Ultrastructure and 28S rDNA Phylogeny of Two Gregarines: Cephaloidophora cf. communis and Heliospora cf. longissima with Remarks on Gregarine Morphology and Phylogenetic Analysis

18S rRNA gene sequences (SSU rDNA) in gregarines are problematic for phylogenetic analysis, mainly due to artifacts related to long branch attraction (LBA). In this study, we sequenced 18S rRNA (SSU rRNA), 5.8S rRNA, and 28S rRNA (LSU rRNA) genes of two gregarine species from crustacean hosts (gregarine superfamily Cephaloidophoroidea): Cephaloidophora cf. communis from a marine cirripedian Balanus balanus (White Sea), and Heliospora cf. longissima from the freshwater amphipods, Eulimnogammarus verrucosus and E. vittatus (Lake Baikal). Phylogenetic analyses of SSU rDNA sequences failed to produce a robust tree topology, for a limited taxon sample (31 operational taxonomic units (OTU), based on 1,604 sites), while LSU (2,869 sites), and concatenated dataset based on SSU, 5.8S, and LSU (4,627 sites) produced more consistent tree topologies for the same taxon sample. Analyses testing for LBA-influence were negative, therefore we suggested that the main reason of the failed topologies in SSU rDNA analyses is insufficient data (insufficient taxon sampling and limited molecular data), rather than LBA. Possible advantages of Bayesian analyses, compared to Maximum Likelihood, and usage of LSU rDNA within the context of apicomplexan phylogenetics were discussed. One of the advantages of LSU is likely its lower rate of evolution in long-branching apicomplexans (e.g., gregarines), relative to other (non-long-branching) apicomplexans, compared to SSU rDNA. Ultrastructure of the epicytic folds was studied. There are 3 to 5 apical arcs (also known as rippled dense structures) and 2 to 5 apical filaments in the tops of the folds. This small number of the apical structures fits into morphological diversity of the epicyte in other Cephaloidophoroidea, but this is not a synapomorphy of the group because this was also detected in several unrelated gregarines. C. cf. communis was found to contain a septum between the epimerite and the protomerite, which has not been reported in other gregarines. More exact terminology, which takes into account number of body sections and septa, is proposed for morphological descriptions of trophozoites and free mature gamonts of gregarines. In accordance with this, C. cf. communis gamonts are tricystid and biseptate, whereas H. cf. longissima gamonts are tricystid and uniseptate, similar to other eugregarines

Dicyemida and Orthonectida: Two Stories of Body Plan Simplification

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

Heterokont predator Develorapax marinus gen. et sp. nov. – a model of the ochrophyte ancestor

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 heteretrophic 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, and has likely occured in the ochrophyte ancestor

Effects of Taxon Sampling in Reconstructions of Intron Evolution

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

Identification of a Divergent Environmental DNA Sequence Clade Using the Phylogeny of Gregarine Parasites (Apicomplexa) from Crustacean Hosts

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 eukaryote

A new view on the morphology and phylogeny of eugregarines suggested by the evidence from the gregarine Ancora sagittata (Leuckart, 1860) Labbé, 1899 (Apicomplexa: Eugregarinida)

International audienceBackground: 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

Apicomplexan-like parasites are polyphyletic and widely but selectively dependent on cryptic plastid organelles

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

Predatory colponemids are the sister group to all other alveolates

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