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18. Abecassis, Z.A., Berceau, B.L., Win, P.H.,
Garcia, D., Xenias, H.S., Cui, Q., Pamukcu, A.,
Cherian, S., Hernandez, V.M., Chon, U., et al.
(2020). Npas1(+)-Nkx2.1(+) neurons are an
integral part of the cortico-pallido-cortical
loop. J. Neurosci. 40, 743–768.
19. Jun, J.J., Steinmetz, N.A., Siegle, J.H.,
Denman, D.J., Bauza, M., Barbarits, B., Lee,
A.K., Anastassiou, C.A., Andrei, A., Aydin, C.,
et al. (2017). Fully integrated silicon probes for
high-density recording of neural activity.
Nature 551, 232–236.
20. Mathis, A., Mamidanna, P., Cury, K.M., Abe,
T., Murthy, V.N., Mathis, M.W., and Bethge, M.
(2018). DeepLabCut: markerless pose
estimation of user-defined body parts
with deep learning. Nat. Neurosci. 21, 1281–
1289.

Evolution: Reconstructing the Timeline

of Eukaryogenesis
Andrew J. Roger1,2,*, Edward Susko1,3, and Michelle M. Leger4
1Centre for Comparative Genomics and Evolutionary Bioinformatics, Dalhousie University, Halifax, NS B3H 4R2, Canada
2Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS B3H 4R2, Canada
3Department of Mathematics and Statistics, Dalhousie University, Halifax, NS B3H 4R2, Canada
4Institute of Evolutionary Biology (CSIC-UPF), Barcelona 08003, Spain

*Correspondence: andrew.roger@dal.ca
https://doi.org/10.1016/j.cub.2020.12.035

Timing the events in the evolution of eukaryotic cells is crucial to understanding this major transition. A recent
study reconstructs the origins of thousands of gene families ancestral to eukaryotes and, using a
controversial approach, aims to order the events of eukaryogenesis.

More than half a century after Stanier
and van Niel delineated the profound
differences between prokaryotic and
eukaryotic cells1, we still lack a clear
understanding of how the latter evolved
from the former2,3. Eukaryogenesis
involved, at minimum, an endosymbiont
related to Alphaproteobacteria that
gave rise to mitochondria, and a host
cell that was related to Archaea.
But the origins of the myriad other
characteristic eukaryotic features and
the thousands of genes underpinning
them remain obscure2. A new paper
by Vosseberg and colleagues4 tackles
this problem by extracting ‘time of
origin’ information from thousands of
phylogenies of proteins sampled from
organisms across the prokaryote–
eukaryote divide.
In the last few decades,
investigations of diverse eukaryotes
have clarified the nature of the last
eukaryotic common ancestor (LECA),
the ‘endpoint’ of eukaryogenesis
(Figure 1). LECA appears to have
had all of the traits of canonical
eukaryotes including a complex
dynamic cytoskeleton, a nucleus,
an endomembrane system, and
mitochondria5. Concepts of the first
eukaryotic common ancestor (FECA)
have also been recently sharpened by
metagenomic discoveries of the asgard
Archaea6. Asgards appear to be the
closest sister lineages to the eukaryote
nucleocytoplasm, and harbour genes
previously thought to be eukaryote-
specific. These include homologs of
eukaryotic cytoskeleton and
endomembrane system components,
suggesting that some asgards may
possess simple precursors of these
eukaryotic cellular systems6–8. Despite
these discoveries, a gulf persists
between an asgard-like FECA and a
fully complex LECA. Dozens of
eukaryotic structures and processes
had to evolve, involving the origination,
duplication and functional
specialization of thousands of genes
(Figure 1). The details of how this
happened, the origins of the genes
involved, and the order of events are
hotly debated.
Some have suggested that the
mitochondrial symbiosis occurred early,
triggering eukaryogenesis and
contributing much more to the proto-
eukaryote lineage than the asgard
archaeal host9. Others maintain that
the mitochondrial symbiosis occurred
later, after phagocytosis and associated
eukaryotic features evolved in the
host lineage10,11. In such scenarios,
the genetic contribution of the
mitochondrial symbiont may not have
been especially large or impactful.
Other bacterial groups might also
have contributed genes to the proto-
eukaryote lineage by lateral gene
transfer11,12. Unfortunately, our limited
information about the FECA to LECA
transition makes it difficult to choose
between these and alternative
eukaryogenesis scenarios.
To address this impasse, Vosseberg
and colleagues4 first attempted to infer
LECA’s protein-domain content and
constructed phylogenies for each one.
They aligned homologs of conserved
protein domains from proteomes of
several hundred representatives of
major eukaryote ‘supergroups’ and
thousands of bacteria and archaea.
After reducing the representation of
closely related sequences,
phylogenies were estimated for
each protein and specific nodes
on each tree were annotated as pre-
LECA gene duplication nodes,
LECA nodes, and post-LECA nodes
(Figure 2). For trees with prokaryotic
sequences, the closest prokaryotic sister
group to the eukaryote clade was
identified.

Current Biology 31, R186–R214, February 22, 2021 Crown Copyright ª 2020 Published by Elsevier Inc. R193
 ll
Dispatches

lack phylogenetic resolution, they

suggest that the genetic contributions
of the mitochondrion and the host
Eukaryotes
lineages may have been similar in
magnitude and that a substantial
proportion of LECA genes originated
from different bacterial donors.
Importantly, asgard families were
most likely to have been duplicated,
whereas alphaproteobacterial families
LECA were the least. Genes of host origin
Gene duplications appear to have contributed more to
Novel genes eukaryogenesis-related innovations and
Gene transfers from prokaryotes genome expansion than those of the
? symbiont.
Cytoskeleton
3 Vosseberg and colleagues then
Microtubular flagellum
attempted to order eukaryogenesis
Nucleus
Mitochondrial events using a method recently
Mitosis & meiosis 2
symbiont developed by Pittis and Gabaldon11. The
Endomembrane system latter suggested that for any protein g and
branch v on its phylogeny, the branch
length Lgv is related to the time span for the
Asgard archaea branch, Tvg , and average substitution rate
Alphaproteobacteria
1 for the protein, Rg , through Lgv = Rg Tvg :
Therefore, if a gene were acquired by the
FECA proto-eukaryote from a prokaryotic
lineage shortly after the latter speciated
from its closest sampled living sister
lineage (Figure 2), the length of the ‘stem’
Current Biology
branch leading to LECA in this
phylogenetic tree could be used to
Figure 1. Eukaryogenesis. determine the relative time of origin of that
The first eukaryotic common ancestor (FECA), which descended from the asgard Archaea, evolved into protein after normalization to remove the
the last eukaryotic common ancestor (LECA) by the sequential acquisition of thousands of traits (box). effect of the protein-specific rate. The
Many intermediate lineages went extinct (represented by crosses). The order in which these traits
normalization factor employed was the
arose, and the relative timing of the mitochondrial endosymbiosis, are unknown. Alternative
‘mitochondria-early’ (1), ‘mitochondria-intermediate’ (2) and ‘mitochondria-late’ (3) scenarios are median of the possible ‘LECA-to-
depicted. The thumbnail illustrations of mitochondria and LECA were provided by Sergio Muñoz Gómez. eukaryote path distances’ (that is, the
sum of consecutive branch lengths on a
path from the LECA node to a eukaryote
Using these annotated trees, overrepresentation of duplicated genes tip sequence) in each phylogenetic tree
Vosseberg and colleagues estimated encoding cytoskeletal, endomembrane, (Figure 2). If Te is the age of LECA, the
that LECA possessed 10,233 protein plasma membrane, and nuclear normalized stem length is then slg =
families (LECA families) and predicted proteins, and an underrepresentation Lgs =medðLge Þ = Rg Tsg =medðRg Te Þ or just:
that LECA had 7,447–21,840 genes. This of genes encoding cytosolic, slg = Tsg =Te (Figure 2). In this case, for two
range was somewhat robust to different peroxisomal, and mitochondrial sets of LECA proteins of different origins A
possible eukaryote root positions and proteins. Overall, eukaryogenesis and B, values of slg will tend to be
fits well with a relatively complex LECA. seems mostly to have involved smaller for group A if its genes have more
Next, they investigated the role of gene innovation in informational, recent origins; that is, group A’s time span
duplications in eukaryogenesis. The endomembrane, signalling and is less than group B’s: TsA < TsB .
total number of LECA families was
1.8 cytoskeletal systems. Vosseberg et al.4 extended this
times the number of novel or acquired A much larger fraction of eukaryotic approach to gene duplication events by
genes, indicating that gene duplication protein families had bacterial affinities calculating normalized ‘duplication
played an important role in (
77%) than had archaeal affinities lengths’ for genes (dlg ) to evaluate the
eukaryogenesis. Duplications were (
16%), congruent with earlier timing of duplication events (Figure 2)
enriched in genes functioning in analyses12. Crucially, however, only relative to the times of origin of genes from
signalling and genes for information 7.2% had alphaproteobacterial affinities prokaryotic sources.
storage and processing but were and 6.8% had asgard origin. Although Vosseberg et al.4 confirmed Pittis and
depleted in metabolic genes. In terms of the latter two numbers are likely Gabaldon’s earlier findings11 that the
cellular localization, there was an underestimates because some families stem-length distributions of archaeal

R194 Current Biology 31, R186–R214, February 22, 2021


Dispatches
origin proteins were systematically larger
than those of bacterial proteins, with
alphaproteobacterial proteins having the
smallest stem lengths of all. Comparing
duplication lengths revealed a similar
pattern, with proteins of bacterial
origin and eukaryote-specific inventions
having the shortest duplication lengths
and archaeal-origin proteins the largest.
Grouping the duplicated proteins into
functional classes revealed that
cytoskeletal, translational, nucleolar
and intracellular trafficking proteins
had the longest duplication lengths,
often longer than the median of
alphaproteobacterial stem-length
distribution. Other classes, such as
the endomembrane system, had
similar duplication-length distribution
medians to the alphaproteobacterial
stem lengths, whereas the shortest
duplication lengths corresponded to
mitochondrial, metabolic, signal
transduction and transcriptional
proteins. Overall, they interpret these
analyses to support a ‘mitochondria-
intermediate’ scenario (Figure 1),
whereby expansions of the cytoskeletal,
nucleolar and membrane trafficking
systems occurred pre-symbiosis, with
the remainder of the systems originating
and complexifying during, or after, the
origin of mitochondria.
But how robust are these
inferences? Pittis and Gabaldon’s
method has been criticized by Martin and
colleagues13 for assuming a molecular
clock whereby substitution rates for
stem and eukaryote branches are equal,
Rgs = Rge , or are a fixed ratio, c = Rgs = Rge ,
for all genes. They pointed to extensive
rate variation within and between
branches of the trees in the original data
set that violates both of these
assumptions13.
Are these assumptions required for
the approach to make sense? No.
Following relaxed molecular clock
theory14, suppose instead that rates
vary throughout the tree according to a
time-dependent stochastic process.
Expressing the rate of substitution as
an overall protein rate, Rg , multiplied
by the relative rate, rvg ðtÞ; for branch
or path v, the length of stem or
duplication branch s in protein g
Z Te + Tsg
becomes Lgs = Rg rsg ðtÞdt15.
Te
ll
Time tree
Asgards
FECA LECA
g
Ts
Eukaryotes
g
Td Te
Gene g
g
med (L e )
L
g
d
g
Ls
Prokaryotic sister group
Protein g tree
g g
Stem length: sl g = L s /med (L e )
g g
Duplication length: dl g = L d /med (L e )
Current Biology
Figure 2. Relating the geological ‘time tree’ (top) to branch lengths on a phylogeny of a
protein (below) acquired from a prokaryote during eukaryogenesis.
Geological timespans for branches, or sets of sequential branches (paths), v in the tree of protein g are Tvg ,
and branch lengths are Lgv . s, stem branch or path; d, duplication branch or path; e, LECA-to-extant-
eukaryote path. Trees are annotated with ‘acquisition nodes’ (green diamond), ‘duplication nodes’
(magenta square) and ‘LECA nodes’ (purple circles). LECA families (purple boxes) and their prokaryotic
sister group (green box) are shown. Expressions for the stem length (slg ) and duplication length (dlg ) are
outlined in orange. Note that Vosseberg et al.4 use the smallest of the possible duplication branches to
calculate stem lengths and duplication lengths. Also, because any gene transfer to the proto-eukaryote
from a prokaryotic source must have occurred after speciation of the donor lineage from its closest
sister group, the stem-branch timespan, Tsg ; is an upper bound on the timespan of the desired stem
g
branch post-acquisition, Ts , as discussed by Susko, Steel, and Roger15. Vosseberg and colleagues4
g
assumed Ts zTsg .
A corresponding expression can be from the same process, with the
given for the eukaryotic path length with same distribution. It is possible to
the complication that the median value, show that, if rvg ðtÞ is bounded away
medðLge Þ; was selected from all possible from 0 and infinity, then Pr½sla > slb > ½
such paths. For two groups of genes, if and only if group A genes have an
A and B, with the same time of earlier time of origin than group B
acquisition or duplication, evolving genes15. This result justifies the use
independently according to the of the Mann-Whitney U-test by
same process, the probability that any Pittis and Gabaldon11 and Vosseberg
gene a in group A has a stem length et al.4 because its null and alternative
greater than that of any gene b in hypotheses are Pr½sla > slb = ½ and
group B is Pr½sla > slb = ½. This is Pr½sla > slb > ½, respectively. In
because stem lengths are independent this framework15, stem lengths are
realizations of random variables coming expected to show variation over
Current Biology 31, R186–R214, February 22, 2021 R195
 ll
genes and no molecular clock is
required.
However, the foregoing assumes all
proteins from a given class have the same
time of origin. For stem lengths of proteins
of asgard or mitochondrial origin, this
assumption is sensible. But for genes
independently acquired from different
prokaryotic groups, times of origin may
show substantial within-class variability.
Similarly, duplication times of proteins of
any origin within functional or localization
classes are not a priori expected to be
clustered in time. In fact, since some
genes in any specific functional class will
end up in more than one localization class
and vice versa, an assumption of similar
times of origin within a class cannot hold
in general. For example, the amino-acid-
metabolism functional class contains
proteins in the cytosol and the
mitochondrion localization classes; these
classes, in turn, contain many proteins not
in the amino-acid-metabolism class (for
example, translation proteins). Although
the framework above can be extended to
cases of varying origin times, rejection of
the null hypothesis then only suggests ‘on
average’ earlier origins for one group
versus the other. Thus, the broad stem-
length and duplication-length
distributions in Vosseberg et al.4 may
reflect not only stochastic variation in
rates, but also large ranges of origin
times.
A second problematic assumption is
that proteins of different origins are all
independently evolving according to the
same probabilistic rate process, rvg ðtÞ.
However, eukaryogenesis must have
involved significant shifts in evolutionary
constraints on proteins as they adopted
new functions. Functional divergence
transiently elevates or permanently
changes substitution rates16 and, if it
differentially affected proteins of
different origins, it will also be reflected
in stem-length differences. To address
this, Vosseberg et al.4 investigated
duplicated proteins and concluded that
functional divergence after gene
duplication did not strongly affect their
conclusions. However, this overlooks
the possibility that functional
divergence could affect non-duplicated
proteins and can occur prior to gene
duplication. For example, important
changes in the translational system
occurred during eukaryogenesis17,18
R196 Current Biology 31, R186–R214, February 22, 2021
that likely induced functional divergence
in translational proteins, as previously
demonstrated for the translation
elongation factor 1 alpha protein19.
Similar functional shifts are likely to
have affected many different proteins as
they acquired new roles in the proto-
eukaryotic lineage. However, groups of
proteins of different origins may have
differentially experienced functional
divergence, shifting their relative stem-
length distributions.
Despite these concerns, Vosseberg
and colleagues have provided important
new insights into the roles of gene
duplication and gene invention in
different cellular systems during
eukaryogenesis, and clarified the relative
contributions of host, symbiont and other
prokaryotes to the genetic makeup of
LECA4. This study, and ongoing
discoveries of microbes that may be even
closer relatives of the eukaryote host and
mitochondrial lineages, will no doubt
spur many future attempts to solve one of
life’s most fundamental evolutionary
puzzles.
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