Microbiomes and metabolomes of dominant coral reef primary producers illustrate a potential role for immunolipids in marine symbioses.

The dominant benthic primary producers in coral reef ecosystems are complex holobionts with diverse microbiomes and metabolomes. In this study, we characterize the tissue metabolomes and microbiomes of corals, macroalgae, and crustose coralline algae via an intensive, replicated synoptic survey of a single coral reef system (Waimea Bay, O'ahu, Hawaii) and use these results to define associations between microbial taxa and metabolites specific to different hosts. Our results quantify and constrain the degree of host specificity of tissue metabolomes and microbiomes at both phylum and genus level. Both microbiome and metabolomes were distinct between calcifiers (corals and CCA) and erect macroalgae. Moreover, our multi-omics investigations highlight common lipid-based immune response pathways across host organisms. In addition, we observed strong covariation among several specific microbial taxa and metabolite classes, suggesting new metabolic roles of symbiosis to further explore.

Hawaiian coral holobionts reveal algal and prokaryotic host specificity, intraspecific variability in bleaching resistance, and common interspecific microbial consortia modulating thermal stress responses.

Historically, Hawai'i had few massive coral bleaching events, until two consecutive heatwaves in 2014-2015. Consequent mortality and thermal stress were observed in Kane'ohe Bay (O'ahu). The two most dominant local species exhibited a phenotypic dichotomy of either bleaching resistance or susceptibility (Montipora capitata and Porites compressa), while the third predominant species (Pocillopora acuta) was broadly susceptible to bleaching. In order to survey shifts in coral microbiomes during bleaching and recovery, 50 colonies were tagged and periodically monitored. Metabarcoding of three genetic markers (16S rRNA gene ITS1 and ITS2) followed by compositional approaches for community structure analysis, differential abundance and correlations for longitudinal data were used to temporally compare Bacteria/Archaea, Fungi and Symbiodiniaceae dynamics. P. compressa corals recovered faster than P. acuta and Montipora capitata. Prokaryotic and algal communities were majorly shaped by host species, and had no apparent pattern of temporal acclimatization. Symbiodiniaceae signatures were identified at the colony scale, and were often related to bleaching susceptibility. Bacterial compositions were practically constant between bleaching phenotypes, and more diverse in P. acuta and M. capitata. P. compressa's prokaryotic community was dominated by a single bacterium. Compositional approaches (via microbial balances) allowed the identification of fine-scale differences in the abundance of a consortium of microbes, driving changes by bleaching susceptibility and time across all hosts. The three major coral reef founder-species in Kane'ohe Bay revealed different phenotypic and microbiome responses after 2014-2015 heatwaves. It is difficult to forecast, a more successful strategy towards future scenarios of global warming. Differentially abundant microbial taxa across time and/or bleaching susceptibility were broadly shared among all hosts, suggesting that locally, the same microbes may modulate stress responses in sympatric coral species. Our study highlights the potential of investigating microbial balances to identify fine-scale microbiome changes, serving as local diagnostic tools of coral reef fitness.

A ridge-to-reef ecosystem microbial census reveals environmental reservoirs for animal and plant microbiomes.

Microbes are found in nearly every habitat and organism on the planet, where they are critical to host health, fitness, and metabolism. In most organisms, few microbes are inherited at birth; instead, acquiring microbiomes generally involves complicated interactions between the environment, hosts, and symbionts. Despite the criticality of microbiome acquisition, we know little about where hosts' microbes reside when not in or on hosts of interest. Because microbes span a continuum ranging from generalists associating with multiple hosts and habitats to specialists with narrower host ranges, identifying potential sources of microbial diversity that can contribute to the microbiomes of unrelated hosts is a gap in our understanding of microbiome assembly. Microbial dispersal attenuates with distance, so identifying sources and sinks requires data from microbiomes that are contemporary and near enough for potential microbial transmission. Here, we characterize microbiomes across adjacent terrestrial and aquatic hosts and habitats throughout an entire watershed, showing that the most species-poor microbiomes are partial subsets of the most species-rich and that microbiomes of plants and animals are nested within those of their environments. Furthermore, we show that the host and habitat range of a microbe within a single ecosystem predicts its global distribution, a relationship with implications for global microbial assembly processes. Thus, the tendency for microbes to occupy multiple habitats and unrelated hosts enables persistent microbiomes, even when host populations are disjunct. Our whole-watershed census demonstrates how a nested distribution of microbes, following the trophic hierarchies of hosts, can shape microbial acquisition.

specificity: an R package for analysis of feature specificity to environmental and higher dimensional variables, applied to microbiome species data.

BACKGROUND: Understanding the factors that influence microbes' environmental distributions is important for determining drivers of microbial community composition. These include environmental variables like temperature and pH, and higher-dimensional variables like geographic distance and host species phylogeny. In microbial ecology, "specificity" is often described in the context of symbiotic or host parasitic interactions, but specificity can be more broadly used to describe the extent to which a species occupies a narrower range of an environmental variable than expected by chance. Using a standardization we describe here, Rao's (Theor Popul Biol, 1982. https://doi.org/10.1016/0040-5809(82)90004-1, Sankhya A, 2010. https://doi.org/10.1007/s13171-010-0016-3 ) Quadratic Entropy can be conveniently applied to calculate specificity of a feature, such as a species, to many different environmental variables. RESULTS: We present our R package specificity for performing the above analyses, and apply it to four real-life microbial data sets to demonstrate its application. We found that many fungi within the leaves of native Hawaiian plants had strong specificity to rainfall and elevation, even though these variables showed minimal importance in a previous analysis of fungal beta-diversity. In Antarctic cryoconite holes, our tool revealed that many bacteria have specificity to co-occurring algal community composition. Similarly, in the human gut microbiome, many bacteria showed specificity to the composition of bile acids. Finally, our analysis of the Earth Microbiome Project data set showed that most bacteria show strong ontological specificity to sample type. Our software performed as expected on synthetic data as well. CONCLUSIONS: specificity is well-suited to analysis of microbiome data, both in synthetic test cases, and across multiple environment types and experimental designs. The analysis and software we present here can reveal patterns in microbial taxa that may not be evident from a community-level perspective. These insights can also be visualized and interactively shared among researchers using specificity's companion package, specificity.shiny.

Hawaiian Fungal Amplicon Sequence Variants Reveal Otherwise Hidden Biogeography.

To study biogeography and other ecological patterns of microorganisms, including fungi, scientists have been using operational taxonomic units (OTUs) as representations of species or species hypotheses. However, when defined by 97% sequence similarity cutoff at an accepted barcode locus such as 16S in bacteria or ITS in fungi, these OTUs can obscure biogeographic patterns, mask taxonomic diversity, and hinder meta-analyses. Amplicon sequence variants (ASVs) have been proposed to alleviate all of these issues and have been shown to do so in bacteria. Analyzing ASVs is just emerging as a common practice among fungal studies, and it is unclear whether the benefits found in bacterial studies of using such an approach carryover to fungi. Here, we conducted a meta-analysis of Hawaiian fungi by analyzing ITS1 amplicon sequencing data as ASVs and exploring ecological patterns. These surveys spanned three island groups and five ecosystems combined into the first comprehensive Hawaiian Mycobiome ASV Database. Our results show that ASVs can be used to combine fungal ITS surveys, increase reproducibility, and maintain the broad ecological patterns observed with OTUs, including diversity orderings. Additionally, the ASVs that comprise some of the most common OTUs in our database reveals some island specialists, indicating that traditional OTU clustering can obscure important biogeographic patterns. We recommend that future fungal studies, especially those aimed at assessing biogeography, analyze ASVs rather than OTUs. We conclude that similar to bacterial studies, ASVs improve reproducibility and data sharing for fungal studies.

Fungi in soil and understory have coupled distribution patterns.

Ecological processes that control fungal distribution are not well understood because many fungi can persist in a wide variety of dissimilar habitats which are seldom sampled simultaneously. Geographic range size is reflective of species' resource usage, and for plants and animals, there is a robust positive correlation between niche-breadth and range-size. It remains unknown whether this pattern is true for fungi. To investigate the fungal niche breadth-range size relationship we identified habitat specialists and generalists from two habitats (plant leaves and soil) and asked whether habitat specialization influenced fungal biogeography. We sampled fungi from the soil and phylloplane of tropical forests in Vanuatu and used DNA metabarcoding of the fungal ITS1 region to examine rarity, range size, and habitat connectivity. Fungal communities from the soil and phylloplane are spatially autocorrelated and the spatial distribution of individual fungal OTU are coupled between habitats. Habitat breadth (generalist fungi) did not result in larger range sizes but did correlate positively with occurrence frequency. Fungi that were frequently found were also found in high abundance, a common observation in similar studies of plants and animals. Fungal abundance-occupancy relationships differed by habitat and habitat-specificity. Soil specialists were found to be locally abundant but restricted geographically. In contrast, phylloplane generalists were found to be abundant over a large range in multiple habitats. These results are discussed in the context of differences between habitat characteristics, stability and spatial distribution. Identifying factors that drive spatial variation is key to understanding the mechanisms that maintain biodiversity in forests.

Evolution and Physiology of Amphibious Yeasts.

Since the emergence of the first fungi some 700 million years ago, unicellular yeast-like forms have emerged multiple times in independent lineages via convergent evolution. While tens to hundreds of millions of years separate the independent evolution of these unicellular organisms, they share remarkable phenotypic and metabolic similarities, and all have streamlined genomes. Yeasts occur in every aquatic environment yet examined. Many species are aquatic; perhaps most are amphibious. How these species have evolved to thrive in aquatic habitats is fundamental to understanding functions and evolutionary mechanisms in this unique group of fungi. Here we review the state of knowledge of the physiological and ecological diversity of amphibious yeasts and their key evolutionary adaptations enabling survival in aquatic habitats. We emphasize some genera previously thought to be exclusively terrestrial. Finally, we discuss the ability of many yeasts to survive in extreme habitats and how this might lend insight into ecological plasticity, including amphibious lifestyles.

Scale-Dependent Influences of Distance and Vegetation on the Composition of Aboveground and Belowground Tropical Fungal Communities.

Fungi provide essential ecosystem services and engage in a variety of symbiotic relationships with trees. In this study, we investigate the spatial relationship of trees and fungi at a community level. We characterized the spatial dynamics for above- and belowground fungi using a series of forest monitoring plots, at nested spatial scales, located in the tropical South Pacific, in Vanuatu. Fungal communities from different habitats were sampled using metagenomic analysis of the nuclear ribosomal ITS1 region. Fungal communities exhibited strong distance-decay of similarity across our entire sampling range (3-110,000 m) and also at small spatial scales (< 50 m). Unexpectedly, this pattern was inverted at an intermediate scale (3.7-26 km). At large scales (80-110 km), belowground and aboveground fungal communities responded inversely to increasing geographic distance. Aboveground fungal community turnover (beta diversity) was best explained, at all scales, by geographic distance. In contrast, belowground fungal community turnover was best explained by geographic distance at small scales and tree community composition at large scales. Fungal communities from various habitats respond differently to the influences of habitat and geographic distance. At large geographic distances (80-110 km), community turnover for aboveground fungi is better explained by spatial distance, whereas community turnover for belowground fungi is better explained by plant community turnover. Future syntheses of spatial dynamics among fungal communities must explicitly consider geographic scale to appropriately contextualize community turnover.

Plant part and a steep environmental gradient predict plant microbial composition in a tropical watershed.

Plant microbiomes are shaped by forces working at different spatial scales. Environmental factors determine a pool of potential symbionts while host physiochemical factors influence how those microbes associate with distinct plant tissues. These scales are seldom considered simultaneously, despite their potential to interact. Here, we analyze epiphytic microbes from nine Hibiscus tiliaceus trees across a steep, but short, environmental gradient within a single Hawaiian watershed. At each location, we sampled eight microhabitats: leaves, petioles, axils, stems, roots, and litter from the plant, as well as surrounding air and soil. The composition of bacterial communities is better explained by microhabitat, while location better predicted compositional variance for fungi. Fungal community compositional dissimilarity increased more rapidly along the gradient than did bacterial composition. Additionally, the rates of fungal community compositional dissimilarity along the gradient differed among plant parts, and these differences influenced the distribution patterns and range size of individual taxa. Within plants, microbes were compositionally nested such that aboveground communities contained a subset of the diversity found belowground. Our findings indicate that both environmental context and microhabitat contribute to microbial compositional variance in our study, but that these contributions are influenced by the domain of microbe and the specific microhabitat in question, suggesting a complicated and potentially interacting dynamic.

Fungal communities living within leaves of native Hawaiian dicots are structured by landscape-scale variables as well as by host plants.

A phylogenetically diverse array of fungi live within healthy leaf tissue of dicotyledonous plants. Many studies have examined these endophytes within a single plant species and/or at small spatial scales, but landscape-scale variables that determine their community composition are not well understood, either across geographic space, across climatic conditions, or in the context of host plant phylogeny. Here, we evaluate the contributions of these variables to endophyte beta diversity using a survey of foliar endophytic fungi in native Hawaiian dicots sampled across the Hawaiian archipelago. We used Illumina technology to sequence fungal ITS1 amplicons to characterize foliar endophyte communities across five islands and 80 host plant genera. We found that communities of foliar endophytic fungi showed strong geographic structuring between distances of 7 and 36 km. Endophyte community structure was most strongly associated with host plant phylogeny and evapotranspiration, and was also significantly associated with NDVI, elevation and solar radiation. Additionally, our bipartite network analysis revealed that the five islands we sampled each harboured significantly specialized endophyte communities. These results demonstrate how the interaction of factors at large and small spatial and phylogenetic scales shapes fungal symbiont communities.

Fungal aerobiota are not affected by time nor environment over a 13-y time series at the Mauna Loa Observatory.

Fungi are ubiquitous and often abundant components of virtually all ecosystems on Earth, serving a diversity of functions. While there is clear evidence that fungal-mediated processes can influence environmental conditions, and in turn select for specific fungi, it is less clear how fungi respond to environmental fluxes over relatively long time frames. Here we set out to examine changes in airborne fungi collected over the course of 13 y, which is the longest sampling time to date. Air filter samples were collected from the Mauna Loa Observatory (MLO) on Hawaii Island, and analyzed using Illumina amplicon sequencing. As a study site, MLO is unique because of its geographic isolation and high elevation, making it an ideal place to capture global trends in climate and aerobiota. We found that the fungal aerobiota sampled at MLO had high species turnover, but compositional similarity did not decrease as a function of time between samples. We attribute these patterns to neutral processes such as idiosyncratic dispersal timing and trajectories. Furthermore, the composition of fungi at any given point was not significantly influenced by any local or global environmental variables we examined. This, and our additional finding of a core set of persistent fungi during our entire sampling period, indicates some degree of stability among fungi in the face of natural environmental fluctuations and human-associated global change. We conclude that the movement of fungi through the atmosphere is a relatively stochastic process.

Plant-microbe specificity varies as a function of elevation.

Specialized associations between interacting species fundamentally determine the diversity and distribution of both partners. How the specialization of guilds of organisms varies along environmental gradients underpins popular theories of biogeography and macroecology, whereas the degree of specialization of a species is typically considered fixed. However, the extent to which environmental context impacts specialization dynamics is seldom examined empirically. In this study, we examine how specialization within a bipartite network consisting of three co-occurring plant species and their foliar fungal endophyte symbionts changes along a 1000-meter elevation gradient where host species were held constant. The gradient, along the slope of Mauna Loa shield volcano, represents almost the entire elevational range of two of the three plants. Network and plant specialization values displayed a parabolic relationship with elevation, and were highest at middle elevations, whereas bipartite associations were least specific at low and high elevations. Shannon's diversity of fungal endophytes correlated negatively with specificity, and was highest at the ends of the transects. Although plant host was a strong determinant of fungal community composition within sites, fungal species turnover was high among sites. There was no evidence of spatial or elevational patterning in fungal community compositon. Our work demonstrates that specificity can be a plastic trait, which is influenced by the environment and centrality of the host within its natural range.

Targeted ITS1 sequencing unravels the mycodiversity of deep-sea sediments from the Gulf of Mexico.

Fungi from marine environments have been significantly less studied than terrestrial fungi. This study describes distribution patterns and associated habitat characteristics of the mycobiota of deep-sea sediments collected from the Mexican exclusive economic zone (EEZ) of the Gulf of Mexico (GoM), ranging between 1000 and > 3500 m depth. Internal Transcribed Spacer 1 (ITS1) amplicons were sequenced by Illumina MiSeq. From 29 stations sampled across three annual campaigns, a total of 4421 operational taxonomic units (OTUs) were obtained, indicating a high fungal richness. Most OTUs assignments corresponded to Ascomycota, unidentified fungi and Basidiomycota. The majority of the stations shared a mere 31 OTUs, including the worldwide reported genera Penicillium, Rhodotorula and Cladosporium. Both a transient and a conserved community were identified, suggesting their dependence on or adaptation to the habitat dynamics, respectively. The differences found in fungal richness and taxonomic compositions were correlated principally with latitude, carbon and carbonates content, and terrigenous content, which could be the potential drivers that delimit fungal distribution. This study represents an expansion of our current knowledge on the biogeography of the fungal community from deep-sea sediments, and identifies the geographic and physicochemical properties that delimit fungal composition and distribution in the GoM.

Phytobiomes are compositionally nested from the ground up.

Plant-associated microbes are critical players in host health, fitness and productivity. Despite microbes' importance in plants, seeds are mostly sterile, and most plant microbes are recruited from an environmental pool. Surprisingly little is known about the processes that govern how environmental microbes assemble on plants in nature. In this study we examine how bacteria are distributed across plant parts, and how these distributions interact with spatial gradients. We sequenced amplicons of bacteria from the surfaces of six plant parts and adjacent soil of Scaevola taccada, a common beach shrub, along a 60 km transect spanning O'ahu island's windward coast, as well as within a single intensively-sampled site. Bacteria are more strongly partitioned by plant part as compared with location. Within S. taccada plants, microbial communities are highly nested: soil and rhizosphere communities contain much of the diversity found elsewhere, whereas reproductive parts fall at the bottom of the nestedness hierarchy. Nestedness patterns suggest either that microbes follow a source/sink gradient from the ground up, or else that assembly processes correlate with other traits, such as tissue persistence, that are vertically stratified. Our work shines light on the origins and determinants of plant-associated microbes across plant and landscape scales.

Marine fungi.

Fungi play a dominant role in terrestrial environments where they thrive in symbiotic associations with plants and animals and are integral to nutrient cycling in diverse ecosystems. Everywhere that moisture and a carbon source coexist in the terrestrial biosphere, fungi are expected to occur. We know that fungi can be devastating to agricultural crops, both in the field and during their storage, and cause mortality in immunocompromised patients in numbers that rival the deaths from malaria. Yet fungi can also be harnessed as sources of food, chemicals and biofuels when humans exploit fungal metabolism. Despite their central role in the health and disease of the terrestrial biosphere, much less is known about the function and potential of marine fungi. Are fungi ubiquitous in marine environments as they are on land? Do they play the same or similar roles in these ecosystems? Here we describe the state of knowledge about the abundance and functions of fungi in the marine environment with a goal to stimulate new inquiry in this very open area.

Synergy among Microbiota and Their Hosts: Leveraging the Hawaiian Archipelago and Local Collaborative Networks To Address Pressing Questions in Microbiome Research.

Despite increasing acknowledgment that microorganisms underpin the healthy functioning of basically all multicellular life, few cross-disciplinary teams address the diversity and function of microbiota across organisms and ecosystems. Our newly formed consortium of junior faculty spanning fields such as ecology and geoscience to mathematics and molecular biology from the University of Hawai'i at Manoa aims to fill this gap. We are united in our mutual interest in advancing a new paradigm for biology that incorporates our modern understanding of the importance of microorganisms. As our first concerted research effort, we will assess the diversity and function of microbes across an entire watershed on the island of Oahu, Hawai'i. Due to its high ecological diversity across tractable areas of land and sea, Hawai'i provides a model system for the study of complex microbial communities and the processes they mediate. Owing to our diverse expertise, we will leverage this study system to advance the field of biology.

Foliar microbiome transplants confer disease resistance in a critically-endangered plant.

There has been very little effort to incorporate foliar microbiomes into plant conservation efforts even though foliar endophytes are critically important to the fitness and function of hosts. Many critically endangered plants that have been extirpated from the wild are dependent on regular fungicidal applications in greenhouses that cannot be maintained for remote out-planted populations, which quickly perish. These fungicides negatively impact potentially beneficial fungal symbionts, which may reduce plant defenses to pathogens once fungicide treatments are stopped. Using the host/parasite system of Phyllostegia kaalaensis and Neoerysiphe galeopsidis, we conducted experiments to test total foliar microbiome transplants from healthy wild relatives onto fungicide-dependent endangered plants in an attempt to mitigate disease and reduce dependency on fungicides. Plants were treated with total microbiome transplants or cultured subsets of this community and monitored for disease severity. High-throughput DNA screening of fungal ITS1 rDNA was used to track the leaf-associated fungal communities and evaluate the effectiveness of transplantation methods. Individuals receiving traditionally isolated fungal treatments showed no improvement, but those receiving applications of a simple leaf slurry containing an uncultured fungal community showed significant disease reduction, to which we partially attribute an increase in the mycoparasitic Pseudozyma aphidis. These results were replicated in two independent experimental rounds. Treated plants have since been moved to a native habitat and, as of this writing, remain disease-free. Our results demonstrate the effectiveness of a simple low-tech method for transferring beneficial microbes from healthy wild plants to greenhouse-raised plants with reduced symbiotic microbiota. This technique was effective at reducing disease, and in conferring increased survival to an out-planted population of critically endangered plants. It was not effective in a closely related plant. Plant conservation efforts should strive to include foliar microbes as part of comprehensive management plans.

Uncovering unseen fungal diversity from plant DNA banks.

Throughout the world DNA banks are used as storage repositories for genetic diversity of organisms ranging from plants to insects to mammals. Designed to preserve the genetic information for organisms of interest, these banks also indirectly preserve organisms' associated microbiomes, including fungi associated with plant tissues. Studies of fungal biodiversity lag far behind those of macroorganisms, such as plants, and estimates of global fungal richness are still widely debated. Utilizing previously collected specimens to study patterns of fungal diversity could significantly increase our understanding of overall patterns of biodiversity from snapshots in time. Here, we investigated the fungi inhabiting the phylloplane among species of the endemic Hawaiian plant genus, Clermontia (Campanulaceae). Utilizing next generation DNA amplicon sequencing, we uncovered approximately 1,780 fungal operational taxonomic units from just 20 DNA bank samples collected throughout the main Hawaiian Islands. Using these historical samples, we tested the macroecological pattern of decreasing community similarity with decreasing geographic proximity. We found a significant distance decay pattern among Clermontia associated fungal communities. This study provides the first insights into elucidating patterns of microbial diversity through the use of DNA bank repository samples.

Fungi associated with mesophotic macroalgae from the 'Au'au Channel, west Maui are differentiated by host and overlap terrestrial communities.

Mesophotic coral ecosystems are an almost entirely unexplored and undocumented environment that likely contains vast reservoirs of undescribed biodiversity. Twenty-four macroalgae samples, representing four genera, were collected from a Hawaiian mesophotic reef at water depths between 65 and 86 m in the 'Au'au Channel, Maui, Hawai'i. Algal tissues were surveyed for the presence and diversity of fungi by sequencing the ITS1 gene using Illumina technology. Fungi from these algae were then compared to previous fungal surveys conducted in Hawaiian terrestrial ecosystems. Twenty-seven percent of the OTUs present on the mesophotic coral ecosystem samples were shared between the marine and terrestrial environment. Subsequent analyses indicated that host species of algae significantly differentiate fungal community composition. This work demonstrates yet another understudied habitat with a moderate diversity of fungi that should be considered when estimating global fungal diversity.

Not just browsing: an animal that grazes phyllosphere microbes facilitates community heterogeneity.

Although grazers have long been recognized as top-down architects of plant communities, animal roles in determining microbial community composition have seldom been examined, particularly in aboveground systems. To determine the extent to which an animal can shape microbial communities, we conducted a controlled mesocosm study in situ to see if introducing mycophageous tree snails changed phyllosphere fungal community composition relative to matched control mesocosms. Fungal community composition and change was determined by Illumina sequencing of DNA collected from leaf surfaces before snails were introduced, daily for 3 days and weekly for 6 weeks thereafter. Scanning electron microscopy was used to confirm that grazing had occurred, and we recorded 3.5 times more cover of fungal hyphae in control mesocosms compared with those containing snails. Snails do not appear to vector novel microbes and despite grazing, a significant proportion of the initial leaf phyllosphere persisted in the mesocosms. Within-mesocosm diversities of fungi were similar regardless of whether or not snails were added. The greatest differences between the snail-treated and control mesocosms was that grazed mesocosms showed greater infiltration of microbes that were not sampled when the experiment commenced and that the variance in fungal community composition (beta diversity) was greater between leaves in snail-treated mesocosms indicating increased community heterogeneity and ecosystem fragmentation.