ANNALS OF THE NEW YORK ACADEMY OF SCIENCES
Volume 108, Art. 2 Pages 339-616
LIFE-LIKE FORMS IN METEORITES AND THE PROBLEMS OF
ENVIRONMENTAL CONTROL ON THE MORPHOLOGY
OF FOSSIL AND RECENT PROTOBIONTA
Consulting Editor
Bartholomew Nagy ^
J. Joseph Lynch, SJ. (Conference Chairman) ^ ^
AUTHORS
E. Anders, E. S. Barghoorn, R. Berger, J. L, Blum, P. Bourrelly, R. E.
Cameron, B. J. Cholnoky, G, Claus, L. Dienes, H, Dombrowski, D. L.
EuROPA, F, W. Fitch, S. W. Fox, D. J. Hennessy, J. H, Johnson,
W. G. Meinschein, B. Nagy, J. Oro, C. M. Palmer, A. Papp, R. Patrick,
R. Ross, A. T. Soldo, P. Tasch, S. A. Tyler, J. R. Vallentyne, S. Yuyama
Editor Managing Editor
Harold E. Whipple Stanley Silverzweig
Q
11
.N4
V. 108
NEW YORK
PUBLISHED BY THE ACADEMY
June 29, 1963
THE NEW YORK ACADEMY OF SCIENCES
(Founded in 1817)
BOARD OF TRUSTEES
BORIS PREGEL, Chairman of the Board
Class of 1960-1963
HENRY C. BRECK
GORDON Y. BILLARD
HILARY KOPROWSKY
LOWELL C. WADMOND
BORIS PREGEL
G. W. MERCK
Class of 1962-1964
FREDERICK A. STAHL
Class of 1962-1965
HARDEN F. TAYLOR
Class of 1963-1966
W. STUART THOMPSON
CHARLES W. MUSHETT, President of the Academy
FREDERICK Y. WISELOGLE, Past President JAMES B. ALLISON, Past President
EUNICE THOMAS MINER, Executive Director
SCIENTIFIC COUNCIL, 1963
CHARLES W. MUSHETT, President
J. JOSEPH LYNCH, S.J., President-Elect
C. CHESTER STOCK, Vice-President KARL MARAMOROSCH, Vice-President
CHARLES R. NOBACK
Recording Secretary
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EMERSON DAY
Elected Councilors
1961-1963
1962-1964
1963-1965
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Corresponding Secretary
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SECTION OF BIOLOGICAL AND MEDICAL SCIENCES
PRESTON L. PERLMAN, Chairman GABRIEL G. NAHAS, Vice-Chairman
DIVISION OF ANTHROPOLOGY
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DIVISION OF INSTRUMENTATION
WALTER E. TOLLES, Chairman CARL BERKLEY, Vice-Chairman
DIVISION OF MICROBIOLOGY
H. CHRISTINE REILLY, Chairman EUGENE L. DULANEY, Vice-Chairman
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SECTION OF CHEMICAL SCIENCES
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DIVISION OF BIOCHEMISTRY
J. J. BURNS, Chairman PAUL GREENGARD, Vice-Chairman
SECTION OF GEOLOGICAL SCIENCES
BARTHOLOMEW S. NAGY, Chairman BRUCE C. HEEZEN, Vice-Chairman
SECTION OF PHYSICAL SCIENCES
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Conferences are also held at irregular intervals at times announced by special programs.
ff J
^ ANNALS OF THE NEW YORK ACADEMY OF SCIENCES K
Volume 108, Art. 2 Pages 339-616
June 29, 1963
Editor Managing Editor
Harold E. Whipple Stanley Silverzweig
LIFE-LIKE FORMS IN METEORITES AND THE PROBLEMS
OF ENVIRONMENTAL CONTROL ON THE MORPHOLOGY
OF FOSSIL AND RECENT PROTOBK
Consulting Editor
Bartholomew Nagy
CONTENTS
itroductory Remarks. By J. Joseph Lynch, S. J 341
nvironmental Biophysics and Microbial Ubiquity. By J. R. Vallentyne 342
"he Influence of Water Currents on the Life Functions of Algae. By John L. Blum. . 353
'he Structure of Diatom Communities under Varying Ecological Conditions. By Ruth
Patrick 359
^'eli Structure and Environment. By B. J. Cholnoky 366
The Morphology of PPLO and Bacterial L Forms. By Louis Dienes 375
Axenic Culture of Paramecium — Some Observations on the Growth Behavior and Nu-
tritional Requirements of a Particle-bearing Strain of Paramecium aurelia 299X.
By Anthony T. Soldo 380
The Effect of Pollution on River Algae. By C. Mervin Palmer 389
Ultrastructure Research as an i\id in the Classification of Diatoms. By R. Ross 396
Morphology of Representative Blue-Green Algae. By Roy E. Cameron 412
Loricae and Cysts in the Chrysophyceae. By Pierre Bourrelly 421
Morphological Trends among Fossil Algae. By J. Harlan Johnson 430
Paleoecological Considerations of Growth and Form of Fossil Protists. 5y Paul Tasch. 437
Fossil Organisms from Precambrian Sediments. By Elso S. Barghoorn and Stanley
A. Tyler 451
Bacteria from Paleozoic Salt Deposits. By Heinz Dombrowski 453
Fossil Protobionta and Their Occurrence. By A. Papp 461
Studies in Experimental Organic Cosmochemistry. By J. Or6 464
Evaluation of Radiation Effects in Space. By Rainer Berger 482
Abiotic Production of Primitive Protein and Formed Microparticles. By Sidney W.
Fox AND Shuhei Yuyama 487
Observations on the Nature of the "Organized Elements" in Carbonaceous Chondrites.
By Frank W. Fitch and Edward Anders 495
On the Origin of Carbonaceous Chondrites. By Edward Anders 514
* This series of papers is the result of a conference on The Problems of Environmental Control
^-^ on the Morphology of Fossil and Recent Protobionta held by The New York .\cademv of Sciences
^ on April 30 and May 1, 1962.
Aqueous, Low Temperature Environment of the Orgueil Meteorite Parent Body. By
Bartholomew Nagy, Warren G. Meinschein, Douglas J. Hennessy 534
Evidence in Meteorites of Former Life: The Organic Compounds in Carbonaceous Chon-
drites Are Similar to Those Found in Marine Sediments. By W. G. Meinschein,
Bartholomew N.\gy, Douglas J. Hennessy 553
Further Observations on the Properties of the "Organized Elements" in Carbonaceous
Chondrites. 5,v George Claus, Bartholomew Nagy, Dominic L. Europa.... 580
Discussion of the Identity of the "Organized Elements." HARf)LD C. Urey, Moderator . 606
^V^NOf».«^^
MBLAJ7HOI
Library
Copyright, 1Q63, by The Neic York Academy of Sciences
INTRODUCTORY REMARKS
J. Joseph Lynch, S. J.
Seismology can contribute nothing to the problem of extraterrestrial life.
One naturally wonders then why a seismologist should be called upon to open
this symposium. Dr. Nagy must be blamed for that. He and I occupy offices
in adjacent buildings and when either of us has a problem in Earth science we
mull it over together. When Dr. Nagy first found evidence of organic fossils
in the Orgueil meteorite he came to me and discussed the evidence with me.
He thought that somehow I had helped him by my encouragement and as an
acknowledgment insisted that I give these opening remarks.
The possibility of life outside of our planet has been a question in man's mind
almost as far back as man himself. The divergence of views on the matter is
about as broad as it could be. Only a century and a half ago the great English
astronomer, Sir William Herschel, first President of the Royal Astronomical
Society and discoverer of the planet Uranus said in one of his Presidential ad-
dresses that he was convinced that life existed within the Sun. Unfortunately
he did not elaborate upon what kind of life he had in mind. The present Secre-
tary of the same Royal Astronomical Society, Michael Ovenden, in his recent
book, Life in ihe Universe, as his view states that life is probably possible any-
where in the universe except within a Sun! It would be hard to imagine two
more divergent views on the same subject by members of the same society. It
has even been suggested that life is older than Earth itself and came to us from
another galaxy. However, confining ourselves to our own solar system, most
thinkers on the subject would restrict the possibility of life — for reasons of tem-
perature — to that part of our solar system between Venus and Mars. Beyond
Venus the temperature would be too hot — beyond Mars and some of the as-
teroids, the temperature would be too cold. Where within this region did the
fossils on the Orgueil meteorite originate?
Dr. Nagy and his co-workers in presenting their evidence for organic fossils
on the Orgueil meteorite have adequately ruled out the possibility of their
origin by contamination since the meteorite fell to Earth. How and where the
organisms — if they were organisms — originated, are questions that this sym-
posium should throw much light on. Did they originate on Earth and later
return to Earth via the moon? Or did they originate on an asteroid or a planet
outside of the Earth? The organizing committee deserves great credit for hav-
ing brought together such a distinguished group of experts. They cover not
only every phase of the subject, but represent the views of almost every coun-
try. Because you are gathered to hear their evidence and not any rambling
conjectures of mine, I shall cut my remarks short and let the session chairman
get the program started.
The which if you with patient ears attend,
Whence came these forms, you'll find out at the end.
{With apologies lo William Shakespeare)
341
ENVIRONMENTAL BIOPHYSICS AND MICROBIAL UBIQUITY
J. R. Vallentyne
Department of Zoology, Cornell University, Ithaca, N.Y.
Since the downfall of the near-collision theory of the origin of the solar sys-
tem and the revival of the dust cloud hypothesis it has generally been assumed
that planetary systems must be common in the universe. There has also been
a strong tendency to regard the formation of life within a planetary system as
the probable outcome of a series of nonbiological events operating within a re-
stricted range of physicochemical conditions. These points of view contrast
markedly with those held even as little as 30 years ago. Few persons today
would attempt to maintain that Earth is the sole place in the universe where
life resides.
In spite of this drastic change in attitude and the recent reports of organized
matter in carbonaceous chondrites (Nagy et al., 1961; Claus and Nagy, 1961),
there are still many who hesitate to beUeve that life within the solar system can
exist beyond the confines of Earth. In relation to the cjuestion of life on Mars,
for example, it is customary to tmd opinions clouded in a mass of delicately
phrased intellectual jargon that is designed to be all inclusive and noncommit-
tal. Much of the criticism levelled against the notion of life on Mars is made
from what the self styled Soviet astrobotanist, G. A. Tikhov (1955), would
term a geocentric point of view. Thus, it is often questioned whether organ-
isms could survive the rigors of a Martian climate: an average temperature
50° C. below that of the earth; daily temperature fluctuations of about 60° C.
at the equator; an atmosphere richer in CO2 , and decidedly lower in O2 and
total pressure than that characteristic of Earth; an environment in which water
is scarce and in which the level of ultraviolet radiation may reach "lethal"
proportions.
This, however, is absolutely the wrong approach to the question. The whole ap-
proach assumes a curious lack of adaptation on the part of the presumed Mar-
tian organisms, almost forcing them to adapt to terrestrial conditions in a
Martian locality. At least two assumptions seem to be involved in the reason-
ing: (1) that a complete body of information exists defining the environmental
limits beyond which life, as known on Earth, is impossible; and (2) that these
geoenvironmental limits of life are not exceeded on a cosmic scale. The first
of these assumptions is clearly erroneous as the present paper will show, and
the second seems rather questionable.
My main purpose here is to summarize current knowledge and ignorance re-
garding the environmental boundaries that delimit the "stability field" of liv-
ing matter. The problem is approached purely on an empirical basis. Most
of the discussion is limited to conditions that permit growth and reproduction
because this is the central cjuestion that has to be faced; however, some remarks
are made concerning survival because of its pertinence to life in fluctuating
environments. The review is not intended to be exhaustive, nor comprehen-
sive in anything other than a qualitative sense; only to serve as a reminder of
forgotten or little known facts concerning some of the extreme types of environ-
ment inhabited by living organisms. Attention is focussed on microorganisms
342
y.
Vallentyne: Environmental Biophysics & Microbial Ubiquity 343
because of their great environmental and physiological diversLty as compared
to the so-called "higher" forms of Hfe. ^~ "^
Temperature
The temperatm-e range for growth and reproduction of different microor-
ganisms extends from —18° to 104° C. These Hmits exceed those defining the
stability field of pure water under one atmosphere of pressure, but they do not
exceed the stabihty field of water in the liquid state when it is impure and under
variable pressure.
Let us first consider some cases of microbial activity at temperatures below
0° C. It is important in this connection to realize that ice does not form in sea
water with a salinity of iS per thousand until the temperature drops below
— 1.9° C, and also that 90 per cent of all sea water has a temperature less than
5° C. It is thus not surprising to find that many marine bacteria will grow at
subzero temperatures. Bedford (1933) was able to culture 65 of 71 marine
bacteria from the north Pacific at subzero temperatures, and ZoBell (1934)
independently showed the same for 76 out of 88 marine bacteria in his collec-
tion. Ten of the taxa cultured by Bedford (1933) were capable of growth and
reproduction in nutrient-enriched salt solutions at — 7.5°C. Twelve others
grew at — 5°C. Horowitz-Wlassowa and Grinberg (1933) found 5 bacteria
that would grow at —5° C, and 14 others that grew at —3° C. Bacteria are
known to multiply in ice cream stored at —10° C. (Weinzirl and Gerdeman,
1929) and on fish stored at -11° C. (Redfort, 1932).
Fungi, and probably algae as well, also multiply at these low temperatures.
Thus, the mold Sporotrichum carnis grows at —7.5° C. and very slowly even
at —10° C. (Haines, 1931). ChoelosLylum fresenii and Horniodendrou cladospo-
roides also grow at — 10°C. (Bidault, 1921). Tchistiakov and Botcharova
(1938) similarly found several different fungi that were capable of growth at
— 8° C., although none of these would grow at —12° C. The flagellate Pyra-
mimonas (Pyramidomonas?) has been observed swimming in saline water at
— 7.7°C. under the cover of ice in Lake Balpash, Kazakh S.S.R. (Zernow,
1944). Populations of 12 other photosynthetic forms were found in the same
water, presumably also alive and metabolizing. Zernow (1944) even observed
swimming Pyramidomonas and Dunaliella in drops of Lake Balpash water de-
rived from soft ice that had formed at —15° C.
The most extreme cases of growth at low temperatures are those referred to
by Borgstrom (1961) who states that some molds and pseudomonads will grow
in concentrated fruit juices and sugar solutions at temperatures of — 18° to
— 20°C. He has also observed the growth of Aspergillus glaucus kept in
glycerol at —18° C. A report of pink yeasts growing on oysters at tempera-
tures of —18° to —30° C. (McCormack, 1950) needs independent verification.
No experiments seem to have been undertaken on the possibility of algal
photosynthesis in saline media at subzero temperatures, but such a result would
not be unexpected. Although slightly out of context, it is worth noting that
some terrestrial plants are able to carry out a limited photosynthesis at —2°
to — 3°C., and respire down to — 7°C. (Zeller, 1951). In the last century,
Jumelle (1891) reported that certain lichens and conifers could photosynthesize
at temperatures between —20° and —40° C., but modern studies have failed
344 Annals New York Academy of Sciences
to corroborate these findings (Rabinowitch, 1945; Zeller, 1951). Before leav-
ing the subject of growth at low temperatures it must be stressed that in all
cases the growth is slow, usually requiring weeks and sometimes months before
definitive results are obtained.
At the upper end of the temperature scale it has long been known that some
bacteria and blue-green algae exist in hot springs with temperatures in the
range of 80° to 88° C. For summaries of existing information the works of
Copeland (1938), Precht et al. (1955), and Allen (1960) should be consulted.
Baker et al. (1955), have cultured a strain of Bacillus stearothermophilus at
80° C. No attempt was made to determine whether growth would still occur
at higher temperatures. According to ZoBell (1958) thermophilic sulfate re-
ducing bacteria isolated from subterranean deposits have been cultured in the
laboratory at temperatures to 65° to 85° C. These forms were originally ob-
tained from depths of 6000 to 12,000 feet, at which temperatures in situ ranged
from 60° to 105° C. and hydrostatic pressures from 200 to 400 atmos. ZoBell
(1958) also states: "The maximum temperature at which the thermophilic cul-
tures are active is increased by compression. At 1000 atmospheres one culture
reproduced and produced HoS at 104° C. No attempt has been made to as-
certain whether bacteria will grow at temperatures higher than 104° C. when
compressed, but indications are highly suggestive of the possibilities in view of
the protective effect of high pressure on the thermal tolerance of bacteria."
The case referred to represents the highest temperature so far recorded for the
growth and reproduction of any organism.
Eh and pH
The best general treatment of the environmental limits of Eh and pH
for growth and reproduction is that given by Baas Becking et al. (1960).
These workers have summarized paired Eh-pH data for the growth of diverse
microorganisms in natural environments and laboratory cultures. Although
the Eh values may in some cases not represent truly reversible potentials they
at least give a reproducible and reasonably accurate picture. Their results
are shown graphically in figure 1. When the data for all microorganisms are
combined and compared to Eh-pH measurements in natural surface waters of
the earth, a complete overlap is observed. This suggests that there is probably
no major aqueous environment that cannot be colonized by some microor-
ganism. The range for growth and reproduction of microorganisms was found
to lie between 850 mv. and —450 mv. on the Eh scale (when expressed as Eh at
the prevailing pH) ; and between values of 1 .0 and 10.2 on the pH scale. These,
however, do not represent the true extremes because the authors considered
only data for which paired measurements of Eh and pH were available.
Some environmental extremes of pH that can be tolerated by reproducing
populations may now be cited. Thiobacilli are well known for their abihty to
grow in acid solutions. In fact, they tend to show optimal growth in the pH
range of 1 to 3, many growing poorly above pH 7. Carbon dioxide is the sole
carbon source, and energy is obtained from the oxidation of reduced forms of
sulfur to sulfate under aerobic conditions. Growth and reproduction can
occur at pH values in the neighborhood of 0, and cultures receiving no initial
Vallentyne: Environmental Biophysics & Microbial Ubiquity 345
supply of H2S()4 can contain concentrations up to 2.08 n H2SO4 at the end of
growth (Starkey, 1925).
Several molds are capable of growth at a pH of 1.7 (Johnson, 1923). The
most acid tolerant fungi known are Acontiuni velatum and fungus D (an un-
identified member of the Dermatiaceae), originally isolated from strong acid
B
H
J K L
Figure 1. Eh-pH characteristics of diverse microorganisms. A, green algae and diatoms;
B, DunalieUa; C, Enteromorpha; D, blue-green algae; E, photosynthetic ])urple bacteria;
F, photosynthetic green bacteria; G, sulfate reducing bacteria; //, thiobacteria; /, iron bac-
teria; J, denitrifying bacteria; A', three species of heterotrophic bacteria; L, methane producing
bacteria. Redrawn from Baas Becking el al. (1960). Eh is expressed in millivolts.
346 Annals New York Academy of Sciences
solutions containing 4 per cent CUSO4 in an industrial plant (Starkey and
Waksman, 1943). These forms grow well when submerged in nutrient-enriched
sulfuric acid solutions at pH values between 0.4 and 7.0. Some growth occurs
at pH (2.5 normal H2SO4) even when solutions are saturated with CUSO4 .
No study was made of the permeability of the cells to copper and hydrogen
ions, but presumably there was little to no penetration.
One alga is notable for its growth in acid solutions, a strain of Cyanidhim
caldarium originally isolated from a hot spring containing 0.1 N H2SO4 . Allen
(1959) has cultured this form in 1 n H2SO4 . No attempt was made to deter-
mine whether growth would still occur in more concentrated solutions or acid
solutions at elevated temperatures.
At the upper end of the pH scale many microorganisms are known to grow
actively at a pH of 10, some at a pH of 11, and a few others possibly at still
higher pH values. Johnson (1923) reported that limiting growth of Penicillium
var labile occurred in the pH range of 10.1 to 11.1. Two other fungi, Fusarium
hullatum and F. oxysporum, were limited by pH values in the range of 9.2 to
11.2. Many alkaline lakes are known with pH values in the range of 9 to 11,
and these are by no means sterile. Jenkin (1936) found populations of 13
algae, 4 rotifers, and 2 copepods living in the alkaline lakes of Kenya. In lakes
Elementeita and Nakuru, in which the pH was commonly in the range of 10 to
11, large concentrations (10^ individuals per ml.) of the blue-green a\a.ga.Arthrop-
sira platensis were found (Jenkin, 1936). Still more extreme cases of growth
at high pH have been reported by Meek and Lipman (1922) for Nitrohacter and
Nitrosomonas. They state that these forms multiplied in solutions with initial
pH values of 13.0, although not when the initial pH was as high as 13.4. These
results, however, seem rather surpising because of the apparent lack of a toxicity
effect due to ammonium hydroxide which would be expected for these forms
under the culture conditions used. Other workers have failed to corroborate
the findings of Meek and Lipman for Nitrobader and Nitrosomonas. Kingsbury
(1954) has reported that the blue-green alga Plectonema nostocorum will grow
in solutions of Ludox (a DuPont 30 per cent SiOo solution) adjusted to an initial
pH of 13, however the growth in this case was apparently limited to the surface.
Salinity
The range of salt concentrations tolerated by microorganisms during growth
and reproduction is enormous. Kalinenko (1957) has shown that some hetero-
trophic bacteria will multiply in double distilled water. (The water in this
case contained only 70 /xg. of organic matter per liter.) On the upper side it is
known that the fungi Aspergillus oryzae and A. terricola will grow in 4.1 m
MgS04 , a concentration equivalent to about 500 g. of salt per liter of solution
(Johnson, 1923). HalophiUc bacteria in nature grow abundantly in salt
Hmans, saturated brines, and on animal hides dried with concentrated salt solu-
tions. Even the Dead Sea with its salinity of 280 to 320 per thousand and high
bromide concentration is not sterile. A small gram negative rod, a yeast-like
form, and a green filamentous form were all found to grow and reproduce in
Dead Sea water enriched with 1 per cent peptone (Wilkansky, 1936). Other
bacteria and algae were also present. Some of the bacteria failed to grow in
Vallentyne: Environmental Biophysics & Microbial Ubiquity 347
media containing less than 15 per cent salt. See Clifton (1958, p. 262) for a
summary of Volcani's study of the Dead Sea biota.
Solar evaporation ponds are often discolored by the growth of halophilic
bacteria and algae. According to Carpelan (cited by Gibor, 1956) photosyn-
thetic production rates in such environments are comparable to those in the
most productive parts of the oceans. Gibor (1956) has shown that the osmo-
tolerant brine flagellate, DunaUella salina, grows well in 10 X concentrated
artificial sea water. Some halophilic bacteria isolated from salt brines fail to
grow in salt solutions containing less than 16 per cent NaCl, and will survive
on dry crystals of salt obtained by the evaporation of brines (Browne, 1922).
According to Gibbons and Payne (1961) the most rapid growth rates of several
halophilic bacteria (Halobacterium spp. and Sarcina littoralis) occur in solutions
containing 20 to 25 per cent NaCl at temperatures in the range of 40° to 45° C.
ZoBell (1958) states that sulfate reducing bacteria grow naturally and can be
cultured in waters with salinities up to 300 per thousand.
Pressure
The effect of varying atmospheric pressure on the growth and reproduction
of microorganisms seems not to have been investigated in much detail. Strug-
hold (1961), however, passingly refers to the cultivation of soil bacteria under
an atmosphere with the composition and total pressure (0.1 Earth atmos.) of
that presumed to exist on Mars. The existence of barophilic bacteria in sub-
terranean deposits and deep sea sediments has been demonstrated by ZoBell
et al. Most organisms living in the surface regions of Earth fail to grow and
are killed by hydrostatic pressures of a few hundred atmospheres. In contrast
to these, barophilic bacteria isolated from the deep sea bottom can be cultured
only under hydrostatic pressures comparable to those in their natural environ-
ment, i.e., pressures of 1000 atmos. or more (ZoBell and Morita, 1956). The
viability of some barophiles is unaffected by alternate compression and decom-
pression between 1 and 1000 atmos. of hydrostatic pressure when applied 10
times within 10 minutes (ZoBell, 1958). ZoBell (personal communication) has
cultured deep sea bacteria under 1400 atmos. of hydrostatic pressure.
Water
Water is the most concentrated single molecule in protoplasm. Its depletion
can therefore be expected to restrict growth and reproduction. Most organ-
isms, microbes included, survive periods of extreme drought in dormant states,
often as spores. On the other hand, in the case of Pleurococcus vulgaris slightly
modified vegetative cells suffice to withstand prolonged drought (Fritch, 1922;
Fritch and Haines, 1923). According to Zeuch (1934) cell division of Pleurococ-
cus vulgaris can still occur at relative humidities of 68 per cent at 1° C, 55 per
cent at 10° C, and 48 per cent at 20° C. Aspergillus glaiicus is well known for
its growth on substrates where the activity of water (a„,) is as low as 0.65 to 0.70
(Scott, 1961). Kordyum and Bobchenko (1959) hold the opinion that many
microorganisms can actually use air as a habitat for growth and reproduction.
The growth of lichens on bare rock surfaces, bacteria and fungi in flour, and
many microorganisms in strongly saline media represent ecological instances of
348 Annals New York Academy of Sciences
growth in environments in which the chemical potential of water is low. Noth-
ing more than speculative attention has been given to the possibility of micro-
bial growth in nonaciueous media. It should not be forgotten, however, that
the water dependent metabolism of all living organisms that are known must
be at least to some extent the end result of selection on a water rich earth. It
is not known whether life could form on a planet on which the predominant
lifjuid was some other compound than water. One should also remember that
under aerobic conditions of metabolism water is one of the main excretory com-
pounds formed by living organisms. Mechanisms for the selective retention of
metabolically formed water might enable some organisms to persist and grow
in liquid media with low water contents.
Other Factors
In relation to natural radiations, direct sunlight is known to be lethal for
many microorganisms, but the effects probably result from dehydration and
high temperatures in most cases. ZoBell and McEwen (1935) were unable to
detect any lethal effect when marine bacteria were exposed in layers of water
greater than 5 mm. in thickness to full noon sunlight on a roof top in La JoUa,
Cahfornia. Two halophilic bacteria isolated by Browne (1922) withstood in-
definite exposure to "the brightest sunlight."
The effect of ultraviolet light on microorganisms has been studied by many
workers; however, most of the data refer to high dosages for short times. It
would be of much interest to know the maximal levels of continuous ultraviolet
radiation that can be tolerated by actively growing cultures. Although ultra-
violet light in high doses is harmful to all organisms, it must be remembered
that deleterious effects are much less pronounced above M)() m/x- than below for
equal energies of incident light (Meier, 1936). There is also a great variation
in the sensitivity of different microorganisms to ultraviolet light. Siliceous
tests of diatoms apparently afford no protection (Ursprung and Bloom, 1917).
Because the possibility of shielding and the well known photoreactivation phe-
nomenon, whereby the lethality of ultraviolet light is partly reversed by later
application of visible light, it is probably incorrect to assume, as many have
done, that an ozone free earth would necessarily be sterile.
The biological effects of gamma- and other types of ionizing radiations have
also been studied by many investigators. Single large doses have usually been
used. Populations of many microorganisms will survive single doses in the
range of 10'' r. (Shields et al., 1961). Saccharomyces cerevisiae has been cultured
under continuous exposure to 50 mr. per day of radium emanations (Maisin et
al., 1960), however, this is doubtlessly far below the maximal level that can be
tolerated. According to Prince (1960) a good place to look for radiation re-
sistant microorganisms would be in nuclear reactors. He states that it is
"common knowledge that some bacteria can adapt even to the water in a swim-
ming-pool-type nuclear reactor."
A few other case histories will serve to round out the picture that has been
presented. Some of these refer to survival rather than to growth and repro-
duction. The cases are as follows.
(1) The growth of several bacteria and fungi in concentrated CuS04 solu-
tions. The subject has been reviewed by Starkey and Waksman (1943).
Vallentyne: Environmental Biophysics & Microbial Ubiquity 349
(2) Bacteria that grow actively in solutions containing 1 g. of phenol per
liter (Putilina, 1959).
(3) Growth of the fungus Aspergillus in a 40 per cent solution of citric acid
(Johnson, 1923).
(4) An aerobic bacterium (Hydrogenomonas?) , originally isolated from sewage
sludge that shows poor growth in air, but develops well in an atmosphere con-
taining 20 per cent by volume O2 and 80 per cent by volume CO (Kistner, 1953).
(5) Heterotrophic growth of algae in lakes during the sunless arctic winter
(Rhodhe, 1955) and reproduction of algae in subterranean caves (Claus, 1955).
(6) The survival of some bacterial spores after 5 hours' immersion in non-
aqueous media at temperatures approaching 140° C. (Rodenbeck, 1932).
(7) The survival of bacterial and fungal spores, and even vegetative cells of
Mycobacterium smegmatis, after 5 days' exposure to ultrahigh vacuum at pres-
sures below 10~^ mm. of Hg. (Portner et al., 1961).
Table 1
Environmental Limits of Temperature, Eh (at the Prevailing pH), pH, Hydrostatic
Pressure, and Salinity for Growth and Reproduction
OF Microorganisms
Factor
Lower limit
Upper limit
Temperature
-18° C. (fungi, bacteria)
104° C. (sulfate reducing bacteria
under 1000 atmos. hydrostatic
pressure)
Eh
-450 mv. at pH 9.5 (sulfate re-
ducing bacteria)
4-850 mv. at pH 3 (iron bacteria)
pH
{Acontium velatum, fungus D,
Tbiobacillus iliiooxidans)
13 (?) (Plectonema nostocorum)
Hydrostatic pres-
Essentially
1400 Atmos. (deep sea and bac-
sure
teria)
Salinity
Double distilled water (heterotro-
Saturated brines {Dunaliella, halo-
phic bacteria)
philic bacteria, etc.)
(8) Survival of many microorganisms after prolonged exposure to tempera-
tures approaching absolute zero (Belehradek, 1935; Becquerel, 1950). Life
may, in some cases, be capable of almost infinite preservation under such condi-
tions.
One could multiply the examples at greater length, but those already pre-
sented suffice to make the point.
General Remarks
In TABLE 1 are summarized the ranges of temperature. Eh, pH, hydrostatic
pressure, and salinity that still permit growth and reproduction of one or more
microorganisms. It is not maintained that growth is anywhere near maximal
under the extreme conditions referred to, merely that it does occur. Selection
and mutation over long periods of time could doubtlessly result in a further
widening of the observed limits. It should also be stressed in this connection
that scientists are inchned to study single factors taken one at a time. When
two or more environmental factors show antagonistic effects, as is the case
with temperature and pressure, one can expect to find an increased tolerance
to each factor using combined action.
350 Annals New York Academy of Sciences
The microorganisms referred to in this paper are pecuhar in that they grow
in environments that are lethal to most other forms of Ufe. One can instruc-
tively reverse the point of view that has been taken here and ask why it is
that most organisms live under "common" conditions. The answer is, of
course, because life as a whole is selectively adapted to growth in common
environments. If the waters of the earth were predominantly acid, growth
at neutral pH values would be regarded as an oddity. Thus, the fact that
most living species conform physiologically and ecologically to average Earth
conditions should not be taken to indicate any inherent environmentally based
physicochemical conservatism of living matter. Adaptation has taken place.
Environments of the Earth that are sterile or nearly so mostly fall into one
of two categories: nonaqueous environments, and noncirculatory aqueous en-
vironments. The first category is so obviously restrictive in a biological sense
that it requires no further comment. The second refers to rock-enclosed waters
that do not readily enter into the hydrological cycle. Oil brines, for example,
that are perfectly sealed in place, seem to be sterile (Shturm, personal communi-
cation), and deeply buried wet sediments usually have low to negligible bac-
terial populations. In small enclosed systems extinction becomes increasingly
probable with time because of the small numbers of organisms involved, the
accumulation of metabolic waste products, and the general decrease in free
energy of the system as a function of time. Continuous circulation negates
these factors and in addition permits occasional injections of diverse micro-
organisms into new environments, to which they may become adapted over
many generations. Given the presence of circulating water, it seems rather
unlikely that any aqueous environment could remain indefinitely sterile over
geologically long periods of time. The powers of microbial reproduction and
variation are so immense in an evolutionary sense as to make this a virtual
impossibility. This assumes, of course, that some energy source is available
for metabolism in the environment concerned; but this is not a restrictive
limitation either biologically or geochemically.
Returning to the cjuestion of extraterrestrial life, the problem involved seems
not so much to be whether organisms could live elsewhere under conditions
that we would regard as unusual on Earth, as it is to account for the origin of
life itself. In relation to the possibility of life on Mars, for example, the ques-
tions should be of two types: (1) whether conditions there were ever favorable
for the origin or introduction of life; and (2) whether subsequent conditions
have been favorable for the persistence of such life as might have been formed.
The second question is far less critical at the present time than is the first.
To appreciate the potentiahties of adaptation one need only contemplate how
an Ordovician observer might have viewed the likelihood of birds flying in the
air, the possibility of an animal maintaining a temperature of 37° ± 1° C. for
virtually all of its lifespan over a period of 100 years, or the existence of plants
that trap and feed on animals. What can the leper know of the scorpion's
sting? And what does the blind man know of the firefly's light?
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THE INFLUENCE OF WATER CURRENTS ON
THE LIFE FUNCTIONS OF ALGAE*
John L. Blum
Canisius College, Bn_ffalo, N. Y.
Selective effects by the current. Of the many habitats on Earth which are
colonized and exploited by sessile organisms, those which are in contact with
a mass of air or water in relatively rapid movement are likely to constitute a
convenience or a necessity to the uptake and excretory systems of the organ-
ism but to represent, at the same time, a major threat to the organism's se-
curity. Metabolizing organisms as we know them are inhabitants of fluids.
These fluids when laden with small cjuantities of nutrients and motionless may
or may not be suitable for successful growth and reproduction. When the
fluid is in unidirectional or turbulent motion and the organism remains in place,
the possibilities for successful growth of many sessile organisms are greatly
enhanced, but security is likely to be threatened by factors like evaporation
or physical buffeting by the current, and by the molar agents which are flung
at the organism. Areas where surface or subsurface currents run in close
proximity to the bottom or other stable objects are successfully exploited by
numerous sessile marine plants and invertebrates; in fresh water currents ses-
sile invertebrates are relatively few and inconspicuous, but the algae have
successfully colonized what to most animals is a peculiarly dangerous spot, the
rapids of streams both large and small. So unicjue is this habitat that some
of the algae which are found in the rapid water habitat are seldom if ever found
anywhere else.
The present paper concerns algae which inhabit and are essentially limited
to fresh water currents, that is, algae which have moving water all around them
or in very close proximity; but inasmuch as the current has varied influences
as well on organisms which are in it only temporarily, I shall make occasional
mention of other river algae. The true current-inhabiting species are not
adequately described by the term "river algae" because the latter category
includes many forms which cannot attach and which are often unable to remain
in place in a strong current. Essentially all surface streams are inhabited by
some such forms, many of which are found as commonly or more commonly in
standing water.
From source to mouth a freshwater stream consists of alternating shallow
(rifHe) areas and pools. These respective habitats differ in many ways and
it is usual to find that each is inhabited by a distinctive assemblage of animals
and plants. Current rate is influenced by a number of well known variables;
in small streams these variables act so as to subject different but adjacent
points to quite different pressures. Such pressures are likely to fluctuate
greatly from moment to moment but minute differences in depth and pre-
sumably in average current rate between points distant by only a few milli-
meters on the stream floor make of each shallow area a mosaic of differing
* This study was aided in part by funds from the National Science Foundation (G-10898).
353
354 Annals New York Academy of Sciences
microhabitats whose existence and individuality is attested by striking differ-
ences in the algal populations which colonize them at certain seasons.
A primary influence of current on algae, therefore, is the exclusion of certain
species from pool areas or other places where current is minimal, or the en-
hancement of growth of such species in the most favorable, frequently the
fastest current. The fact that algae colonize so dangerous a habitat as flowing
water suggests that they can be provided some unique service by this habitat.
The relationship of algal photosynthesis and respiration to water movement
has been discussed by various investigators including Gessner (1937) and
Steeman-Nielsen (1944). Oxygen consumption in the dark and the photo-
synthetic rate are increased in moving water above the respective values for
standing water. More recently, respiratory rate and P uptake by Oedogonium
kurzii Zeller have been studied by Whitford (1961). Radioactive P uptake
in water moving at 18 cm. per second was found to be over 10 times that in
still water. He concludes that the cause for "inherent current demand" by
lotic organisms is the need for rapid exchange of materials with the water and
that the steep diffusion gradient in a current satisfies this demand.
This inherent current demand and the gradients involved may be of sig-
nificance to algae in two ways: for materials which are brought to the algae
by the current and for removal downstream of substances which might be
harmful. At least some algae are known to excrete substances which eventu-
ally retard their own growth rate. That such materials would be flushed
away from an alga growing in a current is evident, and may explain the limita-
tion of at least certain species to rapid water. It may also explain the high
cell density achieved by many current algae.
EJfecls of current on algal size or shape. Precisely how current influences the
structure of an individual algal cell or thallus has received relatively little
attention. Many benthic stream algae are so flexible that the current con-
tinually bends and twists them without visible damage or effect. Unlike a
tree which bends permanently under the influence of prevailing winds, there
is nothing about their structure which would even betray the usual direction
of the current if by some means the current were suddenly averted or brought
to a stop. The same is true of certain less flexible bottom-inhabiting forms.
The Phormidium-Audouinella-Schizothrix community which is known from
streams of the North Temperate Zone (Blum, 1956) does not, in the surface
topography of its crust, show any very evident polarity with respect to the
current. Others — and relatively few cases are known — show by the form or
orientation of their thallus the effects of unidirectional current as in the Phor-
midium community described by Wehrle (1942), a composite community of
Vaucheria and Plectonema described by WaUner (1934), or in the colonies of
Cocconeis growing on a vertical cylindric stake as described by Gessner (1955).
How the current controls the size of certain benthic algae is shown by work
done by Picken on the alga Rivularia. In regions of relatively rapid flow
thallus size was found to be proportional to the size of the stones to which the
thallus was attached. In slower water, however, thallus size was independent
of stone size. The bulk of this alga increases more rapidly than the area of
its attachment, and the current limits the maximal size of the thallus, either
Blum: Water Currents & Algae 355
tearing the thallus away from the stone, or transporting both stone and thallus
to a slower part of the stream (Picken, 1936).
Influence of the current on algal reproduction. In their reproduction current
algae take full advantage of the medium of dispersal which is at their doorstep.
It is commonly observed that many algae which colonize stream bottoms
achieve in certain seasons almost saturation coverage of available and favor-
able sites. Thanks to the mixing done by the current these algae are able to
introduce their reproductive units into what must be a very high percentage
of rock fissures, cracks, scratches, and roughened areas, into enough, at least,
of such depressions to permit subsequent growth from the colonizing cells to
cover close to 100 per cent of the available surface. In southern Michigan
streams which I investigated colonization of rock surfaces is very rapid, and
successful in very high percentages of the space available. The winter dom-
inant diatoms Gomphonema olivaceum and Diatoma vulgare, for example, achieve
good growth in winter on newly submerged rock surfaces in as little as 10 days.
Both of these forms were at the same time colonists and seasonal dominants,
no evidence being found of succession before the establishment of the com-
munities they represent. The period within which G. olivaceum colonized
bare rock surfaces extended from late November to early April, and coloniza-
tion seemed to be possible at any time within this period (Blum, 1954).
Evidence that planktonic forms reproduce as they are carried downstream
has been presented by various workers but there remains the suspicion that
much of the actual cell division occurs on the bottom and that the apparent
increase in phytoplankton downstream is largely the result of more extensive
nutrient beds there and of more dense populations of benthic individuals,
many of which rise every day into the plankton. I observed the vegetative
dissemination of Spirogyra and Oscillatoria communities on warm summer
days in the Saline River in southern Michigan. These communities were
especially characteristic of cjuiet shoals or bays. Here the algae remained
on the bottom in contact with nutrient-rich silt deposits, as masses of filaments
easily visible from a distance. The surface waters of such shoals or bays is
usually in slow circular movement set up by the main current of the stream,
which by-passes the shoal or the bay in a tangent to the circular current which
it produces there. At times of rapid photosynthesis, individual masses of the
algal filaments are detached and buoyed upward by trapped oxygen bubbles.
Once the algal mass has quit the floor of such a shoal, it is carried slowly along
in the eddying surface water. After moving for some time in this circular
manner it may eventually be picked up by the tangential current of the main
stream which removes it definitively from the shoal. As the algal mass travels
downstream, it disseminates live filaments along the way. The progress of
these filaments is arrested on obstructions or on new shoal areas or other sedi-
ments downstream, which in this way are themselves colonized. The elevation
of algal masses by entrapped bubbles can be observed from about noon until
about 2 to 3 p.m. on sunny days in summer and the movement downstream
of these floating masses can be observed throughout an entire afternoon.
The evolution of current-inhabiting algae. I believe the first attempt to clas-
sify the body types of current algae was made by Cedergren (1938). His
356
Annals New York Academy of Sciences
classification included 4 groups, namely (1) richly branched thalli; (2) long,
flexible cylinders, (3) spherical cushions, and (4) simplified platelike forms.
The second of these groups should probably be modified to include forms with
laciniate, reticular or lacunate bodies which float downstream from a point of
attachment; it should also be pointed out that certain algae with short un-
branched filaments, although they indeed qualify as cylinders, nevertheless
have a somewhat unique superficial form since, as in Vaucheria, they frequently
constitute a virtual turf but do not become interwoven to form massive skeins
as in the first group. If body form is a major criterion for these groups, at
Cladophora glomerata
£22^i-tli'.- v,.ii.?jV-t^:„'.-e
Figure 1. Cladophora glomerata. The illustration at bottom represents several algal
thalli X'2 attached to a portion of rock (stippled). The upjier drawings represent increasing
magnifications of small portions of the thallus.
least one other category should probably be added for forms with a rigid,
cylindrical, but pseudoparenchymatous body like Lemanea.
The first 2 groups as outlined by Cedergren can be summarized by the
qualification that they live in the current and permit water to run among their
filaments or at least on more than one side of the thallus. Hence they e.xpose
a large surface area directly to the surrounding water. A common example
of this type is Cladophora glomerata (L.) Kiitz. (figure 1). These groups can
be further subdivided into gelatinous and nongelatinous types. The gelat-
inous types in general have relatively small filament or trichome diameter.
The last 2 groups of Cedergren can be qualified by virtue of their position
mostly below the current — in other words they become a part of the stream
bottom. The current docs not flow among their filaments but only in their
^
^^-., .. "^
^^ ;, '?^
^
Gomphonema ottvaceum
Figure 2. GompJionema olivaceum. The illustration at the bottom represents several
soft thalli attached to and completely covering a rock. The mottled dark area represents
bare rock at a point where an algal thallus has been cut away. The upper drawings represent
successive magnifications of the area cut away. Insect larvae which feed on these diatoms are
shown within the algal mass.
^i-M^^ji'.^^ -^-x
l^ivularici sp
Figure 3. Rivularia sp. Stippled portion represents a rock to which several subspherical
colonies of Rivularia are attached. The upper drawings show increasing magnification of a
single colony or thallus. Calcium carbonate cr)stals are shown as they appear in the gelatin
between adjacent algal filaments.
357
358 Annals New York Academy of Sciences
vicinity. These include massive sheets which cover rocks in the current and
may extend partly into the current as in Phormidium spp.; parenchymatous
or pseudoparenchymatous collections of cells; soft, gelatinous masses that
move slightly in the current (some diatoms such as Gomphonema olivaceum
(Lyngb.), Kiitz. (figure 2); and firmer, spherical, or hemispheric masses which
are frequently gelatinous as in Rivularia spp. (figure 3). The gelatin in these
types serves to lubricate the alga-current interface and to reduce friction and
injury to the plant but it also serves to separate adjacent trichomes or filaments
and to keep, in many algae, a rather precise spatial relationship between fila-
ments as they lie in their intercellular material (figure 3).
When fresh water algae, generally, are compared and contrasted with marine
algae, the essential absence from the former of massive plant bodies, leathery
and foliose types which are so common in the marine Rhodophyta and Phaeo-
phyta is noteworthy. Although the Phaeophyta have proven generally un-
successful in fresh water and would not really be expected to produce such
plant forms in any event in fresh water, the same is not true of the Rhodophyta
or of the Chlorophyta. Nevertheless, the latter groups are not represented
in fresh water by forms more massive than Tuomeya, Lemanea, Chaetophora,
or Monostroma.
The evolution of fresh water algae has thus been successful largely for the
smaller, more delicate forms which are characteristic of standing water rather
than of currents. If we suppose that the rather specialized current algae have
evolved at least in part from their fresh water relatives that are tolerant of
standing water, it must be granted that their form has not been greatly modified
by the change in habitat.
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Cedergren, G. R. 1938. Reofila eller det rinnande vattnets algsamhallen. Svensk. Bot.
Tidskr. 32: 362-373.
Gessner, F. 1955. Hydrobotanik. Vol. 1. VEB Deutscher Verlag der Wissenschaften.
Berlin.
Lastochkin, D. 1945. Achievements in Soviet hydrobiology of continental waters. Ed.
G. E. Hutchinson. Ecology. 26: 320-331.
PiCKEN, L. E. R. 1936. Mechanical factors in the distribution of a blue-green alga, Rivularia
haematites. New Phytol. 35: 221-228.
Steeman-Nielsen, E. 1947. Photosynthesis of aquatic plants with special reference to
the carbon sources. Dansk Botan. Arkiv. 12: 1-71.
Wallner, J. 1934. Beitrag zur Kenntnis der Vaucheria-Tuffe. Zentr. Bakteriol. Parasi-
tenk. 2(90): 150.
Wehrle, E. 1942. Algen in Gebirgsbachen am Sudostrande des Schwarzwaldes. Beitr.
Naturk. Forsch. Oberrheingebiet. 7: 128-286. PI. 1-3.
Whitford, L. a. 1960. The current effect and growth of fresh-water algae. Trans. Am.
Microscop. Soc. 79: 302-309.
THE STRUCTURE OF DIATOM COMMUNITIES UNDER
VARYING ECOLOGICAL CONDITIONS
Ruth Patrick
Department of Limnology, Academy of Natural Sciences of Philadelphia,
Philadelphia, Pa.
During the preceding 15 years we at the Academy of Natural Sciences of
Philadelphia have spent a great deal of time studying the composition of diatom
communities in the eastern and southern United States. The findings from sec-
tions of streams characteristic of this area which have not been adversely
affected by pollution are discussed in this paper. To understand these com-
munities of diatoms not only the species which compose them but also the
sizes of the populations of these species must be known. This necessitates
collecting species from all types of habitats in the community. It also neces-
sitates counting sufiEicient specimens to determine most of the species compos-
ing the community. Obviously, when studying a community which has one
or two species with large numbers of individuals, many more specimens must
be counted to discover the species composed of small populations.
From our studies it is evident that 7000 to 8000 or more specimens usually
must be counted before a reliable picture of a diatom community can be ob-
tained if one wishes to compare the quantitative characteristics of commu-
nities. From TABLE 1 it is evident that only a small percentage of the species
composing the community are seen when only 200 or 500 specimens are counted
and approximately 50 to 75 per cent of the number of species are seen when
1000 specimens are counted when compared with the number seen when several
thousand specimens are counted. Similarly, the percentage of the population
composed of specimens of dominant species in some cases varies greatly when
based upon counts of a few hundred specimens as compared with counts of a
few thousand specimens. As seen in table 2 the numbers of species compos-
ing the diatom community remain fairly similar when similar segments of the
communities are analyzed if no serious change in the environment occurs. As
seen in table 3 the percentage of the population composed of dominant species
does not vary greatly for similar environments when similar segments of the
community are studied.
When the structure of these populations was plotted by representing the
number of species as the ordinate and the number of individuals composing
each species as the abscissa, the data approached the shape of a truncated
normal curve, figure 1. To determine what mathematical formula might
best express the results of these studies several formulae were tried (Patrick
et al., 1959) and the truncated normal curve provided a little better fit than
the other methods investigated. The use of a truncated normal curve to ex-
press the structure of communities of organisms has been supported by the
work of MacArthur el al.
By using this method we objectively compared similar segments of diatom
populations. For example, if enough specimens are counted and enough species
are identified to always place the mode in approximately the same interval,
359
360
Annals New York Academy of Sciences
a similar segment of the community will have been studied regardless of the
dominance of any species that may be present.
We have found in natural rivers which are relatively free from pollution the
communities are composed of many species with most of them having relatively
small populations. These findings support the theory set forth by Thiene-
mann (1939) that optimal environments support many species composed of
relatively small populations. Furthermore, the numbers of species do not
change greatly from season to season in the same area nor do they change very
much from area to area collected at the same time. For example, in table 2
Table 1
The Number of Species and the Percentage of the Specimens in Populations of
Dominant Species Observed when Varying Numbers
OF Specimens are Counted
River
Specimens counted
Number of species
Percentage of
dominance*
Wateree River, South Carolina,
September 22, 1961
200
558
1009
5970t
33
52
81
117
62.5
62.2
51.2
27.2
Assunpink Creek, New Jersey,
September 19, 1959
200
569
1219
12,5841
35
65
97
178
51.5
31.6
61.5
39.5
Potomac River, Maryland, Octo-
ber 18, 1960
206
558
1637
I7,911t
24
37
76
148
89.3
87.3
7t.S
62.2
Sabine River, Texas, October 18,
1960
211
511
1348
7369t
24
39
68
105
75.8
72.2
54.7
60.0
* The percentage of specimens counted composing the dominant species. A dominant
species is one that is represented by 1000 or more specimens when 5000 or more specimens are
counted.
t These are the number of specimens which had to be counted to place the mode in the
second interval when a truncated normal curve is constructed from the data.
are shown the data for these statements derived from studies of the Savannah
River.
When the numbers of species found in different natural soft water rivers, for
example the Savannah River, the Red Clay Creek, and the Wateree River,
are compared, they do not vary greatly. The total number of species for the
Savannah River (South Carolina) was 188; Red Clay Creek (Delaware), 145;
Wateree River (South Carolina), 181. Considering only those species repre-
sented by more than 6 specimens when 7000 or more specimens are counted,
we find Savannah River, 85; Wateree River, 89; Red Clay Creek, 76. The
reason that 6 or more .specimens have been used for estimating that a species
is established in a given area is that if a truncated normal curve is constructed
those species represented by 4 to 8 specimens will have better than a 50 per
Patrick: Structure of Diatom Communities
361
cent chancL- of not shifting their position in the curve (Preston, 1948) and,
therefore, will remain a part of the community.
However, if the kinds of species in similar sections of various rivers are ex-
amined, a great variation as to the kinds of species is seen as described by
Patrick (1961). Also, in studies of the same area of the Savannah River at
about the same season (late August, early September) of the year in different
years, only 34 per cent of the species were common to both studies. A sim-
ilar, but not as great, variation is seen when two different areas in the same
Table 2
Savannah River
Summary of Catherwood Diatometer Readings at Station 1
October 1953 to January 1958
Date
Specimen number
in modal interval
Oct. 1953
Jan. 1954
Apr. 1954
July 1954
Oct. 1954
Jan. 1955
.\l)r. 1955
July 1955
Oct. 1955
Jan. 1956
.Vpr. 1956
July 1956
Oct. 1956
Jan. 1957
.\pr. 1957
July 1957
"Oct. 1957
Jan. 1958
(Apr. 1954-1958 averages)
4-8
2-4
2-4
4-8
4-8
2-4
2-4
2-4
2-4
4-8
2-4
2-4
2-4
2 4
4-8
2-4
2-4
Species in mode
22
19
24
23
21
19
25
20
27
30
35
24
23
29
21
29
25
27
24
Species observed
150
151
169
153
142
132
165
132
171
185
215
147
149
177
132
181
157
152
151
Species in theo-
retical universe
178
181
200
193
168
166
221
180
253
229
252
185
206
233
185
203
232
212
194
Table 3
Dominant Species in Two Areas, Guadalupe River
Station 1
Station 2
9 Sept. 59
9 Sept. 59
Gom plionema affine var. insigne
1272
2850
G. parvulum
4346
2700
Naviciila sp.
1900
N . tripunclatus var. schizonemoides
30,634
23,750
Nitzschia palea
2400
Percentage of total count composed of domi-
95
90
nant species
362
Annals New York Academy of Sciences
river which have the same types of ecological habitats are studied at the same
time. The kinds of species in common are more variable than the numbers.
For example, Stations 1 and 6 on the Savannah River which are about 30 miles
apart were studied in June of 1960 and 187 species were identified at Station 1
and 54 per cent of these were found at Station 6. At Station 6146 species were
identitied and 75 per cent of these were found at Station 1. In October of 1960
when these two areas were studied the number of species at Station 1 was 184
and the number at Station 6 was 185. However, 75 per cent of the species at
Station 1 were at Station 6 and 75 per cent of the species at Station 6 were at
Station 1.
This same principle as to similarity of numbers of species but differences in
kinds of species also holds for the hard water rivers we have studied. Often
the numbers of species are slightly less in natural hard water rivers than in
40 r
INDIVIDUALS = 1-2 2-4 4-8 8-|6 16-32 32-64 64" 128" 256" 512" 1024- 2048-4096" 8192-16384-32768-
128 256 512 1024 2048 4096 8192 16384 32768 65536
INTERVALS =0 1 2 3 4 5 6 7 8 9 1 1 1 12 13 14 15 16
Figure 1. Ridley Creek, Pennsylvania
soft water rivers. For example, in the Potomac River, a hard water river, from
April of 1959 to October of 1960 in one area studied the observed species varied
from 130 to 148 (average 144) as contrasted with a variation from 118 to 185
(average 161) in the Savannah River which is a soft water river, over a similar
period of time.
In brackish waters such as the estuary of the York River the numbers of
species composing a diatom community sometimes are a little less than in a
soft water river. From November of 1956 to May of 1959 the number of
observed species varied from 108 to 147 (average, 130). However, in all of
these three types of rivers — soft, hard, and brackish water — the communities
are made up of many species most of which have relatively small populations
if the rivers are natural and not polluted.
A different picture is found when the structure of diatom communities in
dystrophic streams is examined. In these there is a restricted diatom flora
which can live in these naturally acid streams high in humates. They are
species largely confined to the genera Eunotia and Frustidia and certain species
Patrick: Structure of Diatom Communities
363
of genera such as Pinnularia, Adinella, Anomoeoneis and Surirella. Thus, we
have a community composed of fewer species with populations that are much
more variable in size (figure 2). The truncated normal curve representing
the structure of the community has a much lower mode, fewer observed spe-
40
35
<" 30
UJ
in
^ 20
o
a 15
m
2 10
o
INDIVIDUALS
INTERVALS
1-2 2-4 4-8 8-16 16-32 32-64 64" 128- 256" 512" 1024- 2048-4096" 8192-16384-32768-
128 256 512 1024 2048 4096 B 1 92 16384 32768 65536
= 1
10 II
12
13 14 15 16
Figure 2. Egg Harbor River, N.J.
40
35
<^ 30
UJ
o
Q. 25
"- 20
o
Q: I 5
UJ
CQ
2 10
INDIVIDUALS = 1-2 2-4 4-8 8-16 16-32 32-64 64" 128" 256- 512" 1024- 2048-4096" 8192-16384-32768-
iinuiviuuml;^ l^g 255 512 1024 2048 4096 8192 16384 32768 65536
INTERVALS =0
10 II 12 13 14
15
Figure 3. Back River, Maryland
cies, a much greater a^, which means more variability in the sizes of the diatom
populations, and covers more intervals, because a few species have very large
populations.
This is the type of curve often found for the structure of diatom populations
which are subjected to pollution (figure 3). In both cases one or more eco-
364 Annals New York Academy of Sciences
logical factors have operated to greatly limit the numbers of species which
can survive in these particular kinds of ecological conditions.
A few studies which we have done indicate that in springs in which the en-
vironment is fairly constant the numbers of species composing a diatom com-
munity may be much less than in the very variable environment of an eutrophic
or mesotrophic, natural river. It seems that it is the highly variable, yet con-
tinuously favorable, environment of natural rivers of these types that is largely
responsible for the great diversity of species that make up these communities.
The fact that the numbers of species remain fairly similar, although the kinds
of species vary considerably, suggests that there are a similar number of niches
for diatom species in ecologically similar natural areas and more species are
available than there are niches for them. Thus, each niche is occupied by a
different species. The lack of similarity in kinds of species present is probably
in part due to the highly variable environment in a natural river and the avail-
ability of species which have their best development in different variations of
the environment. Because diatoms have very rapid reproduction under favor-
able conditions the populations of certain species can quickly increase, whereas
populations of other species decrease beyond the limits of collectability or dis-
appear.
Another important consideration in the study of diatom communities is the
kinds of species composing the communities. By careful consideration of the
kinds of species associated together, a qualitative evaluation of many of the
characteristics of the environment can be made. However, because of a lack
of data as to the complete physiological requirements of any species in nature,
it is very dangerous to say that the lack of any species indicates that the spe-
cific characteristic of the environment under consideration is not there, be-
cause the lack of any factor essential for the life of an organism may eliminate
it, although all other factors of the environment may be favorable to it. Also,
it is hazardous to use changes in the population sizes of specific species as a
basis for saying that the quantitative nature of a given environmental factor
has changed. For example, we studied two areas in the Guadalupe River
which were not over 500 yards apart. The structural environmental charac-
teristics of the two areas were very similar. Because no tributaries or pollu-
tion entered the river between these two areas during the time of this study,
the characteristics of the water were very similar. This was substantiated by
chemical analyses. When similar segments of the communities of diatoms
were studied the percentages of the community composed of specimens of dom-
inant species were very similar, 95 and 90 per cent, respectively (table 3).
However, the sizes of the populations of the dominant .species and the kinds
of species varied considerably. At Station 2, the population size of Gompho-
nema affinis var. insigue was twice that found at Station 1. The population
of Gomphonema parvulum was 38 per cent larger at Station 1 than at Station 2.
At Station 2 Navicula sp. had a population of 1900 specimens and Nitzschia
palea had a population of 2400 specimens yet neither of these species were
present at Station 1. Only one of the dominant species, .\avicula Iripunciala
var. schizonemoides had populations of similar size at the two stations.
It is only as a result of thorough and continuous study of an environment
and the species living in it that one can venture to describe the quantitative
Patrick: Structure of Diatom Communities 365
changes in the natural environment of a river by changes in the quantitative
abundance of specific kinds of species.
In conclusion, our studies have shown that diatom communities can be best
characterized by consideration of the kinds of species, the numbers of species,
and the relative sizes of the populations of the species that comprise the com-
munity. An excellent way to consider the relative sizes of the populations of
all the species studied is by the construction of a truncated normal curve. The
presence of certain kinds of species may tell us much as to the quaUtative char-
acteristics of an environment. The best means for determining quantitative
shifts in the environment is by considering the shift in numbers of species and
the ratio of the number of species with small populations to those with large
populations. Perhaps the reasons that the numbers of species do not vary
greatly is that there are similar numbers of niches for species occupancy in
ecologically similar types of streams. Also, at any one time there are prob-
ably more species available to inhabit natural eutrophic or mesotrophic areas of
streams than there are niches available for species occupancy, thus, each niche
is filled with a different species. The reasons that the kinds of species vary con-
siderably in streams of these types are the continually varying yet favorable en-
vironment; the availability of species which have their best development in
different conditions of the environment; and the ability of diatom populations
to quickly expand or contract with changes in the environment.
References
Patrick, R. 1961. A study of the numbers and kinds of species found in rivers in eastern
United States. Acad. Nat. Sci. Phila. 113(10): 215-258.
Patrick, R., M. H. Hohn & J. H. Wallace. 1954. A new method for determining the
pattern of the diatom flora. Acad. Nat. Sci. Phila. No. 259.
Preston, F. W. 1948. The commonness, and rarity, of species. Ecology. 39: 254-283.
Thienemann, a. 1939. Grundzuge einer allgemeinen Okologie. Arch. Hydrobiol. 35;
267-285.
CELL STRUCTURE AND ENVIRONMENT
B. J. Cholnoky
Council for Scientific and Industrial Research, National Institute for Water
Research, Pretoria, South Africa
During the so-called classical period of the study of cells, algal cells were
frequently used in cytological investigations. The great discoveries of Weis-
mann, Biitschli, Ramon y Cajal, and Flemming were made possible with fixed
and stained objects, but as the equipment and the microscopical methods then
available were unsuitable for living and especially for unstained objects, living
algal cells were only rarely used for cytological purposes. Listead, methods
were developed which were supposed to leave the fixed protoplasm unaltered,
and differential staining procedures were used which rendered visible to the
human eye structures which, it was believed, occurred in the living cell. Vio-
lent, but barely scientific controversy which often led to personal insults and
verbal battles ensued, during which the living cell was more and more for-
gotten. This was also due to the exemplary, or not so exemplary preparations
which were made to support sophisticated hypotheses which arose from staining
techniques. These techniques often resulted in works of art rather than
impressions of the living cell.
No matter how perfectly fixation for specific purposes has been accomplished,
the living constituents of the cell must necessarily undergo alteration when
fixed (otherwise they would continue to live), and minute changes in the
protoplasm due to environmental factors cannot, therefore, be detected. The
difficulties were increased because colloidal physics had not yet been developed,
and because the changes were generally of a submicroscopical nature. Investi-
gations of these changes in the living protoplasm, therefore, were only later
tackled.
Seen against this background, the accidental discovery by Benecke (1901)
of the reduction in size and the ultimate disappearance of the chromatophores
in Niizschia putrida (Synedra hyalina Provazek), by means of which he sought
to show a clear connexion between the size of the chromatophores and the
pollution (as he called it) of coastal waters, was surprising and also important.
The approach adopted by Benecke was, however, soon abandoned, and the
observation of living algal cells continued only for the purpose of systematics
and morphology. The observed structures, cell components, etc., were re-
garded as something rigid and unchangeable, or, as we would express it today,
genotypically determined. Consequently, the results of these observations
were used only as characteristics: they were used, and frequently misused, for
describing species, and as a result, plant physiologists and the early ecologists
did not want to do anything with them.
On the other hand, investigations into the causes of adaptation of algae have
begun. These investigations at first pursued a course which was of importance
to Man but not to the algae. Apart from the vague conjectures of the 19th
century, which were mainly concerned with descriptions of the habitat or with
plant geography, the first ecological study of algae was the so-called Saprobic
366
Cholnoky: Cell Structure and Environment 367
System of Kolkwitz. This was, however, not based upon precise observations
or experiments but on an untenable hypothesis (Kolkwitz and Marsson, 1902,
1908, 1909; Kolkwitz, 1950; Liebmann, 1951, etc.). As a basis for the hy-
pothesis it was assumed that the substances (of which no one then bothered to
ascertain the chemical nature) which were responsible for the pollution of
waters could be removed first by reduction and subsec^uently by oxidation.
The so-called reduction phase was called polysaprobic and the oxidation phase
mesosaprobic. Naumann (1932), however, had shown that this hypothesis
was untenable: he demonstrated the nonexistence of a reduction phase and
consequently it was found impossible to judge the quality of waters, let alone
to purify them, on Kolkwitz principles. Under these circumstances it was not
surprising that cytologists found no reason to study protoplasmatic changes
attributable to "pollution" according to Saprobic System concepts.
It was only much later that greater stimulus was given by the ecological
work of Kolbe (1927, 1932), who showed that certain diatom species are better
adapted to a high salt concentration than others. In his opinion it was the
chloride ion of sodium chloride which was responsible for the phenomenon of
adaptation. He also attempted to prove that in the absence of the afore-
mentioned ions (oligohalobic conditions), a moderate concentration (meso-
halobic conditions), or a high concentration (polyhalobic conditions) simulating
salt water was responsible for the distribution and adaptation of certain
species.
Almost at the same time it had been shown (Cholnoky, 1929) that the diatom
associations of the soda lakes of Hungary (which contain carbonates and not
chlorides) were identical with those of Kolbe's mesohalobic waters of Speren-
berg. From these observations it was possible to deduce the fact that pri-
marily it was not the chemical composition of the salt molecules but rather
their concentration which was responsible for the halobic phenomena. In
other words, it was not the chloride ion at all, but the prevailing osmotic
pressure, i.e., molarity. It also became clear very soon after that the prevail-
ing osmotic pressure in the Hungarian soda lakes can be as high as, or even
higher than, that of the sea (a concentration of 2 mol. sodium carbonate is not
exceptional in the lakes), and that these high values do not necessarily give
rise to the growth of typical marine algae. It was recognized that it was not
the absolute salt content or molarity, but the variation of osmotic pressure
which produces the necessary conditions for the so-called brackish water
species; or put more precisely, the ability to withstand the molarity variations
gives advantages to these brackish water species.
Because the variation of osmotic pressure mainly affects the protoplasm of
the brackish water organisms, it was clear that protoplasmic differences must
exist, and that these differences could only be discovered by studying the
living cells.
After the classical studies of de Vries (1871, 1885), one could assume, as a
matter of course, that an increase in osmotic pressure would cause plasmolysis,
and also that plasmolysis could be neutralized by permeance to, or active uptake
of, the plasmolyzing substances. Hofler showed (1918, and more accurate
concept 1931) that de Vries's concept of semipermeability was untenable.
368 Annals New York Academy of Sciences
Thus, the causes of adaptation to the conditions of brackish water, i.e., the
considerable variation of osmotic pressure, were to be found in plasmolysis
which must necessarily and at least temporarily occur.
The experiments which were undertaken (Cholnoky, 1928a, 19306, 1932)
showed that apart from certain fundamental morphological features which
seem to be genotypically determined, and which are characteristic for the
various algal groups during plasmolysis, there are large morphological differ-
ences between the plasmolyses of freshwater and brackish water species.
Among other things, the distribution of viscosity of the protoplasmic colloids
is characteristic for the species. It was ecjually evident that the brackish water
species poses a high degree of permeabihty in regard to the salts in solution
in their habitat. In Hungary the high degree of permeability is confined to
the carbonates, and only to a lesser degree to the chlorides, although the cells
show only slight or nonpermeability to such plasmolytica as nitrates, sugar,
urea, etc. The same species when found along the South African coast are
mostly permeable to the chlorides, whereas when they occur in the South
African sodium carbonate rich waters of the Jakkals River for instance, the
same permeability to carbonates as in Hungary was observed.
These observations forced me to the conclusion that owing to the high degree
of permeability of the protoplasts, the brackish water species are ecologically
favored. It follows that if such an assumption were true, there would be far-
reaching colloid-physical effects. The permeating salt molecules would, under
normal circumstances, alter the electrical charge of the mono- or polymolecular
micelles and thus be the cause of coacervation and ensuing coagulation and
death. The protoplasm of brackish water species appears to be extremely well
protected against such alterations of electrical charge, and further study will
probably provide important information on the submicroscopical structure of
the protoplasm.
Typical freshwater algae which were treated with a plasmolyticum consisting
of some partly evaporated brackish water from another habitat speedily died
as a result of permeation (Cholnoky, 19306, 1931<z, 19316). Others, however,
remained plasmolysed for an extraordinarily long time without showing any
sign of protoplasmatic damage and without the least trace of permeation.
Other chemical compounds for which the protoplasts of the investigated species
were more or less permeable, acted immediately on permeation as poisons,
during which it was seen that the gradual destruction of the protoplasm indi-
cated an unecjual resistance of the protoplasmic components of the cell (Chol-
noky, 1953).
Hofler (1951) obtained similar results and found that Na2C03 acted in a
specific manner on the diverse species of bog algae (Desmidiales). The cells
of some species were slightly permeable, others were barely permeable. The
nonpermeable ones were able to survive plasmolysis lasting several days with-
out sustaining any visible protoplasmatic damage. (It was possible com-
pletely to deplasmoly.se Euastrum after pla.smolysis lasting 72 hours.) I was
able to confirm that certain brackish water algae were even more resistent to
plasmolysis than Euastrum. These species built a superficial inner cell wall
on the site of positive plasmolysis; i.e., at those places where the protoplasm
body had withdrawn from the original cell wall. As a result of the possible
Cholnoky: Cell Structure and Environment 369
repetition of ihis operation, the formation of the so-called inner cell wall is
explained: it arises from an increase in the osmotic pressure of the environment
during the gradual drying up of the waters in the summer (Cholnoky, 19286,
1954; Kamija, 1938; Kuster-Winkelmann, 1949). These phenomena are clear
proof that the otherwise generally fatal plasmolysis does not alter, or alters
only to a Hmited extent, the structure of the protoplasmic colloids in the brack-
ish water species which are adapted to variations in osmotic pressure. These
species not only survive the ordeal but actually build a cell wall during the
process — a procedure which would be hardly thinkable if the metabolism had
been upset.
The repeated reductions in pressure due to variations of osmotic pressure do
not occur without having any side effects. With brackish water diatoms the
reductions in pressure are the triggers for sexual reproduction (Cholnoky,
19296), because the dilution caused by the culture medium always gives rise,
in the diatoms, to sexual propagation, i.e., auxospore formation.
A sudden dilution causes plasmorhexis in brackish water species. This is
evidence for their having comparatively many free salt molecules in the water
mantles of the micelles of their colloids, which can cause an osmotic pressure
(Cholnoky 1928a). Such phenomena do not occur in brackish water species
if the dilution is carefully made. But with marine species, a dilution, no
matter how carefully made, causes plasmorhexis and death. This can be
accepted as proof that the salts causing isotonia, are indispensable to the proto-
plasmic colloids, and are structurally part of, and inseparable from, the micelles.
But further experiments will be necessary to be able to evaluate the position
fully.
Without further experiments it will be equally impossible to explain the
mechanism of the phenomena which Lenk (1953) called Seasonal Variation of
Permeability. Variation of permeability without change of protoplasmic
structure is unthinkable. Consequently it can be assumed that submicro-
scopical protoplasmic structure is also subject to seasonal variation which can
only be due to adaptation to altered conditions of the habitat.
As I have already suggested, the behavior of freshwater algae which have
been killed by the permeation of salt molecules, indicates that they undergo
coacervation and lethal coagulation due to the penetration of the molecules.
These protoplasmic changes are a kind of a poison effect and leads from a study
of adaptation to the important study of resistance (Biebl, 1937, 1952). It
would, however, be inappropriate here to discuss fully all of the hitherto known
cytological resistance phenomena.
From the point of view of cytophysiology, a study of the poisonous effects of
salts and the resulting cytomorphological changes (which are often submicro-
scopical) is all the more important, as far reaching deductions regarding
adaptation phenomena will be possible. The studies on Melosira areiiaria
(Cholnoky, 1934) may be regarded as a beginning; and subsequent work on
cellular changes in other species and other algal groups (Ulothrix, Oedogonium,
Zygnemales, Desmidiales, and Siphonocladiales) led to important ecological
and cytological regularities being discovered. The notes, manuscripts, and
data, however, remained unpublished as they were lost at the end of the war.
The poisonous effects of some salts {e.g., sodium carbonate) could only be
370 Annals New York Academy of Sciences
characterized if the cytomorphological changes which they caused could be
compared with the poisonous effects of other substances. For this purpose
cuhures were utiUzed of which the culture fluids were displaced by cocaine and
colchicine, both of which are known to be cell toxic to a high degree. The
effect of cocaine on Cladophora (Cholnoky, 1930a) showed that this alga was
able to tolerate appreciable concentrations and that it can react in a very
characteristic manner. Without any microscopically visible protoplasmatic
changes occurring, resting stages developed, which were independent of the
seasons, and appeared to be completely resistant to cocaine so that when re-
moved to a normal habitat {i.e., cocaine free) they were able to germinate.
That these observations remained comparatively unknown, may be due to the
title of the paper having been arbitrarily changed by the editor of the journal
to which the paper was sent. The observations made may explain how algae
are able to survive temporary poisoning, as a result, for example, of industrial
effluents.
As is well known, colchicine affects the development of the spindle during
nuclear division and is, therefore, often used for obtaining polyploids. This
substance was also used for culture experiments. Surprisingly, only a high
concentration of colchicine (10 ppm) resulted in damage to the nuclear division,
but no polyploids were obtained. With Cladophora the number of nuclei in
the polyenergid cells was reduced. With Spirogyra, etc. pseudosexual condi-
tions quickly developed which often became lethal after only a lapse of several
weeks. The observed phenomena may explain why certain industrial wastes
produce no poisoning of the cocaine type (certain cells become impermeable to
poisons), but many abnormalities instead. The results of this series of experi-
ments were also entirely lost owing to the war, and as no further opportunity
to repeat them has been given me, it is up to some other researcher to undertake
this work. Nevertheless, they do seem to elucidate the effects of the waste
products produced by human activities as far as the terms "pollution" and
"poisoning" of natural waters are concerned.
I am familiar with only a few of the cytological effects of other poisons:
among these are the studies on aluminium salts and "cramp" plasmolysis
(Weber, 1924, 1933; Hofler, 1958) which clearly show that the salts have ren-
dered impossible the functions of the investigated cells through colloidal
changes etc., and very probably also through interference with the electrical
charge of the micelles. Although the quoted papers do not mention the colloid-
physical significance of the phenomena, it seems to me that they must be due
to coacervation and coagulation.
Colchicine as well as cocaine cause radical changes in the structure of the
protoplasm which are to a certain extent discernible by experiment, but the
mechanism of the effects of poisoning can be better seen microscopically if the
poisons can be seen or can be made to be seen. When the first algal investiga-
tions were started, the results of the experiments which had been made with
the cells of the higher plants led one to suspect that the process known to the
workers during the classical period of cytology as vital staining was actually
a microscopical manifestation of poisoning and destruction of the cells. It was,
therefore, possible without further ado to use stains which were formerly re-
garded as harmless, i.e., which did not kill the protoplasm suddenly.
Cholnoky: Cell Structure and Environment 371
As this exposition is mainly concerned with the efifects of the environment
on the structure of the protoplasm, I shall have to omit a detailed description
of what is known about the general principles of stain uptake and storage or
changes in the stain molecules, e.g., ionization in the cell or its environment.
It would also go beyond the scope of this paper to draw attention to the present
state of our knowledge derived from investigations with the fluorescence micro-
scope. As far as I am aware, those studies have hitherto only been made with
material divorced from its natural habitat, and have, in many cases, degen-
erated merely into a study of stains, without reference to colloidal structure
or the changes it undergoes. Such work often led Biitschli el al., into fruitless
hypothetical discussions.
This scarcely scientific approach is regrettable because even the first experi-
ments on stain uptake in algal cells (Cholnoky, 1934, 1935a, 1935&, 1935c)
showed that uptake and storage of the stain molecules, or the ion gradients in
the protoplasm was far reachingly dependent on the conditions under which
the algae lived before the experiments. When stained with methylene blue or
neutral red, the disassociation of the stain molecules remained dependent upon
the conditions of the culture before the staining experiments were done. Also
in those cases in which the stain fluid (unlike the culture fluid) possessed con-
stant physicochemical characteristics {e.g., stains dissolved in distilled water,
buffer solutions or plasmolytica) the effects on the protoplasm of increased
osmotic pressure, pH, and light conditions could be clearly proved.
The environmental conditions before the staining experiments generally hav-
ing remained neglected; this explains why so many apparently contradictory
results were obtained. The use of fluorochromes increased still further the
complexity of an already complicated position, as conclusions were drawn re-
lating to the storage of stain molecules and ions which incorporated many hypo-
thetical assumptions, such as "full" and "empty" ceU-sap (Hofler and Schindler,
1955), which did not attempt to reconcile observed facts with the environmental
factors under which the algal cells were living before the experiments.
This change of concept became apparent as preliminary work (Cholnoky and
Hofler, 1950) had already been done which went so far as to enable one to dis-
tinguish between the cytological behavior of species (Loub, 1951).
Regarding Loub (1951), it should be remembered that his material came from
ecologically dissimilar environments. After arrival in the laboratory they were
rough cultured and only examined after a more or less lengthy period. Apart
from the fact that the culture conditions were uncontrolled in the rough culture,
Loub did not investigate the natural conditions of the habitat. By his method
he was able to examine only adapted associations. He thus lost the oppor-
tunity to investigate the protoplasmic changes caused by ecological factors.
It will be clear from what has been said that most protoplasmic experiments
(such as plasmolysis and staining) were done without reference to the condi-
tions in which the algae lived in nature or in cultures. Although the cytological
results obtained are of very great value, it is indispensable that the methods
so far used should be thoroughly changed. Ecological studies have shown on
the one hand that not only the conditions of life prevaihng at the time of the
experiments but also their fluctuations must affect protoplasmic structure. On
the other hand, it now seems certain that Naumann's trophic conditions of the
372 Annals New York Academy of Sciences
waters (1932) play a much greater part in cell protoplasm than was formerly
believed. All future experiments must, therefore, take place under rigid control
of the culture conditions. Only in this way shall we discover protoplasmic
adaptation phenomena.
We shall first have to consider the possible effects of changes of pH and the
nitrogen content of waters, the latter having a direct bearing on trophic condi-
tions enabling one to distinguish between autotrophic and heterotrophic algae
(Algeus, 1946; Fogg, 1953; Saubert, 1957).
It seems obvious, finally, that the permeability and uptake of dissolved com-
pounds depends principally upon the structure of the protoplasm, so that one
can no longer think in terms of a specific filter system. Such hypothetical sys-
tems are, however, still accepted by some, although Seifriz (1936) has indicated
that the permeation of the whole protoplasm was responsible.
The correctness of this concept was confirmed by later experiments (Chol-
noky, 1952a, 19526; Hofler, 1959). On this basis, it seems to me highly proba-
ble that the structure of the protoplasm (after obligatory or optional nutrition
of the algal cells) is subject to changes which are also necessarily manifest in
the uptake of stain molecules. As the protoplasm of the purely autotrophic
algae must be adapted to small molecules and even ions, its microstructure
must be very different from that of the nitrogen heterotrophic species, the
protoplasm of which can take up amino acids or even bigger molecules (protein
particles, amino acid groups). These differences in protoplasmic structure,
which are due to the nutritional requirements of the cell and must also be mani-
fest in the uptake and storage of such substances as stains, seem to me so prob-
able that I am presently engaged in appropriate culture experiments. These
experiments will include the uptake of stains and fluorochromes in algae of the
same species which have been given different nutrients and also with algae
which are genotypically different for a study of their metabolism.
Owing to circumstances beyond my control, these experiments have just be-
gun. It has, however, been supposed that the uptake of dissolved substances
represents an active function on the part of the protoplasm, i.e., that it must
be a dynamic process, and not one influenced by static structures such as lamel-
lae or filters. That is why it is hardly likely that the results of these experi-
ments will ever be reconcilable with the static concepts of such researchers as
Frey-Wyssling (1955). Electron microscopical observations cannot be re-
garded as a basis of research on the living substances concerned with the uptake
of dissolved molecules that Frey-Wyssling called "Grundplasma".
I would like to recall what I said when I referred to the classical period of
cytology. The fixing and staining procedures then used could not lead to a
knowledge of protoplasmic structure, let alone changes due to physiological
causes. Electron microscopy must of necessity use similar, if more refined,
methods, as it is technically impossible to study living protoplasm with this
kind of microscope. The images obtained with the electron microscope are
only of static structural elements, and not of dynamic functions and changes in
the protoplasm. More succinctly, fixed protoplasm under the electron micro-
scope is at least partially an artificial product, as otherwise it would continue to
live unchanged.
Cholnoky: Cell Structure and Environment 373
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etc. Botan. Notiser. 1946: 129.
Benecke, W. 1901. tJber farblose Diatomeen der Kieler Fohrde. Pringsheim's Jahrb.
wiss. Botan. 35: 535.
BiEBL, R. 1937. Okologische und zellphvsiologische Studien an Rotalgen der englischen
Sudkiiste. Beih. Bot. Centr, Abt. A. 57: 381.
BiEBL, R. 1952. Ecological and non-environmental constitutional resistance of protoplasts
of marine algae. J. Marine Biol. Assoc. V. K. 31: 307.
Cholnoky, B. J. 1928a. Uber die Wirkung von hyper- und hypotonischen Losungen auf
einige Diatomeen. Intern. Rev. ges. Hydrobioi. Hydrog. 19: 452.
Cholnoky, B. J. 1928i. Uber mehrfache Schalenbil'dungen bei .\nomoenoneis sculpta.
Hedwigia. 68: 297.
Cholnoky, B. J. 1929a. Adnotationes criticae ad floram Bacillariearum Hungariae.
TV. Floristisch-okologische Untersuchungen in den siidlichen Teilen der ungarischen
Tiefebene (Alfold). Magyar Botan. Lapok. 1929: 100.
Cholnoky, B. J. 1930a. Die Dauerorgane von Ciadophora glomerata. Z. wiss. Botan.
22: 545.
Cholnoky, B. J. 19306. Untersuchungen iiber den Plasmolyse-Ort der Algenzellen 1 u.
2. Protoplasma. 11: 278.
Cholnoky, B. J. 1931a. Untersuchungen iiber den Plasmolyse-Ort der Algenzellen. III.
Die Plasmoiyse der ruhenden Zeilen der fadenbildenden Conjugaten. Protoplasma. 12:
321.
Cholnoky, B. J. 19316. Untersuchungen uber den Plasmolyse-Ort der Algenzellen. IV.
Die Plasmoiyse der Gattung Oedogonium. Protoplasma. 12: 510.
Cholnoky, B. J. 1932. Neue Beitrage zur Kenntnis der Plasmoiyse der Diatomeen.
Intern. Rev. ges. Hvdrobiol. Hydrog. 27: 306.
Cholnoky, J. B. 1934.' Plasmoiyse und Lebendfarbung bei Melosira. Protoplasma. 22:
161.
Cholnoky, B. J. 1935a. Protoplasmatische Untersuchungen durch Lebendfarbung und
Plasmoiyse. Mathematikai es Termeszettudomanyi Ertesito. Akad. Wiss. Budapest.
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Cholnoky, B. J. 19356. Farbstoffaufnahme und Farbstoffspeicherung lebender Zeilen
pennater Diatomeen. Osterr. Botan. Z. 84: 91.
Cholnoky, B. J. 1935c. Zur Kenntnis der Cyanophytenzelle. Protoplasma. 28: 524.
Cholnoky, B. J. 1952a. Beobachtungen uber die Wirkung der Kalilauge auf das Proto-
plasma. Protoplasma. 41: 57.
Cholnoky, B. J. 19526. Fin Beitrag zur Kenntnis des Plasmalemmas. Ber. Deut. Botan.
Ges. 65: 369.
Cholnoky, B. J. 1953. Fin Beitrag zur Kenntnis der Oedogonium-Zelle. Osterr. Botan.
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Cholnoky, B. J. & K. Hofler. 1950. Vergleichende Vitalfiirbungsversuche an Hoch-
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DE Vries, H. 1871. Sur le permeabilite du protoplasme de betteraves rouges. Arch.
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Botan. 16: 465.
Fogg, G. E. 1953. The Metabolism of Algae. Methuen's Monographs on Biological
Subjects, I, 1. Catalogue No. 4122/U. London.
Frey-Wyssling, a. 1955. Die submikroskopische Struktur des Cytoplasmas. Proto-
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Hofler, K. 1918. Permeabihtiitsbestimmung nach der plasmometrischen Methode. Ber.
Deut. Botan. Ges. 36: 414.
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Deut. Botan. Ges. 49: 79.
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49: 248.
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KoLKWiTz, R. & M. Marsson. 1902. GrundsJitze fiir die biologische Beurteilung des
Wassers nach seiner Fauna und Flora. Kleine Mittcilungen Kgl. Priifungsanstalt f.
VVasserversorgung u. Abwasserbeseitigung. 1.
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Math.-Natwiss. Kl., Abt. I. 162: 235.
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R. Oldenbourg. Miinchen.
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Seifriz, W. 1936. Protoplasm. McGraw-Hill Book Co., Inc. New York and London.
Weber, F. 1924. Krampfplasmolyse bei Spirog>ra. Arch. ges. Physiol., Pfliigers. 206: 629.
Weber, F. 1933. Aluminiumsalz-Wirkung und Plasmolyse-Permeabilitat. Protoplasma.
17:471.
THE MORPHOLOGY OF PPLO AND BACTERIAL L FORMS*
Louis Dienes
Departments of Medicine and Bacteriology, Massachusetts General Hospital, and the
Robert W . Lovett Memorial Foundation for the Study of Crippling Diseases,
Harvard Medical School, Boston, Mass.
The smallest organisms growing without the help of other cells are found
in the cultures of pleuropneumonia-like organisms (PPLO). Some are as
small as 0.15 to 0.25 /x- The majority of the organisms in the culture is
considerably larger. Size is only one of the distinctive characteristics of
these organisms. Their structure, the appearance of their colonies, their
chemical makeup and their reproductive processes also differ at first sight from
those of other microorganisms. However, many similarities to bacteria are
present. Their organization is as simple as is that of the bacteria. They do
not have distinct nuclei. Their growth requirements, metabolism, and sensi-
tivity to antibiotics are quite similar to those of the bacteria. An important
exception is that the PPLO are not sensitive to penicillin. The basic difference
between PPLO and bacteria is the absence in PPLO of a rigid cell wall, and
most of the distinctive properties of PPLO are the consequence of the lack of
this structural property characteristic of bacteria. The organisms are soft,
fragile, and easily distorted. Their size varies within wide limits from 0.15 /x
to 10 M, or larger. On agar media the structure and appearance of the colonies
of PPLO are characteristic and differ markedly from those of bacteria. Finally,
the method of reproduction seems to be more complex than that of bacteria,
although basically it is probably similar. In the fight of these similarities and
differences some authors propose to create a special class for PPLO,^ while
others regard them as a subdivision of the class of bacteria.-'^
The PPLO were discovered as parasites causing disease in animals or living
on their mucous membranes. They were isolated also from sewage, well water
and soil. The saprophytic strains differ in some respects from the parasitic,
but we have no information to suggest that they are part of the microflora
other than those related to animal organisms.
The suggestion that the PPLO might be an independent subdivision of mi-
croorganisms is made unlikely by the observation that bacteria under certain
conditions assume a growth form which presents aU the distinctive properties
of PPLO.-* These bacterial forms, usually designated as L forms, like the
PPLO are soft and fragile, lack a rigid cell waU, and are considerably smaller
than the usual bacteria. The appearance of the colonies, the morphology of
the organisms, their reproductive processes and their sensitivity to antibiotics
are similar to that of PPLO, and include resistance to penicillin. The best
illustration of the similarity between the two groups is the fact that 15 years
passed before it was generally recognized that the L forms were growth forms
of bacteria and not PPLO mixed with the cultures and thus foreign to the bac-
* The work reported in this paper was supported by a grant from the National Institute
of Arthritis and Metabolic Diseases, Public Health Service, Bethesda, Md. This paper is
pubHcation No. 322 of the Robert W. Lovett Memorial Foundation for the Study of Crip-
pling Diseases.
375
376 Annals New York Academy of Sciences
teria with which they were associated. At present the impression of the ma-
jority of bacteriologists interested in these organisms is that, although they
are fixed in their form of growth, PPLO derived from the bacteria at some time
in the past. This status would correspond to that of the fungi imperfedi. The
impression of some investigators is that PPLO may represent a primitive stage
in the phylogenetic development of bacteria to which under certain conditions
bacteria may return. It should also be mentioned that some authors^ '^'^ re-
gard the similarity between PPLO and bacteria as superficial and without
significance.
Information on morphology and reproductive processes of PPLO has been
confused for a long time, and to some extent it still is today. This confusion
exists not so much because of their small size but because of their fragility and
the ease with which they may be distorted. For these reasons, use of the
electron microscope thus far has yielded hardly more information than a
better definition of the smallest elements in the cultures of PPLO.
In 1935, Turner- gave an excellent description of the morphology of the
organism of bovine pleuropneumonia in broth cultures with dark field illumina-
tion. His basic observations are as follows: "An old broth culture contains
only small granules less than 0.5 /jl in diameter. Transferred to fresh media
these granules increase in size to about 1 n. One or more areas appear on their
peripheries from which short filaments may grow out. The structures thus
formed suggested the first name of the organism "Asterococcus." The ends
of the short filaments grow to a larger size and repeat a similar reproductive
process. The filaments may grow longer and either differentiate into small
granules or develop swellings from which filaments again grow out. In addi-
tion, rather large spherical or irregularly-shaped bodies, several m in diameter
appear in the culture. Under appropriate conditions these reproduce the
granules and filaments. Very long straight filaments, sometimes visible in the
cultures, are apparently artefacts."
The development of colonies of PPLO in agar cultures was carefully studied
by Liebermeister.'^ With the phase microscope he examined several strains.
Like Turner, he observed the extrusion of short filaments from the granules
and the development of new organisms at the end of the filament. It is char-
acteristic that the smallest organisms seem to divide but that the daughter
organisms usually are not closely associated but seem to be at the ends of a
short rod. Liebermeister did not observe the development of multiple fila-
ments from a granule nor the development of long filaments in the strains
which he studied.
The size of the organisms, especially on the surface of agar colonies, varies in
the cultures, and the smallest forms visible with the light microscope usually
make up a very small fraction of the culture. Autolysis and deformation of
the larger forms often produce a bewildering pleomorphism in aging cultures.
Klieneberger^ has published beautiful photographs indicating that the larger
organisms are aggregates of small ones enclosed in a common envelope. This
structure of the large forms is clearly visible in electron micrographs. Under
appropriate conditions granules grow out from the large bodies.
From this short discussion it seems that the morphology and reproductive
Dienes: PPLO & Bacterial L Forms 377
processes of the organisms are very simple. The basic elements are small gran-
ules between 0.15 and 0.3 ^ in diameter that multiply by fission after elonga-
tion. Somewhat larger forms may divide by extruding short filaments. In
addition the granules may form more or less large aggregates enclosed in a
common envelope out of which they again grow. The structure of such large
bodies is essentially similar in cultures of bacteria, L forms, and PPLO. In
the organisms of bovine pleuropneumonia, and possibly in a few other strains,
the granules also can grow into thin filaments. This form of growth was not
observed in most strains.
L forms, like PPLO, do not have rigid cell walls. This lack of a rigid cell
wall is demonstrated in thin sections of L forms examined with the electron
microscope. Chemical studies indicate that the L forms do not have the
chemical complexes that are responsible for the rigidity of bacterial cell walls.
A large part of the similarity of L forms to PPLO is the consequence of this
lack. However, some of the similarities to PPLO do not seem to be the im-
mediate consequence of the absence of a rigid cell wall. One of these is the
small size of both PPLO and the L forms. According to filtration measure-
ments by Klieneberger, the size of viable granules in the L forms of Streptoba-
cillus moniliformis is similar to, or only slightly larger than, the size of PPLO.
The electron microscope shows granules of similar size in both groups. An-
other property not directly connected with the cell wall, common to both
groups, is the tendency of the growing organisms to embed themselves in agar.
Multiplication in agar cultures occurs mainly inside the agar. The charac-
teristic appearance of the colonies in both groups is the consequence of this
tendency. Both groups of organisms invade agar only and not other solid
media. Finally, a remarkable property of both groups is the tendency to grow
into large bodies. This tendency is greater in the L forms than in PPLO.
The L forms in broth or in gelatin multiply only by the growth of granules to
large bodies and by the liberation of granules from the large bodies.
As noted above, bacteria also tend to grow into large bodies. Transforma-
tion of bacteria to L forms is always preceded by the appearance of large
bodies, and the L forms grow out of the large bodies. In a few instances
large bodies were observed during forniation from bacteria,** and like bac-
terial filaments, these bodies developed by multiplication without separation
of the bacteria. In the early stages large bodies disintegrated into a group
of bacteria by the development of cell walls between the constituent organisms.
After this period, the large bodies reproduced bacteria for a certain length of
time. Later, an increasing number lost the ability to develop or they pro-
duced L forms. Some of the L forms so produced, like the large bodies de-
veloping from bacteria, return immediately to bacterial form when the influ-
ence resulting in these transformations, e.g., penicillin, is eliminated. Most L
forms revert to a bacterial form of growth only occasionally and after long
cultivation may lose this ability completely.
The large bodies are formed in these instances, under conditions which in-
hibit the multiplication of the single organisms, by multiplication of organisms
possessing the full potentialities of bacteria. After some time the abihty to
return to bacteria is lost, but the organisms are able on agar media to multiply
378 Annals New York Academy of Sciences
outside the large body. The agar seems to offer a suitable physical environ-
ment similar perhaps to that present in the large bodies and necessary for
multiplication of L forms.
Bacterial large bodies have been known since the beginning of bacteriology
and are usually referred to as involution or dying forms. They are produced
by a great variety of influences on the bacteria that prevent normal multipU-
cation. Large bodies occur in the natural environment of bacteria. In some
cases, in contrast to older opinion, it is apparent that they remain viable and
able to reproduce for a longer period than single bacteria. Hence, the forma-
tion of large bodies is probably a useful process for bacteria in their natural en-
vironment and can be thought of as a phenomenon of adaptation and not
merely the result of degeneration.
At present, L forms can not be regarded in the same light. In most in-
stances they develop and can be propagated only under artificial conditions.
It seems likely that they represent the growth of forms in the laboratory that
naturally occur only in the large bodies derived from bacteria. Small size,
growth into agar, and a tendency to produce large bodies (characteristics of L
forms) may be the result of this natural site of growth.
It is remarkable that bacteria cultivated directly from pathological processes
relatively often have the tendency to grow into large forms and to produce L
colonies. This may be the result of injury to the organism by the defensive
forces of the host. On the other hand, it may be an adaptation of the bac-
teria to parasitism. In one case of peritonitis, for example,^ it seemed that a
bacteroides strain continued to multiply in the L form inside the phagocytic
cells of the host. Such an observation suggests that although L forms may be
produced under artificial conditions, this process might occur naturally and
thus might have been the origin by stabilization of strains of PPLO that have
continued life in this form. The PPLO not only appear to be bacteria without
the usual cell wall but also bacteria that have passed through the processes in-
volved in the growth of the large bodies. The most marked difference between
L forms and PPLO is that PPLO are better adapted to grow in artificial media
and especially to grow in the small granular form. The L forms grow usually
only from large inocula and have a pronounced tendency to grow into large
bodies as well as to undergo autolysis.
At present PPLO do not seem to be of the main stream of phylogenetic
development or to be a link in it. These organisms probably represent the
result of the simplification of the structure of bacteria as a consequence of
parasitism. They are not complex and occasionally are very small but, like
the viruses, they offer no direct clues for the origin of life.
For illustration of the morphology of PPLO and L forms we refer to articles
previously published in the Annals of this Academy.^ ■!"
References
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Bacteriol. Rev. 5: 331.
2. Turner, A. W. 1935. A study on the morphology and life cycles of the organism
of pleuropneumonia contagiosa bovum (Borrelomyces peripneumoniae nov. gen.) by
observation in the living state under dark ground illumination. J. Pathol. Bacteriol.
45: 1.
Dienes: PPLO & Bacterial L Forms 379
3. Freundt, E. a. 1958. The Mycoplasmataceae. Munksgaard. Copenhagen.
4. Dienes, L. & H. J. Weinberger. 1951. The L forms of bacteria. Bacteriol. Rev.
15: 245.
5. Klieneberger-Nobel, E. 1962. Pleuropneumonia-like Organisms (PPLO) Myco-
plasmataceae. Academic Press, Inc. London & New York.
6. 0RSKOV, J. 1942. On the morphology of peripneumonia-virus, agalactia-virus and
Seiffert's microbes. Acta Pathol. Microbiol. Scand. 19: 586.
7. Liebermeister, K. 1953. Untersuchungen zur Morphologic der Pleuropneumonia-
(PPLO-)Gruppe. Z. Naturforsch. 12: 757.
8. Dienes, L. & VV. E. Smith. 1944. The significance of pleomorphism in Bacteroides
strains. J. Bacteriol. 48: 125.
9. Madoff, S. 1960. Isolation and identification of PPLO. Ann. N. Y. Acad. Sci. 79:
383.
10. Dienes, L. 1960. Controversial aspects of the morphology of PPLO. Ann. N.Y.
Acad. Sci. 79: 356.
AXENIC CULTURE OF PARAMECILMSOME OBSERVATIONS ON
THE GROWTH BEHAVIOR AND NUTRITIONAL REQUIRE-
MENTS OF A PARTICLE-BEARING STRAIN OF
PARAMECIUM AURELIA 299X
Anthony T. Soldo*
Department of Cancer Chemotherapy, Schering Corporation, Bloomfield, New Jersey
The genus Paramecium comprises a group of free Uving ciUates noted for
their morphological and genetical complexity. For these reasons and because
the organisms represent an end point in a divergent course of evolution, this
genus has been an object of interest. Certain members of this group exist in
association with self reproducing, intracytoplasmic particles.'- Recent ad-
vances in the knowledge of the nutritional requirements of Paramecium has
made it possible to cultivate these particle-bearing paramecia in sterile medium.
The purpose of this paper is to summarize the present state of knowledge of
the nutrition of Paramecium and to present the results of some detailed studies
on a particle bearing strain, Paramecium aurelia 299X.
Nutrition of Paramecium. In the past, Paramecium was cultivated in a
medium consisting of plant extracts, notably cerophyl and lettuce infusion,
supplemented with living bacteria, usually Aerobacter aerogenes? The first
successful report of axenic cultivation was made by Johnson and Baker in
1942.4 These workers grew Paramecium multimicronucleata in a medium con-
sisting of pressed yeast juice and proteose peptone. Two components of the
pressed yeast juice were recjuired for growth. One proved to be heat labile
which they assumed to be a protein, but was later replaced by a mixture of
ribosidic derivatives of a purine and a pyrimidine; the other was a heat stable
component. In 1949, van Wagtendonk and Hackett successfully established
P. aurelia in a medium composed of equal parts of 0.5 per cent yeast autolysate
and a 24-hour culture of A. aerogenes in lettuce extract.^ This medium could
be heat sterilized and provided the basis for later work which led to the de-
velopment of a more complex bacteria free medium.^ Folic acid, riboflavin,
thiamine, and a steroid proved to be absolute requirements for the growth of
stock 5L7 of P. aurelia; the steroid requirement could be satisfied by ^- and
7-sitosterol, fucosterol, brassicassterol, stigmasterol, and A'*'"-stigmastadie-
none.'^'* Miller and van Wagtendonk found that P. aurelia required 11 amino
acids, nicotinic acid, panothenic acid, and possibly, pyridoxal.'^ Also, one or
more essential growth factors remained in the yeast. Miller and Johnson
studied further the nutrition of P. multimicronucleata, and demonstrated, in
addition to the purine and pyrimidine requirement for that organism, a need
for an exogenous source of a fatty acid. '"■'•' Recently Lilly et al. cultivated
Paramecium caudatuni in a medium chemically defined, except for a single
component.''* Their medium was similar to the one used for the cultivation of
P. aurelia and P. multimicronucleata, except that it was necessary to add mi-
crogram quantities of a protein concentrate obtained from dried green peas.
* Present address: Department of Contractile Proteins, Institute for Muscle Diseases, Inc.,
New York, New York.
380
Soldo : Axenic Culture of Paramecium
381
Purification of the protein factor and subsequent analysis led to the qualitative
identification of 16 amino acids. The nutritional role of this protein has not
been satisfactorily explained. In table 1 is given the composition of a typical
medium which supports the growth of most strains of Paramecium.
Axenic cultivation of X-bearing Paramecium. Lambda particles were dis-
covered in the cytoplasm of stock 299X of P. aurelia by Schneller, in 1958.^^
She noted that animals containing these particles possessed the ability to kill
sensitive or particle free animals when members of the appropriate types were
placed in the same container. In this respect, this particle-protozoan system
is similar to the well known k system.^^
Table 1
Axenic Medium for Paramecium
Amino acids
Mg./ml.
Vitamins
Mg./ml.
L-Alanine
25
Biotin
0.125
*L-Arginine-HCl
100
*Ca-pantothenate
5
L-Aspartic acid
50
*Folic acid
2.5
L-Glutamic acid
75
a-Lipoic acid
0.05
Glycine
25
*Nicotinamide
5
*DL-Histidine
50
*PyridoxaIHCl
5
*DL-Isoleucine
150
Pyridoxamine • HCl
2.5
*DL-Leucine
150
*Riboflavin
5
*L-LysineHCl
125
♦Thiamine -HCl
15
*DL-Methionine
150
Inorganic salts
*L-Phenylalanine
75
Fe(NH4)2(S04)2-6H20
15
L- Proline
50
ZnCla
2
*DL-Serine
200
EDTA
5
*DL-Threonine
150
MnS04-4H20
2
*L-Tryptophan
50
CuS04-5H20
0.3
*L Tyrosine
50
CoS04-7H20
0.5
DL-Valine
75
MgS04-7H20
50
Purines and pyiimidines
Other factors
*Guanylic acid
500
*Stigmasterol
1
*Uridylic acid
500
*Na oleate
40
Na acetate
500
*Yeast factorf
50-500
* Components known to be absolute requirements for the growth of one or more species
of Paramecium.
t For preparation see (9). May be replaced by Pea factor for P. caudatum (14).
Efforts to cultivate X-bearing animals in media used for the growth of parti-
cle free strains were unsuccessful. It was necessary to supplement a crude
medium consisting of proteose peptone, a dialyzable component of hot water
extract of Baker's yeast and salts, with Edamine S, an enzymatic digest of
lactalbumin.'^ This medium supported the growth of the protozoans and main-
tenance of the particles through serial subcultures for a period of 2 years.
Particles of axenically cultivated animals number several hundred per cell,
contain RNA, little or no DNA, and are similar in size to the bacterium,
Escherichia coli.^^ They are gram-negative and may be stained with most
bacteriological dyes. Examination under phase microscope reveals a rod or
diplorod type structure. A furrow which divides the particle into almost
382
Annals New York Academy of Sciences
equal halves suggests that the particles may reproduce by longitudinal divi-
sion; occasionally they appear to be vacuolated.
Particle reproduction is synchronized with the division of the protozoan.'^
Further evidence in support of this view is given in figure 1. Animals re-
SYNCHRONOUS DIVISION
7--
& 5
CL
o
CL
(3 4
o
3--
LAMBDA PARTICLES
Growth Medium
Resting Medium
PARAMECIUM
'Growth Medium
Resting Medium
3 4 5 6
DAYS
Figure 1. Growth medium: see table 1.
jjhosphate buffer, pH 7.0.
7 8 9
Resting medium: isotonic saline, 0.01 M
moved during the log phase of growth, washed to remove all traces of the
original medium and resuspended in a buffered salt solution, "resting medium,"
failed to multiply. Estimates of the particle population revealed that they,
too, did not increase in number. Synchronous division may account for the
ability of the particles to keep pace with the host, although it seems likely that
this may be only a partial answer to the phenomenon. It has been observed
that occasionally one or more of the animals loses all its particles. Clones
Soldo : Axenic Culture of Paramecium
383
derived from these animals are also particle free. It has not yet been possible
to rule out mutation as an explanation for this phenomenon.
The existing synchronism between the particles and the host cell makes it
possible to quantitatively evaluate agents that may selectively inhibit the
particles themselves. Of interest here is the number of antibiotics that possess
this capability (table 2). ID50 values, derived in a manner previously de-
scribed/* reflect the relative effectiveness of these substances to inhibit particle
populations. This selective action correlates with the toxicity produced by
these agents in man. Antibiotics such as penicillin and tetracycline which
exhibit the least toxicity in man prove to be excellent particle inhibitors; those,
Table 2
A- Comparison of the Activity of Antibiotics in the X System
WITH Chronic Toxicity in Man*
Antibiotic tested
Actinomycin D
Actidione
Bacitracin
Neomycin
Polymyxin
Candicidin
Streptomycin
Cephalosporin C
Novobiocin
Oleandomycin
Chloramphenicol
Aureomj'cin
Terramycin
Penicillin
Tetracychne
ID50 ratio
IDso
protozoan
protozoan
particlef
only
X
0.9
1
0.9
> 1,000
0.9
370
1.1
32
1.2
32
0.9
> 1,000
1.8
350
10
> 1,000
10
320
14
450
22
220
39
40
116
370
312
> 1,000
930
220
Toxicity in man
Very high toxicity.
Toxic — fatal to rats— 1 mg./kg. orally.
Nephrotoxicity, proteinuria.
Nephro- and ototoxicity.
Causes renal damage.
Toxic — used topically.
Low toxicit} — damage to eighth cranial nerve on
prolonged therapy.
Low toxicity — mice tolerate 5,000 mg./kg. intra-
venously.
Low loxicit> — 7 mg./kg. intravenously tolerated
in man.
Low toxicity — 40 mg./kg. orally tolerated in
children.
Low toxicity — 30 mg./kg. tolerated in man.
Low toxicity — 15-30 mg./kg. tolerated in man.
Low toxicity — 15-30 mg./kg. tolerated in man.
Verj' low toxicity — very well tolerated.
Very low toxicity — very well tolerated.
* Toxicity data obtained from Spector, W. S. 1957. "Handbook of Toxicology," vol. II.
t IDso ratios of greater than 1.2 indicate selective inhibition.
such as actinomycin and neomycin produce varying degrees of toxicity in man
and are not selectively inhibitory for the particle. Thus, the particle-Pam-
mecium system might be useful in predicting chronic human toxicity of poten-
tially useful antibiotic substances. In figure 2 the effectiveness of penicillin
in reducing the X population is shown. Under the conditions of the experi-
ment complete destruction of the particles is achieved in 1 day at a concen-
tration of 100 units per milliliter of the antibiotic.
Xutrilional requirements of X-beariiig Paramecium. A nutritional study was
made with particle-bearing and particle-free strains. The latter were obtained
by treating axenically cultivated, X-containing animals with penicillin to re-
move the particles. Both require a factor (or factors) present in a nondialyza-
ble aqueous extract of Baker's yeast. Chemical fractionation resulted in a
384
Annals New York Academy of Sciences
partially purified material which is not absorbed on anion or cation exchange
resins; the material may be precipitated with 67 per cent ethanol in the cold,
contains carbohydrate, protein, and no nucleic acid or lipids. Attempts to
replace this fraction with known substances, thus far, have been unsuccessful.
However, it has been possible to demonstrate a purine and pyrimidine require-
ment for the organisms, as well as their need for a number of vitamins, in a
medium (table 1) supplemented with this factor.
Purine requirements for particle containing and particle free animals are
summarized in table 3. The need for exogenous source of a purine derivative
THE EFFECT OF PENICILLIN ON LAMBDA
POPULATION
None
lUvml
7
Figure 2.
is apparent and may be met by guanosine and guanylic acid. The free base,
and adenosine and its derivatives, do not replace the purine. Apparently,
Paramecium converts guanosine to adenosine and its derivatives, whereas
the reverse reactions do not occur. Inosine, its derivatives, and xanthosine
and its derivatives failed to replace guanosine as a growth requirement.
The pyrimidine requirements may be satisfied by uridine, cytidine, uridyhc
and cytidylic acids (table 4). The free bases uracil, cytosine, and thymine,
as well as thymidine and thymidylic acid were not effective in replacing uridine
or cytidine. These data confirm earlier work with P. miillimicronucleala,
P. caudahim, and other strains of P. aurelia.
By means of C^'^-labeled purines, it has been shown that adenosine is in-
corporated into nucleic acid adenine only, whereas exogenously supplied guano-
Soldo: Axenic Culture of Paramecium
385
sine is incorporated into both nucleic acid-guanine and -adenine. ^^ These data
confirm the nutritional findings. Similar data obtained with isotopically la-
beled pyrimidines are in agreement with the nutritional evidence that cytidine
and uridine are interconvertible and serve as precursors for thymidine and thy-
midylic acid. The data further illustrate that similar pathways exist for
Table 3
Purine Requirements of Paramecium
Population den
sity* (No./ml.)
Purines tested (2/iM/ml.)
299X
299 S
Adenine
Guanine
Hvpoxanthine
Xanthine
Adenosine
Guanosine
9200
10,200
Inosine
Xanthosine
Adenylic acid (5')
Guanviic acid (5')
8700
9200
Inosinic acid (5')
* Values obtained after iirst transfer.
Table 4
Pyrisodine Reqltirements of Paramecium
Population density* (No./ml.)
Pyrimidines tested (2/iM/ml.)
299X
299 S
Cvtosine
Uracil
Thymine
Cytidine
6600
5800
Uridine
8200
7600
Thymidine
Cytidylic acid (5')
5200
7200
Uridylic acid (5')
4800
6500
Thymidylic acid (5')
* Values obtained after first transfer.
purine and pyrimidine utilization in both particle free and particle bearing
animals.
Generally, the requirements for vitamins for particle bearing and particle
free animals are similar (table 5). The need for nicotinamide, riboflavin, and
thiamine becomes apparent in the second transfer, whereas the requirement for
pyrido.xal is evident only after three or four serial subcultures. An absolute
requirement for calcium panthentate has not been shown. Some degree of
growth, appro.ximating 10 per cent of the control, remains even after several
transfers. Biotin and lipoic acid are not required. Particle bearing animals,
386
Annals New York Academy of Sciences
in the absence of folic acid, may be subcultured indefinitely. Particle free
animals, on the other hand, show an absolute requirement for this substance,
as judged from their inability to grow beyond the second transfer.
Particles may produce sufficient quantities of folic acid to provide for the
nutritional needs of the protozoan. To test this possibihty, particle bearing
animals were treated with penicillin in the presence and the absence of folic
acid (table 6). As expected, particle free animals did not grow in the con-
Table 5
Vitamin Requirements of Paramecium
Population (% control)
299X
299 S
Serial subculture
Serial subculture
1
2
3
4
5
1
2
3
4
s
Biotin
105
123
94
99
105
110
69
77
112
105
Ca pantothenate
95
38
38
7
12
105
73
74
36
8
Folic acid
100
71
71
69
75
40
a-Lipoic acid
101
103
99
85
95
87
57
96
121
112
Nicotinamide
97
75
Pyridoxal
110
98
82
75
48
37
26
Riboflavin
35
Thiamine
25
12
Table 6
The Effect of Penicillin upon the Folic Acid
Requirement of Paramecium
/
Population density (No. /ml.)
Addition
Medium plus folic acid
Medium minus folic acid
299X
299 S
299X
299 S
None
Penicillin, 1000 U/ml.
5200
9000*
7400
10,200
3800
* Animals particle free.
trols, or in penicillin-treated tubes in the absence of folic acid. Addition of
folic acid to the medium restored the ability of these animals to grow in the
presence or absence of peniciUin. Particle containing animals, on the other
hand, failed to grow in folic acid free medium containing penicillin, whereas
animals under similar conditions retained their particles and grew well in the
absence of penicillin. (Irowth of particle bearing animals in which folic acid
was present in both the control and penicillin-treated tubes was good. Peni-
cillin-treated animals contained no particles. Subsequent deletion of folic
acid from the medium containing these penicillin-treated animals resulted in
\ the death of the protozoan. These data support the view that folic acid pro-
Soldo: Axenic Culture of Paramecium 387
duction is dependent upon the presence of the particle in the cytoplasm; im-
plicit here is that the vitamin is produced by the particles themselves.
Discussion
The symbiotic association between X particles and the host Paramecium is
an example of what is doubtless a widespread phenomenon in nature. Para-
mecium bursaria harbors an alga of the genus Chlorella in its cytoplasm in what
has been described as a symbiotic system.-'' Colicins in bacteria,-^ extranu-
clear particles responsible for cytoplasmic inheritance in yeast," and particle-
Uke inclusions found in many insect tissues"-^ -^ may be further examples. A
well documented case of an endosymbiote has been found for the flagellated
protozoan, Strigomonas}^ This organism, apparently, exists in association
with cytoplasmic bipolar-like bodies. The presence of these particles, as with
Paramecium, alters the nutritional requirements of the host.
A distinctive feature of the A system is the ability of particle bearing animals
to release a toxin which causes the death of certain particle free detector strains,
but is without effect upon the X bearers themselves. In this respect the X
system bears a striking resemblance to colicin producing systems."^ Colicins
are antibiotic substances produced by certain bacteria, notably members of the
family Enterobacteriaceae. The ability of these bacteria to produce these
substances is believed to be due to the presence of a transmissible pathogenic
agent which is regarded as a bacterial virus. The analogy serves to illustrate
the degree to which the X particles may have incorporated themselves into the
genetic structure of the protozoan.
Yet X particles, unlike viruses, are highly complex structures which resemble
bacteria in size, morphology, staining characteristics, chemical composition,
and, possibly, manner of reproduction. Studies concerning the chemistry of
the particles reveal the presence of protein, carbohydrate, phospholipid, and
nucleic acid (W. J. van Wagtendonk and R. Tanguay — personal communica-
tion). Moreover, antibiotics are particularly effective in reducing or eliminat-
ing the particles from the cytoplasm of the protozoan. (These data, obtained
for the first time with axenically cultivated animals, provide the strongest
evidence to date on the action of antibiotics on particles in Paramecium.)
Finally, the finding that X particles produce amounts of folic acid sufficient to
support the growth and reproduction of the protozoan carries with it the im-
plication that the complex enzymatic machinery necessary for the synthesis of
this compound is present in the particles themselves.
A cknowledgments
I wish to express my sincere thanks to Andrea M. Pascale and William E.
Ronca for their excellent technical assistance.
The work described in this article was supported in part by a Government
Contract, SA-43ph-1929, to The Cancer Chemotherapy National Service Cen-
ter, National Cancer Institute, National Institutes of Health, Bethesda, Md.
References
1. SoNNEBORN, T. M. 1959. Kappa and related particles in Paramecium. Adv. Virus
Res. 6: 229.
388 Annals New York Academy of Sciences
2. WiCHTERMAN, R. 1953. The biology of Paramecium. The Blackston Company, Inc.
New York.
3. SoNNEBORN, T. M. 1950. Methods in the general biology and gene genetics of Para-
mecium aurelia. J. Exp. Zool. 113: 87.
4. Johnson, VV. H. & E. G. S. B.aker. 1942. The sterile culture of Paramecium mulli-
nucronucleata. Science. 9: Hi.
5. v.AN Wagtendonk, W. J. & P. L. Hackett. 1949. The culture of Paramecium aurelia
in the absence of other living organisms. Proc. Natl. Acad. Sci., U.S. 35: 155.
6. van Wagtendonk, W.J. , R. L. Conner, C. A. Miller &M. R. R. Rao. 1953. Growth
requirements of Paramecium aurelia var. 4, stock 51.7 sensitives and killers in axenic
medium. Ann. N.Y. Acad. Sci. 56(5): 929.
7. Conner, R. L., W. J. van Wagtendonk & C. A. Miller. 1953. The isolation from
lemon juice of a growth factor of steroid nature required for the growth of a strain of
Paramecium aurelia. J. Gen. Microbiol. 9(3): 434.
8. Conner, R. L. & W. J. VAN W.agtendonk. 1955. Steroid requirements of Paraweciww
aurelia. J. Gen. Microbiol. 12(1): 31.
9. Miller, C. A. & W. J. Wagtendonk. 1956. The essential metabolites of a strain of
Paramecium aurelia (stock 47.8) and a comparison of the growth rate of different
strains of Paramecium aurelia in axenic culture. J. Gen. Microl)iol. 15(2): 280.
10. Johnson, W. H. & C. A. Miller. 1956. A further analysis of the nutrition of Para-
mecium. J. Protozool. 3: 221.
11. Johnson, W. H. & C. A. Miller. 1957. The nitrogen requirements of Paramecium
muliimicronucleatum. Physiol. Zool. 30: 106.
12. Miller, C. A. & W. H. Johnson. 1957. A purine and pyrimidine rec|uirement for
Paramecium muliimicronucleatum. J. Protozool. 4: 200.
13. Miller, C. A. & W. H. Johnson. 1960. Nutrition of Paramecium. A fatty acid re-
quirement. J. Protozool. 7(3): 297.
14. Lilly, D. M. & R. C. Klosek. 1961. A protein factor in the nutrition of Paramecium^
caudatum. J. Gen. Microbiol. 24: 327.
15. Schneller, M. V. 1958. A new type of kiUing action in a stock of ParaweciwiM awre/ja
from Panama. Proc. Indiana Acad. Sci. 67: 302.
16 Sonneborn, T. M. 1938. Mating tvpes in Paramecium aurelia. Proc. Am. Phil.
Soc. 79:411.
17. Soldo, A. T. 1960. Cultivation of two strains of killer Paramecium aurelia in axenic
medium. Proc. Soc. Exp. Biol. Med. 105: 612.
18. Soldo, A. T. 1961. The use of particle-l)earing Paramecium in screening for potential
anti-tumor agents. Trans. N.Y. Acad. Sci. 23(8): 653.
19. Soldo, A. T. & W. J. van Wagtendonk. 1961. Nitrogen metabolism in Paramecium
aurelia. J. Protozool. 8(1): 41.
20. Siegel, R. W. 1960. Hereditary endosymbiosis in Paramecium busaria. Exp. Cell
Research. 19: 239.
21. Eredericq, p. 1950. Rapports entre colicines et bacteriophages. Bull. Acad. Roy.
Med. Belgique. 15:491.
22. Ephrussi, B. 1953. Nucleo-cytoplasmic relations in micro-organisms. Oxford Univ.
Press. London & New York.
23. Steinhaus, E. A. 1946. Insect Microbiology. Cornell Univ. Press (Comstock).
Ithaca.
24. Buchner, p. 1953. Endosymbiose der tiere mit pflanzlichen mikroorganismen.
Birkhauser. Basel.
25. Newton, B. A. & R. W. Horne. 1957. Intracellular structures in Strigomonas onco-
pelti. Exp. Cell Research. 13: 563.
26. Eredericq, P. 1957. Colicins. Ann. Rev. Microbiol. 11: 7.
THE EFFECT OF POLLUTION ON RIVER ALGAE
C. Mervin Palmer
U. S. Department of Health, Education, and Welfare, Public Health. Service,
Robert A. Taft Sanitary Engineering Center, Cincinnati, Ohio
A large majority of algae are affected adversely by the gross pollution of
streams with organic wastes such as domestic sewage. After partial self-purifi-
cation of the stream has occurred, however, the populations and kinds of algae
become much more numerous than are present in the clean portion of the
stream above the area of pollution. This increase is due to the nutrients that
are made available from the decomposing organic wastes.
The undecomposed organic wastes affect the algae by causing chemical and
physical changes in the stream. Increased turbidity reduces the light availa-
ble for photosynthesis. Increased organic content in the water stimulates
saprophytic and saprozoic organisms which then compete for space with the
algae. Certain constituents of the waste are toxic to many algae. Thus,
many factors of the environment that are changed by the organic wastes have
an effect on the algae.
Information on the physiological and morphological effects of organic pollu-
tion on algae is very limited at present. There have been, however, many
studies of the change in the algal tlora as a result of pollution. Gross pollu-
tion causes a great reduction in the number of kinds of algae in the stream.
Those able to remain have frequently been called "indicators" of pollution,
but no specific kinds individually are reliable indicators of grossly polluted
water. Polluted water varies too much to ensure an environment satisfactory
for the growth or persistence of any one particular algal species. Any indi-
vidual species tolerant of pollution may also be found in unpolluted areas of a
stream or may be absent in some areas of pollution.
When a number of the tolerant genera and species are considered, it becomes
likely that a high percentage of these will be present in all areas of streams
grossly polluted with organic wastes. The presence of such a community of
algae in a stream, therefore, is a reliable indicator of the condition of the water.
Many workers have listed the genera and species of algae found in polluted
waters, particularly in the United States and in Europe. The number of
kinds which they have considered to be pollution tolerant is generally quite
limited for any one area or survey, but becomes very large when all of the
results of many investigators are combined.
The lists of pollution-tolerant algae reported by 110 workers have been ex-
amined by the writer to date. The genera and species of algae tolerant to
sewage or to related conditions have been recorded, and a total of more than
600 species and varieties has been compiled.
To tabulate the information, the writer has allotted arbitrary numerical
values to each author's record of an alga. A value of 2 was given to each
alga reported as very highly tolerant, and a value of 1 to each alga highly tolerant
to the presence of organic matter. Lightly tolerant and nontolerant algae
were not recorded in the compilation. The total points from all of the 110
389
390
Annals New York Academy of Sciences
authors were then determined for each genus and species. The algae were
arranged in the order of decreasing emphasis by the authors as a whole as in-
dicated by the comparative total scores for each alga. Theoretically an alga
considered as very highly tolerant by all 110 authors would have had a perfect
score of 110 multiplied by 2, or 220 total points.
For studies in sanitary science the algae are frequently placed into four
groups. All flagellates containing photosynthetic pigments constitute one of
the four groups. The other three groups are the blue-green algae, the diatoms,
and the green algae, the last group including all of the nonflagellated green,
yellow-green, and other related forms.
Table 1
Pollution Algae
Most tolerant genera, by groups
Highest
4
10
50
Blue-greens
Greens
Diatoms
Flagellates
1
1
2
2
3
3
2
8
15
15
12
Table 2
Pollution Algae
Most tolerant species, by groups
Highest
4
10
so
Blue-greens
Greens
Diatoms
Flagellates
1
1
1
1
3
4
1
2
15
10
11
14
All four groups are well represented among the genera and species with high
scores as pollution-tolerant algae. For example, of the 10 genera with the
highest scores, 2 are blue-green algae, 2 are flagellates, 3 are diatoms, and 3 are
green algae (table 1). Of the four species with the four highest scores, each
belongs to a different group. Among the 50 most tolerant species, the range in
number per group is from 10 to 15 (table 2).
The 52 most tolerant genera are listed in table 3. Leading the list, in order
of decreasing total scores, are Euglena, Oscillaloria, Clilamydomonas, Scenedes-
mus, Chlorella, and Xilzschia. The first two were considered as tolerant genera
by 62 and 61 authors and rated 110 and 105 total points, respectively. These
are in contrast with the fiftieth genus, Cocconeis, which was referred to by only
8 authors for a total score of eight.
Palmer : Effect of Pollution on River Algae
391
Table 3
Pollution Tolerant Genera of Algae List of 52 Most Tolerant Genera in
Order of Decreasing Emphasis by 110 Authorities
Genera
Group
No. of authors
Total points*
1
Euglena
F
62
110
2
Oscillatoria
B
61
105
3
Chlamydomonas
F
42
70
4
Scenedesmus
G
40
65
5
C/dorella
G
36
63
6
Nitzschia
D
38
63
7
Navicula
D
35
55
8
Stigeodonium
G
34
50
9
Phormidiiim
B
30
45
10
Synedra
D
25
a
11
F ha ens
F
23
32
12
Ankistrodesmus
G
19
31
13
Gomplionema
D
20
30
14
Spirogyra
G
19
29
15
Cydotella
D
22
29
16
Pandorina
F
18
25
17
Closterium
G
19
25
18
Lepocindis
F
14
24
19
Melosira
D
18
24
20
Chlorogoniiim
F
14
23
21
A nabaena
B
17
23
22
Ulothrix
G
17
23
23
Micractinium
G
13
21
24
FragUaria
D
15
20
25
A nacystis
B
16
20
26
Frachelonwnas
F
16
20
27
Art/irospira
B
11
19
28
Carleria
F
12
19
29
Surirella
D
14
19
30
Cryptomonas
F
15
19
31
AgmeneUiim
B
11
18
32
Lyngbya
B
11
18
33
Eudorina
F
12
18
34
Fediastrum
G
14
18
35
Oocystis
G
12
16
36
Fyrobotiys
F
10
15
37
Cymbella
D
10
14
38
Steplianodisciis
D
10
14
39
Coelaslrum
G
12
14
40
Cladophora
G
13
14
41
Golenkinia
G
9
13
42
Spondylomonim
F
9
13
43
Achnantlies
D
11
13
44
Actinastntm
G
11
13
45
Hanlzschia
D
9
12
46
Spindina
B
9
12
47
Finnitlaria
D
8
11
48
Ski Krone! s
D
9
11
49
Tribonema
G
6
10
50
Coeconeis
D
8
10
51
Selenasfrnm
G
8
10
52
Cosmarium
G
9
10
* Tolerance by author, "Very High," 2 points. Tolerance by author, "High," 1 point.
Table 4
Pollution Tolerant Species of Algae: A List of the 60 Most Tolerant
Species in Order of Decreasing Emphasis by 110 Authorities
Species
Group
No. of
authors
Total points*
1
Euglena viridis
F
34
63
2
Nitzscfiia palea
D
30
46
3
Stigeoclonium lenne
G
17
26
4
Oscillaloria tenuis
B
17
25
5
Oscillaloria limosa
B
14
21
6
Scenedesnms quadricauda
G
12
18
7
Chlorella vulgaris
G
11
17
8
Pandoritia morion
F
12
17
9
Arthrospira jenneri
B
9
16
10
A nkistrodesmiis Jalcatus
G
11
16
11
Cydotella meneghiniana
D
12
16
12
Chlorella pyrenoidosa
G
8
15
13
Gomphonema parvuluni
D
8
15
14
Euglena gracilis
F
9
15
15
Oscillatoria clialybea
B
10
15
16
Synedra ulna
D
12
15
17
Oscillatoria cidorina
B
9
14
18
Nilzschia acicularis
D
10
14
19
Oscillatoria formosa
B
10
14
20
Oscillatoria princeps
B
10
14
21
Oscillatoria putrida
B
8
13
22
Euglena oxyuris
F
9
13
23
Navicula cryptocepliala
D
9
13
24
Flwrmidium uncinatum
B
11
13
25
Agmenellum quadriduplicatum
B
7
12
26
Chlorogonium euchlorum
F
7
12
27
Hantzscliia ampliioxys
D
9
12
28
Phormidium autumnale
B
9
12
29
Surirella ovata
D
9
12
30
Euglena acus
F
10
12
31
LepocincUs ovum
F
7
11
32
Micractinium pusillum
G
7
11
33
Eunorina elegans
F
8
11
34
Euglena deses
F
8
11
35
Oscillaloria splendida
B
9
11
36
Oscillatoria lauterbornii
B
6
10
37
Euglena polymorplta
F
7
10
38
LepocincUs texta
F
7
10
39
Spondylomorum quaternarium
F
7
10
40
A ctinaslru m hantzschi
G
8
10
41
Closterium acerosum
G
8
10
42
A nabaena constricta
B
6
9
43
Anacystis montana
B
6
9
44
Pliacus pyrum
F
6
9
45
Scenedesmus obliquus
G
6
9
46
Cocconeis placentida
D
7
9
47
Achnanthes minutissima
D
8
9
48
Coelastrum micro poruni
G
8
9
49
Melosira varians
D
8
9
50
CItlamydomonas rein liardi
F
5
8
51
Pediastrum horyanum
G
5
8
52
Scenedesmus dimorphus
G
5
8
53
Chlorogonium elongatum
G
6
8
54
Euglena intermedia
F
6
8
55
Euglena pisciformis
F
6
8
56
Phacus pleuronectes
F
6
8
57
Tetraedron mulicum
G
6
8
58
Anacystis cyanea
B
7
8
59
Melosira granulata
D
7
8
60
Phormidium faveolarum
B
8
8
* Tolerance by author, "Very High," 2 points.
392
Tolerance by author, "High," 1 point.
Palmer: Effect of Pollution on River Algae
393
The 60 most tolerant species are given in table 4. Euglena viridis, followed
by Nitzschia palea, are at the top of the list with total scores of 6v^ and 46,
respectively.
The names and total points for the 10 most tolerant species of a genus are
shown for the two leading genera, Euglena and Oscillator ia (tables 5 and 6).
In the former genus, the first species, E. viridis, is far ahead of the other nine
species. In the latter genus there is a more gradual change in total points from
Table 5
Species of Euglena: Ten Most Tolerant of Pollution
Authors
Points
E. viridis
34
63
E. gracilis
9
15
E. oxvuris
9
13
E. aciis
10
12
E. deses
8
11
E. polvmorpha
7
10
E. intermedia
6
8
E. pisciformis
6
8
E. proxima
5
7
E. spirogyra
6
7
Table 6
Species of Oscillatorla: Ten Most Tolerant of Pollution
Authors
Points
0. tenuis
17
25
0. liniosa
14
21
0. clialvhea
10
IS
0. clilorina
9
14
0. fonnosa
10
14
0. prince ps
10
14
0. putrida
8
13
0. splendid a
9
11
0. lauterbornii
6
10
0. brevis
6
7
one species to the next. Eight of the 10 species of Euglena and 9 of Oscillatoria
are among the 60 most tolerant forms as noted in table 4.
It would be interesting to know what species of Chlamydomonas was con-
sidered most tolerant of organic pollution, but unfortunately very few of the
110 investigators have determined and recorded the species for this genus. For
the genus Xavicula, numerous species have been recorded by the investigators,
but there is little indication that there may be one or two species which are
much more tolerant than others that they have named.
Additional records by other workers would undoubtedly change the com-
parative total points and the relative positions of the algae in both the genus and
species lists. This is particularly so for the algae near the low ends of the lists
where a relatively few reports are responsible for their present positions.
394 Annals New York Academy of Sciences
The lists of algae in the tables are meant to be aids for persons engaged in
stream pollution surveys or related projects. They give a general consensus
of opinion as to the relative significance of the many algae tolerant of organic
wastes which have been reported. Particular care can thus be taken in bio-
logical surveys to check for the presence of these genera and species of algae
during the microscopic examination of samples.
The references given represent many of the more exhaustive studies that were
included in the preparation of this report.
References
Blum, J. L. 1956. The ecology of river algae. Botan. Rev. 22: 291-341.
Butcher, R. W. 1949. Pollution and repurification as indicated by the algae. Fourth
International Congress for Microbiology (held) 1947. Report of Proceedings.
Cholnoky, B. J. 1958. Hydrobiologische Untersuchungen in Transvaal. II. Selbstrei-
nigung im Jukskei-Crocodile Flusssystem. Hydrobiologia. 11(3-4): 205-266.
FjERDiNGSTAD, E. 1950. The microflora of the River M^lleaa with special reference to
the relation of the benthal algae to pollution. Folia Limnol. Scand. No. 5.
FoEBES, S. A. & R. E. Richardson. 1913. Studies on the biologj' of the upper Illinois
River. Bull. Illinois State Lab. Nat. Hist. 9(Art. 10): 481-574.
HORNUNG, H. 1959. Floristischokologische Untersuchungen an der Echaz unter be-
sonderer Beriicksichtigung der Verunreinigung durch Abwasser. Arch. Hydrobiol.
55: 52-126.
Hynes, H. B. 1960. The Biology of Polluted Waters. Liverpool Univ. Press. Liverpool.
KoLKWiTZ, R. 1950. Oekologie der Saprobien. Uber die Beziehungen der Wasserorga-
nismen zur Umwelt. Schriftenreihe des Vereins fiir Wasser-, Boden- und Lufthygiene
Berlin-Dahlem. Piscator-Verlag. Stuttgart.
Lackey, J. B. 1941. The significance of plankton in relation to the sanitary condition of
streams. In Symposium on Hydrobiology. : 311-328. LTniv. of Wisconsin, Madison.
Lackey, J. B. 1956. Stream enrichment and microbiota. Public Health Repts. 71: 708-
718.
Liebmann, H. 1951. Handbuch der Frischwasser- und Abwasserbiologie. R. Olden-
bourg. Miinchen.
Mackenthun, K. M., L. a. Lueschow & C. D. McNabb. 1960. A study of the effects
of diverting the effluent from sewage treatment upon the receiving stream. Trans.
Wisconsin Acad. Sci. 49: 51-72.
McGauhey, p. H. & H. F. Eich. 1922. A study of the stream pollution problem in the
Roanoke, Virginia, MetropoHtan District. Part 3. Third portion: The plankton of
the waters and muds. Bull. Va. Polytech. Inst. (Eng. Expt. Stat. Ser. No. 51). 35:
64-88.
Oliff, W. D. 1960. Hydrobiological studies on the Tugela River system. Part II. Or-
ganic pollution in the Bushmans River. Hydrobiologia. 16(2): 137-196.
Palmer, C. M. 1957. Algae as biological indicators of pollution. Biology of Water Pol-
lution: Trans. Seminar on biological j^roblems in water pollution held in 1956. : 60-
69. Robert A. Taft Sanitary Engineering Center. Cincinnati, Ohio.
Palmer, C. M. 1959. Algae in water supplies. U.S. Public Health Service Publ. No. 657.
U.S. Government Print. Off. Washington, D.C.
Palmer, C. M. 1932. Plankton algae of White River in Marion County and Morgan
County, Indiana. Butler Univ. Botan. Studies. 2: 125-131.
Patrick, R. 1948. Factors effecting the distribution of diatoms. Botan. Rev. 14(8):
473-524.
Purdy, W. C. 1930. A study of the pollution and natural purification of the Illinois River.
II. The plankton and related organisms. U.S. Pubhc Health Bull. No. 198. : 1-212.
Silva, p. C. & G. F. PAPENFU.SS. 1953. A systematic study of the algae of sewage oxida-
tion ponds. Calif. State Water PoUution Control Board. Publ. No. 7.
Sramek-Husek, R. 1956. Zur biologischen Charakteristik der hoheren Saprobitatsstufen.
Arch. Hydrobiol. 51: 376-390.
Uherkovich, G. 1961. Limnologia, a tiszai algak a szaprobionta rendszerben. Hidrol.
Kozlony. 1: 85-88.
Weston, R. S. & C. E. Turner. 1917. Studies on the digestion of a sewage-filter effluent
Palmer: Effect of Pollution on River Algae 395
by a small and otherwise unpolluted stream. Contrib. from Sanitary Res. Lab. and
Sewage Expt. Station. Mass. Inst. Technol. Vol. 10.
Whipple, G. C, G. M. Fair & M. C. Whipple. 1948. The Microscopy of Drinking Water.
Ed. (4). J. Wiley & Sons. N. Y.
WiEBE, A. H. 1927. Biological survey of the upper Mississippi River with special refer-
ence to pollution. Document No. 1028. Bull. Bur. Fisheries. 43(2): 137-167.
WiSNiEWSKi, T. F. 1961. The Badtish River before and after diversion of sewage plant
effluent. Algae and MetropoUtan Wastes. Trans. 1960 Seminar. Robert A. Taft
Sanitary Engineering Center, Cincinnati, Ohio, Tech. Rept. W61-3: 118-124.
Wysocka, H. 1961. Periphyton des lamelles en verre comme I'indicateur de la pollution
d'eau. Verhandel. Intern. Verein. Limnol. 14: 1063-1070.
ULTRASTRUCTURE RESEARCH AS AN AID IN
THE CLASSIFICATION OF DIATOMS
R. Ross
British Museum {Natural History), London S.W . 7, England
Present Knowledge of the Ultrastrudure of Diatoms
The frustules of diatoms were among the first biological objects to be exam-
ined with the electron microscope (Mahl, 193^), and in the preceding 20 years
a large number of works dealing with the subject have appeared. These have
been listed comparatively recently by Hendey (1959), and at the time at which
he wrote information about some 300 species was available. Although it is not
important to review the results of these studies in detail, there are two points
about them which need to be emphasized here. The tirst is that none of this
work has been done with any particular taxonomic problem in mind. For the
most part it would seem that investigators took the material which came readily
to hand, mounted drops of it on electron microscope grids, and took pictures of
the forms they found there. This has on occasion led to doubt as to the true
identity of the species studied, as in the case of the illustrations published by
Kolbe (1951, plate 2, tig. 4, plate 3, figs. 5 and 6) as Navicula subtUissima Cleve,
but said by Hustedt (1952, 1955) to be of Anomoeoneis exilis (Kiitz.) Cleve or
A. serians var. brachysira (Breb.) Cleve (Kolbe, 1954, 1956, 1959). A more im-
portant consequence, however, is that there is not any group of supposedly re-
lated species of which more than a small proportion have been studied with the
electron microscope. In no case do we know the patterns of similarity and
difference and the range of ultraslructure to be found within a single genus,
with the possible exception of Pinnularia Ehrenb., of which electron micro-
graphs of some 15 species in a genus totaling at least 250 suggest that the
ultrastructure is as uniform as that revealed by the light microscope.
The other important point is that the interpretation of electron micrographs
is by no means easy, and also that, in some cases, those published do not give
an adequate picture of the structure of the species illustrated, either because the
specimen was damaged in preparation or because the resolution is insutlicient.
Interpretation is ditlftcult because of the great depth of focus of the electron
microscope and the considerable opacity of silica to electrons. Even in pic-
tures of complete frustules, the whole is equally in focus. In the light micro-
scope it is possible to build up a picture in depth from a series of optical sections
obtained by alterations of focus, but this technique is not available to the elec-
tron microscopist. When more than one layer is visible it is often not possible
to tell from single pictures which lies above which. Much of the valve, also, is
completely opaque to electrons, and where this is so there is no information
about differences in thickness from differences in transmission of electrons.
Stereomicrographs accordingly provide much more information than single
prints, as may be seen from the large number published by Helmcke and Krieger
(1953, 1954, Helmcke et al., 1961). These authors have applied stereogram-
metric techniques to the study of their stereoscopic pairs and have produced
models of the structure of a number of species, thus obtaining the maximal
amount of information from the data recorded on the micrographs.
396
Ross: Classification of Diatoms 397
The possibility of being misled by photographs with inadequate resolution
or of damaged specimens is best illustrated by particular examples. Hendey's
(1959) list of the species examined with the electron microscope includes an
indication of the ultrastructure of the valve. Both Stanroneis anceps Ehrenb.
and S. phoenicenteron (Nitzsch) Ehrenb. are said to have laminar valves per-
forated by fully open holes. His information about S. anceps is derived from a
picture published by Helmcke and Krieger (1953, plate 67) and that about
S. phoenicenteron from three pictures pubUshed by Okuno (1949, plate 3, fig. 8,
1952, plate 19, fig. 4, 1955, plate 9, fig. 1). In both species, however, the
striae consist of a series of elongated chambers with a membrane pierced by a
sUt on the outside and a membrane with fine pores in triangular tesselation on
the inside. The outer membrane is visible with a lens on negatives taken at
XlOOO, but not easily so, whereas the inner membrane, in which the repeat
distance of the pores is only about 170 A, can only be seen on negatives taken at
X5000. Helmcke's and Krieger's and Okuno's pictures seem to have been
taken at a much lower magnification than this and enlarged in reproduction.
Recently Helmcke et al. (1961, plates 289-290) have published pictures of S.
phoenicenteron showing the two membranes, but not all the detail described
below (p. 401). The ultrastructure can also be damaged either by chemical
cleaning or in fossilization. Figure 2 (p. 402) of a postpleistocene fossil speci-
men of S. phoenicenteron^ which may be compared with the pictures of the
species published by Helmcke et al., shows an example of this.
When features are misinterpreted or imperfectly understood, and especially
when, in consequence, like things are considered unlike or unlike things are
grouped together, they will not provide satisfactory taxonomic characters. It
is, therefore, necessary to base any taxonomic use of the ultrastructure of
diatoms upon a proper understanding of that structure. Hendey (1959) has
presented a classification of the types of ultrastructure in which the primary
division is into laminar valves, consisting of one layer of siliceous substance,
usually perforate, and locular valves, which are formed of a double layer of
siliceous substance separated by vertical walls. In my opinion, however, such
a distinction cannot be drawn. In most cases, at least, the diatom valves are
pierced by chambers; these may occasionally be completely open on both sides,
when they may properly be described as pores, but more usually they have a
membrane, itself perforate, on one or both sides. In a number of cases, what
were originally thought to be pores have been found, when more critically
examined, to be closed by membranes on one or both sides. This makes it seem
possible that such membranes will be found to be normal throughout the
diatoms, and that only the mucilage pores that occur singly or in small numbers
in some species will prove to be true pores. What Hendey classes as partially
occluded perforations through a single-layered wall are exactly similar in
structure to what he classes as loculi open on one side; the only difference lies
in the closeness of their packing. His failure to realize this may be due in part
to the difficulty of establishing relations in depth from single electron micro-
graphs and his not recognizing in consequence that the membranes occluding
the perforations were at the level of one or other surface of the valve. Ac-
cordingly, the classification of Helmcke et al. (1961), based entirely upon the
structure of the individual chambers, is much more satisfactory. This sepa-
398 Annals New York Academy of Sciences
rates pores, open at both ends, from chambers, with a septum at one or both
ends, and classifies these according to the position and type of perforation of
the septum or septa.
The Use of Diatom Ultrastructure in Taxonomy
In spite of the large amount of information available about the ultrastructure
of diatoms, it has until now been of little use in their taxonomy. Hustedt
(1952, 1955), in the course of an interchange of opinion on the subject with
Kolbe (1954, 1956), maintained that ultrastructure is more uniform than the
features that can be seen with the light microscope, and that its variations
show no correlation with the characters used to distinguish genera; ultrastruc-
ture, accordingly, cannot be regarded as having any taxonomic significance
above the specific level (Hustedt, 1959, pp. V-VI). Hendey (1959) came to a
similar conclusion, but added that when a large number of species have been
examined it may be possible to subdivide the genus NavicuJa Bory. Views
similar to Hustedt 's are presented by Lund (1962) in his recent review of the
criteria adopted in classifying algae.
It is probably not an unfair generalization to suggest that taxonomists are
conservative in their outlook, especially in their views about which characters
are important in classifying a particular group. They do not seize every
opportunity of using a newly discovered set of characters to produce a new
system supplanting the current one. They tend rather to keep alterations to
a minimum, apart from the addition of numerous new species and taxa of lower
rank. One of the most gratifying results of the study of diatom frustules with
the electron microscope has been that it has brought to light nothing really
surprising. Structure too fine to be resolved with the light microscope has been
demonstrated, but this was only to be expected. Nothing which could be seen
with the light microscope has been found to have a structure markedly different
from that which it was thought to have. This represents a great tribute to the
skill and acumen of those who used the light microscope at the limit of its
potentialities to elucidate the structure of the diatom valve, especially O. Miiller
(1889, 1895, 1896a, b, 1898, 1899, 1900, 19()la, b, 1909) and Hustedt (1926o,
h, 1928o, b, 1929a, b, 1935a, b). On the other hand it has meant that no re-
visions of the system have been forced upon diatom taxonomists, and in the
absence of any such pressure they have not actively pursued the question of
how far knowledge gained with the electron microscope could influence classifi-
cation above the specific level.
Although conservatism has played its part in persuading diatomists that
ultrastructure can only play a minor role in the taxonomy of the group, they
have been helped to reach that conclusion by two other factors. Both of
these have already been discussed; they are the inadequate number of species
investigated with the electron microscope and the inadequate information
about the ultrastructure of many of those examined. Thus, Hendey's (1959)
list of the diatoms investigated with the electron microscope includes only 28
identified species of Navicula, out of at least 1000 at present known, and it is
probable that the information about the structure of many of these is as inade-
quate as that which he gives about Stauroneis anceps and S. phoenicenteron
(cf., p. 397). For all other genera fewer species have been investigated, and
Ross: Classification of Diatoms 399
only in Chaetoceros Ehrenb. and Pinnularia of the larger genera is the proportion
studied greater than in Navicula.
There are two parts of the system of classification of the diatoms in which the
currently accepted taxonomy above the specific level is patently unsatisfactory:
the famiUes Navicuiaceae and Biddulphiaceae. In both, species are grouped
with others to which they seem only distantly related and separated from those
which seem close to them. In a taxonomic investigation of a small group
of species in the Navicuiaceae on which I was recently engaged, I decided
that electron stereomicrographs would be useful in elucidating a particular
point about the structures connected with the central nodule. Through the
kindness of K. Little of the Nuffield Orthopaedic Centre, Oxford, England, who
is responsible for all the micrographs illustrating this paper, these were obtained.
They showed not only the details of the central structure but also the ultra-
structure of the perforations through the valve, and the correlations between
these two suggested that ultrastructure might well form a guide to a revision
of the limits of Stauroneis Ehrenb. and possibly certain other genera, and that
the attitude of Hustedt (1959), Hendey (1959), and Lund (1962) to its use for
this purpose was unduly defeatist. These observations are being extended, and
much more needs to be done before any firm conclusions can be reached. This
paper cannot, in consequence, be anything more than a report on progress to
date, but its object will be fulfilled if it dissipates doubts about the value of
ultrastructure as a source of taxonomic characters and stimulates others to
work on similar lines.
Technique
This approach necessitates the accumulation of electron micrographs of a
large proportion of the species in the group under investigation. Many species
of diatom occur only as comparatively rare members of the assemblage con-
tained in a particular gathering. To obtain the electron micrographs needed
in a taxonomic investigation accordingly demands the use of a techniciue similar
to that used in the making of selected slides of individual specimens for the
light microscope. Reliance on serendipity, which has hitherto been the normal
practice when choosing specimens for investigation with the electron micro-
scope, will not suffice.
Each worker who makes selected slides of individual diatoms develops a
technique which suits the resources of his own laboratory and his personal
characteristics, in particular the steadiness of his hand. This account of the
method I have used for selecting individual diatoms for study with the electron
microscope, which is based upon that which I use when making selected slides
for examination with the light microscope, should be taken only as a general
guide and not as a model to be rigidly followed in all of its details.
One starts with a suspension in distilled water of chemically cleaned diatom
frustules (for methods see Hustedt, 1927, 1958, Swatman, 1937, Hendey, 1938,
1951, Leboime, 1952, van der Werff, 1955, Barber, 1962) which is known to
contain the diatom which it is desired to study. A few drops of this are allowed
to evaporate, preferably on a mica surface, to which diatoms adhere less than
they do to glass. Heat should not be used as convection currents cause the
diatoms to clump together. Diatom frustules apparently adsorb some of the
400 Annals New York Academy of Sciences
chemicals used in cleaning and liberate these slowly into the water in which
they are washed or stored. If these chemicals are present, they cause the
diatoms to stick to the mica. It is, therefore, desirable to leave the diatoms in
at least the last two washing waters for a period of 2 days or more, and to pour
off the water in which they have been stored and replace with fresh distilled
water immediately before the preparation of the strews from which specimens
are to be selected.
The actual selecting is most conveniently done under a binocular dissecting
microscope at a magnification of about XlOO. Except with the larger forms,
it is not possible at this magnification to recognize the species to be selected with
certainty. It is, therefore, necessary to locate them under an ordinary micro-
scope and to note their position relative to prominent specimens that can act
as markers. This process is facilitated if a grid is ruled on the back of the slip
of mica with the point of a needle and the scratches filled with India ink. The
micashp can then be mounted with balsam on a microscope slide. It is usually
more convenient to assemble specimens of each species to be investigated in
separate groups near the edge of the mica shp before transferring them to the
grids. When small diatoms are being dealt with, each group can then be
examined under the ordinary microscope to see that all the specimens are of the
correct species.
The necessary number of formvar-coated grids are attached to an ordinary
microscope sUde by tiny drops of gum arable at their edge. They are held
steady by this during mounting but can be readily detached for insertion in the
microscope. A label can be placed at one end of the slide giving a numbered
key to the grids. The diatoms can then be taken up individually on a bristle
from the mica shp and placed on the formvar film over the spaces in the grid.
This can usually be done without tearing the film. When the work is done in a
dry atmosphere, the diatoms at times accjuire an electrostatic charge, which
causes them to fly off the grid when it is lifted off the slide. This trouble can
be obviated by breathing gently on the grids after the diatoms have been trans-
ferred to them. After the thin film of water thus condensed on them has
evaporated, they adhere sufficiently not to fall off when the grid is placed in
the electron microscope, and will normally remain in position through a number
of insertions into and removals from the instrument.
I find it possible to transfer the diatoms freehand, even specimens the major
axis of which is between 10 and 15 n in length. For this I use a bristle mounted
on a cylindrical rod of wood about as thick as a pencil and sharpened like one
to a point at one end. The bristle is stuck to this point with about 2 mm.
protruding. Pelletan (1888) and Hustedt (1927) recommend a pig's eyelash
as the most suitable bristle and I find one very satisfactory.
A number of types of mechanical fingers for the selection of diatoms have
been developed, the most widely used probably being that designed by Meakin
(1939). These could no doubt also be used for transferring diatoms to electron
microscope grids. Stiffer bristles than those used for freehand mounting are,
however, normally used in mechanical fingers and these would be more likely
to tear the formvar films on the grids. When a mechanical finger is used to
mount diatoms for the electron microscope it will probably be advantageous to
replace its normal bristle by a more flexible one.
Ross: Classification of Diatoms 401
It has already been pointed out that stereomicrographs are much more
informative than single ones. The techniques for obtaining these and mount-
ing them for examination have been described by Little (1958, 1962). It is
also important to ensure that the micrographs are taken at a magnification and
with a resolution sufficient to show the true structure of the valve. Low
power micrographs of the specimen, which will enable its identity to be checked,
should also be taken.
New Observations on Diatom Ultrastrudure
The species originally described as Schizostauron crucicula Clrun. ex Cleve and
S. karstenii Zanon are currently placed in the genus Stanroneis, the structure
associated with their central nodule being interpreted as a bifid stauros. Speci-
mens of these two species were recently encountered in some gatherings from
Lake Tanganyika, and in the same material two undescribed species which
seemed related were also found. One of these was very similar to the two
known species, but the other had the asymmetry characteristic of the genus
Amphora Ehrenb., i.e., both its apical and its pervalvar axes were curved. Al-
though it was possible to be reasonably certain under the light microscope that
the structures associated with the central nodule were not very similar to an
ordinary stauros, details of their form could not be made out with certainty.
There was also need to confirm that the asymmetric species differed from the
others only in shape and not in any point of structure. Specimens of Schizo-
stauron crucicula, S. karstenii, and the asymmetric form were therefore examined
in the electron microscope and stereomicrographs of them were obtained.
Specimens of the type species of Stauroneis, S. phoenicenteron, and of 5". anceps
and S. smithii Grun. were also examined for comparison. These observations,
which are reported in detail by Ross (1963), confirmed that the species with a
so-called "bifid stauros" were so different from S. phoenicenteron that they
should be placed in a separate genus, for which the correct name is Caparto-
gramma Kuff. Also, S. phoenicenteron and S. anceps were found to be very
similar, but to differ greatly from 5. smithii. The results may be summarized
as follows.
(1) Stauroneis phoenicenteron (figures 1 and 2) and S. anceps (figures 3 and
4) have a stauros which is a wide but not very deep thickening of the valve.
The chambers that form their striae are elongated along the direction of the
stria, especially near the inner surface, where they are separated by a very
narrow wall. These chambers are closed on their inner side by a membrane
with fine pores in triangular tesselation and on the outer side by a membrane
with a broad slit along the direction of the stria. The length of this slit is
shorter than the length of the main part of the chamber.
(2) Stauroneis smithii (figures 5 and 6) has a deep and narrow thickening
across the valve. Its chambers are not close; they are approximately circular
and are closed on the inner side by a membrane with fine pores in triangular
tesselation and on the outer side by a membrane with a narrowly elliptical
opening of which the major axis is across the direction of the stria and is longer
than the diameter of the main part of the chamber.
(3) All three species of Capartogramma (for illustrations see Ross, 1963) have
on either side of the central nodule two, or occasionally three, deep and very nar-
Figures 1-2. Stauroneis phoenicenleron (Nitzsch) Ehrenb. In figure 2 are shown
artifact structure caused by too rigourous cleaning (r/., Helmcke el al., 1961, plate 289 to 290)
for true structure of this species. Figure 1, X2500. Figure 2, X40,000.
Figures 3-4. Stauroneis anceps Ehrenb. Figure 3, X2000. Figure 4, X40,000.
Figures 5-6. Stauroneis smitliii Grun. These specimens are somewhat eroded but in
figure 6 it is shown that the slits in the outer membrane run across the striae, figure 5,
X2500. Figure 6, X 40,000.
402
Ross : Classification of Diatoms 403
row flanges running from the central nodule to the valve margin, projecting at
right angles to the valve surface but turned towards the apices at their free
edges. Their chambers are not close; they are approximately circular and are
closed on the inner side by a membrane with fine pores in triangular tesselation
and on the outer side by a membrane with a broad slit that runs across the
direction of the stria and is longer than the diameter of the main part of the
chamber.
These observations not only confirmed that it is correct to separate the
species with a "bifid stauros" from Stauroneis and to associate the species with
amphoroid asymmetry and the symmetrical ones; they also suggested that
other species now grouped in Stauroneis might belong to separate genera. To
see whether examination of more species would provide evidence to confirm this,
S. acuta W. Sm., 5*. amphioxys Greg., and S. salina W. Sm. were examined under
the electron microscope, and more species will be as opportunity offers. The
stauros of the first two species appears under the light microscope to be broad
and narrow. The electron micrographs showed their structure to be as follows:
(4) Stauroneis acuta (figures 7 and 8) has a broad and rather shallow stauros,
as in S. phoenicenteron and S. anceps, and the ultrastructure of its striae is
similar to that in those two species.
(5) Stauroneis amphioxys (figures 9 and 10) has a broad and shallow stauros,
which extends for less than two-thirds of the width of the valve. Its striae
consist of distant circular chambers closed on the inner surface by a fine
membrane with pores in triangular tesselation. The chambers taper outwards,
i.e., they have the shape of truncated cones, but they have no membrane on
their outer side.
(6) Stauroneis salina (figures 11 and 12) has a stauros that is rather deep
at the center of the valve and becomes narrower and shallower toward the
margin. The striae consist of distant circular chambers closed on the outer
side by an oblique parallel- sided slit that is slightly longer than the diameter of
the main part of the chamber and on the inner side by a fine membrane with
pores in triangular tesselation. The valve surface is depressed between one-
third and two-thirds of the distance from the raphe to the margin and through-
out this area the chambers in the striae are more distant than elsewhere.
Taxonomic Implications
Attention is here drawn to some similarities and differences in ultrastructure
that may have a taxonomic significance; not only the original observations
recorded above but also published micrographs of various species of Navic-
ulaceae are considered. The present state of our knowledge provides only a
very tenuous basis for taxonomic speculations; the justification for indulging
in these and putting them on record is that others may be stimulated to collect
further data that will tend to confirm or refute them.
Stauroneis. Until recently the presence or absence of pseudosepta has been
treated as a character distinguishing sections within this genus (Cleve-Euler,
195vS). The close similarity which S. acuta, in which these are present, bears
in all other respects to S. phoenicenteron, in which they are absent, confirms the
view put forward by Hustedt (1959) that they are of httle taxonomic signifi-
cance. Also, Hustedt's (1959) contention that S. amphioxys (which he in-
404
Annals New York Academy of Sciences
Figures 7-8. Stauroneis acuta W.Sm. In figure 7 is shown the extension of the cham-
bers along the line of the striae, figure 8 the inner membrane with tine pores in triangular
tesselation and the iiroad slit along the line of the striae. Figure 7, X5000. Figure 8,
X 40,000.
Figures 9-10. Stauroneis amphioxys Greg. Figure 9, X2500. Figure 10, X40,000.
Figures 11-12. Stauroneis salina W.Sm. Figure 11, X2000. Figure 12, X40,000.
Ross : Classification of Diatoms 405
correctly calls S. gregorii Ralfs) and .S". salina are quite distinct species is con-
firmed,
Ultrastructure confirms the view that S. phoenicenteron , S. anceps, and
S. acuta should be placed in the same genus. S. amphioxys, S. saliua, and
S. smithii differ considerably from these and from one another. Meresch-
kowsky (1903a), on the basis of endochrome structure, removed .S\ amphioxys
and .5. salina from Slauroneis and created a new genus, Slaurophora, for the
two species. Although their ultrastructure indicates that they should perhaps
be removed from Slauroneis, it provides no contirmation for grouping them
together. Information about many more species is needed before any firm
conclusions can be drawn about the correct position of these species. S.
smithii, however, seems to be close to Capartogranima both in the structures
associated with the central nodule and in the ultrastructure of the chambers,
and S. salina bears some resemblance. It is noteworthy that Frustulia rhom-
boides var. saxonica (Rabenh.) De Toni (Helmcke et al., 1961, plates 279 to
280) has an ultrastructure almost identical with that of Caparlogramma and
S. smithii, and so also has Scoliopleura tumida (Breb.) Rabenh. (Helmcke and
Krieger, 1954, plate 177), a species grouped by Cleve (1894) not with the
other members of that genus but in his Naviculae Microstigmaticae, in which he
also included Slauroneis. This ultrastructure has certain similarities to that
found in most of the species of Pleurosigma and Gyrosigma examined. Whether
the species that possess this type of ultrastructure in common form a group of
genera more closely related to one another than to the rest of the Naviculaceae
is a question that can only be determined as more knowledge is accumulated,
but it seems that it is a possibiUty.
Amphora. As mentioned, there is a species which differs from the others
placed in the genus Caparlogramma only in shape of frustule; it has that char-
acteristic of the genus Amphora although the other species of the genus are,
like most Naviculaceae, symmetrical about the apical, transapical and pervalvar
planes. Cleve, in 1896, (p. 99) made the suggestion that the species placed in
the asymmetric genera Amphora and Cymbella Ag. were more closely related to
symmetrical species of similar valve structure than they were to one another.
The discovery of this new species of Caparlogramma adds further evidence for
the view that symmetry by itself is not a proper basis for delimiting genera.
The only species of the large and variable genus Amphora the ultrastructure of
which is known are A. cofeiformis (Ag.) Kiitz. (Helmcke and Krieger, 1953,
plate 76), A. deUcalissima Krasske (Helmcke el al., 1961, plate 294) and A.
ovalis (Kiitz.) Kiitz. (Helmcke and Krieger, 1953, plate 77, 1954, plate 181).
In A. cofeiformis and A. ovalis the ultrastructure resembles that found in
Anomoeoneis exilis (Helmcke and Krieger, 1954, plate 169) and A. serians
(Breb.) Cleve (Helmcke and Krieger, 1953, plate 68), which may indicate
relationship. Amphora deUcalissima has a cjuite different structure.
Cymbella. This is another genus which, like Amphora, is distinguished from
Navicula solely on the basis of asymmetry. Cleve (1894, p. 157) considered
that its species were most closely related to those of Navicula subgen. Navicula
(his Naviculae Lineolatae) . As far as ultrastructure is concerned, this is true
of C rabenhorslii Ross (Kolbe and Golz, 1943, plate 1, fig. 3, Helmcke and Krie-
ger, 1953, plate 75, as C. gracilis (Rabenh.) Cleve), C. turgida Greg. (Desika-
406 Annals New York Academy of Sciences
chary, 1952, figs. 17 and 18), and C. venlricosa Ag. (Desikachary, 1952, figs. 19
and 20, Helmcke and Krieger, 1953, plate 75) (c/., Xavicula cryptocephala
Klitz., Helmcke and Krieger, 1953, plate 69, N. digitoradiata (Greg.) A.
Schmidt, Helmcke et al., 1961, plate 292 and 293, N. radiosa Klitz., Helmcke
and Krieger, 1954, plate 172, and A^. viridula (Kiitz.) Kiitz., Helmcke and
Krieger, 1953, plate 73). Cymbella delicatnla Klitz. (Helmcke and Krieger,
1954, plate 180) and C. mexicana (Ehrcnb.) Cleve (Okuno, 1956, plate 21,
fig. 2), however, each have an ultrastructure which is different from that of
these species and from each other's. Electron micrographs of other species of
the genus have been published but none give adequate pictures of the ultra-
structure.
Maslogloia. The ultrastructure of M. braunii Grun. (Helmcke and Krieger,
1953, plates 57 and 58, 1954, plate 159) and M. smitliii Thwaites ex. W. Sm.
(Helmcke and Krieger, 1954, plate 160) is similar and resembles that of the
only two species of Navicula subgen. Lyraneis Freng. of which adequate electron
micrographs are available, viz.: N. forcipata Grev. (Helmcke et al., 1961, plate
291) and N. pygmaea Klitz. (Helmcke and Krieger, 1953, plate 71). Maslogloia
angulata Lewis (Okuno, 1957, plate 7, fig. 2) and M. fimbriata Cleve (Okuno,
1953, plate 1, fig. 3) resemble each other in their ultrastructure, but this is
quite different from that of M. braunii and M. smithii.
Discussion
The principles of taxonomy have recently been much discussed, and from this
discussion it has emerged that the amount of overall similarity is the only basis
for a satisfactory taxonomic classification (Cain, 1962, Sneath, 1962). To
accord overriding importance to a particular character, or to characters derived
from a particular structure, even if there are a priori grounds for considering
these of particular importance, results in an artificial and unsatisfactory system.
Almost without exception, however, diatoms have been classified solely on
the basis of the symmetry and structure of their siliceous frustule as seen under
the light microscope: although this provides comparatively few characters,
some of these, in particular symmetry, have been treated as having an im-
portance overriding that of the others. This concentration of attention on the
frustule has not been based upon any a priori reasoning but purely on con-
venience; in both fossil and recent material the valves are always present and
recognizable, and provide sufficient information for identification at the specific
level.
The current classification of the Naviculaceae rests on such a basis. The
species are separated into genera on the common possession of a single charac-
ter, or a combination of only two or three, all drawn from the structure of the
frustule. Some of the genera so characterized are probably natural groups,
e.g., Diploneis Ehrenb., Neidium Pfitz., and Pinnularia; others contain very
diverse elements, e.g., Amphora and probably Maslogloia Thwaites ex. W. Sm.
and Slauroneis. The species that do not possess any characteristic that has
been seized on as a mark of generic distinction are left in the very large genus,
Navicula, a hotchpotch of species of diverse affinity. The little that we already
know of the ultrastructure of the Naviculaceae shows that it provides a series
Ross: Classification of Diatoms 407
of characters to some extent culling across the present classification. Ultra-
structure, however, provides few characters and a system based solely upon it
would be as open to criticism as one based solely upon the structure of the
valve as seen under the light microscope. All of the information about the
frustule, whether obtainable with the light microscope or the electron micro-
scope, must be taken into consideration with any that can be obtained about
other characters.
A few authors have attempted to use characters from the cell contents, in
particular the form of the chromatophores, for delimiting genera within the
Naviculaceae (Pfitzer, 1871, Mereschkowsky, 1901a,6, 1902, 1903(7,6) or sub-
genera within Navicula (Karsten, 1899). However, except where these groups
could also be readily distinguished by characters of the valve, e.g., Anomoeoneis
Pfitz. and Neidium, they have not been adopted by subsecjuent authors. The
principal reason that there has been no further work along these lines is a matter
of technique. The greatest possible amount of detail in the structure of the
valves of diatoms can be seen most easily under the light microscope if all of
the organic matter is removed and the frustules mounted in a medium of high
refractive index. Diatomists have rarely used any other method of making
preparations and all collections of diatoms consist almost entirely of specimens
treated in this way. They provide information perfectly adecjuate for identifi-
cation, and hence workers on floristics and ecology have had no incentive to
change their technique. These have been the chief fields of work of virtually
all diatomists throughout this century and even when they have turned their
attention to true taxonomy they have not altered their methods. It may be
that it would not have been possible before the phase-contrast microscope was
available to devise a technicjue which made both the fine detail of the valve
structure and the cell contents visible in the same specimen. It would seem,
however, that it was not attempted. The justification for ignoring the cell
contents in taxonomic work has been the contention, also used in connection
with ultrastructure, that a classification by chromatophore number, shape, and
disposition within the cell runs counter to the currently accepted one (Peragallo,
1907). This criticism is valid insofar as it is directed against a classification in
which characters of the chromatophore are accorded overriding importance, but
it is not a reason for ignoring the cell contents completely.
It has been pointed out that the classification of the Naviculaceae is on a very
unsatisfactory basis, at least above the specific level, and there is no reason for
supposing that it is much better in other families of diatoms. Cell contents
and ultrastructure provide characters of which the distribution does not, in
places, accord with the current classification. There is no justification for
arguing from this that variations in these features occur at random and have no
taxonomic significance. To do so is to attach overriding importance to the
particular characters of the frustule on which emphasis is placed in the current
classification; not even a priori grounds have been advanced for this. Instead
of arguing in this way from the lack of correspondence between the current
classification and the distribution of types of cell contents and ultrastructure,
this discrepancy should be regarded as an indication that there is a need for a
new classification based upon the extent of overall resemblance with these
features taken into account.
408 Annals New York Academy of Sciences
Future Developments
At present the data required to construct a classiiication by this method is
not available. Progress in diatom taxonomy depends upon its being obtained.
So far as ultrastructure is concerned, there are techniques for collecting the
data (cf., p. 399). The more difficult problem is to make it available. As can
be seen when Helmcke and Krieger's (1953, 1954, Helmcke et al., 1961) work is
compared with other published electron micrographs of diatoms, the only
method of reproduction that is really adequate is the making of photographic
prints. The cost of pubUcation of sufficient of these to cover most species of
diatoms would be prohibitive. The most feasible method of building up files
of micrographs will be by the exchange of duplicate prints between workers, or
their institutions, in much the same way as herbarium specimens are now ex-
changed. It is to be hoped that diatomists who have the facilities for electron
microscopy will enter into such a scheme. The desirability of stereomicro-
graphs has already been stressed, and also the necessity for adequate resolution.
A low magnification micrograph permitting verification of identity should
accompany those showing the detail of the ultrastructure, and adequate docu-
mentation of the origin of the specimen is essential.
Collection of information about cell contents, on the other hand, depends
upon the development of a technique of preparation that will enable details of
both this and the valve structure to be seen in the same specimen. Now that
the phase-contrast microscope is available, this should be possible. I plan to
attempt it in the immediate future, but, in the words of the old proverb, two
heads are better than one, and there is more likelihood of success if others also
try to find a method. When such a technique is available, the same problem
as with ultrastructure will arise: the examination of large numbers of species
and the dissemination of the resulting information so that, as far as recent
diatoms are concerned, a volume of knowledge about cell contents comparable
to that about valve structure is available. Here again, the quantity involved
is Ukely to make publication impossible and the most satisfactory alternative
will probably be exchange of preparations.
Not until we know the ultrastructure and the cell contents of most of the
species in a group will it be possible to consider whether, and if so in what way,
the taxonomy of the group can be remodeled on sounder lines. At present all
that is pertinent is to suggest that the methods of numerical taxonomy (Sneath
and Sokal, 1962) are likely to be of great use at that stage. As Sneath (1962)
has pointed out, at least 40 or 50 independent characters of each operational
taxonomic unit {e.g., individuals being classified into species or species being
classified into higher groups) need to be taken into consideration when using
the method of numerical taxonomy to construct a natural classification. If, as
has been normal practice, we rely on intuition rather than calculation to eval-
uate overall resemblance, our judgments are likely to be sound only if we take
note of a comparable number of characters. It is this which makes it essential
that diatom taxonomists should no longer confine themselves to studying
cleaned frustules under the light microscope, but should observe the cell con-
tents and the ultrastructure and make use of the information these provide in
their classifications.
Ross: Classification of Diatoms 409
Summary
Our present knowledge of the ultrastructure of diatoms covers only a very
small proportion of the total number of species, and some of the published infor-
mation is inadecjuate or misleading. The variations in types of ultrastructure
found do not, in a number of cases, correspond with the current classification,
which is based almost entirely upon characters of the valve as seen under the
light microscope. On the other hand, the observations made with the light
microscope have not been contradicted by work with the electron microscope.
For these reasons it has been contended that ultrastructure does not provide
information that can be used in diatom taxonomy. This view is criticized.
If the characters of the ultrastructure are to be used in diatom taxonomy,
information about most species in a group is needed. As many species are
often sparsely represented in gatherings, individual specimens need to be
selected and mounted for examination in the electron microscope. A technique
is described.
In a study just completed, electron microscopy has confirmed that a small
group should be removed from Stauroneis and placed in a separate genus. A
continuation of this work now in progress points to the need for further division
of Stauroneis, and there are indications that ultrastructure may provide infor-
mation that will assist in a revision of the present unsatisfactory generic classi-
fication of the Naviculaceae. In such a revision the characters of the frustule
structure as seen under the light microscope, of the ultrastructure, and of the
cell contents should all be given equal weight. It is, therefore, necessary to
obtain information about the ultrastructure and cell contents of a large propor-
tion of the species in the family: a prerequisite for this is the development of
a technique for preparing specimens in such a way that both their cell contents
and the structure of their frustules can be studied.
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MORPHOLOGY OF REPRESENTATIVE BLUE-GREEN ALGAE
Roy E. Cameron
Jel Propulsion Laboratory, California Inslilule of Technology, Pasadena, Calif.
The blue-green algae, of the phylum Gyanophyta or Schizophyta, belong to
the class designated variously as the Cyanophyceae, Schizophyceae, or Myxo-
phyceae, and are plants of a gelatinous, stony, mealy, or leathery nature.
They are tirm or soft, extremely tough, and impregnated with salts or mucus
and easily disrupted when the gelatinous material surrounding them is of slight
viscosity. Their forms vary in size from micro- to macroscopic and in shape
they occur as spheres, cushions, strata, or cyhnders. The growth habit is
frequently centripetal, and depending on the inherent characteristics of the
plant and its environment, the adult plant can be a single cell and of less than
1 /x in diameter or a spreading plant of up to 1 m. in extent. They are cosmo-
politan in nature and are found chiefly on soil and in water but also in a variety
of other habitats wherever moisture, temperature, energy supply (sunlight),
/>H, biogenic salts, respiratory gases (CO2 and O2), and other conditions for
growth and reproduction are favorable. They share with the bacteria a
unique ability to survive, as well as reproduce, at the extreme limits of the
natural environment.
The blue-green algae are considered to be an ancient group of plants ex-
tending back to the Archeozoic (Tilden, 1935) although the geological record
is difficult to determine because they have few hard parts. The evidence of
their presence is attributed frequently to calcareous and sihcious strata and
cushions and very ancient deposits of tufa, marl, travertine, and sinter de-
veloped by activities of mainly filamentous forms (Drouet, in press). The
fossil remains that have been attributed to blue-green algae have not provided
evidence as to their evolutionary sequence (Fritsch, 1942).
Characteristics of blue-green algae show that they resemble nonalgae as well
as other algae. Because they resemble bacteria in some respects, i.e., in
having no organized nuclei or true cell walls and a similar mode of cell division,
they have been classified as coordinate with the bacteria in the Schizophyceae
(Breed el al., 1958). It is also known that both groups contain members that
produce spores, and some have demonstrated the ability to fix atmospheric
nitrogen. Most contain a ,a'-diaminopimehc acid, and their concurrence in
similar ecological habitats and in cultures attests to similarities in certain
physiological characteristics. Sufficient differences, however, are found for
separation of bacteria from blue-green algae. Blue-green algae are rarely
parasitic, pigmentation is not comparable with that of the bacteria, oxygen is
evolved as a result of photosynthesis, movement is of a gUding or oscillating
nature — there are no flagella — and the size range of cells and plants is much
greater. Heterotrophic, colorless forms of blue-green algae usually can be
attributed to bacteria or fungi which have been misinterpreted (Drouet, in
press), unless one accepts an organism such as Beggialoa as a colorless form
(Pringsheim, 1949). Morphologically, the Myxophyceae show a greater
structural complexity and diversity than bacteria, but less so than other algal
412
Cameron: Morphology of Blue-Green Algae 413
groups. It is recognized that the major taxa of algae may show Httle afi&nity
with each other (Papenfuss, 1955), but they are still grouped on the basis of
an "algal-type" of organization, the parallelism cited in the evolution of plant-
body types, the morphology and physiology of the individual cells (Smith,
1950), photosynthates, and especially in regard to the principal protoplasmic
pigments (Dougherty and Allen, 1960). Blue-green algae contain the phyco-
bilins C-phycoerythrin and C-phycocyanin not found in other algae or bacteria
although phycobilins are characteristic for red algae and have also been found
in other groups, e.g., green algae and cryptomonads (Eocha, 1960). Chlo-
rophyll a, and /3-carotene are shared in common with other algal groups, but
certain carotenes and xanthophylls are unique to blue-green algae (Goodwin,
1960). By means of a fluorescence microscope, the pigments are found to show
an orangish red, red, or reddish brown fluorescence in a darkened background.
Photosynthates include polysaccharides and glycoproteins, and cell contents
may become brown when treated with I-KI solution.
In some species cells form reproductive spores which are denoted from
other cells by their larger size, thick walls, and more resistant nature. Color-
less cells, or heterocysts, are also formed in some species. Their function and
necessity are doubtful although they have been observed to germinate (Geit-
ler, 1921), and they have been noted to anchor the trichome to the firm sheath
(Bornet and Flahault, 1886). Endospores, undifferentiated reproductive cells,
are formed by 1 family, the Chamaesiphonaceae, but for filamentous taxa, the
random death of individual cells permits segments of trichomes, or hormogonia,
to propagate the species when moisture is available. Cell division is by fission,
i.e., constriction into two parts, or by centripetal progression of a dividing
membrane through the protoplast. Reproduction is frequently by fragmenta-
tion. Sexual reproduction, although recently reported for a strain of Nostoc
muscorum (Lazaroff and Vishniac, 1961), is not considered characteristic for
the group.
Cytologically, the cells are found to have the aforementioned pigments, pro-
tein granules, pseudovacuoles of a gaseous nature, and occasionally vacuoles,
within a containing membrane. Pseudovacuoles are characteristic of plank-
tonic "water-blooms"; they appear black in transmitted light, red in reflected
fight, and are dissolved when treated with detergent. Vacuoles occur in old
or degenerated cells, particularly as the environment becomes anaerobic. The
protoplast is said to be clearly divisible into two parts (Desikachary, 1959) : the
pigmented, peripheral chromoplasm and the central colorless centroplasm.
It also may be recognized, however, that such a strict differentiation is super-
ficial. Feulgen positive granules are found particularly in the centroplasm
(Cassel and Hutchinson, 1954). Pigments are reported to be in grana-like
lamellae of the chromoplasm according to electron microscope studies (Niklo-
witz and Drews, 1956). Few studies on nucleoproteins of blue-green algae
have been undertaken although it has been reported that these are similar to
those recorded for tissues of other organisms (Biswas, 1961).
Classification
The Myxophyceae have been classified in one or more orders. The classi-
fication followed here considers the blue-green algae to be in a single order.
414 Annals New York Academy of Sciences
the Chroococcales, and 8 families which diverge in morphological characteristics
in a single evolutionary sequence (Drouet, in press). The coccoid families
include the Chroococcaceae, Chamaesiphonaceae and Clastidiaceae. Fila-
mentous famihes consist of the Stigonemataceae, Nostocaceae, Rivulariaceae,
Scytonemataceae, and Oscillatoriaceae. Consideration of the first three
families is given according to a recent comprehensive revision (Drouet and
Daily, 1956), that of the other famihes follows the starting points according
to the International Rules of Nomenclature (Gomont, 1892; Bornet and
Flahault, 1886-1 888a and b).
A representative member of each family is given (figures 1 to 8). These
members are not to be construed as "typical" because there can be wide varia-
tion inter- and intraspecifically in nature as well as in culture. However,
Anacystis montana (figure 1) is the most frequently collected of the coccoid
species (Drouet, 1954). Nostoc musconim (figure 5) is of common occurrence
on soil, and Calothrix parielina (figure 6) is of wide distribution in moist
habitats (Fan, 1956). Scytonema Iwfmannii (figure 7) is also a frequently
encountered species, and Microcoleus vaginatus (figure 8) is an oscillatorioid
member often found on soil as well as in aquatic habitats. These species have
been recently described with others found in the United States north of the
Rio Grande River (Drouet, 1959).
The Chroococcaceae consist of uni- or multicellular, micro- or macroscopic
plants which are subaerial or aerial, free, as cushions or strata. The cells are
spherical, discoid, ovoid, ellipsoid, cylindrical, or pyriform, in regular or ir-
regular order, each cell dividing into 2 ecjual daughter cells which become sepa-
rated from each other by the gelatinous matrix. Reproduction is by fragmen-
tation as for most of the blue-green algae, but in some cases by cell division.
Under most conditions, except for Coccochloris, cells are found in the process
of division. Species of Anacystis, represented here by A. montana (figure 1)
have cells at first hemispherical, later becoming spherical. The cells then di-
vide in 3 planes perpendicular to each other. Coccochloris resembles Anacystis,
but has subspherical to long cylindrical cells and division at right angles to the
long axis. Other genera include Johannesbaptistia which has a linear series of
discoid cells within an elongate gelatinous matrix, and AgmeneUum, Micro-
crocis, and Gomphosphaeria which have cells that divide successively in 2
planes perpendicular to each other. Plants of the first two genera are plate-
like, whereas those of the latter genus are unique in that the cells are frequently
cordiform in division and the remains of individual sheaths form branched
stalks radiating from the center of the plant.
The Chamaesiphonaceae contain one genus, represented here by Entophy-
salis lemaniae (figure 2). Plants of this family are uni- or multicellular,
aquatic, micro-, or macroscopic. The cells are at first solitary and affixed to
the substratum, each dividing serially into first unequal then equal daughter
cells which are not separated by gelatinous material. Subsequently, a stratum
or cushion is developed above the substratum, and branched filaments grow
downward from this into the substratum. Any cell is then capable of enlarg-
ing and dividing internally into a few or many endospores. Reproduction is
by fragmentation as well as by endospores.
Plants of the Clastidiaceae are infrequently collected. The plants consist
1
rdd^ ^
4
A
W
rVii'V
Figure 1. Anacystis montaiia (Lighlfoot) Drouet & Daily.
Figure 2. Entophysalis lemaniae (Agardh) Drouet & Daily.
Figure 3. Slichosiplwn sansibaricus (Hieronymusj Drouet & Dail\-.
Figure 4. Sligonema pani/onne (Agardh) Bornet & Flahault.
Figure 5. Nostoc niuscontm Agardh.
Figure 6. Calothrix parietina (Nageli) Thuret.
Figure 7. Scylonema kofmannii Agardh.
Figure 8. Microcoletis vaginatus (Vaucher) Gomont.
415
416 Annals New York Academy of Sciences
of elongate, epiphytic unicells contained in thin gelatinous sheaths and at-
tached to the substratum by basal developments of the sheath. The entire
protoplast is found to divide into a uniseriate chain of rounded or compressed
spherical cells which usually remain united by their membranes. As the cells
enlarge, the trichome then bursts through the sheath of the mother cell, and
the cells upon dissociation from each other then elongate into a new unicell
and secrete new sheaths. The family is represented by two small genera,
Clastidium and Stichosiphon, each containing one species. 5. sansibarkus
(figure 3), has a smooth apex, whereas plants of C. setigerum terminate in a
spinelike projection of the sheath at the apex.
Plants of the Stigonemataceae are floccose, feltlike, cushion-shaped, or
spherical. The filaments are free or imbedded in a gelatinous matrix, the
trichomes are branched, and the cells are uni- or multiseriate with division
occurring in planes perpendicular to or parallel with the axis of the filament.
Heterocyst formation is random, intercalary or terminal on short branches.
Cell division in planes perpendicular to the axis of the trichome is followed by
a growth in length of cells at filament apices which forms the resulting elongate
and branched filaments. Cell division also occurs in planes parallel to the
axis of older filaments with consequent increase in diameter and in the forma-
tion of subsequent branches. Reproduction is by fragmentation. The family
is represented here by Stigonema panniforme (figure 4). Members of this
genus have filaments which soon develop multiseriate cells connected by proto-
plasmic strands. Other prominent genera include Capsosira which has up-
right and parallel filaments that form compact cushion-shaped plants, Nosto-
chopsis which has radial filaments within a gelatinous matrix of coalesced
sheaths and develops intercalary, pedicellate, or sessile heterocysts, and Hapa-
losiphon and Fischerella which contain trichomes of uniseriate cells except in
the older basal portions of the plant. The latter two genera also exhibit
scytonematoid branching.
The Nostocaceae contain aquatic or terrestrial plants which are free or at-
tached to a substratum. The sheaths are mucous, gelatinous, membranaceous,
or well hydrolyzed and absent. Trichomes are unbranched, frequently twisted
and entangled; all of the cells divide at relatively the same time, and inter-
calary or terminal heterocysts are present. Reproduction is by fragmentation
or by spores that are formed in most species. The trichomes of Anabaena are
free or form a fragile layer; the matrix is composed of hyaline, hydrolyzed
sheaths. Spores are variously situated in relation to the heterocysts. Tri-
chomes of planktonic Raphidiopsis and Aphanizomenon resemble those of
Anabaena except that the end cells are pointed in Raphidiopsis and colorless in
Aphanizomenon. Trichomes of Nostoc, and Wollea develop within a gelatinous
matrix of definite shape; all cells may apparently become spores or hetero-
cysts. In species of Nostoc, e.g., N. muscorum (figure 5), the trichomes
become much contorted, whereas i^ Wollea they are relatively straight. Cylin-
drospermum has comparatively short trichomes with terminal, solitary hetero-
cysts and adjacent spores. Cells and spores of Nodularia are compressed or
disciform in rather straight trichomes. Hydrocoryne, a rarely collected species,
apparently forms no spores and has discrete although readily hydrolyzed cylin-
drical sheaths.
Cameron: Morphology of Blue-Green Algae 417
In the Rivulariaceae plants are aquatic or in moist habitats, spherical, cush-
ion-shaped, crustaceous, velvety, feltlike, or brushlike. The filaments are
branched or unbranched, radiate from the center of the plant outward, or are
parallel and tufthke. Trichomes are unbranched, thick at the base, tapering
above, each ending in a colorless hair. Heterocysts are basal or intercalary,
although absent in some species. Cell division is transverse and primarily in
the middle of the trichome above the heterocyst. Reproduction is by frag-
mentation and spores. Amphithrix is a thin crustaceous plant, which lacks
heterocysts and has terminal ephemeral hairs. Filaments of Calotlirix, as
represented by the most frec^uently collected species, C. parietina (figure 6)
(Fan, 1956) is usually unbranched, whereas the filaments of Dichothrix are
more or less dichotomously branched, the bases of the branches included for a
short distance within the parent sheath. Rkularia and Gloeotrichia have
filaments of coalesced sheaths that develop radially to form spherical or cush-
ion-shaped plants. No spores are formed in Rivularia but in Gloeotrichia they
are thick walled and next to the basal heterocysts.
The Scytonemataceae contain irregularly cushion-shaped or matlike plants
with branched filaments that are single or geminate. The sheaths are firm,
tubular, at first colorless, but later yellow, or brown. Trichomes each consist
of a single row of cells, one or more included in a sheath. Heterocysts and
spores are variously disposed. Cell division primarily occurs behind the tip
of the trichome, resulting in lateral perforation of the sheath by dividing and
elongating cells which then give rise to single or geminate branches. Repro-
duction is usually by fragmentation of the trichome or filament, although one
genus, Aulosira, is unique in that all vegetative cells are capable of forming
thick walled cylindrical spores or heterocysts. Branching varies with the
genera, depending upon its relation to the heterocyst. In species of Scylonema,
e.g., S. hofmannii (figure 7), branches may be single and near a heterocyst,
but commonly arise at a point somewhat remote from the heterocyst and are
geminate. Branches in Tolypothrix are single and arise at the heterocysts.
Branches of Desmonema are included within a common sheath. Filaments of
Fremyella are short, uncommonly branched, and have basal heterocysts.
The Oscillatoriaceae is the largest family of the group. It is comprised of
plants developing as layers or cushions and is differentiated from other families
in that the trichomes do not form spores, heterocysts, or hairs. The cylindri-
cal trichomes consist of 1 row of cells in branched or unbranched filaments;
the broken ends or hormogonia regenerate in a mode characteristic for the
various taxa. In many species, a terminal cell develops a thickened outer
membrane. Cell division occurs throughout the entire trichome and at rela-
tively the same time. Reproduction is by fragmentation. The current divi-
sion of the genera is based largely upon the structure of the sheath (Gomont,
1892) and is in need of further study for clarification. The sheaths of Oscilla-
tor ia, Arthrospira, and Spirulina are seldom discernible even by application of
various staining technicjues. The sheaths of Microcoleiis, e.g., M. vaginatus
(figure 8), and Sckizothrix contain one to many trichomes within diffluent
or firm sheaths. Usually only one trichome is found in firm sheaths of Plec-
lonema, Lyngbya, and Porphyrosiphon. Sheaths of the latter become red or
purple; sheaths of Lyngbya may be hyaline or become yellowish-brown.
418 Annals New York Academy of Sciences
Plectonema may show scytonematoid branching. In Symploca, the sheaths
are discrete and contain one trichome; adhering filaments form fascicles at
the surface of the plant. The sheaths of Phormidium are thin, hyaline, and
become diffluent.
General Ecology
Ecological studies on the Myxophyceae are quite limited. Most attention
has been given to the collection of organisms from a variety of habitats and
some information is available on their geographical distribution. In general,
the blue-green algae occur in all parts of the world where light and water are
available. Individual species may be distributed in the various climatic
zones, but others are found at extreme limits of the environment, from cold
regions such as the Antarctic or in the cryoconite of Greenland (Gerdel and
Drouet, 1960), and from the low elevation of the Dead Sea to mountains over
14,000 feet in altitude. They are a part of the salt marsh flora (Chapman,
1960), occur in extremely saUne Great Salt Lake (Flowers), hard and soft
waters (Palmer, 1959) and hot, dry desert soils (Cameron, 1961; KiUian and
Feher, 1939). Planktonic forms, frequently a single species, may grow pro-
lifically in favorable seasons when nitrates and phosphates are high and in some
cases release obnoxious toxins (Prescott, 1959). Aquatic species have also
been found in the lower subUttoral zone where Ught intensity is low (Ruttner,
1953), and in hot springs where the temperature may reach 86° C. (Kaplan,
1956). Other aquatic habitats can include industrial wastes with a high con-
tent of metals and acids (Palmer, 1959). More exotic habitats include associa-
tions with animals such as sponges, corals, and snails. In barren, eroded soil,
on wood, in sewage, on and under light transmitting rocks, and even in areas
of comparatively recent volcanic activity (Treub, 1888), it has been found that
blue-green algae are able to grow and survive. Furthermore, it has been de-
termined that the Eh range of blue-green algae is from —0.200 to +0.700 volts
and the ^H from 1.5 to 11 (Baas Becking et al., 1960). That they can resist
desiccation for decades has been shown in the revival of species from old, stored
soils (Bristol, 1919). Reproduction can be quite rapid, and oscillatorioid
forms can develop macroscopic growth in a few hours on desert soil which has
remained dry for a number of years. Prolonged resistance to desiccation has
been found in a dried herbarium specimen of nonsporeforming Nostoc commune
previously revived after 88 years of storage (Lipman, 1944), and later revived
after an additional time period of 19 years (Cameron, in press). Resistance is
also found to low temperatures. At —80° C, algae, in combination with
fungi as Uchens have been found to survive, and at —30° C. to even photo-
synthesize slowly (James, 1955). Parasitism of certain species of blue-green
algae by fungi is not uncommon (Drouet, 1954), and where optimal conditions
prevail for one of the organisms, the other is overwhelmed. The association
between the alga and the fungus in forming and maintaining the hchen is ex-
ceedingly complex and although the alga excretes antibiotic substances, the
fungus can have a lethal effect on the alga (Henriksson, 1961).
Environmental conditions which are most favorable for the entire group of
blue-green algae are difficult to determine and correlate. Many species have
been named as distinct on the basis of the kind of environment in which they
Cameron : Morphology of Blue-Green Algae 419
occur. Distinctions have also been made between plants which differ morpho-
logically in some details but are actually only growth forms of the same species
found in a slightly different environment. Microcoleus, for example, has been
considered as a multitrichomatous organism occurring only on soils, and blue-
green algae are said to be more abundant in cultivated than in noncultivated
areas (Tiffany, 1951). Such restrictions have not been found valid upon fur-
ther study. An exhaustive review of specimens and their subsequent enumera-
tion on the basis of pertinent characteristics, as for the coccoid Myxophyceae
(Drouet and Daily, 1956) is needed for the other blue-green algae. Culture
studies, although valuable, are often confusing in that the cultured plant can
lose its identity with more famiUar forms occurring in the natural environ-
ment. Changes in any one of the environmental conditions can result in
plants differing from the original organism in form and structure, as well as
regeneration rate, cell division, size, shape, and contents. Pleomorphism
among the blue-green algae will remain as a confusing factor until an extensive
review has been made of all available material in herbaria and in other collec-
tions, and investigations performed on the growth of organisms in both natu-
ral and induced environments.
A cknowledgment
Appreciation is expressed to Francis Drouet, who has made suggestions,
loaned herbarium specimens and reference materials, and given generously of
his time and assistance.
Rejerences
Baas Becking, L. G. M., I. R. Kaplan & D. Moore. 1960. Limits of the natural en-
vironment in terms of pH and oxidation-reduction potentials. J. Geol. 68: 243.
Biswas, B. B. 1961. Studies on the nucleoproteins of Nostoc muscorum. Trans. Bose
Research Inst. Calcutta. 24: 25.
BoRNET, E. & C. Flahault. 1886. Revision des Nostocacees heterocystees contennes dans
les principaux herbiers de France. Ann. sci. nat. Botan. et Biol, vegetale. 3: 323.
BoRNET, E. & C. Flahault. 1887. Revision des Nostocacees heterocystees contennes
dans les principaux herbiers de France. Ann. sci. nat. Botan. et Biol, vegetale. 4: 343.
BoRNET, E. & C. Flahault. 1888ti. Revision des Nostocacees heterocystees contennes
dans les principaux herbiers de France. Ann. sci. nat. Botan. et Biol, vegetale. 5: 51.
BoRNET, E. & C. Flahault. 1888&. Revision des Nostocacees heterocyste'es contennes
dans les principaux herbiers de France. Ann. sci. nat. Botan. et Biol, vegetale. 7: 177.
Breed, R. S., E. G. D. Murray & N. R. Smith, Eds. 1957. Bergey's Manual of Deter-
minative Bacteriology. Ed. 7. The Williams & VVilkins Co. Baltimore.
Bristol, B. M. 1919. On the retention of vitality by algae from old stored soils. . New
Phytol. 18: 92.
Cameron, R. E. 1961. Algae of the Sonoran Desert in .\rizona. Ph.D. Thesis. Library,
Univ. of Arizona. Tucson.
Cameron, R. E. Species of Nostoc Vaucher occurring in the Sonoran Desert in Arizona.
Trans. Am. Microscop. Soc. In press.
Cassel, W. a. & W. G. Hutchinson. 1954. Nuclear studies on the smaller Myxophyceae.
Exptl. Cell Research. 6: 134.
Chapman, V. J. 1960. Salt Marshes and Salt Deserts of the World. Interscience Pub-
lishers. New York.
Desikachary, T. V. 1958. Cyanophyta. Indian Council of .Agricultural Research. New
Delhi, India.
Dougherty, E. C & M. B. .\llen. 1960. Is pigmentation a clue to protistan phylogeny?
In Comparative Biochemistry of Photoreactive Systems. : 129. M. B. Allen, Ed.
Academic Press. New York.
Drouet, F. 1954. Parasitization by fungi in the coccoid Myxophyceae. Vlllth Int. Bot.
Cong. Paris, Rapp. et Comm. 17: 48.
420 Annals New York Academy of Sciences
Drouet, F. 1959. Myxophyceae. In Fresh-water Biology, Ed. 2. : 95. W. T. Ed-
mondson, Ed. John Wiley & Sons. New York.
Drouet, F. Cyanophyta. Encyclopedia of Science & Technology. McGraw-Hill Book
Co. New York. In press.
Drouet, F. & W. A. D.ailv. 1956. Revision of the coccoid Myxophyceae. Butler Univ.
Botan. Studies. 12: 1.
EocHA, C. 1960. Chemical studies of phycoerythrins and phycocyanins. In Compar-
ative Biochemistry of Photoreactive Systems. : 181. M. B. Allen, Ed. Academic
Press. New York.
Fan, K. C. 1956. Revision of Calotlinx Ag. Rev. Alg. N.S. 2: 154.
Flowers, S. Undated. The blue-green algae of Utah. Mimeograph. Univ. of Utah
Press. Salt Lake City.
Fritsch, F. E. 1942. The interrelations and classification of the Myxophyceae (Cyano-
phyceae). New Phytol. 41: 134.
Gerdel, R. W. & F. Drouet. 1960. The cryoconite of the Thule area, Greenland. Trans.
Am. Microscop. Soc. 79: 256.
Geitler, L. 1921. Versuch einer Losung des Heterocysten-problems. Sitzber. Akad.
VViss. Wien, Mat.-Naturw. Kl. Abt. 1. 130: 223.
Goodwin, T. W. 1960. Algal carotenoids. In Comparative Biochemistry of Photoreac-
tive Systems. : 1. M. B. Allen, Ed. Academic Press. New York.
GoMONT, M. 1892(7. Recherches des Oscillariees (Nostocacees Homocystees). Ann. sci.
nat. Botan. et vegetale. 15: 263.
GoMONT, M. 1892/). Recherches des Oscillariees (Nostocacees Homocystees). Ann. sci.
nat. Botan. et vegetale. 16: 91.
Henriksson, E. 1961. Studies in the phvsiology of the lichen Collema. IV. Physiol.
Plant. 14: 813.
James, P. E. 1955. The limits of life. J. Brit. Inler])lanet. Soc. 14: 265.
Kaplan, I. R. 1956. Evidence of microbiological activity in some of the geothermal
regions of New Zealand. New Zealand J. Tech. 37: 639.
KiLLiAN, C. & D. Feher. 1939. Recherches sur la microbiologic des sols desertiques.
Encvclopt'die periodifjue sci. mc'd-biol. 21: 1.
Lazaroff, N. & VV. ViSHNiAC. 1961. The ])articipation of filament fusion in the develop-
mental cycle of Nostoc musconini. Bacteriol. Proc. 61: i^.
LiPMAN, C. B. 1944. Longevity in microorganisms. /;; Science in the University. : 211.
Univ. of California Press. Berkeley, Calif.
NiKLOwiTz, VV. & G. Drews. 1956. Beitriige zur Cytologic der Blaualgen. Arch. Mikro-
biol. 24: 134.
Palmer, C. M. 1959. Algae in water supplies. Public Health Service Publication No. 657.
U. S. Govt. Print. Off.
Papenfuss, G. F. 1955. Classification of the algae. In A Century of Progress of the
Natural Sciences, 1853-1953. : 115. Cahf. Acad. Sci. San Francisco, Calif.
Prescott, G. W. 1959. Biological disturbances resulting from algal populations in stand-
ing waters. In The Ecology of Algae. : 22-37. Special Publication No. 2. Pyma-
tuning Laboratory of Field Biology. Univ. of Pittsburgh Press. Pittsburgh, Pa.
Pringsheim, E. G. 1949. The relationship between bacteria and Myxophyceae. Bac-
teriol. Rev. 13: 47.
Ruttner, F. (Frey, D. G. & F. E. J. Fry, Trans.). 1953. Fundamentals of Lmnnology.
Ed. 2. Univ. of Toronto Press. Toronto.
Smith, G. M. 1950. The Fresh-water Algae of the United States. Ed. 2. McGraw-Hill
Book Co. New York.
Tiffany, L. H. 1951. Ecology of fresh-water algae. In Manual of Phycology. : 293.
G. M. Smith, Ed. Chronica Botanica Co. Waltham, Mass.
Tilden, J. E. 1935. The Algae and Their Life Relations. Univ. of Minnesota Press.
Minneapolis, Minn.
Treub, M. 1888. Notice sur la nouvelle flora de Krakatau. Ann. Jard. Botan. Buiten-
zorg. 7:221.
LORICAE AND CYSTS IN THE CHRYSOPHYCEAE
Pierre Bourrelly
Museum National d'Hisioire Natiirelle, Paris, France
The unicellular algae, solitary or colonial, often have their cytoplasm enclosed
within shells of various shapes and kinds called loricae or thecae. These
thecae are found in numerous phyla of algae: Euglenales (Trachelomonas,
Strombomonas), Volvocales (Phacotus, Coccomonas), Dinophyceae (Peri-
dinium, Dinophysis, Exuviella), and in numerous Chrysophyceae and Craspedo-
monadinae.*
The Chrysophyceae may have, in addition, a phase of dormancy or of resist-
ance in the form of siliceous cysts or statospores. These cysts always have an
endogenous origin, and may arise from a simple encystment of a vegetative
cell, or, on the contrary, of a zygote resulting from a autogamy or from an
isogamic fusion.
The cysts of the Chrysophyceae are exclusively siliceous, and are of highly
varied forms, but they exhibit a pore closed by a silicopectic plug. The forma-
tion of a siliceous cyst with a pore and plug is the basic characteristic which
enables us to identify the whole Chrysophyceae group without any possibihty
of error.
Certain loricae of the Chrysophyceae {Chrysococcus, for example), are si-
liceous, and have very small pore openings. In the absence of a flagellum and
the plug which closes the pore, one might easily confuse the cyst and the lorica.
In fact, in the Chrysophyceae, the thecae are pierced with a pore opening from
which the flagellum (or the flagella) or the pseudopodia emerge.
Loricae
If we take as an example of loricated Chrysophyceae, the genus Dinobryou
(figure 8) and the kindred genus Hyalobryon, we note that the morphology
and the structure of the loricae vary with the species. In the two genera cited,
the shell is in the form of a conical or cylindroconical horn, more or less flared out
at the apex opening; the cellular body is bound to the lorica by a retractile cysto-
plasmic filament, the epipode. The shell is hyaline, of a cellulose-pectic nature,
with a marked dominance of the cellulose. The outline of this lorica is either
straight or undulating, according to the species The action of coloring agents
(Congo Red) causes the appearance of a very fine heHcoidal striation of the
wall, accompanied at times by a spiral torsion indicated already by the undulat-
ing edge of the theca {Dinobryon divergens (figure 1)). In all of the colonial
Dinobryon which were studied, the basic helicoidal striation has the same direc-
tion of rotation (counter-clockwise), whereas the marginal undulations display
a coiling in the opposite direction.
Dinobryon suecicum (figure 1), a solitary species, free, with a smooth cellu-
lose-pectic lorica, hyaline, with an helicoidal, projecting execresence, brown in
color and of an unknown nature (calcareous substance impregnated with iron
salt?) running throughout the greater part of its length.
An analogous feature is found in some Pseudokephyrion. The solitary fixed
* We will leave out the Silicoflagellates and the family of the Coccolithophoraceae, as
these might constitute the subject of a special stud>-.
421
422
Annals New York Academy of Sciences
Dinobryons: Dinohryon utricidiis (figure 1), have a lorica which is very rich
in pectin, and made up of small elliptical scales, imbricated in helicoidal series.
This structure presages the one which appears in the Synura and the Mallo-
monas.
Figure 1. Loricae after Bourrelly, 1957. 1: Dinohryon cylindncum var. pahistre; 2:
Dinohryon sp.; 3: Lagynion Janei; 4: Dinohryon siiecicuin; 5: Ilyalohryon ramosiim; 6: H.
Borgei; 7: Dinohryon utriculiis; 8: D. sertularia; 9: D. divergens (1, 8, 9: after staining).
O CD
Figure 2. Loricae of Clirysococcus (after Bourrelly). 1: Chrysococcus rufescens; 2:
C. tesselatus; 3: C. ovoides; 4: C. elegans; 5: C. umhonatus; 6: C. porifer; 7: C. minutus; 8: C.
rufescens var. compressa; 9: C. cordiformis; 10: C. rufescens fo. tripora; 11: C. dokidophonts;
12: C. radians; 13: C. 6we/!w; 14: C. spinosus; 15: C. klehsianus; 16: C. heverlensis; 17: C.
ornatus; 18: ('. areolatus; 19: C scidptus.
Finally, a genus very close to Diuobryon: Hyalobryon (figure 1) is charac-
terized by its very long lorica, cellulose-pectic, formed by pieces of encased
cylindrical tubes, of unequal length, the widest one being the one at the base,
and the narrowest one being at the top, presenting a flagellate opening.
Bourrelly: Loricae & Cysts in Chrysophyceae
423
With the genera Chrysococcus and Pseudokephyrion, we have loricae which are
often very much embelhshed and are of a yellow-brown color. These loricae
have a very fine pectic membrane entirely impregnated with calcareous sub-
FiGURE 3. lyoricae of Pseudokephyrion (after Bourrelly). 1-2: conictim; 3; Eulzii; 4, 5,
6: Entzii fo. granulata; 7: lieveiiensis; 8: pocuhim; 9: miniitissimiDu; 10: Rutlneri; 11-12:
cylindricum; 13: depressum; 14: cinctum; 15: obtusum; 16-19*: latum; 20: Skujae; 21: pilidum;
22: Scliilleri; 23: urnula;2i: ehgans; 25: ampullaceum; 26: undulatum; 27: acuiuin; 28: />«/-
cherrimum; 29: lintirniahidum; 30: circumcisum; 31: uiidiilatissimum; 32: spirale; 33: pseudo-
spirale; 34: gallicum; 35: Klarnelii; 36: form os is si mum; 37: ellipsoideum; 3^: ovum; 39: or-
natum; 40-41: circumvallalum.
stance. Acetic acid dissolves the brown and brittle lorica quite well, and there
remains a thin membrane which takes Ruthenium red color admirably.
Along with the numerous Chrysococci (figure 2) with calcareous theca, two
species embellished with spines or needles, have a sihceous wall. We note
that the metabolism of the calcareous type and that of the sihceous type may
co-exist in the same species. Also, some Pseudokephyrion with a calcareous
shell produce siliceous cysts hke the other Chrysophyceae.
424
Annals New York Academy of Sciences
With these calcareous or siHceous impregnations, the lorica becomes thick,
and then presents a stable ornamentation in the same species, but quite variable
from one species to another. Spines, bristles, warts, webs, dots, rings, and
checks decorate the surface of the lorica.
The ma.ximal diversity in ornamentation is obtained in the following two
genera: Pseudokep/iyrion (figure 3) and Kephyrion. Here we find forms with
Figure 4. l.oricae of Kephyrion (after Bourrelly). 1: silla; 2: doUolum; 3-4: nuislign-
phoruni; 5: ciipidijonne; 6: littorale; 7: liltorale var. conslricla; 8: nibri-daustri; 9: ruhri-daus-
tri var. amphora; 10: impletum; 11: cylindricum; 12: hetnispliaericum; 13: petasatum; 14
campantdiforme; 15: amphonda; 16: ovale; 17: cinctum; 18: Valkanovii; 19: globpsum; 20
Starmachii; 21: wo.sqiiensis; 22: spirale; 23: hacillijorme; 24: densalum; 25-29'': as per; 30
Schmidii; 31: imonstans; 32: lalicollis; ii: parvidiim; 34: moniliferum; 35: circnmvallalinn; 36
prismaikum; 37: velatum.
marked calcareous impregnation, and forms with little or no calcification. The
small, more or less calcified cells, such as in Pseudokephyrion undulatum or
Psendokephyrlon latum (figure 3) recall the loricae of the Dinobryons in the
undulating appearance of the edges, but they do not show the hehcoidal torsion.
But the forms with heavily colored, thick lime incrusted walls, have by con-
trast, a more varied ornamentation. One may recognize with them: (1) granu-
lations or striations arranged in regular transversal circles: {Pseudokephyrion
Entzii fo. granulala, Ps. Skujae (figure 3)) or irregular ones (Ps. circum-
Bourrelly: Loricae & Cysts in Chrysophyceae
425
vallalum), (2) helicoidal protruding excrescence (Ps. Klarnelii, Ps. pseudo-
spirale (figure 3)), (3) regular cross checks {Ps. ovum, Ps. ornatum (figure
3)), (4) longitudinally projecting sides {Ps. formosissimuni (figure 3)).
The same remarks may be applied to the genus Kephyriou (figure 4) in
which we note the same diversity in the form of the small cells, but a smaller
variety in the ornamentation of the walls.
In the genus Lagynion (figure 5), the cells do not have flagella, but have
more or less ramified pseudopoda issuing from the oral pole. This genus with
Figure 5. Loricae of Lagynion (after Bourrelly). \: fiihiim; 2: oblongum; 3: arachne;
4: rliizopodicHm; 5: notostomum; 6-7 : rednctiim; %-\\: Seller ffelii; \2:ampullaceiim; 13-15: sub-
ovaium; 16 and 18: maerotraehehim; 17: triangularis; 19 and 20: triangularis var. pyramida-
tiim; 21-23: reflexiini; 24: sphagnieolum; 25: vasieola; 26: Janei; 27-28: cystodinii; 29: globosum
var. undidatum.
calcified pectic lorica does not show any characteristic ornamentation, the
lorica is always brown or yellow, thickened, finely granulated. The forms are
highly varied and the evolutive process comes to bear on the neck terminating
the lorica. In some species {Lagynion Janei (figure 5), for example), the
wall of the theca is double, the inside is thin and hyaline, the outside brown,
thick and calcified. This structure is found in the Diploeca series among the
Craspedomonadines, a large group of collared flagellates related to the Chryso-
phyceae.
In the family of the Stylococcaceae, we note a large variation in the form of
the loricae: along with sessile loricae, there are genera with pediculate shells
{R/iizasler, Slylococcus). We also find genera in which the thecae show numer-
426
Annals New York Academy of Sciences
ous pores, from which issue the pseudopods (Chrysocrinus, Slepkanoporos) for
the sessile forms; Porostylon for the pedicular small cells.
The loricae held by a pedicel are found with other Chrysophyceae belonging
to families very remote from the family of the Stylococcaeae: we cite only the
Figure 6. Loricae of Dcre pyxis (after Bourrelly). 1: Derepyxis amphora; 2-3: ollula;
4: bidbosa; 5: anomala; 6: maxima; 7: tubulosa; 8: dilalata; 9: amphoroides; 10: dispar; 11:
crater; 12: hacchanalis.
Figure 7. Scales of Mallomonas and Synitra (after Bourrelly). 1-2: Mallomonas fasli-
gata var. Kriegeri; 3-6: Mallomonas Leboimei; 7-11: Mallomonas reginae; 12: Mallomonas
tonsitrata; 13-15: Synura Bioretii.
Bourrelly: Loricae & Cysts in Chrysophyceae
427
Derepyxis (figure 6), monads with two flagella and the Lepochromiilina (figure
8), with single flagellum. We mention also, the extraordinary Chrysopyxis of
which the cellulose lorica, in the form of a saddle, attaches itself to the fila-
mentary algae by a thin cellulose cord which completely entwines the support-
ing algae. ^
Alongside of the Chrysophyceae with loricae of homogeneous structure, we
may place the species of the family of the Synuraceae in which the lorica is
replaced by a covering of siliceous scales (figure 7).
Figure 8. Loricae and cysts (after Bourrelly). 1: Dinobryon divergens, lorica and cyto-
plasm; 2: Lepochromulina calyx, lorica and cytoplasm; 3-5: IleterocltromuUna vhipara var.
minor, building of cyst; 6: Dinobryon niriculus, lorica and division; 7: c\st of Chrysostomacea
Outesia; 8: cyst of Ouiesia; 10: cyst of Clericia; 11: cyst of Deflandreia (?). bb: mouth-band;
Cm: muciferous bodies; cv-vc: contractile vacuole; cy: cytoplasm; ep: epipode (contractile
thread); /-/2: flagella; gg: oil-drop; k: membrane of cyst; /: leucosin; Ic: cellulosic lorica; n:
nucleus; p: parabasal body; pi: chromatophore; s-s s2: stigma; sy: symbionts.
The scales arranged in helicoidal series, such as these of the Dinobryon
utriculus (figure 1*), have been the subject of fine studies in electronic mi-
croscopy.
The systematization of the genus Mallomonas (about 100 species) of the genus
Synura (12 species) is almost solely based upon the form of the scales and of the
bristles which adorn them. The observation of a single siliceous scale is some-
times enough to permit the identification of the species. This is not the case
with the true lorica, in which we have a convergence of form to such an extent
that it is impossible in certain instances to decide from the study of an empty
* The scales of Dinobryon utriculus are not siliceous, but pecto-cellulosic.
428
Annals New York Academy of Sciences
lorica whether it is a Chrysophycean, Craspedomonadina, or even one of the
colorless flagellates of the Bicoeca group.
Cysts
The same problem will arise for the Chrysophyceae cysts. We will have, at
all times with the present forms, siliceous cysts with their pore and plug. But
Figure 9. Cysts of Uroglena (after Bourrelly). 1: Uroglena americana; 2: U. Conradi
var. gallica; 3: U. botrys; 4: V. Nygaardii; 5: U. volvox var. uplandica; 6: U. volvox; 7: U.
soniaca; 8: U. Lindii;9: IL marina; 10: IL europaea; 11: U. notabilis.
Figure 10. Cysts of Chrysaslrella furcala (Chr\sostomataceae): polymorphism (after
Bourrelly).
within the same genus, the cysts have a highly varied ornamentation, and the
identity of forms in the cysts does not seem at all related to the organism. The
endogenous cyst is built within the cell, around the nucleus (figure 8). The
cytoplasmic parts left out of the cyst contribute to the external ornamentation
of the cyst wall. The cyst is siliceous, but as was the case with the Diatomae,
a pectic substance remains bound to the silica. The plug which closes the
pore of the cyst is itself siliceous, but with a substantial pectic tendency.
Bourrelly : Loricae & Cysts in Chrysophyceae 429
The genus Uroglena (figure 9) which shows a great structural and cytologic
homogeneity, is an excellent example of the diversity form of the cysts, in fact,
here knowledge of the cyst is indispensable for the determination of the species.
The cysts of many unicellular Chrysophyceae are still unknown. On the
other hand, many cysts are known in which the free vegetative phase is un-
known. This has led the protistologists and the micropaleontologists to give
genus and species names to the cysts of which the vegetative phase is unknown.
It is a convenient method, but these are not true species, only provisional
names without classification value.
The fresh water cysts, both fossils and recent have been placed in the
pseudofamily of the Chrysostoniataceae, whereas the fossil marine cysts make up
the Archaemonadaceae.
The Chrysostomalaceae (figures 8 and 10) are abundant in the present
and fossil peat bogs, and in the Diatomae lacustrine deposits. More than
200 forms have been observed from the Tertiary period to the present time.
The fossil marine forms of the Archaemonadaceae are found in association
with Diatomae from the Cretaceous and Tertiary periods (less than 100 fos-
sil forms are known).
The fossihzation of the cysts is often perfect (the pore plug usually being
missing, however) whereas that of the loricae of Chrysophyceae seems much
more diiBcult, and observations of fossil loricae have been very rare (2 or 3
observations only).
In closing, it must be noted that although the present Chrysophyceae are
well known in fresh waters, the forms of marine nanoplankton are very scant
because their study has been much neglected. There is a vast domain in which
investigation has only begun, and the rare current projects in this field have
already yielded a harvest of interesting and novel facts.
Reference
Bourrelly, P. 1957. Bull. Micr. Appl. n. s. 7(5): 118-124.
MORPHOLOGICAL TRENDS AMONG FOSSIL ALGAE
J. Harlan Johnson
Colorado School of Mines, Golden, Colorado
The algae may be considered as a vast subkingdom of primitive plants that
exhibit an enormous range in structure, reproduction, and life history. Struc-
turally at the base are unicellular forms, often motile, that are indistinguishable
from similar unicellular animals except for the presence in the cell of color spots
or chromatophores, which contain photosynthetic pigments. At the other ex-
treme are tree-sized multicellular plants in which there is some differentiation
of tissue for dilTerent functions.
For convenience in study and classification the algae are divided into a num-
ber of major groups. These groups have been considered as classes by the older
authors but the tendency today is to think of them as phyla. They are named
on the basis of the pigmentation, for example, the Chlorophyta or green algae;
the Rhodophyta or red algae. Nine such groups are recognized in most classi-
fications, 11 in others.
Before considering the morphological trends among fossil algae it will be de-
sirable to review two things regarding recent algae. (1) The structural trends,
and (2) structural parallelism among the major groups of algae.
Algal Morphology
General. The algae show a great range in form, size, and structural develop-
ment. At the bottom are the microscopical unicellular forms. These occur
in all but two of the major groups and in a number of them no higher structural
types have ever developed. A majority of the unicellular forms are motile
flagellate types or at least in their life cycle pass through a flagellate stage.
Structural evolution seems to have followed the steps shown in table 1, with
the first three, either 4 or 6, and 5, forming an evolutionary series.
Parallelism. One of the most striking facts facing a student of algal morphol-
ogy is the evidence of parallel evolution and development among the members
of the various groups (tables 2 and 3).
Marked structural complexity of the plant occurs only in two groups, the
Rhodophyceae and the Phaeophyceae, with some of the green algae (Chloro-
phyceae) reaching a high medium of complexity. It should be emphasized
however, that even in these three groups a majority of the known species have
simple types of structure. The highest structural features have developed
among the brown algae, with some of the reds not far behind. The green algae
probably show the greatest diversity of structural types with, however, the
highest types missing.*
Fossil Algae
General. A review of the structural types and evolutionary trends among
Recent algae, as briefly summarized in the previous section, and a study of
* This has been explained by numerous writers on the basis that the highest types moved
ashore and gave rise to the land plants.
430
Johnson : Morphological Trends among Fossil Algae 431
fossil algae, bring out 2 basic facts. (1) The beginnings of the algae are to be
found in very remote ages of geological time, at or very close to, the origins of
life upon Earth. They were among the earliest forms of life to appear and the
evidence available suggests that each of the major groups started independently,
Table 1
Structural Types
Simple types
Unicell
Palmelloid and dendritic
Coccoid habit
Filamentous habit
Heterotrichous habit (^ creeping basal portion
(an upright portion above
Siphoneous habit
Advanced types
Heterotrichous filaments
Discoid
Crusts or cushions
Elaborately erect type
Compact (uniaxial)
Compact (multiaxial)
Foliose
Tubular
Table 2
Parallelism in Development of Simpler Types of Growth Forms
Algal group
Type of algal structure
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c
c
o
B
o
t-
o
a;
a
"a
u
u
>.
a
o
a;
1)
ca
a;
u
>,
j:
ft
o
-o
o
a
o
J3
J^
^
c
X.
3
-C
u
X
u
X
m
u
p
u
X
X
Ph
Pi
s
Motile holophvtic unicell
X
X
X
X
Motile colorless unicell
X
X
X
X
X
X
X
Encapsuled unicell
X
X
X
Motile colony
X
X
X
X
Dendroid colony
X
X
X
X
X
Palmelloid colonv
X
X
X
X
X
X
Coccoid (zoosporic)
X
X
X
X
X
Coccoid (azoosporic)
X
X
X
X
Simple filament
X
X
X
X
Heterotrichous filament
X
X
X
X
X
X
Siphoneous type
X
X
Holoph\tic amoeboid type
X
X
X
Holozoic amoeboid type
X
X
X
X
Plasmoidial type
X
X
X
X
probably at appro.ximately the same time, and have developed along more or
less parallel courses since. (2) By the beginning of the Paleozoic Era (earliest
Cambrian time) roughly 500 million years ago, the algae had developed to the
point where the algal population was probably equal to that found today, with
432
Annals New York Academy of Sciences
all the major groups present, and even some of the existing orders and families
already present and showing their characteristic features.
Our knowledge of fossil algae is limited and very spotty with many vacant
spaces both in time, and in algal groups. The study is still in its early child-
hood. There are a number of reasons for this. (1) Geologists and paleon-
tologists have only recently become interested in fossil algae, and to begin to
search for and to study them. (2) The nature of the fossils, (table 4); and
(3) the difficulties in accurately identifying and classifying the fossils will be
discussed later.
Thanks to the fact that the oil companies have discovered that algal lime-
stones make good reservoir rocks, petroleum geologists and paleontologists are
becoming interested in fossil algae. However, as yet, very few have the knowl-
edge and experience to use them. I seriously doubt if there are 10 people in
the world with a good working knowledge of the subject. In the Western
Hemisphere there are only 3 people working full time in the field, and 2 of these
Table 3
Parallelism in Development of Advanced Forms
Group of algae
Type of growth form
Chlorophyceae
Phaeophyceae
Rhodophyceae
Heterotrichous filament
X
X
X
Discoid type
X
X
X
Crusts or cushions
X
X
X
Elaborate erect type
X
X
X
Compact (uniaxial)
X
X
Compact multiaxial
X
X
X
Foliose
X
X
X
Tubular
X
X
are interested only in certain groups. However, there are a number who are
learning, and are studying either certain groups or the fossil algae present dur-
ing certain geological periods.
From the very nature of the majority of the algae their chances of being pre-
served as fossils are very slight.
A tiny drop of jelly surrounded by a thin wall of organic material will only be
preserved under very exceptional conditions, and even then the chances of it
iDeing found are very slight. Normally only those microscopical forms which are
encased in a covering of siUca or other mineral material are hkely to be pre-
served, as in the case of diatoms and silicoflagellata. Among the larger forms
it is also true that the chances of the bodies of such soft organisms being pre-
served are almost nil. The only common exceptions are those higher types
which have developed the habit of secreting or depositing calcium carbonate
within or around the plant tissues, and the microscopical forms which are en-
veloped in a siliceous or calcareous covering, or have a hardened encysted stage.
Groups wilh kmmn fossil representatives (tablf, 5). This program deals with
the Protobiota, so emphasis is placed on the microscopical forms. However,
other speakers are giving detailed papers on the diatoms, dinoflagellates, and
Johnson : Morphological Trends among Fossil Algae 433
other types of the chrysophyceae and the siUcoflagellates, and because my work
has been largely with the megascopic limestone building forms, I cannot refrain
from discussing them briefly. (From the point of view of evolutionary trends
Table 4
Methods of Preservation of Fossil Algae
Type
Information given by fossil
Value for accurate
classification
Impressions
Give a general idea of size and
shape, some surface markings.
No internal structure
Very little
Molds and casts
Same as above
Verv little
Preservation in chert
Variable. Some remarkable pres-
Often very good.
ervation of microorganisms and
Probably the best
small megafossils
Carbon films
Size, shape, and surface features
beautifully preserved, some-
times. Rarely traces of internal
structure
Fair
In coal or peat
At times remarkable preservation
of microfossils, and internal
structure of larger ones
Good to very good
Calcareous algae. Original
Good internal structure. Even
Good to very good
material or calcified or silic-
the size, shape, and arrangement
ified
of cells in the tissue in case of
coralline algae
Table 5
Groups with Fossil Representatives
Group
Representatives
Size
Age range
Chlorophyceae
Codiaceae (some
genera)
Dasycladaceae
Mega.
Mega.
Cambrian-recent
Cambrian-recent
Chrysophyceae
Silicoflagellata
Micro.
Miocene-recent
Bacillariophyceae
Diatoms
Micro.
Jurassic ?-recent (pos-
sibly older)
Dinophyceae
Dinoflagellata
Micro.
Ordovician-recent
Rhodophyceae
Solenoporaceae
Corallinaceae
Gymnocodiaceae
Mega.
Mega.
Mega.
Cambrian-cretaceous
Jurassic-recent
Permian-cretaceous
Myxophyceae (Cyano-
phyceae)
Stromatolites
Mega.
Precambrian-recent
these are of interest as they have made much of their development since Cam-
brian time with a fair fossil record to document the development.)
Megascopic fossil algae. The remains of multicellular algae, as well as micro-
scopical ones, may be preserved in a number of ways. The nature of the fossils,
resulting from the way in which they were formed and preserved, is of great
434 Annals New York Academy of Sciences
importance as it controls the amount of information, especially with regard to
structure (table 4).
The most common and the most useful algal fossils are those of calcareous
algae. If not recrystallized these commonly show not only the external form
and surface features but at least some details of the microstructure. In the
case of the coralline algae they actually show the size, shape, and arrangement
of the cells in the tissue and details of the reproductive organs permitting def-
inite, accurate classification.
Identificalion and classification of the fossils. This is the most important and
frequently the most dithcult part of the study of fossil algae. The remarkable
parallelism in structural development and growth form in several of the major
groups and numerous orders and families gives a perplexing choice of possible
assignments for the fossils, which can only definitely be decided on the basis of
internal structure and reproductive organs. As just pointed out, very few of
the fossils can give this information except the calcareous algae.
This means that the calcareous algae are the only groups of megascopic fossil
algae for which we have enough solidly based information to be able to discuss
the evolutionary morphological trends.
Morphological trends. Among the green algae two families, the Dasyclada-
ceae and the Codiaceae have a long fossil record. Both appear in the record
during the Cambrian and continue down to the present.
Dasycladaceae. The general form for most members of this family suggests
a test tube brush, consisting of a central stem from which develop more or less
regularly spaced whorls of primary branches. From the tips of the latter may
arise tufts of secondary branches, which in some genera may produce tertiary
branches. In the earlier, primitive forms the primary branches are not col-
lected in regular whorls, but may be irregularly spaced, or develop in more or
less regular rows which spiral upward around the central stem. However,
genera with regular whorls of primary branches are definitely present during the
Silurian period and characterize most of the genera thereafter. From Silurian
times on the general trend is toward greater structural complexity, involving
greater numbers of whorls, the development of secondary, and tertiary, rarely
even quaternary branches, and the differentiation of the branches into whorls
of purely vegetative branches, and whorls of fertile sporangia bearing branches,
with, in some cases, the modification of certain branches into elaborate holders
of sporangia or spores. This trend toward greater elaboration of structure
reaches its climax during the Jurassic period, after which a tendency toward
simplification begins. This has continued to the present.
Codiaceae. The early Paleozoic record of this family is meager but sufficient
to show that by Ordovician times some members had reached a high structural
level quite close to that of present day types, like Halimeda which thev closely
resemble. Since then "increased structural complexity suggesting evolutionary
changes, such as are seen in the Dasycladaceae, can scarcely be recognized
among the Codiaceae. This fact suggests that, as a consequence of vegetative
differentiation and evolution from primitive plants sometime during the Pre-
cambrian, the family was already well established," (Konishi, 1961, p. 233).
Actually, from the Mississippian up into the Lower Cretaceous various mem-
Johnson : Morphological Trends among Fossil Algae 435
bers of this family are probably numerically the most common fossil algae.
They changed in detail, but the general morphology and structure changed but
Httle.
Red algae. Calcareous red algae were relatively rare throughout the Paleo-
zoic. From the Cambrian to the Pennsylvanian, all found to date appear to
belong to two genera of the family Solenoporaceae. During that time they
show little morphological change. Then, during the Pennsylvanian, several
quite different types of red algae appear. One of these, the genus Archaeolitho-
phyllum, has much higher structural features with the tissue differentiated into
a well developed hypothallus and perithallus, and definite conceptacles. Also
the Pennsylvanian genera show a much greater variety in growth form.
Whether this rapid rise of new types represents an evolutionary surge, or for
some reason long established groups of plants acquired the calcareous habit
and begin to be preserved as fossils, we do not know.
During the Permian another family of calcified red algae, the Gymnocodia-
ceae, appear and in the Late Permian become abundant and widespread, adding
additional morphological types.
The record of Triassic red algae is scanty, but during the upper half of the
Jurassic the group undergoes a strong evolutionary push. Within the family
Solenoporaceae many developments and new growth forms appear, and repre-
sentatives of the family Corallinaceae begin to emerge. The first recorded
articulated corallines appear during the Middle Jurassic, and the earliest known
crustose corallines during the Late Jurassic. By the end of the Cretaceous,
almost all of the common genera of the coralline algae had appeared. They
were well established by the middle Eocene and had developed essentially all
the morphological features known today.
Myxophyceae {Cyanophyceae). The only other important type of calcareous
algae are the stromatolites. These are calcareous masses of distinctive form
and surface markings, commonly showing thin arched laminae, built largely or
entirely by the activity of certain types of glue-green algae. They have been
reported from rocks as old as the late Archaeozoic, and are faily abundant in
the Huronian of few areas. They were the Hmestone building organisms of the
Proterozoic and Early Cambrian. With the appearance of limestone building
animals in the Cambrian and Ordovician their importance decreases greatly,
but they have continued in considerable numbers down to the present day.
However, in morphology and structure, they show practically no change after
Late Cambrian times, consisting of mats or felts of tiny algal filaments which
often trapped some silt or organic debris and was encased in a mold of fine cal-
careous dust precipitated by the algae. Commonly they developed colonies of
a consistent shape, show growth laminae, but little or no microstructure.
Bibliography
Doty, M. S. 1957. Fxology of marine Algae (annotated bibliography). Treatise on marine
ecology and paleoecology. Geol. Soc. America Memoir 67. 1: 1041-1050.
FosLiE, M. & H. Printz. 1929. Contributions to a Monograph of the LUhothamnia.
Royal Norwegian Museum of Natural History. Trondheim.
436 Annals New York Academy of Sciences
Fritsch, F. E. 1956. The Structure and Reproduction of the Algae. Cambridge Univ.
Press. Cambridge, England.
Johnson, J. H. 1960. Paleozoic Solenoporaceae and related red algae. Colorado School
of Mines Quart. 55(3): 77.
Johnson, J. H. 1961. Limestone building algae and algal limestones. Colorado School of
Mines, special ])ubl.
KoNiSHi, K. 1961. Studies of Paleozoic Codiaceae and allied algae. Part I. Codiaceae
(excluding systematic descriptions). Kanazawa Univ. Science Repts. 7(2): 159-261.
PALEOECOLOGICAL CONSIDERATIONS OF GROWTH
AND FORM OF FOSSIL PROTISTS
Paul Tasch
Department of Geology, University of Wichita, Wichita, Kansas
Protists known as fossils range from bacteria (0.5 n in size) lo foraminifers
and tintinnids (from 10 to 1000 n or greater in size). Some protists, for exam-
ple, bacteria and lime secreting algae, are first recorded as fossils in rocks of
Pre-Cambrian age; others, including radiolarians, hystrichosphaerids,* and
foraminifers, apparently make their first appearance in Paleozoic time. Finally,
Mesozoic rocks bear the first record of a dominant element of the living phyto-
plankton, namely, the diatoms, as well as other protists, such as, the coccolitho-
phorids, silicoflagellates, tintinnids, and the Chrysomonadina.
There are numerous studies by protozoologists on variations in size and form,
as well as structure and physiological characteristics of protozoans. They have
found it necessary to distinguish races, varieties, and strains within a given
species to italicize the observed variation.- By contrast, studies on the skeleton
of various protists in which the differential morphology, as well as causative
factors, is considered, are relatively few.
Thompson^ approached protist skeletal morphology and factors influencing
it from a different point of view. With mathematical-physical considerations,
he reached some insightful conclusions. One need but regard the minute mass
of protoplasm that is involved in secreting a protist skeleton as a fluid drop
and subject to all the physical forces known to affect such a drop to explain its
form by the laws of surface tension. It is apparent that many protists tend to
have skeletons of spherical configuration. The sphere, of course, offers the
least surface area for a given volume. Because a chain of such drops is possible,
and any individual drop can be acted on by gravity, the observed variation in
protist skeletal morphology can be simply explained.
Ecology and Paleoecology of Protists
There is a very extensive literature on ecological factors that influence
growth of living phytoplankton.^"^ Nitrogen and phosphorus are primary
nutrient factors.^ Other elements of importance include: silicon, ^'^^ iron,i-'i*
and possibly manganese.^ A sudden increase in vitamin B12 may be the stimu-
lus for certain phytoplankton blooms.^* Among physical factors, tempera-
ture and salinity are effective "selective agents" on the species level. The
species specific salinity response has recently been attributed to "special re-
quirements for the concentration of sodium ions in the medium. "^^ Radiation
is obviousl}^ of primary importance affecting as it does, latitudinal and seasonal
variations in phytoplankton production. In addition, the photic zone must
be replenished by nutrients from deeper waters. This redistribution is attained
in coastal waters by vertical circulation.^'''
* Affinities to dinoflagellates are indicated for several, but not all, hystrichosphaerids.'
Tasch (in press) has found undoubted dinoflagellates in the Permian of Kansas. These were
associated with hystrichosphaerids.
437
438 Annals New York Academy of Sciences
It seems valuable to reiterate, with fossil protists in mind, that "we may
assume that during the period for which we have good fossil evidence, the sea
has remained very much the same in overall chemical composition."^*^ Cer-
tainly, this is applicable to the Tertiary and Mesozoic. By extrapolation, for
protists such as the radiolarians, it may be referred back to the older Paleozoic.
Ecological studies of living marine biotas suggest "dim outlines of food chains
that must have had links similar to those of the present day"' in the geological
past.
In thinking about assemblages of fossil protists, their growth and form in
ancient seas, coastal and inland waters, one can refer to the same or equivalent
physical-chemical factors known to influence living phytoplankton.
The Diatom Frustule and Dinoflagellale Armor
Certain physical realities of the environment have to be satisfied to ensure
survival for various protists including pelagic diatoms and dinoflagellates.
We may speak of these as "fence" or limiting conditions. These restrictions
influence not only distribution but growth and form as well. The first "fence"
is the specific weight of living protoplasm, which is 1.02 to 1.06, and hence
heavier than pure water."* There will be a tendency to sink if the added incre-
ment of a skeleton (test or armor) is superimposed on this naked weight.
Whether the protist is a passive floater like the pelagic diatoms, or capable of
feeble flagellar locomotion like the dinoflagellates, the fence condition will
apply. The second "fence" is established by the requirements of photosynthe-
sis. Pelagic protists need to be physically positioned, or located in a specific
zone of the sea, the photic zone, or both.
Given these fence conditions, a selective advantage will favor individual
pelagic diatoms and dinoflagellates with slight variations in skeletal morphology
that tend to retard the rate of sinking. Natural selection would then become
effective within the available band of skeletal variation characterizing a given
population.
Projecting spines, chains of cells, disc-shaped tests or needle and hair types,
curvature of cells, bevelled ends of tests, are all structural adaptations to resist
the gravitational force. Spines, for example, aid flotation, as do spiral or
flattened chains of cells. This last feature produces more surface area and
hence greater frictional resistance.'^ It should be emphasized that test shape
and modification of the ends of tests do not prevent sinking. Rather, these
features either facilitate a return to the horizontal from a vertical position, or
expand the path of passive descent from a straight line to a zigzag path or a
widely circular one. In this way, removal from the photic zone is slowed down
or delayed.** 'i^''**^
Weight and spination of diatom frustules have been observed to vary ac-
cording to species, season, and habitat. Generally, pelagic species tend to be
thin shelled, whereas bottom and littoral forms are not. Viscosity, which
varies inversely as the temperature, is a factor in flotation of pelagic protists.
Heavier frustules tend to sink under reduced viscosity. It follows then, that
cold water or winter forms will have heavier shells.'' '^'^ In all such instances,
silicon metabolism and the supply of silicon are also involved.'""
Tasch: Growth and P'orm of Fossil Protists 439
The shapes of some nonmarine diatoms can be influenced by other ecological
factors. Individuals of the genus Desmogoniurn were long and had barely
capitate ends in fast flowing water but were short and had broadly capitate
ends in standing water. ^"
Similar considerations also apply to the morphology of armored dinoflagel-
lates.^'^^-^"'''^ Kofoid studied skeletal development {i.e., an armor of loosely
cemented cellulose plates) in the genus Gouyaulax.-^ He found that all modifi-
cations in this genus were variants of the spherical configuration (cf., Lejeune-
Carpentier-' for fossil Gonyaula.x). In turn, this ensures least surface area,
and hence confers an advantage on protists leading a pelagic existence.
Braarud^'^ stresses that form variation is observed in every species. Study
of Schiller's work on dinoflagellates brings this out clearly. Dinophysis
hastata^^ for example, shows a whole spectrum of variation from ovate to sub-
ovate and subelliptical conligurations, and corresponding size and shape varia-
tions in epithecal and hornlike structures. Braarud''' noted that in some
instances, form variation appeared to be "phenotypic" and "tentatively re-
lated" to a whole series of ecological factors such as salinity, temperature,
nutrient salt concentration, and day length. An excellent example of such
infraspecific form variation is found in the fossil record of the dinoflagellate
Nannoceralopsis recovered from beds of Jurassic age.-^
Twenty specimens of N . deflandrei show variations in form from ovate to
subovate hanging drop configurations. These may bear weakly defined an-
tapical horns or lack them. Other forms are broadly and acutely subtriangular
with the base faintly or markedly concave between short horns. This strik-
ingly contrasts with the long horn type, X . pelliicida.^^
It is apparent from our previous discussion that these variants are adaptive
modifications for flotation. Something similar to pelagic diatom adaptation
in thickness of test is found in the armored dinoflagellates. Thus, in colder
waters of the South Ecjuatorial Currents in which viscosity was greater, the
horns of Cerialia were found to be longer than those of equivalents taken in the
warm water of the Guinea stream."*
The short horn, X. deflandrei, may be related to warmer waters, whereas
.V. peUucida, the long horn species, would then indicate colder waters. A third
type tentatively assigned to Xannoceraiopsis has been found in the Permian.
This form is flask-shaped and bears stubs in place of horns (Tasch, in press).
Other structural modifications that have adaptive value in armored dino-
flagellates, include a variety of specializations to ensure suspension or flotation
when the flagella are at rest or swimming is feeble.^ •-^■"•^■-'^ In the genus
Triposolenia, the ends of the antapical horns are deflected. The significance of
this asymmetry has engendered speculation. Kofoid"* thought that the asym-
metry must bear a "profound relationship" to the survival of the forms in
which it appears. It occurs in more than one species and in the genus Amphiso-
lenia also.-'' Still other genera have analogous structures. After a descent of
about 10 times the body length, the asymmetrical horns serve to orient the
long axis horizontally, i.e., the position of greatest resistance to downward
pun.4 19
A few of the morphological variants in armored dinoflagellates include:
round and egg-shaped skeletons (Glenodinium) sometimes bearing spines on the
440 Annals New York Academy of Sciences
hypotheca; hanging-drop configurations to subspherical forms with a tapered
hypotheca and bearing or lacking antapical horns (Peridinium), an eccentric
expression of the same configuration with partly deflected apex and horns
(Heterodinium); bizarre, multihorned Ceratium in which horns may deflect at
all angles and in all directions.^^ -^
Nine varieties of Ceratium hirundella were found in various European
waters.- '^^ Size variants have been reported from different Swiss lakes: 92 ju
in Lake Como, to 707 n in Lake Schwendi.-'' SampHng several ponds in the
vicinity of Darmstadt, Germany at 2-week intervals over a 5-year period,
Ljg|-29a found that population density fluctuated with rise and fall of tempera-
ture. He noted seasonal variation in both horn length and horn number in
C. hirundella. In the summer four-horned type, for example, horns were
shorter during very hot summers than they were during cooler ones. Ap-
parently, in fresh, as well as marine waters, horn development is an adaptive
modification to resist sinking below optimal food and sunUt levels.
Flaring, sail-like, structures from the girdle, inverted umbrella and parachute
type membranes as in Ornithoceras, Diuophysis, and other forms'* ■-'' all tend to
increase the surface area of the anterior over the posterior. In turn, this helps
to orient properly the given protist.
A third group of protists of polyphyletic origin are the hystrichosphaerids.
Many forms classified under this group are apparently dinoflagellate cysts.i -^^
Fossils often show dinoflagellate plates although many forms lack a distinguish-
able plate system. Configuration of the central body is often globular but all
variations are known from subround and ovate to subelUptical. Arising from
the central body are spines or tubular processes, or both, with flattened or
bifurcate terminations. It has become clear that these tubular processes were
originally connected to a circumscribing membrane. A recently found globular
hystrichosphaerid from the Kansas Cretaceous bears a short, tubular process
that terminates in two fine flagella-Hke extensions within the body of the en-
closing membrane. ^^
For those hystrichosphaerids which are definitely dinoflagellate cysts,
morphology was determined by encystment. HystrichospJiaera furcata and
H. speciosa bear an equatorial girdle and dinoflagellate plates, and are good
examples. Generally, hystrichosphaerid form is a variation of the spherical
configuration. Why this is so can best be understood if one observes a cyst
inside a subtriangular-to-buUet-shaped dinoflagellate like Deflandrea phos-
phoritica?- The spherical shape is the most efficient configuration that can be
enclosed in the volume available.
Radiolarian Scleratoma and Tiniinnid Lorica
Radiolarians and tintinnids occur together in the Mesozoic fossil record in
the Mediterranean area^^ and hence it seems desirable to discuss them together.
Both groups have living representatives which occur in great abundance.
Radiolarians found in the fossil record are almost invariably "upper-zone
pelagic types"^^ although abyssal forms are known.
Although radiolarians are incapable of horizontal locomotion, tintinnids can
swim rapidly by the aid of bristles and cilia.^* Both forms had to solve the
problem of resisting passive sinking below optimal levels of the sea.
Tasch: Growth and Form of Fossil Protists 441
The form of radiolarian species seems to be adaptive to environmental
conditions'*'^ although experimental study of factors influencing shell mor-
phology are wanting.'^^ Thompson/'* as noted previously, provided some useful
insights into radiolarian morphology.
Free floating radiolarians, among both fossil and living assemblages, tend to
be spherical and elliptical, with a foamy or spongy appearance. Such forms
occur in the Spumellina, Nasselina, and Acantharina. The shells are delicate,
small, and bear various structures such as, numerous slender apophyses, large
pores, thin bars between pores, and varied spinose development. Inhabitants
of deeper layers (Phaeodarina and some Nassellina) are heavier, more massive,
and tend to bilateral symmetry. They are infrequently burrlike. Structures
found in such forms include: short apophyses and small pores with thick tra-
beculae.^'*'^^ .
In some forms (Semantidae, etc.)'*^ one can observe configurations not too
different from those of the silicoflagellates.^® The shell in the Challengeridae
bears a fine hexagonal mesh resembhng similar structures of the diatom frus-
tule.^^ Some configurations of radiolarians are analogous to those of armored
dinoflagellates, for example, Coelodecas?^ Hexaspyris papilio^^ is reminiscent
of the bizarre spinose development in the dinoflagellate Ceratium.
It is generally agreed that variations in scleratoma configuration and in
skeletal structures found in radiolarians reflect adaptations to retard sinking
below certain depth levels of the sea. Within a given species of course, varia-
tions of shape and structure are merely those of a normal population spread.
The gelatinous or pseudochitinous cuplike or elongate lorica of tintinnids is
frequently agglutinated. Foreign particles encrusted or included in the deli-
cate membranous wall may consist of fine mineral grains, coccoliths, diatoms,
and organic debris.^^'^'^'^^ Shape of skeleton in both fossil and living tintinnids
is extremely diverse. ^^ Surface markings of the lorica include: ribs, ridges,
plications, flutings, shelves, reticulations, fenestrae, and lacunae. Among
aboral structures are apophyses, pedicel, knob, lance, and skirt. ^'"^
Because tintinnids move like squids with oral end directed backwards,
streamline configuration of the aboral tip would offer less frictional resistance
to forward movement. It is also likely that the lorica may aid flotation.^^
The total effect of such configurations is to check descent below optimal levels.
Modification of Shape and Form in Foraminifera
Work on living foraminiferal distribution and ecology has clearly established
characteristic faunal suites in distinct brackish and marine environments. '"'"■*''
Although the majority of foraminifers are vagrant benthos, planktonic forms
that float at or near the surface such as the Hantkennidae, Orbulinidae, and
Globorotalidae have been more closely studied in the past decade.'*" '^^
Bandy^* found a striking correlation in form, structure, and environment in
benthonic foraminiferal assemblages in modern seas. Among the variations
he observed are overall size, shape, and size of chambers, chamberlets, coiled
and uncoiled forms, spinosity, surface sculpture of the test (costa, striae).
These were found to vary with bathymetry (bay, shelf, and bathyal zones).
Phleger'*" believes that the influence of temperature may have been overstated
442 Annals New York Academy of Sciences
in the literature and suggests a whole spectrum of ecological factors that may
have been involved.
In both modern tropical and subtropical waters, spindle-shaped tests seem
to characterize definite depth zones (20 to 80 meters). By extrapolation,
Bandy^^ ascribed equivalent depth zones to fusulinids — an extinct Paleozoic
family — and to the Cretaceous spindle-shaped Loftusia. Similarly, he noted
that deeper water assemblages seem to show a size increase and coarser surface
features.
The planktonic foraminifers show a variety of morphological and structural
adaptations for their floating existence.^^ The variations are ascribed to
temperature and saUnity. Thin walled shells, for example, characterize surface
Orbulina miiversa and Globigerina in contrast to thick walled shells for indi-
viduals living at greater depths. Reduction in the specific gravity of the
planktonic test is also affected by increase in pore size, aperture enlargement,
or the development of supplementary apertures.
Resistance to sinking which is the critical problem facing all pelagic protist
inhabitants, is attained in planktonic foraminifers as follows: flattening of the
body accompanied by a radial test, and elongate or clavate chambers. In the
Orbulinidae and Hantkennidae spinose projections develop. Other adaptations
include: globose chambers that increase in size as added, large primary aper-
tures, and in such forms as Globigerinoides, development of many secondary
openings.
Although all of the above named variations may be related to genetical
events and the operation of natural selection, there are other nongenetical
factors known to influence foraminiferal morphology. On the Argentine shelf,
a depauperate foraminiferal fauna was found to be characterized by its small
size, partial or complete loss of ornamentation, a tendency toward asym-
metry, and growth retardation. Spectrographical study of trace elements in
the shells revealed the presence of lead in depauperate, as compared to, normal
faunas in which it was absent.^^ ■'*^ Study of Allogromia laticoUaris in culture
revealed occasional populations with a large number of flattened discoidal
individuals. In this instance, the flattening was directly attributed to "down-
ward pressure exerted by rapidly multiplying algal filaments."'*^ Dwarfed
foraminifers are reported from poorly ventilated basins.^-
An unusual example of a testate protozoan, Difflugia oblonga, can be cited
here although it belongs to a different order than the foraminifers. A small
pond (10 X 6 meters) in the environs of Prague, Czechoslovakia, contained
numerous individuals of this species. They exhibited an astonishing morpho-
logical variation. Every variant was observed from a globose bowl with a
smooth base, to elongate figures with tapered basal projections variously
curved. Some specimens took on the configuration of an Erlenmeyer flask
with knoblike projections from each basal edge. The heavy discharge of
industrial waste gas (CO2) in the environs was thought to be the causative
factor .^^
Classes Chrysophyceae, Coccolithophorida, and Silicoflagellata
The several flagellates cited in the subtitle of this section, with the diatoms
discussed earlier, constitute the phylum Chrysophyta. Members of the order
Tasch: Growth and Form of Fossil Protists 443
Chrysomonadina are either solitary or colonial. They are widespread in both
fresh- ■'*^ and marine waters'^ and have fossil representatives in the family
Archaemonadaceae Deflandre.^"
Formation of siliceous resting spores or cysts is a "most characteristic feature
of the order. "2 '18 '51 Such cysts have a funnel-shaped opening or neck and
resemble a stoppered or plugged spherical jar. The plug is formed of cyto-
plasm retracted from outside the cyst wall.
In the cyst of Microglena,^^ Conrad has distinguished "numerous minute
lens-shaped masses of silica" embedded in an outer layer of pectic substances.
A delicate, inner smooth layer of cellulose underlies this outer layer. This
genus with other Chromulineae is closely related to the coccolithophorids in
cell structure although it differs from the latter in flagellation and composition
of its cyst.
Cysts are usually spherical but variants from this configuration occur.
Archeomonadopsis, which is flask-shaped, is such a variant. Surficial orna-
mentation finds diverse expression: ridges that may form a reticular network;
encircling equatorial flanges; spine and knob structures on ridges, and peripheral
spines. The size range of cysts is 10 to 25 /x.
Although little is known about the marine Chrysomonadina, it is apparent
that the morphology and small size of the cyst, together with the cytoplasmic
plug, would favor both resistance to sinking below the pelagic zone and wide-
spread passive distribution. Fossil cysts also indicate a broad geographical
spread.^" The same types of adaptive modification found in living representa-
tives occur in fossils.
A third large group of planktonic algae are the Coccolithophoridae.'^'^^"'
52a ,53 ,54 fhey arc typically open sea biflagellates although in places like the
Oslo fjord, they may occasionally occur in such densities, that the water looks
like milk."* Fresh water forms like Hymenomonas are also known.^^
One may study a form like Coccochrysis,^^ Discosphaera,^ or Syracosphaera
and CoccoUlhus^^ and observe a subovate configuration in the first and third
and a more spherical form in the second and fourth. Lohman^- figures several
different species of Pontospliaera, Calyptrosp/iaera, and Coccolilhop/iora, as well
as species of the second and third genera named above. All of these species
show the same trend in configuration. Generally, therefore, the shape of
coccolithophorids are modifications of a sphere.
The formation of the coccolithophorid skeleton is gradually achieved. At
fairly equal intervals, numerous, minute, variously shaped, calcareous discs
(coccoliths) are "imbedded in an investing membrane. "'^^ This envelope of
variable thickness is gelatinous initially. The coccoliths become "rigidly
united when the mucilage calcifies in older individuals."'** Coccoliths have a
central perforation or are imperforate."^ Although living biflagellates commonly
range from 5 to 20 n,'^ sizes can attain 50 )u.^^ Coccoliths found in sediments
range between 2 and 30 fj.}^
Several coccoliths bear anteriorly and medially spinelike processes. Of
interest, is the successive formation of new coccoliths within the old as the old
are gradually dislodged'^ and contribute to oceanic bottom deposits. Although
today coccoliths are but a "minor part" of oceanic carbonate muds, in Miocene
and Oligocene time, for example, they formed "coccolith ooze."^^
444 Annals New York Academy of Sciences
Braarud'^ and others have experimented on variation in salinity and its
influence on the growth of the coccolithophorids, Hymenocaras carterae and
Coccolillnis liuxleyi. For the tirst species, salinity was excluded as an important
environmental influence on growth. This corresponded to the littoral habitat
in which it is most abundant and in which salinities are quite variable. The
second species, C. huxleyi, is distributed worldwide in oceanic waters (35 per
thousand)" and in northern European coastal waters (15 to 20 per thousand).
Experiments have shown that between these ranges of salinity there was good
growth.
Salinity apparently does act as an ecologic fence in excluding C. huxleyi
from brackish waters. A vertical size distribution of coccohthophorids at
equatorial stations has been reported." Small forms were abundant in the
upper 50 meters. Near surface temperatures are also a probable factor in
distribution. It is thought that variety, large size, and abundance of Eocene
coccolithophorids indicate "warmer seas. "•''•''
The life cycle of coccolithophorids has recently been shown to be more
complex than previously thought.'** A motile stage and a cyst stage have been
experimentally demonstrated for Coccolithus pelagiciis}^
From these data, shape, size, and encystment seem to be adaptations similar
to those in the closely related siliceous Chrysomonadina. Coccolilh formation,
shape, their even spacing in the membrane, and spinelike processes arising from
some coccoliths, are all adaptive devices to aid flotation. Abnormal amounts
of calcite in some Tertiary coccoliths are thought to reflect calcium carbonate
rich waters and not a diagenetic effect.''^ Conceivably, this abnormal deposi-
tion may have served to aid buoyancy or to adjust specific gravity.
One can confidently transfer the general interpretation given to Tertiary
coccolithophorids.
The silicoflagellates have a siliceous skeleton which is covered by a delicate
layer of cytoplasm containing chromatophores. This occurs in early develop
ment when the skeleton is internal, whereas in the adult individual it is ex-
ternal.-''^ The skeleton ranging in size from 10 to 150 fx is essentially a "lattice-
work case of hollow siliceous bars."^" Dislephanus ( = DictyocIia) speculum
with 6 radial spines may be taken as an example of the group. In most silico-
flageUates, the spines give the skeleton a stellate appearance. There may also
be accessory and basal spines. The basal body ring may be from 3 to 10-sided
with as many radial spines. Radial spines issue from the point of intersection
of any 2 sides. The basal body ring of .some fossil forms like Mesocena and
Corbisema^^ is 3-sided with a small spine at each angle. Others, like Dictyocha
crux are 4-sided and have longer spines. D. speculum is 6-sided, and D.
flcfonaria is 8-sided .'^^
The siliceous skeleton is most often a complex of 2 rings or polygons joined
by a series of rods."* Dictyocha speculum is a good example of this construc-
tion. The basal body ring of Mesocena forms an ellipse, and in Corbisema, it
forms a triangle.
Silicoflagellates are exclusively marine plankton'^'' and are found in colder
seas. Frequently they occur associated with diatoms and radiolarians in
ancient and modern sediments.^^ Although they are not uncommon in food
I
Tasch: Growth and Form of Fossil Protists 445
vacuoles of tintinnids, quantitatively they are a minor contributor to the food
economy of the sea.^
Thompson's explanation^ of the basket-shaped skeletal units of Dictyocha
envisioned 4 or more vesicles side by side in one plane and separated by a
"polar furrow." The radial spines normal to the main basket or lattice work
were interpreted to be uncompleted portions of a larger basket. This last
interpretation seems unacceptable in light of the work of K. Gemeinhardt.^^
He demonstrated that adult individuals had a smaller skeleton fitted into the
larger one. In this instance, the inner set of radial spines were not the be-
ginnings of a still larger skeleton, but rather parts of the skeleton of a daughter
cell, and its appearance preceded division. Hovasse confirmed this finding, in
1932,^^ and noted that the new skeleton was a mirror-image of the old one.
Thus, the opaline silica lattice work may be envisioned as derived by secre-
tion on a tiny sphere of protoplasm that had a vesicular surface. Open space,
ovate, elliptical, and polygonal skeletal configurations can then be readily
explained. The radial spines which confer a stellate appearance are most
Ukely adaptive modifications to sustain flotation when the single flagellum is
at rest. All other accessory spines and ornamentation, such as beads and pits
on the discs,*^ may constitute minor adjustments of specific gravity of the
skeleton that had selective value.
In the evolution of the silicoflagellates there is a tendency to increase the
number of radial spines from 3 or 4, to 6, 8, and 10. That trend clearly de-
notes the adaptive value of particular skeletal modification.
Miscellaneous Protists
In this section, bacteria and lime secreting algae will be considered from the
special point of view of our discussion. Despite the frequency of pleomorphism,
there are three common or fundamental forms of true bacteria; spherical or
ovoid (coccus), rod-shaped {bacillus), and spiral (spirallum). Spherical forms
may grow in pairs, in fours, or in chains. Rods vary in configuration from
cylindrical to ellipsoidal with rounded-to -flattened ends. In young cultures
and favorable media, bacteria tend to "exhibit characteristic morphology,''
whereas in senescence, there are a decrease in size and considerable form varia-
tion. Other factors influencing shape are: temperature and age of culture,
concentration of substrate, and composition of medium.^^ A barophilic
property (pressure-dependence) has also been reported. Near their threshold
of pressure-tolerance, cells of many bacteria grow into long filaments and
mutations are promoted.""''^
Bacteria are commonly about 0.5 fj, in size but range to 10 fx. Fossil bacteria
are generally identifiable by size, shape, and arrangement alone.*- However,
viable bacteria of Permian age have since been reported from the United States*^
and from Germany (Dombrowski, 1960). In such instances, physiological
activities which distinguish modern bacterial species can also be studied in
ancient populations.
The descriptive literature on lime secreting algae known as fossils is very
extensive. "^"^^ A good review of recent stromatolites and their ancient ana-
logues is given by Ginsburg.*^ Types of stromatoUte configurations include:
446 Annals New York Academy of Sciences
kiminaled algal-mats such as can be observed forming today at Turner and
Price Falls, Oklahoma, or equivalent forms described by Black from the
mudtlats of Andros Island, Bahama; domes, heads, and more extensive digitate
masses. Onkolites (unattached forms) can have the shape of the nucleus or
be variously shaped biscuits or flattened discs. '^''
Factors such as a slight increase in iron above tolerance amounts have long
been known to retard growth, affect size, and ultimately, the shape of several
nonmarine, nonlime secreting, filamentous algae.'"^ Cyclicity in occurrence of
the Cambrian form Cryptozoon nndulatum was attributed to inhibition of
growth due to increasing turbidity caused by transported sediments.'^- Pre-
Cambrian bioherms of Northwestern Montana show different forms — columns,
domes, sheets — which were "apparently" determined by physical conditions
such as water movements.''^ The ultimate external form of Recent algal bis-
cuits is credited to two determinants: stability of the surface on which the
biscuits grow and the strength of attachment to it.*^^
Work in progress (Tasch, unpublished) on newly discovered algal reefs and
onkolites in the Kansas Permian provide some evidence on controls of ultimate
form. An influx of mud over the growing algal mat (stromatolite) inhibited
growth in certain directions only. Turbidity, of course, can exclude or diminish
hght penetration and hence interfere with photosynthesis. If, however, sedi-
ment influx is negligible (4 to 5 mm.), filamentous algae can "move up through
the sediment and reestablish themselves on the surface. '"^^ The topography
of the substrate on which the original filamentous algal mat spread, also can
be a partial determinant of shape of a stromatolite.
Sporadic circular to elliptical perforations of algal blades in the fossil genus
Eugenophyllum appear to represent adaptive modifications. Although these
forms lived below normal wave agitation, the perforations would help to dissi-
pate even gentle current action against the upright blades which are several
inches in height.''"
Among factors influencing growth of stromatolites and onkolites are: sub-
strate, turbidity, amount of light penetration, depth of water, wave and current
action. Influence of metallic cations can also be inferred.
. Terrestrial M icroproblemal ica
Microproblematica are apparent fossils observed in sections of rock suffi-
ciently thin to transmit light. They are primarily of Mesozoic age, but are
also known from the Paleozoic and Pre-Cambrian. Distinctive structure and
form characterize them. However, they cannot confidently be assigned to
any known taxa. Occasionally, additional study and collection permits ulti-
mate resolution of assignment.^' •'"-
The microfossil Xamioconus kanipliicr, 1931, is a good example. The object
ranges from 5 to 50 /u in length, with an average of 15 to 20 fx; width varies
from 5 to 15 /x. It is definitely an "organized object." There is a distinctive
wall composition (spirally arranged calcite wedges, 1 m in thickness). In
longitudinal section, it is either conical, spherical, barrel-, or pear-shaped, or
cylindrical U-shaped. There is an axial canal or a basal cavity, or both, and
2 apertures opposite each other. Through time, it shows apparent speciation.''*
Nannoconus is widespread in distribution, having been reported from the
Tasch: Growth and Form of Fossil Protists 447
Mesozoic (U. Jurassic-Lower Cretaceous) pelagic deposits in the Mediterranean
area, Rumania, Cuba, and Mexico.^^ It is always associated with the pelagic
facies containing radiolarians and tintinnids, and occasionally, with smooth
ammonites. ^
The following affinities have been suggested: (1) the object represents an
embryonic stage of the tiask-shaped foraminifer Lagena; (2) it is a unicellular
chlorophyllous alga; (3) it is of inorganic origin having formed from calcite
crystals in a highly saturated medium; (4) it belongs to the oogonia of certain
Charophyta; and (5) it represents a little known coccolithophorid.''^'^^
There are then a whole set of constants and some variables to explain. Con-
stant factors include: distinctive wall composition and structure, 50 ju or less
in length; persistence of faunal associations; and occurrence in pelagic facies
intermittently deposited over a span of tens of millions of years in different
parts of the globe. Variable factors include: nine species of X annoconus based
upon variations in axial canal and basal cavity, overall shape and size; three
distinct Xaiiiiocoiius faunas in as many zones of the Lower Cretaceous.'^^
In light of our previous discussion on form in many protists, the likelihood
is that configurations in Xannoconus are variants of a sphere.^^ Thus, selective
modification of the sphere gives an elongate type or a cylindrical type. The
basal cavity of circular types have no axial canal when seen in thin section.
Circular types were spherical in life. Elongate, conical, and subovate types
do have an axial canal. The size and configuration of axial canal and basal
cavity could be a function of compression of an original sphere. Although this
can account for the variation in morphology and inner spatial relationships of
the object, it is unclear whether mechanical compaction or genetics was the
active agent.
Although we assume the first of these possibilities, the list of constant fea-
tures still remains to be explained. Colum'^" notes that Xannoconus at times
appears in great numbers in pure limestone lithotopes. Population density is
thus another variable.
What is the likelihood that inorganic precipitation of calcium carbonate and
mechanical distortion alone can account for Xannoconus? The nearest ap-
proach to a regular type of inorganic carbonate deposit is the example of oolites.
These may be radiate in internal structure or bear concentric bands around a
nucleus. In size range, oolites are also restricted wherever they are found.
Mineralogy of the bands tends to be relatively uniform although alterations
are known. There is a definite spherical-to-elliptical configuration. When
compressed, flat, pelletiferous shapes result. Why cannot Xajinoconus be an
object of this type?
The best argument against an inorganic origin is the persistent crystallization
of minute calcite wedges, all of which are perpendicular to, and form a band
about a hollow basal cavity or axial canal. Inorganic origin cannot account
for the discrete thickness of the wall in this case as it can for the successive
bands of oolites. If the calcite wedges were invested in an organic membrane
that surrounded a cavity or canal, both wall thickness and mineral orientation
could be readily explained.
Once the conclusion is reached that Xannoconus is of organic origin, the other
448 Annals New York Academy of Sciences
array of factors readily supports the interpretation that it represents a pelagic
protist of uncertain affinities.
Among other organized micro-objects of uncertain position are Favreina,
Globochaeta, Eothrix, Lomhardia,''^ Pilhonella,^^ and objects described by
Elliott.^i"
Discoaster, an object 3 to 15 /x in diameter, is represented by calcareous,
stellate, or rosette-shaped plates. In many species the central area bears a
stem. These objects are abundant in pelagic sediments of Tertiary age. The
sediments containing discoasters also bear coccohths, Globigerina, and other
pelagic foraminifers.^*'^^ These objects are now thought to be the skeletal
remains of nannoplanktonic organisms of uncertain affinities.
One ecological observation has been made about discoasterids. Across the
Eocene-Oligocene boundary, Riedel found not merely a change of radiolarian
fauna, but "surprisingly," a change in discoasterid assemblage. This is thought
to reflect "some change in surface waters. "^^ The active factor here might be
surface temperature.
A whole series of related forms of uncertain position among the calcareous
nannoplankton include: Claihrolithus, Discoasteroides, Fasciculilhus, Heliolithus,
Tsthmolithus, Polycladolithus, Sphenolithus, and Rhomboasier.^^ Even though
Rhomboasler "is suggestive of some unusual habit of inorganic calcite growth,"
three considerations refer it to the nannoplankton: specimens occur in abun-
dance; they are found only with coccolithophorids; their occurrence in time
is restricted. ^^
Numerous reports of minute sporelike and other types of bodies and "mesh-
work filaments" from Pre-Cambrian algal stromatolites are now at hand from
Russia, Scandinavia, France, West Africa, and the United States (Gunflint
formation of Northern Michigan). The biological organization of Barghoorn's
material "is supported by geochemical evidence" {i.e., the quantity of C'^ per
mil).^^ There is no equivalent verification of pyrite spherules thought to have
replaced microorganisms.^*
Stimmary
Pelagic protists tend to contigurations of least surface area. The sphere and
its modifications is a recurrent shape. The many shapes and structures
(spinosity, for example) of the scleratoma of radiolarians, the lorica in tin-
tinnids, the frustule of pelagic diatoms, the armored skeleton of dinoflagellates,
the test of planktonic foraminifers, the siliceous and calcareous skeleton of
chrysophytes, appear to be adaptations to resist sinking below optimal food
and photic levels of the sea.
Examples of nongenetic factors affecting differential morphology of protists
include: variable oceanic temperature, salinity, depth, and turbidity; presence
of lead, excess iron and copper as well as carbon dioxide; condition of encyst-
ment; nature of substrate, barophilic property, nutrient salt concentration,
and amount of light penetration. Some of these factors apply only to specific
protists.
Fossil microproblematica of Mesozoic age seem to be nannoplankton of un-
certain affinities among the protists (for example, Nannoconus). Micro-
objects of Pre-Cambrian age may represent spores and algae.
Tasch: Growth and Form of Fossil Protists 449
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57. Hasle, G. R. 1959. In Intern. Oceanog. Cong. A.A.A.S. : 156-157. Preprints.
58. Hanna, G. D. 1928. J. Paleontol. 1: 259-263. PI. 41.
59. Salle, A. J. 1954. Fundamental Principles of Bacteriology. : 59-61. McGraw-Hill
Book Co. New York.
60. ZoBell, C. E. 1959. In Intern. Oceanog. Cong. A.A.A.S. : 395-396. Preprints.
61. ZoBell, C. E. & C. H. Oppenheimer. 1950. J. Bacteriol. 60: 771-781.
62. ZoBell, C. E. 1957. Bacteria. In Treatise Marine Ecol. and Paleoecol. G.S.A. Mem.
67: 693-698.
63. Reiser, R. & P. Tasch. 1960. Trans. Kansas Acad. Sci. 63: 31-34.
64. PiA, J. 1926. Pflanzen als Gesteinsbildner. Berlin.
65. PuGH, W. E. 1950. Bibliography of Organic Reefs, Bioherms, and Biostromes. Seis-
mograph Serv. Corp. Tulsa, Okla.
66. Maslov, V. P. 1956. P'ossil Calcareous Algae. Moscow.
67. GiNSBURG, R. N. 1960. /« Proc. 21st Internat. Geol. Cong. Part 22. : 26 35. Copen-
hagen.
68. Tasch, P. 1951. Am. Midland Naturalist. 46: 751-753. Table 1.
69. Fenton, C. L. & M. a. Fenton. 1957. In Treatise Marine Ecol. and Paleoecol. G.S.A.
Mem. 67: 105-106.
70. KoNisHi, K. & J. L. Wray. 1961. J. Paleontol. 35: 659-665. PL 1.
71. Elliott, G. F. 1948. Micropaleontology. 4: 419-428. Pis. 1-3.
72. Elliott, G. F. 1962. Micropaleontology. 8: 29-44. Pis. 1-6.
73. Bronnimann, P. 1955. Micropaleontology. 1: 28-51.
74. Cloud, P. E. & P. H. Abelson. 1961. Proc. Nat. Acad. Sci., U.S. 47: 1705-1712.
FOSSIL ORGANISMS FROM PRECAMBRIAN SEDIMENTS
Elso S. Barghoorn
Department of Biology, Harvard University, Cambridge, Mass.
Stanley A. Tyler
Department of Geology, University of Wisconsin, Madison, Wise.
In widely scattered outcrops of the Gunflint Iron Formation, Lake Superior
region, Ontario, Canada, dense, black, nonferruginous, fossiliferous cherts oc-
cur as thin units in the sedimentary sequence of black shales, argillites, and
dolomites. In its geological setting, the Guntiint Iron Formation is generally
regarded as comprising the middle unit of the Animikie Series (Middle Hu-
ronian equivolent) of the Lake Superior region. Absolute age of the Gunflint
Formation has been determined by P. W. Hurley by measurement of the po-
tassium-argon ratios in authigenic minerals which occur in direct association
with the cherts and interbedded in the Gunflint sedimentary sequence. Repli-
cate determinations have yielded consistent values of 1900 M years (1.9 X 10^
years) .
The cherts have been studied with the use of thin sections, acid maceration,
and a variety of chemical techniques. Thin sections of the chert, when viewed
in transmitted light, reveal that its black color, as seen en masse, is caused by
the abundance of finely disseminated organic matter that appears light amber
to dark brown in color in sections 50 /x or less in thickness. In this respect the
chert behaves petrographically much as a typical bituminous coal, which in
thin section exhibits a range in color of the petrographical components from
light yellow through amber to dark orange red to opaque. In the Gunflint
chert a large fraction of the organic constituents reveal a distinct morphological
organization consisting of filaments, septate and nonseptate, spheroidal or
spherical bodies, and more complex asymmetrical structures. The discrete en-
tities are all microscopical in size and present an appearance analogous to
masses of anastomosing algal filaments in which are enmeshed other microor-
ganisms. The chert matrix in which the organisms are embedded varies from
clear and hyaline to granular and crystalline. In polarized light the chert is
microcrystalline. Crystals of pyrite, calcite, and apatite vary in abundance,
but in no case are more than minor petrographical constituents.
The biological affinities of the organisms preserved in the Gunflint chert
present a curious paleontological problem inasmuch as a number of the distinct
entities or "types" possess a morphology that is quite unlike that in existing
microscopical crganisms, either plant or animal. In this connection it should
be emphasized that the organic structures are 3-dimensionally preserved and
not flattened or unilaterally distorted. They are hence amenable to morpho-
logical and histological study.
The most abundant organisms in the assemblage are filaments ranging in
diameter from 0.6 to 6.0 n. In the most favorably preserved state these are
found to be both septate and nonseptate. The septate types exhibit a form
indistinguishable from that of filamentous blue green algal {vis., Oscillator ia,
Lyngbya, etc.). The nonseptate types are more difficult to interpret in terms
of biological affinities. With exceedingly few exceptions they are unbranched
451
452 Annals New York Academy of Sciences
and visibly devoid of internal structures or inclusions. Whether these repre-
sent coenocytic algae or fungi is not possible to determine, although the general
form and undulating outline of the filaments is more characteristic of algae than
of aquatic fungi. Among the larger nonseptate filaments very occasionally
forms have been observed in which the lumen of the filament contains numerous
spherical sporelike bodies. In living organisms a somewhat comparable
morphology may be found among certain of the iron bacteria {Crenothrix poly-
spora).
The sporelike bodies which are ubiquitous and irregularly distributed
throughout thin sections of the chert vary in size between 1.0 to 16.0 /x in
diameter (measured along the long axis if ellipsoidal). They are predom-
inantly spheroidal and are not appendaged. The range in size, thickness
of wall, and variation in the sculpture pattern of the wall residues indicates that
they comprise an assemblage of forms the morphology of which gives little clue
to phylogenetic aflinity.
A very common and distinct organism in certain facies of the Guntiint chert
is an entity whose closest morphological comparison among living organisms
can be found in certain groups of the phylum Coelenterata. Rather than to
accept the existence of coelenterate animals in an assemblage of such geological
age as the Gunflint sediments exhaustive efforts have been made to compare
these structures with algae, various of the larger colonial bacteria, and protozoa.
It has not been possible, however, to find morphologically comparable struc-
tures in these diverse groups and the authors have been forced to conclude, on
the grounds of morphology, that the organisms most probably represent meta-
zoons, the closest structural affinity of which is among the Coelenterata. A de-
tailed description of these organisms and other microstructures occurring; in the
chert will be made in a forthcoming paper dealing with the detailed geology and
paleontology of the (iunflint chert.
The organic fraction of the darker and more organic samples of the Gunflint
chert varies between 0.2 to 0.6 per cent by dry weight. As previously noted
Si02 comprises the major mineral component and constitutes more than 99 per
cent of the dry weight of much of those chert samples that exhibit the best
preservation of organic structures. The organic residues yield small amounts
of benzol-acetone-methanol soluble substances, probably hydrocarbons of
molecular weights C20 or above. These extracts fluoresce strongly in ultra-
violet light. Upon destructive distillation at 400° C. the insoluble organic resi-
dues yield small amounts of aliphatic hydrocarbons, chiefly methane (87 ppm),
ethane (4 ppm), and propane (0.7 ppm) and traces of aromatic hydrocarbons
(benzene, 0.34 ppm; toluene, 0.15 ppm; xylenes, 0.45 ppm). Degassification
of the chert at room temperature yields methane (6.0 ppm) and butane (0.2
ppm). The chemical data, although limited, are entirely consistent with the
paleontological interpretation that the black chert represents the silicified re-
mains of a biocoenose of microscopical organisms the organic matter of which is
partially retained, although highly modified through time by very low thermal
and metamori)hical alteration. For these reasons the (iunflint chert is uni(|ue
among earlier Precambrian sediments in exhibiting the morphological organi-
zation of an assemblage of very ancient and primitive organisms, some of which
have counterparts among existing primitive group.^
JS.
BACTERIA FROM PALEOZOIC SALT DEPOSITS
Heinz Dombrowski
Justus-Liebig University, Giessen, Germany
Stimulated by the bacteriological tindings in the mineral springs of Bad
Nauheim, which carry salts from Permian deposits, I investigated from a bac-
teriological point of view the Zechstein salts, obtained by means of mining and
drilling. Mliller and Schwartz (1953), Rippel (1945), and Strong (1956) only
achieved the isolation of dead bacteria from Zechstein salts. Reiser and Tasch
(1960) reported the living isolation of a diplococcus from Permian salts. We
now succeeded in isolating living bacteria. Yet, this achievement seemed rather
improbable; for if we had actually extracted living bacteria from Zechstein
salts, then we have to assume that we found creatures of the highest individual
age ever registered.
The following is a description of the isolating technique we used.
In bacteriological work it is obviously very easy to get unwanted secondary
infection. To be sure that this secondary effect would not spoil our results,
we used extraordinary precautions. (1) We chose a small research laboratory
in which an ultraviolet sterilization lamp was kept burning for four days before
the experiment. No one entered this room during these four days. (2) The
two researchers entered the laboratory in sterile clothes and sterile rubber gloves
after thorough disinfection of their hands and arms. (3) The table and neces-
sary tripods were covered with sterile towels. (4) All necessary instruments,
glassware, and apparatus were thoroughly sterilized. (5) The research ma-
terial, i.e., the piece of salt under consideration, was suspended on thin, sterilized
wire from the tripod. (6) This suspended piece of salt was then flamed for one
minute with a hot bunsen flame. (7) Immediately afterwards a glass with a
culture solution was brought under the piece of salt, so that it was suspended
in the solution. (8) The supporting wire was then cut and the glass was closed
after sterilizing the rim and the stopper also with the bunsen flame. (9) The
cultivation was carried out at a temperature of 40° C. (10) As soon as the cul-
ture began to grow, the elaboration to the pure culture proceeded in the usual
bacteriological manner.
To working procedure 6, I must add that the necessary time for the surface
treatment of the salt with the bunsen flame was ascertained in preliminary
experiments. Salt-pieces, which were brought into a fresh suspension of living
Pyocyaneus — about 80,000 per cm.^ — could be sterilized in 45 seconds.
Because salt is a poor heat conductor, the temperature fell rapidly toward the
center of the crystal. We heated the surface for 45 seconds. Then 3 cm. from
the surface, the temperature rose only by 6.2° C. Thus, we achieved a sterility
of the surface and regions close to the surface without producing sterilizing
temperatures in deeper layers. Of course, the crystals must be large enough;
they must have a diameter of at least 6 cm. Such specimens have a weight of
about 250 to 300 gm. A crystal this large saturates about 1 liter of culture
solution; a saturated solution is necessary for the cultivation of halophil and
halotolerant organisms.
453
454 Annals New York Academy of Sciences
For the duration of this work we set up cuhure plates on which germs in the
air coukl germinate, which in most cases did not happen. If the germs of
the air did germinate, however, they were brought into saHne solutions to
prove their tolerance to salt. This test always showed an intolerance to salt,
so that there was no identity to the bacteria that came from the salt specimens.
In counter-checks we sterilized salt crystals for 4 hours at 200° C., before
investigating them bacteriologically in the prementioned manner. These
crystals proved to be sterile. We also examined crystals coming up from a
depth of more than 4300 m.; in the Mesozoic era these salts lay about 1000
meters deeper than today. At this depth the temperature is at least 160° C,
and as expected these salt specimens also showed no sign of life.
Now, how can we find an explanation for the conservation of life over such
an extended period of time, that is for over 180 million years? There are two
possibilities. First, one is reminded of the method for conserving bacteria that
is practiced today, i.e., dehydration at low temperatures. If one extracts al-
most all the water from the protein of micro-organisms, it is possible to preserve
them for years without changing any of their particular characteristics, although
there is no metabolic activity whatsoever. We know of certain germs, which
lived for more than 30 years, although their metabolism was totally inhibited.
Starke and Harrington (1931) consider the vitality of dried bacteria as un-
limited. If this is correct, then the hypothesis of finding living organisms in
Paleozoic layers could not have received better support, and we would then
have found a way of understanding the survival of these organisms over such
long periods of time. Second, there is the possibility of reversibly denaturing
protein by salification. This method can also be used on higher organisms with
good results. For instance, the protein from the eggs of sea urchins can be de-
naturized in a saturated solution of ammonium sulfate. After months, this
process is reversible by simply removing the salts. The eggs retain the ability
to be fertilized. Perhaps in our specific case both methods, that of dehydration
and that of salification, were in effect.
If this interpretation was true, then the method should be reproducible in a
laboratory experiment. For this experimental reproduction we used Pseu-
domonas halocrenaea, which were isolated from Zechstein salts. This bacterium
does not bear spores.
If the nutrient solution in which it started growing is slowly dehydrated,
the bacterium will die. This will not happen if one slowly saturates the
solution by adding 1 gm. of salt per week. This substratum is now slowly de-
hydrated, until all salts are completely dry and crystalline. In this dry state
it can be kept for long periods of time. When bringing these salts into a fresh
nutrient solution again, the original vitality of the bacterium can be re-estab-
lished.
I would like to point out a further peculiarity: the optimal temperature for
many of the germs that we found lies between +45 and +vS5° C, which is
astonishingly high. But, elucidating enough, this temperature corresponds
exactly to that temperature which, geologists say, was present when the Zech-
stein sea was slowly drying up.
I believe that this correspondence of temperatures is certainly not accidental.
Because the bacteria were embedded in the crystals, they were assured against
Dombrowski: Bacteria from Paleozoic Salt Deposits 455
destruction by mechanical pressure. After considering the depth of our find-
ings, we can estimate a maximum of 1400 m. With the normal geothermic
gradient, which gives the temperature at a certain level, we get a maximal
value of +42° C, which the germs were exposed to during their long latent
life. This temperature in no way prevents the preservation of life.
The cjuestion of which geological specimen is to be examined is of foremost
importance. At first I used all sorts of Zechstein salts, while trying out the
bacteriological working procedure. But later, I carefully selected the speci-
mens to be investigated. All specimens, which came from questionable
regions, such as near faults or the upper salt level, were discarded. Specimens
showing signs of recrystallization were also discarded. We used only pieces
which definitely showed signs of being primary Zechstein salts, and of these only
those which came from perfectly undisturbed points in the middle of larger suc-
cessions of rock salt, the layers of which were formed normal-hypidiomorphic
to allot riomorphic. Their grain size lies in the order of millimeters. But even
with this careful selection of specimens, only about every second culture showed
results.
Because it is very probable that the organisms are of primary genesis, we can
undertake an estimation of the age of these isolated living bacteria. Because
pollen grains were isolated, which served as characteristic fossils, it was rela-
tively easy to establish the age of the bacteria.
We also centered our attention on another aspect of the problem: in undis-
turbed geological layers the rock salt has practically no pores, if we disregard
the lye enclosures. If the salt is taken out from its natural environment, it
will not be subject to the pressure of the overlaying strata anymore. It relaxes
and thus increases in volume by a few per cent. Due to this loosening, pores
begin to form and air can automatically enter the salt. This would make
possible the entering of bacterial contamination from the outside. To prove
that this was not happening, we prepared petrographic thin sections of the salt.
In examining these, we found the bacteria to be embedded in the crystalline
structure of the salt and not in the capillary crevices (figure 1).
Contrary to the previously shown Paleozoic microorganisms, this form (fig-
ure 2) is a direct decendent of the Paleozoic germ, which was obtained by cul-
tivation, and identified as Bacillus circulaus. I found this form in three differ-
ent Zechstein formations. It is a very rare specimen, which has been described
only eight times since 1890. A comparison of the Paleozoic and the Recent
representatives of this group is of special interest. When the Recent germs are
compared from an evolutionary point of view they are neither older nor younger
than the Paleozoic ones, but the Recent type has gone through completely
different stages of development. They were not preserved in a latent stage of
life, but have probably gone through an immensely great number of cell divi-
sions. If it were not for the phenomenon of circular migration, which is pecu-
liar to both the Paleozoic and the Recent type, it would be very difficult to find
a relationship between the two.
Comparing them biochemically, we find very distinct differences. Our 3
Paleozoic strains show almost identical biochemical properties. The strain
found by Kienholz lost all its saccharolytic characteristics, which its Paleozoic
relatives had. The only new characteristic is their ability to liquefy gelatine.
456
Annals New York Academy of Sciences
Beyond this fact, a comparison over such long periods of tune gives the
following results: (1) The paleozoic strains of the Bacillus circulans have quite
a lot more biochemical characteristics than those described in the preceding 70
^
^
^
\
i
%
■%
4. m
-"W
ms
Figure 1 (Top). Bacterium in the center of a thin section of a thickness of 15 ^u, enlarge-
ment 3600:1.
Figure 2 (Bottom). Bacillus circulans from the Zechstein salt, enlargement 950:1.
years. (2) It seems that the long, latent life of about 180 million years has
brought about no loss of characteristics for the Paleozoic species. (3) A loss of
characteristics was proved, however, for the Recent representatives of Bacillus
circulans, which have gone through a vast number of cell divisions. (4) Al-
though the differences in biochemical behavior are very distinct, there is an
Dombrowski: Bacteria from Paleozoic Salt Deposits 457
absolute accord in the morphological characteristics between the Paleozoic and
the Recent representatives of the Bacillus circulans. (5) This leads us to be-
lieve that the genes responsible for the morphological differentiation are much
more stable than those leading to the biochemical characteristics of a species.
There is no doubt that this goes for other species as well, but at the moment
we are only considering Bacillus circulans.
We could not have made these statements, if this species did not have the
characteristic of migration. Relying only on the peripherally whipped bac-
terium and its micromorphology, as with Bacillus circulans, any definite deter-
mination would have been impossible. Even biochemical investigations and
comparisons would lead nowhere, because there are great doubts concerning
the cjuestion of whether or not characteristics of the Paleozoic germs came to a
4
Figure 3. Bacterial strain VIII/D from the Middle-Devonian, enlargement 1200:1.
further development in Recent types. Therefore, it should be very difficult to
show the identity of other types of bacteria, isolated in mineral salts, with
Recent species beyond the probable affinity to a species.
If all of these considerations were true, then it should be possible to cultivate
bacteria from salts of even older origin than those of the Permian age, provided
that these salts come from regions where no tectonic movement had occurred
since their original formation. These experiments had positive results. In
FIGURE 3 are shown bacteria from Middle-Devonian salts from Saskatchewan.
All in all we achieved the isolation of six different species from Middle-Devonian
salts. We were also fortunate to be able to isolate three different species from
Silurian salts, coming from Meyers, New York (figure 4).
Because it was possible to cultivate 2 bacterial species out of Precambrian salt
specimens from Irkutsk, we have reached a sort of absolute level of research.
It is highly improbable that scientists will find even older individual life than
Precambrian, alread}^ approximately 650 million years old.
In FIGURE 5 is shown a bacterium from the Precambrian salt after silver
458
Annals New York Academy of Sciences
impregnation by the method of Zettnow. Both bacteria found in the Pre-
cambrian seem to be closely related to each other.
A list of biochemical data of the isolated germs from paleozoic salts is given
in TABLE 1.
#
8^ •wi^^^^-
«♦• •
'4^'
^%'~^ >•
W%Bi
I V
Figure 4 (Top). Bacterium from the Silurian, strain XV/1, enlargement 1200:1.
Figure 5 (Bottom). Bacterium from the Precambrian salt, strain XXX/1, enlargement
1200:1. (The pictured bacteria are probably the oldest known living organisms with their
approximate age of 650 million years.)
I have not yet examined salts from the Carboniferous. The bacteria from the
Precambrian, Silurian, and some from the Devonian show only few biochemical
properties. The "younger" these germs are, the more they are able to perform
biochemically, only to lose this ability in later life, as shown in the comparison
'S
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Morphology
Spore forming
Motile with flagella
Gram
Physiology
Starch hydrolysis
Nitrate reduction
Indol production
Pigment production
Gelatin liquefaction
H2S production
Salt tolerance
Methyl red test
Voges-Proskauer test
Hemolysis:
Acid from:
Glucose
Laevulose
Sucrose
Maltose
Lactose
Raffinose-hydrate
Rhamnose-hydrate
1-arabinose
Salicin
Inulin
Xylose
Trehalose
Dulcitol
Inositol
Mannitol
O
459
460 Annals New York Academy of Sciences
with Bacillus circulans. A final proof of my findings is now in preparation.
Because it is now possible to find the bacteria in thin sections of the salts, I
want to isolate each bacterium individually with a micromanipulator and let
it grow in a microculture. During this process it will be kept under constant
observation until it shows the germination of spores, or until it starts the first
cell division after being dormant for more than 650 million years. I hope soon
to be able to show this exciting moment in a motion picture film.
Other institutes are now doing research on the coenzymes and proteins of
these Paleozoic bacteria.
Summary
For the first time it became possible to isolate and cultivate bacteria from
Permian deposits. The methods of isolation are described in detail and the
arguments, which lead to the assumption that the discovered microbes are
living representatives of the oldest known individual ages, are sunmiarized.
(1) Only such salt deposits were investigated, which showed indications of being
of primary genesis. (2) From these salt specimens pollen grains were isolated,
which served as characteristic fossils for establishing the age of the deposit.
(3) None of the geological prerequisites, such as tectonics, orogenesis, and geo-
thermic gradients, proved to be contrary to the findings. (4) The method of
isolation, as well as the precautionary measures and the controlling experiments,
are discussed in detail. (5) The results of dehydration at low temperatures
and the reversible method of denaturation by salification are pointed out. (6)
The embedded bacteria are shown optically in thin sections of the examined
salts.
Studies on other salt deposits were made, and living bacteria were isolated
from salt deposits from the Middle-Devonian, the Silurian, and the Precam-
brian. A comparison of the biological characteristics of the Paleozoic germs
with Recent bacteria was carried out.
References
DoMBROWSKi, H. 1960(;. Fundamental balneobiokim. 1: H3.
DOMBROWSKI, H. 1960/). Zentr. Bakteriol. Parasitenk. 178: 83.
DoMBROwsKi, H. 1960c. Munch. Med. Wochschr. 102: 526.
DOMBROWSKI, H. 1960(/. Arztl. Mitt. 4: 143.
DOMBROWSKI, H. 1961(7. Arch. Phys. Therapie. 13(H2): 191.
DoMBROWSKi, H. 19616. Monatsh. iirztl. Forlbild. 11: 78.
DOMBROWSKI, H. 1961c. Zentr. Bakteriol. Parasitenk. 183: 173.
DOMBROWSKI, H. 1961(/. Therap. Gegenw. 100(H9): 442.
DOMBROWSKI, H. Wiss. Arheits. Burgenld. In press.
DoMBROwsKi, H. 1962(1. Kosmos. 58: H3.
DOMBROWSKI, H. 19626. Heilbad u. Kurort. 14: S50.
MtiLLER, A. & W. Schwartz. 1953. Z. Geol. Ges. 105:
Reiser, R. & P. Tasch. 1960. Trans. Kansas Acad. Sci. 63: 31.
RIPPEL, A. 1945. Arch. Mikrobiol. 6: 350.
Starke, C. N. & B. L. Harrington. 1931. J. Bacteriol. 21: 13.
Strong, M. W. 1956. Adv. Sci. 12(49): 583.
FOSSIL PROTOBIONTA AND THEIR OCCURRENCE
A. Papp
Department of Paleontology, University of Vienna, Vienna, Austria
The body of paleontological research consists essentially of knowledge of
organisms having a preservable skeleton. Therefore, one cannot expect that
the oldest organisms will have been preserved. Amino acids, but not the
organisms themselves, have been found in lower Precambrian limestone. The
earliest phase — origin of the basic building blocks, their development into
primitive organisms, as well as the primary evolutionary forms — is beyond the
scope of present paleontological basic research.
By the Cambrian (600 million years ago) many highly diversified skeleton-
forming organisms had developed. Therefore these organisms are within the
focus of paleontological research. At that time life had already attained a
considerable level of evolution, with almost all the invertebrate families present.
In spite of this fact, our knowledge of fossil protobionta is still incomplete; it is
quite possible that a whole array of different organisms is still unknown. How-
ever, the following groups may be classified: (1) bacteria; (2) protobionta with
a preservable outer skeleton of organic material; (3) protobionta with an outer
skeleton of inorganic material; and (4) sporomorpha and spores.
(1) The presence of fossil bacteria has been revealed by different techniques.
Since 1960 H. Dombrowsky's observations regarding bacteria from paleozoic
salt deposits have shown, however, that paleobacteriology is still in its infancy.
The role of anaerobic or sulfate-reducing bacteria in the development of Ufe
lies outside the field of paleontology.
(2) In the group of protobionta with preservable outer skeleton those com-
posed of organic material are believed to be the older species. In this group
only the already relatively complicated structures are known, among them such
Dinoflagellate forms as Chitinozoa and Hystrichosphaeridae. Fossil Hystri-
chosphaeridae with shells of material resembling cutin, which developed in the
Cambrian, cannot be readily distinguished from later forms. In fact, their
close resemblance to spores of fungi (Zygospores) is noteworthy. Skeletons of
fossil protobionta show remarkable resistance under suitable conditions of
fossilization.
(3) Microorganisms of simple structure, such as Archaemonadidae, Sili-
coflagellatae, Diatoms, and Radiolaria, deposit silica in their shells. Fora-
minifers built shells by cementing stone particles with calcite, or occasionally
of calcite alone. In rare instances they employed chitin-like substances. In
contrast, Dinoflagellata seusu lata, undoubtedly represent a later phase in
evolution and offer a vast amount of material for research. Coccolitho-
phoridean skeletons show small calcite particles which may form rock strata
under favorable conditions of fossilization.
(4) The terms sporomorpha and spores imply a state of reproduction con-
siderably different from the fully developed organism. Their resistant outer
layer (exine) composed of sporopoUinin ensures preservation under favorable
conditions. Sporomorpha and spores result from reduction division, which
461
462 Annals New York Academy of Sciences
may take place in a limited number of ways of which the following are known:
(a) tetrahedral — resulting in trilete spores; (b) abortive — same as (a) except
that only one spore develops fully; and (c) rhomboidal — resulting in monolet
spores.
The most common form, tetrahedral meiotic division, necessarily results in
trilate spores with three-sided symmetry. Some of the organized elements
from carbonaceous chondrites described by G. Claus and B. Nagy appear to
resemble such trilete spores. It should be emphasized that in CirciiUna sp.
of the upper Trias — although similar to the above-mentioned organized ele-
ments — the tetradic (trilete) mark is not evident.
The existence of truly multicellular organisms is allied to the function of
reduction division; otherwise, polyploidy would result. We know, however,
that in terrestrial conditions tetraploidy may cause sterility.
A so-called Dauerstadimn is usually linked with the formation of spores or
sporomorpha. Protecting the plasma is a strong hull which consists of the
exine or the sporoderm and the intine. The sporoderm is made up of sporopol-
linin, a terpene derivative, which can become soluble in the presence of oxygen
but is very resistant and capable of fossilization in the absence of oxygen when
minerals are present. It can bind iodine, bromine, and chlorine. During coal
and peat formation, where bacterial activity is reduced because of the acidic
environment, spore preservation is enhanced. Under suitable conditions rich
deposits of sporoderms may occur (fimminit).
The Dauerstadmm allows the organisms to live through highly unfavorable
periods — an especially important consideration if they are subjected to wide
variations in environment, such as extremes of cold or drought.
The majority of skeleton-forming fossil protobionta lived in the oceans of
primeval tmies, although sporomorpha and spores form in marine, limnetic,
and terrestrial biotopes. Adequate preservation of all residues of organisms
depends upon the particular fossilization process. Skeleton-forming proto-
bionta have been described mainly in marine sediments. Sporomorpha and
spores occur in both marine and limnetic deposits and very exceptionally in
terrestrial deposits. Quick embedding in all instances is favorable to the
preservation of fossils. Concentration of residues depends upon the following:
(1) mass of the organisms; (2) mass of the inorganic material involved in the
sedimentation process; {?>) resistance of the organic substance; and (4) destruc-
tive factors before and during fossilization (diagenesis). Ciranting factors 1 and
v3 even relatively small organisms may affect the mineral composition of rocks,
e.g., enrichment of Coccolithophoridae will affect the lime content of marl.
The rule for concentration of fossil spores or pollen is: 20,000 to 40,000 exines
per gram represents the accumulation of normal flora in a given sediment. A
larger number per gram is positive proof of autochthonous flora. However,
the occurrence of fewer and scattered exines points to contamination.
Minute fossilia, except for spores and pollen, are found principally in the
marine biotope. An aquatic medium is usually necessary for preservation and
fossilization of such organisms.
Theories of extraterrestrial life are based on existing conditions on earth.
Each organism, wherever it occurs, must fultill certain regular functions in line
with a given physical law in order to remain alive. The most simple organism
Papp: Fossil Protobionta 463
is a single cell whose plasma is protected by a resistant cell wall. The stronger
the wall, the more likely it is to have perforations (pores or marks) which allow
the plasma to come in contact with the surrounding environment. Only the
most primitive organisms reproduce by simple cell division. All higher forms
of life depend upon sporomorpha to survive hostile periods and to reproduce.
According to G. Erdtmann, sporomorpha in the broadest sense are spores whose
position in the system in unknown. Although they do not always exhibit
trilete markings, their three-sided symmetry may indicate that reduction
division has taken place. One of the criteria of survival is that during the
Dauerstadium substances needed to maintain life be reduced to a minimum.
The basic importance of reduction division (meiosis) to genetic propagation
has already been mentioned. It should also be noted that tetrahedral meiotic
division results in spores with three-sided symmetry. However, three-sided
symmetry is the rule with the widespread trilete spores and the exception with
protobionta and, in fact, with the total animal and plant kingdom. From the
above definition of sporomorpha, it is reasonable to apply this term to the
organized elements of extraterrestrial life having three-sided symmetry, i.e.,
the triporate or trilete forms. This does not specify their position in the sys-
tem, nor does it suggest that an organism similar to an organized element is
equivalent to it. The possibility that organized elements with three-sided
symmetry result from reduction division may not be excluded in the case of
extraterrestrial life. The function of such division is also a possibility in an
extraterrestrial environment .
Residues of extraterrestrial organisms could not be preserved at all except
for a process which may be called fossilization. The following rule holds in all
circumstances: the more residues, the more favorable were the conditions of
fossilization. This requirement is undoubtedly best fulfilled on earth in the
aquatic medium which offers conditions for suitable embedding.
The organized elements w'ith resistant exines or organic material must have
depended on the functions of protein molecules. In this event the extrater-
restrial temperature range of the organized elements' environment would have
to be similar to that on the earth.
A cknowledgments
I wish to thank Dr. W. Klaus, Dr. A. Freisinger, and Dr. K. Turnovsky for
many stimulating discussions on this subject.
STUDIES IN EXPERIMENTAL ORGANIC COSMOCHEMISTRY
J. Oro
Chemistry Department, University of Houston, Houston, Tex.
The four most abundant elements in the universe, with the exception of
the noble gases, are hydrogen, oxygen, carbon and nitrogen,'"'^ which are also
precisely the four major constituent elements of organic compounds and of
living matter. Indeed, as has recently been said, "the composition of living
matter turns out to be a better sample of the universe than the dead earth."''
These four elements exist mainly as atoms and diatomical combinations,
such as CN, CH, C2 , CO, NH, and OH, in the atmospheres of relatively cool
stars,^'^ including the sun,^ and in interstellar or circumstellar space.^'^-^ They
also exist as di- and polyatomic combinations in planets,^"" comets,^-'i^ and
meteorites. ^^'^^ Thus, simple and complex compounds of carbon are found
widely distributed in the universe. In principle, these compounds will exist
wherever the prevailing temperatures are compatible with the stability of the
bonds between carbon and the other elements. If the carbon containing
diatomic combinations, CN, CH, C2 , and CO, are considered, it is observed
that their thermal stability ranges from the low temperatures of interstellar
space to the relatively high temperatures prevailing above the surface of stars.
In fact, such diatomical combinations have been detected in the atmospheres of
supergiant carbon stars at temperatures of the order of 6000° K. at which some
of the most thermally stable oxides, namely titanium and zirconium oxides,
are dissociated into their metallic ions.'
Observations bearing on the distribution of simple and complex compounds
of carbon in cosmic bodies and on the natural formation of these compounds,
form part of a space science which may be called "organic cosmochemistry."
Because of the limited observational data so far obtained and the importance
of the fundamental problems involved ,-'''^^ attempts have been made to follow
an experimental approach in this study. As a result of the initial experiments
of Garrison et al.p Miller,^" '^^ and the more recent ones carried out in this^- and
other laboratories,^^ it has become apparent that processes of organic synthesis
which may have occurred in the primitive Earth's atmosphere, or may be
occurring in certain cosmic bodies such as comets, can be partially reproduced
in the laboratory. These experiments have opened a field of investigation
for which the name "experimental organic cosmochemistry" is proposed.
Models for Organic Synthesis
Any experimental approach to duplicate an incompletely known natural proc-
ess requires the formulation of assumptions about the experimental model to
be used. It is recognized that it would be difficult to determine with certainty
all the conditions applying to a cosmic model for organic synthesis. However,
if it is understood that organic reactions pathways are determined by rather
general laws, then it becomes possible to obtain significant knowledge about
natural organic synthetic processes even with only partially complete models.
We have focused our attention on a cometary modeP" primarily because
464
Oro: Experimental Organic Cosmochemistry 465
comets are supposed to contain large amounts of reactive carbon compounds
and because it is considered that their composition reflects approximately the
composition of the primordial solar nebula and protoplanets.'*'* Indeed, a
recent model for the protoplanets of the solar system,-''^ as suggested by Fowler,
is almost identical to a cometary model proposed some time ago by Whipple^^
and recently revised by the same author.'*^ Cn the basis of this physical and
chemical similarity it is reasonable to assume that the chemical processes which
occur in comets by the action of solar radiation, when these bodies are at dis-
tances of less than 3 A.U. from the sun may have also occurred, but in a much
larger scale in the Earth protoplanet.
Furthermore, it is possible that the conditions for organic synthesis were
quite favorable during the transformation of the gravitationally undiffer-
entiated protoplanet into the primitive planet. This would result from the
mixing of the reactive precursors of organic compounds with inorganic particles,
such as silicate and metallic grains, which could have acted as surface catalysts.
Due to the low density of the synthesized organic compounds, these compounds
would migrate toward the exterior of the planet during the process of gravita-
tional differentiation. The nonvolatile combinations, ionic or high molecular
weight compounds, would accumulate on the surface of Earth, whereas the
gases and the compounds volatile at the prevailing temperatures, would be
evaporated into the outer region of the solar system where comets originate
presently .^^ The difiicult problem of the escape of gases and volatile com-
pounds from primitive planetary atmospheres has been discussed mainly by
Suess'^ and Urey.-'-^"
With regard to the composition of the model, it is known that the spectra of
comets show fluorescence emission bands corresponding to the molecules or
radicals CN, CH, CH. , C2 , C3 , NH, NHo , and OH, to the radical ions CH+,
0H+, C0+, N2+, and CO2+ and to the atoms of Fe, Ni, Cr, and other ele-
ments. '-'^^ ■■*"•*' These emission bands are observed in the heads or in the tails
of comets when these bodies are at less than 3 A.U. from the sun. The band
corresponding to the CN radical is generally the first emission band to appear
on the tails of comets during the travel of these bodies toward the sun, and it is
also the band with the largest degree of extension into the comets' heads fol-
lowed in intensity by the C2 (Swan) and C-i bands.
The above compounds exist in the nuclei of comets either as frozen free
radicals,^--^^ or as "ices"'*'^-'*^ (or crystalline clathrate type hydrates^^) of mole-
cules, which are vaporized and dissociated into radicals by the solar radiation.
In general, it is considered that the parent molecules of CN, NH2 , and OH are
hydrogen, cyanide or cyanogen, ammonia, and water, respectively. The
parent molecules of the carbon radicals are supposed to be methane, acetylene,
and other hydrocarbons. Therefore, a simplified experimental model could be
made of hydrogen cyanide, ammonia, and water. A slightly more complex
model could contain, in addition, cyanogen, acetylene, carbon monoxide, carbon
suboxide, and other compounds. There are certain relations between this
model and the 2 atmospheric models which have been studied previously,
namely, the "primitive planetary atmosphere" model,-^ and the "volcanic
atmosphere" model. ■*'^'^' These models should not be considered as providing
466 Annals New York Academy of Sciences
alternative, but rather complementary, approaches to the study of the forma-
tion of organic compounds on the abiotic earth.^^'** In fact, they represent
progressive stages in the development of the earth. An important condition
which is common to all of these models is thai they are essentially reducing or
at least nonoxidizing in character, of which we have cosmochemical^"-^' and
geochemical^- evidence. Additional evidence for the reducing conditions of
the atmosphere of magmatic origin is provided by the fact that the terrestrial
rate of oxygen production by photolysis of water is less than the rate of vol-
canic carbon monoxide production.*^
Energy Sources
Several sources of energy were available for the synthesis of organic com-
pounds during the transformation of the Earth from protoplanet into planet.
The main source was, of course, the sun providing ultraviolet light and ion-
izing radiation at a rate 10^ times as high as that observed at the present time.^*
A second source was the earth itself with its natural radioactivity^^ ■*■* and the
heat derived from gravitational compression and radioactivity.^^
However, I wish to emphasize that if, as indicated above, some of the primor-
dial constituents of the earth protoplanet were radicals or reactive chemical
compounds, then organic synthesis could have occurred spontaneously at
relatively low temperatures during the melting of the protoplanetary ices in
the absence of highly activating forms of energy. It is surmised that these
spontaneous syntheses were responsible for the formation of substantial
amounts of organic and biochemical compounds. Furthermore, due to the rela-
tively low prevailing temperatures and the reducing conditions of the proto-
planetary environment, the compounds thus formed would have been pre-
served for very long times.
During the further stages of geological development additional sources of
energy were available on the surface and atmosphere of the earth. It is
likely that in addition to ultraviolet light and ionizing radiation, electric
discharges and the heat from plutonic processes contributed also to the forma-
tion of organic compounds.
Synlliesis of Amino Acids and Hydroxy Acids
The synthesis of amino acids and hydroxy acids under possible primitive
Earth conditions has been accomplished by several investigators who used
electrical discharges, ultraviolet light, and ionizing radiation. Moreover,
when some of the reactive carbon compounds detected in comets were used, the
formation of amino acids and hydroxy acids was observed to occur spon-
taneously at moderate temperatures.
(/) By electric discharges. In particular, Loeb,** Miller, *^"*^ Hough and
Rogers,*^ Abelson,^'' Heyns e/ a/.,''^ Pavlovskaya and Pasynskii,*^- Franck,^^ and
Oro and Engberg,^'' applied silent and spark discharges to aqueous mixtures of
totally reduced (CH4 , NH3) or partially oxidized carbon and nitrogen com-
pounds. The products obtained include the amino acids glycine, alanine,
(3-alanine, sarcosine, a-amino-n-butyric acid, a-aminoisobutyric acid, glutamic
acid, aspartic acid, valine, and leucines, and the hydroxy acids glycolic, lactic,
succinic and hydroxybutyric.
Oro: Experimental Organic Cosmochemistry 467
The yield of total amino acids in these experiments was usually less than 5
per cent of the theoretical and the relative yield of each individual amino acid
was approximately inversely proportional to the number of carbon atoms in the
molecule. When methane was used the amino acids formed contained almost
exclusively from 2 to 4 carbon atoms. When methane was replaced partially
by ethane or higher hydrocarbons, valine and leucines were formed in addition
to the other amino acids. ^^ Aside from these and other small variations, the
overall qualitative composition of amino acids obtained in different experiments
by several investigators is very similar, if not identical.
Although the mechanisms of synthesis have not been studied in detail, it
seems that the first phase of one of the possible mechanisms involves the forma-
tion of radicals which recombine to form many compounds including hydrogen
cyanide, aldehydes, amines, nitriles, and aliphatic hydrocarbons. The primary
formation of methyl radicals has been suggested by the experiments of Franck,^^
with either isooctane or methanol in the presence of ammonia and water.
When methanol was used, the observed amino acid yield was increased more
than 50 per cent as compared to that obtained from methane. This is in
line with the fact that 20 per cent less energy is required to form a methyl
radical from methanol than from methane. ^^ That methyl radicals are formed
can also be deduced from a study of the products formed by the action of
electrical discharges upon methane, ^^ and upon mixtures of methane and
ammonia. ^^ Because of the high thermal stability of the triple bonded radical
C2H derived from acetylene®* one would expect that this radical should act as a
trap for other radicals giving rise to the formation of methyl, ethyl, vinyl, and
ethynyl derivatives of acetylene. In fact, these compounds were precisely
the products identified in the aforementioned experiments.*® In a similar
manner the nitrile analogues of the above compounds, namely, acetonitrile,
propionitrile, acrylonitrile, and cyanogen should also be expected to be formed
from the thermally stable triple bonded CN radical derived from hydrogen
cyanide. And in fact some of these compounds were detected by Sagan and
Miller" in model experiments with Jovian atmospheres.
The second phase of this mechanism of amino acid synthesis does not seem to
occur in the gas phase, but rather in aqueous solution. It involves a Strecker
condensation of aldehydes with hydrogen cyanide in the presence of
ammonia.^® '^^ The resulting a-amino acid nitriles which can be detected during
the first hours®'* are progressively hydrolyzed into the corresponding amides
and acids.
In addition to a-amino nitriles, (8-aminonitriles have also been detected in the
reaction product. In particular, |S-aminopropionitrile which is a precursor of
/3-alanine and of pyrimidines has been detected by paper chromatography,®^
This nitrile gives a characteristic green derivative when it reacts with ninhydrin.
An alternative mechanism for the formation of amino acids in the experiments
with electrical discharges is suggested by the presence in the reaction product of
polymers of hydrogen cyanide which are known to be converted into amino
acids (section (4)).
(2) By ultraviolet light. Studies on the photochemical synthesis of amino
acids in aqueous systems were reported some time ago by several investigators.
Baudisch®^ claimed the formation of amino acids from potassium nitrite, carbon
468 Annals New York Academy of Sciences
monoxide, and ferric chloride. Dhar and Mukherjee observed the formation of
glycine from glycol, and of arginine from glucose. Nitrates were used as a
source of nitrogen and titanium dioxide or ferrous sulfate as catalyst. More
recently, Bahadur ef a/.,^^"'^ also with the use of nitrates and ferric chloride
have observed the formation of serine, aspartic acid, and asparagine from
paraformaldehyde. Other amino acids formed in these experiments as detected
by paper chromatography (without previous separation from other ninhydrin
positive compounds by ion exchange) were glycine, alanine, and threonine and
in particular C5 and Ce amino acids which are formed with difficulty in the
experiments with electric discharges. These include valine, ornithine, arginine,
proline, glutamic acid, histidine, leucine, isoleucine, and lysine. The above
amino acids comprise essentially all the building blocks of proteins with the
exception of the aromatic and sulfur containing amino acids.
It would be difficult to visualize the presence of nitrates in a primitive Earth
environment or in a cosmic body. However, the nitrate ion per se should not
be considered as the immediate precursor of the amino group of amino acids.
It is clear that the nitrates must be reduced at the expense of the oxidation of
part of the carbon compounds, such as formaldehyde, which are always present
in a large excess in these experiments. In fact it is known that in the presence
of metallic ions and partiahy reduced carbon compounds, nitrates,'^^ and ni-
trites^^ are rapidly reduced by the action of light to some nitrogen compound of
a lower oxidation level.
Hydroxylamine was suggested by Oro et al.^^ as one of the nitrogen com-
pounds which may be involved more directly in the formation of amino acids.
In fact, this could also be deduced from the synthesis of amino acids from
formhydroxamic acid and formaldehyde by Baly et alP The preferred partici-
pation of hydroxylamine in the comparative photochemical synthesis of amino
acids from formaldehyde and either nitrates, nitrites, hydroxylamine hydro-
chloride, or ammonium chloride has been coniirmed in our laboratory .^^ The
same conclusion has been arrived at by Ferrari^^ •^'' from similar comparative
photochemical experiments but with more complex carbon compounds instead
of formaldehyde.
From a conceptual point of view, ammonia and ammonium chloride are
perhaps the most logical precursors of the amino group of amino acids in a
primitive Earth environment. Experiments carried out by Miller,^** and by
Groth and von Weyssenhoff ,*i '^^ have given evidence that the amino acids
glycine and alanine can be synthesized by irradiating wjth short wave ultra-
violet light (Krypton 1165, 1235 A, Xenon 1295, 1470 A, and mercury vapor
1850 A), aqueous mixtures containing ammonia as the nitrogen source and
either methane or ethane as the carbon source. A higher amino acid yield was
obtained when ethane was used instead of methane. On exposing a mixture
of methane, ammonia, carbon monoxide, and water to the radiation of a hy-
drogen lamp through a thin LiF window, Terenin**^ observed the formation of
the alanines and of several other amino acids.
On the basis of the experimental quantum yields obtained by Groth and
recent theories of solar evolution, Sagan^'' has calculated that the synthesized
organic compounds in the contemporary atmospheres of the Jovian planets.
Oro: Experimental Organic Cosmochemistry 469
and in the primitive reducing atmospheres of the terrestrial planets is of the
order of 1000 g. per cm.^ of planetary surface.
Experiments carried out by Pavlovskaya and Pasynskii"- and also in this
laboratory/^ have shown that several amino acids can be synthesized by irradia-
tion with ultraviolet light of aqueous mixtures containing formaldehyde and
ammonium salts. The synthesized amino acids, which were separated by ion
exchange resins and detected by paper chromatography, include glycine, serine,
alanine, and glutamic acid. The Russian investigators found also vaUne,
isoleucine, phenylalanine, and basic amino acids.
With regard to the mechanism of photochemical synthesis of amino acids it
has been pointed out previously, that the amino group may be derived from
either ammonia or hydroxylamine. However, very little is known about the
mechanism of formation of the hydrocarbon chain. Perhaps monosaccharides
of 2 to 6 carbons are lirst formed photochemically and then transformed by
redox processes into a-keto acids which upon transamination are converted into
amino acids.
That hexoses and hydroxy acids or their lactides are formed by the irradiation
of formaldehyde solutions with ultraviolet light was shown by Baly^^ and Irvine
and Francis.'^'' Moreover, when the syrupy product, thus obtained, was
heated with a trace of acid at 100° C. it was found to resinify into a polymeric
material. This suggested the additional presence in the reaction product of
furfuryl alcohols or polyhydroxyphenols. If phenolic compounds were formed
from formaldehyde these compounds may be the precursors of the aromatic
amino acids.
That hydroxy acids and also keto acids and dicarboxylic acids react photo-
chemically with ammonia, ammonium salts, or other nitrogen compounds to
produce amino acids has been shown by Deschreider*^ and by Cultrera and
Ferrari.^^'*^ Nonphotochemical transamination reactions are also well known.
The synthesis of amino acids containing straight chains with 5 or 6 carbon
atoms could be explained by the intermediate formation of Cs or Ce mono-
saccharides, respectively. These compounds become stabilized by the forma-
tion of furanose and pyranose cyclic structures, stopping the growth of the
monosaccharide chain by preventing the condensation of additional formalde-
hyde molecules. Therefore, essentially no monosaccharides and amino acids
with a linear chain of more than 6 carbon atoms are formed. Branched chain
amino acids could be derived from branched chain monosaccharides such as
dendroketose.
It is of interest that the same maximal amino acid chain length is observed in
these photochemical experiments as in the experiments with electric discharges.
Whereas in the present case the maximal chain length may be determined by
the stability of cyclic structures, in the experiments with electrical discharges it
may be the result of the decreased probability of formation of long chains by
processes of methyl radical recombination.
(3) By ionizing radiations. The synthesis of organic compounds by ionizing
radiation was reviewed by Swallow.'^* After the pioneering investigations in this
area by Garrison et al.,-^ the formation of amino acids by the action of ionizing
radiations has been studied by several investigators. Hasselstrom et al.,^^
470 Annals New York Academy of Sciences
obtained glycine, aspartic acid and possibly diaminosuccinic by irradiating
with j8-rays an aqueous solution of ammonium acetate. Paschke et al.,^^
irradiated solid ammonium carbonate with the 7-rays from a cobalt-60 source
and obtained glycine, 2 other ninhydrin-positive compounds, 1 of which was
tentatively identified as alanine, and ammonium formate.
It is known that formic acid and simple aldehydes are formed by the action
of ionizing radiation over aqueous solutions of carbonic acid.-^-^^ It is also
known the glycolic acid is produced by the irradiation of formic acid.''^ There-
fore, it is conceivable that glycine and other amino acids could also be obtained
by the irradiation of aqueous solutions of ammonium carbonate.
Although from the above experiments it is evident that amino acids can be
synthesized from partially oxidized compounds such as ammonium carbonate,
it would seem more logical, on the basis of theoretical considerations,^^ to study
the irradiation of aqueous mixtures of reduced carbon and nitrogen compounds,
such as methane and ammonia. This has been done by Dose et o/.,^'*'^^ and a
larger number of amino acids and bases have thus been obtained. More
recently, Calvin^® and Palm and Calvin^'' have irradiated mixtures containing
C^*-methane, ammonia and water, among other compounds, with 5 MeV elec-
trons and have obtained a number of amino acids including glycine, alanine,
and aspartic acid. Radiochemical and nonradiochemical mechanisms of syn-
thesis may be involved in this case because hydrogen cyanide, which is known
to condense into products which yield amino acids, was also formed in sub-
stantial amounts in these experiments.
Apart from these amino acid syntheses, it may be added that the 7-irradia-
tion of mixtures of carbon dioxide and ethylene at room temperature yields
significant amounts of long chain carboxylic acids containing as many as 40
carbon atoms. ^^ Also, high energy proton or electron irradiation of methane,
ammonia, and water at 77° K., in a simulated cometary model, yields a number
of organic compounds. ^^
(4) From reactive precursors. As pointed out earlier it is known from astro-
nomical observations that in the atmospheres of carbon stars, very reactive
diatomic combinations of carbon, nitrogen, oxygen and hydrogen are formed.
These combinations are presumed to diffuse out and eventually become part of
interstellar matter, cosmic bodies and protoplanets, being converted in the
process into simple but reactive compounds. These may include hydrogen
cyanide, acetylene, carbon monoxide, formaldehyde, acetaldehyde, ammonia,
hydrazine, and hydroxylamine among others. Some of these compounds have
also been produced in the laboratory from aqueous ammonia-methane mixtures.
Thus, it was considered of interest to discover whether some of these com-
pounds are sufficiently reactive to yield amino acids, and other biochemical
compounds in the absence of electrical discharges, ultraviolet light, or ionizing
radiation.
It was first shown in our laboratory^® that aqueous mixtures of formaldehyde
and hydroxylamine hydrochloride at moderate temperatures and under slightly
acidic conditions yield large amounts of glycine and smaller amounts of alanine,
(3-alanine, serine, threonine, and aspartic acid, the last 3 having been only
identified by paper chromatography. Amino acid amides, glycinamide in
Oro: Experimental Organic Cosmochemistry 471
particular, were found as intermediates, and formic, lactic, and glycolic acids
as side products.
It was found^'' that the mechanism of synthesis involves the initial formation
of formaldoxime and its dehydration into hydrogen cyanide. Strecker and
cyanohydrin condensations yield nitriles which are hydrolyzed first into amides
and then into acids. Condensation of formaldehyde with glycinamide is
presumed to yield serinamide which can be converted into serine and alanine.'""
A similar formation of serine and threonine involving aldol type condensations
of formaldehyde and acetaldehyde with methylene-activated glycine deriva-
tives, such as glycine chelates or polyglycine, was also shown by Akabori
et a/.i"'"i"^ It may be added here that when the formaldehyde-hydroxylamine
hydrochloride mixtures were made slightly basic, pyridines were also formed
in addition to amino acids.
A subsequent study in our laboratory of the products formed by refluxing
aqueous mixtures of formaldehyde and hydrazine revealed the formation of
glycine, vaHne, and lysine as detected by paper chromatography.'"* The
mechanism of lysine formation is thought to involve the intermediate formation
of hexoses and their reduction-oxidation by hydrazine. It is well known that
hexoses are formed from formaldehyde by base catalysis, that hydrazine is
formed by the action of electric discharges on ammonia,'"^ and that hydrazines
can be both reducing and oxidizing reactants.
As mentioned earlier, 3 of the major compounds which are supposed to
exist in comets are hydrogen cyanide, ammonia, and water. For this reason, a
study of the products formed with mixtures of these 3 compounds was subse-
quently undertaken in our laboratory. It was observed that the amino acids
glycine, alanine, and aspartic acid, and other biochemical compounds were
formed spontaneously at moderate temperatures in these mixtures.'*"^ Oli-
gomers of hydrogen cyanide are presumed to be the intermediates of the amino
acids. In fact, tetrameric hydrogen cyanide was observed to be one of the
first products formed in the above mixtures,'"^ and it is known that tetrameric
hydrogen cyanide can be hydrolytically degraded into glycine.'"*''"^ Two
possible degradation mechanisms of tetrameric hydrogen cyanide into glycine
have been suggested by Loquin"" and Ruske."' Other mechanisms involving
processes of reductive deamination can be postulated for the formation of
alanine and aspartic acid.
The formation of amino acids in the hydrogen cyanide-ammonia-water
mixtures has been confirmed and extended by Lowe et al}^'^ In addition to the
above 3 amino acids, Lowe and co-workers have also detected the presence of
(8-alanine, a,(8-diaminopropionic, a-aminoisobutyric, glutamic acid, arginine,
leucine, and isoleucine in the reaction product. The formation of hydroxy
amino acids could conceivably take place in these mixtures if aldehydes were
present, because it is known that formaldehyde and acetaldehyde condense
with methyleneaminoacetonitrile to form serine and threonine, respectively."^
It can thus be seen that, with the exception of the aromatic and sulfur con-
taining amino acids, most of the building blocks of proteins can be synthesized
nonenzymatically in aqueous sytems from very simple precursors in the absence
of highly activating forms of energy.
472 Annals New York Academy of Sciences
With regard to the formation of sulfur containing amino acids, simple
nonenzymatic pathways can also be visualized. Cysteine could be formed in a
similar manner as serine by condensation of thioformaldehyde"'* with a methyl-
ene-activated glycine derivative, such as glycine nitrile, glycinamide, poly-
glycine or a metal chelate of glycine. Methionine could be formed by the addi-
tion of methyl mercaptan to acrolein, followed by the condensation of the
resulting methional"'^ with hydrogen cyanide and subsequent hydrolysis of the
nitrile. One of the possible pathways for the synthesis of aromatic amino acids
could be through monosaccharides or similar compounds obtained from form-
aldehyde.**^
Synthesis of Monosaccharides
Since the early studies of Butlerow,"'"' Loew,"^ and Fischer"* it has been
known that formaldehyde in aqueous solutions condenses into sugars by the
action of basic catalysts. As a result of the work of Fischer"* and others,"^-'-"
fructose, sorbose, xylulose, and glycolaldehyde were identilied among other
compounds in the formaldehyde reaction product.
Relatively recently, Mariani and Torraca'^^ analyzed by two-dimensional
paper chromatography the product of the base catalyzed condensation of
formaldehyde and confirmed and extended the previous results. They detected
the presence of the hexoses galactose, glucose, mannose, fructose and sorbose,
and the pentoses arabinose, ribose, ribulose, xylose, xylulose, and lyxose in
addition to 10 more unidentified monosaccharides. More recent studies by
Mayer and Jaschke^- and by Pfeil and Ruckert^-^ have shown the formation of
glycolaldehyde, glyceraldehyde, dihydroxyacetone and tetroses in addition to
pentoses and hexoses. Dendroketose was also obtained as the product of the
condensation of two moles of dihydrox3^acetone.
The reaction is supposed to be initiated by the condensation of two moles of
formaldehyde into glycolaldehyde which occurs at a very slow rate (induction
phase) .1-* This is followed by aldol condensations which lead to the formation
of trioses, tetroses, pentoses, and hexoses and use up all the formaldehyde in a
very short time (autocatalytic phase) .^-^ The overall reaction is catalyzed by
calcium carbonate, calcium oxide, and other bases.
Because no attempts had been reported on the synthesis of 2-deoxypentoses.
in particular 2-deoxyribose, we undertook the synthesis of this compound,^"
which is known to be one of the essential building blocks of deoxyribonucleic
acid. This deoxypentose and its isomer, 2-deoxyxylose, were obtained in
yields of about 5 per cent by the condensation of acetaldehyde with glyceral-
dehyde in aqueous systems. The reaction occurs very rapidly at room tem-
perature when catalyzed by calcium, magnesium and other divalent metallic
oxides. Results from our laboratory have shown that the reaction is also
catalyzed by ammonia and other simple nitrogen bases which may have been
the predominant bases in the primitive Earth's environment. In contrast to
the fast reaction which divalent metallic oxides catalyze, the reaction occurs
in a slow and controllable manner when ammonium hydroxide is used as cata-
lyst. In fact, the continuous synthesis of this compound was observed for an
uninterrupted period of more than 2 months. 2-Deoxyribose was also obtained
Oro: Experimental Organic Cosmochemistry 473
in smaller yields from aqueous solutions of formaldehyde and acetaldehyde in
the presence of calcium oxide.^-^
Synthesis of Purines and Purine Intermediates
The formation of purines on the primitive Earth or in cosmic bodies pose^
a priori a difficult conceptual problem because it requires the formation of two
fused heterocyclic structures, an imidazole and a pyrimidine.
In principle, there are, however, two relatively simple mechanisms or path-
ways which can be visualized for the formation of the purine ring. One involves
condensation of a 3-carbon compound with a 1-carbon reactant to form a 4,5-
disubstituted imidazole and the other involves condensation of a C3 compound
with a Ci reactant to form a 4,5-disubstituted pyrimidine. The reaction
terminates by cyclization of either the disubstituted imidazole or the disubsti-
tuted pyrimidine with another mole of the Ci reactant.
It is known that the formation of purines in living organisms occurs by a
pathway involving 4,5-disubstituted imidazole derivatives,^-'' and it has also
been observed that the acid degradation of adenine yields 4-aminoimidazole-5-
carboxamidine as an intermediate.'-^ On one hand we have the very mild
conditions of enzymatic synthesis and on the other hand the very drastic
conditions of acid hydrolysis, yet in both cases a 4,5-disubstituted imidazole
shows as an intermediate. Shortly after these observations were made it
became apparent to the author that if a nonenzymatic synthesis of purines
under possible primitive Earth conditions was discovered, it may likely proceed
through the imidazole pathway. The first demonstration of the spontaneous
synthesis of adenine from hydrogen cyanide under conditions presumed to
have existed on the primitive Earth was made relatively recently in our labora-
tory,^^* and in line with the above reasoning 4,5-disubstituted imidazoles were
found in the reaction product as intermediates.
Adenine was synthesized in substantial amounts by heating a solution of
hydrogen cyanide (1 to 15 m) in aqueous ammonia for 1 or several days at
moderate temperatures (27 to 100°). The insoluble black polymer of hydrogen
cyanide was removed by centrifugation and adenine was isolated from the
red-brown supernatant solution by chromatographic methods. The main
ultraviolet absorbing compound of the reaction product was identified as
adenine by a number of different procedures including ultraviolet spectro-
photometry and the melting point of its picrate derivative. The synthesis was
found linear with time at room temperature, and in a typical experiment at the
end of 4 days more than 100 mg. of adenine per liter of reaction mixture were
obtained.^'^^
Since adenine is an essential building block of nucleic acids and of the most
important coenzymes, and since hydrogen cyanide, ammonia, and water are
presumed to be common natural constituents of the solar system, these findings
were considered to be of special significance in relation to the problem of the
origin of life.
In addition to adenine several purine precursors, namely 4-aminoimidazole-
5-carboxamide (AICA), 4-aminoimidazole-5-carboxamidine (AICAI), form-
amide, and formamidine were also found in the reaction product.'^^'^^' The
474 Annals New York Academy of Sciences
mechanism of adenine synthesis is supposed to be initiated by the base catalyzed
polymerization of hydrogen cyanide into nitriles.^^- The role played by am-
monia in this synthesis is 2-fold. It acts as a basic catalyst and it causes the
ammonolysisof hydrogen cyanide into formamidine and of nitriles into amidines.
One of the resuhing nitriles, possibly aminomalonodinitrile, condenses either
directly or after transformation to its mono- or diamidine with formamidine to
form AICAI. In the last step, AICAI condenses with another mole of formami-
dine to yield adenine. This last step has been confirmed in a separate experi-
ment in our laboratory. ^^^
The other purines were postulated to be formed from 4-aminoimidazole-5-
carboxamide.^^^ Recent experiments in our laboratory have confirmed this
assumption. 1^* It has been observed that AICA and guanidine condense in
aqueous ammonia systems to yield guanine. Moreover, when AICA is allowed
to react with urea under similar conditions, guanine and xanthine are formed. '^^
The formation of the 1-carbon reactants, guanidine and urea, in the absence of
free oxygen, poses no special problem because compounds of this oxidation
level, such as urea, were detected by Miller, ^^ Berger,^* and Palm and Cal-
vin,^^ in their respective experiments with electric discharges, high energy
protons, and high energy electrons, which were carried under reducing condi-
tions. Other workers have also observed the formation of guanidine^^- and
urea^^^'^^^'^^^ from cyanides, cyanogen, or cyanates.
The above experiments on the synthesis of adenine from mixtures of hydrogen
cyanide, ammonia, and water have been confirmed by Lowe et al}^^ who have
found an additional purine, hypoxanthine, among the reaction products. A
significant extension of these experiments has been carried out recently by
Calvin, ^^ and Palm and Calvin," who have observed the formation of adenine
by irradiating with 5 MeV electrons a mixture containing methane, ammonia,
and water among other reduced compounds. In summary, it seems to be well
established that the 4 major biological purines can be synthesized, from very
simple precursors, in aqueous systems under possible primitive Earth conditions.
From a historical point of view, it should be said that at the turn of the last
century, cyanogen^^^ and hydrocyanic acid^^^'^^^ were thought to be involved in
the synthesis of proteins and purines in living organisms. These have since
been found to be erroneous concepts. Nevertheless, it is of interest that such
early ideas may apply to the abiogenic formation of these compounds. Studies
on the polymerization of hydrocyanic acid were initially carried out more than
150 years ago,'^- and, therefore, it is highly probable that purines, purine inter-
mediates, and other compounds of biological significance were synthesized in
the laboratory many times since then, yet have remained unidentified until the
present time. Interesting observations bearing on the synthesis of purines from
hydrogen cyanide were made by Gautier,''"* Fischer,'*'' Salomone,^'" and Johnson
and Nicolet,'*- and they are discussed in some detail in a recent paper from our
laboratory.'^' Aside from these early unsuccessful attempts on the synthesis
of purines from hydrogen cyanide, it should be added that uric acid was syn-
thesized from glycine and urea by Horbaczewski,''*^ and purine from formamide
and other simple compounds by Bredereck et «/.'■" '^^ However, none of the
biochemical purines found in nucleic acids was isolated or identified in these
experiments.
Oro: Experimental Organic Cosmochemistry 475
Synthesis of Pyrimidines
With regard to the formation of pyrimidines it was proposed recently^- that
derivatives from the C3 molecular species found in comets could be the source
of these heterocyclic compounds. One of these C3 derivatives is malonamide
semialdimine or its isomer /3-aminoacrylamide which by condensation with urea
could be expected to yield uracil.
Because (S-aminoacrylamide was not available to us, we tested some of the
C3 compounds which are formed in the experiments with electric discharges
and which are considered to be intermediates in the formation of i3-alanine.
These intermediates are acrylonitrile, /3-aminopropionitrile, and /3-aminopro-
pionanide. When each of these compounds was allowed to react with urea in
aqueous ammonia systems at 130° C, the formation of small amounts of uracil
was observed in each case.''*'^ Uracil was characterized by paper and ion
exchange column chromatography and by ultraviolet spectrophotometry. The
yields obtained from /3-aminopropionanide were approximately 2 and 5 times
higher than those obtained from /(i-aminopropionitrile and acrylonitrile, respec-
tively. This is what would be expected if acrylonitrile has to undergo first
amination into /3-aminopropionitrile and this, in turn, has to undergo hydrolysis
into /3-aminopropionanide. Because this amide is, in fact, the dihydroderiva-
tive of /3-aminoacrylamide it is obvious that the mechanism of the reaction
must involve a dehydrogenation step either before or after the cyclization.
The mechanism of uracil formation involving |S-aminoacrylamide or its
isomer, malonamide semialdimire, is in line with the well known chemical
synthesis of uracil from malic acid and urea in the presence of a strong mineral
acid.^''^''^'^ A strong mineral acid transforms malic acid into malonic semialde-
hyde which then condenses with urea to form uracil. '^^ Also, in line with the
above mechanism, it is known from the work of Bredereck et al.,^'^^ that the
pyrimidine ring can be formed in good yield from either aminoacrolein or
malonodialdehyde. In theory the 3 pyrimidines found in nucleic acids could
conceivably be formed in aqueous systems under possible primitive earth
conditions by the mechanism described above. In addition to /3-aminoacryl-
amide yielding uracil, /3-aminoacrylamidine could be expected to condense with
urea into cytosine, and a-methyl-;3-aminoacrylamide into thymine.
A possible pathway for the conversion of the symmetrical C3 species of comets
into ;8-aminoacrylamide or malonamide semialdimine is through the formation
of carbon suboxide (C3O2), which has been suggested to exist in several cosmic
bodies.''^*' By the addition of hydrogen and ammonia to carbon suboxide,
malonamide semialdehyde or malonamide semialdimine might be obtained. In
fact, malonic acid derivatives have been obtained recently in the laboratory
from carbon suboxide.^^^ In addition to purines and pyrimidines, preliminary
data have been obtained on the synthesis of other heterocyclic compounds and
fluorescent pigments. ^•^-
Synlhesis of Polypeptides
The early literature on the direct polymerization of unsubstituted amino
acids has been previously reviewed in some detail. '•^^"^'•^ Current studies on
the synthesis of peptides and of polymers containing amino acids, under condi-
tions presumed to have existed on the primitive Earth were initiated by Fox and
476 Annals New York Academy of Sciences
Middlebrook/^^ and by Akabori.^^^ This work has been reviewed recently'^*"^^^
and has been extended by other workers. As a result of these investigations a
number of different pathways for the formation of polypeptides in a cosmic
body or on the primitive Earth seems possible.
Polymers containing many of the amino acids found in proteins can be pre-
pared by heating a mixture of these amino acids in the presence of an excess of
dicarboxylic"'- ■^'^^ or diamine amino acids.^^^ This synthesis requires anhydrous
conditions and heating at high temperatures for relatively short periods of
time.
The formation of homo- and heteropolypeptides can occur also under aqueous
conditions and at moderate temperatures, as shown by other workers. Thus,
unsubstituted amino acids'^' '^^^ and their corresponding amides^ *''^"'^^ and
nitriles'®^'^^'^^^ have been observed to polymerize directly, or by the action of
basic (ammonia) or surface (silicates) catalysts.
A pathway which seems to be particularly good for the formation of poly-
peptides containing hydroxy acids is that of Akabori et al.,^^^ which is based
upon the condensation of aldehydes (also olefins) with polyglycine. The
natural occurrence of this process would be quite probable because, as has been
shown in our laboratory, polyglycines are readily formed from glycine in
aqueous ammonia systems. Furthermore, in practically all of the abiogenic
synthesis of amino acids studied, glycine has been found to be the predominant
amino acid formed.
Another interesting pathway has been described recently by Schramm
et al™ Polyarginine (mol. wt. 4000 to 5000) was prepared from arginine
with the help of polyphosphate esters. Using the same method, polyleucine,
polyvaline, and polyserine were prepared in our laboratory. '^^
In addition to the above pathways of polypeptide formation other obser-
vations have been made which indicate that peptides or polymers containing
amino acids can also be obtained by the action of ultraviolet light^^- and
electric discharges."^ It should be added that some of the products obtained
by thermal polymerization have the ability to form microspheres with internal
structure,"* and of displaying some catalytic activity.^^^
Finally, a very significant recent development is the isolation of polymers
containing several amino acids from the reaction product of mixtures of hy-
drogen cyanide, ammonia, and water. ^'^ This is the same reaction mixture
that has been shown to give rise to the formation of amino acids, purines, purine
intermediates, and fluorescent pigments among other compounds. Because
nitriles are formed in this system it is possible that the above polymers result
from nitrile condensation reactions. Hydrogen cyanide has been suggested as
an amino acid condensing agent by Calvin."^ Hydrogen cyanide and also
cyanamide (formed by combination of CN and NHo radicals), were probably
abundant in the primordial cosmic bodies of the solar system. It is quite
possible that these reactants were responsible for the formation of a number of
polymeric compounds including polypeptides. In fact, it is known that un-
substituted cyanamide can be used for the synthesis of peptides."^
Synthesis of Polymicleotides
A possible abiogenic mechanism for the formation of a high energy phos-
phate compound, carbamyl phosphate, was proposed some time ago."^ F'orm-
Oro: Experimental Organic Cosmochemistry 477
iminyl phosphate, obtained by condensation of hydrogen cyanide with mono-
hydrogen phosphate, is suggested here as another possibiUty of a primitive
high energy phosphate compound. More recently, Schramm et al.,^'^ have
shown that mononucleosides, mononucleotides, and polynucleotides can be
synthesized at moderate temperatures, from their building monomeric blocks,
with the help of polyphosphate esters. The polymers obtained seem to have
the v3',5'-phosphate diester linkages which are common to RNA and DNA.
Strand complementarity, which is the principle of molecular self duplication,
and autocatalytic activity, have also been observed in the above polynucleo-
tides. The role that nucleic acids and other macromolecules may have played
in directing prebiochemical evolution has been discussed in some detail by
several authors.'''^ •^^*"^*''
Conclusion
There is no doubt that carbon compounds exist widely distributed in the
universe. Whether the more complex biochemical compounds described in
this paper are present in cosmic bodies other than the earth will only be
answered with certainty by space probes. Probes to the moon. Mars, and
Venus are feasible and should provide valuable information about the organic
and inorganic chemistry in these bodies. However, more information about
the chemistry prevailing during the beginning of the solar system would be
obtained by sending probes to Jupiter and to comets passing sufficiently close
to the earth's orbit.
From the experimental studies presented here it is reasonable to say that if
the Earth protoplanet had some of the simple organic constituents of comets,
a large number of biochemical compounds (including carbohydrates, amino
acids, purines, pyrimidines, and polymers containing amino acids) would have
been spontaneously synthesized during the development of this cosmic body.
The formation of complex biochemical compounds from simple organic mole-
cules is not in disagreement with thermodynamic principles. In fact, these
syntheses can occur because the initial precursors (nitriles, aldehydes, olefins,
etc.) are compounds of high energy content which, in their tendency to acquire
lower energy states and to become stabilized, react and are ipso facto trans-
formed into biochemical compounds.
The possibility that organic chemical synthesis may have occurred in inter-
stellar dust and planetesimal bodies before the Earth was formed has also been
suggested by Lederberg and Cowie^^' and Fowler, Greenstein and Hoyle.^^^
Acknowledgment
Some of the work from our laboratory reported in this paper was supported
in part by research grants from the National Science Foundation (G-13117)
and the National Aeronautics and Space Administration (NsG-257-62).
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amino Acids. M. Stahmann, Ed. Univ. of Wisconsin. Madison.
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EVALUATION OF RADIATION EFFECTS IN SPACE
Rainer Berger
Lockheed-California Company, Biirbank, California
In addition to such radiation effects in space as nuclear transformations, the
breakage of chemical bonds and other physical phenomena, the formation of
chemical compounds by radiation synthesis is of great importance.
The aspects of the synthesis of more complicated organic compounds from
simple predecessors are primarily to be discussed in this paper, because they
offer clues to the evolution of organic compounds and to some degree to ques-
tions connected with studies on the origin of life.
Figure 1 illustrates the overlapping successive evolutions which occurred
ever since the body of the Earth accreted. It is evident how the span of chem-
ical evolution interacts with that of organic evolution, the period in which
somewhere life began.
Radiation reactions of the kind discussed here are believed to have occurred
on the primitive Earth. They proceeded in the past and still do on planets,
their satellites, comets, meteors, and even particles of the smallness of inter-
stellar grains. However, each type of reaction may not be applicable every-
where in space.
One of the first experiments carried out in this area of research is the dis-
charge experiment of Miller.' Theories of Oparin- and also Urey^ held for
some time that the atmosphere of the primitive Earth was essentially com-
posed of methane, water, ammonia, and hydrogen. When these compounds
were subjected to an electrical discharge in the laboratory to simulate condi-
tions in nature during a thunderstorm or in the proximity of corona discharges,
a host of different biologically important compounds was detected in the reac-
tion mixture. A number of the resulting compounds are listed in table 1.
The most interesting species are the synthesized amino acids, which as is
generally known are the building blocks of all proteins. It is significant that
none of the complicated amino acids such as tryptophane or serine are produced
in this way (figure 2).
A number of similar confirming experiments were performed by Abelson'*
who used various mixtures of H2 , CHi , CO2 , NH3 , N2 , O2 and H2O. Heyns,
Walter, and Meyer'^ in addition to confirming Miller's work used also HoS in
their investigations and obtained ammonium thiocyanate, thiourea, and thio-
acetamide. Pavlovskaya and Passynsky® equally checked the discharge experi-
ments.
Generally speaking, amino acids were obtained from reducing mixtures only
containing an excess of either H2 , CH4 , CO, or NH;j . No amino acids could
be obtained from an oxidative environment. The mechanism of amino acid
production follows essentially the path of a Strecker synthesis. First HCN
and aldehydes are obtained in the gas phase by the action of the electrical
discharge, then these compounds give amino nitrilcs in the aqueous phase.
Finally, hydrolysis leads to the amino acids.
The experiments on the reaction mechanism show that special conditions of
482
Berger: Radiation Effects in Space
483
the electron bombardment are not necessary, which make it the more plausible
that these radiation syntheses were responsible for the occurrence of amino
acids in the oceans of the primitive earth." It was there that they could be
used to further evolution.
EVOLUTIONS
ORGANIC EVOLUTION CHEMICAL EVOLUTION FORMATION OF THE EARTH
TODAY
a'llO^ 3'«I03 4«I0^ YEARS AGO
Figure 1.
Table 1
Yields! from Sparking a Mixture of CH4 , NH3 , H3O , and H2 ; 710 mg. of Carbon
Was .\dded as CH4
Compound
Yield (moles (XIO^))
Glycine
Glycolic acid
Sarcosine
63
56
5
Alanine
Lactic acid
N-Methylaianine
34
31
1
a-Amino-«-l)Ut\ric acid
a-Aminoisobutjric acid
a-Hydroxybutyric acid
5
0.1
5
)3-Alanine
Succinic acid
15
4
Aspartic acid
Glutamic acid
Iminodiacetic acid
Iminoacetic-propionic acid
0.4
0.6
5.5
1.5
Formic acid
Acetic acid
Propionic acid
233
15
13
Urea
N-Methyl urea
2.0
1.5
Because the composition of gas mixtures may be varied at will in the labora-
tory, conditions in the gas envelope of other planets may be approximated.
Sagan and Miller^ used hydrogen, methane and ammonia resembling the at-
mosphere of Jupiter. The reaction mixture contained afterward a number of
different lower hydrocarbons and acetonitrile. Recent investigations on mag-
netic fields around Jupiter indicate that very strong ones are indeed present.^
Therefore, Jupiter may contain currents of molten material which cause the
fields. It may be deduced that the possibility of warmer zones on Jupiter has
484
Annals New York Academy of Sciences
to be reckoned with. In such zones further chemical reactions are entirely
possible to yield more compUcated systems.
The effects of ultraviolet light on a mixture of methane, water, and ammonia
have been studied also by Miller. ^° Only a very small yield of amino acids
could be obtained. Groth, and separately, Terenin, examined similar reac-
tions.'^ Ellenbogen irradiated a reaction mixture containing FeS, NH4CI,
H2O, and CH4 with ultraviolet light and observed the formation of a substance
the infrared absorption spectrum of which indicated peptide bonds. ''^ Ap-
parently other similar experiments to synthesize amino acid sequences held
together by peptide bonds have not yet been successful.
The effects of visible light on the formation of many different organic com-
pounds have been examined extensively in the literature on photochemistry
and need not be discussed in this paper.
AMINOACID AND PROTEIN STRUCTURE
R R R
— NH — CH— CO— NH — CH — CO— NH — CH-
CO —
V.
H
I
/
Y
H
I
^N —
-C C-
II
H
II
H
/
c4_
-N-
C
II
Figure 2.
In a number of experiments high energy particle radiation as well as x- and
7-rays were utilized. Dose and Rajewsky obtained amino acids and amines
from gaseous mixtures of NH3 , N2 , H2O, CH4 , and CO2 with X-rays.'^ The
action of 2 Mev electrons on CH4 , H2O, and NH3 yielded also amino acids.''*
Calvin irradiated CH4 , NH3 , H2O, and PH3 in the gas phase with electrons.'^
Radiochemical analysis showed the presence of small quantities of nucleic acid
bases, substances which are of vast importance in genetic material (figure 3).
Garrison et al.,^^ used 40 Mev helium ions to obtain formic acid and formal-
dehyde from carbon dioxide. Aqueous formic acid yielded formaldehyde and
oxalic acid.""' Hasselstrom and Henry also obtained oxalic acid from Ca-
(HC03)2 and NH4HCO3.'' Succinic, tricarbolic, malic, citric, and malonic
acid were isolated from the reaction of aqueous acetic acid with helium ions.'*
Also, glycine and aspartic acid were the products in the 2 Mev electron bom-
bardment of acjueous ammonium acetate.'^
All of the previously mentioned radiation reactions occur in gaseous or liquid
systems, but even reactions in the solid state may be carried out. For example,
high doses of 7-rays on solid (NH4)2C03 yielded formic acid and glycine.^^
Berger: Radiation Effects in Space
485
In another experiment, methane, water, and ammonia were condensed to a
solid icy mixture and irradiated with 12 Mev protons.''^ These conditions
approximate the environment thought to exist on comet heads. Analysis of
the reaction mixture indicated the presence of urea, acetamide, and acetone.
The mechanism of this reaction proceeds presumably through a free radical
stage. Either the radicals react with each other in the cold when radical
concentrations become too high, or reaction takes place when the reaction site
warms up to a higher temperature. Similar reactions may not only occur on
comets and icy meteors, but also on the colder outer planets of the solar system
and their satellites.
Based upon astronomical, chemical, and physical observations, it is clear
that not all reactions apply to the same body in space; rather certain reac-
tions will not occur in some instances but play a major role in others. There-
fore, it is essential to consider carefully the environment of the object in
NUCLEIC ACID STRUCTURE
OH OH °V/°"
-0 P^— — CH-CH-CH— — ^^P — — CH-CH — > —
2
ADENINE N
/ \
CM2
H "CH^
/ \
^ I
THYMINE
H.
H
/•^-c-^°
/ \
CH» O
N C
GUANINE
/C^^/'^N^"
"^ ^c^ ^c-
CYTOSINE
H >• ' O'' Nh2
CH,
\ /
-0— ^P.— 0-CH,-CH — CH_o — P — CHi-CH-CH— 0-^--0 —
OH
0' OH
Figure 3.
OH ^0
space before assigning which reactions may predominate. Glasel bombarded
solid D2O with electrons and observed the liberation of considerable amounts
of D2 .^^ Because the bond energy of the D — O bond is higher than that of the
C — H bond in organic compounds, it is to be expected that over periods of
time unshielded organic compounds will be destroyed in space. Therefore,
organic material initially produced on cosmic grains will not remain intact.
Similar destructive radiation effects will occur elsewhere; it is only there, where
shielding from damaging radiation comes into play, that organic compounds
will be available for further reactions.
If I may speculate a little, it may very well be that initially radiation may
have been the agent which at least in part built up molecules to such systems,
which finally were able to handle in a controlled manner radiation or rather
light quanta. The first such successful system to use radiation energy for the
synthesis of organic compounds was the beginning of photosynthesis.
In conclusion, let me say that with the aid of radiation as a form of energy,
one can synthesize chemical species which are the building blocks of proteins,
nucleic acids and other important biological compounds.
486 Annals New York Academy of Sciences
It is reasonable to assume that vast quantities of organic material are or
were formed in space from which a fraction under special circumstances was
the substrate for the evolution of life. To what extent radiation was involved
is hard to assess quantitatively at the moment but the experimental evidence
points to a major role in the processes leading to the creation and functioning
of life.
References
1. Miller, S. L. 1953. Science. 117: 528. 1955. J. Am. Chem. Soc. 77: 2351. 1957-
Biochim. et Biophys. Acta. 23: 480.
2. Oparin, a. I. 1957. The Origin of Life. Academic Press. New York.
3. Urey, H. C. 1952. The Planets. Yale Univ. Press. New Haven, Conn.
4. Abelson, P. H. 1956. Science. 124: 935.
5. Heyns, K., W. Walter & E. Meyer. 1957. Naturwissenschaften. 44: 385.
6. Pavlovskay'a, T. E. & A. G. Passynsky. 1957. Reports of the Moscow Symposium
on the Origin of Life.
7. Miller, S. L. & H. C. Urey. 1959. Science. 130: 245.
8. Sagan, C. & S. L. Miller. 1960. \m. .Astronom. Soc. Meeting, August 1960. : 106.
1960. Astronomical J. 65: 499.
9. Morris, D. & G. L. Berge. Astrophvs. J. In press.
10. Miller, S. L. 1957. Ann. N.Y. Acad. Sci. 69: 260.
11. Groth, W. 1957. Angew. Chem. 69: 68T.
12. Ellenbogen, E. 1958. Abstract of Am. Chem. Soc. Meeting, Chicago.
13. Dose, K. & B. Rajewsky. 1957. Biochim. et Biophys. Acta. 25: 225.
14. Miller, S. L. Unpublished experiments.
15. Calvin, M. In press.
16. Garrison, W. M. c/ (3/. 1951. Science. 114: 416. 1952. J. Am. Chem. Soc. 74: 4216.
17. Hasselstrom, T. & M. C. Henry. 1956. Science. 123: 1038.
18. Garrison, W. M. et al. 1953. J. Am. Chem. Soc. 75: 2459.
19. Hasselstrom, T., M. C. Henry & B. Murr. 1957. Science. 125: 350.
20. Paschke, R., R. Ch.ang & D. Young. 1957. Science. 125: 881.
21. Berger, R. 1961. Proc. Natl. Acad. Sci., U.S. 47 (9): 1434.
22. Gl.\sel, J. A. In press.
ABIOTIC PRODUCTION OF PRIMITIVE PROTEIN AND FORMED
MICROPARTICLES*
Sidney W. Fox and Shuhei Yuyama
Institute for Space Biosciences, The Florida State I'niversity, Tallahassee, Florida
This presentation of results with a thermal model of origins will focus par-
ticularly on some of the properties of the microparticles which emerge there-
from. The more purely chemical aspects of the model have been treated else-
where.'"^ Although the significance of the particles found is much ditTerent
in this context than in others, salient features of the experimentally derived
scheme of origins will, however, again be reviewed here.
One of the popular assumptions which had to be abandoned before the re-
search could proceed was the widely held belief that heating amino acids above
the boiling point of water will yield only dark, unworkable products. This
evaluation has been a common one in the experience of many organic and
biological chemists and has been documented many times.** If, however, one
follows the suggestions from analyses of evolution at the molecular leveF it
becomes possible simultaneously to condense thermally all of the amino acids
common to protein. The products contain each of these amino acids and
have many of the properties of protein. The necessary conditions are the use
of a sufficient proportion of aspartic acid or lysine and an initially dry state.
Heating can be at 170° for 3 hours. '^•' The product is a light amber in color
when sufficient aspartic acid is used, and, like protein, it may then be further
purified by dialysis and reprecipitation by salting out the polymer from aque-
ous solution.
A second heresy concerns the belief that heat has generally been thought to
be a reliable agent for denaturation of protein. Not so generally known is the
fact that this process is "extraordinarily sensitive'"^ to the amount of water
present.^ Also, enzymes are more stable when dry}'* Accordingly, the pro-
duction of biologically significant polymers by heating amino acids is not
precluded.
After extensive study of thermal copolymerization of simple combinations of
amino acids, initial evidence that these processes could be effected simultane-
ously was obtained by chromatography.'' End group assay'' '^ showed that
molecular weights were above that of insulin' (6000 for insulin, or approxi-
mately 3000 per end group). With lysine, thermal polymers of mean molecu-
lar weight over 300,000 have been demonstrated in the ultracentrifuge.^^ The
two criteria of qualitative composition and molecular weight are common to
the only two textbook definitions of protein that we have found." ■'-
Of particular interest is the fact that polymerization is aided by phosphoric
acid,'5 7.i3 polyphosphoric acid, or ATP,!^!^ and especially, that the minimal
*The work reported in this paper was supported in part by Grant no. C-3971(04) of the
National Institutes of Health, U.S. Puljlic Health Service, and Grant no. NsG- 173-62 of the
National Aeronautics and Space Administration. Presented in part at the Symposium on
Extraterrestrial Biochemistry and Biology, American Association for the Advancement of
Science meeting, December 27, 1961, Denver, Colorado. Contribution no. 5 of the Institute
for Space Biosciences.
487
488 Annals New York Academy of Sciences
temperature for polymerization is lowered by addition of polyphosphoric acid
to about 70°/ '^^ as well as its contribution to the formation of uracil.^*^ The
recent report of Schramm/^ in which is claimed the polymerization of nucleo-
tides as earlier proposed in a thermal mode/ occurs under similar conditions.
A principal difference is Schramm's use of the ethyl ester of polyphosphoric
acid.
The polyamino acids obtained are referred to as proteinoids because of
molecular weif^ht and (jualitative composition, but they have in addition many
properties in common with protein.^ '-"
Two properties of most interest are those of catalytic activity and morpho-
genicity. Catalytic activity has been found and studied for the hydrolysis of
/»-nitrophenyl acetate. This is an unnatural substrate popularly used in studies
by enzyme model chemists.-' This substrate is unstable and hydrolyzes spon-
taneously over a large range of pH. Histidine, which has been implicated as
part of the active site of many enzymes/" catalyzes this hydrolysis. Simple
derivatives of histidine also have this effect and some which are several times
as active as histidine have been reported, e.g., carbobenzoxyhistidine.-- Pro-
teinoids have been found to be many times as active as that, and in fact 2 of
them are more than 15 times as active.
Of more interest is the fact that the catalytically active proteinoids are in-
activated by heat at 100° for 20 minutes in aqueous buffer solution at pH 6.8.
This effect has been observed in numerous repetitions and the percentage of
inactivation has been found to be greatest for those proteinoids possessing the
highest relative activity.
In an overall view, one interesting relationship involves the fact that cat-
alytically powerful macromolecules are formed under almost dry conditions
by heating and that this activity is later lost also by heating, but the loss
occurs in acjueous solution. The signiiicance of understanding the intimate
and subtle effects of water is emphasized by this relationship. Also demon-
strated is the fact that very elaborate molecules, approximately as complex as
protein molecules, can be produced by a process which, although mechanis-
tically complicated, is remarkably simple in operation.
The kind of morphogenicity observed also depends upon the intrusion of
water into the system, under conditions different from those for inducing loss
of catalytic activity. Acid proteinoid is typically heated in boiling water or
salt solution (1 part of solid to 2000 parts of aqueous phase) for 10 seconds, the
hot supernatant decanted and allowed to cool. There result, for each milli-
gram of solid, approximately 10^ to lO'^ microspheres of the kind shown in
FIGURE 1. The fact that intrusion of water is required for formation of spher-
ules demands a relative absence of water from the system before the macro-
molecules are organized into supramolecular entities.
These formed units are of interest as precell models alternative to Oparin's
coacervate droplets, also studied as precell models.-^ They and derivatives
are of interest also for their morphological similarity to some microfossils-^
and to formed elements found in meteorites.''-^ Interesting differences between
microspheres and coacervate droplets are known; for example, both the micro-
spheres and bacteria retain their integrity on centrifugation, whereas the
coacervate droi)lets coalesce easily.-^ The microspheres also emerge from a
/ Fox & Yuyama : Abiotic Production of Primitive Protein 489
continu um of condit ions which can explain the origins of enzymes and of
"^-inetaBblism, whereas the coacervates are fabricated from such materials as
gelatin and gum arabic, which arose late in evolution.
The units in figure 1 are slightly less than 2.0 ^u in diameter. They have
the size and shape of the cocci, which have been thought of as the most primi-
tive of the bacteria.-^
In FIGURE 2 are microspheres which have been transferred to a solution
saturated with proteinoid and containing 38 per cent calcium chloride. Two
boundaries can be seen. The effects are not optical, as indicated by acentricity
in some of the units. It was later learned that double boundaries could be
'1
f\
D
O
Figure 1. Microspheres. Photomicrograph courtesy of Dr. K. Harada. Lhiits are
approximately 2 fi in diameter.
more easily produced by raising the pH, as from 3.0 to 5.5. Time lapse photo-
micrographic studies demonstrate that the interior can be completely dissolved,
yet the outer membrane remains. This behavior poses the provocative ques-
tion of the difference between the nature of the outer membrane and the inner
material.
In FIGURE 3 is seen a field in which appears a form resembling a cell in divi-
sion. In fact, this one is very similar to an object carefully referred to by
Claus and Nagy in figure 5 of their paper as an organized element resembling
cell division. Preliminary time lapse studies suggest neither division nor
fusion is occurring in the majority or all of these units. The appearance of
such phenomena, however, is provocative in the sense of the properties and
behavior found in the units. An additional field of twinned microspheres is
490
Annals New York Academy of Sciences
seen in figure 4. This figure also shows filamentous structures which arise
from proteinoid.
In FrcuRE 5 are seen the effects of pressure on the microspheres. This seg-
mentation resulted from digital pressure on the coverglass.
In FIGURE 6 are seen algal-like associations of microspheres. These were
produced by making them under a coverglass on the microscope slide. The
resemblance is to Auaboeiia or Xostocr^ We are indebted to Dr. Chester S.
Nielsen for aid in verifying the superficial, albeit incomplete, resemblance.
The resemblance of alleged fossils of this type is also imperfect.
#
U'
Figure 2. Microspheres with double boundaries following increase in pH. Larger figures
are approximately 10 yu in length.
The microspheres are also found to be birefringent, indicating internal order.
When we review the results of almost a decade of experimental studies of
models of biochemical origins we can perceive: (1) amino acids have been pro-
duced by many workers under many laboratory conditions and from many
reactants that plausibly existed on or in the prebiological Earth; (2) in a
majority of such experimental reports, the key aspartic acid appears as a prod-
uct; (3) the polymerization of amino acids has now been accomplished in
hundreds of variations over a range of conditions; and (4) similarly, the forma-
tion of spherular forms has been accomplished in thousands of variations in the
laboratory. We now regard processes 3 and 4 as so rugged and so inexorable
as to believe that they could and should have occurred on many occasions
in many places in the universe. Also, the origin of the necessary amino acids
seems to be inexorable, by one process or another.-^
Fox & Yuyama: Abiotic Production of Primitive Protein 491
-*«
Figure 3. Twinned microspheres produced by rise in pH. Size as in figure 2.
^
J :mm
W
I
Figure 4. An additional field of twinned microspheres. Size as in figure 2.
492
Annals New York Academy of Sciences
C
Figure 5. I'^ffect of digital pressure on microspheres. Size as in figure 2.
■k^ "'•-%;
3»g'
^^c^"^^
-^-r
-2X
Figure 6. Associations of microspheres with resemblance to algae. Size as in figure 2.
<\
Fox & Yuyama : Abiotic Production of Primitive Protein 493
In the context of the orighi of livhig units, one inference is that nature had
almost endless opportunities to experiment with precellular forms until the
necessary apparatus for repHcation was included by chance.
In the context of the present conference, the presumed protobionta observed
in fossils and meteorites may actually be prebionta. If they are, they would be V
in one sense more significant than if they are protobionta. A third possibility ^
is that they are meaningless artifacts, easy for nature to come by because of the
simpUcity of the processes leading to their formation. This point of view has^x
a semantic flavor, because of the position that no natural experiment is truly
without meaning. Also of interest is the fact that Dr. Philip Morrison inde- J)
pendent ly reached the same conclusion from data presented at the Denver
meeting of the American Association for the Advancement of Science.^" In
essence, however, and either with or without regard to the difficult questions
of terrestrial contamination of meteorites, the conclusion at present is that
there cannot yet be a conclusion on the cjuestion of whether the inclusions in
meteorites are protobionta.
References
1. Fox, S. W. 1960. How did life begin? Science. 132: 200-208.
2. Fox, S. W. & K. Harada. In press. Experiments related to the chemical origins of
protein. G. Bourne, Ed. Space Flight. : 261-270. Academic Press. New York.
3. Harada, K. 1961. On the formation of primordial protein and the thermal theory
(Title transl.). Proteins, Nucleic Acids, Enzj-mes (Tokyo). 6: 65-75.
4. Fox, S. W., K. Harada & A. Vegotsky. 1959. Thermal polymerization of amino
acids and a theory of biochemical origins. Exjjerientia. 15: 81-84.
5. Fox, S. \V. 1956. Evolution of protein molecules and thermal synthesis of biochemical
substances. Am. Scientist. 44: 347-359.
6. Fox, S. W. & K. Har.ada. 1958. Thermal copolymerization of amino acids to a product
resembling protein. Science. 128: 1214.
7. Fox, S. \V. & K. Harada. 1960. The thermal copolymerization of amino acids com-
mon to protein. J. .\m. Chem. Soc. 82: 3745-3751.
8. Altman, R. L. & S. W. Benson. 1960. The etYect of water upon the rate of heat de-
naturation of egg albumin. J. Am. Chem. Soc. 82: 3852-3857.
9. Barker, H. A. 1933. The effect of water content upon the rate of heat denaturation
of crj'Stallizable egg albumin. J. Gen. Physiol. 17: 21-34.
10. Dixon, M. & E. C. Webb. 1958. Enzymes. : 153. Academic Press. New York.
11. Fruton, J. S. & S. SiMMONDS. 1958. General biochemistry. : 16. John Wiley and
Sons. New York.
12. Mitchell, P. H. 1948. A textbook of general physiology. : 245. McGraw-Hill Book
Co. New York.
13. Fox, S. W. & K. H.AR.A^DA. 1960. Thermal copolymerization of amino acids in the
presence of phosphoric acid. .\rch. Biochem. Biophys. 86: 281-285.
14. Vegotsky, A. & S. W. Fox. 1959. Pyropolymerization of amino acids to proteinoids
with phosphoric acid or polyphosphoric acid. Federation Proc. 18: 343.
15. Vegotsky, .4. 1961. Thermal copolymers of amino acids. Ph.D. dissertation. Florida
State University.
16. Harada, K. & S. W. Fox. 1960. Thermal copolymerization of amino acids at tem-
peratures below 100°. : 28C-29C. .\merican Chemical Society meeting, Cleveland.
Ohio. Abstracts.
17. Genaux, C. & S. W. Fox. Unpubhshed experiments.
18. Fox, S. W. & K. H.ARADA. 1961. Synthesis of uracil under conditions of a thermal
model of prebiological chemistry. Science. 133: 1923-1924.
19. Schramm, G. 1962. Nicht-enzymatische s}nthese von polysacchariden, nucleosiden
und nucleinsauren. .\ngew. Chem. 74: 53-59.
20. Fox, S. W., K. Harada & D. L. Rohlfing. 1962. The thermal copolymerization of
a-amino acids. : 47-54. M. Stahmann, Ed. Polyamino Acids, Polypeptides and
Proteins. Univ. of Wisconsin Press. Madison.
21. Bender, M. L. 1960. Mechanisms of catalysis of nucleophilic reactions of carboxylic
acid derivatives. Chem. Revs. 60: 53 113.
494 Annals New York Academy of Sciences
22. NoGUCHi, j. & T. Saito. 1962. Studies on the catalytic activity of synthetic poly-
amino acids having an imidazole group in the active site. : 313-327. M. Stahmann,
Ed. Polyamino Acids, Polypeptides and Proteins. Univ. of Wisconsin Press. Madi-
son.
23. Oparin, a. I. 1961. Life: Its Nature, Origin and Development. Oliver and Boyd.
Edinburgh.
24. Barghoorn, E. In P. E. Cloud, Jr. & P. H. Abelson. 1961. Woodring conference on
major l)iological innovations and the geologic record. Proc. Natl. Acad. Sci. U.S. 47:
1705-1712.
25. Claus, G. & B. Nagv. 1961. A microlnological examination of some carbonaceous
chondrites. Nature. 192: 594-596.
26. Bungenbf.rg DeJong, H. G. 1949. Morphology of coacervates. 433-482. In
Colloid Science. II. H. R. Kruyt, Ed. Elsevier Publishing Co. New York.
27. Lamanna, C. & M. F. Mallette. 1959. Basic Bacteriology. : 44-47. The Williams
and Wilkins Co. Baltimore.
28. Smith, G. M. 1950. The Fresh-water Algae of the United States. McGraw-Hill
Book Co. New York.
29. Fox, S. W. 1957. The chemical problem of spontaneous generation. J. Chem. Educ.
34: 472-479.
30. Morrison, P. 1962. Carbonaceous snowflakes and the origin of life. Science. 135: 663-
664.
OBSERVATIONS ON THE NATURE OF THE "ORGANIZED
ELEMENTS" IN CARBONACEOUS CHONDRITES
Frank W. Fitch
Department of Pathology, University of Chicago, Chicago, III.
Edward Anders
Enrico Fermi Institute for Nuclear Studies, Departments of Chemistry and Geophysical
Sciences, University of Chicago, Chicago, III.
Our interest in the morphological study of carbonaceous chondrites was
stimulated by reports of Claus and Nagy^ and of Nagy et al.,- describing a
variety of "organized elements" found in Class I carbonaceous chondrites.
The organized elements had been classified by Claus and Nagy into 5 types on
the basis, primarily, of morphology. The various properties of the organized
elements are tabulated in table 1. Types I and II which were circular or
spherical were most numerous; the other types were much less abundant. A
total of about 1700 organized elements per milligram were reported.' ■"'
In an attempt to confirm these observations and to characterize further the
composition of the organized elements, we examined samples of the carbona-
ceous chondrites Orgueil and Ivuna. One sample of Orgueil was obtained
several years ago from the Musee d'Histoire Naturelle, Paris. Another sample
was obtained through the courtesy of Henderson of the U.S. National Museum,
and was from the same fragment given to Nagy. A sample of Ivuna was ob-
tained through the courtesy of Roy of the Chicago Natural History Museum.
Conventional brightfield, phase contrast and fluorescence microscopy were
used. Other methods included staining with biological stains, and the use of
x-ray diffraction and electron microprobe analysis.
Microscopical Observations
Both samples of Orgueil and the single sample of Ivuna had crumbled apart
and consisted of fragments ranging in size from a fine dust to several millimeters
in diameter. Fragments were inspected visually to be certain that they were
free from fusion crust, paint markings, and other visible contaminants. To
minimize sampling errors, observations were made on the fine dust as well as
fragments broken from larger pieces. This dust that had accumulated at the
bottom of the sealed glass containers came from the surface of many individual
fragments and should, therefore, be fairly representative of the meteorite as a
whole. Because of the friable nature and the porosity of the carbonaceous
chondrites, it is not feasible to clean the meteorite surface. For microscopy,
samples of the meteorite weighing about 1 mg. were placed in a drop of glycerin
on a microscope slide which had been cleaned with 95 per cent ethanol. The
sample was gently crushed with a glass rod cleaned with ethanol. Samples
subjected to density separation were lightly crushed in an alcohol-cleaned agate
mortar.
Initially, particles were sought which had the general morphological charac-
teristics of the organized elements. Because Types I and II elements were
circular or spherical, particles with this morphology were sought. As reported
495
496
Annals New York Academy of Sciences
in an earlier paper,^ the most conspicuous particles with this shape and oc-
curring in the abundance of several thousand per milligram were opacjue and
highly magnetic. They could be concentrated by density separation in the
fraction with a density greater than 3.33. Although opaque, many had trans-
parent, yellow-brown mineral fragments attached to the surface. When viewed
with phase-contrast microscopy, the diffraction pattern around the particles
frequently gave a false impression of a double outer wall, especially when the
particles were slightly out of focus. X-ray diffraction and electron microprobe
studies of isolated particles of this type indicated that they were composed of
troilite or magnetite.^ Although possessing several characteristics of the or-
ganized elements, these troilite and magnetite particles were opacjue. Subse-
Table 1
Reported Properties of Organized Elements*
T3 J-.
V a
O
Shape
Surface
Color
size
Abundance
I
Circular
Double wall, thickening
Yellow-
4-10
Abundant
II
III
Circular
Shield-shaped
and sculpturing
Spines, appendages, furrows
Thickening and sculpturing
green
8-30
15
Abundant
Less com-
mon
IV
Cylindrical
Thick wall, sculpturing
10-12 X 20
Less com-
mon
V
Hexagonal
Appendages
20
Rare
Other reported general properties:
Fluorescence in ultraviolet light
Staining with biological stains
Appearance suggesting cell division occasionally
Resistance to HF treatment
* From Claus and Nagy' and Nagy et al.^
quently, Nagy et al.^^ emphasized several differences between these particles
and the "organized elements."
Other spherical particles were found in some samples of the meteorite which
had been subjected to a density separation with organic liquids. These ranged
in size from about 1 to 20 fx and were transparent and colorless or yellow. Some
appeared to have a double wall. These had a bluish fluorescence of the outer
portion when viewed with ultraviolet light. The smaller particles had uniform
bluish fluorescence. A number of tests indicated that these were hydrocarbon
droplets and droplets of supercooled liquid sulfur coated with hydrocarbon.^
They could be removed by repeated washing of the sample with chloroform
or acetone and therefore did not seem to be organized elements.
A variety of hexagonal particles varying in size from about 2 to 20 yu were
also found. Some hexagonal particles were transparent and yellow-brown
with an opaque, irregular central area; these particles were highly magnetic.
They may be goethite pseudomorphs after troilite, probably formed by pre-
terrestrial oxidation of troihte. Other hexagonal particles were quite
Fitch & Anders: "Organized Elements" in Chondrites 497
small, colorless, and transparent. These were probably silicate or carbonate
minerals. Other hexagonal particles were opac|ue and nonmagnetic. These
were probably one form of troilite which is non-magnetic. None of these
hexagonal particles had appendages cjuite like those found in the type V
hexagonal particle illustrated by Claus and Nagy.^ It should be noted, how-
ever, that the type V organized element is quite rare; only two and a fragment
of a third were found by them in Orgueil.
No other particles of distinctly spheroidal shape could be found. The bulk
of the meteorite consists of a brownish-yellow hydrated silicate (Orgueil LM).*
Most of the silicate particles had a very irregular shape, but a few were roughly
spherical (figure 1). However, even these ovoid to spherical fragments had
at least a partially irregular surface, and none had any definite internal struc-
ture or double walls. They were not magnetic. Although some variation in
color and refractility was noted, the spheroidal particles had numerous irregular
counterparts which matched them in every way except shape. It seems likely
that all of these particles were mineral fragments.
Although each of these types of particles had some of the characteristics of
the organized elements, none seemed to possess all of the primary morphological
properties. However, other properties of the organized elements have been
described.''^ These include fluorescence in ultraviolet light, staining with
biological stains, and insolubility in hydrofluoric acid. Particles with these
characteristics were then sought.
Fluorescence in Ultraviolet Light
Crushed, but otherwise untreated, Orgueil from the U. S. National Museum
was examined with the fluorescence microscope and all of the tkiorescent
particles seen were photographed in visible and ultraviolet light. Based upon
the information of Claus and Nagy,' 39 organized elements should have been
found in the area of the slide examined. Actually, 15 fluorescent particles
were found, but they did not seem to resemble the published illustrations or
descriptions of the organized elements (table 2). They were quite irregular
and when viewed with ordinary illumination were colorless or slightly yellow.
Two typical particles selected from the 14 photographed are illustrated in
FIGURE 2.
Biological Stains
Since the organized elements have been reported to stain with various
biological staining reactions including Feulgen and PAS, these as well as other
staining procedures were used on samples of the meteorite. Many of the
irregular yellow-brown grains stained slightly with the PAS and Feulgen reac-
tions. Although many particles stained slightly, none stained the brilliant
magenta usually achieved in biological materials, and many of the rounded
grains did not appear to stain at all (figure 1). Similar results were obtained
with the Feulgen reaction.
To interpret these staining results it is necessary to examine the nature of the
PAS and Feulgen reactions. The color in both reactions is produced by using
Schiff's reagent, prepared by decolorizing basic fuchsin with sulfurous acid.^
498
Annals New York Academy of Sciences
4
■■■"" «*
"■10 ■
f
%
imtHmtm
Fitch & Anders: "Organized Elements" in Chondrites 499
If aldehydes are reacted with Schiff's reagent, a red- violet color develops that
is different from the original fuchsln. In addition to aldehydes, certain ketones,
certain unsaturated compounds, and various oxidants can colorize Schiff's
reagent.^'* The solution must be fairly freshly prepared; oxidation, aging,
exposure to air, and sunlight can recolorize Schiff's reagent stored in the
laboratory/
In addition to any aldehyde groups present initially, Schiff's reagent will
react with any artificially produced aldehyde groups. For example, periodic
acid oxidizes 1,2 glycol linkages to aldehyde groups. If one of the hydroxyl
groups is substituted with amino alcohol, alkylamino alcohol or carbonyl, it is
also oxidized to give a positive reaction. In biological materials, the reaction
is relatively specific for carbohydrates, mucoproteins and glycolipids. Un-
saturated lipids which can also react are usually removed from biological
samples during preparation for microscopical examination.'''^
Table 2
UV Fluorescence in Orgueil
Color
Size range
Number of particles
Regular
Irregular
Yellow
Bluish
Bluish
Bluish
2-10
2-10
10-50
>50
5
3
5
2
In biological tissues the Feulgen reaction is usually considered to be specific
for desoxyribonucleic acid (DNA).^'^ As the first step in the procedure, DNA
is partially hydrolyzed by 1 n HCl to produce the aldehyde form of desoxyribose
phosphate. The aldehyde groups then react with Schiff's reagent to produce
the same magenta color found in the PAS reaction. In biological samples,
substances which will react directly with Schiff's reagent are usually not
present. With meteorite samples it is essential to determine whether or not
materials are present that will react directly with Schiff's reagent. Such
substances would give a false positive Feulgen reaction and simulate the pres-
ence of DNA. To correctly interpret the results of the staining reactions on
the meteorite samples, proper controls are necessary.
To control the staining reactions, sections of rat spleen tissue fixed in Carnoy's
solution and embedded in paraffin, as well as samples of kimberlite and Orgueil
were studied. Kimberlite, the diamond-bearing rock usually believed to have
come from deep within the earth, was chosen becaues it is perhaps more similar
to Orgueil in mineral composition than other terrestrial rocks. Both Orgueil
and kimberlite consist primarily of serpentine-like hydrated silicates produced
from olivine by alteration under aqueous, reducing conditions. For the stain-
ing reactions, samples of Orgueil and kimberhte were suspended in 6 per cent
gelatin and the mixture was spread on microscope slides and allowed to dry.
500
Annals New York Academy of Sciences
4J Ov
I"
r
J-l '-'
-4—' Ct
c -0
nj O
U "*-
O en
O
U
>^ I-
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c
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oj ra JJ
JJ O r-
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Fitch & Anders: "Organized Elements" in Chondrites 501
Gelatin was used to adhere the samples to the slides because preliminary experi-
ments showed that there was little staining of the gelatin.
The Feulgen reaction was carried out in the routine manner on these samples
with the usual hydrolysis with 1 n HCl at 60° C. for 8 minutes followed by
Figure 3. Orgueil, rat spleen, and kimberlite stained with the Feulgen reaction. (.4)
Orgueil, Feulgen reaction. Most particles stain irregularly. (B) Rat spleen, Feulgen reac-
tion. Nuclei have brilliant magenta staining of DNA. Other tissue comi)onents do not
stain. (C) Kimberlite, Feulgen reaction. Most particles stain irregularly. Some of the
sample was dissolved by the HCl treatment. (Z?) Orgueil, Schiff's reagent only. Staining is
as intense as with the Feulgen reaction. (E) Rat spleen, Schiff's reagent only. There is no
staining of nuclear DNA. (F) Kimberlite, Schiff's reagent only. Staining is as intense as
with the Feulgen reaction.
treatment with Schiff's reagent for 1 hour.^ As a control procedure, samples
were reacted with Schiff's reagent for 1 hour without previous treatment with
acid. In the rat spleen sections, nuclear DNA stained brilliantly after acid
hydrolysis (figure S, B). In sections treated with Schiff's reagent alone, no
staining occurred (figure 3, E). However, samples of Orgueil and kimberlite
stained equally well whether treated with acid or not (figure 3; A, D, C, F).
Something is present in the meteorite and in kimberlite which reacts directly
502 Annals New York Academy of Sciences
with the Schiff's reagent. Therefore, the development of a magenta color
with the Feulgen reaction is, in this instance, not specific for DNA.
Similar results were obtained with the PAS reaction. There seemed to be
no additional staining produced when samples were treated with periodic acid
before reaction with Schiff's reagent, as compared with reaction with Schiff's
reagent alone. Attempts to inhibit the staining produced by Schiff's reagent
by previous treatment of samples with aniline chloride and hydroxylamine, to
block the aldehyde groups,'^ were only partly successful in the samples and in
periodic acid treated starch controls. Hence, the nature of the reactive groups
is not known at present.
The presence of DNA in the organized elements would be powerful evidence
of their biologic origin. Because the results of the Feulgen reaction had been
interpreted in published reports as indicating the presence of nucleic acid in
the meteorite,^-* it was desirable to confirm this interpretation with another
histochemical test for DNA. Methyl green is freciuently used for this purpose.^'
The characteristic reaction of DNA with this stain is thought to be the result
of binding of the dye by phosphoric acid radicals in the intact, polymerized
DNA.* Thus, the mechanism of this reaction is altogether different from that
of the Feulgen reaction.
Samples of rat spleen, Orgueil and kimberlite were stained with methyl
green.* As a control procedure, samples were treated with 10 per cent per-
chloric acid for 4 hours and 30 minutes, a procedure which depolymerizes and
extracts DNA from biological samples.'-' In rat spleen sections stained directly
with methyl green, there was brilliant green staining of the nuclei (figure 4, B).
In spleen sections treated with perchloric acid to remove DNA before reaction
with methyl green, there was no nuclear staining (figure 4, E). However,
the samples of Orgueil and kimberlite stained brilliantly with methyl green
whether treated previously with perchloric acid or not (figure 4; A, D, C, F).
It is evident that when biological staining reactions are applied to nonbiolog-
ical materials, great care is necessary in the interpretation of results. Because
of the presence of other reactive groups the usual tests for DNA are no longer
specific. Positive or negative reactions of any DNA present would be masked
by the intense, nonspecific staining due to other groups. Under these condi-
tions, the staining tests cannot be regarded as evidence for the presence of
DNA in the meteorite.
Treatment with Hydrofluoric Acid
The "organized elements" were reported by Nagy el at., not to be seriously
affected morphologically by treatment with boiling hydrofluoric acid (HF) for
15 minutes, whereas silicate minerals should be dissolved.- We treated a
sample of Orgueil with boiling HF for 15 minutes; 49 per cent of the sample
remained (tabi.e 3). Because the carbon content of the meteorite is only 3.1
per cent, the bulk of this residue must have been inorganic. Consideration of
the pertinent solubility products indicates that calcium, magnesium, and
possibly other major constituents of the meteorite should remain as insoluble
fluorides or fluosilicates. Thus, persistence after HF treatment is not a suffi-
cient criterion for the organic nature of a particle.
To dissolve the mineral residue, the sample was first treated with HF for 17
Fitch & Anders: "Organized Elements" in Chondrites 503
hours at 60° C. and whh 6 N HCl for 18 hours at 25° C. Treatment with
HF-HCl is a standard palynological technique which leaves organic materials
of biological origin, including various pollen grains, morphologically unaf-
fected.'" After this treatment only 3 per cent of the sample remained. X-ray
Figure 4. Orgueil, rat spleen, and kimberlite stained with methyl green. (.4) Orgueil
methyl green stain. Many particles stain irregularly. (B) Rat spleen, methyl green stain
Nuclei are stained a dark green. Other tissue components do not stain. (C) Kimberlite
methyl green stain. Many particles stain irregularly. (D) Orgueil, methyl green stain after
HCIO4 treatment. Staining is as intense as before extraction. (E) Rat spleen, methyl
green stain after HCIO4 treatment. There is no staining of nuclei; DNA has been depoly-
merized and extracted. iF) Kimberlite, methyl green stain after HCIO4 treatment. Stain-
ing is as intense as before extraction.
diffraction and infrared spectrophotometry indicate that this residue is mainly
amorphous carbon with traces of MgFo and organic matter. Microscopical
examination of the residue showed finely granular, black to brown material
virtually devoid of any structure (figure 5, C, D). Often, it was present in
large irregular aggregates (figure 5, B). Very rarely, spherical transparent
particles were seen (figure 5, A), but only 2 were found in an area where
several thousand organized elements should have been present. Granular
504
Annals New York Academy of Sciences
material was adherent to their surface, and httle structural detail could be
resolved with either phase-contrast or brightfield microscopy. The possible
nature of these particles will be discussed in the following section.
Table 3
Orgueil Meteorite: Treatment with HF
Reagent
Temperature
Time
Residue
Composition of residue
HF 24 M
HF24M
HF24M
HC16M
75°
100°
60°
25°
hoius
24
18/
%
>50
49
3
MgF2, CaFo, Fe304, FeS, or-
ganic matter
MgF2 , CaF2 , organic matter
Carbon + organic matter
1
*
_ .^y
-^r
V
.Bjr-%
r,. '
'^
M
m. J'
#
D
Figure 5. Orgueil after HF-HCl treatment. (.4) One of 2 transparent spherical par-
ticles seen. Irregular black-brown material is adherent to the surface. (B, C, D) Amorphous
residue remaining after HI-'-HCl treatment. Most of this material is amorphous carbon with
traces of MgF2 and organic matter. The line is 20 n in length.
A ttempls at Identification of Some Organized Elements
It is evident that there are discrepancies between our findings and those of
Nagy et al. In an attempt to resolve these differences, we visited the labora-
tories of Claus and Nagy at their invitation. They examined our material
and we examined their material. It became evident that there were several
reasons for the differences.
4
I
Fitch & Anders: "Organized Elements" in Chondrites 505
First, their material contained a few particles of striking morphology which
we had not found and which they did not find in our material. Examples of
such particles found in their material are shown in figure 6, A and B, and
FIGURE 7, A and B. These were classified by Claus and Nagy as type II
organized elements with double wall and spiny surface. Particles of strik-
ingly similar morphology are illustrated in figure 6, C and D, and figure 7,
C and D. These are common ragweed pollen grains. The particles in figures
6, A and B, and 7, A and B, were suggested by them to be extraterrestrial life
Figure 6. (A and B) Organized element from preparation of Claus and Nagy. The
different levels of focus indicate double wall structure and spin\- surface. (C and D) Ragweed
pollen grain. Double wall and surface spines are shown at different levels of focus. The
line is 20 n in length.
forms resembling hystrichospherids, spiny fossil algae. The appearance of
these algae and some pollen grains may be similar. It seems that in this in-
stance, morphological criteria alone may not be a sufficient basis for identifica-
tion.
Two other particles from their material identified by them as type II or-
ganized elements are illustrated in figure 8, A and B. A third organized
element of similar appearance was also seen in their material. All 3 particles
were found on a slide reportedly stained with the Feulgen reaction. They
show a resemblance to starch grains (figure 8, C and D), stained with the
PAS reaction. The difference between the Feulgen and PAS reactions may
not be of significance in this instance, since we have noted that Schiff's reagent
Figure 7. (A and B) Another organized element from preparation of Claus and Nagy
(C and D) Ragweed pollen grain. The line is 20 m in length.
^ir
B
D
Figure 8. (.1 and B) 'F'wo different organized elements from prej)aration of Claus and
Nagy stained with i'eulgen reaction. (C and D) Starch grains stained with PAS reaction.
See text for discussion of significance of staining. The line is 20 /u in length.
506
1
Fitch & Anders: "Organized Elements" in Chondrites 507
alone will stain some starch grains. This staining was more pronounced when
an aged batch of Schiff's reagent was used, and was somewhat stronger for
"Biosorb" (modified starch prepared by Ethicon Laboratories) than for potato
starch. We cannot exclude the possibility that the particle in figure 8, A is
actually a juniper pollen grain. Again, morphological criteria seem to be in-
adequate to establish the identity of a given particle.
Another organized element, classified by Claus as a type II element resem-
bling a Thecamoeba, is shown in figure 9, A. Illustrated in figure 9, B is an
object with similar morphology found in the airborne pollen sample collected
on July 20, 1961 by Siegel at the Jewish Hospital in Brooklyn, N.Y. These
microscope slides, prepared for the New York City annual pollen survey, were
t'iGURE 9. (.4) Organized element from preparation of Claus and Nagy. (B) Particles
with similar appearance found in pollen survey slide. See text for discussion.
kindly loaned to us by Siegel. We are not certain as to the identity of this
object, but the resemblance between the organized element from the meteorite
and the airborne particle is evident.* More recently, we have found several
similar particles in dust samples from the American Museum of Natural His-
tory.
Pollen, mold, and fungus spores, and a variety of other objects are present in
large numbers in the atmosphere at certain seasons, with daily totals of up to
100 ragweed pollen grains per cm.- " and up to 363 mold spores per cm.- ^~ being
* Gregory (i)rivate communication) has suggested that these particles might be furnace
ash spheres.
508
Annals New York Academy of Sciences
reported for New York City. Several of these objects are illustrated in figure
10. It is extremely difficult to prevent contamination by this type of ma-
terial. These types of particles are often present in great abundance in the
air and are deposited as dust that later forms a secondary source for con-
tamination.
Siegel has pointed out in personal communication that he had found it
extremely difficult during the summer and fall to prepare Vaseline-coated
slides free of pollen contamination, although working in a dust free, "sterile"
Figure 10. Objects found in pollen survey slides. (A) Unidentified object. (B)
Ragweed pollen grains. (C'j Oak pollen grain. (D) Unidentified object. The line is 20 n
in length.
room. Also, ragweed pollen grains were occasionally seen by Siegel in pollen
study slides exposed long after the period of bloom, and probably represent
contamination from the laboratory or other sources.
Thin Sedions
Organized elements embedded in mineral veins in thin sections of the Orgueil
meteorite have been described and illustrated.- It is extremely important to
characterize these particles because they are undoubtedly indigenous to the
meteorite. However, the nature of the thin sections makes adecjuate morpho-
logical study difficult. The sections are relatively thick, 10 to 25 ^i, and al-
though the veins are composed of transparent minerals, there are irregularities
and impurities which cause optical distortion. It is difficult to be certain of
tine surface detail because the practical limit of resolution for the microscope
Fitch & Anders: "Organized Elements" in Chondrites 509
under ideal conditions is only 0.2 to about 0.3 fx for the objectives that must be
used with this sort of preparation. Akhough the organized element illustrated
by Nagy et al.~ had to be viewed through a layer of optically imperfect mag-
nesium sulfate, the presumed spines illustrated in the drawing were spaced
only 0.3 yu apart.
Judging from both visual inspection and the published illustration [tigure 4d
in reference 2] this organized element appears to be opaque. Previously, it
was emphasized that all organized elements in crushed preparations were
transparent.^ - Also, none of the particles in the thin sections seems to have
the highly structured morphology, although about 8000 organized elements
should have been present in a thin section I4 inch in diameter and 20 yu in
thickness.
Some organized elements in the thin sections were described as having pink
fluorescence [tigure 5 in reference 2]. We encountered occasional particles in
crushed preparations which appeared red against the dark background when
illuminated with ultraviolet light. However, this did not prove to be true
fluorescence. These particles when viewed with polarized visible light were
doubly refractile. The fluorescence microscopes commonly used in biological
investigations use darkfield illumination. The usual light source is a high
pressure mercury arc with various filters placed in the light path to absorb the
visible light. All of the 5 filters commonly used transmit ultraviolet and some
blue light but they have an appreciable transmittance in the red portion of the
spectrum as well.'^ Hence, doubly refractile particles should be expected to
appear red when viewed with ultraviolet light in the fluorescence microscope.
Perhaps additional study of thin sections will reveal particles with a more
conclusive combination of properties.* In our opinion the present evidence is
inadequate to suggest a biological origin for the indigenous particles.
Discussion
Several features make it difficult to accept the highly structured particles as
extraterrestrial in origin. They are absent from our preparations of Orgueil,
although material from the same stone was used. They have not been ob-
served in thin sections, and they often show a morphological resemblance to
common airborne contaminants. Although a strong case can be made for the
biological origin of some of these structures, the probability of a terrestrial
contamination has not been ruled out in their case.
The situation is altogether different in the case of the small, brownish-yellow,
somewhat irregular, roughly spherical grains which apparently make up most
of the 1700 particles per milligram reported previously by Claus and Nagy^
and Nagy ei air Although our own experience suggests that this number
represents an appreciable overestimation, there is no doubt that such particles
do exist.
They are undoubtedly indigenous to the meteorite, but their morphology is
so featureless that an inorganic origin cannot be ruled out. None of the other
* Additional observations on thin sections are reported in another paper (Anders and
Fitch, Science, in press).
510 Annals New York Academy of Sciences
criteria for a biological origin seems to hold for these particles. They do not
fluoresce and they do not take biological stains in a manner that will distinguish
them from irregular silicate fragments in Orgueil and in kimberlite. Because
they disappear after treatment with acids, we believe that they are most Ukey
grains of minerals, although they are classilied as organized elements by Nagy
et al. The 2 particles remaining in our sample after HF-HCl treatment re-
semble terrestrial contaminants. Moreover, it must be emphasized that only
2 were seen where several thousand should have been found.
Even if organic particles should be found, a biological origin need not be
inferred. Both the polypeptide particles of Fox'* and the hydrocarbon poly-
mer particles of Wilson'^ have an appearance at least as organized as the less
structured organized elements. These materials are produced in vitro, by dry
polymerization of amino acids,^* and the Miller-Urey type synthesis,'*'''^ re-
spectively. In FIGURE 11 is illustrated a preparation obtained through the
courtesy of Wilson in which most of the polymer occurred in the form of sheets
containing thickened, round spots about 10 /x in diameter. Much of the ma-
terial was fluorescent, but some of the larger spots were not.
It may well be that life did exist in meteorites, but we feel that the present
evidence is not adequate to suggest an extraterrestrial biological origin for the
particles found in the carbonaceous chondrites.
Criteria for Identification of Life Forms
If the present data are inadequate, what kind of information is needed to
decide whether or not a particle is, in fact, a life form? This requires an initial
definition of life. Life has three essential qualities. Life requires reproduc-
tion of itself with the possibility of mutations developing along the way. Regu-
lated and integrated anabolical and catabolical chemical processes are a second
feature of life. Structural organization at the molecular and supramolecular
levels is a third feature. Probably for simple, small organisms, it is necessary
to demonstrate all of these features — reproduction, metabolism, and organiza-
tion — to establish the presence of life.
What is needed to establish that life had been present at some time in the
past? Ideally, remnants of all these features should be found. In reproduc-
tion of all terrestrial forms, nucleic acids carry information from one genera-
tion to the next. Nucleic acids or breakdown products from them may remain
after life has ceased. Evidence of metabolic processes frequently remains.
Many carbohydrates and lipids are rather resistant and persist for long periods.
Persistence of the organization of any organism forms the basis for terrestrial
paleontology. This morphology may be the result of partial or complete
replacement of biological materials with nonbiogenic compounds. If replace-
ment has been complete, probably one can never be entirely certain that a
given structure was originally of biological origin. In terrestrial materials,
this is occasionally an important question but it is never a critical one. For
nonterrestrial materials it is a critical question.
If "fossilization" or replacement has been incomplete, then metabolical
products of various sorts will remain. In pre-Cambrian rocks containing ap-
parent fossil forms, there are, in fact, substances that resist the acid treatments
used to remove the mineral materials.'" With cytochemical as well as other
Fitch & Anders: "Organized Elements" in Chondrites 511
microscopical techniciues, il should be possible to gain considerable information
about the composition of these substances. Once characterized at the micro-
scopical level, the substances can be isolated in larger quantities and other
parameters including optical activity and isotopic composition can be measured.
•
G
t'
!»».
O
1B3p
^ i
1%"^
y •
% %
A* A \
,^
J*
^^
♦
IT •
Figure 11. Hytlrocailmn polymer prepared by Wilson (1960). Thickened sjjots are
present in the sheet. Viewed in ultraviolet light, the spots and the sheet had a yellowish
fluorescence. The line is 20 /x in length.
512 Annals New York Academy of Sciences
The observed properties of the resistant material can be compared with proper-
ties of biological compounds as well as with those of various synthetic materials
including polypeptide particles prepared by Fox^'' and hydrocarbon polymer
particles prepared by Wilson. ^^ It is evident from their work as well as that
of Miller,"^ '^^ Palm and Calvin/- '^^ Or6,-° Berger'-^ and others that complex
organic materials can be prepared through nonbiological processes.
This approach assumes to some extent at least that extraterrestrial life re-
sembles terrestrial life chemically. This may be a provincial idea, but com-
parison of unknown materials with terrestrial forms would seem to be a good
starting place. It may be that even after this information is gathered and
analyzed, no detinite conclusions can be drawn. However, this information
should provide a broader basis for critical evaluation than morphology alone.
Summary
"Organized elements" described by Claus and Nagy^ and by Nagy et air
are a heterogeneous group of particles which, in our opinion, are best classified
into two types: those that have a highly structured morphology and those
that have a much simpler appearance. The particles with highly structured
morphology are less numerous than the simpler type. They have not been
seen in thin sections and many appear to have a strong resemblance to com-
mon terrestrial contaminants. The particles of simpler morphology which
do not fluoresce, which either do not stain or stain atypically with biological
stains, and which are soluble in acids seem to be of an inorganic composition
and origin. It is possible that life did exist in meteorites, but we think that
the present evidence is not adequate to suggest an extraterrestrial biological
origin for the particles found in the carbonaceous chondrites.
A cknowledgments
The authors express their gratitude to the Argonne Cancer Research Hospital
for allowing the use of its facilities for some of the experiments, and to the
staff of the Allergy Laboratory of the Jewish Hospital of Brooklyn for the loan
of pollen slides. We are also indebted to Dr. George Claus and Prof. Bartholo-
mew Nagy for permission to study and photograph their samples, in exchange
for our preparations which they described in reference 2.
This work was supported in part by the U.S. Atomic Energy Commission.
References
1. Claus, G. & B. Nagy. 1961. A microbiological examination of some carbonaceous
chondrites. Nature. 192: 594.
2. Nagy, B., G. Claus & D. J. Hennessy. 1962. Organic particles embedded in minerals
in the Orgueil and Ivuna carbonaceous chondrites. Nature. 193: 1129.
3. Fitch, F., H. P. Schwarcz & E. Anders. 1962. "Organized elements" in carbona-
ceous chondrites. Nature. 193: 1123.
4. DuFresne, E. R. & E. Anders. 1962. On the chemical evolution of the carbonaceous
chondrites. Geochim. et Cosmochim. Acta. 26: 1085.
5. Pearse, a. G. E. 1960. Histochemistry, Theoretical and Applied. Little, Brown &
Co. Boston.
6. LisoN, L. 1960. Histochemie et Cytochemie Animates, Principes et Methodes. Vol.
I. Gauthier-Villar. Paris.
7. McManus, J. F. A. 1961. Periodate oxidation techniques. In General Cytochemical
Methods. 2. : 171. J. F. DanieUi, Ed. Academic Press. New York.
Fitch & Anders: "Organized Elements" in Chondrites 513
8. Bernal, J. D. 1962. Comments. Nature. 193: 1127.
9. Seshachar, B. R. & E. VV. Flick. 1949. Application of perchloric acid technique to
protozoa. Science. 110: 639.
10. FuNKHOUSE, J. VV. & W. R. EviTT. 1959. Preparation techniques tor acid-insoluble
microfossils. Micropaleontology. 5: 369.
11. DuRH.AM, O. C. 1950. Report of the Pollen Survey Committee of the .\merican Acad-
emy of Allergy for the season of 1949. J. Allergy. 21: 442.
12. DuRH.AM, 0. C. 1938. Incidence of air-borne fungus spores. II. Hormodendnim,
Aliertiaria and rust spores. J. Allergy. 10: 40.
13. Richards, O. W. 1955. Fluorescence microscopy. In Analytical Cytology. Ed. 1 :
5/1. R. C. Mellors, Ed. Blakiston Diy., McGraw-Hill Book Co! New York.
14. Fox, S. VV. & S. YuYAM.A. 1963. Abiotic production of primitiye protein and formed
microparticles. Ann. N.V. Acad. Sci. 108(2): 487-494.
15. Wilson, A. T. 1960. S\nthesis of macromolecules vmder j)ossible primeval Earth
conditions. Nature. 188: 1007.
16. Miller, S. L. 1953. A production cf amino acids under possible primitive Earth
conditions. Science. 117: 528.
17. Miller, S. L. 1955. Production of some organic compounds under possible primitive
Earth conditions. J. Am. Chem. Soc. 77: 2351.
18. Palm, C. & M. Calvin. 1961. Primordial Organic Chemistry. I. Compounds re-
sulting from electron irradiation of C'^H4 . J. .\m. Chem. Soc.
19. Palm, C. & M. Calvin. 1961. Electron irradiation of aqueous solutions of HCN. : 65.
Bio-Organic Chemistry Quarterly Report UCRL-9900.
20. Oro, J. 1963. Studies in experimental organic cosmochemistry. Ann. N.Y. Acad. Sci.
108(2): 464-481.
21. Berger, R. 1963. Evaluation of radiation effects in space. .\nn. N.Y. Acad. Sci.
108(2): 482-486.
ON THE ORIGIN OF CARBONACEOUS CHONDRITES*
Edward Anders
Enrico Fermi Institute for Nuclear Studies, and Departments of Chemistry and Geophysical
Sciences, University of Chicago, Chicago, III.
Carbonaceous chondrites are related to other classes of meteorites in many
ways, and much of what has been said about the origin of meteorites, in gen-
eral, appUes to carbonaceous chondrites as well. Like all other meteorites,
they are fragments of larger bodies. To reconstruct their history, we must
try to learn more about the nature of these bodies, that is, their size, number,
and location, and the chemical and physical processes that produced the de-
tailed structural and compositional features of the meteorites.
Some of the principal hypotheses on the origin of meteorites are outlined
in TABLE 1. (A more complete review of the subject has been given by Anders
and Goles, 1961.) Each of these hypotheses can account for some 90 to 95
per cent of the properties of the meteorites, and it is only the last 5 to 10 per
cent that causes difficulties. There is just as much disagreement on the origin
of the carbonaceous chondrites (table 2). Mason (1960, 1961) and Ring-
wood (1961) assume that they represent some of the primitive material from
which the solar system formed; Urey (1961) believes that they are alteration
products of the high iron group chondrites, which are themselves several steps
removed from primitive material. Finally, Wood (1958, 1962) and others
believe that they are alteration products of a hypothetical, primitive chon-
drite, similar to Renazzo or Ornans (Fish et al., 1960; DuFresne and Anders,
1962a).
Clues to the Origin of Carbonaceous Chondrites
Mineralogy. Some clues to the origin of the carbonaceous chondrites can
be obtained from a study of their mineralogy. Results for 9 of these meteor-
ites are shown in table 3 (DuFresne and Anders, 1962a). The estimated
relative abundances are expressed as negative logarithms of 2; the entry 3,
for example, stands for 2"^ or 1/8. The minerals found can be divided into
three classes: conventional, "high-temperature" minerals; "characteristic"
minerals pecuhar to this class of meteorites; and trace minerals. In addition,
these meteorites also contain appreciable amounts of sulfur, hydrated MgS04,t
elemental carbon, and organic compounds. On the basis of their mineral
composition, the carbonaceous chondrites can be divided into 5 subclasses.
These show a fair degree of correspondence with Wiik's (1956) three classes,
established on the basis of chemical composition only.
One can prove rather convincingly that the characteristic minerals are al-
teration products of the high-temperature minerals, rather than vice versa.
* This work was supported in part by the U.S. Atomic Energy Commission.
t The state of hydration varies with the temperature and the relative humidity at the
time of measurement. Very probably, the MgS04 was present as the anhydrous salt or as
the monohydrate at the time of fall, and became hydrated after exposure to atmospheric
moisture. Boato's (1954) measurements show that the water released below 180° C. has a
normal D/H ratio, and is probably of terrestrial origin.
514
Anders: Origin of Carbonaceous Chondrites
515
X-ray diffraction and optical studies of composite grains of olivine and Murray
F (a hydrated silicate), show that the olivine sometimes occurs in thin parallel
plates of the same crystallographic orientation, although the individual plates
are separated by a thin layer of exceedingly finely grained, randomly oriented,
Murray F mineral. The common orientation of the olivine plates can be
understood only if single crystal olivine served as the starting material (Du-
Fresne and Anders, 1962a). Still, one cannot exclude the possibility that some
fraction of the characteristic minerals is primordial, rather than being derived
from the olivine.
Many of the other characteristic minerals, too, seem to be hydrated silicates.
This fact, and particularly the occurrence of MgS04 in distinct veins (figure 1)
Table 1
Properties of Meteorite Parent Bodies
Size
Location
Number
Heat source
Lovering (1957)
Planetary
2-5 a.u.
One
Long-lived
radioactivity
Urey (1959)
Lunar
1 a.u.
One
Chemical reac-
tions; adiabatic
compression
of gases
Fish et al. (1960);
Wood (1958, 1962)
Asteroidal
2-5 a.u.
Several
Extinct radio-
activity
Ringwood (1961)
Lunar
2-5 a.u.
Several
Radioactivity
Table 2
Origin of Carbonaceous Chondrites
\. High-iron group chondrites altered by infiltration of water, carbonaceous matter, and
hydrogen sulfide from some other source (Urey, 1961).
2. Primitive material accreted at low temperatures from solar nebula (Mason, 1960, 1961;
Ringwood, 1961). Other chondrites were derived from this material by heating and
reduction.
3. Primitive material expelled from the sun at high temperatures (Wood, 1958), accreted at
low temperatures into asteroidal-sized bodies (Wood, 1958, 1962; Fish et al., 1960),
altered by liquid water and sulfur compounds (DuFresne & Anders, 1962a).
suggests that licjuid water must once have acted on these meteorites. This
raises three interesting questions. First, what were the chemical and phys-
ical conditions (pH, reduction potential, and temperature) during this aqueous
stage, and how long did it last? Second, what was the source material of the
carbonaceous chondrites, i.e., where did the high temperature minerals come
from? And third, in what setting did this aqueous stage occur?
Former environment. To answer the first question, one can turn to the sta-
bility diagrams of Garrels (1960), which give the stabihty regions for various
minerals and ions as a function of pH and reduction potential (Eh) . In figure
2 is shown a composite diagram based upon Garrels' data. Looking up the
stability regions of the principal constituents of carbonaceous chondrites on
this diagram, one finds that nearly all of them [Fe304 , (Mg,Fe)C03 , MgS04 ,
S, organic matter] can coexist under equilibrium conditions at pH 8 to 10 and
516
Annals New York Academy of Sciences
Eh > —0.2 V. This conclusion was reached independently by Nagy el al.
(19626). The only exception is FeS, in place of which one would expect FeSa .
It is not too difficult to find an ad hoc assumption that accounts for this dis-
crepancy. For example, one can argue that the FeS was first made under
conditions in which it was stable, possibly even at high temperatures, and
that it was then brought in contact with solid sulfur at such low temperatures
that the rate of reaction was very slow.
It is quite remarkable that the carbonaceous chondrites are so close to chem-
ical equilibrium, because intuitively one would think of an assemblage of highly
Table 3
Mineralogy of Carbonaceoits Chondrites*!
Orgueil
Ivuna
Hari-
pura
Cold
Bok.
Mighei
Murray
Ornans
Lance
Mokoia
Wiik's class
I
I
II
II
11
II
III
III
III
Subclass
A
A
B
C
C
C
D
D
E
Clinopyroxene
Olivine
a-Iron
7-Iron
Magnetic troilile
Orgueil LM
Magnetite
Murray F
Haripura M
Mokoia HT and SW
Epsomite
Sulfur
Dolomite
Breunnerite
Pentlandite
Higli Temperature Minerals
3
?
3
3
1
1
0-1
0-1
9
10
10
7
5
10
5
5
5
"C
liaracle
nslic" j1
lineraL
f
1
1
1
1
3
1
3
1
lit
1
I
?
?
3
3
6
6
6
6
>16
10
6
6
6
9
9
9
>20
13
5
6
6
Trace Minerals
9
10
8
11
* After DuFresne and Anders (1962a).
t Estimated abundances are given as negative logarithms of 2. Thus Mighei is about 50
per cent olivine and 50 per cent "Murray F" mineral, with mere traces of iron, pent-
landite, magnetite, epsomite, and sulfur. Italicized values are of lower accuracy.
I Trace associated with metallic iron.
oxidized (S04=, Fe.s04 , CO,r) and reduced (S, FeS, C, organic matter) species
as being far from chemical equilibrium. The source for the basic pH might
be ammonia, and for the negative Eh, hydrogen (< 10""^ atmos.). Both would
conveniently disappear as the water evaporated.
The temperature at which the aqueous stage occurred is a little harder to
determine. A lower limit near 0° C. is implied by the condition that the water
was liquid; an upper limit of 200° to 400° C. is provided by various other ob-
servations, e.g., the strained glass found in the Mighei carbonaceous chondrite
(DuFresne and Anders, 1961). As shown in figure 3, the strain disappears
after annealing for 48 hours at 206° C, so that after the incorporation of this
Anders: Origin of Carbonaceous Chondrites
517
glass into the meteorite the temperature of Mighei could never have exceeded
206° C. for as long as 48 hours. Other time-temperature combinations can be
read off the graph, although it is doubtful whether any extrapolation beyond
the measured points is valid. One can infer that temperatures were much lower
from the fact that the characteristic minerals are quite finely grained, judging
from the diffuseness of their x-ray diffraction patterns. It seems likely that
the aqueous stage occurred at approximately room temperature. There is
hope of obtaining a more accurate value by measuring the O'YO^'' fractiona-
tion between carbonate and magnetite (Clayton, 1962). Presumably the
Figure 1. A fragment of Orgueil, showing white vein of magnesium sulfate running hori-
zontally across specimen. This vein must have deposited from water solution, thus offering
evidence of the onetime presence of liquid water in the meteorite parent body. (Reproduced
from DuFresne and Anders, 1962a, with permission of the editor.)
carbonate was made during the aqueous stage, by the action of CO2 on basic
oxides. The CO2 was, in turn, probably evolved from the interior of the body
during reduction of iron oxides to metallic iron. If the carbonate and mag-
netite reached isotopic ecjuiUbrium during the aqueous stage, the temperature
of this stage may be determined by means of Urey's paleotemperature method.
A clue to the duration of the aqueous stage is given by the relatively high
degree of ordering of the Ca++ and Mg++ ions in the dolomite from Orgueil
and Ivuna. From a comparison with terrestrial dolomites. Goldsmith has
estimated a formation time of > 10^ years.
Ancestral material of carbonaceous chondrites. It is a little harder to get an
answer to the second question, concerning the origin of the high temperature
minerals. Edwards and Urey (1955) and Urey (1961) have pointed out that
518
Annals New York Academy of Sciences
the carbonaceous chondrites have a variable, and frequently lower, content of
Na and K than the ordinary chondrites. In the most extreme case, Nogoya,
this depletion amounts to a factor of ^^4. Urey, therefore, suggested that the
carbonaceous chondrites were derived from the ordinary chondrites [specif-
0.8-
FiGURE 2. Stability relations among some of the important constituents of carbonaceous
chondrites, as a function of reduction potential and hydrogen ion concentration. Solid lines
show boundaries between solids and aqueous species at an activity of the latter of 10"" m;
dashed boundaries, those between aqueous species at 1:1 ratios. Temperature = 298° K.;
total pressure = 1 atmos. Total activity of dissolved sulfur species = 0.1; of carbonate
species, 0.01. Most of the constituents of carl)onaceous chondrites could coexist under equi-
librium conditions at Eh -^ —0.2 and pH 6 to 10. The exceptions are FeS (in place of which
FeSo would be expected) and (Mg,Fe)CO:! . The absence of FeS.> was discussed in the text.
The presence of (Mg,Fe)CO:j is not surprising: although i)ure FeCOn is unstable under the
particular conditions indicated, magnesium-rich breunnerite is likely to be stable. Also, an
increase in the total carbonate, and a decrease in the total sulfur activity will make FeCOs
stable in the triangular field bounded bv the dotted line. This figure has been adapted from
Garrets (1960), figures 6.11, 6.18, 6.19,' 6.20, and 6.21. (Rejjroduced from DuFresne and
Anders, 1962a, with permission of the editor.)
ically, the high iron group, Fe/Si ^ 0.85, Urey and Craig (1953)], by an altera-
tion process that depleted the alkalis while introducing S, C, and a few other
elements in free or combined form.
This picture has become less satisfactory now that the abundances of various
trace elements in meteorites have been determined. Most elements occur in
meteorites in approximately their "cosmic" abundances, as given by the semi-
Anders: Origin of Carbonaceous Chondrites
519
empirical abundance curves of Suess and Urey (1956) and Cameron (1959).
Other trace elements, including most chalcophile ones, do not conform to this
pattern. They occur in approximately their predicted abundances in car-
bonaceous chondrites, but are depleted by factors of up to 1000 in ordinary
chondrites (figure 4). If the carbonaceous chondrites were derived from
ordinary chondrites, as suggested by Urey, one would have to assume that
the depleted elements were somehow added to the carbonaceous chondrites
during the alteration process. In that case, it would be a remarkable coinci-
dence if 6 of the 7 elements happened to be restored to just their cosmic abun-
dances. (The seventh, mercury, may be exceptional because of its high
Annealing of Mighei Glass
• Almost Complete Anneal
— I Completion Observed
X Discontinued
300
o
260 I
0.1
10 100
Annealing Time (hrs)
1,000
Figure 3. .\nnealing of strained glass from Mighei carbonaceous chondrite. .\fter the
incorporation of the glass, the meteorite cannot have been heated to temperatures as high as
206° for as long as 48 hours, or the strain would have disappeared. (After DuFresne and
Anders, 1961.)
volatility, but it should be noted that the point in figure 4 is based upon a
single measurement.)
The olivine in carbonaceous chondrites has a highly variable iron content
(Ringwood, 1961), whereas it is of nearly constant composition in ordinary
chondrites (Mason, 1962). This factor, too, makes it difficult to derive car-
bonaceous chondrites from ordinary chondrites by any simple process.
Another clue comes from the primordial noble gases which seem to be present
in all carbonaceous chondrites (figure 5). All meteorites contain noble gases
produced by cosmic rays or the decay of long lived radioactivities, but the car-
bonaceous chondrites also contain primordial noble gases that can be distin-
guished from cosmogenic or radiogenic noble gases by their isotopic and ele-
mental composition (Stauffer, 1961 ; Anders, 19626). With the exception of He^
and Ar^", most of which is radiogenic, the noble gases in an ordinary chondrite
520
Annals New York Academy of Sciences
are produced chielly by the action of cosmic rays on iron, silicon, and other sta-
ble elements in the meteorite. For example, the 3 neon isotopes are made in
nearly equal amounts in this process (Eberhardt and Eberhardt, 1961) whereas
in primordial neon (represented by neon in the earth's atmosphere) the ratio
Ne^o/Ne^VNe-- is 90.8/0.26/8.9. The elemental ratios differ too, as can be
seen in figure 5. The bulk of the primordial noble gases once associated
with the matter of the terrestrial planets and the asteroids seems to have been
lost at a very early stage in the history of the solar system. It is not very
plausible to assume that these gases were first lost from the ordinary chon-
100
.10
c
o
-o
c
■D
XI
<
"(J
e
(/>
o
o
■ Carbonaceous Chondrites
n Ordinary Chondrites
Pb
Pb
Tl
Bi
10
10
In
Ls_
-4
10'^ 10' "^
Observed Abundance (atoms/ 10 atoms Si )
Figure 4. Trace element abundances in carbonaceous chontlrites and ordinary chondrites.
x\lthough strongly depleted in ordinary chondrites, most of these trace elements occur in
carbonaceous chondrites in nearly their "cosmic" abundances. This suggests that carbona-
ceous chondrites are more closely related to primordial matter than the ordinary chondrites.
[Data were taken from the following sources: Bi, Hg, Pb, and Tl, Reed et al. (1960), and
Ehmann and Huizenga (1959); Cd, Schmitt (1961); I and Te, Goles and Anders (1962); In,
Schindevvolf and Wahlgren (1960); Sb, Anders (1960).]
drites, then stored somewhere, and finally incorporated somehow in the car-
bonaceous chondrites.
The spheroidal troilite and magnetite particles found in Orgueil also suggest
a high-temperature stage (Fitch et al., 1962). Their chemical identilacation was
confirmed by electron microprobe analysis (Smith, 1962). Spheroidal par-
ticles might be expected from the condensation of vapors in the liquid field,
but in the presence of cosmic proportions of hydrogen, metalUc iron rather
than FeS or Fe;j04 would result (Urey, 1952). Such "primary" metal spherules
might be transformed to FeS or Fe304 by the action of HoS or HoO at lower
temperatures. It is interesting that Sztrokay et al. (1961) have observed
spherical, opaque particles in olivine chondrules from the Kaba carbonaceous
chondrite. Similar particles are found in chondrules of many ordinary chon-
Anders: Origin of Carbonaceous Chondrites
521
drites as well (Fredriksson, 1%2). Alteration of the olivine by water would
release these spherules, possibly in altered form, from their chondrule matrix.
But it is also possible that the spherules formed at a later stage. The particles
in Orgueil are quite similar to the troilite globules in meteorite veins (Anders
and Goles, 1961) and may well be of similar origin. The association of many
#-
• Carbonaceous Chondrite (Murray)
o Ordinary Chondrite (Holbroolt)
»20
.21
.22
.36
.38
He He Ne'^"' Ne"' Ne'^'" Ar'^ Ar""
FiGURE 5. Noble gases in a cariionaceous and an ordinary chondrite. In Holhrook, these
gases (except for radiogenic He-") are produced by cosmic-ray induced spallation reactions on
iron and other stable nuclides. The 3 neon isotopes are made in nearly ef|ual aliundance.
In Murray, the isotopic abundances resemble those in Earth's atmosphere, suggesting that
these gases, too, are of primordial origin. A small amount of cosmogenic gas is present in
Murray as indicated l)y the increased abundances of He^ and Ne-^ relative to their atmospheric
abundances.
of the Orgueil spherules with firmly attached silicate fragments is consistent
with either hypothesis.
The trace element abundances, the variations in the olivine composition, and
the primordial gas content are most easily e.xplained by assuming that both
the carbonaceous chondrites and the ordinary chondrites were derived from
still more primitive ancestral matter. Perhaps the most embarrassing require-
ment for this material is that some of it at least must have passed through an
earlier, high-temperature stage without losing its primordial gases completely.
It is possible to accomplish this in the meteorite parent body, but some
special assumptions are required (DuFresne and Anders, 19626). A more
522 Annals New York Academy of Sciences
attractive possibility is offered by Wood's (1958, 1962) hypothesis, according to
which planetary matter, expelled from the sun at high initial temperatures,
cooled by adiabatic expansion, so that progressive expansion could take place.
The least volatile constituents would condense to high-temperature minerals
(olivine, pyroxene, nickel-iron, and later, magnetite), which would trap some of
the surrounding primordial gas. Other substances, e.g., H2O, NH3 , and carbon
compounds, would condense on temperature drop. The further accretion of
the (now cold) dust into solid bodies, and the separation of the solids from the
noncondensable gas would proceed along the path outlined by Urey (1952,
1954, 1956, 1957, 1958) or Fish et al. (1960). Incidentally, if such a high-tem-
perature stage ever took place, then cometary matter, too, must have passed
through it. This raises some new possibilities in regard to the mineral com-
position of comets. In particular, the presence in comet tails of metal (or mag-
netite?) spherules, inferred from scattered light and polarization measurements
(Liller, 1960), is somewhat easier to understand if part of the cometary material
had a high temperature history, even though its final accretion occurred at low
temperatures. This view gains further support from the discovery in cosmic
dust of metal flakes with amorphous organic attachments. The fall dates of
these particles seem to be correlated with several meteor showers of cometary
origin (Parkin, Hunter, and Brownlow, 1962). Perhaps Herbig's (1961) sug-
gestion that the carbonaceous chondrites were derived from comets should be
re-examined in the hght of this possibility.
Aqueous stage and the prerequisites for life. What about the third question,
the setting in which the aqueous stage took place? This is one point in which
the large planet hypothesis has an advantage over all others. A planet of
terrestrial size can hold water vapor gravitationally, and can maintain bodies of
liquid water, from ponds to oceans. Surely, the surface temperature must be
high enough to allow liquid water to exist, but the temperature is controlled
not only by the distance from the sun, but also by the composition of the
atmosphere. If Venus, with its CO^-rich atmosphere, were located in the
asteroidal belt, it would have a comfortable surface temperature near 300° K.,
instead of the 600° K. prevailing at its present location. If it were not for the
fact that the planetary hypothesis runs into so many other ditficulties (Anders
and Goles, 1961), one could stop here.
Of all the parent bodies discussed, the asteroids are least likely to retain
liquid water at their surfaces, owing to their small size and consequent low
escape velocities. But there is a way in which they could retain liquid water in
their interiors. If the asteroids were ever heated by an internal heat source
{e.g., extinct radioactivity), some temperature distribution resembling the
curves in figure 6 would result. The surface temperature of the body would
be controlled by the amount of solar radiation reaching it, and might be around
100 to 200° K. Farther inward, the temperature would rise until the melting
point of ice was reached. Liquid water could exist in this zone, down to a
depth at which the boiling point at the prevailing pressure was reached. In
FIGURE 7 is shown the location of this zone of liquid water for a body with a
central temperature of 1900° K. In this case, some 5 per cent of the volume of
the body will contain liquid water.
The water will not last forever, of course. Above the zone of liquid water,
Anders: Origin of Carbonaceous Chondrites
523
there will be a permafrost zone,* and the ice from this zone will evaporate at a
rate determined by its vapor pressure (Watson et al., 1961). The vapor pres-
sure depends upon the temperature, which in turn depends on the distance from
the sun. For a body with 100-km. radius, with an initial water content of 10%,
these times are indicated in table 4.
Unfortunately, this water zone is located in a dark, underground region,
where photosynthetic organisms could not grow or reproduce. To support
4000
0.00
Relative Fractional Volume
Figure 6. Temperature distribution of asteroids heated by radioactivit}- or some other
uniformly distributed internal heat source. The 2 solid curves are calculated for different
heating rates, assuming heat transport by conduction only; the daslied cu 
