Contributed papers to the spring 2002 BSSP meeting held at Bristol University

Fusions of vegetative cells in naked amoebae: a step forward, or a side branch of evolution?
Alexey V. Smirnov

Dept. of Invertebrate Zoology, Fac. of Biology & Soil Sci., St.Petersburg State University, Universitetskaja nab. 7/9, 199034 St.Petersburg, Russia.

Fusions of vegetative cells are well known in many groups of amoeboid potists and may be, or may be not related with the sexual process. In the latter case nuclei of all participating cells remain intact (agamous fusions). Literary data indicate that agamous fusions are widely distributed among naked amoebae, but remains fragmentary and poorly documented. Many interesting records existing in older literature still are neither confirmed nor denied. Few well-studied cases of agamous fusions - in remarkable heterolobosean Euhyperamoeba fallax and in several species of leptomyxids demonstrate especially interesting and diverse types of fusions, in some cases related with the complex feeding behaviour. However, summarising, we have to recognise that fusions of vegetative cells in each particular case have more or less evident adaptive significance, and among naked amoebae there are no direct indications of the subsequent evolution from agamous fusions to sexuality. Perhaps, fusions of vegetative cells is a side phenomena that independently appeared in various phylogenetic branches of protists and is related more with the interesting peculiarities of the cell biology rather than represent a step toward the sexuality or to the alternative ways of genetic exchange in agamous organisms.

Size matters....Problems for the Cell Biology of Gregarine Protozoans
Conrad King and Susan Le,

Biology Dept, University College London, Gower Street, London, WC1E 6BT

The genus Gregarina is found in the gut of many invertebrates where it can reach a length of up to 300 mm. These large uninucleate cells can be described as trophozoites or gamonts. Each trophozoite originates from a single sporozoite which enters an intestinal epithelial cell. This brief intracellular phase is followed by the egress of the apicomplexan cell into the gut lumen. There follows a spectacular increase in cell size from sporozoite to trophozoite (in the region of 4 orders of magnitude). It might be expected that a well developed intracellular transport system would occur. No evidence for cytoplasmic streaming at the microscope level was found. Following syzygy each gamont undergoes nuclear division to produce several thousand gametes.

The genus Porospora can be found in the gut of the lobster Homarus us where trophozoites having a length of 15000mm have been recorded (in our experience lengths of 1000- 3000 mm are more common). Again no cytoplasmic streaming was detected.

One possibility is that the biological motors powering gliding motility over a substratum could be used for intracellular transport within these large masses of cytoplasm.

Abundance of Naked Amoebae in Sandy Sediments of Kames Bay, Isle of Cumbrae, Scotland.
Phillip Cowie & Fiona Hannah

University Marine Biological Station, Millport, Isle of Cumbrae, KA28 OEG.

Core samples were obtained from aerobic sand in the upper intertidal (2.4 m above chart datum), lower intertidal (1.4 m above chart datum) and subtidal (6 m depth) zones of Kames Bay, Isle of Cumbrae, Scotland. Sampling was carried out from May 2000 until July 2001 and included 10 time points. Amoebae were categorized using the 4 morphotype scheme of Anderson & Rogerson (1995) and 19 morphotype scheme of Smirnov & Goodkov (1999). Analysis of the 4-morphotype scheme data indicated that the abundance of total naked amoebae was low, and ranged from 3 to 40 cells/g dry sediment. Amoebae morphotypes 1 and 4 dominated throughout the year. Temporal and spatial variations in the amoebae communities were complex and will be discussed in relation to variability in other food-web components e.g. meiofauna, bacteria, heterotrophic protozoa and abiotic factors (temperature, pH, salinity, grain size, particulate organic carbon). The higher resolution data obtained from use of the 19 morphotype scheme, was analyzed using PRIMER in order to obtain a better understanding of the changes occurring in the amoebae communities throughout the year. Results from these analyses are also presented.

Amoeboid locomotion at the water:air interface
Terry M. Preston & Conrad A. King

Biology Dept., University College London, London WC 1E 6BT

In order to move progressively amoebae need to exert traction on a suitable substratum 1. In nature this is usually provided by a solid surface, though in the laboratory a water: fluorocarbon interface will suffice 2. Examination of environ-mental isolates from the surface microlayers of freshwater bodies shows that several small amoebae, including Acanthamoeba, Naegleria & Vannella, whose motile phenotypes differ, occupy this ephemeral niche 3 exploiting the microbial food source there. The motile behaviour exhibited by these amoebae at the water:air interface is identical to that expressed on a water:plastic or water:glass interface. Furthermore the speed of amoeboid movement of Naegleria at the water:air interface is modulated by the ionic strength of the experimental medium, which accords with our results from previous experiments using planar glass, plastic & agar surfaces.

To test the hypothesis that surface tension alone can account for the ability of the water:air interface to resist the tractional forces exerted during amoeboid locomotion we present an analysis of the physical forces involved here.

  1. Preston,T.M. & King,C.A. (1978) Cell-substrate associations during the amoeboid locomotion of Naegleria. Journal of General Microbiology 104, 347-351
  2. King,C.A., Davies,A.H. & Preston,T.M. (1981) Lack of substrate-specificity on the speed of amoeboid locomotion in Naegleria gruberi. Experientia 37, 709-710.
  3. Preston,T.M., Richards,H. & Wotton,R.S. (2001) Locomotion and feeding of Acanthamoeba at the water-air interface of ponds. FEMS Microbiology Letters 194, 143-147.
The potential for interactions between protozoa and coliform bacteria in biofilms
Joanna English2, Jackie Parry1 and Roger Pickup1.
  1. Dept Biological Sciences, Lancaster University, Lancaster, LA1 4YQ, UK.
  2. CEH Windermere, The Ferry House, Far Sawrey, Ambleside, Cumbria, LA22 OLP, UK.

Biofilms in aquatic environments have been shown to comprise of all the major groups of microorganisms. Certain protozoa can utilise the bacteria associated with surfaces as a prey source. Other studies have shown that protozoa, especially amoebae, will ingest some bacteria, but once inside the cell they can then replicate and exchange genetic material. Examples of bacterial species that can do this are Legionella pneumophila and Vibrio cholera. This has given rise to the nickname "Trojan Horses of the Microbial World". Coliform organisms have also been shown to survive within protozoan hosts, but not replicate.

An experiment was carried out to ascertain whether coliform organisms, including E. coli, are available in natural biofilms to protozoan predators. A three week study was undertaken growing biofilms from a virgin substratum in the River Conder, Lancashire, UK. Direct counts and enrichment experiments were carried out to enumerate and identify the protozoa present in the system. The bacterial composition of the biofilm was also studied.

Protozoan diversity, temperature relations and Eukaryotic stimulation of oil biodegradation in permeable pavement structures.
Stephen J Coupe and Humphrey G Smith

School of Science and the Environment, Coventry University, Coventry, CV1 5FB, UK.

Permeable Pavement Structures (PPS) consist of concrete, gravel and polypropylene geotextile over a granite base. They have been demonstrated to function as effective in-situ bioreactors to degrade oil. Investigation of initial rig substrates showed the presence of bacterial, protozoan, fungal and metazoan taxa. These indigenous organisms degraded oil at as fast a rate as those from a pre-adapted oil degrading inoculum. To assess the contribution of protozoa to oil degradation, PPS organisms were cultured in oil, slow release fertiliser and distilled water.The eukaryotes were filtered and diluted out to leave a bacterial only culture.

Fungi from the full assemblage isolated onto oil agar were added to bacterial cultures to study the effects of protozoa and metazoa in isolation on oil degradation. The effectiveness of the full assemblage against the bacterial and fungal mixture showed that the complete PPS community had a considerably larger degradation capacity than the bacteria and fungi alone.

Because filtering and diluting the original culture might have removed important bacterial oil degraders, it was decided to chemically remove bacteria with tetracyclin and eukaryotes with cycloheximide; once again the full community degraded the most oil. Oil degradation within cultures was found to be strongly temperature dependent with a optimum of 22-25°C.

The granite base was shown to have the highest protozoan diversity of air dried PPS materials but the geotextile, exposed to deposition from the air only, produced the highest cell count; whilst the concrete and gravel produced relatively low numbers and diversity. In order to determine if enhanced oil degradation due to the presence of protozoa operates in vivo as well as in vitro, experiments are underway, using full sized PPS laboratory models drip fed with antibiotics or cycloheximide to remove prokaryotes or eukaryotes.

Processing of food vacuoles in protozoa
Parry, JD, Thurman, J, Gedney, S, Phillips, S and J Drinkall

Dept Biological Sciences, Lancaster University, Lancaster LA1 4YQ

Current knowledge on protozoan food vacuole dynamics is limited, as most work has centred on the ingestion of prey rather than on the fate of that prey after ingestion. This study examined the maximum time a protozoan cell might have, to digest any prey trapped inside its food vacuoles i.e. the length of time a vacuole exists within a cell before its contents are defecated. The Vacuole Passage Times (VPTs) of one flagellate Paraphysomonas imperforata and two ciliates, Tetrahymena pyriformis and Cyclidium glaucoma, were determined using single- and multiple-pulse-chase experiments. The results showed that (i) food vacuole processing occurs in an orderly fashion i.e. the first vacuole formed is the first vacuole defecated, and that (ii) the rate of defecation is slower than the rate of ingestion i.e. a specific period of time is required for the defecation of a single food vacuole. This would mean that some food vacuoles might have to queue at the cytoproct of ciliates (and at a currently unidentified cytoplasmic area of Paraphysomonas) before they can be defecated and would go some way in explaining why estimates of VPT for a single protozoan species are often highly variable.

An investigation of predator-induced defense responses in ciliated protozoa
Janusz Fyda2, Justyna Wolinska2 and Alan Warren1

  1. Jagiellonian University, Krakow, Poland.
  2. Dept of Zoology, Natural History Museum, Cromwell Road, London, SW7 5BD, UK.

The phenomenon of predator-induced defense is well known in a wide range of organisms including cyanobacteria, protists, corals, rotifers, cladocerans, bryozoans and even fish. The defense response can be morphological, behavioural, chemical or be related to the life cycle. Among ciliates, such responses have been found in 15 species representing three groups: hypotrichs, stichotrichs and hymenostomesStenostomum sphagnoretum) and an oligochaete worm (Chaetogaster sp.). In each experiment, one potential predator species was incubated for 24 hours with one potential prey species. One new example of predator-induced morphological change was recorded (Euplotes viridis) and detailed observations were made for one poorly-known example (E. eurystomus). Both species significantly increased their width (by about 35% and 23% respectively) in the presence of the turbellarian S. sphagnoretum. An induced life cycle change was recorded for the first time among hypotrichs, with E. muscorum exhibiting significantly increased rates of encystation when in the presence of Dileptus anser or Spathidium sp. Finally, Euplotes patella, Euplotes sp. and Stylonychia pustulata, which are usually regarded as omnivorous rather than predatory ciliates, all induced morphological change in Colpidium kleini, the C. kleini cells becoming significantly shorter and wider. No examples of induced defense response were found among groups other than hypotrichs and hymenostomes.

Identification of two Toxoplasma gondii proteins that share domains of the dense granule protein, GRA3
Henriquez, F.L., Lyons, R.E., Lyons, K. and Roberts, C.W.

Department of Immunology, Strathclyde Institute of Biomedical Sciences, University of Strathclyde, Glasgow, G4 0NR.

We have identified a T. gondii EST with identity to GRA3 at the c-terminal portion and a unique n-terminal region (GRA3b). The consensus of toxoplasma quality sequence database was searched for further possible polymorphic forms of GRA3. A further protein that shares the n-terminal region of GRA3, but has a unique c-terminal region was identified (GRA3c). The common n-terminal region of GRA3/GRA3c and the unique n-terminal domain of GRA3b were expressed in E. coli and Abs raised in mice. Antibodies raised to the n-terminal region of GRA3b recognised a protein in T. gondii lysate of 70kDa, whereas antibodies raised to the n-terminal domain of GRA3c recognised a protein of 28kDa. The entire GRA3c was amplified by PCR and sequenced. The ORF is 792 nucleotides and codes for a protein of 264 amino acids with a predicted molecular weight of 28.5kDa. GRA3c has been cloned into a number of GATEWAYTM vectors and optimisation of protein expression is currently being performed. In addition, this gene was transferred into pVAX and used to obtain Abs by DNA vaccination. Antibodies raised against these proteins may be used in immunolocalisation studies.

Variation in a Ribosomal Internal Transcribed Spacer (ITS1) among Choanoflagellate taxa
Ruhana Hassan and Barry Leadbeater

School of Biosciences, The University of Birmingham, Edgbaston, Birmingham, B15 2TT

Choanoflagellates are colourless monads with a single flagellum and cone-shaped collar. In evolutionary terms, they are considered to be part of a 'crown group' from which animals and fungi have evolved. At present, based on morphology, the choanoflagellates are divided into three families. Two families, the Codosigidae and Salpingoecidae are typified by having organic coverings and the third family, the Acanthoecidae, has a highly characteristic, silica basket-like covering. Within the latter group, two patterns of division can be detected, tectiform and nudiform. This project is designed to establish the possibility of using some gene sequences, other than 18S SSU rDNA, in order to investigate the relationships between taxa in the Acanthoecidae. The internal transcribed spacer region 1 (ITS1) of the nuclear ribosomal DNA is a non-coding gene, that evolves rapidly and has been used elsewhere to resolve phylogenetic relationships between closely related species. In this study the ITS1 gene has been sequenced from five choanoflagellate taxa belonging to the Acanthoecidae. The length of ITS1 from choanoflagellates varies between 334- 495 nucleotides. This region exists in multiple copies which display considerable variation. A detailed examination of ITS1 from Stephanoeca diplocostata showed that copies of this gene can be separated into two distinct groupings - within each grouping there is between 85-100% similarity. However between the two groupings there is only 38-39% similarity. Two clones of Stephanoeca diplocostata, which were isolated from widely separated localities (France and Australia), have been investigated so far. The possibility of using ITS1 in biogeographical and phylogenetic studies will be discussed.

Global Coccolithophorid Populations in a Changing Ocean
M. Débora Iglesias-Rodríguez1, 4 Christopher W. Brown2, Scott C. Doney3, Joan Kleypas3, Dorota Kolber1, Zbigniew Kolber1, Paul K. Hayes4 and Paul G. Falkowski1,5.

  1. Environmental Biophysics and Molecular Ecology Program, Institute of Marine and Coastal Sciences, Rutgers University, 71 Dudley Road, New Brunswick, NJ 08901 U.S.A.
  2. Office of Research and Applications, National Oceanographic and Atmospheric Administration, 5200 Auth Road, Camp Springs, MD 20746-4304.
  3. National Center for Atmospheric Research, Climate and Global Dynamics, PO Box 3000, Boulder, CO 80307-3000.
  4. School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, U.K.
  5. Department of Geology, Rutgers University.

In this paper we address the physical and chemical processes that select for coccolithophorid blooms detected in SeaWiFS ocean color imagery. Our primary goal is to develop both diagnostic and prognostic models that represent the spatial and temporal dynamics of coccolithophorid blooms in order to improve our knowledge of the role of these organisms in mediating fluxes of carbon between the ocean, the atmosphere, and the lithosphere. Based on monthly composite images of classified coccolithophorid blooms and global climatological maps of physical variables and nutrient fields, we developed a probability density function that accounts for the physical chemical variables that predict the spatio-temporal distribution of coccolithophorids in the world oceans. Our analysis revealed that areas with sea surface temperatures (SST) between 3 and 15oC, a critical irradiance between 25 and 150 µmol quanta m-2 s-1, and decreasing nitrate concentrations (DN/Dt < 0) are selective for upper ocean large-scale coccolithophorid blooms. While these conditions favor both Northern and Southern Hemisphere blooms of the most abundant coccolithophorid in the modern oceans, Emiliania huxleyi, the northern and southern hemisphere populations of this organism are genetically distinct. Applying amplified fragment length polymorphism as a marker of genetic diversity, we identified two major taxonomic clades of E. huxleyi; one is associated with the Northern Hemisphere blooms, while the other is found in the Southern Hemisphere. We suggest a rule of `universal distribution and local selection', i.e. coccolithophorids can be considered cosmopolitan taxa, but their genetic plasticity provides physiological accommodation to local environmental selection pressure. Sea surface temperature, critical irradiance, and DN/Dt were predicted for the years 2060-2070 using the NCAR Community Climate System Model to generate future monthly probability distributions of coccolithophorids based upon the relationships observed between the environmental variables and coccolithophorid blooms in modern oceans. Our projected probability distribution analysis suggests that in the North Atlantic, the largest habitat for coccolithophorids on Earth, the areal extent of blooms will decrease by up to 50% by the middle of this century. We discuss how the magnitude of carbon fluxes may be affected by the evolutionary success of coccolithophorids in future climate scenarios.

Protozoa-bacteria interactions in a model system
William Gaze1, Nigel Burroughs2, Elizabeth MH Wellington1 & Maurice P Gallagher3

  1. Biological Sciences, University of Warwick, Coventry, CV4 7AL.
  2. Institute of Mathematics, University of Warwick, Coventry, CV4 7AL.
  3. Institute of Cell & Molecular Biology, Biology Division, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR.

Acanthamoeba polyphaga feeding on Salmonella typhimurium were observed by light microscopy and a detailed record of interactions kept by digital image capture and image analysis. Images matrices and time lapse sequences (100x magnification) were collected at daily intervals for analysis of population level interactions. A strain of S. typhimurium SL1344 carrying a fis:gfp reporter construct (pPDT105) was used to assess intracellular growth in A. polyphaga on non nutrient agar (NNA) plates. Invasion of the contractile vacuole was observed as a rare phenomenon in 1:100-1000 acanthamoebae. The salmonellae contained in contractile vacuoles illustrated significant up-regulation of fis relative to extracellular bacteria indicating that they were in the early stages of exponential growth, and reached numbers of 100-200 cells per vacuole after 4 days.

Data analysis was performed giving an extensive statistical characterisation of interaction, migration and growth events in the ecosystem at both the individual and population levels. Automated image analysis techniques were used to locate and count amoebae, cysts and bacteria in a series of spatial images and videos. Most algorithms were based on thresholding, or a modification of this idea with probabilistic models for noise. Our strategy was two tiered, we performed an automated analysis for classification and counting followed by user intervention/reclassification using custom written graphical user interfaces (GUIs) in MATLAB.

The evolution of Autralian trypanosomes
Patrick B. Hamilton*, W.C. Gibson*, Dr J.R. Stevens** and J.M. Gidley*

  1. *School of Biological Sciences, Woodland Road, University of Bristol, Bristol, BS8 1UG
  2. ** School of Biological Sciences, University of Exeter, Hatherly Laboratories, Price of Wales Road, Exeter, EX4 4PS

Trypanosomes are protozoan parasites of which several species cause important diseases of humans and domestic livestock. Little is known about the trypanosomes of Australian mammals. We have undertaken a survey of trypanosomes in Australian vertebrates and invertebrates. Nested PCR using primers specific to the ssu rRNA gene of trypanosomes detected a high prevalence in marsupials including kangaroos, wallabies, wombats and possums. Trypanosomes isolated from three species of marsupial (a kangaroo Macropus giganteus giganteus, a wallaby Wallabia bicolor bicolor and the common wombat Vombatus ursinus ursinus) were included within in a phylogeny of trypanosomes based on the ssu rRNA gene. These trypanosomes from marsupials fell in different clades, being more closely related to mammalian trypanosomes from placental mammals outside Australia than they are to each other suggesting divergent evolutionary origins of these parasites. Trypanosomes detected in haemadipsid leeches were closely related to the wallaby trypanosome, suggesting they may be important vectors of marsupial trypanosomes.

The Evolutionary Origins of Vertical Transmission in Microsporidian Parasites
Judith E Smith, Rebecca S Terry, Joe Ironside, Alison Dunn, Tim Littlewood and David Rollinson.

School of Biology, Leeds University and Natural History Museum, London.

Microsporidian parasites frequently make use of vertical transmission (VT) during their complex life cycles, yet its importance has never been specifically investigated. We have previously described a microsporidian, Nosema granulosis, which appears to be exclusively vertically transmitted and feminises its crustacean host G.duebeni. We have initiated screening program to investigate the diversity and distribution of VT microsporidia and to test to what extent vertical transmission is associated with traits such as reduced virulence and host sex ratio distortion (SRD). We sampled fifteen species of crustacea, via PCR of gonadal tissue/eggs with microsporidia specific small subunit rDNA primers, and found that VT microsporidia were ubiquitous in these hosts. Eleven VT parasites were detected in the crustacean survey and these fell into diverse lineages of the phylum Microspora. Evidence of host sex ratio distortion has been found for three out of four of these parasites tested to date. The novel sequence data was used to reconstruct the parasite phylogeny. Mapping of VT and SRD traits show that these have multiple origins and lead us to propose, that vertical transmission may be an ancestral transmission route, and that it is associated with host sex ratio distortion. If our observations on VT microsporidia in the crustacea are borne out within other host phyla these parasites may come to rival the bacterial endosymbionts (Wohlbachia) in their importance as sex ratio distorters. We are currently extending our VT screen to a wider range of hosts including snails, insects and fish.

Nosema-like microsporidia in bryozoans: need for new genera
Canning, E.U.1; Refardt, D.2; Okamura, B.3; Vossbrinck, C.R.4 and Curry, A.5

  1. Department of Biological Sciences, Imperial College, London, UK
  2. Department of Biology, University of Fribourg, Switzerland
  3. School of Animal & Microbial Sciences, University of Reading, Reading, UK
  4. Connecticut Agricultural Experiment Station, New Haven, U.S.A
  5. Public Health Laboratory, Withington Hospital, Manchester, UK
Nosema is the most species rich genus in the phylum Microsporidia. It is characterised as diplokaryotic (all stages with paired, closely apposed nuclei), disporoblastic (two sporoblasts formed from each sporont) and with development in direct contact with host cell cytoplasm. Several species have already been transferred from this genus after analysis of their 16S rDNA sequences had shown them to be unrelated to the type species Nosema bombycis. Ultrastructural and 16S rDNA data are presented on three microsporidia from bryozoans: Nosema cristatellae Canning, Okamura and Curry 1996, infecting the epithelium of Cristatella mucedo and two new species found within hypertrophic host cells, free floating in the coelom of Plumatella nitens and Pectinatella magnifica. All three species resemble Nosema as characterised above but each requires a new generic assignment. In the species infecting P. magnifica, the exospore layer of the spore wall bears a dense coat of hair-like processes and in the species in P. nitens there is a dense spherical body located near the Golgi system in the spores. Otherwise the three species show no differentiating morphological characters. The 16S rDNA sequences show 73-79% identity.

The large scale phylogeny and classification of Protozoa.
T. Cavalier-Smith

Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS

I shall outline a somewhat revised higher-level classification of the kingdom Protozoa into 13 phyla* and discuss the phylogenetic evidence for key innovations. The phyla are grouped into four infrakingdoms and two subkingdoms. There is now compelling evidence that the infrakingdom Alveolata (Ciliophora, Sporozoa, dinoflagellates and protalveolates) had a photosynthetic common ancestor and that they are sisters of the kingdom Chromista. Alveolates and chromists form a clade (chromalveolates) that arose by the symbiogenetic uptake of a red alga. The new infrakingdom Excavata, ancestrally with three microtubular ciliary roots, comprises Metamonada, Parabasalia, Euglenozoa, Percolozoa and Loukozoa. Excavata are probably most closely related to plants or to chromalveolates or to their common ancestor. They are grouped with Alveolata as the derived protozoan subkingdom Corticata. The more basal protozoan subkingdom, Gymnomyxa, comprises the probably ancestrally uniciliate infrakingdom Sarcomastigota (phyla Choanozoa, Amoebozoa) and the new ancestrally biciliate infrakingdom Rhizaria. Rhizaria comprise Cercozoa (now including Ascetospora), Retaria (Radiolaria and Foraminifera), Heliozoa, and Apusozoa. Choanozoa, animals, and fungi together comprise a robust opisthokont clade. Rhizaria and Corticata are informally grouped together with the kingdoms Chromista and Plantae as the bikonts, a major eukaryotic clade supported by concatenated protein trees and by the derived gene fusion involving dihydrofolate reductase and thymidylate synthase that probably occurred at or close to the base of the bikonts. Amoebozoa comprise Mycetozoa, Archamoebae and Lobosa and may be ancestrally uniciliate and unicentriolar; the relatively few Mycetozoa that are biciliate may have doubled their centrioles independently of bikonts, as their anterior cilia are older. In contrast, all well-studied bikonts have a pattern of ciliary transformation with a younger anterior cilium. Amoebozoa either branch below the opisthokont/bikont bifurcation or are sisters of the opisthokonts or (more likely) bikonts. The new rooting of the tree, coupled with the probably holophyly of the bikonts, means that the earlier grouping of Choanozoa and Apusozoa as phylum Neomonada was incorrect. Metamonada and Archezoa (collectively treated as superphylum Archezoa) are secondarily amitochondrial and may be sisters of Percolozoa; these three tetrakont phyla may have had a photosynthetic common ancestor shared with that of Euglenozoa. Thus losses of mitochondria, plastids and cilia all occurred several times in excavates, as in chromalveolates.

*Cavalier-Smith, T. (2002). The phagotrophic origin of eukaryotes and phylogenetic classification of Protozoa. Int. J. Syst. Evol. Microbiol. 52, 297-354.