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

Biodiversity And Phylogeny Of The Phylum Cercozoa (protozoa)

David Bass, Ema Chao and Thomas Cavalier-Smith
Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS

The novel phylum Cercozoa Cavalier-Smith 1998 (created by renaming a radically modified phylum Rhizopoda) comprises several groups previously of obscure affinity, including Cercomonadida, Thaumatomonadida, chlorarachniophyte algae, euglyphid testate amoebae, and plasmodiophorid plant pathogens. Their relationship was initially shown by 18S rRNA phylogeny. Sequences from a much greater variety of Cercozoa now show that many previously unclassified genera also belong to this phylum, including Helkesimastix, Sainouron, and Bodomorpha. Furthermore, when the analysis uses a more realistic substitution model (general time reversible: GTR) and allows for variation in evolutionary rate along the molecule by a gamma distribution, it seems that the parasitic Ascetospora (haplosporidia and Paramyxa) are related to the Cercozoa, in which they are now placed as a class. Cercozoa exhibit a wide range of morphologically and ecologically diverse forms, including most soft-surfaced biciliate zooflagellates (e.g. Cercomonas, Heteromita), zooflagellates with silicious scales and emergent pseudopodia (e.g. Thaumatomonas), amoeboflagellate algae (e.g. Chlorarachnion), syncytial parasites (e.g. Plasmodiophora, Haplosporidium), most filose amoebae, typically testate (e.g. Euglypha), some reticulose amoebae (e.g. Gymnophrys, Biomyxa), colonial flagellates (e.g. Spongomonas), and numerous small gliding flagellates (e.g. Allantion and many not yet named).

Cercozoa are currently more clearly defined by this rRNA phylogeny than phenotypic characters, although morphological homogeneity within the major sub-branches is generally preserved. However, many phylogenetic issues remain, for example whether or not the two clades containing Cercomonas are really separate. The molecular divergence among Cercomonas species is as great as among all fungi or land plants. Ribosomal RNA trees suggest weakly that Cercozoa are related to Foraminifera (also supported by actin trees) and Radiolaria (new grouped as phylum Retaria Cavalier-Smith 1999). Another recently recognised zooflagellate phylum, Apusozoa, has a tendency to extrude ventral pseudopods (like many Cercozoa) but differs in having a rigid cortical layer. Recently, Cercozoa have been grouped with Retaria, Apusozoa, and Heliozoa as a new infrakingdom Rhizaria; however their relative branching order remains unclear and the grouping requires further investigation.

In order to investigate the ecological and geographic distribution of Cercozoa, which include many of the most important soil Protozoa, we have developed group-specific primers for screening environmental samples for Cercozoa from a wide range of sites. This poster presents some preliminary results of this study and micrographs of a diversity of interesting Cercozoa.

The sequence of colonisation of a glass surface by protozoa from lake water under laminar flow

K. Heaton and J. Parry
Dept Biological Science, Lancaster University, Lancaster LA1 4YQ

Although it has been established that protozoa are always present in aquatic biofilms, and that they play an active role in microbial dynamics, to date, there is very little known about the order of protozoan colonisation. This research investigates the sequence of colonisation of a virgin glass coverslip in a flow cell by ciliates, flagellates and amoebae from lake water. The water was pumped through the flow cell under laminar flow and the surface of the coverslip examined microscopically daily and filmed using a CCD camera attached to the microscope. The effect of removing different size fractions of protozoa by filtering the water through a range of sizes of nylon mesh (3µm, 10µm, 30µm, 100µm) on the protist species presence and their abundance compared to full lake water over a 10 day experimental period was also examined.

The effect of flow rate on biofilm development and stability

Moodie, J1, Bevan, K2, Elliot, A3 and JD Parry1

  1. Dept Biological Science, Lancaster University, Lancaster LA1 4YQ.
  2. Environmental Science, Lancaster University, Lancaster LA1 4YQ.
  3. CEH Windermere, Far Sawray, Ambleside, Cumbria.

The majority of microbial activity in riverine systems is located on surfaces, These surface-associated communities (biofilms) are generally considered hot-spots' of microbial activity and important sites for biogeochemical cycling. They comprise all microbial groups, including the primary predators, protozoa. The effect of flow rate, particularly spates of extremely high flow, on the development and persistence of biofilm communities has not been studied in any great depth to date. We plan to use biofilms as the model communities with which to test the Intermediate Disturbance Hypothesis. We hypothesise that occasional spates in a previously low flow environment result in a high level of disturbance to resident biofilms which may lead to a complete change in community structure within those biofilms. Frequent spates in areas of constant high flow have led to the development of 'flow resistant' biofilms which, when a spate occurs and disturbs the biofilms, they always revert back to the original 'flow resistant' community structure. It is envisaged that the protozoan communities within each biofilm 'type' will be very different.

Zooflagellate and heliozoan phylogeny and early eukaryote evolution

Thomas Cavalier-Smith and Ema E. Chao
Department of Zoology, University of Oxford, UK, OX14 5HS

Zooflagellates are phagotrophic flagellates without plastids. They are basal to virtually all derived eukaryotic lineages. The first eukaryote was probably a zooflagellate. To clarify the basal branches of the eukaryotic tree we sequenced 18S RNA genes from scores of previously unclassified zooflagellates of unknown evolutionary affinities (all but one aerobic). They all prove to belong to one or other of nine established phyla. There are no totally novel major lineages. The great majority belong in the phylum Cercozoa Cavalier-Smith 1998, evidently a major previously unrecognised eukaryotic lineage. Most others are heterokont chromists (mainly in the phylum Sagenista (1995), where some have lost the usual distinguishing ciliary hairs) or members of five protozoan phyla: Euglenozoa (1981), Choanozoa (1981), Apusozoa (1986), Miozoa (1999: Protalveolata, Dinoflagellata and Sporozoa) or Loukozoa (1999: Jakobea plus Anaeromonadea). Phalansterium, with a single cilium and centriole, possibly therefore more primitive than most zooflagellates, seems to belong in Amoebozoa (also predominantly unicentriolar). Heliozoa are phagotrophic protists that trap prey with fine radiating axopodia supported internally by an axoneme of microtubules. We sequenced 18S rRNA of four heliozoa with axonemes radiating from the centrosome (centrohelids) and four superficially similar protists with radiating axopodia or filopodia. Centrohelids are a monophyletic novel lineage on the plant/chromist side of the eukaryotic tree. The other four protists all evolved independently from zooflagellates in three separate phyla: The axo/filopodial'Dimorpha-like' and Massisteria are both Cercozoa. The filopodial Ministeria evolved from choanoflagellates in Choanozoa. The filopodial Pinaciophora is a secondarily non-flagellate heterokont (phylum Sagenista; class Bicoecea). Gamma-corrected minimum evolution distance trees (which allow for substitution rate variation along the molecule) with hundreds of taxa are necessary to get a reasonable ribosomal RNA phylogeny for eukaryotes. These and a signature sequence show that two groups of plasmodial parasites (Haplosporidia and Paramyxida) are not separate phyla but really Cercozoa, possibly related to Phytomyxea, also plasmodial parasites. Gamma trees also support the grouping of Radiolaria (including Acantharea) and Foraminifera as phylum Retaria (1999) and of Retaria and Cercozoa as infrakingdom Rhizaria. Centrohelid heliozoa may be Rhizaria.

The ecological significance of diatom grazing by Prymnesium parvum

M. Martin-Cereceda and G. Novarino
Dept. of Zoology, The Natural History Museum, London SW7 5BD.

Small diatoms (<5 µm) are extremely abundant and widespread in world oceans; however, they are not grazed by "traditional" zooplankton grazers (i.e. copepods). This, in addition to their size, make the small diatoms very suitable potential prey for nanoflagellates. In this poster, we present the first evidence of the grazing of Prymnesium parvum over the nanoplaktonic diatoms Minidiscus trioculatus and Thalassiosira sp. Flagellate grazing and diatom-species preference were quantitatively determined in monoxenic batch cultures, and high resolution video-microscopy was used to visualize the ingestion process. Prey switching mechanisms have also been investigated using bacteria as an alternative diet and compared with flagellate growth dynamics in absence of any prey. Our results indicate that Prymnesium parvum may totally remove prey-populations of the small diatoms in a 48 hours period, and that the grazing starts 10 minutes after the contact with the prey. Diatom capture usually occurs at the posterior cell end, and implies the development of a pseudopodium-like structure.

These results suggest that nanoflagellate predation may be an important factor to regulate populations of small diatoms in marine planktonic environments. Further investigations testing a variety of heterotrophic and (theoretically) autotrophic nanoflagellates are currently being undertaken.

The DHFR-TS fusion protein suggests that the eukaryote root is near the Amoebozoa/ opisthokont divergence

Alexandra Stechmann and Thomas Cavalier-Smith
Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK

One of the most challenging evolutionary problems is locating the root of the eukaryote tree. We know that the root lies somewhere within the kingdom Protozoa, but the widespread view that early eukaryotes were amitochondrial has recently been dramatically overturned. Systematic biases in sequence evolution prevent the reliable inference of the eukaryote root from single-gene trees. Concatenated sequence trees should be more reliable, but leave many possibilities open. In principle deletions/insertions or gene fusions should be superior for this purpose whenever ancestral and derived states are clearly distinguishable. Therefore, we have used a derived gene fusion between dihydrofolate reductase (DHFR) and thymidylate synthase (TS) genes to greatly narrow down the position of the root. These genes are translated into separate proteins in bacteria, animals and fungi, which is thus the ancestral condition. It was previously known that in plants, alveolate Protozoa, and Euglenozoa the DHFR and TS coding regions are translated as a single polypeptide. Thus the genes have become fused together after inverting their order compared with that in the bacterial operon. This novel fused gene must be a derived condition compared with the separate genes of the bacteria that were ancestral to eukaryotes. We looked by pcr for the presence or absence of the fusion gene in various protist groups where data were lacking. Sequencing revealed the derived fusion gene in chromists and representatives of all major protozoan groups except the uniciliate Choanozoa and Amoebozoa. We were able to amplify the TS gene alone from Choanozoa and Amoebozoa; thus it is probable (but not yet certain) that they have separate genes like animals and fungi. As heliozoa and all ancestrally biciliate protozoa and their chromist and plant descendants (collectively called bikonts) have the fusion gene - the derived condition - the root of the eukaryote tree cannot lie among them, unless the fusion originally took place in the ancestral eukaryote and the genes were secondarily separated again in animals and fungi (and probably Choanozoa and Amoebozoa). As opisthokonts (Choanozoa, animals, fungi) are definitely derived, Amoebozoa are the only possible early diverging eukaryote lineage. Ciliary evolution suggests that even they may not be truly early diverging and that the root may lie between opisthokonts and all other eukaryotes (bikonts plus Amoebozoa).

Cloning of a partial cDNA encoding Acanthamoeba castellani alternative oxidase, by degenerate PCR

McBride, J., Tinney, S., Lyons, R.E., Roberts, F. & Roberts, C.W.
Department of Immunology, Strathclyde Institute of Biomedical Sciences, University of Strathclyde, Glasgow, G4 0NR

Acanthamoeba (species) is a major cause of ocular keratitis and loss of vision. Current therapies are inadequate and new more effective anti-microbials are urgently required. Acanthamoeba has been demonstrated to possess a cyanide insensitive alternative oxidase (AOX). As this is absent from mammals it may be a useful target for novel therapeutic agents. Herein, we describe studies to obtain the coding sequence of the Acanthamoeba castellani AOX. Multiple sequence alignments of a AOXs from diverse species were used to identify areas of conservation. Degnerate PCR primers were designed using these areas and incorporating an Acanthamoeba castellani codon bias. Degenerate PCR using A. castellani cDNA as template yielded a product within the expected size range, which was gel-purified and cloned into pDRIVE for sequencing. The cloned, PCR-amplified product has homology with the AOXs of a number of diverse species. The full length sequence is now being sought by 5' and 3' RACE.

Assessing Bias in Environmental Profiling using DNA Analysis

Ruth Cordero Peters1, Nozomi Ytow2 & Dave Roberts3

  1. School of Biomedical & Life Sciences, University of Surrey, Guildford, Surrey, GU2 7XH
  2. Institute of Biological Sciences, University of Tsukuba, Tsukuba 305-8572, Japan.
  3. Dept. Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD.

The use of nucleic acid extraction, amplification and analysis to determine a profile of diversity in environmental samples has become the method of choice in microbial ecology because it avoids the biases inherent in traditional cultivation techniques and gives access to information about strains which are reluctant to grow in culture. DNA was extracted from freshwater sediment through a series of washes using the common phenol-cholroform methodology. Up to 12 washes could be completed in one day through to lyophilisation and high molecular weight (genomic) DNA was recovered in significant quantities from all washes. PCR amplification with universal primers to eukaryotic small-subunit ribosomal DNA yielded no amplified product, but universal primers to all domains gave good product. The results were examined by DGGE analysis using eukaryote-specific primers and revealed a changing pattern of bands across the washes. Use of the hydroxyapatite extraction method resulted in more bands being visible and the difference between washes disappearing.

These results suggest that lysis methods are probably not taxonomically biased through the wash cycle but that DNA-binding material is co-isolated with the DNA and inhibits the PCR reaction, either in a random or in a non-specific stochastic manner. These results suggest that the hydroxyapatite method should be the routine method of choice.

Group-specific primers for studying bodonid and heliozoan biodiversity and dispersal

Sophie Herden and Thomas Cavalier-Smith
Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK.

Macroscopic animal and plant species are differentially distributed among the major biogeographic regions of the world, because natural barriers prevent their free dispersal. Does this also apply to Protozoa? The theory of microbial cosmopolitanism states that it does not. Instead all free-living micro-organisms below a certain size (possibly in the range of 100 to 300 µm) should be found growing in suitable habitats in every geographic region of the world; this is because there are no effective barriers to their free global dispersal and their astronomic populations will ensure that every region of the world is frequently seeded with viable propagules. If this is true, a single sampling site should contain all the globally described species for which it is ecologically suitable and a similar site anywhere in the world should have the very same species.

To test both these predictions we are beginning to identify and measure the morphospecies of bodonid flagellates and heliozoans in Priest Pot, a one-hectare lake in the Lake District and a comparable one in Western Australia, as comprehensively as possible. But a single morphospecies might include many physiologically, ecologically or genetically different variants, especially in protozoan groups like zooflagellates that have been poorly studied and which lack enough morphological characters for making fine taxonomic distinctions. Therefore we are also culturing as many strains from these groups as we can and sequencing their 18S rRNA genes in order to study the geographical distribution of different genotypes. For selected bodonid species that are both geographically and ecologically ubiquitous and can live in freshwater, oceans and soils, e.g. Bodo designis and Rhynchomonas nasuta, we shall sample other geographical locations also and determine whether isolates from the different habitats have preferences for different growth conditions.

To enable more comprehensive and speedier sampling of the genotypes of both groups, and perhaps reveal novel lineages, we shall randomly sequence 18S rRNA gene libraries following pcr from DNA extracted directly from environmental samples at different sites. This poster describes the design and testing of group-specific pcr primers for bodonids and centrohelid heliozoa needed for making the libraries, and outlines the overall aims of the project.

NOMENCURATOR: a database to manage protistan names under multiple taxonomic views

Nozomi Ytow 1, David R. Morse2 & Dave Roberts3

  1. Institute of Biological Sciences, University of Tsukuba, Tsukuba 305-8572, Japan.
  2. Computing Department, Faculty of Mathematics and Computing, The Open University, Walton Hall, Milton Keynes MK7 6AA.
  3. Dept. Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD.

Interconnecting databases of names, taxa or collections requires that they interoperate. This includes providing support for multiple taxonomic views because it is inconceivable that all databases should use the same, single taxonomic view of the data. A wide coverage of data, a diversity of intended audiences or even a long period of development can result in multiple taxonomic views being provided in a single database. Therefore, the ability to support multiple taxonomic views is a core requirement for global-scale interoperability of databases.

Several data models have been developed and proposed independently in recent years. These models include FGDC (ITIS, 2000), Nomencurator (Ytow et al., 2001), Pisces-II (Pyle, 2000) and Prometheus (Pullan et al., 2000). All of them support multiple taxonomic views, which demonstrates an increasing recognition of the necessity to provide such a facility in order to assist taxonomic work. Each of these models inherits some of the customs and practices from the background taxa for which the model was developed and only Nomencurator was conceived to handle ambiregnal taxa. This suggests that the models may be lacking in their ability to support multiple taxonomic views of a global-scale federation of databases. Finally, each of the models also contains elements of the potential taxon concept (Berendsohn, 1995 and 1997).

Temporal succession and diversty of biofilm cilitates in a RBC plant.

B. Pérez-Uz, M. Martín-Cereceda, S. Serrano & A. Guinea
Dept. Microbiología III. Facultad CC. Biológicas. Universidad Complutense de Madrid. Spain.

Biofilms of a wastewater treatment plant by rotating biological contactors (Boadilla, Madrid-Spain) have been studied during a year. Sampling was carried out in three stages along the biological treatment. Spatial and temporal microbial succession in this variable physical-chemical background was explored to prove the differential distribution of the protists populations.

Protists communities of these biofilms consisted mainly of ciliates and diatoms. Diatoms represented a high proportion along the system, reaching in the last stages of the treatment over 50% of protist abundance. Therefore, they were an important biological factor in the biofilm cohesion, in contrast with that observed in other RBC plants studied. A low ciliate diversity and abundance in the first stage sampled of the biological system was found; only species of the genus Epistylis sp. and Vorticella convallaria appeared in high numbers along the whole year. A significant increment on both diversity and abundance of ciliates was observed in the intermediate stage sampled, where peritrich ciliates showed a clear seasonal succession. Opercularia coarctata appeared in spring and was substituted by Epistylis species during autumn and summer, and then in winter by V. convallaria. The last stage showed a decrease of peritrich ciliates and only Zoothamnium procerius appeared with a high density along the year. A seasonal succession of non-sessile groups was observed in this stage; populations of Aspidisca lynceus and A. cicada appeared in spring and were replaced by Litonotus lamella, Dexiotricha sp. and Uronema nigricans during summer and autumn while in winter the dominant species was Trochilia minuta. These results suggest that seasonal species succession allow the same functional groups to occupy the same trophic niche in the biofilms.

Dissecting the sexual cycle of Trypanosoma brucei.

A. MacLeod 1, A. Tweedie1, S. Taylor2, S. McLellan2, C.M.R.Turner2 and A. Tait1

  1. Wellcome Centre for Molecular Parasitology, Anderson College,University of Glasgow, 56, Dumbarton Road, Glasgow, G11 6NU.
  2. Division of Infection & Immunity, IBLS, Joseph Black Building,University of Glasgow, Glasgow, G12 8QQ.

Genetic exchange is a fundamental biological process which results in the generation of diversity. The process by which T. brucei undergoes genetic exchange has been the subject of debate for some time with several different models of mating being proposed.

Here we describe the genetic analysis of a large number of progeny clones from three laboratory crosses and present evidence that genetic exchange in T. brucei is Mendelian.

We demonstrate sexual recombination (in agreement with Mendel's first law), independent segregation (Mendel's second law), crossing over between homologous chromosomes and a low frequency of triploidy and trisomy. The evidence unequivocally supports a standard diploid meiotic Mendelian system of genetic exchange.

Highlighting the development of Trypanosoma brucei within the vector using GFP

Wendy Gibson and Mick Bailey
School of Biological Sciences and Dept of Clinical Veterinary Science, University of Bristol, Bristol, BS8 1UG

Although the developmental cycle of Trypanosoma brucei within its vector, the tsetse fly, was described nearly a century ago, certain aspects remain obscure. When the fly takes up trypanosomes with a bloodmeal, the trypanosomes first differentiate and multiply within the lumen of the gut, before penetrating into the ectoperitrophic space. From here they proceed anteriorly, penetrating back through the peritrophic matrix so they can enter the lumen of the gut again and make their way up the oesophagus to the salivary glands for further rounds of differentiation and multiplication. We have recently transfected the gene for Green Fluorescent Protein (GFP) into trypanosomes. When this gene is expressed, the trypanosomes become intensely fluorescent and even single cells can be detected easily within the organs of the fly. By following the process of infection by dissecting infected flies every 24 hours, we hope to build up a picture of the developmental process.

Sex in parasitic and free living protists

Sex in malaria parasites, and its implications for control of the disease.
David Walliker
Institute of Cell, Animal and Population Biology, University of Edinburgh, UK.

The compulsory sexual stage of the malaria parasite in mosquitoes means that the organism has a highly efficient means of generating novel genotypes by genetic recombination. However this process depends on ingestion by mosquitoes of gametocytes of at least two genetically distinct clones, so that cross-mating between gametes of each clone can take place. In highly malaria-endemic regions of the world, multiclonal infections are common, while elsewhere a more clonal population structure is apparent.

Information on the genetic structure of natural populations of Plasmodium falciparum is now being obtained by

Such studies are important if we are to predict the likelihood of parasites being produced which contain new combinations of genes determining, for example, polymorphic antigens. The work is also providing important insights into the spread of recently evolved genes, such as those determining drug-resistance, from one country to another.

Genetic hybridisation in Trypanosoma cruzi

Michael A Miles, Matthew Yeo, +Iain Frame, Russell Stothard, *Hernan Carrasco, Martin C. Taylor and Michael Gaunt
Pathogen Molecular Biology and Biochemistry Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, UK
+ now at the Wellcome Trust
* now at Facultad de Medicina, Universidad Central de Caracas, Venezuela.

The species T. cruzi is genetically diverse. Two principal sub-specific groups have been identified and named T. cruzi I and T. cruzi II, the latter with five subgroups (a-e). T. cruzi I predominates in the Amazon basin and in endemic countries north of the Amazon, whilst T. cruzi II is the predominant cause of Chagas disease throughout the southern cone countries of South America. We have generated T. cruzi I hybrids in the laboratory from clonal parental genotypes, proving for the first time that T. cruzi has an extant capacity for genetic exchange. Two biological clones of T. cruzi I, derived from putative parental Amazonian strains, were genetically transformed to carry different drug resistance markers. The resultant episomal transformants were passaged, singly or together, axenically and through the entire life cycle in vitro and in vivo. Six double drug resistant clones were recovered. The clones were analysed by isoenzymes, random amplificaton of polymorphic DNA (RAPDs), karyotypes, microsatellites and by DNA sequencing. Results demonstrated:

  1. genetic fusion of parental genotypes within T. cruzi I progeny
  2. loss of alleles in the polyploid progeny
  3. some homologous recombination
  4. apparent uniparental inheritance of kinetoplast maxicircle DNA.

There were strong genetic parallels between the hybrids and some natural isolates of T. cruzi, in evidence of both recombination and of polyploid genotypes (such as T. cruzi IId). Thus aneuploidy appears to be an important feature of T. cruzi populations.
We thank the Wellcome Trust for financial support.

Genetic exchange in Trypanosoma brucei


School of Biological Sciences, University of Bristol, Bristol, BS8 1UG, UK.

It was only proved relatively recently (1986) by Jenni and colleagues that trypanosomes can undergo some form of genetic exchange. Since then various labs have been trying to unravel details of the process. For Trypanosoma brucei, Jenni's experiment already showed that genetic exchange took place in the tsetse fly vector. Subsequently the location within the fly has been narrowed down to the salivary glands, where epimastigote and metacyclic stages occur. The process of genetic exchange is not an obligatory part of the trypanosome lifecycle and the factors that trigger the event are unknown. Crosses of several different trypanosome strains have been successful and there is no evidence for mating types; a single clone can also undergo selfing. Comparison of parental and hybrid genotypes strongly suggests that meiosis occurs at some stage, but haploid gametes have not been detected. The fact that stable triploid (as well as diploid) hybrid progeny can be produced also supports the idea of meiosis. One remarkable feature of genetic exchange in trypanosomes is the inheritance of the mitochondrial DNA, which is contained within an organelle, the kinetoplast. Analysis of kinetoplast DNA of parents and hybrids reveals a complex process whereby the mini- and maxi-circles are inherited bi parentally or uniparentally respectively. The observation that mini-circles are inherited biparentally implies that the parental mitochondria, and hence the trypanosome cells, fuse during genetic exchange.

Ciliate pheromones: from sex factors to growth factors

Piero Luporini
Dipartimento di Biologia Molecolare, Cellulare e Animale, University of Camerino, 62032 Camerino, Italy.

Sex in ciliates is a phenomenon with unique evolutionary and biological features. It has inappropriately been called conjugation, causing confusion with unrelated phenomena. Somatic (diploid) cells, whether carrying different or identical genomes, temporarily leave their reproductive (asexual) life and unite in (non-reproductive) mating pairs to eventually exchange genes and cytoplasm and generate new genomes in substitution of the old ones before resuming vegetative growth. Activation of the cell to conjugate has been found associated with cell type-specific signal molecules, now usually called pheromones (earlier, mating type factors or gamones). Pheromones have been purified and characterized along with their coding (macronuclear) genes from species of Blepharisma and Euplotes, which constitutively secrete them into the environment. We showed that pheromones of E. raikovi were homologous members of a numerous family of small proteins which, regardless of their low levels of amino acid sequence similarity, have a common structure based on a compact bundle of exclusively a-helix motifs. Because of this similarity in conformation among different pheromones, the cell surface pheromone-receptors (that in each cell arise from a splicing mechanism of the transcripts of the same pheromone genes) are exposed to a competitive binding between their homologous (partner) pheromone (autocrine binding) and other, heterologous pheromones (paracrine binding). The cell response will vary according to which binding prevails: activation to grow in association with prevalence of autocrine binding, activation to mate when the reverse occurs. The activity of ciliate pheromones thus appears to be context-dependent, and the autocrine (mitogenic) activity of these molecules is likely the ancestral one.

Testing theories of the evolution of sex using protists

Arjan de Visser
Dept. of Genetics, Wageningen University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands

Why sex and recombination have evolved as a means of reproduction is the subject of (sometimes intense) debate. Asexual reproduction is fast and mutation seems a flexible means to generate genetic variation that serves as raw material for adaptation. A whole array of theories has been brought forward over the years to solve this enigma. Two dominant ideas think of sex and recombination as a means either (i) of more efficient selection against the numerous deleterious mutations, or (ii) to combine advantageous mutations and accelerate adaptation. I will present experimental tests with the unicellular alga Chlamydomonas and the fungus Aspergillus to test the first idea. Next, I will present data from a laboratory evolution experiment with the bacterium E. coli that indicate a particular disadvantage of clonal reproduction, and suggest that sex may have evolved to allow fast adaptation.

Origins and evolution of meiosis in protists: complex machinery for sex

John M. Logsdon, Jr.*, S.-Banoo Malik and Marilee A. Ramesh
Department of Biology, Emory University, Atlanta GA 30322 USA

The origin and evolution of sexual reproduction in eukaryotes is a major unsolved puzzle for biology. We have begun molecular phylogenetic studies of meiotic genes obtained from a wide range of species, focusing mainly on protozoa, which comprise most of eukaryotic phylogenetic diversity. The major eukaryotic homologs of recA, RAD51 and DMC1 (the latter, a meiosis-specific paralog), have been isolated from a diversity of protists. The RAD51/DMC1 gene duplication is a probable marker for the origin of meiosis and the presence of DMC1 may indicate sexuality -- with its absence suggesting asexuality. Our results show that the RAD51/DMC1 gene duplication occurred early in eukaryotic evolution; if coincident with the origin of meiosis, this indicates that either meiosis is a process ancestral to all extant eukaryotes or that some key protist species representing early-diverging lineage(s) have not been sampled. Giardia lamblia, a putatively deep-branching, possibly asexual species, is perhaps the best candidate for a primitively ameiotic protist. Surprisingly, it encodes two versions of DMC1 but apparently does not encode RAD51. The presence of DMC1 genes indicates that G. lamblia may be cryptically sexual. Starting from the partially-sequenced G. lamblia genome and using a combination of bioinformatic and directed isolation methods, we have isolated and sequenced a number of additional meiotic genes from G. lamblia. These and other results have allowed us to begin describing a conserved "core" meiotic machinery for eukaryotes.