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Phylogenetic Position of Lophomonas striata


Btschli (Parabasalia) from the Hindgut of the
Cockroach Periplaneta americana
Article in Protist August 2011
DOI: 10.1016/j.protis.2011.07.002 Source: PubMed

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Gillian H Gile

Claudio Slamovits

Arizona State University

Dalhousie University

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Available from: Gillian H Gile


Retrieved on: 12 October 2016

Protist, Vol. 163, 274283, March 2012


http://www.elsevier.de/protis
Published online date 12 August 2011

ORIGINAL PAPER

Phylogenetic Position of Lophomonas striata


Btschli (Parabasalia) from the Hindgut of the
Cockroach Periplaneta americana
Gillian H. Gile 1 , and Claudio H. Slamovits
Department of Biochemistry and Molecular Biology, Dalhousie University, 5850 College Street, room 9-G1,
Halifax, NS, B3H 1X5, Canada
Submitted February 25, 2011; Accepted June 26, 2011
Monitoring Editor: Michael Melkonian

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

Corresponding author; fax: +1 902-494-1355


e-mail g.gile@dal.ca (G.H. Gile).

2011 Elsevier GmbH. All rights reserved.


doi:10.1016/j.protis.2011.07.002

approximately 20 to 60 m in length, are exible but


generally ovoid to pear-shaped, and bear a tuft of at
least 50 agella at the anterior extremity (Brugerolle
and Lee 2000; Grass 1952; Janicki 1910; Kudo
1926). Two more Lophomonas species have been
described since, L. striata Btschli (1878), also
from B. orientalis, and L. sulcata Schuster (1898),
though they were later synonymized (Kudo 1926).
Lophomonas striata is similar in size to L. blattarum (about 30-40 m long and 8-15 m wide,
Hollande and Carruette-Valentin 1972) and shares
the anterior tuft of agella, but differs by having

Phylogenetic Position of Lophomonas 275

a more rigid, spindle-shaped cell whose internal


contents are obscured by faintly helical longitudinal banding (Btschli 1878). Years later it was
suggested that these characteristics are due to
fusiform bacteria covering the cell, and this interpretation was conrmed by electron microscopy
(Beams et al. 1960; Grass 1926; Kudo 1954). Both
species have also been observed inhabiting the gut
of another common, omnivorous cockroach, Periplaneta americana (Semans 1943).
The taxonomic position of Lophomonas has
varied over the years, at rst mainly due to
new genera being established, but later due to
more detailed observations of cell division and
ultrastructure. In an early taxonomic scheme,
Lophomonas was grouped with Trichomonas and
Tetramitus (Kent 1880). When Joenia and Microjoenia were established as new genera (Grassi 1885;
Grassi and Sandias 1894), their characteristic apical tufts of agella linked them to Lophomonas.
These three genera were soon included in the
new order Hypermastigida (Grassi and Fo 1911)
along with the trichonymphids Trichonympha and
Pseudotrichonympha and the spirotrichonymphids
Spirotrichonympha, Holomastigotes, and Holomastigotoides. Fundamental differences in cellular
organization and mitosis between the lophomonads and other hypermastigotes were soon noted,
however (Dogiel 1922; Kirby 1949; Light 1926a).
In Lophomonas and Joenia, there is a single agellar area that degenerates upon division (except
for the privileged basal bodies), while in the trichonymphids and spirotrichonymphids, there are
two (or sometimes four) agellar regions, each
with its own set of privileged basal bodies. Upon
mitosis, each daughter cell receives one agellar region and later regenerates the bilateral
symmetry (Hollande and Carruette-Valentin 1971,
1972). Such observations established the general concept of lophomonads as a distinct group
within the hypermastigotes. This concept has
largely endured: a recent morphological classication of the parabasalids places Lophomonas in
its own family within the order Lophomonadida
(class Hypermastigida) along with four other families, Joeniidae, Rhizonymphidae, Microjoeniidae,
and Kofoidiidae; all except Joeniidae are monogeneric (Brugerolle and Lee 2000). Interestingly,
all except Lophomonas are found exclusively in
the hindguts of lower termites (Brugerolle and Lee
2000). Consensus has not been reached as to the
closest relative of Lophomonas, however. Kofoidia
(Light 1926b), Joenia (Grassi 1885), Microjoenia
(Kirby 1949, though now considered a spirotrichonymphid, Brugerolle 2001; Yamin 1979), and

even Polymastix (now known to be an oxymonad,


MacKinnon 1913) have each been proposed as the
sister genus of Lophomonas.
Molecular phylogenetics have upset the traditional concept of lophomonads as a lineage
of hypermastigotes. One of the rst points to
emerge from such studies was the reliably recovered union of lophomonads with devescovinids and
calonymphids (Frlich and Knig 1999; Gerbod
et al. 2000, Keeling et al. 1998), a grouping that
was soon acknowledged as consistent with ultrastructural evidence and ofcially established as the
order Cristamonadida (Brugerolle and Patterson
2001). The remaining (non-lophomonad) hypermastigotes were shown to be polyphyletic, falling
into two supported clades, now called Trichonymphida and Spirotrichonymphida (Adl et al.
2005; Carpenter et al. 2009; Carpenter and
Keeling 2007; Cepicka et al. 2010; Ohkuma et al.
2000, 2005). Finally, and most signicantly for
the lophomonads, the sampled Joeniidae genera (Joenia, Joenina, and Joenoides) were shown
to branch separately from each other and from
the deltotrichonymphids Deltotrichonympha and
Koruga (Frlich and Knig 1999; Noda et al.
2009). Due to the lack of consistent support
for any sub-groupings within the Cristamonadida,
all cristamonads were recently included within a
single family, Lophomonadidae (Cepicka et al.
2010). Thus, the currently recognized relatives of
Lophomonas are nearly the inverse of the traditional grouping: Lophomonadidae now includes
many of the traditional trichomonads and neither contains, nor belongs to, any hypermastigote
grouping. Molecular data are still lacking from many
lophomonad genera, so we have contributed to
addressing this deciency by determining the SSU
rRNA sequence of L. striata. We nd, unexpectedly,
that it does not branch within the Cristamonadida,
as other sampled lophomonads have to date, but
instead forms a new lineage sister to the Trichonymphida.

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

276 G.H. Gile and C.H. Slamovits

never observed L. blattarum, though it has been


reported to occasionally co-occur with L. striata in
the hindgut of P. americana (Kudo 1926; Semans
1943). Genomic DNA from a single isolated L. striata cell and from a pool of 50 isolated cells yielded
SSU PCR products of approximately 1600 bp. Six
clones from the single cell PCR product and three
clones from the 50 cell pool PCR product were
sequenced, assembled into a contig, and found to
differ at only 17 positions in total, nine of which were
evenly distributed among the single cell clones. No
clone from the single cell PCR differed by more than
two positions from the consensus, and all polymorphisms occurred within variable or highly variable
regions.
Our phylogenetic analysis (Fig. 1A) includes
representatives of every parabasalid clade for
which data are available, with the exception of
the extremely divergent Trichonympha spp. from
Cryptocercus punctulatus (Carpenter et al. 2009),
which in preliminary analyses had a disruptive
effect on the topology within the Trichonymphida.
It is displayed as if rooted as nearly as the topology will allow to the position determined previously
by midpoint and enforced molecular clock rooting
methods (Hampl et al. 2004). In maximum likelihood (ML) and Bayesian phylogenetic analyses
(Fig. 1A), L. striata branches with the Trichonymphida with strong support (100% ML bootstrap,
99% logDet distance bootstrap, and 1.0 posterior
probability). Because its exclusion from within the
Trichonymphida is less well supported (89/74/0.97),
we carried out approximately unbiased (AU) tests
to assess the level of condence in alternative
trees in which L. striata branches within the Trichonymphida. We also tested topologies in which
L. striata branches with other traditional lophomonads or with Monocercomonas, which was the top
BLAST hit for L. striata SSU (Table 1). All alternate
positions for L. striata were rejected at the 5% level.
To investigate the possibility that L. striata SSU
branches sister to the Trichonymphida due to long
branch attraction artifact (though the L. striata
branch is not particularly long), we carried out
phylogenetic analyses in which all Trichonymphida
sequences were removed from the alignment (not
shown). In both PhyloBayes and RAxML trees, the
topology of the remaining taxa did not change,
and L. striata branched weakly (52% ML BS, 0.52
PP) with a clade of Hexamastix and its unidentied parabasalid relatives (putatively Tricercomitus
spp., Hampl et al. 2004; Ohkuma et al. 2000).
Notably, this is the branching position of L. striata and the Trichonymphida in our full analysis.
Because Hexamastix and its sister taxon Crypto-

cercus punctulatus symbiont AB443599 are also


long branches, we performed an additional analysis
with these two sequences as well as the Trichonymphida omitted. Once again, the topology of
the remaining tree did not change, and L. striata branched weakly (47% ML bootstrap support)
with the unidentied parabasalid relatives of Hexamastix or in a tritomy with Hexamastix relatives
and the clade of Monotrichomonas, Ditrichomonas,
and Honigbergiella (0.76 posterior probability); both
are consistent with the branch point of L. striata
in the full analysis. These analyses suggest the
long branches of the Trichonymphida clade are not
attracting L. striata away from a better-supported
position.
Nucleotide composition bias is another potentially confounding factor in phylogenetic analyses.
We performed a chi-square test of base composition heterogeneity, and found that both L.
striata and the Trichonymphida SSU sequences
fail at the 5% level. Accordingly, we performed
a distance-based phylogenetic analysis using the
logDet transformation (Lockhart et al. 1994; Tamura
and Kumar 2002) which was designed to overcome
the inuence of biased nucleotide composition on
phylogenetic inference. The topology we recovered
was similar to the ML topology. The position of
L. striata at the base of the Trichonymphida was
recovered with 99% bootstrap support, and the
branching order within the Trichonymphida differed
only in the placement of Leptospironympha sister
to the Barbulanympha/Urinympha clade. Our distance analysis therefore suggests that nucleotide
composition bias is not the main cause of L. striata branching sister to the Trichonymphida. One
notable difference in the distance topology was the
sisterhood of Spirotrichonymphida and the L. striata
plus Trichonymphida clade, though this relationship
was very weakly supported (51%).
Though supported, the position of L. striata
remains technically ambiguous due to the lack of
a root in our analysis. The root of the parabasalid
SSU tree is currently unknown and likely impossible
to determine, according to previous comprehensive
analyses (Hampl et al. 2004). The extremely long
branch leading to parabasalids makes any rooting
unreliable (Gunderson et al. 1995; Huelsenbeck
et al. 2002; Keeling and Palmer, 2000). Because
the placement of L. striata reduces the length of
the Trichonymphida branch, we might expect the
artifactual attraction of the outgroup to the Trichonymphida to be reduced, though the extreme
length of the outgroup branch would likely still preclude a robust placement. Nevertheless, we carried
out phylogenetic analyses using the shortest-

Phylogenetic Position of Lophomonas 277


AB262486 Pseudotrichonympha grassii
HQ683705 Pseudotrichonympha paulistana
HQ683706 Pseudotrichonympha hertwigi
92/-/1.0
AB032235 Eucomonympha sp.
AB183876 Teranympha mirabilis
76/-/0.99
GQ168515 Leptospironympha sp.
0.1
AB443594 Barbulanympha sp.
-/70/98/61/1.0
ML/logDet/PB
AB443592 Urinympha talea
71/80/AB443595 Barbulanympha ufalula
AB183879 Hoplonympha sp.
89/74/0.97
84/99/AB434784 Trichonympha sphaerica
AB434800 Trichonympha sp.
AF052711 Trichonympha magna
75/-/0.93
AB183882 Staurojoenina assimilis
JN088049
Lophomonas
striata
77/-/0.99
AY321149 Hexamastix kirbyi
89/90/0.99
AB443599 Cryptocercus punctulatus symbiont
100/71/0.99
AB443597
Cryptocercus
punctulatus
symbiont
63/-/0.99
-/-/0.99
AB032215 Cryptotermes domesticus symbiont
92/70/0.99
AF052700 Cryptotermes brevis symbiont
90/99/0.99
AF072905 Monotrichomonas sp.
DHU17505 Ditrichomonas honigbergi
AY319280 Honigbergiella ruminantium
90/100/AY247745 Cochlosoma anatis
90/-/0.99
-/-/0.99
X87131 Pentatrichomonoides scroa
GQ254639 Tetratrichomonas undula
AY338473 Trichomonas vaginalis
94/-/0.99
AF479642 Trichomitopsis termopsidis
AF052706 Pseudotrypanosoma giganteum
AB032206 Reticulitermes speratus symbiont
AF124609 Pentatrichomonas hominis
94/81/0.98
HM581663 Pseudotrichomonas keilini
HM748759 Lacusteria cypriaca
AF076958 Trichomitus batrachorum
AF076959 Hypotrichomonas acosta
AB032204 Reticulitermes speratus symbiont
76/-/0.99
73/-/AB032226 Spirotrichonympha sp.
70/98/AB183881 Spirotrichonymphella sp.
AF052715 Porotermes adamsoni symbiont
AB032213 Spirotrichonympha leidyi
AB032212 Holomastigotoides mirabile
87/99/0.96
DQ855405 Caduceia versatilis
HQ215839 Devescovina sp.
-/-/0.91
FN377766 Devescovina sp.
U17506 Metadevescovina polyspira
FM160644 Metadevescovina modica
AB458860 Foaina nana
FJ986221 Coronympha koidzumia
FJ986222 Coronympha mackinnonia
82/57/1.0
FN377802 Cryptotermes havilandi symbiont
95/96/0.94
AB458862 Macrotrichomonas sp.
AB458854 Joenia annectens
AB458861 Gigantomonas herculea
74/62/HQ593144 Snyderella yamini
74/70/0.94
HQ215838 Snyderella swezyae
78/56/0.97
AY063294 Calonympha grassii
HQ215836 Calonympha chia
AB458857 Stephanonympha sp.
-/68/AJ583377 Mixotricha paradoxa
AJ583378 Deltotrichonympha nana
AJ132467 Koruga bonita
AB458855 Joenina pulchella
AB458856 Joenoides intermedia
80/-/0.99
AJ920323 Histomonas meleagridis
U37461 Dientamoeba fragilis
AY055803 Tritrichomonas nonconforma
GQ254637 Simplicimonas similis
-/96/HQ215840 Neotermes castaneus symbiont
U17507 Monocercomonas sp. ATCC 50210
DQ174297 Monocercomonas colubrorum

-/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.

278 G.H. Gile and C.H. Slamovits


Table 1. Approximately unbiased (AU) test results for phylogenetic positions of Lophomonas striata.
Clade

p-value

Trichonymphida (ML and Bayesian topology)


Pseudotrichonympha, Eucomonympha, Teranympha (PET)
PET plus Leptospironympha (PETL)
PETL plus Barbulanympha and Urinympha (PETLBU)
PETLBU plus Hoplonympha
Barbulanympha and Urinympha
Trichonympha
Trichonympha and Staurojoenina
Joenia
Joenina
Joenoides
Koruga and Deltotrichonympha
Cristamonadida
Monocercomonas
Hexamastix and Cryptocercus and Cryptotermes symbionts
Histomonas and Dientamoeba

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

branching available Fornicata and Oxymonadida


sequences available as an outgroup to examine the
effects on the placement of L. striata (not shown).
The ML tree was rooted on the branch of Snyderella
swezyae, and the PhyloBayes consensus tree was
rooted on a 5-way polytomy in which the Cristamonadida were split into two clades with Joenina
pulchella, Joenoides intermedia, and the rest of
Parabasalia forming the other three clades. Neither of these root placements were supported, and
neither agree with morphological evolutionary interpretations of the group, but in both analyses L.
striata maintained a supported position sister to
Trichonymphida.

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

of the parabasalid tree pointed to Trichonymphida


plus Spirotrichonymphida (Keeling et al. 1998)
or Pseudotrichonympha (Ohkuma et al. 2000) as
the earliest-diverging lineage, though both concluded that the root could not be determined
with condence. Later analyses including more
representatives of Trichonymphida and Spirotrichonymphida found that the root position varied
according to the method of analysis, and that no
position for the root was signicantly more likely
than any other (Hampl et al. 2004; Ohkuma et al.
2005). Furthermore, a long branch taxon-exclusion
analysis strongly suggested that the most commonly recovered root position, on or within the
Trichonymphidae, is due to long branch attraction
(LBA) artifact (Hampl et al. 2004). Protein phylogenies also tend to root the parabasalid tree
on Trichonympha (Gerbod et al. 2004) or Trichonymphida when Eucomonympha is included
(Ohkuma et al. 2007), though the -tubulin phylogeny is rooted instead on Tetratrichomonas
(Gerbod et al. 2004). While this has not been
examined specically, the long branches of Trichonymphida in protein phylogenies suggest LBA is
likely to be the culprit here as well. Despite many
attempts, phylogenetic analyses have failed to support a root position for parabasalids that is not
discredited by phylogenetic artifact.
If the root of Parabasalia were to fall within
or at the base of the Trichonymphida, L. striata
would have to be considered sister to the rest of
Parabasalia, rather than sister to Trichonymphida.
A root position within the Trichonymphida would
seem unlikely due to their shared morphological

Phylogenetic Position of Lophomonas 279

characteristics (a true rostrum, bilateral symmetry,


and mitosis in which each daughter cell inherits one
agellar region, Brugerolle and Lee 2000). Given
that molecular phylogenetics have failed to nd a
well-supported root, and that morphology-based
evolutionary scenarios consider simpler cells such
as Monocercomonas to be the ancestral state of
parabasalids (Honigberg 1963; Kirby 1947), we
consider the root unlikely to fall within or at the base
of Trichonymphida, and L. striata is therefore a new
parabasalid lineage sister to the Trichonymphida.
The unexpected sisterhood of L. striata and Trichonymphida raises the question of whether shared
morphological or ultrastructural characters exist
that have been previously overlooked. Members
of the Trichonymphida share a bilateral symmetry
with two agellar areas that are each donated to
a daughter cell upon mitosis (Brugerolle and Lee
2000). The agella in Lophomonas were reported
to occur in a ring in light microscopical studies (Janicki 1910; Kudo 1926), suggesting the
possibility of bilateral symmetry, but transmission
electron microscopy revealed that the basal bodies
of the apical tuft instead form a single asymmetrical, loosely helical cluster, likened in shape to an
ear (Beams et al. 1960; Hollande and CarruetteValentin 1972). Mitosis in Lophomonas is said
to proceed exactly as observed in Joenia (agella are resorbed during prophase), though the
unusual persistence of the mitotic spindle during interphase in Lophomonas has also been
noticed occasionally in Staurojoenina, Idionympha,
and Holomastigotoides (Hollande and CarruetteValentin 1972). Overall, the general body plan and
details of mitosis do not suggest a close relationship
between Lophomonas and Trichonymphida. Likewise, ultrastructural details of the agellar area in
Lophomonas are more easily compared with Joeniidae than Trichonymphida. In Trichonymphida, a
set of one, two, or three privileged basal bodies is typically associated with each of the two
agellar areas, but the morphology of the cell
apex (rostrum) is complex and varies considerably among the genera, making the arrangement
of privileged basal bodies difcult to compare
with other parabasalids (Hollande and CarruetteValentin 1971). In Joeniidae, the arrangement of
the privileged basal bodies and their associated
bres is strikingly similar to Monocercomonas and
other simple parabasalids: basal bodies 1 and 3
each bear a hooked lamina, basal body 2 bears
a sigmoid bre, and the fourth basal body is perpendicular to the other three and subtends the
recurrent agellum (Brugerolle 1991). The basal
bodies of the agellar tuft each bear a hooked

lamina, suggesting they derive from multiplication


of basal body 1 and/or 3 (Brugerolle 1991, 2001;
Brugerolle and Bordereau 2005, Lavette 1970;
Radek and Hausmann 1994). Lophomonas differs from the joeniid arrangement in two ways.
First, the basal body of the recurrent agellum
is parallel rather than perpendicular to the other
privileged basal bodies. Second, and more interestingly, the basal bodies of the agellar tuft do not
bear a hooked lamina (Brugerolle 1991; Brugerolle
and Lee 2000; Hollande and Carruette-Valentin
1972), suggesting they may derive from multiplication of the unadorned basal body of the recurrent
agellum. The apical agellar tuft of Lophomonas
could therefore have arisen independently from the
agellar tufts of Joeniidae. Although the available
ultrastructural data do not reveal shared characters
between Lophomonas and Trichonymphida, the differences between Lophomonas and Joeniidae are
consistent with their distant positions in our molecular phylogeny.
The sisterhood of L. striata and Trichonymphida
also raises the possibility that L. striata, from cockroaches, and Trichonymphida, from wood roaches
and termites, were inherited vertically from a common, gut symbiotic ancestor. Because termites
and the wood roach Cryptocercus are sister taxa
(Inward et al. 2007; Legendre et al. 2008) and harbour related parabasalid and oxymonad symbionts,
it is now accepted that these symbionts were inherited vertically from the insects common ancestor.
This has long been suggested for the genus Trichonympha, which occurs in both Cryptocercus
and termites (Kirby 1932, 1949). Although Trichonympha spp. from Cryptocercus may be better
considered a distinct genus due to their morphological and ultrastructural differences (Carpenter et al.
2009; Hollande and Carruette-Valentin 1971), the
Trichonympha clade from Cryptocercus branches
sister to the Trichonympha clade from termites
(Carpenter et al. 2009; Ohkuma et al. 2009). For
Lophomonas and the Trichonymphida to share
a common, gut-symbiotic ancestor, however, the
ancestral host insect would have to have been the
common ancestor of cockroaches, wood roaches,
and termites. This is not unreasonable considering the insect phylogeny: Blattidae, which includes
P. americana and B. orientalis, is the sister group
to the Cryptocercus/termites clade (Inward et al.
2007).
A related, though more distant possibility is that
Lophomonas or its close relatives may not only
have been inherited vertically from a more ancient
common ancestor than previously thought, but may
inhabit Cryptocercus and/or certain termites to

280 G.H. Gile and C.H. Slamovits

this day. Putative close relatives of Lophomonas


exist in the literature: the genus Eulophomonas
was described from Kalotermes avicollis (Grassi
and Fo 1911), though this organism has not
been observed since and may have been a multiple ssion form of Monocercomonas (Cleveland
et al. 1934), and Prolophomonas topocola was
described from certain populations of Cryptocercus punctulatus (Cleveland et al. 1934), though it
may properly belong to Lophomonas (Brugerolle
and Lee 2000). It should be noted in this context
that Lophomonas can encyst (Grass 1952; Janicki
1910; Kudo 1926), and may therefore have the ability to colonize different hosts in addition to the more
typical vertical transmission route of termite and
wood roach symbionts. In order to conrm or refute
these possibilities, it would be interesting to determine whether Lophomonas or related organisms
inhabit the hindguts of other members of the Blattidae, such as Eurycotis or Deropeltis spp., and to
nd and acquire molecular data from the putative
genera Prolophomonas and Eulophomonas.
Another implication of L. striata being sister
to Trichonymphida is taxonomic. Previous analyses have shown that the Lophomonadida of
Brugerolle and Lee (2000) is not monophyletic,
though the species sampled before now, Joenia
annectens, Joenina pulchella, Joenoides intermedia, Deltotrichonympha nana, Deltotrichonympha
operculata, and Koruga bonita, all form deep
branches within the Cristamonadida (Frlich and
Knig 1999; Ikeda-Ohtsubo et al. 2007; Noda
et al. 2009). The common term lophomonads
remains useful, however, in order to designate
non-trichonymphid, non-spirotrichonymphid, multiagellate parabasalids with a single nucleus.
Because neither lophomonads nor devescovinids
nor calonymphids are monophyletic, Cepicka et al.
(2010) lumped all cristamonads together in one
family, called the Lophomonadidae. This family
name can now only properly include Lophomonas,
though this will ultimately depend on the position
of the type species, L. blattarum. There is no reason to doubt that L. blattarum and L. striata are
closely related, as they share morphological and
ultrastructural characteristics (Beams et al. 1960,
1961; Btschli 1898; Janicki 1910). However, L.
blattarum is the type species, so L. striata would
require a new genus name if the two were found not
to be sisters. Whether a future Lophomonadidae
will include genera besides Lophomonas remains
to be determined; several lophomonad taxa still
lack molecular data. However, since Lophomonas
is alone among the lophomonads both in inhabiting
the guts of omnivorous roaches and in not branch-

ing among the Cristamonadida, perhaps a better


place to look for relatives of L. striata would be the
uninvestigated guts of other blattid roaches.

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

Phylogenetic Position of Lophomonas 281


with the long-branching Trichonymphida omitted from one, and
Trichonymphida, Hexamastix kirbyi, and Cryptocercus punctulatus symbiont AB443599 omitted from the other. Condence
in alternate topologies was assessed using approximately unbiased (AU) tests (Shimodaira 2002, results in Table 1) on a set of
1000 trees from the ML bootstrap analysis plus 16 constrained
maximum likelihood trees. Constraint trees were computed in
RAxML with only the relevant grouping specied; the topologies
both within and outside of the constrained clades were allowed
to vary. AU tests were performed using the program CONSEL
1.19 (Shimodaira and Hasegawa 2001) from site-likelihoods
computed in RAxML. A Chi-square test of base composition
heterogeneity was performed in TREE-PUZZLE 5.2 (Schmidt
et al. 2002), and a distance-based phylogenetic analysis using
a modied logDet transformation (Lockhart et al. 1994; Tamura
and Kumar 2002) was carried out on the full, unrooted dataset
in MEGA5 (Tamura et al. 2011).

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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