Plastid origin and evolution: new models provide insights into old problems.

Algae are defined by their photosynthetic organelles (plastids) that have had multiple independent origins in different phyla. These instances of organelle transfer significantly complicate inference of the tree of life for eukaryotes because the intracellular gene transfer (endosymbiotic gene transfer, EGT) associated with each round of endosymbiosis generates highly chimeric algal nuclear genomes. In this Update we review the current state in the field of endosymbiosis research with a focus on the use of the photosynthetic amoeba Paulinella to advance our knowledge of plastid evolution and current ideas about the origin of the plastid translocons. These research areas have been revolutionized by the advent of modern genomic approaches. ALGAE AND PLANTS IN THE EVOLVING TREE OF LIFE Plastids (e.g., chloroplasts in plants) are chlorophyll containing, membranebound organelles that are the sites of photosynthesis in a cell. The first plastid is thought to have originated through primary endosymbiosis, in which a photosynthetic cyanobacterium was captured by a heterotrophic protist and eventually transformed into an intracellular organelle. Molecular clock analysis suggests this key event in eukaryote evolution occurred ca. 1.5 billion years ago (Yoon et al., 2004; see Douzery et al. 2004 for another perspective). This chimeric eukaryote was an evolutionary success story Plant Physiology Preview. Published on February 22, 2011, as DOI:10.1104/pp.111.173500 Copyright 2011 by the American Society of Plant Biologists www.plantphysiol.org on July 15, 2017 Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved. 2 and gave rise to the three major photosynthetic lineages, the red, green (including land plants), and glaucophyte algae (Bhattacharya and Medlin, 1995; Delwiche et al., 1995; McFadden, 2001; Bhattacharya et al., 2004). The supergroup Plantae or Archaeplastida (Adl et al., 2005) was established to include these three primary plastid-containing groups that contain a double-membrane-bound photosynthetic organelle lying free in the cytosol (Cavalier-Smith, 1998). Plantae monophyly has been recovered when inferring trees with plastid genes (Yoon et al., 2002; Rodríguez-Ezpeleta et al., 2005; Kim and Archibald, 2010), nuclear genes that encode plastid-targeted proteins (Li et al., 2006; Nosenko et al., 2006; Deschamps and Moreira, 2009), and rarely, single nuclear genes (Moreira et al., 2000). The strongest support for Plantae monophyly comes from a 143-gene phylogeny with 34 taxa (Rodríguez-Ezpeleta et al., 2005), a 16-gene tree with 46 taxa that included all of the eukaryotic supergroups (Hackett et al., 2007), and trees inferred from >125 protein data sets (Burki et al., 2007; Burki et al., 2008). However, recent multi-gene studies based on genome data have often shown Plantae to be polyphyletic (Nozaki et al., 2009; Baurain et al., 2010). Some of these uncertainties likely stem from methodological limitations in phylogenetic inference (Stiller, 2007) and the impact of endosymbiotic or horizontal gene transfer (E/HGT) on the evolution of microbial eukaryote genomes (Keeling and Palmer, 2008). Nevertheless, this situation is also likely explained by the absence of broadly sampled red algal genes for phylogenomic analysis, and limited availability of expressed sequence tag (EST) data from the glaucophytes. All of the existing analyses have for example relied on complete genome data from the thermoacidophilic Cyanidiales red alga Cyanidioschyzon merolae (Matsuzaki et al., 2004) and partial genome data from its sister Galdieria sulphuraria (http://genomics.msu.edu/galdieria/). The Cyanidiales have reduced and specialized genomes and includes only seven described species. These highly derived taxa do not represent the taxonomic breadth (ca. 6,000 species) of Rhodophyta, most of which are mesophilic (Reeb and Bhattacharya, 2010; Yoon et al., 2006a; Yoon et al., 2010). In light of this issue, a recent analysis of genome data from two mesophilic red algae, Porphyridium cruentum (extensive ESTs) and Calliarthron tuberculosum (draft nuclear genome assembly) uncovered a strong phylogenetic signal for monophyly of the www.plantphysiol.org on July 15, 2017 Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved. 3 red + green lineages that is 4-fold greater than when only C. merolae is used to represent the rhodophytes (Chan et al., 2011). The addition of 60,000 novel genes from P. cruentum and C. tuberculosum in the phylogenomic analysis also showed that ca. 50% of red algal genes are shared with other eukaryotes and prokaryotes putatively via E/HGT (Fig. 1). This level of gene sharing can potentially complicate resolution of the Plantae supergroup and of lineages that are recipients of Plantae genes (see below). These genome-wide data do however go a long way toward substantiating the Plantae hypothesis, at least for the red + green lineages (glaucophyte complete genome is currently being analyzed [D. Bhattacharya, J. Boore et al., work in progress]). The evolution of many key synapomorphies shared by Plantae that are often associated with photosynthesis or other plastid functions; e.g., origin of Calvin cycle enzymes (ReyesPrieto and Bhattacharya, 2007), plastid translocon components (Weber et al., 2006), solute transporters (Colleoni et al., 2010), and dozens of unique Chlamydiae gene transfers (Huang and Gogarten, 2007; Moustafa et al., 2008) can now be interpreted with some confidence under the parsimonious scenario of a single origin in the Plantae ancestor. The difficulties faced in resolving the issue of Plantae monophyly pale however in comparison to the phylogeny of algal groups that harbor a red algal derived plastid that contains chlorophyll-c. This supergroup was united by Cavalier-Smith (1998) under the moniker “Chromalveolata” and originally contained the alveolate (ciliates, apicomplexans, dinoflagellates) and chromist (Stramenopiles, cryptophytes, haptophytes) protist lineages. These taxa were hypothesized to share a single, ancient red algal secondary endosymbiosis that gave rise to the chromophyte plastid present in many photosynthetic chromalveolates such as diatoms and haptophytes and lost secondarily in groups like ciliates and the plastid-lacking cryptophyte lineage Goniomonas. Recently several other protist groups such as telonemids (ShalchianTabrizi et al., 2006), katablepharids, centrohelids (Okamoto and Inouye, 2005; Okamoto et al., 2009), picobiliphytes (Not et al., 2007), and rappemonads (Kim et al., 2011) have been suggested to share an affiliation with the chromalveolates. Undoubtedly the quickening pace of environmental genomics research will turn up other candidate taxa affiliated with the ever-growing assemblage of “chromalveolates” that no longer bears www.plantphysiol.org on July 15, 2017 Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved. 4 any resemblance to the taxonomic scheme that was originally envisaged by CavalierSmith (1998). The monophyly of this lineage has been tested by many groups with initial multi-gene analyses supporting the supergroup but more recently, a number of papers have provided evidence against its existence or specifically, the hypothesis of a single red algal secondary endosymbiosis among all chlorophyll c-containing chromalveolates (e.g., Baurain et al., 2010; Parfrey et al., 2010). The question of chromalveolate monophyly, the branching order of its constituent taxa, and the history of plastid endosymbiosis in its photosynthetic members (Baurain et al., 2010; Janouškovec et al., 2010) remain as challenging issues that are yet to be clarified. More important than “getting the tree right” is of course why it has proven so difficult in the first place when other similarly ancient groups such as opisthokonts and Amoebozoa appear monophyletic with strong bootstrap support in many multi-gene trees (e.g., Parfrey et al., 2006). The reason for this dichotomy may be explained by a complex history of reticulate gene evolution in chromalveolates that has not yet been fully understood (Stiller, 2007). E/HGT can produce a reticulate origin of gene fragments (in addition to whole genes) that would mislead inference of phylogenetic relationships (Chan et al., 2009). The Chan et al. (2011) analysis of mesophilic red algal genome data shows how widespread “red” genes are in the tree of life due to E/HGT. The role of red and green algal genomes as donors of genes means that unless carefully controlled for, many protist groups that have been recipients of Plantae genes may group with these taxa in phylogenies not due to vertical ancestry but rather E/HGT (Fig. 1), that may or may not be recognized in all single gene trees. A striking example of unanticipated, large-scale E/HGT is provided by a recent phylogenomic analysis of predicted proteins in the completed genomes of the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum that showed hundreds of genes in these taxa to be derived from green algae (in particular prasinophytes; Moustafa et al., 2009). These data significantly complicate our understanding of chromalveolate evolution and may be explained by a cryptic green algal endosymbiosis that predated the canonical red algal capture. Under this scenario, the presence of a red alga-derived plastid in many chromalveolates conceals a past endosymbiosis, with the “green” E/HGTs acting as footprints of this ancient event (Moustafa et al., 2009; for www.plantphysiol.org on July 15, 2017 Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved. 5 discussion, see Elias and Archibald, 2009). Similarly, whereas the chlorarachniophyte Bigelowiella natans contains a large number of green alga-derived nuclear genes to support the function of its “green” plastid, a significant number of red algal-derived, plastid-targeted proteins also exist in this lineage (Archibald et al., 2003). A similar situation was recently described for the dinoflagellate Lepidodinium chlorophanum that contains a green alga-derived plastid but has collected nuclear genes that encode plastid–targeted proteins from multiple different algal lineages (Minge et al., 2010). These data suggest a more complex history of E/HGT in chlorarachniophytes and L. chlorophanum than would be expected based on plastid identity. Another potential source of confusion wrought by endosymbiosis is plastid loss. The absence of a plastid in chromalveolates such as telonemids and katablepharids does not necessarily mean these genomes have been protected from EGT. As suggested for ciliates (Archibald, 2008; Reyes-Prieto et al., 2008), other heterotrophic lineages such as katablepharids and telonemids with phylogenetic affiliations to photosynthetic chromalveolates (Okamoto and Inouye, 2005; Okamoto et al., 2009) may have retained algal derived genes that were introduced by EGT in their plastidbearing ancestor, and survived due to a plastid-independent function. These unanticipated levels of genome complexity may partly explain the widely divergent views on Plantae and chromalveolate phylogeny that have regularly appeared in recent years (Burki et al., 2007; Nozaki et al., 2007; Patron et al., 2007; Kim and Graham, 2008; Baurain et al., 2010). Rather than a continued focus on currently unresolved controversies regarding the structure of the algal and plant tree of life, we devote the remainder of the update to recent advances in the understanding of plastid evolution that provide novel insights into the process of endosymbiosis. The two major areas to be covered are: (i) use of the photosynthetic amoeba Paulinella to understand primary plastid origin, and (ii) models to explain the establishment of host-control of plastid metabolism and evolution of the plastid machinery for protein import (the organelle translocons). www.plantphysiol.org on July 15, 2017 Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved. 6 PAULINELLA AND PRIMARY PLASTID ORIGIN Paulinella chromatophora is a thecate, filose amoeba (Bhattacharya et al., 1995) with blue-green chromatophores (Fig. 2) that was first described in 1895 by Robert Lauterborn (Lauterborn, 1895). With respect to Plantae, this species has undergone an independent primary (cyanobacterial) plastid acquisition (Kies, 1974; Marin et al., 2005; Melkonian and Mollenhauer, 2005; Rodríguez-Ezpeleta and Philippe, 2006; ReyesPrieto et al., 2010) and consequently, P. chromatophora is an outstanding model for understanding plastid establishment. Sequence analysis of the nuclear small subunit rDNA gene shows the photosynthetic amoeba to be closely related to species in the Order Euglyphida (Phylum Cercozoa, Supergroup Rhizaria), whereas the Paulinella plastid rDNA is most closely related to Synechococcus WH5701 within a larger clade of Prochlorococcusand Synechococcus-type cyanobacteria (Yoon et al., 2006b; Yoon et al., 2009; Nowack et al., 2011), i.e., PS-clade shown in Fig. 2. The P. chromatophora plastid, also known as a cyanelle, retains cyanobacterial features such as a peptidoglycan wall and carboxysomes. Nonetheless several lines of evidence suggest the cyanelle is a bona fide endosymbiotic-derived organelle. The cyanelle is not bound by a vacuolar membrane and lies free in the cytoplasm, its number is regulated (i.e., two cyanelles per mature host cell) implying genetic integration, the cyanelle cannot be cultured without the host (Kies, 1974; Kies and Kremer, 1979; Johnson et al., 1988), and its genome is reduced in size to ca. 1 Mbp, that is about 1/3 of the size of the putative cyanobacterial donor genome (Nowack et al., 2007; Reyes-Prieto et al., 2010), demonstrating significant decay of genetic potential of the endosymbiont. The complete plastid genome of P. chromatophora M0880/a was recently published (Nowack et al., 2008). Following this, the plastid genome from another photosynthetic strain isolated in Japan, P. microporus FK01 (Yoon et al., 2009) was determined by pyrosequencing whole-genome amplified DNA isolated via flow cytometric single-plastid sorting (Reyes-Prieto et al., 2010). Although greatly reduced in size, these plastid genomes show strong conservation in gene order when compared to the donor PS clade. The size of the Paulinella plastid genome however far exceeds the typical genome sizes for plastids (ca. 100-200 Kbp) in algae and plants indicating an instance of “work-in-progress” with regard to gene loss. Based on the mode and tempo www.plantphysiol.org on July 15, 2017 Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved. 7 of plastid genome reduction, Nowack et al. (2008) postulated the minimum age of primary endosymbiosis in photosynthetic Paulinella to be 60 million years. Alignment of P. microporus and P. chromatophora plastid DNA indicates strong conservation of gene order with only five inversions involving fragments of sizes 110.3 Kbp, 24.9 Kbp, 3.9 Kb, 2.1 Kbp, and 569 bp, as well as a single 9.2 Kbp translocation that distinguishes them. Comparison of gene inventories shows that 27 genes encoded in P. microporus are absent from P. chromatophora, whereas 39 genes in the latter are absent from P. microporus. These 66 genes are present in PS clade members, suggesting lineage-specific losses in these amoebae. The inspection of 681 pairwise alignments of plastid protein-encoding genes reveal Ka/Ks ratios <1 in the vast majority, consistent with strong purifying selection (Reyes-Prieto et al., 2010). These data highlight the evolutionary forces that impact organelle genomes and provide empirical evidence that differential gene losses, as well as selection to maintain protein function, are key characteristics of plastid genome evolution. The Paulinella model also provides an excellent opportunity to study the phenomenon of EGT and to investigate its fundamental role in organellogenesis (see Gross and Bhattacharya, 2009a). The marked plastid genome reduction in Paulinella species, suggests that ca. 2 Mbp of cyanobacterial sequence has either been lost outright or transferred to the nuclear genome of the amoeba. Demonstration of differential gene loss suggests that both forces have clearly impacted this system. Evidence for EGT in Paulinella was initially reported for psaE and psaI (Nakayama and Ishida, 2009; Reyes-Prieto et al., 2010). Cyanobacterial psaI that encodes subunit VIII of photosystem I was silenced by two non-sense mutations in the P. microporus plastid genome, whereas an intact copy with a 198-nt spliceosomal intron was found in the amoeba nuclear genome. This suggests that the plastid copy of psaI likely become a pseudogene after activation of the transferred nuclear gene. In contrast, P. chromatophora retains psaI in the plastid genome. A more-recent analysis of 32,012 ESTs from P. chromatophora (Nowack et al., 2011) provide 32 examples of EGT, (a majority of the implicated genes are involved in photosynthesis), and a recent example of intron insertion in different regions of psaE that distinguish P. chromatophora from P. microporus. These authors also reported two expressed genes (hli and psaK) out of 19 www.plantphysiol.org on July 15, 2017 Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved. 8 that are plastid-encoded in P. microporus but absent in the P. chromatophora organelle, suggesting differential EGT after the split of these taxa. Given these exciting observations of EGT, a key area of future research with Paulinella will be to understand how nuclear-encoded proteins are targeted to the nascent plastid. One possibility is that delivery into and across the cyanelle outer membrane (OM) may involve vesicular transport via the endomembrane system (see Bhattacharya et al., 2007). This is observed for α-carbonic anhydrase in Arabidopsis (Villarejo et al., 2005) and is the standard pathway for targeting proteins to the complex plastids of euglenophytes, Apicomplexa, and peridinin-containing dinoflagellates (Bolte et al., 2009). Interestingly, the conspicuous absence in the reduced cyanelle genome of many transport components that control the flux of metabolites across the cyanobacterial cell membranes suggests that an irreversible, long-term metabolic and cell biological connection, between host and plastid in P. chromatophora has already developed (Nowack et al., 2008). This could be primarily controlled by key host-derived metabolite transporters integrated into the OM of the endosymbiont, such as PfoTPT in Plasmodium (Mullin et al., 2006), or a wholesale reprogramming of the inner membrane (IM) permeome of the new organelle with host transporters (Weber et al., 2006; Tyra et al., 2007). Such a phenomenon is predicted by our recently postulated outsiders’ hypothesis that posits an outside-to-inside host-guided establishment of protein sorting components in an endosymbiotic-derived organelle; i.e., initially in the OM and then gradually inwards to the organelle lumen (Gross and Bhattacharya, 2009a). The model predicts that EGT of molecular components that act in the organelle interior is explained by an advanced stage in the evolution of organelle protein topogenesis. EGT has already occurred for key genes that control photosynthesis and cyanelle function in Paulinella, and determining the final inventory of transfers and gene activations (expression) in this system is currently under way using genome-wide approaches (H.S. Yoon, D. Bhattacharya unpublished data). A fascinating addition to the Paulinella story is provided by its closely related sister taxon P. ovalis. This protist feeds actively on cyanobacteria and other prey that have been identified in food vacuoles in its cytoplasm (Johnson et al., 1988). Comparison of these amoebal genomes may allow the identification of genetic www.plantphysiol.org on July 15, 2017 Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved. 9 innovations (e.g., HGT events and gene duplication of loci potentially involved in plastid maintenance) that underlie the critical transition from heterotrophy to obligate autotrophy (Moustafa et al., 2008). Recent work in our lab has identified cyanobacterial DNA fragments that are putatively derived from prey ingested by P. ovalis cells captured in nature. Extensive sequencing of these single-cell derived genomic DNAs using nextgeneration sequencing has shown that many cyanobacterial fragments are derived from PS clade taxa and their cyanophages (D. Price, H.S. Yoon, and D. Bhattacharya, unpublished data). This result suggests a possible link between feeding behavior in a phagotroph and the source of a novel plastid in its photosynthetic sister. EVOLUTION OF PLASTID PROTEIN SORTING As an intracellular compartment in eukaryotes, plastids rely on thousands of nuclear-encoded proteins that are translated by cytosolic ribosomes and then moved and assembled into the organelle subcompartments (Gould et al., 2008; Gross and Bhattacharya, 2009b; Li and Chiu, 2010). However plastids derive from a cyanobacterial endosymbiont that was presumably free from nuclear control during early the stages of the intracellular association with its host. Therefore the progressive transformation of the endosymbiont into an organelle includes an increasing governance of the host nucleus over cyanobacterial functions. This must have been made possible by the evolution of a regulated system to transport (translocate) nuclear-encoded proteins into the endosymbiont-derived compartment (Gross and Bhattacharya, 2009a; 2009b). In extant algae and plants, this task is fulfilled by specialized multisubunit machines that constitute the translocons at the outer and inner envelope membranes of chloroplasts (Toc and Tic, respectively; Reumann et al., 1999; Gould et al., 2008; Kalanon and McFadden, 2008). Studies of the Toc and Tic protein import systems have primarily been done in land plants, yielding a complex picture in which more than 15 subunits serve as protein-conducting pores, receptors, chaperones, co-chaperones, and regulatory subunits (Li and Chiu, 2010; see Fig. 3). This advanced level of complexity provides a challenge to evolutionary biologists interested in identifying the putative minimal set of protein sorting subunits that supported early organelle evolution. www.plantphysiol.org on July 15, 2017 Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved. 10 To improve our understanding of incipient plastid evolution we recently postulated a simple ancestral protein sorting system in plastids (Gross and Bhattacharya, 2009a, 2009b). This system was composed solely of the Toc75 and Tic110 protein channels (at the outer [OM] and inner membrane [IM] of the plastids, respectively), associated chaperones, and the Toc34 receptor at the plastid entrance (Fig. 3). The remainder of modern-day Toc and Tic subunits presumably accrued later to the complexes, during the evolution of green algae and land plants to improve or regulate translocon activity. Many of these subunits are members of protein families (mostly cyanobacterium-derived) and likely arose via gene duplications (Kalanon and McFadden, 2008). In some cases the new paralog apparently retained the original preendosymbiotic properties (e.g., Tic55, Tic32 and Tic62 might be bona fide metabolic enzymes, and Tic20, Tic21 are putative solute channels) (Hormann et al., 2004; Gray et al., 2004; Balsera et al., 2007; Duy et al., 2007; Gross and Bhattacharya, 2009b) (Gross and Bhattacharya, 2009b). This may mean that the new paralog was (i) recruited to the translocon complexes while still maintaining its previous function in a different system (multifunctionality), or alternatively (ii) co-opted to a new exclusive function at the translocon while still preserving its ancestral enzymatic property. Some of the Tic subunits that appear to be functional recruitments are traditionally interpreted under the view that they represent regulatory subunits of the translocon (e.g., Tic55, Tic32, and Tic62) which modulate protein import activities according to intracellular cues; e.g., redox status, calcium levels (Balsera et al., 2010). Under our view, the metabolic proteins Tic55, Tic32 and Tic62, as well as the putative channels Tic20 and Tic21, and chaperone Tic22, might act as integrative subunits (Gross and Bhattacharya, 2009b). As multifunctional proteins they could circumstantially interact with the Tic110 channel to regulate protein import according to the status of a biological process in which the protein is primarily associated. Reciprocally, their primary function might be regulated by protein import rates. For example, Tic55 is putatively involved in chlorophyll breakdown (Gray et al., 2004; Bhattacharya and Gross, 2009b). Hypothetically, its association with Tic110 might modulate protein import activity according to the status of chlorophyll catabolism in plastids. In contrast, Tic55 enzymatic turnover could be reciprocally finetuned by information on protein import rates relayed by the Tic110 channel. Although www.plantphysiol.org on July 15, 2017 Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved. 11 speculative, this “integrator hypothesis” offers a novel perspective on the functions of proteins such as Tic55, Tic32, Tic62, Tic20, Tic21, and Tic22, which still lack a defined mechanistic role in protein translocation. These include (i) the possibility of multifunctionality (e.g., Teng et al., 2006; Duy et al., 2007); which implies (ii) possible pleiotropic effects in gene knockout studies (e.g., Duy et al., 2007); (iii) a family-wide perspective to understand original and derived functions of the tested subunits (e.g., Balsera et al., 2007); (iv) the use of cyanobacteria to investigate conserved ancestral properties of many translocon subunits (e.g., Lv et al., 2009); and (v) a system biology understanding of protein translocation in plastids. Knowledge of the ancestral functions of the Toc and Tic subunits is also crucial to reconstruct the formative stages of plastid evolution. That the OM pore Toc75 is derived from a cyanobacterial Omp85-type of beta-barrel assembly protein suggests the onset of plastid organellogenesis was marked by the host cell, thus gaining control over OM activities of the endosymbiont (Gross and Bhattacharya, 2009b). The Toc75 homolog OEP80 could still in modern plastids, retain this ancestral function in the biogenesis of beta-barrel proteins (Patel et al., 2008). In addition, the development of plastid organellogenesis included the establishment of an IM pore to reprogram the cyanobacterial permeome predominantly with host-derived solute carriers (Tyra et al., 2007). To illuminate this crucial step in plastid evolution it is important to investigate whether special Tic subcomplexes (e.g., involving or not Tic20, Tic21, and/or Tic110) or a still unknown pore-forming complex might control the IM permeome assembly in modern plastids (Firlej-Kwoka et al., 2009). OUTLOOK Developments and discoveries made in the fields of plastid origin and algal evolution have come rapidly in recent years. The foreseeable future of these fields is rife with uncertainties and challenges, largely depending on the advancement of techniques in molecular and computational biology. There are many genome projects nearing completion; e.g., Chondrus crispus (florideophyte red alga), Calliarthron tuberculosum (coralline red alga), Porphyra umbilicalis (Bangiales red alga; see Blouin et al., 2011), Guillardia theta (cryptophyte), and Bigelowiella natans (chlorarachniophyte). The rapid www.plantphysiol.org on July 15, 2017 Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved. 12 advances in sequencing, e.g., single-cell environmental genomics (Woyke et al., 2009) and functional genomics have opened up new windows into genome evolution and gene function in non-model taxa that were unimaginable only a decade ago. The ideas highlighted in this update are therefore destined to change in the near future, as new data are analyzed and old hypotheses re-interpreted. In particular, work on Paulinella by different research groups will be crucial to test existing ideas about the evolution of organelle protein import and more generally the process of organellogenesis. We also expect the eukaryote tree of life to undergo some adjustments as investigators begin to understand better the dynamics of protist genome evolution and the limitations these forces place on phylogenetic approaches. Rather than being discouraged by this rapid pace of research we are excited to recognize that algal and plastid research have become center stage for both academic pursuits and for their key role in applied uses such as biofuel development. ACKNOWLEDGMENTS This research was supported by grants from the National Science Foundation awarded to HSY and DB (EF 08-27023, DEB 09-36884) and MCB 09-46528 awarded to DB. 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BMC Evol Biol 9: 98 Yoon HS, Ciniglia C, Wu M, Comeron JM, Pinto G, Pollio A, Bhattacharya D (2006a) Establishment of endolithic populations of extremophilic Cyanidiales (Rhodophyta). BMC Evol Biol 6: 78 Yoon HS, Reyes-Prieto A, Melkonian M, Bhattacharya D (2006b) Minimal plastid genome evolution in the Paulinella endosymbiont. Curr Biol 16: R670-R672 Yoon HS, Zuccarello GC, Bhattacharya D (2010) Evolutionary history and taxonomy of red algae. In J Seckbach, D Chapman, eds, Red Algae in the Genomic Age, Vol 13, Cellular Origins, Life in Extreme Habitats and Astrobiology. Springer, New York, pp 25-42 www.plantphysiol.org on July 15, 2017 Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved. 19 FIGURE LEGENDS Figure 1. Evolutionary history of red algal genes as determined in Chan et al. (2011). (A) Output from an automated phylogenetic pipeline was analyzed to identify the percentage of maximum likelihood (RAxML) trees that support different groupings of red and green (RG) algae with other eukaryotes at bootstrap support ≥90%. The impact of increasing the number of terminal taxa in each RAxML tree (N) on the percentage of shared genes is shown for taxa 4→10→20→30→40. The category RG-exclusive are trees in which RG algae and another phylum form an exclusive clade, whereas RGshared are trees in which RG monophyly is recovered but other phyla are positioned within this clade (i.e., due to gene “sharing” explained by E/HGT). Surprisingly, nearly 50% of RG genes are shared with other eukaryotes due to E/HGT. Due to the expectation of gene transfer associated with algal endosymbiosis we do not interpret this result as evidence for monophyly of RG algae with groups such as alveolates. Rather, it is more likely that RG genes have been transferred to the nucleus of some alveolates and the signal of algal ancestry is recovered in the phylogenies. (B) Schematic representation of the tree of life showing the extent of RG sharing with other eukaryotes and with prokaryotes indicated by the numbers to the right of the phylum names. The branch shown as a dashed line represents ambiguous relationships among the lineages to the right. As would be predicted, the “chromalveolates” (monophyly still in question) shown in the gray field are recipients of many RG genes due to endosymbiosis (see Moustafa et al., 2009), whereas the Euglenozoa share many algal genes due to their green alga-derived plastid and associated EGT (e.g., Durnford and Gray, 2006). Significant gene sharing however extends between these recipient lineages. See Chan et al. (2011) for more details. Figure 2. Evolution of photosynthetic Paulinella species. (A) Schematic maximum likelihood (RAxML) phylogenetic tree of plastid-derived 16S rDNA from P. chromatophora, P. microporus, algal and plant (Plantae) plastids, and the cyanobacterial donors of these organelles. Note the clear independent origins of Plantae and photosynthetic Paulinella plastids. The numbers at the nodes show support values derived from a RAxML bootstrap analysis followed by those from a PhyML www.plantphysiol.org on July 15, 2017 Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved. 20 analysis. When same, this is marked with an asterisk and when a node is not resolved with a method than this is denoted with dashes. Only bootstrap values ≥50% are shown. Branch lengths are proportional to the number of substitutions per site (see scale bar). See Yoon et al. (2009) for details. (B) Light micrograph images of P. chromatophora (top two images) and P. microporus (bottom two images). The scale bar indicates 5 μm. Figure 3. Schematic representation of The Toc/Tic translocons embedded in the chloroplast membranes of land plants (image on right) in the process of importing a preprotein from the cytosol (figure based on Gross and Bhattacharya, 2009b). The unfolded cytosolic protein contains a transit peptide (in red) that is initially recognized by the receptors Toc34 and Toc159 and then imported across the outer and inner envelope membranes (OEM and IEM, respectively) via Toc75 and Tic110. The energy dependent translocation process is driven by heat shock-type molecular chaperones (Hsp70 and Hsp93) at the cytosolic inter membrane space (IMS), and stromal sides of the organelle. The stromal processing peptidase (SPP) removes the transit peptide after translocation. The subunits of the translocon of cyanobacterial (endosymbiont) origin are shown in green, those of eukaryote origin are in red, whereas those in yellow are of unknown phylogenetic affiliation. The proposed ancestral state of the complex plant translocon is shown at the left and is composed of the Toc34, Toc75, and Tic110 subunits and the Hsp70 and Hsp93 chaperones. Tic20 and Tic21 are depicted by broken lines, indicating their possible participation in the ancestral translocon (discussed in Gross and Bhattacharya, 2009b). Evolution of the plant translocons required the addition of subunits to fulfil an exclusive new function in protein import (Toc12, Toc159, and Tic40) and subunits recruited to the translocon but maintaining ancestral properties (below the arrow). We tentatively classified Toc64 in the latter group because it shows sequence conservation with plant amidases (67% amino acid similarity over the full sequence of functional Amidase 1 in Arabidopsis). The potential correspondence of protein import-related anion channel (PIRAC) to Tic20 is discussed in detail in Gross and Bhattacharya (2009b). www.plantphysiol.org on July 15, 2017 Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved.