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.     

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.     

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.     

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).     

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.     

MOLECULAR EVOLUTION OF EUGLENOZOAN PARAXONEMAL ROD GENES par1 AND par2 COINCIDES WITH PHYLOGENETIC RECONSTRUCTION BASED ON SMALL SUBUNIT rDNA DATA1

Emergent flagella of Euglenozoa consist of two prominent structural elements: the axoneme built by microtubules with motor proteins to enable the movement of the flagellum and a highly organized protein structure of unknown function, called the paraxonemal rod (PAR), which consists of two major proteins paralleling the axoneme of euglenid and kinetoplastid emergent flagella. These flagellar structures are considered apomorphic characters of Euglenozoa. We examined the evolution of the genes par1 and par2 encoding the two major proteins, where we could show that these proteins are encoded by two very similar genes found in kinetoplastids and euglenids. The branching pattern indicated a gene duplication before the diversification into euglenids and kinetoplastids. In the clades of the genes, subtrees of euglenid and kinetoplastid monophyla arose. Both genes showed strong genetic diversity with biased GC content at taxon rather than at gene level. We also examined phylogenies inferred from PAR genes that are well in agreement with established small subunit rDNA analyses. Both showed further separation of the euglenid subtree into primary osmotrophs and a phototrophic clade, including secondarily derived osmotrophs.     

Unusual semi‐extractability as a hallmark of nuclear body‐associated architectural noncoding RNAs

NEAT1_2 long noncoding RNA (lncRNA) is the molecular scaffold of paraspeckle nuclear bodies. Here, we report an improved RNA extraction method: extensive needle shearing or heating of cell lysate in RNA extraction reagent improved NEAT1_2 extraction by 20‐fold (a property we term “semi‐extractability”), whereas using a conventional method NEAT1_2 was trapped in the protein phase. The improved extraction method enabled us to estimate that approximately 50 NEAT1_2 molecules are present in a single paraspeckle. Another architectural lncRNA, IGS16, also exhibited similar semi‐extractability. A comparison of RNA‐seq data from needle‐sheared and control samples revealed the existence of multiple semi‐extractable RNAs, many of which were localized in subnuclear granule‐like structures. The semi‐extractability of NEAT1_2 correlated with its association with paraspeckle proteins and required the prion‐like domain of the RNA‐binding protein FUS. This observation suggests that tenacious RNA–protein and protein–protein interactions, which drive nuclear body formation, are responsible for semi‐extractability. Our findings provide a foundation for the discovery of the architectural RNAs that constitute nuclear bodies.     

Evolutionary History of the Enzymes Involved in the Calvin–Benson Cycle in Euglenids

Euglenids are an ancient lineage that may have existed as early as 2 billion years ago. A mere 65 years ago, Melvin Calvin and Andrew A. Benson performed experiments on Euglena gracilis and elucidated the series of reactions by which carbon was fixed and reduced during photosynthesis. However, the evolutionary history of this pathway (Calvin–Benson cycle) in euglenids was more complex than Calvin and Benson could have imagined. The chloroplast present today in euglenophytes arose from a secondary endosymbiosis between a phagotrophic euglenid and a prasinophyte green alga. A long period of evolutionary time existed before this secondary endosymbiotic event took place, which allowed for other endosymbiotic events or gene transfers to occur prior to the establishment of the green chloroplast. This research revealed the evolutionary history of the major enzymes of the Calvin–Benson cycle throughout the euglenid lineage and showed that the majority of genes for Calvin–Benson cycle enzymes shared an ancestry with red algae and/or chromophytes suggesting they may have been transferred to the nucleus prior to the acquisition of the green chloroplast.     

TRENDS IN THE EVOLUTION OF THE EUGLENID PELLICLE

Abstract Trends in the evolution of the euglenid pellicle were described using phylogenetic methods on 18S rDNA, morphological, and combined data from 25 mostly phototrophic taxa. The tree topology from a total‐evidence analysis formed a template for a synthetic tree that took into account conflicting results derived from the partitioned datasets. Pellicle character states that can only be observed with the assistance of transmission and scanning electron microscopy were phylogenetically mapped onto the synthetic tree to test a set of previously established homology statements (inferences made independently from a cladogram). The results permitted us to more confidently infer the ancestral‐derived polarities of character state transformations and provided a framework for understanding the key cytoskeletal innovations associated with the evolution of phototrophic euglenids. We specifically addressed the character evolution of (1) the maximum number of pellicle strips around the cell periphery; (2) the patterns of terminating strips near the cell posterior end; (3) the substructural morphology of pellicle strips; (4) the morphology of the cell posterior tip; and (5) patterns of pellicle pores on the cell surface.     

Influence of different substrates on the evolution of morphology and life‐history traits of azooxanthellate solitary corals (Scleractinia: Flabellidae)

Sessile organisms are influenced considerably by their substrate conditions, and their adaptive strategies are key to understanding their morphologic evolution and traits of life history. The family Flabellidae (Cnidaria: Scleractinia) is composed of the representative azooxanthellate solitary corals that live on both soft and hard substrates using various adaptive strategies. We reconstructed the phylogenetic tree and ancestral character states of this family from the mitochondrial 16S and nuclear 28S ribosomal DNA sequences of ten flabellids aiming to infer the evolution of their adaptive strategies. The Javania lineage branched off first and adapted to hard substrates by using a tectura‐reinforced base. The extant free‐living flabellids, including Flabellum and Truncatoflabellum, invaded soft substrates and acquired the flabellate corallum morphology of their common ancestor, followed by a remarkable radiation with the exploitation of adaptive strategies, such as external soft tissue [e.g. Flabellum (Ulocyathus)], thecal edge spine, and transverse division (e.g. Placotrochus and Truncatoflabellum). Subsequently, the free‐living ancestors of two genera (Rhizotrochus and Monomyces) invaded hard substrates independently by exploiting distinct attachment apparatuses such as tube‐like and massive rootlets, respectively. In conclusion, flabellids developed various morphology and life‐history traits according to the differences in substrate conditions during the course of their evolution. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 184–192.     

Coexpression of Escherichia coli RNase III in silkworm cells improves the efficiency of RNA interference induced by long hairpin dsRNAs

Abstract Long hairpin dsRNA transcribed from chromosomal DNA can induce RNA interference in Bombyx mori cells, although its gene silencing efficiency is lower than that of exogenously introduced double‐stranded RNAs (dsRNAs). To solve this problem, we monitored the nuclear cytoplasmic translocation of the transcribed hairpin dsRNA and analyzed the processing efficiency into mature small interfering RNA (siRNA). Northern blot analysis revealed that the transcribed hairpin dsRNAs were spliced and transported into the cytoplasm, but were not effectively diced into siRNAs. Interestingly, RNAi with hairpin dsRNAs from genome‐integrated IR transgene was stimulated by the coexpression of Escherichia coli RNase III, although this exogenous enzyme seemed to bring about nonspecific cleavage of cellular mRNA.     

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.     

Primate-specific spliced <Emphasis Type="Italic">PMCHL</Emphasis> RNAs are non-protein coding in human and macaque tissues

Background  

Brain-expressed genes that were created in primate lineage represent obvious candidates to investigate molecular mechanisms that contributed to neural reorganization and emergence of new behavioural functions in Homo sapiens. PMCHL1 arose from retroposition of a pro-melanin-concentrating hormone (PMCH) antisense mRNA on the ancestral human chromosome 5p14 when platyrrhines and catarrhines diverged. Mutations before divergence of hylobatidae led to creation of new exons and finally PMCHL1 duplicated in an ancestor of hominids to generate PMCHL2 at the human chromosome 5q13. A complex pattern of spliced and unspliced PMCHL RNAs were found in human brain and testis.     

The dynamic loss and gain of introns during the evolution of the Brassicaceae

Sequence comparison allows the detailed analysis of evolution at the nucleotide and amino acid levels, but much less information is known about the structural evolution of genes, i.e. how the number, length and distribution of introns change over time. We constructed a parsimonious model for the evolutionary rate of intron loss (IL) and intron gain (IG) within the Brassicaceae and found that IL/IG has been highly dynamic, with substantial differences between and even within lineages. The divergence of the Brassicaceae lineages I and II marked a dramatic change in the IL rate, with the common ancestor of lineage I losing introns three times more rapidly than the common ancestor of lineage II. Our data also indicate a subsequent declining trend in the rate of IL, although in Arabidopsis thaliana introns continue to be lost at approximately the ancestral rate. Variations in the rate of IL/IG within lineage II have been even more remarkable. Brassica rapa appears to have lost introns approximately 15 times more rapidly than the common ancestor of B. rapa and Schenkiella parvula, and approximately 25 times more rapidly than its sister species Eutrema salsugineum. Microhomology was detected at the splice sites of several dynamic introns suggesting that the non‐homologous end‐joining and double‐strand break repair is a common pathway underlying IL/IG in these species. We also detected molecular signatures typical of mRNA‐mediated IL, but only in B. rapa.     

Parallel evolution of site‐specific changes in divergent caribou lineages

The parallel evolution of phenotypes or traits within or between species provides important insight into the basic mechanisms of evolution. Genetic and genomic advances have allowed investigations into the genetic underpinnings of parallel evolution and the independent evolution of similar traits in sympatric species. Parallel evolution may best be exemplified among species where multiple genetic lineages, descended from a common ancestor, colonized analogous environmental niches, and converged on a genotypic or phenotypic trait. Modern North American caribou (Rangifer tarandus) originated from three ancestral sources separated during the Last Glacial Maximum (LGM): the Beringian–Eurasian lineage (BEL), the North American lineage (NAL), and the High Arctic lineage (HAL). Historical introgression between the NAL and the BEL has been found throughout Ontario and eastern Manitoba. In this study, we first characterized the functional differentiation in the cytochrome‐b (cytB) gene by identifying nonsynonymous changes. Second, the caribou lineages were used as a direct means to assess site‐specific parallel changes among lineages. There was greater functional diversity within the NAL despite the BEL having greater neutral diversity. The patterns of amino acid substitutions occurring within different lineages supported the parallel evolution of cytB amino acid substitutions suggesting different selective pressures among lineages. This study highlights the independent evolution of identical amino acid substitutions within a wide‐ranging mammal species that have diversified from different ancestral haplogroups and where ecological niches can invoke parallel evolution.     

Preadaptation and post‐introduction evolution facilitate the invasion of Phragmites australis in North America

Compared with non‐invasive species, invasive plant species may benefit from certain advantageous traits, for example, higher photosynthesis capacity and resource/energy‐use efficiency. These traits can be preadapted prior to introduction, but can also be acquired through evolution following introduction to the new range. Disentangling the origins of these advantageous traits is a fundamental and emerging question in invasion ecology. We conducted a multiple comparative experiment under identical environmental condition with the invasive haplotype M lineage of the wetland grass Phragmites australis and compared the ecophysiological traits of this invasive haplotype M in North America with those of the European ancestor and the conspecific North American native haplotype E lineage, P. australis ssp. americanus. The invasive haplotype M differed significantly from the native North American conspecific haplotype E in several ecophysiological and morphological traits, and the European haplotype M had a more efficient photosynthetic apparatus than the native North American P. australis ssp. americanus. Within the haplotype M lineage, the introduced North American P. australis exhibited different biomass allocation patterns and resource/energy‐use strategies compared to its European ancestor group. A discriminant analysis of principal components separated the haplotype M and the haplotype E lineages completely along the first canonical axis, highly related to photosynthetic gas‐exchange parameters, photosynthetic energy‐use efficiency and payback time. The second canonical axis, highly related to photosynthetic nitrogen use efficiency and construction costs, significantly separated the introduced P. australis in North America from its European ancestor. Synthesis. We conclude that the European P. australis lineage was preadapted to be invasive prior to its introduction, and that the invasion in North America is further stimulated by rapid post‐introduction evolution in several advantageous traits. The multicomparison approach used in this study could be an effective approach for distinguishing preadaptation and post‐introduction evolution of invasive species. Further research is needed to link the observed changes in invasive traits to the genetic variation and the interaction with the environment.