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Should I stay or should I go? Retention and loss of components in vestigial endosymbiotic organelles
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ScienceDirect Should I stay or should I go? Retention and loss of components in vestigial endosymbiotic organelles Kacper Maciszewski and Anna Karnkowska Our knowledge on the variability of the reduced forms of endosymbiotic organelles – mitochondria and plastids – is expanding rapidly, thanks to growing interest in peculiar microbial eukaryotes, along with the availability of the methods used in modern genomics and transcriptomics. The aim of this work is to highlight the most recent advances in understanding these organelles’ diversity, physiology and evolution. We also outline the known mechanisms behind the convergence of traits between organelles which have undergone reduction independently, the importance of the earliest evolutionary events in determining the vestigial organelles’ eventual fate, and a proposed classification of nonphotosynthetic plastids. Address Department of Molecular Phylogenetics and Evolution, Biological and Chemical Research Centre, Faculty of Biology, University of Warsaw, ul. _ Zwirki i Wigury 101, 02-089, Warsaw, Poland Corresponding author: Karnkowska, Anna ([email protected])
Current Opinion in Genetics & Development 2019, 59:33–39 This review comes from a themed issue on Evolutionary genetics Edited by Jeremy Wideman and Thomas Richards
https://doi.org/10.1016/j.gde.2019.07.013 0959-437X/ã 2019 Elsevier Ltd. All rights reserved.
Introduction Simplification sensu lato, including such phenomena as differential loss or convergent loss among others, is one of the main processes shaping diversity, but its role in the evolution has been appreciated only recently [1]. Great examples of simplification come from the evolution of endosymbiotic organelles: mitochondria and plastids. They underwent dramatic reduction early in their evolutionary history through partial loss of contents, from genes to metabolic pathways, becoming fully dependent on their host—the eukaryotic cell. In both cases, further reduction happened many times across the tree of life. Vestigial plastids (leucoplasts) and reduced mitochondria organelles, MROs) were (mitochondrion-related described decades ago, but thanks to high-throughput sequencing techniques, and growing interest in the www.sciencedirect.com
diversity of microbial eukaryotes, we are now discovering the range of reduced forms of these organelles.
Reduction is widespread Many distinct eukaryotic lineages have adapted to living in low oxygen conditions independently, which is reflected on the patchy distribution of MROs on the tree of eukaryotes. MROs are present in all supergroups except Archaeplastida [2]; in some lineages they are rare (e.g. Euglenozoa, Apicomplexa), while in others, such as Metamonada, they are ubiquitous [3]. Their diverse forms, such as mitosomes (e.g. Giardia, Entamoeba), hydrogenosomes (e.g. some metamonads, ciliates, archamoebae), and hydrogen-producing mitochondria (e.g. Blastocystis, Nyctotherus), can be found in endobiotic parasites, commensals, or mutualistic symbionts. Moreover, the number of examples of free-living MRO-bearing organisms is growing: these include the amoebozoa, breviates, heteroloboseans, stramenopiles, jakobids, metamonads and flagellates related to diplomonads [4], and multicellular animals from the phylum Loricifera [5]. Every group that acquired plastids, except for haptophytes, has secondarily nonphotosynthetic representatives. Colorless red algae and plants are almost always parasitic, but among colorless secondary plastid-bearing algae, there are free-living phagotrophs and osmotrophs as well [6–9]. Nonphotosynthetic groups are often sister lineages to the mixotrophic ones, suggesting that loss of photosynthesis might be promoted in mixotrophs if the energy costs of maintaining the photosynthetic apparatus outweigh the benefits of its products [10], especially considering that phototrophs employ detoxification mechanisms to counteract reactive oxygen species generated by photosynthesis [11].
Convergence in the evolution of reduced organelles MROs in unrelated eukaryotes show strikingly convergent features, suggesting a limited range of possible mitochondrial adaptations to hypoxia. In most of the known examples, the electron transport chain (ETC) has been truncated or lost, and the organisms fulfil their energetic requirements with ATP produced by substrate-level phosphorylation [4]. In order to explain the early steps of reductive evolution of mitochondria, intermediate stages in transition to the anaerobic lifestyle must be investigated. Among the organisms postulated to possess these intermediate traits are Brevimastigomonas motovehiculus [12], Current Opinion in Genetics & Development 2019, 58:33–39
34 Evolutionary genetics
Blastocystis, and Nyctotherus [2], whose mitochondria are modified to function under hypoxic conditions, but without concurrent loss of some typically mitochondrial features. Brevimastigomonas’s MROs retained mitochondrial metabolic pathways, such as the TCA cycle and ETC-driven ATP synthesis, but also pyruvate metabolism and substrate-level phosphorylation (Figure 1). In contrast, its mitochondrial genome is degenerate, with protein-coding contents including ETC complexes I, III, IV, and V (complex II is nucleusencoded), but complexes III and IV are highly divergent and possibly incapable of efficient electron transport. Degeneration or complete loss of the oxidative phosphorylation pathway (OXPHOS) is also reflected in the reduction of mitochondrial cristae, as demonstrated by transmission electron microscopy and molecular analyses showing that the loss of OXPHOS co-occurs with the loss of the cristae organizing complex MICOS [13]. This example suggests that the free-living organisms most likely first evolved the capacity to survive in anaerobic environments by gaining new metabolic pathways and establishing anaerobic mitochondria,
which eventually underwent reduction and evolved into hydrogenosomes and mitosomes (Figure 1). Following the loss of the ETC, all hydrogenosomes and mitosomes lost their genomes, and consequently, all proteins functioning in those organelles are encoded by the nucleus. Surprisingly, mitochondrial genome loss has recently been documented in the aerobic, OXPHOS-capable dinoflagellate Amoebophrya ceratii [14], which indicates that such occurrence is not exclusive to anaerobic MROs, therefore subverting the previously proposed hypothesis that the functional ETC is a universal constraint against mtDNA loss [15]. Still, the remnant mitochondria, regardless of the extent of their reduction, always possess mitochondrial transport machinery, proteins involved in the production or import of ATP and, with the exception of Entamoeba, the ISC (iron–sulfur cluster) machinery (Figure 1)—a single operon-encoded pathway of Fe–S cluster assembly [16]. In case of plastids, the loss of photosynthetic functions is also reflected in the organellar morphology,
Figure 1
Brevimastigomonas-like MRO
LGT
Mitochondrion
Bacteria Anaerobic mitochondrion
Amoebophrya-like mitochondrion
Hydrogenosome
Mitosome
genes encoding Fe-S cluster formation system ISC
LGT
genes encoding other mitochondrion-targeted proteins genes encoding proteins involved in oxidative phosphorylation (OxPhos)
Bacteria genes encoding mitochondrial transport membrane proteins genes encoding proteins involved in substrate phosphorylation
Amitochondriate
proteins
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Schematic representation of the stages of reductive evolution of mitochondrion-related organelles, documented in the available case studies.
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Reductive evolution of endosymbiotic organelles Maciszewski and Karnkowska 35
where thylakoids are degenerate [17] or absent [18,19] (Figure 2). The vestigial plastids are incapable of photosynthesis and usually lack all genes involved in this process, although the recently discovered retention of the chlorophyll biosynthesis gene cluster in corallicoid apicoplasts is a prominent exception [20]. Nonetheless, a large part of the overall plastid genetic repertoire may remain, specifically: ribosomal protein genes (rps, rpl), rRNAs, tRNAs, usually rpo, and a number of other genes, which most commonly include accD, clpC, sufB, tufA and/or infA. This pattern has been observed in a variety of organisms, such as mycoheterotrophic plants [21–25], apicomplexans [26,27], and parasitic red and green algae [28,29,30]. One of the repeatedly occurring exceptions is rbcL—a crucial photosynthesis-related gene, present in a number of leucoplast genomes, both in plants [31] and microbial eukaryotes of diverse ancestry [32–34]. As shown for one of the latter, the euglenid Euglena longa, rbcL has a low level of expression, the full RuBisCO molecules are likely not assembled at all, and its function remains under investigation [34,35]. Despite their vestigial character, leucoplasts are far from being disposable. The retained genes encode mostly housekeeping proteins, allowing transcription and translation in the organelle, but others are involved in the indispensable processes performed in the apochlorotic plastids, including biosynthesis of isoprenoids, tetrapyrroles, heme, Fe–S clusters, and fatty acids (Figure 2) [8,9]. The
sets of retained pathways and their plastid-encoded components can be diverse between lineages: sufB and clpC genes are often among the only non-housekeeping genes in the red lineage-derived leucoplasts [9], but in plants, it is usually accD and clpP [8]. Although fatty acid biosynthesis is the fundamental leucoplast function in plants [8] and apicomplexans [18], it is the one most commonly lost in nonphotosynthetic dinoflagellates [7] and chrysophytes [36]. Curiously, land plants are prone to loss of plastid rpo genes probably because they possess nuclear-encoded, phage-derived NEP polymerase, which can compensate for the loss of rpo [8]. In contrast to MROs, almost all known colorless plastids retain at least a rudimentary genome. Plastids without a genome are rare, yet some documented cases exist (Figure 2), for example: a green alga Polytomella [37], a parasitic plant Rafflesia [38,39], and a colpodellid Voromonas pontica [40].
The range of reduced forms MROs represent various outcomes and transitional stages of a gradual evolutionary process, which greatly complicates the efforts to systematize them. Most often they are classified according to their metabolic capacity: into anaerobic mitochondria, hydrogen-producing mitochondria (HMPs), hydrogenosomes and mitosomes [2] (Figure 1). This classification does not reflect the evolutionary history, as related taxa often possess different
Figure 2
genome-deprived leucoplast loss of plastid genome
loss of photosynthesis
leucoplast
cytosolic variant loss
hypothetical intermediate stage
photosynthetic plastid
plastid variant loss
stage after the acquisition of the plastid nuclear/plastid pathway redundancy unsolved loss of photosynthesis
hypothetical intermediate stage genes encoding proteins involved in photosynthesis genes encoding proteins involved in other metabolic pathways plastid completely lost
genes encoding plastid transport membrane proteins
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Schematic representation of the stages of reductive evolution of nonphotosynthetic plastids. For the sake of clarity, hypothetical intermediate stages are included.
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36 Evolutionary genetics
MROs, and each MRO type is distributed across unrelated lineages. Differences in components, functions, and morphological features among MROs exist, which may have been generated by a secondary gene loss and/or lateral gene transfer [41]. In metamonads, which are a showcase for the diversity of MROs, additional data coming from bacteriovorous, freeliving Carpediemonas-like organisms allowed deeper analysis of lineage-specific gains and losses [3]. Several conserved mitochondrial enzymes have very scarce distribution: cardiolipin synthase is present only in Carpediemonas and aminoadipate-semialdehyde dehydrogenase—in Trimastix marina, indicating that they have probably been vertically inherited from the last eukaryotic common ancestor, but later lost in majority of MROs. Another interesting evolutionary pattern emerges from the distribution of glycine cleavage system (GCS): parasitic and secondarily free-living lineages always lack a complete GCS, whereas ancestrally free-living lineages possess the complete system. Even more complicated is the evolutionary history of acetyl-CoA synthase (ACS), which generates ATP directly from the conversion of acetyl-CoA to acetate. Two types of ACS were identified in metamonads: the cytosolic ACS1, proposed to be acquired through a single LGT event into the last common ancestor of Chilomastix, Kipferlia, Dysnectes and diplomonads, and the other ACS homologue (ACS2) of different origin, yet it remains unclear if it was ancestrally present in metamonads [3]. Furthermore, the diplomonad Spironucleus most likely possesses both ACS1 and ACS2, but the latter is localized in its hydrogenosome and was probably acquired through LGT [42]. Comparative studies of the fornicates allowed a detailed reconstruction of intermediate stages of MROs. The MRO of the freeliving Kipferlia bialata with more complex suite of metabolic pathways involved in ATP generation such as succinyl-CoA synthase (SCS) and two subtypes of acetate: succinyl-CoA transferase (ASCT), might resemble the common ancestor of the hydrogenosome of Spironucleus and the mitosome of Giardia [43]. MRO of another fornicate Dysnectes may represent an evolutionarily intermediate state between hydrogenosomes and mitosomes. Dysnectes produces ATP through the cytosolic ACS-based pathway, but surprisingly, its MRO still produces hydrogen. In this case, hydrogen production functions as an electron acceptor for amino acid metabolism and not, as in hydrogenosomes, for pyruvate metabolism. The evolutionary transition to parasitism leads to further functional reduction of MRO and allows the loss of amino acid metabolism and hydrogen production, resulting in formation of mitosomes [3]. New findings from closely related apochlorotic species show that the vestigial plastids may possess diverse traits, even in organisms of shared ancestry. The first case is the diatom genus Nitzschia, encompassing a Current Opinion in Genetics & Development 2019, 58:33–39
number of nonphotosynthetic strains [17,44]. Plastids of these strains lack genes for all core photosynthesisrelated processes, but despite that, they retain ATP synthase complex genes, suggesting that there is a general evolutionary constraint against the loss of ATP synthase in nonphotosynthetic plastids, previously observed only in orchids [45]. In contrast, each of the investigated strains differentially lost few functionally diverse genes (ycf, sec, rps and rpl), different ones in every case [44]; as it was shown that the loss of photosynthesis occurred only once in Nitzschia, the differential gene loss must have taken place after the divergence of the described strains [46]. A second example comes from two pathogenic green algal genera. Phylogenetic analyses strongly suggest at least three independent losses of photosynthesis in Prototheca and its close relative Helicosporidium. Prototheca plastid genomes have a quite vast range of size – from 28.7 to 55.6 kb – and contents – from 47 to 72 genes. Comparative analyses of the plastid and nuclear genomes revealed that the gene content for plastid functions was highly conserved among these nonphotosynthetic lineages, and the photosynthesisrelated genes have mostly disappeared, indicating concurrent elimination of photosynthetic apparatus in both plastid and nuclear genomes [29,47]. Nevertheless, while Prototheca zopfii additionally lost the rpo genes [29], Prototheca cutis and Prototheca wickerhamii retained the complete set of six genes encoding plastid ATP synthase. Furthermore, the gene order in these two species’ plastid genomes was almost identical, suggesting that they have independently eliminated the same set of genes, while retaining the genome structure. In contrast, the plastid genomes of Prototheca stagnora and Helicosporidium sp. were highly rearranged, probably due to differences in the evolutionary time during which respective lineages lost their photosynthetic ability [47]. Many authors attempted to outline discrete steps in reductive evolution of plastids and construct universal models, especially for plants [8,48]. It is particularly challenging due to vast variety of possible evolutionary roads leading to the loss of photosynthesis. We propose CHER as the acronymic name for such classification, which would include four classes, ranging from an almost intact plastid genome as in Cornospumella [36], through ones with only photosynthesis-related genes absent as in Helicosporidium [28] and with majority of non-photosynthesis-related genes absent as in Eimeria [49], to complete loss of genome as in Rafflesia [39] (Table 1, Figure 2).
Metabolic redundancy – towards the loss of organelle Despite the drastic reduction, including loss of the genome and majority of functions, remnant organelles are in most cases retained, as they have become indispensable for the cell due to metabolic processes they www.sciencedirect.com
Reductive evolution of endosymbiotic organelles Maciszewski and Karnkowska 37
Table 1 The proposed CHER classification of secondarily nonphotosynthetic plastids, based on genetic contents of their autonomous genomes. The four classes described below correspond to four representative organisms: Cornospumella, Helicosporidium, Eimeria and Rafflesia. Note: presence of specific genes involved in secondary metabolite biosynthesis pathways may vary with lineage Cornospumella-like
Helicosporidium-like
Eimeria-like
Rafflesia-like
Photosynthesis-related genes ( psa, psb, atp, rbc, chl) Housekeeping genes (rpo, rps, rpl, rrn, trn)
Present in the plastid, partially pseudogenized Present in the plastid
All pseudogenized or lost
Lost
Lost
Partially transferred to the nucleus
All transferred to the nucleus or lost
Secondary metabolite biosynthesis pathways (accD, acpP, clpC, clpP . . . )
Partially transferred to the nucleus
Partially transferred to the nucleus
Mostly transferred to the nucleus, some pseudogenized or lost Almost all transferred to the nucleus
perform. Following the endosymbiotic gain of new pathways, the evolution acts against metabolic redundancy: if metabolic pathways serving the same function exist both in the cytosol and the organelle, one of those will likely be lost. Therefore, loss of even one crucial cytosolic pathway may make the organelle indispensable (Figures 1, and 2) [9,50]. There are very few exceptions, as complete loss of the plastid occurred only in several lineages of Apicomplexa [9,27], and the lineage of dinoflagellate parasites encompassing genera Hematodinium and Amoebophrya [14,51], all of which are obligatory parasites. Thorough search for the nucleus-encoded plastid genes in Hematodinium did not yield any candidates for plastid-targeted proteins, strongly suggesting complete loss of the plastid. In other alveolates, the remnant plastids are commonly retained due to fatty acid, tetrapyrrole, IPP and heme synthesis. In Hematodinium and Amoebophrya, however, fatty acids are generated by using cytosolic type I, and not plastid type II, fatty acid synthase. Similarly, the tetrapyrrole biosynthesis-related HemB–E enzymes and the DAP pathway of lysine biosynthesis both occur in the cytosol of this organism; although the HemD gene and DAP pathway are of plastid origin, they underwent relocation to the cytosol. Finally, isoprenoids are scavenged from the host. In brief, a rather unique combination of pathway relocation events and parasitic lifestyle of these two dinoflagellates allowed the complete loss of the remnant plastid [14,51]. The cryptomonad Goniomonas avonlea has been postulated to represent the unique case of a free-living organism that lost its plastid completely [52]. There is no clear evidence that G. avonlea evolved from a plastidbearing ancestor—its cells do not have any apparent plastid-like internal structures on the transmission electron micrographs, and thorough investigation of its nuclear genome and transcriptome did not reveal any possible plastid-targeted candidate proteins, as in Hematodinium. Nonetheless, several enzymes, such as GT28 and GWD, which are almost exclusively found in plastid-bearing organisms, have been identified in this enigmatic protist [52]. www.sciencedirect.com
All transferred to the nucleus or lost
Compared with plastids, the complete loss of mitochondria is even more rare and it has been shown only for the endobiotic preaxostylan Monocercomonoides exilis [50]. The final step of MRO loss for this metamonad was the replacement of the ISC pathway of Fe–S cluster assembly by the horizontally transferred, prokaryotederived cytosolic SUF system, and that the gain of the SUF pathway preceded the loss of an ISC pathway (Figure 1) [50]. Interestingly, the lack of ISC pathway in Monocercomonoides’s relatives and presence of the SUF pathway suggests that the latter most likely has been acquired by the common ancestor of all Preaxostyla [53].
Conclusions The multitude of case studies outlined in this work shows that the reductive evolution of endosymbiotic organelles is widespread, especially among microbial eukaryotes. The reduction of organelles is most often related to the change of ecological niche (for example, to an anaerobic environment or host tissues), where some functions of organelles (such as oxidative phosphorylation or photosynthesis) might become obsolete and their loss reduces the energetic cost of the organelle’s maintenance. A growing number of identified reduced organelles from different lineages resulted in the discovery of intermediate stages between already well-described forms. Those examples make it more apparent that reductive evolution is a step-wise process, and more importantly, that the major steps are usually similar, as most of the reduced organelles lose the same pathways and, eventually, the entire genome. In contrast, the evidence from closely related organisms proves that the details of the process might differ and the order of steps is not always the same, which may result in slightly different genome contents or range of metabolic capabilities. Some of the significant differences come from unprecedented horizontal gene transfers, which might have a massive impact on the evolutionary fate of the organelles, including loss of the entire mitochondrial organelle. It is, however, noteworthy that the complete loss of the endosymbiotic Current Opinion in Genetics & Development 2019, 58:33–39
38 Evolutionary genetics
organelle is sporadic and limited only to the cells that overcame metabolic dependence on the organelle. Evidence from newly described lineages, including freeliving organisms, greatly expanded our understanding of the reductive evolution of endosymbiotic organelles, but as we fill the white spots on the map of global biodiversity, we will inevitably find more examples of vestigial organelles following evolutionary roads converging to similar points. The patchy distribution of known examples makes backtracking their evolutionary history challenging, and the differential loss of traits often blurs the picture even further. Cases of extreme reduction, including loss of genomes and organelles, are still scarce; exploring their cell biology, physiology and evolution might shed new light on the nature of tight relationship between the endosymbiotic organelles and their eukaryotic hosts.
Conflict of interest statement Nothing declared.
Acknowledgements Funding: This work was supported by the National Science Centre, Poland; [SONATA grant number 2016/21/D/NZ8/01288 to A.K.]; EMBO Installation Grant to A.K.; and Polish Ministry of Science and Higher Education scholarship for outstanding young researchers to A.K.
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34. Za´honova´ K, Fu¨ssy Z, Obornı´k M, Elia´sˇ M, Yurchenko V: RuBisCO in non-photosynthetic alga Euglena longa: divergent features, transcriptomic analysis and regulation of complex formation. PLoS One 2016, 11:e0158790. 35. Za´honova´ K, Fu¨ssy Z, Bir9 ca´k E, Nova´k Vanclova´ AMG, Klimesˇ V, Vesteg M, Kraj9covi9c J, Obornı´k M, Elia´sˇ M: Peculiar features of the plastids of the colourless alga Euglena longa and photosynthetic euglenophytes unveiled by transcriptome analyses. Sci Rep 2018, 8:17012. 36. Dorrell RG, Azuma T, Nomura M, Audren de Kerdrel G, Paoli L, Yang S, Bowler C, Ishii K, Miyashita H, Gile GH et al.: Principles of plastid reductive evolution illuminated by nonphotosynthetic chrysophytes. Proc Natl Acad Sci U S A 2019, 116:6914-6923. This work provides important insights on the diversity of traits in secondarily non-photosynthetic plastids, both within and between lineages. The authors also highlight the unobvious tendency for divergence of features in closely related organisms and convergence in distantly related ones. 37. Smith DR, Lee RW: A plastid without a genome: evidence from the nonphotosynthetic green algal genus Polytomella. PLANT Physiol 2014, 164:1812-1819. 38. Molina J, Hazzouri KM, Nickrent D, Geisler M, Meyer RS, Pentony MM, Flowers JM, Pelser P, Barcelona J, Inovejas SA et al.: Possible loss of the chloroplast genome in the parasitic flowering plant Rafflesia lagascae (Rafflesiaceae). Mol Biol Evol 2014, 31:793-803. 39. Ng SM, Lee XW, Mat-Isa MN, Aizat-Juhari MA, Adam JH, Mohamed R, Wan KL, Firdaus-Raih M: Comparative analysis of nucleus-encoded plastid-targeting proteins in Rafflesia cantleyi against photosynthetic and non-photosynthetic representatives reveals orthologous systems with potentially divergent functions. Sci Rep 2018, 8:17258. A comprehensive study of the unexpected abundance of operational metabolic pathways in the genome-deprived plastids of Rafflesia
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47. Suzuki S, Endoh R, Manabe RI, Ohkuma M, Hirakawa Y: Multiple losses of photosynthesis and convergent reductive genome evolution in the colourless green algae Prototheca. Sci Rep 2018, 8:940. 48. Graham SW, Lam VKY, Merckx VSFT: Plastomes on the edge: the evolutionary breakdown of mycoheterotroph plastid genomes. New Phytol 2017, 214:48-55. 49. Cai X, Fuller AL, McDougald LR, Zhu G: Apicoplast genome of the coccidian Eimeria tenella. Gene 2003, 321:39-46. 50. Karnkowska A, Vacek V, Zuba´c9 ova´ Z, Treitli SC, Petr9zelkova´ R, Eme L, Nova´k L, a´rsky´ V, Barlow LD, Herman EK et al.: A eukaryote without a mitochondrial organelle. Curr Biol 2016, 26:1274-1284. 51. Gornik SG, Febrimarsa, Cassin AM, MacRae JI, Ramaprasad A, Rchiad Z, McConville MJ, Bacic A, McFadden GI, Pain A et al.: Endosymbiosis undone by stepwise elimination of the plastid in a parasitic dinoflagellate. Proc Natl Acad Sci U S A 2015, 112:5767-5772. 52. Cenci U, Sibbald SJ, Curtis BA, Kamikawa R, Eme L, Moog D, Henrissat B, Mare´chal E, Chabi M, Djemiel C et al.: Nuclear genome sequence of the plastid-lacking cryptomonad Goniomonas avonlea provides insights into the evolution of secondary plastids. BMC Biol 2018, 16:137. This paper investigates the first case of complete plastid loss in a freeliving protist and thoroughly explores the challenge of understanding the evolution of an organism with uncertain ancestry. cka I, Kolı´sko M, 53. Vacek V, Nova´k LVF, Treitli SC, Ta´borsky´ P, Cepi9 Keeling PJ, Hampl V: Fe-S cluster assembly in oxymonads and related protists. Mol Biol Evol 2018, 35:2712-2718. Authors have shown that the crucial evolutionary step of the acquisition of the SUF Fe–S cluster assembly pathway from prokaryotes, which makes the mitochondrial ISC pathway dispensable, is not unique for the amitochondriate Monocercomonoides exilis, but it is widespread in Oxymonads.
Current Opinion in Genetics & Development 2019, 58:33–39
ScienceDirect Should I stay or should I go? Retention and loss of components in vestigial endosymbiotic organelles Kacper Maciszewski and Anna Karnkowska Our knowledge on the variability of the reduced forms of endosymbiotic organelles – mitochondria and plastids – is expanding rapidly, thanks to growing interest in peculiar microbial eukaryotes, along with the availability of the methods used in modern genomics and transcriptomics. The aim of this work is to highlight the most recent advances in understanding these organelles’ diversity, physiology and evolution. We also outline the known mechanisms behind the convergence of traits between organelles which have undergone reduction independently, the importance of the earliest evolutionary events in determining the vestigial organelles’ eventual fate, and a proposed classification of nonphotosynthetic plastids. Address Department of Molecular Phylogenetics and Evolution, Biological and Chemical Research Centre, Faculty of Biology, University of Warsaw, ul. _ Zwirki i Wigury 101, 02-089, Warsaw, Poland Corresponding author: Karnkowska, Anna ([email protected])
Current Opinion in Genetics & Development 2019, 59:33–39 This review comes from a themed issue on Evolutionary genetics Edited by Jeremy Wideman and Thomas Richards
https://doi.org/10.1016/j.gde.2019.07.013 0959-437X/ã 2019 Elsevier Ltd. All rights reserved.
Introduction Simplification sensu lato, including such phenomena as differential loss or convergent loss among others, is one of the main processes shaping diversity, but its role in the evolution has been appreciated only recently [1]. Great examples of simplification come from the evolution of endosymbiotic organelles: mitochondria and plastids. They underwent dramatic reduction early in their evolutionary history through partial loss of contents, from genes to metabolic pathways, becoming fully dependent on their host—the eukaryotic cell. In both cases, further reduction happened many times across the tree of life. Vestigial plastids (leucoplasts) and reduced mitochondria organelles, MROs) were (mitochondrion-related described decades ago, but thanks to high-throughput sequencing techniques, and growing interest in the www.sciencedirect.com
diversity of microbial eukaryotes, we are now discovering the range of reduced forms of these organelles.
Reduction is widespread Many distinct eukaryotic lineages have adapted to living in low oxygen conditions independently, which is reflected on the patchy distribution of MROs on the tree of eukaryotes. MROs are present in all supergroups except Archaeplastida [2]; in some lineages they are rare (e.g. Euglenozoa, Apicomplexa), while in others, such as Metamonada, they are ubiquitous [3]. Their diverse forms, such as mitosomes (e.g. Giardia, Entamoeba), hydrogenosomes (e.g. some metamonads, ciliates, archamoebae), and hydrogen-producing mitochondria (e.g. Blastocystis, Nyctotherus), can be found in endobiotic parasites, commensals, or mutualistic symbionts. Moreover, the number of examples of free-living MRO-bearing organisms is growing: these include the amoebozoa, breviates, heteroloboseans, stramenopiles, jakobids, metamonads and flagellates related to diplomonads [4], and multicellular animals from the phylum Loricifera [5]. Every group that acquired plastids, except for haptophytes, has secondarily nonphotosynthetic representatives. Colorless red algae and plants are almost always parasitic, but among colorless secondary plastid-bearing algae, there are free-living phagotrophs and osmotrophs as well [6–9]. Nonphotosynthetic groups are often sister lineages to the mixotrophic ones, suggesting that loss of photosynthesis might be promoted in mixotrophs if the energy costs of maintaining the photosynthetic apparatus outweigh the benefits of its products [10], especially considering that phototrophs employ detoxification mechanisms to counteract reactive oxygen species generated by photosynthesis [11].
Convergence in the evolution of reduced organelles MROs in unrelated eukaryotes show strikingly convergent features, suggesting a limited range of possible mitochondrial adaptations to hypoxia. In most of the known examples, the electron transport chain (ETC) has been truncated or lost, and the organisms fulfil their energetic requirements with ATP produced by substrate-level phosphorylation [4]. In order to explain the early steps of reductive evolution of mitochondria, intermediate stages in transition to the anaerobic lifestyle must be investigated. Among the organisms postulated to possess these intermediate traits are Brevimastigomonas motovehiculus [12], Current Opinion in Genetics & Development 2019, 58:33–39
34 Evolutionary genetics
Blastocystis, and Nyctotherus [2], whose mitochondria are modified to function under hypoxic conditions, but without concurrent loss of some typically mitochondrial features. Brevimastigomonas’s MROs retained mitochondrial metabolic pathways, such as the TCA cycle and ETC-driven ATP synthesis, but also pyruvate metabolism and substrate-level phosphorylation (Figure 1). In contrast, its mitochondrial genome is degenerate, with protein-coding contents including ETC complexes I, III, IV, and V (complex II is nucleusencoded), but complexes III and IV are highly divergent and possibly incapable of efficient electron transport. Degeneration or complete loss of the oxidative phosphorylation pathway (OXPHOS) is also reflected in the reduction of mitochondrial cristae, as demonstrated by transmission electron microscopy and molecular analyses showing that the loss of OXPHOS co-occurs with the loss of the cristae organizing complex MICOS [13]. This example suggests that the free-living organisms most likely first evolved the capacity to survive in anaerobic environments by gaining new metabolic pathways and establishing anaerobic mitochondria,
which eventually underwent reduction and evolved into hydrogenosomes and mitosomes (Figure 1). Following the loss of the ETC, all hydrogenosomes and mitosomes lost their genomes, and consequently, all proteins functioning in those organelles are encoded by the nucleus. Surprisingly, mitochondrial genome loss has recently been documented in the aerobic, OXPHOS-capable dinoflagellate Amoebophrya ceratii [14], which indicates that such occurrence is not exclusive to anaerobic MROs, therefore subverting the previously proposed hypothesis that the functional ETC is a universal constraint against mtDNA loss [15]. Still, the remnant mitochondria, regardless of the extent of their reduction, always possess mitochondrial transport machinery, proteins involved in the production or import of ATP and, with the exception of Entamoeba, the ISC (iron–sulfur cluster) machinery (Figure 1)—a single operon-encoded pathway of Fe–S cluster assembly [16]. In case of plastids, the loss of photosynthetic functions is also reflected in the organellar morphology,
Figure 1
Brevimastigomonas-like MRO
LGT
Mitochondrion
Bacteria Anaerobic mitochondrion
Amoebophrya-like mitochondrion
Hydrogenosome
Mitosome
genes encoding Fe-S cluster formation system ISC
LGT
genes encoding other mitochondrion-targeted proteins genes encoding proteins involved in oxidative phosphorylation (OxPhos)
Bacteria genes encoding mitochondrial transport membrane proteins genes encoding proteins involved in substrate phosphorylation
Amitochondriate
proteins
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Schematic representation of the stages of reductive evolution of mitochondrion-related organelles, documented in the available case studies.
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Reductive evolution of endosymbiotic organelles Maciszewski and Karnkowska 35
where thylakoids are degenerate [17] or absent [18,19] (Figure 2). The vestigial plastids are incapable of photosynthesis and usually lack all genes involved in this process, although the recently discovered retention of the chlorophyll biosynthesis gene cluster in corallicoid apicoplasts is a prominent exception [20]. Nonetheless, a large part of the overall plastid genetic repertoire may remain, specifically: ribosomal protein genes (rps, rpl), rRNAs, tRNAs, usually rpo, and a number of other genes, which most commonly include accD, clpC, sufB, tufA and/or infA. This pattern has been observed in a variety of organisms, such as mycoheterotrophic plants [21–25], apicomplexans [26,27], and parasitic red and green algae [28,29,30]. One of the repeatedly occurring exceptions is rbcL—a crucial photosynthesis-related gene, present in a number of leucoplast genomes, both in plants [31] and microbial eukaryotes of diverse ancestry [32–34]. As shown for one of the latter, the euglenid Euglena longa, rbcL has a low level of expression, the full RuBisCO molecules are likely not assembled at all, and its function remains under investigation [34,35]. Despite their vestigial character, leucoplasts are far from being disposable. The retained genes encode mostly housekeeping proteins, allowing transcription and translation in the organelle, but others are involved in the indispensable processes performed in the apochlorotic plastids, including biosynthesis of isoprenoids, tetrapyrroles, heme, Fe–S clusters, and fatty acids (Figure 2) [8,9]. The
sets of retained pathways and their plastid-encoded components can be diverse between lineages: sufB and clpC genes are often among the only non-housekeeping genes in the red lineage-derived leucoplasts [9], but in plants, it is usually accD and clpP [8]. Although fatty acid biosynthesis is the fundamental leucoplast function in plants [8] and apicomplexans [18], it is the one most commonly lost in nonphotosynthetic dinoflagellates [7] and chrysophytes [36]. Curiously, land plants are prone to loss of plastid rpo genes probably because they possess nuclear-encoded, phage-derived NEP polymerase, which can compensate for the loss of rpo [8]. In contrast to MROs, almost all known colorless plastids retain at least a rudimentary genome. Plastids without a genome are rare, yet some documented cases exist (Figure 2), for example: a green alga Polytomella [37], a parasitic plant Rafflesia [38,39], and a colpodellid Voromonas pontica [40].
The range of reduced forms MROs represent various outcomes and transitional stages of a gradual evolutionary process, which greatly complicates the efforts to systematize them. Most often they are classified according to their metabolic capacity: into anaerobic mitochondria, hydrogen-producing mitochondria (HMPs), hydrogenosomes and mitosomes [2] (Figure 1). This classification does not reflect the evolutionary history, as related taxa often possess different
Figure 2
genome-deprived leucoplast loss of plastid genome
loss of photosynthesis
leucoplast
cytosolic variant loss
hypothetical intermediate stage
photosynthetic plastid
plastid variant loss
stage after the acquisition of the plastid nuclear/plastid pathway redundancy unsolved loss of photosynthesis
hypothetical intermediate stage genes encoding proteins involved in photosynthesis genes encoding proteins involved in other metabolic pathways plastid completely lost
genes encoding plastid transport membrane proteins
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Schematic representation of the stages of reductive evolution of nonphotosynthetic plastids. For the sake of clarity, hypothetical intermediate stages are included.
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36 Evolutionary genetics
MROs, and each MRO type is distributed across unrelated lineages. Differences in components, functions, and morphological features among MROs exist, which may have been generated by a secondary gene loss and/or lateral gene transfer [41]. In metamonads, which are a showcase for the diversity of MROs, additional data coming from bacteriovorous, freeliving Carpediemonas-like organisms allowed deeper analysis of lineage-specific gains and losses [3]. Several conserved mitochondrial enzymes have very scarce distribution: cardiolipin synthase is present only in Carpediemonas and aminoadipate-semialdehyde dehydrogenase—in Trimastix marina, indicating that they have probably been vertically inherited from the last eukaryotic common ancestor, but later lost in majority of MROs. Another interesting evolutionary pattern emerges from the distribution of glycine cleavage system (GCS): parasitic and secondarily free-living lineages always lack a complete GCS, whereas ancestrally free-living lineages possess the complete system. Even more complicated is the evolutionary history of acetyl-CoA synthase (ACS), which generates ATP directly from the conversion of acetyl-CoA to acetate. Two types of ACS were identified in metamonads: the cytosolic ACS1, proposed to be acquired through a single LGT event into the last common ancestor of Chilomastix, Kipferlia, Dysnectes and diplomonads, and the other ACS homologue (ACS2) of different origin, yet it remains unclear if it was ancestrally present in metamonads [3]. Furthermore, the diplomonad Spironucleus most likely possesses both ACS1 and ACS2, but the latter is localized in its hydrogenosome and was probably acquired through LGT [42]. Comparative studies of the fornicates allowed a detailed reconstruction of intermediate stages of MROs. The MRO of the freeliving Kipferlia bialata with more complex suite of metabolic pathways involved in ATP generation such as succinyl-CoA synthase (SCS) and two subtypes of acetate: succinyl-CoA transferase (ASCT), might resemble the common ancestor of the hydrogenosome of Spironucleus and the mitosome of Giardia [43]. MRO of another fornicate Dysnectes may represent an evolutionarily intermediate state between hydrogenosomes and mitosomes. Dysnectes produces ATP through the cytosolic ACS-based pathway, but surprisingly, its MRO still produces hydrogen. In this case, hydrogen production functions as an electron acceptor for amino acid metabolism and not, as in hydrogenosomes, for pyruvate metabolism. The evolutionary transition to parasitism leads to further functional reduction of MRO and allows the loss of amino acid metabolism and hydrogen production, resulting in formation of mitosomes [3]. New findings from closely related apochlorotic species show that the vestigial plastids may possess diverse traits, even in organisms of shared ancestry. The first case is the diatom genus Nitzschia, encompassing a Current Opinion in Genetics & Development 2019, 58:33–39
number of nonphotosynthetic strains [17,44]. Plastids of these strains lack genes for all core photosynthesisrelated processes, but despite that, they retain ATP synthase complex genes, suggesting that there is a general evolutionary constraint against the loss of ATP synthase in nonphotosynthetic plastids, previously observed only in orchids [45]. In contrast, each of the investigated strains differentially lost few functionally diverse genes (ycf, sec, rps and rpl), different ones in every case [44]; as it was shown that the loss of photosynthesis occurred only once in Nitzschia, the differential gene loss must have taken place after the divergence of the described strains [46]. A second example comes from two pathogenic green algal genera. Phylogenetic analyses strongly suggest at least three independent losses of photosynthesis in Prototheca and its close relative Helicosporidium. Prototheca plastid genomes have a quite vast range of size – from 28.7 to 55.6 kb – and contents – from 47 to 72 genes. Comparative analyses of the plastid and nuclear genomes revealed that the gene content for plastid functions was highly conserved among these nonphotosynthetic lineages, and the photosynthesisrelated genes have mostly disappeared, indicating concurrent elimination of photosynthetic apparatus in both plastid and nuclear genomes [29,47]. Nevertheless, while Prototheca zopfii additionally lost the rpo genes [29], Prototheca cutis and Prototheca wickerhamii retained the complete set of six genes encoding plastid ATP synthase. Furthermore, the gene order in these two species’ plastid genomes was almost identical, suggesting that they have independently eliminated the same set of genes, while retaining the genome structure. In contrast, the plastid genomes of Prototheca stagnora and Helicosporidium sp. were highly rearranged, probably due to differences in the evolutionary time during which respective lineages lost their photosynthetic ability [47]. Many authors attempted to outline discrete steps in reductive evolution of plastids and construct universal models, especially for plants [8,48]. It is particularly challenging due to vast variety of possible evolutionary roads leading to the loss of photosynthesis. We propose CHER as the acronymic name for such classification, which would include four classes, ranging from an almost intact plastid genome as in Cornospumella [36], through ones with only photosynthesis-related genes absent as in Helicosporidium [28] and with majority of non-photosynthesis-related genes absent as in Eimeria [49], to complete loss of genome as in Rafflesia [39] (Table 1, Figure 2).
Metabolic redundancy – towards the loss of organelle Despite the drastic reduction, including loss of the genome and majority of functions, remnant organelles are in most cases retained, as they have become indispensable for the cell due to metabolic processes they www.sciencedirect.com
Reductive evolution of endosymbiotic organelles Maciszewski and Karnkowska 37
Table 1 The proposed CHER classification of secondarily nonphotosynthetic plastids, based on genetic contents of their autonomous genomes. The four classes described below correspond to four representative organisms: Cornospumella, Helicosporidium, Eimeria and Rafflesia. Note: presence of specific genes involved in secondary metabolite biosynthesis pathways may vary with lineage Cornospumella-like
Helicosporidium-like
Eimeria-like
Rafflesia-like
Photosynthesis-related genes ( psa, psb, atp, rbc, chl) Housekeeping genes (rpo, rps, rpl, rrn, trn)
Present in the plastid, partially pseudogenized Present in the plastid
All pseudogenized or lost
Lost
Lost
Partially transferred to the nucleus
All transferred to the nucleus or lost
Secondary metabolite biosynthesis pathways (accD, acpP, clpC, clpP . . . )
Partially transferred to the nucleus
Partially transferred to the nucleus
Mostly transferred to the nucleus, some pseudogenized or lost Almost all transferred to the nucleus
perform. Following the endosymbiotic gain of new pathways, the evolution acts against metabolic redundancy: if metabolic pathways serving the same function exist both in the cytosol and the organelle, one of those will likely be lost. Therefore, loss of even one crucial cytosolic pathway may make the organelle indispensable (Figures 1, and 2) [9,50]. There are very few exceptions, as complete loss of the plastid occurred only in several lineages of Apicomplexa [9,27], and the lineage of dinoflagellate parasites encompassing genera Hematodinium and Amoebophrya [14,51], all of which are obligatory parasites. Thorough search for the nucleus-encoded plastid genes in Hematodinium did not yield any candidates for plastid-targeted proteins, strongly suggesting complete loss of the plastid. In other alveolates, the remnant plastids are commonly retained due to fatty acid, tetrapyrrole, IPP and heme synthesis. In Hematodinium and Amoebophrya, however, fatty acids are generated by using cytosolic type I, and not plastid type II, fatty acid synthase. Similarly, the tetrapyrrole biosynthesis-related HemB–E enzymes and the DAP pathway of lysine biosynthesis both occur in the cytosol of this organism; although the HemD gene and DAP pathway are of plastid origin, they underwent relocation to the cytosol. Finally, isoprenoids are scavenged from the host. In brief, a rather unique combination of pathway relocation events and parasitic lifestyle of these two dinoflagellates allowed the complete loss of the remnant plastid [14,51]. The cryptomonad Goniomonas avonlea has been postulated to represent the unique case of a free-living organism that lost its plastid completely [52]. There is no clear evidence that G. avonlea evolved from a plastidbearing ancestor—its cells do not have any apparent plastid-like internal structures on the transmission electron micrographs, and thorough investigation of its nuclear genome and transcriptome did not reveal any possible plastid-targeted candidate proteins, as in Hematodinium. Nonetheless, several enzymes, such as GT28 and GWD, which are almost exclusively found in plastid-bearing organisms, have been identified in this enigmatic protist [52]. www.sciencedirect.com
All transferred to the nucleus or lost
Compared with plastids, the complete loss of mitochondria is even more rare and it has been shown only for the endobiotic preaxostylan Monocercomonoides exilis [50]. The final step of MRO loss for this metamonad was the replacement of the ISC pathway of Fe–S cluster assembly by the horizontally transferred, prokaryotederived cytosolic SUF system, and that the gain of the SUF pathway preceded the loss of an ISC pathway (Figure 1) [50]. Interestingly, the lack of ISC pathway in Monocercomonoides’s relatives and presence of the SUF pathway suggests that the latter most likely has been acquired by the common ancestor of all Preaxostyla [53].
Conclusions The multitude of case studies outlined in this work shows that the reductive evolution of endosymbiotic organelles is widespread, especially among microbial eukaryotes. The reduction of organelles is most often related to the change of ecological niche (for example, to an anaerobic environment or host tissues), where some functions of organelles (such as oxidative phosphorylation or photosynthesis) might become obsolete and their loss reduces the energetic cost of the organelle’s maintenance. A growing number of identified reduced organelles from different lineages resulted in the discovery of intermediate stages between already well-described forms. Those examples make it more apparent that reductive evolution is a step-wise process, and more importantly, that the major steps are usually similar, as most of the reduced organelles lose the same pathways and, eventually, the entire genome. In contrast, the evidence from closely related organisms proves that the details of the process might differ and the order of steps is not always the same, which may result in slightly different genome contents or range of metabolic capabilities. Some of the significant differences come from unprecedented horizontal gene transfers, which might have a massive impact on the evolutionary fate of the organelles, including loss of the entire mitochondrial organelle. It is, however, noteworthy that the complete loss of the endosymbiotic Current Opinion in Genetics & Development 2019, 58:33–39
38 Evolutionary genetics
organelle is sporadic and limited only to the cells that overcame metabolic dependence on the organelle. Evidence from newly described lineages, including freeliving organisms, greatly expanded our understanding of the reductive evolution of endosymbiotic organelles, but as we fill the white spots on the map of global biodiversity, we will inevitably find more examples of vestigial organelles following evolutionary roads converging to similar points. The patchy distribution of known examples makes backtracking their evolutionary history challenging, and the differential loss of traits often blurs the picture even further. Cases of extreme reduction, including loss of genomes and organelles, are still scarce; exploring their cell biology, physiology and evolution might shed new light on the nature of tight relationship between the endosymbiotic organelles and their eukaryotic hosts.
Conflict of interest statement Nothing declared.
Acknowledgements Funding: This work was supported by the National Science Centre, Poland; [SONATA grant number 2016/21/D/NZ8/01288 to A.K.]; EMBO Installation Grant to A.K.; and Polish Ministry of Science and Higher Education scholarship for outstanding young researchers to A.K.
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Current Opinion in Genetics & Development 2019, 58:33–39