios for sustainable development. The story on how the ecology and evolution of coffee within its indigenous distribution shape biodiversity conservation from genes to social-ecological systems can inspire us to view other landscapes with fresh eyes. The ubiquitous presence of human-nature interactions demands proactive, creative solutions to foster biodiversity conservation not only in remote protected areas but across entire landscapes inhabited by people.

Local climate, air quality and leaf litter cover shape foliar fungal communities on an urban tree.

Foliar fungi on urban trees are important for tree health, biodiversity and ecosystem functioning. Yet, we lack insights into how urbanization influences foliar fungal communities. We created detailed maps of Stockholm region's climate and air quality and characterized foliar fungi from mature oaks (Quercus robur) across climatic, air quality and local habitat gradients. Fungal richness was higher in locations with high growing season relative humidity, and fungal community composition was structured by growing season maximum temperature, NO2 concentration and leaf litter cover. The relative abundance of mycoparasites and endophytes increased with temperature. The relative abundance of pathogens was lowest with high concentrations of NO2 and particulate matter (PM2.5), while saprotrophs increased with leaf litter cover. Our findings show that urbanization influences foliar fungi, providing insights for developing management guidelines to promote tree health, prevent disease outbreaks and maintain biodiversity within urban landscapes.

Temperature and water availability drive insect seasonality across a temperate and a tropical region.

The more insects there are, the more food there is for insectivores and the higher the likelihood for insect-associated ecosystem services. Yet, we lack insights into the drivers of insect biomass over space and seasons, for both tropical and temperate zones. We used 245 Malaise traps, managed by 191 volunteers and park guards, to characterize year-round flying insect biomass in a temperate (Sweden) and a tropical (Madagascar) country. Surprisingly, we found that local insect biomass was similar across zones. In Sweden, local insect biomass increased with accumulated heat and varied across habitats, while biomass in Madagascar was unrelated to the environmental predictors measured. Drivers behind seasonality partly converged: In both countries, the seasonality of insect biomass differed between warmer and colder sites, and wetter and drier sites. In Sweden, short-term deviations from expected season-specific biomass were explained by week-to-week fluctuations in accumulated heat, rainfall and soil moisture, whereas in Madagascar, weeks with higher soil moisture had higher insect biomass. Overall, our study identifies key drivers of the seasonal distribution of flying insect biomass in a temperate and a tropical climate. This knowledge is key to understanding the spatial and seasonal availability of insects-as well as predicting future scenarios of insect biomass change.

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The effect of local habitat and spatial connectivity on urban seed predation.

PREMISE: During the last centuries, the area covered by urban landscapes is increasing all over the world. Urbanization can change local habitats and decrease connectivity among these habitats, with important consequences for species interactions. While several studies have found a major imprint of urbanization on plant-insect interactions, the effects of urbanization on seed predation remain largely unexplored. METHODS: We investigated the relative impact of sunlight exposure, leaf litter, and spatial connectivity on predation by moth and weevil larvae on acorns of the pedunculate oak across an urban landscape during 2018 and 2020. We also examined whether infestations by moths and weevils were independent of each other. RESULTS: While seed predation varied strongly among trees, seed predation was not related to differences in sunlight exposure, leaf litter, or spatial connectivity. Seed predation by moths and weevils was negatively correlated at the level of individual acorns in 2018, but positively correlated at the acorn and the tree level in 2020. CONCLUSIONS: Our study sets the baseline expectation that urban seed predators are unaffected by differences in sunlight exposure, leaf litter, and spatial connectivity. Overall, our findings suggest that the impact of local and spatial factors on insects within an urban context may depend on the species guild. Understanding the impact of local and spatial factors on biodiversity, food web structure, and ecosystem functioning can provide valuable insights for urban planning and management strategies aimed at promoting urban insect diversity.

From climate perceptions to actions: A case study on coffee farms in Ethiopia.

Increasing temperatures and shifting precipitation patterns have major consequences for smallholder farmers, especially in the Global South. Our study examined spatial patterns and climatic drivers of farmers' perceptions of climate change, and how these perceptions translated into adaptation actions. We interviewed 56 farmers in southwestern Ethiopia and analyzed ERA5-Land reanalysis climate data from 1971 to 2020. The majority of farmers perceived the recorded temperature increase as well as a decrease and shift in the timing of rainfall. Perceived climate change varied with local climate factors and not with the rate of climate change itself. Farmers' adaptation practices showed associations with local temperature, but not with farmers' perceptions of climate change. Our findings highlight that even if farmers perceive climate change, perceptions are most common in areas where climate action is already urgent, and perceptions may not translate into adaptation. Thus, targeted and timely information and extension programs are crucial.

Parasitism rate differs between herbivore generations in the univoltine, but not bivoltine, range.

With climate change, plant-feeding insects increase their number of annual generations (voltinism). However, to what degree the emergence of a new herbivore generation affects the parasitism rate has not been explored. We performed a field experiment to test whether the parasitism rate differs between the first and the second generations of a specialist leaf miner (Tischeria ekebladella), both in the naturally univoltine and bivoltine parts of the leaf miner's distribution. We found an interactive effect between herbivore generation and geographical range on the parasitism rate. The parasitism rate was higher in the first compared to the second host generation in the part of the range that is naturally univoltine, whereas it did not differ between generations in the bivoltine range. Our experiment highlights that shifts in herbivore voltinism might release top-down control, with potential consequences for natural and applied systems.

Optimizing insect metabarcoding using replicated mock communities.

1: Metabarcoding (high-throughput sequencing of marker gene amplicons) has emerged as a promising and cost-effective method for characterizing insect community samples. Yet, the methodology varies greatly among studies and its performance has not been systematically evaluated to date. In particular, it is unclear how accurately metabarcoding can resolve species communities in terms of presence-absence, abundances, and biomass. 2: Here we use mock community experiments and a simple probabilistic model to evaluate the effect of different DNA extraction protocols on metabarcoding performance. Specifically, we ask four questions: (Q1) How consistent are the recovered community profiles across replicate mock communities?; (Q2) How does the choice of lysis buffer affect the recovery of the original community?; (Q3) How are community estimates affected by differing lysis times and homogenization?; and (Q4) Is it possible to obtain adequate species abundance estimates through the use of biological spike-ins? 3: We show that estimates are quite variable across community replicates. In general, a mild lysis protocol is better at reconstructing species lists and approximate counts, while homogenization is better at retrieving biomass composition. Small insects are more likely to be detected in lysates, while some tough species require homogenization to be detected. Results are less consistent across biological replicates for lysates than for homogenates. Some species are associated with strong PCR amplification bias, which complicates the reconstruction of species counts. Yet, with adequate spike-in data, species abundance can be determined with roughly 40% standard error for homogenates, and with roughly 50% standard error for lysates, under ideal conditions. In the latter case, however, this often requires species-specific reference data, while spike-in data generalizes better across species for homogenates. 4: We conclude that a non-destructive, mild lysis approach shows the highest promise for presence/absence description of the community, while also allowing future morphological or molecular work on the material. However, homogenization protocols perform better for characterizing community composition, in particular in terms of biomass.

Reciprocal influence of soil, phyllosphere, and aphid microbiomes.

BACKGROUND: The effect of soil on the plant microbiome is well-studied. However, less is known about the impact of the soil microbiome in multitrophic systems. Here we examined the effect of soil on plant and aphid microbiomes, and the reciprocal effect of aphid herbivory on the plant and soil microbiomes. We designed microcosms, which separate below and aboveground compartments, to grow oak seedlings with and without aphid herbivory in soils with three different microbiomes. We used amplicon sequencing and qPCR to characterize the bacterial and fungal communities in soils, phyllospheres, and aphids. RESULTS: Soil microbiomes significantly affected the microbial communities of phyllospheres and, to a lesser extent, aphid microbiomes, indicating plant-mediated assembly processes from soil to aphids. While aphid herbivory significantly decreased microbial diversity in phyllospheres independent of soil microbiomes, the effect of aphid herbivory on the community composition in soil varied among the three soils. CONCLUSIONS: This study provides experimental evidence for the reciprocal influence of soil, plant, and aphid microbiomes, with the potential for the development of new microbiome-based pest management strategies.

FAVIS: Fast and versatile protocol for non-destructive metabarcoding of bulk insect samples.

Insects are diverse and sustain essential ecosystem functions, yet remain understudied. Recent reports about declines in insect abundance and diversity have highlighted a pressing need for comprehensive large-scale monitoring. Metabarcoding (high-throughput bulk sequencing of marker gene amplicons) offers a cost-effective and relatively fast method for characterizing insect community samples. However, the methodology applied varies greatly among studies, thus complicating the design of large-scale and repeatable monitoring schemes. Here we describe a non-destructive metabarcoding protocol that is optimized for high-throughput processing of Malaise trap samples and other bulk insect samples. The protocol details the process from obtaining bulk samples up to submitting libraries for sequencing. It is divided into four sections: 1) Laboratory workspace preparation; 2) Sample processing-decanting ethanol, measuring the wet-weight biomass and the concentration of the preservative ethanol, performing non-destructive lysis and preserving the insect material for future work; 3) DNA extraction and purification; and 4) Library preparation and sequencing. The protocol relies on readily available reagents and materials. For steps that require expensive infrastructure, such as the DNA purification robots, we suggest alternative low-cost solutions. The use of this protocol yields a comprehensive assessment of the number of species present in a given sample, their relative read abundances and the overall insect biomass. To date, we have successfully applied the protocol to more than 7000 Malaise trap samples obtained from Sweden and Madagascar. We demonstrate the data yield from the protocol using a small subset of these samples.

Different spatial structure of plant-associated fungal communities above- and belowground.

The distribution and community assembly of above- and belowground microbial communities associated with individual plants remain poorly understood, despite its consequences for plant-microbe interactions and plant health. Depending on how microbial communities are structured, we can expect different effects of the microbial community on the health of individual plants and on ecosystem processes. Importantly, the relative role of different factors will likely differ with the scale examined. Here, we address the driving factors at a landscape level, where each individual unit (oak trees) is accessible to a joint species pool. This allowed to quantify the relative effect of environmental factors and dispersal on the distribution of two types of fungal communities: those associated with the leaves and those associated with the soil of Quercus robur trees in a landscape in southwestern Finland. Within each community type, we compared the role of microclimatic, phenological, and spatial variables, and across community types, we examined the degree of association between the respective communities. Most of the variation in the foliar fungal community was found within trees, whereas soil fungal community composition showed positive spatial autocorrelation up to 50 m. Microclimate, tree phenology, and tree spatial connectivity explained little variation in the foliar and soil fungal communities. Foliar and soil fungal communities differed strongly in community structure, with no significant concordance detected between them. We provide evidence that foliar and soil fungal communities assemble independent of each other and are structured by different ecological processes.

Genetic composition and diversity of Arabica coffee in the crop's centre of origin and its impact on four major fungal diseases.

Conventional wisdom states that genetic variation reduces disease levels in plant populations. Nevertheless, crop species have been subject to a gradual loss of genetic variation through selection for specific traits during breeding, thereby increasing their vulnerability to biotic stresses such as pathogens. We explored how genetic variation in Arabica coffee sites in southwestern Ethiopia was related to the incidence of four major fungal diseases. Sixty sites were selected along a gradient of management intensity, ranging from nearly wild to intensively managed coffee stands. We used genotyping-by-sequencing of pooled leaf samples (pool-GBS) derived from 16 individual coffee shrubs in each of the 60 sites to assess the variation in genetic composition (multivariate: reference allele frequency) and genetic diversity (univariate: mean expected heterozygosity) between sites. We found that genetic composition had a clear spatial pattern and that genetic diversity was higher in less managed sites. The incidence of the four fungal diseases was related to the genetic composition of the coffee stands, but in a specific way for each disease. In contrast, genetic diversity was only related to the within-site variation of coffee berry disease, but not to the mean incidence of any of the four diseases across sites. Given that fungal diseases are major challenges of Arabica coffee in its native range, our findings that genetic composition of coffee sites impacted the major fungal diseases may serve as baseline information to study the molecular basis of disease resistance in coffee. Overall, our study illustrates the need to consider both host genetic composition and genetic diversity when investigating the genetic basis for variation in disease levels.

From seed to seed: the role of microbial inheritance in the assembly of the plant microbiome.

Despite evidence that the microbiome extends host genetic and phenotypic traits, information on how the microbiome is transmitted and maintained across generations remains fragmented. For seed-bearing plants, seeds harbor a distinct microbiome and play a unique role by linking one generation to the next. Studies on microbial inheritance, a process we suggest including both vertical transmission and the subsequent migration of seed microorganisms to the new plant, thus become essential for our understanding of host evolutionary potential and host-microbiome coevolution. We propose dividing the inheritance process into three stages: (i) plant to seed, (ii) seed dormancy, and (iii) seed to seedling. We discuss the factors affecting the assembly of the microbiome during the three stages, highlight future research directions, and emphasize the implications of microbial inheritance for fundamental science and society.

Spring phenology and pathogen infection affect multigenerational plant attackers throughout the growing season.

Climate change has been shown to advance spring phenology, increase the number of insect generations per year (multivoltinism) and increase pathogen infection levels. However, we lack insights into the effects of plant spring phenology and the biotic environment on the preference and performance of multivoltine herbivores and whether such effects extend into the later part of the growing season. To this aim, we used a multifactorial growth chamber experiment to examine the influence of spring phenology on plant pathogen infection, and how the independent and interactive effects of spring phenology and plant pathogen infection affect the preference and performance of multigenerational attackers (the leaf miner Tischeria ekebladella and the aphid Tuberculatus annulatus) on the pedunculate oak in the early, mid and late parts of the plant growing season. Pathogen infection was highest on late phenology plants, irrespective of whether inoculations were conducted in the early, mid or late season. The leaf miner consistently preferred to oviposit on middle and late phenology plants, as well as healthy plants, during all parts of the growing season, whereas we detected an interactive effect between spring phenology and pathogen infection on the performance of the leaf miner. Aphids preferred healthy, late phenology plants during the early season, healthy plants during the mid season, and middle phenology plants during the late season, whereas aphid performance was consistently higher on healthy plants during all parts of the growing season. Our findings highlight that the impact of spring phenology on pathogen infection and the preference and performance of insect herbivores is not restricted to the early season, but that its imprint is still present - and sometimes equally strong - during the peak and end of the growing season. Plant pathogens generally negatively affected herbivore preference and performance, and modulated the effects of spring phenology. We conclude that spring phenology and pathogen infection are two important factors shaping the preference and performance of multigenerational plant attackers, which is particularly relevant given the current advance in spring phenology, pathogen outbreaks and increase in voltinism with climate change.

Single, but not dual, attack by a biotrophic pathogen and a sap-sucking insect affects the oak leaf metabolome.

Plants interact with a multitude of microorganisms and insects, both below- and above ground, which might influence plant metabolism. Despite this, we lack knowledge of the impact of natural soil communities and multiple aboveground attackers on the metabolic responses of plants, and whether plant metabolic responses to single attack can predict responses to dual attack. We used untargeted metabolic fingerprinting (gas chromatography-mass spectrometry, GC-MS) on leaves of the pedunculate oak, Quercus robur, to assess the metabolic response to different soil microbiomes and aboveground single and dual attack by oak powdery mildew (Erysiphe alphitoides) and the common oak aphid (Tuberculatus annulatus). Distinct soil microbiomes were not associated with differences in the metabolic profile of oak seedling leaves. Single attacks by aphids or mildew had pronounced but different effects on the oak leaf metabolome, but we detected no difference between the metabolomes of healthy seedlings and seedlings attacked by both aphids and powdery mildew. Our findings show that aboveground attackers can have species-specific and non-additive effects on the leaf metabolome of oak. The lack of a metabolic signature detected by GC-MS upon dual attack might suggest the existence of a potential negative feedback, and highlights the importance of considering the impacts of multiple attackers to gain mechanistic insights into the ecology and evolution of species interactions and the structure of plant-associated communities, as well as for the development of sustainable strategies to control agricultural pests and diseases and plant breeding.

Elevation and plant species identity jointly shape a diverse arbuscular mycorrhizal fungal community in the High Arctic.

Knowledge about the distribution and local diversity patterns of arbuscular mycorrhizal (AM) fungi are limited for extreme environments such as the Arctic, where most studies have focused on spore morphology or root colonization. We here studied the joint effects of plant species identity and elevation on AM fungal distribution and diversity. We sampled roots of 19 plant species in 18 locations in Northeast Greenland, using next generation sequencing to identify AM fungi. We studied the joint effect of plant species, elevation and selected abiotic conditions on AM fungal presence, richness and composition. We identified 29 AM fungal virtual taxa (VT), of which six represent putatively new VT. Arbuscular mycorrhizal fungal presence increased with elevation, and as vegetation cover and the active soil layer decreased. Arbuscular mycorrhizal fungal composition was shaped jointly by elevation and plant species identity. We demonstrate that the Arctic harbours a relatively species-rich and nonrandomly distributed diversity of AM fungi. Given the high diversity and general lack of knowledge exposed herein, we encourage further research into the diversity, drivers and functional role of AM fungi in the Arctic. Such insight is urgently needed for an area with some of the globally highest rates of climate change.

The co-existence of multiple oak leaf flushes contributes to the large within-tree variation in chemistry, insect attack and pathogen infection.

Many plant species produce multiple leaf flushes during the growing season, which might have major consequences for within-plant variation in chemistry and species interactions. Yet, we lack a theoretical or empirical framework for how differences among leaf flushes might shape variation in damage by insects and diseases. We assessed the impact of leaf flush identity on leaf chemistry, insect attack and pathogen infection on the pedunculate oak Quercus robur by sampling leaves from each leaf flush in 20 populations across seven European countries during an entire growing season. The first leaf flush had higher levels of primary compounds, and lower levels of secondary compounds, than the second flush, whereas plant chemistry was highly variable in the third flush. Insect attack decreased from the first to the third flush, whereas infection by oak powdery mildew was lowest on leaves from the first flush. The relationship between plant chemistry, insect attack and pathogen infection varied strongly among leaf flushes and seasons. Our findings demonstrate the importance of considering differences among leaf flushes for our understanding of within-tree variation in chemistry, insect attack and disease levels, something particularly relevant given the expected increase in the number of leaf flushes with climate change.

Climate variability and aridity modulate the role of leaf shelters for arthropods: A global experiment.

Current climate change is disrupting biotic interactions and eroding biodiversity worldwide. However, species sensitive to aridity, high temperatures, and climate variability might find shelter in microclimatic refuges, such as leaf rolls built by arthropods. To explore how the importance of leaf shelters for terrestrial arthropods changes with latitude, elevation, and climate, we conducted a distributed experiment comparing arthropods in leaf rolls versus control leaves across 52 sites along an 11,790 km latitudinal gradient. We then probed the impact of short- versus long-term climatic impacts on roll use, by comparing the relative impact of conditions during the experiment versus average, baseline conditions at the site. Leaf shelters supported larger organisms and higher arthropod biomass and species diversity than non-rolled control leaves. However, the magnitude of the leaf rolls' effect differed between long- and short-term climate conditions, metrics (species richness, biomass, and body size), and trophic groups (predators vs. herbivores). The effect of leaf rolls on predator richness was influenced only by baseline climate, increasing in magnitude in regions experiencing increased long-term aridity, regardless of latitude, elevation, and weather during the experiment. This suggests that shelter use by predators may be innate, and thus, driven by natural selection. In contrast, the effect of leaf rolls on predator biomass and predator body size decreased with increasing temperature, and increased with increasing precipitation, respectively, during the experiment. The magnitude of shelter usage by herbivores increased with the abundance of predators and decreased with increasing temperature during the experiment. Taken together, these results highlight that leaf roll use may have both proximal and ultimate causes. Projected increases in climate variability and aridity are, therefore, likely to increase the importance of biotic refugia in mitigating the effects of climate change on species persistence.

Herbivory in a changing climate-Effects of plant genotype and experimentally induced variation in plant phenology on two summer-active lepidopteran herbivores and one fungal pathogen.

With climate change, spring warming tends to advance plant leaf-out. While the timing of leaf-out has been shown to affect the quality of leaves for herbivores in spring, it is unclear whether such effects extend to herbivores active in summer. In this study, we first examined how spring and autumn phenology of seven Quercus robur genotypes responded to elevated temperatures in spring. We then tested whether the performance of two summer-active insect herbivores (Orthosia gothica and Polia nebulosa) and infection by a pathogen (Erysiphe alphitoides) were influenced by plant phenology, traits associated with genotype or the interaction between these two. Warm spring temperatures advanced both bud development and leaf senescence in Q. robur. Plants of different genotype differed in terms of both spring and autumn phenology. Plant phenology did not influence the performance of two insect herbivores and a pathogen, while traits associated with oak genotype had an effect on herbivore performance. Weight gain for O. gothica and ingestion for P. nebulosa differed by a factor of 4.38 and 2.23 among genotypes, respectively. Herbivore species active in summer were influenced by traits associated with plant genotype but not by phenology. This suggest that plant attackers active in summer may prove tolerant to shifts in host plant phenology-a pattern contrasting with previously documented effects on plant attackers active in spring and autumn.

The relationship between pathogen life-history traits and metapopulation dynamics.

Plant pathogen traits, such as transmission mode and overwintering strategy, may have important effects on dispersal and persistence, and drive disease dynamics. Still, we lack insights into how life-history traits influence spatiotemporal disease dynamics. We adopted a multifaceted approach, combining experimental assays, theory and field surveys, to investigate whether information about two pathogen life-history traits - infectivity and overwintering strategy - can predict pathogen metapopulation dynamics in natural systems. For this, we focused on four fungal pathogens (two rust fungi, one chytrid fungus and one smut fungus) on the forest herb Anemone nemorosa. Pathogens infecting new plants mostly via spores (the chytrid and smut fungi) had higher patch occupancies and colonization rates than pathogens causing mainly systemic infections and overwintering in the rhizomes (the two rust fungi). Although the rust fungi more often occupied well-connected plant patches, the chytrid and smut fungi were equally or more common in isolated patches. Host patch size was positively related to patch occupancy and colonization rates for all pathogens. Predicting disease dynamics is crucial for understanding the ecological and evolutionary dynamics of host-pathogen interactions, and to prevent disease outbreaks. Our study shows that combining experiments, theory and field observations is a useful way to predict disease dynamics.