Apusomonadida

Aaron A. Heiss, Matthew W. Brown, and Alastair G. B. Simpson

Contents Summary Classification ...... 2 Introduction ...... 3 General Characteristics ...... 3 Occurrence ...... 8 Literature and History of Knowledge ...... 8 Practical Importance ...... 9 Habitats and Ecology ...... 9 Characterization and Recognition ...... 10 General Appearance ...... 10 Ultrastructure ...... 12 Life Cycle...... 13 Systematics ...... 14 Maintenance and Cultivation ...... 14 Evolutionary History ...... 14 Internal Relationships ...... 14 Overall Phylogenetic Position ...... 15 Implications for Evolution ...... 16 Coda: Breviates and Ancyromonads ...... 17 References ...... 22

A.A. Heiss (*) Department of Invertebrate Zoology and RGGS, American Museum of Natural History, New York, NY, USA e-mail: [email protected] M.W. Brown (*) Department of Biological Sciences, Mississippi State University, Mississippi State, MS, USA e-mail: [email protected] A.G.B. Simpson (*) Department of Biology, Dalhousie University, Halifax, NS, Canada e-mail: [email protected]

# Springer International Publishing Switzerland 2016 1 J.M. Archibald et al. (eds.), Handbook of the , DOI 10.1007/978-3-319-32669-6_15-1 2 A.A. Heiss et al.

Abstract is a small group of free-living heterotrophic flagellates. Apusomonads are small (~5–20 μm long) gliding aerobes with two flagella. The dorsal cell membrane is underlain by a pellicle, which also supports a “skirt” of folded membrane that extends laterally/ventrally. The anterior flagellum is enclosed by a sleeve-like extension of the skirt system, forming a flexible proboscis. itself is a rounded cell with an anterior extension, the mastigophore, that contains the flagellar apparatus. All other apusomonads (usu- ally now assigned to the genera Amastigomonas, Chelonemonas, Manchomonas, Multimonas, Podomonas, and Thecamonas) are elongated and plastic and may form ventral pseudopodia. Apusomonas is a soil flagellate. Most other apusomonads that have been cultured to date are marine. Apusomonads are closely related to (e.g., and fungi), making them an impor- tant group for examining, for example, the origins of multicellularity. The genome of Thecamonas trahens encodes several proteins and pathways previ- ously considered specific to animals, including much of the integrin system, which functions in cell-cell communication and adhesion in metazoa. This chapter also briefly reviews breviates and ancyromonads, two groups of surface-associating flagellates that are (or may be) closely related to apusomonads and are of similar evolutionary significance. Breviates comprise three genera of small (~10–15 μm long) anaerobic cells that produce fine pseudopodia. Ancyromonads (synonym planomonads) comprise four genera of tiny (~5 μm long) flattened cells with an inflexible pellicle underlying most of the cell membrane and a battery of extrusomes in a lateral rostrum.

Keywords Aerobe • Anaerobe • Ancyromonad • Apusomonad • Bacterivore • Breviate • • Integrin • Opisthokonts • • Thecamonas

Summary Classification

• Apusomonadida ••Apusomonadidae ••• Apusomonadinae (Apusomonas, Manchomonas) ••• Thecamonadinae (Thecamonas, Chelonemonas) ••• Amastigomonas (Amastigomonas) ••• Multimonas (Multimonas) ••• Podomonas (Podomonas) [Other Apusomonadida: “Thecamonas” oxoniensis] • Breviatea (, Subulatomonas, Pygsuia, Lenisia) • (= Planomonadida) •• Ancyromonadidae (, Nutomonas) Apusomonadida 3

•• Planomonas (Planomonas) •• Fabomonas (Fabomonas)

Introduction

General Characteristics

Apusomonadida is a group of small free-living heterotrophic flagellates that glide on surfaces. All known apusomonads have two flagella, with the anterior flagellum surrounded by a membranous “sleeve” that extends from the main cell body. The combined flagellum-sleeve apparatus forms a highly mobile proboscis, which is a primary characteristic of the group (Karpov and Myľnikov 1989). The posterior flagellum runs underneath the cell venter (ventral face), on the left side of the cell. Pseudopodia, which are used for feeding, are produced from the ventral region of the cell in some members of the group. The dorsal cell membrane is underlain by a pellicle, which continues into a ventrally projecting “skirt” on the sides of the cell, and which is continuous with the proboscis sleeve (Fig. 1). Apusomonads are currently divided into at least five main phylogenetic groups, based on molecular and morphological data of cultured strains (Cavalier-Smith and Chao 2010; Heiss et al. 2015): (i) Apusomonadinae, containing the genera Apusomonas and Manchomonas; (ii) the genus Podomonas; (iii) the genus Multimonas; (iv) Thecamonadinae, including the genus Chelonemonas and the majority of members of the genus Thecamonas; and (v) the single freshwater species “Thecamonas” oxoniensis (Figs. 1 and 2; Table 1). Another genus, Amastigomonas sensu stricto (see below), is of uncertain position relative to other apusomonads. The most distinctive genus is Apusomonas, which has an inflexible, rounded body and an extended “mastigophore” that contains both the proboscis and the flagellar apparatus (Karpov and Myľnikov 1989; Vickerman et al. 1974; Figs. 1a and 2d). The other genera contain more elongate, flexible cells, with the flagellar apparatus positioned within the anterior end of the main cell body. The morphological differences between them are often subtle, and until recently all apusomonads other than Apusomonas were assigned to the genus Amastigomonas (a practice continued by some authors: Karpov 2011;Myľnikov and Myľnikova 2012). Apusomonads have an important phylogenetic position within the eukaryote tree of life. They are amongst the closest relatives of Opisthokonta, the “supergroup” that includes both animals and fungi (Brown et al. 2013; Burki et al. 2016; Cavalier-Smith and Chao 1995;Cavalier-Smithetal.2014; Derelle and Lang 2012;Heetal.2014; Kim et al. 2006;Papsetal.2013; Torruella et al. 2012, 2015). This suggests that apusomonads are important for understanding the origins of multicellularity in animals and fungi. In particular, the genome of the apusomonad Thecamonas trahens encodes most components of the integrin machinery critical to cell adhesion in animals (Seb- é-Pedrós et al. 2010). Thecamonas also has a more complex flagellar apparatus cytoskeleton than that seen in opisthokonts, and this sheds light on the deep-level evolution of the cytoskeletal architecture in extant (Heiss et al. 2013b). 4 A.A. Heiss et al.

abacroneme anterior flagellum sleeve acroneme tusk mastigophore anterior flagellum sleeve proboscis proboscis skirt posterior flagellum

cell lateral body pseudopodium

posterior flagellum

acroneme

trailing pseudopodium

Fig. 1 Appearance by light microscopy of living apusomonads. (a) Apusomonas proboscidea; (b) Thecamonas trahens. Nuclei are light grey; mitochondria are dark grey. Scale bar in (b) = 2 μm for both drawings

Occurrence

The majority of known apusomonads are marine; however, Apusomonas occurs in soil and “Thecamonas” oxoniensis was isolated from the surface of a terres- trial , both being essentially freshwater organisms (Cavalier-Smith and Chao 2010). The original account of Amastigomonas (see below) was also of a freshwater organism (de Saedeleer 1931). Apusomonads are one of the most frequently encountered groups of heterotrophic flagellates in microscopy studies of marine sediments (Patterson and Lee 2000), though almost always low in cell number.

Literature and History of Knowledge

The scientific history of apusomonads extends back a century, although the group was united less than three decades ago. The first described apusomonad was originally called Rhynchomonas mutabilis (Griessmann 1913), although it was not recognized as an apusomonad until almost 80 years later (Larsen and Patterson 1990; Apusomonadida 5

Fig. 2 Light (a–d) and scanning electron (e–i) micrographs of apusomonads. Panels (a–d) are differential interference contrast images of living cells: (a) Thecamonas trahens without prominent pseudopodia but with visible “tusk” (T); (b) Thecamonas trahens with prominent trailing 6 A.A. Heiss et al. true Rhynchomonas organisms are kinetoplastids – see chapter “▶ Kinetoplastea”). Apusomonas itself was known from an unpublished account in 1917 (Vickerman et al. 1974) and formally described a few years later (Aléxéieff 1924). Shortly after this, the genus Amastigomonas was established (de Saedeleer 1931) for Amastigomonas debrunyei, a gliding organism with a prominent proboscis but no visible flagella (hence the name), although the cells were probably biflagellated in reality (see below). Apusomonads remained little studied for the next 40 years, until the redescription of Apusomonas proboscidea by Vickerman et al. (1974). Addi- tional new species were assigned to Amastigomonas from the 1970s onwards (Ekelund and Patterson 1997; Hamar 1979; Larsen and Patterson 1990;Myľnikov 1999;Myľnikov and Myľnikova 2012; Zhukov 1975). Ultrastructural studies in the 1980s led to the recognition that Apusomonas was related to the various organisms described as Amastigomonas species, and to the proposal of the taxon Apusomonadida, containing both types of organisms (Karpov and Myľnikov 1989). The monophyly of Apusomonadida has since been confirmed by SSU rRNA gene phylogenies (e.g., Cavalier-Smith and Chao 2003, 2010; Cavalier- Smith et al. 2004, 2008; Heiss et al. 2015; Nikolaev et al. 2006; Walker et al. 2006). Recently there has been a considerable expansion in the number of described genera and species of apusomonads. Larsen and Patterson (1990) introduced the new genus Thecamonas for certain small apusomonads. It was soon recognized, however, that these organisms were very similar to those previously known as Amastigomonas,andThecamonas was temporarily considered a junior synonym (Molina and Nerad 1991). Until 2010, additional information on Amastigomonas- like apusomonads continued to be referred to the genus Amastigomonas (Cavalier- Smith 2002; Ekelund and Patterson 1997; Lee 2002; Lee et al. 2005; Molina and Nerad 1991; Patterson and Simpson 1996; Tikhonenkov et al. 2006;Vørs1993). In 2010, however, the first broad molecular phylogenetic study of apusomonads dem- onstrated that the “amastigomonad”-type apusomonads were genetically diverse

and represented a paraphyletic group within apusomonads (Cavalier-Smith and ä

Fig. 2 (continued) pseudopodium; (c) Podomonas magna;(d) Apusomonas sp. Panels (e–i) are scanning electron micrographs of cells fixed with osmium tetroxide: (e) Thecamonas trahens, dorsal view, showing continuity of proboscis sleeve (Pr) with dorsal cell covering; (f) Thecamonas trahens, ventral view, showing different texture between “skirt” (sides of cell) and ventral surface (center), “tusk” (T) protruding from near origin of proboscis (Pr), and posterior flagellum (PF) tucked between cell body and “skirt”;(g) Chelonemonas geobuk, dorsal view, showing hexagonal “tortoise-shell” patterning on dorsum; (h) Multimonas media, dorsal view, showing numerous discharged extrusomes (Ex) and reduced proboscis (Pr) with exposed anterior flagellum (AF); (i) Multimonas media, ventral view, showing “frilled” margin of cell “skirt” (open arrowheads). Closed arrowheads – acroneme at end of anterior flagellum; open arrowheads –“frilled” margin of “skirt”; AF anterior flagellum, Ex extrusomes, Ms mastigophore, PF posterior flagellum, Pr proboscis, Ps pseudopodium, T “tusk.” Scale bar in (d) = 5 μm for (a–d); scale bar in (g) = 1 μmin (e–g); scale bar in (i) = 1 μmin(h) and (i) (Images a and b reproduced from originals used for Heiss et al. 2013b; image c by AAH; image d courtesy of Yana Eglit (Dalhousie University); images e–i reproduced from originals used for Heiss et al. 2015) Table 1 Summary of published species/strains of apusomonads. Type species is listed 7 first for each genus Apusomonadida Molecular Deposited data Comments Taxon Species names Synonym(s) Authority Cultures (selected) SEM TEM [v] Apusomonadinae Apusomonas Aléxéieff (1924) CCAP SSU rDNA, * * [y] from soil; has proboscidea 1905/1 some mastigophore proteins Apusomonas Ekelund and Patterson from soil; has australiensis (1997) mastigophore Manchomonas Amastigomonas Molina and Nerad (1991), ATCC SSU rDNA; ** bermudensis bermudensis Cavalier-Smith (2010) [w] 50234 ESTs [x] Podomonas Podomonas Cavalier-Smith (2010) [w] CCAP SSU rDNA magna 1901/4 Podomonas Cavalier-Smith (2010) [w] prev. in SSU rDNA * * capensis ATCC Podomonas Am. gigantea Myľnikov (1999), gigantea Cavalier-Smith (2010) [w] Podomonas Am. griebenis Myľnikov (1999), griebenis Cavalier-Smith (2010) [w] Podomonas Am. klosteris Myľnikov (1999), klosteris Cavalier-Smith (2010) [w] Multimonas Multimonas Cavalier-Smith (2010) [w] CCAP SSU rDNA; forms media 1901/3 454 “ESTs” syncytia Multimonas Cercomonas Myľnikov (1989a), marina marina, Cavalier-Smith (2010) [w] Am. marina Multimonas Heiss et al. (2015) SSU rDNA * koreensis (continued) Table 1 (continued) 8 Molecular Deposited data Comments Taxon Species names Synonym(s) Authority Cultures (selected) SEM TEM [v] Thecamonadinae Thecamonas Am. trahens Larsen and Patterson ATCC Genome * * [z] trahens (1990) 50062 Thecamonas Larsen and Patterson filosa (1990) Thecamonas Am. muscula Myľnikov (1999), muscula Cavalier-Smith (2010) [w] Thecamonas Rhynchomonas Griessmann (1913), mutabilis mutabilis, Larsen and Patterson Am. mutabilis (1990) Chelonemonas Heiss et al. (2015) SSU rDNA * geobuk Chelonemonas Heiss et al. (2015) SSU rDNA * masanensis “Thecamonas” “Thecamonas” Cavalier-Smith (2010) [w] CCAP SSU rDNA; freshwater; oxoniensis oxoniensis 1901/2 454 “ESTs” forms cysts Amastigomonas Amastigomonas de Saedeleer (1931) freshwater debruynei Amastigomonas Hamar (1979) freshwater borokensis Amastigomonas Zhukov (1975) freshwater caudata

Amastigomonas Myľnikov and Myľnikova * al. et Heiss A.A. marisrubri (2012) [v]: Unless otherwise specified, all strains are marine [w]: Description in Cavalier-Smith and Chao (2010) [x]: Unpublished (B. Franz Lang) [y]: Including 3D reconstuction of the flagellar apparatus [z]: Including 3D reconstuction of the entire cell Apusomonadida 9

Chao 2010). As a consequence, these were divided into several genera, including the reestablished genus Thecamonas and the new genera Manchomonas, Multimonas, and Podomonas (Cavalier-Smith and Chao 2010), with an additional “amastigomonad”-like genus, Chelonemonas, being described recently (Heiss et al. 2015). Under this scheme, the genus Amastigomonas has been retained to encompass only freshwater organisms closely resembling the original account of Amastigomonas debruynei (de Saedeleer 1931). As mentioned above, however, this scheme has not been universally accepted, with some authorities continuing to use Amastigomonas for all non-Apusomonas-type apusomonads (Karpov 2011; Myľnikov and Myľnikova 2012). Regardless, there are neither molecular nor ultra- structural data for Amastigomonas sensu stricto at present, and consequently its identity as a relative of other apusomonads is in some doubt (Cavalier-Smith and Chao 2010). There are several cursory accounts of the ultrastructure of apusomonads (Cavalier-Smith and Chao 2010; Karpov and Myľnikov 1989; Karpov and Zhukov 1984, 1986; Molina and Nerad 1991;Myľnikov 1989b), with detailed reconstruc- tions of the flagellar apparatus completed for Apusomonas proboscidea (Karpov 2007)andThecamonas trahens (Heiss et al. 2013b). At the time of writing, most sequence data is from Thecamonas trahens, for which there is a genome project (Ruiz-Trillo et al. 2007).

Practical Importance

All known apusomonads are free-living. One species, “Thecamonas” oxoniensis, was isolated from the surface of a leaf of English ivy, but it has not been established whether this species is a true epibiont (Cavalier-Smith and Chao 2010). The eco- logical importance of apusomonads is essentially unknown (see below). They have not been exploited commercially. Their primary scientific relevance at present is their importance for understanding the deep evolutionary history of eukaryotes and the evolution of multicellularity (see below).

Habitats and Ecology

All known apusomonads are gliding organisms, and thus primarily surface- associated. All are heterotrophic, and primarily or exclusively bacterivorous (Karpov and Zhukov 1984; Cavalier-Smith and Chao 2010). Apusomonads appear to be ubiquitous: apusomonad cells or SSU rRNA sequences have been detected in samples from fresh water (Lee et al. 2005; Scheckenbach et al. 2006), marine material (Larsen and Patterson 1990; Lee and Patterson 2000; Massana et al. 2011; Myľnikov and Myľnikova 2012; al-Qassab et al. 2002; Tong 1997; Tong et al. 1998; Vørs 1993), soil samples (Ekelund and Patterson 1997; Vickerman et al. 1974), and from at least moderately hypersaline environments (Patterson and Simpson 1996). They have been recovered from surface waters (Massana and Pedrós-Alió 2008; 10 A.A. Heiss et al.

Scheckenbach et al. 2005), littoral sediments (al-Qassab et al. 2002; Massana et al. 2015; Tikhonenkov et al. 2006), and the deep sea (López-García et al. 2003; Scheckenbach et al. 2005; Takishita et al. 2007, 2010). The marine apusomonads (currently understood as the genera Thecamonas sensu stricto, Chelonemonas, Multimonas, Podomonas, and Manchomonas) are collectively among the 20 most- encountered varieties of heterotrophic flagellates in microscopy studies of marine sediment samples (Patterson and Lee 2000), although always in low densities (Arndt et al. 2000). Freshwater apusomonads (“Thecamonas” oxoniensis and Amastigomonas sensu stricto) have only rarely been encountered (Cavalier-Smith and Chao 2010). Soil-dwelling apusomonads are widely distributed and can be abundant (Ekelund and Patterson 1997; Foissner 1991). It is important to note that all apusomonads other than Apusomonas appear rather similar and that their recognition as different genera was a recent proposal, which has not been adopted universally (see above and below). Consequently, these apusomonads have been recorded as one member or another of the genus Amastigomonas in almost every ecological survey published to date. Because of this, the true distribution across habitats is unknown for all genus-level taxa of apusomonads other than Apusomonas.

Characterization and Recognition

General Appearance

Apusomonads are all small, usually 5–10 μm in length, though some species may approach 20 μm. Most have an ovoid main cell body (though the main cell body of Apusomonas has a subcircular profile), with a characteristic highly mobile anterior proboscis that includes the anterior flagellum (reported cell lengths generally refer to the main cell body, without the proboscis). The proboscis has a smooth anterosinistral motion and often curves along its length as it moves (Cavalier- Smith and Chao 2010; Heiss et al. 2013b; Karpov and Myľnikov 1989; Vickerman et al. 1974). An acroneme (from the anterior flagellum) may emerge from the tip of the proboscis, and may be distinguishable by light microscopy. The posterior flagellum runs under the cell body, along its left side, and (depending on the taxon) may extend beyond the cell outline to trail behind the cell (Figs. 1 and 2). The cell is generally two to three times as long as it is wide, though most taxa are relatively flexible. Most genera produce pseudopodia, which emerge from the cell venter and may extend in any direction, though rarely more than half the cell length (Figs. 1b, 2a, b, e, f, and 3b; Cavalier-Smith and Chao 2010; Heiss et al. 2013b; Karpov and Myľnikov 1989), except in the case of the trailing pseudopodium (see below). All genera except for Apusomonas (see below) appear quite similar under the light microscope. Podomonas is larger than other apusomonads (12–20 μm) and has Apusomonadida 11

Fig. 3 Intracellular features of the apusomonad Thecamonas trahens. Panels (a–h) are transmis- sion electron micrographs: (a) longitudinal section of whole cell, showing general cell features; (b) cross section roughly one third through whole cell, showing principal organelles as well as pseudopodium (Ps) and components of posterior flagellar apparatus; (c) mitochondrion, showing tubular cristae and nucleoid (arrowhead); (d) cross section of cell showing pellicle (dark layer under plasma membrane) and subpellicular layer (arrowhead); (e) cross section of proboscis, showing anterior flagellum (AR) surrounded by double layer of cell membrane; (f) internal structure of ‘tusk’ (T); (g) arrangement of posterior roots near distal end of posterior basal body (PB), with split (arrowhead) in right root between 2-membered (RR2) and 6-membered (RR6) subparts, and associated electron-dense rod (EDR). (h) Three-dimensional reconstruction of anterior (proximal) flagellar apparatus, with basal bodies represented as large cylinders (arrows begin at transition zone and indicate direction of flagellum) and individual microtubules as small cylinders. AB anterior basal body; AF anterior flagellum; AR anterior root; FV food vacuoles; G Golgi apparatus; LR left posterior root; M mitochondrion; MF bundles of microfibrils; N nucleus; No nucleolus; PB posterior basal body; PF posterior flagellum; Ps pseudopodium; Rb right band of microtubules; RR right posterior root; RR2 2-membered part of right root; RR6 6-membered part of right root; SR singlet root; T ‘tusk’. Scale bars in (a)&(b) = 500 nm; scale bars in (c–g) = 200 nm (All micrographs and reconstruction reproduced from originals used for Heiss at al. (2013b)) lines of refractile granules running in parallel to the posterior flagellum, a reduced proboscis sleeve, and more-prominent pseudopodia (Fig. 2c). Multimonas occasion- ally forms syncytia. Multimonas, Thecamonas, and Chelonemonas will often have a prominent trailing pseudopodium that may be up to twice the length of the cell body 12 A.A. Heiss et al.

(Fig. 2b). “Thecamonas” oxoniensis is somewhat leaf-shaped, though the cell body can “fold” longitudinally and has tiny refractile granules and a contractile vacuole (consistent with its being a freshwater organism). Manchomonas has neither acronemes nor conspicuous pseudopodia and a more leftward than anteriorly ori- ented proboscis (Cavalier-Smith and Chao 2010; Heiss et al. 2015; Molina and Nerad 1991). Amastigomonas itself has a contractile vacuole and supposedly lacks a visible posterior flagellum (Cavalier-Smith and Chao 2010). Apusomonas differs in having a relatively inflexible main cell body, from which emerges an anterior extension called the mastigophore. The mastigophore forms the proximal part of the proboscis (Figs. 1a and 2d). It contains the basal bodies of both flagella as well as the proximal portions of the posterior flagellum and of the flagellar roots (see below). It is therefore significantly thicker than the distal flagellum-and- sleeve portion of the proboscis. The proboscis of Apusomonas thus has a tripartite appearance (from proximal to distal): mastigophore, anterior flagellum and sleeve, and acroneme (Karpov 2007; Karpov and Myľnikov 1989; Karpov and Zhukov 1984, 1986).

Ultrastructure

Apusomonads have characteristic folds that emerge from the lateral edges of the cell body and extend ventrally, forming a “skirt” about the cell body that is most easily resolved using scanning or transmission electron microscopy (SEM; TEM). The “skirt” is continuous with the sleeve that extends around the anterior flagellum to form the proboscis (Figs. 1 and 2; Heiss et al. 2013b; Vickerman et al. 1974). The posterior flagellum runs between the left lip of the skirt and the main cell body for at least half of the cell length (Figs. 1, 2, and 3a, b). The dorsal cell membrane is underlain by a thin pellicle (sometimes called a “theca,” a term that usually denotes an extracellular covering) that extends into and supports the skirt. As a consequence, the dorsal cell membrane is smoother than the unsupported ventral cell membrane when viewed by SEM or TEM (Figs. 2e–i and 3a, b). The pellicle has a polygonal substructure in Chelonemonas that is visible on the dorsal surface of the cell in SEM preparations (Heiss et al. 2015; Fig. 2g). Scanning electron microscopy images of the dorsal surface of Multimonas strains have shown small knobs and elongate strands that may represent undischarged and discharged extrusomes, respectively (Heiss et al. 2015; Fig. 2h). Some apusomonads have an anterior projection of the cell body, the “tusk,” which is rigid, and contains complex supporting material (Figs. 1b, 2a, f, and 3f; Heiss et al. 2013b). It is known to be present in at least some strains of Thecamonas, Chelonemonas, and Podomonas and to be absent in Manchomonas and Apusomonas. The tusk is under 1 μm long in Thecamonas and is just barely visible using light microscopy under optimal conditions (Fig. 2a; Heiss et al. 2013b, 2015). Internally, apusomonads have a dorsally positioned nucleus with a distinct central nucleolus. The nucleus is usually but not always circular; in Apusomonas,itis strongly reniform (Figs. 1a and 2d). The single Golgi body is found near the anterior Apusomonadida 13 of the cell. The multiple mitochondria have tubular cristae (Fig. 2c). At least some taxa have conspicuous, densely-staining microbodies; in Thecamonas trahens, there is usually one per cell (Heiss et al. 2013b). Food vacuoles are most often found in the ventral half to two-thirds of the cell (Fig. 3a, b; Cavalier-Smith and Chao 2010; Heiss et al. 2013b; Karpov 2007). The flagellar apparatus has been reconstructed in detail for Apusomonas (Karpov 2007) and Thecamonas trahens (Fig. 3h; Heiss et al. 2013b). The flagellar apparatus comprises the two basal bodies, which are joined by at least two (probably three) fibrous connectives, plus three posterior microtubular roots, one anterior microtu- bular root, a “ribbon” of microtubules associated with the anterior basal body, and a number of nonmicrotubular accessory structures (Fig. 3b, f–h). Apusomonads appear to show the typical eukaryotic pattern of flagellar transformation during the cell cycle, with the anterior basal body younger and the posterior elder (Cavalier- Smith and Chao 2010). The posterior right root (“RR”; equivalent to R2 in the universal terminology of Moestrup 2000) comprises several microtubules (eight to sixteen have been reported: Heiss et al. 2013b; Karpov 2007; Karpov and Myľnikov 1989; Molina and Nerad 1991); the leftmost two of which split off from the remainder (Fig. 3g, h). The posterior left root (“LR,” equivalent to R1) generally contains two microtubules, and a singlet root arises between the other posterior roots. The anterior microtubular root (“AR,” equivalent to R3) is a doublet that runs across the dorsal cell surface posteriorly and to the left (Fig. 3f, h). The “ribbon” has sometimes been identified as a flagellar microtubular root (Cavalier-Smith and Chao 2010; Karpov 2007; Molina and Nerad 1991) but may instead be homologous to the systems of secondary peripheral microutubules in other eukaryotes (Heiss et al. 2013b). It originates alongside the anterior basal body in association with a non-microtubular sheet. In the posterior half of T. trahens at least, the posterior roots reorganize into two structures, both on the left side of the cell; (i) a dorsally displaced root comprising most of the right root microtubules (“RR7”) and (ii) a ventral “left band” made of the left root, the singlet root, and one microtubule from the right root (Fig. 3b, h; Heiss et al. 2013b). The left band extends to the posterior end of the cell, likely into the base of the trailing pseudopodium. The right root runs opposite the left lip of the skirt, and in a similar fashion, the ribbon runs opposite the right lip of the skirt, likely reinforcing the cell outline.

Life Cycle

Only a single cell type has been observed in the majority of apusomonads, that of gliding flagellates. Cells divide by mitotic binary fission; sex has not been observed (Karpov and Myľnikov 1989). Cysts are known for “Thecamonas” oxoniensis (Cavalier-Smith and Chao 2010). Apusomonas can recover following seasonal desiccation, but a true cyst form appears to be absent (Cavalier-Smith and Chao 2010; Karpov and Myľnikov 1989). Vickerman et al. (1974) reported a wall-less cryptobiotic stage in Apusomonas but also indicated that this form does not survive total desiccation. 14 A.A. Heiss et al.

Systematics

The formal taxon for apusomonads is the family Apusomonadidae Karpov and Myľnikov (1989), the sole member of order Apusomonadida Karpov and Myľnikov (1989). The clade comprising Apusomonas and Manchomonas has been recognized as the subfamily Apusomonadinae Karpov and Myľnikov (1989) (sensu Cavalier- Smith and Chao 2010) and that comprising Chelonemonas and marine Thecamonas as Thecamonadinae Larsen and Patterson (1990) (sensu Heiss et al. 2015).

Maintenance and Cultivation

Members of five apusomonad lineages have been maintained in monoprotistan but not axenic laboratory culture (see Table 1). Such cultures were generally established through serial dilution (e.g., Cavalier-Smith and Chao 2010), although cell migration hasalsobeenused(Heissetal.2015; Molina and Nerad 1991). They have been grown in standard laboratory media, generally water of appropriate salinity (distilled water, sterilized bottled mineral water, or artificial or natural seawater, often diluted) with a plant-based carbon source (either a sterile cereal grain or an infusion of such material, e.g., Cerophyl). Live prey bacteria (e.g., Pseudomonas) can be added instead of the carbon source. When grown in standard culture tubes or tissue culture flasks, cultures of most strains can last for >2 months but are always sparse; cultures generally last longer and grow to higher density in tissue culture flasks than in tubes (AAH, pers. obs.). When grown in Petri plates, the same strains can form visible plaques of very high density within days of inoculation, but die within ~2 weeks (AAH, pers. obs.). Cultures can be maintained at 14 C (e.g., Cavalier-Smith and Chao 2010) but often are more robust at 16–21 C or room temperature (AAH, pers. obs.).

Evolutionary History

Internal Relationships

As discussed above, five clades of apusomonads with cultured representatives have been delimited using phylogenies of SSU rRNA genes (Cavalier-Smith and Chao 2010; Heiss et al. 2015). Two of these are Apusomonadinae (comprising the genera Apusomonas and Manchomonas) and Thecamonadinae (comprising the genus Chelonemonas and the marine members of the genus Thecamonas). Another two lineages are represented by individual genera (Podomonas and Multimonas). The final lineage comprises the single freshwater species “Thecamonas” oxoniensis, a species with no specific relationship to the marine members of the genus Thecamonas (Cavalier- Smith et al. 2014; Heiss et al. 2015). No stable relationships between the five lineages have been established to date (Cavalier-Smith and Chao 2010; Heiss et al. 2015). At least four additional lineages are known from environmental sequences only; nothing is known about the biology of the organisms corresponding to those sequences. Apusomonadida 15

Overall Phylogenetic Position

The first molecular phylogenetic study to include an apusomonad identified the group as a possible relative of opisthokonts (Cavalier-Smith and Chao 1995). Some of the first multigene phylogenies also weakly supported this relationship (Kim et al. 2006). Recent multigene analyses (Katz et al. 2011; Paps et al. 2013) and phylogenomic studies (Brown et al. 2013; Burki et al. 2016; Cavalier-Smith et al. 2014; Derelle and Lang 2012; Torruella et al. 2012, 2015; Zhao et al. 2013) have lent increasing support to the placement of apusomonads as a sister group to opisthokonts. Some studies have also demonstrated that another enigmatic group of organisms, the breviates, (see Coda and Fig. 4a) is most closely related to apusomonads and opisthokonts, and this relationship has been formalized by the erection of the taxon , which encompasses all three groups (Brown et al. 2013). However, it is not clearly resolved whether apusomonads alone represent the closest relatives to opisthokonts or whether it is an apusomonad-breviate clade that represents the sister group to opisthokonts. The most detailed phylogenomic analysis to date, with relatively limited taxon sampling, found that the preferred phylogeny depended on the evolutionary model used for phylogenetic inference (Brown et al. 2013): more complex evolutionary models incorporating among-site model heterogeneity, such as CAT-GTR (Le et al. 2008), favor an apusomonad- clade, to which breviates are the sister group (Brown et al. 2013). Recent phylogenomic studies with expanded taxon sampling have not clearly resolved between these hypotheses (Cavalier-Smith et al. 2014); it is hoped that additional taxon and gene sampling data will more precisely resolve the position of the apusomonads in the near future. Meanwhile, Cavalier-Smith (2002) proposed that the pellicle of apusomonads was homologus to that of another “non-supergroup” lineage, the ancyromonads (see Coda and Fig. 4b), and suggested a common evolutionary history for the two groups. Interestingly, some early SSU rRNA phylogenies including ancyromonads suggested a close relationship with opisthokonts (Atkins et al. 2000b; Cavalier- Smith and Chao 2003), similarly to the early SSU rRNA phylogenies of apusomonads (see above). This arrangement has been loosely supported by recent multigene phylogenetic and phlyogenomic analyses, although it is unclear at present whether ancyromonads fall within Obazoa like apusomonads, or are a sister to Obazoa, or are more distantly related (Cavalier-Smith et al. 2014; Katz et al. 2011; Paps et al. 2013).

Implications for Eukaryote Evolution

Apusomonads are important for our understanding of eukaryote evolution for at least two reasons. One of these concerns the evolution of the flagellar apparatus cytoskel- eton at the “supergroup” level. Apusomonads possess a complex flagellar apparatus with multiple posterior microtubular roots, including an R2 root that splits into two parts and a “supernumerary” singlet microtubular root, as well as a posteriorly 16 A.A. Heiss et al. directed array of secondary microtubules (the ribbon). These structures are also found together in acyromonads and breviates (Heiss et al. 2011, 2013a), in “typical excavates” (Simpson 2003), and in other taxa such as some stramenopiles (Moestrup and Thomsen 1976; Yubuki et al. 2010), suggesting that these specific features may have been ancestral to the majority of major eukaryote lineages (Cavalier-Smith 2013; Heiss et al. 2013b; Leander and Yubuki 2013). Since apusomonads are most closely related to opisthokonts and (less so) to amoebozoans, this suggests that the simple flagellar apparatus cytoskeletons seen in opisthokonts and many flagellated amoebozoans (e.g., pelobionts) are not primitive ancestral systems but in fact could represent independent secondary simplifications from a complex ancestral form. The other area of importance concerns the evolution of multicellularity. The supergroup Opisthokonta includes two substantial lineages that have evolved multicellularity independently of one another: animals and fungi. Each lineage has established key systems associated with multicellularity, including cell-cell commu- nication and adhesion (Grosberg and Strathmann 2007). Interestingly, elements of some of these systems are encoded in the genome of Thecamonas trahens, indicating that such pathways were present in the common ancestor of apusomonads and opisthokonts, and substantially predate the evolution of multicellularity in both animals and fungi. For example, the Thecamonas genome encodes most components of the integrin system (Sebé-Pedrós et al. 2010). In animals, integrins span the cell membrane, connecting to the actin cytoskeleton on the cytoplasmic side (via a series of associated proteins, mostly present in Thecamonas) and binding to the laminin and collagen of the extracellular matrix. Sodium-channel (Cai 2012) and calcium- signalling (Cai and Clapham 2012) genes involved in cell communication in ani- mals, and absent from fungi, are also present in the Thecamonas genome. The cyclin dependent kinase 4/6 and cyclin D subfamilies are also thought to play a part in the development of -type multicellularity and are also found in Thecamonas,as well as in amoebozoans (Cao et al. 2014).

Coda: Breviates and Ancyromonads

Apusomonads are not the only organisms to have been suggested to have a close relationship to opisthokonts. Molecular phylogenetic evidence indicates that several more obscure lineages of small protozoa, mostly heterotrophic flagellates, may also be closely related to opisthokonts and/or apusomonads. The best known of these understudied groups are breviates and ancyromonads. Breviates (Cavalier-Smith et al. 2004) are a group of anaerobic or microaerophilic amoeboid flagellates with an apical anterior flagellum and either a posterior flagel- lum or a nonflagellated posterior basal body (Figs. 4a and 5). There are four described genera, each with a single species: the freshwater Breviata anathema and the marine Subulatomonas tetraspora, Pygsuia biforma and Lenisia limosa. They are surface-associated gliding organisms, although a distinct swimming stage is also known in Pygsuia (Figs. 5a, c; Brown et al. 2013). Breviates produce fine pseudopodia that typically form at near-regular intervals from a point at the anterior Apusomonadida 17

Fig. 4 Appearance by light a b microscopy of living breviates and ancyromonads. (a) Breviata anathema;(b) acroneme Ancyromonas sigmoides. of anterior Nuclei are light grey; flagellum mitochondria (Ancyromonas) or mitochondrion-related rostrum organelles (Breviata) are dark grey. Scale bars = 2 μm for extrusomes each drawing

anterior flagellum

cell body

posterior pseudopodia flagellum

end of the cell, thus forming a series down the cell as it glides forward (Figs. 4a and 5b). The cells engulf bacteria with these pseudopodia (Heiss et al. 2013a). The cells have a moderately complex cytoskeleton including several flagellar microtubular roots, and some unusual non-microtubular elements (Figs. 5g-k; see Heiss et al. 2013a for details). At least some breviates are reported to form cysts, though this is not well documented (Katz et al. 2011; Walker et al. 2006). No sexual stages have been observed. Cultures and/or SSU rRNA sequences have been obtained from environmental samples taken from Europe, North America, and Japan (Katz et al. 2011; Brown et al. 2013). All investigated breviates have a large mitochondrion-related organelle (MRO; Fig. 5e), which in Breviata has occasionally been found to contain a few tubular cristae (Fig. 5f; Heiss et al. 2013a). However, all cultured breviates are maintained exclusively under anaerobic or suboxic conditions (Brown et al. 2013; Heiss et al. 2013a; Katz et al. 2011; Walker et al. 2006). The biochemical capacity of these MROs has been inferred primarily from transcriptome data from Pygsuia (Stairs et al. 2014), with some additional information from Breviata (Minge et al. 2009). The MRO does not produce energy using classical oxidative 18 A.A. Heiss et al.

Fig. 5 Images of breviates. Panels (a and b) are differential interference contrast images of live cells: (a) Pygsuia biforma, showing both flagella; (b) Breviata anathema, showing its single flagellum. Panel (c) is a scanning electron micrograph of Pygsuia biforma. Panels (d–j) are transmission electron micrographs of Breviata anathema:(d) longitudinal section of whole cell showing general cell features; (e) longitudinal section of whole cell showing size of mitochondrion- related organelle (M) and its proximity to anterior basal body (AB); (f) mitochondrion-related organelle with tubular cristae; (g) longitudinal section through flagellar apparatus showing rela- tionship between flagellated anterior (AB) and nonflagellated posterior (PB) basal bodies, as well as longitudinal section through “semicone” structure (SC); (h) section through posterior basal body Apusomonadida 19 phosphorylation (electron transport chain complexes I, III–V are absent) but instead acts as a hydrogenosome that generates ATP anaerobically via substrate-level phos- phorylation (Stairs et al. 2014). The Pygsuia MRO has several highly unusual features, the most notable being an archaeal-related “SUF” system for Fe-S cluster assembly, which appears to have replaced the “ISC” system that is found in virtually all other mitochondria and MROs across eukaryotes (Stairs et al. 2014). The first known breviate (Breviata) was originally identified as a member of the pelobiont genus Mastigamoeba (see chapter “▶ ”), a situation that led to Mastigamoeba appearing to be polyphyletic when molecular phylogenies included multiple organisms attributed to it (Edgcomb et al. 2002; Stiller and Hall 1999). Resolution of this misidentification led to the recognition of a new lineage of eukaryotes (Cavalier-Smith et al. 2004) and the new genus Breviata for this single species (Walker et al. 2006). The first phylogenomic analysis including (relatively sparse) data from this strain suggested that breviates were basal to or branched within (Minge et al. 2009). However, several environmental SSU rRNA sequences have been identified as belonging to breviates (summarized in Katz et al. 2011), and strains from additional lineages have now been cultivated in the laboratory, including the recently described Subulatomonas tetraspora (Katz et al. 2011), Pygsuia biforma (Brown et al. 2013) and Lenisia limosa (Hamann et al. 2016). Recent SSU rRNA gene trees, multigene phylogenies, and phylogenomic analyses that include more breviate species show that breviates are actually most closely related to apusomonads and/or opisthokonts (Brown et al. 2013; Burki et al. 2016; Cavalier-Smith et al. 2014; Katz et al. 2011; see above). Interestingly, as with the apusomonad Thecamonas trahens, a large com- plement of genes encoding integrin complex proteins is present in the breviate Pygsuia (Brown et al. 2013). Ancyromonads (Atkins et al. 2000b; Cavalier-Smith 1997; also called planomonads: see Cavalier-Smith et al. 2008; Heiss et al. 2010) are a molecularly diverse but morphologically conservative group of small bacterivorous flagellates

(Figs. 4b and 6). There are about 15 nominal species in four genera. Ancyromonads ä

Fig. 5 (continued) (PB) showing roots near point of origin; (i) longitudinal section through anterior end of cell showing paths of posterior roots, as well as cross-section of “semicone” (SC); (j) section through anterior basal body (AB) showing short anterior root (AR) and dorsal fan. (k) Three- dimensional reconstruction of flagellar apparatus, with basal bodies represented by large cylinders (arrow begins at transition zone and points in direction of flagellum) and individual microtubules by small cylinders. AB anterior basal body, AF anterior flagellum, AR anterior root, DS “double sandwich” structure between basal bodies, F food (bacteria), fan dorsal microtubular fan, G Golgi apparatus, LR left posterior root, M mitochondrion-related organelle, MR middle posterior root, N nucleus, PF posterior flagellum, Ps pseudopodium, RR right posterior root, RRa left part of right root, RRb right part of right root, SC “semicone” structure. Scale bar in (b) = 10 μm for (a and b); scale bar in (c) = 2 μm; scale bars in (d and e) = 1 μm; scale bars in (f, g, i) = 500 nm; scale bars in (h, j) = 200 nm (Micrograph in (a) by MWB; micrograph in (b) by AAH; scanning electron micrograph in (c) reproduced from original used for Brown et al. (2013); transmission electron micrographs and reconstruction in (d–k) reproduced from originals used for Heiss et al. (2013a)) 20 A.A. Heiss et al. have a rounded cell body, typically 4–6 μm long, which is dorsoventrally com- pressed and essentially inflexible (Figs. 4b and 6a–c). The anterior-left portion of the cell forms a laterally/posteriorly directed rostrum that contains extrusomes (Figs. 4b and 6e, f). The anterior flagellum is generally short and often either terminates at the cell outline or is almost entirely an acroneme (Figs. 4b and 6c, d) and thus difficult to detect by light microscopy. The posterior flagellum is about two to three times the length of the cell (4B, 6A, 6C). The cell adheres to the substrate using the distal portion of the posterior flagellum and either glides or tethers to one location. The cell body nods rapidly due to flexure of the proximal part of the flagellum (Glücksman et al. 2013; Heiss et al. 2010). Like apusomonads, ancyromonads have a pellicle (Fig. 6h), in this case underlying almost all of the cell surface. Unlike apusomonads, ancyromonads have flat mitochondrial cristae (Fig. 6d, j). The microtubular cyto- skeleton is complex, with five distinct flagellar microtubular roots (Figs. 6i-k; see Heiss et al. 2011 for details). Neither sexual stages nor cysts are known. Ancyromonads are as widely distributed as apusomonads, though generally more locally abundant (Atkins et al. 2000a; Chen et al. 2008; Ekelund and Patterson 1997; Hänel 1979; Larsen and Patterson 1990; Lee 2002; Lee and Patterson 2000; Lee et al. 2005; Patterson and Simpson 1996; Patterson and Zölffel 1991; al-Qassab et al. 2002; Scheckenbach et al. 2005; 2006; Stock et al. 2009; Tikhonenkov et al. 2006; Tong 1997; Tong et al. 1997, 1998;Vørs1993), and have been cultured under the same conditions (Cavalier-Smith et al. 2008; Glücksman et al. 2013; Heiss et al. 2010;Myľnikov 1990). Ancyromonads were first identified over 130 years ago (Saville Kent 1882) but received little mention until phylogenetic analysis identified them as an independent lineage with opisthokont affinities (Atkins et al. 2000b; Cavalier-Smith 1997). The most recent taxonomic scheme for ancyromonads (Glücksman et al. 2013) is based on SSU rRNA gene phylogenies, which distinguish five clades. Three of these are marine and correspond to the genera Ancyromonas, Planomonas, and Fabomonas. The other two clades are known from fresh water and have both been placed in the genus Nutomonas, as they are sister taxa; they have been separated into the subgenera Striomonas (containing N. longa) and Nutomonas (containing all remaining species in the genus). Ancyromonas and Nutomonas are sisters in published phylogenies (Cavalier-Smith et al. 2014; Glücksman et al. 2013), com- prising a clade that has been given the name Ancyromonadidae (Glücksman et al. 2013). The other genera (Planomonas and Fabomonas) may or may not be a clade; the name Planomonadidae has been proposed for such a grouping. Another fresh water genus, Phyllomonas (Klebs 1893), has been regarded as an ancyromonad by some researchers (Lemmermann 1914; Patterson and Simpson 1996; Tong et al. 1998) but not by others (Cavalier-Smith et al. 2008; Hänel 1979; Patterson and Zölffel 1991; Patterson et al. 2000); its actual status awaits a modern study (Cavalier-Smith et al. 2008; Heiss et al. 2010). As stated above, the phylogenetic positions of breviates and ancyromonads are not fully resolved (the latter especially). However, both groups have complex flagellar apparatus cytoskeletons with most of the potentially ancestral features for eukaryotes discussed above for apusomonads (Figs. 3h, 5k, and 6k; Heiss et al. 2011, Apusomonadida 21

Fig. 6 Images of Ancyromonas sigmoides. Panels (a and b) are phase-contrast micrographs of living cells, viewed from dorsal (a) and lateral (b) aspects. Panel (c) is a scanning electron micrograph of a fixed cell. Panels (d–j) are transmission electron micrographs: (d) section through both basal bodies, showing full extent of anterior flagellum (AF); (e) longitudinal section through cell, with cross-section of extrusomes (Ex); (f) longitudinal section through both Golgi apparatus (G) and extrusomes (Ex), with immature extrusome material (IEx) in transition from Golgi apparatus to “firing” position; (g) cross section through Golgi apparatus (G) and section through stacked membrane structures (SM); (h) closeup of pellicle; (i) anterior root (AR) and anterior singlet (AS) on either side of anterior flagellum (AF)inflagellar pocket; (j) arrangement of roots around posterior flagellum (PF)inflagellar pocket, as well as mitochondrion (M) with flat cristae. (k) Three-dimensional reconstruction of flagellar apparatus, including full extent of anterior basal body (AB) and flagellum, as well as various peripheral microtubular structures (X, Y, Z; the latter two are possible homologues to the dorsal fan of breviates and the right band of apusomonads). AB anterior basal body, AF anterior flagellum, AR anterior root, AS anterior singlet, CMT crescent microtubules (part of posterior left root), Ex extrusome, G Golgi apparatus, I electron-lucent inclusion, IEx immature extrusome material, L1 posterior left root, L2, L3 parts of posterior 22 A.A. Heiss et al.

2013a, b). Irrespective of the precise phylogenetic positions of ancyromonads and breviates, this reinforces the notion that the ancestors of the opisthokonts and Amoebozoa each had complex cytoskeletons.

Acknowledgments Thanks are due to WonJe Lee (Kyungnam University, South Korea) for discussion and scanning electron micrographs of Ancyromonas sigmoides, to Yana Eglit (Dalhousie University) for translations of the Russian literature and for providing light micrographs of Apusomonas proboscidea, to Courtney Stairs (Dalhousie University) and Giselle Walker (Charles University in Prague) for comments, and to Ping Li and Patricia Scallion (Dalhousie University) for assistance with electron microscopy.

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Apusomonadida

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Fig. 6 (continued) right root, M mitochondrion, N nucleus, PB posterior basal body, PF posterior flagellum, PS posterior singlet root, SM stacked membrane structure, X, Y, Z peripheral microtubular systems “X,”“Y, ”“Z.” Scale bars in (b) = 5 μm for (a and b); scale bar in (c) = 2 μm; scale bars in (d and e) = 500 nm; scale bars in (f, g, i, j) = 200 nm; scale bar in (h) = 50 nm (Micrographs in a and b reproduced from originals used for Heiss et al. (2010). Micrograph in c courtesy of Won Je Lee (Kyungnam University, South Korea). All transmission electron micrographs and reconstruc- tion reproduced from originals used for Heiss et al. (2011)) Apusomonadida 23

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