Population-level analyses indirectly reveal cryptic sex and life history traits of Pseudoperkinsus tapetis (Ichthyosporea, Opisthokonta): a unicellular relative of the animals.

We use population genetics to detect the molecular footprint of a sexual cycle, of a haploid vegetative state, and of lack of host specificity in Pseudoperkinsus tapetis, a marine unicellular relative of the animals. Prior to this study, complete life cycles were not known for any of the unicellular lineages sharing common ancestry with multicellular animals and fungi. We established the first collection of conspecific cultures of any member from the unicellular opisthokont lineage ichthyosporea, isolating 126 cultures of P. tapetis from guts of marine invertebrates ranging from clams to sea cucumbers. We sequenced fragments of the elongation factor alpha-like (EFL) and heat-shock protein 70 (HSP70) genes for a subset of our isolates. Absence of heterozygotes from the EFL locus in 52 isolates provided evidence for haploidy. Phylogenetic incongruence and a lack of support for linkage between two loci from 34 sequenced isolates signified a history of recombination consistent with a sexual cycle. Shared haplotypes in different invertebrate species showed that P. tapetis was not host specific. Based on estimates of the frequency of sex and on observations of cultures, we propose that P. tapetis is transmitted between hosts via asexual endospores. New protists are continually being discovered, and, as this study illustrates, analysis of culturable collections from natural habitats can transform a species from a near unknown to a model system for better understanding the evolution of life histories.

Labyrinthulomycetes phylogeny and its implications for the evolutionary loss of chloroplasts and gain of ectoplasmic gliding.

The labyrinthulomycetes, also known as the 'Labyrinthulomycota' are saprotrophic or less frequently parasitic stramenopilan protists, usually in marine ecosystems. Their distinguishing feature is an 'ectoplasmic net,' an external cytoplasmic network secreted by a specialized organelle that attaches the cell to its substrate and secretes digestive enzymes for absorptive nutrition. In this study, one of our aims was to infer the phylogenetic position of the labyrinthulomycetes relative to the non-photosynthetic bicoeceans and oomycetes and the photosynthetic ochrophytes and thereby evaluate patterns of change from photosynthesis to saprotrophism among the stramenopiles. For the labyrinthulomycetes, we determined sequences of the actin, beta-tubulin, and elongation factor 1-alpha gene fragments and where necessary, ribosomal small subunit (SSU) genes. Multilocus analysis using standard tree construction techniques not only strongly supported the oomycetes as the sister group to the phototrophic stramenopiles, but also, for the first time with moderate statistical support, showed that the labyrinthulomycetes and the bicoecean as sister groups. The paraphyly of the non-photosynthetic groups was consistent with independent loss of photosynthesis in labyrinthulomycetes and oomycetes. We also wished to develop a phylogenetically based hypothesis for the origin of the gliding cell bodies and the ectoplasmic net found in some labyrinthulomycetes. The cells of species in Labyrinthula and Aplanochytrium share a specialized form of motility involving gliding on ectoplasmic tracks. Before our study, only ribosomal DNA genes had been determined for these genera and their phylogenetic position in the labyrinthulomycetes was equivocal. Multilocus phylogenies applying our newly determined protein-coding sequences divided the labyrinthulomycetes between sister clades 'A' and 'B' and showed that the monophyletic group containing all of the gliding species was nested among non-gliding species in clade B. This phylogeny suggested that species that glide via an ectoplasm evolved from species that had used the ectoplasm mainly for anchorage and assimilation rather than motility.

Multiple isolations of a culturable, motile Ichthyosporean (Mesomycetozoa, Opisthokonta), Creolimax fragrantissima n. gen., n. sp., from marine invertebrate digestive tracts.

A fragrant, spherical, osmotrophic eukaryote was isolated 27 times from the digestive tracts of marine invertebrates collected from the Northeast Pacific. The isolates were cultured from 7 animal collections over a 2-year period, most from the peanut worm, Phascolosoma agassizii. A small subunit ribosomal DNA phylogeny placed the spherical organism within the ichthyosporea, closest to Sphaeroforma arctica and Pseudoperkinsus tapetis. Supporting the close relationship of isolates, the sequences of ribosomal gene internal transcribed spacers determined for 26 isolates were identical, as were the elongation factor 1-alpha-like gene fragments from 7 isolates. Dispersal via amoeboid cells distinguished this species from its closest relatives and led to the erection of a new genus and species, "Creolimax fragrantissima." Vegetative cells reproduced asexually in vitro after they reached 30-60 microm in diameter by producing amoebae or endospores, which escaped through openings in the parent cell wall. Ultrathin sections of vegetative cells prepared by high-pressure-freeze substitution provided some of the first images of ichthyosporean spindle pole bodies and document, for the first time, tubular extensions of the plasma membrane into an electron-translucent inner layer of the cell wall. Ichthyosporeans are parasites and commensals of animals and culturable species are few. Because "C. fragrantissima" can be isolated regularly and repeatedly from nature and then grown easily through cycles of asexual reproduction, it has the potential to serve as a model organism for further research into marine ichthyosporeans.

Phylogeny of marine Gregarines (Apicomplexa)--Pterospora, Lithocystis and Lankesteria--and the origin(s) of coelomic parasitism.

Gregarines constitute a large group of apicomplexans with diverse modes of nutrition and locomotion that are associated with different host compartments (e.g. intestinal lumena and coelomic cavities). A broad molecular phylogenetic framework for gregarines is needed to infer the early evolutionary history of apicomplexans as a whole and the evolutionary relationships between the diverse ultrastructural and behavioral characteristics found in intestinal and coelomic gregarines. To this end, we sequenced the SSU rRNA gene from (1) Lankesteria abbotti from the intestines of two Pacific appendicularians, (2) Pterospora schizosoma from the coelom of a Pacific maldanid polychaete, (3) Pterospora floridiensis from the coelom of a Gulf Atlantic maldanid polychaete and (4) Lithocystis sp. from the coelom of a Pacific heart urchin. Molecular phylogenetic analyses including the new sequences demonstrated that several environmental and misattributed sequences are derived from gregarines. The analyses also demonstrated a clade of environmental sequences that was affiliated with gregarines, but as yet none of the constituent organisms have been described at the ultrastructural level (apicomplexan clade I). Lankesteria spp. (intestinal parasites of appendicularians) grouped closely with other marine intestinal eugregarines, particularly Lecudina tuzetae, from polychaetes. The sequences from all three coelomic gregarines branched within a larger clade of intestinal eugregarines and were similarly highly divergent. A close relationship between Pterospora schizosoma (Pacific) and Pterospora floridiensis (Gulf Atlantic) was strongly supported by the data. Lithocystis sp. was more closely related to a clade of marine intestinal gregarines consisting of Lankesteria spp. and Lecudina spp. than it was to the Pterospora clade. These data suggested that coelomic parasitism evolved more than once from different marine intestinal eugregarines, although a larger taxon sample is needed to further explore this inference.

Current ecological understanding of fungal-like pathogens of fish: what lies beneath?

Despite increasingly sophisticated microbiological techniques, and long after the first discovery of microbes, basic knowledge is still lacking to fully appreciate the ecological importance of microbial parasites in fish. This is likely due to the nature of their habitats as many species of fish suffer from living beneath turbid water away from easy recording. However, fishes represent key ecosystem services for millions of people around the world and the absence of a functional ecological understanding of viruses, prokaryotes, and small eukaryotes in the maintenance of fish populations and of their diversity represents an inherent barrier to aquatic conservation and food security. Among recent emerging infectious diseases responsible for severe population declines in plant and animal taxa, fungal and fungal-like microbes have emerged as significant contributors. Here, we review the current knowledge gaps of fungal and fungal-like parasites and pathogens in fish and put them into an ecological perspective with direct implications for the monitoring of fungal fish pathogens in the wild, their phylogeography as well as their associated ecological impact on fish populations. With increasing fish movement around the world for farming, releases into the wild for sport fishing and human-driven habitat changes, it is expected, along with improved environmental monitoring of fungal and fungal-like infections, that the full extent of the impact of these pathogens on wild fish populations will soon emerge as a major threat to freshwater biodiversity.

Comparative morphology and genealogical delimitation of cryptic species of sympatric isolates of Sphaeroforma (Ichthyosporea, Opisthokonta).

Of the ancient clades of unicellular relatives of the multicellular animals, ichthyosporea are among the easiest to collect, cultivate, and analyze at the population level. Once identified, species can be correlated with their animal hosts and geographical ranges. However, the spherical stages common to many ichthyosporea provide little basis for morphological species identification. This study of the genus Sphaeroforma is the first to apply patterns of genetic discontinuity to delimit species among any of the unicellular 'holozoa.' Sequences of three loci from 148 sympatric isolates, along with type cultures, provided concordant support for new species "Sphaeroforma nootkatensis" and "Sphaeroforma gastrica," and for formally describing 'Pseudoperkinsus tapetis,' as "Sphaeroforma tapetis". We document light and electron microscopic characters that distinguish the genus but not its species. "S. tapetis" sometimes had brief amoeboid or plasmodial motile stages and endospore release through pores. Unlike closely related Creolimax, Sphaeroforma lacked a central vacuole but had multiple peripheral nucleoli. Like distantly related eccrinales, Sphaeroforma cell walls had pores and a calyx. Analyses of allele frequencies in "S. tapetis" indicated geographical differentiation but no host specificity. Accurate molecular identification of species will increase the feasibility and reliability of further studies of Sphaeroforma in its natural habit.

Molecular phylogeny of unikonts: new insights into the position of apusomonads and ancyromonads and the internal relationships of opisthokonts.

The eukaryotic supergroup Opisthokonta includes animals (Metazoa), fungi, and choanoflagellates, as well as the lesser known unicellular lineages Nucleariidae, Fonticula alba, Ichthyosporea, Filasterea and Corallochytrium limacisporum. Whereas the evolutionary positions of the well-known opisthokonts are mostly resolved, the phylogenetic relationships among the more obscure lineages are not. Within the Unikonta (Opisthokonta and Amoebozoa), it has not been determined whether the Apusozoa (apusomonads and ancyromonads) or the Amoebozoa form the sister group to opisthokonts, nor to which side of the hypothesized unikont/bikont divide the Apusozoa belong. Aiming at elucidating the evolutionary tree of the unikonts, we have assembled a dataset with a large sampling of both organisms and genes, including representatives from all known opisthokont lineages. In addition, we include new molecular data from an additional ichthyosporean (Creolimax fragrantissima) and choanoflagellate (Codosiga botrytis). Our analyses show the Apusozoa as a paraphyletic assemblage within the unikonts, with the Apusomonadida forming a sister group to the opisthokonts. Within the Holozoa, the Ichthyosporea diverge first, followed by C. limacisporum, the Filasterea, the Choanoflagellata, and the Metazoa. With our data-enriched tree, it is possible to pinpoint the origin and evolution of morphological characters. As an example, we discuss the evolution of the unikont kinetid.

Facing unknowns: living cultures (Pirum gemmata gen. nov., sp. nov., and Abeoforma whisleri, gen. nov., sp. nov.) from invertebrate digestive tracts represent an undescribed clade within the unicellular Opisthokont lineage ichthyosporea (Mesomycetozoea).

During a culture-based survey of opisthokonts living in marine invertebrate digestive tracts, we isolated two new eukaryotes that differed from described taxa by more than 10% in their small subunit ribosomal DNA sequences. Phylogenetic analysis showed that the two isolates represented a divergent clade of ichthyosporeans known previously only from environmental clone sequences. We used light and electron microscopy to describe the isolates as new genera and species Pirum gemmata and Abeoforma whisleri. A. whislerihad a complex life cycle that remains incompletely known but involved walled spherical cells, plasmodia and amoebae. Asexual reproduction occurred via dispersal amoebae, endospores, binary fission and budding. In contrast P. gemmatahad a less complex life cycle with no amoeboid or plasmodial stages. Both species had membrane-bound tubular extensions of the cytoplasm embedded in the inner layers of their cell walls. By comparing P. gemmata and A. whislerito other ichthyosporea we speculate on the characters that may have been present in the ancestral ichthyosporean. P. gemmata and A. whisleri illustrate the unique and diverse forms that can be found by capturing taxa belonging to divergent and uncultured lineages.