PROBLEMS & PARADIGMS
Prospects & Overviews
Debating Eukaryogenesis—Part 1: Does Eukaryogenesis
Presuppose Symbiosis Before Uptake?
Dave Speijer
Eukaryotic origins are heavily debated. The author as well as others have
proposed that they are inextricably linked with the arrival of a
pre-mitochondrion of alphaproteobacterial-like ancestry, in a so-called
symbiogenic scenario. The ensuing mutual adaptation of archaeal host and
endosymbiont seems to have been a defining influence during the processes
leading to the last eukaryotic common ancestor. An unresolved question in
this scenario deals with the means by which the bacterium ends up inside.
Older hypotheses revolve around the application of known antagonistic
interactions, the bacterium being prey or parasite. Here, in reviewing the field,
the author argues that such models share flaws, hence making them less
likely, and that a “pre-symbiotic stage” would have eased ongoing metabolic
integration. Based on this the author will speculate about the nature of the
(endo) symbiosis that started eukaryotic evolution–in the context of bacterial
entry being a relatively “early” event–and stress the differences between this
uptake and subsequent ones. He will also briefly discuss how the mutual
adaptation following the merger progressed and how many eukaryotic
hallmarks can be understood in light of coadaptation. Also see the video
abstract here https://youtu.be/ekqtNleVJpU
…mitochondria originated when some sort of single-celled or-
ganism… swallowed a bacterium and then failed to digest it,
or became infected by a bacterium and then failed to cure
itself, or was otherwise entered by a bacterium and allowed
the thing to stay. This signal event happened only once. David
Quammen—The Tangled Tree[1]
1. Introduction
Most scientists, myself included, seem to live in a bubble when it
comes to contentious issues in our field of study, engaging in too
Dr. D. Speijer
Department of Medical Biochemistry, AmsterdamUMC
University of Amsterdam
Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands
E-mail: d.speijer@amsterdamumc.nl
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/bies.201900157
© 2020 The Authors. BioEssays published by Wiley Periodicals, Inc.
This is an open access article under the terms of the Creative Commons
Attribution License, which permits use, distribution and reproduction in
any medium, provided the original work is properly cited.
DOI: 10.1002/bies.201900157
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little real open-minded exchange of ideas.
This is certainly true with regard to one of
the great conundrums in biology: “How
did eukaryotes come about?” I will try to
set out what aspects of eukaryogenesis
most researchers agree upon and what
are the “bones of contention,” in a two-
part contribution. The focus will be on
a comparison of the two main models
(“symbiogenic/chimeric” vs “autogenic/
incremental,” see Table 1), which can be
seen as opposite positions on a spectrum of
hybrid views. I will try to show that a sym-
biotic relationship between prokaryotes,
characterized by metabolic interdepen-
dency well before uptake (referred to as
pre-uptake symbiosis or “pre-symbiosis”)
seems essential for eukaryogenesis. The
second article will attempt to highlight
the many ways in which anachronistic
reasoning (using examples from highly
derived present-day eukaryotes to ex-
plain their origins) keep on clouding the
debate.
I will try to carefully elucidate the reasoning behind my own,
symbiogenic position. An extensive description of last eukaryotic
common ancestor’s (LECA) complexity and a less biased overview
of different ideas regarding its evolution can be found in a rel-
atively up-to-date review by Koumandou et al.[2] Let me stress
here that reconstructing interactions occurring more than 1.5
billion years ago, under unknown ecological conditions, involv-
ing organisms that have to be reconstructed from their present-
day relatives is daunting. Thus all our statements should be cau-
tious and conditional. When lacking such caution in what fol-
lows, please remember this proviso.
2. Symbiogenesis Versus Autogenesis: Did
Eukaryogenesis Start with Bacterial Uptake or Not?
Many biologists agree that there were two (and only two) differ-
ent cell types directly involved in the processes that gave rise to
the evolution of the eukaryotes (one archaeon-related, the other
a bacterium); see ref. [3] and references therein, though some al-
ternatives have been proposed as well (e.g., Part III of ref. [1] and
below). How and why the bacterium ended up in the archaeon
is debated. An important aspect to point out is that most re-
searchers that have wrestled with the mechanisms involved in eu-
karyogenesis either come from a metabolic/biochemical or a cell
biology/protistology background. The first group tends to focus
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Table 1. Two basic models of eukaryogenesis.
Symbiogenesis Autogenesis
Characteristic:
Mitochondria Early Late
a)
Phagocytosis Late Early
Driving force(s) Integration of metabolism/ROS ? 2) Symbiogenesis, starting with the merger of two prokaryotes
Intermediate states “Fleeting” ? (archaeon and bacterium) by an unknown, “primitive” mech-
Timespan Short Long
b)
First uptake mechanism ? Phagocytic
Occurrence Very rare Likely?
Relation of organisms Merger (chimera) Host acquisition
a) WhetherLECA was phagocytic is debated; b) Uptake in accordance with the inside-
out model?[ 58 ] Essential differences in the two competing models in the form of
catchphrases.
on the challenges of getting an integrated metabolism, starting
with two separate, self-contained, biochemical units; while not
worrying too much about either the physical mechanism of up-
take or how defining characteristics of the eukaryotic cell evolved.
Overall, the second group is more interested in these aspects,
and metabolic integration takes the back seat. Taken very broadly,
this is reflected in their respective preferences. Overall, the bio-
chemists favor symbiogenesis, liking the process to a merger af-
ter which developments possibly occurred rapidly. The cell biol-
ogists favor a more protracted process in which the “host” cell
incrementally develops many defining eukaryotic traits in the ab-
sence of the future endosymbiont, which will allow its uptake by
phagocytic-like mechanisms: autogenesis.[3–6] By “autogenesis” I
do not mean that all organelles are inventions of the eukaryotic
cell (the mitochondrion clearly is not), but that most, if not all,
of its defining cellular structures evolved in the absence of selec-
tive pressures derived from endosymbiont entry. To avoid confu-
sion, let me stress the following. Using phylogenomic analysis,
archaea and eukaryotes end up in the same group, giving rise to a
robustly supported two-domains tree of life.[7] However, eukary-
otes and archaea are so fundamentally different that the need for
three domains can still be defended; see, for example, ref. [8]. In
the following discussion I use “pre-eukaryote” whenever I refer
to a pre-LECA, more complex, cellular entity derived from an ar-
chaeal progenitor.
Thus, the basically shared idea is that we begin with ei-
ther specific archaea or (archaeon derived) pre-eukaryotes, and
alphaproteobacterium-like bacteria. The first are “hosts,” the sec-
ond are pre-endosymbionts that will give rise to mitochondria.
Filling in the details, many points of disagreement come into
focus. An important point of contention revolves around pref-
erences for either an archaeon or a pre-eukaryote. Those who
prefer the term “pre-eukaryote” generally envisage at least some
eukaryotic characteristics developing in the absence of the en-
dosymbiont. Of specific importance in the latter framework is
the development of endo- or phagocytosis. If this capacity devel-
oped in the absence of the endosymbiont it can be used to explain
its entry. Proponents of this line of thought have the “host” de-
velop a minimally efficient endocytotic machinery and undergo
at least some initial growth allowing for the uptake of an integral
prokaryotic organism, either by aborted phagocytosis or aborted
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digestion in the absence of a “pre-mitochondrion.” The two main
contending theories,
1) Autogenesis, in which small steps lead to a pre-eukaryote
making a living by use of endo/phagocytic capabilities, allow-
ing uptake of organisms, one of which will become the mito-
chondrion, the other,
anism, postulating that most, if not all, eukaryotic character-
istics arose as adaptations to challenges the resulting chimera
faced, are compared in Figure 1 and Table 1.
Of course, several researchers have championed hybrid the-
ories. Two extensive, well written, reviews give an overview of
current thinking, one from researchers sharing the symbiogenic
point of view,[9] the other tending toward “phagocytosis early.”[5]
2.1. The First Autogenic Hypothesis Had To Be Abandoned
As indicated, the older, more gradualist vision hypothesizes that
eukaryotic features such as internal membrane structures, a nu-
cleus, and phagocytosis developed early on. The phagocytic ca-
pability in turn allowed uptake of a bacterium related to present-
day alphaproteobacteria that gave rise to mitochondria. Its oldest
incarnation, the archezoan hypothesis, saw certain amitochon-
driate eukaryotes (“Archezoa”) as “living fossils,” reflecting the
stage before endosymbiont uptake.[10] However, all amitochon-
driate groups are now known to have evolved from mitochondri-
ate ancestors, so they are not seen as representing early branch-
ing clades anymore. Though the Archezoa have been abandoned,
the idea that endo/phagocytic capabilities were a precondition for
uptake of the endosymbiont is still thriving (see ref. [5] and refer-
ences therein), because it is hard to envisage alternative mecha-
nisms.
2.2. How Do Cells Merge in the Absence of Phagocytic
Mechanisms?
Indeed, a weakness of a symbiogenic theory, with a form of
phagocytosis developing after uptake, is the merger by an un-
known mechanism. In this light, the very recent, first success-
ful cultivation of an Asgard archaeon, Prometheoarchaeum, (re-
sembling the “hosts” involved in eukaryogenesis,[11–13] but not
belonging to the group most related to the “host”) shows it to
have extensions, demonstrating increased curvature and pos-
sibilities for membrane–membrane interactions.[14] Further re-
search could show whether these archaea might occasionally take
up their symbiotic partners. Even before this finding, some prob-
lems with the proposals based on full blown phagocytosis were
obvious. The two main proposals for entry by established phago-
cytic mechanisms are entry as a pathogenic agent, for example,
a facultative intracellular energy parasite, or engulfment by a
predatory pre-eukaryote. Based on phylogenetic considerations,
known non-phagocytic methods of entry by parasites (such as
used by Bdellovibrio bacteria[15] ) are unlikely to have played a
role. As mentioned, phylogenetic analyses have shown the “host”
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Figure 1. Alternative scenarios for eukaryogenesis. A) Phagocytotic engulfment or parasitic invasion. The further programmed development is inter-
rupted and somehow endosymbiosis ensues. The “host” cell is a pre-eukaryote of archaeal origin (brown). The timing of many inventions as well as the
driving forces responsible are uncertain. Only some further increase in complexity is seen to be dependent on bacterial uptake. B) Presymbiosis leading
to symbiogenesis via many intermediate steps. Enhanced internal ROS formation is probably crucial in the symbiogenic process. Internal ROS leads to
bacterial DNA (black circles) reduction and host DNA increase (thicker circles) plus possible membrane protection by bacterial OMVs (blue; forming a
pre-ER?). Retargeting of FA oxidation enzymes to the pre-mitochondrion and (related) OMVs, combined with insertion of catalase, part of the prokaryotic
secretome, gives rise to peroxisomes (olive). The archaeal outer membrane (brown) is replaced and a nuclear membrane (red) and full-blown ER (green)
and Golgi (purple) evolve. The specific sequence of events here is again uncertain. For reasons of simplicity, mitochondria (blue with thin DNA circle) are
depicted with a single membrane structure. The double nuclear membrane (derived from the stacking of OMVs?) is also depicted as a single structure
(with ER and Golgi as connected “extensions”). Phago- or syntrophic modes refer to the use of these terms in ref. [86].

lineage leading to the eukaryotes to be most related to recently
discovered groups of archaea, the Asgards, engendering specula-
tions regarding their phagocytic capacities.[11,12] However, work
from Burns and co-workers based on the analysis of gene sets
predictive of trophic mode seems to suggest that Asgard archaea
are not phagocytic.[16] Their findings highlight that eukaryotic
phagocytosis is a capability based on a combination of archaeal
and bacterial gene products. However, they cannot distinguish
between a scenario in which the bacterial genes were picked up
in slow progression before endosymbiont uptake or were (partly)
derived from the pre-mitochondrion itself, after entry, instead. I
will leave out further discussion of the highly contentious phylo-
genetic debates surrounding these uptake proposals (“who swal-
lowed/infected whom”) and just focus on some aspects making
the whole model of an efficiently phagocytosing amitochondri-
ate Asgard archaeon (relative) at the start of eukaryogenesis less
likely. Here, it should be pointed out that there is an example of
a phagocytosing amitochondriate prokaryote, though completely
unrelated: a planctomycete bacterium. It demonstrates that the
presence of a mitochondrion is not an absolute requirement for
a form of phagocytosis to occur.[17]
Indeed, in autogenous scenarios a more complex, larger,
phagocytic cell is presupposed to have come about before ef-
ficient endogenous ATP generation (using O2 as the final ac-
ceptor) became available from multitudes of (pre)mitochondria.
Although precise reasoning vis-a-vis an energetic barrier to or-
ganismal complexity is difficult, there seems to be a correla-
tion between increasing complexity levels and (mitochondrial)
oxidation.[18–20] Also, for the spoils of phagocytosis to be used
efficiently, catabolic pathways linking proteins, carbohydrates,
and fats to the oxidative electron transport chains (ETCs) of
(pre)mitochondria seems necessary; the large majority of these
came with the endosymbiont. The existence of anaerobic phago-
cytic eukaryotes (which are derived from aerobic, “normal” an-
cestors) does not invalidate these arguments; see, for example,
ref. [3] and Part 2 of this paper (How anachronistic reasoning
can lure us into inventing intermediates). Based on these inher-
ent problems, I[21] and others opted for a non-phagocytic mecha-
nism of entry, though inefficient phagocytosis, boosted by getting
stuck with the endosymbiont, cannot be ruled out, especially in
the light of the recent example of a phagocytosing prokaryote.[17]
An extensive overview of the most persuasive arguments can be
found in ref. [22]. The absence of all intermediate forms prior to
LECA is a difficulty for both theories of eukaryogenesis, but es-
pecially for the more gradual one. Autogenesis also seems to lack
credible driving forces for many of the eukaryotic “inventions.”
Many of these do make sense in the context of symbiogenesis
(see below).
3. Predator or Parasite Models of Eukaryogenesis
Have Conceptual Problems
Let us leave aside the problem of the development of a larger
and more complex cell capable of uptake of complete prokaryotes
without efficient ATP generation from breakdown of its main
components for now. Many of the eukaryotic traits also come
from mergers of the capabilities present in either participant,

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and eukaryotic endo/phagocytosis is no exception, highlighting
a temporal difficulty.
Eörs Szathmáry and John Maynard Smith make an important
observation in their illuminating review of “The major evolution-
ary transitions.”[23] They point out the real danger of selection at
lower levels (in this case the individual prokaryotes) disrupting
integration at the higher level (the eukaryote to be). To make such
a difficult transition work (of course in the absence of foresight),
starting with two essentially antagonistic cells does look less
promising. A gradually developing symbiosis of groups of cells
in a changing environment leading to mutual dependency seems
a superior spot to look for the birthplace of the eukaryotes, with
“pre-adapted” (consortia of) bacteria having better chances of
surviving an “accidental” uptake,[20,24,25] because of a pre-existing
metabolic integration. Here, it is worthwhile to be reminded
that the successful primordial uptake of the mitochondrial
forerunner depended on two aspects: entry and functional
retention. Though the focus has been on the first, it might be
that the second aspect represents the biggest hurdle. Retention
is problematic because of the really challenging molecular and
biochemical puzzle that arises upon entry and not because of
“host defences”[26] as the host cells were not primed by evolution
for relatively rare events (see also below). Still, one can imagine
inadvertent uptake of a bacterium in symbiotic populations of ar-
chaea and bacteria, several of which have been shown to exist so
far.[27]
Symbiotic interactions involving archaea and bacteria are
probably very ancient. With the aid of secondary ion mass spec-
troscopy analysis, Schopf and co-workers found indications for
interactions between CH4 producing and CH4 metabolizing
prokaryotes already present ≈0.5 × 109 years after the late heavy
bombardment ended.[28] Rapid evolution of prokaryotic trade,
starting with the use of the waste product of one participant, but
ending with populations in which both partners secrete costly
products, has even been observed in laboratory settings.[29–31] An
important precondition for these stable symbioses to take hold
is that they occur in spatially structured environments. There
are different scenarios for the primordial symbiosis giving rise
to eukaryogenesis, such as the “hydrogen hypothesis”[24] (named
after the waste product involved) or alternating cellular electron-
acceptors operating in the presence of varying amounts of oxygen
(with intermediate metabolites as waste).[20] Both occur in highly
structured environments.
Some archaea are capable of cell fusion; see ref. [32] and ref-
erences therein. Bacteria can take up other bacteria (e.g., found
in Mealybugs, in which insect cells contain betaproteobacteria,
in turn harboring gammaproteobacteria[33,34] ). The last example
shows such uptake is best understood as the end result of an in-
creasingly close pre-symbiosis. However, we should be wary of
modern examples, as they occur in the context of full-fledged
modern eukaryotic phagocytosis.
4. Are Modern-Day Examples of Bacterial Entry
Evolutionarily Misleading?
In a previous article I argued that the lessons modern-day
uptake examples had to offer with regard to how the process
played out early in life history, might be severely limited.[6,35] In
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the context of eukaryogenesis, many instances of interpreting
current life forms or biological processes as representative of
ancient stages have turned out to be misleading or might be
so. The most notorious example was already mentioned: that
the amitochondriate eukaryotes were living fossils, representing
the complex phagocytosing stage before bacterium uptake.
However, they are products of reductive evolution, having lost
mitochondria; see, for example, ref. [36]. The current debate
whether eukaryotic hydrogenosomes are reflective of ancient
mitochondrial capabilities or late adaptations to new ecological
niches following horizontal gene transfer (HGT; compare refs.
[24, 36–40]) illustrates the difficulties involved; see also Part 2.
But looking for the closest relative of the original endosymbiont
among modern intracellular alphaproteobacterial parasites[26]
might be misguided as well. The idea that mitochondria are
derived from intracellular parasites, tricking the host into phago-
cytosis, got a boost from the presence of genes encoding proteins
associated with the dynamic eukaryotic architecture (including
phagocytosis) in archaeal “Asgard” metagenomes.[11,12] This
induced Ball and co-workers to revisit the hypothesis that the
parasite in question was “a Rickettsiales-like” organism.[26] Much
is made of the fact that Rickettsia can multiply in the host cytosol
without the use of inclusion vesicles, because this demonstrates
an ability to cope with “host defences.” However, the scenario
of rare, accidental, uptake, does not have to contend with such
mechanisms being in place. These authors then suggest a “tran-
sition from endocytosis of nutrients to a full-fledged phagocytic
lifestyle” powered by the mitochondrion. Here a conceptual flaw
seems to present itself. The “endocytosis of nutrients” by what
seems to be primitive/simple phagocytosis, resembling pinocy-
tosis, functions as a construct to solve the paradox of the uptake
and catabolic use of a full-size bacterium in the absence of mito-
chondrial power and metabolism. As pointed out by Sven Gould,
all Rickettsia are obligate intracellular pathogens depending
on eukaryotes for survival.[41,42] They represent highly derived
adaptations to ecological niches that only became available after
the flowering of eukaryotes, and co-evolved with them. Thus it
becomes really difficult to deduce whether a free-living ancestor
could have been involved. At this juncture it is appropriate to
point out that many bacterial taxons contain organisms capable
of intracellular parasitism of eukaryotes. Just looking at human
pathogens: Mycobacterium tuberculosis (actinobacteria), Legionella
pneumophila, Salmonella enterica, Yersinia pseudotuberculosis (all
gammaproteobacteria), and Listeria monocytogenes (firmicutes) to
name but a few. Even if alphaproteobacteria are overrepresented
among the intracellular parasites of eukaryotes, this, first of all,
reflects the fact that Wolbachia species can infect large amounts
of arthropods (especially insects) and nematodes.[43] Crucially,
the most recent phylogenetic evidence points to mitochondria
evolving from a sister lineage splitting before the divergence of
all sampled alphaproteobacteria, barring a meaningful choice
between free-living or parasitic mitochondrial precursors (as-
suming such existed back then).[44] However, accidental uptake
of an alphaproteobacterium-like organism that morphs into mi-
tochondria would of course enable their modern-day relatives to
escape eukaryotic host defences more easily. The recent cultiva-
tion of an Asgard representative without indications for endocytic
mechanisms, but with membrane extensions makes the whole
“parasite precursor for mitochondria” model less likely.[14]
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5. Predator/Parasite Models of Eukaryogenesis
Can Be Challenged by Physical Considerations
LECA, likely capable of phagocytosis, was a highly complex
organism, using the specialized “new” functions enabled by
repeated gene duplications to perform well-regulated endocytic
processes.[2] Even the Asgard archaea do not come close to this
level of complexity. The cultivated representatives, though en-
coding quite a few “eukaryotic specific proteins” remain archaea.
Burns and co-workers, analyzing trophic mode predictive gene
sets, conclude that these archaea are not phagocytic,[16] Dey
et al. convincingly show that even claiming a “primitive” form
is premature at best.[25] Such claims are based on the genomes
encoding small GTPases, resembling those involved in vesicle
transport, as well as COP (vesicle coat proteins) and TRAnsport
protein particle (TRAPP) homologues. First of all: “homologous
genes” do not automatically have “homologous functions” (see
also Part 2). More importantly, for these GTPases to function in
membrane traffic, proper localization is needed. In eukaryotes,
most of them are anchored to membranes via hydrophobic
groups. Interestingly, prenylated and palmitoylated GTPases
are both involved in membrane/vesicle transport regulation,
again stressing the hybrid nature of these processes, using both
archaeal (isoprene building blocks) and bacterial (fatty acid build-
ing blocks) anchors. The Asgard GTPases miss canonical recog-
nition sequences for the attachment of the hydrophobic groups
and the metagenomes do not encode the necessary enzymes
either.[25] Asgard metagenomes miss other endocytosis com-
ponents, mediating inward membrane curvature as well as the
enzymes capable of membrane scission: dynamins. These seem
to be of (mitochondrial) endosymbiont origin as well.[45] One
also has to consider that, though mixing of the two membrane
types turns out to be easily feasible,[46] we do not know whether
endocytosis with archaeal membranes is easy as well. In light of
their analyses, Dey et al. stress the importance of an increasingly
close pre-symbiosis.[25] This conclusion was born out upon the
cultivation of an Asgard representative mentioned above.[14] Fi-
nally, as the “Asgard” metagenomes seem to predict operational
ETCs, even pinocytosis could disturb their membrane potential.
Thus, endo/phagocytic models need to explain adequate ATP
generation in the absence of adequately functioning ETCs.
This brings us to the question of driving forces for eukaryotic
inventions (e.g., the greatly increased eukaryotic gene set) begin-
ning with the merger of two prokaryotes and why the “mitochon-
dria late” model[47] is less likely to be correct. Let us expand upon
symbiogenic theories,[13,20,24,48] explaining their “revolutionary”
character.[3,6]
6. How Symbiogenesis, the “Ultimate
Coadaptation” Model, Gave Rise to the Last
Eukaryotic Common Ancestor
Symbiogenic theories, explaining eukaryotic characteristics as
mutual adaptations of two prokaryotes, started with the work of
Martin and Muller[24] and Moreira and Lopez-Garcia.[48] Though
Schopf and co-workers found indications for such interactions
(possibly between archaea and 𝛾-proteobacteria) dating back
≈3.5 × 109 years,[28] eukaryotes appeared on the scene much
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later (between 2.1 × 109 and 1.6 × 109 years ago[49] ). This time
lag in eukaryotic evolution seems to correlate with the slow in-
crease of O2 due to cyanobacterial activity (and illustrated by
the great oxygenation event or “GOE”; 2.3 × 109 years ago). In
light of such considerations, several alternative symbiotic sce-
narios have been proposed (see e.g. the reviews in refs. [5, 50]
and references therein). I favour a primordial symbiosis based
on intermediate carbohydrate metabolite exchange necessitated
by recurring changes in oxygen availability alternatively hamper-
ing the different terminal electron chain complexes of the two
prokaryotes involved.[20] This seems to be in line with observa-
tions in the cultivated Asgard archaeon that lives in symbioses
and has metabolic pathways that could be enhanced by transfer-
ring metabolites to bacteria capable of using molecular oxygen
as the final electron-acceptor.[14] The essence of symbiogenesis
is nicely described by Nick Lane: “The dominant selective forces
driving eukaryotic evolution arose from within the cell, not the ex-
ternal environment.”[3] However, I disagree when he downplays
the role of O2 by stressing that even after the GOE many envi-
ronments were still anoxic (which is hardly relevant). Also the
existence of anaerobic eukaryotes does not mean that these in
anyway reflect what might have happened during evolution to-
wards LECA or that LECA might have had mitochondria with
anaerobic capabilities. It is surprising that he correctly dismisses
phagothrophic anaerobic eukaryotes as misleading later develop-
ments (stressing the difference between “origin” and “retention”
of phagocytosis; see also Part 2, while he does not consider this
possibility for anaerobic eukaryotes as such.[3] I stress this fact
because eukaryotic evolution seems to me to only make sense in
the light of an aerobic bacterium as one of the partners in the
primordial merger (see below).
Whatever the specific model, symbiogenesis has turned out to
be a fruitful concept, its many new insights making it a likely
description of events (see ref. [6] and references therein). Let us
try to reconstruct eukaryogenesis starting with our merger: we
will see eukaryotic features make much more sense when seen
through the prism of symbiogenic theory.
Starting out with a symbiosis between archaea and bacteria,
the bacteria using oxygen when available, the archaea taking care
of business when not, one can envisage cell/cell contacts aris-
ing for efficient exchange of intermediate metabolites.[20] A re-
lated model bases the symbiosis on “reverse flow” involving elec-
tron or hydrogen uptake by the bacterium.[13] Closer symbio-
sis led to more intimate interdependency. Rather early under
these kind of circumstances “cheaters” will appear[30] and such
archaeal cheaters might, for example, have lost their membrane
potential. Such cells could have more easily survived increases
in [O2 ] and accidental uptake of the ever more interacting bacte-
ria. Whatever the details, such a merger is not unlikely to have
occurred in the Asgard communities described in.[14]
7. Essential Eukaryotic Characteristics Can Best Be
Explained by Symbiogenesis and Internal ROS
Formation
A merger thus envisaged allows the development of many
quintessential eukaryotic characteristics, because it led to a
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constellation in which the internal mitochondrial membranes
generated copious ATP for eukaryotic inventions most likely re-
quiring larger amounts of energy[51] and the driving force of
highly damaging internal reactive oxygen species (ROS) forma-
tion; see, for example, refs. [52–55] and references therein. Why
is internal ROS formation crucial?
1) ROS is an intrinsic by-product of oxidative phosphorylation
(OXPHOS). The merger led to oxidation of many different
substrates (reflecting modern-day eukaryotic metabolic ver-
satility): carbohydrates (sugars), amino acids and fatty acids
(FAs). Alternating between them leads to enhanced ROS
formation.[54,56]
2) Eukaryotes often contain myriads of mitochondrial mem-
branes, multiplying ROS effects.
3) The endosymbionts used to produce ROS at their outer mem-
branes, now these are produced smack in the middle of the
pre-eukaryote.
4) ROS need metal ions (mostly Fe) for full reactive oxidative ca-
pacity, and these are concentrated inside as essential enzyme
cofactors.
I will list the most important eukaryotic characteristics to be
explained along these lines.
7.1. Endomembrane System, Nuclei, Peroxisomes,
Autophagosomes, and Phagocytosis
The highly complex, diverse, eukaryotic endomembrane system
possibly arose out of bacterial outer membrane vesicles (OMVs)
released by the mitochondrial ancestor into the cytosol.[57] How-
ever, some kind of internal membrane structure could have
arisen prior to, or with, uptake in archaeal “hosts” by an inside-
out mechanism.[14,58] Alternative scenarios focussing more on
the evolution of the machinery involved can be found in refs.
[2, 59]. An interesting aspect of the endosymbiont OMV theory
is that diluting out archaeal plasma membranes would nicely ex-
plain eukaryotes ending up with bacterial ones. In this respect
the theory is superior to the ad hoc explanation by the bacterium-
archaeon fusion model of Forterre,[60] which was invoked to deal
with phylogenetic challenges (see Section 8).
Nuclei, peroxisomes, autophagosomes, and phagocytosis are
best understood as evolving in response to, or in the context
of, pre-mitochondrial entry. The nucleus might have evolved
from incremental OMV stacking around the host chromosomes,
protecting against internal ROS formation of endosymbiont
origin, and, after reaching a certain level of complexity, separat-
ing splicing from translation in the wake of migration of intron
containing genes from mitochondrial ancestors to hosts.[61] This
argument is sometimes countered with the observation that “the
nuclear membrane would be a poor ROS defence as they can
pass freely through nuclear pores.” However, a typical mem-
brane has ≈1000 pores with a diameter of 120 nm, and an actual
opening of ≈5–10 nm. Such openings are negligible compared
to the complete surface surrounding the chromosomes, so the
membrane would still be a formidable barrier indeed. Even small
interposing vesicles would already reduce ROS reaching DNA
behind it, so this could constitute a viable scenario for incremen-
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tal evolution of the nucleus. OMVs enriched in sterols, another
eukaryotic invention that makes sense in the light of endogenous
ROS,[62] would be effective barriers. Descriptions of the use of
membranes in ROS containment can be found in ref. [63].
Histone evolution also seems to have links to ROS protection.[6]
Autophagy and autophagosomes, already “complete” in LECA,[2]
could have evolved from mitophagy, the process of disposing
of oxidatively damaged mitochondria. Phagocytosis might be
seen as a variation on these mechanisms. Peroxisomes can be
perfectly understood as organelles that evolved as a system to alle-
viate internal ROS formation associated with pre-mitochondrial
FA oxidation and are indeed build from endosymbiont building
blocks.[21,52,64,65] However, to illustrate the range of scenarios
invoked to explain eukaryotic cell structures: Mans et al. specu-
lated that nuclei emerged in an autogenic fashion in primitive
eukaryotic ancestors, during compartmentalization triggered by
archaeo-bacterial symbiosis.[66]
7.2. Meiotic Sex, Mitochondrial Genome Reduction, and Levels
of Repair
The evolution of meiotic sex can be illuminated by internal ROS
formation as well,[67–70] as can gradual mitochondrial genome re-
duction (see e.g. the effect of mitochondrial mutation rates[71] ),
bringing endosymbiont genes to places of greater safety. A revo-
lutionary rapid period of adjustment characterized by a high mu-
tation rate, as previously proposed, see, for example, refs. [6, 72]
and references therein, is most easily understood in light of en-
hanced internal ROS formation. The combination of ROS and
meiotic sex must have hugely sped up evolution rates, which
should warn us against too easily using the normal phylogenetic
algorithms (see below). Can we find traces of this “mutation bom-
bardment”? Recently, it was found that large amounts of destabi-
lizing mutations in mitoribosomal RNAs and the genes encoding
hydrophobic core subunits of OXPHOS complexes were coun-
tered by the addition of extra (≈75) protein subunits in the mature
structures. This process was completed in LECA, while no such
process was found to occur later on for chloroplast ribosomes.[73]
Thus, mitochondrial gene migration and targeting of mito-
chondrial proteins developed under natural selection, first dis-
tancing them from direct ROS generating complexes, second be-
cause behind the (beginnings of) protective nuclear membrane
other beneficial effects of the selective force of “battling ROS”
were evolving, such as chromosomes, extended anti-oxidant
mechanisms, and superior DNA repair. A recent model looks at
whether “mitochondrial complementation” could also have been
a factor favoring the development of archaeal/early eukaryote cell
fusion: a necessary ingredient of eukaryotic sex.[74]
7.3. Metabolic Restructuring and ROS Signaling
The long evolution of eukaryotes can be seen as a history of cop-
ing with internal ROS formation. It is reflected in, for example,
the way in which low amounts of ROS are actually beneficial, as
they invoke important antioxidant responses: mitohormesis. It
is found in a number of examples of mitochondrial ROS acting
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www.advancedsciencenews.com
as a cellular differentiation mechanism in complex multicellular
organisms and the invention of uncoupling proteins (UCPs).
Examples are given in refs. [54, 56] and further details can be
found in references therein. Here I will only highlight one
striking example. Myeloid cells confronted with pathogens
and/or cellular stress can respond with NLRP3 inflammasome
activation. The NLRP3 complex is highly versatile and can be
activated by a range of diverse stimuli.[75] It has been suggested
that at least three NLRP3 activating mechanisms (ROS increase,
K+ efflux, and damage to lysosomes) all signal via the induction
of oxidized mitochondrial DNA release, which then binds and
activates NLRP3.[76] Recently it was demonstrated that this pro-
cess depends on enhanced synthesis of mitochondrial DNA and
that the newly made mitochondrial DNA is crucial. The authors
of the study hypothesize that the new DNA, not packaged in
condensed nucleoid structures yet, would be more susceptible
to oxidation and subsequent fragmentation by nuclease(s).[77]
These fragments are then released via membrane pores, opened
by NLRP3 activators. This convergence of many signals on
oxidative damage in mitochondria seems indicative of an old,
evolutionarily basic, eukaryotic pathway.
8. Defining “Pre-Symbiosis” and Asking Whether
the Endosymbiont Arrived Early or Late
The reconstruction of eukaryogenesis just presented seems
rather convincing. Are there no major problems that have to be
resolved? One difficulty resides in the fact that most phyloge-
netic analyses give unexpected results. Reconstructing LECA, we
naturally find archaeal (Asgard archaea containing most) “host”
and bacterial (“alphaproteobacterium-like”) genes. However,
“eukaryotic inventions” (genes that probably evolved so quickly,
they can only be classified as eukaryotic), together with bacterial
genes seemingly coming from quite diverse bacteria, unrelated
to the endosymbiont, form a very sizeable group. Looking at the
mitochondrial proteome (i.e., the genes encoding the proteins
that will end up in the mitochondrion) we encounter the same
difficulty. Stating that at most 20% of the “mitoproteome” is of the
“correct” bacterial clade, Gray proposed the pre-endosymbiont
hypothesis, invoking an autologous organelle before uptake of
the pre-mitochondrion.[78] Phylogenetic challenges like these
recently led Gabaldón to valiantly propose another alternative, re-
lated, theory: the “pre-mitochondrial symbioses” hypothesis.[79]
He presumes eukaryogenesis to have involved multiple symbi-
otic interactions that predate the acquisition of mitochondria.
This proposal seems nicely compatible with the latest models
invoked upon studying a cultivated Asgard archaeon.[14] The
multiple symbioses, invoked to explain the large diversity of
organisms that seemed to have contributed to the mitoproteome,
could have existed when the uptake of the “mitochondrion-to
be” took place, for example, in the context of a temporary cellular
syncytium as proposed in ref. [80]. However, based on an earlier
analysis[47] Gabaldón proposes the late arrival of the endosym-
biont, that is, after many of the eukaryotic inventions occurred.
Before we discuss these proposals, an important point has to
be made. I stress the importance of pre-symbiosis because it
explains metabolic routes crucial for the eukaryotes and makes
the step toward a successful merger less unlikely, while Gray
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and Gabaldón come up with the idea to explain perceived phylo-
genetic difficulties (Gray with a pre-existing organelle, Gabaldón
with a broader scenario of extensive gene uptake).
8.1. Phylogenetic Analysis of Eukaryogenesis Is Fraught with
Difficulties
Phylogenetic analyses (especially with the availability of complete
genomes and metagenomes) is a powerful technique to probe
(even deep) evolution. However, it is clear that rampant HGT, the
complexity of algorithms to choose from, and the degrees of free-
dom regarding dataset inclusion/exclusion as well as mutation
rates easily lead to conflicting results. The analysis regarding the
timing of endosymbiont entry is a case in point. Whether the “late
arrival analysis” withstands subsequent critiques I leave up to the
reader.[5,47,81,82] However, a few aspects should be pointed out.
In the analysis of Degli Esposti the effects of differing grouping
criteria are nicely illustrated: a well-reasoned grouping of “aer-
obic proteobacteria” gets rid of most of the bacterial outliers in
the phylogenetic trees of 81 bioenergetic proteins; for details see
ref. [81]. He also hints at distorting effects inherent to the mito-
chondrial clade, as they contain a lot of information derived from
rapidly evolving eukaryotic parasite lineages. The recently pro-
posed reordering of mitochondria as derived from a sister lin-
eage of the alphaproteobacteria further complicates matters,[44]
as does the possibility of more “bacterial” genes in the archaeal
contribution to eukaryotic ancestry than previously imagined.[83]
In his “pre-mitochondrial symbioses” hypothesis,[79] Gabaldón
uses a nice metaphor to describe the phylogenetic problems. Ob-
serving that a long stem separates all the eukaryotes from their
prokaryotic ancestors, he introduces the “palm tree of eukary-
otes.” We encounter a massive radiation in the absence of sur-
viving lineages reflecting intermediate stages (the trunk of the
palm tree). This image can be used to illustrate some of the dif-
ferences between his model and the strict symbiogenesis that is
defended here.[3,6,84] If one uses a rather constant rate of change
(a constant molecular clock), we are indeed missing branches. If,
instead, one assumes a “revolutionary” period or bottleneck of
rapid adaptions (with selection for new functions in many pro-
tein families, on populations of mitochondria inside a cell[3] with
mutating ROS producing endosymbionts on their way to becom-
ing mitochondria,[52] clearly a situation never encountered be-
fore) the trunk grew so rapidly, there was no time for branch-
ing. Can I give some extra arguments for this scenario, which
will also highlight some of the phylogenetic pitfalls? If we indeed
start out with two different genomes, only partially integrated,
still separate, metabolisms and a “mutator” in the form of strong
internal ROS formation we can imagine a rapid acceleration in
the rate of change. Also, separate, partly overlapping genomes al-
low large scale gene loss from mitochondria and make readjust-
ment of enzymatic activities more easily feasible (using redun-
dant copies[2] ). Even HGT could have been more extensive due
to oxidative membrane damage, though this is speculative.
To determine the relative influence of HGT on the findings
with regard to levels of relatedness and timing of events, we
should better distinguish between easily transferred genes and
more hardwired ones. The latter ones encode proteins or RNAs
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www.advancedsciencenews.com
that are so central that they are involved in myriads of molecular
interactions; these “hubs” are not easily swapped or lost. Such an
“indexed HGT” algorithm might make the phylogenetic conclu-
sions more reliable. Other problems have to do with the mito-
proteome. The original genome being next to ROS forming sites
of the readjusting ETC and the skewing effects of more rapidly
evolving mitochondria (e.g., in parasites) have already been men-
tioned. But the novel circumstance of large-scale migration of
genes to another genomic environment, and the combination of
gene products from different genomes in molecular machines of
an organelle in the making, encountering completely new stres-
sors, might make the mitoproteome difficult to analyse with stan-
dard approaches.
Here we have been engaged in some speculation. Let us now
instead use Occam’s razor. What are the things we really know?
We know that the endosymbiont was present in LECA and we
know that increased amounts of ATP[51] were needed to be able
to make such a complicated organism as LECA.[2] I just referred
to Lane and Martin,[51] who point out that eukaryotic gene com-
plexity is expensive, and paid for by mitochondrial power. How-
ever, they explicitly state that oxygen is not essential for this power
(referring to anaerobic eukaryotes), though they also mention
that: “By enabling oxidative phosphorylation across a wide area
of internal membranes, mitochondrial genes enabled a roughly
200000-fold rise in genome size compared with bacteria” [my ital-
ics]. This tension disappears when one recognizes that invok-
ing anaerobic eukaryotes here might constitute an example of
anachronistic reasoning, see Part 2. The role of oxygen (both as fi-
nal electron acceptor in OXPHOS and as a motor driving change
in its endogenous ROS form), can be considered essential.[6,20]
We have explanations for many novel eukaryotic features that rely
on the presence of the endosymbiont. It also seems clear that
the entry of the first endosymbiont was different from all later
ones (even of the cyanobacterium that gave rise to the chloro-
plast) because the later ones were uptakes by full-fledged eukary-
otes with mitochondria (mostly?) using phagocytosis (as can be
seen by extra membranes surrounding some of these entities).
They also required less extensive adaptations in both host and
endosymbiont.[6] Thus, strict symbiogenesis is our best bet to
understand the evolution of the eukaryotes. The final picture of
LECA: a completely new kind of organism based on a merger
of an archaeon and a bacterium,[8] the evolution of which was
strongly influenced by the presence of oxygen, the integration of
metabolic pathways, HGT from other prokaryotes present in its
complex ecological niche,[14] and the development of meiotic sex.
9. Is This Indeed How Eukaryotes Evolved (Future
Experiments and Conclusions)?
I argued against an autologous model of eukaryotic inventions
in the absence of endosymbiont influences with “late” phagocy-
tosis, because merging metabolic routes is more difficult in the
absence of pre-symbiosis. The driving forces for eukaryotic in-
ventions also seem less clear. However, the symbiogenic model
seems to explain the how, why and when. How? Accidental en-
try of the (respiring) bacterium in the archaeon during pre-
symbiosis, which could coevolve into an ever-increasing source
of highly efficient ATP generation, more easily allowing eukary-
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otic complexity. Indeed, the curved extensions of Prometheoar-
chaeum as part of a syntrophic consortium with multiple bacte-
rial species conjures up an image of accidental uptake in such
consortia.[14,58,85] Why? The inescapable by-product of mitochon-
drial OXPHOS, fluctuating internal ROS, both speeds up evolu-
tion because of higher mutation rates and represents acute se-
lective forces for eukaryotic inventions. When? After the GOE,
when more O2 became available. How can we study whether this
relatively simple scenario is correct? Harcombe, Marx, and co-
workers have been performing experiments to define parameters
conductive to symbiosis, using different bacterial strains.[29,31]
What would happen during the co-culture of non-O2 using ar-
chaea and oxidizing bacteria under fluctuating O2 ? In other
model systems, what will happen to membrane potentials and
ROS formation in mitochondria missing UCP “safety valves”?
Could archaeal membranes be more susceptible to oxidative
damage than bacterial ones? I eagerly await further findings.
Acknowledgements
The author would like to thank generations of students for asking difficult
questions, several outstanding anonymous reviewers for their input, and
Andrew Moore for continued support.
Conflict of Interest
The author declares no conflict of interest.
Keywords
Asgard archaea, eukaryogenesis, mitochondria, origin of sex, reactive oxy-
gen species, symbiogenesis
Received: September 2, 2019
Revised: January 26, 2020
Published online: February 20, 2020
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