Phylogenetic placement of the protosteloid amoeba Microglomus paxillus identifies another case of sporocarpic fruiting in Discosea (Amoebozoa).

Protosteloid amoebae are a paraphyletic assemblage of amoeboid protists found exclusively in the eukaryotic assemblage Amoebozoa. These amoebae can facultatively form a dispersal structure known as a fruiting body, or more specifically, a sporocarp, from a single amoeboid cell. Sporocarps consist of one to a few spores atop a noncellular stalk. Protosteloid amoebae are known in two out of three well-established major assemblages of Amoebozoa. Amoebae with a protosteloid life cycle are known in the major Amoebozoa lineages Discosea and Evosea but not in Tubulinea. To date, only one genus, which is monotypic, lacks sequence data and, therefore, remains phylogenetically homeless. To further clarify the evolutionary milieu of sporocarpic fruiting we used single-cell transcriptomics to obtain data from individual sporocarps of isolates of the protosteloid amoeba Microglomus paxillus. Our phylogenomic analyses using 229 protein coding markers suggest that M. paxillus is a member of the Discosea lineage of Amoebozoa most closely related to Mycamoeba gemmipara. Due to the hypervariable nature of the SSU rRNA sequence we were unable to further resolve the phylogenetic position of M. paxillus in taxon rich datasets using only this marker. Regardless, our results widen the known distribution of sporocarpy in Discosea and stimulate the debate between a single or multiple origins of sporocarpic fruiting in Amoebozoa.

Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes.

This revision of the classification of eukaryotes follows that of Adl et al., 2012 [J. Euk. Microbiol. 59(5)] and retains an emphasis on protists. Changes since have improved the resolution of many nodes in phylogenetic analyses. For some clades even families are being clearly resolved. As we had predicted, environmental sampling in the intervening years has massively increased the genetic information at hand. Consequently, we have discovered novel clades, exciting new genera and uncovered a massive species level diversity beyond the morphological species descriptions. Several clades known from environmental samples only have now found their home. Sampling soils, deeper marine waters and the deep sea will continue to fill us with surprises. The main changes in this revision are the confirmation that eukaryotes form at least two domains, the loss of monophyly in the Excavata, robust support for the Haptista and Cryptista. We provide suggested primer sets for DNA sequences from environmental samples that are effective for each clade. We have provided a guide to trophic functional guilds in an appendix, to facilitate the interpretation of environmental samples, and a standardized taxonomic guide for East Asian users.

Between a Pod and a Hard Test: The Deep Evolution of Amoebae.

Amoebozoa is the eukaryotic supergroup sister to Obazoa, the lineage that contains the animals and Fungi, as well as their protistan relatives, and the breviate and apusomonad flagellates. Amoebozoa is extraordinarily diverse, encompassing important model organisms and significant pathogens. Although amoebozoans are integral to global nutrient cycles and present in nearly all environments, they remain vastly understudied. We present a robust phylogeny of Amoebozoa based on broad representative set of taxa in a phylogenomic framework (325 genes). By sampling 61 taxa using culture-based and single-cell transcriptomics, our analyses show two major clades of Amoebozoa, Discosea, and Tevosa. This phylogeny refutes previous studies in major respects. Our results support the hypothesis that the last common ancestor of Amoebozoa was sexual and flagellated, it also may have had the ability to disperse propagules from a sporocarp-type fruiting body. Overall, the main macroevolutionary patterns in Amoebozoa appear to result from the parallel losses of homologous characters of a multiphase life cycle that included flagella, sex, and sporocarps rather than independent acquisition of convergent features.

Variation in the SSUrDNA of the Genus Protostelium Leads to a New Phylogenetic Understanding of the Genus and of the Species Concept for Protostelium mycophaga (Protosteliida, Amoebozoa).

Members of the genus Protostelium (including P. mycophaga, P. nocturnum, and P. okumukumu) are protosteloid amoebae commonly found in terrestrial habitats on dead plant matter. They, along with the closely allied nominal genus Planoprotostelium, containing the single species Pl. aurantium, all have an amoeboid trophic stage with acutely pointed subpseudopodia and orange lipid droplets in the granuloplasm. These amoebae form stalked fruiting bodies topped with a single, usually deciduous spore. The species are identified based on their fruiting body morphologies except for Pl. aurantium which looks similar to P. mycophaga in fruiting morphology, but has amoebae that can make flagella in liquid medium. We built phylogenetic trees using nuclear small subunit ribosomal DNA sequences of 35 isolates from the genera Protostelium and Planoprotostelium and found that (1) the nonflagellated P. nocturnum and P. okumukumu branch basally in the genus Protostelium, (2) the flagellate, Pl. aurantium falls within the genus Protostelium in a monophyletic clade with the nominal variety, P. mycophaga var. crassipes, (3) the cultures initially identified as Protostelium mycophaga can be divided into at least three morphologically recognizable taxa, P. aurantium n. comb., P. apiculatum n. sp., and P. m. rodmani n. subsp., as well as a paraphyletic assemblage that includes the remainder of the P. mycophaga morphotype. These findings have implications for understanding the ecology, evolution, and diversity of these amoeboid organisms and for using these amoebae as models for other amoeboid groups.

Sorodiplophrys stercorea: Another Novel Lineage of Sorocarpic Multicellularity.

Sorodiplophrys stercorea is a sorocarpic organism that utilizes filose pseudopodia for locomotion and absorptive nutrition. It has traditionally been considered to be a member of the Labyrinthulae based on its morphology. Its closest relatives were thought to be species in the taxon Diplophrys. Since the genus Diplophrys has been shown to be paraphyletic and S. stercorea has pseudopodia similar to some members of Rhizaria, we examined its relationship with other eukaryotes. We obtained four isolates from the dung of cow and horse, brought each into monoeukaryotic culture, and sequenced their SSU rRNA gene for phylogenetic analysis. All our isolates were shown to form a monophyletic group in the Labyrinthulae, nested in the Amphifiloidea clade. Our results demonstrate that Sorodiplophrys is more closely related to species of the genus Amphifila than to Diplophrys and represents an additional independent origin of sorocarpic multicellularity among eukaryotes. This study represents the first confirmed sorocarpic lifestyle in the Stramenopiles.

Contractile vacuoles: a rapidly expanding (and occasionally diminishing?) understanding.

Osmoregulation is the homeostatic mechanism essential for the survival of organisms in hypoosmotic and hyperosmotic conditions. In freshwater or soil dwelling protists this is frequently achieved through the action of an osmoregulatory organelle, the contractile vacuole. This endomembrane organelle responds to the osmotic challenges and compensates by collecting and expelling the excess water to maintain the cellular osmolarity. As compared with other endomembrane organelles, this organelle is underappreciated and under-studied. Here we review the reported presence or absence of contractile vacuoles across eukaryotic diversity, as well as the observed variability in the structure, function, and molecular machinery of this organelle. Our findings highlight the challenges and opportunities for constructing cellular and evolutionary models for this intriguing organelle.

The revised classification of eukaryotes.

This revision of the classification of eukaryotes, which updates that of Adl et al. [J. Eukaryot. Microbiol. 52 (2005) 399], retains an emphasis on the protists and incorporates changes since 2005 that have resolved nodes and branches in phylogenetic trees. Whereas the previous revision was successful in re-introducing name stability to the classification, this revision provides a classification for lineages that were then still unresolved. The supergroups have withstood phylogenetic hypothesis testing with some modifications, but despite some progress, problematic nodes at the base of the eukaryotic tree still remain to be statistically resolved. Looking forward, subsequent transformations to our understanding of the diversity of life will be from the discovery of novel lineages in previously under-sampled areas and from environmental genomic information.

Microhabitat and climatic preferences of protosteloid amoebae in a region with a Mediterranean climate.

The role of microhabitat and climate variation in structuring protosteloid amoebae communities has been investigated for the first time in the Mediterranean Basin, a biodiversity hotspot for plants and animals and the largest of the world's five areas with a Mediterranean climate. Abundance data were obtained from natural substrates collected in 13 localities from central Spain, and a total of 1,504 colonies and 18 species were recorded. For this new area, it has been carried out an optimization of the culturing effort based on rarefaction analyses, thus making possible to adapt the protocol to the objectives in future research. Canonical correspondence analysis and generalized linear models showed that microhabitat type was the most important factor for differentiating the niches of the species studied, but climatic variables, especially minimum temperature of the coldest month, precipitation seasonality, and temperature range, had secondary but also important effects. Bark inhabitants tend to be more abundant in localities with high temperature range and low annual precipitation. Aerial litter was the microhabitat with the highest species richness, abundance, and evenness. Species typical of this microhabitat are more abundant when there is high precipitation, low temperature of the warmest month, and low minimum temperature of the coldest month.

Phylogeny of the "forgotten" cellular slime mold, Fonticula alba, reveals a key evolutionary branch within Opisthokonta.

The shared ancestry between Fungi and animals has been unequivocally demonstrated by abundant molecular and morphological data for well over a decade. Along with the animals and Fungi, multiple protists have been placed in the supergroup Opisthokonta making it exceptionally diverse. In an effort to place the cellular slime mold Fonticula alba, an amoeboid protist with aggregative, multicellular fruiting, we sequenced five nuclear encoded genes; small subunit ribosomal RNA, actin, beta-tubulin, elongation factor 1-alpha, and the cytosolic isoform of heat shock protein 70 for phylogenetic analyses. Molecular trees demonstrate that Fonticula is an opisthokont that branches sister to filose amoebae in the genus Nuclearia. Fonticula plus Nuclearia are sister to Fungi. We propose a new name for this well-supported clade, Nucletmycea, incorporating Nuclearia, Fonticula, and Fungi. Fonticula represents the first example of a cellular slime mold morphology within Opisthokonta. Thus, there are four types of multicellularity in the supergroup-animal, fungal, colonial, and now aggregative. Our data indicate that multicellularity in Fonticula evolved independent of that found in the fungal and animal radiations. With the rapidly expanding sequence and genomic data becoming available from many opisthokont lineages, Fonticula may be fundamental to understanding opisthokont evolution as well as any possible commonalities involved with the evolution of multicellularity.

A morphologically simple species of Acrasis (Heterolobosea, Excavata), Acrasis helenhemmesae n. sp.

In the course of a large-scale global survey of mycetozoans, amoeboid organisms that form fruiting bodies, a new species of Acrasis was discovered from several subtropical locales in Hawaii, Australia, Bermuda, and South Africa. We isolated four strains from dead, still attached, plant material, and one strain from attached bark of a tree. Each isolate forms simple uniseriate multicellular fruiting bodies typically consisting of two bottle-shaped, basal stalk cells and a chain of <20 spores. The isolate from Bermuda often forms dichotomous simple branches, each consisting of <10 spores. Amoebae from these new isolates are limax with eruptive pseudopodial formation and display rapid locomotion-characters indicative of amoebae in the excavate taxon Heterolobosea. These isolates form simpler fruiting bodies than is typical of the well-known Acrasis rosea. Although in the original description, A. rosea is known to form uniseriate fruiting bodies similar to our isolates, A. rosea isolates typically form more complex fruiting structures along side simple ones, but never strictly simple ones. Nuclear-encoded 18S rRNA gene phylogenies demonstrate that our five isolates form a highly supported clade that is sister to A. rosea. Given the differences both in gene sequences and fruiting body morphology between our isolates and A. rosea, we propose the new species, Acrasis helenhemmesae n. sp.

First records and microhabitat assessment of protostelids in the Aberdare Region, Central Kenya.

A rapid assessment survey on the occurrence and distribution of protosteloid amoebae was carried out in central Kenya. Samples of dead plant materials were collected from 46 study sites (each 20 x 20 m) situated along an elevation gradient (1,785-3,396 m) that encompassed five major land use/cover types. Twenty-four species and subspecific taxa were recovered and included 23 protostelids and one minute myxomycete, often included in surveys for protostelids. All of these were the first records for Kenya, and six were new for Africa. Numbers of taxa were highest in ground litter and aerial litter microhabitats (20 taxa each) and lowest on aerial bark (10) and ground bark (7). Relative species abundance was greatest in aerial litter, moderate in ground litter, and low on aerial and ground bark microhabitats. The most frequently occurring species on ground litter were Schizoplasmodiopsis pseudoendospora, Schizoplasmodiopsis amoeboidea, and Protostelium mycophaga var. mycophaga, whereas the most common species on aerial litter were P. mycophaga var. mycophaga and Soliformovum irregularis. Species richness and abundance decreased with increasing elevation.

Distribution and ecology of protostelids in Great Smoky Mountains National Park.

In Oct. 2000 and Nov. 2003 samples for isolation of protostelids were collected from throughout Great Smoky Mountains National Park. Collecting sites included examples of all major forest types found in the park along with a number of non-forest vegetation types at 460-1920 m above sea level. Most samples consisted of aerial litter (dead but still attached plant parts) and ground litter, but some samples of the bark from living trees and coarse woody debris also were collected. Twenty-six of the 32 described species of protostelids were recovered, along with five species that appear to be new to science. Of the 205 cultures analyzed, 177 (86.5%) yielded protostelid fruiting bodies with an average of 3.22 species per sample. Protostelium mycophaga was the most frequently observed species followed by Soliformovum irregularis and Schizoplasmodiopsis pseudoendospora. Differences in protostelid community diversity and species richness were found between the aerial litter and ground litter microhabitats of primary plant tissues, between primary tissues and bark of living trees, between bark from angiosperms and bark from gymnosperms, and between primary tissues and coarse woody debris.

Soil protists: a fertile frontier in soil biology research.

Protists include all eukaryotes except plants, fungi and animals. They are an essential, yet often forgotten, component of the soil microbiome. Method developments have now furthered our understanding of the real taxonomic and functional diversity of soil protists. They occupy key roles in microbial foodwebs as consumers of bacteria, fungi and other small eukaryotes. As parasites of plants, animals and even of larger protists, they regulate populations and shape communities. Pathogenic forms play a major role in public health issues as human parasites, or act as agricultural pests. Predatory soil protists release nutrients enhancing plant growth. Soil protists are of key importance for our understanding of eukaryotic evolution and microbial biogeography. Soil protists are also useful in applied research as bioindicators of soil quality, as models in ecotoxicology and as potential biofertilizers and biocontrol agents. In this review, we provide an overview of the enormous morphological, taxonomical and functional diversity of soil protists, and discuss current challenges and opportunities in soil protistology. Research in soil biology would clearly benefit from incorporating more protistology alongside the study of bacteria, fungi and animals.

A New Classification of the Dictyostelids.

Traditional morphology-based taxonomy of dictyostelids is rejected by molecular phylogeny. A new classification is presented based on monophyletic entities with consistent and strong molecular phylogenetic support and that are, as far as possible, morphologically recognizable. All newly named clades are diagnosed with small subunit ribosomal RNA (18S rRNA) sequence signatures plus morphological synapomorphies where possible. The two major molecular clades are given the rank of order, as Acytosteliales ord. nov. and Dictyosteliales. The two major clades within each of these orders are recognized and given the rank of family as, respectively, Acytosteliaceae and Cavenderiaceae fam. nov. in Acytosteliales, and Dictyosteliaceae and Raperosteliaceae fam. nov. in Dictyosteliales. Twelve genera are recognized: Cavenderia gen. nov. in Cavenderiaceae, Acytostelium, Rostrostelium gen. nov. and Heterostelium gen. nov. in Acytosteliaceae, Tieghemostelium gen. nov., Hagiwaraea gen. nov., Raperostelium gen. nov. and Speleostelium gen. nov. in Raperosteliaceae, and Dictyostelium and Polysphondylium in Dictyosteliaceae. The "polycephalum" complex is treated as Coremiostelium gen. nov. (not assigned to family) and the "polycarpum" complex as Synstelium gen. nov. (not assigned to order and family). Coenonia, which may not be a dictyostelid, is treated as a genus incertae sedis. Eighty-eight new combinations are made at species and variety level, and Dictyostelium ammophilum is validated.

Protostelids and myxomycetes isolated from aquatic habitats.

Protostelids and myxomycetes have been isolated from dead plant parts in many different habitats, including tropical rain forests and deserts. However underwater habitats largely have been overlooked. The purpose of this study was to determine whether protostelids do occur in aquatic habitats and to survey the myxomycetes associated with these habitats. Protostelids and myxomycetes were isolated from substrates collected from just above and just below the surface of the water. Several species of both groups were present, and their distributions above and below the water were different. It is not surprising that the trophic cells of slime molds occur in ponds because they are known to grow in films of water. However these findings are significant because this is the first study to demonstrate clearly the occurrence of protostelids in underwater environments and one of the few surveys of myxomycetes from aquatic systems.

Expansion of the molecular and morphological diversity of Acanthamoebidae (Centramoebida, Amoebozoa) and identification of a novel life cycle type within the group.

BACKGROUND: Acanthamoebidae is a "family" level amoebozoan group composed of the genera Acanthamoeba, Protacanthamoeba, and very recently Luapeleamoeba. This clade of amoebozoans has received considerable attention from the broader scientific community as Acanthamoeba spp. represent both model organisms and human pathogens. While the classical composition of the group (Acanthamoeba + Protacanthamoeba) has been well accepted due to the morphological and ultrastructural similarities of its members, the Acanthamoebidae has never been highly statistically supported in single gene phylogenetic reconstructions of Amoebozoa either by maximum likelihood (ML) or Bayesian analyses. RESULTS: Here we show using a phylogenomic approach that the Acanthamoebidae is a fully supported monophyletic group within Amoebozoa with both ML and Bayesian analyses. We also expand the known range of morphological and life cycle diversity found in the Acanthamoebidae by demonstrating that the amoebozoans "Protostelium" arachisporum, Dracoamoeba jormungandri n. g. n. sp., and Vacuolamoeba acanthoformis n.g. n.sp., belong within the group. We also found that "Protostelium" pyriformis is clearly a species of Acanthamoeba making it the first reported sporocarpic member of the genus, that is, an amoeba that individually forms a walled, dormant propagule elevated by a non-cellular stalk. Our phylogenetic analyses recover a fully supported Acanthamoebidae composed of five genera. Two of these genera (Acanthamoeba and Luapeleameoba) have members that are sporocarpic. CONCLUSIONS: Our results provide high statistical support for an Acanthamoebidae that is composed of five distinct genera. This study increases the known morphological diversity of this group and shows that species of Acanthamoeba can include spore-bearing stages. This further illustrates the widespread nature of spore-bearing stages across the tree of Amoebozoa. REVIEWERS: This article was reviewed by Drs. Eugene Koonin, Purificacion Lopez-Garcia and Sandra Baldauf. Sandra Baldauf was nominated by Purificacion Lopez-Garcia, an Editorial Board member.

Taxonomy, slime molds, and the questions we ask.

Taxonomic treatments often influence the way we both ask and attempt to answer certain biological questions. The classical taxonomy of the dictyostelid cellular slime molds (Dictyosteliales) involves a convenient set of categories that were developed independent of phylogeny. In order to test whether the characters supporting the classical taxonomy hold any phylogenetic signal, we subjected 19 described taxa belonging to two families (Acytosteliaceae and Dictyosteliaceae) and three genera (Acytostelium, Dictyostelium, and Polysphondylium) to rooted cladistic analyses using PAUP* v 4.0b4a. Neither family nor any of the three genera were found to represent monophyletic groups. These results confirm that the classical taxonomy used to delineate families and genera within these slime molds carries very little phylogenetic signal. Taxonomic character sets should be scrutinized phylogenetically in order to determine what information they provide about the relatedness of taxa within a group. Because taxonomy often drives the nature of biological inquiry, caution should be exercised when drawing conclusions regarding the evolution of developmental systems in Dictyostelium.

Ecological distribution of protosteloid amoebae in New Zealand.

During the period of March 2004 to December 2007, samples of aerial litter (dead but still attached plant parts) and ground litter (dead plant material on the ground) were collected from 81 study sites representing a wide range of latitudes (34°S to 50°S) and a variety of different types of habitats throughout New Zealand (including Stewart Island and the Auckland Islands). The objective was to survey the assemblages of protosteloid amoebae present in this region of the world. Twenty-nine described species of protosteloid amoebae were recorded by making morphological identifications of protosteloid amoebae fruiting bodies on cultured substrates. Of the species observed, Protostelium mycophaga was by far the most abundant and was found in more than half of all samples. Most species were found in fewer than 10% of the samples collected. Seven abundant or common species were found to display significantly increased likelihood for detection in aerial litter or ground litter microhabitats. There was some evidence of a general correlation between environmental factors - annual precipitation, elevation, and distance from the equator (latitude) - and the abundance and richness of protosteloid amoebae. An increase in each of these three factors correlated with a decrease in both abundance and richness. This study provides a thorough survey of the protosteloid amoebae present in New Zealand and adds to a growing body of evidence which suggests several correlations between their broad distributional patterns and environmental factors.

A revision of the order Pellitida Smirnov et al., 2011 (Amoebozoa, Discosea) based on ultrastructural and molecular evidence, with description of Endostelium crystalliferum n. sp.

We present the results of an ultrastructural re-investigation of two amoebae strains that can be identified morphologically as previously described species of the genus Pellita, as well as the first molecular phylogenetic analysis of these amoebae based on SSU rRNA and actin gene sequences. The results obtained show close relationships between the genera Pellita, Gocevia, and Endostelium. These relationships are further supported by the description of Endostelium crystalliferum n. sp., which shares morphological characters simultaneously with Pellita spp. and Endostelium zonatum. The three genera form a robust clade that branches deeply within Amoebozoa, among either Flabellinia, or Longamoebia, depending on taxon sampling. The results suggest that Gocevia and Endostelium should not be included in the Himatismenida; therefore, we transfer the family Goceviidae into Pellitida. The type of cell organisation that was considered to be typical of Himatismenida (a lens-shaped cell covered dorsally with a flexible layer of organic material) has most probably evolved in some of these amoebae independently of Cochliopodiidae and Parvamoebidae. The robustness of the Pellitida clade in the molecular trees is consistent with the fine structure cytoplasmic evidence for these taxa, in particular, the presence of a centrosphere (a dictyosome-associated lamellar MTOC) in all these genera.