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ORIGINAL ARTICLE
Not just browsing: an animal that grazes
phyllosphere microbes facilitates community
heterogeneity
Richard O’Rorke
1,2
, Leah Tooman
1
, Kapono Gaughen
1
, Brenden S Holland
3
and Anthony S Amend
1
1
Department of Botany, University of Hawaii, Honolulu, HI, USA;
2
Environmental Futures Research Institute,
Griffith University, Nathan, Queensland, Australia and
3
Center for Conservation Research and Training,
Pacific Biosciences Research Center, University of Hawaii, Honolulu, HI, USA
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.
The ISME Journal (2017) 11, 1788–1798; doi:10.1038/ismej.2017.52; published online 28 April 2017
Introduction
Animal activity, such as grazing, may be as impor-
tant a factor in determining environmental microbial
community assembly as climatic, geographic, physi-
cal, nutrient or other chemical factors that are
observed routinely in microbial ecology studies. If
heterogeneity is defined as the extent to which
species are aggregated (Taylor, 1961), then animals
may either increase or decrease microbial commu-
nity heterogeneity depending on context and the
nature of the interaction in question (Wardle et al.,
2004; Tylianakis et al., 2008). For example, animals
may promote homogeneity among microbial com-
munities by active gardening. This type of animal
microbial mutualistic interaction has arisen multiple
times in numerous microbial and animal lineages,
including textbook examples in insects such as
termites (Aanen et al., 2002), ambrosia beetles
(Beaver, 1989; Farrell et al., 2001) and leaf cutter
ants (Poulsen and Boomsma, 2005). By vectoring
microbes into an ecosystem, animals shape micro-
bial communities to become more similar between
geographically distinct locations (Aizenberg-
Gershtein et al., 2013; Lax et al., 2014). Animals
may also indirectly perturb microbial composition
by modifying substrate availability or site physio-
chemistry. For example by foraging or burrowing,
mammals may enable an alternative state in which
microbial substrates are simplified by mixing
(Eldridge et al., 2015; Pansu et al., 2015). Alterna-
tively, animals can eat the hosts of microbes making
their substrate range more complex (Bråthen et al.,
2015).
Less studied are cases where animals are directly
impacting wild non-cultivated microbial commu-
nities via direct grazing of microbes. Analogous
literature from vegetation ecology suggests that, at
least for plant communities, the impacts of grazing
depend largely on factors such as productivity,
grazing pressure and on the degree of diet selectivity
of the grazers (Proulx and Mazumder, 1998). At
Correspondence: R O’Rorke, Department of Botany, University of
Hawaii, Room 101, 3190 Maile Way, Honolulu, HI 96822, USA.
E-mail: roro002@aucklanduni.ac.nz
Received 17 July 2016; revised 18 February 2017; accepted
3 March 2017; published online 28 April 2017
The ISME Journal (2017) 11, 1788 –1798
© 2017 International Society for Microbial Ecology All rights reserved 1751-7362/17
www.nature.com/ismej
opposite ends of the spectrum, grazing may result in
either reduced alpha diversity (by selecting for one
or a few dense weedy species), or alternatively,
animals may create gaps in microbial communities
and facilitate novel introductions and variability at
small spatial scales (Denslow, 1985). It is unknown
how these dynamics relate to aboveground microbial
systems in which diversity, growth and dispersal
rates are elevated relative to plants.
Here we determine whether presence of mycopha-
geous tree snails impacts phyllosphere microbial
community homogeneity. Hawaiian tree snails were
once species rich and abundant across the archipe-
lago, but they have succumbed to habitat loss,
predation by exotic animals and a European fad for
collecting numerous beautiful shells into cabinets
(Hadfield, 1986). Tree snails feed continuously and
voraciously throughout the night (O'Rorke et al.,
2016), scraping phyllosphere microbes off of the leaf
surface with a specialized radula. Although caution
must be taken when making generalizations from
mesocosm studies, we tested whether the reintro-
duction of tree snails is likely to increase or decrease
species diversity at a fine scale, here sampled at the
scale of a leaf, by reducing dominant taxa or,
alternatively, by promoting the growth of particular
fungal species. We also assess if snail introduction
will change fungal heterogeneity across a coarser
grain, which is the range over which snails can graze
(that is, by measuring the variance of local alpha
diversity within mesocosms). To test this, we
translocated tree snails to five mesocosms and
measured changes in fungal community composition
over time. These mesocosms were compared with
five matched control mesocosms that remained free
of snails. As many of the fungi consumed by snails
are still viable after passage through their gastro-
intestinal tract (O'Rorke et al., 2016), we also
hypothesized that tree snails engage in microbial
‘farming’by vectoring preferred fungus into the
phyllosphere through fecal deposits as is observed
in other snail systems (Silliman and Newell, 2011).
Materials and methods
Study design
We collected individuals of the non-listed, endemic
O’ahu tree-snail Auriculella diaphana (Acahtinelli-
dae), at a mid-elevation site, from a non-native host
plant Cestrum nocturnum (Solanaceae; night-
blooming jasmine), upon which they subsist in the
study area (Holland et al., 2016), and translocated
them to Metrosideros polymorpha (Myrtaceae), a
native Hawaiian tree species on which arboreal
snails have been observed (Hadfield, 1986; Meyer
et al., 2014; Price et al., 2017). It is highly likely that
mycophageous organisms other than snails can
potentially interact with the fungal operational
taxonomic units (OTUs) inside either control or
snail-treated mesocosms. Although the recipient site
is located within the snails’historical range (Pilsbry
and Cooke, 1912–1914), multiple surveys over the
past three decades indicate that snails have been
locally extirpated. Therefore, the new host plant had
never hosted snails and is 42 km from the remnant
snail population (study site location co-ordinates
available from the Hawaii Department of Land and
Natural Resources upon request). We placed 10 adult
snails on living tree branches inside each of five
mesocosms (50 snails total). Mesocosms were ~ 36 l
cylinders constructed from fiberglass mesh sewn at
the edges and reinforced with polyethelyne tape
(Figure 1). The mesh size (1 mm
2
) reduced light to
the branch by 30% (measured by lux, Light Sensor
LS-BTA (Vernier, Beaverton, OR, USA)). We paired
each of these snail-treated mesocosms with a
mesocosm that was free of snails but otherwise
9µm9µm3µm3µm
Grazed by snails. No-snail control
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.
Phyllosphere microbes
RO’Rorke et al
1789
The ISME Journal
identical, so that the experiment had a total of 10
mesocosms distributed into five blocks. Mesocosms
blocks were located on the same tree (to minimize
host genotype effects) and spaced at least 80 cm
apart. Mesocosms were placed over leaves of the
same age (new season leaves of o4 months). We
transported snails to the mesocosms in sterile 10 cm
polypropylene Petri dishes (Falcon, Corning, NY,
USA). We collected snail feces deposited in transit
(n= 10) and stored them in hexadecyltrimethylam-
monium bromide (CTAB) buffer for subsequent DNA
extraction and Illumina sequencing, these were used
to determine if the snails do vector fungus into new
habitats. Two vials of CTAB buffer were taken into
the field, opened and then stored until the DNA was
extracted for PCR amplification and sequencing,
these served as negative controls for field contam-
ination during the sampling process.
We sampled mesocosms at 1300 hours Hawaiian
standard time before the snails were introduced (T
0
)
and then near this time (±30 min) each day for the
subsequent 3 days and then weekly thereafter for a
total of 6 weeks. This duration is sufficient to detect
community change in other studies (Lax et al., 2014).
For each sampling event, we swabbed the entire top
and bottom surfaces of three leaves (44cm in
length) from each mesocosm and stored the swabs
in CTAB buffer until DNA extraction and Illumina
sequencing. Triplicate swabs of individual leaves
(that is, biological replicates, not triplicate technical
replicates of the same leaves) were taken from
mesocosms at T
final
, and T
0
to determine extent of
heterogeneity within mesocosms. Leaves were also
collected at T
final
and stored in scanning electron
microscopy (SEM) fixative (2.5% gluteraldehyde
buffered in 0.1 Msodium cacodylate) for SEM
analysis.
Amplicon sequencing
We determined the composition of fungal commu-
nities on leaves via Illumina sequencing of DNA
amplicons following the methods outlined in
O’Rorke et al. (2015). Briefly, we extracted DNA
from feces using the 96-well PowerPlant Pro DNA
Isolation Kit (Mo Bio Laboratories, Jefferson City,
MO, USA) and then PCR amplified for 20 cycles with
primers that amplified the ITS1 region of fungi
(Gardes and Bruns, 1993). The ITS1 region is well
represented in public DNA sequence databases and
has been demonstrated to work well in previous
studies of the Hawaiian phyllosphere (Zimmerman
and Vitousek, 2012; O’Rorke et al., 2015; Price et al.,
2017) and the ITS1f primer is, in our experience, the
only primer that will not co-amplify plant DNA
when fungi are present in low biomass. The primers
had a 5’universal sequence as used in the standard
Nextera protocol (Illumina, 2012; PCR master mixes
and thermocycler protocol in Supplementary
Materials). PCR amplicons were transferred to a
second 22-cycle reaction in which unique barcodes
were added to each sample, as well as the Illumina i5
and i7 adapters (Supplementary Data S1). Reactions
were cleaned using a SPRI plate (Beckman Coulter,
Brea, CA, USA) and Sera-Mag Speedbeads (Fish-
erSci, Pittsburgh, PA, USA) in an amplicon:bead
ratio of 1:0.7, were eluted in water, made equimolar
in SequalPrep Normalization plates (Invitrogen,
Grand Island, NY, USA) and subsequently pooled.
Pooled samples were cleaned again with magnetic
beads, and submitted to Beckman Coulter Genomics
(Danvers, MA, USA) for sequencing on an Illumina
MiSeq sequencer using the MiSeq Reagent v3 600-
cycle chemistry (Illumina, San Diego, CA, USA).
PCR-negative controls were also amplified and
sequenced; contents of these controls were identical
to the samples and were PCR amplified at the same
time, except that only molecular grade water was
added to the PCR reaction mix.
Microscopy
We used SEM to quantify differences between snail-
treated and control mesocosms. We sectioned gluter-
aldehyde fixed leaves, then dehydrated them by serial
washing in increasing concentrations of ethanol,
critical point dried and then sputter coated with
gold-palladium after mounting on metal stubs. Sam-
ples were viewed on a Hitachi S-4800 (Hitachi,
Tokyo, Japan) field emission SEM at × 1400 magnifi-
cation. SEM images were taken of the top and bottom
of a leaf from each of the 10 mesocosms. Transects
were run at the widest part of the leaf between the
central vein (midrib) and the leaf’sedgeat1mm
intervals. We quantified filamentous microbes across
these transects by measuring their surface area as a
total of the proportion of pixels in Adobe Photoshop,
and then converted this into area coverage (mm
2
).
Bioinformatics and analyses
Forward and reverse Illumina sequences were
merged using PEAR (Zhang et al., 2013), demulti-
plexed in QIIME (Caporaso et al., 2010) and
clustered into OTUs at 97% similarity using
UPARSE (Edgar, 2013). Subsequent analyses were
performed in R 3.1.3 (www.R-project.org). Subsam-
pling was performed to standardize read number to
the lowest number of reads that would enable the
majority of samples to be included in the data set.
The impact of grazing on the predictability and
uniformity of fungal species abundance at a local
scale was calculated using Shannon’s index H
(Shannon and Weaver, 1949) in R using the vegan
package, as was species richness (Dixon, 2009).
Whether richness and H varied significantly between
treatment and control groups was assessed by
analysis of variance. We determined whether snails
preferentially feed on particular fungus using Paine’s
per capita effect index: (E–C)/(C × P), where E is the
experimental food density, C is the control density
and P refers to predator density (Paine, 1992). This
Phyllosphere microbes
RO’Rorke et al
1790
The ISME Journal
index gives a value of –1 when a consumer has a
strong interaction with a prey item (that is, removes
it) relative to the control, a value of 0 for no
interaction and any positive value is interpreted as
the consumer favoring the continued existence or
recruitment of a species.
Temporal analyses of microbial community
composition
We hypothesized that the addition of grazers to an
ecosystem will change the microbial community
composition over time. Consequently, we tested
whether the model y ~ time+treatment+(time × treat-
ment) explained changes in OTU abundances by
permutational analysis of variances after running the
manyglm command in the mvabund package (Wang
et al., 2012; Warton, 2008; Szöcs et al., 2015) to
ascertain the effect of interaction term. The ‘treat-
ment’term refers to whether the mesocosm con-
tained snails or not, but includes measurements
made before introduction of snails. To reflect the
repeated-measures experimental design, the null
hypothesis was tested by permutation of OTU values
at any time points within each mesocosm to see if the
random order deviated from the actual order of OTU
values (R script in Supplementary Files; Dixon,
2009). We also graph the raw compositional abun-
dance data in ggplot (Warton, 2008; Wickham, 2009)
to visualize how the mean–variance relationship of
the fungus in snail-treated mesocosms differed from
that of controls. The mean–variance relationship can
be taken as an indicator of system heterogeneity, in
which systems with greater intercepts and slopes can
be considered to have greater heterogeneity (Taylor,
1961; Fairweather, 1988).
We used the Bayesian statistical program Source-
Tracker v 1.0 (Knights et al., 2011) to determine if the
composition of fungal communities was consistent
over time, if snail feces vector microbes into an
environment, and if so, how long these microbes
persist in the community. For this analysis, we
included three initial groups of fungus: the initial
phyllosphere (measured at T
0
in all mesocosms
before snails were introduced), fungus from snail
feces (suggestive of snail ‘vectoring’) and unex-
plained variance attributable to the environment,
aerobiota or sampling error. All subsequent sam-
plings of the fungus community were then compared
with these initial three groups to see which initial
group became more abundant over time. In a
community with high resistance (sensu Pimm,
1984; for a glossary refer de Vries and Shade,
2013), all composition would be identical to initial
composition and would be classified as ‘phyllo-
sphere’. If feces were a source of fungus, then ‘fecal’
OTUs would come to be a significant part of the
microbiota and if the snails grazing increased the
infiltration of microbes from the regional species
pool, then this ‘environmental’group would become
more dominant in the snail-treated mesocosms than
in the controls. A likelihood ratio test was used to
evaluate if a mixed effect model that included
‘treatment’significantly explained the data. In this
model, ‘treatment’was nested inside ‘block’, which
was treated as a random factor.
Results
Sequencing results
Miseq amplicon sequencing yielded a total of
3 217 880 reads from this study that passed quality
control (mean = 20 364 ± 1116 s.e.). As there were
two samples that yielded fewer than 1000 reads (767
and 557 reads), all samples were randomly sub-
sampled to a depth of 750 reads. The four negative
PCR controls yielded 1, 1, 1 and 0 reads and controls
for field collection and extraction negatives yielded
293 and 23 reads, respectively, suggesting that
contamination did not contribute much noise to
our data or analyses.
Temporal analyses of changes in microbial composition
Does snail treatment disturb the phyllosphere
through time?. The interaction term snail treatment
× time is significant (Po0.05) indicating that snails
modify the abundances of fungal OTUs over time
(Table 1). Generally, the changes to fungal OTUs in
the snail-treated mesocosms were either small
changes in abundances over time (Table 2 and
Figure 2) or erratic invasions and there was greater
heterogeneity in the snail-treated mesocosms
(Figure 3). Paine’s per capita effect index demon-
strated that no fungal OTUs had a strong negative or
positive effect as a result of grazing (Table 2).
Sources of microbes
The Bayesian software Sourcetracker (Knights et al.,
2011) found that the composition of the fungal
communities in the mesocosms was dominated by
microbes that could be traced back to the initial
Table 1 Analysis of deviance for modeling changes in OTU
abundances against snail treatment over time
Resid. Df Df.dif Dev P-value
Treatment 68 1 3396.774 0.795
Time 62 6 11 964.259 0.005
Time:Treatment 56 6 3272.256 0.005
Abbreviation: OTU, operational taxonomic unit.
The change in abundances of each fungal OTU in the phyllosphere (y)
as a function of sampling dates (time) and the presence or absence of
snail grazers (treatment) is modeled by: y ~ time+treatment+(time x
treatment).
Fungal OTU abundances changed significantly over time. The
interaction term was also significant, showing that the introduction of
snails does change fungal communities. The data modeled includes
time points before the experimental treatment. Therefore, the
‘treatment’term was not significant indicates that there were no
significant differences between control and treatment mesocosms
before the experiment commenced and that the change observed in
snail-treated mesocosms was not an artifact of differences that already
existed between the communities.
Phyllosphere microbes
RO’Rorke et al
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The ISME Journal
phyllosphere community (Figures 3a and b). How-
ever, fungal OTUs from the environmental species
pool and were significantly greater in snail-treated
mesocosms than controls (χ
2
(1) = 14.43, P= 0.00015),
contributing 25.5% (±3.2% s.e.) of fungus counted in
the snail-treated mesocosm after 1 week and 11.3%
(±1.1% s.e.) to the control after 1 week. Microbes
assigned to feces were only a small component of the
mesocosm assemblages with the exception of three
mesocosms: G, I and J. Mesocosm G had an elevated
proportion of fungus assigning to feces on days 10
and 17 (Figure 3).
End-point analyses: differences in absolute abundances
and alpha diversity
Alpha diversity and evenness for fungi were rela-
tively similar between mesocosms for snail treat-
ments (H = 3.37 ± 0.05 s.e.) and control mesocosms
(H = 3.45 ± 0.04 s.e.). At the end of the experiment,
the mean Shannon alpha diversity of fungal mes-
cosms was similar for snail treatments (3.36 ±0.11
s.e.) and controls (3.57 ±0.09 s.e.) and were not
significantly different (F(1, 28) = 1.41, P= 0.25). How-
ever, mesocosms treated with snails had substan-
tially greater variation in the s.d. of the mean alpha
diversity when multiple samples were measured
within the mesocosm (Figure 4). In other words, snail
addition increased the patchiness of the relative
abundances of microbes. Species richness did
significantly decline in snail-grazed mesocosms
(F(1, 28) = 7.03, P= 0.01, values in Table 3). Micro-
scopy was used to compare the absolute cover of
fungal hyphae between snail treatments and controls
(Figure 5). Fungal hyphae was 3.59 times more dense
on control mesocosm leaves than it was on snail
leaves, indicating that snails significantly reduce
fungal cover (Figure 5).
Discussion
Our study found that snail introductions impacted
microbial communities by promoting infiltrations of
fungal OTUs from the environmental species pool. We
hypothesized that fungal community assemblages
would be changed by mycophagous tree snails being
reintroduced into a habitat. This was the case, because
not only do fungal communities change over time, but
communities with snails change differently compared
with those without snails. It was not possible to predict
aprioriif the presence of snails would increase or
decrease community homogeneity, because studies of
other grazing systems suggest that snail grazing could
do either. Our comparison of mesocosms treated with
snails with those without snails demonstrated that
snails alter microbial assembly by clearing fungal
patches (which is likely to reduce resource limitation).
Spatial heterogeneity and patch size
In this study, the mean alpha diversity was the same
for control and treatment mesocosms at the scale of
the leaf, most likely due to changes in the relative
abundances of fungal OTUs. However, this study
found that there was much greater deviation from the
mean of alpha diversity within mesocosms treated
with snails compared with controls (Figure 4). This
is explained by the way that disturbances disrupt
community homogeneity and creates patches of
habitat favorable to diverse environmental microbes
found elsewhere (Pickett and White, 1985). Grazing
snails rescale the size of patches in their environ-
ment, making them considerably smaller and more
numerous than the single mesocosm inside which
they are enclosed. By contrast, in the control
mesocosms, the patch size remains identical to the
mesocosm size as evidenced by high similarity of
samples. The leaves grazed by snails do experience a
Table 2 Impact of snails on the most abundant OTUs
OTU Temporal
interaction
strength ( ±s.e.)
Final
interaction
strength ( ±s.e.)
Temporal Final Genus
Mean (%) Var Mean (%) Var
OTU_1 −0.046 (±0.01582) −0.004 (±0.01758) 6.1 4.31 11.3 16.04 Teratosphaeria
OTU_3 −0.007 (±0.02939) −0.041 (±0.02718) 4.55 8.78 4.9 5.43 Capnobotryella
OTU_4 0.051 (±0.0481) 0.092 (±0.07222) 6.36 25.85 4.2 3.02 Mycosphaerella
OTU_5 0.017 (±0.03676) −0.028 (±0.04211) 6.57 18.3 5.6 9.88 Capnobotryella
OTU_8 −0.004 (±0.03358) 0.011 (±0.03869) 2.42 1.89 2.52 2.74 Capnobotryella
OTU_10 −0.03 (±0.43219) −0.036 (±0.25918) 1.76 5.02 2.5 15.34 Capnobotryella
OTU_13 −0.027 (±0.02481) −0.053 (±0.03182) 4.08 5.44 5.59 7.61 Capnobotryella
OTU_18 0.007 (±0.03391) −0.01 (±0.04709) 1.81 0.99 1.7 1.21 Capnobotryella
OTU_936 −0.062 (±0.01723) −0.042 (±0.02199) 1.84 0.82 4.89 3.13 Ramichloridium
OTU_1091 −0.001 (±0.01931) 0.05 (±0.02334) 5.97 6.22 6.03 3.99 Capnobotryella
OTU_2701 −0.02 (±0.03206) −0.005 (±0.04955) 2.19 2.87 2.75 2.4 Taphrina
OTU_3851 −0.06 (±0.03078) −0.041 (±0.03729) 1.55 1.93 3.86 5.32 Capnobotryella
Abbreviation: OTU, operational taxonomic unit.
Twelve OTUs accounted for 50% of the reads detected in the mesocosms. The strength of the interaction of snails with each of these OTUs was
determined using Paine’s per capita effect index, which ranges from −1 for strong interactions (that is, a preferred food) to 0 for no preference to
any positive number when the grazing of the animal favors the fungus (for example, by facilitating fungal recruitment). Standard errors were
determined by bootstrapping. Means refer to the mean percentage of the fungal OTU.
Phyllosphere microbes
RO’Rorke et al
1792
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small, but statistically significant, decrease in fungal
richness (Table 3). A recent study examining the
impacts of foraging animals on soil microbiology by
Eldridge et al. (2015) found that there were no
differences in fungal or bacterial richness between
grazed and undisturbed sites, despite finding differ-
ences in beta diversity. The contrasting response of
richness to grazing in this study is most likely a
reflection of this study and the soil system having
contrasting productivity or disturbance frequencies
(Huston, 2014), but the both this study and
Eldridge’set al. (2015) indicate how animals can
modify the structure of microbial communities at a
broader scale.
OTU_5637 Paecilomyces OTU_1 Teratosphaeria OTU_10 Capnobotryella
OTU_14 Capnobotryella OTU_3985 Passalora OTU_936 Ramichloridium
OTU_7708 Capnobotryella OTU_13 Capnobotryella OTU_1091 Capnobotryella
OTU_2701 Taphrina OTU_3851 Capnobotryella OTU_4 Mycosphaerella
OTU_18 Capnobotryella OTU_3 Capnobotryella OTU_15 Cystocoleus
OTU_8 Capnobotryella OTU_5 Capnobotryella
1
10
100
1
10
100
1
10
100
1
10
100
1
10
100
1
10
100
0 10203040 010203040
Time (days)
010203040
Time
(
da
y
s
)
Treatment
Control
Snail
Log(Abundance +1)
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).
Phyllosphere microbes
RO’Rorke et al
1793
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Temporal heterogeneity
Stochasticity tends to dominate assembly processes
before a habitat reaches carrying capacity, after
which point deterministic environmental filters
become more important for community assemblage
(Fukami, 2010). The grazing action of snails prolongs
this stochastic phase, and because snails reduce
microbial biomass the mechanism for prolonging
stochasticity is likely to be through competitor
removal. Crowther et al. (2013) found that grazing
of soil fungi by isopods removed the mycelia of
dominant Basidomycetes and diversified the func-
tional profile of the fungal assemblage.
Our results indicate snails do not target a parti-
cular fungal OTU as food, but instead have a minor
Sources
Initial Phyllosphere
Environmental Donor Feces
1.0
0.0
1.0
0.0
1.0
0.0
1.0
0.0
1.0
0.0
JI
HG
FE
DC
BA
0 20 40 0 20 40
Grazed by snails No-snail controls
Proportion of fungus OTUs
Days
Log(variance of OTU abundance)
Log(mean of OTU abundances)
-1 0 1
2
1
0
3
-1
-2
Control (no snails)Snails present
Heterogeneity in mesocosms across time
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.
Variability of alpha diversity (H)
At T0Control Snail Treated
0.6
0.2
0.1
0.3
0.4
0.5
Figure 4 Snails increase within-system variability. Each meso-
cosm was sampled in triplicate before the experiment commenced
(T
0
) 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.
Table 3 Mean Shannon diversity (H) and richness (S) of leaves
within mesocosms and their standard deviations
Mesocosm Mean H
(n=3)
s.d. Mean S
(n=3)
s.d.
Snail
A 3.28 0.27 108.67 16.29
C 3.29 0.31 103.67 34.21
E 3.60 0.58 119.67 45.01
G 3.38 0.59 112.67 10.02
I 3.34 0.34 109.00 16.00
Control
B 3.28 0.05 113.67 9.29
D 3.52 0.11 119.33 18.90
F 4.06 0.09 154.00 12.29
H 3.72 0.08 146.33 9.02
J 3.12 0.19 125.67 9.29
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impact on a broad range of OTUs (Table 2). Visual
inspection of leaves at the close of the experiment
also shows that snails clear the leaf surfaces in a
general and not in a ‘picky’manner. Consistent with
our results (Figure 3c), Berlow (1999) argued that
consumers that weakly interact with food species
tend to increase the variance within the trophic
group on which they predate. This is because
consumers that strongly interact with prey groups
should consistently remove the same food items and
reduce variance (Berlow, 1999). Conversely, the
absence of a predator can lead to a loss of diversity
in the lower trophic levels because of the unchecked
growth of individual prey species (Paine, 1966). The
response of fungus to grazing, or snail selectivity
require further research, but it does appear that
snails do not specifically target dominant fungi (for
example, isopods Crowther et al., 2013; and collem-
bola Jørgensen et al., 2005) or uncompetitive fungi
(for example, millipedes Crowther et al., 2011), but
instead serve to clear areas of leaf surfaces and
prolong the initial phase of assembly.
Snails increased the occurrence of fungal OTUs
from the environmental species pool (Figures 3a and b),
where that species pool consists of fungi from the
aerobiota, endophytes or OTUs whose abundance
was below detection at the sampling depth used in
the study, diminishing the role that plant hosts may
otherwise exert on phyllosphere communities. These
spikes in abundance are likely to be the result of
opportunistic fungi infiltrating spaces cleared by the
snails, which is likely to account for the significant
treatment × time interaction that was detected
(Table 1). In a previous study conducted across the
extent of the Waianae mountain range, we showed
that the phyllosphere composition of trees contain-
ing Achatinella snails was more greatly influenced
by location than by host tree identity (O'Rorke et al.,
2015). Plant host identity strongly impacts phyllo-
sphere community composition, and although phyl-
losphere communities are geographically structured,
the plant host can be a stronger determining factor in
phyllosphere composition (Kim et al., 2012)—even
at the continental scale (Redford et al., 2010; Ludlow
et al., 2016). The results of this study indicate that
grazing pressure may reduce the specificity of the
relationship between host plant and phyllosphere
community as grazing keeps a high proportion of the
microbial community in a sustained transitory state.
The similarity of microbial community composition
in the phyllosphere over time is evidence for the
selectivity of plants to determine which microbes
persist (Lindow and Brandl, 2003), despite substantial
removal of microbes by grazing. SourceTracker analyses
show that there is a substantial consortium of fungi that
are a persistent part of the phyllosphere in both
treatments (Figure 3). It is plausible that some of the
OTUs that persisted are epiphytes that also occur in the
intercellular space inside leaves, which is consistent
with a ‘refugia’response to predation (Belyea and
Lancaster, 1999). Phyllosphere microbes often originate
from within the plant (Whipps et al., 2008), and if the
cells that recolonize cleared leaf patches arise from
within the leaf stomata, then this would explain some
consistency in the assemblage composition.
Empirical studies of how grazing pressure affects
richness (Proulx and Mazumder, 1998) have shown
variable results. This variance has been explained, in
part, by the nutrient status of the environment (Proulx
and Mazumder, 1998), where decreased plant diver-
sity is typical in grazed habitats with low nutrients.
The phyllosphere is characteristically oligotrophic
(Vorholt, 2012), so our finding that grazing does not
impact alpha diversity contradicts this trend. That
phyllosphere microbes are host associated is one
important distinction between our study and the
majority of those considering the grazing impacts of
macro-organisms. In our case, the community com-
position of fungi may be constrained by the plant
host, which maintains more homogenous diversity
levels compared with an inert substrate. In this study,
we controlled for the effects of plant genotype by
using single host, but further research is required to
determine whether the effects that we observe vary
between different tree hosts. We also used a constant
number of snails in each of our treatments, but it is
likely that fungal relative abundances will respond
differently under different grazing intensities (Grime,
1973; Connell, 1978; Huston, 2014). Therefore, further
experiments are required to shed light on whether
host association or other factors contribute to differ-
ences in grazing impacts on microbial assemblages.
Mesocosms
Proportion of hyphal cover
0.00
0.35
0.30
0.25
0.20
0.15
0.10
0.05
ABCDEF GHI J
Figure 5 Assessment of grazing impacts on microbial abun-
dances 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.
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Feces are not a source for novel OTUs
We hypothesized that snails are vectors for microbes,
because they not only excrete viable fungi, but when
they do so they deposit them within nutrient-rich
feces that should facilitate rapid and vigorous growth
(O'Rorke et al., 2015; Price et al., 2017). The results
of this study do not support this hypothesis, and
although there are brief pulses when the microbes
found in translocated snail feces can be detected on
the surfaces of leaves, these microbes subsequently
become a minor component of the phyllosphere.
This is surprising, but is concordant with our
personal observations that snail feces persisted in
snail mesocosms for weeks at a time and did not
appear to be degraded by viable microbes within.
The pulses when a fecal signature is detected in
SourceTracker analyses may be the result of co-
sampling feces along with the phyllosphere. That
feces are not a substrate for rampant microbial
growth could be due to physical properties of the
feces. Tree snail feces are compacted with mucus,
which is profoundly hydrophobic in A. diaphana
(personal observation, see Supplementary
Materials), and therefore not particularly conducive
to microbial growth—despite containing viable
microbes (O’Rorke et al., 2016). In addition, inspec-
tion of SEM images and leaf surfaces give no
indication that A. diaphana snails damage the leaf
cuticle when grazing microbial films. They are
therefore very different from the marsh snail Littor-
aria irrorata, which damages live leaf surfaces of salt
marsh cordgrass (Spartina alterniflora) when feeding
and deposits fecal pellets on leaf wounds, encoura-
ging fungal growth (Silliman and Newell, 2011).
The SourceTracker results of this study make it
clear that snails are not homogenizing their environ-
ments by seeding microbes as we had hypothesized,
but instead that snails increase the influence of
environmental microbes via grazing. The net result
of native tree snail grazing and feces deposition was
enhancement of microbial diversity in the phyllo-
sphere. Snails therefore change microbial commu-
nities by reducing the host plants’top–down
controls on the community structure while facilitat-
ing greater community turnover than in environ-
ments without snails.
Conclusion
A. diaphana grazing of vegetative fungi off leaf
surfaces increases ecosystem patchiness and the
incidence of colonization from the wider environ-
mental species pool, but they do not seed fungus into
the ecosystem through their feces. The disruptive
manner in which these generalist feeders modify
microbial assembly is therefore different from that
observed in mycophageous animals that are specia-
lists. We conclude that it is important for microbial
ecologists to observe whether animals could account
for any of the unexplained variance in their
community assembly models and to observe if these
animals are specialists, generalists or simply foragers
that modify assembly processes. The results of this
study are also in accord with other recent studies
with a conservation focus (for example, Clarke et al.,
2015) that point out that management of species of
conservation interest needs to take into account how
animal extinctions and reintroductions will impact
on the composition and ecosystem services of the
microbiota.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements
We acknowledge the help of David Sischo, Jenny Prior and
Cynthia King at the DNLR. Chloë Heiniemi, Talia Sellars,
Casey Jones and Gerry Cobian for volunteering their time
and help in the field, and Tina Carvahlo for electron
microscopy at the BEMF at the University of Hawai’i. This
project was funded through the US Army cooperative
agreement W9126G-11-2-0066 with Pacific Cooperative
Studies Unit, University of Hawaii through the Pacific
International Center for High Technology Research, State
of Hawaii, DLNR, Special Funds and NSF grant #1255972.
FASTQ files have been deposited in NCBI’s short read
archive (SRA) under project number PRJNA357010. All
research was conducted under the Department of Land and
Natural Resources Native Invertebrate Research and
Collecting permit # FHM15-T&E-16.
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