BioSystems 204 (2021) 104408

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BioSystems
journal homepage: http://www.elsevier.com/locate/biosystems

Prokaryotic symbiotic consortia and the origin of nucleated cells: A critical

review of Lynn Margulis hypothesis
Antonio Lazcano a, b, **, Juli Peretó c, d, *
a
Facultad de Ciencias, Universidad Nacional Autónoma de México, Mexico
b
Miembro de El Colegio Nacional, Mexico
c
Department of Biochemistry and Molecular Biology, Universitat de València, C. Dr. Moliner 50, 46100, Burjassot, Spain
d
Institute for Integrative Systems Biology (I2SysBio), Universitat de València-CSIC, C. José Beltrán 2, 46980, Paterna, Spain

A R T I C L E I N F O A B S T R A C T

Keywords: The publication in the late 1960s of Lynn Margulis endosymbiotic proposal is a scientific milestone that brought
Endosymbiosis to the fore of evolutionary discussions the issue of the origin of nucleated cells. Although it is true that the times
Gene transfer were ripe, the timely publication of Lynn Margulis’ original paper was the product of an intellectually bold 29-
Asgard archaea
years old scientist, who based on the critical analysis of the available scientific information produced an all-
Non-mendelian inheritance
Structural heredity
encompassing, sophisticated narrative scheme on the origin of eukaryotic cells as a result of the evolution of
Microbial consortia prokaryotic consortia and, in bold intellectual stroke, put it all in the context of planetary evolution. A critical
historical reassessment of her original proposal demonstrates that her hypothesis was not a simple archival
outline of past schemes, but a renewed historical narrative of prokaryotic evolution and the role of endosym­
biosis in the origin of eukaryotes. Although it is now accepted that the closest bacterial relatives of mitochondria
and plastids are α-proteobacteria and cyanobacteria, respectively, comparative genomics demonstrates the
mosaic character of the organelle genomes. The available evidence has completely refuted Margulis’ proposal of
an exogenous origin for eukaryotic flagella, the (9 + 2) basal bodies, and centromeres, but we discuss in detail
the reasons that led her to devote considerable efforts to argue for a symbiotic origin of the eukaryotic motility.
An analysis of the arguments successfully employed by Margulis in her persuasive advocacy of endosymbiosis,
combined with the discussions of her flaws and the scientific atmosphere during the period in which she
formulated her proposals, are critical for a proper appraisal of the historical conditions that shaped her theory
and its acceptance.

1. Introduction Margulis’ lifelong advocacy of the biological significance of symbi­

In March 1967 the Journal of Theoretical Biology published the article
“On the origin of mitosing cells”, by Lynn Margulis (who at the time
signed her work as Lynn Sagan), in which she famously proposed that
the symbiotic association of once-free living prokaryotes had given rise
to mitochondria, plastids and the basal bodies present in nucleated cells.
Although it is true that the hypothesis of organelle-producing symbiosis
is part of a storied series of theoretical schemes that begun in the late
19th century (Khakhina, 1993; Lazcano and Peretó, 2017; Sapp, 1994),
the publication of Margulis’ paper was a milestone that brought to the
forefront of scientific discussions the issue of the role of endosymbiosis
in the origin of eukaryotes.
osis, which for a long time was considered by many restricted to lichens
and few other textbooks examples, contributed to its recognition as part
of the conceptual frameworks of issues ranging from the role of the
microbiome in human health, to the discussions on the evolution of
mutualism, morphogenesis, and the emergence of evolutionary
innovations.
The chimeric character of eukaryotes is firmly established. Although
the recognition that mitochondria and plastids are descendants from
free-living prokaryotes is supported in full by phylogenetic, genomic,
metabolic and cellular evidence, how symbiogenesis actually occurred is
far from being understood in full (McInerney et al., 2014). Although the
available evidence has finally refuted Margulis’ proposal of an

* Corresponding author. Institute for Integrative Systems Biology (I2SysBio), Universitat de València-CSIC, C. José Beltrán 2, 46980, Paterna, Spain.
** Corresponding author. Facultad de Ciencias, Universidad Nacional Autónoma de México, Mexico.
E-mail addresses: alar@ciencias.unam.mx (A. Lazcano), pereto@uv.es (J. Peretó).

https://doi.org/10.1016/j.biosystems.2021.104408
Received 1 March 2021; Accepted 11 March 2021
Available online 17 March 2021
0303-2647/© 2021 Elsevier B.V. All rights reserved.
A. Lazcano and J. Peretó
exogenous origin for eukaryotic flagella, the (9 + 2) basal bodies, and
centromeres, the association of spirochaetes with protists demonstrates
that, at least in some eukaryotic microbes, motility is due to symbiotic
consortia. As reviewed here, an analysis of the arguments successfully
employed by Margulis in her persuasive advocacy of endosymbiosis,
combined with the discussions of the flaws of her original ideas and the
scientific atmosphere of the period in which she formulated her pro­
posals, are critical for a proper appraisal of the historical conditions that
shaped her theory and its acceptance.
2. Endosymbiosis: from a bad penny to academic respectability
Few, if any, scientific theories or hypothesis are born without his­
torical precedents, and indeed few researchers can claim that their ideas
are free from proposals of their intellectual forerunners. The same is
true, of course, of Margulis’ endosymbiotic theory. The discovery of
chloroplasts and their mode of reproduction was rapidly interpreted as
evidence of their bacterial origin, and from the end of the 19th century
onwards a number of proposals on endosymbiosis where published,
including some which suggested symbiosis as a teleological alternative
to natural selection (Khakhina, 1993; Lazcano and Peretó, 2017;
O’Malley, 2015; Sapp, 1994).
The recognition that a number of endosymbiotic proposals had been
made since the late 19th century has led some to question Lynn Margulis
intellectual originality. These petty criticisms should not be taken at face
value. As underlined elsewhere, her original proposal was not a mere
archival summary of past hypothesis or a simple listing the available
evidence of the traits shared by free-living prokaryotes and eukaryotic
organelles, but a renewed evolutionary historical narrative of the role of
endosymbiosis in the origin of nucleated cells (Lazcano and Peretó,
2017). It is often forgotten that she began her paper by quoting the work
of those she always recognized as her scientific predecessors. As she
wrote in the beginning of her rather lengthy paper, “[a]lthough these
ideas are not new [Merezhkovsky (1910) & Minchin (1915) in Wilson
(1925), Wallin (1927), Lederger (1952), Haldane (1954), Ris and Plaut
(1962)], in this paper they have been synthesized in such a way as to be
consistent with recent data on the biochemistry and cytology of sub­
cellular organelles” (Sagan, 1967).
Margulis was quick to acknowledge the significance of ideas that
were on the margins of scientific respectability, and vigorously pro­
moted the work of almost forgotten scholars like the American anato­
mist Ivan Wallin, and of scientists like Konstantin S. Merezhkovsky
(Kowallik and Martin, 2021), Andrei S. Famintsyn, and Boris M.
Kozo-Polyanski, who represented the sophisticated Russian school of
symbiosis that had been largely ignored in the USA and other Western
countries. It is fair to say that the current familiarity that the community
has with these authors who, with few exceptions, were largely unknown
outside the circle of historians of science, is the outcome, to a consid­
erable extent, of Margulis promotion of their work (see for, instance,
Khakhina, 1993; Kozo-Polyansky, 2010).
Margulis proposal was not a simple remake of previous hypothesis.
As noted elsewhere, she rejected Wallin’s scheme according to which
plastids had evolved from mitochondria, and refused Merezhkovsky
ideas on a polyphyletic origin of life and his suggestion that the
eukaryotic nucleus had evolved from a mitochondria-like microor­
ganism. Although her rejection of the orthodox population genetic
theory sometimes put her in odds with neo-Darwinian premises
(O’Malley, 2015), she was quick to acknowledge that endosymbiotic
entities and prokaryotic consortia, like conjugation and meiotic sex
followed by fertilization, represented a combinatorial mode of evolu­
tionary change over which natural selection acts (Lazcano and Peretó,
2017).
Margulis proposed a coherent, step-wise but not necessarily slow
process in based in microscopic observations, ultrastructural evidence,
biochemical and molecular biology information, and microbial ecology
that recognized the role of endosymbiosis, and connected prokaryotic
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BioSystems 204 (2021) 104408
evolution and eukaryogenesis with the geochemical evolution of the
planet. As documented by Sapp (1994), by the late 1960s advances in
molecular biology had led to the isolation of chloroplasts and mito­
chondrial DNA, and there was an increased interest by cell biologists and
geneticists on the endosymbiotic origin of chloroplast. It can be argued
that the time was ripe for the acceptance of endosymbiosis, but in fact
Margulis’ ideas encountered harsh opposition due to (i) the strict sep­
aration of life sciences into a number of unconnected fields; (ii) the
microbial theory of disease that view prokaryotes as pathogens; and (iii)
the stiffening of neo-Darwinism, that assumed that point mutations were
the only source of biological diversity. Although she always remained
reluctant to admit it, the fact that for many she was a young non-tenured
female professor with a rather small publication record, spoke against
her views (Lazcano and Peretó, 2017).
3. Planetary sciences and cell biology
In the wake of the 1957 launching of the first Soviet satellite and as a
result of a complex mixture of social, political, military and scientific
interests, the USA academic world received an extraordinary support
that led to the strengthening of research in universities and employment
opportunities (Dick and Strick, 2004). The National Aeronautics and
Space Act of 1958 created NASA, and with this unexpected opportunities
and support for planetary sciences rapidly developed. Thanks to the
critical insights of Joshua Lederberg and other life scientists, NASA
stopped defining life-sciences as a “man in space” program, with space
biology as a branch of space medicine or physiology at high altitude or
contamination, and became committed to exobiology, which was
defined as the study the origin, evolution and distribution of life in the
Universe (Wolfe, 2002).
NASA’s lavish support to the study of the origin and evolution of life,
changed forever the research environment. Space exploration led to a
renewed, vigorous interest of the geochemical history of the Earth,
which facilitated the recognition of microbes as agents of geological
change. Stimulated by the financial backing of NASA, a scientific com­
munity with different backgrounds started to develop a new framework
for the study of planetary sciences. Their work was supported by a
number of senior researchers like the geophysicist Lloyd V. Berkner, who
chaired an advisory board that assisted NASA and whose work in the
modelling of the evolution of the terrestrial atmosphere recognized the
key role of cyanobacteria, and of paleontologists like Elso Barghoorn
and Preston Cloud, who had extended the microbial paleontological
record into the early Precambrian. For the first time in the USA academic
environment, prokaryotes were seen not only as agents of disease but
also as active components of the evolution of the planet.
This new scientific environment clearly favored the acceptance of
Margulis endosymbiotic proposal, that linked prokaryotic evolution and
the emergence of eukaryotes to planetary development. It is true that the
times were ripe, but the timely publication of Lynn Margulis’ original
paper was the product of an intellectually bold 29-years old scientist,
who based on the critical analysis of the available scientific information
produced an all-encompassing, sophisticated narrative scheme on the
origin of eukaryotic cells as a result of the evolution of increasingly in­
tegrated prokaryotic consortia.
4. Heredity and endosymbiosis
Genetic variation in a population can be due to point mutations,
recombination, genetic drift, lateral gene transfer, symbiosis, conjuga­
tion and meiotic sex followed by fertilization. As Maynard-Smith once
remarked, “the relevance of symbiosis is that it affords a mechanism
whereby genetic material from very distantly related organism can be
brought together in a single descendant … and the possibility that new
evolutionary potentialities may arise suddenly if genetic material that
has been programmed by selection in different ancestral lineages is
brought together by symbiosis” (Maynard Smith, 1991). One can pursue
A. Lazcano and J. Peretó
the comparison beyond the mere accretion of genetic sequences as
mediated by plasmids or viruses. Highly integrated symbiotic associa­
tions can be compared to lateral gene transfer (Archibald, 2014; Lazcano
and Peretó, 2017). First of all, as discussed elsewhere in detail, the
components of prokaryotic symbiotic consortia have all been shaped by
a previous evolutionary history in which selection acted upon their
different ancestral lineages. Secondly, the symbiotic association be­
tween different species is the outcome of natural selection acting on an
originally nonadaptive partnership, i.e., the symbiotic partners are first
acquired independently of the selective advantage they may eventually
confer to the newly formed consortium in a given environment (Lazcano
and Peretó, 2017).
Symbiotic consortia can lead to the evolutionary integration of
diverse genomes. Depending on the degree of independence of the
different components of a symbiotic consortium, a wide variety of non-
Mendelian, i.e., non-chromosomal mechanisms have evolved that
guarantee the transmission of the partnerships. Beyond the explanatory
power of a definition of evolution based on a mere change in gene fre­
quencies, as in classical population genetics, for Margulis (as for many
other life scientists) the full range of biological consequences of sym­
biosis cannot be reduced to the mixing of genetic material from different
species in a descendant. This includes, of course, the striking examples of
non-Mendelian behavior-dependent inheritance of symbionts, as the
anus-to-mouth feeding of pupae by adult termites, and in the licking of
neonates that have been described in many different mammal species,
that guarantee the transmission of microbiomes.
At a cellular level, the inventory of non-Mendelian mechanisms
comprises cytoplasmic heredity, as in uniparental chloroplast inheri­
tance and the well-known maternal mitochondrial transmission. An
exemplary case is the microaerophilic amitotic multinucleated giant
amoeba Pelomyxa palustris, where the perinuclear distribution of its
different bacterial symbionts increases the likelihood that the amoeba
offspring will receive at least one full copy of its prokaryotic genomes.
The listing of the multiple cases of microbial consortia associated
with a variety of protist species exhibits is an extraordinary array of
evolutionary strategies that guarantee the continuity of the symbiont
associations, although the detailed mechanisms are largely unknown.
One of the most striking examples, to which Margulis devoted consid­
erable attention because of her hypothesis of the exogenous origin for
eukaryotic flagella, are the different motility symbiotic partnerships of
spirochaetes and nonmotile bacteria with different flagellate protists,
including the trichomonad Mixotricha paradoxa, the oxymonad Pyrso­
nympha, and several devesconivid species, which are found in a variety
of termites and wood roaches (cf. (Smith and Douglas, 1987). The
complex history of these motility associations is revealed by the poly­
phyletic modifications of the attachment sites of both the exosymbionts
and the protist surfaces revealed by ultrastructural analyses (Margulis,
1981; Tamm, 1980). The synchronous beating of the spirochaetes is a
hydrodynamical issue that requires no genetic explanation (Machin and
E, 1963) (Lighthill, 1976), but the nature of biochemical and molecular
signals involved in the maintenance and coordination of the reproduc­
tion strategies that maintain the consortia.
5. Mitotic components do not have an exogenous origin
The most innovative proposal of Margulis endosymbiotic scheme
was the hypothesis that the nucleated cell motility structures, i.e., the
eukaryotic flagellum and the related structures including centromeres,
centrioles and the (9 + 2) basal bodies, had originated, as she wrote,
when “some ancestral amoeboid acquired a motile parasite, perhaps
spirochaete-like, by ingestion. The genes of the parasite, of coded for its
characteristic morphology, (9 + 2) fibrils in cross section. Amoeboid
host cells which retained their endosymbionts had the immense selec­
tive advantage of motility long before mitosis evolved; they could
actively pursue their food” (Sagan, 1967). The core of her postulate was
that the eukaryotic flagellum (undulipodium, in her usage) and mitotic
3
BioSystems 204 (2021) 104408
apparatus originated from an endosymbiotic, spirochaete-like organism,
leading to a chromosome partition mechanism dependent on
DNA-microtubule binding, which had originated from a hypothetical
replicative centriole-DNA complex.
The search for evidence supporting her view on the phylogenetic
connection between undulipodia, mitosis and meiosis defined her pro­
fessional career. She rapidly recognized that the paraphyletic nature of
protists, in which an extraordinary variety of mitotic strategies can be
observed, could provide clues on the numerous variations, by-ways and
dead-ends which must have taken place during the evolution of mitosis.
Hence, she suggested an evolutionary sequence, which she accompanied
with a number of naive drawings that she prepared herself, that started
with the establishment of the consortia formed by the spirochaete-like
endosymbiont and the ancestral nucleocytoplasm, followed by “at
least two series of mutational steps were required: one leading to the
development of some attraction between nucleic acid of the host and
that of the symbiont, and eventually, to permanent connections of
daughter endosymbionts precisely to daughter chromosomes of the host
in the formation of chromosomal centromeres, and a second one leading
to the segregation of the replicated daughter endosymbionts to opposite
poles of the host cells … [insuring] continuing selection pressure for
improved mechanisms of host nucleic acid segregation” (Sagan 1967).
Margulis (1981) recognized the spirochaete origin of the eukaryotic
motility apparatus was “the least accepted, most original, and most
questionable aspect of the SET [Serial Endosymbiotic Theory]” (Mar­
gulis 1981). What led her to suggest and insist an exogenous origin of the
components of the mitotic machinery? She was keenly aware of the
morphological similarities between eukaryotes flagella and spiro­
chaetes, and of their association with unicellular flagellated eukaryotes
where “have been mistaken for additional flagella”, as components of
motility symbiosis. In fact, the morphological similarities and the com­
parable staining properties of spirochaetes and eukaryotic cilia had led
the latter to be described as “spirochaetes or pseudo-spirochaetes” since
the late 1910s (Adams et al., 1925; Kuhn and Steiner, 1917; May and
Goodner, 1926).
As Margulis wrote, “[b]asal bodies” (found at the base of eukaryotic
flagella of these motile centrioles, or some other submicroscopic ho­
mologues are considered to be the repository of the nucleic acid of the
replicating system and, thus, the descendants of the genes of the original
symbiont, although, as she continued, the DNA associated with (9 + 2)
bodies “has evaded detection because it has very little metabolic re­
sponsibility … identification of (9 + 2) homologue-specific DNA …
should eventually be possible” (Sagan 1967). This was not a whimsical
proposition. Her suggestion of an exogenous origin of the centriole was
based on (i) the superficial morphological similarity of spirochaetes with
eukaryotic cilia discussed above; (ii) the striking examples of the
motility symbiotic partnerships that led to surprising morphological
holdfasts of spirochaetes and the protists’ outer surface; and (iii) the
experimental work of Sonneborn and his associates that suggested that
the cilia-rich cortex of a number of ciliates, including Paramecium,
Euplotes, and Tetrahymena are endowed with an genetic system with
considerable autonomy from the nuclear control (Beisson and Sonne­
born, 1965).
Centrioles, kinetosomes, microtubule organizing centers (MTOCs)
and (9 + 2) bodies are all part of motility systems of eukaryotic cells.
They are generally surrounded by the so-called periocentriolar material
that nucleates the microtubules of both the mitotic spindle and the
cytoskeleton (Marshall and Rosenbaum 2000). Centrioles are the paired
constituents of mitotic centrosomes in animal cells and, as seen in pro­
tists, during mitosis become kinetosomes (basal bodies) when their 9
(2)+2 microtubular axonemes grow outwards (Chapman et al., 2000).
The awareness that during the cell cycle centrioles double from two to
four per cell and segregate during mitosis, combined with observations
of ciliate kinetosome replication (Lwoff, 1950), were interpreted by
many as evidence of their genetic autonomy, prompting a search first of
DNA and then of RNA associated with them. However, the significance
A. Lazcano and J. Peretó
of spirochaete genes to eukaryotic genomes is quite modest (Rochette
et al., 2014). In fact, it is now recognized that the de novo formation of
centrioles is an example of structural inheritance, i.e., a non-DNA in­
heritance of cytotaxis or cortical inheritance somewhat equivalent to
prion duplication (Beisson, 2008). Neither centrioles or centromeres
contain DNA, and the RNA found in them does not play a role in neither
in their assembly or function (Johnson and Rosenbaum, 1991; Marshall
and Rosenbaum, 2000).
6. The quest for the host
The nature of the prokaryotic phagocyte that originally hosted the
ancestor of the mitochondria remained over twenty years an open
problem. The issue is complicated by the fact that phagotrophy has not
been observed in contemporary bacteria, most of which are walled.
Questions on the origin of the host are, in fact, inquiries on the nature of
the origin and appearance of the entire nucleocytoplasm. Although the
presence of nuclear specific molecules and structures such as the nuclear
pore complex, LINC complexes, and the nuclear envelope itself is well
documented (Devos et al., 2014), the topological continuity between the
nucleus, the endoplasmic reticulum and the cell plasma membrane is
well-established. Suggestion of a viral origin of the eukaryotic nucleus
arguing that it is the product of the invasion of a protoeukaryote by a
DNA virus (Bell, 2001, 2020; Takemura, 2001) fail to recognize that the
nucleocytoplasm is an anatomic, functional and ontogenetic unit and
that the issue of the origin of the nuclear envelope cannot be separated
from the nature of the prokaryote ancestral to the host.
Although the complex endomembrane system of the heterotrophic
aerobic Gemmata obscuriglobus is a demonstration of the extraordinary
potential of bacteria to develop complex intracellular structures, no life
forms are known that correspond to an intermediate stage between
prokaryotes and eukaryotes.
Following Margulis proposal that “eukaryotic cell arose by a specific
series of endosymbioses”, a diversity of events and microbial compo­
nents in these ancient symbioses have been postulated. The most explicit
models of eukaryogenesis underline metabolic complementation or
syntrophy as the driving force behind the association between pro­
karyotes. For Margulis the cohesive force for the association of pro­
karyotic microorganisms en route to eukaryotic complexity was survival
in an oxygen-containing atmosphere, represented by the origin of the
“first aerobic amitotic amoeboid organisms” endowed with respiratory
abilities. In her updated version of the model, Margulis proposed that
the host of this primordial association was a Themoplasma-like archaeal
cell (Margulis, 1996 and references therein). Since she modified her
original scheme to argue that the association of spirochaetes had pre­
ceded the engulfing of the mitochondrial ancestor, in later years she
became intellectually enticed by the “Thiodendron” consortium, which is
actually a non-stable association between aerotolerant spirochetes and
motile sulfidogens, to postulate a primitive consortium of spirochetes
and archaea as precursors of the nucleocytoplasm (Grabovich et al.,
2021).
Phylogenetic methods and functional inference from genomes reveal
new microbial lineages, among them candidate descendants of those
involved in the process of eukaryogenesis. In 2015, Ettema and co­
workers described a new phylum of archaea (Lokiarchaeota) by meta­
genomic sequencing (Spang et al., 2015). Phylogenomic analysis of the
sequences recovered from marine sediments revealed that the
Lokiarchaeota form with eukaryotes a monophyletic group. As shown by
the eocyte model (Rivera and Lake, 1992), the notion of a paraphyletic
archaea challenging the classical three-domain tree of life is not new, but
functional analysis unveiled that Lokiarchaeota genomes harbor a di­
versity of genes encoding cytoskeleton components including tubulin
and actin, proteins involved in vesicle trafficking and in the ubiquitin
pathway, and other eukaryotic signature proteins, i.e., ESPs. Further
phylogenomic and functional analyses (Zaremba-Niedzwiedzka et al.,
2017) expanded the repertoire of ESPs associated to the new
4
BioSystems 204 (2021) 104408
superphylum of Argard archaea that now include Lokiarchaeota, Thor­
archaeota, Odinarchaeota and Heimdallarchaeota, which at the time
being appears to be the closest lineage to eukaryotes.
Environmental genomics thus suggest that the nucleocytoplasm
lineage is monophyletic and is nested within the archaeal domain.
Available information supports the contention that Heimdallarchaeota
are the closest modern kin of the original host that engulfed the pro­
teobacterial ancestor of mitochondria. A decade of laboratory work led
to the isolation from a deep marine sediment of ‘Candidatus Prom­
etheoarchaeum syntrophicum’, a small Asgard archaea related to
Lokiarchaeota (Imachi et al., 2020) with a complex morphology pro­
ducing membrane vesicles and protrusions resembling eukaryotic filo­
podia and microvilli. Its genome encodes for a typical array of ESPs,
including members of a dynamic cytoskeleton system, like actin, profi­
lin, and gelsolins. A remarkable observation was that P. syntrophicum has
a reduced metabolic network, with restricted biosynthetic capacities and
a highly specialized heterotrophic amino acid catabolism, and that
grows in a symbiotic association with a sulfate-reducing deltaproteo­
bacterium and a methanogenic archaeon.
This cultured Asgard archaeon, due to its remarkable cell charac­
teristics, is the first experimental model that can be used in more inte­
grative biological approaches beyond the genome and proteome level
(Akıl et al., 2021). Remarkably, the obligatory syntrophic lifestyle of
P. syntrophicum has implications for constraining and testing alternative
models of eukaryogenesis (López-García and Moreira, 2020), as well as
the metabolic phenotypes inferred for the closest described relatives of
eukaryotes. Since the Heimdallarchaeota genomes encode not only for
traits involved in heterotrophic fermentation and in anaerobic and
aerobic respiratory chains (Spang et al., 2019), but also for the kynur­
enine pathway for the aerobic biosynthesis of NAD+ (Bulzu et al., 2019)
they may colonize both oxic and anoxic niches, supporting the possi­
bility that the primitive eukaryotic host was a facultative aerobe.
7. The search for the mitochondrial ancestor
Our partial sampling of biodiversity has also shaped the debate about
which alphaproteobacterial lineage is the extant closest relative to the
mitochondrial ancestor. Classic phylogenomic approaches pointed out
to Rickettsiales, an alphaproteobacterial order with members exhibiting
a diversity of symbiotic lifestyles (Wang and Wu, 2015) and references
therein). A recent survey of ocean metagenomes identified thirteen new
alphaproteobacterial clades, one of them being basal to the rest of
Alphaproteobacteria (Martijn et al., 2018). Methods addressing issues of
the phylogenetic reconstruction such as long branch attraction and
compositional bias allowed the authors to propose an early divergence
of mitochondria that appeared before the emergence of all the currently
known clades. However important might be the phylogenetic position of
the mitochondrial ancestor as an early branching alphaproteobacterial
lineage, for any eukaryogenesis model it is necessary to know the
metabolic features of its closest extant relatives and contrast with the
results of the top-down approaches reconstructing the metabolic map of
the protomitochondrion (Gabaldón and Huynen, 2003, 2007).
The search for the identity of (at least) two partners involved in the
primitive consortium during eukaryogenesis has been complemented
with questions that include the issue of whether mitochondria (i) were
established early or late during the emergence of nucleated cells and (ii)
the chimeric nature of the partner genomes participating in the primi­
tive consortia. Based on phylogenetic distances (Pittis and Gabaldón,
2016), proposed that proteins of alphaproteobacterial origin were
latecomers during the complexification process of eukaryotic cell or­
ganization. A recent updated study using ancient gene duplications
(Vosseberg et al., 2021) has shown an intermediate incorporation of
mitochondria during eukaryogenesis and, at the same time, confirms
that the early archaeal host likely was endowed with some
eukaryote-like traits, such as cytoskeleton and vesicle trafficking com­
ponents. This observation is consistent with the identification of ESTs in
A. Lazcano and J. Peretó
Asgard metagenomes (Zaremba-Niedzwiedzka et al., 2017), as well as
the phenotypic traits of the first cultivated lokiarchaeon (Imachi et al.,
2020) and the biochemical characterization of Asgard profilins (Akıl and
Robinson, 2018) and gelsolins (Akıl et al., 2020), which indicate the
presence of a dynamic actin system in these archaea.
As for the issue of the chimeric nature of genomes of the partners
participating in the eukaryogenic consortia Gabaldón (2018) has dis­
cussed a model of “premitochondrial symbioses”, going beyond
simplistic consortia, and trying to explain the genomic heterogeneity of
the primitive archaeal host, which apparently harbored a diversity of
bacterial genes different from the alphaproteobacterial contribution in
the origin of mitochondria (Pittis and Gabaldón, 2016). This appears to
be the case, for instance, of an enzymatic machinery involved in the
hydrogen metabolism of anaerobically-adapted eukaryotes that was
acquired from an ancient lineage close to extant Anoxychlamydiales
(Stairs et al., 2020). The most parsimonious scenario is that this meta­
bolic innovation took place before or concomitant with the mitochon­
drial advent. These and many other gene exchanges contributed to the
mosaic evolution of eukaryotic metabolic pathways already postulated
by Margulis in her original work (Sagan, 1967).
8. Conclusions
Although it is true that some of Lynn Margulis original proposals
were incomplete or mistaken, the 1967 publication of her seminal assay
represented a major contribution to evolutionary theory. Her demon­
stration that the symbiotic association of once-free living prokaryotes
had given rise to nucleated cells sparked a debate on the key forces that
shaped the origin and early stages of eukaryotic evolution. Her lifelong
work on eukaryogenesis and the role of symbiosis in ecological and
evolutionary relationships stand a valid explanatory contribution to cell
evolution. Lynn Margulis did not simply made a remake of previous
ideas. Almost single-handed she was able to bring the idea of symbiosis
into mainstream scientific discussion.
The chimeric nature of eukaryotes is firmly established. In spite of
the spirited discussions on origin of organelles that took place following
the publication of Margulis’s first article, advances in genomics have
demonstrated that two independent monophyletic processes gave rise to
plastids and mitochondria. Why did Margulis found so enticing the
possibility that spirochaetes had played a role in the origin of eukaryotic
motility? Because, as underlined elsewhere (Lazcano and Peretó, 2017),
she as a naturalist with eyes set on the microbial world, and was keenly
aware of the complex behavior of the multiple cases of microbial
motility consortia involving spirochaetes and protists. She was wrong,
but she contributed to bring the discussion on the origin of eukaryotic
motility and mitosis to the forefront of evolutionary discussions.
What we find intellectually fascinating is the connection that Lynn
Margulis established between cell evolution and planetary change. As
reviewed elsewhere (Lazcano and Peretó 2017), she was very much
aware that the work of Berkner, Barghoorn, Cloud and others had
demonstrated that after the origin of life the major biogeochemical
change had been the accumulation of free atmospheric oxygen during
the Precambrian. This allowed her to stablish a relative timescale which
included not only for the origin of eukaryotes but also, as she wrote in
her 1970 book, published few years after the publication of her 1967
paper, that “the enormously varied responses of the prokaryotes as a
whole to gaseous oxygen, convinces me that aerobiosis itself evolved
within the prokaryotic group …. mechanisms of oxygen toleration pre­
sumably preceded respiratory and other mechanisms of oxygen utiliza­
tion … and that the mitochondrial-host symbiosis resulted in the
biosynthetic steps required to form the typical eukaryotic phospholipid
membranes, probably steroid synthesis, and, in particular, the formation
of a nuclear membrane and endoplasmic reticulum” (cf. Lazcano and
Peretó, 2017). This short paragraph is indicative of her ability to review
and summarize information from disparate disciplines led her to
establish a connection between microbial evolution and planetary
5
BioSystems 204 (2021) 104408
changes. That was, in itself, a major intellectual feat that worth
remembering.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
The results discussed here are based on a previously published paper
Lazcano and Peretó (2017). We are indebted to Drs. Jose Alberto Cam­
pillo Balderas and Ricardo Hernandez-Morales for their help in different
aspects of the work reported here. Support from DGAPA-PAPIME
(PE204921) and DGAPA-PAPIIT (IN214421) to A. L., and Agencia
Estatal de Investigación (AEI), Ministerio de Ciencia e Innovación (grant
SETH ref. RTI 2018-095584-B-C41-42-43-44 co-financed by ERDF) and
the European Union H2020 (BioRobooST project ID 210491758;
MIPLACE project ref. PCI 2019-111845-2, Programación Conjunta
Internacional 2019, AEI) to J. P. is gratefully acknowledged.
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