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The phylum Nematoda remains very poorly sampled for mtDNA, with a strong bias toward parasitic, economically important or model species of the Chromadoria lineage. Most chromadorian mitogenomes share a specific order of genes encoded on one mtDNA strand. However, the few sequenced representatives of the Dorylaimia lineage exhibit a variable order of mtDNA genes encoded on both strands. While the ancestral arrangement of nematode mitogenome remains undefined, no evidence has been reported for Enoplia, the phylum's third early divergent major lineage. We describe the first mitogenome of an enoplian nematode, Campydora demonstrans, and contend that the complete 37-gene repertoire and both-strand gene encoding are ancestral states preserved in Enoplia and Dorylaimia versus the derived mitogenome arrangement in some Chromadoria. The C. demonstrans mitogenome is 17,018 bp in size and contains a noncoding perfect inverted repeat with 2013 bp-long arms, subdividing the mitogenome into two coding regions. This mtDNA arrangement is very rare among animals and instead resembles that of chloroplast genomes in land plants. Our report broadens mtDNA taxonomic sampling of the phylum Nematoda and adds support to the applicability of cox1 gene as a phylogenetic marker for establishing nematode relationships within higher taxa., (© 2024 Wiley Periodicals LLC.)

Nematomorpha (hairworms) is a phylum of parasitic ecdysozoans, best known for infecting arthropods and guiding their hosts toward water, where the parasite can complete its life cycle. Over 350 species of nematomorphs have been described, yet molecular data for the group remain scarce. The few available mitochondrial genomes of nematomorphs are enriched with long inverted repeats, which are embedded in the coding sequences of their genes-a remarkably unusual feature exclusive to this phylum. Here, we obtain and annotate the repeats in the mitochondrial genome of another nematomorph species- Parachordodes pustulosus . Using genomic and transcriptomic libraries, we investigate the impact of inverted repeats on the read coverage of the mitochondrial genome. Pronounced drops in the read coverage coincide with regions containing long inverted repeats, denoting the 'blind spots' of short-fragment sequencing libraries. Phylogenetic inference with the novel data reveals multiple disagreements between the traditional system of Nematomorpha and molecular data, rendering several genera paraphyletic, including Parachordodes .

The phylum Cnidaria consists of several morphologically diverse classes including Anthozoa, Cubozoa, Hydrozoa, Polypodiozoa, Scyphozoa, Staurozoa, and Myxozoa. Myxozoa comprises two subclasses of obligate parasites-Myxosporea and Malacosporea, which demonstrate various degrees of simplification. Myxosporea were previously reported to lack the majority of core protein domains of apoptotic proteins including caspases, Bcl-2, and APAF-1 homologs. Other sequenced Cnidaria, including the parasite Polypodium hydriforme from Polypodiozoa do not share this genetic feature. Whether this loss of core apoptotic proteins is unique to Myxosporea or also present in its sister subclass Malacosporea was not previously investigated. We show that the presence of core apoptotic proteins gradually diminishes from free-living Cnidaria to Polypodium to Malacosporea to Myxosporea. This observation does not favor the hypothesis of catastrophic simplification of Myxosporea at the genetic level, but rather supports a stepwise adaptation to parasitism that likely started from early parasitic ancestors that gave rise to Myxozoa., (© 2023. The Author(s).)

Molecular phylogenetics of microbial eukaryotes has reshaped the tree of life by establishing broad taxonomic divisions, termed supergroups, that supersede the traditional kingdoms of animals, fungi and plants, and encompass a much greater breadth of eukaryotic diversity 1 . The vast majority of newly discovered species fall into a small number of known supergroups. Recently, however, a handful of species with no clear relationship to other supergroups have been described 2-4 , raising questions about the nature and degree of undiscovered diversity, and exposing the limitations of strictly molecular-based exploration. Here we report ten previously undescribed strains of microbial predators isolated through culture that collectively form a diverse new supergroup of eukaryotes, termed Provora. The Provora supergroup is genetically, morphologically and behaviourally distinct from other eukaryotes, and comprises two divergent clades of predators-Nebulidia and Nibbleridia-that are superficially similar to each other, but differ fundamentally in ultrastructure, behaviour and gene content. These predators are globally distributed in marine and freshwater environments, but are numerically rare and have consequently been overlooked by molecular-diversity surveys. In the age of high-throughput analyses, investigation of eukaryotic diversity through culture remains indispensable for the discovery of rare but ecologically and evolutionarily important eukaryotes., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Over the past decade, molecular phylogenetics has reshaped our understanding of the fungal tree of life by unraveling a hitherto elusive diversity of the protistan relatives of Fungi. Aphelida constitutes one of these novel deep branches that precede the emergence of osmotrophic fungal lifestyle and hold particular significance as the pathogens of algae. Here, we obtain and analyze the genomes of aphelid species Amoeboaphelidium protococcarum and Amoeboaphelidium occidentale. Genomic data unmask the vast divergence between these species, hidden behind their morphological similarity, and reveal hybrid genomes with a complex evolutionary history in two strains of A. protococcarum. We confirm the proposed sister relationship between Aphelida and Fungi using phylogenomic analysis and chart the reduction of characteristic proteins involved in phagocytic activity in the evolution of Holomycota. Annotation of aphelid genomes demonstrates the retention of actin nucleation-promoting complexes associated with phagocytosis and amoeboid motility and also reveals a conspicuous expansion of receptor-like protein kinases, uncharacteristic of fungal lineages. We find that aphelids possess multiple carbohydrate-processing enzymes that are involved in fungal cell wall synthesis but do not display rich complements of algal cell-wall-processing enzymes, suggesting an independent origin of fungal plant-degrading capabilities. Aphelid genomes show that the emergence of Fungi from phagotrophic ancestors relied on a common cell wall synthetic machinery but required a different set of proteins for digestion and interaction with the environment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

There are more than 350 species of amphipods (Crustacea) in Lake Baikal, which have emerged predominantly through the course of endemic radiation. This group represents a remarkable model for studying various aspects of evolution, one of which is the evolution of mitochondrial (mt) genome architectures. We sequenced and assembled the mt genome of a pelagic Baikalian amphipod species Macrohectopus branickii. The mt genome is revealed to have an extraordinary length (42,256 bp), deviating significantly from the genomes of other amphipod species and the majority of animals. The mt genome of M. branickii has a unique gene order within amphipods, duplications of the four tRNA genes and Cox2 , and a long non-coding region, that makes up about two thirds of the genome's size. The extension of the mt genome was most likely caused by multiple duplications and inversions of regions harboring ribosomal RNA genes. In this study, we analyzed the patterns of mt genome length changes in amphipods and other animal phyla. Through a statistical analysis, we demonstrated that the variability in the mt genome length may be a characteristic of certain phyla and is primarily conferred by expansions of non-coding regions.

Background: Gregarines are a major group of apicomplexan parasites of invertebrates. The gregarine classification is largely incomplete because it relies primarily on light microscopy, while electron microscopy and molecular data in the group are fragmentary and often do not overlap. A key characteristic in gregarine taxonomy is the structure and function of their attachment organelles (AOs). AOs have been commonly classified as "mucrons" or "epimerites" based on their association with other cellular traits such as septation. An alternative proposal focused on the AOs structure, functional role, and developmental fate has recently restricted the terms "mucron" to archigregarines and "epimerite" to eugregarines., Methods: Light microscopy and scanning and transmission electron microscopy, molecular phylogenetic analyses of ribosomal RNA genes., Results: We obtained the first data on fine morphology of aseptate eugregarines Polyrhabdina pygospionis and Polyrhabdina cf. spionis , the type species. We demonstrate that their AOs differ from the mucron in archigregarines and represent an epimerite structurally resembling that in other eugregarines examined using electron microscopy. We then used the concatenated ribosomal operon DNA sequences (SSU, 5.8S, and LSU rDNA) of P. pygospionis to explore the phylogeny of eugregarines with a resolution superior to SSU rDNA alone. The obtained phylogenies show that the Polyrhabdina clade represents an independent, deep-branching family in the Ancoroidea clade within eugregarines. Combined, these results lend strong support to the hypothesis that the epimerite is a synapomorphic innovation of eugregarines. Based on these findings, we resurrect the family Polyrhabdinidae Kamm, 1922 and erect and diagnose the family Trollidiidae fam. n. within the superfamily Ancoroidea Simdyanov et al., 2017. Additionally, we re-describe the characteristics of P. pygospionis , emend the diagnoses of the genus Polyrhabdina , the family Polyrhabdinidae, and the superfamily Ancoroidea., Competing Interests: The authors declare there are no competing interests., (©2021 Paskerova et al.)

The origin of animals is one of the most intensely studied evolutionary events, and our understanding of this transition was greatly advanced by analyses of unicellular relatives of animals, which have shown many "animal-specific" genes actually arose in protistan ancestors long before the emergence of animals [1-3]. These genes have complex distributions, and the protists have diverse lifestyles, so understanding their evolutionary significance requires both a robust phylogeny of animal relatives and a detailed understanding of their biology [4, 5]. But discoveries of new animal-related lineages are rare and historically biased to bacteriovores and parasites. Here, we characterize the morphology and transcriptome content of a new animal-related lineage, predatory flagellate Tunicaraptor unikontum. Tunicaraptor is an extremely small (3-5 μm) and morphologically simple cell superficially resembling some fungal zoospores, but it survives by preying on other eukaryotes, possibly using a dedicated but transient "mouth," which is unique for unicellular opisthokonts. The Tunicaraptor transcriptome encodes a full complement of flagellar genes and the flagella-associated calcium channel, which is only common to predatory animal relatives and missing in microbial parasites and grazers. Tunicaraptor also encodes several major classes of animal cell adhesion molecules, as well as transcription factors and homologs of proteins involved in neurodevelopment that have not been found in other animal-related lineages. Phylogenomics, including Tunicaraptor, challenges the existing framework used to reconstruct the evolution of animal-specific genes and emphasizes that the diversity of animal-related lineages may be better understood only once the smaller, more inconspicuous animal-related lineages are better studied. VIDEO ABSTRACT., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Agamococcidians are enigmatic and poorly studied parasites of marine invertebrates with unexplored diversity and unclear relationships to other sporozoans such as the human pathogens Plasmodium and Toxoplasma. It is believed that agamococcidians are not capable of sexual reproduction, which is essential for life cycle completion in all well studied parasitic apicomplexans. Here, we describe three new species of agamococcidians belonging to the genus Rhytidocystis. We examined their cell morphology and ultrastructure, resolved their phylogenetic position by using near-complete rRNA operon sequences, and searched for genes associated with meiosis and oocyst wall formation in two rhytidocystid transcriptomes. Phylogenetic analyses consistently recovered rhytidocystids as basal coccidiomorphs and away from the corallicolids, demonstrating that the order Agamococcidiorida Levine, 1979 is polyphyletic. Light and transmission electron microscopy revealed that the development of rhytidocystids begins inside the gut epithelial cells, a characteristic which links them specifically with other coccidiomorphs to the exclusion of gregarines and suggests that intracellular invasion evolved early in the coccidiomorphs. We propose a new superorder Eococcidia for early coccidiomorphs. Transcriptomic analysis demonstrated that both the meiotic machinery and oocyst wall proteins are preserved in rhytidocystids. The conservation of meiotic genes and ultrastructural similarity of rhytidocystid trophozoites to macrogamonts of true coccidians point to an undescribed, cryptic sexual process in the group.

Alveolates are a major supergroup of eukaryotes encompassing more than ten thousand free-living and parasitic species, including medically, ecologically, and economically important apicomplexans, dinoflagellates, and ciliates. These three groups are among the most widespread eukaryotes on Earth, and their environmental success can be linked to unique innovations that emerged early in each group. Understanding the emergence of these well-studied and diverse groups and their innovations has relied heavily on the discovery and characterization of early-branching relatives, which allow ancestral states to be inferred with much greater confidence. Here we report the phylogenomic analyses of 313 eukaryote protein-coding genes from transcriptomes of three members of one such group, the colponemids (Colponemidia), which support their monophyly and position as the sister lineage to all other known alveolates. Colponemid-related sequences from environmental surveys and our microscopical observations show that colponemids are not common in nature, but they are diverse and widespread in freshwater habitats around the world. Studied colponemids possess two types of extrusive organelles (trichocysts or toxicysts) for active hunting of other unicellular eukaryotes and potentially play an important role in microbial food webs. Colponemids have generally plesiomorphic morphology and illustrate the ancestral state of Alveolata. We further discuss their importance in understanding the evolution of alveolates and the origin of myzocytosis and plastids., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Labyrinthulomycetes are mostly fungus-like heterotrophic protists that absorb nutrients in an osmotrophic or phagotrophic manner. Members of order Labyrinthulida produce unique membrane-bound ectoplasmic networks for movement and feeding. Among the various types of labyrinthulids' food substrates, diatoms play an important role due to their ubiquitous distribution and abundant biomass. We isolated and cultivated new diatom consuming Labyrinthulida strains from shallow coastal marine sediments. We described Labyrinthula diatomea n. sp. that differs from all known labyrinthulids in both molecular and morphological features. We provided strain delimitation within the genus Labyrinthula based on ITS sequences via haplotype network construction and compared it with previous phylogenetic surveys., (© 2020 International Society of Protistologists.)

The evolution of tRNA genes in mitochondrial (mt) genomes is a complex process that includes duplications, degenerations, and transpositions, as well as a specific process of identity change through mutations in the anticodon (tRNA gene remolding or tRNA gene recruitment). Using amphipod-specific tRNA models for annotation, we show that tRNA duplications are more common in the mt genomes of amphipods than what was revealed by previous annotations. Seventeen cases of tRNA gene duplications were detected in the mt genomes of amphipods, and ten of them were tRNA genes that underwent remolding. The additional tRNA gene findings were verified using phylogenetic analysis and genetic distance analysis. The majority of remolded tRNA genes (seven out of ten cases) were found in the mt genomes of endemic amphipod species from Lake Baikal. All additional mt tRNA genes arose independently in the Baikalian amphipods, indicating the unusual plasticity of tRNA gene evolution in these species assemblages. The possible reasons for the unusual abundance of additional tRNA genes in the mt genomes of Baikalian amphipods are discussed. The amphipod-specific tRNA models developed for MiTFi refine existing predictions of tRNA genes in amphipods and reveal additional cases of duplicated tRNA genes overlooked by using less specific Metazoa-wide models. The application of these models for mt tRNA gene prediction will be useful for the correct annotation of mt genomes of amphipods and probably other crustaceans., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The phylum Apicomplexa comprises human pathogens such as Plasmodium but is also an under-explored hotspot of evolutionary diversity central to understanding the origins of parasitism and non-photosynthetic plastids. We generated single-cell transcriptomes for all major apicomplexan groups lacking large-scale sequence data. Phylogenetic analysis reveals that apicomplexan-like parasites are polyphyletic and their similar morphologies emerged convergently at least three times. Gregarines and eugregarines are monophyletic, against most expectations, and rhytidocystids and Eleutheroschizon are sister lineages to medically important taxa. Although previously unrecognized, plastids in deep-branching apicomplexans are common, and they contain some of the most divergent and AT-rich genomes ever found. In eugregarines, however, plastids are either abnormally reduced or absent, thus increasing known plastid losses in eukaryotes from two to four. Environmental sequences of ten novel plastid lineages and structural innovations in plastid proteins confirm that plastids in apicomplexans and their relatives are widespread and share a common, photosynthetic origin., Competing Interests: JJ, GP, TM, KM, TB, VA, TS No competing interests declared, (© 2019, Janouškovec et al.)

Inverted repeats are common DNA elements, but they rarely overlap with protein-coding sequences due to the ensuing conflict with the structure and function of the encoded protein. We discovered numerous perfect inverted repeats of considerable length (up to 284 bp) embedded within the protein-coding genes in mitochondrial genomes of four Nematomorpha species. Strikingly, both arms of the inverted repeats encode conserved regions of the amino acid sequence. We confirmed enzymatic activity of the respiratory complex I encoded by inverted repeat-containing genes. The nucleotide composition of inverted repeats suggests strong selection at the amino acid level in these regions. We conclude that the inverted repeat-containing genes are transcribed and translated into functional proteins. The survey of available mitochondrial genomes reveals that several other organisms possess similar albeit shorter embedded repeats. Mitochondrial genomes of Nematomorpha demonstrate an extraordinary evolutionary compromise where protein function and stringent secondary structure elements within the coding regions are preserved simultaneously., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Molecular phylogenetic analysis of 18S rRNA gene sequences of nearly any species of Chytridiomycota has typically challenged traditional classification and triggered taxonomic revision. This has often led to the establishment of new taxa which, normally, appears well supported by zoospore ultrastructure, which provides diagnostic characters. To construct a meaningful and comprehensive classification of Chytridiomycota, the combination of molecular phylogenies and morphological studies of traditionally defined chytrid species is needed. In this work, we have studied morphological and ultrastructural features based on light and transmission electron microscopy as well as molecular phylogenetic analysis of a parasite (strain X-124 CCPP ZIN RAS) morphologically similar to Rhizophydium granulosporum living on the yellow-green alga Tribonema gayanum. Phylogenetic analysis of the 18S rRNA gene sequence of this strain supports that it represents a new genus and species affiliated to the recently established order Gromochytriales. The ultrastructure of X-124 confirms its phylogenetic position sister to Gromochytrium and serves as the basis for the description of the new genus and species Apiochytrium granulosporum. The 18S rRNA gene of A. granulosporum contains a S943 group I intron that carries a homing endonuclease pseudogene., (© 2018 International Society of Protistologists.)

Two enigmatic groups of morphologically simple parasites of invertebrates, the Dicyemida (syn. Rhombozoa) and the Orthonectida, since the 19th century have been usually considered as two classes of the phylum Mesozoa. Early molecular evidence suggested their relationship within the Spiralia (=Lophotrochozoa), however, high rates of dicyemid and orthonectid sequence evolution led to contradicting phylogeny reconstructions. Genomic data for orthonectids revealed that they are highly simplified spiralians and possess a reduced set of genes involved in metazoan development and body patterning. Acquiring genomic data for dicyemids, however, remains a challenge due to complex genome rearrangements including chromatin diminution and generation of extrachromosomal circular DNAs, which are reported to occur during the development of somatic cells. We performed genomic sequencing of one species of Dicyema , and obtained transcriptomic data for two Dicyema spp. Homeodomain (homeobox) transcription factors, G-protein-coupled receptors, and many other protein families have undergone a massive reduction in dicyemids compared to other animals. There is also apparent reduction of the bilaterian gene complements encoding components of the neuromuscular systems. We constructed and analyzed a large dataset of predicted orthologous proteins from three species of Dicyema and a set of spiralian animals including the newly sequenced genome of the orthonectid Intoshia linei . Bayesian analyses recovered the orthonectid lineage within the Annelida. In contrast, dicyemids form a separate clade with weak affinity to the Rouphozoa (Platyhelminthes plus Gastrotricha) or (Entoprocta plus Cycliophora) suggesting that the historically proposed Mesozoa is a polyphyletic taxon. Thus, dramatic simplification of body plans in dicyemids and orthonectids, as well as their intricate life cycles that combine metagenesis and heterogony, evolved independently in these two lineages.

ᅟ: Some tumor cells can evolve into transmissible parasites. Notable examples include the Tasmanian devil facial tumor disease, the canine transmissible venereal tumor and transmissible cancers of mollusks. We present a hypothesis that such transmissible tumors existed in the past and that some modern animal taxa are descendants of these tumors. We expect potential candidates for SCANDALs (speciated by cancer development animals) to be simplified relatives of more complex metazoans and have genomic alterations typical for cancer progression (such as deletions of universal apoptosis genes). We considered several taxa of simplified animals for our hypothesis: dicyemida, orthonectida, myxosporea and trichoplax. Based on genomic analysis we conclude that Myxosporea appear to be the most suitable candidates for a tumor ancestry. They are simplified parasitic cnidarians that universally lack major genes implicated in cancer progression including all genes with Caspase and BCL2 domains as well as any p53 and apoptotic protease activating factor - 1 (Apaf-1) homologs, suggesting the disruption of main apoptotic pathways in their early evolutionary history. Further comparative genomics and single-cell transcriptomic studies may be helpful to test our hypothesis of speciation via a cancerous stage. REVIEWERS: This article was reviewed by Eugene Koonin, Mikhail Gelfand and Gregory M Woods.

Archigregarines are a key group for understanding the early evolution of Apicomplexa. Here we report morphological, ultrastructural, and molecular phylogenetic evidence from two archigregarine species: Selenidium pygospionis sp. n. and S. pherusae sp. n. They exhibited typical features of archigregarines. Additionally, an axial row of vacuoles of a presumably nutrient distribution system was revealed in S. pygospionis. Intracellular stages of S. pygospionis found in the host intestinal epithelium may point to the initial intracellular localization in the course of parasite development. Available archigregarine SSU (18S) rDNA sequences formed four major lineages fitting the taxonomical affiliations of their hosts, but not the morphological or biological features used for the taxonomical revision by Levine (1971). Consequently, the genus Selenidioides Levine, 1971 should be abolished. The branching order of these lineages was unresolved; topology tests rejected neither para- nor monophyly of archigregarines. We provided phylogenies based on LSU (28S) rDNA and near-complete ribosomal operon (concatenated SSU, 5.8S, LSU rDNAs) sequences including S. pygospionis sequences. Although being preliminary, they nevertheless revealed the monophyly of gregarines previously challenged by many molecular phylogenetic studies. Despite their molecular-phylogenetic heterogeneity, archigregarines exhibit an extremely conservative plesiomorphic structure; their ultrastructural key features appear to be symplesiomorphies rather than synapomorphies., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Blastogregarines are poorly studied parasites of polychaetes superficially resembling gregarines, but lacking syzygy and gametocyst stages in the life cycle. Furthermore, their permanent multinuclearity and gametogenesis by means of budding considerably distinguish them from other parasitic Apicomplexa such as coccidians and hematozoans. The affiliation of blastogregarines has been uncertain: different authors considered them highly modified gregarines, an intermediate apicomplexan lineage between gregarines and coccidians, or an isolated group of eukaryotes altogether. Here, we report the ultrastructure of two blastogregarine species, Siedleckia nematoides and Chattonaria mesnili, and provide the first molecular data on their phylogeny based on SSU, 5.8S, and LSU rDNA sequences. Morphological analysis reveals that blastogregarines possess both gregarine and coccidian features. Several traits shared with archigregarines likely represent the ancestral states of the corresponding cell structures for parasitic apicomplexans: a distinctive tegument structure and myzocytotic feeding with a well-developed apical complex. Unlike gregarines but similar to coccidians however, the nuclei of male blastogregarine gametes are associated with two kinetosomes. Molecular phylogenetic analyses reveal that blastogregarines are an independent, early diverging lineage of apicomplexans. Overall, the morphological and molecular evidence congruently suggests that blastogregarines represent a separate class of Apicomplexa., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

The Monoblepharidomycetes is the sister class to the Chytridiomycetes in the phylum Chytridiomycota. The six known genera have thalli that are either monocentric and without rhizoids or produce hyphae with an independent evolutionary origin from the hyphae of higher fungi. On the basis of morphological characters and phylogenetic evidence from the small and large subunits of nuclear ribosomal RNA, we established two new genera, Sanchytrium and Telasphaerula, each with a single species. We re-analyzed intergeneric relationships within the monoblephs, and established two new families. The new genera significantly expand the known morphological and ecological diversity of the Monoblepharidomycetes by adding a monocentric, epibiotic, algal parasitic species and a rhizomycelial, saprotrophic species. Based on the presence of environmental sequences related to Sanchytrium strains, the Monoblepharidomycetes contain previously unsuspected diversity. The ribosomal DNA of the new genera contains an unusually high density of group I introns. We found 20 intron insertion positions including six that are new for rRNA genes (S1053, L803, L829, L961, L1844, and L2281)., (Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Background: Gregarines are a group of early branching Apicomplexa parasitizing invertebrate animals. Despite their wide distribution and relevance to the understanding the phylogenesis of apicomplexans, gregarines remain understudied: light microscopy data are insufficient for classification, and electron microscopy and molecular data are fragmentary and overlap only partially., Methods: Scanning and transmission electron microscopy, PCR, DNA cloning and sequencing (Sanger and NGS), molecular phylogenetic analyses using ribosomal RNA genes (18S (SSU), 5.8S, and 28S (LSU) ribosomal DNAs (rDNAs))., Results and Discussion: We present the results of an ultrastructural and molecular phylogenetic study on the marine gregarine Ancora sagittata from the polychaete Capitella capitata followed by evolutionary and taxonomic synthesis of the morphological and molecular phylogenetic evidence on eugregarines. The ultrastructure of Ancora sagittata generally corresponds to that of other eugregarines, but reveals some differences in epicytic folds (crests) and attachment apparatus to gregarines in the family Lecudinidae, where Ancora sagittata has been classified. Molecular phylogenetic trees based on SSU (18S) rDNA reveal several robust clades (superfamilies) of eugregarines, including Ancoroidea superfam. nov., which comprises two families (Ancoridae fam. nov. and Polyplicariidae) and branches separately from the Lecudinidae; thus, all representatives of Ancoroidea are here officially removed from the Lecudinidae. Analysis of sequence data also points to possible cryptic species within Ancora sagittata and the inclusion of numerous environmental sequences from anoxic habitats within the Ancoroidea. LSU (28S) rDNA phylogenies, unlike the analysis of SSU rDNA alone, recover a well-supported monophyly of the gregarines involved (eugregarines), although this conclusion is currently limited by sparse taxon sampling and the presence of fast-evolving sequences in some species. Comparative morphological analyses of gregarine teguments and attachment organelles lead us to revise their terminology. The terms "longitudinal folds" and "mucron" are restricted to archigregarines, whereas the terms "epicystic crests" and "epimerite" are proposed to describe the candidate synapomorphies of eugregarines, which, consequently, are considered as a monophyletic group. Abolishing the suborders Aseptata and Septata, incorporating neogregarines into the Eugregarinida, and treating the major molecular phylogenetic lineages of eugregarines as superfamilies appear as the best way of reconciling recent morphological and molecular evidence. Accordingly, the diagnosis of the order Eugregarinida Léger, 1900 is updated., Competing Interests: The authors declare that they have no competing interests.

Metchnikovellidae are a group of unusual microsporidians that lack some of the defining ultrastructural features characteristic of derived Microsporidia and are thought to be one of their earliest-branching lineages. The basal position of metchnikovellids was never confirmed by molecular phylogeny in published research, and thus far no genomic data for this group were available. In this work, we obtain a partial genome of metchnikovellid Amphiamblys sp. using multiple displacement amplification, next-generation sequencing, and metagenomic binning approaches. The partial genome, which we estimate to be close to 90% complete, displays genome compaction on par with gene-dense microsporidian genomes, but contains an unusual repertoire of unique repeat elements. Phylogenetic analyses of multigene datasets place Amphiamblys sp. as the first branch of the microsporidian lineage following the divergence of a mitochondriate microsporidian Mitosporidium. We find evidence for a mitochondrial remnant presumably functionally equivalent to a mitosome in Amphiamblys sp. and the common enzymatic complement for microsporidian anaerobic metabolism. Comparative genomic analyses identify the conservation of components for clathrin vesicle formation as one of the key features distinguishing the metchnikovellid from its highly derived relatives. The presented data confirm the notion of Metchnikovellidae as a less derived microsporidian group, and provide an additional stepping stone for reconstruction of an evolutionary transition from the early diverging parasitic fungi to derived Microsporidia., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: Amphipods (Crustacea) of Lake Baikal are a very numerous and diverse group of invertebrates generally believed to have originated by adaptive radiation. The evolutionary history and phylogenetic relationships in Baikalian amphipods still remain poorly understood. Sequencing of mitochondrial genomes is a relatively feasible way for obtaining a set of gene sequences suitable for robust phylogenetic inferences. The architecture of mitochondrial genomes also may provide additional information on the mechanisms of evolution of amphipods in Lake Baikal., Results: Three complete and four nearly complete mitochondrial genomes of Baikalian amphipods were obtained by high-throughput sequencing using the Illumina platform. A phylogenetic inference based on the nucleotide sequences of all mitochondrial protein coding genes revealed the Baikalian species to be a monophyletic group relative to the nearest non-Baikalian species with a completely sequenced mitochondrial genome - Gammarus duebeni. The phylogeny of Baikalian amphipods also suggests that the shallow-water species Eulimnogammarus has likely evolved from a deep-water ancestor, however many other species have to be added to the analysis to test this hypothesis. The gene order in all mitochondrial genomes of studied Baikalian amphipods differs from the pancrustacean ground pattern. Mitochondrial genomes of four species possess 23 tRNA genes, and in three genomes the extra tRNA gene copies have likely undergone remolding. Widely varying lengths of putative control regions and other intergenic spacers are typical for the mitochondrial genomes of Baikalian amphipods., Conclusions: The mitochondrial genomes of Baikalian amphipods display varying organization suggesting an intense rearrangement process during their evolution. Comparison of complete mitochondrial genomes is a potent approach for studying the amphipod evolution in Lake Baikal.

In this study, we present a complete mitochondrial genome of a deep-water amphipoda Brachyuropus grewingkii (Dybowsky, 1874) from Lake Baikal. A circular mitochondrial DNA has 17,118 bp in length and contains 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, a putative control region, and five intergenic spacers. An extended control region and altered positions of some tRNA genes distinguish mitochondrial genome of B. grewingkii from the mitochondrial genomes described for other Baikalian amphipoda species.

Many features of mitochondrial genomes of animals, such as patterns of gene arrangement, nucleotide content and substitution rate variation are extensively used in evolutionary and phylogenetic studies. Nearly 6,000 mitochondrial genomes of animals have already been sequenced, covering the majority of animal phyla. One of the groups that escaped mitogenome sequencing is phylum Kinorhyncha-an isolated taxon of microscopic worm-like ecdysozoans. The kinorhynchs are thought to be one of the early-branching lineages of Ecdysozoa, and their mitochondrial genomes may be important for resolving evolutionary relations between major animal taxa. Here we present the results of sequencing and analysis of mitochondrial genomes from two members of Kinorhyncha, Echinoderes svetlanae (Cyclorhagida) and Pycnophyes kielensis (Allomalorhagida). Their mitochondrial genomes are circular molecules approximately 15 Kbp in size. The kinorhynch mitochondrial gene sequences are highly divergent, which precludes accurate phylogenetic inference. The mitogenomes of both species encode a typical metazoan complement of 37 genes, which are all positioned on the major strand, but the gene order is distinct and unique among Ecdysozoa or animals as a whole. We predict four types of start codons for protein-coding genes in E. svetlanae and five in P. kielensis with a consensus DTD in single letter code. The mitochondrial genomes of E. svetlanae and P. kielensis encode duplicated methionine tRNA genes that display compensatory nucleotide substitutions. Two distant species of Kinorhyncha demonstrate similar patterns of gene arrangements in their mitogenomes. Both genomes have duplicated methionine tRNA genes; the duplication predates the divergence of two species. The kinorhynchs share a few features pertaining to gene order that align them with Priapulida. Gene order analysis reveals that gene arrangement specific of Priapulida may be ancestral for Scalidophora, Ecdysozoa, and even Protostomia., Competing Interests: The authors have declared that no competing interests exist.

Heterotrophic lineages of Heterokonta (or stramenopiles), in contrast to a single monophyletic group of autotrophs, Ochrophyta, form several clades that independently branch off the heterokont stem lineage. The nearest neighbors of Ochrophyta in the phylogenetic tree appear to be almost exclusively bacterivorous, whereas the hypothesis of plastid acquisition by the ancestors of the ochrophyte lineage suggests an ability to engulf eukaryotic alga. In line with this hypothesis, the heterotrophic predator at the base of the ochrophyte lineage may be regarded as a model for the ochrophyte ancestor. Here, we present a new genus and species of marine free-living heterotrophic heterokont Develorapax marinus, which falls into an isolated heterokont cluster, along with the marine flagellate Developayella elegans, and is able to engulf eukaryotic cells. Together with environmental sequences D. marinus and D. elegans form a class-level clade Developea nom. nov. represented by species adapted to different environmental conditions and with a wide geographical distribution. The position of Developea among Heterokonta in large-scale phylogenetic tree is discussed. We propose that members of the Developea clade represent a model for transition from bacterivory to a predatory feeding mode by selection for larger prey. Presumably, such transition in the grazing strategy is possible in the presence of bacterial biofilms or aggregates expected in eutrophic environment, and has likely occurred in the ochrophyte ancestor.

Orthonectids are rare parasites of marine invertebrates [1] that are commonly treated in textbooks as a taxon of uncertain affinity [2]. Trophic forms of orthonectids reside in the tissues of their hosts as multinucleated plasmodia, generating short-lived, worm-like ciliated female and male organisms that exit into the environment for copulation [3]. These ephemeral males and females are composed of just several hundred somatic cells and are deprived of digestive, circulatory, or excretory systems. Since their discovery in the 19(th) century, the orthonectids were described as organisms with no differentiated cell types and considered as part of Mesozoa, a putative link between multicellular animals and their unicellular relatives. More recently, this view was challenged as the new data suggested that orthonectids are animals that became simplified due to their parasitic way of life [3, 4]. Here, we report the genomic sequence of Intoshia linei, one of about 20 known species of orthonectids. The genomic data confirm recent morphological analysis asserting that orthonectids are members of Spiralia and possess muscular and nervous systems [5]. The 43-Mbp genome of I. linei encodes about 9,000 genes and retains those essential for the development and activity of muscular and nervous systems. The simplification of orthonectid body plan is associated with considerable reduction of metazoan developmental genes, leaving what might be viewed as the minimal gene set necessary to retain critical bilaterian features., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Urosporids (Apicomplexa: Urosporidae) are eugregarines that parasitise marine invertebrates, such as annelids, molluscs, nemerteans and echinoderms, inhabiting their coelom and intestine. Urosporids exhibit considerable morphological plasticity, which correlates with their different modes of motility and variations in structure of their cortical zone, according to the localisation within the host. The gregarines Urospora ovalis and U. travisiae from the marine polychaete Travisia forbesii were investigated with an emphasis on their general morphology and phylogenetic position. Solitary ovoid trophozoites and syzygies of U. ovalis were located free in the host coelom and showed metabolic activity, a non-progressive movement with periodic changes of the cell shape. Solitary trophozoites of U. travisiae, attached to the host tissue or free floating in the coelom, were V-shaped. Detached trophozoites demonstrated gliding motility, a progressive movement without observable cell body changes. In both gregarines, the cortex formed numerous epicytic folds, but superfolds appeared exclusively on the surface of U. ovalis during metabolic activity. SSU rDNA sequences obtained from U. ovalis and U. travisiae revealed that they belong to the Lecudinoidea clade; however, they are not affiliated with other coelomic urosporids (Pterospora spp. and Lithocystis spp.), but surprisingly with intestinal lecudinids (Difficilina spp.) parasitising nemerteans., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

A complete mitochondrial genome sequence of amphipoda Eulimnogammarus vittatus Dybowsky, 1874 from Lake Baikal was obtained using next-generation sequencing approach. Mitochondrial DNA with the length of 15,534 bp contains 13 protein-coding genes, 2 ribosomal RNA, 23 transfer RNA and non-coding sequences: a putative control region and 7 intergenic spacers. A brief comparative analysis of mitochondrial genomes of E. vittatus and its sister species Eulimnogammarus verrucosus was performed.

During the last decade several new orders were established in the class Chytridiomycetes on the basis of zoospore ultrastructure and molecular phylogeny. Here we present the ultrastructure and molecular phylogeny of strain x-51 CALU - a parasite of the alga Tribonema gayanum, originally described as Rhizophydium sp. based on light microscopy. Detailed investigation revealed that the zoospore ultrastructure of this strain has unique characters not found in any order of Chytridiomycetes: posterior ribosomal core unbounded by the endoplasmic reticulum and detached from the nucleus or microbody-lipid complex, and kinetosome composed of microtubular doublets. An isolated phylogenetic position of x-51 is further confirmed by the analysis of 18S and 28S rRNA sequences, and motivates the description of a new genus and species Gromochytrium mamkaevae. The sister position of G. mamkaevae branch relative to Mesochytrium and a cluster of environmental sequences, as well as the ultrastructural differences between Gromochytrium and Mesochytrium zoospores prompted us to establish two new orders: Gromochytriales and Mesochytriales.

The evolutionary and ecological importance of predatory flagellates are too often overlooked. This is not only a gap in our understanding of microbial diversity, but also impacts how we interpret their better-studied relatives. A prime example of these problems is found in the alveolates. All well-studied species belong to three large clades (apicomplexans, dinoflagellates, and ciliates), but the predatory colponemid flagellates are also alveolates that are rare in nature and seldom cultured, but potentially important to our understanding of alveolate evolution. Recently we reported the first cultivation and molecular analysis of several colponemid-like organisms representing two novel clades in molecular trees. Here we provide ultrastructural analysis and formal species descriptions for both new species, Colponema vietnamica n. sp. and Acavomonas peruviana n. gen. n. sp. Morphological and feeding characteristics concur with molecular data that both species are distinct members of alveolates, with Acavomonas lacking the longitudinal phagocytotic groove, a defining feature of Colponema. Based on ultrastructure and molecular phylogenies, which both provide concrete rationale for a taxonomic reclassification of Alveolata, we establish the new phyla Colponemidia nom. nov. for the genus Colponema and its close relatives, and Acavomonidia nom. nov. for the genus Acavomonas and its close relatives. The morphological data presented here suggests that colponemids are central to our understanding of early alveolate evolution, and suggest they also retain features of the common ancestor of all eukaryotes.

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.

The alveolates comprise three well-studied protist lineages of significant environmental, medical, and economical importance: apicomplexans (e.g., Plasmodium), dinoflagellates (e.g., Symbiodinium), and ciliates (e.g., Tetrahymena). These major lineages have evolved distinct and unusual characteristics, the origins of which have proved to be difficult evolutionary puzzles. Mitochondrial genomes are a prime example: all three groups depart from canonical form and content, but in different ways. Reconstructing such ancient transitions is difficult without deep-branching lineages that retain ancestral characteristics. Here we describe two such lineages and how they illuminate the ancestral state of alveolate mitochondrial genomes. We established five clonal cultures of colponemids, predatory alveolates without cultured representatives and molecular data. Colponemids represent at least two independent lineages at the phylum level in multilocus phylogenetic analysis; one sister to apicomplexans and dinoflagellates, and the other at a deeper position. A genome survey from one strain showed that ancestral state of the mitochondrial genomes in the three major alveolate lineages consisted of an unusual linear chromosome with telomeres and a substantially larger gene set than known alveolates. Colponemid sequences also identified several environmental lineages as colponemids, altogether suggesting an untapped potential for understanding the origin and evolution of apicomplexans, dinoflagellates, and ciliates., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Introns comprise a considerable portion of eukaryotic genomes; however, their evolution is understudied. Numerous works of the last years largely disagree on many aspects of intron evolution. Interpretation of these differences is hindered because different algorithms and taxon sampling strategies were used. Here, we present the first attempt of a systematic evaluation of the effects of taxon sampling on popular intron evolution estimation algorithms. Using the "taxon jackknife" method, we compared the effect of taxon sampling on the behavior of intron evolution inferring algorithms. We show that taxon sampling can dramatically affect the inferences and identify conditions where algorithms are prone to systematic errors. Presence or absence of some key species is often more important than the taxon sampling size alone. Criteria of representativeness of the taxonomic sampling for reliable reconstructions are outlined. Presence of the deep-branching species with relatively high intron density is more important than sheer number of species. According to these criteria, currently available genomic databases are representative enough to provide reliable inferences of the intron evolution in animals, land plants, and fungi, but they underrepresent many groups of unicellular eukaryotes, including the well-studied Alveolata.

Background: Environmental SSU rDNA surveys have significantly improved our understanding of microeukaryotic diversity. Many of the sequences acquired using this approach are closely related to lineages previously characterized at both morphological and molecular levels, making interpretation of these data relatively straightforward. Some sequences, by contrast, appear to be phylogenetic orphans and are sometimes inferred to represent "novel lineages" of unknown cellular identity. Consequently, interpretation of environmental DNA surveys of cellular diversity rely on an adequately comprehensive database of DNA sequences derived from identified species. Several major taxa of microeukaryotes, however, are still very poorly represented in these databases, and this is especially true for diverse groups of single-celled parasites, such as gregarine apicomplexans., Methodology/principal Findings: This study attempts to address this paucity of dna sequence data by characterizing four different gregarine species, isolated from the intestines of crustaceans, at both morphological and molecular levels: Thiriotia pugettiae sp. n. from the graceful kelp crab (Pugettia gracilis), Cephaloidophora cf. communis from two different species of barnacles (Balanus glandula and B. balanus), Heliospora cf. longissima from two different species of freshwater amphipods (Eulimnogammarus verrucosus and E. vittatus), and Heliospora caprellae comb. n. from a skeleton shrimp (Caprella alaskana). SSU rDNA sequences were acquired from isolates of these gregarine species and added to a global apicomplexan alignment containing all major groups of gregarines characterized so far. Molecular phylogenetic analyses of these data demonstrated that all of the gregarines collected from crustacean hosts formed a very strongly supported clade with 48 previously unidentified environmental DNA sequences., Conclusions/significance: This expanded molecular phylogenetic context enabled us to establish a major clade of intestinal gregarine parasites and infer the cellular identities of several previously unidentified environmental SSU rDNA sequences, including several sequences that have formerly been discussed broadly in the literature as a suspected "novel" lineage of eukaryotes.

For over a century, Haeckel's Gastraea theory remained a dominant theory to explain the origin of multicellular animals. According to this theory, the animal ancestor was a blastula-like colony of uniform cells that gradually evolved cell differentiation. Today, however, genes that typically control metazoan development, cell differentiation, cell-to-cell adhesion, and cell-to-matrix adhesion are found in various unicellular relatives of the Metazoa, which suggests the origin of the genetic programs of cell differentiation and adhesion in the root of the Opisthokonta. Multicellular stages occurring in the complex life cycles of opisthokont protists (mesomycetozoeans and choanoflagellates) never resemble a blastula. Here, we discuss a more realistic scenario of transition to multicellularity through integration of pre-existing transient cell types into the body of an early metazoon, which possessed a complex life cycle with a differentiated sedentary filter-feeding trophic stage and a non-feeding blastula-like larva, the synzoospore. Choanoflagellates are considered as forms with secondarily simplified life cycles.

The pLF1311 natural plasmid from Lactobacillus fermentum 1311 was used to construct a single-replicon vector suitable for rapid cloning in a wide range of gram-positive hosts and Escherichia coli. The new vector is capable of conjugative mobilization from E. coli to various hosts by conjugal transfer. The final vector (3.4 kb) showed a high segregational and structural stability and a high copy number. Glutamyl endopeptidase genes from Bacillus licheniformis (gseBL) and B. intermedius (gseBI) were cloned in both pLF9 and pLF14 vectors and introduced to B. subtilis. The yield of enzymes in the pLF-derived producers was 6- to 30-fold more than in the natural producers and reached 100-150 mg/L of mature protease., (Copyright 2000 Academic Press.)