266
BioSystems 7 (1975) 266--292
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
THE MICROBES' CONTRIBUTION TO EVOLUTION*
LYNN MARGULIS
Department of Biology, Boston University, 2 Cummington Street, Boston, Mass. 02215, USA
1. I n t r o d u c t i o n
We light microscopists, w h e t h e r we call our-
selves microbiologists, p r o t o z o o l o g i s t s , cell
biologists, m i c r o p a l e o n t o l o g i s t s , c y t o g e n e t i c -
ists, histologists or m e m b e r s o f some o t h e r
artificial discipline are direct d e s c e n d a n t s o f
the D u t c h school o f m i c r o b i o l o g y and its' great
ancestor f r o m Delft, A n t o n i van L e e u w e n h o e k .
Insatiably curious, he c o n s i d e r e d all o f n a t u r e
and especially its " a n i m a l c u l e s " w o r t h y o f
a t t e n t i o n . T o van L e e u w e n h o e k the world was
an organic whole e x t e n d i n g into smaller places;
it was never divided into p e r m i t t e d and forbid-
den territories--botanical, zoological, proto-
zoological or bacteriological. In this p a p e r I
h o p e to revive van L e e u w e n h o e k ' s view t h a t
e v e r y t h i n g small is w o r t h y o f study.
It is m y belief t h a t if o n e follows the exam-
ple of van L e e u w e n h o e k and excludes n o n e o f
the living m i c r o b e s f r o m c o n s i d e r a t i o n b u t
recognizes all as p r o d u c t s o f the neo-Darwin-
* On March 26 and 27th, 1975, The Royal Academy
of the Netherlands (Koninklijke Akademie van Weten-
schappen) celebrated the 300th anniversary of the
work of their illustrious countryman, Antoni van
Leeuwenhoek. This contribution to that occasion was
the only one that, unfortunately, was not delivered in
the Dutch language. Because of the nature of the
meeting, which called together an active, diverse
group of biologists and physicians, the paper is a
review of the present status of the symbiosis theory
of cell evolution, some aspects of which have ap-
peared before (Margulis, 1975a and b). The entire
symposium ("De microbiologie drie eeuwen na An-
toni van Leeuwenhoek") will be published by Pudoc,
Wageningen.
ian e v o l u t i o n a r y process, o n e is led d i r e c t l y t o
the realization t h a t the organelles o f e u k a r y o t i c
cells originated t h r o u g h a s e q u e n c e o f heredi-
tary e n d o s y m b i o t i c association. One reason is
simple: the processes p o s t u l a t e d to have oc-
curred during the e v o l u t i o n o f e u k a r y o t e s re-
curred m a n y times and gave rise to m a n y
strange microbeasts, some which were first
witnessed b y t h e draper. C o n t i n u e d s t u d y , van
L e e u w e n h o e k style, has led to r e o b s e r v a t i o n o f
these organisms with far finer tools than he had
available, and t o a r e a w a k e n i n g o f interest in
the t h e o r y I am advocating.
This p a p e r presents the "serial e n d o s y m b i o -
sis t h e o r y " ( T a y l o r , 1974) in its m o s t c u r r e n t
f o r m and shows h o w r e e x a m i n a t i o n o f these
microbial e c o s y s t e m s o f van L e e u w e n h o e k
(e.g., microbes o f the m o u t h , sea water and
ponds, d e c a y i n g m a t t e r ) leads to discoveries o f
p r e c e d e n t s and analogs for all o f the processes
h y p o t h e s i z e d as steps in the origin of n u c l e a t e d
cells {Margulis, 1 9 7 0 ) .
It has t a k e n until this second half o f the 2 0 t h
C e n t u r y to bring van L e e u w e n h o e k ' s observa-
tions into a c o h e r e n t whole. He described
h u m a n and animal sperm, placoid scales o f
e l a s m o b r a n c h fish, insect appendages, oral and
fecal spirochaetes, fossil f o r a m i n f e r o u s lime-
stone, cell division and sexual c o n j u g a t i o n in
ciliates. Indeed, m o s t m o d e m biologists have
never gazed u p o n m a n y o f the objects he saw. If
t h e y had, t h e y would perhaps have n o t f o u n d it
so startling t h a t c o m p l e x i n t e r a c t i o n b e t w e e n
species is a fact o f life, t h a t m u c h i n t e r a c t i o n
leads to symbioses and t h a t symbioses include
t h o s e o f the h e r e d i t a r y t y p e . T h e h e r e d i t a r y or

cyclical type of symbiosis is a relationship in
which two partners of different species are
regularly associated throughout the life cycle
of both.
Some 200 years after the death of van
Leeuwenhoek came the publication of the little
book by Kluyver and van Niel (1956) in con-
tinuation of the Dutch school of microbiology.
This may have been the most important step
toward the integration of the bacterial "animal-
cules" into the conceptual framework of bio-
logical thought. Followed by van Niel and
Stanier's classic paper {1962), it led to the firm
recognition of the prokaryotic nature of bac-
teria and blue green algae. Although the word
" p r o k a r y o t e " was probably first coined by
Chatton*, the concept of the prokaryote dis-
continuity was developed and brought to the
rest of Europe and the USA by the descendants
of the Dutch school (Stanier and van Niel,
1962; Stanier, 19,34).
In the late 1950's, with the publication of
The Microbial World (Stanier et al., 1957) and,
more recently, Brock's {1970) elegant tome
Biology of Microorganisms, it became clear
what the "animalcules" are and how they differ
from each other and from larger, more familiar
organisms. However, the knowledge that mi-
crobes, like metazoans and green plants, are
products of neo-Darwinian evolution is still in
process of realization. That evolution in mi-
crobes has happened by changes of gene fre-
quencies in natu::al populations with time at
specific places and periods in the history of the
Earth is a concept still under development. The
field of evolutionary microbiology is right now
emerging, and we are witnessing an enormously
energetic intellectual synthesis in this direc-
tion. However, :.1o one yet calls himself a
microbial evolutionist.
It is possible that the first seven-eighths of
earth's history (from about 3.5 to about 0.6
billion years ago) was the "Age of the Prokary-
ote Microbes" (Barghoorn, 1974). Entire fields
* This is reputed to be in E. Chatton, 1937, Titres et
travaux scientifique. E. Sottano sete Quatro. 407 p.,
but I have been unable to locate the book.
267
of study are being seen as relevant to the
evolution of microorganisms on the early
Earth. For some references to these develop-
ments see Barghoorn (1971); Ponnamperuma
(1971); Ponnamperuma and Buvet (1971);
Schopf (1974); Dose et al. (1974); Walters
(1976). In spite of fantastic new methodologi-
cal advances -- transmission and scanning elec-
tron microscopy; gas chromatograph-mass
spectrometry; the ultracentrifuge; the use of
radioisotopes; column chromatography and
electrophoresis; and so forth -- it is the tool of
van Leeuwenhoek himself that still occupies
the most central role in the analysis of objects
seen for the first time by him. For comparisons
of live microorganisms, for the study of micro-
bial fossils trapped in cherts (e.g., Tyler and
Barghoorn, 1954; Schopf and Blacic, 1971; see
Schopf, 1974 for review) and for the study of
chromosomes, the light microscope has no
equal.
2. Outline of the theory of the origin of euka-
ryotic cells by hereditary serial endosymbiosis
What follows is admittedly the " e x t r e m e "
version of the serial endosymbiotic theory
(S.E.T.) in that all three classes of organelles
(mitochondria, photosynthetic plastids and fla-
gella/cilia, i.e. 9 + 2 homologues made of micro-
tubules) are hypothesized to have originated by
hereditary endosymbiosis. For critical discus-
sion of these points see Taylor (1974). Al-
though many are willing to accept the S.E.T.
for plastids and mitochondria (e.g., Schnepf
and Brown, 1971; Lipmann, 1974; Taylor,
1974; John and W~atley, 1975), others feel that
the evidence for the symbiotic origin of mito-
chondria is inconclusive or unconvincing (Raff
and Mahler, 1972; Avers, 1974; Uzell and
Spolsky, 1974; Raff, 1975) and very few (if
any) serious scientists share my belief that the
eukaryote cilia-flagella system of microtubules
originated by hereditary endosymbiosis. Even
though it may not be correct, the extreme
version of the S.E.T. is both explicit and
testable and will therefore be included in the
268
subsequent discussion on the status of germane
evidence.
All organisms on earth have anaerobic, fer-
mentative, heterotrophic, prokaryotic ances-
tors. The triplet nonoverlapping nucleotide
base sequence that serves as the universal ge-
netic code determining amino acid sequences in
proteins was present in the bacterial population
ancestral to all extant forms of life. In the early
precambrian, selection pressures led to exten-
sive adaptive radiation among prokaryotes,
primarily on the metabolic level. Among oth-
ers, the following cell types evolved: myco-
plasm-like fermenters (catabolizing glucose to
pyruvate via anaerobic glycolysis using the
Embden Meyerhof metabolic pathway); spiro-
chaetes; photosynthetic oxygen-eliminating
prokaryotes (coccoid blue green algae); and
aerobic gram negative eubacteria that oxidized
small organic acids via the Krebs cycle (Sagan,
1967; Margulis, 1970; 1971b; 1972a,b;
1975a,b).
According to the S.E.T., the first step in the
origin of eukaryotes from prokaryotes was the
acquisition of protomitochondria. This event
could have occurred when a fermentative an-
aerobe (host) was invaded by Krebs cycle-con-
taining gram negative eubacteria (protomito-
chondria); stabilization of this initially pre-
datory association led to the formation of the
mitochondria-containing amoeboids, from
which all other eukaryotes derive (Hall, 1973).
Because pinocytosis and phagocytosis are vir-
tually unknown in prokaryotes (Stanier, 1970)
and because recently characterized Bdellovi-
brio-like organisms provide such a fine model
for the penetration of bacterial hosts by aero-
bic gram negative eubacteria, I now feel the
first prokaryotic-prokaryotic symbiotic step
probably occurred by invasion, modification
and stabilization of Bdellovibrio-like behavior.
(See Starr and Seidler, 1971, and Starr, 1975,
for details concerning BdeUovibrio.) Alterna-
tively because the electron transport chain of
Paracoccus denitrificans is so similar to that of
mitochondria, the first step could have been
the acquisition of an intracellular endosym-
biont comparable to the facultative aerobe
Paracoccus ( Micrococcus) denitrifricans. The
general similarity of the cytochromes, qui-
nones, inhibitor sensitivities and electron trans-
port chain between Paracoccus and mitochon-
dria is overwhelming (John and Whatley,
1975}. The differences between them can be
interpreted as adaptations to an intracellular
role (e.g., loss of the constitutive nitrate reduc-
tase pathway; transport of ATP to the sur-
roundings; and so forth}. Perhaps a Paracoccus
with predatory behavior might be sought as
ancestor to mitochondria. The nucleus and
other endoplasmic membranes probably
evolved autogenously, after the presence of
protomitochondria provided the way for the
steroid biosynthetic pathway. Selection pres-
sures for the formation of the nuclear mem-
brane probably involved the segregation of
newly synthesized DNA on endomembrane
(Leibowitz and Schaechter, 1974} and/or the
sequestering of nucleoplasm DNA to protect it
from the more oxidizing conditions of the
cytoplasm surrounding the mitochondria.
The second symbiotic step is hypothesized
to be the acquisition by the mitochondria-con-
taining amoeboids of highly motile anaerobic
surface bacteria (very likely spirochaetes}.
Many mutations and intracellular transfer of
genes from bacteria to host preceded the origin
of amoeboflagellates, ciliates and many other
protists. The surface spirochaete bacteria
evolved into the "9 + 2" homologues: basal
bodies, cilia, flagella, and other microtubule-
based structures. Mitosis, as a process, evolved
in many lines of organisms as the motile bacte-
ria merged with the amoeboid host cell, and the
morphogenetic processes of the bacteria even-
tually were utilized in the formation of the
"achromatic apparatus" (mitotic spindle) of
mitosis. (See Margulis, 1970, 1974b, and
Pickett-Heaps, 1974, for detailed but different
expositions of the possible steps involved in the
origin of mitosis.)
The final symbiotic step involved the acqui-
sition of photosynthesis by different popula-
tions of eukaryotic heterotrophs. Ingestion,
without digestion, of blue green algae by vari-
ous protist hosts under nutrient-poor condi-
 269

tions led to the establishment of stable, herita-
ble, intracellular symbioses. The prokaryotic
algae eventually became the obligatory sym-
biotic photosynt:netic plastids in the origin of
various lines of nucleated algae and, eventually,
chlorophytes anti archegoniate multicellular
green plants.
Thus, according to the S.E.T., all eukaryotes
are at least digenomic (contain two indepen-
dently derived genomes: host and mitochon-
dria); according to the extreme S.E.T., most
heterotrophic eukaryotes were originally tri-
genomic (host, mitochondria, 9 + 2 homol-
ogue and plastids) as illustrated in Fig. 1.
I agree with Taylor's (1974) contention that
"cell" is an inadequate term, and follow his
suggestion that in referring to cells we use
words that reflect the number of membrane
bound protein synthesizing units; for example,
the use of "monad" for prokaryote, "dyad" for
heterotrophic eukaryotes and "triad" for pho-
tosynthetic eukaryotes. (If the spirochaete ori-
gin for the flagellar-mitotic system is correct, it
is clear that the once independent protein
synthesizing system of the motile bacteria must
have become completely integrated into the
complex eukaryotic host system. Thus Taylor's
terminology can be applied to eukaryotes no
matter which version of the serial endosym-
biotic theory is finally accepted.) Multicellular

O
L~~ '~~p . (~
b--~ PLANTS FUNGI ANIMALS

AMOEBOFLAGELLATE

I-
i O

m AMOEBO
<I,
/
/
;Z
C~ BLUE GREEN AEROBIC FERMENTING SPIROCHETES
ALGAl:_ BACTERIA ANAEROBIC
BACTERIA
Fig. 1. Model for the origin of eukaryotic organelles by symbiosis (see Margulis, 1970 for details).
270
units, then (according to Taylor, 1974) be-
come: polymonads (e.g., filamentous and my-
celial prokaryotes such as Gardnerula, Poly-
angium or Myxococcus); polydyads (e.g., slime
molds, ascomycetes, metazoa); and polytriads
(e.g., bryophytes, tracheophytes, brown and
red eukaryotic seaweeds); and so forth. Appli-
cation of Taylor's analyses to both cells and
extant associations has already proven useful
(Margulis, 1975b).
3. Historical theories: on the nature of the
evidence
It is never possible to rigorously prove after
the fact that a unique series of events did occur
in any historical context. Evolutionary biolo-
gists, like historians, deal with series of com-
plex, irreversible phenomena. They can only
present arguments based on the assumption
that of all the plausible historical sequences,
one is more likely to be a correct description of
past events than another. The probability that
one particular historical reconstruction is more
accurate than another increases as the number
of explanations for existing observations in-
crease. An evolutionary theory can also be
judged by its predictive power. In this con-
text, some experimental studies already gener-
ated by the extreme S.E.T. are described be-
low, and a number of potential areas of investi-
gation are presented in Section 5. These studies
and their predicted conclusions do not neces-
sarily follow from alternative monophyletic
and/or partial symbiotic concepts for the origin
of eukaryotic cells.
In his remarkably complete and well-argued
paper Taylor (1974) explores several alterna-
tives to the endosymbiotic view of the origin of
mitochondria and plastids. He enumerates the
logical problems with most endogenous or
"pinching o f f " hypotheses and considers new
data from molecular and cellular biology in the
light of nonsymbiotic theories (Taylor, 1976).
Since his presentation of non-S.E.T, alterna-
tives is far less impassioned and biased than
mine, the reader is referred to his reviews. In
my opinion, the strongest advocates of the
nonsymbiotic view (Allsopp, 1969; Raff and
Mahler, 1972 and Raff, 1975; Perlman and
Mahler, 1970; Uzzell and Spolsky, 1974) ig-
nore certain critical bodies of literature and
facts (see Margulis, 1975a, for discussion). The
burden of detailing the nonsymbiotic theory
predictions must lie with its advocates.
4. Experimental studies and explanations
generated by the serial endosymbiotic theory
4.1. Genetic behavior of organelles
Three distinct patterns of inheritance can be
detected in the ciliate protist Paramecium aure-
l/a: nuclear (Mendelian); caryonidal (a special-
ized modified nuclear mode); and cytoplasmic
(Sonneborn 1947; Barnett, 1966). For many
years the inheritance of the killer trait has been
the standard example of the behavior of cyto-
plasmic genes (Sonnenborn, 1959). The killer
p h e n o t y p e was found to depend on the pres-
ence of cytoplasmic kappa particles. Traits
correlated with the presence of kappa particles
are inherited independently of the nucleus, yet
kappa requires several Mendelian genes for its
maintenance (K genes) and is negatively af-
fected by several other nuclear alleles (S genes).
The complete solution of the problem is at
hand. Kappa (Fig. 2) is a class (only one of
many, Beale et al., 1969) of hereditary endo-
symbiotic bacteria that harbors virus (Preer et
al., 1974). The similarity between the genetic
behavior of kappa and similar endosymbiotic
bacteria and mitochondria and photosynthetic
plastids is striking. An obvious explanation is
that both kappa and these organelles began as
free-living prokaryotes and, with time, have
become hereditary endosymbionts more and
more dependent on products of nuclear genes.
In fact, the hypothesis that mitochondria and
plastids originated as prokaryotic symbionts
has generated some fine experiments and an
immense literature (see, for example, Nass,
M.M.K., 1969, 1971; Nass, S., 1969; Cohen,
1970, 1973 for review; Pigott and Carr, 1972;
Ebringer, 1972). Let us take just one limited
 271

b m

'r '7 ,'2

b'o~, 7, t - _~o,.~*l "

~ii! •
(2
C un n n nnnn

cl

Fig. 2. Examples of bacterial symbionts in the ciliate, Paramecium aurelia. (a) Osmium-lacto-orcein preparation
of stock 239 (syngen 4), whole Paramecium containing "lambda" symbionts, the numerous dark-stained rods
filling the cell. ([Park phase contrast X500, courtesy of Dr. John Preer, University of Indiana, Dept. of Zoology,
Bloomington, Indiana.) (b) Unfixed purified preparation of kappa particles (bacteria) isolated from stock 51
(syngen 4) paramecia. The rods with uniform color are non-bright kappas; those containing a light spherical
refractile (R) body are very slightly unrolled due to coverslip pressure, taking a characteristic V shape. It is
thought that kal:.Pa particles with the ability to kill noninfected paramecia harbor refractile bodies in various
stages of development and that these bodies are a manifestation of the phage (virus) particles untimately responsi-
ble for the killing action. (Bright phase contrast X1650, courtesy of Dr. C. Kung, University of California, Santa
Barbara.) (c) Electron micrograph of stock 7 (syngen 2) Paramecium showing longitudinal section through a
bright kappa particle (bacterium). Note dark staining spherical phage-like structures inside the coiled R body.
Surrounding the P~ body and extending beyond it on either side is a fine membrane, the sheath. (d) Electron
micrograph of stock 1039 (syngen 2) unrolled R body of kappa. The end at the bottom terminates in an acute
angle while the end at top is blunt and irregular (X 30,000).
272
but extremely exciting example of the useful-
ness of the S.E.T. in explaining the recently
discovered "sexual" behavior of mitochondria
and chloroplasts.
The evidence that recombination of non-
Mendelian (cytoplasmic) markers occurs be-
tween differently marked mitochondria enter-
ing a cross in yeast is now overwhelming
(Mounolou et al., 1967; Thomas and Wilkie,
1968; Wilkie and Thomas, 1973; see Kujon et
al., 1974 and Gillham, 1974, 1976 for details.)
Comparable organellar sexual recombination
phenomena are probably occurring after zy-
gote formation in Chlamydomonas chloro-
plasts as well (Sager, 1972; Gillham, 1974). The
best model for the nature of the recombination
process in organelles is a potentially multipar-
ent, unidirectional (polarized) model {Gillham
et al., 1974; Gillham, 1975). Thus, the detailed
comparison of the genetic behavior of organel-
les (mitochondria and plastids) with prokary-
otic recombination systems has proven fruitful.
If these organelles had originated by differen-
tiation of cytoplasm within a primitive eukary-
ote, the independent origin of prokaryote-type
recombination in organelles and free-living bac-
teria and their viruses is difficult to account for.
Genetic interaction between organisms that
are clearly recognizable symbiotic partners is
not well understood. There is no doubt that
heritable modification of symbionts occurs
with time. The gradual development of depen-
dence of the host nuclei on symbiont products
has been directly observed (by Jeon, 1972, for
example) in new bacterial-amoebae associa-
tions (Fig. 3). Because of the usefulness of such
information in understanding the processes
that have led to the evolution of organelles
from free-living symbionts, I have recently
reviewed this literature (see Margulis, 1975b).
4.2. Protist phylogeny
The contradictory classification systems
devised by zoologists and botanists for the
"lower eukaryotes" are notorious. The recogni-
tion of "anastomosing phylogenies," i.e., that
certain groups such as lichens and chlorophyte
algae are products of symbioses, has begun to
clarify the relationships among these orga-
nisms. Single consistent phylogenies and taxo-
nomic schemes for the "lower eukaryotes" can
now be drawn. Although symbiosis may not be
a factor in the origin of most higher taxa, it has
been decidedly significant in many cases
{Fig. 4). (See Whittaker, 1969, 1975; Margulis,
1974a,c for details and Leedale, 1974, for
discussion.)
4.3 The microfossiliferous Preca m brian
It has long been recognized that there is no
catastrophic environmental gap in the fossil
record between the precambrian and the Pha-
nerozoic. The "sudden appearance" of animal
fossils at the base of the Cambrian has been
puzzling, but the recognition that, fundamen-
tally, the precambrian was the "Age of the
Prokaryote Microbes" and the Phanerozoic the
"age of Eukaryotes" clarifies this discrepancy
(Barghoorn, 1974; Margulis, 1972c). Of course
this is an oversimplification not addressed to
the origin of shelled metazoans (Cloud, 1968)
and the role of oxygen and other environmen-
tal variables shaping the selection pressures on
the biota(Cloud, 1974). However, the symbiot-
ic theory is consistent with the idea that pro-
karyotic microorganisms, including oxygen-
eliminating photosynthesizers, preceded by
hundreds of millions of years the origin of the
eukaryotic metazoan animals and green plants
(senso stricto ).
The recognition that precambrian times
were dominated by prokaryotes has led to
fruitful collaboration between blue green algal
mat ecologists and precambrian sedimentary
geologists (e.g., Waiters, 1976; Golubic, 1973),
the identification of Entophysalis-like coccoid
blue green algae in 2500 million year old rocks
from Belcher Island, Canada for example (Go-
lubic and Hofmann, 1975).
4.4. Additional examples
The recognition that microbes are products
of neo-Darwinian evolution, with selection act-
 273

¢,

"2"

Fig. 3. The effect o:[ nuclear transplant on the maintenance of symbiotic microorganisms in amoebae. These
amoebae contain mitochondria, the "DNA containing bodies", and a second population of more recently
acquired endosymbiotic bacteria. (a) Electron micrograph of normal Amoeba discoides showing the normal
morphology of the DNA-containing bodies that happen to be clustered together, individually surrounded by
membranes. (X 37,000) (b) Electron micrograph of Ameoba discoides infected with microorganisms of two
kinds. Shown are the more recent symbionts which, unlike the "DNA-containing bodies" and the mitochondria,
tend to look less adversely affected by intraspecific nuclear transplants. (c) Electron micrograph of nucleocyto-
plasmic hybrid (A. proteus nucleus and A. discoides cytoplasm} showing abnormal morphology of a mitochon-
drion and that of DNA-containing bodies. Since proteus nucleus is incompatible with cytoplasm of discoides, this
hybrid is not viable. Courtesy of K. Jeon, University of Tennessee.

ing m a i n l y on m e t a b o l i c r a t h e r t h a n m o r p h o l - a t m o s p h e r e (Cloud, 1 9 7 4 ; L o v e l o c k and Mar-

ogic traits, has aided in placing in a t e m p o r a l gulis, 1 9 7 4 a , b ; Margulis and L o v e l o c k , 1 9 7 4 ;
sequence certain steps in t h e h i s t o r y o f t h e Walker, 1976).
274

!I
I METAPHYTE PHYLA
? ,.,
' ¢ / FUNGI PHYLA I
:
~" ANIMAL PHYLA / /

\ : ..¢.~o.l.,e.. k /'~ z~o-..oo- ,,~ i \ "Y//-

' \\: : "~, ~ " ',.t ", or besidiospores l " ', ~, l.~''r"

,, . \'..,.\-!- \ : . .,, -,.. -..:\\ ,,

, ' -~"-~--- . . . . ~ ...... ~ ..... :--~_" ~'--" 5~-~-.
', "'" ~ .... car-yobleetea}_. . PROTIST I~'IYLA " : : : ~ ? ~
i

I~--- ]'t
i
1-- ~
_
I

M O N E R A N PHYLA

e naerOJOlc fermenting
prokeryote

Fig. 4. Diagram of the role of symbiosis in origin of higher taxa. (From Margulis, 1975a).

The concept that complex (9 + 2) micro- Of course, the concept that mitochondria
tubule-based flagella regeneration is a process and plastids originated as bacteria has led to
homologous to the movement of chromosomes widespread use of specific antibiotics (e.g.,
in mitosis has led to the discovery of mitotic streptomycin and other aminoglycosides,
arrest by melatonin and other specific actions chloramphenicol, cycloheximide) in investi-
of certain microtubule inhibitors (Banerjee and gating the role of the organellar protein syn-
Margulis, 1973; Margulis, 1973; Banerjee et al., thetic system relative to the " h o s t " eukaryote
1975). The awareness of the homology be- (Ebringer, 1972). The assumption that plant
tween the flagella and cilia systems of eukary- cells are the products of hereditary endosym-
otes has suggested a new theory of sensory biosis explains the presence, for example, of
transduction {primarily olfactory, gustatory redundant chloroplast and nonplastid metab-
and auditory, Atema, 1973). olic pathways (e.g., polyunsaturated fatty

acids, Jacobson et al., 1973). It also clarifies the
discovery of a very skewed distribution of
qualitatively recoverable types of mutants in
plants and Chlamydomonas relative to animal
cells and fungi (L: et al., 1967).
5. Some predicted results of potential experi-
ments generated by the S.E.T.
The serial endosymbiosis theory has the
advantage of generating a large number of
experimentally verifiable predictions. Some
are listed here.
5.1. Lack of mitosis in blue green algae
No blue green algae that show trends to-
wards the evolu;ion of mitosis will ever be
found (e.g., with centriotes, microtubules or
histone-containing chromosomes). All blue
green algal developmental and sexual systems
will prove to be similar to those of bacteria.
5.2. Free-living homologues: plastids
Since coccoid blue green algae are hypothe-
sized to be ancestral to photosynthetic plastids,
the primary sequence of many proteins in the
photosynthetic a:3paratus of plastids will turn
out to be homologous to those from the appro-
priate coccoid algae.
5.3. Free-living homologues: mitochondria
Since certain aerobic bacteria (e.g., Paracoc-
cus denitrificans; John and Whatley, 1975) are
hypothesized to be ancestral to mitochondria,
the primary sequences of many of the proteins
in the mitochondria will show homologies to
those from the appropriate respiring bacteria
(e.g., superoxide dismutase, Fredovich, 1975;
c y t o c h r o m e 550 and c y t o c h r o m e oxidase).
5.4. Free-living homologues: spirochetes
Since certain ,;pirochaetes (e.g., Pillotinas,
Hollande and Gharagozlou, 1967) are hypothe-
275
sized to be ancestral to the 9 + 2 flagella/cilia
homologues, the primary sequence of some
spirochaete proteins will show homologies to
those of the flagellar/ciliary axonemes and/or
matrix. It is predicted that spirochaetes that
contain authentic microtubes with colchicine-
binding microtubule protein (perhaps even in a
9 + 2 array) will be found.
Primary amino acid sequence of the eukary-
otic flagella proteins (tubulins) will show great-
er similarities to whatever protein(s) is/are
intrinsically motile in spirochaetes than to any
other prokaryotic proteins.
5. 5. Basal body RNA and spirochetes
RNA of the lumen of eukaryotic basal
bodies will be found homologous to RNA of
appropriate free-living spirochaetes. The repli-
cation (polymerization) of this RNA (Hartman
et al., 1974} will be required for new basal body
formation, whether in centrioles, kinetosomes
or other de novo production of 9 + O's (Sonne-
born, 1974). This RNA should also be homol-
ogous to "division centre" RNA in Physarum
(Laane and Haugli, 1974).
5. 6. Phylogenetic significance of mitotic varia-
tions
Striking variations on the theme of mitosis
will be found in "lower eukaryotes." In opposi-
tion to statements in the classical literature
(Wilson, 1925), these will turn out to be of
phylogenetic significance and generally inde-
pendent of the presence of photosynthetic
plastid-related traits. (Pickett-Heaps, 1974, has
already shown the phylogenetic significance of
mitotic variations in certain green algae; Kubai,
1973, and Heath, 1974, have done the same for
protists and flagellated fungi.) Meiosis will be
shown to have originated independently in
several preadapted lines of protists (e.g., cili-
ates, slime molds, hypermastigotes) just as
h o m e o t h e r m y originated independently in sev-
eral preadapted vertebrate lines.
276
5.7. Derivation of the red algae thallus from a
mycelium with cyanelles
The photosynthetic plastids of red algae will
be found to have many metabolic pathways
and primary amino acid sequences in common
with blue green algae; non-plastid cytoplasmic
proteins in red algae will turn out to be more
like certain heterotrophic protists and primi-
tive zygomycetes or ascomycetes (amastigote
conjugating organisms).
5.8. Lack of autonomy as the advanced state
Organisms containing well developed Men-
delian genetic systems (e.g., yeasts, metazoans,
metaphytes) will be found to have less auton-
omous protein synthetic systems in their
mitochondria and photosynthetic plastids.
Conversely, certain "lower eukaryotes" that
have no eumitotic sexual systems will be shown
to contain organelles with greater autonomy.
Attempts to "culture" organelles will be more
successful in organisms that show no or idio-
syncratic sexuality (e.g., euglenids, dinoflagel-
lates, etc; see Margulis, 1974b and Pickett-
Heaps, 1974 for discussion of some of these
forms).
5. 9. Bdellovibrio-like symbionts
Bdellovibrio-like bacteria will be found that
maintain stable relationships with their "prey."
5.10. Tubulins
Because of their hypothesized common ori-
gin, ultimately from the ancestral spirochaetes,
the amino acid sequence of mitotic spindle
tubulin proteins from all eumitotic organisms
will show striking homologies with those from
the (9 + 2) flagellar and ciliary axonemes.
It will be shown that flagellar proteins (a and
fl tubulins) are used directly in the formation of
the mitotic spindle microtubes in many if not
all eumitotic eukaryotes. This protein will not
be found in blue green algae. However, homol-
ogous tubulin proteins will be detected in
extant forms of red algae, fungi and other
eukaryotes that have mitosis but lack flagella.
5.11. Eukaryote transformation and polyge-
nomic control of metabolic pathways
In certain intracellular associations, gene
transfer from symbionts (bacteria) to hosts
(eukaryotes) will be proved. With respect to
organelles, the particular genes transferred
from the mitochondria and/or plastids in any
lineage will tend to be the same within that
lineage and tend to be different outside that
lineage (see Fig. 4). Many gene products (e.g.,
proteins) will be cytoplasmic recombinant
products requiring two or more non-homol-
ogous genomes for their formation (e.g., of
ribolosediphosphocarboxylase and leghemo-
globin -- the former requires both nucleocyto-
plasmic and chloroplast protein synthesizing
systems, the latter requires both symbiotic
bacteroid and plant cell systems.)
End products of high selective advantage
produced by complex metabolic pathways
{e.g., certain alkaloids, toxins, steroids, lichenic
acids) will tend to be products of more than
one genome; that is, the nucleic acids coding
for the polypeptides of the proteins of these
pathways will reside in part in the organelle(s)
as well as in the nucleus. The "biological
clocks," or circadian rhythms so characteristic
of and limited to eukaryotic organisms may
well turn out to be the result of interaction
between gene products and metabolites of the
multigenomic systems making up eukaryotic
cells (e.g., dyads and triads).
5.12. In vivo growth of organelles
Photosynthetic plastids and mitochondria
will be cultured in vitro. The growth require-
ments for their culture will tend to be gene
products (proteins, peptides or coenzymes)
and/or metabolites supplied to them by the in
vivo cycloheximide sensitive 80S ribosomal sys-
tem. Other organeUes (those that presumably
had an endogenous rather than exogenous ori-
gin, e.g., Golgi apparatus; pigment granules;

lysosomes; nucle:i) will not be culturable. Or-
ganelles that originated by hereditary endo-
symbiosis will piove to be intrinsically repli-
cating units, unlike the endogenous structures,
which presumably differentiated from the rest
of the cell.
5.13. Protein homologies of the 80S ribosome
By amino acid sequence analysis, eukaryotic
80S ribosomes will show protein homologies,
indicating that they have more than one pro-
karyotic ancestor (e.g., anaerobic fermenter
and heterotrophic eubacteria, host and proto-
mitochondrion, respectively; anaerobic fer-
menter and spirochaete, host and flagellum,
respectively; and so forth). Perhaps proteins of
the 80S ribosomes have direct ancestry in
certain spirochaete ribosomal proteins.
5.14. Anastomosing phylogenies
Only after acceptance of the symbiotic theo-
ry for the origin of organelles, will we be able to
construct consis'~ent phylogenies at higher
taxonomic levels {such as those available for
tracheophytes and chordates) that are accept-
able to botanists, zoologists and microbiolo-
gists.
5.15. Hybridization between organelles and
free-living microorganism DNAs
Direct nucleic acid hybridization studies will
reveal homologies between organisms and or-
ganelles as outlined in Fig. 4. The a m o u n t of
unique sequence transcribable DNA per or-
ganelle will turn out to be an approximate
inverse function of the age of the association.
This hypothesis can also be applied to the
nucleic acids of l:,artners in clear-cut examples
of symbioses.
5.16. Organellar genetics and physiology: mod-
els for infection
Understanding of the genetic interactions in
symbioses and pathogenic relations will be
277
greatly enhanced by an understanding of euka-
ryotic organellar genetics.
6. Implications of the S.E.T. for the overall
classification of living organisms into five
kingdoms
Whittaker (1959, 1969), on ecological
grounds, has devised a sound scheme for the
classification of all living organisms into five
groups, one prokaryote and four eukaryote.
The five-kingdom system of Whittaker was
originally published with only the inclusion of
the highest taxa: kingdoms and phyla. By
applying the concepts generated by the sym-
biotic theory, Whittaker's scheme could be
modified to form an overall classification con-
sistent with information generated by genetic
and developmental studies. This detailed clas-
sification as published in Margulis ( 1 9 7 4 c ) a n d
in more detail in Margulis (1974a) is schemati-
cally drawn in Fig. 1, which outlines the hypo-
thesized role of symbiosis in the origin of
higher taxa as well as other familiar groups that
have traits strongly influenced by regular sym-
biotic associations. Although Wallin {1927)
certainly overstated the case, it is obvious that
"symbionticism" has been a mechanism in the
origin of new taxa; examples may be found on
every level from species to phylum.
The basic modified Whittaker five-kingdom
scheme can be summarized as follows: mone-
rans (all prokaryotes, or monads); protists
{dyads and triads, diverse groups of asexual and
eumitotic eukaryotes, ploidy levels and mitotic
systems vary); fungi (dyads: amastigote hap-
loid or dikaryotic forms, zygo, asco- and basidi-
ospore formers that grow by absorptive nutri-
tion); animals (dyads: diploid metazoans that
develop from blastulas and grow by ingestive
nutrition); plants (archegoniate e m b r y o p h y t e
triad photoautotrophs). (For discussion of the
monad, dyad and triad terminology, see Taylor
1974 and Margulis, 1975b.)
This is not the place to detail the modified
Whittaker classification but only to urge you to
look up your organism in it. Is the system
278
consistent with y o u r own experience with the
living form? Any comments would be appre-
ciated.
An alternative logical two-kingdom, pro-
karyote and eu k ar yot e , system has been sug-
gested by Taylor (1974). This and several other
possibilities have been discussed by Leedale
(1974). Following Whitehouse (1969), how-
ever, it seems to me both logical and respectful
of systematic tradition to keep the p r o k a r y o t e
(chromonemal) versus eukaryote (chromo-
somal) distinction at the superkingdom level.
Thus the plant and animal kingdoms would
remain in a restricted form supplemented by
three other kingdoms: monerans, protists and
fungi. For the first time the mycologists them-
selves have suggested the t r e a t m e n t of fungi as a
kingdom (or at least a subkingdom, they have
hedged a bit) in their most recent t a x o n o m i c
treatise (Ainsworth et al., 1973), although they
have not separated out flagellated " l o w e r "
forms from the amastigote line.
7. Evolutionary convergence in the microbial
world
7. 1. The lesson o f Mesodinium: acquisition of
photosyn thesis by eukaryotes
It is a well know observation in evolutionary
biology that severe environmental restrictions
produce, by convergence, similar structures in
r e m o tely related organisms, for example: wings
for aerial flight in bats, birds and insects; spores
resistant to desiccation in clostridia, m y x o c o c -
ci, cellular slime molds and ascomycetes; spines
and thorns for p r o t ect i on in roses and lobsters;
protective bold black and white color patterns
in clownfish and zebras; stromatolites by fila-
mentous blue green and green algae; and so
forth.
Two o f the most significant sources of selec-
tion pressure in nature are (1) lack of carbohy-
drate and other organic food sources and (2)
lack o f sufficient motility to escape potential
predators and to migrate from inadequate to
optimal habitats. Those organisms best
equipped to photosynthesize are often the least
equipped to move. The formation of a motile
p h o t o s y n t h e t i c unit by the joining of a fast-
moving organism to a p h o t o a u t o t r o p h has re-
peatedly happened t h r o u g h o u t the course of
evolution. In certain cases, such as the photo-
synthetic paramecia, hydroids or mollusks
(Fig. 5), it is obvious that the host is fundamen-
tally heterotrophic, and the motile-photosyn-
thetic complex is a p r o d u c t of symbiosis. In
other cases, such as Cyanidium, Cyanophora
and all eukaryotic algae, the m et hod of origin
of the motile photosynthesizer is not so clear.
What has becom e increasingly evident in recent
years, primarily due to ultrastructural analyses,
is the prevalence of the p h e n o m e n o n of the
acquisition of photosynthesis in motile orga-
nisms by symbiosis in unrelated forms. Table 1
summarizes some of this information.
Mesodinium (Fig. 5b), a functionally photo-
synthetic ciliate, provides us with an example•
Since it reddens the sea water and its distribu-
tion is ubiquitous, it has often been seen. This
highly successful microorganism was noted by
Charles Darwin off the coast of Chile in about
1839. It is possible that it was first seen by van
Leeuwenhoek at Scheveningen, July 27, 1676
(Blackbourn et al., 1973).
• . . I went to the seaside at S c h e v e n i n g e n . . .
and viewing some of the Sea-water very
attentively . . I saw in it a little animal that
was blackish, looking as if it had been made
up of two globules• This creature had a
peculiar motion, after the manner as when
we see a very little flea leaping upon a white
paper (yet at every leap it moved only about
the length of a coarse sand grain); so that it
might very well be called a Water-flea; but it
was by far not so great as the eye of that little
animal which Dr. Swammerdam calls the
Water-flea [Daphnia]. Letter 18, 1676,
Dobell (1958)
Unlike Paramecium bursaria, Stentor poly-
morphous and Paraeuplotes tortugensis,
(which are all photosynthesizing ciliates),
Mesodinium does not contain chlorella-algae.
Rather, every individual of the species rubrum
contains what appear to be partial crypto-

monad photosynthetic symbionts. Mesodi-
nium rubrum fu:¢thermore seems to be swim-
ming backwards and eating little (compared to
other mesodinia), suggesting that the associa-
tion with the provident photosynthesizer has
led to hereditary alterations in the lifestyle of
M. rubrum. The reduction of the ingestion
apparatus and t:ne tendency towards greater
autotrophy (relative to their nonphotosyn-
thetic relatives} has happened convergently
(where known) in most of the organism listed
in Table 2.
The acquisition of photosynthesis by hetero-
trophic flagellates that subsequently became
279
the various groups of eukaryotic algae may be
seen as one example of a c o m m o n process. The
evolutionary origin of Mesodinium (as well as
the other entries in Table 1) are further exam-
ples. The merging of photosynthetic and motile
organisms to form new complexes has hap-
pened at many times and places from the
precambrian until recent times.
7.2. Natural history repeats itself: bacterial
attachment sites
The basal b o d y and root system of cilia and
flagella has been an enigma since its discovery
280

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Fig. 5. Functionally photosynthetic organism derived from nonphotosynthetic motile and photosynthetic immo-
tile ancestors. (a) Pelochromatium, bacterial consortium in which large flagellated heterotrophic bacteria are
covered with small photosynthetic bacteria. (structure at the limit of resolution of the light microscope) (Cour-
tesy of James Staley) (b) Mesodinium rubrum, a ciliate, harboring "incomplete photosynthetic cryptomonads."
(Courtesy of F.J.R. Taylor, see Taylor, 1974). (c) Elysia viridis, a gastropod mollusk, harboring functional
chloroplasts (below) ultimately derived from siphoneous food algae. (Courtesy of Robert Trench).

in t h e late 1 9 t h c e n t u r y (see Wilson, 1925 f o r 1 9 7 5 ; a m o n g o t h e r s ) has s h o w n w i t h o u t ques-

review o f early w o r k ) . All e u k a r y o t e cilia and
flagella, w h e t h e r o f s e n s o r y cells, ciliates or
s p e r m , originate f r o m these characteristic 9 + 0
basal bodies. L w o f f {1950) t h o u g h t t h a t these
organelles divide a u t o n o m o u s l y . Careful elec-
t r o n m i c r o s c o p y (Allen, 1 9 6 9 ; Dippell, 1 9 6 8 ,
tion t h a t m o s t basal b o d i e s d o n o t divide. T h e y
are d i f f e r e n t i a t e d f r o m a m o r p h o u s " g r a n u l a r -
fibrillar" m a t e r i a l in various f o r m s a n d n u m -
bers, d e p e n d i n g o n the o r g a n i s m . In s o m e cases
(e.g., a m o e b o f l a g e l l a t e s ) basal b o d i e s a p p e a r
" d e n o v o , " t h a t is, f r o m no p r e e x i s t i n g recog-
 283

'FABLE 2
E v o l u t i o n a r y s t r u c t u r a l and f u n c t i o n a l c o n v e r g e n c e s in microbial systems.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Convergence E x a m p l e s of Type of Environ- Selection Pressures References

Organisms merit
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

spores myxoeocci
clostridia; bacilli;
slime molds; s p o r o z o a ;
a p i c o m p l e x a ; fungi
sporebearing strut- m y x o c o c c i (e.u:.,
tures Polya ngiu m ) ;
colonial ciliate; fungi
bacterial attach- spirochaetes; uniden-
m e n t sites tified b a c t e r i a with
o t h e r bacteria or
protists
mycelia slime m o l d s ; actino-
m y c e t e s fungi
stromatolites blue green algae;
c h l o r o p h y t e and o t h e r
e u k a r y o t i c algae
a c q u i s i t i o n of bacterial c o n s o r t i a ;
photosynthesis P a r a m e c i u m bursaria,
soil; tissues and cells desiccation; nutrient Brock, 1970
of o t h e r organisms depletion
soil desiccation and Olive, 1 9 7 5 ;
dispersal Brock, 1970
hindl~uts o f roaches hold fasts ( s e p a r a t i o n Raikov, 197,t;
and t e r m i t e s ; sand from h o s t ) B h ) o d g o o d , 1974
grains; ( p s a m m o -
phile) ciliates; w o r m
cuticles
soil; cells and tissues p e n e t r a t i o n ; in-
of o t h e r organisms crease of surface area
intertidal p h o t i c s t a b i l i z a t i o n of local G o l u b i c , 1973;
z o n e e n v i r o n m e n t against A w r a m i e et al., 1975
t u r b u l e n c e , etc.
lake m u d s ; o p e n nutrient depletion Table 1
o c e a n

by motile organ- Glaucocystis: Meso-

isms diniu m
sessile " a d u l t " b u t Caulobacter; s u c t o r i a n surfaces altering
motile "larva" ciliates; c h y t r i d s , (e.g. e n v i r o n m e n t a l con-
(hold fast.s) Blast oclad icl la ) d i t i o n s (larval dis-
mollusks ; t u n i c a t e s persion to o p t i m a l
e n v i r o n m e n t for
adult development)

nizable structure. In others they appear at The basal bodies and their axonemes (cilia
regular positions in regular relations to preex- and flagella shafts) have been hypothesized to
isting basal bodies (e.g., the ciliate cortex). In have originated from free-living spirochaete-
some cases there is one old basal body for each like microbes; it is this postulate that distin-
newly emerging c ne. In other cases they appear guishes the "extreme S.E.T." from all other
as multiples; for example, in the formation of versions of the cell symbiotic theory. The evi-
some plant sperm hundreds to thousands may dence for this concept has been stated previ-
be made at once. There is no evidence that the ously (Sagan, 1967; Margulis, 1971) and will
"reproduction" of basal bodies is an event that not be repeated here.
requires DNA synthesis (Younger et al., 1972); This discussion centers on the principle that
however, new RNA synthesis is required, and it nature repeats itself, that given the appropriate
is thought by some that a specific class of basal environmental selection pressures, comparable
body RNA exists (For discussion of these convergent structures will evolve. Based on this
complex issues see Hartman et al., 1974; principle, one would expect that organelles of
Younger et al., 1972 and Frankel, 1974). motility have evolved from free-living spire-
284

chaete bacteria by convergence several times, mitosis and the typical basal body (9 + 2) com-
although perhaps in just one ancient popula- plex originate. Many protists form associations
tion of microbes ancestral to eukaryotes did of greater or lesser intimacy with motile bac-

:4,
 285

Fig. 6. Bacterial att~chments to protists. (a) Unidentified bacteria on the surface of Urinympha, a hypermastigote
from the hindgut of a termite. (Courtesy of Robert A. Bloodgood, Yale University) (b) Bacteria on the surface of
Pyrsonympha, a polymastigote from the termite Reticulitermes flavipes. (Courtesy of Robert A. BIoodgood, Yale
University) (c) Unidentified spirochaete attached to the surface of Pyrsonympha. (Courtesy of Robert A. Blood-
good, Yale University) (d) Rootlet of the spirochaete, thought to anchor it to Pyrsonyrnpha. Part o f t h e micro-
tubular axostyle of the host can be seen. (Courtesy of Harriet Smith, University of Chicago) (e) Spirochaete
attached to surface of Pyrsonympha from Reticulitermes tibialis showing the specialized attachment region (A).
(Courtesy of Robert A. BIoodgood, Yale University) (f) Bacterial attachment sites on the surface of host protist
Barbulanympha. (g) Detail of Fig. 6f. (Courtesy of Dr. A. Hollande, Laboratoire de l'Evolution des etres organi-
ses, Paris) (h) Unidentified bacteria attached to the sand ciliate Kentrophoros (Courtesy of I.B. Raikov, Institute
of Cytology, Leningrad)

teria. Examples of bacterial attachment struc- ancestral basal bodies. In this connection, the
tures (which clearly evolved by convergence) similarity between the spirochaete attachment
are shown in Fig. 6. These may be analogous to "rootlets" on Pyrsonympha (Fig. 6 c and d)
286
and eukaryotic cilia rootlets (e.g., gill cilia
Fig. 7 a,b) is striking. Basal bodies (which are
t h o u g h t to have subsequently evolved into
mitotic centrioles for reasons outlined in Mar-
gulis, 1970) began perhaps as bacterial attach-
ment sites. In some groups of organisms (e.g.,
Mixotricha) continued association led to the
acquisition o f motility, conferred on the host
by the symbionts. In the line of organism
leading to eukaryotes, it is postulated that this
association went much farther: the microtu-
bules of the microtubule-containing spiro-
chaete were utilized for axopod, axonem e and
axostyle structure, sensory behavior, mitosis,
intracellular pigment transport and many other
microtubule-based eukaryotic cell prosesses.
Again, the first description of symbiotic
spirochaetes was given by van L eeuw e nhoe k:
My e x c r e m e n t being so thin, I was at divers
times persuaded to examine it; and at each
time I kept in mind what f ood I had eaten,
and what drink I had drunk, and what I
found afterwards: but to tell all my observa-
tions here would make all too long a story... I
have also seen a sort of animalcule that had
the figure of the river eels: these were in very
great plenty, and so small withal, that I
deemed 500 or 600 of 'em laid out end to
end would not reach to the length of a full
grown eel such as there are in vinegar. These
had a very nimble motion, and bent their
bodies serpentwise, and shot through the
stuff as quick as a pike does through the
water. Letter 34, 1681, Dobell (1958)
Accordingly, I t o o k (with the help of a
magnifying mirror) the stuff of f and from
between the teeth further back in my m o u t h
where the heat o f the coffee c o u l d n ' t get at
it. This stuff I mixt with a little spit out of
my m o u t h (in which there were no air
bubbles), and I did all this in the way I've
always done: and then I saw with as great a
w o n d e r m e n t as ever before, an inconceiv-
ably great n u mbe r of little animalcules.., the
whole stuff seemed alive and a-moving...
And I saw, too, sundry animalcules... These
moved their bodies in great bends so swift a
motion, in swimming first forwards and then
backwards, and particularly with rolling
around on their long axis, that I coul dn't but
behold t hem again with great w o n d e r and
delight: the more so because I hadn't been
able to find t hem for several years, as l've
already said. Letter 75, 1692, Dobell (1958)
The hypothesis that basal bodies originated as
spirochaete a t t a c h m e n t sites and that the cili-
ary root l et systems arose from bacterial root l et
systems analogous to those of the spirochetes
of Pyrsonympha (Smith and Arnott, 1974) is
certainly testable (Fig. 7) as noted above. For
example, I predict that the RNA of basal bodies
will show hom ol ogy in the primary sequence of
nucleotides to one of the RNAs of the appro-
priately chosen population of spirochaetes.
7.3. Evolutionary convergences: cysts, spores
and fruiting bodies
The many mechanisms by which groups of
organisms resist desiccation and insure disper-
sion are well known. That only very distantly
related microorganisms arrive by convergence
at the same sorts of "fruiting b o d y " structures
can be seen directly by examination of Fig. 8.
Again van Leeuwenhoek was the first to
observe the adaptions o f microbial life to dry-
ing and rewetting.
Now since we see that these animalcules can
lie bedded so long in dry matter, as before
described, and then on coming into water
can sweel out their bodies, and swim off, we
may therefore conclude that in all pools and
marshes, which have water standing in them
in winter but which dry up in summer, many
kinds of animalcules ought to be found; and
even though there were none at first in such
waters, they would be brought thither by
water fowl, by way of the mud or water
sticking to their feet and feathers.
Letter 147, April 1702, Dobell (1958)
The discussion in this section illustrates that
under the same sorts of selection pressures,
convergent structures, behaviors and com-
plexes of organisms originate. The microbial
world contains processes precisely analogous
to those occurring among larger organisms,
 287

Fig. 7. Electron micrographs of cilia and ciliary rootlets from the gill of the fresh water mollusk Elliptio (a) X
51,000 (b) X 90,000. (Courtesy of Fred D. Warner, Dept. of Biology, Syracuse University)
288

Fig. 8. Convergent evolution of fruiting structures in microorganisms. Light microscope views of live material.
(a) Rhizopus sporangium (zygomycetous fungus, eukaryote). (b) Myxococcus fulvus (fruiting bacterium, prokary-
ote). (Courtesy M. Dworkin and H. Reichenbach, University of Minnesota) (c) Chondromyces apiculatus (fruiting
bacterium, prokaryote). (Courtesy M. Dworkin, University of Minnesota) (d) Unidentified colonial ciliate (eu-
karyote). (Courtesy Lindsay Olive, University of North Carolina).

including precedents for those events hypothe-
sized to occur in the serial endosymbiotic ori-
gin of eukaryotes.
8. Summary
The purpose of a scientific theory is to unite
apparently disparate observations into accurate
generalizations with predictive power. Histori-
cal theories, which necessarily treat complex,
irreversible events, can never be tested directly.
However, they certainly can lead to predic-
tions. The " e x t r e m e " version of the serial
endosymbiotic theory states that three classes
of eukaryotic organelles: mitochondria; basal
bodies/flagella/cilia [(9 + 2) homologues] ; and
photosynthetic plastids all were derived from
free-living ancestors. This hypothesis is sup-
ported by microbial precedents. The symbiotic
origin of photosynthesis in motile heterotro-
phic microorganisms is hypothesized to have
happened convergently several times, as did the
development of specialized attachment sites
for motile spirochaetes. Even if the cell symbio-
sis theory turns out to be wrong, it has the
advantage of generating a large number of
unique and experimentally verifiable predic-
tions. The collection of such new information
can do no less than add to our knowledge of the
evolution of microorganisms, many of which
were first seen by van Leeuwenhoek.

Acknowledgements
M a n y ideas here were d e v e l o p e d in discus-
sions with J.K. Kelleher, S. G o l u b i c , C. Har-
w o o d , L. To, R o b e r t T r e n c h and m e m b e r s o f
m y symbiosis class. I t h a n k M. Karakashian f o r
excellent c o m m e n t s on the m a n u s c r i p t and
J.R. Williams and B. Miranda f o r editorial
assistance. I a c k n o w l e d g e with gratitude re-
search s u p p o r t f::om N A S A ( N G R - 0 0 4 - 0 2 5 )
and travel s u p p o r t to a t t e n d the van Leeuwen-
h o e k S y m b o s i u m w h i c h c a m e f r o m the Ko-
ninklijke A k a d e m i e van W e t e n s c h a p p e n , Am-
sterdam.
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