Red Algal Endosymbiont Research Articles

Gene loss, pseudogenization, and independent genome reduction in non-photosynthetic species of Cryptomonas (Cryptophyceae) revealed by comparative nucleomorph genomics

BackgroundCryptophytes are ecologically important algae of interest to evolutionary cell biologists because of the convoluted history of their plastids and nucleomorphs, which are derived from red algal secondary endosymbionts. To better understand the evolution of the cryptophyte nucleomorph, we sequenced nucleomorph genomes from two photosynthetic and two non-photosynthetic species in the genus Cryptomonas. We performed a comparative analysis of these four genomes and the previously published genome of the non-photosynthetic species Cryptomonas paramecium CCAP977/2a.ResultsAll five nucleomorph genomes are similar in terms of their general architecture, gene content, and gene order and, in the non-photosynthetic strains, loss of photosynthesis-related genes. Interestingly, in terms of size and coding capacity, the nucleomorph genome of the non-photosynthetic species Cryptomonas sp. CCAC1634B is much more similar to that of the photosynthetic C. curvata species than to the non-photosynthetic species C. paramecium.ConclusionsOur results reveal fine-scale nucleomorph genome variation between distantly related congeneric taxa containing photosynthetic and non-photosynthetic species, including recent pseudogene formation, and provide a first glimpse into the possible impacts of the loss of photosynthesis on nucleomorph genome coding capacity and structure in independently evolved colorless strains.

Secondary Plastids of Euglenids and Chlorarachniophytes Function with a Mix of Genes of Red and Green Algal Ancestry.

Endosymbiosis has been common all along eukaryotic evolution, providing opportunities for genomic and organellar innovation. Plastids are a prominent example. After the primary endosymbiosis of the cyanobacterial plastid ancestor, photosynthesis spread in many eukaryotic lineages via secondary endosymbioses involving red or green algal endosymbionts and diverse heterotrophic hosts. However, the number of secondary endosymbioses and how they occurred remain poorly understood. In particular, contrasting patterns of endosymbiotic gene transfer have been detected and subjected to various interpretations. In this context, accurate detection of endosymbiotic gene transfers is essential to avoid wrong evolutionary conclusions. We have assembled a strictly selected set of markers that provides robust phylogenomic evidence suggesting that nuclear genes involved in the function and maintenance of green secondary plastids in chlorarachniophytes and euglenids have unexpected mixed red and green algal origins. This mixed ancestry contrasts with the clear red algal origin of most nuclear genes carrying similar functions in secondary algae with red plastids.

A functional zeaxanthin epoxidase from red algae shedding light on the evolution of light‐harvesting carotenoids and the xanthophyll cycle in photosynthetic eukaryotes

The epoxy-xanthophylls antheraxanthin and violaxanthin are key precursors of light-harvesting carotenoids and participate in the photoprotective xanthophyll cycle. Thus, the invention of zeaxanthin epoxidase (ZEP) catalyzing their formation from zeaxanthin has been a fundamental step in the evolution of photosynthetic eukaryotes. ZEP genes have only been found in Viridiplantae and chromalveolate algae with secondary plastids of red algal ancestry, suggesting that ZEP evolved in the Viridiplantae and spread to chromalveolates by lateral gene transfer. By searching publicly available sequence data from 11 red algae covering all currently recognized red algal classes we identified ZEP candidates in three species. Phylogenetic analyses showed that the red algal ZEP is most closely related to ZEP proteins from photosynthetic chromalveolates possessing secondary plastids of red algal origin. Its enzymatic activity was assessed by high performance liquid chromatography (HPLC) analyses of red algal pigment extracts and by cloning and functional expression of the ZEP gene from Madagascaria erythrocladioides in leaves of the ZEP-deficient aba2 mutant of Nicotiana plumbaginifolia. Unlike other ZEP enzymes examined so far, the red algal ZEP introduces only a single epoxy group into zeaxanthin, yielding antheraxanthin instead of violaxanthin. The results indicate that ZEP evolved before the split of Rhodophyta and Viridiplantae and that chromalveolates acquired ZEP from the red algal endosymbiont and not by lateral gene transfer. Moreover, the red algal ZEP enables engineering of transgenic plants incorporating antheraxanthin instead of violaxanthin in their photosynthetic machinery.

Re-analyses of "Algal" Genes Suggest a Complex Evolutionary History of Oomycetes.

The spread of photosynthesis is one of the most important but constantly debated topics in eukaryotic evolution. Various hypotheses have been proposed to explain the plastid distribution in extant eukaryotes. Notably, the chromalveolate hypothesis suggested that multiple eukaryotic lineages were derived from a photosynthetic ancestor that had a red algal endosymbiont. As such, genes of plastid/algal origin in aplastidic chromalveolates, such as oomycetes, were considered to be important supporting evidence. Although the chromalveolate hypothesis has been seriously challenged, some of its supporting evidence has not been carefully investigated. In this study, we re-evaluate the “algal” genes from oomycetes with a larger sampling and careful phylogenetic analyses. Our data provide no conclusive support for a common photosynthetic ancestry of stramenopiles, but show that the initial estimate of “algal” genes in oomycetes was drastically inflated due to limited genome data available then for certain eukaryotic lineages. These findings also suggest that the evolutionary histories of these “algal” genes might be attributed to complex scenarios such as differential gene loss, serial endosymbioses, or horizontal gene transfer.

A Plastid Protein That Evolved from Ubiquitin and Is Required for Apicoplast Protein Import in Toxoplasma gondii.

ABSTRACTApicomplexan parasites cause a variety of important infectious diseases, including malaria, toxoplasma encephalitis, and severe diarrhea due to Cryptosporidium. Most apicomplexans depend on an organelle called the apicoplast which is derived from a red algal endosymbiont. The apicoplast is essential for the parasite as the compartment of fatty acid, heme, and isoprenoid biosynthesis. The majority of the approximate 500 apicoplast proteins are nucleus encoded and have to be imported across the four membranes that surround the apicoplast. Import across the second outermost membrane of the apicoplast, the periplastid membrane, depends on an apicoplast-specific endoplasmic reticulum-associated protein degradation (ERAD) complex and on enzymes of the associated ubiquitination cascade. However, identification of an apicoplast ubiquitin associated with this machinery has long been elusive. Here we identify a plastid ubiquitin-like protein (PUBL), an apicoplast protein that is derived from a ubiquitin ancestor but that has significantly changed in its primary sequence. PUBL is distinct from known ubiquitin-like proteins, and phylogenomic analyses suggest a clade specific to apicomplexans. We demonstrate that PUBL and the AAA ATPase CDC48AP both act to translocate apicoplast proteins across the periplastid membrane during protein import. Conditional null mutants and genetic complementation show that both proteins are critical for this process and for parasite survival. PUBL residues homologous to those that are required for ubiquitin conjugation onto target proteins are essential for this function, while those required for polyubiquitination and preprotein processing are dispensable. Our experiments provide a mechanistic understanding of the molecular machinery that drives protein import across the membranes of the apicoplast.

Regulation of chloroplast and nucleomorph replication by the cell cycle in the cryptophyte Guillardia theta

The chloroplasts of cryptophytes arose through a secondary endosymbiotic event in which a red algal endosymbiont was integrated into a previously nonphotosynthetic eukaryote. The cryptophytes retain a remnant of the endosymbiont nucleus (nucleomorph) that is replicated once in the cell cycle along with the chloroplast. To understand how the chloroplast, nucleomorph and host cell divide in a coordinated manner, we examined the expression of genes/proteins that are related to nucleomorph replication and chloroplast division as well as the timing of nuclear and nucleomorph DNA synthesis in the cryptophyte Guillardia theta. Nucleus-encoded nucleomorph HISTONE H2A mRNA specifically accumulated during the nuclear S phase. In contrast, nucleomorph-encoded genes/proteins that are related to nucleomorph replication and chloroplast division (FtsZ) are constantly expressed throughout the cell cycle. The results of this study and previous studies on chlorarachniophytes suggest that there was a common evolutionary pattern in which an endosymbiont lost its replication cycle-dependent transcription while cell-cycle-dependent transcriptional regulation of host nuclear genes came to restrict the timing of nucleomorph replication and chloroplast division.

Nucleomorph Genome Sequences of Two Chlorarachniophytes, Amorphochlora amoebiformis and Lotharella vacuolata.

Many algal groups acquired complex plastids by the uptake of green and red algae through multiple secondary endosymbioses. As a result of gene loss and transfer during the endosymbiotic processes, algal endosymbiont nuclei disappeared in most cases. However, chlorarachniophytes and cryptophytes still possess a relict nucleus, so-called the nucleomorph, of the green and red algal endosymbiont, respectively. Nucleomorph genomes are an interesting and suitable model to study the reductive evolution of endosymbiotically derived genomes. To date, nucleomorph genomes have been sequenced in four cryptophyte species and two chlorarachniophyte species, including Bigelowiella natans (373 kb) and Lotharella oceanica (610 kb). In this study, we report complete nucleomorph genome sequences of two chlorarachniophytes, Amorphochlora amoebiformis and Lotharella vacuolata, to gain insight into the reductive evolution of nucleomorph genomes in the chlorarachniophytes. The nucleomorph genomes consist of three chromosomes totaling 374 and 432 kb in size in A. amoebiformis and L. vacuolata, respectively. Comparative analyses among four chlorarachniophyte nucleomorph genomes revealed that these sequences share 171 function-predicted genes (86% of total 198 function-predicted nucleomorph genes), including the same set of genes encoding 17 plastid-associated proteins, and no evidence of a recent nucleomorph-to-nucleus gene transfer was found. This suggests that chlorarachniophyte nucleomorph genomes underwent most of their reductive evolution prior to the radiation of extent members of the group. However, there are slight variations in genome size, GC content, duplicated gene number, and subtelomeric regions among the four nucleomorph genomes, suggesting that the genomes might be undergoing changes that do not affect the core functions in each species.

Ancient origin of the biosynthesis of lignin precursors.

BackgroundLignin plays an important role in plant structural support and water transport, and is considered one of the hallmarks of land plants. The recent discovery of lignin or its precursors in various algae has raised questions on the evolution of its biosynthetic pathway, which could be much more ancient than previously thought. To determine the taxonomic distribution of the lignin biosynthesis genes, we screened all publicly available genomes of algae and their closest non-photosynthetic relatives, as well as representative land plants. We also performed phylogenetic analysis of these genes to decipher the evolution and origin(s) of lignin biosynthesis.ResultsEnzymes involved in making p-coumaryl alcohol, the simplest lignin monomer, are found in a variety of photosynthetic eukaryotes, including diatoms, dinoflagellates, haptophytes, cryptophytes as well as green and red algae. Phylogenetic analysis of these enzymes suggests that they are ancient and spread to some secondarily photosynthetic lineages when they acquired red and/or green algal endosymbionts. In some cases, one or more of these enzymes was likely acquired through lateral gene transfer (LGT) from bacteria.ConclusionsGenes associated with p-coumaryl alcohol biosynthesis are likely to have evolved long before the transition of photosynthetic eukaryotes to land. The original function of this lignin precursor is therefore unlikely to have been related to water transport. We suggest that it participates in the biological defense of some unicellular and multicellular algae.ReviewersThis article was reviewed by Mark Ragan, Uri Gophna, Philippe Deschamps.

What was the real contribution of endosymbionts to the eukaryotic nucleus? Insights from photosynthetic eukaryotes.

Eukaryotic genomes are composed of genes of different evolutionary origins. This is especially true in the case of photosynthetic eukaryotes, which, in addition to typical eukaryotic genes and genes of mitochondrial origin, also contain genes coming from the primary plastids and, in the case of secondary photosynthetic eukaryotes, many genes provided by the nuclei of red or green algal endosymbionts. Phylogenomic analyses have been applied to detect those genes and, in some cases, have led to proposing the existence of cryptic, no longer visible endosymbionts. However, detecting them is a very difficult task because, most often, those genes were acquired a long time ago and their phylogenetic signal has been heavily erased. We revisit here two examples, the putative cryptic endosymbiosis of green algae in diatoms and chromerids and of Chlamydiae in the first photosynthetic eukaryotes. We show that the evidence sustaining them has been largely overestimated, and we insist on the necessity of careful, accurate phylogenetic analyses to obtain reliable results.

Polyploidy of Endosymbiotically Derived Genomes in Complex Algae

Chlorarachniophyte and cryptophyte algae have complex plastids that were acquired by the uptake of a green or red algal endosymbiont via secondary endosymbiosis. The plastid is surrounded by four membranes, and a relict nucleus, called the nucleomorph, remains in the periplastidal compartment that is the remnant cytoplasm of the endosymbiont. Thus, these two algae possess four different genomes in a cell: Nuclear, nucleomorph, plastid, and mitochondrial. Recently, sequencing of the nuclear genomes of the chlorarachniophyte Bigelowiella natans and the cryptophyte Guillardia theta has been completed, and all four genomes have been made available. However, the copy number of each genome has never been investigated. It is important to know the actual DNA content of each genome, especially the highly reduced nucleomorph genome, for studies on genome evolution. In this study, we calculated genomic copy numbers in B. natans and G. theta using a real-time quantitative polymerase chain reaction approach. The nuclear genomes were haploid in both species, whereas the nucleomorph genomes were estimated to be diploid and tetraploid, respectively. Mitochondria and plastids contained a large copy number of genomic DNA in each cell. In the secondary endosymbioses of chlorarachniophytes and cryptophytes, the endosymbiont nuclear genomes were highly reduced in size and in the number of coding genes, whereas the chromosomal copy number was increased, as in bacterial endosymbiont genomes. This suggests that polyploidization is a general characteristic of highly reduced genomes in broad prokaryotic and eukaryotic endosymbionts.

Overexpression of Molecular Chaperone Genes in Nucleomorph Genomes

Chlorarachniophytes and cryptophytes possess complex plastids that were acquired by the ingestion of a green and red algal endosymbiont, respectively. The plastids are surrounded by four membranes, and a relict nucleus, called the nucleomorph, remains in the periplastidal compartment, which corresponds to the remnant cytoplasm of the endosymbiont. Nucleomorphs contain a greatly reduced genome that possesses only several hundred genes with high evolutionary rates. We examined the relative transcription levels of the genes of all proteins encoded by the nucleomorph genomes of two chlorarachniophytes and three cryptophytes using an RNA-seq transcriptomic approach. The genes of two heat shock proteins, Hsp70 and Hsp90, were highly expressed under normal conditions. It has been shown that molecular chaperone overexpression allows an accumulation of genetic mutations in bacteria. Our results suggest that overexpression of heat shock proteins in nucleomorph genomes may play a role in buffering the mutational destabilization of proteins, which might allow the high evolutionary rates of nucleomorph-encoded proteins.

Cell Cycle and Nucleomorph Division in Pyrenomonas helgolandii (Cryptophyta)

The cells of cryptophycean and chlorarachniophycean algae contain a nucleomorph, a vestigial nucleus derived from red and green algal endosymbionts respectively. The origin of the nucleomorph is therefore different from that of cellular organelles such as mitochondria and chloroplasts. In this study, we sought to determine whether cell cycle regulation of the nucleomorph in the cryptophycean alga Pyrenomonas helgolandii is functionally similar to that of the cell nucleus. We performed an ultrastructural analysis of nucleomorph division in cells prepared by rapid freezing fixation - freeze substitution and also carried out BrdU labeling experiments to determine the timing of nucleomorph DNA synthesis in relation to that of the cell nucleus. In cells cultured under 16 hours light: 8 hours dark conditions, BrdU labeling experiments showed that DNA synthesis in the nucleomorph occurred during a limited period from 2 hr to 4 hr after the beginning of the dark period. The S phase in the nucleomorph started just after completion of the nuclear S phase. Thus, DNA synthesis in the nucleomorph occurred at a defined period of the cell cycle. By contrast, our BrdU experiments showed that the nucleoids of mitochondria and chloroplasts could perform DNA synthesis throughout the whole cell cycle.

What do human parasites with a chloroplast anyway?

Apicomplexan parasites are a major threat to human health causing malaria and a variety of AIDS associated opportunistic infections. One of the most promising targets for chemotherapeutic intervention for this group of pathogens is the apicoplast. The apicoplast is a unique parasite organelle that was derived from a red algal endosymbiont. Genome analyses suggest that the apicoplast is engaged in a variety of biosynthetic activities that could be targeted for drug development. Using Toxoplasma gondii as a genetic model organism we are dissecting the apicoplast metabolism to identify the most effective choke points. Taking a broader biological view we are interested to understand which of the endosymbiont's functions is most critical and the reason for the continued presence of a plastid long after the loss of photosynthesis. Our current work is focused on the mechanisms and importance of parasite lipid synthesis in particular that of fatty acids and isoprenoids.

A Systematic Screen to Discover and Analyze Apicoplast Proteins Identifies a Conserved and Essential Protein Import Factor

Parasites of the phylum Apicomplexa cause diseases that impact global health and economy. These unicellular eukaryotes possess a relict plastid, the apicoplast, which is an essential organelle and a validated drug target. However, much of its biology remains poorly understood, in particular its elaborate compartmentalization: four membranes defining four different spaces. Only a small number of organellar proteins have been identified in particular few proteins are known for non-luminal apicoplast compartments. We hypothesized that enlarging the catalogue of apicoplast proteins will contribute toward identifying new organellar functions and expand the realm of targets beyond a limited set of characterized pathways. We developed a bioinformatic screen based on mRNA abundance over the cell cycle and on phyletic distribution. We experimentally assessed 57 genes, and of 30 successful epitope tagged candidates eleven novel apicoplast proteins were identified. Of those, seven appear to target to the lumen of the organelle, and four localize to peripheral compartments. To address their function we then developed a robust system for the construction of conditional mutants via a promoter replacement strategy. We confirm the feasibility of this system by establishing conditional mutants for two selected genes – a luminal and a peripheral apicoplast protein. The latter is particularly intriguing as it encodes a hypothetical protein that is conserved in and unique to Apicomplexan parasites and other related organisms that maintain a red algal endosymbiont. Our studies suggest that this peripheral plastid protein, PPP1, is likely localized to the periplastid compartment. Conditional disruption of PPP1 demonstrated that it is essential for parasite survival. Phenotypic analysis of this mutant is consistent with a role of the PPP1 protein in apicoplast biogenesis, specifically in import of nuclear-encoded proteins into the organelle.

The apicoplast: a red alga in human parasites

Surprisingly, some of the world's most dangerous parasites appear to have had a benign photosynthetic past in the ocean. The phylum Apicomplexa includes the causative agents of malaria and a number of additional human and animal diseases. These diseases threaten the life and health of hundreds of millions each year and pose a tremendous challenge to public health. Recent findings suggest that Apicomplexa share their ancestry with diatoms and kelps, and that a key event in their evolution was the acquisition of a red algal endosymbiont. A remnant of this endosymbiont is still present today, albeit reduced to a small chloroplast-like organelle, the apicoplast. In the present chapter, I introduce the remarkably complex biology of this organelle. The apicoplast is bounded by four membranes, and these membranes trace their ancestry to three different organisms. Intriguingly, this divergent ancestry is still reflected in their molecular makeup and function. We also pursue the raison d'être of the apicoplast. Why did Apicomplexa retain a chloroplast when they abandoned photosynthesis for a life as obligate parasites? The answer to this question appears to lie in the profound metabolic dependence of the parasite on its endosymbiont. This dependence may prove to be a liability to the parasite. As humans lack chloroplasts, the apicoplast has become one of the prime targets for the development of parasite-specific drugs.

Evolution and Functional Diversification of Fructose Bisphosphate Aldolase Genes in Photosynthetic Marine Diatoms

Diatoms and other chlorophyll-c containing, or chromalveolate, algae are among the most productive and diverse phytoplankton in the ocean. Evolutionarily, chlorophyll-c algae are linked through common, although not necessarily monophyletic, acquisition of plastid endosymbionts of red as well as most likely green algal origin. There is also strong evidence for a relatively high level of lineage-specific bacterial gene acquisition within chromalveolates. Therefore, analyses of gene content and derivation in chromalveolate taxa have indicated particularly diverse origins of their overall gene repertoire. As a single group of functionally related enzymes spanning two distinct gene families, fructose 1,6-bisphosphate aldolases (FBAs) illustrate the influence on core biochemical pathways of specific evolutionary associations among diatoms and other chromalveolates with various plastid-bearing and bacterial endosymbionts. Protein localization and activity, gene expression, and phylogenetic analyses indicate that the pennate diatom Phaeodactylum tricornutum contains five FBA genes with very little overall functional overlap. Three P. tricornutum FBAs, one class I and two class II, are plastid localized, and each appears to have a distinct evolutionary origin as well as function. Class I plastid FBA appears to have been acquired by chromalveolates from a red algal endosymbiont, whereas one copy of class II plastid FBA is likely to have originated from an ancient green algal endosymbiont. The other copy appears to be the result of a chromalveolate-specific gene duplication. Plastid FBA I and chromalveolate-specific class II plastid FBA are localized in the pyrenoid region of the chloroplast where they are associated with β-carbonic anhydrase, which is known to play a significant role in regulation of the diatom carbon concentrating mechanism. The two pyrenoid-associated FBAs are distinguished by contrasting gene expression profiles under nutrient limiting compared with optimal CO2 fixation conditions, suggestive of a distinct specialized function for each. Cytosolically localized FBAs in P. tricornutum likely play a role in glycolysis and cytoskeleton function and seem to have originated from the stramenopile host cell and from diatom-specific bacterial gene transfer, respectively.

Complete Nucleomorph Genome Sequence of the Nonphotosynthetic Alga Cryptomonas paramecium Reveals a Core Nucleomorph Gene Set

Nucleomorphs are the remnant nuclei of algal endosymbionts that were engulfed by nonphotosynthetic host eukaryotes. These peculiar organelles are found in cryptomonad and chlorarachniophyte algae, where they evolved from red and green algal endosymbionts, respectively. Despite their independent origins, cryptomonad and chlorarachniophyte nucleomorph genomes are similar in size and structure: they are both <1 million base pairs in size (the smallest nuclear genomes known), comprised three chromosomes, and possess subtelomeric ribosomal DNA operons. Here, we report the complete sequence of one of the smallest cryptomonad nucleomorph genomes known, that of the secondarily nonphotosynthetic cryptomonad Cryptomonas paramecium. The genome is 486 kbp in size and contains 518 predicted genes, 466 of which are protein coding. Although C. paramecium lacks photosynthetic ability, its nucleomorph genome still encodes 18 plastid-associated proteins. More than 90% of the “conserved” protein genes in C. paramecium (i.e., those with clear homologs in other eukaryotes) are also present in the nucleomorph genomes of the cryptomonads Guillardia theta and Hemiselmis andersenii. In contrast, 143 of 466 predicted C. paramecium proteins (30.7%) showed no obvious similarity to proteins encoded in any other genome, including G. theta and H. andersenii. Significantly, however, many of these “nucleomorph ORFans” are conserved in position and size between the three genomes, suggesting that they are in fact homologous to one another. Finally, our analyses reveal an unexpected degree of overlap in the genes present in the independently evolved chlorarachniophyte and cryptomonad nucleomorph genomes: ∼80% of a set of 120 conserved nucleomorph genes in the chlorarachniophyte Bigelowiella natans were also present in all three cryptomonad nucleomorph genomes. This result suggests that similar reductive processes have taken place in unrelated lineages of nucleomorph-containing algae.

The cell wall polysaccharide metabolism of the brown alga Ectocarpus siliculosus. Insights into the evolution of extracellular matrix polysaccharides in Eukaryotes.

• Brown algal cell walls share some components with plants (cellulose) and animals (sulfated fucans), but they also contain some unique polysaccharides (alginates). Analysis of the Ectocarpus genome provides a unique opportunity to decipher the molecular bases of these crucial metabolisms. • An extensive bioinformatic census of the enzymes potentially involved in the biogenesis and remodeling of cellulose, alginate and fucans was performed, and completed by phylogenetic analyses of key enzymes. • The routes for the biosynthesis of cellulose, alginates and sulfated fucans were reconstructed. Surprisingly, known families of cellulases, expansins and alginate lyases are absent in Ectocarpus, suggesting the existence of novel mechanisms and/or proteins for cell wall expansion in brown algae. • Altogether, our data depict a complex evolutionary history for the main components of brown algal cell walls. Cellulose synthesis was inherited from the ancestral red algal endosymbiont, whereas the terminal steps for alginate biosynthesis were acquired by horizontal gene transfer from an Actinobacterium. This horizontal gene transfer event also contributed genes for hemicellulose biosynthesis. By contrast, the biosynthetic route for sulfated fucans is an ancestral pathway, conserved with animals. These findings shine a new light on the origin and evolution of cell wall polysaccharides in other Eukaryotes.

Central and storage carbon metabolism of the brown alga Ectocarpus siliculosus: insights into the origin and evolution of storage carbohydrates in Eukaryotes

• Brown algae exhibit a unique carbon (C) storage metabolism. The photoassimilate D-fructose 6-phosphate is not used to produce sucrose but is converted into D-mannitol. These seaweeds also store C as β-1,3-glucan (laminarin), thus markedly departing from most living organisms, which use α-1,4-glucans (glycogen or starch). • Using a combination of bioinformatic and phylogenetic approaches, we identified the candidate genes for the enzymes involved in C storage in the genome of the brown alga Ectocarpus siliculosus and traced their evolutionary origins. • Ectocarpus possesses a complete set of enzymes for synthesis of mannitol, laminarin and trehalose. By contrast, the pathways for sucrose, starch and glycogen are completely absent. • The synthesis of β-1,3-glucans appears to be a very ancient eukaryotic pathway. Brown algae inherited the trehalose pathway from the red algal progenitor of phaeoplasts, while the mannitol pathway was acquired by lateral gene transfer from Actinobacteria. The starch metabolism of the red algal endosymbiont was entirely lost in the ancestor of Stramenopiles. In light of these novel findings we question the validity of the 'Chromalveolate hypothesis'.

Actin Gene Family Dynamics in Cryptomonads and Red Algae

Here we present evidence for a complex evolutionary history of actin genes in red algae and cryptomonads, a group that acquired photosynthesis secondarily through the engulfment of a red algal endosymbiont. Four actin genes were found in the nuclear genome of the cryptomonad, Guillardia theta, and in the genome of the red alga, Galdieria sulphuraria, a member of the Cyanidiophytina. Phylogenetic analyses reveal that the both organisms possess two distinct sequence types, designated "type-1" and "type-2." A weak but consistent phylogenetic affinity between the cryptomonad type-2 sequences and the type-2 sequences of G. sulphuraria and red algae belonging to the Rhodophytina was observed. This is consistent with the possibility that the cryptomonad type-2 sequences are derived from the red algal endosymbiont that gave rise to the cryptomonad nucleomorph and plastid. Red algae as a whole possess two very different actin sequence types, with G. sulphuraria being the only organism thus far known to possess both. The common ancestor of Rhodophytina and Cyanidiophytina may have had two actin genes, with differential loss explaining the distribution of these genes in modern-day groups. Our study provides new insight into the evolution and divergence of actin genes in cryptomonads and red algae, and in doing so underscores the challenges associated with heterogeneity in actin sequence evolution and ortholog/paralog detection.