Alexander P. Mylnikov

Untangling the early diversification of eukaryotes: a phylogenomic study of the evolutionary origins of Centrohelida, Haptophyta and Cryptista.

Assembling the global eukaryotic tree of life has long been a major effort of Biology. In recent years, pushed by the new availability of genome-scale data for microbial eukaryotes, it has become possible to revisit many evolutionary enigmas. However, some of the most ancient nodes, which are essential for inferring a stable tree, have remained highly controversial. Among other reasons, the lack of adequate genomic datasets for key taxa has prevented the robust reconstruction of early diversification events. In this context, the centrohelid heliozoans are particularly relevant for reconstructing the tree of eukaryotes because they represent one of the last substantial groups that was missing large and diverse genomic data. Here, we filled this gap by sequencing high-quality transcriptomes for four centrohelid lineages, each corresponding to a different family. Combining these new data with a broad eukaryotic sampling, we produced a gene-rich taxon-rich phylogenomic dataset that enabled us to refine the structure of the tree. Specifically, we show that (i) centrohelids relate to haptophytes, confirming Haptista; (ii) Haptista relates to SAR; (iii) Cryptista share strong affinity with Archaeplastida; and (iv) Haptista + SAR is sister to Cryptista + Archaeplastida. The implications of this topology are discussed in the broader context of plastid evolution.

The phylogeny of colpodellids (Alveolata) using small subunit rRNA gene sequences suggests they are the free-living sister group to apicomplexans

Abstract In an attempt to reconstruct early alveolate evolution, we have examined the phylogenetic position of colpodellids by analyzing small subunit rDNA sequences from Colpodella pontica Myl’nikov 2000 and Colpodella sp. (American Type Culture Collection 50594). All phylogenetic analyses grouped the colpodellid sequences together with strong support and placed them strongly within the Alveolata. Most analyses showed colpodellids as the sister group to an apicomplexan clade, albeit with weak support. Sequences from two perkinsids, Perkinsus and Parvilucifera, clustered together and consistently branched as the sister group to dinoflagellates as shown previously. These data demonstrate that colpodellids and perkinsids are plesiomorphically similar in morphology and help provide a phylogenetic framework for inferring the combination of character states present in the last common ancestor of dinoflagellates and apicomplexans. We can infer that this ancestor was probably a myzocytotic predator with two heterodynamic flagella, micropores, trichocysts, rhoptries, micronemes, a polar ring, and a coiled open-sided conoid. This ancestor also very likely contained a plastid, but it is presently not certain whether it was photosynthetic, and it is not clear whether extant perkinsids or colpodellids have retained the organelle.

Factors mediating plastid dependency and the origins of parasitism in apicomplexans and their close relatives

Apicomplexans are a major lineage of parasites, including causative agents of malaria and toxoplasmosis. How such highly adapted parasites evolved from free-living ancestors is poorly understood, particularly because they contain nonphotosynthetic plastids with which they have a complex metabolic dependency. Here, we examine the origin of apicomplexan parasitism by resolving the evolutionary distribution of several key characteristics in their closest free-living relatives, photosynthetic chromerids and predatory colpodellids. Using environmental sequence data, we describe the diversity of these apicomplexan-related lineages and select five species that represent this diversity for transcriptome sequencing. Phylogenomic analysis recovered a monophyletic lineage of chromerids and colpodellids as the sister group to apicomplexans, and a complex distribution of retention versus loss for photosynthesis, plastid genomes, and plastid organelles. Reconstructing the evolution of all plastid and cytosolic metabolic pathways related to apicomplexan plastid function revealed an ancient dependency on plastid isoprenoid biosynthesis, predating the divergence of apicomplexan and dinoflagellates. Similarly, plastid genome retention is strongly linked to the retention of two genes in the plastid genome, sufB and clpC, altogether suggesting a relatively simple model for plastid retention and loss. Lastly, we examine the broader distribution of a suite of molecular characteristics previously linked to the origins of apicomplexan parasitism and find that virtually all are present in their free-living relatives. The emergence of parasitism may not be driven by acquisition of novel components, but rather by loss and modification of the existing, conserved traits.

Molecular Phylogeny and Surface Morphology of Colpodella edax (Alveolata): Insights into the Phagotrophic Ancestry of Apicomplexans

Abstract The molecular phylogeny of colpodellids provides a framework for inferences about the earliest stages in apicomplexan evolution and the characteristics of the last common ancestor of apicomplexans and dinoflagellates. We extended this research by presenting phylogenetic analyses of small subunit rRNA gene sequences from Colpodella edax and three unidentified eukaryotes published from molecular phylogenetic surveys of anoxic environments. Phylogenetic analyses consistently showed C. edax and the environmental sequences nested within a colpodellid clade, which formed the sister group to (eu)apicomplexans. We also presented surface details of C. edax using scanning electron microscopy in order to supplement previous ultrastructural investigations of this species using transmission electron microscopy and to provide morphological context for interpreting environmental sequences. The microscopical data confirmed a sparse distribution of micropores, an amphiesma consisting of small polygonal alveoli, flagellar hairs on the anterior flagellum, and a rostrum molded by the underlying (open-sided) conoid. Three flagella were present in some individuals, a peculiar feature also found in the microgametes of some apicomplexans.

The origin of Metazoa: a transition from temporal to spatial cell differentiation†

For over a century, Haeckels 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.

Molecular Comparisons of Freshwater and Marine Isolates of the Same Morphospecies of Heterotrophic Flagellates

ABSTRACT Heterotrophic flagellates are key components of all ecosystems. Understanding the patterns of biodiversity of these organisms is thus particularly important. Here we analyzed the intraspecific diversity of 10 morphospecies of heterotrophic flagellates comprising representatives of the Apusozoa (2 morphospecies) and Kinetoplastea (8 morphospecies), all belonging to the most common flagellates with worldwide distribution. Most morphospecies showed a mixing of lineages isolated from diverse habitats, indicating that some lineages of these morphospecies had been able to colonize different habitats several times. Furthermore, our results revealed remarkable levels of genetic divergence within most of the morphospecies studied, underlining the difficulty of correctly determining species by means of morphology alone. Many cryptic or pseudocryptic species seem to occur. Our results revealed clear divergence between marine and freshwater lineages of the morphospecies Ancyromonas sigmoides, showing that freshwater lineages have not been able to colonize marine environments and marine lineages have not been able to colonize freshwater environments for a long time.

Distribution and Phylogenetic Relationships of Freshwater Thaumatomonads with a Description of the New Species Thaumatomonas coloniensis n. sp.

ABSTRACT. The order Thaumatomonadida includes biflagellated heterotrophic flagellates that form filopodia and typically possess siliceous surface scales. We found thaumatomonads to contribute on average about 5%–10% to flagellate abundance in different benthic habitats. A new species of thaumatomonads, Thaumatomonas coloniensis n. sp., is described on the basis of morphological and molecular biological features. This new species was isolated both from groundwater at Appeldorn near Rees (Germany) and from the Rhine River at Cologne (Germany). We have sequenced the small subunit rRNA (ssu rRNA) gene and a fragment of the large subunit rRNA (lsu rRNA) gene (D3–D5 region) from the isolates of the new species, including the first sequence of a representative of the thaumatomonad genus Gyromitus. In agreement with previous studies, the differences in ribosomal genes of different thaumatomonad species are very small. For understanding the phylogenetic relationships of Thaumatomonadida and to explore their sister group relationships, we have created three sequence data sets (ssu rRNA, partial lsu rRNA, concatenated alignment of both) with the same composition of isolates (from Thaumatomonadida, Euglyphida, Cercomonadidae, and Heteromitidae). According to a Kishino–Hasegawa test, Thaumatomonadida evolved within the Cercozoa as a sister taxon to the Heteromitidae. A possibly close relationship to the Euglyphida, recently grouped together with the Thaumatomonadida in the class Imbricatea/Silicofilosea based on the rRNA data sets was not supported by our analyses.

An Evaluation of the Use of the LSU rRNA D1-D5 Domain for DNA-based Taxonomy of Eukaryotic Protists

Diagnostic signature DNA sequences are important tools for the identification of species. There is an active debate in the literature on the choice of the best markers applicable for a broad range of organisms. Protists have seldom been included in these evaluations. Mitochondrial gene sequences are inappropriate for protists since several groups do not possess mitochondria. Here we studied the application of the large subunit (LSU) rRNA gene fragments (D1-D5) regarding their usefulness to discriminate between a wide range of heterotrophic nanoflagellates. Phylogenetic analyses based on the LSU rRNA fragments showed similar results compared to phylogenetic trees based on the small subunit (SSU) rRNA. The data set indicates the power of the use of the D1-D5 region as a marker for a DNA-based taxonomy. Our results, together with the available sequences in Genbank, form a comprehensive database for unicellular eukaryotes, especially heterotrophic flagellates. It is now possible to assign new sequences to the different groups of heterotrophic flagellates which we have tested for different closely related Cercomonas and Paracercomonas strains from groundwater.

Description of Colponema vietnamica sp.n. and Acavomonas peruviana n. gen. n. sp., two new alveolate phyla (Colponemidia nom. nov. and Acavomonidia nom. nov.) and their contributions to reconstructing the ancestral state of alveolates and eukaryotes.

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.

FEEDING APPARATUS OF THE COLORLESS FLAGELLATE KATABLEPHARIS (CRYPTOPHYCEAE)1

The feeding apparatus of Kalablepharis ovalis (isolated from a freshwater impoundment in Colorado) and Katablepharis clone G‐2 (isolated from the littoral of the Black Sea near Yalta in the Crimea) consists of inner and outer oval‐shaped arrays of microtubules that begin at the anterior end of the cell and pass into the posterior of the cell. Each array of microtubules contains groups of microtubules with two to eight microtubules per group depending on the position of the array in the cell. A specialized area of the plasma membrane, the mouth, occurs at the anterior end of the cell. The mouth is oval with the long axis oriented dorsoventrally and consists of a raised ridge surrounding a central depression. The anterior end of the microtubules of the inner and outer arrays supports the raised ridge of the mouth. In freeze‐fracture replicas, the protoplasmic face of the plasma membrane contains intramembrane particles on the raised ridge of the mouth. Three small membrane‐cisternae occur on the protoplasmic side of the plasma membrane in the area of the mouth. Katablepharis clone G‐2 also has five or six additional large membrane‐cisternae associated with the inner microtubular array in the anterior portion of the cell. These larger membrane‐cisternae do not occur in K. ovalis. Vesicles with electron‐dense contents occur in association with the microtubular arrays. Katablepharis ovalis has a second type of vesicle containing a single‐membrane profile associated with the microtubule arrays. The structure of the microtubular arrays in Katablepharis is compared with similar structures in suctorian ciliates and dinoflagellates.