Phylogeny of phagotrophic euglenids (Euglenozoa) as inferred from hsp90 gene sequences

Molecular phylogenies of euglenids are usually based on ribosomal RNA genes that do not resolve the branching order among the deeper lineages. We addressed deep euglenid phylogeny using the cytosolic form of the heat-shock protein 90 gene (hsp90), which has already been employed with some success in other groups of euglenozoans and eukaryotes in general. Hsp90 sequences were generated from three taxa of euglenids representing different degrees of ultrastructural complexity, namely Petalomonas cantuscygni and wild isolates of Entosiphon sulcatum, and Peranema trichophorum. The hsp90 gene sequence of P. trichophorum contained three short introns (ranging from 27 to 31 bp), two of which had non-canonical borders GG-GG and GG-TG and two 10-bp inverted repeats, suggesting a structure similar to that of the non-canonical introns described in Euglena gracilis. Phylogenetic analyses confirmed a closer relationship between kinetoplastids and diplonemids than to euglenids, and supported previous views regarding the branching order among primarily bacteriovorous, primarily eukaryovorous, and photosynthetic euglenids. The position of P. cantuscygni within Euglenozoa, as well as the relative support for the nodes including it were strongly dependent on outgroup selection. The results were most consistent when the jakobid Reclinomonas americana was used as the outgroup. The most robust phylogenies place P. cantuscygni as the most basal branch within the euglenid clade. However, the presence of a kinetoplast-like mitochondrial inclusion in P. cantuscygni deviates from the currently accepted apomorphy-based definition of the kinetoplastid clade and highlights the necessity of detailed studies addressing the molecular nature of the euglenid and diplonemid mitochondrial genome.     

Protein phylogenies robustly resolve the deep-level relationships within Euglenozoa

The deepest-level relationships amongst Euglenozoa remain poorly resolved, despite a rich history of morphological examination and numerous molecular phylogenetic studies of small subunit ribosomal RNA (SSU rRNA) data. We address this question using two nuclear-encoded proteins, the cytosolic isoforms of heat shock protein 90 (hsp90) and heat shock protein 70 (hsp70). For both proteins we examined sequences from the three primary groups within Euglenozoa (euglenids, diplonemids, and kinetoplastids), and from their close relatives, Heterolobosea. Maximum likelihood (ML) and ML distance analyses of these proteins support a close relationship between diplonemids and kinetoplastids to the exclusion of the euglenid Euglena gracilis. In hsp90 and combined protein analyses bootstrap support is very strong and alternative topologies are generally rejected by 'approximately unbiased' (AU) tests. This result is consistent with recent molecular biological and morphological data, but contradicts early structural accounts and many SSU rRNA analyses that favour a closer relationship between diplonemids and euglenids. However, a re-examination of an important SSU rRNA data set highlights the instability of the inferences from this marker. The protein analyses also suggest that bodonids are paraphyletic, with trypanosomatids grouping with 'clade 2' and 'clade 3' bodonids to the exclusion of 'clade 1' bodonids.     

Unique mitochondrial genome structure in diplonemids, the sister group of kinetoplastids

Kinetoplastid flagellates are characterized by uniquely massed mitochondrial DNAs (mtDNAs), the kinetoplasts. Kinetoplastids of the trypanosomatid group possess two types of mtDNA molecules: maxicircles bearing protein and mitoribosomal genes and minicircles specifying guide RNAs, which mediate uridine insertion/deletion RNA editing. These circles are interlocked with one another to form dense networks. Whether these peculiar mtDNA features are restricted to kinetoplastids or prevail throughout Euglenozoa (euglenids, diplonemids, and kinetoplastids) is unknown. Here, we describe the mitochondrial genome and the mitochondrial ultrastructure of Diplonema papillatum, a member of the diplonemid flagellates, the sister group of kinetoplastids. Fluorescence and electron microscopy show a single mitochondrion per cell with an ultrastructure atypical for Euglenozoa. In addition, DNA is evenly distributed throughout the organelle rather than compacted. Molecular and electron microscopy studies distinguish numerous 6- and 7-kbp-sized mitochondrial chromosomes of monomeric circular topology and relaxed conformation in vivo. Remarkably, the cox1 gene (and probably other mitochondrial genes) is fragmented, with separate gene pieces encoded on different chromosomes. Generation of the contiguous cox1 mRNA requires trans-splicing, the precise mechanism of which remains to be determined. Taken together, the mitochondrial gene/genome structure of Diplonema is not only different from that of kinetoplastids but unique among eukaryotes as a whole.     

PHYLOGENY OF PHAGOTROPHIC EUGLENIDS (EUGLENOZOA): A MOLECULAR APPROACH BASED ON CULTURE MATERIAL AND ENVIRONMENTAL SAMPLES1

Molecular studies based on small subunit (SSU) rDNA sequences addressing euglenid phylogeny hitherto suffered from the lack of available data about phagotrophic species. To extend the taxon sampling, SSU rRNA genes from species of seven genera of phagotrophic euglenids were investigated. Sequence analyses revealed an increasing genetic diversity among euglenid SSU rDNA sequences compared with other well‐known eukaryotic groups, reflecting an equally broad diversity of morphological characters among euglenid phagotrophs. Phylogenetic inference using standard parsimony and likelihood approaches as well as Bayesian inference and spectral analyses revealed no clear support for euglenid monophyly. Among phagotrophs, monophyly of Petalomonas cantuscygni and Notosolenus ostium, both comprising simple ingestion apparatuses, is strongly supported. A moderately supported clade comprises phototrophic euglenids and primary osmotrophic euglenids together with phagotrophs, exhibiting a primarily flexible pellicle composed of numerous helically arranged strips and a complex ingestion apparatus with two supporting rods and four curved vanes. Comparison of molecular and morphological data is used to demonstrate the difficulties to formulate a hypothesis about how the ingestion apparatus evolved in this group.     

Did trypanosomatid parasites have photosynthetic ancestors?

Some molecular phylogenies of plastid-like genes suggest that chloroplasts (the structures responsible for photosynthesis in plants and algae) might have been secondarily lost in trypanosomatid parasites. Chloroplasts are present in some euglenids, which are closely related to trypanosomatids, and it has been argued that chloroplasts arose early in the diversification of the lineage Euglenozoa, to which trypanosomatids and euglenids belong (plastids-early hypothesis). This article reviews how euglenid ultrastructural systems are functionally integrated and phylogenetically correlated. I argue that chloroplast acquisition profoundly altered the structure of certain euglenids, and that the complete absence of these modifications in other euglenozoans is most consistent with their never having had a chloroplast. Ultrastructural evidence suggests that chloroplasts arose relatively recently within a specific subgroup of euglenids and that trypanosomatids are not secondarily non-photosynthetic (plastids-recent hypothesis).     

Cryptic paraflagellar rod in endosymbiont-containing kinetoplastid protozoa

Cilia and flagella are central to many biological processes in a diverse range of organisms. The kinetoplastid protozoa are very appealing models for the study of flagellar function, particularly in the light of the availability of extensive trypanosomatid genome information. In addition to the highly conserved 9 + 2 axoneme, the kinetoplastid flagellum contains a characteristic paraflagellar rod structure (PFR). The PFR is necessary for full motility and provides support for metabolic regulators that may influence flagellar beating. However, there is an intriguing puzzle: one clade of endosymbiont-containing kinetoplastids apparently lack a PFR yet are as motile as species that possess a PFR and are able to attach to the invertebrate host epithelia. We investigated how these organisms are able to locomote despite the apparent lack of PFR. Here we have identified a PFR1 gene in the endosymbiont-bearing trypanosome Crithidia deanei. This gene is expressed in C. deanei and is able to partially complement a pfr1 null mutation in Leishmania mexicana cells, demonstrating that the encoded protein is functional. Careful reexamination of C. deanei flagellar ultrastructure revealed a greatly reduced PFR missed by many previous analyses. This affirms the PFR as a canonical organelle of kinetoplastids. Moreover, although PFR proteins have been conserved in evolution, primary sequence differences contribute to particular PFR morphotypes characteristic of different kinetoplastid species.     

Combining Molecular Data with Classical Morphology for Uncultured Phagotrophic Euglenids (Excavata): A Single‐Cell Approach

Phagotrophic euglenids are one of the most diverse and important forms of heterotrophic flagellates in sediment systems, and are key to understanding the evolution of photosynthetic euglenids and ‘primary osmotrophs’, yet relatively little is known about their biodiversity and phylogenetic relationships. A wealth of light microscopy‐based information is available, but little progress has been made in associating this with molecular sequence data. We established a protocol to obtain light microscopy data and molecular data from single euglenid cells isolated from environmental samples. Individual cells from freshwater and marine benthic samples were isolated and rinsed by micropipetting, documented using high‐resolution photomicroscopy, then subjected to single‐cell nested PCR using taxon‐specific primers in combination with universal eukaryotic primers, generating > 75% or full‐length SSU rDNA sequences. As a proof‐of‐principle eight individuals were characterised and subjected to phylogenetic analyses. Many of these cells were identified as Anisonema or Dinema, and grouped with existing sequences assigned to these taxa, and with a ‘Peranema sp.’ sequence that we could now clearly demonstrate was misidentified or misannotated. Another cell is Heteronema c.f. exaratum, the first ‘skidding heteronemid’ for which sequence data are available. This is not closely related to Heteronema scaphurum, and intriguingly, branches as the sister group to primary osmotrophs. A cell similar to Ploeotia vitrea (the type of this genus), shows no particular phylogenetic affinity to Ploeotia costata, the best studied Ploeotia species. Our experimental protocol provides a useful starting point for future analyses on euglenid biodiversity (including environmental sequence surveys), and their evolution and systematics.     

Ultrastructure and molecular phylogeny of Calkinsia aureus: cellular identity of a novel clade of deep-sea euglenozoans with epibiotic bacteria

Background  

The Euglenozoa is a large group of eukaryotic flagellates with diverse modes of nutrition. The group consists of three main subclades - euglenids, kinetoplastids and diplonemids - that have been confirmed with both molecular phylogenetic analyses and a combination of shared ultrastructural characteristics. Several poorly understood lineages of putative euglenozoans live in anoxic environments, such as Calkinsia aureus, and have yet to be characterized at the molecular and ultrastructural levels. Improved understanding of these lineages is expected to shed considerable light onto the ultrastructure of prokaryote-eukaryote symbioses and the associated cellular innovations found within the Euglenozoa and beyond.     

Evolutionary analysis of synteny and gene fusion for pyrimidine biosynthetic enzymes in Euglenozoa: an extraordinary gap between kinetoplastids and diplonemids

A unique feature of the genome architecture in the parasitic trypanosomatid protists is large-scale synteny. We addressed the evolutionary trait of synteny in the eukaryotic group, Euglenozoa, which consists of euglenoids (earliest branching), diplonemids, and kinetoplastids (trypanosomatids and bodonids). Synteny of the pyrimidine biosynthetic (pyr) gene cluster, which constitutes part of a large syntenic cluster in trypanosomatids and includes four separate genes (pyr1-pyr4) and one fused gene (pyr6/pyr5 fusion), was conserved in the bodonid, Parabodo caudatus. In the diplonemid, Diplonema papillatum, we identified pyr4 and pyr6 genes. Phylogenetic analyses of pyr4 and pyr6 showed the separate origin of each in kinetoplastids and euglenoids/diplonemids and suggested that kinetoplastids have acquired these genes via lateral gene transfer (LGT). Because replacement of genes by non-orthologs within the syntenic cluster is highly unlikely, we concluded that, after separation of the line leading to diplonemids, the syntenic pyr gene cluster was established in the common ancestor of kinetoplastids, preceded by their acquisition via LGT. Notably, we found that diplonemid pyr6 is a stand-alone gene, inconsistent with both euglenoid pyr5/pyr6 and kinetoplastid pyr6/pyr5 fusions. Our findings provide insights into the evolutionary gaps within Euglenozoa and the evolutionary trait of rearrangement of gene fusion in this lineage.     

Ultrastructure and Molecular Phylogenetic Position of Heteronema scaphurum: A Eukaryovorous Euglenid with a Cytoproct

Euglenids comprise a distinct clade of flagellates with diverse modes of nutrition, including phagotrophy, osmotrophy and phototrophy. Much of the previous research on euglenids has focused on phototrophic species because of their ecological abundance and significance as indicators for the health of aquatic ecosystems. Although largely understudied, phagotrophic species probably represent the majority of euglenid diversity. Phagotrophic euglenids tend to be either bacterivorous or eukaryovorous and use an elaborate feeding apparatus for capturing prey cells. We characterized the ultrastructure and molecular phylogenetic position of Heteronema scaphurum, a eukaryovorous euglenid collected in freshwater. This species was equipped with a distinct cytoproct through which waste products were eliminated in the form of faecal pellets; a cytoproct has not been reported in any other member of the Euglenida. Heteronema scaphurum also had a novel predatory mode of feeding. The euglenid ensnared and corralled several green algal prey cells (i.e. Chlamydomonas) with hook‐like flagella covered in mucous before engulfing the bundle of prey cells whole. Molecular phylogenetic analyses inferred from small subunit rDNA sequences placed this species with other eukaryovorous euglenids, which was consistent with ultrastructural features associated with the feeding apparatus, flagellar apparatus, extrusomes, and pellicle.     

Phylogenetic analyses of various euglenoid taxa (euglenozoa) based on 18s rdna sequence data

18S rRNA genes (SSU rDNA) of five newly sequenced species were used as molecular markers to infer phylogenetic relationships within the euglenoids. Two members of the order Euglenales ( Lepocinclis ovata Playfair , Phacus similis Christen), two of the order Eutreptiales ( Distigma proteus Ehrenberg, , D. curvata Pringsheim) and Gyropaigne lefévrei Bourelly et Georges of the order Rhabdomonadales were used in parsimony, maximum likelihood, and distance analyses. All trees derived from SSU rRNA data strongly supported the monophyletic origin of the Euglenozoa, with kinetoplastids as sister clade to the euglenoids and Petalomonas cantuscygni Cann et Pennick diverging at the base of the monophyletic euglenoid lineage. The data also supported the theory that phagotrophic euglenoids arose prior to osmotrophs and phototrophs. A lineage of Peranema trichophorum Ehrenberg and all sequenced Euglenales formed a sister clade to the osmotrophs. This suggests that the evolution of phototrophy within the euglenoids radiated from a single event.     

Phylogenetic position of Rhynchopus sp. and Diplonema ambulator as indicated by analyses of euglenozoan small subunit ribosomal DNA

The taxa Rhynchopus Skuja and Diplonema Griessmann were first described as remarkable protists with euglenid affinities. Later on, the placement of Diplonema within the Euglenozoa was confirmed by molecular data. For this study two new sequences were added to the euglenozoan data set. The uncertainly placed Rhynchopus can be identified as a close relative to Diplonema by small subunit ribosomal DNA (SSU rDNA) analysis. The new sequence of Diplonema ambulator is in close relationship to two other Diplonema species. Our molecular analyses clearly support the monophyly of the diplonemids comprising Rhynchopus and Diplonema. Yet the topology at the base of the euglenozoan tree remains unresolved, and especially the monophyly of the euglenids is arguable. SSU rDNA sequence analyses suggest that significantly different GC contents, high mutational saturation in the euglenids, and different evolutionary rates in the euglenozoan clades make it difficult to identify any sister group to the diplonemids.     

PHYLOGENETIC RELATIONSHIPS OF SELECTED EUGLENOID GENERA BASED ON MORPHOLOGICAL AND MOLECULAR DATA1

The small subunit rRNA (SSU rRNA) coding regions sequenced from the euglenoids Petalomonas cantuscygni, Peranema trichophorum, and Khawkinea quartana were used to assess the phylogenetic relationships of these genera within the Euglenozoa. Phylogenies derived from distance, parsimony, and maximum likelihood methods infer that the euglenoids and kinetoplastids form sister clades within a monophyletic assemblage. Distances representative of closely related lineages separate the genera within the Kinetoplastida, whereas larger distance values separate genera within the euglenoid assemblage. The results of the morphological and molecular studies suggest that phagotrophy arose early in the euglenozoan lineage with the subsequent acquisition of phototrophy, osmotrophy, and parasitism. Phagotrophic euglenoids with a pellicle composed of longitudinal strips appear to have diverged prior to genera with helically arranged strips. This study suggests that the hypothetical ancestor to the Euglenozoa was a phagotroph with two flagella, both containing paraxonemal rods. Furthermore, its basal bodies contained proximal cartwheels, were connected by a prominent fiber, and were anchored with three asymmetrically arranged flagellar roots.     

Phylogenetic Relationships and Morphological Character Evolution of Photosynthetic Euglenids (Excavata) Inferred from Taxon‐rich Analyses of Five Genes

Photosynthetic euglenids acquired chloroplasts by secondary endosymbiosis, which resulted in changes to their mode of nutrition and affected the evolution of their morphological characters. Mapping morphological characters onto a reliable molecular tree could elucidate major trends of those changes. We analyzed nucleotide sequence data from regions of three nuclear‐encoded genes (nSSU, nLSU, hsp90), one chloroplast‐encoded gene (cpSSU) and one nuclear‐encoded chloroplast gene (psbO) to estimate phylogenetic relationships among 59 photosynthetic euglenid species. Our results were consistent with previous works; most genera were monophyletic, except for the polyphyletic genus Euglena, and the paraphyletic genus Phacus. We also analyzed character evolution in photosynthetic euglenids using our phylogenetic tree and eight morphological traits commonly used for generic and species diagnoses, including: characters corresponding to well‐defined clades, apomorphies like presence of lorica and mucilaginous stalks, and homoplastic characters like rigid cells and presence of large paramylon grains. This research indicated that pyrenoids were lost twice during the evolution of phototrophic euglenids, and that mucocysts, which only occur in the genus Euglena, evolved independently at least twice. In contrast, the evolution of cell shape and chloroplast morphology was difficult to elucidate, and could not be unambiguously reconstructed in our analyses.     

Morphological and Molecular Characterisation of Notosolenus urceolatus Larsen and Patterson 1990, a Member of an Understudied Deep‐branching Euglenid Group (Petalomonads)

Petalomonads are particularly important for understanding the early evolution of euglenids, but are arguably the least studied major group within this taxon. We have established a culture of the biflagellate petalomonad Notosolenus urceolatus, and conducted electron microscopy observations and molecular phylogenetic analysis. Notosolenus urceolatus has eight pellicular strips bordered by grooves and underlain by close‐set microtubules. There are ventral and dorsal Golgi bodies. Mitochondria apparently contain fibrous inclusions, as in Petalomonas cantuscygni. A previously undocumented type of large, globular extrusome is present instead of the tubular extrusomes characteristic of Euglenozoa. The feeding apparatus lacks rods and vanes, and is partly supported by an “MTR”. The flagella have complex transition zones that are extremely elongated but unswollen. Only the emergent portion of the anterior flagellum has an organised paraxonemal rod, and also has very fine mastigonemes. The basal bodies are offset and lack connecting fibres. 18S rRNA gene phylogenies show that N. urceolatus is closely related to Petalomonas sphagnophila and P. cantuscygni, not Notosolenus ostium, confirming that current generic assignments based on the number of emergent flagella are phylogenetically unreliable, and making it difficult to infer whether features shared by N. urceolatus and P. cantuscygni (for example) are general for petalomonads.     

Occurrence of multiple, independent gene fusion events for the fifth and sixth enzymes of pyrimidine biosynthesis in different eukaryotic groups

The genes encoding orotate phosphoribosyltransferase (OPRT) and orotidine-5'-monophosphate decarboxylase (OMPDC), the fifth and sixth enzymes in the de novo pyrimidine biosynthetic pathway, are fused as OPRT-OMPDC in most eukaryotic groups. On the other hand, the inversely linked OMPDC-OPRT fusion is present in trypanosomatids, belonging to kinetoplastids together with bodonids in a supergroup, Euglenozoa. Here, we show the presence of OMPDC-OPRT in the bodonid, Bodo caudatus, while OPRT-OMPDC in Euglena gracilis, another euglenozoan species belonging to euglenoids. These results suggest that the OMPDC-OPRT fusion event occurred in a common ancestor of kinetoplastids. Genome sequence database searches further revealed the presence of OMPDC-OPRT in stramenopiles and cyanobacteria. Phylogenetic reconstruction of OPRT and OMPDC rejected statistically the monophyly of the OPRT domains of stramenopile and kinetoplastid OMPDC-OPRT, demonstrating that these gene fusions do not share a common evolutionary origin, despite the identical gene order. Thus, the OMPDC-OPRT fusion is likely to have emerged independently in these eukaryotic groups. Phylogenetic analyses also suggested that cyanobacterial OMPDC-OPRT arose via lateral transfer. We conclude that gene fusion events occur more frequently than previously thought and that lateral gene transfer has made a marked contribution to establishment of the rearranged structure of OPRT and OMPDC genes in eukaryotes.