Golgi apparatus in parasitic protists (review of the literature)

This review summarizes modem data on Golgi apparatus of parasitic protists and demonstrates how the parasitic lifestyle determines functional and structural peculiarities of secretory systems in unrelated groups of unicellular parasites, in comparison to ones of "model systems", mammalian and yeast cells. The review covers the most well-studied protists, predominantly of high medical importance, belonging to following taxons: Parabasalia (Trichomonas), Diplomonada (Giardia), Entamoebidae (Entamoeba), parasitic Alveolata of the phyllum Apicomplexa (Toxoplasma and Plasmodium), and Kinetoplastida (Trypanosoma and Leishmania). Numerous recent publications demonstrated that studies on intracellular traffic in the mentioned above parasites essentially advanced our knowledge of Golgi function, traditionally based on research of cultured mammalian and yeast cells. Morphology of Golgi organelle in eukaryotes from various taxonomic groups has been compared. Within three of total six the highest taxons of Eukatyota (Adl et al., 2005) there exist at minimum eight groups represented by species lacking Golgi dictiosomes. However, biochemical and (or) molecular (genomic) evidences indicate that the organelle with functions of Golgi was present in every studied so far lineage of eukaryotes. Loss of Golgi organelle is a secondary event, which has been proven by identification of Golgi genes in the genomes of Golgi-lacking lineages. This loss might have occurred independently several times in the course of evolution. Neither the number of stacks, nor the size of the organelle correlates with intensity of secretion, or the position of the species on the evolutionary tree (in terms of presumably early/lately diverged lineages).     

Biogenesis of iron-sulphur clusters in amitochondriate and apicomplexan protists

During the last 4 years there has been an enormous interest in the question how iron-sulphur ([Fe-S]) clusters, which are essential building blocks for life, are synthesised and assembled into apo-proteins, both in prokaryotes and in eukaryotes. The emerging picture is that the basic mechanism of this pathway has been well conserved during evolution. In yeast and probably all other eukaryotes the mitochondrion is the place where [Fe-S] clusters are synthesised, even for extramitochondrial [Fe-S] cluster-containing proteins, and a number of proteins have been functionally characterised to a certain extent within this pathway. However, almost nothing is known about this aspect in parasitic protists, although recent studies of amitochondriate protists and on the plastid-like organelle of apicomplexan parasites, the apicoplast, have started to change this. In this article I will summarise the current view of [Fe-S] cluster biogenesis in eukaryotes and discuss its implications for amitochondriate protists and for the plastid-like organelle of apicomplexan parasites.     

Discrimination Experiments in Entamoeba and Evidence from Other Protists Suggest Pathogenic Amebas Cooperate with Kin to Colonize Hosts and Deter Rivals

Entamoeba histolytica is one of the least understood protists in terms of taxa, clone, and kin discrimination/recognition ability. However, the capacity to tell apart same or self (clone/kin) from different or nonself (nonclone/nonkin) has long been demonstrated in pathogenic eukaryotes like Trypanosoma and Plasmodium, free‐living social amebas (Dictyostelium, Polysphondylium), budding yeast (Saccharomyces), and in numerous bacteria and archaea (prokaryotes). Kin discrimination/recognition is explained under inclusive fitness theory; that is, the reproductive advantage that genetically closely related organisms (kin) can gain by cooperating preferably with one another (rather than with distantly related or unrelated individuals), minimizing antagonism and competition with kin, and excluding genetic strangers (or cheaters = noncooperators that benefit from others’ investments in altruistic cooperation). In this review, we rely on the outcomes of in vitro pairwise discrimination/recognition encounters between seven Entamoeba lineages to discuss the biological significance of taxa, clone, and kin discrimination/recognition in a range of generalist and specialist species (close or distantly related phylogenetically). We then focus our discussion on the importance of these laboratory observations for E. histolytica's life cycle, host infestation, and implications of these features of the amebas’ natural history for human health (including mitigation of amebiasis).     

The secretory pathway of protists: spatial and functional organization and evolution.

All cells secrete a diversity of macromolecules to modify their environment or to protect themselves. Eukaryotic cells have evolved a complex secretory pathway consisting of several membrane-bound compartments which contain specific sets of proteins. Experimental work on the secretory pathway has focused mainly on mammalian cell lines or on yeasts. Now, some general principles of the secretory pathway have become clear, and most components of the secretory pathway are conserved between yeast cells and mammalian cells. However, the structure and function of the secretory system in protists have been less extensively studied. In this review, we summarize the current knowledge about the secretory pathway of five different groups of protists: Giardia lamblia, one of the earliest lines of eukaryotic evolution, kinetoplastids, the slime mold Dictyostelium discoideum, and two lineages within the "crown" of eukaryotic cell evolution, the alveolates (ciliates and Plasmodium species) and the green algae. Comparison of these systems with the mammalian and yeast system shows that most elements of the secretory pathway were presumably present in the earliest eukaryotic organisms. However, one element of the secretory pathway shows considerable variation: the presence of a Golgi stack and the number of cisternae within a stack. We suggest that the functional separation of the plasma membrane from the nucleus-endoplasmic reticulum system during evolution required a sorting compartment, which became the Golgi apparatus. Once a Golgi apparatus was established, it was adapted to the various needs of the different organisms.     

Evidence of taxa-, clone-, and kin-discrimination in protists: ecological and evolutionary implications

Unicellular eukaryotes, or protists, are among the most ancient organisms on Earth. Protists belong to multiple taxonomic groups; they are widely distributed geographically and in all environments. Their ability to discriminate among con- and heterospecifics has been documented during the past decade. Here we discuss exemplar cases of taxa-, clone-, and possible kin-discrimination in five major lineages: Mycetozoa (Dictyostelium, Polysphondylium), Dikarya (Saccharomyces), Ciliophora (Tetrahymena), Apicomplexa (Plasmodium) and Archamoebae (Entamoeba). We summarize the proposed genetic mechanisms involved in discrimination-mediated aggregation (self vs. different), including the csA, FLO and trg (formerly lag) genes, and the Proliferation Activation Factors, which facilitate clustering in some protistan taxa. We caution about the experimental challenges intrinsic to studying recognition in protists, and highlight the opportunities for exploring the ecology and evolution of complex forms of cell–cell communication, including social behavior, in a polyphyletic, still superficially understood group of organisms. Because unicellular eukaryotes are the evolutionary precursors of multicellular life, we infer that their mechanisms of taxa-, clone-, and possible kin-discrimination gave origin to the complex diversification and sophistication of traits associated with species and kin recognition in plants, fungi, invertebrates and vertebrates.     

Divergent Molecular Evolution of the Mitochondrial Sulfhydryl:Cytochrome c Oxidoreductase Erv in Opisthokonts and Parasitic Protists

Mia40 and the sulfhydryl:cytochrome c oxidoreductase Erv1/ALR are essential for oxidative protein import into the mitochondrial intermembrane space in yeast and mammals. Although mitochondrial protein import is functionally conserved in the course of evolution, many organisms seem to lack Mia40. Moreover, except for in organello import studies and in silico analyses, nothing is known about the function and properties of protist Erv homologues. Here we compared Erv homologues from yeast, the kinetoplastid parasite Leishmania tarentolae, and the non-related malaria parasite Plasmodium falciparum. Both parasite proteins have altered cysteine motifs, formed intermolecular disulfide bonds in vitro and in vivo, and could not replace Erv1 from yeast despite successful mitochondrial protein import in vivo. To analyze its enzymatic activity, we established the expression and purification of recombinant full-length L. tarentolae Erv and compared the mechanism with related and non-related flavoproteins. Enzyme assays indeed confirmed an electron transferase activity with equine and yeast cytochrome c, suggesting a conservation of the enzymatic activity in different eukaryotic lineages. However, although Erv and non-related flavoproteins are intriguing examples of convergent molecular evolution resulting in similar enzyme properties, the mechanisms of Erv homologues from parasitic protists and opisthokonts differ significantly. In summary, the Erv-mediated reduction of cytochrome c might be highly conserved throughout evolution despite the apparent absence of Mia40 in many eukaryotes. Nevertheless, the knowledge on mitochondrial protein import in yeast and mammals cannot be generally transferred to all other eukaryotes, and the corresponding pathways, components, and mechanisms remain to be analyzed.     

Evidence for Golgi bodies in proposed 'Golgi-lacking' lineages

Golgi bodies are nearly ubiquitous in eukaryotic cells. The apparent lack of such structures in certain eukaryotic lineages might be taken to mean that these protists evolved prior to the acquisition of the Golgi, and it raises questions of how these organisms function in the absence of this crucial organelle. Here, we report gene sequences from five proposed 'Golgi-lacking' organisms (Giardia intestinalis, Spironucleus barkhanus, Entamoeba histolytica, Naegleria gruberi and Mastigamoeba balamuthi). BLAST and phylogenetic analyses show these genes to be homologous to those encoding components of the retromer, coatomer and adaptin complexes, all of which have Golgi-related functions in mammals and yeast. This is, to our knowledge, the first molecular evidence for Golgi bodies in two major eukaryotic lineages (the pelobionts and heteroloboseids). This substantiates the suggestion that there are no extant primitively 'Golgi-lacking' lineages, and that this apparatus was present in the last common eukaryotic ancestor, but has been altered beyond recognition several times.     

Cellular responses to endoplasmic reticulum stress and apoptosis

The endoplasmic reticulum (ER) is the cell organelle where secretory and membrane proteins are synthesized and folded. Correctly folded proteins exit the ER and are transported to the Golgi and other destinations within the cell, but proteins that fail to fold properly—misfolded proteins—are retained in the ER and their accumulation may constitute a form of stress to the cell—ER stress. Several signaling pathways, collectively known as unfolded protein response (UPR), have evolved to detect the accumulation of misfolded proteins in the ER and activate a cellular response that attempts to maintain homeostasis and a normal flux of proteins in the ER. In certain severe situations of ER stress, however, the protective mechanisms activated by the UPR are not sufficient to restore normal ER function and cells die by apoptosis. Most research on the UPR used yeast or mammalian model systems and only recently Drosophila has emerged as a system to study the molecular and cellular mechanisms of the UPR. Here, we review recent advances in Drosophila UPR research, in the broad context of mammalian and yeast literature.     

Eukaryotes devoid of the most important cellular organelles (flagella, Golgi apparatus, mitochondria) and the main task of organellology]

Comparative evidence on the lack of three important organelles (flagella, Golgi-complex, mitochondria) in cells and organisms at the cellular level of organization has been summarized for all the four eukaryotic kingdoms--Protista, Fungi, Plantae and Animalia (Metazoa). It is established that in the course of evolution these organelles may undergo the total reduction. There is no cellular organelle to be regarded as universal, indispensable. There are only three main obligatory cell components--the plasmalemma, nucleus and cytoplasm (with applied cytoskeleton, cytomembranes and ribosomes). The reduction of flagella (cilia) is occurring in different taxa independent of the transition of protists from the flagellate type of locomotion to the amoeboid, gliding of metabolizing ones, and in the number of metazoan cells. The members of Protista and Fungi, which line in microaerobic or anaerobic conditions, nearly inevitably lose their mitochondria. The tendency to lose Golgi-complex is demonstrated in protists with parasitic mode of life, especially in combination with anaerobiosis. There is so far no satisfied morphological criterium that could say with certainty whether the lacking of flagella, Golgi complex or mitochondria in the low eukaryotes may be primary or secondary (as the result of reduction). Data on the composition, structure and RNA nucleotide sequences cannot be either the straight evidence. A comparative analysis of these data shows that the ribosomes of the primary eukaryotes were, presumably, of a prokaryotic type. Their eukaryotization was carried out for a long time during the evolution of the low eukaryotes (Protista and Fungi), probably, independently in different phylogenetic lines. It is unknown at what steps and in what main phylogenetic lines the three above mentioned organelles may have appeared. It is proposed to single out a special division of cytology--organellology (organoidology)--as an individual science whose main purpose may be investigation of the origination, evolution and disappearance of organelles.     

Mock communities highlight the diversity of host‐associated eukaryotes

Host‐associated microbes are ubiquitous. Every multicellular eukaryote, and even many unicellular eukaryotes (protists), hosts a diverse community of microbes. High‐throughput sequencing (HTS) tools have illuminated the vast diversity of host‐associated microbes and shown that they have widespread influence on host biology, ecology and evolution (McFall‐Ngai et al. 2013 ). Bacteria receive most of the attention, but protists are also important components of microbial communities associated with humans (Parfrey et al. 2011 ) and other hosts. As HTS tools are increasingly used to study eukaryotes, the presence of numerous and diverse host‐associated eukaryotes is emerging as a common theme across ecosystems. Indeed, HTS studies demonstrate that host‐associated lineages account for between 2 and 12% of overall eukaryotic sequences detected in soil, marine and freshwater data sets, with much higher relative abundances observed in some samples (Ramirez et al. 2014 ; Simon et al. 2015 ; de Vargas et al. 2015 ). Previous studies in soil detected large numbers of predominantly parasitic lineages such as Apicomplexa, but did not delve into their origin [e.g. (Ramirez et al. 2014 )]. In this issue of Molecular Ecology, Geisen et al. ( 2015 ) use mock communities to show that many of the eukaryotic organisms detected by environmental sequencing in soils are potentially associated with animal hosts rather than free‐living. By isolating the host‐associated fraction of soil microbial communities, Geisen and colleagues help explain the surprisingly high diversity of parasitic eukaryotic lineages often detected in soil/terrestrial studies using high‐throughput sequencing (HTS) and reinforce the ubiquity of these host‐associated microbes. It is clear that we can no longer assume that organisms detected in bulk environmental sequencing are free‐living, but instead need to design studies that specifically enumerate the diversity and function of host‐associated eukaryotes. Doing so will allow the field to determine the role host‐associated eukaryotes play in soils and other environments and to evaluate hypotheses on assembly of host‐associated communities, disease ecology and more.     

Genetic diversity of microbial eukaryotes in anoxic sediment around fumaroles on a submarine caldera floor based on the small-subunit rDNA phylogeny

Recent culture-independent molecular analyses have shown the diversity and ecological importance of microbial eukaryotes (protists) in various marine environments. In the present study we directly extracted DNA from anoxic sediment near active fumaroles on a submarine caldera floor at a depth of 200 m and constructed genetic libraries of PCR-amplified eukaryotic small-subunit (SSU) rDNA. By sequencing cloned SSU rDNA of the libraries and their phylogenetic analyses, it was shown that most sequences have affiliations with known major lineages of eukaryotes (Cercozoa, Alveolata, stramenopiles and Opisthokonta). In particular, some sequences were closely related to those of representatives of eukaryotic parasites, such as Phagomyxa and Cryothecomonas of Cercozoa, Pirsonia of stramenopiles and Ichthyosporea of Opisthokonta, although it is not clear whether the organisms occur in free-living or parasitic forms. In addition, other sequences did not seem to be related to any described eukaryotic lineages suggesting the existence of novel eukaryotes at a high-taxonomic level in the sediment. The community composition of microbial eukaryotes in the sediment we surveyed was different overall from those of other anoxic marine environments previously investigated.     

Evolution and diversity of the Golgi

The Golgi is an ancient and fundamental eukaryotic organelle. Evolutionary cell biological studies have begun establishing the repertoire, processes, and level of complexity of membrane-trafficking machinery present in early eukaryotic cells. This article serves as a review of the literature on the topic of Golgi evolution and diversity and reports a novel comparative genomic survey addressing Golgi machinery in the widest taxonomic diversity of eukaryotes sampled to date. Finally, the article is meant to serve as a primer on the rationale and design of evolutionary cell biological studies, hopefully encouraging readers to consider this approach as an addition to their cell biological toolbox. It is clear that the major machinery involved in vesicle trafficking to and from the Golgi was already in place by the time of the divergence of the major eukaryotic lineages, nearly 2 billion years ago. Much of this complexity was likely generated by an evolutionary process involving gene duplication and coevolution of specificity encoding membrane-trafficking proteins. There have also been clear cases of loss of Golgi machinery in some lineages as well as innovation of novel machinery. The Golgi is a wonderfully complex and diverse organelle and its continued exploration promises insight into the evolutionary history of the eukaryotic cell.     

A comparative analysis of trypanosomatid SNARE proteins

The Kinetoplastida are flagellated protozoa evolutionary distant and divergent from yeast and humans. Kinetoplastida include trypanosomatids, and a number of important pathogens. Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. inflict significant morbidity and mortality on humans and livestock as the etiological agents of human African trypanosomiasis, Chagas' disease and leishmaniasis respectively. For all of these organisms, intracellular trafficking is vital for maintenance of the host–pathogen interface, modulation/evasion of host immune system responses and nutrient uptake. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are critical components of the intracellular trafficking machinery in eukaryotes, mediating membrane fusion and contributing to organelle specificity. We asked how the SNARE complement evolved across the trypanosomatids. An in silico search of the predicted proteomes of T. b. brucei and T. cruzi was used to identify candidate SNARE sequences. Phylogenetic analysis, including comparisons with yeast and human SNAREs, allowed assignment of trypanosomatid SNAREs to the Q or R subclass, as well as identification of several SNAREs orthologous with those of opisthokonts. Only limited variation in number and identity of SNAREs was found, with Leishmania major having 27 and T. brucei 26, suggesting a stable SNARE complement post-speciation. Expression analysis of T. brucei SNAREs revealed significant differential expression between mammalian and insect infective forms, especially within R and Qb-SNARE subclasses, suggesting possible roles in adaptation to different environments. For trypanosome SNAREs with clear orthologs in opisthokonts, the subcellular localization of TbVAMP7C is endosomal while both TbSyn5 and TbSyn16B are at the Golgi complex, which suggests conservation of localization and possibly also function. Despite highly distinct life styles, the complement of trypanosomatid SNAREs is quite stable between the three pathogenic lineages, suggesting establishment in the last common ancestor of trypanosomes and Leishmania. Developmental changes to SNARE mRNA levels between blood steam and procyclic life stages suggest that trypanosomes modulate SNARE functions via expression. Finally, the locations of some conserved SNAREs have been retained across the eukaryotic lineage.     

Conserved function of the lysine-based KXD/E motif in Golgi retention for endomembrane proteins among different organisms

We recently identified a new COPI-interacting KXD/E motif in the C-terminal cytosolic tail (CT) of Arabidopsis endomembrane protein 12 (AtEMP12) as being a crucial Golgi retention mechanism for AtEMP12. This KXD/E motif is conserved in CTs of all EMPs found in plants, yeast, and humans and is also present in hundreds of other membrane proteins. Here, by cloning selective EMP isoforms from plants, yeast, and mammals, we study the localizations of EMPs in different expression systems, since there are contradictory reports on the localizations of EMPs. We show that the N-terminal and C-terminal GFP-tagged EMP fusions are localized to Golgi and post-Golgi compartments, respectively, in plant, yeast, and mammalian cells. In vitro pull-down assay further proves the interaction of the KXD/E motif with COPI coatomer in yeast. COPI loss of function in yeast and plants causes mislocalization of EMPs or KXD/E motif–containing proteins to vacuole. Ultrastructural studies further show that RNA interference (RNAi) knockdown of coatomer expression in transgenic Arabidopsis plants causes severe morphological changes in the Golgi. Taken together, our results demonstrate that N-terminal GFP fusions reflect the real localization of EMPs, and KXD/E is a conserved motif in COPI interaction and Golgi retention in eukaryotes.     

Structural-functional organization of Golgi apparatus

This review is dedicated to the structure and function of Golgi apparatus (GA). It summarizes contemporary data published in numerous experimental papers and in several reviews. Possible ways of intra-Golgi transport of proteins, existent models of structural and functional organization of Golgi organelle, as well as the issues of its biogenesis, posttranslational modification and sorting of proteins and lipids, and mechanisms of their trafficking are discussed. Special attention is paid to the role of coatomer proteins (COPI, COPII and clathrin), fusion proteins (SNAREs), and small GTPases (ARF, SARI) in the secretory pathway. In addition, the phenomena of ultrastructural alterations of GA due to various functional conditions and physiological stimuli are specifically accented. We included in this review our original data on a probable involvement of GA in water transport, and on the organization of atypical GA in microsporidia--intracellular parasitic protists.     

Structural-functional organization of Golgi apparatus

This review is dedicated to the structure and function of Golgi apparatus (GA). It summarizes contemporary data published in numerous experimental papers and in several reviews. Possible ways of intra-Golgi transport of proteins, existent models of structural and functional organization of Golgi organelle, as well as the issues of its biogenesis, posttranslational modification and sorting of proteins and lipids, and mechanisms of their traffic-king are discussed. Special attention is paid to the role of coatomer proteins (COPI, COPII and clathrin), fusion proteins (SNAREs), and small GTPases (ARF, SARI) in the secretory pathway. In addition, the phenomena of ultrastructural alterations of GA due to various functional conditions and physiological stimuli are specifically accented. We included in this review our original data on a probable involvement of GA in water transport, and on the organization of atypical GA in microsporidia--intracellular parasitic protists.     

Rewiring and regulation of cross-compartmentalized metabolism in protists

Plastid acquisition, endosymbiotic associations, lateral gene transfer, organelle degeneracy or even organelle loss influence metabolic capabilities in many different protists. Thus, metabolic diversity is sculpted through the gain of new metabolic functions and moderation or loss of pathways that are often essential in the majority of eukaryotes. What is perhaps less apparent to the casual observer is that the sub-compartmentalization of ubiquitous pathways has been repeatedly remodelled during eukaryotic evolution, and the textbook pictures of intermediary metabolism established for animals, yeast and plants are not conserved in many protists. Moreover, metabolic remodelling can strongly influence the regulatory mechanisms that control carbon flux through the major metabolic pathways. Here, we provide an overview of how core metabolism has been reorganized in various unicellular eukaryotes, focusing in particular on one near universal catabolic pathway (glycolysis) and one ancient anabolic pathway (isoprenoid biosynthesis). For the example of isoprenoid biosynthesis, the compartmentalization of this process in protists often appears to have been influenced by plastid acquisition and loss, whereas for glycolysis several unexpected modes of compartmentalization have emerged. Significantly, the example of trypanosomatid glycolysis illustrates nicely how mathematical modelling and systems biology can be used to uncover or understand novel modes of pathway regulation.     

Trophic and Non-Trophic Interactions in a Biodiversity Experiment Assessed by Next-Generation Sequencing

Plant diversity affects species richness and abundance of taxa at higher trophic levels. However, plant diversity effects on omnivores (feeding on multiple trophic levels) and their trophic and non-trophic interactions are not yet studied because appropriate methods were lacking. A promising approach is the DNA-based analysis of gut contents using next generation sequencing (NGS) technologies. Here, we integrate NGS-based analysis into the framework of a biodiversity experiment where plant taxonomic and functional diversity were manipulated to directly assess environmental interactions involving the omnivorous ground beetle Pterostichus melanarius. Beetle regurgitates were used for NGS-based analysis with universal 18S rDNA primers for eukaryotes. We detected a wide range of taxa with the NGS approach in regurgitates, including organisms representing trophic, phoretic, parasitic, and neutral interactions with P. melanarius. Our findings suggest that the frequency of (i) trophic interactions increased with plant diversity and vegetation cover; (ii) intraguild predation increased with vegetation cover, and (iii) neutral interactions with organisms such as fungi and protists increased with vegetation cover. Experimentally manipulated plant diversity likely affects multitrophic interactions involving omnivorous consumers. Our study therefore shows that trophic and non-trophic interactions can be assessed via NGS to address fundamental questions in biodiversity research.     

Biochemistry and Evolution of Anaerobic Energy Metabolism in Eukaryotes

Summary: Major insights into the phylogenetic distribution, biochemistry, and evolutionary significance of organelles involved in ATP synthesis (energy metabolism) in eukaryotes that thrive in anaerobic environments for all or part of their life cycles have accrued in recent years. All known eukaryotic groups possess an organelle of mitochondrial origin, mapping the origin of mitochondria to the eukaryotic common ancestor, and genome sequence data are rapidly accumulating for eukaryotes that possess anaerobic mitochondria, hydrogenosomes, or mitosomes. Here we review the available biochemical data on the enzymes and pathways that eukaryotes use in anaerobic energy metabolism and summarize the metabolic end products that they generate in their anaerobic habitats, focusing on the biochemical roles that their mitochondria play in anaerobic ATP synthesis. We present metabolic maps of compartmentalized energy metabolism for 16 well-studied species. There are currently no enzymes of core anaerobic energy metabolism that are specific to any of the six eukaryotic supergroup lineages; genes present in one supergroup are also found in at least one other supergroup. The gene distribution across lineages thus reflects the presence of anaerobic energy metabolism in the eukaryote common ancestor and differential loss during the specialization of some lineages to oxic niches, just as oxphos capabilities have been differentially lost in specialization to anoxic niches and the parasitic life-style. Some facultative anaerobes have retained both aerobic and anaerobic pathways. Diversified eukaryotic lineages have retained the same enzymes of anaerobic ATP synthesis, in line with geochemical data indicating low environmental oxygen levels while eukaryotes arose and diversified.