Preaxostyla

Abstract

Preaxostyla comprises Oxymonadida, containing 14 genera of gut endosymbionts plus two genera of free-living bacterivorous flagellates from low oxygen sediments (Trimastix and Paratrimastix). The group was recognized on the 18S rRNA phylogenies, and ultrastructural investigations have revealed a synapomorphy in the organization of the “I” fiber that supports microtubular root R2. Trimastix and Paratrimastix are typical excavates with three anterior/lateral flagella and the recurrent flagellum passing through a conspicuous ventral feeding groove. The cellular structure of oxymonads is more derived, and a particularly striking diversity of large cellular forms is observed in genera inhabiting guts of lower termites and wood-eating cockroaches. Here the large oxymonad species and their bacterial ecto- and endosymbionts are probably involved in the cellulose digestion, similarly to the large species of parabasalids. All Preaxostyla live in low oxygen environments, and this has affected their metabolism and organelle complement. Glycolysis is apparently the main source of cellular ATP and mitochondria are either reduced to hydrogenosome-like compartments (in Trimastix and Paratrimastix) or lost completely (in oxymonads). Peroxisomes are absent in the whole group. Stacked Golgi bodies are unknown in oxymonads; however, genes encoding proteins functional in Golgi are present, indicating the existence of a cryptic Golgi. Phylogenomic analyses have shown that Preaxostyla represent one of the three main lineages of Metamonada (within Excavata). Because oxymonads are the only known eukaryotes that have completely lost the mitochondrial organelle, they may serve as models for studies of amitochondriality and mitochondrial evolution.

Full text

Preaxostyla
Vladimir Hampl
Contents
Summary Classification ........................................................................... 2
Introduction ....................................................................................... 3
General Characteristics ........................................................................ 3
Occurrence . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Literature and History of Knowledge . . . ...................................................... 3
Practical Importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Habitats and Ecology . . . . . . . . ..................................................................... 5
Characterization and Recognition . . . . . . . .. . .. . . . .. .. . . . . .. .. . . . .. . . . .. . .. . . . .. . .. . . . .. .. . . . .. . .. . 11
Organization of Cytoskeleton . . . .............................................................. 11
Sex and Reproduction ......................................................................... 13
Taxonomy . . . ...................................................................................... 15
Trimastigidae . ................................................................................. 15
Paratrimastigidae . . . ........................................................................... 15
Oxymonadida . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . 16
Polymastigidae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . .. .. . . . .. . .. . . . .. .. . . . .. . .. . . . .. . .. . . . .. . 16
Streblomastigidae . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . 19
Pyrsonymphidae ............................................................................... 19
Saccinobaculidae . ............................................................................. 21
Oxymonadidae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . .. . . .. .. . . . .. .. . . .. . .. . . .. .. . . . .. .. . . .. . .. 22
Opisthomitus Duboscq & Grassé 1934 ....................................................... 24
Maintenance and Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . 24
Phylogeny and Evolution . . . . . . . . . . .. . .. . . .. . .. . . . .. .. . . . .. . .. . . .. . .. . . . .. .. . . . .. .. . . . .. . .. . . . 25
References . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Abstract
Preaxostyla comprises Oxymonadida, containing 14 genera of gut endosymbi-
onts plus two genera of free-living bacterivorous flagellates from low oxygen
V. Hampl (*)
Department of Parasitology, Charles University in Prague, Prague, Czech Republic
e-mail: vlada@natur.cuni.cz
#Springer International Publishing Switzerland 2016
J.M. Archibald et al. (eds.), Handbook of the Protists,
DOI 10.1007/978-3-319-32669-6_8-1
1

sediments (Trimastix and Paratrimastix). The group was recognized on the 18S
rRNA phylogenies, and ultrastructural investigations have revealed a synapo-
morphy in the organization of the “I”fiber that supports microtubular root R2.
Trimastix and Paratrimastix are typical excavates with three anterior/lateral
flagella and the recurrent flagellum passing through a conspicuous ventral feeding
groove. The cellular structure of oxymonads is more derived, and a particularly
striking diversity of large cellular forms is observed in genera inhabiting guts of
lower termites and wood-eating cockroaches. Here the large oxymonad species
and their bacterial ecto- and endosymbionts are probably involved in the cellulose
digestion, similarly to the large species of parabasalids. All Preaxostyla live in
low oxygen environments, and this has affected their metabolism and organelle
complement. Glycolysis is apparently the main source of cellular ATP and
mitochondria are either reduced to hydrogenosome-like compartments
(in Trimastix and Paratrimastix) or lost completely (in oxymonads). Peroxisomes
are absent in the whole group. Stacked Golgi bodies are unknown in oxymonads;
however, genes encoding proteins functional in Golgi are present, indicating the
existence of a cryptic Golgi. Phylogenomic analyses have shown that Preaxostyla
represent one of the three main lineages of Metamonada (within Excavata).
Because oxymonads are the only known eukaryotes that have completely lost
the mitochondrial organelle, they may serve as models for studies of amitochon-
driality and mitochondrial evolution.
Keywords
Bacterivore •Endosymbionts •Termites •Excavata •Trimastix •Paratrimastix •
Oxymonads •Amitochondriate
Summary Classification
•Preaxostyla
•• Trimastigidae (Trimastix)
•• Paratrimastigidae (Paratrimastix)
•• Oxymonadida
•••Polymastigidae (Monocercomonoides,Polymastix,Tubulimonoides,
Paranotila)
•••Streblomastigidae (Streblomastix)
•••Pyrsonymphidae (Pyrsonympha,Dinenympha,Pyrsonymphites†,
Dinenymphites†)
••• Saccinobaculidae (Saccinobaculus,Notila)
•••Oxymonadidae (Oxymonas,Microrhopalodina,Barroella,Sauromonas,
Oxymonites†,Microrhopalodites†,Sauromonites†)
••• Opisthomitus
2 V. Hampl

Introduction
General Characteristics
Preaxostyla are heterotrophic protists, typically bearing four flagella. Trimastix and
Paratrimastix have a typical excavate morphology, with a hunched appearance and
conspicuous excavate ventral groove. Flagella originate subapically, and the poste-
rior flagellum trails through the cytostome and bears two vanes. Oxymonadida
Grassé 1952 are morphologically diverse group, and they never form cytostomes.
In oxymonads, flagella are arranged in two separated pairs, and their number can
multiply to eight in Pyrsonympha and to eight or 12 in Saccinobaculus or increase to
many in, e.g., Microrhopalodina and Sauromonas. Nuclei or whole karyomastigonts
(nucleus, flagella, basal bodies, preaxostyle, axostyle, and microtubular roots) are
multiplied in the oxymonad genera Microrhopalodina and Barroella. Cells of
Trimastix and Paratrimastix contain hydrogenosome-like derivates of mitochondria
and, usually, stacked Golgi bodies. Neither mitochondria nor peroxisomes nor Golgi
bodies were reported in oxymonads with the potential exception of Saccinobaculus
doroaxostylus. Several oxymonad species have developed a microfibrillar organelle
for attachment to the intestinal wall (holdfast) often situated on an anterior extension
of the cell (rostellum). Preaxostyla are divided by binary fission and have either open
mitosis (Paratrimastix) or mitosis of a closed type with an intranuclear spindle
(Oxymonadida). Trophozoites are the dominant life stages of the cell cycle; forma-
tion of gametes and cysts has been demonstrated in only a few species.
Occurrence
Trimastix and Paratrimastix are free-living inhabitants of hypoxic sediments in
marine or freshwater habitats, respectively. The typical habitat of oxymonads is
the gut of insects; the exceptions are several species of Monocercomonoides that
inhabit intestines of vertebrates. The largest diversity of oxymonads, in terms of both
species count and morphology, is found in the hindgut of lower termites and the
cockroach genus Cryptocercus.
Literature and History of Knowledge
Light microscopy of Trimastix and Paratrimastix was studied by Saville Kent
(1880), Grasse (1952), and Bernard et al. (2000). Light microscopy and ultrastruc-
ture was studied by Brugerolle and Patterson (1997), O’Kelly et al. (1999), Simpson
et al. (2000), and, most recently, Zhang et al. (2015), who also revised the taxonomy
and created genus Paratrimastix for two species originally classified as Trimastix.
Preaxostyla 3

Transcriptomic and cell biological studies, all on Paratrimastix pyriformis, were
performed by Stechmann et al. (2006), and Zubáčová et al. (2013). The evolutionary
history of the group has been investigated by Dacks et al. (2001), Hampl
et al. (2009), and Zhang et al. (2015).
Oxymonads (Pyrsonympha vertens and Dinenympha gracilis)werefirst
observed by Leidy (1877). During the first half of the twentieth century, all genera
and most species were described using light microscopy. Between 1960 and 1990,
the ultrastructure of the most important genera was reconstructed using electron
microscopy, with a particular focus on the structureofaxostyleandthemechanism
of its movement (e.g., McIntosh et al. 1973; Brugerolle and Joyon 1973;
Bloodgood et al. 1974). The first papers that employed molecular methods to
study the diversity and evolutionary history of oxymonads and their bacterial
symbionts were published at the very end of the twentieth century (Moriya
et al. 1998;Iidaetal.2000;Tokuraetal.2000; Dacks et al. 2001). Fragmentary
information on oxymonad biochemistry, molecular genetics, and cellular biology
became available from 2003 onward (e.g., Keeling and Leander 2003; Liapounova
et al. 2006). The genome project of Monocercomonoides sp. was finished in 2016
(Karnkowska et al. 2016).
The earliest light microscopic observations of oxymonads were performed by
Porter (1897), Kofoid and Swezy (1919,1926), Kidder (1929), Powell (1928),
Kirby (1928), Jírovec (1929), Georgevitch (1932), Cleveland et al. (1934), Cross
(1939,1946), Kirby and Honigberg (1949), Nie (1950), Cleveland (1950a,b,c,
1966), Moskowitz (1951), Gabel (1954), and Jensen and Hammond (1964). The
most comprehensive light microscopic tract is in Grassé (1952). The fossils of
oxymonads have been studied by Poinar (2009a,b). Oxymonad ultrastructure was
studiedwithelectronmicroscopybyGrimstoneandCleveland(1965), Hollande
and Carruette-Valentin (1970a,b), Brugerolle (1970), Smith and Arnott (1973a),
Mcintosh et al. (1973), Lavette (1973), Brugerolle and Joyon (1973), Bloodgood
et al. (1974), Kulda and Nohýnková (1978), Cochrane et al. (1979), Brugerolle
(1981), Radek (1994), Brugerolle and König (1997), Rother et al. (1999), Simpson
et al. (2002), Brugerolle et al. (2003), Leander and Keeling (2004), Maass and
Radek (2006), Carpenter et al. (2008), and Tamschick and Radek (2013). Physi-
ological and electron microscopic studies regarding axostyle motility were
performed by Mcintosh et al. (1973), Mcintosh (1973,1974), Bloodgood and
Fitzharris (1978), Heuser (1986), and Jensen and Smaill (1986). The symbiotic
bacteria of oxymonads were studied by Smith and Arnott (1974b), Iida
et al. (2000), Tokura et al. (2000), Noda et al. (2003,2006), Stingl et al. (2005),
Yang e t a l . ( 2005), and Hongoh et al. (2007). The cell biology and biochemistry of
oxymonads have been studied by Keeling and Leander (2003), Slamovits and
Keeling (2006a,b), Liapounova et al. (2006), de Koning et al. (2008), and
Dacks et al. (2008). The first genomic project was carried out by Karnkowska
et al. (2016). The evolutionary history of oxymonads has been studied by Moriya
et al. (1998,2001,2003), Dacks et al. (2001),Hampletal.(2005,2009), Heiss and
Keeling (2006), Carpenter et al. (2008), and Radek et al. (2014).
4 V. Hampl

Practical Importance
Oxymonads are of indirect practical importance because of their obligate association
with their wood-destroying hosts, the dry wood and subterranean lower termites, and
the closely related wood-feeding cockroach Cryptocercus (Lo et al. 2000; Inward
et al. 2007). Because of their large microtubular axostyles, pyrsonymphids, and
saccinobaculids have been useful subjects for research into microtubule function.
Oxymonads represent the only known group of eukaryotes containing
amitochondriate representatives (Karnkowska et al. 2016).
Habitats and Ecology
Trimastix and Paratrimastix are small free-living bacterivorous flagellates inhabiting
marine and freshwater sediments that are low in oxygen, where they presumably
play a role in grazing bacteria, creating a food-web link between the bacterial
biomass and larger organisms.
All oxymonads are endobiotic, and most representatives inhabit the hindgut of
lower termites and the intestine of the wood-feeding cockroaches. Several species
live in the intestine of larvae of the crane fly and Scarabaeoidea beetles, myriapods,
and the intestine of vertebrates. The list of oxymonad species and their hosts is given
in Table 1. There are no known pathogenic species.
Oxymonads are often involved in symbiotic relationships. Oxymonads of ter-
mites and wood-feeding cockroaches are members of large communities of bacteria,
archaea, and anaerobic protists (especially parabasalids) in the hindgut of the host
(Brune and Ohkuma 2011; Ohkuma and Brune 2011). The community is essential
for cellulose digestion, and if the microorganisms are killed, the insect dies within a
few weeks (Cleveland 1924). The exact role of the flagellates (oxymonads and
parabasalids) in cellulose digestion is not clear (for review, see Radek (1999), Li
et al. (2006), Brugerolle and Radek (2006), Brune and Ohkuma (2011)). Micro-
scopic observations clearly show that large oxymonads (Pyrsonympha,Oxymonas,
Microrhopalodina), similarly to large parabasalids (e.g., Trichonympha), internalize
and digest large pieces of wood. High-resolution imaging mass spectrometry
(NanoSIMS) gave direct evidence for the flow of organic carbon from
13
C-enriched
cellulose to the cell interior of Oxymonas dimorpha (Carpenter et al. 2013). The
smaller species are probably not involved in cellulose digestion (Cleveland 1925;
Radek 1999). Production of cellulolytic enzymes has been reported in several
parabasalid species (Yamin 1981; Nakashima et al. 2002; Zhou et al. 2007) but
not, so far, in any oxymonad. The association of oxymonads with termites and
roaches was observed in 97–110 mya old Cretaceous fossils (Poinar 2009a,b).
The surface and cytoplasm of most oxymonads are colonized by prokaryotic
symbionts. The surface bacteria belong to the groups Spirochaetes (Iida et al. 2000;
Noda et al. 2003)andBacteroidales (Noda et al. 2006;Hongohetal.2007). Protists
and/or bacteria often form special attachment structures (Bloodgood et al. 1974; Smith
Preaxostyla 5

Table 1 List of species of Preaxostyla and their hosts. The older synonyms are given in
brackets. †Extinct species
Species Host
Barroella coronaria Cross 1946 Postelectrotermes [Neotermes] howa
Barroella zeteki (Zeliff 1930)Calcaritermes brevicollis
Dinenympha aculeata Georgevitch 1951 Reticulitermes lucifugus
Dinenympha aviformis Georgevitch 1951 Reticulitermes lucifugus
Dinenympha exilis Koidzumi 1921 Reticulitermes [Frontotermes] speratus
Dinenympha fimbriata Kirby 1924 Reticulitermes lucifugus,Reticulitermes
flavipes. Reticulitermes hageni,Reticulitermes
hesperus,Reticulitermes virginicus
Dinenympha gracilis Leidy 1877 Reticulitermes lucifugus,Reticulitermes
flavipes,Reticulitermes hesperus,
Reticulitermes tibialis
Dinenympha leidyi Koidzumi 1921 Reticulitermes speratus
Dinenympha mukundia Mukherjee and Maiti
1989
Reticulitermes tirapi
Dinenympha nobilis Koidzumi 1921 Reticulitermes speratus
Dinenympha parva Koidzumi 1921 Reticulitermes speratus
Dinenympha porteri Koidzumi 1921 Reticulitermes speratus
Dinenympha rayi Mukherjee and Maiti 1989 Reticulitermes tirapi
Dinenympha rugosa Koidzumi 1921 Reticulitermes speratus
Dinenymphites spiris Poinar 2009a †Kalotermes burmensis
Microrhopalodina hofmanni (De Mello and De
Mello 1944)
“Indian Cryptotermes”
Microrhopalodina inflata (Grassi and Foà
1911)
Kalotermes flavicollis
Microrhopalodina multinucleata (Kofoid and
Swezy 1926)
Cryptotermes dudleyi
Microrhopalodina occidentis (Lewis 1933) Pterotermes [Kalotermes]occidentis
Microrhopalodites polynucleatis Poinar 2009a
†
Kalotermes burmensis
Monocercomonoides adarshii Mali et al. 2001 Oryctes rhinoceros
Monocercomonoides aurangabadae Mali and
Patil 2003
Blattella germanica
Monocercomonoides blattae Blatta sp.
Monocercomonoides bovis Jensen and
Hammond 1964
Bos taurus
Monocercomonoides caprae (Das Gupta 1935)
[Monocercomonoides sayeedi Abraham 1961]
Capra hircus
Monocercomonoides caviae daCunha and
Muniz 1921 [Monocercomonoides hassalli
daCunha and Muniz 1927]
Cavia aperea var. porcellus
Monocercomonoides chakravartii
Krishnamurthy and Sultana 1976
Polyphaga indica
Monocercomonoides cunhai (daFonseca 1939) Cuniculus paca
Monocercomonoides digranula (Crouch 1933)Marmota monax
(continued)
6 V. Hampl

Table 1 (continued)
Species Host
Monocercomonoides dobelli Krishnamurthy
and Madre 1979
Amphibians (Bufo melanostictus)
Monocercomonoides exilis Nie 1950 Cavia aperea var. porcellus
Monocercomonoides filamentum Janakidevi
1961 maybe identical with
Monocercomonoides lacertae (Tanabe 1933)
Testudo elegans
Monocercomonoides ganapatii Rao 1969 Gryllotalpa africana
Monocercomonoides garnhami Rao 1969 Periplaneta americana
Monocercomonoides globus Cleveland
et al. 1934
Cryptocercus punctulatus
Monocercomonoides gryllusae Sultana and
Krishnamurthy 1978
Gryllus bimaculatus
Monocercomonoides hausmanni Radek 1996/
1997
Kalotermes sinaicus
Monocercomonoides indica Navarathnam
1970
Tatera indica
Monocercomonoides khultabadae Mali and
Mali 2004
Pycnoscelus surinamensis
Monocercomonoides krishnamurthii Sultana
1976
Pycnoscelus surinamensis
Monocercomonoides lacertae (Tanabe 1933)
[? Monocercomonoides filamentum Janakidevi
1961,Monocercomonoides mehdii
Krishnamurthy 1967,Monocercomonoides
singhi Krishnamurthy 1967]
Lizards, snakes, tortoises (Erimias argus)
Monocercomonoides lepusi Todd 1963 Lepus nigricollis
Monocercomonoides marathwadensis
Krishnamurthy and Sultana 1976
Periplaneta americana
Monocercomonoides mehdii Krishnamurthy
1967 maybe identical with
Monocercomonoides lacertae (Tanabe 1933)
Calotes versicolor
Monocercomonoides melolonthae Grassi 1879
[Monocercomonoides cetoniae (Jollos) Travis
1932,Monocercomonoides ligrodis Travis
1932]
Coleoptera larvae, Tipula larvae (Tipula sp.)
Monocercomonoides nimiei Ray 1949 Cavia cutleri
Monocercomonoides omergae Mali et al. 2001 Oryctes rhinoceros
Monocercomonoides orthopterorum Parisi
1910
Ectobius lapponicus,Periplaneta orientalis,
Periplaneta americana,Tipula abdominalis
larvae
Monocercomonoides oryctesae Krishnamurthy
and Sultana 1977
Oryctes rhinoceros
Monocercomonoides panesthiae Kidder 1937 Panesthia sp.
Monocercomonoides pileata Kirby and
Honigberg 1949
Citellus beecheyi,Citellus beldingi,Citellus
lateralis chrysoideus,Citellus leucurus,
Citellus tridecemlineatus,Peromyscus
maniculatus
(continued)
Preaxostyla 7

Table 1 (continued)
Species Host
Monocercomonoides polyphagae
Krishnamurthy and Sultana 1976
Polyphaga indica
Monocercomonoides qadrii Rao 1969 Oryctes rhinoceros
Monocercomonoides quadrifunilis Nie 1950 Cavia aperea var. porcellus
Monocercomonoides robustus Gabel 1954 Marmota monax
Monocercomonoides rotunda (Bishop 1932) Anuran amphibians
Monocercomonoides sayeedi Abraham 1961 Capra aegagrus hircus
Monocercomonoides segoviae Perez Reyes
1966
?
Monocercomonoides shortii Navarathnam
1970
Rattus rattus frugivorus
Monocercomonoides singhi Krishnamurthy
1967 maybe identical with
Monocercomonoides lacertae (Tanabe 1933)
Chameleon zeylanicus
Monocercomonoides spirostreptae
Krishnamurthy and Sultana 1980
Spirostreptus sp.
Monocercomonoides viperae Mandrae and
Krishnamurthy 1976
Vipera russelli
Monocercomonoides termitis Krishnamurthy
and Sultana 1977
“Indian termite”
Monocercomonoides tipulae Grassé 1926 Tipula larvae
Monocercomonoides wenrichi Nie 1950 Cavia aperea var. porcellus
Notila proteus Cleveland 1950c Cryptocercus punctulatus
Notila proteus ussuriensis Bobyleva 1973 Cryptocercus relictus
Opisthomitus avicularis Duboscq and Grasse
1934
Kalotermes flavicollis
Opisthomitus longiflagellatus Radek
et al. 2014
Neotermes jouteli
Opisthomitus flagellae Hollande and
Carruette-Valentin 1970b
Kalotermes dispar
Opisthomitus brasiliensis De Mello 1953 Cryptotermes brevis
Oxymonas barbouri Zeliff 1930 Glyptotermes angustus [barbouri]
Oxymonas bastiensis Tiwari 2005 Neotermes bosei
Oxymonas bengalensis Das, 1974 Cryptotermes havilandi
Oxymonas bosei Das 1974 Neotermes bosei
Oxymonas brevis Zeliff 1930 Cryptotermes brevis
Oxymonas caudata Cross 1946 maybe
identical with Oxymonas panamae Zeliff 1930
Proneotermes [Calotermes]perezi
Oxymonas chilensis Guzman 1961 Calotermes chilensis
Oxymonas clevelandi Zeliff 1930 Incisitermes immigrans [Kalotermes
clevelandi], Incisitermes [Kalotermes]
tabogae,Incisitermes fruticavus
Oxymonas dimorpha Connell 1930 Paraneotermes simplicicornis
Oxymonas diundulata Nurse 1945 Kalotermes brouni
Oxymonas gigantea Poinar 2009b †Blattellidae
(continued)
8 V. Hampl

Table 1 (continued)
Species Host
Oxymonas gracilis Kofoid and Swezy 1926 Rugitermes [Kalotermes] magninotus
Oxymonas grandis Cleveland 1935 Neotermes dalbergiae,Neotermes tectonae,
Neotermes bosei
Oxymonas granulosa Janicki 1915 Incisitermes marginipennis,Neotermes
connexus
Oxymonas hirtelli Mello 1954 Neotermes hirtellus
Oxymonas hubbardi Zeliff, 1930 Incisitermes marginipennis,Marginitermes
[Kalotermes] hubbardi
Oxymonas janicki Zeliff 1930 Kalotermitidae
Oxymonas jouteli Zeliff 1930 Neotermes [Kalotermes] jouteli
Oxymonas kirbyi Zeliff 1930 Rugitermes kirbyi
Oxymonas megakaryosoma Cross 1946 Glyptotermes sp.
Oxymonas megarostelata Bala and Bhagat
1993
Odontotermes obesus
Oxymonas minor Zeliff 1930 Incisitermes [Kalotermes] minor
Oxymonas notabilis Cross 1946 Postelectrotermes [Neotermes] howa
Oxymonas ovata Zeliff 1930 Calcaritermes brevicollis
Oxymonas panamae Zeliff 1930 maybe
identical with Oxymonas caudata Cross 1946
Rugitermes panamae
Oxymonas parvula Kirby 1926 Cryptotermes domesticus [hermsi]
Oxymonas pediculosa Kofoid and Swezy 1926 Calcaritermes [Kalotermes] nigriceps,
Rugitermes panamae
Oxymonas projector Kofoid and Swezy 1926 Incisitermes seeversi [Kalotermes perparvus]
Oxymonas protus Poinar 2009a †Kalotermes burmensis
Oxymonas rotunda Cross 1946 [Oxymonas
ovata Zeliff 1930]
Calcaritermes emarginicollis,Incisitermes
marginipennis
Oxymonas synderi Zeliff 1930 Cryptotermes breviarticulatus
Oxymonas tenuicollis Grassé and Hollande Neotermes aburiensis
Oxymonites gerus Poinar 2009a †Kalotermes burmensis
Paranotila lata Cleveland 1966 Cryptocercus punctulatus
Paratrimastix eleionoma Zhang et al. 2015 Free-living, freshwater
Paratrimastix pyriformis (convexa) (Klebs
1892) Zhang et al. 2015
Free-living, freshwater
Polymastix ganapatii Sultana 1976 Scarabeid larvae
Polymastix hystrix Grassé 1952 Neotermes aburiensis
Polymastix indica Krishnamurthy and Sultana
1978
Polyphaga indica
Polymastix jadhavii Mali 1993 Periplaneta americana
Polymastix legeri Grassé 1926 Glomeris
Polymastix melolonthae Grassi 1879 maybe
identical with Polymastix wenrichi Geiman
1933
Coleoptera larvae, Tipula larvae
Polymastix nitidus Hasselmann 1928 Rhizocrinus
Polymastix periplanetae Qadri and Rao 1963 Periplaneta americana
(continued)
Preaxostyla 9

Table 1 (continued)
Species Host
Polymastix phyllophagae Travis and Becker
1931
Larvae of Phyllophaga
Polymastix rayi Sultana 1976 Periplaneta americana
Polymastix wenrichi Geiman 1933 maybe
identical with Polymastix melolonthae Grassi
1879
Tipula abdominalis
Pyrsonympha affinis Fedorowa 1923 Coptotermes sp.
Pyrsonympha elongata Georgevitch 1932 Reticulitermes lucifugus
Pyrsonympha flagellata Grassi and Sandias
1893
Reticulitermes lucifugus
Pyrsonympha grandis Koidzumi 1921 Reticulitermes speratus
Pyrsonympha granulata Powell 1928 Reticulitermes lucifugus,Reticulitermes
hesperus
Pyrsonympha havilandi Das 1972 Cryptotermes havilandi
Pyrsonympha major Powell 1928 Reticulitermes flavipes,Reticulitermes
lucifugus,Reticulitermes tibialis,
Reticulitermes hesperus
Pyrsonympha minor Powell 1928 Reticulitermes lucifugus,Reticulitermes
hageni,Reticulitermes tibialis,Reticulitermes
hesperus
Pyrsonympha modesta Koidzumi 1921 Reticulitermes speratus
Pyrsonympha omblensis Georgevitch 1951 Reticulitermes lucifugus
Pyrsonympha rostrata Mukherjee and Maiti
1988
Reticulitermes tirapi
Pyrsonympha tirapi Mukherjee and Maiti
1988
Reticulitermes tirapi
Pyrsonympha vertens Leidy 1877 Reticulitermes flavipes
Pyrsonymphites cordylinus Poinar 2009a †Kalotermitidae
Saccinobaculus ambloaxostylus Cleveland
et al. 1934
Cryptocercus punctulatus
Saccinobaculus doroaxostylus Cleveland
et al. 1934 [Oxymonas doroaxostylus emend.
Cleveland 1950a]
Cryptocercus punctulatus
Saccinobaculus gloriosus Bobyleva 1973 Cryptocercus relictus
Saccinobaculus minor Cleveland et al. 1934
[Oxymonas nana emend. Cleveland 1950a]
Cryptocercus punctulatus
Saccinobaculus lata Cleveland 1950b Cryptocercus punctulatus
Saccinobaculus scabiosus Bobyleva 1973 Cryptocercus relictus
Saccinobaculus spatiatus Bobyleva 1973 Cryptocercus relictus
Sauromonas m’baikiensis Grassé et Hollande
1952
Glyptotermes boukoko
Sauromonites katatonis Poinar 2009a Kalotermitidae
Streblomastix strix Kofoid and Swezy 1919 Zootermopsis angusticollis,Zootermopsis
nevadensis
Trimastix elaverinus Dumas 1930 Free-living, freshwater
(continued)
10 V. Hampl

and Arnott 1974b; Rother et al. 1999;Brugerolle1981;LeanderandKeeling2004).
Ectobiotic bacteria are occasionally phagocytosed by the host (Brugerolle 1981;
Leander and Keeling 2004;Nodaetal.2006). The prokaryotes in the cytoplasm of
oxymonads belong to the groups Endomicrobia (TG-1), which are specifictothis
environment (Stingl et al. 2005;Yangetal.2005), methanogens (Tokura et al. 2000),
and mycoplasmas (Yang et al. 2005). Verrucomicrobial symbionts have been reported
from the nuclei (Sato et al. 2014). The essence of the oxymonad-bacterial relationship
is unclear, although some metabolite transfers have been proposed between
parabasalid protists and their endosymbionts living in the same environment (Hongoh
2010). The association of protists with prokaryotes is not strictly one-to-one specific, i.
e., unrelated protists are associated with closely related bacteria and several types of
bacteria are associated with a single oxymonad.
Characterization and Recognition
Organization of Cytoskeleton
The organization of the Trimastix and Paratrimastix cytoskeleton closely follows the
basic scheme known from other typical excavates (Simpson 2003; Yubuki
et al. 2013), and it likely represents the ancestral organization of the group. Four
basal bodies are arranged in a cruciate pattern. Left (R1) and right (R2) microtubular
roots are connected to a recurrent basal body B1 and support the margins of the
cytostome. The right root (R2) is associated with a thick I fiber with a lattice-work
substructure (see below). From the anterior basal body B2 originates the anterior root
(R3), which is associated with the dorsal fan of microtubules (F) supporting the
dorsal side of the cell. Differences between Trimastix and Paratrimastix are subtle.
Common features of both, which distinguish them from other typical excavates, can
be found in the organization of the supportive fibers B, C, and I (Zhang et al. 2015).
In particular, the I fiber forms one thin sheet connected to R2 by lattice-like structure,
which resembles the structure of the paracrystalline part of the preaxostyle in
oxymonads. These two cytoskeletal components are regarded as homologous, and
similarity of their fine structure is the defining synapomorphy of Preaxostyla
(Simpson 2003).
Table 1 (continued)
Species Host
Trimastix inaequalis, Bernard et al. 2000 Free-living, marine
Trimastix marina Saville Kent 1880–1882 Free-living, marine
Tubulimonoides aurangabadae Mali
et al. 2003
Oryctes rhinoceros
Tubulimonoides gryllotalpae Krishnamurthy
and Sultana 1976
Gryllotalpa africana
Tubulimonoides shivamurthi
Mal and Sultana 1993
Oryctes rhinoceros
Preaxostyla 11

The structure of the oxymonad cytoskeleton has diverged from the canonical
excavate form. Here it will be described using the genus Monocercomonoides, which
probably resembles the ancestral state in oxymonads, employing terminology
according to Radek (1994) (Figs. 1and 2). Each oxymonad cell contains one
karyomastigont (as in the case of Monocercomonoides) or sometimes more than
one. Each karyomastigont consists of a nucleus, four basal bodies with flagella that
are organized in two pairs, and a preaxostyle that connects the pairs of basal bodies.
The preaxostyle (=“primary row”in older works) is made of two layers. The layer
facing the nucleus consists of a single row of microtubules (homologous to R2 in
excavates), and this attaches to a second layer made of non-microtubular material
(homologous to the I fiber in excavates). The preaxostylar region is rich in polysac-
charide granules. The cell’s anterior-posterior axis is formed by an axostyle that
consists of parallel rows of microtubules that are interconnected by bridges. In the
nuclear region, the axostyle is associated with the preaxostyle by the single row of
microtubules that is continuous between both structures. The axostyle is contractile
in Pyrsonymphidae, Saccinobaculidae, and Oxymonadidae, where it serves as the
organelle for locomotion. Microtubular root R1 or funis (fully developed in
Monocercomonoides) is connected to the basal body of the recurrent flagellum
(basal body 1) and underlies this flagellum. In Monocercomonoides, the most
Fig. 1 Transmission electron micrographs of Monocercomonoides sp. from Parasphaeria
boleiriana.(a) Transverse section of the nuclear region, (b) longitudinal section, and (c) transverse
section of the axostyle composed of microtubular rows connected by bridges. 1, 2, 4 basal bodies
1, 2, 4, Ax axostyle, Bbacterium, BB basal body, DV digestive vacuole, hhook-like fiber, Nnucleus,
Pax preaxostyle, Pe pelta, RER rough endoplasmic reticulum; bars 200 nm. Terminology according
to Radek (1994) (Courtesy of Guy Brugerolle)
12 V. Hampl

anterior basal body (4) is associated with a microtubular root (not shown in Figures),
which underlies the pelta. This pelta is a microtubular sheet that covers the nucleus
and that is homologous to the dorsal fan of typical excavates. Simpson et al. (2002)
suggested homologies between the oxymonad cytoskeleton and cytoskeleton of
typical excavates; the excavate terminology for cytoskeletal structures according to
Yubuki and Leander (2013) is given in Fig. 2in brackets.
Sex and Reproduction
Preaxostyla reproduce by binary fission. Paratrimastix use an open mitosis, while
mitosis is of a closed type in oxymonads. A characteristic migration of nuclei
through the cell is typical for family Oxymonadidae and will be described in more
detail below. Sexual processes comprising gametogenesis, fertilization, and meiosis
were reported in the oxymonads of the wood-feeding cockroach Cryptocercus,
namely, Notila,Saccinobaculus, and Paranotila (Cleveland 1950b,c; Cleveland
1966). Cleveland also described sexual processes in two Oxymonas species from
Cryptocercus (O. doroaxostylus and O. nana) (Cleveland 1950a); however, these
species are currently regarded as members of the genus Saccinobaculus
Fig. 2 Ultrastructure of Monocercomonoides.Terminology follows Radek (1994); terminology
according to Yubuki and Leander (2013) is given in brackets. 1, 2, 3, 4 basal bodies 1–4, AFl
anterior flagella, Ax axostyle, DV digestive vacuole, Ffan, Pax preaxostyle, Pe pelta, PPG
perinuclear polysaccharide granules, R1 microtubular root R1, R2 microtubular root R2, RFl
recurrent flagellum, RER rough endoplasmic reticulum, SR striated root. The axostyle in its distal
part is artificially interrupted to show the organization of microtubules (Courtesy of Eva
Nohýnková, adopted)
Preaxostyla 13

(S. doroaxostylus,S. minor; Heiss and Keeling 2006). Synaptonemal complex
characteristic for meiosis was reported from Pyrsonympha flagellata (Hollande
and Carruette-Valentin 1970a). Encystation has been reported in Monocerco-
monoides,Saccinobaculus, and Sauromonas, as well as in Paratrimastix (Cleveland
1950a; Grassé 1952;O’Kelly et al. 1999). The developmental cycles of flagellates
and the sexual cycles (where present) are synchronized with the molting cycle of the
insect host and are governed by the molting hormone ecdysone (May 1941; Grassé
1952; Cleveland 1956; Cleveland et al. 1960). Termites lose all intestinal protozoa
during nymphal molt; both young termites, and post-molt adult termites must
establish their protozoan biota by proctodeal feeding from adults (Brugerolle and
Radek 2006; Brune and Ohkuma 2011).
Molecular Genetics and Biochemistry
Due to the impossibility of axenic cultivation, our knowledge on molecular genetics
and biochemistry is very fragmentary, and the studies are restricted to transcriptomic
and genomic surveys and gene fishing from genomic DNA and cDNA. The only
sequenced genome of the group (Monocercomonoides sp.) is ~75 MB in size and
36.8 % GC and contains 16,629 predicted protein coding genes (Karnkowska
et al. 2016).
The cytoplasm of Trimastix and Paratrimastix contains electron-dense mito-
chondrion-like organelles with poorly known biochemistry. In Paratrimastix
pyriformis, the only protein localized into these organelles is the enzyme of the
glycine cleavage system, part of amino acid metabolism. Transcriptome studies
in P. pyriformis indicate the presence of pyruvate:ferredoxin oxidoreductase and
[FeFe]hydrogenase, suggesting an extended glycolysis in this organism
(Zubacova et al. 2013). Peroxisomes have not been reported in Trimastix and
Paratrimastix.
Energetic metabolism of oxymonads seems to be broadly similar to other studied
anaerobes such as Trichomonas,Giardia,orEntamoeba (Reeves et al. 1977; Upcroft
and Upcroft 1998;M
€
uller 1992). Among the glycolytic enzymes of Monocerco-
monoides, several were acquired by lateral gene transfer from prokaryotes,
including the ATP-efficient alternatives pyrophosphate fructose-6-phosphate
phosphotransferase and pyruvate orthophosphate dikinase (Liapounova et al. 2006;
Slamovits and Keeling 2006b). Pyruvate is probably oxidatively decarboxylated by
pyruvate:ferredoxin oxidoreductase (PFO) in the cytosol, and the resulting acetyl-CoA
is further fermented to ethanol (Karnkowska et al. 2016). Transcripts of [FeFe]hydrog-
enase are abundant (Karnkowska et al. 2016), but the production of hydrogen has not
been established. Dacks et al. (2008) found that the expression of cathepsin B cysteine
proteases in Monocercomonoides is relatively high and comparable to housekeeping
genes. Unlike other metamonads (Giardia,Trichomonas), the oxymonad genome is
relatively intron rich (1.1 and 1.9 introns per gene in Streblomastix and Monocerco-
monoides, respectively) (Slamovits and Keeling 2006a;Karnkowskaetal.2016). Some
oxymonads (Streblomastix,someMonocercomonoides) use a noncanonical genetic
code, in which the codons TAA and TAG encode the amino acid glutamine (Keeling
and Leander 2003; de Koning et al. 2008).
14 V. Hampl

Mitochondria, stacked Golgi, and peroxisomes have not been clearly demonstrated
in oxymonads. Electron-dense organelles of uncertain nature, but resembling mito-
chondria, were reported from Saccinobaculus doroaxostylus however (Carpenter
et al. 2008). In the case of Monocercomonoides sp. strain from chinchilla, no genes
for mitochondrion-specific proteins have been detected in the fully sequenced genome,
confirming the absence of any mitochondrion suggested by electron microscopy
(Karnkowska et al. 2016). The same applies to peroxisomes, but in the case of the
Golgi apparatus, a full set of genes coding for “Golgi-associated”proteins was found.
The cellular localization of their protein products is unknown.
Taxonomy
Preaxostyla are classified within the phylum Metamonada (Cavalier-Smith 2003), a
subgroup of the taxon Excavata (Cavalier-Smith 2002; Simpson 2003; Adl
et al. 2005; Hampl et al. 2009; Adl et al. 2012). Preaxostyla contains three described
species of Trimastigidae, two of Paratrimastigidae (Zhang et al. 2015), and approx-
imately 140 described species of oxymonads, divided into five families –
Polymastigidae, Saccinobaculidae, Pyrsonymphidae, Streblomastigidae, and
Oxymonadidae (Brugerolle and Lee 2000), plus the isolated genus Opisthomitus
(Fig. 3). List of described species is given in Table 1.
Trimastigidae
The family contains a single genus Trimastix Saville Kent. Cells bear four flagella
stretched roughly in the anterior, right, left, and posterior directions. The posterior
flagellum passes through a suspension-feeding groove and bears two vanes. Vane
margins are not thickened. The genus contains two marine species T. marina and
T. inaequalis and one freshwater species T. elaverinus with uncertain status. Light
microscopy of Trimastix was studied by Saville Kent (1880), Dumas (1930), Grasse
(1952), and Bernard et al. (2000). Light microscopy and ultrastructural observations
were reported by Zhang et al. (2015).
Paratrimastigidae
The family contains a single genus Paratrimastix Zhang, Taborsky, Silberman,
Panek, Čepička, and Simpson. Cells bear four flagella directed anteriorly, to the
right and left, and posteriorly. The posterior flagellum passes through a suspension-
feeding groove and bears two vanes with thickened margins. The genus contains two
species P. pyriformis (syn. convexa) and P. eleionoma from freshwater habitats
around the globe (Fig. 3). Light microscopy and ultrastructure of Paratrimastix
was studied by Brugerolle and Patterson (1997), O’Kelly et al. (1999), and Simpson
Preaxostyla 15

et al. (2000). In the literature between years 1997 and 2013, these two species are
referred to as Trimastix pyriformis and Trimastix marina, respectively.
Oxymonadida
More than 140 described species of oxymonads (Table 1and Fig. 4) are all gut
endobionts. They are classified into five families (Polymastigidae,
Streblomastigidae, Pyrsonymphidae, Saccinobaculidae, and Oxymonadidae) and a
genus Opisthomitus.
Polymastigidae
There are four described genera of small tetraflagellates with pelta and slender
noncontractile axostyle and without attachment organelles.
Fig. 3 DIC images of Paratrimastix. P. pyriformis (a,b) and P. eleionoma (c,d); bars 10 μm
16 V. Hampl

Fig. 4 DIC images and protargol preparations of oxymonads. (a,b) DIC images and (c)
protargol preparations of Monocercomonoides sp. from Chinchilla,(d)DICimageofPolymastix
sp. from Parasphaeria boleiriana,(e) protargol preparation of Polymastix melolonthae from crane fly
larva, (f) protargol preparation and (j)DICimageofStreblomastix strix from Zootermopsis
angusticollis,(g) protargol preparation of Dinenympha gracilis from Reticulitermes lucifugus,(h)
DIC image of Pyrsonympha vertens from Reticulitermes flavipes,(i) protargol preparation of
Pyrsonympha sp. from Reticulitermes lucifugus,(k)DICimageofDinenympha fimbriata from
Reticulitermes lucifugus,(l)DICimageofDinenympha sp. from Reticulitermes lucifugus,(m)DIC
image of Saccinobaculus ambloaxostylus from Cryptocercus punctulatus,(n) DIC image of nuclear
region, and (o) whole cell of Oxymonas sp. from Cryptocercus punctulatus;bars10μm. (d) was kindly
provided by Guy Brugerolle; (m–o) were kindly provided by Patrick Keeling and Kevin Carpenter
Preaxostyla 17

Monocercomonoides Travis
Monocercomonoides Travis has small oval to pyriform body (5–15 μm in length)
and four flagella arranged in two pairs, with one which is recurrent and attached to
the body (Fig. 4a–c). The organization of the Monocercomonoides cytoskeleton was
described above and is depicted in Fig. 2. Over 40 species have been described
(Table 1), but the validity of some of them is uncertain. About half of the species
inhabit the posterior part of the digestive tract of wood-eating insect imagoes (the
cockroaches Cryptocercus and Parasphaeria and lower termites), insect larvae
(Tipula, Coleoptera), or millipedes, while the rest live in the gut of vertebrates
(rodents, bovids, reptiles, and amphibians). The ultrastructure was studied by
Brugerolle and Joyon (1973), Kulda and Nohýnková (1978), Radek (1994),
Simpson et al. (2002), and Brugerolle et al. (2003).
Tubulimonoides Krishnamurthy and Sultana
The genus Tubulimonoides described from the gut of Gryllotalpa africana (African
mole cricket) is very similar to Monocercomonoides but differs from it by its tubular
axostyle. In the type species (Tubulimonoides gryllotalpae) the flagella are report-
edly organized into groups of three and one unlike all other oxymonads. The other
two species have the flagella organized in a typical 2:2 fashion. Because of these
discrepancies and in the absence of electron microscopic study, the validity of this
genus is questionable. Light microscopy was carried out by Krishnamurthy and
Sultana (1976), Mali and Sultana (1993), and Mali et al. (2003).
Polymastix Bütschli
Spindle-shaped tetramastigotes (5–22 μm in length) differ from Monocerco-
monoides by the absence of a recurrent (cell-adhering) flagellum, very short or no
fiber R1, the presence of a microfibrillar bundle connecting the nucleus to the first
pair of basal bodies, a narrow and grooved preaxostyle, a slender axostyle composed
of about 10 microtubules, a small pelta, and, most strikingly, the presence of long
symbiotic Fusiformis bacteria on the surface (Fig. 4d, e). Up to 11 species are
currently recognized (Table 1); another as-yet undescribed species has been
observed in the cockroach Parasphaeria (Brugerolle et al. 2003). Polymastix was
found in the gut of larvae of Scarabaeoidea beetles and crane flies, myriapods
(Glomeris and Rhizocrinus), cockroaches, and termites. EM studies were conducted
by Brugerolle (1981) and Brugerolle et al. (2003).
Paranotila Cleveland
A single species P. lata was described from the gut of Cryptocercus punctulatus.
On the basis of morphology, Brugerolle and Lee (2000)classified Paranotila
among polymastigids. The uninuclear cell is larger than Monocercomonoides
(15–25 μm) and has four flagella only slightly adhering to the cell and directed
laterally and a single axostyle that does not protrude from the cell. Under the
influence of molting hormone ecdysone, Paranotila undergoes a sexual cycle that
involves automixis. During the nuclear division without cytokinesis, the cell
transforms to a gametocyte containing eight male and eight female gametic nuclei.
18 V. Hampl

The nuclei fuse to form eight-nuclear zygote that breaks gradually into eight
uninuclear cells. A single morphological study was conducted by Cleveland
(1966); no EM study has been published.
Streblomastigidae
The family contains a single genus with one described species, Streblomastix strix
Kofoid and Swezy 1919, that inhabits the hindgut of termopsid termites, e.g.,
Zootermopsis angusticollis (Fig. 4f, j). Noda et al. (2006) report undescribed
Streblomastix sp. from Archotermopsis sp. The relatively rigid spindle-shaped
cells of S. strix are typically 15–50 μm long, but rare giant forms can be as long as
300 μm. Four flagella are inserted subapically and do not adhere to the cell. The
anterior tip of the cell forms a thin rostellum with a cup-like holdfast. This structure
can be lengthened and retracted, and it serves for attachment to the gut epithelium. In
many individuals (probably recently divided cells), the rostellum is small or absent.
The surface of the cell (besides the very anterior tip) is covered by 100–200 long
rod-shaped epibiotic bacteria of at least three morphotypes, and the sequencing of
16S rRNA revealed three closely related phylotypes related to Bacteroides (Leander
and Keeling 2004; Noda et al. 2006). In transverse section, Streblomastix shows a
stellate organization with the cytoplasm reduced to a dense central core from which
radiate 6–7 thin vanes. The ridges between vanes are apparent in the light micro-
scope and typically show torsion from left to right starting at the anterior end. The
ragged cell shape is probably an adaptation to accommodate bacterial epibionts and
naked cells, produced by antibiotic treatment, shift to a teardrop shape (Leander and
Keeling 2004). The nucleus is a dense thin spiral rod. The microtubular cytoskeleton
consists of axostyle, pelta, and preaxostyle. In the prenuclear region, the microtu-
bules of the axostyle are organized in several parallel rows (syn. “rhizoplast”in
Kidder (1929)); in the nuclear region, microtubules form a single row that envelops
the nucleus, and in the post-nuclear region, the axostyle consists of a loose bundle of
microtubules. The pelta helically encircles the prenuclear axostyle and covers the
anterior part of the nucleus. The cell divides by binary fission, and the cell cycle is
probably affected by the molting cycle of the termite. No cysts have been reported.
Morphology was studied by Kofoid and Swezy (1919) and Kidder (1929). Electron
microscopy was conducted by Hollande and Carruette-Valentin (1970b) and Lean-
der and Keeling (2004).
Pyrsonymphidae
All 25 described species in two genera are hindgut symbionts of the lower termites
Reticulitermes (Table 1). The nucleus is situated anteriorly. Four or eight flagella are
organized in two or four pairs separated by preaxostyle(s). Flagella emerge at the
anterior end of the cell, bend posteriorly, insert into grooves on the cell surface, wind
around the cell in left-handed spirals, and trail posteriorly. The contractile axostyle is
Preaxostyla 19

the main motile organelle. It extends the entire length of the organism and consists of
thousands of microtubules arranged in many parallel rows connected by bridges.
Most, if not all, pyrsonymphids contain endobiotic bacteria in the cytosol, and many
species also harbor epibiotic bacteria on the surface (Smith and Arnott 1974b; Iida
et al. 2000; Tokura et al. 2000; Stingl et al. 2005; Yang et al. 2005; Hongoh
et al. 2007). The bacteria are often attached to the cell by specialized structures
developed by both bacteria and protists (Smith and Arnott 1974b). Two extant and
two fossil genera are currently recognized (Poinar 2009a,b).
Pyrsonympha Leidy
Representatives of this genus (13 described species; Table 1) are relatively large cells
(up to 150 μm) and show a pyriform, sack-like appearance (Fig. 4h,i). The broader
posterior end of the cell is filled with phagocytic vesicles containing wood pieces.
Pyrsonympha often develops an attachment organelle (holdfast) at the anterior pole
of the cell (Cochrane et al. 1979). Many individuals of Pyrsonympha vertens have
eight flagella and two preaxostylar fibers (Bloodgood et al. 1974). These individuals
likely represent a prolonged stage in the life cycle prior to cell division. The axostyle
of Pyrsonympha can be isolated and movement reactivated in vitro (Bloodgood
et al. 1974). In P. vertens, a loose bundle of microtubules (paraxostyle) runs parallel
to the axostyle from the basal bodies region (Brugerolle 1970). The pelta is reduced
to several microtubules (=solénolemme in Hollande and Carruette-Valentin
(1970b)). The surface of Pyrsonympha is covered by fine scales of unknown
function and composition (Smith and Arnott 1973). Ring-like structures were
reported on the surface of an undetermined pyrsonymphid from the gut of Neotermes
cubanus (Maass and Radek 2006). Hollande and Carruette-Valentin (1970a)
reported synaptonemal complexes in P. flagellata, suggesting the existence of
meiosis.
Dinenympha Leidy
Dinenympha Leidy are smaller (tens of μm) freely motile cells with four flagella,
which are characterized by a screw-like shape. If not associated with epibiotic
bacteria like D. fimbriata, the cells exhibit distinctive wiggly movement
(D. gracilis). Twelve species have been described (Fig. 4g, k,land Table 1).
The long-lasting debate as to whether Pyrsonympha and Dinenympha represent
separate genera or life-cycle stages of the same genus was apparently resolved by
molecular studies (Moriya et al. 2003; Stingl and Brune 2003) showing that the
sequences of Dinenympha and Pyrsonympha form separate groups and, importantly,
that specific DNA probes hybridize exclusively to one genus but not the other and
vice versa (see Fig. 1in Moriya et al. (2003)). Light microscopic observations of
pyrsonymphids were carried out by Porter (1897), Powell (1928), Jírovec (1929),
Georgevitch (1932), and Grassé (1952) and electron microscopy by Brugerolle
(1970), Hollande and Carruette-Valentin (1970a,b), Smith and Arnott (1973,
1974a,b), Smith et al. (1975), Bloodgood et al. (1974), Cochrane et al. (1979),
and Maass and Radek (2006). Two fossil species of Pyrsonymphidae –
Dinenymphites spiris and Pyrsonymphites cordylinis –have been described from
20 V. Hampl

Cretaceous amber from Burma. The age of the amber was dated between 97 and
110 mya (Poinar 2009a,b). The protists were found in association with a fossil
termite, Kalotermes burmensis.
Saccinobaculidae
Saccinobaculidae are hindgut symbionts of the wood-feeding cockroaches
Cryptocercus punctulatus and C. relictus. The four, eight, or 12 flagella do not
adhere to the body except, in some cases, in the proximal part (Fig. 4m). No
attachment organelle has been observed. The large axostyle is contractile and is
responsible for cell locomotion. It undulates vigorously inside the cell, like “a snake
in a bag,”causing rapid and dramatic changes in the cell shape (Cleveland
et al. 1934). The waves originate at the anterior end and propagate posteriorly in a
single plane –they are sinusoidal rather than helical (Mcintosh 1973; Mcintosh
et al. 1973). As in Pyrsonympha, the movement of the isolated axostyle can be
reactivated in vitro (Mooseker and Tilney, 1973). A sexual process was reported in
this family (Cleveland 1950a,b,c). The family contains two morphologically very
similar genera.
Saccinobaculus Cleveland
As in other oxymonads, the basic unit of the mastigont consists of two pairs of basal
bodies associated with a preaxostyle, and multiplication of flagella is accompanied
by the multiplication of preaxostyles. The microtubules of the preaxostyle continue
to form the first row of axostylar microtubules facing away from the nucleus. In the
prenuclear region, similar but shorter rows of microtubules gradually attach to this
primary row, forming the axostyle that contains more than 8000 microtubules in the
largest sections. The axostyle forms an arch anteriorly to the nucleus and then runs
posteriorly, twisting and forming a crescent that almost closes to a circle or spiral to
the distal end, where it protrudes from the cell. The number of microtubules
decreases significantly toward the posterior end. The nucleus is tightly associated
with the axostyle by its dorsal side. The region of the nucleus and preaxostyle is
wrapped from the posterior and ventral side in a thin single layer of microtubular
sheet, the pelta (=thin lamina in Mcintosh et al. (1973)). Conspicuous electron-
dense granules were reported from the cytoplasm of Saccinobaculus (Mcintosh
et al. 1973; Carpenter et al. 2008), which may represent peroxisomes or a modified
mitochondrion (Carpenter et al. 2008). The surface of the cell is covered by circular
concavities that sometimes show circular pits in the center (Carpenter et al. 2008).
These are similar to those reported from pyrsonymphids (Maass and Radek 2006).
Their function is unknown, but the presence of what appears to be clathrin coating in
these pits suggests they may play a role in endocytosis. Epibiotic bacteria are present
only rarely. Seven species of Saccinobaculus are currently recognized (Table 1).
They differ in size and presence of granules in the axostyle or cytoplasm (Cleveland
et al. 1934; Heiss and Keeling 2006). Cleveland (1950b) transferred S. doroaxostylus
and S. minor into the genus Oxymonas as O. doroaxostylus and O. nana, but the
Preaxostyla 21

molecular phylogenetic study by Heiss and Keeling (2006) showed that they should
be classified as Saccinobaculus. Light microscopy studies were performed by
Cleveland et al. (1934), Cleveland (1950b,c), and Heiss and Keeling (2006) electron
microscopy by Grimstone and Cleveland (1965), Mcintosh et al. (1973), Mcintosh
(1973), and Carpenter et al. (2008).
Notila Cleveland
Cleveland (1950c) distinguished Notila from Saccinobaculus on the basis of differ-
ences in their sexual cycles. The major difference is that both trophozoites and
“gametes”of Notila are diploid. As late as after fusion of two diploid “gametic”
cells, their nuclei undergo single-step meiosis to form four haploid gametic nuclei.
The gametic nuclei fuse to form a double zygote that soon undergoes cytokinesis.
Morphologically, Notila differs from Saccinobaculus by its axostyle that does not
protrude, has no terminal sheath, and contains granules. The validity of the genus has
yet to be confirmed. A single species, Notila proteus, was studied using light
microscopy by Cleveland (1950c), Grassé (1952), and Bobyleva (1973); no EM
study has been done.
Oxymonadidae
All described species are hindgut symbionts of termites, specifically Kalotermitidae.
They can either take the form of free-swimming flagellates or attach to the intestinal
wall by a microfibrillar holdfast situated at the tip of a cellular extension –the
rostellum. In some cases the rostellum may be several times longer than the cell
(Fig. 4o). It is probably able to contract or extend by a slow passive movement. The
stout axostyle is contractile, but does not undulate as violently as in Saccinobaculus.
Locomotion probably results from the combined activity of the axostyle and flagella.
Oxymonadidae may have single or multiple nuclei. Nuclei migrate posteriorly
during mitosis and travel back after telophase. The surface of the cell (including
the rostellum of most species) is densely covered by epibiotic rod-shaped bacteria,
oriented perpendicularly to the cell. Four extant and three fossil genera of
Oxymonadidae are currently recognized (Table 1).
Oxymonas Janicki
Oxymonas Janicki are club-shaped cells, usually containing a single nucleus, two
pairs of flagella, and a single axostyle (Fig. 4n, o). Amoeboid forms have been also
reported (Tamschick and Radek 2013). Over 30 species have been described
(Table 1), including two fossil species. The length of the reported species varies
between 5 and 240 μm and the width between 4 and 165 μm. The rostellum of
Oxymonas is supported by a paraxostyle (homology to the paraxostyle of
Pyrsonympha is unclear) and a bundle of free microtubules. The paraxostyle orig-
inates at the dense microtubule-organizing center at the tip of the rostellum and
extends posteriorly to the cell body. It consists of microtubules organized in convo-
luted ribbons. Free microtubules originate at various positions in the trunk of the
22 V. Hampl

rostellum, extend posteriorly, and continue to the axostyle. The stout axostyle
consists of parallel, stacked rows containing thousands of microtubules. It originates
at the base of the rostellum by inserting new microtubules among the free microtu-
bules continuing from the rostellum. The microtubules in the axostyle are
interconnected by cross-bridges. The axostyle is tightly adpressed to the nucleus,
continues posteriorly, splits into smaller bundles, and often enrolls at the posterior
end. In some cells, the axostyle protrudes posteriorly. The preaxostyle that connects
the pairs of basal bodies is situated close to the origin of the axostyle, but studies do
not show any connection between the two structures. A dense plate adjacent to the
preaxostyle underlies a region in the flagellar area where long spirochetes attach
(Cross 1946; Brugerolle and König 1997; Rother et al. 1999).
The surface of Oxymonas, under the epibiotic bacteria, is densely covered by
external surface structures that form a honeycomb-like pattern. They are formed by a
cylindrical base and are covered by a lid. Pits with a coat resembling clathrin are
formed from the bottom. The surface structures are composed of carbohydrates and
likely function in pinocytosis. The lid also serves as an attachment place for bacteria
(Rother et al. 1999). Light microscopic observations of Oxymonas were conducted
by Kofoid and Swezy (1926) and Cross (1939,1946). Fossils were studied by Poinar
(2009a,b). Studies using EM were conducted by Brugerolle and König (1997),
Rother et al. (1999), Tamschick and Radek (2013), and Radek et al. (2014).
Microrhopalodina (syn. Proboscidiella) Grassi and Foa
Four species are described (Table 1). Cell dimensions range from 23 to 165 μmin
length and 11–113 μm in width and contain multiple karyomastigonts. The number
of karyomastigonts varies from four to 50, but the common numbers are four, eight,
and 12. The karyomastigonts are arranged in a collar at the base of the rostellum.
Every karyomastigont associates to its own axostyle. Posterior to the nuclei,
axostyles extend independently as bands composed of parallel microtubular rows
connected by electron-dense bridges. The bands are strongly curved at the posterior
end. In the region of the nucleus, at least one row of axostylar microtubules splits
from the band and laterally encircles the nucleus, forming a calyx. In the anterior
direction, the microtubules lose the periodic organization, and microtubules from all
axostyles join into a single loose bundle that extends into the rostellum. One lamella
of microtubules encircles this loose bundle. Similarly to Oxymonas, the rostellum
contains microtubules of the paraxostyle that originate in the holdfast and extend
into the cell as convoluted ribbons. These ribbons are less developed than in
Oxymonas. The cell body contains numerous vesicles filled with digested material.
The surface of the cell is covered by external surface structures and bacteria, as in
Oxymonas (Rother et al. 1999). Light microscopy was carried out by Kofoid and
Swezy (1926), Kirby (1928), Cross (1946), and Rother et al. (1999) and EM by
Lavette (1973) and Rother et al. (1999).
Barroella (syn. Kirbyella) Zeliff
Only two species are described (Table 1), with cell dimensions ranging between
27 and 224 μm in length and 11–80 μm in width (Cross 1946). The mature cell has
Preaxostyla 23

club-like shape, no flagella, and multiple nuclei (2–114), which are scattered
throughout the body. Slender axostyles are tortuously curved and much longer
than the body. Axostyles and nuclei are rarely equal in number. Immature cells are
similar to Microrhopalodina, with a collar of flagella and shorter axostyles. They
originate by budding from larger cells that are distinguished by formation of multiple
karyomastigont coronas (Cross 1946). No EM study has been done.
Sauromonas Grassé and Hollande
The single species, Sauromonas m’baikiensis, is a symbiont of the termite
Glyptotermes boukoko. In the attached form, the cell is organized like Oxymonas
and possesses a single nucleus, four flagella, and a single axostyle. The rostellum of
Sauromonas contains a recurvent fibrillar bundle, which may in fact correspond to
the paraxostyle of Oxymonas and Microrhopalodina. When the termite molts, the
organism detaches from the intestinal wall and undergoes series of transformations
resulting in a polyflagellated cell, which then loses the flagella and encysts. Light
microscopy was carried out by Grassé (1952). No EM study has been done.
Three fossil species of Oxymonadidae –Oxymonites gerus,Microrhopalodites
polynucleatis, and Sauromonites katatonis –have been described in association with
a fossil termite species Kalotermes burmensis from Cretaceous amber from Burma,
97–110 mya (Poinar 2009a,b).
Opisthomitus Duboscq & Grassé 1934
Opisthomitus Duboscq and Grassé 1934 are small oxymonads bearing four flagella.
The anterior end of the cell is pointed and forms a conspicuous lappet that may be
homologous to a rostellum; however, the attachment of the cells to the gut wall has
never been observed. The organization of the cytoskeleton resembles Monocerco-
monoides, including the presence of a pelta supported by a microtubular root
associated with anterior basal body nr. four. The surface of the body is covered by
numerous ring-like bulges resembling the concavities in Saccinobacullus. Light
microscopy was studied by Duboscq and Grassé (1934), De Mello (1953), Hollande
and Carruette-Valentin (1970b), and Radek et al. (2014). An EM study was
performed by Radek et al. (2014). Two valid species, Opisthomitus avicularis and
O. longiflagellatus, and two species with uncertain status, O. brasiliensis and
O. flagellae, have been described. The genus is not classified into any oxymonad
family, and the phylogeny based on 18S rRNA suggests its affiliation to
Pyrsonymphidae (Radek et al. 2014).
Maintenance and Cultivation
Stable cultures have been established so far only for representatives of Trimastix,
Paratrimastix, and Monocercomonoides. The cultures are monoeukaryotic but
polyxenic (with admixed bacteria). Trimastix grows on ATCC 1525 medium that
24 V. Hampl

should, for some strains, be supplemented by 1 ml of simplified ATCC 1034 medium
(without folic acid and yeast nucleic acid added; Zhang et al. 2015). Paratrimastix
grows well on bacterized ATCC 802 (Sonneborn’sParamecium medium).
Monocercomonoides grows on TYSGM (Diamond 1982) or Dobell-Laidlaw
two-phase medium (Dobell and Laidlaw 1926; Hampl et al. 2005). Cultures are
maintained in 22 Cor37C, if from a mammalian host, and are transferred every
4–7 days. Insect oxymonads can be maintained in the lab in their hosts.
Phylogeny and Evolution
The close relationship between endobiotic oxymonads and free-living Paratrimastix
was first realized through phylogenetic analyses of 18S rRNA genes (Dacks
et al. 2001). Based on this finding and on ultrastructural comparisons, the taxon
Preaxostyla was established and defined by ultrastructural synapomorphy –a char-
acteristic appearance of the I fiber in Paratrimastix and its homologue, the
paracrystalline part of preaxostyle, in oxymonads (Simpson 2003). The name
Anaeromonada has also been used for this grouping (Cavalier-Smith 2003). The
“typical excavate”morphology of Paratrimastix justified inclusion of Preaxostyla
into the supergroup Excavata (Cavalier-Smith 2002; Simpson 2003). Within
Excavata, Preaxostyla are regarded as members of Metamonada –a commonly
recognized group containing most of the other anaerobic Excavata (i.e., parabasalids
and fornicates) (Cavalier-Smith 2003; Hampl et al. 2009). Both Metamonada and
Excavata represent reasonable taxonomic hypotheses based on data available today,
but the statistical support specifically for Excavata is never strong in molecular
phylogenetic/phylogenomic analyses (Hampl et al. 2005,2009; Simpson
et al. 2006; Rodriguez-Ezpeleta et al. 2007a,b; Parfrey et al. 2010; Grant and Katz
2014; Kamikawa et al. 2014). The validity of all Excavata as a clade has been
strongly challenged by a potential rooting of eukaryotes “within”Excavata, with
Malawimonas on one side of the root and other examined Excavata on the other
(Derelle et al. 2015); however, these analyses have not included Metamonada. The
position of Metamonada relative to this proposed root therefore remains to be
established.
The internal phylogeny of Preaxostyla recovered using 18S rRNA genes by
Zhang et al. (2015) is schematically depicted in Fig. 5. It suggests that the common
ancestor of Preaxostyla was a typical excavate with four flagella resembling the
extant genera Trimastix and Paratrimastix. The morphology of oxymonads is
derived and probably affected by their endobiotic way of life. A striking evolution-
ary explosion of morphological diversity is apparent in oxymonads from cockroach
and termite guts. Fossils resembling some current genera of oxymonads have been
reported in association with Kalotermes burmensis and a blattellid cockroach found
in early Cretaceous amber (97–110 mya) from a mine in the Hukawng Valley,
southwest of Maingkhwan, Burma (Table 1, Poinar 2009a,b). Sequence data have
been obtained from the oxymonad genera Pyrsonympha,Dinenympha,Oxymonas,
Streblomastix,Monocercomonoides,Saccinobaculus, and Opisthomitus
Preaxostyla 25

Fig. 5 Genera of oxymonads, their division into families, and probable relationships between
the families. Ax axostyle, Cv contractile vacuole, EB ectosymbiotic bacteria, Fg feeding
groove, Fl flagellum, FM free microtubules, Ho holdfast, Nu nucleus, MR microtubular ribbons,
Pe pelta, Pax preaxostyle, Ro rostellum, Sp spirochaetes, UM undulating membrane, Va vacuole.
bar: 10 μm for Polymastigidae and Streblomastigidae; 20 μm for Pyrsonymphidae,
26 V. Hampl

representing all five families. The relationships within oxymonads are not well
resolved, but all recent analyses generally agree on the relatively robust clade of
Polymastigidae + Streblomastigidae and a weakly supported clade of the remaining
oxymonads (Hampl et al. 2005; Heiss and Keeling 2006; de Koning et al. 2008;
Radek et al. 2014).
Acknowledgments The author would like to thank Guy Brugerolle, Patrick Keeling, Kevin
Carpenter, and Eva Nohýnková for kindly providing figures; Joel B Dacks, Jaroslav Kulda, Naoji
Yubuki, Alastair Simpson, and an anonymous reviewer for proofreading the manuscript and helpful
comments; Ivan Čepička for providing protargol preparations; and Ivan Hrdý for providing
termites. Support for the author’s salary came from the project of the Ministry of Education,
Youth, and Sports of CR within the National Sustainability Program II (Project BIOCEV-FAR)
LQ1604 and by the project “BIOCEV”(CZ.1.05/1.1.00/02.0109).
References
Abraham, R. (1961). A description of Monocercomonoides sayeedi n. sp., from the rumen of an
Indian goat. Zeitschrift f€
ur Parasitenkunde, 20, 558–562.
Adl, S. M., Simpson, A. G. B., Farmer, M. A., Andersen, R. A., Anderson, O. R., Barta, J. R.,
Browser, S. S., Brugerolle, G., Fensome, R. A., Fredericq, S., James, T. Y., Karpov, S., Kugrens,
P., Krug, J., Lane, C. E., Lewis, L. A., Lodge, J., Lynn, D. H., Mann, D. G., McCourt, R. M.,
Mendoza, L., Moestrup, O., Mozley-Standridge, S. E., Nerad, T. A., Shearer, C. A., Smirnov,
A. V., Spiegel, F. W., & Taylor, M. F. J. R. (2005). The new higher level classification of
eukaryotes with emphasis on the taxonomy of protists. Journal of Eukaryotic Microbiology, 52,
399–451.
Adl, S. M., Simpson, A. G., Lane, C. E., Lukeš, J., Bass, D., Bowser, S. S., Brown, M. W., Burki, F.,
Dunthorn, M., Hampl, V., Heiss, A., Hoppenrath, M., Lara, E., Le Gall, L., Lynn, D. H.,
McManus, H., Mitchell, E. A., Mozley-Stanridge, S. E., Parfrey, L. W., Pawlowski, J., Rueckert,
S., Shadwick, L., Schoch, C. L., Smirnov, A., & Spiegel, F. W. (2012). The revised classification
of eukaryotes. Journal of Eukaryotic Microbiology, 59, 429–493.
Bala, P., & Bhagat, R. C. (1993). The intestinal protozoans of the termite, Odontotermes obesus
(Rambur). Indian Journal of Parasitology, 17, 179–187.
Bernard, C., Simpson, A. G. B., & Patterson, D. J. (2000). Some free-living flagellates (protista)
from anoxic habitats. Ophelia, 52,113–142.
Bhaskar Rao, T. (1969). The morphology and incidence of the genus Monocercononoides (Grassi,
1879) Travis, 1932 of insects found in Andhra Pradesh. Proceedings of the Indian Academy of
Science (B), 70, 208–214.
Bishop, A. (1932). A note upon Retortamonas rotunda n. sp. an intestinal flagellate in Bufo
vulgaris. Parasitology, 24, 233–237
Bloodgood, R. A., & Fitzharris, T. P. (1978). Initiation of bends in the microtubular axostyle of
Pyrsonympha. Cytobios, 23, 109–117.
ä
Fig. 5 (continued) Saccinobaculidae, and Oxymonas; and 40 μm for Sauromonas,Microrho-
palodina, and Barroella. Drawing of Barroella after Cross (1946), Oxymonas after Brugerolle
and König (1997), Microrhopalodina after Kirby (1928), Sauromonas after Grassé (1952),
Saccinobaculus after Cleveland (1950b), Notila after Cleveland (1950c), and Paranotila after
Cleveland (1966); remaining figures are original
Preaxostyla 27

Bloodgood, R. A., Miller, K. R., Fitzharris, T. P., & McIntosh, J. R. (1974). The Ultrastructure of
Pyrsonympha and its associated microorganisms. Journal of Morphology, 143,77–105.
Bobyleva, N. N. (1973). The mastigophora fauna from the hind-gut of the far-eastern woodroach
Cryptocercus relictus. Parazitologiya, 7, 201–213.
Brugerolle, G. (1970). Sur l’ultrastructure et la position systématique de Pyrsonympha vertens
(Zooflagellate Pyrsonymphina). Comptes Rendus de l’Académie des Sciences, Paris, 270,
3474–3478.
Brugerolle, G. (1981). Ultrastructural-study of the parasitic flagellate Polymastix melolonthae
(Oxymonadida). Protistologica, 17, 139–145.
Brugerolle, G., & Joyon, L. (1973). Ultrastructure du genre Monocercomonoides (Travis).
Zooflagellata, Oxymonadida. Protistologica, 9,1–80.
Brugerolle, G., & König, H. (1997). Ultrastructure and organization of the cytoskeleton in
Oxymonas, an intestinal flagellate of termites. Journal of Eukaryotic Microbiology, 44,
305–313.
Brugerolle, G., & Lee, J. J. (2000). Order Oxymonadida. In J. J. Lee, G. F. Leedale, & P. Bradbury
(Eds.), The illustrated guide to the protozoa (Vol. 2, pp. 1186–1195). Lawrence: Allen Press.
Brugerolle, G., & Patterson, D. (1997). Ultrastructure of Trimastix convexa Hollande, an
amitochondriate anaerobic flagellate with a previously undescribed organization. European
Journal of Protistology, 33, 121–130.
Brugerolle, G., & Radek, R. (2006). Symbiotic protozoa of termites. In H. König & A. Varma
(Eds.), Intestinal microorganisms of termites and other invertebrates (pp. 243–269). Berlin/
Heidelberg: Springer.
Brugerolle, G., Silva-Neto, I. D., Pellens, R., & Grandcolas, P. (2003). Electron microscopic
identification of the intestinal protozoan flagellates of the xylophagous cockroach Parasphaeria
boleiriana from Brazil. Parasitology Research, 90, 249–256.
Brune, A., & Ohkuma, M. (2011). Role of the termite gut microbiota in symbiotic digestion. In
D. E. Bignell, Y. Roisin, & N. Lo (Eds.), Biology of termites: A modern synthesis (pp. 439–475).
Dordrecht: Springer.
Carpenter, K. J., Waller, R. F., & Keeling, P. J. (2008). Surface morphology of Saccinobaculus
(Oxymonadida): Implications for character evolution and function in oxymonads. Protist, 159,
209–221.
Carpenter, K. J., Weber, P. K., Davisson, M. L., Pett-Ridge, J., Haverty, M. I., & Keeling, P. J.
(2013). Correlated SEM, FIB-SEM, TEM, and NanoSIMS imaging of microbes from the
hindgut of a lower termite: Methods for in situ functional and ecological studies of uncultivable
microbes. Microscopy and Microanalysis, 19, 1490–1501.
Cavalier-Smith, T. (2002). The phagotrophic origin of eukaryotes and phylogenetic classification of
protozoa. International Journal of Systematic and Evolutionary Microbiology, 52, 297–354.
Cavalier-Smith, T. (2003). The excavate protozoan phyla Metamonada Grasse emend.
(Anaeromonadea, Parabasalia, Carpediemonas, Eopharyngia) and Loukozoa emend. (Jakobea,
Malawimonas): Their evolutionary affinities and new higher taxa. International Journal of
Systematic and Evolutionary Microbiology, 53, 1741–1758.
Cleveland, L. R. (1924). The physiological and symbiotic relationships between the intestinal
protozoa of termites and their host, with special reference to Reticulitermes flavipes Kollar.
The Biological Bulletin, 46, 203–227.
Cleveland, L. R. (1925). The effects of oxygenation and starvation on the symbiosis between the
termite Termopsis and its intestinal flagellates. The Biological Bulletin, 48, 309–325.
Cleveland, L. R. (1935). The intranuclear achromatic figure of Oxymonas grandis sp. nov. Biolog-
ical Bulletin, 69,54–65.
Cleveland, L. R. (1950a). Hormone-induced sexual cycles of flagellates: II. Gametogenesis, fertil-
ization, and one-division meiosis in Oxymonas. Journal of Morphology, 86, 185–214.
Cleveland, L. R. (1950b). Hormone-induced sexual cycles of flagellates: III. Gametogenesis,
fertilization, and one-division meiosis in Saccinobaculus. Journal of Morphology, 86, 215–228.
28 V. Hampl

Cleveland, L. R. (1950c). Hormone-induced sexual cycles of flagellates: IV. Meiosis after syngamy
and before nuclear fusion in Notila. Journal of Morphology, 87, 317–348.
Cleveland, L. R. (1956). Brief accounts of the sexual cycles of the flagellates of Cryptocercus. The
Journal of Protozoology, 3, 161–180.
Cleveland, L. R. (1966). Nuclear division without cytokinesis followed by fusion of pronuclei in
Paranotila lata gen. et sp. nov. The Journal of Protozoology, 13, 132–136.
Cleveland, L. R., Hall, S. R., Sanders, E. P., & Collier, J. (1934). The wood-feeding roach
Cryptocercus, its protozoa, and the symbiosis between protozoa and roach. Memoirs of the
American Academy of Arts and Sciences, 17, 185–342.
Cleveland, L. R., Arthur, W., Burke, J. R., & Karlson, P. (1960). Ecdysone induced modifications in
the sexual cycles of the protozoa of Cryptocercus. Journal of Eukaryotic Microbiology, 7,
229–239.
Cochrane, S. M., Smith, H. E., Buhse, H. E., & Scammell, J. G. (1979). Structure of the attached
stage of Pyrsonympha in the termite Reticulitermes flavipes Kollar. Protistologica, 15, 259–270.
Connell, F. H. (1930). The morphology and life-cycle of Oxymonas dimorpha sp. nov., from
Neotermes simplicicornis (Banks). University of California Publications in Zoology, 36,51–66.
Corliss, J. O. (1994). An interim utilitarian (users-friendly) hierarchical- classification and charac-
terization of the protists. Acta Protozoologica, 33,1–51.
Cross, J. B. (1939). A study on Oxymonas minor Zeliff from the termite Kalotermes minor Hagen.
University of California Publications in Zoology, 43, 379–404.
Cross, J. B. (1946). The flagellate subfamily oxymonadidae. University of California Publications
in Zoology, 53,67–162.
Crouch, H. B. (1933). Four new species of Trichomonas from the Woodchuck (Marmota monax
Linn.). The Journal of Parasitology, 19, 293–301.
da Cunha, A. M., & Muniz, J. (1921). Sobre flagellados parasitas. I. Monocercomononas caviae n.
sp. Brazil-Medicine, 35, 379–380.
da Cunha, A. M., & Muniz, J. (1927). Sur les flagellés intestinaux; description de trois especes
novelles. Comptes Rendus de la Société de Biologie, 96, 496–498.
Dacks, J. B., Silberman, J. D., Simpson, A. G. B., Moriya, S., Kudo, T., Ohkuma, M., & Redfield,
R. J. (2001). Oxymonads are closely related to the excavate taxon Trimastix. Molecular Biology
and Evolution, 18, 1034–1044.
Dacks, J. B., Kuru, T., Liapounova, N. A., & Gedamu, L. (2008). Phylogenetic and primary
sequence characterization of cathepsin B cysteine proteases from the oxymonad flagellate
Monocercomonoides. Journal of Eukaryotic Microbiology, 55,9–17.
Das, A. K. (1974). On the genus Oxymonas Janicki (Pyrsonymphidae: Mastigophora) from Indian
termites. Acta Protozoologica, 12, 335–344.
Das Gupta, M. (1935). Preliminary observations on the protozoan fauna of the rumen of the Indian
goat, Capra hircus Linn. Archiv fur Protistenkunde, 85, 153–172.
de Koning, A. P., Noble, G. P., Heiss, A. A., Wong, J., & Keeling, P. J. (2008). Environmental PCR
survey to determine the distribution of a non-canonical genetic code in uncultivable oxymonads.
Environmental Microbiology, 10,65–74.
De Mello, I. F. de. (1953). Sur une oxymonade de l’intestin du termite africain Cryptotermes
havilandi Sjos-tedt, recolte a Santos (Bresil). Revista Brasileira de Biologia, 13,65–72.
Derelle, R., Torruella, G., Klimeš, V., Brinkmann, H., Kim, E., Vlček, Č., Lang, B. F., & Eliáš,
M. (2015). Bacterial proteins pinpoint a single eukaryotic root. Proceedings of the National
Academy of Sciences of the United States of America, 112, E693–E699.
Diamond, L. S. (1982). A new liquid medium for xenic cultivation of Entamoeba histolytica and
other lumen dwelling Protozoa. The Journal of Parasitology, 68, 958–959.
Dobell, C., & Laidlaw, P. P. (1926). On the cultivation of Entamoeba histolytica and some other
entozoic amoebae. Parasitology, 18, 283–318.
Duboscq, O., & Grassé, P. P. (1934). Sur Microrhopalodina inflata(Grassi). Archives de zoologie
expérimentale et générale, 75, 615–637.
Preaxostyla 29

Dumas, E. (1930). Les microzoaires ou infusoires proprementdits. Faune du centre. 2e Fascicule.
Moulins (‘les imprimeriesreunies’), 166 p.
Gabel, J. R. (1954). The morphology and taxonomy of the intestinal Protozoa of the american
woodchuck, Marmota monax Linnaeus. Journal of Morphology, 94, 473–549.
Geiman, Q. M. (1933). The intestinal protozoa of the larvae of the crane fly Tipula abdominalis.
Journal of Parasitology, 19, 173.
Georgevitch, J. (1932). Recherches sur les flagellés des termites de Yougoslavie. Archives de
zoologie expérimentale et générale, 74,81–109.
Georgevitch, J. (1951). Etude des flagelles d’un termite de Dalmatie Reticulitermes lucifugus.
Glasn Acad Serbe Sci NS, 200,95–108.
Grant, J. R., & Katz, L. A. (2014). Building a phylogenomic pipeline for the eukaryotic tree of life –
Addressing deep phylogenies with genome-scale data. PLoS Currents, 2,6.
Grassé, P. P. (1926). Contribution à l’étude des Flagellés parasites. Archives de zoologie
expérimentale et générale, 65, 342–602.
Grassé, P. P. (1952). Traité de Zoologie. Tome I, Fascicule 1: Phylogénie. Protozoaires: généralités.
Flagellés. Paris: Masson et Cie.
Grassi, B. (1879). Dei protozoi parassiti specialmente di quelli che sono nell’uomo. Gaz Ital
Lombardi, 39, 445–448.
Grassi, B., & Foá, A. (1911). Intorno ai protozoi dei termitidi. Atti Reale Accad Lincei, 20, 725–741.
Grassi, B., & Sandias, A. (1893). Constitutione e svillupo della societa dei termitidi. Atti Accad.
Gioenia di scienze naturali in Catania, 6, 150–155.
Grimstone, A. V., & Cleveland, L. R. (1965). The fine structure and function of the contractile
axostyles of certain flagellates. The Journal of Cell Biology, 24, 387–400.
Guzmen, R. S. (1961). Oxymonas chilensis n. sp., flagelado simbionte del termite Calotermes
chilensis (Blanchard). Investigaciones Zoologicas Chilenas, 7,83–95.
Hampl, V., & Simpson, A. G. B. (2008). Possible mitochondria-related organelles in poorly-studied
‘amitochondriate’eukaryotes. In J. Tachezy (Ed.), Hydrogenosomes and mitosomes of the
amitochondrial protists (Microbiology monographs). Heidelberg: Springer.
Hampl, V., Horner, D. S., Dyal, P., Kulda, J., Flegr, J., Foster, P. G., & Embley, T. M. (2005).
Inference of the phylogenetic position of oxymonads based on nine genes: Support for
Metamonada and Excavata. Molecular Biology and Evolution, 22, 2508–2518.
Hampl, V., Hug, L., Leigh, J. W., Dacks, J. B., Lang, B. F., Simpson, A. G. B., & Roger, A. J.
(2009). Phylogenomic analyses support the monophyly of excavata and resolve relationships
among eukaryotic “supergroups”.Proceedings of the National Academy of Sciences of the
United States of America, 106, 3859–3864.
Hasselmann, G. (1928). Novo proeesso de divisao no genero Polymastix-descriptao de Polymastix
nitidus, nov. sp. de flagellado. Boletim do Instituto Brasileiro de Sciencias Rio de Janeiro, 3,
40–46.
Heiss, A. A., & Keeling, P. J. (2006). The phylogenetic position of the oxymonad Saccinobaculus
based on SSU rRNA. Protist, 157, 335–344.
Heuser, J. E. (1986). Different structural states of a microtubule cross-linking molecule, captured by
quick-freezing motile axostyles in Protozoa. The Journal of Cell Biology, 103, 2209–2227.
Hollande, A., & Carruette-Valentin, J. (1970a). Appariement chromosomique et complexes
synaptonématiques dans les noyaux de dépolyploidisation chez Pyrsonympha flagellata:le
cycle évolutif des Pyrsonymphines symbiotiques de Reticulitermes lucifugus. Comptes Rendus
de l’Académie des Sciences, Paris, D, 270, 2250–2255.
Hollande, A., & Carruette-Valentin, J. (1970b). La lignée des Pyrsonymphines et les caracteres
infrastructuraux commus aux autres genres Opisthomitus,Oxymonas,Saccinobaculus,
Pyrsonympha et Streblomastix. Comptes Rendus de l’Académie des Sciences, Paris, D, 270,
1587–1590.
Hongoh, Y. (2010). Diversity and genomes of uncultured microbial symbionts in the termite gut.
Bioscience Biotechnology and Biochemistry, 74, 1145–1151.
30 V. Hampl

Hongoh, Y., Sato, T., Noda, S., Ui, S., Kudo, T., & Ohkuma, M. (2007). Candidatus Symbiothrix
dinenymphae: Bristle-like Bacteroidales ectosymbionts of termite gut protists. Environmental
Microbiology, 9, 2631–2635.
Iida, T., Ohkuma, M., Ohtoko, K., & Kudo, T. (2000). Symbiotic spirochetes in the termite hindgut:
Phylogenetic identification of ectosymbiotic spirochetes of oxymonad protists. FEMS Microbi-
ology Ecology, 34,17–26.
Inward, D., Beccaloni, G., & Eggleton, P. (2007). Death of an order: A comprehensive molecular
phylogenetic study confirms that termites are eusocial cockroaches. Biology Letters, 3,
331–335.
Janakidevi, K. (1961). The morphology of Monocercomonoides filamentum n. sp., parasite of the
indian starred tortoise. Archiv fur Protistenkunde, 106,37–40.
Janicki, C. (1915). Untersuchungen an parasitischen Flagellaten. II. Teil: Die Gattungen
Devescovina, Parajoenia, Stephanonympha, Calonympha. Uber den Parabasalapparat.-Uber
Kernkonstitution und Kernteilung. Zeitschrift f€
ur wissenschaftliche Zoologie, 112, 573.
Jensen, E. A., & Hammond, D. M. (1964). A morphological study of trichomonads and related
flagellates from the bovine digestive tract. The Journal of Protozoology, 11, 386–394.
Jensen, C. G., & Smaill, B. H. (1986). Analysis of the spatial organization of microtubule associated
proteins. The Journal of Cell Biology, 103, 559–569.
Jírovec, O. (1929). La faune du tube digestif du Calotermes lucifugus récolté en Gréce. Bulletin
international de l’Académie des Sciences de Boheme, 39,1–15.
Kamikawa, R., Kolisko, M., Nishimura, Y., Yabuki, A., Brown, M. W., Ishikawa, S. A., Ishida, K.,
Roger, A. J., Hashimoto, T., & Inagaki, Y. (2014). Gene content evolution in Discobid
mitochondria deduced from the phylogenetic position and complete mitochondrial genome of
Tsukubamonas globosa. Genome Biology and Evolution, 6, 306–315.
Karnkowska, A., Vacek, V., Zubáčová, Z., Treitli, S. C., Petrželková, R., Eme, L., Novák, L.,
Žárský, V., Barlow, L. D., Herman, E. K., Soukal, P., Hroudová, M., Doležal, P., Stairs, C. W.,
Roger, A. J., Eliáš, M., Dacks, J. B., Vlček, Č., & Hampl, V. (2016). A eukaryote without a
mitochondrial organelle. Current Biology. doi:10.1016/j.cub.2016.03.053.
Keeling, P. J., & Leander, B. S. (2003). Characterisation of a non-canonical genetic code in the
oxymonad Streblomastix strix. Journal of Molecular Biology, 326, 1337–1349.
Kent, W. S. (1880). Manual of the Infusoria. London: David Bogue.
Kidder, G. W. (1929). Streblomastix strix, morphology and mitosis. University of California
Publications in Zoology, 33, 109–124.
Kidder, G. W. (1937). The intestinal protozoa of the wood-feeding roach Panesthia. Parasitology,
29, 163–205.
Kirby, H., Jr. (1924). Morphology and mitosis of Dinenympha fimbriata sp. nov. University of
California Press.
Kirby, H., Jr. (1926). The intestinal flagellates of the termite, Cryptotermes hermsi Kirby. University
of California Publications in Zoology, 29, 103–120.
Kirby, H. (1928). A species of Proboscidiella from Kalotermes (Cryptotermes)dudleyi Banks, a
termite of Central America, with remarks on the oxymonad flagellates. The Quarterly Journal of
Microscopical Science, 72, 355–386.
Kirby, H., & Honigberg, B. M. (1949). Flagellates of the caecum of ground squirrels. University of
California Publications in Zoology, 53, 315–366.
Klebs, G. (1892). Flagellatenstudien. Zeitschrift f€ur wissenschaftliche Zoologie, 55, 262–445.
Kofoid, C. A., & Swezy, O. (1919). Studies on the parasites of the termites I. On Streblomastix strix,
a polymastigote flagellate with a linear plasmodial phase. University of California Publications
in Zoology, 20,1–20.
Kofoid, C. A., & Swezy, O. (1926). On Oxymonas,aflagellate with an extensile and retractile
proboscis from Kalotermes from British Guiana. University of California Publications in
Zoology, 28, 285–300.
Koidzumi, M. (1921). Studies on the intestinal protozoa found in the termites of Japan. Parasitol-
ogy, 13, 235–309.
Preaxostyla 31

Krishnamurthy, R. (1967). Two new species of the genus Monocercomonoides Travis, 1932 (Pro-
tozoa: Mastigophora) from reptiles. Proceedings of the Indian Academy of Science, 66,
184–191.
Krishnamurthy, R., & Madre, V. E. (1979). Studies on two flagellates of the genus Monocerco-
monoides Travis, 1932 (Mastigophora: Polymastigina) from amphibian and reptiles in India.
Acta Protozoologica, 18, 251–257.
Krishnamurthy, R., & Sultana, T. (1976). Tubulimonoides gryllotalpae n. g., n. sp. (Mastigophora:
Oxymonadida) from cricket in India. Proceedings of the Indiana Academy of Sciences, 84(B),
137–140.
Krishnamurthy, R., & Sultana, T. (1977). Studies on two flagellates of the genus Monocerco-
monoides Travis, 1932 from the gut of the dung beetle larva (Oryctes rhinoceros) in India.
Archiv fur Protistenkunde, 119, 121–126.
Krishnamurthy, R., & Sultana, T. (1977). The flagellates of the genus Monocercomonoides Travis,
1932 (Mastigophora: Oxymonadida) from insects in India –A review, with a key to the species.
Proceedings of the Zoological Society. Calcutta, 32,51–55.
Krishnamurthy, R., & Sultana, T. (1978). A new species of the genus Polymastix Butschli, 1884
from an insect Polyphaga indica in India. Archiv fur Protistenkunde, 120, 301–303.
Krishnamurthy, R., & Sultana, T. (1980). The description of new flagellate Monocercomonoides
spirostreptae sp. n. (Mastigophora: Oxymonadida), from the millipedes in Maharashtra, India.
Acta Parasitologica Polonica, 27, 257–260.
Kulda, J., & Nohýnková, E. (1978). Flagellates of the human intestine and of intestines of other
species. In J. P. Kreier (Ed.), Parasitic protozoa (pp. 1–138). New York: Academic.
Lavette, A. (1973). Ultrastructure and systematic affinities of Microrhopalodina inflata, symbiotic
flagellate of Calotermes flavicollis. Comptes Rendus de l’Académie des Sciences, Paris, D, 276,
1309–1311.
Leander, B. S., & Keeling, P. J. (2004). Symbiotic innovation in the oxymonad Streblomastix strix.
Journal of Eukaryotic Microbiology, 51, 291–300.
Leidy, J. (1877). On intestinal parasites of Termes flavipes. Proceedings of the National Academy of
Sciences, Philadelphia, 29, 146–149.
Li, L., Fröhlich, J., & König, H. (2006). Cellulose digestion in the termite gut. In H. König &
A. Varma (Eds.), Intestinal microorganisms of termites and other invertebrates (pp. 221–241).
Berlin/Heidelberg: Springer.
Liapounova, N. A., Hampl, V., Gordon, P. M., Sensen, C. W., Gedamu, L., & Dacks, J. B. (2006).
Reconstructing the mosaic glycolytic pathway of the anaerobic eukaryote Monocercomonoides.
Eukaryotic Cell, 5, 2138–2146.
Lo, N., Tokuda, G., Watanabe, H., Rose, H., Slaytor, M., Maekawa, K., Bandi, C., & Noda,
H. (2000). Evidence from multiple gene sequences indicates that termites evolved from
wood-feeding cockroaches. Current Biology, 10, 801–804.
Maass, A., & Radek, R. (2006). The gut flagellate community of the termite Neotermes cubanus
with special reference to Staurojoenina and Trichocovina hrdyi nov. gen. nov. sp. European
Journal of Protistology, 42, 125–141.
Madre, V. E., & Krishnamurthy, R. (1976). Studies on two flagellates from the rectum of the viper,
Vipera russeli in Aurangabad. National Science Journal of Marathwada University, 15,
143–147.
Mali, M. S. (1993). Studies on the Polymastix jadhavii, a new flagellate from the gut of the
cockroach Periplareta americana in India. Geobios New Reports, 14, 189–191.
Mali, M., Kulkarni, S., & Mali, S. (2001). Two species of flagellates of the genus Monocerco-
monoides Travis, 1932 from the gut of dung beetle larva (Oryctes rhinoceros) in India. Geobios
(Jodhpur), 28, 201–204.
Mali, M., & Mali, S. (2004). Monocercomonoides khultabadae n.sp., a new flagellate from the gut
of Pycnoscelus surinamensis. Uttar Pradesh Journal Zoology, 24,55–58.
Mali, M., & Patil, D. (2003). The morphology of Monocercomonoides aurangabadae n. sp. a
flagellata from the gut of Blatta germanica. Uttar Pradesh Journal of Zoology, 23,117–119.
32 V. Hampl

Mali, M., & Sultana, T. (1993). The morphology of Tubulimonoides shivamurthi n. sp. –A new
flagellate from the gut of Oryctes rhinoceros. Geobios New Reports, 12,30–32.
Mali, M., Kulkarni, S., & Mali, S. (2003). Tubulimonoides aurangabadae n. sp. (Mastigophora:
Oxymonadida), a new flagellate from the gut of Oryctes rhinoceros. Geobios, 30, 291–292.
May, E. (1941). The behavior of the intestinal protozoa of termites at the time of the last ecdysis.
Transactions of the American Microscopical Society, 60, 281–292.
Mcintosh, J. R. (1973). Axostyle of Saccinobaculus. 2. Motion of microtubule bundle and a
structural comparison of straight and bent axostyles. The Journal of Cell Biology, 56, 324–339.
Mcintosh, J. R. (1974). Bridges between microtubules. The Journal of Cell Biology, 61, 166–187.
Mcintosh, J. R., Ogata, E. S., & Landis, S. C. (1973). Axostyle of Saccinobaculus. 1. Structure of
organism and its microtubule bundle. The Journal of Cell Biology, 56, 304–323.
Mooseker, M. S., & Tilney, L. G. (1973). Isolation and reactivation of the axostyle. Evidence for a
dynein-like ATPase in the axostyle. The Journal of Cell Biology, 56,13–26.
Moriya, S., Ohkuma, M., & Kudo, T. (1998). Phylogenetic position of symbiotic protist
Dinenympha [correction of Dinemympha]exilis in the hindgut of the termite Reticulitermes
speratus inferred from the protein phylogeny of elongation factor 1 alpha. Gene, 210, 221–227.
Moriya, S., Tanaka, K., Ohkuma, M., Sugano, S., & Kudo, T. (2001). Diversification of the
microtubule system in the early stage of eukaryote evolution: Elongation factor 1 alpha and
alpha-tubulin protein phylogeny of termite symbiotic oxymonad and hypermastigote protists.
Journal of Molecular Evolution, 52,6–16.
Moriya, S., Dacks, J. B., Takagi, A., Noda, S., Ohkuma, M., Doolittle, W. F., & Kudo, T. (2003).
Molecular phylogeny of three oxymonad genera: Pyrsonympha,Dinenympha and Oxymonas.
Journal of Eukaryotic Microbiology, 50, 190–197.
Moskowitz, N. (1951). Observations on some intestinal flagellates from reptilian host (Squamata).
Journal of Morphology, 89, 257–321.
Mukherjee, P., & Maiti, P. K. (1988). Two new species of flagellates of the genus Pyrsonympha
Leidy (Mastigophora: Protozoa) from Reticulitermes tirapi Chhotani and Das (Isoptera:
Insecta). Proceedings of the Zoological Society, Calcutta, 38,37–45.
Mukherjee, P., & Maiti, P. K. (1989). Description of two new species of flagellates of the genus
Dineympha Leidy (Mastigophora: Polymastigida) from Reticulitermes tirapi Chhotani & Das
(Isoptera). Archiv fur Protistenkunde, 137,95–100.
M€
uller, M. (1992). Energy metabolism of ancestral eukaryotes: A hypothesis based on the bio-
chemistry of amitochondriate parasitic protists. Biosystems, 28,33–40.
Nakashima, K. I., Watanabe, H., & Azuma, J. I. (2002). Cellulase genes from the parabasalian
symbiont Pseudotrichonympha grassii in the hindgut of the wood-feeding termite Coptotermes
formosanus. Cellular and Molecular Life Sciences, 59, 1554–1560.
Navarathnam, E. S. (1970). Intestinal flagellates of the common Indian rat Rattus rattus frugivorous.
Acta Protozoologica, 8, 155–165.
Nie, D. (1950). Morphology and taxonomy of the intestinal Protozoa of the guinea-pig, Cavia
porcella. Journal of Morphology, 86, 381–494.
Noda, S., Ohkuma, M., Yamada, A., Hongoh, Y., & Kudo, T. (2003). Phylogenetic position and in
situ identification of ectosymbiotic spirochetes on protists in the termite gut. Applied and
Environmental Microbiology, 69, 625–633.
Noda, S., Inoue, T., Hongoh, Y., Kawai, M., Nalepa, C. A., Vongkaluang, C., Kudo, T., & Ohkuma,
M. (2006). Identification and characterization of ectosymbionts of distinct lineages in
Bacteroidales attached to flagellated protists in the gut of termites and a wood-feeding cock-
roach. Environmental Microbiology, 8,11–20.
Nurse, F. R. (1945). Protozoa from New Zealand Termites. Transactions of the Royal Society of
New Zealand, 74, 305–314.
Ohkuma, M., & Brune, A. (2011). Diversity, structure, and evolution of the termite gut microbial
community. In D. E. Bignell, Y. Roisin, & N. Lo (Eds.), Biology of termites: A modern synthesis
(pp. 439–475). Dordrecht: Springer.
Preaxostyla 33

O’Kelly, C. J., Farmer, M. A., & Nerad, T. A. (1999). Ultrastructure of Trimastix pyriformis (Klebs)
Bernard et al.: Similarities of Trimastix species with retortamonad and jakobid flagellates.
Protist, 150, 149–162.
Parfrey, L. W., Grant, J., Tekle, Y. I., Lasek-Nesselquist, E., Morrison, H. G., Sogin, M. L.,
Patterson, D. J., & Katz, L. A. (2010). Broadly sampled multigene analyses yield a well-
resolved eukaryotic tree of life. Systematic Biology, 59, 518–533.
Poinar, G. O., Jr. (2009a). Description of an early Cretaceous termite (Isoptera: Kalotermitidae) and its
associated intestinal protozoa, with comments on their co-evolution. Parasites & Vectors, 2,12.
Poinar, G. O., Jr. (2009b). Early Cretaceous protist flagellates (Parabasalia: Hypermastigida:
Oxymonada) of cockroaches (Insecta:Blattaria) in Burmese amber. Cretaceous Research, 30,
1066–1072.
Porter, J. F. (1897). Trichonympha, and other parasites of Termes flavipes. Bulletin of the Museum of
Comparative Zoo¨logy, 31,47–63.
Powell, W. N. (1928). On the morphology of Pyrsonympha with a description of three new species
from Reticulitermes hesperus Banks. University of California Publications in Zoology, 31,
179–200.
Qadri, S. S., & Rao, T. B. (1963). On a new flagellate Polymastix periplanetae from the common
cockroach, Periplaneta americana. Rivista Parasitologica, 24, 153–158.
Radek, R. (1994). Monocercomonoides termitis n. sp, an oxymonad from the lower termite
Kalotermes sinaicus. Archiv f€
ur Protistenkunde, 144, 373–382.
Radek, R. (1997). Monocercomonoides hausmanni nom. nov, a new species name for M. termitis
Radek, 1994. Archiv f€
ur Protistenkunde, 147,411.
Radek, R. (1999). Flagellates, bacteria, and fungi associated with termites: Diversity and function in
nutrition –A review. Ecotropica, 5, 183–196.
Radek, R., Strassert, J. F., Kr€
uger, J., Meuser, K., Scheffrahn, R. H., & Brune, A. (2014). Phylogeny
and ultrastructure of Oxymonas jouteli, a rostellum-free species, and Opisthomitus longifla-
gellatus sp. nov., Oxymonadida flagellates from the gut of Neotermes jouteli. Protist, 165(3),
384–399.
Ray, D. K. (1949). On a Monocercomonoides nimiei n. sp., from the caecum of Indian guinea pig,
Cavia cutleri Bennet. Proceedings of the Indian Science Congress, 36, 155.
Reeves, R. E., Warren, L. G., Susskind, B., & Loi, H. S. (1977). Energy conserving pyruvate to
acetate pathway in Entamoeba histolytica –Pyruvate synthase and a new acetate thiokinase. The
Journal of Biological Chemistry, 252, 726–731.
Rodriguez-Ezpeleta, N., Brinkmann, H., Burger, G., Roger, A. J., Gray, M. W., Philippe, H., &
Lang, B. F. (2007a). Toward Resolving the eukaryotic tree: The phylogenetic positions of
jakobids and cercozoans. Current Biology, 17, 1420–1425.
Rodriguez-Ezpeleta, N., Brinkmann, H., Roure, B., Lartillot, N., Lang, B. F., & Philippe,
H. (2007b). Detecting and overcoming systematic errors in genome-scale phylogenies. System-
atic Biology, 56, 389–399.
Rother, A., Radek, R., & Hausmann, K. (1999). Characterization of surface structures covering
termite flagellates of the family oxymonadidae and ultrastructure of two oxymonad species,
Microrhopalodina multinucleata and Oxymonas sp. European Journal of Protistology, 35,
1–16.
Sato, T., Kuwahara, H., Fujita, K., Noda, S., Kihara, K., Yamada, A., Ohkuma, M., & Hongoh,
Y. (2014). Intranuclear verrucomicrobial symbionts and evidence of lateral gene transfer to the
host protist in the termite gut. ISME Journal, 8(5), 1008–1019.
Simpson, A. G. B. (2003). Cytoskeletal organization, phylogenetic affinities and systematics in the
contentious taxon Excavata (Eukaryota). International Journal of Systematic and Evolutionary
Microbiology, 53, 1759–1777.
Simpson, A. G. B., Bernard, C., & Patterson, D. J. (2000). The ultrastructure of Trimastix marina
Kent, 1880. European Journal of Protistology, 36, 229–251.
34 V. Hampl

Simpson, A. G. B., Radek, R., Dacks, J. B., & O’Kelly, C. J. (2002). How oxymonads lost their
groove: An ultrastructural comparison of Monocercomonoides and excavate taxa. Journal of
Eukaryotic Microbiology, 49, 239–248.
Simpson, A. G. B., Inagaki, Y., & Roger, A. J. (2006). Comprehensive multigene phylogenies of
excavate protists reveal the evolutionary positions of “primitive”eukaryotes. Molecular Biology
and Evolution, 23, 615–625.
Slamovits, C. H., & Keeling, P. J. (2006a). A high density of ancient spliceosomal introns in
oxymonad excavates. BMC Evolutionary Biology, 6, 34.
Slamovits, C. H., & Keeling, P. J. (2006b). Pyruvate-phosphate dikinase of oxymonads and
parabasalia and the evolution of pyrophosphate-dependent glycolysis in anaerobic eukaryotes.
Eukaryotic Cell, 5, 148–154.
Smith, H. S., & Arnott, H. J. (1973). Scales associated with external surface of Pyrsonympha
vertens. Transactions of the American Microscopical Society, 92, 670–677.
Smith, H. E., & Arnott, H. J. (1974a). Axostyle structure in termite protozoan Pyrsonympha vertens.
Tissue & Cell, 6, 193–207.
Smith, H. E., & Arnott, H. J. (1974b). Epibiotic and endobiotic bacteria associated with
Pyrsonympha vertens –Symbiotic protozoan of termite Reticulitermes flavipes. Transactions
of the American Microscopical Society, 93, 180–194.
Smith, H. E., Stamler, S. J., & Buhse, B. E., Jr. (1975). A scanning electron microscope survey of
the surface features of polymastigote flagellates from Reticulitermes flavipes. Transactions of
the American Microscopical Society, 94, 401–410.
Stechmann, A., Baumgartner, M., Silberman, J. D., & Roger, A. J. (2006). The glycolytic pathway
of Trimastix pyriformis is an evolutionary mosaic. BMC Evolutionary Biology, 6, 101.
Stingl, U., & Brune, A. (2003). Phylogenetic diversity and whole-cell hybridization of oxymonad
flagellates from the hindgut of the wood-feeding lower termite Reticulitermes flavipes. Protist,
154, 147–155.
Stingl, U., Radek, R., Yang, H., & Brune, A. (2005). “Endomicrobia”: Cytoplasmic symbionts of
termite gut protozoa form a separate phylum of prokaryotes. Applied and Environmental
Microbiology, 71, 1473–1479.
Sultana, T. (1975). A redescription of Monocercomonoides ganapatii Bhaskar Rao, 1969 from
Gryllotalpa africana. Natural Science. Journal of Marathwada University, 14, 229–232.
Sultana, T. (1976). Monocercomonoides krishnamurthii n. sp., a new flagellate (Protozoa:
Plastigophora) from the gut of a blattid in India. Natural Science. Journal of Marathwada
University, 15, 149–152.
Sultana, T. (1976). Studies on two new species of flagellates of the genus Polymastix Butschli, 1884
from insects in India. Acta Protozoologica, 15,1–8.
Sultana, T., & Krishnamurthy, R. (1978). Monocercomonoides gryllusae n. sp. (Mastigophora:
Oxymonadida) from Gryllus bimaculatus. Geobios, 6,114–115.
Tamschick, S., & Radek, R. (2013). Colonization of termite hindgut walls by oxymonad flagellates
and prokaryotes in Incisitermes tabogae,I. marginipennis and Reticulitermes flavipes.
European Journal of Protistology, 49,1–14.
Tanabe, M. (1933). The morphology and division of Monocercomonas lacertae, n. sp. from lizards.
Keijo Journal of Medicine, 4, 367–377.
Tiwari, D. N. (2005). Oxymonas bastiensis sp.nov. (Oxymonadidae) a new flagellate from the
termite Neotermes bosei synder of Uttar Pradesh, India. Journal of Advanced Zoology, 26,
50–51.
Todd, S. R. (1963). Studies on some parasitic flagellates of certain wild mammals of Hyderabad.
Archiv fur Protistenkunde, 107,1–116.
Tokura, M., Ohkuma, M., & Kudo, T. (2000). Molecular phylogeny of methanogens associated
with flagellated protists in the gut and with the gut epithelium of termites. FEMS Microbiology
Ecology, 33, 233–240.
Preaxostyla 35

Travis, B. V. (1932). A discussion of synonymy in the nomenclature of certain insect flagellates,
with the description of a new flagellate from the larvae of Ligyrodes relictus Say (Coleoptera-
Scarabaeidae). Iowa State College Journal of Science, 6, 317–323.
Travis, B. V., & Becker, E. R. (1931). A preliminary report on the intestinal protozoa of white grubs.
(Phyllophaga sp. Coleoptera). Iowa State Journal of Science, 5, 223–235.
Upcroft, J., & Upcroft, P. (1998). My favorite cell: Giardia. Bioessays, 20, 256–263.
Yamin, M. A. (1981). Cellulose metabolism by the flagellate Trichonympha from a termite is
independent of endosymbiotic bacteria. Science, 211,58–59.
Yang, H., Schmitt-Wagner, D., Stingl, U., & Brune, A. (2005). Niche heterogeneity determines
bacterial community structure in the termite gut (Reticulitermes santonensis). Environmental
Microbiology, 7, 916–932.
Yubuki, N., & Leander, B. S. (2013). Evolution of microtubule organizing centers across the tree of
eukaryotes. The Plant Journal, 75(2), 230–244.
Yubuki, N., Simpson, A. G., & Leander, B. S. (2013). Comprehensive ultrastructure of Kipferlia
bialata provides evidence for character evolution within the Fornicata (Excavata). Protist, 164
(3), 423–439.
Zeliff, C. C. (1930). Kirbyella zeteki, a new genus and species of protozoa from Kalotermes
(Calcaritermes) brevicollis from the canal zone. American Journal of Epidemiology, 11,
740–742.
Zeliff, C. C. (1930). A cytological study of Oxymonas,aflagellate, including descriptions of new
species. American Journal of Epidemiology, 11, 714–739.
Zhang, Q., Táborský, P., Silberman, J. D., Pánek, T., Čepička, I., & Simpson, A. G. B. (2015).
Marine Isolates of Trimastix marina form a plesiomorphic deep-branching lineage within
Preaxostyla, separate from other known trimastigids (Paratrimastix n. gen.). Protist, 166(4),
468–491.
Zhou, X., Smith, J. A., Oi, F. M., Koehler, P. G., Bennett, G. W., & Scharf, M. E. (2007). Correlation
of cellulase gene expression and cellulolytic activity throughout the gut of the termite
Reticulitermes flavipes. Gene, 395(1–2), 29–39.
Zubáčová, Z., Novák, L., Bublíková, J., Vacek, V., Fousek, J., Rídl, J., Tachezy, J., Doležal, P.,
Vlček, C., & Hampl, V. (2013). The mitochondrion-like organelle of Trimastix pyriformis
contains the complete glycine cleavage system. PLoS One, 8, e55417.
36 V. Hampl