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The social amoeba Dictyostelium discoideum is a versatile organism that is unusual in alternating between single-celled and multi-celled forms. It possesses highly-developed systems for cell motility and chemotaxis, phagocytosis, and developmental pattern formation. As a soil amoeba growing on microorganisms, it is exposed to many potential pathogens; it thus provides fruitful ways of investigating host-pathogen interactions and is emerging as an influential model for biomedical research.

Aggregative multicellularity has evolved multiple times in diverse groups of eukaryotes, exemplified by the well-studied development of dictyostelid social amoebas, for example, Dictyostelium discoideum However, it is still poorly understood why multicellularity emerged in these amoebas while the majority of other members of Amoebozoa are unicellular. Previously, a novel type of noncoding RNA, Class I RNAs, was identified in D. discoideum and shown to be important for normal multicellular development. Here, we investigated Class I RNA evolution and its connection to multicellular development. We identified a large number of new Class I RNA genes by constructing a covariance model combined with a scoring system based on conserved upstream sequences. Multiple genes were predicted in representatives of each major group of Dictyostelia and expression analysis confirmed that our search approach identifies expressed Class I RNA genes with high accuracy and sensitivity and that the RNAs are developmentally regulated. Further studies showed that Class I RNAs are ubiquitous in Dictyostelia and share highly conserved structure and sequence motifs. In addition, Class I RNA genes appear to be unique to dictyostelid social amoebas because they could not be identified in outgroup genomes, including their closest known relatives. Our results show that Class I RNA is an ancient class of ncRNAs, likely to have been present in the last common ancestor of Dictyostelia dating back at least 600 million years. Based on previous functional analyses and the presented evolutionary investigation, we hypothesize that Class I RNAs were involved in evolution of multicellularity in Dictyostelia., (© 2021 Kjellin et al.; Published by Cold Spring Harbor Laboratory Press.)

Unicellular protozoa that encyst individually upon starvation evolved at least eight times into organisms that instead form multicellular fruiting bodies with spores. The Dictyostelia are the largest and most complex group of such organisms. They can be subdivided into 4 major groups, with many species in groups 1-3 having additionally retained encystment. To understand fitness differences between spores and cysts, we measured long-term survival of spores and cysts under climate-mimicking conditions, investigated spore and cyst ultrastructure, and related fitness characteristics to species ecology. We found that spores and cysts survived 22 °C equally well, but that spores survived wet and dry frost better than cysts, with group 4 spores being most resilient. Spore walls consist of three layers and those of cysts of maximally two, while spores were also more compacted than cysts, with group 4 spores being the most compacted. Group 4 species were frequently isolated from arctic and alpine zones, which was rarely the case for group 1-3 species. We inferred a fossil-calibrated phylogeny of Dictyostelia, which showed that its two major branches diverged 0.52 billion years ago, following several global glaciations. Our results suggest that Dictyostelium multicellular sporulation was a likely adaptation to a cold climate.

Dictyostelium discoideum and the other dictyostelid slime moulds ('social amoebae') are popular model organisms best known for their demonstration of sorocarpic development. In this process, many cells aggregate to form a multicellular unit that ultimately becomes a fruiting body bearing asexual spores. Several other unrelated microorganisms undergo comparable processes, and in some it is evident that their multicellular development evolved from the differentiation process of encystation. While it has been argued that the dictyostelid fruiting body had similar origins, it has also been proposed that dictyostelid sorocarpy evolved from the unicellular fruiting process found in other amoebozoan slime moulds. This paper reviews the developmental biology of the dictyostelids and other relevant organisms and reassesses the two hypotheses on the evolutionary origins of dictyostelid development. Recent advances in phylogeny, genetics, and genomics and transcriptomics indicate that further research is necessary to determine whether or not the fruiting bodies of the dictyostelids and their closest relatives, the myxomycetes and protosporangids, are homologous., (© 2019 Cambridge Philosophical Society.)

The Dictyostelid social amoebas are a popular model system for cell- and developmental biology and for evolution of sociality. Small subunit (SSU) ribosomal DNA-based phylogenies subdivide the known 150 species into four major and some minor groups, but lack resolution within groups, particularly group 4, and, as shown by genome-based phylogenies of 11 species, showed errors in the position of the root and nodes separating major clades. We are interested in the evolution of cell-type specialization, which particularly expanded in group 4. To construct a more robust phylogeny, we first included 7 recently sequenced genomes in the genome-based phylogeny of 47 functionally divergent proteins and next selected 6 proteins (Agl, AmdA, PurD, PurL, RpaA, SmdA) that independently or in sets of two fully reproduced the core-phylogeny. We amplified their coding regions from 34 Dictyostelium species and combined their concatenated sequences with those identified in the 18 genomes to generate a fully resolved phylogeny. The new AAPPRS based phylogeny (after the acronym of the 6 proteins) subdivides group 4 into 2 branches. These branches further resolve into 5 clades, rather than the progressively nested group 4 topology of the SSU rDNA tree, and also re-orders taxa in the other major groups. Ancestral state reconstruction of 25 phenotypic traits returned higher "goodness of fit" metrics for evolution of 19 of those traits over the AAPPRS tree, than over the SSU rDNA tree. The novel tree provides a solid framework for studying the evolution of cell-type specialization, signalling and other cellular processes in particularly group 4, which contains the model Dictyostelid D. discoideum., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Dictyostelid cellular slime molds (dictyostelids) are microscopic throughout their entire life cycle. The vegetative phase consists of single-celled amoeboid forms which live in the soil/leaf litter microhabitat of fields and forests along with animal dung, where they feed upon bacteria and other microbes, grow, and multiply until the available food supply is exhausted. When this happens, the amoeboid forms aggregate together in large numbers to form multi-celled pseudoplasmodia, which then give rise to fruiting bodies (sorocarps) that consist of supportive stalks and unwalled sori containing propagative spores., Results: Dictyostelium purpureum var. pseudosessile, a new variant of dictyostelid, is described herein, based on morphological features and molecular data. This new variant was isolated from soil samples collected in two tropical areas of China. The complete spore-to-spore life cycle of this species, which required 50 h, including spore germination, myxamoebae, cell aggregation, pseudoplasmodium, and sorocarp formation, was documented. Descriptions and illustrations are provided for this species based on our collections. Data from ontogeny, morphology and phylogeny analyses (SSU) of D. purpureum var. pseudosessile confirm that it is a Group 4 species according to the newly proposed classification of dictyostelids., Conclusions: Our results suggest that the violet sori, widens at the midpoint of sorophore and simple recurved sorophore bases represent the prominent features for the new variant D. purpureum var. pseudosessile. The latter is a Group 4 species now known from two tropical areas of China where dictyostelids remains understudied.

The establishment of symbioses between eukaryotic hosts and bacterial symbionts in nature is a dynamic process. The formation of such relationships depends on the life history of both partners. Bacterial symbionts of amoebae may have unique evolutionary trajectories to the symbiont lifestyle, because bacteria are typically ingested as prey. To persist after ingestion, bacteria must first survive phagocytosis. In the social amoeba Dictyostelium discoideum, certain strains of Burkholderia bacteria are able to resist amoebal digestion and maintain a persistent relationship that includes carriage throughout the amoeba's social cycle that culminates in spore formation. Some Burkholderia strains allow their host to carry other bacteria, as food. This carried food is released in new environments in a trait called farming. To better understand the diversity and prevalence of Burkholderia symbionts and the traits they impart to their amoebae hosts, we first screened 700 natural isolates of D. discoideum and found 25% infected with Burkholderia. We next used a multilocus phylogenetic analysis and identified two independent transitions by Burkholderia to the symbiotic lifestyle. Finally, we tested the ability of 38 strains of Burkholderia from D. discoideum, as well as strains isolated from other sources, for traits relevant to symbiosis in D. discoideum. Only D. discoideum native isolates belonging to the Burkholderia agricolaris, B. hayleyella, and B. bonniea species were able to form persistent symbiotic associations with D. discoideum. The Burkholderia-Dictyostelium relationship provides a promising arena for further studies of the pathway to symbiosis in a unique system., (© 2019 John Wiley & Sons Ltd.)

Aggregative multicellularity requires the ability of cells to recognise conspecifics. Social amoebae are among the best studied of such organisms, but the mechanism and evolutionary background of species recognition remained to be investigated. Here we show that heterologous expression of a single Dictyostelium purpureum gene is sufficient for D. discoideum cells to efficiently make chimaeric fruiting bodies with D. purpureum cells. This gene forms a bidirectional pair with another gene on the D. purpureum genome, and they are both highly polymorphic among independent wild isolates of the same mating group that do not form chimaeric fruiting bodies with each other. These paired genes are both structurally similar to D. discoideum tgrB1/C1 pair, which is responsible for clonal discrimination within that species, suggesting that these tgr genes constitute the species recognition system that has attained a level of precision capable of discriminating between clones within a species. Analysis of the available genome sequences of social amoebae revealed that such gene pairs exist only within the clade composed of species that produce precursors of sterile stalk cells (prestalk cells), suggesting concurrent evolution of a precise allorecognition system and a new 'worker' cell-type dedicated to transporting and supporting the reproductive cells.

Dictyostelia is a monophyletic group of transiently multicellular (sorocarpic) amoebae, whose study is currently limited to laboratory culture. This tends to favour faster growing species with robust sorocarps, while species with smaller more delicate sorocarps constitute most of the group's taxonomic breadth. The number of known species is also small (∼150) given Dictyostelia's molecular depth and apparent antiquity (>600 myr). Nonetheless, dictyostelid sequences are rarely recovered in culture independent sampling (ciPCR) surveys. We developed ciPCR primers to specifically target dictyostelid small subunit (SSU or 18S) rDNA and tested them on total DNAs extracted from a wide range of soils from five continents. The resulting clone libraries show mostly dictyostelid sequences (∼90%), and phylogenetic analyses of these sequences indicate novel lineages in all four dictyostelid families and most genera. This is especially true for the species-rich Heterostelium and Dictyosteliaceae but also the less species-rich Raperosteliaceae. However, the most novel deep branches are found in two very species-poor taxa, including the deepest branch yet seen in the highly divergent Cavenderiaceae. These results confirm a deep hidden diversity of Dictyostelia, potentially including novel morphologies and developmental schemes. The primers and protocols presented here should also enable more comprehensive studies of dictyostelid ecology., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Amino acid repeats, or homorepeats, are low complexity protein motifs consisting of tandem repetitions of a single amino acid. Their presence and relative number vary in different proteomes, and some studies have tried to address this variation, proteome by proteome. In this work, we present a full characterization of amino acid homorepeats across evolution. We studied the presence and differential usage of each possible homorepeat in proteomes from various taxonomic groups, using clusters of very similar proteins to eliminate redundancy. The position of each amino acid repeat within proteins, and the order of co-occurring amino acid repeats were also addressed. As a result, we present evidence about the unevenly evolution of homorepeats, as well as the functional implications of their relative position in proteins. We discuss some of these cases in their taxonomic context. Collectively, our results show evolutionary and positional signals that suggest that homorepeats have biological function, likely creating unspecific protein interactions or modulating specific interactions in a context dependent manner. In conclusion, our work supports the functional importance of homorepeats and establishes a basis for the study of other low complexity repeats. Proteins 2017; 85:709-719. © 2016 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

The polyketide MPBD (4-methyl-5-pentylbenzene-1, 3-diol) is produced by the polyketide synthase SteelyA (StlA) in Dictyostelium discoideum. MPBD is required for appropriate expression of cAMP signalling genes involved in cell aggregation and additionally induces the spore maturation at the fruiting body stage. The MPBD signalling pathway for regulation of cell aggregation is unknown, but MPBD effects on sporulation were reported to be mediated by the G-protein coupled receptor CrlA in D. discoideum KAx3. In this study, we deleted the crlA gene from the same parental strain (Ax2) that was used to generate the MPBD-less mutant. We found that unlike the MPBD-less mutant, Ax2-derived crlA- mutants exhibited normal cell aggregation, indicating that in Ax2 MPBD effects on early development do not require CrlA. We also found that the Ax2/crlA- mutant formed normal spores in fruiting bodies. When transformed with PkaC, both Ax2 and Ax2/crlA- similarly responded to MPBD in vitro with spore encapsulation. Our data make it doubtful that CrlA acts as the receptor for MPBD signalling during the development of D. discoideum Ax2., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Amoeba often use cell movement as a mechanism to find food, such as bacteria, in their environment. The chemotactic movement of the soil amoeba Dictyostelium to folate or other pterin compounds released by bacteria is a well-documented foraging mechanism. Acanthamoeba can also feed on bacteria but relatively little is known about the mechanism(s) by which this amoeba locates bacteria. Acanthamoeba movement in the presence of folate or bacteria was analyzed in above agar assays and compared to that observed for Dictyostelium. The overall mobility of Acanthamoeba was robust like that of Dictyostelium but Acanthamoeba did not display a chemotactic response to folate. In the presence of bacteria, Acanthamoeba only showed a marginal bias in directed movement whereas Dictyostelium displayed a strong chemotactic response. A comparison of genomes revealed that Acanthamoeba and Dictyostelium share some similarities in G protein signaling components but that specific G proteins used in Dictyostelium chemotactic responses were not present in current Acanthamoeba genome sequence data. The results of this study suggest that Acanthamoeba does not use chemotaxis as the primary mechanism to find bacterial food sources and that the chemotactic responses of Dictyostelium to bacteria may have co-evolved with chemotactic responses that facilitate multicellular development., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Background: Dictyostelia are a well-studied group of organisms with colonial multicellularity, which are members of the mostly unicellular Amoebozoa. A phylogeny based on SSU rDNA data subdivided all Dictyostelia into four major groups, but left the position of the root and of six group-intermediate taxa unresolved. Recent phylogenies inferred from 30 or 213 proteins from sequenced genomes, positioned the root between two branches, each containing two major groups, but lacked data to position the group-intermediate taxa. Since the positions of these early diverging taxa are crucial for understanding the evolution of phenotypic complexity in Dictyostelia, we sequenced six representative genomes of early diverging taxa., Results: We retrieved orthologs of 47 housekeeping proteins with an average size of 890 amino acids from six newly sequenced and eight published genomes of Dictyostelia and unicellular Amoebozoa and inferred phylogenies from single and concatenated protein sequence alignments. Concatenated alignments of all 47 proteins, and four out of five subsets of nine concatenated proteins all produced the same consensus phylogeny with 100% statistical support. Trees inferred from just two out of the 47 proteins, individually reproduced the consensus phylogeny, highlighting that single gene phylogenies will rarely reflect correct species relationships. However, sets of two or three concatenated proteins again reproduced the consensus phylogeny, indicating that a small selection of genes suffices for low cost classification of as yet unincorporated or newly discovered dictyostelid and amoebozoan taxa by gene amplification., Conclusions: The multi-locus consensus phylogeny shows that groups 1 and 2 are sister clades in branch I, with the group-intermediate taxon D. polycarpum positioned as outgroup to group 2. Branch II consists of groups 3 and 4, with the group-intermediate taxon Polysphondylium violaceum positioned as sister to group 4, and the group-intermediate taxon Dictyostelium polycephalum branching at the base of that whole clade. Given the data, the approximately unbiased test rejects all alternative topologies favoured by SSU rDNA and individual proteins with high statistical support. The test also rejects monophyletic origins for the genera Acytostelium, Polysphondylium and Dictyostelium. The current position of Acytostelium ellipticum in the consensus phylogeny indicates that somatic cells were lost twice in Dictyostelia.

Terpenes are structurally diverse natural products involved in many ecological interactions. The pivotal enzymes for terpene biosynthesis, terpene synthases (TPSs), had been described only in plants and fungi in the eukaryotic domain. In this report, we systematically analyzed the genome sequences of a broad range of nonplant/nonfungus eukaryotes and identified putative TPS genes in six species of amoebae, five of which are multicellular social amoebae from the order of Dictyosteliida. A phylogenetic analysis revealed that amoebal TPSs are evolutionarily more closely related to fungal TPSs than to bacterial TPSs. The social amoeba Dictyostelium discoideum was selected for functional study of the identified TPSs. D. discoideum grows as a unicellular organism when food is abundant and switches from vegetative growth to multicellular development upon starvation. We found that expression of most D. discoideum TPS genes was induced during development. Upon heterologous expression, all nine TPSs from D. discoideum showed sesquiterpene synthase activities. Some also exhibited monoterpene and/or diterpene synthase activities. Direct measurement of volatile terpenes in cultures of D. discoideum revealed essentially no emission at an early stage of development. In contrast, a bouquet of terpenes, dominated by sesquiterpenes including β-barbatene and (E,E)-α-farnesene, was detected at the middle and late stages of development, suggesting a development-specific function of volatile terpenes in D. discoideum. The patchy distribution of TPS genes in the eukaryotic domain and the evidence for TPS function in D. discoideum indicate that the TPS genes mediate lineage-specific adaptations., Competing Interests: The authors declare no conflict of interest.

Background: Spliceosomal introns are a common feature of eukaryotic genomes. To approach a comprehensive understanding of intron evolution on Earth, studies should look beyond repeatedly studied groups such as animals, plants, and fungi. The slime mold Dictyostelium belongs to a supergroup of eukaryotes not covered in previous studies., Results: We found 441 precise intron losses in Dictyostelium discoideum and 202 precise intron losses in Dictyostelium purpureum. Consistent with these observations, Dictyostelium discoideum was found to have significantly more copies of reverse transcriptase genes than Dictyostelium purpureum. We also found that the lost introns are significantly further from the 5' end of genes than the conserved introns. Adjacent introns were prone to be lost simultaneously in Dictyostelium discoideum. In both Dictyostelium species, the exonic sequences flanking lost introns were found to have a significantly higher GC content than those flanking conserved introns. Together, these observations support a reverse-transcription model of intron loss in which intron losses were caused by gene conversion between genomic DNA and cDNA reverse transcribed from mature mRNA. We also identified two imprecise intron losses in Dictyostelium discoideum that may have resulted from genomic deletions. Ninety-eight putative intron gains were also observed. Consistent with previous studies of other lineages, the source sequences were found in only a small number of cases, with only two instances of intron gain identified in Dictyostelium discoideum., Conclusions: Although they diverged very early from animals and fungi, Dictyostelium species have similar mechanisms of intron loss.

The allantoicase (allC) gene of Dictyostelium discoideum allC RNAi mutant strain was silenced using the RNA interference technique. The mutant strain is motile, aggregated, and could not undergo further morphological development. The growth rate is high and the cells show a shortened cell cycle comparing with wild-type D. discoideum. However, the mechanisms regarding these actions remain unclear. mRNA differential display was used in this study to identify genetic differences. A novel D. discoideum gene (GenBank accession number: KC759140) encoding a new zinc protease was cloned. The amino acid sequence of the novel gene exhibited a conserved zinc-binding domain (HEX2HX18E) that allowed its classification into the M1 family of metallopeptidases. The gene encoded a 345-amino acid protein with a theoretical molecular mass of 39.69 kDa and a theoretical pI of 6.05. This protein showed strong homology with leukotriene A4 (LTA4) hydrolase of Homo sapiens (41% identity and 60% similarity at the amino acid level). By analyzing quantitative reverse transcription-polymerase chain reaction data, this zinc protease gene was more highly expressed in D. discoideum allC RNAi mutant type than in wild-type KAx-3 cells during the trophophase. The novel zinc protease gene may function as an LTA4 hydrolase and contribute to the shortening of the allC RNAi mutant cell cycle.

Dictyostelia are common soil microbes that can aggregate when starved to form multicellular fruiting bodies, a characteristic that has also led to their long history of study and widespread use as model systems. Ribosomal RNA phylogeny of Dictyostelia identified four major divisions (Groups 1-4), none of which correspond to traditional genera. Group 1 was also tentatively identified as sister lineage to the other three Groups, although not consistently or with strong support. We tested the dictyostelid root using universal protein-coding genes identified by exhaustive comparison of six completely sequenced dictyostelid genomes, which include representatives of all four major molecular Groups. A set of 213 genes are low-copy number in all genomes, present in at least one amoebozoan outgroup taxon (Acanthamoeba castellanii or Physarum polycephalum), and phylogenetically congruent. Phylogenetic analysis of a concatenation of the deduced protein sequences produces a single topology dividing Dictyostelia into two major divisions: Groups 1+2 and Groups 3+4. All clades in the tree are fully supported by maximum likelihood and Bayesian inference, and all alternative roots are unambiguously rejected by the approximately unbiased (AU) test. The 1+2, 3+4 root is also fully supported even after deleting clusters with strong individual support for this root, or concatenating all clusters with low support for alternative roots. The 213 putatively ancestral amoebozoan proteins encode a wide variety of functions including 21 KOG categories out of a total of 25. These comprehensive analyses and consistent results indicate that it is time for full taxonomic revision of Dictyostelia, which will also enable more effective exploitation of its unique potential as an evolutionary model system., (Copyright © 2015 Elsevier Inc. All rights reserved.)

P2X receptors are calcium permeable ligand-gated ion channels activated by ATP. Their role as cell surface receptors for extracellular ATP released physiologically by mammalian cells is well established. However, the cellular function of P2X receptor subtypes that populate the membranes of intracellular compartments is not defined. An initial report described how intracellular P2X receptors control the function of the contractile vacuole, an osmoregulatory organelle in Dictyostelium and other protists, and that genetic disruption of P2X receptors severely impaired cell volume control during hypotonic stress. However, later studies refuted a functional role of intracellular P2X receptors in Dictyostelium. Here we provide evidence that the discrepancies reported between the studies are due to the laboratory strain of Dictyostelium employed, which display different phenotypes in response to hypotonic stress and a varied dependency upon P2X receptors for osmoregulation. We use the recent discovery that intracellular P2X receptors are novel calcium release channels to provide some mechanistic insight in an effort to explain why the strain variance may exist.

The social amoeba Dictyostelium discoideum is a commonly used model organism for the study of social evolution, multicellularity, and cell biology. But the boundaries and structure of the species have not been explored. The lack of morphological traits to distinguish D. discoideum makes even knowing whether a given clone is D. discoideum a challenge. We address this with a phylogeny of a widespread collection of clones from a range of locations and including clones identified previously as potential cryptic species. We sequenced portions of nuclear ribosomal DNA and mitochondrial DNA, analyzing approximately 5500 and 2500 base pairs from the two regions respectively. We compared these sequences to known reference sequences for both D. discoideum and other closely related Dictyostelium species to create Bayesian and neighbor-joining phylogenetic trees representing the evolutionary relationships among the clones. We identified 51 unique D. discoideum concatenated sequences based on the combined mitochondrial and ribosomal sequence data. We also identified four unique D. citrinum concatenated sequences, three of which were previously classified as D. discoideum clones. Our analysis of the data revealed that all D. discoideum clones form a monophyletic group, but there are several well-supported subclades and pronounced genetic differentiation among locations (F(ST)=0.242, P=0.011), suggesting the presence of geographic or other barriers between populations. Our results reveal the need for further investigation into potential tropical cryptic species., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Three species of dictyostelid cellular slime molds were isolated from forest soil, meadow soil and leaf litter collected from Yalta, Crimea, Ukraine. Dictyostelium globisporum is new to science; D. crassicaule and D. sphaerocephalum are new records from Ukraine. Descriptions and illustrations are given based on the Ukraine specimens.

Dictyostelium discoideum belongs to a group of multicellular life forms that can also exist for long periods as single cells. This ability to shift between uni- and multicellularity makes the group ideal for studying the genetic changes that occurred at the crossroads between uni- and multicellular life. In this Primer, I discuss the mechanisms that control multicellular development in Dictyostelium discoideum and reconstruct how some of these mechanisms evolved from a stress response in the unicellular ancestor.

In late Jan and early Feb 2005 samples for isolation of dictyostelid cellular slime molds (dictyostelids) were collected in five different provinces and from six national parks (all located 39-55°S) in Patagonia and Tierra del Fuego, Argentina. Southern beech (Nothofagus) forests represented the primary vegetation type investigated, but some samples were obtained from Patagonian steppe, alpine meadows, Valdivian temperate rainforests and coniferous forests dominated by Araucaria, Austrocedrus and Fitzroya. Among the dictyostelids isolated from the samples we collected were seven species new to science. These species (Dictyostelium austroandinum, D. chordatum, D. fasciculoideum, D. gargantuum, D. leptosomopsis, D. valdivianum and Polysphondylium patagonicum) are described herein on the basis of both morphology and molecular (SSU rDNA) data. One of the new species, D. gargantuum, is one of the largest representatives of the group reported to date. Another unusual species, D. chordatum, produces long interwoven sorocarps that do not appear to respond to a spacing gas similar to the condition first noted in D. implicatum.

Dictyostelid cellular slime molds (dictyostelids) associated with grassland ecosystems of the central and western United States were investigated at nine sites that included examples of the three major ecological types of grasslands (tall grass, mixed grass and short grass) generally recognized for the region. Samples of soil/humus collected from each site were examined with the Cavender method of isolating dictyostelids. For each of those six sites with well developed gallery forests present, an additional set of forest soil/humus samples was collected. A more intensive sampling effort was carried out at one site (Konza LTER) to assess the possible effects of burning and grazing on dictyostelid diversity and density. Twelve species of dictyostelids were recovered from grassland sites, whereas gallery forest sites yielded only nine species. Four cosmopolitan species (Dictyostelium giganteum, D. mucoroides, D. sphaerocephalum and Polysphondylium pallidum) were represented by the greatest densities of clones, with D. sphaerocephalum particularly common. The general pattern across all sites was that both species richness and density of dictyostelids decreased with decreasing precipitation. Samples collected from ungrazed grassland plots yielded higher numbers of both species and clones as compared to grazed plots, and the general pattern was for both values to increase as the interval between fires increased. For numbers of clones this correlation was statistically significant.

Dictyostelium discoideum is a eukaryotic microbial model system for multicellular development, cell-cell signaling, and social behavior. Key models of social evolution require an understanding of genetic relationships between individuals across the genome or possibly at specific genes, but the nature of variation within D. discoideum is largely unknown. We re-sequenced 137 gene fragments in wild North American strains of D. discoideum and examined the levels and patterns of nucleotide variation in this social microbial species. We observe surprisingly low levels of nucleotide variation in D. discoideum across these strains, with a mean nucleotide diversity (pi) of 0.08%, and no strong population stratification among North American strains. We also do not find any clear relationship between nucleotide divergence between strains and levels of social dominance and kin discrimination. Kin discrimination experiments, however, show that strains collected from the same location show greater ability to distinguish self from non-self than do strains from different geographic areas. This suggests that a greater ability to recognize self versus non-self may arise among strains that are more likely to encounter each other in nature, which would lead to preferential formation of fruiting bodies with clonemates and may prevent the evolution of cheating behaviors within D. discoideum populations. Finally, despite the fact that sex has rarely been observed in this species, we document a rapid decay of linkage disequilibrium between SNPs, the presence of recombinant genotypes among natural strains, and high estimates of the population recombination parameter rho. The SNP data indicate that recombination is widespread within D. discoideum and that sex as a form of social interaction is likely to be an important aspect of the life cycle., Competing Interests: The authors have declared that no competing interests exist.

Centrins are a family of proteins within the calcium-binding EF-hand superfamily. In addition to their archetypical role at the microtubule organizing center (MTOC), centrins have acquired multiple functionalities throughout the course of evolution. For example, centrins have been linked to different nuclear activities, including mRNA export and DNA repair. Dictyostelium discoideum centrin B is a divergent member of the centrin family. At the amino acid level, DdCenB shows 51% identity with its closest relative and only paralog, DdCenA. Phylogenetic analysis revealed that DdCenB and DdCenA form a well-supported monophyletic and divergent group within the centrin family of proteins. Interestingly, fluorescently tagged versions of DdCenB were not found at the centrosome (in whole cells or in isolated centrosomes). Instead, DdCenB localized to the nuclei of interphase cells. This localization disappeared as the cells entered mitosis, although Dictyostelium cells undergo a closed mitosis in which the nuclear envelope (NE) does not break down. DdCenB knockout cells exhibited aberrant nuclear architecture, characterized by enlarged and deformed nuclei and loss of proper centrosome-nucleus anchoring (observed as NE protrusions). At the centrosome, loss of DdCenB resulted in defects in the organization and morphology of the MTOC and supernumerary centrosomes and centrosome-related bodies. The multiple defects that the loss of DdCenB generated at the centrosome can be explained by its atypical division cycle, transitioning into the NE as it divides at mitosis. On the basis of these findings, we propose that DdCenB is required at interphase to maintain proper nuclear architecture, and before delocalizing from the nucleus, DdCenB is part of the centrosome duplication machinery.

Sampling soils to look for dictyostelids in southern Portugal we found an isolate that has a morphology that differed from any previously described species of the group. We sequenced the internally transcribed spacer (ITS) and small subunit (SSU) genes of the nuclear ribosomal RNA and found that both sequences are distinct from all previously described sequences. Phylogenetic analyses place the new species in dictyostelid Group 3 (Rhizostelids) together with D. potamoides, with which it shares 65.8% identity for ITS and 96.6% for SSU. In this paper we describe a new species of cellular slime mold, Dictyostelium ibericum, based on morphological and molecular characters. It is a small species with polar granules in its spores.

Little is known about the population structure of social microorganisms, yet such studies are particularly interesting for the ways that genetic variation impacts their social evolution. Dictyostelium, a eukaryotic microbe widely used as a developmental model, has a social fruiting stage in which some formerly independent individuals die to help others. To assess genetic variation within the social amoeba Dictyostelium purpureum, we sequenced approximately 4000 base pairs of ribosomal DNA (rDNA) from 37 isolates collected in Texas, Virginia, and Japan. Our analysis showed extensive genetic variation between populations and clear evidence of phylogenetic structure. We identified three major phylogenetic groups that were more different than other accepted species pairs. Tests using pairs of clones showed that both sexual macrocyst and asexual fruiting body formation were influenced by genetic divergence. Macrocysts were less likely to form between pairs of clones from different groups than from the same group. There was also a correlation between the genetic divergence of a pair of clones and their degree of mixing within fruiting bodies. These observations suggest that cryptic species might occur within D. purpureum and, more importantly, reveal how genetic variation impacts social interactions.

Transcription factor Dd-STATa, a functional Dictyostelium homologue of metazoan signal transducers and activators of transcription proteins, is necessary for culmination during development. We have isolated more than 18 putative multicopy suppressors of Dd-STATa using genetic screening. One was hssA gene, whose expression is known to be G-box-binding-factor-dependent and which was specific to prestalk A (pstA) cells, where Dd-STATa is activated. Also, hssA mRNA was expressed in pstA cells in the Dd-STATa-null mutant. At least 40 hssA-related genes are present in the genome and constitute a multigene family. The tagged HssA protein was translated; hssA encodes an unusually high-glycine-serine-rich small protein (8.37 kDa), which has strong homology to previously reported cyclic-adenosine-monophosphate-inducible 2C and 7E proteins. Overexpression of hssA mRNA as well as frame-shifted versions of hssA RNA suppressed the phenotype of the partially active Dd-STATa strain, suggesting that translation is not necessary for suppression. Although overexpression of prespore-specific genes among the family did not suppress the parental phenotype, prestalk-specific family members did. Although overexpression of the hssA did not revert the expression of Dd-STATa target genes, and although its suppression mechanism remains unknown, morphological reversion implies functional relationships between Dd-STATa and hssA.

The variability and adaptability of the amoebae from the class Dictyosteliomycetes greatly complicate their systematics. The nucleotide sequences of the ribosomal internal transcribed spacers and the 5.8S ribosomal DNA gene have been determined for 28 isolates, and their utility to discriminate between different species and genera has been shown.

The social amoebas (Dictyostelia) display conditional multicellularity in a wide variety of forms. Despite widespread interest in Dictyostelium discoideum as a model system, almost no molecular data exist from the rest of the group. We constructed the first molecular phylogeny of the Dictyostelia with parallel small subunit ribosomal RNA and a-tubulin data sets, and we found that dictyostelid taxonomy requires complete revision. A mapping of characters onto the phylogeny shows that the dominant trend in dictyostelid evolution is increased size and cell type specialization of fruiting structures, with some complex morphologies evolving several times independently. Thus, the latter may be controlled by only a few genes, making their underlying mechanisms relatively easy to unravel.

Kin recognition helps cooperation to evolve in many animals, but it is uncertain whether microorganisms can also use it to focus altruistic behaviour on relatives. Here we show that the social amoeba Dictyostelium purpureum prefers to form groups with its own kin in situations where some individuals die to assist others. By directing altruism towards kin, D. purpureum should generally avoid the costs of chimaerism experienced by the related D. discoideum.

In Dictyostelium discoideum Ax2 l-erythro-tetrahydrobiopterin (BH4) is produced in much smaller amount than its stereoisomer d-threo-tetrahydrobiopterin (DH4), both of which are catalyzed by sepiapterin reductase (SR) at the terminal steps. In order to investigate their putative function and biosynthetic regulation, we performed quantitative analysis of not only the intracellular pteridines by HPLC but also the biosynthetic enzymes (GTP cyclohydrolase I, 6-pyruvoyltetrahydropterin synthase, SR, and aldose reductase-like enzyme) by Northern blot analysis and activity assay. We found that both SR transcript and activity increased in parallel with a remarkable decline in aldose reductase-like enzyme activity when BH4 increased transiently in the early development. Through in vitro assay of BH4/DH4 synthesis and in vivo rescue experiment of SR knockout mutant, we demonstrated that Dictyostelium SR favors DH4 synthesis while human SR does BH4 synthesis. The results suggest that Dictyostelium SR prefers 1'-oxo-2'-d-hydroxypropyl-tetrahydropterin to 6-pyruvoyltetrahydropterin as a substrate, thereby maintaining dominant production of DH4 over BH4 in sufficient supply of AR-like enzyme, while allowing increase of BH4 when SR prevails quantitatively over aldose reductase-like enzyme. On the other hand, a transient increase of BH4 may imply that BH4 has an independent function from DH4 in Dictyostelium.

The Amoebozoa are a sister clade to the fungi and the animals, but are poorly sampled for completely sequenced genomes. The social amoeba Dictyostelium discoideum and amitochondriate pathogen Entamoeba histolytica are the first Amoebozoa with genomes completely sequenced. Both organisms are classified under the Conosa subphylum. To identify Amoebozoa-specific genomic elements, we compared these two genomes to each other and to other eukaryotic genomes. An expanded phylogenetic tree built from the complete predicted proteomes of 23 eukaryotes places the two amoebae in the same lineage, although the divergence is estimated to be greater than that between animals and fungi, and probably happened shortly after the Amoebozoa split from the opisthokont lineage. Most of the 1,500 orthologous gene families shared between the two amoebae are also shared with plant, animal, and fungal genomes. We found that only 42 gene families are distinct to the amoeba lineage; among these are a large number of proteins that contain repeats of the FNIP domain, and a putative transcription factor essential for proper cell type differentiation in D. discoideum. These Amoebozoa-specific genes may be useful in the design of novel diagnostics and therapies for amoebal pathologies.

The biosynthesis of D-threo-tetrahydrobiopterin (DH4, tetrahydrodictyopterin) in Dictyostelium discoideum Ax2 was investigated through the mutant disrupted in the gene encoding sepiapterin reductase (SR) by insertional inactivation. The mutant cells, being completely devoid of SR protein, showed 18.1% of L-erythro-tetrahydrobiopterin (BH4) and 0.6% of DH4 productions in the wild type cells. The mutant cells were also identified to excrete D- and L-sepiapterin, which were presumed to originate from intracellular 1'-oxo-2'-D-hydroxypropyl- and 1'-oxo-2'-L-hydroxypropyl-tetrahydropterin (H4-pterin), respectively. Furthermore, in a coupled assay with Dictyostelium SR, the mutant cell extract exhibited a novel enzyme activity converting 6-pyruvoyltetrahydropterin to 1'-oxo-2'-D-hydroxypropyl-H4-pterin. These results are clear demonstration of the in vivo synthesis of DH4 via 1'-oxo-2'-D-hydroxypropyl-H4-pterin as well as an alternative synthesis of BH4 and DH4 in the complete absence of SR.

Dictyostelium discoideum is a powerful and genetically tractable model system used for the study of numerous cellular molecular mechanisms including chemotaxis, phagocytosis and signal transduction. The past 2 years have seen a significant expansion in the scope and accessibility of online resources for Dictyostelium. Recent advances have focused on the development of a new comprehensive online resource called dictyBase (http://dictybase.org). This database not only provides access to genomic data including functional annotation of genes, gene products and chromosomal mapping, but also to extensive biological information such as mutant phenotypes and corresponding reference material. In conjunction with additional sites (http://genome. imb-jena.de/dictyostelium/, http://dictyensembl. bioch.bcm.tmc.edu and http://www.sanger.ac.uk/Projects/D_discoideum/) from the genome sequencing and assembly centers, these improvements have expanded the scope of the Dictyostelium databases making them accessible and useful to any researcher interested in comparative and functional genomics in metazoan organisms.

Dictyostelium produces spores at the end of its developmental cycle to propagate the lineage. The spore coat is an essential feature of spore biology contributing a semipermeable chemical and physical barrier to protect the enclosed amoeba. The coat is assembled from secreted proteins and a polysaccharide, and from cellulose produced at the cell surface. They are organized into a polarized molecular sandwich with proteins forming layers surrounding the microfibrillar cellulose core. Genetic and biochemical studies are beginning to provide insight into how the deliveries of protein and cellulose to the cell surface are coordinated and how cysteine-rich domains of the proteins interact to form the layers. A multidomain inner layer protein, SP85/PsB, seems to have a central role in regulating coat assembly and contributing to a core structural module that bridges proteins to cellulose. Coat formation and structure have many parallels in walls from plant, algal, yeast, protist, and animal cells.

The genome of the lower eukaryote Dictyostelium discoideum comprises six chromosomes. Here we report the sequence of the largest, chromosome 2, which at 8 megabases (Mb) represents about 25% of the genome. Despite an A + T content of nearly 80%, the chromosome codes for 2,799 predicted protein coding genes and 73 transfer RNA genes. This gene density, about 1 gene per 2.6 kilobases (kb), is surpassed only by Saccharomyces cerevisiae (one per 2 kb) and is similar to that of Schizosaccharomyces pombe (one per 2.5 kb). If we assume that the other chromosomes have a similar gene density, we can expect around 11,000 genes in the D. discoideum genome. A significant number of the genes show higher similarities to genes of vertebrates than to those of other fully sequenced eukaryotes. This analysis strengthens the view that the evolutionary position of D. discoideum is located before the branching of metazoa and fungi but after the divergence of the plant kingdom, placing it close to the base of metazoan evolution.

The phylogenetic relationships of amoebae are poorly resolved. To address this difficult question, we have sequenced 1,280 expressed sequence tags from Mastigamoeba balamuthi and assembled a large data set containing 123 genes for representatives of three phenotypically highly divergent major amoeboid lineages: Pelobionta, Entamoebidae, and Mycetozoa. Phylogenetic reconstruction was performed on approximately 25,000 aa positions for 30 species by using maximum-likelihood approaches. All well-established eukaryotic groups were recovered with high statistical support, validating our approach. Interestingly, the three amoeboid lineages strongly clustered together in agreement with the Conosa hypothesis [as defined by T. Cavalier-Smith (1998) Biol. Rev. Cambridge Philos. Soc. 73, 203-266]. Two amitochondriate amoebae, the free-living Mastigamoeba and the human parasite Entamoeba, formed a significant sister group to the exclusion of the mycetozoan Dictyostelium. This result suggested that a part of the reductive process in the evolution of Entamoeba (e.g., loss of typical mitochondria) occurred in its free-living ancestors. Applying this inexpensive expressed sequence tag approach to many other lineages will surely improve our understanding of eukaryotic evolution.

Rho GTPases are ubiquitously expressed across the eukaryotes where they act as molecular switches, cycling between an active GTP-bound state and an inactive GDP-bound state. Activation enables Rho GTPases to interact with a multitude of effectors that relay upstream signals to cytoskeletal and other components, eliciting rearrangements of the actin cytoskeleton and diverse other cellular responses. In Dictyostelium the Rho family comprises 15 members. Some of them (Rac1a/b/c, RacF1/F2, RacB) are members of the Rac subfamily, and one, RacA, belongs to the RhoBTB subfamily, however the Rho and Cdc42 subfamilies are not represented. Dictyostelium Rho GTPases regulate actin polymerization, cell morphology, endocytosis, cytokinesis, cell polarity and chemotaxis. Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) modulate the activation/inactivation cycle of the GTPases. In addition, guanine nucleotide-dissociation inhibitors (GDIs) regulate cycling of the GTPases between membranes and cytosol. Members of these three classes of regulatory molecules along with some effectors have been identified in Dictyostelium during the last years and their role in Rho signaling pathways has been investigated.

For the elucidation of the diversity of secondary metabolites of Dictyostelium cellular slime molds, we investigate the constituent of three species of slime molds. From the methanol extract of their fruit bodies, we obtained three novel compounds, dictyopyrone A (1) and B (2) from D. discoideum and D. rhizoposium and dictyopyrone C (3) from D. longosporum. They possess a unique alpha-pyrone moiety with a side chain at the C-3 position. Their relative structures were elucidated by spectral means, and the absolute configuration was confirmed by asymmetric synthesis of 1. Since these compounds were obtained from different species of Dictyostelium slime molds, they may be a type of compound common to this genus.

A variety of extracellular signals lead to the accumulation of cAMP which can act as a second message within cells by activating protein kinase A (PKA). Expression of many of the essential developmental genes in Dictyostelium discoideum are known to depend on PKA activity. Cells in which the receptor-coupled adenylyl cyclase gene, acaA, is genetically inactivated grow well but are unable to develop. Surprisingly, acaA(-) mutant cells can be rescued by developing them in mixtures with wild-type cells, suggesting that another adenylyl cyclase is present in developing cells that can provide the internal cAMP necessary to activate PKA. However, the only other known adenylyl cyclase gene in Dictyostelium, acgA, is only expressed during germination of spores and plays no role in the formation of fruiting bodies. By screening morphological mutants generated by Restriction Enzyme Mediated Integration (REMI) we discovered a novel adenylyl cyclase gene, acrA, that is expressed at low levels in growing cells and at more than 25-fold higher levels during development. Growth and development up to the slug stage are unaffected in acrA(-) mutant strains but the cells make almost no viable spores and produce unnaturally long stalks. Adenylyl cyclase activity increases during aggregation, plateaus during the slug stage and then increases considerably during terminal differentiation. The increase in activity following aggregation fails to occur in acrA(-) cells. As long as ACA is fully active, ACR is not required until culmination but then plays a critical role in sporulation and construction of the stalk.

The phylogenetic relationships between major slime mould groups and the identification of their unicellular relatives has been a subject of controversy for many years. Traditionally, it has been assumed that two slime mould groups, the acrasids and the dictyostelids were related by virtue of their cellular slime mould habit; a view still endorsed by at least one current classification scheme. However, a decade ago, on the basis of detailed ultrastructural resemblances it was proposed that acrasids of the family Acrasidae were not relatives of other slime moulds but instead related to a group of mostly free-living unicellular amoebae, the Schizopyrenida. The class Heterolobosea was created to contain these organisms and has since figured in many discussions of protist evolution. We sought to test the validity of Heterolobosea by characterizing homologs of the highly conserved glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from an acrasid, Acrasis rosea; a dictyostelid, Dictyostelium discoideum; and the schizopyrenid Naegleria andersoni. Phylogenetic analysis of these and other GAPDH sequences, using maximum parsimony, neighbour-joining distance and maximum likelihood methods strongly supports the Heterolobosea hypothesis and discredits the concept of a cellular slime mould grouping. Moreover, all of our analyses place Dictyostelium discoideum as a relatively recently originating lineage, most closely related to the Metazoa, similar to other recently published phylogenies of protein-coding genes. However, GAPDH phylogenies do not show robust branching orders for most of the relationships between major groups. We propose that several of the incongruencies observed between GAPDH and other molecular phylogenies are artifacts resulting from substitutional saturation of this enzyme.

Chlorarachniophytes are amoeboid algae with chlorophyll a and b containing plastids that are surrounded by four membranes instead of two as in plants and green algae. These extra membranes form important support for the hypothesis that chlorarachniophytes have acquired their plastids by the ingestion of another eukaryotic plastid-containing alga. Chlorarachniophytes also contain a small nucleus-like structure called the nucleomorph situated between the two inner and the two outer membranes surrounding the plastid. This nucleomorph is a remnant of the endosymbiont's nucleus and encodes, among other molecules, small subunit ribosomal RNA. Previous phylogenetic analyses on the basis of this molecule provided unexpected and contradictory evidence for the origin of the chlorarachniophyte endosymbiont. We developed a new method for measuring the substitution rates of the individual nucleotides of small subunit ribosomal RNA. From the resulting substitution rate distribution, we derived an equation that gives a more realistic relationship between sequence dissimilarity and evolutionary distance than equations previously available. Phylogenetic trees constructed on the basis of evolutionary distances computed by this new method clearly situate the chlorarachniophyte nucleomorphs among the green algae. Moreover, this relationship is confirmed by transversion analysis of the Chlorarachnion plastid small subunit ribosomal RNA.

The evolutionary relationship of Dictyostelium discoideum to the yeasts, fungi, plants, and animals is considered on the basis of physiological, morphological and molecular characteristics. Previous analyses of five proteins indicated that Dictyostelium diverged after the yeasts but before the metazoan radiation. However, analyses of the small ribosomal subunit RNA indicated divergence prior to the yeasts. We have extended the molecular phylogenetic analyses to six more proteins and find consistent evidence for a more recent common ancestor with metazoans than yeast. A consensus phylogeny generated from these new results by both distance matrix and parsimony analyses establishes Dictyostelum's place in evolution between the yeasts Saccharomyces cerevisiae and Schizzosaccharomyces pombe and the worm Caenorhabditis elegans.

The phylogenetic position of Dictyostelium inferred from 18S rRNA data contradicts that from protein data. Protein trees always show the close affinity of Dictyostelium with animals, fungi, and plants, whereas in 18S rRNA trees the branching of Dictyostelium is placed at a position before the massive radiation of protist groups including the divergence of the three kingdoms. To settle this controversial issue and to determine the correct position of Dictyostelium, we inferred the phylogenetic relationship among Dictyostelium and the three kingdoms Animalia, Fungi, and Plantae by a maximum-likelihood method using 19 different protein data sets. It was shown at the significance level of 1 SE that the branching of Dictyostelium antedates the divergence of Animalia and Fungi, and Plantae is an outgroup of the Animalia-Fungi-Dictyostelium clade.