EEF2 analysis challenges the monophyly of Archaeplastida and Chromalveolata

Background

Classification of eukaryotes provides a fundamental phylogenetic framework for ecological, medical, and industrial research. In recent years eukaryotes have been classified into six major supergroups: Amoebozoa, Archaeplastida, Chromalveolata, Excavata, Opisthokonta, and Rhizaria. According to this supergroup classification, Archaeplastida and Chromalveolata each arose from a single plastid-generating endosymbiotic event involving a cyanobacterium (Archaeplastida) or red alga (Chromalveolata). Although the plastids within members of the Archaeplastida and Chromalveolata share some features, no nucleocytoplasmic synapomorphies supporting these supergroups are currently known.

Methodology/Principal Findings

This study was designed to test the validity of the Archaeplastida and Chromalveolata through the analysis of nucleus-encoded eukaryotic translation elongation factor 2 (EEF2) and cytosolic heat-shock protein of 70 kDa (HSP70) sequences generated from the glaucophyte Cyanophora paradoxa, the cryptophytes Goniomonas truncata and Guillardia theta, the katablepharid Leucocryptos marina, the rhizarian Thaumatomonas sp. and the green alga Mesostigma viride. The HSP70 phylogeny was largely unresolved except for certain well-established groups. In contrast, EEF2 phylogeny recovered many well-established eukaryotic groups and, most interestingly, revealed a well-supported clade composed of cryptophytes, katablepharids, haptophytes, rhodophytes, and Viridiplantae (green algae and land plants). This clade is further supported by the presence of a two amino acid signature within EEF2, which appears to have arisen from amino acid replacement before the common origin of these eukaryotic groups.

Conclusions/Significance

Our EEF2 analysis strongly refutes the monophyly of the Archaeplastida and the Chromalveolata, adding to a growing body of evidence that limits the utility of these supergroups. In view of EEF2 phylogeny and other morphological evidence, we discuss the possibility of an alternative eukaryotic supergroup.     

When Less is More: Red Algae as Models for Studying Gene Loss and Genome Evolution in Eukaryotes

Genome evolution is usually viewed through the lens of growth in size and complexity over time, exemplified by plants and animals. In contrast, genome reduction is associated with a narrowing of ecological potential, such as in parasites and endosymbionts. But, can nuclear genome reduction also occur in, and potentially underpin a major radiation of free-living eukaryotes? An intriguing example of this phenomenon is provided by the red algae (Rhodophyta) that have lost many conserved pathways such as for flagellar motility, macroautophagy regulation, and phytochrome based light sensing. This anciently diverged, species-rich, and ecologically important algal lineage has undergone at least two rounds of large-scale genome reduction during its >1 billion-year evolutionary history. Here, using recent analyses of genome data, we review knowledge about the evolutionary trajectory of red algal nuclear and organelle gene inventories and plastid encoded autocatalytic introns. We compare and contrast Rhodophyta genome evolution to Viridiplantae (green algae and plants), both of which are members of the Archaeplastida, and highlight their divergent paths. We also discuss evidence for the speculative hypothesis that reduction in red algal plastid genome size through endosymbiotic gene transfer is counteracted by ongoing selection for compact nuclear genomes in red algae. Finally, we describe how the spliceosomal intron splicing apparatus provides an example of “evolution in action” in Rhodophyta and how the overall constraints on genome size in this lineage has left significant imprints on this key step in RNA maturation. Our review reveals the red algae to be an exciting, yet under-studied model that offers numerous novel insights as well as many unanswered questions that remain to be explored using modern genomic, genetic, and biochemical methods. The fact that a speciose lineage of free-living eukaryotes has spread throughout many aquatic habitats after having lost about 25% of its primordial gene inventory challenges us to elucidate the mechanisms underlying this remarkable feat.     

Classification,Naming and Evolutionary History of Glycosyltransferases from Sequenced Green and Red Algal Genomes

The Archaeplastida consists of three lineages, Rhodophyta, Virideplantae and Glaucophyta. The extracellular matrix of most members of the Rhodophyta and Viridiplantae consists of carbohydrate-based or a highly glycosylated protein-based cell wall while the Glaucophyte covering is poorly resolved. In order to elucidate possible evolutionary links between the three advanced lineages in Archaeplastida, a genomic analysis was initiated. Fully sequenced genomes from the Rhodophyta and Virideplantae and the well-defined CAZy database on glycosyltransferases were included in the analysis. The number of glycosyltransferases found in the Rhodophyta and Chlorophyta are generally much lower then in land plants (Embryophyta). Three specific features exhibited by land plants increase the number of glycosyltransferases in their genomes: (1) cell wall biosynthesis, the more complex land plant cell walls require a larger number of glycosyltransferases for biosynthesis, (2) a richer set of protein glycosylation, and (3) glycosylation of secondary metabolites, demonstrated by a large proportion of family GT1 being involved in secondary metabolite biosynthesis. In a comparative analysis of polysaccharide biosynthesis amongst the taxa of this study, clear distinctions or similarities were observed in (1) N-linked protein glycosylation, i.e., Chlorophyta has different mannosylation and glucosylation patterns, (2) GPI anchor biosynthesis, which is apparently missing in the Rhodophyta and truncated in the Chlorophyta, (3) cell wall biosynthesis, where the land plants have unique cell wall related polymers not found in green and red algae, and (4) O-linked glycosylation where comprehensive orthology was observed in glycosylation between the Chlorophyta and land plants but not between the target proteins.     

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.     

Origin and evolution of green plants in the light of key evolutionary events

Green plants (Viridiplantae) are ancient photosynthetic organisms that thrive both in aquatic and terrestrial ecosystems, greatly contributing to the changes in global climates and ecosystems. Significant progress has been made toward understanding the origin and evolution of green plants, and plant biologists have arrived at the consensus that green plants first originated in marine deep-water environments and later colonized fresh water and dry land. The origin of green plants, colonization of land by plants and rapid radiation of angiosperms are three key evolutionary events during the long history of green plants. However, the comprehensive understanding of evolutionary features and molecular innovations that enabled green plants to adapt to complex and changeable environments are still limited. Here, we review current knowledge of phylogenetic relationships and divergence times of green plants, and discuss key morphological innovations and distinct drivers in the evolution of green plants. Ultimately, we highlight fundamental questions to advance our understanding of the phenotypic novelty, environmental adaptation, and domestication of green plants.     

Reverse Evolution: Driving Forces Behind the Loss of Acquired Photosynthetic Traits

Background

The loss of photosynthesis has occurred often in eukaryotic evolution, even more than its acquisition, which occurred at least nine times independently and which generated the evolution of the supergroups Archaeplastida, Rhizaria, Chromalveolata and Excavata. This secondary loss of autotrophic capability is essential to explain the evolution of eukaryotes and the high diversity of protists, which has been severely underestimated until recently. However, the ecological and evolutionary scenarios behind this evolutionary “step back” are still largely unknown.

Methodology/Principal Findings

Using a dynamic model of heterotrophic and mixotrophic flagellates and two types of prey, large bacteria and ultramicrobacteria, we examine the influence of DOC concentration, mixotroph''s photosynthetic growth rate, and external limitations of photosynthesis on the coexistence of both types of flagellates. Our key premises are: large bacteria grow faster than small ones at high DOC concentrations, and vice versa; and heterotrophic flagellates are more efficient than the mixotrophs grazing small bacteria (both empirically supported). We show that differential efficiency in bacteria grazing, which strongly depends on cell size, is a key factor to explain the loss of photosynthesis in mixotrophs (which combine photosynthesis and bacterivory) leading to purely heterotrophic lineages. Further, we show in what conditions an heterotroph mutant can coexist, or even out-compete, its mixotrophic ancestor, suggesting that bacterivory and cell size reduction may have been major triggers for the diversification of eukaryotes.

Conclusions/Significance

Our results suggest that, provided the mixotroph''s photosynthetic advantage is not too large, the (small) heterotroph will also dominate in nutrient-poor environments and will readily invade a community of mixotrophs and bacteria, due to its higher efficiency exploiting the ultramicrobacteria. As carbon-limited conditions were presumably widespread throughout Earth history, such a scenario may explain the numerous transitions from phototrophy to mixotrophy and further to heterotrophy within virtually all major algal lineages. We challenge prevailing concepts that affiliated the evolution of phagotrophy with eutrophic or strongly light-limited environments only.     

Algae obscura: The potential of rare species as model systems

Model organism research has provided invaluable knowledge about foundational biological principles. However, most of these studies have focused on species that are in high abundance, easy to cultivate in the lab, and represent only a small fraction of extant biodiversity. Here, we present three examples of rare algae with unusual features that we refer to as “algae obscura.” The Cyanidiophyceae (Rhodophyta), Glaucophyta, and Paulinella (rhizarian) lineages have all transitioned out of obscurity to become models for fundamental evolutionary research. Insights have been gained into the prevalence and importance of eukaryotic horizontal gene transfer, early Earth microbial community dynamics, primary plastid endosymbiosis, and the origin of Archaeplastida. By reviewing the research that has come from the exploration of these organisms, we demonstrate that underappreciated algae have the potential to help us formulate, refine, and substantiate core hypotheses and that such organisms should be considered when establishing future model systems.     

Hypothesis: Gene‐rich plastid genomes in red algae may be an outcome of nuclear genome reduction

Red algae (Rhodophyta) putatively diverged from the eukaryote tree of life >1.2 billion years ago and are the source of plastids in the ecologically important diatoms, haptophytes, and dinoflagellates. In general, red algae contain the largest plastid gene inventory among all such organelles derived from primary, secondary, or additional rounds of endosymbiosis. In contrast, their nuclear gene inventory is reduced when compared to their putative sister lineage, the Viridiplantae, and other photosynthetic lineages. The latter is thought to have resulted from a phase of genome reduction that occurred in the stem lineage of Rhodophyta. A recent comparative analysis of a taxonomically broad collection of red algal and Viridiplantae plastid genomes demonstrates that the red algal ancestor encoded ~1.5× more plastid genes than Viridiplantae. This difference is primarily explained by more extensive endosymbiotic gene transfer (EGT) in the stem lineage of Viridiplantae, when compared to red algae. We postulate that limited EGT in Rhodophytes resulted from the countervailing force of ancient, and likely recurrent, nuclear genome reduction. In other words, the propensity for nuclear gene loss led to the retention of red algal plastid genes that would otherwise have undergone intracellular gene transfer to the nucleus. This hypothesis recognizes the primacy of nuclear genome evolution over that of plastids, which have no inherent control of their gene inventory and can change dramatically (e.g., secondarily non‐photosynthetic eukaryotes, dinoflagellates) in response to selection acting on the host lineage.     

The Origin of Primary Plastids: A Pas de Deux or a Ménage à Trois?

The idea of an endosymbiotic origin of plastids has become incontrovertible, but many important aspects of plastid origins remain obscured in the mists of more than a billion years of evolutionary history. This commentary provides a critical summary of a recent proposal that primary plastid endosymbiosis was facilitated by the secretion into the host cytosol of effector proteins from intracellular Chlamydiales pathogens that allowed the host to utilize carbohydrates exported from the incipient plastid. Although not without flaws, the model provides an explanation for why primary plastids have evolved so rarely and why Archaeplastida, among all phagotrophic eukaryotes, succeeded in establishing primary plastids.It was over a century ago in 1905 that Mereschkowsky proposed that plastids derived from engulfed cyanobacteria (Martin and Kowallik, 1999), and more than 40 years since this idea entered the biological mainstream, primarily through the work of Lynn Margulis (e.g., Sagan, 1967; Margulis, 1970, 1981). The basic idea of an endosymbiotic origin of plastids is supported by abundant lines of evidence, especially by phylogenetic analyses showing that plastid genomes represent a particular branch of the cyanobacteria. Nonetheless, many important aspects of plastid origins remain. Ball et al. (2013) have now contributed to our understanding of primary endosymbiosis, exploring the possible role of Chlamydieae, intracellular parasitic bacteria, in allowing for the integration of cyanobacterial and host carbohydrate metabolism.Looking across the entire tree of life, we see many instances in which a lineage has made a remarkable transition in its way of life, often resulting in a major adaptive radiation. In seeking to explain these major transitions, evolutionary biologists generally start by identifying the traits needed for survival in the new adaptive zone and documenting the order in which they were acquired. However, a full evolutionary narrative should also explain why, if an evolutionary adaptive zone transition is so beneficial, did it not occur more frequently? And also why, among all of life’s diversity, did one specific lineage make the leap? For example, the reason that among all the many lineages of multicellular algae it was just the charophytes that succeeded in invading land (as land plants) has been suggested to be due to specific preadaptations of these algae, such as being specialized for freshwater (Graham, 1993; Becker and Marin, 2009) and having particular developmental (Graham et al., 2000) or biochemical characteristics (Sørensen et al., 2011). Ball et al. (2013) attempt to provide such a narrative to help explain why one particular clade of eukaryotes, the Archaeplastida, came to host the endosymbiotic cyanobacteria that evolved into plastids.Phylogenies of plastid-encoded genes confirm that plastids form a single clade embedded within the Cyanobacteria, consistent with a single event of plastid origination. The data from the nuclear genome is also consistent with this inference: Viridiplantae (which includes green algae and land plants), rhodophytes, and glaucophytes, the three eukaryotic lineages that are inferred on anatomical grounds to have plastids that were derived directly from engulfed cyanobacteria (primary plastids), form a clade, Archaeplastida (Keeling, 2004, 2010; Price et al., 2012). Evolution of primary plastids has indeed been an evolutionary rarity of the highest degree.The lack of additional origins of primary plastids is not simply because photoendosymbiosis is difficult. At least seven independent eukaryotic lineages have taken up single-celled eukaryotic algae as plastids, including ancestors of such successful groups as euglenoids, brown algae/diatoms, and dinoflagellates (Keeling, 2010). By contrast, there is only one other instance where a heterotrophic eukaryote evolved a stable association with an endosymbiotic cyanobacterium, the cercozoan genus Paulinella (Marin et al., 2005; Nowack et al., 2008), and the status of this case as a true plastid has been subject to debate (Theissen and Martin, 2006). The obvious conclusion from the rarity of primary plastid origins is that it is more difficult to integrate a prokaryotic endosymbiont into the host’s metabolism than another eukaryote. But what might the problem be, and how might the ancestor of Archaeplastida have overcome that impediment? Ball et al. (2013) argue that one of the main challenges during plastid origin is meshing host and plastid carbohydrate metabolism and that Archaeplastida were uniquely able to accommodate a cyanobacterial endosymbiont because they were subject to coinfections by chlamydial endoparasites.The idea that Chlamydiae have been important in the evolutionary history of Archaeplastida was first hinted at by the discovery that a surprisingly large number of genes in the Chlamydia trachomatis genome have closely related genes in plants (Stephens et al., 1998). Subsequent analyses have suggested that this is the result of direct horizontal gene transfer from Chlamydiae into ancestral Archaeplastida (Greub and Raoult, 2003; Huang and Gogarten, 2007; Becker et al., 2008; Moustafa et al., 2008). Phylogenomic analyses have identified between 21 and 55 specific genes that appeared to have been acquired by plants from Chlamydiae (Huang and Gogarten, 2007; Becker et al., 2008; Moustafa et al., 2008). Ball et al. (2013) corroborate this conclusion with their own analysis of currently available sequence data. Even using rather stringent criteria, they show that there are many more cases of chlamydial genes being sister to archaeplastidial genes than is the case for other eukaryotic clades, even clades such as fungi and animals, which are overrepresented in sequence databases.The conclusion that chlamydial genes’ presence in plants is a result of direct horizontal gene transfer is not universally accepted. Some suspect that these genes are the result of more complex histories entailing gene transfers among different prokaryotic lineages, including the cyanobacterial ancestors of plastids, perhaps combined with gene transfers into Archaeplastida later in their evolution and/or transfers from Archaeplastida into Chlamydiae (Brinkman et al., 2002; Dagan and Martin, 2009; Martin et al., 2012). However, the relatively large number of chlamydial genes in plant genomes points to some history of stable association between Chlamydiae and Archaeplastida that allowed abundant gene transfer. This leaves open the possibility that Chlamydiae were present in the host cell at the time that primary plastids evolved (Huang and Gogarten, 2007). If that were so, might chlamydial genes have facilitated plastid evolution?Following on from the group’s prior work on carbohydrate metabolism in Archaeplastida (Deschamps et al., 2008), Ball et al. (2013) explore the possibility that key genes were acquired from Chlamydiae. They focused on genes that would have been needed for sugar nucleotides exported from the cyanobacterium to be converted into glycogen, the original storage polysaccharide, and those that would be needed to effectively use stored glycogen. Cyanobacteria are assumed to have exported ADP-Glc. This would have posed a problem, however, since eukaryotic glucan synthases use UDP-Glc as substrate, rather than ADP-Glc. Ball et al. (2013) argued that the archaeplastidial glucan synthases that are most likely to have had ADP-Glc activity in solution are members of the class III or class IV starch synthases. A phylogenetic analysis of glucan synthases identified a well-supported (98% bootstrap) clade composed only of archaeplastidial class III and class IV sequences and a number of bacterial sequences (see Supplemental Figure 1 online in Ball et al., 2013). As shown in an unrooted version of this subtree (Figure 1A), the chlamydial sequences would end up sister to Archaeplastida under plausible rooting scenarios. Notably, this tree rules out a cyanobacterial origin of the archaeplastidial genes. Similarly, Ball et al. (2013) argued that effective use of glycogen stores required direct debranching activity, which is provided in modern Archaeplastida by isoamylase, which is closely related to chlamydial GlgX proteins (Huang and Gogarten, 2007). Phylogenetic analysis of GlgX/isoamylase sequences yields an unrooted tree on which plausible roots imply, once again, that archaeplastidial genes have chlamydial ancestry (Figure 1B).Open in a separate windowFigure 1.Unrooted Phylogenetic Trees for Gene Families Involved in Carbohydrate Metabolism.Both trees are heavily pruned and have branches with bootstrap percentages below 50% collapsed. Branches are coded by taxon: green = Viridiplantae; red = red algae; brown = glaucophytes; pink = Chlamydiae; cyan = cyanobacteria; black = other bacteria.(A) The starch synthase III-IV tree based on Supplemental Figure 1 from Ball et al. (2013). In the original analysis, this subtree was rooted on the branch marked with an arrow, but a more plausible rooting would be on one of the black (bacterial) branches.(B) Glycogen direct debranching genes based on Figure 5 and Supplemental Figure 2 from Ball et al. (2013). This tree is unrooted, but the true root most likely resides on one of the black branches.However, the inference that genes needed for proper interactions between a eukaryotic host and a prokaryotic endosymbiont were acquired from Chlamydiae poses a problem. It is unlikely that these genes entered the archaeplastidial genome before plastid endosymbiosis because they would have had no function, and genes do not persist in genomes for long if they lack a function. But equally, if these enzymes were critical for the establishment of the plastid, then they had to be present at the time of endosymbiosis. Ball et al. (2013) propose a resolution of this paradox: Suitable glucan synthases (GlgA) and direct glycogen debranching enzymes (GlgX) were present in the cytoplasm of the host cell because they were being secreted into the cytoplasm by coinfecting Chlamydiae. In support of this hypothesis, Ball et al. showed that many extant chlamydial proteins involved in carbohydrate metabolism, including GlgX, have sequences consistent with being effector proteins released into the host cell by a type III secretion system. Furthermore, an in vivo assay using Shigella flexneri as a model confirmed that the N-terminal peptide of several of proteins, including GlgX and GlgA, allows secretion through the type III system, suggesting that they could act as effector proteins in Chlamydiae.Putting the pieces together, Ball et al. (2013) propose a narrative in which the ancestral Archaeplastida were subject to infection by Chlamydiae, which injected a suite of proteins into host cells. These effector proteins served to manipulate carbohydrate metabolism to the parasite’s advantage. The presence of these enzymes in the cytoplasm had the beneficial side effect of allowing the host and an incipient plastid to enter into a mutualistic association that would otherwise have been impossible because of the inability of the host to synthesize and use storage carbohydrates from ADP-Glc. Only later, after horizontal gene transfer introduced the enzyme-coding genes into the nucleus, could the mutualism persist without Chlamydiae. Only at that point would selection favor Archaeplastida that resisted chlamydial infection. Or, to put it simplistically, rather than viewing the endosymbiotic origin of plastids as a dance of two partners, Ball et al. suggest it was a more complex, ménage à trois.The Ball et al. model has some compelling features. It provides an explanation for why primary plastids have evolved so rarely and why Archaeplastida, among all phagotrophic eukaryotes, succeeded in establishing primary plastids. However, for all its appeal, the hypothesis is not without its flaws. For example, many groups of eukaryotes are or have been subject to infection by Chlamydiae, so the model does not fully explain why there were no other origins of primary plastids. Also, there remains a possibility that the 50 or so genes transferred from Chlamydiae to Viridiplantae were acquired at different times in the course of simple parasitism, with the apparent enrichment of carbohydrate metabolism genes in the list being coincidental. Nonetheless, by combining new insights into the evolution of carbohydrate metabolism in Archaeplastida with phylogenomics and experimental studies of potential metabolic effector proteins, Ball et al. (2013) have contributed to our understanding of one of the most important events in the origin of life on earth: the origin of plastids.     

An ancient evolutionary origin of genes associated with human genetic diseases

Several thousand genes in the human genome have been linked to a heritable genetic disease. The majority of these appear to be nonessential genes (i.e., are not embryonically lethal when inactivated), and one could therefore speculate that they are late additions in the evolutionary lineage toward humans. Contrary to this expectation, we find that they are in fact significantly overrepresented among the genes that have emerged during the early evolution of the metazoa. Using a phylostratigraphic approach, we have studied the evolutionary emergence of such genes at 19 phylogenetic levels. The majority of disease genes was already present in the eukaryotic ancestor, and the second largest number has arisen around the time of evolution of multicellularity. Conversely, genes specific to the mammalian lineage are highly underrepresented. Hence, genes involved in genetic diseases are not simply a random subset of all genes in the genome but are biased toward ancient genes.     

The macroecology of sustainability

The discipline of sustainability science has emerged in response to concerns of natural and social scientists, policymakers, and lay people about whether the Earth can continue to support human population growth and economic prosperity. Yet, sustainability science has developed largely independently from and with little reference to key ecological principles that govern life on Earth. A macroecological perspective highlights three principles that should be integral to sustainability science: 1) physical conservation laws govern the flows of energy and materials between human systems and the environment, 2) smaller systems are connected by these flows to larger systems in which they are embedded, and 3) global constraints ultimately limit flows at smaller scales. Over the past few decades, decreasing per capita rates of consumption of petroleum, phosphate, agricultural land, fresh water, fish, and wood indicate that the growing human population has surpassed the capacity of the Earth to supply enough of these essential resources to sustain even the current population and level of socioeconomic development.     

Isolation of <Emphasis Type="Italic">Serratia marcescens</Emphasis> involved in chitin degradation in the bulb mite <Emphasis Type="Italic">Rhizoglyphus robini</Emphasis>

Cymbomonas tetramitiformis is a peculiar green alga that unites in one cell the abilities of photosynthesis and phagocytosis, which makes it a very useful model for the study of the evolution of plastid endosymbiosis. We have pondered over this issue and propose an evolutionary scenario of trophic strategies in eukaryotes, including primary and secondary plastid endosymbioses. C. tetramitiformis is a prototroph, just like the common ancestor of Archaeplastida was, and can synthesize most small organic molecules contrary to other eukaryotic phagotrophs, e.g. some metazoans, amoebozoans, and ciliates, which have not evolved tight endosymbiotic relationships. In order to establish a permanent photosynthetic endosymbiont they do not have to become prototrophs, but have to acquire the genes necessary for plastid retention via horizontal (including endosymbiotic) gene transfer. Such processes occurred successfully in the ancestors of eukaryotes with permanent secondary plastids and thus led to their great diversification. The preservation of phagocytosis in Cymbomonas (and some other prasinophytes as well) seems to result from nutrient deficiency in their oligotrophic habitats. This forces them to supplement their diet with phagocytized prey, in contrasts to the thecate amoeba Paulinella chromatophora, which also successfully transformed cyanobacteria into permanent organelles. Although Paulinella endosymbionts were acquired very recently in comparison to primary plastids, Paulinella has lost the ability to phagocytose, most probably due to the fact that it inhabits nutrient-rich environments, which renders the phagotrophy nonessential.     

The new higher level classification of eukaryotes with emphasis on the taxonomy of protists

This revision of the classification of unicellular eukaryotes updates that of Levine et al. (1980) for the protozoa and expands it to include other protists. Whereas the previous revision was primarily to incorporate the results of ultrastructural studies, this revision incorporates results from both ultrastructural research since 1980 and molecular phylogenetic studies. We propose a scheme that is based on nameless ranked systematics. The vocabulary of the taxonomy is updated, particularly to clarify the naming of groups that have been repositioned. We recognize six clusters of eukaryotes that may represent the basic groupings similar to traditional "kingdoms." The multicellular lineages emerged from within monophyletic protist lineages: animals and fungi from Opisthokonta, plants from Archaeplastida, and brown algae from Stramenopiles.     

Planetary Organic Chemistry and the Origins of Biomolecules

Organic chemistry on a planetary scale is likely to have transformed carbon dioxide and reduced carbon species delivered to an accreting Earth. According to various models for the origin of life on Earth, biological molecules that jump-started Darwinian evolution arose via this planetary chemistry. The grandest of these models assumes that ribonucleic acid (RNA) arose prebiotically, together with components for compartments that held it and a primitive metabolism that nourished it. Unfortunately, it has been challenging to identify possible prebiotic chemistry that might have created RNA. Organic molecules, given energy, have a well-known propensity to form multiple products, sometimes referred to collectively as “tar” or “tholin.” These mixtures appear to be unsuited to support Darwinian processes, and certainly have never been observed to spontaneously yield a homochiral genetic polymer. To date, proposed solutions to this challenge either involve too much direct human intervention to satisfy many in the community, or generate molecules that are unreactive “dead ends” under standard conditions of temperature and pressure. Carbohydrates, organic species having carbon, hydrogen, and oxygen atoms in a ratio of 1:2:1 and an aldehyde or ketone group, conspicuously embody this challenge. They are components of RNA and their reactivity can support both interesting spontaneous chemistry as part of a “carbohydrate world,” but they also easily form mixtures, polymers and tars. We describe here the latest thoughts on how on this challenge, focusing on how it might be resolved using minerals containing borate, silicate, and molybdate, inter alia.Interesting organic chemistry occurs throughout the cosmos, including in presolar nebulae (see the article in this collection by Pascale Erhrenfreund), asteroidal bodies (see the article in this collection by Sandra Pizzarello) and icy bodies near the outer boundary of our solar system (Bernstein et al. 2002). Although organic molecules made in off-Earth locales almost certainly contributed to the reduced carbon inventory on Earth before life emerged, planetary processing on Earth undoubtedly also contributed to the inventory of prebiotic molecules that were available to life as it originated (assuming that Earth was the site of life''s origin). Indeed, in the RNA first model for the origin of life on Earth (Joyce and Orgel 1999)(Benner 2009), it is often proposed that terran-based chemistry produced RNA in oligomeric form to initiate Darwinian evolution.How are we to constrain models for planetary processing to converge on a model for what actually happened on Earth four billion years ago? Today, atmospheric dioxygen (O₂) readily converts organic materials to carbon dioxide, making it essentially impossible to observe such processing on the surface of Earth. Furthermore, the ubiquity of life on modern Earth means that any organic processing is more likely to reflect biology than prebiology. The closest we may come today to observe organic transformations absent biology on a planetary scale might be on Titan, a moon of Saturn whose atmosphere and surface is rich in reduced carbon.Nevertheless, it is possible to apply a general understanding of organic chemical reactivity to suggest chemical reactions that might have occurred on early Earth and the products that they might have produced. These suggestions are constrained by models for the atmosphere and mineralogy of early Earth, although these constraints might change as models improve.In this article, we assume that the atmosphere of early Earth was less oxidizing than today''s atmosphere, although not as rich in methane as the simulated atmosphere used in the classic experiments of Stanley Miller (Miller 1955). Further, we assume that the atmosphere on early Earth had access to many sources of energy. These include electrical discharge, ultraviolet and visible light (although the Sun was almost certainly dimmer then than now, a Titan-like haze may have prevented high energy photons from reacting the Earth''s surface), volcanism (providing not only heat but also reactive species and mixtures not at thermodynamic equilibrium), ionizing radiation, and impacts. (See Pizzarello and Shock 2010 for a discussion of such energy sources.)We also assume that life emerged after the planet underwent a geological fractionation in which heavier minerals and elemental iron sank towards the core, leaving lighter rocks to form the crust. Open questions concern the inventory of water relative to the surface of early Earth, an inventory that determined whether planetary organic transformations might have occurred on dry land or below water on a planetary surface that was totally submerged.     

How life began

Study of the origin of life has become a legitimate scientific inquiry, with an international, multidisciplinary membership and a cogent body of data. Experiments involving plausible early Earth conditions and biogeochemical analyses of carbonaceous meteorites imply a variety of available starting molecules. Biogeological evidence indicates microbial beginnings about 3800 million years (3.8 aeons) ago. By then the known universe had been in existence for perhaps 15 aeons and galaxies abundant for ten. Conditions suitable for the origin of life may require a long prior cosmic evolution. The natural origin of life on the early Earth is now widely agreed upon but not the pathways. The beginnings of catalysis, replication and a functional cell remain moot. Much discussion has centered on the templating role that crystals such as clays and zeolites might have played in prebiotic evolution. Recent discovery of the catalytic and replicative functions of RNA recommend it as the key molecule in the transition from chemical to biological evolution.     

Survival of the fittest before the beginning of life: selection of the first oligonucleotide-like polymers by UV light

Background  

A key event in the origin of life on this planet has been formation of self-replicating RNA-type molecules, which were complex enough to undergo a Darwinian-type evolution (origin of the "RNA world"). However, so far there has been no explanation of how the first RNA-like biopolymers could originate and survive on the primordial Earth.     

Phylogeny of galactolipid synthase homologs together with their enzymatic analyses revealed a possible origin and divergence time for photosynthetic membrane biogenesis

The photosynthetic membranes of cyanobacteria and chloroplasts of higher plants have remarkably similar lipid compositions. In particular, thylakoid membranes of both cyanobacteria and chloroplasts are composed of galactolipids, of which monogalactosyldiacylglycerol (MGDG) is the most abundant, although MGDG biosynthetic pathways are different in these organisms. Comprehensive phylogenetic analysis revealed that MGDG synthase (MGD) homologs of filamentous anoxygenic phototrophs Chloroflexi have a close relationship with MGDs of Viridiplantae (green algae and land plants). Furthermore, analyses for the sugar specificity and anomeric configuration of the sugar head groups revealed that one of the MGD homologs exhibited a true MGDG synthetic activity. We therefore presumed that higher plant MGDs are derived from this ancestral type of MGD genes, and genes involved in membrane biogenesis and photosystems have been already functionally associated at least at the time of Chloroflexi divergence. As MGD gene duplication is an important event during plastid evolution, we also estimated the divergence time of type A and B MGDs. Our analysis indicated that these genes diverged -323 million years ago, when Spermatophyta (seed plants) were appearing. Galactolipid synthesis is required to produce photosynthetic membranes; based on MGD gene sequences and activities, we have proposed a novel evolutionary model that has increased our understanding of photosynthesis evolution.     

Green land: Multiple perspectives on green algal evolution and the earliest land plants

Green plants, broadly defined as green algae and the land plants (together, Viridiplantae), constitute the primary eukaryotic lineage that successfully colonized Earth's emergent landscape. Members of various clades of green plants have independently made the transition from fully aquatic to subaerial habitats many times throughout Earth's history. The transition, from unicells or simple filaments to complex multicellular plant bodies with functionally differentiated tissues and organs, was accompanied by innovations built upon a genetic and phenotypic toolkit that have served aquatic green phototrophs successfully for at least a billion years. These innovations opened an enormous array of new, drier places to live on the planet and resulted in a huge diversity of land plants that have dominated terrestrial ecosystems over the past 500 million years. This review examines the greening of the land from several perspectives, from paleontology to phylogenomics, to water stress responses and the genetic toolkit shared by green algae and plants, to the genomic evolution of the sporophyte generation. We summarize advances on disparate fronts in elucidating this important event in the evolution of the biosphere and the lacunae in our understanding of it. We present the process not as a step-by-step advancement from primitive green cells to an inevitable success of embryophytes, but rather as a process of adaptations and exaptations that allowed multiple clades of green plants, with various combinations of morphological and physiological terrestrialized traits, to become diverse and successful inhabitants of the land habitats of Earth.     

Flourishing in water: the early evolution and diversification of plant receptor-like kinases

Receptor-like kinases (RLKs) play significant roles in mediating innate immunity and development of plants. The evolution of plant RLKs has been characterized by extensive variation in copy numbers and domain configurations. However, much remains unknown about the origin, evolution, and early diversification of plant RLKs. Here, we perform phylogenomic analyses of RLKs across plants (Archaeplastida), including embryophytes, charophytes, chlorophytes, prasinodermophytes, glaucophytes, and rhodophytes. We identify the presence of RLKs in all the streptophytes (land plants and charophytes), nine out of 18 chlorophytes, one prasinodermophyte, and one glaucophyte, but not in rhodophytes. Interestingly, the copy number of RLKs increased drastically in streptophytes after the split of the clade of Mesostigmatophyceae and Chlorokybophyceae and other streptophytes. Moreover, phylogenetic analyses suggest RLKs from charophytes form diverse distinct clusters, and are dispersed along the diversity of land plant RLKs, indicating that RLKs have extensively diversified in charophytes and charophyte RLKs seeded the major diversity of land plant RLKs. We identify at least 81 and 76 different kinase-associated domains for charophyte and land plant RLKs, 23 of which are shared, suggesting that RLKs might have evolved in a modular fashion through frequent domain gains or losses. We also detect signatures of positive selection for many charophyte RLK groups, indicating potential functions in host–microbe interaction. Taken together, our findings provide significant insights into the early evolution and diversification of plant RLKs and the ancient evolution of plant–microbe symbiosis.     

Origin and early evolution of photosynthetic eukaryotes in freshwater environments: reinterpreting proterozoic paleobiology and biogeochemical processes in light of trait evolution

Phylogenetic analyses were performed on concatenated data sets of 31 genes and 11,789 unambiguously alignable characters from 37 cyanobacterial and 35 chloroplast genomes. The plastid lineage emerged somewhat early in the cyanobacterial tree, at a time when Cyanobacteria were likely unicellular and restricted to freshwater ecosystems. Using relaxed molecular clocks and 22 age constraints spanning cyanobacterial and eukaryote nodes, the common ancestor to the photosynthetic eukaryotes was predicted to have also inhabited freshwater environments around the time that oxygen appeared in the atmosphere (2.0–2.3 Ga). Early diversifications within each of the three major plastid clades were also inferred to have occurred in freshwater environments, through the late Paleoproterozoic and into the middle Mesoproterozoic. The colonization of marine environments by photosynthetic eukaryotes may not have occurred until after the middle Mesoproterozoic (1.2–1.5 Ga). The evolutionary hypotheses proposed here predict that early photosynthetic eukaryotes may have never experienced the widespread anoxia or euxinia suggested to have characterized marine environments in the Paleoproterozoic to early Mesoproterozoic. It also proposes that earliest acritarchs (1.5–1.7 Ga) may have been produced by freshwater taxa. This study highlights how the early evolution of habitat preference in photosynthetic eukaryotes, along with Cyanobacteria, could have contributed to changing biogeochemical conditions on the early Earth.