An important missing piece in the puzzle of how plastids spread across the eukaryotic tree of life is a robust evolutionary framework for the host lineages. Four assemblages are known to harbour plastids derived from red algae and, according to the controversial chromalveolate hypothesis, these all share a common ancestry. Phylogenomic analyses have consistently shown that stramenopiles and alveolates are closely related, but haptophytes and cryptophytes remain contentious; they have been proposed to branch together with several heterotrophic groups in the newly erected Hacrobia. Here, we tested this question by producing a large expressed sequence tag dataset for the katablepharid Roombia truncata, one of the last hacrobian lineages for which genome-level data are unavailable, and combined this dataset with the recently completed genome of the cryptophyte Guillardia theta to build an alignment composed of 258 genes. Our analyses strongly support haptophytes as sister to the SAR group, possibly together with telonemids and centrohelids. We also confirmed the common origin of katablepharids and cryptophytes, but these lineages were not related to other hacrobians; instead, they branch with plants. Our study resolves the evolutionary position of haptophytes, an ecologically critical component of the oceans, and proposes a new hypothesis for the origin of cryptophytes.

One major lineage of green plants, the Chlorophyta, is represented by the green algal classes Prasinophyceae, Ulvophyceae, Trebouxiophyceae, and Chlorophyceae. The Prasinophyceae occupies the most basal position in the Chlorophyta, but the branching order of the Ulvophyceae, Trebouxiophyceae, and Chlorophyceae remains unresolved. The chloroplast genome sequences currently available for representatives of three chlorophyte classes have revealed that this genome is highly plastic, with Chlamydomonas (Chlorophyceae) and Chlorella (Trebouxiophyceae) showing fewer ancestral features than Nephroselmis (Prasinophyceae). We report the 195,867-bp chloroplast DNA (cpDNA) sequence of Pseudendoclonium akinetum (Ulvophyceae), a member of the class that has not been previously examined for detailed cpDNA analysis. This genome shares common evolutionary trends with its Chlorella and Chlamydomonas homologs. The gene content, number of ancestral gene clusters, and abundance of short dispersed repeats in Pseudendoclonium cpDNA are intermediate between those observed for Chlorella and Chlamydomonas cpDNAs. Although Pseudendoclonium cpDNA features a large inverted repeat, its quadripartite structure is unusual in displaying an rRNA operon transcribed toward the large single-copy (LSC) region and a small single-copy region containing 14 genes that are normally found in the LSC region. Twenty-seven group I introns lie in nine genes and fall within four subgroups (IA1, IA2, IA3, and IB); 19 encode putative homing endonucleases, and 7 have homologs at identical insertion sites in other chlorophyte or streptophyte organelle genomes. The high similarity observed among the 14 IA1 and 7 IA2 introns and their encoded endonucleases suggests that many introns arose from intragenomic proliferation of a few founding introns in the lineage leading to Pseudendoclonium. Interestingly, one intron (in atpA) and some of the dispersed repeats also reside in Pseudendoclonium mitochondria, providing strong evidence for interorganellar lateral transfer of these genetic elements. Phylogenetic analyses of 58 cpDNA-encoded proteins and genes support the hypothesis that the Ulvophyceae is sister to the Trebouxiophyceae but cannot eliminate the hypothesis that the Ulvophyceae is sister to the Chlorophyceae. We favor the latter hypothesis because it is strongly supported by phylogenetic analyses of gene order data and by independent structural evidence based on shared gene losses and rearrangement break points within ancestrally conserved gene clusters.

The genome of the microsporidia Encephalitozoon cuniculi is widely recognized as a model for extreme reduction and compaction. At only 2.9 Mbp, the genome encodes approximately 2,000 densely packed genes and little else. However, the nuclear genome of its sister, Encephalitozoon intestinalis, is even more reduced; at 2.3 Mbp, it represents a 20% reduction from an already severely compacted genome, raising the question, what else can be lost? In this paper, we describe the complete sequence of the E. intestinalis genome and its comparison with that of E. cuniculi. The two species share a conserved gene content, order and density over most of their genomes. The exceptions are the subtelomeric regions, where E. intestinalis chromosomes are missing large gene blocks of sequence found in E. cuniculi. In the remaining gene-dense chromosome 'cores', the diminutive intergenic sequences and introns are actually more highly conserved than the genes themselves, suggesting that they have reached the limits of reduction for a fully functional genome.

Significance Intracellular obligate parasitism results in extreme adaptations, whose evolutionary history is difficult to understand, because intermediate forms are hardly ever found. Microsporidia are highly derived intracellular parasites that are related to fungi. We describe the evolutionary history of a new microsporidian parasite found in the hindgut epithelium of the crustacean Daphnia and conclude that the new species has retained ancestral features that were lost in other microsporidia, whose hallmarks are the evolution of a unique infection apparatus, extreme genome reduction, and loss of mitochondrial respiration. The first evolutionary steps leading to the extreme metabolic and genomic simplification of microsporidia involved the adoption of a parasitic lifestyle, the development of a specialized infection apparatus, and the loss of diverse regulatory proteins.

Microsporidia of the genus Encephalitozoon are widespread pathogens of animals that harbor the smallest known nuclear genomes. Complete sequences from Encephalitozoon intestinalis (2.3 Mbp) and Encephalitozoon cuniculi (2.9 Mbp) revealed massive gene losses and reduction of intergenic regions as factors leading to their drastically reduced genome size. However, microsporidian genomes also have gained genes through horizontal gene transfers (HGT), a process that could allow the parasites to exploit their hosts more fully. Here, we describe the complete sequences of two intermediate-sized genomes (2.5 Mbp), from Encephalitozoon hellem and Encephalitozoon romaleae. Overall, the E. hellem and E. romaleae genomes are strikingly similar to those of Encephalitozoon cuniculi and Encephalitozoon intestinalis in both form and content. However, in addition to the expected expansions and contractions of known gene families in subtelomeric regions, both species also were found to harbor a number of protein-coding genes that are not found in any other microsporidian. All these genes are functionally related to the metabolism of folate and purines but appear to have originated by several independent HGT events from different eukaryotic and prokaryotic donors. Surprisingly, the genes are all intact in E. hellem, but in E. romaleae those involved in de novo synthesis of folate are all pseudogenes. Overall, these data suggest that a recent common ancestor of E. hellem and E. romaleae assembled a complete metabolic pathway from multiple independent HGT events and that one descendent already is dispensing with much of this new functionality, highlighting the transient nature of transferred genes.evolution | genomics | parasitology

BackgroundPhotosynthetic euglenids acquired their plastid by secondary endosymbiosis of a prasinophyte-like green alga. But unlike its prasinophyte counterparts, the plastid genome of the euglenid Euglena gracilis is riddled with introns that interrupt almost every protein-encoding gene. The atypical group II introns and twintrons (introns-within-introns) found in the E. gracilis plastid have been hypothesized to have been acquired late in the evolution of euglenids, implying that massive numbers of introns may be lacking in other taxa. This late emergence was recently corroborated by the plastid genome sequences of the two basal euglenids, Eutreptiella gymnastica and Eutreptia viridis, which were found to contain fewer introns.Methodology/Principal FindingsTo gain further insights into the proliferation of introns in euglenid plastids, we have characterized the complete plastid genome sequence of Monomorphina aenigmatica, a freshwater species occupying an intermediate phylogenetic position between early and late branching euglenids. The M. aenigmatica UTEX 1284 plastid genome (74,746 bp, 70.6% A+T, 87 genes) contains 53 intron insertion sites, of which 41 were found to be shared with other euglenids including 12 of the 15 twintron insertion sites reported in E. gracilis.ConclusionsThe pattern of insertion sites suggests an ongoing but uneven process of intron gain in the lineage, with perhaps a minimum of two bursts of rapid intron proliferation. We also identified several sites that represent intermediates in the process of twintron evolution, where the external intron is in place, but not the internal one, offering a glimpse into how these convoluted molecular contraptions originate.

BackgroundIn one small group of dinoflagellates, photosynthesis is carried out by a tertiary endosymbiont derived from a diatom, giving rise to a complex cell that we collectively refer to as a ‘dinotom’. The endosymbiont is separated from its host by a single membrane and retains plastids, mitochondria, a large nucleus, and many other eukaryotic organelles and structures, a level of complexity suggesting an early stage of integration. Although the evolution of these endosymbionts has attracted considerable interest, the plastid genome has not been examined in detail, and indeed no tertiary plastid genome has yet been sequenced.Methodology/Principal FindingsHere we describe the complete plastid genomes of two closely related dinotoms, Durinskia baltica and Kryptoperidinium foliaceum. The D. baltica (116470 bp) and K. foliaceum (140426 bp) plastid genomes map as circular molecules featuring two large inverted repeats that separate distinct single copy regions. The organization and gene content of the D. baltica plastid closely resemble those of the pennate diatom Phaeodactylum tricornutum. The K. foliaceum plastid genome is much larger, has undergone more reorganization, and encodes a putative tyrosine recombinase (tyrC) also found in the plastid genome of the heterokont Heterosigma akashiwo, and two putative serine recombinases (serC1 and serC2) homologous to recombinases encoded by plasmids pCf1 and pCf2 in another pennate diatom, Cylindrotheca fusiformis. The K. foliaceum plastid genome also contains an additional copy of serC1, two degenerate copies of another plasmid-encoded ORF, and two non-coding regions whose sequences closely resemble portions of the pCf1 and pCf2 plasmids.Conclusions/SignificanceThese results suggest that while the plastid genomes of two dinotoms share very similar gene content and genome organization with that of the free-living pennate diatom P. tricornutum, the K. folicaeum plastid genome has absorbed two exogenous plasmids. Whether this took place before or after the tertiary endosymbiosis is not clear.

The mitochondrial genome has undergone radical changes in both the Chlorophyta and Streptophyta, yet little is known about the dynamics of mtDNA evolution in either of these lineages. In the Chlorophyta, which comprises four of the five recognized classes of green algae (Prasinophyceae, Trebouxiophyceae, Ulvophyceae, and Chlorophyceae), the mitochondrial genome varies from 16 to 55 kb. This genome has retained a compact gene organization and a relatively complex gene repertoire ("ancestral" pattern) in the basal lineages represented by the Trebouxiophyceae and Prasinophyceae, whereas it has been reduced in size and gene complement and tends to evolve much more rapidly at the sequence level ("reduced-derived" pattern of evolution) in the Chlorophyceae and the lineage leading to the enigmatic chlorophyte Pedinomonas. To gain information about the evolutionary trends of mtDNA in the Ulvophyceae and also to gain insights into the phylogenetic relationships between ulvophytes and other chlorophytes, we have determined the mtDNA sequence of Pseudendoclonium akinetum. At 95,880 bp, Pseudendoclonium mtDNA is the largest green-algal mitochondrial genome sequenced to date and has the lowest gene density. These derived features are reminiscent of the "expanded" pattern exhibited by embryophyte mtDNAs, indicating that convergent evolution towards genome expansion has occurred independently in the Chlorophyta and Streptophyta. With 57 conserved genes, the gene repertoire of Pseudendoclonium mtDNA is slightly smaller than those of the prasinophyte Nephroselmis olivacea and the trebouxiophyte Prototheca wickerhamii. This ulvophyte mtDNA contains seven group I introns, four of which have homologs in green-algal mtDNAs displaying an "ancestral" or a "reduced-derived" pattern of evolution. Like its counterpart in the chlorophycean green alga Scenedesmus obliquus, it features numerous small, dispersed repeats in intergenic regions and introns. Its overall rate of sequence evolution appears to be accelerated to an intermediary level as compared with the rates observed in "ancestral" and "reduced-derived" mtDNAs. In agreement with the finding that Pseudendoclonium mtDNA exhibits features typical of both the "ancestral" and "reduced-derived" patterns of evolution, phylogenetic analyses of seven mtDNA-encoded proteins revealed a sister-group relationship between this ulvophyte and chlorophytes displaying "reduced-derived" mtDNAs.