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What Was the Real Contribution of to the Eukaryotic Nucleus? Insights from Photosynthetic

David Moreira and Philippe Deschamps

Unite´ d’Ecologie, Syste´matique et Evolution, UMR CNRS 8079, Universite´ Paris-Sud, 91405 Orsay Cedex, France Correspondence: [email protected]

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

oday it is widely accepted that photosynthe- filose that hosts a cyanobacterium with Tsis originated in eukaryotes by the endosym- a reduced genome that has been described as biosis of a cyanobacterium within a heterotro- “a plastid in the making” (Marin et al. 2005; phic eukaryotic . This occurred in a line- Keeling and Archibald 2008; Nowack et al. age that subsequently diversified to give rise to 2008). Primary endosymbioses resulted in the the three contemporary groups of primary pho- establishment of plastids with two membranes. tosynthetic eukaryotes: Viridiplantae (includ- However, a vast variety of eukaryotes possess ing and land ), Rhodophyta plastids with three or more membranes. They and Glaucophyta, grouped collectively within a derive from the endosymbioses of primary pho- unique eukaryotic superphylum called Archae- tosynthetic eukaryotes within other eukaryotic plastida (Adl et al. 2005) or Plantae (Cavalier- cells (Delwiche 1999; Keeling 2013). Such sec- Smith 1982). Recently, a second case of primary ondary endosymbioses have spread photosyn- endosymbioses has been unveiled thanks to the thesis across the eukaryotic tree, either by the characterization of Paulinella chromatophora,a endosymbiosis of red or of green algae. Whereas

Editors: Patrick J. Keeling and Eugene V. Koonin Additional Perspectives on The Origin and Evolution of Eukaryotes available at www.cshperspectives.org. Copyright # 2014 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a016014 Cite this article as Cold Spring Harb Perspect Biol 2014;6:a016014

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D. Moreira and P. Deschamps

it is almost certain that secondary endosymbi- origin in their nuclear genomes. This has been oses of green algae occurred twice (in euglenids shown for a variety of nonphotosynthetic eu- and chlorarachniophytes), secondary red algal karyotes, such as, for example, apicomplexan plastids are found in a variety of , stra- parasites (Fast et al. 2001; Roos et al. 2002; Wil- menopiles, cryptophytes, and , and liams and Keeling 2003; Huang et al. 2004), the number of red algal endosymbioses at the perkinsids (Stelter et al. 2007; Matsuzaki et al. origin of these groups has been matter of intense 2008; Ferna´ndez Robledo et al. 2011) or non- debate (Baurain et al. 2010; Keeling 2010, 2013; photosynthetic dinoflagellates (Sanchez-Puerta Burki et al. 2012b). Moreover, the existence of et al. 2007; Slamovits and Keeling 2008), and tertiary endosymbioses (namely, the green algae (de Koning and Keeling 2004). Al- of a secondary photosynthetic within though much more controversial, potential another eukaryotic ) and of plastid replace- EGTs have also been used to propose a photo- ments makes the picture of plastid evolution synthetic ancestry for (Reyes-Prieto et al. in eukaryotes even more complex. Dinoflagel- 2008) or that algae with secondary plastids of lates, some of which have replaced their ances- red algal origin, such as diatoms and chromer- tral red algal plastids by green algae, diatoms, ids, may have contained green algal endosymbi- haptophytes, or cryptophytes, are paradigmatic onts in their past (Moustafa et al. 2009; Woehle examples of such complex situations (Keeling et al. 2011). Likewise, several dozens of potential 2013). EGTs have been detected in algae and plants The evolution of plastids has been studied that appear to have been acquired from Chla- using genes from the plastid genome as well as mydiae, a group of parasitic (Huang typical eukaryotic nuclear genes, which allow and Gogarten 2007; Becker et al. 2008; Moustafa inferring the phylogenies of both the plastids et al. 2008), which led to proposing that cryp- and their hosts. The use of those markers has tic chlamydial endosymbionts may have helped led to interesting discoveries, such as the mono- to establish the first plastids, in particular, by phyly of the (Moreira et al. 2000; providing essential functions for plastid activity Rodrı´guez-Ezpeleta et al. 2005) or the difficul- (Greub and Raoult 2003; Ball et al. 2013; Baum ties in reconciling the plastid and host histories 2013). in eukaryotes with red algal plastids (Baurain Werevise here some of these cases of cryptic et al. 2010; Burki et al. 2012b). However, a third endosymbiosis, with special attention on the of genes can also provide useful comple- difficulties in accurately detecting EGT and the mentary information: the genes of plastid ori- importance of proper phylogenetic analysis and gin retrieved within the nuclear genome of the of an adequate taxonomic sampling to achieve host. In fact, contemporary plastids have small that task. genomes, which is due to the fact that most of the original cyanobacterial symbiont genes CRYPTIC GREEN ALGAL ENDOSYMBIOSES were lost or transferred to the host nucleus (by IN DIATOMS AND CHROMERIDS a process called endosymbiotic gene transfer, EGT) during the evolution of plastids (Weeden Diatoms are a speciose group of unicellular al- 1981; Martin et al. 1998). These transfer events gae belonging to the Stramenopila or are not restricted to plastid endosymbioses— Heterokonta, whereas chromerids are a recently the same phenomenon occurred during the en- discovered group of algae closely related to the dosymbiosis that gave rise to the mitochondria (Moore et al. 2008). Thanks to the (Gray et al. 1999; Burger et al. 2003). availability of complete genome sequences or of EGT genes may serve to study the evolution- data, recent studies have tried to ary history of plastids and, in particular, the identify EGTs in these . In the case of presence of cryptic endosymbioses. In fact, spe- diatoms, a phylogenomic survey performed by cies that had a plastid in the past but lost pho- Moustafa et al. (2009) detected 4956 putative tosynthesis may have conserved genes of plastid EGTs in the genomes of the species Thalassiosira

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Endosymbiotic Gene Transfer in Photosynthetic Eukaryotes

pseudonana (2533 cases) and Phaeodactylum the idea that the green signal did not reveal true tricornutum (2423 cases). Such a large number green algal ancestry but just the lack of a sam- of EGTs was not necessarily surprising because, pling of red algal genes as rich as the one avail- for example, thousands of genes of presumed able for green algae and plants. Woehle et al. cyanobacterial origin have been seen in Arabi- (2011) thus concluded that improving the tax- dopsis thaliana (Martin et al. 2002). However, onomic sampling of should continue the origin of most of those EGTs in diatoms to erase the green signal observed in Chromera was completely unexpected. More than 70% of and, likely, also in diatoms. them appeared to be more closely related to green algal and homologs than to red algal A PROBLEM OF TAXONOMIC SAMPLING ... ones. This was astonishing because diatom plas- AND METHODS tids are widely accepted to be derived from a red algal , and, thus, EGTs should be The work by Woehle et al. showed the impor- related to red algal homologs. To explain their tance of taxonomic sampling for the accurate results, Moustafa et al. proposed that diatoms, characterization of EGTevents. This was a clear and perhaps other related phyla, originally ac- problem in the work of Moustafa and colleagues quired a plastid by the endosymbiosis of a green on diatoms. They compared all proteins en- alga, which was secondarily replaced by the red coded by two diatom genomes (T. pseudonana algal plastid found today. and P.tricornutum) against a local sequence da- In the case of chromerids, Woehle et al. tabase that, concerning the Archaeplastida, con- (2011) analyzed expressed sequence tags (ESTs) tained 11,356 protein sequences of red algae from the species Chromera velia. From 3151 and 193,394 from green algae and plants, name- ESTs, 513 appeared to be of EGT origin and ly, an overwhelming 1:17 red:green ratio. This had homologs in red and green algae. Two hun- disproportion could explain many of the cases dred sixty-three of them were more similar to in which diatom sequences were close to green red algal sequences, as expected, because chro- ones just because of the absence of red algal merids, as the closely related Apicomplexa, are homologs, an absence that could reflect incom- supposed to have secondary plastids of red algal plete sampling (i.e., the red algal genomes con- origin (Moore et al. 2008). However, a similar taining the corresponding genes have simply number of EGTs, 250, appeared to have an un- not been sequenced yet) rather than a true expected green algal ancestry. Although inferior general absence in all red algal species. More- to the 70% found in diatoms, this represented over, balanced taxonomic sampling is essential 50% of putative “green” EGT genes in chro- for accurate phylogenetic reconstruction (Le- merids. However, Woehle et al. interpreted cointre et al. 1993), and many trees where dia- their results in a very different way from Mou- toms branched with green sequences even in the stafa and coworkers. In fact, they considered the presence of red algal homologs should be tested green signal found in Chromera not as evidence with a richer sampling of red algal species. This is of a cryptic green algal endosymbiosis (which, what we tried in a study in which we reanalyzed in their own words, “leads to worryingly com- all of the putative EGTs in diatoms using a data- plicated evolutionary scenarios”) but most like- base with as many red algal sequences as possible ly as phylogenetic error, probably caused by (Deschamps and Moreira 2012). Wemanaged to poor taxonomic sampling for red algae. In fact, double the number of red algal sequences, and, when Woehle et al. repeated their analysis elim- even if the red:green ratio remained extremely inating all of the red algal sequences with the biased, this had a great impact on the inference exception of only one species (the highly de- of EGTevents (see below). rived thermophile Cyanidioschyzon merolae, in- In addition to the problem of unbalanced cidentally the single one used by Moustafa and taxonomic sampling, the diatom and Chromera coworkers), the number of potential green EGTs studies had a second source of potential bias: in Chromera slightly increased. This supported the use of automatic tools to filter phylogenetic

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D. Moreira and P. Deschamps

trees. In both cases, trees were screened to look These studies weakened the support for the for the nearest neighbor of diatoms and Chro- hypothesis of cryptic endosymbioses of green mera using PhyloSort (Moustafa and Bhatta- algae in diatoms and chromerids. They also, as charya 2008) and the Newick Utilities package Burkietal.(2012a)clearlystated,“emphasizethe (Junier and Zdobnov 2010), respectively. This lack of congruence and the subjectivity result- approach was very limited because it only con- ing from independent phylogenomic screens siders a pair of branches (the target—diatoms for EGT, which appear to call for extreme cau- or Chromera—and its closest relative) without tion when drawing conclusions for major evo- taking into account the general topology of trees lutionary events.” and their global support or the taxonomic rep- resentation of the different proteins across the eukaryotic diversity. In fact, that simplistic ap- DID CRYPTIC CHLAMYDIAE PLAY A ROLE AT THE ORIGIN OF PHOTOSYNTHETIC proach ignores very common problems such as EUKARYOTES? unresolved phylogenies (typical for single- marker phylogenetic analyses), hidden paralo- Plastids import ATPfrom the cell cytosol thanks gies, or very unbalanced taxonomic samplings. to their ATP/ADP translocases, which are en- The incidence of these problems on the infer- zymes that catalyze the transport of ATP across ence of EGTs in diatoms and Chromera was well a membrane in exchange with ADP. Among illustrated by the reanalyses conducted using a , these enzymes are very rare and manual inspection of phylogenetic trees instead have only been identified in a few obligate in- of the automatic filtering. In the case of Chro- tracellular bacteria belonging to the Chlamy- mera, Burki et al. (2012a) showed that, from the diales and the Rickettsiales. Both are parasitic 513 genes reported to have originated from red bacteria that steal ATPfrom their infected hosts. and green algae in an 1:1 ratio, only 51 ap- The first phylogenetic analysis of ATP/ADP peared to actually have an EGT origin, and, translocases that included a good representation among them, 23 were more closely related to of these bacteria suggested that these trans- red algae, only nine supported a green algal or- locases originated from a chlamydial ancestor igin, and 19 had an ambiguous signal. Using a and were transferred horizontally to rickettsiae similar manual approach to the study of diatom and plants (Schmitz-Esser et al. 2004). This was EGTs, we observed that only 286 cases (,10% in agreement with an earlier study showing a of the EGTs originally proposed by Moustafa high proportion of chlamydial proteins simi- and coworkers) actually supported an EGTori- lar to plant proteins, which was interpreted as gin (Deschamps and Moreira 2012). Moreover, the reflection of an unappreciated evolution- only 30 of these 286 cases appeared to have a ary relationship between the Chlamydiae and well-supported green algal origin, with more theCyanobacteria-chloroplastlineage(Brinkman cases supporting a red algal origin or just an et al. 2002). Some years subsequently, a massive ambiguous attribution. It is remarkable that phylogenomic analysis of the red alga Cyanidio- the two manual reanalyses (Burki et al. 2012a; schyzon merolae identified at least 21 additional Deschamps and Moreira 2012) converged to a genes that appeared to have been transferred similar degree of validation of only 10% of the to the primary photosynthetic eukaryotes from EGTs detected by automatic filtering. For dia- Chlamydiae similar to the genus Protochlamy- toms, among the .90% of cases rejected, half dia (Huang and Gogarten 2007). A similar anal- of them corresponded to genes with a very poor ysis including more genomes (C. merolae as representation of eukaryotic species, and the well as the green algae rein- other half to genes that produced phylogenet- hardtii, lucimarinus, and Ostreo- ic trees compatible with a vertical inheritance coccus tauri, and the diatoms Phaeodactylum from an ancient eukaryotic ancestor rather tricornutum and Thalassiosira pseudonana)re- than a more recent EGT from a green or red algal trieved 39 proteins of putative chlamydial ori- donor. gin, not only in primary photosynthetic eukary-

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Endosymbiotic Gene Transfer in Photosynthetic Eukaryotes

otes but also spreading through secondary plas- those 17 trees did not support the monophyly of tid endosymbioses to other photosynthetic eu- the Archaeplastida, with green algae and plants karyotes (Becker et al. 2008). The number of branching close to the Chlamydiae but red algae genes of potential chlamydial origin continued branching with . Tentrees showed to increase when a similar analysis with a larger a surprising pattern because Archaeplastida taxonomic sampling of photosynthetic eukary- emerged in a group containing the Chlamydiae otes led to the detection of 55 candidate genes in and the Cyanobacteria and, in some cases, a few algae and plants. Moreover, 37 were putatively other bacteria (e.g., Fig. 1A). A similar case, but plastid-targeted, reinforcing the potential role including Archaeplastida, Chlamydiae, and Al- of these genes in plastid functioning (Moustafa phaproteobacteria, was supported by two pro- et al. 2008). Those studies agreed to hypothesize teins (Fig. 1B). Nevertheless, the most common that a chlamydial endosymbiont or parasite may class of trees was the one in which Chlamydiae have facilitated the subsequent establishment of branched close to a variety of eukaryotes, not the cyanobacterial endosymbiont that give rise only including Archaeplastida but also nonpho- to the plastids. tosynthetic eukaryotes that are supposed to nev- More recently, it has been proposed that er have hosted plastids. In five cases, this con- chlamydial cells infecting the host of that prima- cerned just a single eukaryotic lineage (such as ry cyanobacterial endosymbiont secreted into the Diplomonadida—Giardia and Hexamita the host cytosol effector proteins that allowed [Fig. 1C] or the Mycetozoa—Dictyostelium) the host to use carbohydrates exported from the and in 14 other cases a larger eukaryotic diver- incipient plastid (Ball et al. 2013). Thus, Chla- sity,with , fungi, and other lineages (e.g., mydiae would have been essential not only to Fig. 1D). Thus, in .50% of the putative chla- stabilize the plastid endosymbiosis through the mydial genes in Archaeplastida, we either did long term by providing the ATP/ADP translo- not observe a real phylogenetic relationship cases, but also to allow the very first installation with this group or it concerned also nonphoto- of the cyanobacterial endosymbiont thanks to synthetic eukaryotes. those effector proteins. Such necessity of the Among the proteins that appeared to sup- presence of chlamydial cells would also explain port a putative chlamydial origin of the archae- why primary plastids have evolved so rarely plastid sequences, the 12 phylogenies showing (Ball et al. 2013; Baum 2013). Although this is a relationship also with cyanobacterial or al- a very interesting hypothesis, one potential ca- phaproteobacterial sequences were difficult to veat is that Chlamydiae have been detected as interpret. This was notably the case for the chla- parasites of a variety of phyla and differ- mydial-like ADP-Glc–specific starch synthases ent amoebae, but never of plants or algae (Horn postulated to have been crucial to the installa- 2008). tion of the first plastids in eukaryotes (see Fig. 2 We have addressed this question by reana- in Ball et al. 2013). Phylogenetic analyses have lyzing the largest set of Chlamydiae-like pro- suggested that Chlamydiae are closely related teins, 55 sequences, using Bayesian inference to , , and some phylogenetic reconstruction, which is less sen- other less-known lineages, forming the “PVC sitive than other methods to several tree recon- superphylum” (see Wagner and Horn 2006), struction artifacts. As in the case of the study of which has no particular affinity with the Cya- the putative diatom green EGTs, we retrieved a nobacteria or the . Thus, the variety of situations (P Deschamps and D Mo- close relationship of the Archaeplastida, Chla- reira, unpubl.). Whereas seven proteins did not mydiae, and Cyanobacteria and/or Alphapro- retrieve any close relationship between Chla- teobacteria observed in 12 of our phylogenetic mydiae and primary photosynthetic eukary- trees did not reflect the expected bacterial phy- otes, 17 others appeared to support a Chlamy- logeny (i.e., the PVC relationship). Twoalterna- diae–Archaeplastida relationship (two of them tive possibilities may explain those 12 phyloge- being found only in these two lineages). Two of nies: either that Chlamydiae are not the donors

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D. Moreira and P. Deschamps

0.79 mobile Rhodopseudomonas palustris ABStreptococcus agalactiae Bartonella henselae Coprococcus eutactus 1 1 Sinorhizobium meliloti kluyveri 0.86 Burkholderia ambifaria 0.58 1 Bacillus cereus Methylobacillus flagellatus maritima 1 Gloeobacter violaceus 1 Neisseria meningitidis 0.92 Prochlorococcus marinus 0.81 Polaromonas naphthalenivorans 1 Synechococcus sp. 1 Bordetella petrii 1 Synechococcus elongatus 1 1 Ralstonia metallidurans Nostoc punctiforme 1 0.71 Burkholderia phytofirmans 1 Synechocystis sp. Nitrococcus mobilis 1 Cyanothece sp. 0.75 Francisella tularensis trachomatis Pseudomonas putida 1 Chlamydia trachomatis Marinobacter sp. 1 pneumoniae 0.87 harveyi Chlamydophila caviae Haemophilus ducreyi 0.83 Chlamydophila abortus 1 Haemophilus influenzae 1 1 Chlamydophila felis Yersinia bercovieri Nitrosococcus oceani Escherichia coli 1 Methylococcus capsulatus 1 Klebsiella pneumoniae 1 1 Cyanothece sp. Planctomyces maris 0.76 Microcystis aeruginosa 0.77 biflexa 1 Cyanothece sp. 0.73 Nostoc sp. 1 1 Synechococcus sp. 1 Leptospira borgpetersenii 1 Physcomitrella patens 1 Rickettsiella grylli Physcomitrella patens (1/3) Zymomonas mobilis 1 Roseovarius sp. 1 Arabidopsis thaliana 1 Paracoccus denitrificans 1 Oryza sativa 0.87 Triticum aestivum 1 0.79 0.86 Rhodobacter sphaeroides 1 Chlamydomonas reinhardtii 0.87 Chlamydia trachomatis Ostreococcus lucimarinus 1 Chlamydomonas reinhardtii 0.72 1 Chlamydophila pneumoniae Ostreococcus lucimarinus Chlamydophila abortus 0.82 Arabidopsis tha/iana 1 1 Chlamydophila felis Sorghum bicolor 0.85 1 1 Zea mays 1 Chlamydophila caviae 0.88 Triticum aestivum Protochlamydia amoebophila 1 0.79 1 Oryza sativa Physcomitrella patens Chlamydomonas reinhardtii 0.89 1 Physcomitrella patens Ostreococcus lucimarinus Picea sitchensis 0.77 0.76 pusilla 1 Arabidopsis thaliana 0.85 Vitis vinifera 0.3 sp. 1 1 Vitis vinifera Chlamydomonas reinhardtii 0.85 0.79 1 Vitis vinifera Zea mays 1 Zea mays 1 Oryza sativa 0.3 Oryza sativa 1 1 Oryza sativa C thermophila burgdorferi 1 0.84 denticola 1 Galdieria sulphuraria 0.91 1 Cyanidioschyzon merolae Emiliania huxleyi D Flavobacteria bacterium Giardia Iamblia 0.89 Cytophaga hutchinsonii 0.76 1 Hexamita inflata Amoebophilus asiaticus Chlorella sp. Desulfococcus oleovorans 1 Bartonella quintana Arabidopsis thaliana Roseobacter sp. 1 1 1 Oryza sativa 1 Clostridium novyi 1 Bacteroides capillosus Physcomitrella patens 1 Physcomitrella patens Ruminococcus torques 1 0.81 Thermobifida fusca (1/3) berghei ulcerans Plasmodium vivax 1 0.72 1 agalactiae 1 Plasmodium yoelii 1 Bacillus thuringiensis 0.86 Physcomitrella patens 1 0.85 1 Physcomitrella patens 1 Listeria monocytogenes faecalis Arabidopsis thaliana 1 1 1 1 Bacillus cereus Citrus sinensis Chromobacterium violaceum 1 Oryza sativa Neisseria meningitidis 0.81 Oryza sativa Escherichia coli 1 0.83 Oryza sativa 0.99 enterica 1 Marinomonas sp. 1 Oryza sativa 0.88 Vibrio cholerae Protochlamydia amoebophila Dictyostelium discoideum 0.83 0.86 Chlamydophila pneumoniae Saccharomyces cerevisiae Chlamydia muridarum 1 Pichia stipitis Drosophila melanogaster 1 1 Chlamydia trachomatis 1 1 Anopheles gambiae 0.74 Chlamydophila caviae 1 Homo sapiens 0.83 Chlamydophila abortus Mus musculus Chlamydophila felis 1 Rattus norvegicus Paramecium tetraurelia Chlamydia trachomatis 0.85 1 Euglena gracilis 1 Chlamydia muridarum 0.84 1 Chlamydophila pneumoniae Cryptosporidium hominis 1 Chlamydophila pneumoniae Micromonas pusilla Chlamydophila abortus Babesia bovis 1 Chlamydophila felis 0.76 1 0.89 1 Theileria annulata 1 1 Chlamydophila caviae Cryptosporidium hominis Protochlamydia amoebophila 1 Cyanidioschyzon merolae 0.85 Plasmodium berghei 1 1 Galdieria sulphuraria Chlamydophila pneumoniae Ostreococcus lucimarinus Chlamydia muridarum 1 Chlamydomonas reinhardtii 1 1 Chlamydia trachomatis 1 Ostreococcus tauri Chlamydophila abortus Physcomitrella patens 0.81 Arabidopsis thaliana 0.3 1 Chlamydophila caviae 0.3 1 Oryza sativa Chlamydophila felis 1 0.85 0.84 Vitis vinifera

Figure 1. Bayesian phylogenetic trees of proteins suggested to have been acquired by photosynthetic eukaryotes from Chlamydiae. (A) Starch synthase/glycogen synthase. (B) Glycosyltransferase. (C) Pyrophosphate-depen- dent phosphofructokinase. (D) Tyrosyl-tRNA synthase. (Bold orange) Species names correspond to chlamydiae; (blue) cyanobacteria; (brown) ; (green) green algae and plants and eukaryotes with sec- ondary green algal plastids; (red) red algae; (pink) eukaryotes with secondary red algal plastids. (Bold black) Species are nonphotosynthetic eukaryotes. Some branches shortened to one-third of their actual length are indicated by (1/3). Numbers at branches are posterior probabilities. The scale bar indicates the number of substitutions per position.

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Endosymbiotic Gene Transfer in Photosynthetic Eukaryotes

of those genes but the recipients in hypothetical synthetic eukaryotes. The extent of the contri- Archaeplastida-to-Chlamydiae horizontal gene bution of these different endosymbionts to the transfer (HGT) events, or that the ancestral cy- host genome remains uncertain, but some re- anobacterium at the origin of the first plastid searchers consider that it may have been massive had acquired those genes from chlamydial do- with, for example, 75% of Saccharomyces cer- nors and then transferred them to the eukary- evisiae genes of putative bacterial origin (Esser otic host by EGT (Wolf et al. 1999). The fact et al. 2004) and 18% of A. thaliana genes of that, as explained before, no chlamydial species potential cyanobacterial origin (Martin et al. is known to infect plants or algae could be seen 2002). However, most of these analyses were per- as evidence against the first hypothesis. In con- formed several years ago with the limited taxo- trast, the second hypothesis could also explain nomic sampling available at that time, which the observed Archaeplastida–Chlamydiae–Al- may have induced an overestimation of the phaproteobacteria relationships by Chlamydi- number of EGTevents. ae-to-Alphaproteobacteria HGTs followed by In addition, these are very ancient events EGTs to eukaryotes through the mitochondrial that took place many millions of years ago, so endosymbiosis. This same idea has been pro- that the phylogenetic signal conserved in in- posed in more general grounds to explain the dividual genes has been considerably erased. many non-alphaproteobacterial genes of bacte- In addition, the genes acquired by EGT had rial origin found in a phylogenomic study of the to adapt to a new genomic environment and yeast nuclear genome (Esser et al. 2004). Like- then, often, accelerated their evolutionary rates wise, the cases in which Viridiplantae branched (Baurain et al. 2010). The combination of these close to Chlamydiae but not the other archae- two factors makes the identification of those plastid lineages (which branched, as expected, EGT genes very difficult. This is especially the close to Cyanobacteria) could also argue against case for secondaryendosymbiotic events involv- the idea of a very early implication of Chlamyd- ing red and green algae. These two groups of iae in the origin of Archaeplastida. This phylo- algae are separated by a single node and a very genetic pattern has already been observed for short evolutionary distance in most phylogenet- the 4-hydroxy-3-methylbut-2-en-1-yl diphos- ic trees, so even small biases in tree reconstruc- phate synthase, involved in carotenoid biosyn- tion can be enough to shift a sequence from one thesis (Frommolt et al. 2008). Thus, the real group to the other. Moreover, the distance be- contribution of Chlamydiae to the acquisition tween the Archaeplastida and other eukaryotic of the first plastids has to be seen as a not yet groups seems also to be very short, so that tree close, difficult evolutionary question. reconstruction artifacts can easily misplace ar- tificially a sequence close to or far from them, not to speak about the numerous gene, or even CONCLUDING REMARKS complete genome, duplications that most eu- Comparative genomic and phylogenomic anal- karyotes have experienced during their evolu- yses have shown that eukaryotic genomes are tion and that very frequently lead to hidden intricate patchworks of genes of different evo- paralogy problems. Because of all these prob- lutionary origins. This is a general situation lems, it is naı¨ve to believe that rigid automatic because all known eukaryotes have, at least, in- procedures can accurately identify the complete tegrated genes from the mitochondrial endo- set of EGT genes in a given genome. On the symbiont into their nuclear genomes. The case contrary, these methods have been shown to of photosynthetic eukaryotes is certainly the greatly overestimate EGT to levels approaching most complex one, because their nuclear ge- not less than 90% of false positives (Burki nomes have been bombarded by bacterial genes et al. 2012a; Deschamps and Moreira 2012). coming from the primary plastids but also by Not only careful analyses trying to control for eukaryotic genes provided by the nuclei of red as many of those biases as possible are unavoid- or green algae in the case of secondary photo- able to address these questions, but, repeating

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D. Moreira and P. Deschamps

Burki et al.’s words, it is also important to avoid Deschamps P,Moreira D. 2012. Reevaluating the green con- as much as possible the subjectivity in those tribution to diatom genomes. Genome Biol Evol 4: 683– 688. analyses and remember to keep “extreme cau- Esser C, Ahmadinejad N, Wiegand C, Rotte C, Sebastiani F, tion when drawing conclusions for major evo- Gelius-Dietrich G, Henze K, Kretschmann E, Richly E, lutionary events.” Leister D, et al. 2004. A genome phylogeny for mitochon- dria among a-proteobacteria and a predominantly eu- bacterial ancestry of yeast nuclear genes. Mol Biol Evol 21: 1643–1660. ACKNOWLEDGMENTS Fast NM, Kissinger JC, Roos DS, Keeling PJ. 2001. Nuclear- encoded, plastid-targeted genes suggest a single common We thank the French Centre National de la Re- origin for apicomplexan and dinoflagellate plastids. Mol cherche Scientifique (CNRS) for support. Biol Evol 18: 418–426. 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What Was the Real Contribution of Endosymbionts to the Eukaryotic Nucleus? Insights from Photosynthetic Eukaryotes

David Moreira and Philippe Deschamps

Cold Spring Harb Perspect Biol 2014; doi: 10.1101/cshperspect.a016014

Subject Collection The Origin and Evolution of Eukaryotes

The Persistent Contributions of RNA to Eukaryotic Origins: How and When Was the Eukaryotic Gen(om)e Architecture and Cellular Mitochondrion Acquired? Function Anthony M. Poole and Simonetta Gribaldo Jürgen Brosius Green Algae and the Origins of Multicellularity in Bacterial Influences on Animal Origins the Plant Rosanna A. Alegado and Nicole King James G. Umen The Archaeal Legacy of Eukaryotes: A Missing Pieces of an Ancient Puzzle: Evolution of Phylogenomic Perspective the Eukaryotic Membrane-Trafficking System Lionel Guy, Jimmy H. Saw and Thijs J.G. Ettema Alexander Schlacht, Emily K. Herman, Mary J. Klute, et al. Origin and Evolution of the Self-Organizing The Neomuran Revolution and Phagotrophic Cytoskeleton in the Network of Eukaryotic Origin of Eukaryotes and Cilia in the Light of Organelles Intracellular Coevolution and a Revised Tree of Gáspár Jékely Life Thomas Cavalier-Smith On the Age of Eukaryotes: Evaluating Evidence Protein Targeting and Transport as a Necessary from Fossils and Molecular Clocks Consequence of Increased Cellular Complexity Laura Eme, Susan C. Sharpe, Matthew W. Brown, Maik S. Sommer and Enrico Schleiff et al. Origin of Spliceosomal Introns and Alternative How Natural a Kind Is ''Eukaryote?'' Splicing W. Ford Doolittle Manuel Irimia and Scott William Roy

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Protein and DNA Modifications: Evolutionary What Was the Real Contribution of Imprints of Bacterial Biochemical Diversification Endosymbionts to the Eukaryotic Nucleus? and Geochemistry on the Provenance of Insights from Photosynthetic Eukaryotes Eukaryotic Epigenetics David Moreira and Philippe Deschamps L. Aravind, A. Maxwell Burroughs, Dapeng Zhang, et al. The Eukaryotic Tree of Life from a Global Bioenergetic Constraints on the Evolution of Phylogenomic Perspective Complex Life Fabien Burki Nick Lane

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Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved