Sergey A. Karpov

Morphology, phylogeny, and ecology of the aphelids (Aphelidea, Opisthokonta) and proposal for the new superphylum Opisthosporidia

The aphelids are a small group of intracellular parasitoids of common species of eukaryotic phytoplankton with three known genera Aphelidium, Amoeboaphelidium, and Pseudaphelidium, and 10 valid species, which form along with related environmental sequences a very diversified group. The phyla Microsporidia and Cryptomycota, and the class Aphelidea have recently been considered to be a deep branch of the Holomycota lineage forming the so called the ARM-clade which is sister to the fungi. In this review we reorganize the taxonomy of ARM-clade, and establish a new superphylum the Opisthosporidia with three phyla: Aphelida phyl. nov., Cryptomycota and Microsporidia. We discuss here all aspects of aphelid investigations: history of our knowledge, life cycle peculiarities, the morphology (including the ultrastructure), molecular phylogeny, ecology, and provide a taxonomic revision of the phylum supplied with a list of species. We compare the aphelids with their nearest relatives, the species of Rozella, and improve the diagnosis of the phylum Cryptomycota.

Obligately phagotrophic aphelids turned out to branch with the earliest-diverging fungi.

Reconstructing the early evolution of fungi and metazoans, two of the kingdoms of multicellular eukaryotes thriving on earth, is a challenging task for biologists. Among extant organisms having characters intermediate between fungi and hypothetical protistan ancestors, from which both fungi and metazoans are believed to have evolved, aphelids are unfairly neglected. The phylogenetic position of these microalgal endoparasites remained uncertain, since no nucleotide sequence data have been reported to date. Aphelids resemble some primitive zoosporic fungi in life cycle, but, unlike fungi, they live by phagotrophy. Here we present a phylogeny, in which a cultured aphelid species, Amoeboaphelidium protococcarum, forms a monophyletic group with Rozella and microsporidia as a sister group to Fungi. We also report a non-canonical nuclear genetic code in A. protococcarum.

Dinomyces arenysensis gen. et sp. nov. (Rhizophydiales, Dinomycetaceae fam. nov.), a chytrid infecting marine dinoflagellates.

Environmental 18S rRNA gene surveys of microbial eukaryotes have recently revealed the diversity of major parasitic agents in pelagic freshwater systems, consisting primarily of chytrid fungi. To date, only a few studies have reported the presence of chydrids in the marine environment and a limited number of marine chytrids have been properly identified and characterized. Here, we report the isolation and cultivation of a marine chytrid from samples taken during a bloom of the toxic dinoflagellate Alexandrium minutum in the Arenys de Mar harbour (Mediterranean Sea, Spain). Cross-infections using cultures and natural phytoplankton communities revealed that this chytrid is only able to infect certain species of dinoflagellates, with a rather wide host range but with a relative preference for Alexandrium species. Phylogenetic analyses showed that it belongs to the order Rhizophydiales, but cannot be included in any of the existing families within this order. Several ultrastructural characters confirmed the placement of this taxon within the Rhizophydiales as well its novelty notably in terms of zoospore structure. This marine chytridial parasitoid is described as a new genus and species, Dinomyces arenysensis, within the Dinomycetaceae fam. nov.

The Parasitic Dinoflagellates Blastodinium spp. Inhabiting the Gut of Marine, Planktonic Copepods: Morphology, Ecology, and Unrecognized Species Diversity

Blastodinium is a genus of dinoflagellates that live as parasites in the gut of marine, planktonic copepods in the World’s oceans and coastal waters. The taxonomy, phylogeny, and physiology of the genus have only been explored to a limited degree and, based on recent investigations, we hypothesize that the morphological and genetic diversity within this genus may be considerably larger than presently recognized. To address these issues, we obtained 18S rDNA and ITS gene sequences for Blastodinium specimens of different geographical origins, including representatives of the type species. This genetic information was in some cases complemented with new morphological, ultrastructural, physiological, and ecological data. Because most current knowledge about Blastodinium and its effects on copepod hosts stem from publications more than half a century old, we here summarize and discuss the existing knowledge in relation to the new data generated. Most Blastodinium species possess functional chloroplasts, but the parasitic stage, the trophocyte, has etioplasts and probably a limited photosynthetic activity. Sporocytes and swarmer cells have well-developed plastids and plausibly acquire part of their organic carbon needs through photosynthesis. A few species are nearly colorless with no functional chloroplasts. The photosynthetic species are almost exclusively found in warm, oligotrophic waters, indicating a life strategy that may benefit from copepods as microhabitats for acquiring nutrients in a nutrient-limited environment. As reported in the literature, monophyly of the genus is moderately supported, but the three main groups proposed by Chatton in 1920 are consistent with molecular data. However, we demonstrate an important genetic diversity within the genus and provide evidences for new groups and the presence of cryptic species. Finally, we discuss the current knowledge on the occurrence of Blastodinium spp. and their potential impact on natural copepod populations.

Parvilucifera rostrata sp. nov. (Perkinsozoa), a Novel Parasitoid that Infects Planktonic Dinoflagellates

The diversity and ecological roles of protists in marine plankton are still poorly known. In 2011, we made a substantial effort to isolate parasites into cultures during the course of blooms of the toxic microalga Alexandrium minutum (Dinophyceae) in two estuaries (the Penzé and the Rance, Brittany coast, north-west of France). In total, 99 parasitic strains were obtained. Screening of ribosomal internal transcribed spacer regions (including ITS1, 5.8S and ITS2) revealed the existence of two ribotypes. Small subunit and partial large subunit rRNA genes revealed that these two ribotypes belong to different species of the genus Parvilucifera. The first ribotype was tentatively affiliated to the species Parvilucifera infectans, whilst the second represents a new species, Parvilucifera rostrata sp. nov. The new species has several distinct morphological features in the general organization of its zoospore and in the shape and size of processes covering the sporangium. Both Parvilucifera species are generalist parasitoids with similar generation times, and this study thus raises the question of how two parasitoids exploiting similar ecological resources and infection strategies can coexist in the same ecosystem. Taxonomic relationships between Parvilucifera spp. and other closely related marine parasitoids, such as syndinians, are discussed.

Paracercomonas kinetid ultrastructure, origins of the body plan of Cercomonadida, and cytoskeleton evolution in Cercozoa.

Serial section reconstruction shows that kinetid ultrastructure in two genetically divergent Paracercomonas (P. virgaria, P. metabolica) is basically similar, differing somewhat from clade A cercomonads. Paracercomonas (Paracercomonadidae fam. n.) have a posterior root (dp1) attached to the posterior centriole, unlike Cercomonadidae (here revised to include only Eocercomonas, Cercomonas, Filomonas gen. n., and Neocercomonas), which belong in clade A (new suborder Cercomonadina) with Cavernomonas (Cavernomonadidae fam. n.). Whether dp1 is serially homologous to anterior root da is unclear. The common ancestor of Cercomonadida probably had five microtubular roots, two fibrillar microtubule-nucleating centres generating microtubular cones, and striated connectors between obtusely angled centrioles. Our new data leave the question of holophyly versus polyphyly of Cercomonadida unresolved, but clarify cercozoan root diversity and homologies. Ventral root vp1 is throughout Cercozoa; vp2 might be restricted to the new superclass Ventrifilosa plus Sarcomonadea. Though cercozoan microtubular arrangements differ substantially from others within the kingdom Chromista, the microtubular root numbering system used for other chromists and Plantae is applicable to them; in doing this we found that the single anterior root of excavates (probably ancestral to Chromista, Plantae and unikonts) and Euglenozoa corresponds with R3 (not R4 as previously thought) of corticate eukaryotes (Chromista plus Plantae).

Molecular phylogeny and ultrastructure of Aphelidium aff. melosirae (Aphelida, Opisthosporidia)

Aphelids are a poorly known group of parasitoids of algae that have raised considerable interest due to their pivotal phylogenetic position. Together with Cryptomycota and the highly derived Microsporidia, they have been recently re-classified as Opisthosporidia, being the sister group to fungi. Despite their huge diversity, as revealed by molecular environmental studies, and their phylogenetic interest, only three genera have been described (Aphelidium, Amoeboaphelidium, and Pseudaphelidium), from which 18S rRNA gene sequences exist only for Amoeboaphelidium species. Here, we describe the life cycle and ultrastructure of Aphelidium aff. melosirae, and provide the first 18S rRNA gene sequence obtained for this genus. Molecular phylogeny analysis indicates that Aphelidium is very distantly related to Amoebaphelidium, highlighting the wide genetic diversity of the aphelids. The parasitoid encysts and penetrates the host alga, Tribonema gayanum through an infection tube. Cyst germination leads to a young trophont that phagocytes the algal cell content and progressively develops a plasmodium, which becomes a zoospore-producing sporangium. Aphelidium aff. melosirae has amoeboflagellate zoospores, tubular/lamellar mitochondrial cristae, a metazoan type of centrosome, and closed orthomitosis with an intranuclear spindle. These features together with trophont phagocytosis distinguish Aphelidium from fungi and support the erection of the new superphylum Opisthosporidia as sister to fungi.

Mechanism of male gamete motility in araphid pennate diatoms from the genus Tabularia (Bacillariophyta).

During sexual reproduction, araphid pennate diatoms of the genus Tabularia (Kützing) D. M. Williams and Round released male gametes directly into the medium, sometimes at a considerable distance from the female gametes. This raised the question of how male gametes, suspended in water, manage to reach female ones, given that no locomotive organelles have been described in gametes of pennate diatoms. Optical microscopic investigation revealed cytoplasmic projections produced by male gametes of Tabularia tabulata (C. A. Agardh) Snoeijs and T. fasciculata (C. A. Agardh) D. M. Williams and Round. Morphology and behavior of these projections is consistent with pseudopodia, however, which specific type of pseudopodia they may be, remains inconclusive. The growth and retraction of the pseudopodia coincided with gamete motility and so we postulate that it explains the otherwise apparent random movement of male gametes. Spinning, shuffling and chaotic patterns of motility were documented. In theory, gamete mobility increases the probability of gamete encounter thus enhancing the probability of syngamy. This is the first known case where cytoplasmic projections have been described in diatom gametes, and possibly in mature gametes in general.

Phylogenetic position of the genus Mesochytrium (Chytridiomycota) based on zoospore ultrastructure and sequences from the 18S and 28S rRNA gene

Ultrastructural features and a molecular phylogeny of Mesochytrium penetrans (strain X-10 CALU) based on 18S and 28S rRNA gene sequences were investigated for the first time. The parasite is strongly specific for the green alga Chlorococcum minutum (Chlorococcales) and did not grow on another 29 strains of algae from the CALU collection. Most attention was paid to the zoospore ultrastructure. Ribosomes are dispersed through the cytoplasm. The nucleus and microbodylipid globule complex (MLC) is surrounded by rough endoplasmic reticulum. The MLC is composed of a single mitochondrion and a single lipid globule partially covered with a microbody and a fenestrated cisterna, which is most posterior. The non-flagellated centriole is shorter than the kinetosome and lies at an angle of approximately 30° to the latter, the two being connected with a broad, dense fibrillar bridge. The flagellar transition zone contains a spiral fiber. The non-flagellated centriole is surrounded by a thin fiber, and has a veil. The features that distinguish Mesochytrium are the partial penetration of the sporangium into the host cell, and a zoospore with unique ultrastructural configuration. In our phylogeny, M. penetrans was nested within Lobulomycetales and the Polychytrium clade, but its phylogenetic position is tenuous because of marginal support, and thus its family and order status are considered incertae sedis. Zoospores of M. penetrans are most similar to those of Synchytrium macrosporum and Rozella allomycis, which belong to different clades. These ultrastructural similarities may be a result of the relative simplicity of zoospore morphology, and do not reflect a phylogenetic relationship between these genera.

Ecologically relevant choanoflagellates collected from hypoxic water masses of the Baltic Sea have untypical mitochondrial cristae

BackgroundProtist communities inhabiting oxygen depleted waters have so far been characterized through both microscopical observations and sequence based techniques. However, the lack of cultures for abundant taxa severely hampers our knowledge on the morphology, ecology and energy metabolism of hypoxic protists. Cultivation of such protists has been unsuccessful in most cases, and has never yet succeeded for choanoflagellates, even though these small bacterivorous flagellates are known to be ecologically relevant components of aquatic protist communities.ResultsQuantitative data for choanoflagellates and the vertical distribution of Codosiga spp. at Gotland and Landsort Deep (Baltic Sea) indicate its preference for oxygen-depleted zones. Strains isolated and cultivated from these habitats revealed ultrastructural peculiarities such as mitochondria showing tubular cristae never seen before for choanoflagellates, and the first observation of intracellular prokaryotes in choanoflagellates. Analysis of their partial 28S rRNA gene sequence complements the description of two new species, Codosiga minima n. sp. and C. balthica n. sp. These are closely related with but well separated from C. gracilis (C. balthica and C. minima p-distance to C. gracilis 4.8% and 11.6%, respectively). In phylogenetic analyses the 18S rRNA gene sequences branch off together with environmental sequences from hypoxic habitats resulting in a wide cluster of hypoxic Codosiga relatives so far only known from environmental sequencing approaches.ConclusionsHere, we establish the morphological and ultrastructural identity of an environmental choanoflagellate lineage. Data from microscopical observations, supplemented by findings from previous culture-independent methods, indicate that C. balthica is likely an ecologically relevant player of Baltic Sea hypoxic waters. The possession of derived mitochondria could be an adaptation to life in hypoxic environments periodically influenced by small-scale mixing events and changing oxygen content allowing the reduction of oxygen consuming components. In view of the intricacy of isolating and cultivating choanoflagellates, the two new cultured species represent an important advance to the understanding of the ecology of this group, and mechanisms of adaptations to hypoxia in protists in general.