The document summarizes a study investigating the factors influencing the assembly of microbial communities within the pitchers of three Nepenthes plant species in Singapore. The study finds that bacterial and eukaryotic community composition in the pitchers is most influenced by the host plant species and pH level. While collection site and pitcher volume also have significant effects, bacterial and eukaryotic diversity are strongly correlated, suggesting similar responses to environmental conditions rather than interactions between organisms shaping the communities. Deterministic factors like biochemical characteristics have a stronger influence on community assembly than stochastic factors like chance colonization.

1. Do pitcher plants control the assembly
of pitcher microbiomes?
Leonora S. Bittleston, Anne Pringle, and Naomi E. Pierce.
Department of Organismic and Evolutionary Biology
Harvard University, Cambridge, MA USA
Figure 1.
Fluid collected in Singapore from
3 Nepenthes species (a) N. gracilis,
(b) N. rafflesiana, and (c) N.
ampullaria each in 3 sites: UPR =
Upper Peirce Reservoir; BTNP =
Bukit Timah Nature Preserve; KRP
= Kent Ridge Park.
References
1. Srivastava, et al, (2004). Are natural microcosms useful model sys-
tems for ecology? TREE 19: 379-384
2. Adlassnig, et al, (2011). Traps of carnivorous pitcher plants as a
habitat: composition of the fluid, biodiversity and mutualistic activi-
ties. Ann. Bot. 107: 181-194.
3. Earth Microbiome Project, http://www.earthmicrobiome.org
4. Caporaso, et al, (2010). QIIME allows analysis of high-throughput
community sequencing data. Nature Methods 7: 335-336.
5. Oksanen, et al, (2013). vegan: Community Ecology Package. R
package version 2.0-10. http://CRAN.R-project.org/package= vegan
Contact: lbittles@fas.harvard.edu
Methods
We collected approximately one hundred samples from three
Nepenthes species in three sites in Singapore in 2012 and 2013
(Fig. 1). Total pitcher volume and pH were recorded in 2013. We
used a phenol-chloroform method to extract DNA, and
sequenced 16S and 18S rRNA using Illumina MiSeq technology
with Earth Microbiome Project protocols (3). Sequences were
processed with QIIME (4), using USEARCH for initial chimera
removal and UCLUST for additional quality checking. We
clustered Operational Taxonomic Units (OTUs) with open
reference clustering to the Greengenes and SILVA databases, and
assigned taxonomy with UCLUST and BLAST. Additional statistical
analyses were computed using the vegan package in R (5).
Introduction
What forces control microbial colonization of a host, and how do
bacteria and eukaryotes interact in a microbiome? Carnivorous
pitcher plants are ideal models for microbial ecology, as the
aquatic microcosms within the pitchers are small and
self-contained (1). Once a pitcher opens it forms an entire food
web of arthropods, bacteria, fungi, and protists. The pitcher
microbiome is hypothesized to assist the plant in digestion and
nutrient acquisition from prey. By changing the environment, a
host species may restrict habitat colonization. Plants can excrete
hydrogen ions or O2 into internal fluids, affecting acidity and
oxygen levels (2). Our objective is to investigate if host species,
collecting site, pH, or volume influence the community
composition of bacteria and eukaryotes within the pitchers of
Nepenthes plants in Singapore (Fig. 1). Multivariate analyses
indicate host species and pH have the largest influence on
composition of the communities (Fig. 2). Phylogenetic diversity
of bacterial and eukaryotic communities are strongly correlated
(Fig. 3), suggesting interactions among taxa or similar reactions
to environmental factors.
Assembly of food webs in pitchers is shaped by
deterministic and stochastic processes. Multivariate
analyses reveal that bacterial and eukaryotic community
composition is most influenced by host species and pH,
though site and volume also have significant effects.
Phylogenetic diversity and Unifrac dissimilarity matrices
of bacteria and eukaryotes are strongly correlated.
Bacteria may be introduced into pitchers primarily via
arthropod guts; however, if this were the case we would
expect a higher correlation between bacterial and
arthropod diversity. The organisms within pitchers are
likely interacting via predation, competition, mutualism
and parasitism. Environmental filtering due to the effects
of pH or other biochemical factors may determine
bacterial community composition in pitchers, which
could drive the colonization of eukaryotes. Alternatively,
bacterial and eukaryotic diversity may be structured by
similar environmental factors, and not determined by
interactions among organisms. In future experiments, we
will grow pitcher plants in controlled conditions in order
to manipulate variables, such as pH or the organisms
present, and distinguish between the effects of
biochemical factors and interspecific interactions.
Deterministic and stochastic factors influencing
community composition
Deterministic Stochastic
HOST SPECIES COLLECTION SITE OTHER
Biochemical
factors:
e.g. pH, O2,
enzymes, nu-
trients, cues
Physical
factors:
e.g. age,
volume,
viscocity
Chance
colonization
Variability: e.g.
weather, temporal
changes, nutrient
pulses
BACTERIA EUKARYOTA
COMMUNITY COMPOSITION
Interactions: e.g. predation,
competition, mutualism
Bacterial and eukaryotic communities cluster by host species, and to a lesser extent by site
Figure 2.
NMDS plots displaying axes 1 and 2 of
Bacterial (A) and Eukaryotic (B) commu-
nity composition by Host Species (i),
Collecting Site (ii), and pH (iii). We
tested significance using permutational
MANOVAs (Adonis in R) and Mantel
tests. Eukaryotes by pH (not shown),
Mantel: R2 = 0.15, P = 0.0045.
Each dot represents the bacterial or eu-
karyotic community in one pitcher.
Communities from the same host spe-
cies tend to be more similar, even when
we control for pH, volume, and site.
Bacteria by Host Species Bacteria by Site Bacteria by pH
Eukaryotes by Host Species Eukaryotes by Site
Adonis: R2 = 0.26, P = 0.0001
Adonis: R2 = 0.15, P = 0.0001 Adonis: R2 = 0.10, P = 0.0001
Mantel: R2 = 0.54, P = 0.0001Adonis: R2 = 0.14, P = 0.0001
Purple = measured in this study
Phylogenetic alpha and beta diversity of bacterial and eukaryotic communities are correlated
Figure 3.
Phylogenetic diversity (PD) of bacte-
rial and eukaryotic communities are
significantly correlated (A), as are
community dissimilarity matrices
(DMs) using the Unifrac phylogenetic
metric (B). Pitchers with more diverse
bacterial communities tend to have
more diverse eukaryotic communi-
ties. The trend does not appear to be
driven by arthropods, as bacteria ex-
plain a lower proportion of arthro-
pod variation than total eukaryote
variation.
Phylogenetic diversity Unifrac community dissimilarity
PD Bacterial Communities
PDEukaryoticCommunities
UnifracEukaryoticDM
Unifrac Bacterial DM
Linear model: Adj. R2 = 0.55, P = 5.06e-15 Mantel: R2 = 0.45, P = 0.0001
Discussion
UPR
KRP
BTNP
a
b
c
Acknowledgements
Thank you to the Pringle and Pierce Labs, in particular Jon Sanders
and Chris Baker for useful suggestions, Lila Strominger for laboratory
help, and Kadeem Gilbert for collecting help. Collecting permits
were issued by Singapore NParks. Gregory Jedd at TLL provided
laboratory space in Singapore. We greatly appreciate Steven
Worthington’s statistical advice. Funding was provided by an NSF
Graduate Research Fellowship, OEB, and a Putnam Expedition Grant
from the Museum of Zoology.
A(i) A(ii) A(iii)
B(i) B(ii)
A B