Ancient Origin of Four-Domain Voltage-gated Na<Superscript>+</Superscript> Channels Predates the Divergence of Animals and Fungi

The four-domain voltage-gated Na⁺ channels are believed to have arisen in multicellular animals, possibly during the evolution of the nervous system. Recent genomic studies reveal that many ion channels, including Na⁺ channels and Ca²⁺ channels previously thought to be restricted to animals, can be traced back to one of the unicellular ancestors of animals, Monosiga brevicollis. The eukaryotic supergroup Opisthokonta contains animals, fungi, and a diverse group of their unicellular relatives including M. brevicollis. Here, we demonstrate the presence of a putative voltage-gated Na⁺ channel homolog (TtrNa_V) in the apusozoan protist Thecamonas trahens, which belongs to the unicellular sister group to Opisthokonta. TtrNa_V displays a unique selectivity motif distinct from most animal voltage-gated Na⁺ channels. The identification of TtrNa_V suggests that voltage-gated Na⁺ channels might have evolved before the divergence of animals and fungi. Furthermore, our analyses reveal that Na_V channels have been lost independently in the amoeboid holozoan Capsaspora owczarzaki of the animal lineage and in several basal fungi. These findings provide novel insights into the evolution of four-domain voltage-gated ion channels, ion selectivity, and membrane excitability in the Opisthokonta lineage.     

Are Ichthyosporea animals or fungi? Bayesian phylogenetic analysis of elongation factor 1alpha of Ichthyophonus irregularis

Ichthyosporea is a recently recognized group of morphologically simple eukaryotes, many of which cause disease in aquatic organisms. Ribosomal RNA sequence analyses place Ichthyosporea near the divergence of the animal and fungal lineages, but do not allow resolution of its exact phylogenetic position. Some of the best evidence for a specific grouping of animals and fungi (Opisthokonta) has come from elongation factor 1alpha, not only phylogenetic analysis of sequences but also the presence or absence of short insertions and deletions. We sequenced the EF-1alpha gene from the ichthyosporean parasite Ichthyophonus irregularis and determined its phylogenetic position using neighbor-joining, parsimony and Bayesian methods. We also sequenced EF-1alpha genes from four chytrids to provide broader representation within fungi. Sequence analyses and the presence of a characteristic 12 amino acid insertion strongly indicate that I. irregularis is a member of Opisthokonta, but do not resolve whether I. irregularis is a specific relative of animals or of fungi. However, the EF-1alpha of I. irregularis exhibits a two amino acid deletion heretofore reported only among fungi.     

Unicellular Ca2+ signaling 'toolkit' at the origin of metazoa

Ca(2+) signaling pathways control many physiological processes in almost all types of animal cells such as fertilization, muscle contraction, hormone release, and learning and memory. Each animal cell type expresses a unique group of molecules from the Ca(2+) signaling 'toolkit' to control spatiotemporal patterns of Ca(2+) signaling. It is generally believed that the complex Ca(2+) signaling 'toolkit' has arisen from the ancestral multicellular organisms to fit unique physiological roles of specialized cell types. Here, we demonstrate for the first time the presence of an extensive Ca(2+) signaling 'toolkit' in the unicellular choanoflagellate Monosiga brevicollis. Choanoflagellates possess homologues of various types of animal plasma membrane Ca(2+) channels including the store-operated channel, ligand-operated channels, voltage-operated channels, second messenger-operated channels, and 5 out of 6 animal transient receptor potential channel families. Choanoflagellates also contain homologues of inositol 1,4,5-trisphosphate receptors. Furthermore, choanoflagellates master a complete set of Ca(2+) removal systems including plasma membrane and sarco/endoplasmic reticulum Ca(2+) ATPases and homologues of 3 animal cation/Ca(2+) exchanger families. Therefore, a complex Ca(2+) signaling 'toolkit' might have evolved before the emergence of multicellular animals.     

Emerging genomic and proteomic evidence on relationships among the animal, plant and fungal kingdoms

Sequence-based molecular phylogenies have provided new models of early eukaryotic evolution. This includes the widely accepted hypothesis that animals are related most closely to fungi, and that the two should be grouped together as the Opisthokonta. Although most published phylogenies have supported an opisthokont relationship, a number of genes contain a tree-building signal that clusters animal and green plant sequences, to the exclusion of fungi. The alternative tree-building signal is especially intriguing in light of emerging data from genomic and proteomic studies that indicate striking and potentially synapomorphic similarities between plants and animals. This paper reviews these new lines of evidence, which have yet to be incorporated into models of broad scale eukaryotic evolution.     

Acidic calcium stores of Saccharomyces cerevisiae

Fungi and animals constitute sister kingdoms in the eukaryotic domain of life. The major classes of transporters, channels, sensors, and effectors that move and respond to calcium ions were already highly networked in the common ancestor of fungi and animals. Since that time, some key components of the network have been moved, altered, relocalized, lost, or duplicated in the fungal and animal lineages and at the same time some of the regulatory circuitry has been dramatically rewired. Today the calcium transport and signaling networks in fungi provide a fresh perspective on the scene that has emerged from studies of the network in animal cells. This review provides an overview of calcium signaling networks in fungi, particularly the model yeast Saccharomyces cerevisiae, with special attention to the dominant roles of acidic calcium stores in fungal cell physiology.     

Are Ichthyosporea animals or fungi? Bayesian phylogenetic analysis of elongation factor 1α of Ichthyophonus irregularis

Ichthyosporea is a recently recognized group of morphologically simple eukaryotes, many of which cause disease in aquatic organisms. Ribosomal RNA sequence analyses place Ichthyosporea near the divergence of the animal and fungal lineages, but do not allow resolution of its exact phylogenetic position. Some of the best evidence for a specific grouping of animals and fungi (Opisthokonta) has come from elongation factor 1α, not only phylogenetic analysis of sequences but also the presence or absence of short insertions and deletions. We sequenced the EF-1α gene from the ichthyosporean parasite Ichthyophonus irregularis and determined its phylogenetic position using neighbor-joining, parsimony and Bayesian methods. We also sequenced EF-1α genes from four chytrids to provide broader representation within fungi. Sequence analyses and the presence of a characteristic 12 amino acid insertion strongly indicate that I. irregularis is a member of Opisthokonta, but do not resolve whether I. irregularis is a specific relative of animals or of fungi. However, the EF-1α of I. irregularis exhibits a two amino acid deletion heretofore reported only among fungi.     

Evidence for an early evolutionary emergence of γ-type carbonic anhydrases as components of mitochondrial respiratory complex I

Background  

The complexity of mitochondrial complex I (CI; NADH:ubiquinone oxidoreductase) has increased considerably relative to the homologous complex in bacteria. Comparative analyses of CI composition in animals, fungi and land plants/green algae suggest that novel components of mitochondrial CI include a set of 18 proteins common to all eukaryotes and a variable number of lineage-specific subunits. In plants and green algae, several purportedly plant-specific proteins homologous to γ-type carbonic anhydrases (γCA) have been identified as components of CI. However, relatively little is known about CI composition in the unicellular protists, the characterizations of which are essential to our understanding of CI evolution.     

Microbial Pathogens in the Fungal Kingdom

The fungal kingdom is vast, spanning ~1.5 to as many as 5 million species diverse as unicellular yeasts, filamentous fungi, mushrooms, lichens, and both plant and animal pathogens. The fungi are closely aligned with animals in one of the six to eight supergroups of eukaryotes, the opisthokonts. The animal and fungal kingdoms last shared a common ancestor ~1 billion years ago, more recently than other groups of eukaryotes. As a consequence of their close evolutionary history and shared cellular machinery with metazoans, fungi are exceptional models for mammalian biology, but prove more difficult to treat in infected animals. The last common ancestor to the fungal/metazoan lineages is thought to have been unicellular, aquatic, and motile with a posterior flagellum, and certain extant species closely resemble this hypothesized ancestor. Species within the fungal kingdom were traditionally assigned to four phyla, including the basal fungi (Chytridiomycota, Zygomycota) and the more recently derived monophyletic lineage, the dikarya (Ascomycota, Basidiomycota). The fungal tree of life project has revealed that the basal lineages are polyphyletic, and thus there are as many as eight to ten fungal phyla. Fungi that infect vertebrates are found in all of the major lineages, and virulence arose multiple times independently. A sobering recent development involves the species Batrachochytrium dendrobatidis from the basal fungal phylum, the Chytridiomycota, which has emerged to cause global amphibian declines and extinctions. Genomics is revolutionizing our view of the fungal kingdom, and genome sequences for zygomycete pathogens (Rhizopus, Mucor), skin-associated fungi (dermatophytes, Malassezia), and the Candida pathogenic species clade promise to provide insights into the origins of virulence. Here we survey the diversity of fungal pathogens and illustrate key principles revealed by genomics involving sexual reproduction and sex determination, loss of conserved pathways in derived fungal lineages that are retained in basal fungi, and shared and divergent virulence strategies of successful human pathogens, including dimorphic and trimorphic transitions in form. The overarching conclusion is that fungal pathogens of animals have arisen repeatedly and independently throughout the fungal tree of life, and while they share general properties, there are also unique features to the virulence strategies of each successful microbial pathogen.     

真菌激素研究进展

真菌种类繁多,与人类健康、工农业生产和生态系统物质循环的关系非常密切。真菌合成多种性激素、植物激素和动物激素,这些内源激素以及来自动植物产生的外源激素能够被真菌感知,并影响真菌的生长发育、子实体形成、代谢、致病性和共生性等。然而,对真菌激素的合成和信号转导途径,及其起源和进化还知之不多。建立真菌激素学将极大地促进对真菌激素的研究和应用。     

Genome analysis of the unicellular green alga Chlamydomonas reinhardtii Indicates an ancient evolutionary origin for key pattern recognition and cell-signaling protein families

The evolution of specific cell signaling and adhesion domains may have played an important role in the transition to a multicellular existence in the metazoans. Genomic analysis indicates that several signaling domains predominately found in animals are also present in the unicellular green alga, Chlamydomonas reinhardtii. A large group of proteins is present, containing scavenger receptor cysteine-rich (SRCR) and C-type lectin domains, which function in ligand binding and play key roles in the innate immune system of animals. Chlamydomonas also contains a large family of putative tyrosine kinases, suggesting an important role for phosphotyrosine signaling in the green algae. These important signaling domains may therefore be widespread among eukaryotes and most probably evolved in ancestral eukaryotes before the divergence of the Opisthokonts (the animal and fungal lineage).     

Comparison of morphogenetic networks of filamentous fungi and yeast.

Fungi generally display either of two growth modes, yeast-like or filamentous, whereas dimorphic fungi, upon environmental stimuli, are able to switch between the yeast-like and the filamentous growth mode. Signal transduction pathways have been elucidated in the budding yeast Saccharomyces cerevisiae, establishing a morphogenetic network that links cell-cycle events with cellular morphogenesis. Recent molecular genetic studies in several filamentous fungal model systems revealed key components required for distinct steps from fungal spore germination to the maintenance of polar hyphal growth, mycelium formation, and nuclear division. This allows a mechanistic comparison of yeast-like and hyphal growth and the establishment of a core model morphogenetic network for filamentous growth including signaling via the cAMP pathway, Rho modules, and cell cycle kinases. Appreciating similarities between morphogenetic networks of the unicellular yeasts and the multicellular filamentous fungi will open new research directions, help in isolating the central network components, and ultimately pave the way to elucidate the central differences (of many) that distinguish, e.g., the growth mode of filamentous fungi from that of their yeast-like relatives, the role of cAMP signaling, and nuclear division.     

The protistan origins of animals and fungi

Recent molecular studies suggest that Opisthokonta, the eukaryotic supergroup including animals and fungi, should be expanded to include a diverse collection of primitively single-celled eukaryotes previously classified as Protozoa. These taxa include corallochytreans, nucleariids, ministeriids, choanoflagellates, and ichthyosporeans. Assignment of many of these taxa to Opisthokonta remains uncorroborated as it is based solely on small subunit ribosomal RNA trees lacking resolution and significant bootstrap support for critical nodes. Therefore, important details of the phylogenetic relationships of these putative opisthokonts with each other and with animals and fungi remain unclear. We have sequenced elongation factor 1-alpha (EF-1alpha), actin, beta-tubulin, and HSP70, and/or alpha-tubulin from representatives of each of the proposed protistan opisthokont lineages, constituting the first protein-coding gene data for some of them. Our results show that members of all opisthokont protist groups encode a approximately 12-amino acid insertion in EF-1alpha, previously found exclusively in animals and fungi. Phylogenetic analyses of combined multigene data sets including a diverse set of opisthokont and nonopisthokont taxa place all of the proposed opisthokont protists unequivocally in an exclusive clade with animals and fungi. Within this clade, the nucleariid appears as the closest sister taxon to fungi, while the corallochytrean and ichthyosporean form a group which, together with the ministeriid and choanoflagellates, form two to three separate sister lineages to animals. These results further establish Opisthokonta as a bona fide taxonomic group and suggest that any further testing of the legitimacy of this taxon should, at the least, include data from opisthokont protists. Our results also underline the critical position of these "animal-fungal allies" with respect to the origin and early evolution of animals and fungi.     

Molecular evidence of fungal signatures in the marine protist Corallochytrium limacisporum and its implications in the evolution of animals and fungi

Fungi, animals, and single-celled organisms belonging to the choanozoans together constitute the supergroup Opisthokonta. The latter are considered crucial in understanding the evolutionary origin of animals and fungi. The choanozoan Corallochytrium limacisporum is an enigmatic marine protist of considerable interest in opisthokontan evolution. Several isolates of the organism were obtained from a coral reef lagoon in the Lakshadweep group of islands of the Arabian Sea. The capability of these cultures to grow on media containing inorganic nitrogen sources prompted us to examine the possible presence of fungal signatures, namely the enzyme alpha-aminoadipate reductase (alpha-AAR) involved in the alpha-aminoadipate (AAA) pathway for synthesizing lysine and ergosterol, in one of the isolates. These features, as well as the sterol C-14 reductase gene involved in the sterol pathway of animals and fungi, were detected in the organism. Phylogenetic trees based on the alpha-AAR gene suggested that Corallochytrium limacisporum is a sister clade to fungi, while those based on the C-14 reductase gene did not adequately resolve whether the organism was more closely related to fungi or animals. While many studies indicate that Corallochytrium is a sister clade to animals, we suggest that further studies are required to examine whether this protist is in fact more closely related to fungi rather than to animals.     

Ca2+-sensing transgenic mice: postsynaptic signaling in smooth muscle

Genetically encoded signaling proteins provide remarkable opportunities to design and target the expression of molecules that can be used to report critical cellular events in vivo, thereby markedly extending the scope and physiological relevance of studies of cell function. Here we report the development of a transgenic mouse expressing such a reporter and its use to examine postsynaptic signaling in smooth muscle. The circularly permutated, Ca2+-sensing molecule G-CaMP (Nakai, J., Ohkura, M., and Imoto, K. (2001) Nat. Biotechnol. 19, 137-141) was expressed in vascular and non-vascular smooth muscle and functioned as a lineage-specific intracellular Ca2+ reporter. Detrusor tissue from these mice was used to identify two separate types of postsynaptic Ca2+ signals, mediated by distinct neurotransmitters. Intrinsic nerve stimulation evoked rapid, whole-cell Ca2+ transients, or "Ca2+ flashes," and slowly propagating Ca2+ waves. We show that Ca2+ flashes occur through P2X receptor stimulation and ryanodine receptor-mediated Ca2+ release, whereas Ca2+ waves arise from muscarinic receptor stimulation and inositol trisphosphate-mediated Ca2+ release. The distinct ionotropic and metabotropic postsynaptic Ca2+ signals are related at the level of Ca2+ release. Importantly, individual myocytes are capable of both postsynaptic responses, and a transition between Ca2+ -induced Ca2+ release and inositol trisphosphate waves occurs at higher synaptic inputs. Ca2+ signaling mice should provide significant advantages in the study of processive biological signaling.     

The evolution of the Wnt pathway

Wnt genes are important regulators of embryogenesis and cell differentiation in vertebrates and insects. New data revealed by comparative genomics have now shown that members of the Wnt signaling pathway can be found in all clades of metazoans, but not in fungi, plants, or unicellular eukaryotes. This article focuses on new data from recent genomic analyses of several basal metazoan organisms, providing evidence that the Wnt pathway was a primordial signaling pathway during evolution. The formation of a Wnt signaling center at the site of gastrulation was instrumental for the formation of a primary, anterior-posterior body axis, which can be traced throughout animal evolution.     

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.     

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.     

Expression of the Cameleon calcium biosensor in fungi reveals distinct Ca(2+) signatures associated with polarized growth, development, and pathogenesis

Calcium is a universal messenger that translates diverse environmental stimuli and developmental cues into specific cellular and developmental responses. While individual fungal species have evolved complex and often unique biochemical and structural mechanisms to exploit specific ecological niches and to adjust growth and development in response to external stimuli, one universal feature to all is that Ca(2+)-mediated signaling is involved. The lack of a robust method for imaging spatial and temporal dynamics of subcellular Ca(2+) (i.e., "Ca(2+) signature"), readily available in the plant and animal systems, has severely limited studies on how this signaling pathway controls fungal growth, development, and pathogenesis. Here, we report the first successful expression of a FRET (F?rster Resonance Energy Transfer)-based Ca(2+) biosensor in fungi. Time-lapse imaging of Magnaporthe oryzae, Fusarium oxysporum, and Fusarium graminearum expressing this sensor showed that instead of a continuous gradient, the cytoplasmic Ca(2+) ([Ca(2+)](c)) change occurred in a pulsatile manner with no discernable gradient between pulses, and each species exhibited a distinct Ca(2+) signature. Furthermore, occurrence of pulsatile Ca(2+) signatures was age and development dependent, and major [Ca(2+)](c) transients were observed during hyphal branching, septum formation, differentiation into specialized plant infection structures, cell-cell contact and in planta growth. In combination with the sequenced genomes and ease of targeted gene manipulation of these and many other fungal species, the data, materials and methods developed here will help understand the mechanism underpinning Ca(2+)-mediated control of cellular and developmental changes, its role in polarized growth forms and the evolution of Ca(2+) signaling across eukaryotic kingdoms.     

Phylogeny of the CDC25 homology domain reveals rapid differentiation of Ras pathways between early animals and fungi

The members of the Ras-like superfamily of small GTP-binding proteins are molecular switches that are in general regulated in time and space by guanine nucleotide exchange factors and GTPase activating proteins. The Ras-like G-proteins Ras, Rap and Ral are regulated by a variety of guanine nucleotide exchange factors that are characterized by a CDC25 homology domain. Here we study the evolution of the Ras pathway by determining the evolutionary history of CDC25 homology domain coding sequences. We identified CDC25 homology domain coding sequences in animals, fungi and a wide range of protists, but not in plants. This suggests that the CDC25 homology domain originated in or before the last eukaryotic ancestor but was subsequently lost in plant. We provide evidence that at least seven different ancestral Ras guanine nucleotide exchange factors were present in the ancestor of fungi and animals. Differences between present day fungi and animals are the result of loss of ancestral Ras guanine nucleotide exchange factors early in fungal and animal evolution combined with lineage specific duplications and domain acquisitions. In addition, we identify Ral guanine exchange factors and Ral in early diverged fungi, dating the origin of Ral signaling back to before the divergence of animals and fungi. We conclude that the Ras signaling pathway evolved by gradual change as well as through differential sampling of the ancestral CDC25 homology domain repertoire by both fungi and animals. Finally, a comparison of the domain composition of the Ras guanine nucleotide exchange factors shows that domain addition and diversification occurred both prior to and after the fungal–animal split.