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Review
Defining eukaryotes to dissect eukaryogenesis
} si3,4,5, Anna Nenarokova1, Edmund R.R. Moody1,
Philip C.J. Donoghue1,*, Chris Kay1, Anja Spang2, Gergely Szöllo
Davide Pisani1,6,*, and Tom A. Williams6,*
1Bristol Palaeobiology Group, School of Earth Sciences, Life Sciences Building, University of Bristol, Bristol BS8 1TQ, UK
2Department of Marine Microbiology and Biogeochemistry, NIOZ, Royal Netherlands Institute for Sea Research, Utrecht University, Den Burg
1790 AB, The Netherlands
3Department of Biological Physics, Eötvös Lorand University, H-1117 Budapest, Hungary
4MTA-ELTE ‘‘Lendü let’’ Evolutionary Genomics Research Group, H-1117 Budapest, Hungary
5Institute of Evolution, Centre for Ecological Research, H-1113 Budapest, Hungary
6Bristol Palaeobiology Group, School of Biological Sciences, Life Sciences Building, University of Bristol, Bristol BS8 1TQ, UK

*Correspondence: Phil.Donoghue@bristol.ac.uk (P.C.J.D.), davide.pisani@bristol.ac.uk (D.P.), tom.a.williams@bristol.ac.uk (T.A.W.)

https://doi.org/10.1016/j.cub.2023.07.048

SUMMARY

The origin of eukaryotes is among the most contentious debates in evolutionary biology, attracting multiple
seemingly incompatible theories seeking to explain the sequence in which eukaryotic characteristics were
acquired. Much of the controversy arises from differing views on the defining characteristics of eukaryotes.
We argue that eukaryotes should be defined phylogenetically, and that doing so clarifies where competing
hypotheses of eukaryogenesis agree and how we may test among aspects of disagreement. Some hypoth-
eses make predictions about the phylogenetic origins of eukaryotic genes and are distinguishable on that ba-
sis. However, other hypotheses differ only in the order of key evolutionary steps, like mitochondrial endosym-
biosis and nuclear assembly, which cannot currently be distinguished phylogenetically. Stages within
eukaryogenesis may be made identifiable through the absolute dating of gene duplicates that map to eukary-
otic traits, such as in genes of host or mitochondrial origin that duplicated and diverged functionally prior to
emergence of the last eukaryotic common ancestor. In this way, it may finally be possible to distinguish heat
from light in the debate over eukaryogenesis.

Introduction and discriminating conceptual confusion from genuine disagree-

The origin of eukaryotes is among the most formative episodes in
evolutionary history, underpinning a step change in organismal
complexity1, facilitating the emergence of organisms as dispa-
rate as dinoflagellates and dugongs. Research into eukaryogen-
esis is a glorious nexus of philosophy, theory, and empirical sci-
ence. Nevertheless, almost every aspect of eukaryogenesis is
controversial, from its dramatis personae to the timing and
sequence of assembly of diagnostic eukaryote characteristics.
In part, this reflects genuine disagreement, conflicting results
arising from different methods, conflicting interpretations of
similar data, or competing hypotheses that have not been, or
cannot be, distinguished given the available data. However,
there is no doubt that a lack of conceptual clarity has contributed
significantly to a stalemate in the debate over eukaryogenesis,
leading to research groups effectively working within mutually
exclusive philosophies that are based on different views of clade
relationships, sequences of character evolution, definitions of
what it is to be a eukaryote (for example, see Fournier and Poole2
and Spang3), and even whether the overall history of life can be
adequately represented by a tree4–6. Such a state of affairs is
neither necessary nor conducive to elucidating this fundamental
episode in evolutionary history. Even the most conflicting hy-
potheses of eukaryogenesis (Box 1) have elements in common,
often obscured by semantic differences on what constitutes a
eukaryote. Our aim here is to outline a framework with which to
compare competing hypotheses, exposing their commonalities
ment. We anticipate that such a framework will serve to
clarify the differences between hypotheses of eukaryogenesis
and help researchers to devise experiments to discriminate
among them.
What is a eukaryote?
Much of the debate over eukaryogenesis has been rooted in
essentialist definitions of what constitutes a eukaryote — a sub-
ject on which opinions vary based on researchers’ views con-
cerning the primacy of metabolism, ecology, cytology, or phy-
logeny. Any biology undergraduate should be able to reel off a
list of the characteristics that distinguish eukaryotes from their
prokaryote kin: a cell with a nucleus, endoplasmic reticulum,
Golgi apparatus, cytoskeleton, centriole-based cilium, as well
as membrane-bound vesicles (phagosomes and autophago-
somes) and organelles (endosomes, lysosomes and mitochon-
dria). These features are often used to define eukaryotes and
every scenario for eukaryogenesis entails their sequential evolu-
tionary acquisition. But at what stage during eukaryogenesis did
these organisms achieve the upgrade from prokaryote to
eukaryote? Was it after the acquisition of one, two, or all of these
features, or are some of these evolutionary novelties more
important than others? Many researchers assert the primacy of
the eponymous nucleus7, whereas some favour the actin cyto-
skeleton8, and yet others insist that mitochondrial endosymbi-
osis was the key step that catalysed the subsequent evolution

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Box 1. Contemporary hypotheses of eukaryogenesis.

There is a long and distinguished history of models to explain the evolutionary assembly of the eukaryotic cell. Current hypotheses
(see Box 1 figure) minimally implicate an archaeon and an a-proteobacterium, but they differ on the nature of their antecedent me-
tabolisms, the mechanism by which endosymbiosis was achieved, the sequence within which crown-eukaryote innovations were
accrued, or some combination thereof. Some hypotheses implicate additional partners, but they can all generally be classified ac-
cording to the proposed timing of mitochondrial endosymbiosis relative to the acquisition of other crown-eukaryote characteris-
tics, that is, as mitochondria-early or mitochondria-late. Most hypotheses focus on specific aspects of eukaryogenesis and are
neutral with respect to others. The hypotheses are often modular, with partially separable answers to each of the above questions,
and so it is possible that some aspects of a hypothesis will prove correct whereas others will prove to be incorrect.

Hydrogen hypothesis

" Reverse-flow model

Inside-out model

E³ model

Phagocytosing archaeon
model

Syntrophy hypothesis

" " " Serial endosymbiosis

model
LECA

Archaeal-derived ∂-protobacterial symbiont Archaeal-derived nucleus

host cell
Other bacterial symbionts Autogenous nucleus
∂-protobacterial-
derived host cell D-proteobacterial-derived endo/symbiont/mitochondrion
Current Biology

Hydrogen hypothesis: In this model, a symbiosis (followed by endosymbiosis) between an anaerobic, hydrogen-dependent auto-
trophic archaeon and a heterotrophic, hydrogen-generating a-proteobacterium45 precipitated the evolution of all other eukaryotic
characters. In this sense, the hydrogen hypothesis is best defined as a mitochondria-first hypothesis. The nucleus, Golgi and endo-
plasmic reticulum are interpreted to have evolved as neomorphisms from lipid vesicles generated by the a-proteobacterium, which
also gradually displaced archaeal membrane phospholipids72.

The reverse-flow model47: suggests that the archaeal host was an organoheterotrophic organism that depended on a-proteo-
bacterial partners as electron sinks. While the reverse flow model did not discuss the relative timing of nucleus evolution, the au-
thors speculated that the archaeal host possessed actin-based cellular protrusions that, as in the inside-out hypothesis (see
below), were suggested to have surrounded bacterial partners, allowing for more efficient electron transfer. Accordingly, the
reverse flow hypothesis is a mitochondria-intermediate scenario11.

The inside-out model: focuses on the process by which the free-living proto-mitochondrion was engulfed, envisaging the endo-
membrane system emerging as a vestige of membrane extensions that, over evolutionary time, develop to surround and encom-
pass proto-mitochondrial symbionts48,73. The inside-out hypothesis, like the E3 model (see below) and reverse-flow model (see
above), requires the establishment of a relatively complex cytoskeleton and endomembrane system prior to symbiosis and is
accordingly most compatible with a mitochondria-intermediate scenario.

E3 model: This hypothesis was based on observations from the first cultured asgard archeon, Candidatus Prometheoarchaeum syn-
trophicum strain MK-D132. The E3 model proposes that a syntrophic/fermentative archaeon (the future host) entered into symbiotic
(Continued on next page)

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Box 1. Continued

relationships with a sulphate-reducing bacterium (perhaps a v-proteobacterium35) and an aerobic organotrophic a-proteobacterium
(the proto-mitochondrion). The a-proteobacterium was engulfed in a process where membrane extensions representing antecedents
of the endomembrane system developed to surround and encompass the proto-mitochondrion. The symbiosis with the sulphate-
reducing bacterium was transient and subsequently lost. The E3 model implies that the cytoskeleton and endomembrane system
predate mitochondrion endosymbiosis and is therefore also consistent with a mitochondria-intermediate scenario.

The phagocytosing archaeon model (PhAT): envisages that a phagocytic heterotrophic archaeon (future host cell) captured a
free-living ancestral mitochondrion and, rather than digesting it, established an endosymbiotic relationship. This model requires
that the ancestor of the host cell must have already evolved a complex cytoskeleton and endomembrane system46. The original
PhAT scenario advocated for the origin of a nucleus prior to mitochondrial endosymbiosis, serving as a barrier against phagocy-
tosed prey DNA. However, this order of events is not a logical requirement of the hypothesis, in that mitochondrial acquisition could
have occurred at any point following the evolution of phagocytosis. Nevertheless, as originally proposed, the PhAT model is clearly
a mitochondria-late hypothesis.

The syntrophy hypothesis: proposes that an H2-producing archaeon (future nucleus) was engulfed by a sulphate-reducing v-pro-
teobacterium (future host cell), which subsequently also took up a facultatively aerobic, sulphide-oxidizing, a-proteobacterium
(future mitochondrion)35,38,74. Given that it envisages mitochondrial acquisition post-dating endogenization of an archaeon
through phagocytosis in a v-proteobacterium as well as the origin of a nucleus, the syntrophy hypothesis is very clearly a mitochon-
dria-late hypothesis.

Serial endosymbiosis model: There have been many incarnations of the serial endosymbiosis model, but the latest is evidenced
by contributions of genes from multiple bacterial sources, interpreted as vestiges of a sequence of ultimately unstable endosym-
bioses prior to mitochondrial endosymbiosis39,75. Details are lacking, but the serial endosymbioses imply early acquisition of a
complex cytoskeleton, endomembrane system, and the nucleus, and a late acquisition of a mitochondrion, making for a mitochon-
dria-late hypothesis.

These hypotheses allow for different predictions, yet they all share similarities and address different aspects of eukaryogenesis. In
their emphasis on syntrophic interactions among prokaryotes in anaerobic settings, the hydrogen, syntrophy, reverse flow, and E3
hypotheses are closely related, although they differ in the nature of the interactions among different prokaryotic lineages and the
precise timing of the mitochondrial endosymbiosis. The inside-out hypothesis is an account of how a bacterium could be endo-
genized by an archaeon without phagocytosis but is neutral on other aspects of eukaryogenesis and, as such, it could be ration-
alised with any mitochondria-intermediate scenario. In terms of the organisms involved and the nature of their initial interactions,
mitochondria-late scenarios such as PhAT are reminiscent of the Archezoa hypothesis76, substituting a complex phagocytosing
archaeon for an ancestrally amitochondriate eukaryote, but it clearly makes contrasting predictions about the phylogenetic posi-
tions of the cells involved and the timing of mitochondrial endosymbiosis (prior to LECA in PhAT versus post-LECA in the Archezoa
hypothesis). Interestingly, although the largest proportion of prokaryote-derived genes in eukaryotes appear to be of asgard
archaeal and a-proteobacterial ancestry, large fractions of genes seem to trace their ancestry to other prokaryotes. The ‘fluid chro-
mosome’ model49 proposes that eukaryote genomes received two injections of prokaryotic genes, one from the archaeal host and
the other from the bacterial endosymbiont. However, horizontal gene transfer in the ancestors of these lineages and ongoing hor-
izontal gene transfer among prokaryotes since the time of the endosymbiosis has created the apparent jumble of donor groups77.
Hypotheses invoking more than two prokaryotic partners in eukaryogenesis explain some of these genes via these additional sour-
ces, although it also seems possible that some of this signal might be the result of individual horizontal gene transfer events into
eukaryotes from various sources (for example, including phagocytosed prey bacteria46) prior to LECA, as well as limitations on the
resolution and accuracy of phylogenetic reconstruction.

of eukaryotic cellular complexity9 (Box 1 figure). Indeed, evolu-
tionary scenarios have been classified as ‘mitochondrion-early’
versus ‘mitochondrion-late’, based on the relative timing and
importance assigned to the origin of mitochondria10,11. The dis-
covery of Asgardacheota archaea (henceforth: asgards) intro-
duced ‘mitochondrion-intermediate’ scenarios11, and scenarios
that propose that mitochondrial acquisition preceded and
precipitated every other step of eukaryotic evolution9 could be
considered ‘mitochondrion-first’.
However, character-based taxonomic definitions are prob-
lematic, as would be laid bare by attempts to classify fossil
species that preserve a subset of these defining characteristics,
perhaps reflecting now extinct lineages that branched from the
main lineage leading to living eukaryotes partway through eukar-
yogenesis. Are these fossils eukaryotes? Perhaps we do not
need to agree on a eukaryote definition simply to accommodate
fossil classification. But how should we rationalise the discovery
of asgard archaea, which appear to possess a condensed and
spatially distinct genome12 and an actin-based cytoskeleton13,14
that supports cellular protrusions in cultivated representa-
tives15? There seem to be two possibilities from the perspective
of character-based definitions, neither of them entirely

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Box 2. Phylogenetic taxonomy.

p
ou
gr
n
ow
Cr
Time

To
ta
lg
ro
up

p
ou
gr
em
St
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Phylogenetic taxonomy was developed, in part, to control for the subjective nature of taxonomic definitions based on apomor-
phies, which change with the discovery of phylogenetic intermediates, living and extinct. Classic examples include the modifica-
tion of the diagnostic characteristics of Mammalia following the discovery of monotremes78, which possess some of the ‘primitive’
characters of reptiles (for example, egg laying) as well as the ‘advanced’ characters of mammals (such as fur). Fossil taxa inter-
mediate between traditional clades are encountered more commonly with their mosaic of primitive and derived characteristics,
such as with the discovery of feathered non-avian dinosaurs, which demonstrated that feathers are not exclusive to the living clade
of birds21, or the discovery that placoderms are jawed vertebrates that lie outside the living clade of gnathostomes79.

These cases are informative of evolutionary history since they reveal the timing and sequence of assembly of the body plan of living
clades21, but revising taxonomic concepts with the discovery of every new organism (living or extinct) impedes scientific commu-
nication. Phylogenetic taxonomy provides the solution by defining clades on the basis of their living membership, from which it is
possible to obtain all possible evidence informative of phylogenetic relationships; apomorphies are demoted to being merely diag-
nostic of clade membership. Living clades define the crown concept of a clade that encompasses not only all living members but
their last common ancestor and all of its descendants living or extinct (Box 2 figure); this is usually equivalent to the traditional
concept of a taxonomic group. All extinct species that are more closely related to the crown group than to its nearest living sister
clade are assigned to the crown clade’s stem group. The crown group and the stem group comprise the total group, which is a
useful taxonomic concept for classifying fossil taxa when their membership in the stem or crown group cannot be discriminated,
perhaps because of incomplete preservation. Assignment to the total group alone effectively represents a statement of qualified
ignorance with regard to the taxonomic affinity of a fossil organism.

Phylogenetic taxonomy has generally applied phylogenetic definitions to traditional clade concepts and so the eukaryote crown
group is composed of all living eukaryotes, their last common ancestor (LECA) and all of its descendants, living or extinct. The dis-
covery of new eukaryote species can be readily accommodated if they are descendants of LECA, but they would require modifi-
cation (after agreement among the research community) upon discovery of lineages that lie outside of this clade. The discovery of
asgard achaea that possess some but not all of the characteristics of eukaryotes (for example, an actin cytoskeleton) represented
an implicit challenge to the phylogenetic definition of Eukarya2. Were they extinct, the asgards would be classified as stem eukary-
otes. The research community appears to have accepted that the asgard archaea are non-eukaryote Archaea, but this is not the
only taxonomic possibility.

Phylogenetic definitions are no less arbitrary than apomorphy-based definitions, but they are objective; communication is aided
because there can be no equivocation over the meaning of a taxonomic concept rooted within a phylogeny, that is, except where
phylogenies differ. This raises another attractive quality of phylogenetic definitions; they are readily falsifiable and therefore scien-
tific.

satisfactory: either we should reinterpret asgards as eukaryotes2
or we could remove these aspects of genome cytoskeletal
biology from our list of defining eukaryotic characteristics. And
what of lineages that have lost some of these defining character-
istics, such as mitochondria16, centrioles17 or a cilium18? Are
these no longer eukaryotes? All of this matters because unless
researchers can agree on what constitutes a eukaryote, we
can never agree on an evolutionary scenario that seeks to
explain their origins, or design and interpret the results of exper-
iments to test those scenarios3. Conceptual confusion results
in seemingly competing hypotheses that effectively explain
different phenomena.
Phylogenetic definitions and character diagnoses
Conceptual confusion arising from trait-based taxonomic defini-
tions is not a new problem and it has been solved previously

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Box 3. The root of crown eukaryotes and the nature of the last eukaryotic common ancestor (LECA).

Multicellular organisms such as animals, plants, seaweeds, and some fungi are the most well-known eukaryotes, but the genetic
diversity of extant eukaryotes is largely unicellular, comprising many unfamiliar, fascinating, and poorly studied lineages (reviewed
by Burki et al.80). Phylogenomic analyses have suggested that the root of crown eukaryotes lies either: between two major clades,
Amorphea (animals, fungi, amoebozoans, and their protist relatives) and Diaphoretickes (plants, algae, and their protist relatives)81;
on a branch between Opisthokonta and all other eukaryotes82; or within the Excavates60,83,84, a group of single-celled eukaryotes
that share a common cellular architecture but that may not form a monophyletic group85–87.

Nevertheless, the uncertain position of the root within eukaryote phylogeny has not precipitated much debate over the nature of
LECA because many core cellular and genomic features of crown eukaryotes are conserved across all of the major groups. Pub-
lished analyses agree that LECA was already a fully-fledged eukaryote similar in complexity to modern protists88–91. It was capable
of meiosis and sexual reproduction92,93. Genome reconstructions suggest LECA had a large, intron-rich genome consisting of
linear chromosomes and encoding roughly as many genes (likely >10,000) as modern protists40,94, although these inferences
are sensitive to still-debated assumptions about rates of molecular sequence evolution in eukaryotes55,69,95. In terms of cell
biology, LECA possessed both a nucleus and a mitochondrion (though see Al Jewari and Baldauf84). In some anaerobic and para-
sitic lineages, mitochondria lost the ability to respire oxygen but were retained to perform other key metabolic functions, including
forms of anaerobic metabolism96 and iron-sulphur cluster biogenesis97; these modified mitochondria are called hydrogenosomes,
mitosomes, or mitochondria-related organelles98. In at least one instance, a mitochondrion has been lost entirely16. Other cell bio-
logical traits that can uncontroversially be mapped to LECA include the endomembrane system and intracellular trafficking99,100,
Golgi, and flagella69. Based on the broad distribution of the ‘excavate’ cell architecture across the eukaryotic tree87, it seems
possible that LECA was a suspension feeding organism that pumped food particles into a ventral feeding groove via the action
of posteriorly oriented flagella (reviewed in Burki et al.80).

through the introduction of phylogenetic taxonomy (Box 2),
where conceptual clarity is achieved through the demotion of
apomorphies (defining characteristics) to simply diagnostic
features of clades otherwise defined on the basis of their
evolutionary relationships19,20. For example, the discovery of
feathered dinosaurs challenged conceptions of the defining
characteristics of birds and led to confusion as to what consti-
tutes a bird versus a dinosaur21; instead, this was resolved by
defining Aves phylogenetically. In this way, clade concepts are
robust to changing views on the evolution of characters, such
as can occur with the discovery of new taxa with new com-
binations of characters. This is an apt approach for a time
when metagenomic surveys are revealing fundamentally new
branches of the tree of life22–25, or in attempting to classify fossils
that exhibit chimaeric subsets of characters seen in their living
relatives. It also provides a stable framework in which to formu-
late, compare, and test scenarios of character evolution.
All that is required is a phylogenetic framework in which to
apply these taxonomic concepts. This is not uncontroversial in
itself since there remains a diversity of opinion on both the posi-
tion of the ‘root’ within eukaryote phylogeny (that is, the position
of the last eukaryotic common ancestor — LECA) and the near-
est living relatives of the eukaryotes (see Box 3 for a summary of
views on the nature of LECA). Furthermore, when applied to
chimaeric lineages such as the eukaryotes, phylogenetic taxo-
nomic concepts need to be modified to account for the existence
of multiple ancestral lineages. Most researchers accept that
Archaea and Eukarya comprise a clade to the exclusion of Bac-
teria, differing only in terms of whether they recognise Archaea
as a clade separate from Eukarya in the three-domain tree26
or comprising a grade from which Eukarya emerges in the
two-domain tree27. The two-domain tree concept has found
increasing support over the past decade, due to better taxon
sampling and testing using increasingly sophisticated
evolutionary models that better capture complex processes of
molecular sequence evolution28–31. This consensus has been
further strengthened by the discovery of asgard archaea and
their recognition as the closest living archaeal relatives of Eu-
karya25, exhibiting a number of molecular3 and cell-morpholog-
ical15,32 characteristics hitherto considered exclusive to eu-
karyotes.
Defining eukaryotes following the principles of phylogenetic
systematics, the reference framework is the extant clade that
comprises the eukaryote crown group: the crown group is the
clade comprised of all living eukaryotes, their last common
ancestor (LECA) and all of its extinct descendants33,34 (see Box
2 figure). The eukaryote stem group is composed of all extinct lin-
eages more closely related to crown eukaryotes than to their
nearest living relatives; the eukaryote total group is composed
of the eukaryote stem and crown groups. So far, so simple. How-
ever, applying the stem- and total-group concepts to eukaryotes
is unusual since, as a consequence of mitochondrial endosymbi-
osis prior to LECA, eukaryotes have at least two distinct stem
groups (Figure 1). One stem relates to the ancestry of the host
cell35 from among the asgard archaea25 and another from the
ancestry of mitochondria among a-proteobacteria36,37. Some
hypotheses of eukaryogenesis invoke additional stems (Figure 2),
such as the syntrophy hypothesis that also envisages a v-proteo-
bacterial origin of the host cell while the archaeon evolves into the
nucleus35,38. The serial endosymbiosis hypothesis envisages a
series of sequential (but ultimately unstable) endosymbioses
with distinct bacterial partners, effectively invoking a still unde-
fined number of stems in addition to those of the host cell and
mitochondrion39. Indeed, a spectrum of contributions to the
crown eukaryote have been invoked40,41, ranging from cells, to
organelles, to individual genes, and it may seem intuitive to
extend the stem-group concept to every contribution to the
genome of LECA. However, in taxonomy, stem groups represent

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Figure 1. Phylogenetic definition of
Bacterial crown group eukaryotes.
Archaeal crown group Eukaryotes are defined in relation to a crown group
comprised of all living members, their last common
Eukaryote crown group ancestor (LECA) and all of its descendants,
living and extinct. The eukaryote stem group is
comprised of all extinct taxa more related to crown
eukaryotes than any other living group. Since eu-
karyotes are derived from a-proteobacteria and
archaea, we must recognise two FECAs and two
stem lineages that coalesced with mitochondrial
LPCA endosymbiosis. The same principle applies to the
plastid lineage, which coalesced with its host in
the archaeplastid stem lineage.

LECA
FPCA
articulated in reference to this framework
and, in terms of evolutionary grade, it
Endosymbiosis is possible to recognise the organisms
that populate these stems as being of
prokaryotic grade or eukaryotic grade
(whether archaeal or bacterial derived),
or anywhere along the spectrum between
Host — FECAs — Mito
these concepts. However, although con-
cepts of evolutionary grade might have a
utility in describing ancestral eukaryotes
and their physiology44, they are not help-
Host/nucleus stem lineage ful in discussing and discriminating
Mitochondrial stem lineage alternative scenarios for the origin of eu-
Plastid stem lineage karyotes as they recall essentialist apo-
LUCA
morphy-based definitions that few can
Current Biology
agree upon. In terms of classification,
they are all stem eukaryotes, related by
organismal lineages, and so it is the evolutionary merger and degree to the extant (crown) eukaryotes. Instead, our focus
accompanying loss of individuality of lineages, not only the ge- should be on trying to constrain the relative order in which eu-
netic contribution, that identifies eukaryotic stems. That is, karyotic innovations were acquired relative to the archaea-
gene transfer or larger scale gene flows provide evidence for derived FECA, the a-proteobacteria-derived FECA, the mito-
stem groups but are not themselves stem groups. This provides chondrial endosymbiosis, and the LECA.
a framework of organismal relationships within which other Within this framework, it is possible to rationalize a hierarchical
important genetic contributions to LECA can be rationalised. relationship between the principal hypotheses of eukaryogen-
esis (Figure 2). The hydrogen hypothesis45, phagocytosing ar-
Implications of a phylogenetic definition for eukaryotes chaeon46, reverse flow47, inside-out48 and E3 32 models (Box 1)
and eukaryogenesis all invoke the same two archaeal- and a-proteobacterial-derived
The consequence of bringing together concepts of endosym- stem lineages, and so are not distinguishable on the basis
biosis and crown-group classification is that for each stem group of species relationships. The syntrophy hypothesis35 is distinct
we can recognise a first eukaryotic common ancestor (FECA)42. because it also invokes an additional v-proteobacterial-derived
To be clear, FECA is the first member of a eukaryote lineage after stem lineage; the serial endosymbiosis model39 expands upon
separation from its nearest living sister lineage; it need not this to invoke multiple additional stem lineages, but does not spe-
possess any characters of the crown clade. Thus, we can recog- cifically define them (Box 1 figure).
nise an archaea-derived FECA and an a-proteobacteria-derived
FECA. Although each of the stems extend as a lineage from their Testing hypotheses of eukaryogenesis
respective FECAs to a singular LECA, it is also possible to The various models of eukaryogenesis differ in terms of the
discriminate between portions of these stems before and after relative timing of the symbiotic events invoked, the way
their merger at the point of mitochondrial endosymbiosis. those symbioses contributed to eukaryogenesis, and in some
Thus, stem eukaryotes can be distinguished as archaeal-derived cases the phylogenetic origin of particular genes in eukaryotes.
stem eukaryotes or a-proteobacterial-derived stem eukaryotes, However, only in regards to this third aspect can the predic-
and both types can be further discriminated as branching prior tions of the different models be directly tested by phyloge-
to, or following, endosymbiosis. Indeed, if one accepts that mito- netics. For example, the hydrogen hypothesis45 predicts that
chondrial endosymbiosis represents a transition in individual- genes involved in hydrogen production were inherited from
ity43, it could be considered that only one stem lineage emerges the mitochondrial endosymbiont, whereas the syntrophy hy-
from endosymbiosis. Hypotheses of eukaryogenesis can be pothesis35 predicts an additional genetic contribution from

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A Hydrogen hypothesis, E³, inside-out, phago- B Syntrophy hypothesis

cytosing archaeon and reverse-flow models

Bacteria

Bacteria
FE FE
ria CA ria CA
te te
Bac Bac
FE

Eukarya

Eukarya
CA
LUCA LECA LUCA LECA

Ar Ar
ch ch

Archaea
ae

Archaea
ae
a CA a CA
FE FE

C Serial endosymbiosis model

Cyto/nuclear stem (from Archaea)
Bacteria

Misc stem (from Bacteria)

FE F Mito stem (from α-proteobacteria)
ria CA ECA
a cte Cyto stem (from ∂-proteobacteria)
B F FE
EC
Eukarya

CA Eukaryote crown
A
LUCA LECA

Ar
ch
Archaea

ae
a
CA
FE
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Figure 2. Rationalising the phylogenetic definition of eukaryotes with competing models of eukaryogenesis.
(A) The hydrogen hypothesis, E3, inside-out, phagocytosing-archaeon, and reverse-flow models are phylogenetically indistinguishable. (B) The syntrophy hy-
pothesis differs from those described in panel A only in that it invokes an additional stem lineage. (C) The serial endosymbiosis model is a further elaboration of the
models in panel A, invoking a series of additional endosymbiosis episodes during eukaryogenesis.

v-proteobacteria prior to LECA. In practice, testing these hy-
potheses has proven difficult due to gene transfer before and af-
ter eukaryogenesis, which complicates the identification of
donor lineages in single-gene trees49,50.
However, much of the current debate is focussed on the
sequence of acquisition of eukaryotic characters, where the
challenge is to devise means of discriminating among time or-
ders so as to render them testable in terms other than perceived
plausibility. Although it remains formally possible that the a-pro-
teobacteria- and archaea-derived eukaryotic stems united
through endosymbiosis quickly following a-FECA, it is more
likely that early members of this stem-mitochondrial lineage ex-
isted as free-living microbes of a-proteobacterial grade, prior to
endosymbiosis51. However, these considerations have no rele-
vance to whether the origin of eukaryotes is better represented
by a mitochondria-first9, mitochondria-early45, or a mitochon-
dria-late scenario39. These terms refer to the relative timing of
acquisition of eukaryotic characters, rather than to the absolute
time that elapsed between the emergence of the a-proteobacte-
ria-derived eukaryotic stem lineage and the time of mitochon-
drial endosymbiosis. Yet, knowledge of the absolute age of
key events in eukaryotic evolution is important because it could
be used to constrain the timing of acquisition of eukaryote nov-
elties relative to mitochondrial endosymbiosis. Molecular-clock
analyses invoke a temporally extensive mitochondrial stem line-
age emerging from the a-proteobacteria-derived FECA and an
even longer stem arising from the archaea-derived FECA52,53.
Constraining the absolute timing of mitochondrial endosymbi-
osis is problematic since this node, which many evolutionary
scenarios envisage to be the key cytological and metabolic event
in eukaryogenesis, is not identifiable in molecular phylogenies.
This is because the first mitochondriate eukaryote has left only
one living descendent lineage (Figure 1); if it had two descendent
lineages, their last common ancestor would be represented by a
node in molecular phylogenies. This is the central difficulty with
testing eukaryogenesis scenarios: all of the key characters
evolved somewhere along one of the eukaryotic stems and we
currently lack an approach for resolving their order of acquisition.
Genomic and, more recently, cultivation-based analyses of
asgard archaea have provided vital new data for the
eukaryogenesis debate. In particular, the genomic and cellular
complexity of sampled asgards now renders mitochondria-first
scenarios unlikely15,32 given accumulating phylogenomic evi-
dence for the placement of the archaeal derived stem within
the asgard clade30,54. Instead, it seems that some key features
of eukaryotes, such as an actin-based cytoskeleton, were likely
in place prior to mitochondrial endosymbiosis, suggesting mito-
chondria-intermediate scenarios. However, this encompasses

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 ll
the great majority of possible sequences of character evolution,
raising the question of whether comparative-genomic ap-
proaches might be able to differentiate between the alterna-
tives based on the data at hand. This has been attempted by
comparing the stem lengths of genes that contributed to the
eukaryotic genome with different sister groups among modern
Archaea and Bacteria39,40. The logic of the approach is that dif-
ferences in stem lengths associated with different prokaryotic
sister groups might reflect gene acquisition from distinct pro-
karyotic clades more or less closely related to eukaryotes on
the tree of life; all else being equal, longer stems were inter-
preted as evidence for earlier acquisition events. Eukaryotic
genes of archaeal origin had longer stem lengths, which these
authors interpreted as consistent with either a mitochondria-in-
termediate40 or mitochondria-late39 scenario in which some,
and perhaps much, of the eukaryotic cell complexity was ac-
quired prior to mitochondrial endosymbiosis. This approach is
promising but has limitations55–57. Stem lengths are measured
by degree of genetic change and so estimating the relative
ages of ancestral nodes requires assumptions about changes
in the rate of molecular evolution over time55. Further, the
stem length of a gene is measured from the common ancestor
with its closest extant bacterial or archaeal relative, not neces-
sarily the donor lineage that contributed these genes to the
eukaryote stem; closer relatives may be unsampled or
extinct55,56,58. Despite these potentially confounding factors,
stem-length analyses can provide some information about the
relative ages of common ancestors39. Within Fungi, stem
lengths of Ascomycota genes were shorter for genes shared
with Basidiomycota than for genes shared only with more
distantly related eukaryotes, reflecting the more recent com-
mon ancestry of Ascomycota and Basidiomycota59. However,
there is no node within gene or species trees that corresponds
to mitochondrial endosymbiosis. As such, stem lengths cannot
be used to estimate whether genes were acquired before or af-
ter that event. Longer stems for eukaryotic genes of archaeal
origin compared to genes of bacterial origin59 are consistent
with a scenario in which the eukaryotic nuclear lineage
diverged from its (extant, sampled) archaeal relatives earlier
than the mitochondrial lineage diverged from (extant, sampled)
a-proteobacteria. However, the relative divergence times of the
eukaryotic stems from their closest prokaryotic relatives do not
directly distinguish mitochondria-early versus mitochondria-
late, because these hypotheses concern the relative order in
which eukaryotic traits evolved; gene and lineage divergence
times provide a maximum age for key eukaryotic features,
but the traits in question might have evolved at any point along
the stem right up until LECA.
More recent analyses have instead focused on genes that
have inter-FECA–LECA nodes because they underwent duplica-
tion along the archaeal derived or mitochondrial stem lineage.
With knowledge of their function, they have the potential to
inform on the timing of origin of eukaryotic traits40,60. Although
existing studies have focussed on stem length, it is possible to
estimate the absolute age of these gene duplications using mo-
lecular-clock methodology. Molecular clocks are usually em-
ployed to date species’ phylogenies but they can also be used
to date gene trees, which contain nodes reflecting both species
divergences and gene duplications. Calibrated on species
R926 Current Biology 33, R919–R929, September 11, 2023
Review
divergences, it is possible to date gene duplications with some
precision61,62. Thus, where duplicated genes differ in function
or subcellular localization, the inferred age of the duplication
event can be used to establish at least a maximum age of func-
tional or subcellular differentiation. For example, multiple gene
families associated with the endomembrane system underwent
duplication within the archaeal derived eukaryote stem line-
age63,64. These include BAR-domain and ESCRT-III proteins
for membrane deformation65, dynamins for scission66, adaptins
for trafficking67, SNARE proteins for membrane fusion, along
with other proteins necessary for their function68. However, care-
ful interpretation is required because functional change associ-
ated with duplications might alter evolutionary rates and compli-
cate clock analyses69, but also because it is often difficult to
relate single gene families to organism-level characters. For
example, SNARE-protein duplicates might be involved in sexual
reproduction, autophagy70, or moonlight in other processes
such as mitosis71.
Beyond constraining the timing of endosymbiosis, it should
also be possible to dissect the development of the mitochondrial
endosymbiont into a primarily metabolic organelle through
dating the massive expansion of gene families like the mitochon-
drial carrier family, ATP synthase, and the oxidative phosphory-
lation complexes. Testing among hypotheses will require
methods that can account for evolutionary-rate changes during
eukaryogenesis but also, crucially, an unambiguous framework
indicating which nodes on gene and species trees bear on spe-
cific hypotheses (Figure 1). As we have outlined, phylogenetic
taxonomy can provide this framework.
Conclusions
Current debate about the origin of eukaryotes derives from
several sources. First, there is conflict among hypotheses that
make contrasting predictions about the events and processes
of eukaryogenesis. For example, was a v-proteobacterial partner
involved at the start of eukaryogenesis? Next, there is debate
about how to interpret the available evidence and which hypoth-
eses remain within the space of possibilities given what is
currently known. For example, do diverse prokaryotic sister
groups of eukaryotic genes provide evidence for many genetic
contributions along the eukaryotic stems? Finally, we argue,
these questions are obscured by conceptual confusion about
what constitutes a eukaryote and, therefore, the evolutionary
events we are seeking to explain. Here we have outlined how
phylogenetic taxonomy can provide a framework within which
hypotheses can be compared with each other and that reveals
which aspects can be tested phylogenetically. Within this frame-
work, it is clear that key aspects of contemporary hypotheses of
eukaryogenesis are phylogenetically indistinguishable, differing
principally in terms of the evolutionary narratives woven to bridge
phylogenetic gaps. As such, the relative timing of eukaryogenic
events cannot be inferred directly from nodes in gene or species
trees, leaving hypotheses about the relative timing of events
effectively untestable by any measure other than plausibility.
Better methods for determining the prokaryotic donor lineages
of important eukaryotic genes and further exploration of extant
asgard and proteobacterial diversity will help to refine the possi-
bility space of hypotheses. Meanwhile nodes in gene and spe-
cies trees that can be linked to shifts in function prior to LECA,

Review
such as gene duplications of core eukaryotic machinery40,60, as
well as comparisons of intron positions between paralogs40, may
provide promising routes for inferring the order of key events in
eukaryogenesis.
ACKNOWLEDGMENTS
We thank Andrew Roger and an anonymous reviewer for their comments on an
earlier draft of this manuscript. This study benefited from funding from Gordon
and Betty Moore Foundation (Grant GBMF9741 to T.A.W., P.C.J.D., D.P., A.S.,
G.S.), the John Templeton Foundation (Grant 62220 to P.C.J.D., D.P., T.A.W.,
A.S., G.S.; the opinions expressed in this publication are those of the authors
and do not necessarily reflect the views of the John Templeton Foundation),
the Biotechnology and Biological Sciences Research Council (Grant
BB/T012773/1 to P.C.J.D.) and the Leverhulme Trust (RF-2022-167 to
P.C.J.D. and RPG-2020-199 to P.C.J.D. and T.A.W.).
DECLARATION OF INTERESTS
The authors declare no competing interests.
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