Protozoan community structure in a fractal soil environment.

Protozoan abundance was quantified, and 365 protozoan species were recorded, in 150 soil samples from an upland grassland in Scotland. Across the entire size range (2-200 pm) protozoan species richness varied by a factor of two, whereas abundance increased by a factor of 20 with decreasing body size. As the soil had fractal structure, the relatively flat species curve can be explained if spatial heterogeneity determines species number--for in a fractal environment, heterogeneity will be the same at all spatial scales. Community structure appeared to approach a temporary steady-state about six days after re-hydration of dried soil. A simple model based on combining the fractal character of increasing habitat area at smaller spatial scales, with the weight-specific energy requirements of protozoa, provided theoretical curves of abundance and biovolume on body size which provide a reasonable fit to real data. We suggest two possibilities--that the apparent competence of the theoretical model is fortuitous and the product of poorly understood dynamic elements of the trophic structure in the community; or that key elements of protozoan community structure in a fractal soil environment may be largely explained in terms of habitat space and energy requirements.

Redescription of Psilotricha acuminata Stein, 1859 and revisions of the genera Psilotricha and Urospinula (Ciliophora, Hypotrichida).

Psilotricha acuminata was described by Stein in 1859 as the type species of the ciliate genus Psilotricha Stein, 1859. The ciliate has rarely been found since, and its infraciliature has never been described with the aid of silver-impregnation techniques. We have found P. acuminata Stein, 1859 in soil samples from upland grassland in Scotland (U.K.). Living and healthy organisms of P. acuminata are oblong in outline, and dorso-ventrally compressed. They closely resemble ciliates of the genus Euplotes. The main morphological features used for identification of P. acuminata are the very long and sparse cirri, and the two macronuclei. When the ciliate crawls, the cirri appear stiff and directed backwards. Specimens observed from the ventral side have a protruding anterior end, a rounded or acuminate posterior pole, and a "beak-like" projection to the left side of the posterior end. The ciliate shares characters with the Euplotidae (body shape and reduced ciliature) and with the Oxytrichidae (marginal rows, macronuclei, reduced number of transverse cirri). Because the arrangement of the silver-impregnated infraciliature was unknown, and as the only description of the ciliate was that of Stein (1859a, b), the genus Psilotricha became confused with other hypotrich genera, especially Urospinula Corliss, 1960. Here we provide a full redescription of P. acuminata based on living and silver-impregnated specimens, and a revision of the genera Psilotricha Stein, 1859 and Urospinula Corliss, 1960. We resurrect the genus Urospinula, and give an emended diagnosis for both genera. The species now included within the genus Psilotricha are P. acuminata Stein, 1859 (type species); Psilotricha viridis (Penard, 1922) Kahl, 1932; and Psilotricha geleii (Gelei, 1954) Stiller, 1974. Psilotricha viridis sensu Kahl, 1932 and Psilotricha dragescoi Grolière, 1975 are considered incertae sedis.

Exploring Leeuwenhoek's legacy: the abundance and diversity of protozoa.

Towards the end of the 17th century, Leeuwenhoek built "magnifying glasses" that enabled him to see and describe protozoa for the first time. Continued exploration of the natural history of protozoa during the past 300 years has progressed far beyond simply documenting morphospecies (global total probably <20,000). We now realize that protozoan 'biodiversity' is multi-faceted (e.g. sibling species, variant genotypes and syntrophic consortia). Realization of their extraordinary abundance has secured for protozoa the position of dominant phagotrophs and regenerators of nutrients within microbial food webs. And studies of protozoa in the natural environment have done much to effect a paradigm shift in our understanding of why specific microbes live where they do and how they got there in the first place. In particular, the hypothesis of ubiquitous dispersal of protozoan species does seem to be supported by the evidence provided by morphospecies, sibling species and even individual genotypes.

Biodiversity of terrestrial protozoa appears homogeneous across local and global spatial scales.

Free-living microbes are by far the most abundant group of organisms in the biosphere, yet estimates of global species richness remain nebulous, and there is no consensus regarding the likely geographical distribution of species. Both uncertainties are addressed by the suggestion that the vast abundance of microbes may drive their ubiquitous random dispersal; for this would also make it likely that global species richness is relatively low. Here we test the idea of ubiquitous dispersal of testate amoebae and ciliates living in soil. We analysed their abundance and species richness in 150 soil samples collected from the one-hectare grassland site at Sourhope in Scotland, and in comparable published data from 1500 soil samples collected worldwide. Following taxonomic revision and removal of synonyms, there remained a total of 186 taxa (91 testate and 95 ciliate) recorded from both Sourhope and other places in the world. A fundamental pattern of random spatial distribution of species was revealed in species that are relatively rare. This probably arises from random dispersal, for when localised population growth occurs, the distributions become aggregated, as in virtually all metazoan species. We find no evidence for geographically-restricted protozoan morphospecies at spatial scales of 4 m2, 10,000 m2, or worldwide. Species that are locally rare or abundant are similarly rare or abundant on a global scale. Approximately one third of the global diversity of soil protozoa was found at the one-hectare grassland site in Scotland, but this is a minimum figure, for recorded species richness is proportional to sampling effort, as shown here.

Estimating the growth potential of the soil protozoan community.

We have developed a method for determining the potential abundance of free-living protozoa in soil. The method permits enumeration of four major functional groups (flagellates, naked amoebae, testate amoebae, and ciliates) and it overcomes some limitations and problems of the usual 'direct' and 'most probable number' methods. Potential abundance is determined using light microscopy, at specific time intervals, after quantitative re-wetting of air-dried soil with rain water. No exogenous carbon substrates or mineral nutrients are employed, so the protozoan community that develops is a function of the resources and inhibitors present in the original field sample. The method was applied to 100 soil samples (25 plots x 4 seasons) from an upland grassland (Sourhope, Southern Scotland) in the UK. Median abundances for all four functional groups lie close to those derived from the literature on protozoa living in diverse soil types. Flagellates are the most abundant group in soil, followed by the naked amoebae, then the testate amoebae and ciliates. This order is inversely related to typical organism size in each group. Moreover, preliminary evidence indicates that each functional group contains roughly the same number of species. All of these observations would be consistent with soil having fractal structure across the size-scale perceived by protozoa. The method described will be useful for comparing the effects on the soil protozoan community of different soil treatments (e.g. liming and biocides).

Apparent global ubiquity of species in the protist genus Paraphysomonas.

Evidence is presented for the ubiquity of protist species. Using the example of protists that leave traces (siliceous scales) of their recent population growth, we show that most - perhaps all species in the genus Paraphysomonas, are ubiquitous. Of the species recorded in surveys carried out worldwide, we have identified 78% of their number in 0.1 cm2 of sediment collected from a freshwater pond (total area 10(8) cm2) in England. Moreover, the pond appears to act like a microcosm of aquatic environments in general, for species that are globally rare or abundant, are likewise rare or abundant in the pond. We assume that the rate of neutral migration to the pond is greatest for the globally abundant species. As these species are probably capable of growth in a broad range of conditions, they will more frequently encounter the environment they require for population growth. Thus globally abundant species are also locally abundant in the pond - a pattern that will be amplified by periodic cyst production. Ubiquitous dispersal is probably driven by very high absolute abundance of individuals, and the water column of the pond was estimated to support >10(14) Paraphysomonas individuals. Ubiquity will dampen rates of speciation, and the evidence presented here indicates that global species richness of Paraphysomonas is indeed modest - perhaps close to what is already known.

The global diversity of protozoa and other small species.

It is widely believed that the number of species of micro-organisms in the world is extremely large. Here, we offer the contrasting view--that the number may be quite modest. Most of the work reviewed refers to the ciliated protozoa. As with all microbial groups, we must define our concept of "species", and for ciliates, the "morphospecies" concept appears to be at least as robust as any other. Critical examination of published descriptions of ciliates provides a "best estimate" of 3744 for the global number of free-living morphospecies. Of these, 793 are associated with marine sediments, and 1370 with freshwater sediments. In an independent analysis based on extrapolation (assuming the ubiquity of species) from ecological datasets, we estimate the numbers of species in marine and freshwater sediments as 597 and 732, respectively (i.e. within a factor of two of the figures obtained from taxonomic analysis). This apparent convergence of independent estimates will strengthen if, as is likely, the number of nominal species is further reduced by taxonomic revision. These relatively low numbers of species are consistent with (a) the vast amount of published information indicating typically cosmopolitan distributions for ciliates and other microbes, and (b) recent experimental evidence that most free-living ciliates are rare or cryptic--seldom detectable, but present, and "waiting" for suitable conditions to arrive. In summary, most ciliates (and other micro-organisms) are probably ubiquitous, endemics are rare, global species richness is relatively low, and, at least in the case of the ciliates, most species have already been discovered.

Protozoan diversity: converging estimates of the global number of free-living ciliate species.

Protozoa are the most abundant phagotrophs in the biosphere, but no scientific strategy has emerged that might allow accurate definition of the dimensions of protozoan diversity on a global scale. We have begun this task by searching for the common ground between taxonomy and ecology. We have used two methods - taxonomic analysis, and extrapolation from ecological datasets - to estimate the global species richness of free-living ciliated protozoa in the marine interstitial and freshwater benthos. The methods provide estimates that agree within a factor of two, and it is apparent that the species-area curves for ciliates must be almost flat (the slope z takes the very low value of 0.043 in the equation: [number of species] = [constant][area](z)). Insofar as independent ecological datasets can be extrapolated to show similiar, flat, species-area relations, and that these converge with an independent estimate from taxonomic analysis, we conclude that the great majority of freeliving ciliates are ubiquitous. This strengthens our recent claim that the global species richness of free-living ciliated protozoa is relatively low (∼3000).

Planktonic ciliate species diversity as an integral component of ecosystem function in a freshwater pond.

A diverse and dynamic community of ciliated protozoa lives in the stratified water column of the productive freshwater pond known as 'Priest Pot'. As part of a long-term continuous monitoring programme, this community was examined with 10 cm-scale vertical sampling in August 1995 and June 1997, and found to be dominated by species with endosymbiotic algae (1995), or by a quite different set of species, feeding on the dinoflagellate Peridinium (1997). On both occasions, the community structure was comprehensible in terms of the preceding sequence of reciprocal interactions involving microbiological, physical and chemical factors (e.g. oxygen depletion, thermal gradient, essential nutrient concentrations). In this one pond, very different ciliate communities appear at different times, yet each community may be nothing more than a transient bi-product of dynamic ecosystem functions. The facility with which the ciliate community (or any other microbial community) transforms in a continuously changing environment probably depends on a large local diversity of rare and encysted species and the rapidity with which these species fill vacant niches.

Carbohydrate and Amino Acid Fermentation in the Free-Living Primitive Protozoon Hexamita sp.

Hexamita sp. is an amitochondriate free-living diplomonad which inhabits O(2)-limited environments, such as the deep waters and sediments of lakes and marine basins. C nuclear magnetic resonance spectroscopy reveals ethanol, lactate, acetate, and alanine as products of glucose fermentation under microaerobic conditions (23 to 34 muM O(2)). Propionic acid and butyric acid were also detected and are believed to be the result of fermentation of alternative substrates. Production of organic acids was greatest under microaerobic conditions (15 muM O(2)) and decreased under anaerobic (<0.25 muM O(2)) and aerobic (200 to 250 muM O(2)) conditions. Microaerobic incubation resulted in the production of high levels of oxidized end products (70% acetate) compared to that produced under anoxic conditions (20% acetate). In addition, data suggest that Hexamita cells contain the arginine dihydrolase pathway, generating energy from the catabolism of arginine to citrulline, ornithine, NH(4), and CO(2). The rate of arginine catabolism was higher under anoxic conditions than under microaerobic conditions. Hexamita cells were able to grow in the absence of a carbohydrate source, albeit with a lower growth rate and yield.

Evolution of the hydrogenosome.

Since its discovery almost 25 years ago the enigmatic hydrogenosome, a redox organelle of anaerobic unicellular eukaryotes, has puzzled evolutionists as to its origin and function. Synthesis of recent molecular, physiological and morphological studies now favours the hypothesis that hydrogenosomes derived from a modification of pre-existing mitochondria, and argues against the previously held view that the hydrogenosome had a polyphyletic origin. These data provide evidence for a more ancient origin of mitochondria than hitherto thought.

Oxygen uptake and antioxidant responses of the free-living diplomonad Hexamita sp.

The free-living anaerobic flagellate Hexamita sp. was observed to actively consume O2 with a K(m) O2 of 13 microM. Oxygen consumption increased linearly with O2 tension up to a threshold level of 100 microM, above which it was inhibited. Oxygen uptake was supported by a number of substrates but probably not coupled to energy conservation as cytochromes could not be detected spectro-photometrically. In addition, inhibitors specific for respiratory chain components did not significantly affect O2 uptake. Respiration was however, partially inhibited by flavoprotein and iron-sulfur protein inhibitors. NAD(P)H supported O2 consumption was measured in both particulate and soluble fractions; this activity was partially inhibited by quinacrine. A chemosensory response was observed in cells exposed to air, however no response was observed in the presence of superoxide dismutase plus catalase. Catalase and nonspecific peroxidase activity could not be detected, but superoxide dismutase plus catalase. Catalase and nonspecific peroxidase activity could not be detected, but superoxide dismutase activity was present. Superoxide dismutase was sensitive to NaN3, and H2O2 but not KCN, suggesting a Fe prosthetic group. Flow cytometric analysis revealed that thiol levels in live cells were depleted in the presence of t-butyl H2O2. The observed NADPH-driven glutathione reductase activity is believed to recycle oxidized thiols in order to re-establish reduced thiol levels in the cell. The corresponding thiol cycling enzyme glutathione peroxidase could not be detected. The ability to withstand high O2 tensions (100 microM) would enable Hexamita to spend short periods in a wider range of habitats. Prolonged exposure to O2 tensions higher than 100 microM leads to irreversible damage and cell death.

Multiple origins of anaerobic ciliates with hydrogenosomes within the radiation of aerobic ciliates.

Some ciliates live anaerobically and lack mitochondria, but possess hydrogenosomes: organelles that contain hydrogenase and produce hydrogen. The origin of hydrogenosomes has been explained by two competing hypotheses: (i) they are biochemically modified mitochondria; or (ii) they are derived from endosymbiotic association(s) of ciliates and anaerobic eubacteria that possessed the hydrogenosome biochemistry. Phylogenetic analyses of representative aerobic, and anaerobic hydrogenosomal ciliates using host nuclear SSU rDNA sequences indicate a minimum of three, but more likely four, separate origins of hydrogenosomes. Whereas this does not refute either hypothesis, the implausibility of multiple convergent endosymbioses gives further support to the view that hydrogenosomes in ciliates derive from an existing organelle, which ultrastructural evidence suggests is the mitochondrion. Our results indicate a considerable potential for physiological-biochemical plasticity among a group of predominantly aerobic eucaryotes, and provide a phylogenetic framework to further refine and test hypotheses of the origins of the hydrogenosomal enzymes.

Phylogenetic relationships among karyorelictids and heterotrichs inferred from small subunit rRNA sequences: resolution at the base of the ciliate tree.

Ciliate protozoa are among the most diverse and complex cells that have been described. Ciliates are characterized by nuclear dimorphism, possessing a macronucleus and a micronucleus which share the same cytoplasm. An understanding of the evolution of ciliate diversity depends upon knowledge of their phylogeny. In this study we attempted to resolve some of the relationships at the base of the ciliate tree by determining the phylogenetic position of a sample of heterotrich and hypothesized primitive karyorelictid ciliates. Karyorelictids are considered primitive because they possess a "simple" form of nuclear dualism whereby the macronucleus does not divide once it has differentiated from a micronucleus. We micromanipulated cells of two heterotrichs, Spirostomum ambiguum and Gruberia sp., and two karyorelictids, Loxodes magnus and Tracheloraphis sp., and amplified their small subunit (SSU) rDNA using PCR. The primary structure of the SSU rDNA was determined for each species and used to infer their positions in the ciliate phylogenetic tree. The results indicate, with strong support, that the aerobic heterotrichs and the karyorelictids sampled constitute a monophyletic group. The most parsimonious interpretation of the form of nuclear dimorphism in karyorelictids is that it is derived from the general condition as found in its sister group the aerobic heterotrichs. The two anaerobic heterotrichs, Metopus contortus and Metopus palaeformis, comprise a distinct clade, so that the subclass Heterotrichia, as currently conceived, is not a monophyletic group. The complex mouth architecture which characterizes all heterotrichs must be reassessed in light of this finding.

Some rumen ciliates have endosymbiotic methanogens.

Most of the small ciliate protozoa, including Dasytricha ruminantium and Entodinium spp. living in the rumen of sheep, were found to have intracellular bacteria. These bacteria were not present in digestive vacuoles. They showed characteristic coenzyme F420 autofluorescence and they were detected with a rhodamine-labelled Archaea-specific oligonucleotide probe. The measured volume percent of autofluorescing bacteria (1%) was close to the total volume of intracellular bacteria estimated from TEM stereology. Thus it is likely that all of the bacteria living in the cytoplasm of these ciliates were endosymbiotic methanogens, using H2 evolved by the host ciliate to form methane. Intracellular methanogens appear to be much more numerous than those attached to the external cell surface of ciliates.

A new polymorphic methanogen, closely related to Methanocorpusculum parvum, living in stable symbiosis within the anaerobic ciliate Trimyema sp.

A new anaerobic microbial consortium has been discovered: the partners are the ciliated protozoon Trimyema sp. and a single species of methanogen. The consortium has been maintained in culture for more than four years. Each ciliate contains up to 300 symbiotic bacteria; many are relatively small and irregularly disc-shaped, and these are distributed throughout the host's cytoplasm, whereas those which are attached to the ciliate's hydrogenosomes are significantly larger and profusely dentate. This attachment is interpreted as an adaptation to maximize capture by the bacteria of the H2 escaping from hydrogenosomes. The 16S rRNA gene of the symbionts has been partially sequenced, and fluorescent oligonucleotide probes have been constructed and used to detect the different morphotypes of the symbiont within the ciliate. The symbionts belong to a new species of archaeobacterium which is a close relative of the free-living methanogen Methanocorpusculum parvum.

Systematic and morphological diversity of endosymbiotic methanogens in anaerobic ciliates.

The identities and taxonomic diversity of the endosymbiotic methanogens from the anaerobic protozoa Metopus contortus, Metopus striatus, Metopus palaeformis, Trimyema sp. and Pelomyxa palustris were determined by comparative analysis of their 16S ribosomal RNA sequences. Fluorescent oligonucleotide probes were designed to bind to the symbiont rRNA sequences and to provide direct visual evidence of their origins from methanogenic archaea contained within the host cells. Confocal microscopy was used to analyze the morphology of the endosymbionts in whole cells of Metopus palaeformis, Metopus contortus, Trimyema sp, and Cyclidium porcatum. The endosymbionts are taxonomically diverse and are drawn from three different genera; Methanobacterium, Methanocorpusculum and Methanoplanus. In every case the symbionts are closely related to, but different from, free-living methanogens for which sequences are available. It is thus apparent that symbioses have been formed repeatedly and independently. Ciliates which are unrelated to each other (Trimyema sp. and Metopus contortus) may contain symbionts which are closely related, and congeneric ciliates (Metopus palaeformis and M. contortus) may contain symbionts which are distantly related to each other. This suggests that some of the symbiotic associations must be relatively recent. For example, at least one of the symbioses in Metopus must postdate the speciation of M. palaeformis and M. contortus. Despite this, Metopus contortus, Trimyema sp., Cyclidium porcatum and their respective endosymbionts show sophisticated morphological interactions which probably facilitate the exchange of materials between the partners.

Cyclidium porcatum n. sp.: a Free-living anaerobic scuticociliate containing a stable complex of hydrogenosomes, eubacteria and archaeobacteria.

A new ciliate species (Cyclidium porcatum) is the first freshwater anaerobic scuticociliate to be cultured and described. It contains a unique tripartite structure consisting of hydrogenosomes (confirmed by cytochemical staining for hydrogenase), interspersed with methanogens (confirmed by auto fluorescence and in situ hybridisation with an archaeobacterial 16S rRNA-specific probe) and unidentified eubacteria (confirmed with a eubacterial 16S rRNA-specific probe). This complex structure is stable and persistent, indicating that it is an anaerobic symbiotic consortium incorporating three functional partners.