A molecular timeline for the origin of photosynthetic eukaryotes

The appearance of photosynthetic eukaryotes (algae and plants) dramatically altered the Earth's ecosystem, making possible all vertebrate life on land, including humans. Dating algal origin is, however, frustrated by a meager fossil record. We generated a plastid multi-gene phylogeny with Bayesian inference and then used maximum likelihood molecular clock methods to estimate algal divergence times. The plastid tree was used as a surrogate for algal host evolution because of recent phylogenetic evidence supporting the vertical ancestry of the plastid in the red, green, and glaucophyte algae. Nodes in the plastid tree were constrained with six reliable fossil dates and a maximum age of 3,500 MYA based on the earliest known eubacterial fossil. Our analyses support an ancient (late Paleoproterozoic) origin of photosynthetic eukaryotes with the primary endosymbiosis that gave rise to the first alga having occurred after the split of the Plantae (i.e., red, green, and glaucophyte algae plus land plants) from the opisthokonts sometime before 1,558 MYA. The split of the red and green algae is calculated to have occurred about 1,500 MYA, and the putative single red algal secondary endosymbiosis that gave rise to the plastid in the cryptophyte, haptophyte, and stramenopile algae (chromists) occurred about 1,300 MYA. These dates, which are consistent with fossil evidence for putative marine algae (i.e., acritarchs) from the early Mesoproterozoic (1,500 MYA) and with a major eukaryotic diversification in the very late Mesoproterozoic and Neoproterozoic, provide a molecular timeline for understanding algal evolution.     

Visualization of microbodies in Chlamydomonas reinhardtii

In Chlorophycean algal cells, these organelles are generally called microbodies because they lack the enzymes found in the peroxisomes of higher plants. Microbodies in some algae contain fewer enzymes than the peroxisomes of higher plants, and some unicellular green algae in Chlorophyceae such as Chlamydomonas reinhardtii do not possess catalase, an enzyme commonly found in peroxisomes. Thus, whether microbodies in Chlorophycean algae are similar to the peroxisomes of higher plants, and whether they use a similar transport mechanism for the peroxisomal targeting signal (PTS), remain unclear. To determine whether the PTS is present in the microbodies of Chlorophycean algae, and to visualize the microbodies in Chlamydomonas cells, we examined the sub-cellular localization of green fluorescent proteins (GFP) fused to several PTS-like sequences. We detected GFP compartments that were spherical with a diameter of 0.3-1.0?μm in transgenic Chlamydomonas. Comparative analysis of the character of GFP-compartments observed by fluorescence microscopy and that of microbodies by electron microscopy indicated that the compartments were one and the same. The result also showed that the microbodies in Chlorophycean cells have a similar transport mechanism to that of peroxisomes of higher plants.     

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).     

Chlororespiration in Green Algae Isolated From Desert Crusts

Photosynthetic organisms enduring extreme temperatures, low water availability, or high light require photoprotective mechanisms to prevent sustained damage to photosynthetic machinery. Green microalgae living in desert crust communities of the south‐western US experience all these environmental stresses, yet photophysiological studies of green algae in the literature have focused on only a handful of common aquatic and marine species. We are examining the variation in green algal photoprotective mechanisms that is the result of natural selection acting independently in multiple lineages of highly diverse desert green algae (Chlorophyta) within the classes Chlorophyceae and Trebouxiophyceae. We have found that unusually extensive dark reduction of the plastoquinone pool is a prominent photophysiological feature among these desert algae; this reduction may be linked with enhanced chlororespiration. Recently, chlororespiration in higher plants has been linked through mutant analysis to control of the carotenoid synthetic pathway, heat stress, and starch metabolism among other pathways, though the function of chlororespiration remains controversial. Given that green algae and higher plants are monophyletic, analysis of potential chlororespiration in desert green algae may help decipher the evolution of the chlororespiratory process as well as its potential role in photoprotection in desert habitats.     

Characterization of the diversification of phospholipid:diacylglycerol acyltransferases in the green lineage

Triacylglycerols have important physiological roles in photosynthetic organisms, and are widely used as food, feed and industrial materials in our daily life. Phospholipid:diacylglycerol acyltransferase (PDAT) is the pivotal enzyme catalyzing the acyl‐CoA‐independent biosynthesis of triacylglycerols, which is unique in plants, algae and fungi, but not in animals, and has essential functions in plant and algal growth, development and stress responses. Currently, this enzyme has yet to be examined in an evolutionary context at the level of the green lineage. Some fundamental questions remain unanswered, such as how PDATs evolved in photosynthetic organisms and whether the evolution of terrestrial plant PDATs from a lineage of charophyte green algae diverges in enzyme function. As such, we used molecular evolutionary analysis and biochemical assays to address these questions. Our results indicated that PDAT underwent divergent evolution in the green lineage: PDATs exist in a wide range of plants and algae, but not in cyanobacteria. Although PDATs exhibit the conservation of several features, phylogenetic and selection‐pressure analyses revealed that overall they evolved to be highly divergent, driven by different selection constraints. Positive selection, as one major driving force, may have resulted in enzymes with a higher functional importance in land plants than green algae. Further structural and mutagenesis analyses demonstrated that some amino acid sites under positive selection are critically important to PDAT structure and function, and may be central in lecithin:cholesterol acyltransferase family enzymes in general.     

Chloroplast gene arrangement variation within a closely related group of green algae (Trebouxiophyceae, Chlorophyta)

The 22 published chloroplast genomes of green algae, representing sparse taxonomic sampling of diverse lineages that span over one billion years of evolution, each possess a unique gene arrangement. In contrast, many of the >190 published embryophyte (land plant) chloroplast genomes have relatively conserved architectures. To determine the phylogenetic depth at which chloroplast gene rearrangements occur in green algae, a 1.5-4 kb segment of the chloroplast genome was compared across nine species in three closely related genera of Trebouxiophyceae (Chlorophyta). In total, four distinct gene arrangements were obtained for the three genera Elliptochloris, Hemichloris, and Coccomyxa. In Elliptochloris, three distinct chloroplast gene arrangements were detected, one of which is shared with members of its sister genus Hemichloris. Both species of Coccomyxa examined share the fourth arrangement of this genome region, one characterized by very long spacers. Next, the order of genes found in this segment of the chloroplast genome was compared across green algae and land plants. As taxonomic ranks are not equivalent among different groups of organisms, the maximum molecular divergence among taxa sharing a common gene arrangement in this genome segment was compared. Well-supported clades possessing a single gene order had similar phylogenetic depth in green algae and embryophytes. When the dominant gene order of this chloroplast segment in embryophytes was assumed to be ancestral for land plants, the maximum molecular divergence was found to be over two times greater in embryophytes than in trebouxiophyte green algae. This study greatly expands information about chloroplast genome variation in green algae, is the first to demonstrate such variation among congeneric green algae, and further illustrates the fluidity of green algal chloroplast genome architecture in comparison to that of many embryophytes.     

Nucleus-encoded, plastid-targeted acetolactate synthase genes in two closely related chlorophytes, Chlamydomonas reinhardtii and Volvox carteri: phylogenetic origins and recent insertion of introns

Acetolactate synthase (ALS) catalyzes the first committed step in the synthesis of branched-chain amino acids. In green plants and fungi, ALS is encoded by a nuclear gene whose product is targeted to plastids (in plants) or to mitochondria (in fungi). In red algae, the gene is plastid-encoded. We have determined the complete sequence of nucleus-encoded ALS genes from the green algae Chlamydomonas reinhardtii and Volvox carteri. Phylogenetic analyses of the ALS gene family indicate that the ALS genes of green algae and plants are closely related, sharing a recent common ancestor. Furthermore, although these genes are clearly of eubacterial origin, a relationship to the ALS genes of red algae and cyanobacteria (endosymbiotic precursors of plastids) is only weakly indicated. The algal ALS genes are distinguished from their homologs in higher plants by the fact that they are interrupted by numerous spliceosomal introns; plant ALS genes completely lack introns. The restricted phylogenetic distribution of these introns suggests that they were inserted recently, after the divergence of these green algae from plants. Two introns in the Volvox ALS gene, not found in the Chlamydomonas gene, are positioned precisely at sites which resemble “proto-splice” sequences in the Chlamydomonas gene.     

Resolution of 16 to 20 chlorophyll-protein complexes using a low ionic strength native green gel system

Conventional native "green gel" systems resolve at most 10 chlorophyll-protein complexes from thylakoid membranes of higher plants and green algae. Such analyses suggest a simplicity of the thylakoid membrane that is not supported by a growing body of evidence on the heterogeneity of photosystems I and II (PSI and PSII) and their associated antennae (LHCI and LHCII). We report here the development and characterization of a low ionic strength native "green gel" system that resolves from 16 to 20, mostly large chlorophyll-protein complexes from a variety of higher plant and green algal species with very little release of free pigment. In Chlamydomonas, this system resolves multiple PSI-LHCI complexes, multiple PSII-LHCII complexes, four oligomeric LHCII complexes, as well as several low electrophoretic mobility reaction center complexes, and a number of small complexes. We have obtained similar resolution with a large number of higher plant and green algal species. We also demonstrate how this system can be used as a sort of "fingerprinting" technique to distinguish thylakoids of different species, and for the analysis of photosynthetic mutants, using the chlorophyll b-less chlorina f2 mutant of barley as an example.     

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.     

Functional characterization of the plastidic phosphate translocator gene family from the thermo-acidophilic red alga Galdieria sulphuraria reveals specific adaptations of primary carbon partitioning in green plants and red algae

In chloroplasts of green plants and algae, CO₂ is assimilated into triose-phosphates (TPs); a large part of these TPs is exported to the cytosol by a TP/phosphate translocator (TPT), whereas some is stored in the plastid as starch. Plastidial phosphate translocators have evolved from transport proteins of the host endomembrane system shortly after the origin of chloroplasts by endosymbiosis. The red microalga Galdieria sulphuraria shares three conserved putative orthologous transport proteins with the distantly related seed plants and green algae. However, red algae, in contrast to green plants, store starch in their cytosol, not inside plastids. Hence, due to the lack of a plastidic starch pool, a larger share of recently assimilated CO₂ needs to be exported to the cytosol. We thus hypothesized that red algal transporters have distinct substrate specificity in comparison to their green orthologs. This hypothesis was tested by expression of the red algal genes in yeast (Saccharomyces cerevisiae) and assessment of their substrate specificities and kinetic constants. Indeed, two of the three red algal phosphate translocator candidate orthologs have clearly distinct substrate specificities when compared to their green homologs. GsTPT (for G. sulphuraria TPT) displays very narrow substrate specificity and high affinity; in contrast to green plant TPTs, 3-phosphoglyceric acid is poorly transported and thus not able to serve as a TP/3-phosphoglyceric acid redox shuttle in vivo. Apparently, the specific features of red algal primary carbon metabolism promoted the evolution of a highly efficient export system with high affinities for its substrates. The low-affinity TPT of plants maintains TP levels sufficient for starch biosynthesis inside of chloroplasts, whereas the red algal TPT is optimized for efficient export of TP from the chloroplast.     

Succession of soil algae on dumps from strip coal-mining in the Most region (Czechoslovakia)

In the years 1985–1986, the primary succession of algae on dumps from brown coal mining was studied in the Most region (North Bohemia, Czechoslovakia). The colonization of sterile clayic substrates by algae (several coccal species of chlorophytes and heteroconts) and mosses (protonemata) starts before the first ecesis of higher plants (in deposits about 3 months old). Diatoms and, later, cyanophytes accompany the algal community after 1 year. Filamentous types of heteroconts and green algae occur first on 7 year old dumps. Green algae represent the commonest group throughout the succession. The number of species continuously increases with the age of the dumps and finally reaches about 40 species from the 18th to 30th year. The species composition is similar to that in grassland biotopes in Czechoslovakia.     

DISCONTINUOUS MITOCHONDRIAL AND CHLOROPLAST LARGE SUBUNIT RIBOSOMAL RNAs AMONG GREEN ALGAE: PHYLOGENETIC IMPLICATIONS1

To provide insights into the occurrence, evolution, and phylogenetic distribution of discontinuous mitochondrial and chloroplast large subunit ribosomal RNAs (LSU rRNAs) among green algae, we surveyed 12 taxa representing three classes of green algae: the Chlorophyceae, Pleurastrophyceae, and Micromonadophyceae (sensu Mattox and Stewart 1984). We present evidence that discontinuous mitochondrial and chloroplast LSU rRNAs are quite widespread among green algae. Mitochondrial LSU rRNAs appear discontinuous in zoosporic chlorophycean lineages displaying a clockwise or directly opposed configuration in their flagellar apparatus, as well as in chlorococcalean autosporic taxa phylogenetically related to them, but are continuous among zoosporic green algal lineages with a counterclockwise flagellar apparatus configuration, as well as among chlorococcalean autosporic taxa phylogenetically related to them. Chloroplast LSU rRNAs appear discontinuous in all of the lineages investigated. Discontinuous mitochondrial LSU rRNA represents a molecular trait that might have originated at or near the base of Chlorophyceae, whereas discontinuous chloroplast LSU rRNA might have developed very early in the evolutionary history of the green algal group itself. We suggest, therefore, that the presence of discontinuous mitochondrial but not chloroplast LSU rRNA can be used as an additional character in assessing phylogenetic affiliations among green algae.     

Tracing the evolution of the light-harvesting antennae in chlorophyll a/b-containing organisms

The light-harvesting complexes (LHCs) of land plants and green algae have essential roles in light capture and photoprotection. Though the functional diversity of the individual LHC proteins are well described in many land plants, the extent of this family in the majority of green algal groups is unknown. To examine the evolution of the chlorophyll a/b antennae system and to infer its ancestral state, we initiated several expressed sequence tag projects from a taxonomically broad range of chlorophyll a/b-containing protists. This included representatives from the Ulvophyceae (Acetabularia acetabulum), the Mesostigmatophyceae (Mesostigma viride), and the Prasinophyceae (Micromonas sp.), as well as one representative from each of the Euglenozoa (Euglena gracilis) and Chlorarachniophyta (Bigelowiella natans), whose plastids evolved secondarily from a green alga. It is clear that the core antenna system was well developed prior to green algal diversification and likely consisted of the CP29 (Lhcb4) and CP26 (Lhcb5) proteins associated with photosystem II plus a photosystem I antenna composed of proteins encoded by at least Lhca3 and two green algal-specific proteins encoded by the Lhca2 and 9 genes. In organisms containing secondary plastids, we found no evidence for orthologs to the plant/algal antennae with the exception of CP29. We also identified PsbS homologs in the Ulvophyceae and the Prasinophyceae, indicating that this distinctive protein appeared prior to green algal diversification. This analysis provides a snapshot of the antenna systems in diverse green algae, and allows us to infer the changing complexity of the antenna system during green algal evolution.     

Some properties and amino acid sequence of plastocyanin from a green alga, Ulva arasakii

Plastocyanin was purified from a multicellular, marine green alga, Ulva arasakii, by conventional methods to homogeneity. The oxidized plastocyanin showed absorption maxima at 252, 276.8, 460, 595.3, and 775 nm, and shoulders at 259, 265, 269, and 282.5 nm; the ratio A276.8/A595.3 was 1.5. The midpoint redox potential was determined to be 0.356 V at pH 7.0 with a ferri- and ferrocyanide system. The molecular weight was estimated to be 10,200 and 11,000 by SDS-PAGE and by gel filtration, respectively. U. arasakii also has a small amount of cytochrome c6, like Enteromorpha prolifera. The amino acid sequence of U. arasakii plastocyanin was determined by Edman degradation and by carboxypeptidase digestion of the plastocyanin, six tryptic peptides, and five staphylococcal protease peptides. The plastocyanin contained 98 amino acid residues, giving a molecular weight of 10,236 including one copper atom. The complete sequence is as follows: AQIVKLGGDDGALAFVPSKISVAAGEAIEFVNNAGFPHNIVFDEDAVPAGVDADAISYDDYLNSKGETV VRKLSTPGVY G VYCEPHAGAGMKMTITVQ. The sequence of U. arasakii plastocyanin is closet to that of the E. prolifera protein (85% homology). A phylogenetic tree of five algal and two higher plant plastocyanins was constructed by comparing the amino acid differences. The branching order is considered to be as follows: a blue-green alga, unicellular green algae, multicellular green algae, and higher plants.     

Nucleus-encoded, plastid-targeted acetolactate synthase genes in two closely related chlorophytes, Chlamydomonas reinhardtii and Volvox carteri : phylogenetic origins and recent insertion of introns

Acetolactate synthase (ALS) catalyzes the first committed step in the synthesis of branched-chain amino acids. In green plants and fungi, ALS is encoded by a nuclear gene whose product is targeted to plastids (in plants) or to mitochondria (in fungi). In red algae, the gene is plastid-encoded. We have determined the complete sequence of nucleus-encoded ALS genes from the green algae Chlamydomonas reinhardtii and Volvox carteri. Phylogenetic analyses of the ALS gene family indicate that the ALS genes of green algae and plants are closely related, sharing a recent common ancestor. Furthermore, although these genes are clearly of eubacterial origin, a relationship to the ALS genes of red algae and cyanobacteria (endosymbiotic precursors of plastids) is only weakly indicated. The algal ALS genes are distinguished from their homologs in higher plants by the fact that they are interrupted by numerous spliceosomal introns; plant ALS genes completely lack introns. The restricted phylogenetic distribution of these introns suggests that they were inserted recently, after the divergence of these green algae from plants. Two introns in the Volvox ALS gene, not found in the Chlamydomonas gene, are positioned precisely at sites which resemble “proto-splice” sequences in the Chlamydomonas gene. Received: 27 November 1998 / Accepted: 21 April 1999     

Plant Proteus: brown algal morphological plasticity and underlying developmental mechanisms

Brown algae are multicellular photosynthetic marine organisms, ubiquitous on rocky intertidal shores at cold and temperate latitudes. Nevertheless, little is known about many aspects of their biology, particularly their development. Given their phylogenetic distance (1.6 billion years) from other plant organisms (land plants, and green and red algae), brown algae harbor a high, as-yet undiscovered diversity of biological mechanisms governing their development. They also show great morphological plasticity, responding to specific environmental constraints, such as sea currents, reduced light availability, grazer attacks, desiccation and UV exposure. Here, we show that brown algal morphogenesis is rather simple and flexible, and review recent genomic data on the cellular and molecular mechanisms known to date that can possibly account for this developmental strategy.     

Phylogenetic analyses of nuclear, mitochondrial, and plastid multigene data sets support the placement of Mesostigma in the Streptophyta

All extant green plants belong to 1 of 2 major lineages, commonly known as the Chlorophyta (most of the green algae) and the Streptophyta (land plants and their closest green algal relatives). The scaly green flagellate Mesostigma viride has an important place in the debate on the origin of green plants. However, there have been conflicting results from molecular systematics as to whether Mesostigma diverges before the Chlorophyta/Streptophyta split or is an early diverging flagellate member of the Streptophyta. Previous studies employed either a limited taxon sampling (plastid and mitochondrial genomes) or a small number of phylogenetically informative sites (single nuclear genes). Here, we use large data sets from the nuclear (125 proteins; 29,319 positions), mitochondrial (33 proteins; 6,622 positions), and plastid (50 proteins; 10,137 positions) genomes with an expanded taxon sampling (21, 13, and 28 species, respectively) to reevaluate the phylogenetic position of Mesostigma. Our study supports the placement of Mesostigma in the Streptophyta (as an early diverging lineage) and provides evidence that systematic biases have played a role in generating some of the previous conflicting results. Importantly, we demonstrate that using an increased taxon sampling as well as more realistic models of evolution allows increasing congruence among the nuclear, mitochondrial, and plastid data sets.     

Phyllosphere eukaryotic microalgal communities in rainforests: Drivers and diversity

Phyllosphere algae are common in tropical rainforests, forming visible biofilms or spots on plant leaf surfaces. However, knowledge of phyllosphere algal diversity and the environmental factors that drive that diversity is limited. The aim of this study is to identify the environmental factors that drive phyllosphere algal community composition and diversity in rainforests. For this purpose, we used single molecule real-time sequencing of full-length 18S rDNA to characterize the composition of phyllosphere microalgal communities growing on four host tree species (Ficus tikoua, Caryota mitis, Arenga pinnata, and Musa acuminata) common to three types of forest over four months at the Xishuangbanna Tropical Botanical Garden, Yunnan Province, China. Environmental 18S rDNA sequences revealed that the green algae orders Watanabeales and Trentepohliales were dominant in almost all algal communities and that phyllosphere algal species richness and biomass were lower in planted forest than in primeval and reserve rainforest. In addition, algal community composition differed significantly between planted forest and primeval rainforest. We also found that algal communities were affected by soluble reactive phosphorous, total nitrogen, and ammonium contents. Our findings indicate that algal community structure is significantly related to forest type and host tree species. Furthermore, this study is the first to identify environmental factors that affect phyllosphere algal communities, significantly contributing to future taxonomic research, especially for the green algae orders Watanabeales and Trentepohliales. This research also serves as an important reference for molecular diversity analysis of algae in other specific habitats, such as epiphytic algae and soil algae.     

Gene replacement of fructose-1,6-bisphosphate aldolase supports the hypothesis of a single photosynthetic ancestor of chromalveolates

Plastids (photosynthetic organelles of plants and algae) are known to have spread between eukaryotic lineages by secondary endosymbiosis, that is, by the uptake of a eukaryotic alga by another eukaryote. But the number of times this has taken place is controversial. This is particularly so in the case of eukaryotes with plastids derived from red algae, which are numerous and diverse. Despite their diversity, it has been suggested that all these eukaryotes share a recent common ancestor and that their plastids originated in a single endosymbiosis, the so-called "chromalveolate hypothesis." Here we describe a novel molecular character that supports the chromalveolate hypothesis. Fructose-1,6-bisphosphate aldolase (FBA) is a glycolytic and Calvin cycle enzyme that exists as two nonhomologous types, class I and class II. Red algal plastid-targeted FBA is a class I enzyme related to homologues from plants and green algae, and it would be predicted that the plastid-targeted FBA from algae with red algal secondary endosymbionts should be related to this class I enzyme. However, we show that plastid-targeted FBA of heterokonts, cryptomonads, haptophytes, and dinoflagellates (all photosynthetic chromalveolates) are class II plastid-targeted enzymes, completely unlike those of red algal plastids. The chromalveolate enzymes form a strongly supported group in FBA phylogeny, and their common possession of this unexpected plastid characteristic provides new evidence for their close relationship and a common origin for their plastids.