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

Abstract

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.

Figure 1

(a) We placed mycophageous tree snails into five mesh mesocosms of ~36 l. A matched snail-free control mesocosm was placed adjacent to each snail-treated mesocosm, making up ten mesocosms in total. (b) To determine how snails changed the fungal community composition of the phyllosphere we swabbed leaves before we introduced the snails, then daily (3 days) and weekly thereafter for 6 weeks. We extracted DNA from the swabs and Illumina sequenced the ITS1 intergenic region to identify which fungus were present at each time point. To confirm whether grazing by snails reduced microbial abundance we took scanning electron microscope images of leaf surfaces from (c) snail-treated and (d) control mesocosms on the final day of the experiment.

Figure 2

Raw abundance data over time. The abundances of the 17 most abundant fungal taxa from within the mesocosms (which together comprised two-thirds of the total read count) are shown. The plots are ordered by effect size, with OTU_5637, OTU_1 and OTU_10 having the greatest effect size under the treatment × time interaction and OTU_15, OTU_8 and OTU_5 having lower effects. Data are log transformed and lines are fitted by local polynomial regression (loss).

Figure 3

Sources of phyllosphere fungi and the heterogeneity of the assemblage. There was considerable community inertia as fungi persisted through the course of the experiment and were present in the initial phyllosphere. However, fungi from the environmental species pool (blue area of figure) also infiltrated the mesocosm, but this was more so in the snail-treated mesocosm, (a) than the control mesocosms (b). The microbes in the feces of snails was also determined before snails were introduced into the mesocosms to determine if snail feces would be a source for fungus being vectored into the new community. These fecal samples were not a significant part of the community assemblage, occurring in a large part in only one sample point in mesocosm G, where it is likely that a fecal sample adhering to a leaf as the commencement of the experiment was inadvertently sampled. (c) Despite the persistence of certain OTUs inside the mesocosms, the heterogeneity of these OTUs was greater in the snail-treated mesocosms, where heterogeneity is measured as degree of variance as a function of mean (as per Taylor, 1961), then snail-treated mesocosms all have greater heterogeneity than any control mesocosm, which is represented by the slopes and intercepts of all lines fitting the log (variance)~log (mean) being greater for the snail-treated mesocosms than control mesocosms.

Figure 4

Snails increase within-system variability. Each mesocosm was sampled in triplicate before the experiment commenced (T₀) and 6 weeks after the introduction of snails to half of the mesocosms. The within-mesocosm variability of fungal diversity increased if treated with snails, but slightly decreased in the no-snail control mesocosms over the time course of the experiment. Whiskers are 1.5 times the interquartile range±the first and third quartile.

Figure 5

Assessment of grazing impacts on microbial abundances using scanning electron microscopy. Fungal hyphal abundance is reduced by grazing as snail-grazed mesocosms (open boxes) have 3.5 times less fungal biomass than ungrazed controls (striped boxes). Abundances were determined by measuring the surface area occupied by fungal hyphae across transect of leaves at the widest part of the leaf, so that the number of counts was at least 12 along each transect. Whiskers are 1.5 times the interquartile range±the first and third quartile.