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||Proc. Natl. Acad. Sci. USA. 2000 June
20; 97 (13): 6954–6959|
The chimeric eukaryote: Origin of the nucleus from the
karyomastigont in amitochondriate protists
*Department of Geosciences, Organismic and
Evolutionary Biology Graduate Program, University of Massachusetts,
Amherst, MA 01003; and ‡Department of Microbiology, and Special
Research Center Complex Systems (Microbiology Group), University of
Barcelona, 08028 Barcelona, Spain
| Abstract |
We present a testable model for the origin of the nucleus, the
membrane-bounded organelle that defines eukaryotes. A chimeric cell
evolved via symbiogenesis by syntrophic merger between an
archaebacterium and a eubacterium. The archaebacterium, a
thermoacidophil resembling extant Thermoplasma, generated
hydrogen sulfide to protect the eubacterium, a heterotrophic swimmer
comparable to Spirochaeta or Hollandina that oxidized
sulfide to sulfur. Selection pressure for speed swimming and oxygen
avoidance led to an ancient analogue of the extant cosmopolitan
bacterial consortium “Thiodendron latens.” By
eubacterial-archaebacterial genetic integration, the chimera, an
amitochondriate heterotroph, evolved. This “earliest branching
protist” that formed by permanent DNA recombination generated the
nucleus as a component of the karyomastigont, an intracellular
complex that assured genetic continuity of the former symbionts. The
karyomastigont organellar system, common in extant amitochondriate
protists as well as in presumed mitochondriate ancestors, minimally
consists of a single nucleus, a single kinetosome and their protein
connector. As predecessor of standard mitosis, the karyomastigont
preceded free (unattached) nuclei. The nucleus evolved in
karyomastigont ancestors by detachment at least five times
(archamoebae, calonymphids, chlorophyte green algae, ciliates,
foraminifera). This specific model of syntrophic chimeric fusion can
be proved by sequence comparison of functional domains of motility
proteins isolated from candidate taxa.Archaeprotists | spirochetes | sulfur syntrophy |
| Two Domains, Not
All living beings are composed of cells and are unambiguously
classifiable into one of two categories: prokaryote (bacteria) or
eukaryote (nucleated organisms). Here we outline the origin of the
nucleus, the membrane-bounded organelle that defines eukaryotes. The
common ancestor of all eukaryotes by genome fusion of two or more
different prokaryotes became “chimeras” via symbiogenesis (1).
Long term physical association between metabolically dependent
consortia bacteria led, by genetic fusion, to this chimera. The
chimera originated when an archaebacterium (a thermoacidophil) and a
motile eubacterium emerged under selective pressure: oxygen threat
and scarcity both of carbon compounds and electron acceptors. The
nucleus evolved in the chimera. The earliest descendant of this
momentous merger, if alive today, would be recognized as an
amitochondriate protist. An advantage of our model includes its
simultaneous consistency in the evolutionary scenario across fields
of science: cell biology, developmental biology, ecology, genetics,
microbiology, molecular evolution, paleontology, protistology.
Environmentally plausible habitats and modern taxa are easily
comprehensible as legacies of the fusion event. The scheme that
generates predictions demonstrable by molecular biology, especially
motile protein sequence comparisons (2),
provides insight into the structure, physiology, and classification
Our analysis requires the two- (Bacteria/Eukarya) not the three-
(Archaea/Eubacteria/Eukarya) domain system (3).
The prokaryote vs. eukaryote that replaced the animal vs. plant
dichotomy so far has resisted every challenge. Microbiologist's
molecular biology-based threat to the prokaryote vs. eukaryote
evolutionary distinction seems idle (4).
In a history of contradictory classifications of microorganisms
since 1820, Scamardella (5)
noted that Woese's entirely nonmorphological system ignores
symbioses. But bacterial consortia and protist endosymbioses
irreducibly underlie evolutionary transitions from prokaryotes to
eukaryotes. Although some prokaryotes [certain Gram-positive
are intermediate between eubacteria and archaebacteria, no organisms
intermediate between prokaryotes and eukaryotes exist. These facts
render the 16S rRNA and other nonmorphological taxonomies of Woese
and others inadequate. Only all-inclusive taxonomy, based on the
work of thousands of investigators over more than 200 years on live
suffices for detailed evolutionary reconstruction (4).
When Woese (8)
insists “there are actually three, not two, primary phylogenetic
groupings of organisms on this planet” and claims that they, the
“Archaebacteria” (or, in his term that tries to deny their bacterial
nature, the “Archaea”) and the “Eubacteria” are “each no more like
the other than they are like eukaryotes,” he denies intracellular
motility, including that of the mitotic nucleus. He minimizes these
and other cell biological data, sexual life histories including
cyclical cell fusion, fossil record correlation (9),
and protein-based molecular comparisons (10,
The tacit, uninformed assumption of Woese and other molecular
biologists that all heredity resides in nuclear genes is patently
contradicted by embryological, cytological, and cytoplasmic heredity
The tubulin-actin motility systems of feeding and sexual cell fusion
facilitate frequent viable incorporation of heterologous nucleic
acid. Many eukaryotes, but no prokaryotes, regularly ingest entire
cells, including, of course, their genomes, in a single phagocytotic
event. This invalidates any single measure alone, including
ribosomal RNA gene sequences, to represent the evolutionary history
of a lineage.
As chimeras, eukaryotes that evolved by integration of more than
a single prokaryotic genome (6)
differ qualitatively from prokaryotes. Because prokaryotes are not
directly comparable to symbiotically generated eukaryotes, we must
reject Woese's three-domain interpretation. Yet our model greatly
appreciates his archaebacterial-eubacterial distinction: the very
first anaerobic eukaryotes derived from both of these prokaryotic
lineages. The enzymes of protein synthesis in eukaryotes come
primarily from archaebacteria whereas in the motility system
(microtubules and their organizing centers), many soluble heat-shock
and other proteins originated from eubacteria (9).
Here we apply Gupta's idea (from protein sequences) (10)
to comparative protist data (13)
to show how two kinds of prokaryotes made the first chimeric
eukaryote. We reconstruct the fusion event that produced the
| The Chimera:
Archaebacterium/Eubacterium Merger |
Study of conserved protein sequences [a far larger data set than
that used by Woese et al. (3)]
led Gupta (10)
to conclude “all eukaryotic cells, including amitochondriate and
aplastidic cells received major genetic contributions to the nuclear
genome from both an archaebacterium (very probably of the eocyte,
i.e., thermoacidophil group and a Gram-negative bacterium … [t]he
ancestral eukaryotic cell never directly descended from
archaebacteria but instead was a chimera formed by fusion and
integration of the genomes of an archaebacerium and a Gram-negative
bacterium” (p. 1487). The eubacterium ancestor has yet to be
identified; Gupta rejects our spirochete hypothesis. In answer to
which microbe provided the eubacterial contribution, he claims: “the
sequence data … . suggest that the archaebacteria are polyphyletic
and are close relatives of the Gram-positive bacteria” (p. 1485).
The archaebacterial sequences, we posit, following Searcy (14),
come from a Thermoplasma acidophilum-like thermoacidophilic
(eocyte) prokaryote. This archaebacterial ancestor lived in warm,
acidic, and sporadically sulfurous waters, where it used either
elemental sulfur (generating H2S) or less than 5% oxygen
(generating H2O) as terminal electron acceptor. As does
its extant descendant, the ancient archaebacterium survived
acid-hydrolysis environmental conditions by nucleosome-style
histone-like protein coating of its DNA (14)
and actin-like stress-protein synthesis (15).
The wall-less archaebacterium was remarkably pleiomorphic; it tended
into tight physical association with globules of elemental sulfur by
use of its rudimentary cytoskeletal system (16).
The second member of the consortium, an obligate anaerobe, required
for growth the highly reduced conditions provided by sulfur and
sulfate reduction to hydrogen sulfide. Degradation of carbohydrate
(e.g., starch, sugars such as cellobiose) and oxidation of the
sulfide to elemental sulfur by the eubacterium generated carbon-rich
fermentation products and electron acceptors for the
archaebacterium. When swimming eubacteria attached to the
archaebacterium, the likelihood that the consortium efficiently
reached its carbon sources was enhanced. This hypothetical
consortium, before the integration to form a chimera (Fig. 1),
differs little from the widespread and geochemically important
“Thiodendron” Stage |
The “Thiodendron” stage refers to an extant bacterial
consortium that models our idea of an archaebacteria-eubacteria
sulfur syntrophic motility symbiosis. The partners in our view
merged to become the chimeric predecessor to archaeprotists. The
membrane-bounded nucleus, by hypothesis, is the morphological
manifestation of the chimera genetic system that evolved from a
Thiodendron-type consortium. Each phenomenon we suggest, from
free-living bacteria to integrated association, enjoys extant
Study of marine microbial mats revealed relevant bacterial
consortia in more than six geographically separate locations.
Isolations from Staraya Russa mineral spring 8, mineral spring
Serebryani, Lake Nizhnee, mud-baths; littoral zone at the White Sea
strait near Veliky Island, Gulf of Nilma; Pacific Ocean hydrothermal
habitats at the Kurile Islands and Kraternaya Bay; Matupi Harbor
Bay, Papua New Guinea, etc. (17)
all yielded “Thiodendron latens” or very similar bacteria.
Samples were taken from just below oxygen-sulfide interface in
anoxic waters (17,
Laboratory work showed it necessary to abolish the genus
Thiodendron because it is a sulfur syntrophy. A stable
ectosymbiotic association of two bacterial types grows as an
anaerobic consortium between 4 and 32°C at marine pH values and
salinities. Starch, cellobiose, and other carbohydrates (not
cellulose, amino acids, organic acids, or alcohol) supplemented by
heterotrophic CO2 fixation provide it carbon.
Thiodendron appears as bluish-white spherical gelatinous
colonies, concentric in structure within a slimy matrix produced by
the consortium bacteria. The dominant partner invariably is a
distinctive strain of pleiomorphic spirochetes: they vary from the
typical walled Spirochaeta 1:2:1 morphology to large
membranous spheres, sulfur-studded threads, gliding or nonmotile
cells of variable width (0.09–0.45 μm) and lengths to millimeters.
The other partner, a small, morphologically stable vibrioid,
Desulfobacter sp., requires organic carbon, primarily
acetate, from spirochetal carbohydrate degradation. The spirochetal
Escherichia coli-like formic acid fermentation generates
energy and food. Desulfobacter sp. cells that reduce both
sulfate and sulfur to sulfide are always present in the natural
consortium but in far less abundance than the spirochetes. We
envision the Thiodendron consortium of “free-living
spirochetes in geochemical sulfur cycle” (ref. 18,
p. 456) and spirochete motility symbioses (19)
as preadaptations for chimera evolution. Thiodendron differs
from the archaebacterium-eubacterium association we hypothesize; the
marine Desulfobacter would have been replaced with a
pleiomorphic wall-less, sulfuric-acid tolerant soil
Thermoplasma-like archaebacterium. New thermoplasmas are
under study. We predict strains that participate in spirochete
consortia in less saline, more acidic, and higher temperature
sulfurous habitats than Thiodendron will be found.
When “pure cultures” that survived low oxygen were first
described [by B. V. Perfil'ev in 1969, in Russian (see refs. 17
a complex life history of vibrioids, spheroids, threads and helices
was attributed to “Thiodendron latens”. We now know these
morphologies are artifacts of environmental selection pressure:
Dubinina et al. (ref. 17,
p. 435), reported that “the pattern of bacterial growth changes
drastically when the redox potential of the medium is brought down
by addition of 500 mg/l of sodium sulfide.” The differential growth
of the two tightly associated partners in the consortium imitates
the purported Thiodendron bacterial developmental patterns.
The syntrophy is maintained by lowering the level of oxygen enough
for spirochete growth. The processes of sulfur oxidation-reduction
and oxygen removal from oxygen-sensitive enzymes, we suggest, were
internalized by the chimera and retained by their protist
descendants as developmental cues.
Metabolic interaction, in particular syntrophy under anoxia,
retained the integrated prokaryotes as emphasized by Martin and
However, we reject their concept, for which no evidence exists, that
the archaebacterial partner was a methanogen. Our sulfur syntrophy
idea, by contrast, is bolstered by observations that hydrogen
sulfide is still generated in amitochondriate, anucleate eukaryotic
cells (mammalian erythrocytes) (21).
T. acidophilum in pure culture attach to suspended
elemental sulfur. When sulfur is available, they generate hydrogen
Although severely hindered by ambient oxygen, they are
microaerophilic in the presence of small quantities (<5%) of
oxygen. The Thermoplasma partner thus would be expected to
produce sulfide and scrub small quantities of oxygen to maintain low
redox potential in the spirochete association. The syntrophic
predecessors to the chimera is metabolically analogous to
Thiodendron where Desulfobacter reduces sulfur and
sulfate producing sulfide at levels that permit the spirochetes to
grow. We simply suggest the replacement of the marine sulfidogen
with Thermoplasma. In both the theoretical and actual case,
the spirochetes would supply oxidized sulfur as terminal electron
acceptor to the sulfidogen.
The DNA of the Thermoplasma-like archaebacterium
permanently recombined with that of the eubacterial swimmer. A
precedent exists for our suggestion that membrane hypertrophies
around DNA to form a stable vesicle in some prokaryotes: the
membrane-bounded nucleoid in the eubacterium Gemmata
The joint Thermoplasma-like archaebacterial DNA package that
began as the consortium nucleoid became the chimera's nucleus.
The two unlike prokaryotes together produced a persistent protein
exudate package. This step in the origin of the nucleus—the genetic
integration of the two-membered consortium to form the chimera—is
traceable by its morphological legacy: the karyomastigont. The
attached swimmer partner, precursor to mitotic microtubule system,
belonged to genera like the nearly ubiquitous consortium-former
Spirochaeta or the cytoplasmic tubule-maker Hollandina
The swimmer's attachment structures hypertrophied as typically they
do in extant motility symbioses (19).
The archaebacterium-eubacterium swimmer attachment system became the
karyomastigont. The proteinaceous karyomastigont that united partner
DNA in a membrane-bounded, jointly produced package, assured
stability to the chimera. All of the DNA of the former prokaryotes
recombined inside the membrane to become nuclear DNA while the
protein-based motility system of the eubacterium, from the moment of
fusion until the present, segregated the chimeric DNA. During the
lower Proterozoic eon (2,500–1,800 million years ago), many
interactions inside the chimera generated protists in which mitosis
and eventually meiotic sexuality evolved. The key concept here is
that the karyomastigont, retained by amitochondriate protists and
later by their mitochondriate descendants, is the morphological
manifestation of the original archaebacterial-eubacterial fused
genetic system. Free (unattached) nuclei evolved many times by
disassociation from the rest of the karyomastigont. The
karyomastigont, therefore, was the first microtubule-organizing
Preceded Nuclei |
The term “karyomastigont” was coined by Janicki (23)
to refer to a conspicuous organellar system he observed in certain
protists: the mastigont (“cell whip,” eukaryotic flagellum, or
undulipodium, the [9 (2)
microtubular axoneme underlain by its [9 (3)
+ 0)] kinetosome) attached by a “nuclear connector” or “rhizoplast”
to a nucleus. The need for a term came from Janicki's work on highly
motile trichomonad symbionts in the intestines of termites where
karyomastigonts dominate the cells. When kinetosomes, nuclear
connector, and other components were present but the nucleus was
absent from its predictable position, Janicki called the organelle
system an “akaryomastigont.” In the Calonymphidae, one family of
entirely multinucleate trichomonads, numerous karyomastigonts, and
akaryomastigonts are simultaneously present in the same cell (e.g.,
Calonympha grassii) (24).
The karyomastigont, an ancestral feature of eukaryotes, is
present in “early branching protists” (25–27).
Archaeprotists, a large inclusive taxon (phylum of Kingdom
are heterotrophic unicells that inhabit anoxic environments. All
lack mitochondria. At least 28 families are placed in the phylum
Archaeprotista. Examples include archaemoebae (Pelomyxa and
Mastigamoeba), metamonads (Retortamonas), diplomonads
(Giardia), oxymonads (Pyrsonympha), and the two orders
of Parabasalia: Trichomonadida [Devescovina,
Mixotricha, Monocercomonas, Trichomonas, and
calonymphids (Coronympha, Snyderella)] and
Hypermastigida (Lophomonas, Staurojoenina, and
Trichonympha). These cells either bear karyomastigonts or
derive by differential organelle reproduction (simple morphological
steps) from those that do (Table 1).
When, during evolution of these protists, nuclei were severed from
their karyomastigonts, akaryomastigonts were generated (31).
Nuclei, unattached, at least temporarily, to undulipodia were freed
to proliferate and occupy central positions in cells. Undulipodia,
also freed to proliferate, generated larger, faster-swimming cells
in the same evolutionary step.
The karyomastigont is the conspicuous central cytoskeleton in
basal members of virtually all archaeprotist lineages [three
classes: Archamoeba, Metamonads, and Parabasalia (32)]
In trichomonads, the karyomastigont, which includes a parabasal body
(Golgi complex), coordinates the placement of hydrogenosomes
(membrane-bounded bacterial-sized cell inclusions that generate
hydrogen). The karyomastigont reproduces as a unit structure.
Typically, four attached kinetosomes with rolled sheets of
microtubules (the axostyle and its extension the pelta) reproduce as
their morphological relationships are retained. Kinetosomes
reproduce first, the nucleus divides, and the two groups of
kinetosomes separate at the poles of a thin microtubule spindle
called the paradesmose. Kinetosomes and associated structures are
partitioned to one of the two new karyomastigonts. The other
produces components it lacks such as the Golgi complex and axostyle.
Nuclear α-proteobacterial genes were interpreted to have
originated from lost or degenerate mitochondria in at least two
archaeprotist species [Giardia lamblia (33);
Trichomonas vaginalis (34,
and in a microsporidian (36).
Hydrogenosomes, at least some types, share common origin with
mitochondria. In the hydrogen hypothesis (20),
hydrogenosomes are claimed to be the source of eubacterial genes in
amitochondriates. That mitochondria were never acquired in the
ancestors we consider more likely than that they were lost in every
species of these anaerobic protists. Eubacterial genes in the
nucleus that are not from the original spirochete probably were
acquired in amitochondriate protists from proteobacterial symbionts
other than those of the mitochondrial lineage. Gram-negative
bacteria, some of which may be related to ancestors of
hydrogenosomes, are rampant as epibionts, endobionts, and even
endonuclear symbionts—for example, in Caduceia versatilis (37).
Karyomastigonts freed (detached from) nuclei independently in
many lineages both before and after the acquisition of mitochondria.
Calonymphid ancestors of Snyderella released free nuclei
before the mitochondrial symbiosis (13),
and Chlamydomonas-like ancestors of other chlorophytes such
as Acetabularia released the nuclei after the lineage was
fully aerobic (38).
In trophic forms of protists that lack mastigote stages, the
karyomastigont is generally absent. An exception is
Histomonas, an amoeboid trichomonad cell that lacks an
axoneme but bears enough of the remnant karyomastigont structure to
permit its classification with parabasalids rather than with
rhizopod amoebae (39).
This organellar system appears in the zoospores, motile trophic
forms, or sperm of many organisms, suggesting the relative ease of
karyomastigont development. The karyomastigont, apparently in some
cells, is easily lost, suppressed, and regained. In many taxa of
multinucleate or multicellular protists (foraminifera, green algae)
and even in plants, the karyomastigont persists only in the
zoospores or gametes.
In yeast, nematode, insect, and mammalian cells,
nonkaryomastigont microtubule-organizing centers are “required to
position nuclei at specific locations in the cytoplasm” (40).
The link between the microtubule organizing center and the nuclei
“is mysterious” (40).
To us, the link is an evolutionary legacy, a remnant of the original
archaebacterial-eubacterial connector. The modern organelles (i.e.,
centriole-kinetosomes, untethered nuclei, Golgi, and axostyles)
derive from what first ensured genetic continuity of the chimera's
components: the karyomastigont, a structure that would have been
much more conspicuous to Proterozoic investigators than to
| Acknowledgments |
We thank our colleagues Ray Bradley, Michael Chapman, Floyd
Craft, Kathryn Delisle (for figures), Ugo d'Ambrosio, Donna Reppard,
Dennis Searcy, and Andrew Wier. We acknowledge research assistance
from the University of Massachusetts Graduate School via Linda
Slakey, Dean of Natural Science and Mathematics, from the Richard
Lounsbery Foundation, and from the American Museum of Natural
History Department of Invertebrates (New York). Our research is
supported by National Aeronautics and Space Administration Space
Sciences and Comision Interministerial de Ciencia y Tecnologia
Project No. AMB98-0338 (to R.G.).
| Footnotes |
|This paper was presented at the National
Academy of Sciences colloquium “Variation and Evolution in Plants
and Microorganisms: Toward a New Synthesis 50 Years After Stebbins,”
held January 27–29, 2000, at the Arnold and Mabel Beckman Center in
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