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2 authors:
Gillian H Gile
Claudio Slamovits
Dalhousie University
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ORIGINAL PAPER
Lophomonas striata is a multiagellate parabasalid commensal in the hindgut of the omnivorous cockroaches Blatta orientalis and Periplaneta americana. Its closest relatives were traditionally thought
to include similar multiagellate parabasalids with a single agellar area that degenerates during
mitosis, such as Joenia and Kofoidia. However, molecular phylogenetic analyses have shown that
lophomonads are not monophyletic. We have determined the SSU rRNA sequence of L. striata and
we nd that it branches sister to the Trichonymphida with strong support. This is surprising because all
other lophomonads sampled to date branch within the Cristamonadida, and the order Trichonymphida
(e.g. Trichonympha, Pseudotrichonympha, and Hoplonympha) is both morphologically coherent and
monophyletic in SSU rRNA phylogenies. Trichonymphida, unlike the lophomonads, share a bilateral
symmetry, in which their multiple agella occur in two (or sometimes four) regions, and instead of
degenerating upon mitosis, half of the agella are passed to each daughter cell. The single apical agellar region characteristic of lophomonads is therefore either plesiomorphic or it has arisen multiple
times in parabasalids; our phylogenetic analyses and available ultrastructural evidence suggest the
latter. Our results also suggest that parabasalid gut symbionts may have been vertically transmitted in
cockroaches before the common ancestor of Cryptocercus and termites.
2011 Elsevier GmbH. All rights reserved.
Key words: Cristamonadida; gut symbiont; hypermastigote; Joeniidae; Lophomonadidae; termite.
Introduction
Lophomonas blattarum Stein was rst described
in 1860 from the gut of the common omnivorous cockroach, Blatta orientalis (Stein 1860).
Lophomonas is therefore one of the earliest
described parabasalid genera, predating the rst
termite-gut specic genus Trichonympha by 17
years (Leidy 1877). Cells of L. blattarum range from
1
Results
We examined ten individuals of Periplaneta americana for the presence of parabasalid symbionts.
Nematodes and Nyctotherus ovalis Leidy were
abundant, but L. striata was apparent in only ve,
all of which were females. The abundance of L. striata varied considerably, with only a few individuals
apparent in certain roaches, while many hundreds
of L. striata individuals were visible in just a few
microlitres of gut contents from other roaches. We
-/100/-
Trichonymphida
Honigbergiellida
Trichomonadida
Hypotrichomonadida
Spirotrichonymphida
Cristamonadida
Tritrichomonadida
Figure 1. A, Maximum likelihood phylogenetic tree of SSU rRNA gene sequences, including representatives of
each parabasalid clade for which molecular data is available. Lophomonad taxa are indicated by white text on
a black background. Numbers at nodes indicate maximum likelihood and logDet distance bootstrap support as
percentages in agreement out of 1000 replicates and Bayesian posterior probability values, respectively. Only
nodes with 60/50/0.90 or greater support are indicated. Filled circles at nodes indicate full support (100/100/1.0).
Hollow circles at nodes indicate support levels of 98/96/1.0 or greater. B, Differential interference contrast light
micrograph of Lophomonas striata, showing the anterior tuft of agella and longitudinal striations on the cell
body. Scale bar 20 m.
p-value
0.988
0.006
0.000
0.000
0.000
0.000
0.000
0.000
0.001
0.000
0.000
0.003
0.000
0.010
0.000
0.000
Discussion
In our phylogenetic analyses, L. striata branches
at the base of the Trichonymphida, a position
compatible with L. striata being either a basal trichonymphid or a sister lineage, but since Lophomonas
does not t the morphological criteria of the Trichonymphida, we consider it a sister lineage.
This interpretation, however, is complicated by the
unknown position of the root of the parabasalian
tree. Early phylogenetic analyses of parabasalid
SSU rRNA placed the root either on the spirotrichonymphids branch (represented only by U17508
Reticulitermes avipes symbiont 1, Dacks and
Redeld 1998; Gunderson et al. 1995) or on the
trichonymphids branch (represented only by Trichonympha spp., Frlich and Knig 1999; Ohkuma
et al. 1998). Analyses specically seeking the root
Methods
Cell isolation, DNA extraction, PCR, and sequencing: A
colony of Periplaneta americana Linnaeus was maintained in
the laboratory of Drs. Pivi Torkkeli and Andrew French, Department of Physiology and Biophysics, Dalhousie University, at
22 C on a 13:11 hour light:dark cycle. Cockroaches were dissected and hindgut contents were suspended in Trager medium
U (Trager 1934). Individual Lophomonas striata Btschli cells
were isolated by micropipette using a Zeiss Axiovert 200 M
inverted microscope, and photographed with a Hamamatsu
Orca R2 camera. A single cell and a pool of 50 cells were
washed twice in clean medium U, after which DNA was
extracted using the Masterpure Complete DNA and RNA Purication Kit (Epicentre, Madison, WI, USA). Lophomonas striata
SSU rRNA gene sequence was amplied with GoTaq Green
Master Mix (Promega, Madison, WI, USA) using the universal
eukaryotic primers PF1 5 - TGC GCT ACC TGG TTG ATC CTG
CC-3 (Keeling 2002) and FAD4 5 - TGA TCC TTC TGC AGG
TTC ACC TAC-3 (Deane et al. 1998; Medlin et al. 1988). PCR
conditions included a 3 minute denaturation at 95 C followed
by 30 cycles of 95 C for 30 seconds, 55 C for 30 seconds, and
72 C for 1 minute 30 seconds, then an additional 7 minutes at
72 C. The identity of P. americana was conrmed by amplifying
its SSU rDNA using the same primers and reaction conditions (not shown). PCR products were electrophoresed and
gel-puried using the UltraClean15 PCR purication kit (MoBio,
Carlsbad, CA, USA) and cloned with the pGEM-T Easy Vector
System and competent cells (Promega, Madison, WI, USA).
Six clones from the single cell and three clones from the 50 cell
sample were sequenced on both strands using BigDye Terminator v 3.1 (Applied Biosystems, Carlsbad, CA, USA). One clone
sequence from the single cell PCR (which was identical to the
consensus sequence) was chosen for phylogenetic analyses
and was deposited in GenBank under accession JN088049.
Phylogenetic analyses: Selected parabasalid SSU
sequences spanning the phylogenetic diversity of the group
were aligned with MAFFT (Katoh et al. 2002). Alignments were
visually inspected, and the highly variable and ambiguously
aligned sites were masked in MacClade 4.08 (Maddison and
Maddison 2003) for a nal alignment of 70 taxa and 1337 sites
(approximately 80% of the raw alignment). Phylogenetic trees
were inferred using maximum likelihood (ML) and Bayesian
methods in the programs RAxML 7.0.4 (Stamatakis 2006)
and PhyloBayes 3.2 (Lartillot et al. 2009), under the GTR++I
model as selected by all four criteria in Modelgenerator
0.85 (Keane et al. 2006), with four discrete rate categories.
Support for ML topologies was assessed from 1000 bootstrap
replicates. Bayesian analyses included two independent runs
with one tree saved every ten cycles. The rst 2500 saved trees
from each run were discarded as burn-in, and consensus trees
and posterior probabilities were computed from the 15,000
pooled nal trees from both runs. The maximum discrepancy
across bipartitions between the two runs (maxdiff value) was
0.03.
In order to test the robustness of Lophomonas position,
additional analyses (not shown) were performed on different
combinations of taxa using the same parameters as above.
These included a rooted analysis, using Fornicata and Oxymonadida spp. as an outgroup, and two unrooted analyses
Acknowledgements
We wish to thank Shannon Meisner and Pivi
Torkkeli for providing cockroaches and assistance
with dissection and Alastair Simpson for a critical reading of this manuscript. Thanks are also
due to two anonymous reviewers for their insightful comments and suggestions. GHG is supported
by a Natural Sciences and Engineering Research
Council of Canada postdoctoral fellowship. CHS is
supported by the Canadian Institute for Advanced
Research and the Tula Foundation.
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