Phylogenomic analysis identifies red algal genes of endosymbiotic origin in the chromalveolates

Endosymbiosis has spread photosynthesis to many branches of the eukaryotic tree; however, the history of photosynthetic organelle (plastid) gain and loss remains controversial. Fortuitously, endosymbiosis may leave a genomic footprint through the transfer of endosymbiont genes to the "host" nucleus (endosymbiotic gene transfer, EGT). EGT can be detected through comparison of host genomes to uncover the history of past plastid acquisitions. Here we focus on a lineage of chlorophyll c-containing algae and protists ("chromalveolates") that are postulated to share a common red algal secondary endosymbiont. This plastid is originally of cyanobacterial origin through primary endosymbiosis and is closely related among the Plantae (i.e., red, green, and glaucophyte algae). To test these ideas, an automated phylogenomics pipeline was used with a novel unigene data set of 5,081 expressed sequence tags (ESTs) from the haptophyte alga Emiliania huxleyi and genome or EST data from other chromalveolates, red algae, plants, animals, fungi, and bacteria. We focused on nuclear-encoded proteins that are targeted to the plastid to express their function because this group of genes is expected to have phylogenies that are relatively easy to interpret. A total of 708 genes were identified in E. huxleyi that had a significant Blast hit to at least one other taxon in our data set. Forty-six of the alignments that were derived from the 708 genes contained at least one other chromalveolate (i.e., besides E. huxleyi), red and/or green algae (or land plants), and one or more cyanobacteria, whereas 15 alignments contained E. huxleyi, one or more other chromalveolates, and only cyanobacteria. Detailed phylogenetic analyses of these data sets turned up 19 cases of EGT that did not contain significant paralogy and had strong bootstrap support at the internal nodes, allowing us to confidently identify the source of the plastid-targeted gene in E. huxleyi. A total of 17 genes originated from the red algal lineage, whereas 2 genes were of green algal origin. Our data demonstrate the existence of multiple red algal genes that are shared among different chromalveolates, suggesting that at least a subset of this group may share a common origin.     

Red and green algal origin of diatom membrane transporters: insights into environmental adaptation and cell evolution

Membrane transporters (MTs) facilitate the movement of molecules between cellular compartments. The evolutionary history of these key components of eukaryote genomes remains unclear. Many photosynthetic microbial eukaryotes (e.g., diatoms, haptophytes, and dinoflagellates) appear to have undergone serial endosymbiosis and thereby recruited foreign genes through endosymbiotic/horizontal gene transfer (E/HGT). Here we used the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum as models to examine the evolutionary origin of MTs in this important group of marine primary producers. Using phylogenomics, we used 1,014 diatom MTs as query against a broadly sampled protein sequence database that includes novel genome data from the mesophilic red algae Porphyridium cruentum and Calliarthron tuberculosum, and the stramenopile Ectocarpus siliculosus. Our conservative approach resulted in 879 maximum likelihood trees of which 399 genes show a non-lineal history between diatoms and other eukaryotes and prokaryotes (at the bootstrap value ≥70%). Of the eukaryote-derived MTs, 172 (ca. 25% of 697 examined phylogenies) have members of both red/green algae as sister groups, with 103 putatively arising from green algae, 19 from red algae, and 50 have an unresolved affiliation to red and/or green algae. We used topology tests to analyze the most convincing cases of non-lineal gene history in which red and/or green algae were nested within stramenopiles. This analysis showed that ca. 6% of all trees (our most conservative estimate) support an algal origin of MTs in stramenopiles with the majority derived from green algae. Our findings demonstrate the complex evolutionary history of photosynthetic eukaryotes and indicate a reticulate origin of MT genes in diatoms. We postulate that the algal-derived MTs acquired via E/HGT provided diatoms and other related microbial eukaryotes the ability to persist under conditions of fluctuating ocean chemistry, likely contributing to their great success in marine environments.     

The phylogenetic position of red algae revealed by multiple nuclear genes from mitochondria-containing eukaryotes and an alternative hypothesis on the origin of plastids

Abstract Red algae are one of the main photosynthetic eukaryotic lineages and are characterized by primitive features, such as a lack of flagella and the presence of phycobiliproteins in the chloroplast. Recent molecular phylogenetic studies using nuclear gene sequences suggest two conflicting hypotheses (monophyly versus non-monophyly) regarding the relationships between red algae and green plants. Although kingdom-level phylogenetic analyses using multiple nuclear genes from a wide-range of eukaryotic lineages were very recently carried out, they used highly divergent gene sequences of the cryptomonad nucleomorph (as the red algal taxon) or incomplete red algal gene sequences. In addition, previous eukaryotic phylogenies based on nuclear genes generally included very distant archaebacterial sequences (designated as the outgroup) and/or amitochondrial organisms, which may carry unusual gene substitutions due to parasitism or the absence of mitochondria. Here, we carried out phylogenetic analyses of various lineages of mitochondria-containing eukaryotic organisms using nuclear multigene sequences, including the complete sequences from the primitive red alga Cyanidioschyzon merolae. Amino acid sequence data for two concatenated paralogous genes (α- and β-tubulin) from mitochondria-containing organisms robustly resolved the basal position of the cellular slime molds, which were designated as the outgroup in our phylogenetic analyses. Phylogenetic analyses of 53 operational taxonomic units (OTUs) based on a 1525-amino-acid sequence of four concatenated nuclear genes (actin, elongation factor-1α, α-tubulin, and β-tubulin) reliably resolved the phylogeny only in the maximum parsimonious (MP) analysis, which indicated the presence of two large robust monophyletic groups (Groups A and B) and the basal eukaryotic lineages (red algae, true slime molds, and amoebae). Group A corresponded to the Opisthokonta (Metazoa and Fungi), whereas Group B included various primary and secondary plastid-containing lineages (green plants, glaucophytes, euglenoids, heterokonts, and apicomplexans), Ciliophora, Kinetoplastida, and Heterolobosea. The red algae represented the sister lineage to Group B. Using 34 OTUs for which essentially the entire amino acid sequences of the four genes are known, MP, distance, quartet puzzling, and two types of maximum likelihood (ML) calculations all robustly resolved the monophyly of Group B, as well as the basal position of red algae within eukaryotic organisms. In addition, phylogenetic analyses of a concatenated 4639-amino-acid sequence for 12 nuclear genes (excluding the EF-2 gene) of 12 mitochondria-containing OTUs (including C. merolae) resolved a robust non-sister relationship between green plants and red algae within a robust monophyletic group composed of red algae and the eukaryotic organisms belonging to Group B. A new scenario for the origin and evolution of plastids is suggested, based on the basal phylogenetic position of the red algae within the large clade (Group B plus red algae). The primary plastid endosymbiosis likely occurred once in the common ancestor of this large clade, and the primary plastids were subsequently lost in the ancestor(s) of the Discicristata (euglenoids, Kinetoplastida, and Heterolobosea), Heterokontophyta, and Alveolata (apicomplexans and Ciliophora). In addition, a new concept of “Plantae” is proposed for phototrophic and nonphototrophic organisms belonging to Group B and red algae, on the basis of the common history of the primary plastid endosymbiosis. The Plantae include primary plastid-containing phototrophs and nonphototrophic eukaryotes that possibly contain genes of cyanobacterial origin acquired in the primary endosymbiosis.     

Localization and evolution of septins in algae

Septins are a group of GTP‐binding proteins that are multi‐functional, with a well‐known role in cytokinesis in animals and fungi. Although the functions of septins have been thoroughly studied in opisthokonts (fungi and animals), the function and evolution of plant/algal septins are not as well characterized. Here we describe septin localization and expression in the green algae Nannochloris bacillaris and Marvania geminata. The present data suggest that septins localize at the division site when cytokinesis occurs. In addition, we show that septin homologs may be found only in green algae, but not in other major plant lineages, such as land plants, red algae and glaucophytes. We also found other septin homolog‐possessing organisms among the diatoms, Rhizaria and cryptomonad/haptophyte lineages. Our study reveals the potential role of algal septins in cytokinesis and/or cell elongation, and confirms that septin genes appear to have been lost in the Plantae lineage, except in some green algae.     

Evolutionary origins of the eukaryotic shikimate pathway: gene fusions, horizontal gene transfer, and endosymbiotic replacements

Currently the shikimate pathway is reported as a metabolic feature of prokaryotes, ascomycete fungi, apicomplexans, and plants. The plant shikimate pathway enzymes have similarities to prokaryote homologues and are largely active in chloroplasts, suggesting ancestry from the plastid progenitor genome. Toxoplasma gondii, which also possesses an alga-derived plastid organelle, encodes a shikimate pathway with similarities to ascomycete genes, including a five-enzyme pentafunctional arom. These data suggests that the shikimate pathway and the pentafunctional arom either had an ancient origin in the eukaryotes or was conveyed by eukaryote-to-eukaryote horizontal gene transfer (HGT). We expand sampling and analyses of the shikimate pathway genes to include the oomycetes, ciliates, diatoms, basidiomycetes, zygomycetes, and the green and red algae. Sequencing of cDNA from Tetrahymena thermophila confirmed the presence of a pentafused arom, as in fungi and T. gondii. Phylogenies and taxon distribution suggest that the arom gene fusion event may be an ancient eukaryotic innovation. Conversely, the Plantae lineage (represented here by both Viridaeplantae and the red algae) acquired different prokaryotic genes for all seven steps of the shikimate pathway. Two of the phylogenies suggest a derivation of the Plantae genes from the cyanobacterial plastid progenitor genome, but if the full Plantae pathway was originally of cyanobacterial origin, then the five other shikimate pathway genes were obtained from a minimum of two other eubacterial genomes. Thus, the phylogenies demonstrate both separate HGTs and shared derived HGTs within the Plantae clade either by primary HGT transfer or secondarily via the plastid progenitor genome. The shared derived characters support the holophyly of the Plantae lineage and a single ancestral primary plastid endosymbiosis. Our analyses also pinpoints a minimum of 50 gene/domain loss events, demonstrating that loss and replacement events have been an important process in eukaryote genome evolution.     

Ancient recruitment by chromists of green algal genes encoding enzymes for carotenoid biosynthesis

Chromist algae (stramenopiles, cryptophytes, and haptophytes) are major contributors to marine primary productivity. These eukaryotes acquired their plastid via secondary endosymbiosis, whereby an early-diverging red alga was engulfed by a protist and the plastid was retained and its associated nuclear-encoded genes were transferred to the host genome. Current data suggest, however, that chromists are paraphyletic; therefore, it remains unclear whether their plastids trace back to a single secondary endosymbiosis or, alternatively, this organelle has resulted from multiple independent events in the different chromist lineages. Both scenarios, however, predict that plastid-targeted, nucleus-encoded chromist proteins should be most closely related to their red algal homologs. Here we analyzed the biosynthetic pathway of carotenoids that are essential components of all photosynthetic eukaryotes and find a mosaic evolutionary origin of these enzymes in chromists. Surprisingly, about one-third (5/16) of the proteins are most closely related to green algal homologs with three branching within or sister to the early-diverging Prasinophyceae. This phylogenetic association is corroborated by shared diagnostic indels and the syntenic arrangement of a specific gene pair involved in the photoprotective xanthophyll cycle. The combined data suggest that the prasinophyte genes may have been acquired before the ancient split of stramenopiles, haptophytes, cryptophytes, and putatively also dinoflagellates. The latter point is supported by the observed monophyly of alveolates and stramenopiles in most molecular trees. One possible explanation for our results is that the green genes are remnants of a cryptic endosymbiosis that occurred early in chromalveolate evolution; that is, prior to the postulated split of stramenopiles, alveolates, haptophytes, and cryptophytes. The subsequent red algal capture would have led to the loss or replacement of most green genes via intracellular gene transfer from the new endosymbiont. We argue that the prasinophyte genes were retained because they enhance photosynthetic performance in chromalveolates, thus extending the niches available to these organisms. The alternate explanation of green gene origin via serial endosymbiotic or horizontal gene transfers is also plausible, but the latter would require the independent origins of the same five genes in some or all the different chromalveolate lineages.     

Nuclear DNA content estimates in green algal lineages: chlorophyta and streptophyta

BACKGROUND AND AIMS: Consensus higher-level molecular phylogenies present a compelling case that an ancient divergence separates eukaryotic green algae into two major monophyletic lineages, Chlorophyta and Streptophyta, and a residuum of green algae, which have been referred to prasinophytes or micromonadophytes. Nuclear DNA content estimates have been published for less than 1% of the described green algal members of Chlorophyta, which includes multicellular green marine algae and freshwater flagellates (e.g. Chlamydomonas and Volvox). The present investigation summarizes the state of our knowledge and adds substantially to our database of C-values, especially for the streptophyte charophycean lineage which is the sister group of the land plants. A recent list of 2C nuclear DNA contents for isolates and species of green algae is expanded by 72 to 157. METHODS: The DNA-localizing fluorochrome DAPI (4',6-diamidino-2-phenylindole) and red blood cell (chicken erythrocytes) standard were used to estimate 2C values with static microspectrophotometry. Key RESULTS: In Chlorophyta, including Chlorophyceae, Prasinophyceae, Trebouxiophyceae and Ulvophyceae, 2C DNA estimates range from 0.01 to 5.8 pg. Nuclear DNA content variation trends are noted and discussed for specific problematic taxon pairs, including Ulotrichales-Ulvales, and Cladophorales-Siphonocladales. For Streptophyta, 2C nuclear DNA contents range from 0.2 to 6.4 pg, excluding the highly polyploid Charales and Desmidiales, which have genome sizes of up to 14.8 and 46.8 pg, respectively. Nuclear DNA content data for Streptophyta superimposed on a contemporary molecular phylogeny indicate that early diverging lineages, including some members of Chlorokybales, Coleochaetales and Klebsormidiales, have genomes as small as 0.1-0.5 pg. It is proposed that the streptophyte ancestral nuclear genome common to both the charophyte and the embryophyte lineages can be characterized as 1C = 0.2 pg and 1n = 6. CONCLUSIONS: These data will help pre-screen candidate species for the on-going construction of bacterial artificial chromosome nuclear genome libraries for land plant ancestors. Data for the prasinophyte Mesostigma are of particular interest as this alga reportedly most closely resembles the 'ancestral green flagellate'. Both mechanistic and ecological processes are discussed that could have produced the observed C-value increase of >100-fold in the charophyte green algae whereas the ancestral genome was conserved in the embryophytes.     

A new scenario of plastid evolution: plastid primary endosymbiosis before the divergence of the “Plantae,” emended

A recent hypothesis on the origin of eukaryotic phototrophs proposes that red algae, green plants (land plants plus green algae), and glaucophytes constitute the primary photosynthetic eukaryotes, whose plastids may have originated directly from a cyanobacterium-like prokaryote via primary endosymbiosis, whereas the plastids of other lineages of eukaryotic phototrophs appear to be the result of secondary endosymbiotic events involving a phototrophic eukaryote and a host cell. However, the phylogenetic relationships among the three lineages of primary photosynthetic eukaryotes remained unresolved because previous nuclear multigene phylogenies used incomplete red algal gene sequences derived mainly from Porphyra (Rhodophyceae, one of the two lineages of the Rhodophyta), and lacked sequences from the Cyanidiophyceae (the other red algal lineage). Recently, the complete nuclear genome sequences from the red alga Cyanidioschyzon merolae 10D of the Cyanidiophyceae were determined. Using this genomic information, nuclear multigene phylogenetic analyses of various lineages of mitochondrion-containing eukaryotes were conducted. Since bacterial and amitochondrial eukaryotic genes present serious problems to eukaryotic phylogenies, basal eukaryotes were deduced based on the paralogous comparison of the concatenated - and -tubulin. The comparison demonstrated that cellular slime molds (Amoebozoa) represent the most basal position within the mitochondrion-containing organisms. With the cellular slime molds as the outgroup, phylogenetic analyses based on a 1,525-amino acid sequence of four concatenated nuclear genes [actin, elongation factor-1( EF-1), -tubulin, and -tubulin] resolved the presence of two large, robust monophyletic groups and the basal eukaryotic lineages (Amoebozoa). One of the two groups corresponded to the Opisthokonta (Metazoa and Fungi), whereas the other included various lineages containing primary and secondary plastids (red algae, green plants, glaucophytes, euglenoids, heterokonts, and apicomplexans), Ciliophora, Kinetoplastida, dinoflagellates, and Heterolobosea, for which the red algae represented the most basal lineage. Therefore, the plastid primary endosymbiosis likely occurred once in the common ancestor of the latter group, and the primary plastids were subsequently lost in the ancestor(s) of organisms within the group that now lacks primary plastids. A new concept of Plantae was proposed for phototrophic and nonphototrophic organisms belonging to this group on the basis of their common history of plastid primary endosymbiosis. This new scenario of plastid evolution is discussed here, and is compared with recent genome information and findings on the secondary endosymbiosis of the Euglena plastid.     

Chlorophyll c-containing plastid relationships based on analyses of a multigene data set with all four chromalveolate lineages

The chlorophyll c-containing algae comprise four major lineages: dinoflagellates, haptophytes, heterokonts, and cryptophytes. These four lineages have sometimes been grouped together based on their pigmentation, but cytological and rRNA data had suggested that they were not a monophyletic lineage. Some molecular data support monophyly of the plastids, while other plastid and host data suggest different relationships. It is uncontroversial that these groups have all acquired plastids from another eukaryote, probably from the red algal lineage, in a secondary endosymbiotic event, but the number and sequence of such event(s) remain controversial. Understanding chlorophyll c-containing plastid relationships is a first step towards determining the number of endosymbiotic events within the chromalveolates. We report here phylogenetic analyses using 10 plastid genes with representatives of all four chromalveolate lineages. This is the first organellar genome-scale analysis to include both haptophytes and dinoflagellates. Concatenated analyses support the monophyly of the chlorophyll c-containing plastids and suggest that cryptophyte plastids are the basal member of the chlorophyll c-containing plastid lineage. The gene psbA, which has at times been used for phylogenetic purposes, was found to differ from the other genes in its placement of the dinoflagellates and the haptophytes, and in its lack of support for monophyly of the green and red plastid lineages. Overall, the concatenated data are consistent with a single origin of chlorophyll c-containing plastids from red algae. However, these data cannot test several key hypothesis concerning chromalveolate host monophyly, and do not preclude the possibility of serial transfer of chlorophyll c-containing plastids among distantly related hosts.     

Are red algae plants?

For 200 years prior to the 1938 publication of H. F. Copeland, all authorities (with one exception) classified red algae (Rhodophyta) within Kingdom Plantae or its equivalent. Copeland's reclassification of red algae within Kingdom Protista or Protoctista drew from an alternative tradition, dating to Cohn in 1867, in which red algae were viewed as the earliest or simplest eukaryotes. Analyses of ribosomal RNA (rRNA) sequence data initially favoured Copeland's reclassification. Many more rRNA gene (rDNA) sequences are now available from the eukaryote lineages most closely related to red algae, and based on these data, the hypothesis that red algae and green plants are sister groups cannot be rejected. An increasing body of sequence, intron-location and functional data from nuclear- and mitochondrially encoded proteins likewise supports a sister-group relationship between red algae and green plants. Submerging Kingdoms Plantae, Animalia and Fungi into Eukarya would provide a more natural framework for the eventual resolution of whether red algae are plants or prorists.     

Probing the Monophyly of the Sphaeropleales (Chlorophyceae) Using Data From Five Genes

Molecular phylogenetic analyses have had a major impact on the classification of the green algal class Chlorophyceae, corroborating some previous evolutionary hypotheses, but primarily promoting new interpretations of morphological evolution. One set of morphological traits that feature prominently in green algal systematics is the absolute orientation of the flagellar apparatus in motile cells, which correlates strongly with taxonomic classes and orders. The order Sphaeropleales includes diverse green algae sharing the directly opposite (DO) flagellar apparatus orientation of their biflagellate motile cells. However, algae across sphaeroplealean families differ in specific components of the DO flagellar apparatus, and molecular phylogenetic studies often have failed to provide strong support for the monophyly of the order. To test the monophyly of Sphaeropleales and of taxa with the DO flagellar apparatus, we conducted a molecular phylogenetic study of 16 accessions representing all known families and diverse affiliated lineages within the order, with data from four plastid genes (psaA, psaB, psbC, rbcL) and one nuclear ribosomal gene (18S). Although single‐gene analyses varied in topology and support values, analysis of combined data strongly supported a monophyletic Sphaeropleales. Our results also corroborated previous phylogenetic hypotheses that were based on chloroplast genome data from relatively few taxa. Specifically, our data resolved Volvocales, algae possessing predominantly biflagellate motile cells with clockwise (CW) flagellar orientation, as the monophyletic sister lineage to Sphaeropleales, and an alliance of Chaetopeltidales, Chaetophorales, and Oedogoniales, orders having multiflagellate motile cells with distinct flagellar orientations involving the DO and CW forms.     

Cyanobacterial contribution to algal nuclear genomes is primarily limited to plastid functions

A single cyanobacterial primary endosymbiosis that occurred approximately 1.5 billion years ago is believed to have given rise to the plastid in the common ancestor of the Plantae or Archaeplastida--the eukaryotic supergroup comprising red, green (including land plants), and glaucophyte algae. Critical to plastid establishment was the transfer of endosymbiont genes to the host nucleus (i.e., endosymbiotic gene transfer [EGT]). It has been postulated that plastid-derived EGT played a significant role in plant nuclear-genome evolution, with 18% (or 4,500) of all nuclear genes in Arabidopsis thaliana having a cyanobacterial origin with about one-half of these recruited for nonplastid functions. Here, we determine whether the level of cyanobacterial gene recruitment proposed for Arabidopsis is of the same magnitude in the algal sisters of plants by analyzing expressed-sequence tag (EST) data from the glaucophyte alga Cyanophora paradoxa. Bioinformatic analysis of 3,576 Cyanophora nuclear genes shows that 10.8% of these with significant database hits are of cyanobacterial origin and one-ninth of these have nonplastid functions. Our data indicate that unlike plants, early-diverging algal groups appear to retain a smaller number of endosymbiont genes in their nucleus, with only a minor proportion of these recruited for nonplastid functions.     

Extrinsic proteins of photosystem II: an intermediate member of PsbQ protein family in red algal PS II.

The oxygen-evolving photosystem II (PS II) complex of red algae contains four extrinsic proteins of 12 kDa, 20 kDa, 33 kDa and cyt c-550, among which the 20 kDa protein is unique in that it is not found in other organisms. We cloned the gene for the 20-kDa protein from a red alga Cyanidium caldarium. The gene consists of a leader sequence which can be divided into two parts: one for transfer across the plastid envelope and the other for transfer into thylakoid lumen, indicating that the gene is encoded by the nuclear genome. The sequence of the mature 20-kDa protein has low but significant homology with the extrinsic 17-kDa (PsbQ) protein of PS II from green algae Volvox Carteri and Chlamydomonas reinhardtii, as well as the PsbQ protein of higher plants and PsbQ-like protein from cyanobacteria. Cross-reconstitution experiments with combinations of the extrinsic proteins and PS IIs from the red alga Cy. caldarium and green alga Ch. reinhardtii showed that the extrinsic 20-kDa protein was functional in place of the green algal 17-kDa protein on binding to the green algal PS II and restoration of oxygen evolution. From these results, we conclude that the 20-kDa protein is the ancestral form of the extrinsic 17-kDa protein in green algal and higher plant PS IIs. This provides an important clue to the evolution of the oxygen-evolving complex from prokaryotic cyanobacteria to eukaryotic higher plants. The gene coding for the extrinsic 20-kDa protein was named psbQ' (prime).     

Membrane heredity and early chloroplast evolution

Membrane heredity was central to the unique symbiogenetic origin from cyanobacteria of chloroplasts in the ancestor of Plantae (green plants, red algae, glaucophytes) and to subsequent lateral transfers of plastids to form even more complex photosynthetic chimeras. Each symbiogenesis integrated disparate genomes and several radically different genetic membranes into a more complex cell. The common ancestor of Plantae evolved transit machinery for plastid protein import. In later secondary symbiogeneses, signal sequences were added to target proteins across host perialgal membranes: independently into green algal plastids (euglenoids, chlorarachneans) and red algal plastids (alveolates, chromists). Conservatism and innovation during early plastid diversification are discussed.     

Multiple genes of apparent algal origin suggest ciliates may once have been photosynthetic

Plantae (as defined by Cavalier-Smith, 1981) plastids evolved via primary endosymbiosis whereby a heterotrophic protist enslaved a photosynthetic cyanobacterium. This "primary" plastid spread into other eukaryotes via secondary endosymbiosis. An important but contentious theory in algal evolution is the chromalveolate hypothesis that posits chromists (cryptophytes, haptophytes, and stramenopiles) and alveolates (ciliates, apicomplexans, and dinoflagellates) share a common ancestor that contained a red-algal-derived "secondary" plastid. Under this view, the existence of several later-diverging plastid-lacking chromalveolates such as ciliates and oomycetes would be explained by plastid loss in these lineages. To test the idea of a photosynthetic ancestry for ciliates, we used the 27,446 predicted proteins from the macronuclear genome of Tetrahymena thermophila to query prokaryotic and eukaryotic genomes. We identified 16 proteins of possible algal origin in the ciliates Tetrahymena and Paramecium tetraurelia. Fourteen of these are present in other chromalveolates. Here we compare and contrast the likely scenarios for algal-gene origin in ciliates either via multiple rounds of horizontal gene transfer (HGT) from algal prey or symbionts, or through endosymbiotic gene transfer (EGT) during a putative photosynthetic phase in their evolution.     

The tetrapyrrole synthesis pathway as a model of horizontal gene transfer in euglenoids

The history of euglenoids may have begun as early as ~2 bya. These early phagotrophs ate cyanobacteria, archaea, and eubacteria, and the subsequent appearance of red algae and chromalveolates provided euglenoids with additional food sources. Following the appearance of green algae, euglenoids acquired a chloroplast via a secondary endosymbiotic event with a green algal ancestor. This endosymbiosis also involved a massive transfer of nuclear‐encoded genes from the symbiont nucleus to the host. Expecting these genes to have a green algal origin, this research has shown, through the use of DNA‐sequences and the analysis of phylogenetic relationships, that many housekeeping genes have a red algal/chromalveolate ancestry. This suggested that many other endosymbiotic/horizontal gene transfers, which brought genes from chromalveolates to euglenoids, may have been taking place long before the acquisition of the chloroplast. The investigation of the origin of the enzymes involved in the tetrapyrrole synthesis pathway provided insights into horizontal gene transfer in euglenoids and demonstrated that the euglenoid nuclear genome is a mosaic comprised of genes from the ancestral lineage plus genes transferred endosymbiotically/horizontally from green, red, and chromalveolates lineages.     

Assessing red algal supraordinal diversity and taxonomy in the context of contemporary systematic data

The wondrously diverse eukaryotes that constitute the red algae have been the focus of numerous recent molecular surveys and remain a rich source of undescribed and little known species for the traditional taxonomist. Molecular studies place the red algae in the kingdom Plantae; however, supraordinal classification has been largely confined to debate on subclass vs. class level status for the two recognized subgroups, one of which is widely acknowledged as paraphyletic. This narrow focus has generally masked the extent to which red algal classification needs modification. We provide a comprehensive review of the literature pertaining to the antiquity, diversity, and systematics of the red algae and propose a contemporary classification based on recent and traditional evidence.     

Expression of the nucleus-encoded chloroplast division genes and proteins regulated by the algal cell cycle

Chloroplasts have evolved from a cyanobacterial endosymbiont and their continuity has been maintained by chloroplast division, which is performed by the constriction of a ring-like division complex at the division site. It is believed that the synchronization of the endosymbiotic and host cell division events was a critical step in establishing a permanent endosymbiotic relationship, such as is commonly seen in existing algae. In the majority of algal species, chloroplasts divide once per specific period of the host cell division cycle. In order to understand both the regulation of the timing of chloroplast division in algal cells and how the system evolved, we examined the expression of chloroplast division genes and proteins in the cell cycle of algae containing chloroplasts of cyanobacterial primary endosymbiotic origin (glaucophyte, red, green, and streptophyte algae). The results show that the nucleus-encoded chloroplast division genes and proteins of both cyanobacterial and eukaryotic host origin are expressed specifically during the S phase, except for FtsZ in one graucophyte alga. In this glaucophyte alga, FtsZ is persistently expressed throughout the cell cycle, whereas the expression of the nucleus-encoded MinD and MinE as well as FtsZ ring formation are regulated by the phases of the cell cycle. In contrast to the nucleus-encoded division genes, it has been shown that the expression of chloroplast-encoded division genes is not regulated by the host cell cycle. The endosymbiotic gene transfer of minE and minD from the chloroplast to the nuclear genome occurred independently on multiple occasions in distinct lineages, whereas the expression of nucleus-encoded MIND and MINE is regulated by the cell cycle in all lineages examined in this study. These results suggest that the timing of chloroplast division in algal cell cycle is restricted by the cell cycle-regulated expression of some but not all of the chloroplast division genes. In addition, it is suggested that the regulation of each division-related gene was established shortly after the endosymbiotic gene transfer, and this event occurred multiple times independently in distinct genes and in distinct lineages.     

Gene-rich plastid genomes of two parasitic red algal species,Laurencia australis and L. verruciformis (Rhodomelaceae,Ceramiales), and a taxonomic revision of Janczewskia

Parasitic red algae are an interesting system for investigating the genetic changes that occur in parasites. These parasites have evolved independently multiple times within the red algae. The functional loss of plastid genomes can be investigated in these multiple independent examples, and fine-scale patterns may be discerned. The only plastid genomes from red algal parasites known so far are highly reduced and missing almost all photosynthetic genes. Our study assembled and annotated plastid genomes from the parasites Janczewskia tasmanica and its two Laurencia host species (Laurencia elata and one unidentified Laurencia sp. A25) from Australia and Janczewskia verruciformis, its host species (Laurencia catarinensis), and the closest known free-living relative (Laurencia obtusa) from the Canary Islands (Spain). For the first time we show parasitic red algal plastid genomes that are similar in size and gene content to free-living host species without any gene loss or genome reduction. The only exception was two pseudogenes (moeB and ycf46) found in the plastid genome of both isolates of J. tasmanica, indicating potential for future loss of these genes. Further comparative analyses with the three highly reduced plastid genomes showed possible gene loss patterns, in which photosynthetic gene categories were lost followed by other gene categories. Phylogenetic analyses did not confirm monophyly of Janczewskia, and the genus was subsumed into Laurencia. Further investigations will determine if any convergent small-scale patterns of gene loss exist in parasitic red algae and how these are applicable to other parasitic systems.