Do we need many genes for phylogenetic inference?

Fifty-six nuclear protein coding genes from Taxonomically Broad EST Database and other databases were selected for phylogenomic-based examination of alternative phylogenetic hypotheses concerning intergroup relationship between multicellular animals (Metazoa) and other representatives of Opisthokonta. The results of this work support sister group relationship between Metazoa and Choanoflagellata. Both of these groups form the taxon Holozoa along with the monophyletic Ichthyosporea or Mesomycetozoea (a group that includes Amoebidium parasiticum, Sphaeroforma arctica, and Capsaspora owczarzaki). These phylogenetic hypotheses receive high statistical support both when utilizing whole alignment and when only 5000 randomly selected alignment positions are used. The presented results suggest subdivision of Fungi into Eumycota and lower fungi, Chytridiomycota. The latter form a monophyletic group that comprises Chytridiales+Spizellomycetales+Blastocladiales (Batrachochytrium, Spizellomyces, Allomyces, Blastocladiella), contrary to the earlier reports based on the analysis of 18S rRNA and a limited set of protein coding genes. The phylogenetic distribution of genes coding for a ubiquitin-fused ribosomal protein S30 implies at least three independent cases of gene fusion: in the ancestors of Holozoa, in heterotrophic Heterokonta (Oomycetes and Blastocystis) and in the ancestors of Cryptophyta and Glaucophyta. Ubiquitin-like sequences fused with ribosomal protein S30 outside of Holozoa are not FUBI orthologs. Two independent events of FUBI replacement by the ubiquitin sequence were detected in the lineage of C. owczarzaki and in the monophyletic group of nematode worms Tylenchomorpha+Cephalobidae. Bursaphelenchus xylophilus (Aphelenchoidoidea) retains a state typical of the rest of the Metazoa. The data emphasize the fact that the reliability of phylogenetic reconstructions depends on the number of analyzed genes to a lesser extent than on our ability to recognize reconstruction artifacts.     

Phylogenetic relationships within the Opisthokonta based on phylogenomic analyses of conserved single-copy protein domains

Many of the eukaryotic phylogenomic analyses published to date were based on alignments of hundreds to thousands of genes. Frequently, in such analyses, the most realistic evolutionary models currently available are often used to minimize the impact of systematic error. However, controversy remains over whether or not idiosyncratic gene family dynamics (i.e., gene duplications and losses) and incorrect orthology assignments are always appropriately taken into account. In this paper, we present an innovative strategy for overcoming orthology assignment problems. Rather than identifying and eliminating genes with paralogy problems, we have constructed a data set comprised exclusively of conserved single-copy protein domains that, unlike most of the commonly used phylogenomic data sets, should be less confounded by orthology miss-assignments. To evaluate the power of this approach, we performed maximum likelihood and Bayesian analyses to infer the evolutionary relationships within the opisthokonts (which includes Metazoa, Fungi, and related unicellular lineages). We used this approach to test 1) whether Filasterea and Ichthyosporea form a clade, 2) the interrelationships of early-branching metazoans, and 3) the relationships among early-branching fungi. We also assessed the impact of some methods that are known to minimize systematic error, including reducing the distance between the outgroup and ingroup taxa or using the CAT evolutionary model. Overall, our analyses support the Filozoa hypothesis in which Ichthyosporea are the first holozoan lineage to emerge followed by Filasterea, Choanoflagellata, and Metazoa. Blastocladiomycota appears as a lineage separate from Chytridiomycota, although this result is not strongly supported. These results represent independent tests of previous phylogenetic hypotheses, highlighting the importance of sophisticated approaches for orthology assignment in phylogenomic analyses.     

Multigene phylogeny of choanozoa and the origin of animals

Animals are evolutionarily related to fungi and to the predominantly unicellular protozoan phylum Choanozoa, together known as opisthokonts. To establish the sequence of events when animals evolved from unicellular ancestors, and understand those key evolutionary transitions, we need to establish which choanozoans are most closely related to animals and also the evolutionary position of each choanozoan group within the opisthokont phylogenetic tree. Here we focus on Ministeria vibrans, a minute bacteria-eating cell with slender radiating tentacles. Single-gene trees suggested that it is either the closest unicellular relative of animals or else sister to choanoflagellates, traditionally considered likely animal ancestors. Sequencing thousands of Ministeria protein genes now reveals about 14 with domains of key significance for animal cell biology, including several previously unknown from deeply diverging Choanozoa, e.g. domains involved in hedgehog, Notch and tyrosine kinase signaling or cell adhesion (cadherin). Phylogenetic trees using 78 proteins show that Ministeria is not sister to animals or choanoflagellates (themselves sisters to animals), but to Capsaspora, another protozoan with thread-like (filose) tentacles. The Ministeria/Capsaspora clade (new class Filasterea) is sister to animals and choanoflagellates, these three groups forming a novel clade (filozoa) whose ancestor presumably evolved filose tentacles well before they aggregated as a periciliary collar in the choanoflagellate/sponge common ancestor. Our trees show ichthyosporean choanozoans as sisters to filozoa; a fusion between ubiquitin and ribosomal small subunit S30 protein genes unifies all holozoa (filozoa plus Ichthyosporea), being absent in earlier branching eukaryotes. Thus, several successive evolutionary innovations occurred among their unicellular closest relatives prior to the origin of the multicellular body-plan of animals.     

Insights into the evolutionary origin and genome architecture of the unicellular opisthokonts Capsaspora owczarzaki and Sphaeroforma arctica

Molecular phylogenetic analyses have recently shown that the unicellular amoeboid protist Capsaspora owczarzaki is unlikely to be a nucleariid or an ichthyosporean as previously described, but is more closely related to Metazoa, Choanoflagellata, and Ichthyosporea. However, the specific phylogenetic relationship of Capsaspora to other protist opisthokont lineages was poorly resolved. To test these earlier results we have expanded both the taxonomic sampling and the number of genes from opisthokont unicellular lineages. We have sequenced the protein-coding genes elongation factor 1-alpha (EF1-alpha) and heat shock protein 70 (Hsp70) from C. owczarzaki and the ichthyosporean Sphaeroforma arctica. Our maximum likelihood (ML) and Bayesian analyses of a concatenated alignment of EF1-alpha, Hsp70, and actin protein sequences with a better sampling of opisthokont-related protist lineages indicate that C. owczarzaki is not clearly allied with any of the unicellular opisthokonts, but represents an independent unicellular lineage closely related to animals and choanoflagellates. Moreover, we have found that the ichthyosporean S. arctica possesses an EF-like (EFL) gene copy instead of the canonical EF1-alpha, the first so far described in an ichthyosporean. A maximum likelihood phylogenetic analysis shows that the EF-like gene of S. arctica strongly groups with the EF-like genes from choanoflagellates. Finally, to begin characterizing the Capsaspora genome, we have performed pulsed-field gel electrophoresis (PFGE) analyses, which indicate that its genome has at least 12 chromosomes with a total genome size in the range of 22-25 Mb.     

Evolutionary relationships of Metazoa within the eukaryotes based on molecular data from Porifera

Recent molecular data provide strong support for the view that all metazoan phyla, including Porifera, are of monophyletic origin. The relationship of Metazoa, including the Porifera, to Plantae, Fungi and unicellular eukaryotes has only rarely been studied by using cDNAs coding for proteins. Sequence data from rDNA suggested a relationship of Porifera to unicellular eukaryotes (choanoflagellates). However, ultrastructural studies of choanocytes did not support these findings. In the present study, we compared amino acid sequences that are found in a variety of metazoans (including sponges) with those of Plantae, Fungi and unicellular eukaryotes, to obtain an answer to this question. We used the four sequences from 70 kDa heat-shock proteins, the serine-threonine kinase domain found in protein kinases, beta-tubulin and calmodulin. The latter two sequences were deduced from cDNAs, isolated from the sponge Geodia cydonium for the phylogenetic analyses presented. These revealed that the sponge molecules were grouped into the same branch as the Metazoa, which is statistically (significantly) separated from those branches that comprise the sequences from Fungi, Plantae and unicellular eukaryotes. From our molecular data it seems evident that the unicellular eukaryotes existed at an earlier stage of evolution, and the Plantae and especially the Fungi and the Metazoa only appeared later.     

Primary structure of theParamecium tetraurelia small-subunit rRNA coding region: Phylogenetic relationships within the caliophora

We have sequenced the coding region for the small-subunit rRNA gene from Paramecium tetraurelia. Similarity comparisons between small-subunit rRNAs from representatives of the Metazoa, the Plantae, the Fungi and four other members of the Ciliophora were used to construct phylogenetic trees. In these phylogenies the Ciliophora diverged from the eukaryotic line of descent as a loose phylogenetic grouping during a radiative period that gave rise to the Fungi, the Plantae and the Metazoa.     

High‐throughput environmental sequencing reveals high diversity of litter and moss associated protist communities along a gradient of drainage and tree productivity

Although previous studies, mostly based on microscopy analyses of a few groups of protists, have suggested that protists are abundant and diverse in litter and moss habitats, the overall diversity of moss and litter associated protists remains elusive. Here, high‐throughput environmental sequencing was used to characterize the diversity and community structure of litter‐ and moss‐associated protists along a gradient of soil drainage and forest primary productivity in a temperate rainforest in British Columbia. We identified 3262 distinct protist OTUs from 36 sites. Protists were strongly structured along the landscape gradient, with a significant increase in alpha diversity from the blanket bog ecosystem to the zonal forest ecosystem. Among all investigated environmental variables, calcium content was the most strongly associated with the community composition of protists, but substrate composition, plant cover and other edaphic factors were also significantly correlated with these communities. Furthermore, a detailed phylogenetic analysis of unicellular opisthokonts identified OTUs covering most lineages, including novel OTUs branching with Discicristoidea, the sister group of Fungi, and with Filasterea, one of the closest unicellular relatives to animals. Altogether, this study provides unprecedented insight into the community composition of moss‐ and litter‐associated protists.     

The Mitochondrial Genome of Protists

The data on the structure and functions of the mitochondrial genomes of protists (Protozoa and unicellular red and green algae) are reviewed. It is emphasized that mitochondrial gene structure and composition, as well as organization of mitochondrial genomes in protists are more diverse than in multicellular eukaryotes. The gene content of mitochondrial genomes of protists are closer to those of plants than animals or fungi. In the protist mitochondrial DNA, both the universal (as in higher plants) and modified (as in animals and fungi) genetic codes are used. In the overwhelming majority of cases, protist mitochondrial genomes code for the major and minor rRNA components, some tRNAs, and about 30 proteins of the respiratory chain and ribosomes. Based on comparison of the mitochondrial genomes of various protists, the origin and evolution of mitochondria are briefly discussed.     

The mitochondrial genome of protists

The data on the structure and functions of the mitochondrial genomes of protists (Protozoa and unicellular red and green algae) are reviewed. It is emphasized that mitochondrial gene structure and composition, as well as organization of mitochondrial genomes in protists are more diverse than in multicellular eukaryotes. The gene content of mitochondrial genomes of protists are closer to those of plants than animals or fungi. In the protist mitochondrial DNA, both the universal (as in higher plants) and modified (as in animals and fungi) genetic codes are used. In the overwhelming majority of cases, protist mitochondrial genomes code for the major and minor rRNA components, some tRNAs, and about 30 proteins of the respiratory chain and ribosomes. Based on comparison of the mitochondrial genomes of various protists, the origin and evolution of mitochondria are briefly discussed.     

Structure of the choanocyte kinetid in the sponge <Emphasis Type="Italic">Haliclona</Emphasis> sp. (Demospongiae: Haplosclerida) and its implication for taxonomy and phylogeny of Demospongiae

Sponges are a group of primitive animals that are of particular interest in terms of evolutionary theory. Molecular and phylogenic studies have revealed the common origin of sponges and Metazoa and the relationship of Metazoa to Choanoflagellata. Comparative studies of the morphology have allowed reconstruction of the structure of ancestors common to Metazoa and Choanoflagellata, as well as for Porifera and other Metazoa. For this purpose, a comparative analysis of phylogenically informative structures, such as the kinetid, is needed. This article presents data on the structure of the choanocyte kinetid in Haliclona sp., which was investigated for the first time. The reconstruction of the kinetid based on ultrathin serial sections shows that it lacks an accessory centriole; it has fibrillar roots despite the basal position of the nucleus; two-to-three triplets of the kinetosome are longer than the others; bundles of lateral microtubules arise from fibrillar roots and reach the cytoplasmic membrane in the region of intercellular contact. The studies of the kinetid in the few representatives of Demospongiae investigated showed the compatibility of the data on the ultrastructure and molecular phylogeny.     

Mitochondrial carrier homolog 2 (MTCH2): the recruitment and evolution of a mitochondrial carrier protein to a critical player in apoptosis

Recent studies report mitochondrial carrier homolog 2 (MTCH2) as a novel and uncharacterized protein that acts as a receptor-like protein for the truncated BH3-interacting domain death agonist (tBID) protein in the outer membrane of mitochondria. These studies, using mouse embryonic stem cells and fibroblasts as well as mice with a conditional knockout of MTCH2 in the liver, showed that deletion of MTCH2 hindered recruitment of tBID to the mitochondria with subsequent reductions in the activation of pro-apoptotic proteins, mitochondrial outer membrane permeabilization and apoptosis. Sequence analysis shows that MTCH2 is present in all examined multicellular Metazoa as well as unicellular Choanoflagellata, and is a highly derived member of the mitochondrial carrier family. Mitochondrial carriers are monomeric transport proteins that are usually found in the inner mitochondrial membrane, where they exchange small substrates between the mitochondrial matrix and intermembrane space. There are extensive differences between the protein sequences of MTCH2 and other mitochondrial carriers that may explain the ability of MTCH2 to associate with tBID and thus its role in apoptosis. We review the experimental evidence for the role of MTCH2 in apoptosis and suggest that the original transport function of the ancestral MTCH2 mitochondrial carrier has been co-opted by the apoptotic machinery to provide a receptor and signaling mechanism.     

The backbone of the post-synaptic density originated in a unicellular ancestor of choanoflagellates and metazoans

Background  

Comparative genomics of the early diverging metazoan lineages and of their unicellular sister-groups opens new window to reconstructing the genetic changes which preceded or accompanied the evolution of multicellular body plans. A recent analysis found that the genome of the nerve-less sponges encodes the homologues of most vertebrate post-synaptic proteins. In vertebrate excitatory synapses, these proteins assemble to form the post-synaptic density, a complex molecular platform linking membrane receptors, components of their signalling pathways, and the cytoskeleton. Newly available genomes from Monosiga brevicollis (a member of Choanoflagellata, the closest unicellular relatives of animals) and Trichoplax adhaerens (a member of Placozoa: besides sponges, the only nerve-less metazoans) offer an opportunity to refine our understanding of post-synaptic protein evolution.     

A phylogenomic investigation into the origin of metazoa

The evolution of multicellular animals (Metazoa) from theirunicellular ancestors was a key transition that was accompaniedby the emergence and diversification of gene families associatedwith multicellularity. To clarify the timing and order of specificevents in this transition, we conducted expressed sequence tagsurveys on 4 putative protistan relatives of Metazoa includingthe choanoflagellate Monosiga ovata, the ichthyosporeans Sphaeroformaarctica and Amoebidium parasiticum, and the amoeba Capsasporaowczarzaki, and 2 members of Amoebozoa, Acanthamoeba castellaniiand Mastigamoeba balamuthi. We find that homologs of genes involvedin metazoan multicellularity exist in several of these unicellularorganisms, including 1 encoding a membrane-associated guanylatekinase with an inverted arrangement of protein-protein interactiondomains (MAGI) in Capsaspora. In Metazoa, MAGI regulates tightjunctions involved in cell-cell communication. By phylogenomicanalyses of genes encoded in nuclear and mitochondrial genomes,we show that the choanoflagellates are the closest relativesof the Metazoa, followed by the Capsaspora and Ichthyosporealineages, although the branching order between the latter 2groups remains unclear. Understanding the function of "metazoan-specific"proteins we have identified in these protists will clarify theevolutionary steps that led to the emergence of the Metazoa.     

Evolution of the 14-3-3 Protein Family: Does the Large Number of Isoforms in Multicellular Organisms Reflect Functional Specificity?

14-3-3 proteins constitute a family of eukaryotic proteins that are key regulators of a large number of processes ranging from mitosis to apoptosis. 14-3-3s function as dimers and bind to particular motifs in their target proteins. To date, 14-3-3s have been implicated in regulation or stabilization of more than 35 different proteins. This number is probably only a fraction of the number of proteins that 14-3-3s bind to, as reports of new target proteins have become more frequent. An examination of 14-3-3 entries in the public databases reveals 153 isoforms, including alleloforms, reported in 48 different species. The number of isoforms range from 2, in the unicellular organism Saccharomyces cerevisiae, to 12 in the multicellular organism Arabidopsis thaliana. A phylogenetic analysis reveals that there are four major evolutionary lineages: Viridiplantae (plants), Fungi, Alveolata, and Metazoa (animals). A close examination of the aligned amino acid sequences identifies conserved amino acid residues and regions of importance for monomer stabilization, dimer formation, target protein binding, and the nuclear export function. Given the fact that 53% of the protein is conserved, including all amino acid residues in the target binding groove of the 14-3-3 monomer, one might expect little to no isoform specificity for target protein binding. However, using surface plasmon resonance we show that there are large differences in affinity between nine 14-3-3 isoforms of A. thaliana and a target peptide representing a novel binding motif present in the C terminus of the plant plasma membrane H⁺ATPase. Thus, our data suggest that one reason for the large number of isoforms found in multicellular organisms is isoform-specific functions. Received: 19 April 2000 / Accepted: 24 July 2000     

Distinct Protein Classes in Human Red Cell Proteome Revealed by Similarity of Phylogenetic Profiles

The minimal set of proteins necessary to maintain a vertebrate cell forms an interesting core of cellular machinery. The known proteome of human red blood cell consists of about 1400 proteins. We treated this protein complement of one of the simplest human cells as a model and asked the questions on its function and origins. The proteome was mapped onto phylogenetic profiles, i.e. vectors of species possessing homologues of human proteins. A novel clustering approach was devised, utilising similarity in the phylogenetic spread of homologues as distance measure. The clustering based on phylogenetic profiles yielded several distinct protein classes differing in phylogenetic taxonomic spread, presumed evolutionary history and functional properties. Notably, small clusters of proteins common to vertebrates or Metazoa and other multicellular eukaryotes involve biological functions specific to multicellular organisms, such as apoptosis or cell-cell signaling, respectively. Also, a eukaryote-specific cluster is identified, featuring GTP-ase signalling and ubiquitination. Another cluster, made up of proteins found in most organisms, including bacteria and archaea, involves basic molecular functions such as oxidation-reduction and glycolysis. Approximately one third of erythrocyte proteins do not fall in any of the clusters, reflecting the complexity of protein evolution in comparison to our simple model. Basically, the clustering obtained divides the proteome into old and new parts, the former originating from bacterial ancestors, the latter from inventions within multicellular eukaryotes. Thus, the model human cell proteome appears to be made up of protein sets distinct in their history and biological roles. The current work shows that phylogenetic profiles concept allows protein clustering in a way relevant both to biological function and evolutionary history.     

Evolution of the MAGUK protein gene family in premetazoan lineages

Background  

Cell-to-cell communication is a key process in multicellular organisms. In multicellular animals, scaffolding proteins belonging to the family of membrane-associated guanylate kinases (MAGUK) are involved in the regulation and formation of cell junctions. These MAGUK proteins were believed to be exclusive to Metazoa. However, a MAGUK gene was recently identified in an EST survey of Capsaspora owczarzaki, an unicellular organism that branches off near the metazoan clade. To further investigate the evolutionary history of MAGUK, we have undertook a broader search for this gene family using available genomic sequences of different opisthokont taxa.     

Social Amoebae: Environmental Factors Influencing Their Distribution and Diversity Across South-Western Europe

The social amoebae (dictyostelids) are the only truly multicellular lineage within the superkingdom Amoebozoa, the sister group to Ophistokonts (Metazoa+Fungi). Despite the exceptional phylogenetic and evolutionary value of this taxon, the environmental factors that determine their distribution and diversity are largely unknown. We have applied statistical modeling to a set of data obtained from an extensive and detailed survey in the south-western of Europe (The Iberian Peninsula including Spain and Portugal) in order to estimate some of the main environmental factors influencing the distribution and diversity of dictyostelid in temperate climates. It is the first time that this methodology is applied to the study of this unique group of soil microorganisms. Our results show that a combination of climatic (temperature, water availability), physical (pH) and vegetation (species richness) factors favor dictyostelid species richness. In the Iberian Peninsula, dictyostelid diversity is highest in colder and wet environments, indicating that this group has likely diversified in relatively cold places with high levels of water availability.     

Tyrosine kinase signaling and the emergence of multicellularity

Tyrosine phosphorylation is an essential element of signal transduction in multicellular animals. Although tyrosine kinases were originally regarded as specific to the metazoan lineage, it is now clear that they evolved prior to the split between unicellular and multicellular eukaryotes (≈600million years ago). Genome analyses of choanoflagellates and other protists show an abundance of tyrosine kinases that rivals the most complex animals. Some of these kinases are orthologs of metazoan enzymes (e.g., Src), but others display unique domain compositions not seen in any metazoan. Biochemical experiments have highlighted similarities and differences between the unicellular and multicellular tyrosine kinases. In particular, it appears that the complex systems of kinase autoregulation may have evolved later in the metazoan lineage.     

The new higher level classification of eukaryotes with emphasis on the taxonomy of protists

This revision of the classification of unicellular eukaryotes updates that of Levine et al. (1980) for the protozoa and expands it to include other protists. Whereas the previous revision was primarily to incorporate the results of ultrastructural studies, this revision incorporates results from both ultrastructural research since 1980 and molecular phylogenetic studies. We propose a scheme that is based on nameless ranked systematics. The vocabulary of the taxonomy is updated, particularly to clarify the naming of groups that have been repositioned. We recognize six clusters of eukaryotes that may represent the basic groupings similar to traditional "kingdoms." The multicellular lineages emerged from within monophyletic protist lineages: animals and fungi from Opisthokonta, plants from Archaeplastida, and brown algae from Stramenopiles.