Gillian H. Gile

CBOL Protist Working Group: Barcoding Eukaryotic Richness beyond the Animal, Plant, and Fungal Kingdoms

A group of protist experts proposes a two-step DNA barcoding approach, comprising a universal eukaryotic pre-barcode followed by group-specific barcodes, to unveil the hidden biodiversity of microbial eukaryotes.

Algal genomes reveal evolutionary mosaicism and the fate of nucleomorphs

Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote–eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have >21,000 protein genes and are intron rich, and B. natans exhibits unprecedented alternative splicing for a single-celled organism. Phylogenomic analyses and subcellular targeting predictions reveal extensive genetic and biochemical mosaicism, with both host- and endosymbiont-derived genes servicing the mitochondrion, the host cell cytosol, the plastid and the remnant endosymbiont cytosol of both algae. Mitochondrion-to-nucleus gene transfer still occurs in both organisms but plastid-to-nucleus and nucleomorph-to-nucleus transfers do not, which explains why a small residue of essential genes remains locked in each nucleomorph.

The Inadequacy of Morphology for Species and Genus Delineation in Microbial Eukaryotes: An Example from the Parabasalian Termite Symbiont Coronympha

Background For the majority of microbial eukaryotes (protists, algae), there is no clearly superior species concept that is consistently applied. In the absence of a practical biological species concept, most species and genus level delineations have historically been based on morphology, which may lead to an underestimate of the diversity of microbial eukaryotes. Indeed, a growing body of molecular evidence, such as barcoding surveys, is beginning to support the conclusion that significant cryptic species diversity exists. This underestimate of diversity appears to be due to a combination of using morphology as the sole basis for assessing diversity and our inability to culture the vast majority of microbial life. Here we have used molecular markers to assess the species delineations in two related but morphologically distinct genera of uncultivated symbionts found in the hindgut of termites. Methodology/Principal Findings Using single-cell isolation and environmental PCR, we have used a barcoding approach to characterize the diversity of Coronympha and Metacoronympha symbionts in four species of Incisitermes termites, which were also examined using scanning electron microscopy and light microcopy. Despite the fact that these genera are significantly different in morphological complexity and structural organisation, we find they are two life history stages of the same species. At the same time, we show that the symbionts from different termite hosts show an equal or greater level of sequence diversity than do the hosts, despite the fact that the symbionts are all classified as one species. Conclusions/Significance The morphological information used to describe the diversity of these microbial symbionts is misleading at both the genus and species levels, and led to an underestimate of species level diversity as well as an overestimate of genus level diversity. The genus ‘Metacoronympha’ is invalid and appears to be a life history stage of Coronympha, while the single recognized species of Coronympha octonaria inhabiting these four termites is better described as four distinct species.

Characterization of Periplastidal Compartment–Targeting Signals in Chlorarachniophytes

Secondary plastids are acquired by the engulfment and retention of eukaryotic algae, which results in an additional surrounding membrane or pair of membranes relative to the more familiar primary plastids of land plants. In most cases, the endocytosed alga loses its eukaryotic genome as it becomes integrated, but in two algal groups, the cryptophytes and chlorarachniophytes, the secondary plastids retain a vestigial nucleus in the periplastidal compartment (PPC), the remnant eukaryotic cytoplasm between the inner and the outer membrane pairs. Many essential housekeeping genes are missing from these reduced genomes, suggesting that they are now encoded in the host nucleus and their products are targeted to the PPC. One such nucleus-encoded, PPC-targeted protein, the translation elongation factor like (EFL) was recently identified in chlorarachniophytes. It bears an N-terminal-targeting sequence comprising a signal peptide and a transit peptide-like sequence (TPL) similar to the plastid-targeted proteins of chlorarachniophytes as well as a hydrophilic C-terminal extension rich in lysine and aspartic acid. Here, we characterize the function of the N- and C-terminal extensions of PPC-targeted EFL in transformed chlorarachniophyte cells. Using green fluorescent protein as a reporter molecule, we demonstrate that several negatively charged amino acids within the TPL are essential for accurate targeting to the PPC. Our findings further reveal that the C-terminal extension functions as a PPC retention signal in combination with an N-terminal plastid-targeting peptide, which suggests that plastid and PPC proteins may be sorted in the PPC.

Complex phylogenetic distribution of a non-canonical genetic code in green algae

BackgroundA non-canonical nuclear genetic code, in which TAG and TAA have been reassigned from stop codons to glutamine, has evolved independently in several eukaryotic lineages, including the ulvophycean green algal orders Dasycladales and Cladophorales. To study the phylogenetic distribution of the standard and non-canonical genetic codes, we generated sequence data of a representative set of ulvophycean green algae and used a robust green algal phylogeny to evaluate different evolutionary scenarios that may account for the origin of the non-canonical code.ResultsThis study demonstrates that the Dasycladales and Cladophorales share this alternative genetic code with the related order Trentepohliales and the genus Blastophysa, but not with the Bryopsidales, which is sister to the Dasycladales. This complex phylogenetic distribution whereby all but one representative of a single natural lineage possesses an identical deviant genetic code is unique.ConclusionsWe compare different evolutionary scenarios for the complex phylogenetic distribution of this non-canonical genetic code. A single transition to the non-canonical code followed by a reversal to the canonical code in the Bryopsidales is highly improbable due to the profound genetic changes that coincide with codon reassignment. Multiple independent gains of the non-canonical code, as hypothesized for ciliates, are also unlikely because the same deviant code has evolved in all lineages. Instead we favor a stepwise acquisition model, congruent with the ambiguous intermediate model, whereby the non-canonical code observed in these green algal orders has a single origin. We suggest that the final steps from an ambiguous intermediate situation to a non-canonical code have been completed in the Trentepohliales, Dasycladales, Cladophorales and Blastophysa but not in the Bryopsidales. We hypothesize that in the latter lineage an initial stage characterized by translational ambiguity was not followed by final reassignment of both stop codons to glutamine. Instead the standard code was retained by the disappearance of the ambiguously decoding tRNAs from the genome. We correlate the emergence of a non-canonical genetic code in the Ulvophyceae to their multinucleate nature.

Distribution and Phylogeny of EFL and EF-1α in Euglenozoa Suggest Ancestral Co-Occurrence Followed by Differential Loss

Background The eukaryotic elongation factor EF-1α (also known as EF1A) catalyzes aminoacyl-tRNA binding by the ribosome during translation. Homologs of this essential protein occur in all domains of life, and it was previously thought to be ubiquitous in eukaryotes. Recently, however, a number of eukaryotes were found to lack EF-1α and instead encode a related protein called EFL (for EF-Like). EFL-encoding organisms are scattered widely across the tree of eukaryotes, and all have close relatives that encode EF-1α. This intriguingly complex distribution has been attributed to multiple lateral transfers because EFLs near mutual exclusivity with EF-1α makes an extended period of co-occurrence seem unlikely. However, differential loss may play a role in EFL evolution, and this possibility has been less widely discussed. Methodology/Principal Findings We have undertaken an EST- and PCR-based survey to determine the distribution of these two proteins in a previously under-sampled group, the Euglenozoa. EF-1α was found to be widespread and monophyletic, suggesting it is ancestral in this group. EFL was found in some species belonging to each of the three euglenozoan lineages, diplonemids, kinetoplastids, and euglenids. Conclusions/Significance Interestingly, the kinetoplastid EFL sequences are specifically related despite the fact that the lineages in which they are found are not sisters to one another, suggesting that EFL and EF-1α co-occurred in an early ancestor of kinetoplastids. This represents the strongest phylogenetic evidence to date that differential loss has contributed to the complex distribution of EFL and EF-1α.

The Distribution of Elongation Factor‐1 Alpha (EF‐1α), Elongation Factor‐Like (EFL), and a Non‐Canonical Genetic Code in the Ulvophyceae: Discrete Genetic Characters Support a Consistent Phylogenetic Framework

ABSTRACT. The systematics of the green algal class Ulvophyceae have been difficult to resolve with ultrastructural and molecular phylogenetic analyses. Therefore, we investigated relationships among ulvophycean orders by determining the distribution of two discrete genetic characters previously identified only in the order Dasycladales. First, Acetabularia acetabulum uses the core translation GTPase Elongation Factor 1α (EF‐1α) while most Chlorophyta instead possess the related GTPase Elongation Factor‐Like (EFL). Second, the nuclear genomes of dasycladaleans A. acetabulum and Batophora oerstedii use a rare non‐canonical genetic code in which the canonical termination codons TAA and TAG instead encode glutamine. Representatives of Ulvales and Ulotrichales were found to encode EFL, while Caulerpales, Dasycladales, Siphonocladales, and Ignatius tetrasporus were found to encode EF‐1α, in congruence with the two major lineages previously proposed for the Ulvophyceae. The EF‐1α of I. tetrasporus supports its relationship with Caulerpales/Dasycladales/Siphonocladales, in agreement with ultrastructural evidence, but contrary to certain small subunit rRNA analyses that place it with Ulvales/Ulotrichales. The same non‐canonical genetic code previously described in A. acetabulum was observed in EF‐1α sequences from Parvocaulis pusillus (Dasycladales), Chaetomorpha coliformis, and Cladophora cf. crinalis (Siphonocladales), whereas Caulerpales use the universal code. This supports a sister relationship between Siphonocladales and Dasycladales and further refines our understanding of ulvophycean phylogeny.

Nucleus-encoded periplastid-targeted EFL in chlorarachniophytes

Chlorarachniophytes are cercozoan amoeboflagellates that acquired photosynthesis by enslaving a green alga, which has retained a highly reduced nucleus called a nucleomorph. The nucleomorph lacks many genes necessary for its own maintenance and expression, suggesting that some genes have been moved to the host nucleus and their products are now targeted back to the periplastid compartment (PPC), the reduced eukaryotic cytoplasm of the endosymbiont. Protein trafficking in chlorarachniophytes is therefore complex, including nucleus-encoded plastid-targeted proteins, nucleomorph-encoded plastid-targeted proteins, and nucleus-encoded periplastid-targeted proteins. A major gap in our understanding of this system is the PPC-targeted proteins because none have been described in any chlorarachniophytes. Here we describe the first such protein, the GTPase EFL. EFL was characterized from 7 chlorarachniophytes, and 2 distinct types were found. One is related to foraminiferan EFL and lacks an amino-terminal extension. The second, distantly related, type encodes an amino-terminal extension consisting of a signal peptide followed by sequence sharing many characteristics with transit peptides from nucleus-encoded plastid-targeted proteins and which we conclude is most likely PPC targeted. Western blotting with antibodies specific to putative host and PPC-targeted EFL from the chlorarachniophytes Bigelowiella natans and Gymnochlora stellata is consistent with posttranslational cleavage of the leaders from PPC-targeted proteins. Immunolocalization of both proteins in B. natans confirmed the cytosolic location of the leaderless EFL and a distinct localization pattern for the PPC-targeted protein but could not rule out a plastid location (albeit very unlikely). We sought other proteins with a similar leader and identified a eukaryotic translation initiation factor 1 encoding a bipartite extension with the same properties. Transit peptide sequences were characterized from all 3 classes of targeted protein by comparing all examples of each class from expressed sequence tag surveys of B. natans and G. stellata. No recognizable difference between plastid- and PPC-targeted proteins was observed, but nucleomorph-encoded transit peptides differ, likely reflecting high AT content of nucleomorph genomes. Taken together, the data suggest that the system that directs proteins to the PPC in chlorarachniophytes uses a bipartite targeting sequence, as does the PPC-targeting system that evolved independently in cryptomonads.

Evolutionary origins of rhizarian parasites

The SAR group (Stramenopila, Alveolata, Rhizaria) is one of the largest clades in the tree of eukaryotes and includes a great number of parasitic lineages. Rhizarian parasites are obligate and have devastating effects on commercially important plants and animals but despite this fact, our knowledge of their biology and evolution is limited. Here, we present rhizarian transcriptomes from all major parasitic lineages in order to elucidate their evolutionary relationships using a phylogenomic approach. Our results suggest that Ascetosporea, parasites of marine invertebrates, are sister to the novel clade Apofilosa. The phytomyxean plant parasites branch sister to the vampyrellid algal ectoparasites in the novel clade Phytorhiza. They also show that Ascetosporea + Apofilosa + Retaria + Filosa + Phytorhiza form a monophyletic clade, although the branching pattern within this clade is difficult to resolve and appears to be model-dependent. Our study does not support the monophyly of the rhizarian parasitic lineages (Endomyxa), suggesting independent origins for rhizarian animal and plant parasites.

Transcriptomic Analysis Reveals Evidence for a Cryptic Plastid in the Colpodellid Voromonas pontica, a Close Relative of Chromerids and Apicomplexan Parasites

Colpodellids are free-living, predatory flagellates, but their close relationship to photosynthetic chromerids and plastid-bearing apicomplexan parasites suggests they were ancestrally photosynthetic. Colpodellids may therefore retain a cryptic plastid, or they may have lost their plastids entirely, like the apicomplexan Cryptosporidium. To find out, we generated transcriptomic data from Voromonas pontica ATCC 50640 and searched for homologs of genes encoding proteins known to function in the apicoplast, the non-photosynthetic plastid of apicomplexans. We found candidate genes from multiple plastid-associated pathways including iron-sulfur cluster assembly, isoprenoid biosynthesis, and tetrapyrrole biosynthesis, along with a plastid-type phosphate transporter gene. Four of these sequences include the 5′ end of the coding region and are predicted to encode a signal peptide and a transit peptide-like region. This is highly suggestive of targeting to a cryptic plastid. We also performed a taxon-rich phylogenetic analysis of small subunit ribosomal RNA sequences from colpodellids and their relatives, which suggests that photosynthesis was lost more than once in colpodellids, and independently in V. pontica and apicomplexans. Colpodellids therefore represent a valuable source of comparative data for understanding the process of plastid reduction in humanitys most deadly parasite.