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Full text of "Life-like forms in meteorites and the problems of environmental control on the morphology of fossil and recent protobionta"

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 



JACOB FELD 
EMERSON DAY 



Elected Councilors 
1961-1963 

1962-1964 

1963-1965 



ROSS F. NIGRELLI 
Corresponding Secretary 



ANDRES FERRARI 
HARRY EAGLE 
L. WILLIAM MAX 



RHODES W. FAIRBRIDGE 

EUNICE THOMAS MINER, Executive Director 

SECTION OF BIOLOGICAL AND MEDICAL SCIENCES 

PRESTON L. PERLMAN, Chairman GABRIEL G. NAHAS, Vice-Chairman 

DIVISION OF ANTHROPOLOGY 
ROBERT HECKEL, Chairman JEROME BRIGGS, Vice-Chairman 

DIVISION OF INSTRUMENTATION 
WALTER E. TOLLES, Chairman CARL BERKLEY, Vice-Chairman 

DIVISION OF MICROBIOLOGY 
H. CHRISTINE REILLY, Chairman EUGENE L. DULANEY, Vice-Chairman 

DIVISION OF PSYCHOLOGY 
GEORGE KETTNER BENNETT, Chairman DAVID M. LEVY, Vice-Chairman 

SECTION OF CHEMICAL SCIENCES 
MINORU TSUTSUI, Chairman GEORGE de STEVENS, Vice-Chairman 

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 
M. H. KALOS, Chairman GERALD GOERTZEL, Vice-Chairman 

DIVISION OF BIOPHYSICS 
JOHN S. LAUGHLIN, Chairman ROSALYN S. YALOW, Vice-Chairman 

DIVISION OF ENGINEERING 
JOSEPH F. SKELLY, Chairman JULIAN D. TEBO, Vice-Chairman 

DIVISION OF MATHEMATICS 
MARY P. DOLCIANI, Chairman J. P. RUSSELL, Vice-Chairman 

SECTION OF PLANETARY SCIENCES 
RICHARD L. PFEFFER, Chairman HAROLD L. STOLOV, Vice-Chairman 

Past Presidents 
FREDERICK Y. WISELOGLE JAMES B. ALLISON 

The Sections and Divisions hold meetings regularly, one evening each month, during the 
academic year, October to May, inclusive. All meetings are held at the building of The New 
York Academy of Sciences, 2 East Sixty-third Street, New York 21, New York. 

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. 

References 

Blum, J. L. 19vS4. Two winter diatom communities of Michigan streams. Pap. Mich. 

Acad. Sci. Arts, Lett. 39: 3-7. 
Blum, J. L. 1956. The ecology of river algae. Bot. Rev. 22: 291-341. 
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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einige Diatomeen. Intern. Rev. ges. Hydrobioi. Hydrog. 19: 452. 
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Tiefebene (Alfold). Magyar Botan. Lapok. 1929: 100. 
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Cholnoky, B. J. 19306. Untersuchungen iiber den Plasmolyse-Ort der Algenzellen 1 u. 

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321. 
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Die Plasmoiyse der Gattung Oedogonium. Protoplasma. 12: 510. 
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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. 

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Cholnoky, B. J. 1953. Fin Beitrag zur Kenntnis der Oedogonium-Zelle. Osterr. Botan. 

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Hofler, K. 1918. Permeabihtiitsbestimmung nach der plasmometrischen Methode. Ber. 

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173. 
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Wassers nach seiner Fauna und Flora. Kleine Mittcilungen Kgl. Priifungsanstalt f. 

VVasserversorgung u. Abwasserbeseitigung. 1. 
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Natur- u. Heilk. Giessen. Naturw. Abt. 24: 34. 
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Math.-Natwiss. Kl., Abt. I. 162: 235. 
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R. Oldenbourg. Miinchen. 
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bijugattis. S. African J. Sci. 53: 335. 
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Weber, F. 1924. Krampfplasmolyse bei Spirog>ra. Arch. ges. Physiol., Pfliigers. 206: 629. 
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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 

1. Sabin, a. B. 1941. The filterable microorganisms of the pleuropneumonia group. 

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 
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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- 

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14. Lilly, D. M. & R. C. Klosek. 1961. A protein factor in the nutrition of Paramecium^ 

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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. 
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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 


1) 

<u 
u 
>> 

J= 

a 

£ 

o 


u 

>. 

J= 
a 
o 

c 


u 
o 


u 

u 

>. 

D. 
— 


ca 
i> 
u 
>> 

j: 
p. 
o 

ft 


a 

O 


ca 
u 

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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20. KoFOiD, C. A. 1911. Univ. Calif. Publ. 8(4): 187-286. Pis. 9-17. 

21. Schiller, J. 1933. Dinoflagellatae. Rabenhsrst's Kr\'ptogamen- Flora von Deutsch, 

Osterr., und der Schweiz. Ed. 2. 10: abt. 3. Teif 1. 617 p. 631 figs. 1937. 
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22. Graham, H. W. 1942. Carnegie Inst. Wash. Publ. 542. Biology-III. 1-129. 

23. Bra.arud, T. 1945. Avhandl. Norske Videnskaps-Akad. Oslo. 11: 1-18. Pis. 1-4. 

24. Hesse, R., W. C. Allee & K. P. Schmidt. 1947. Ecological Animal Ecology. John 

Wiley & Sons. New York. 

25. Deflandre, G. 1952. /« Traite de Zoologie. I. : 391-404. P. P. Grasse, Ed. Masson 

26. Chatton, E. 1952. In Traite de Zoologie. I. : 310-390. P. P. Grasse, Ed. Masson 

27. Lejeune-Carpentier, M. 1938. .\nn. see. geol. belg. Bull. 62: 525-529. Figs. 1-2. 

28. EviTT, W. R. 1961. Micropaleontology. 7: 305-316. 

29. Deflandre, G. 1938. Travaux de la Station Zoologique de Wimereux. XIII. : 198 p. 

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30. Deflandre, G. 1947. Bull. inst. oceanog. 918: 1-23. 

31. Tasch, P., K. McClure & 0. Oftendahl. 1962. Biostratigraphy of a hystrichosphae- 

rid-dinoflagellate assemblage from the Kansas Cretaceous (Albian). Intern. Palynol- 
ogy Conf. Tucson, Arizona (Abstract). Micropaleontology. In press. 

32. Eisenack, A. 1959. Arch. Protistenk. 104: 43-47. PI. 2. Figs. 3-5. 
ii. COLUM, G. 1948. J. Paleontology. 22: 233-263. PL 33-35. 

34. Campbell, A. S. 1954a. Radiolaria. In Protista. Part D. : D 11-D 160. Treatise 

on Invertebrate Paleontology. R. C. Moore, Ed. G.S.A. & K. U. Press. Lawrence, 
Kansas. 

35. RiEDEL, W. R. & C. A. Holm. 1957. In Treat. Mar. Ecol. and Paleoecol. G.S.A. Mem. 

67. 1: 1069-1072. 

36. Tynan, E. J. 1957. Micropaleontology. 3: 127-136. 

37. RiEDEL, W. R. 1959. Micropaleontology. 5: 285-302. 

38. Campbell, A. S. 1954b. Tintinnia. In Protista. Part D. : D 166-D 180. Treatise 

on Invertebrate Paleontology. R. C. Moore, Ed. G.S.A. & K.U. Press. Lawrence, 

39. CoLUM, G. 1955. Micropaleontology. 1: 109-124. PI. 1-5. Text-figs. 1-4. 

40. Phleger, F. B. 1960. Ecology and Distribution of Recent Foraminifera. The Johns 

Hopkins Press. Baltimore. 



450 Annals New York Academy of Sciences 

41. Bandy, O. L. & R. E. Arnal. 1960. A.A.P.G. Bull. 44: 1921-1932. 

42. Bandy, O. L. 1962. Micropaleontology. 7: 1-26. 

43. LoEBLiCH, A. R. et al. 1957. U.S. Nat. Museum Bull. 215: 1-235. 

44. Bandy, O. L. 1960. Intern. Geol. Cong. XXI. Session. Norden. Part 2. : 7-19. 

Copenhagen. 

45. BoLTOVSKOY, E. 1956. Micropaleontology. 2: 321-326. 

46. Tasch, p. 1953. J. Paleontol. 27: 356-444. 

47. Arnold, Z. M. 1953. Contrib. Cushman Found, for Foraminifera Research. IV(1): 

24-26. 

48. PoKORNY, V. 1958. Grundziige der Zoologischen Mikropaleont. Vol. 1. D.V.W. 

Berlin. 

49. Borradaile, L. A. & F. A. Potts. The Invertebrata. Ed. 3. Rev. by G. A. Kerkut. 

: 54-58. Fig. 29. Cambridge Univ. Press. London. 

50. Tynan, E. J. 1960. Micropaleontology. 6: 33-39. 

51. Hyman, L. H. 1940. The Invertebrates: Protozoa through Ctenophora. : 30-32. Fig. 

23:90. Fig. 23n. McGraw-Hill Book Co. New York. 

52. LoHMAN, H. 1902. Arch. Protistenk. 1: 89 165. Taf. 4-6. 

53. Braarud, T. et al. 1955. Micropaleontology. 1: 157-159. 

54. Bramlette, M. N. & F. R. Sullivan. 1961. Micropaleontology. 7: 129-188. Pis. 

1-14. 

55. Bramlette, M. N. & W. R. Riedel. 1954. J. Paleontol. 28: 385-403. Pis. 38 39. 

Figs. 1-3. 

56. Bramlette, M. N. 1961. In Oceanographv. A.A.A.S. Publ. 67: 345-366. 

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 

VI 

u 

V 

N 

c 

a> 

PL, 


ex: 




+ 1 -H +11 1 + + + 1 1 +<«- ++-H+ 1 1 i+ + 4ll-Hl 1 -H 


t— 


+ +-H +1 1 !+ + + +! 1 ++ + + 1 1 I+ + 41I + I ij 


o 


+ +-H +111 1 + + + 1 i + + +l+4^l l + l^l 1 1 1 


lo 


+ +-W +* 1 1 1 + + + 1 1 ++-H + 4^-Hl + i 1 -H 1 1 1 1 


■^ 


1 + 1 1 * + + + + + 1 + + ^ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 


fo 


++ 1 ++ 1 1 1 ++ 1 1 +^ ++ 1 1 1 1 1 ++ 1 1 + 1 1 + 


fN 


+ + + 1 + 1 1 1 ++ 1 1 1 41-H 1 1 1 1 1 1 1 1 1 1 1 1 1 


C 
C 

o 

> 

9 

."2 


- 


++ 1 i + i I++++I 1 4^^^++l 1 I-H4^l5+I i + 


O 


+ + -H ++ 1 I+ + +I + I 1 -H 1 + 1 1 li+-H^4^l 1 + 


0^ 


++-H +++ 1 +++ 1 + 1 +5+1 1 1 i^^^i"^' ' + 


OO 


+ +-H +IIIM + III Ili^lllMlJllll 


i'- 


1 ++ 1 + 1 1 +++ III 1 1 1 1 1 1 1 1 1 1 5 1 1 1 1 


>o 


III +111 +++ 1 1 1 1 1 1 1 1 1 1 5 1 1 5 1 1 1 1 


c 
.2 

3 


lO 


+ 1+ +1! 1 + + + + 1 +^ -H+ 1 1 1 1 1 1 + 1 1 1 1 1! 


-* 


+1+ +11 l++++l+^ +5 1 1 M 1 1 + 1 1 1 1 II 


ro 


+++ +1 1 1++++!+^ +1111111+11^111 


c 
.2 

i 

u 

|1< 


r^ 


+ + + + 1 1 + + + + 1 ++ e ^ 1 1 1 1 1 1 1 II 1 1 1 1 1 


+ + + +T 1 i + + + 1 ++ a -H 1 1 1 1 1 1 1 


"5 

o 


1 


c 

b 
u 
o 
c 




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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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- 



•^ o 



c 

>- i; i:^ 
oj ra JJ 

JJ O r- 



o 

r2 <U 1^ 
-to ^_rt 

OH l^ 



c 









B 6 









u C 



(J <u 



<u rt <u 

o Q. > 

f^ ^ — ^ S 

° E_ 

n tn -; 



H <= fi 

=« E 

• OJ 

'^ S N 

►1 ra d 

^ -. ??. 



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 curve includes an allow- 
ance for convective heat transport as well. In all 3 cases, some 5 per cent of the body will 
find itself in the temiierature range 273° to ^400° K., in which liquid water can exist. Melt- 
ing points of important meteorite minerals are indicated by horizontal lines. (Reproduced 
from fish el al., 1960, with permission of the editor. Copyright, 1960 by the University of 
Chicago.) 



life, some source of free energy must be available. Sunlight could provide this 
free energy indirectly, if some mechanism existed for bringing photosynthetic 
products from the surface to the interior. It is hard to see how this might be 
accomplished without a liquid vehicle. Hence, the principal remaining possi- 
bility is to derive the free energy from a local source, as first suggested by Sagan 
(1961). A nonequilibrium assemblage of minerals might provide such a source. 

* This permafrost zone can serve to retain an "internal atmosphere" within the meteorite 
parent bodN', and mav have played a role in the retention of noble gases (DuFresne and Anders, 
1962a,b). 



524 



Annals New York Academy of Sciences 



The free energy change in the conversion of high-temperature minerals to char- 
acteristic minerals cannot be calculated with any accuracy, because no thermo- 
dynamic data exist for the latter or their terrestrial counterparts, the serpentine 
and chlorite minerals. As a crude approximation, the following reaction may 
be considered: 

MgoSiOj + H,0 (1) -> MgSiOs + Mg(OH)o 

for which AFoys is —20 kcal. per mole. This corresponds to about 0.1 kcal. 
per gram of olivine, and because the products in this reaction are capable of 

rZ^ Wholly Molten (>I620°K) 

^ Zone of Fe-FeS Eutectic ( 1260° < T<I620°) 

H Zone of Liquid Water 




Figure 7. Temperature distribution in an internally heated asteroid, for a central tem- 
perature of 1900° K. The location of the zone of liquid water is indicated. 

Table 4 
Times for Water Loss from Asteroids 




reacting further to give hydrated siUcates, this value is probably conservative. 
A chondrite of the type suggested as a possible precursor of the carbonaceous 
chondrites, e.g., Ornans or Warrenton, contains more than 75 per cent olivine 
on a normative basis. Thus, although possible contributions by the other 
minerals are neglected, the average amount of free energy released in the forma- 
tion of the characteristic minerals is likely to be close to 0.1 kcal. per gram. 

The extractablc organic matter in Orgueil comprises about 10 per cent of the 
total carbon content (3.1 percent, Wiik, 1956). Thus, approximately 3 X 10"* 
calories would be available for each gram of organic matter, assuming that none 



Anders: Origin of Carbonaceous Chondrites 525 

of this energy is wasted by direct reactions between the minerals. At most, 
only a few thousand calories per gram would be required to produce biochemical 
compounds from simpler starting materials. If some form of life arose at this 
point, the remaining chemical energy could sustain it for many generations. 

Any such life form would be doomed from the outset, because its energy 
supply, once exhausted, would no longer be replenished. But the total amount 
of energy available from this source is appreciable, f'or a liquid water zone 
comprising 5 per cent of the volume of a 100-km. body, as much as 8 X 10'^ cal. 
could be stored in this manner. At a typical asteroidal distance of 2.8 a.u., 
this corresponds to the total solar energy received by the body in 2 X 10^ years. 

Of course, the futility of a doomed subterranean life form based upon a finite 
supply of energy makes it less appealing to the human mind than a photosyn- 
thetic form with a life expectancy approaching that of the planet or its central 
star. But if life arose by a spontaneous event, without guidance from above, 
then the probability of this event would have depended upon the chemical and 
physical conditions in the environment only, and not upon the perpetuity of 
the energy supply. 

The suitability of asteroidal bodies as abodes of life would thus seem to 
hinge mainly on three questions. First, were the times for water retention 
(table 4) long enough for life to arise spontaneously? All we known about this 
"induction period" for the origin of life is that it lasted less than 0.5 AE on 
Earth (Kulp, 1961). Hence the asteroids cannot be disqualified on this count 
alone. Second, were the necessary organic compounds present? From the 
work of Calvin and Vaughn (1960), and Briggs (1961), it seems that this ques- 
tion can be answered in the atfirmative, although Degens and Bajor's (1962) 
observations on the bacterial production of some of these compounds may 
require a reevaluation of the evidence. Third, could the initial hfe forms learn 
to utilize the particular inorganic energy sources present (e.g., reactions of HoO 
with olivine, Fe°, etc.)? No definite answer to this question is possible, al- 
though it is perhaps relevant to point out the known, high adaptability of 
modern terrestrial microorganisms.* 

Thus, one cannot conclude a priori that the asteroids were never capable of 
supporting life. The question of whether life ever existed in meteorites may, 
therefore, be examined on its own merits, because the size of the parent body 
does not impose any major limitations. 

Isotope measurements. Further clues to the history of these meteorites come 
from isotope measurements, although the interpretation of the data is not 
always free from ambiguities. If we assume a simple, monotonic cooling 
history for the meteorites, the K'^'VAr^'^ ages in table 5 give the time at which 
the temperature of the meteorite fell to a low enough value to permit the 
retention of radiogenic Ar'*" from the decay of K'"'. Judged from the heating 
experiments of Stauffer (1961), interpreted according to the model of Goles et al. 
(1960), this temperature probably lies near 200° K. Of course, short K-Ar 
ages would also result if the meteorite were reheated at some later stage in its 

* If such subterranean life forms ever arose on the meteorite parent bodies, they are likeh' 
to have arisen on Earth and on the moon as well. This would somewhat reduce the chances 
of finding prebiotic organic matter on the moon (Sagan, 1961). Moreover, much of the 
Earth's initial endowment of organic matter would have been transformed by biological 
activity at a very early stage in its history. 



526 



Annals New York Academy of Sciences 



history (e.g., during close approaches to the Sun), or if its parent body happened 
to remain at a temperature somewhat above, say, 200° K., where sHght, but 
continuous argon losses by diffusion would occur. 

That the short exposure ages are not due to diffusion losses at perihelion has 
been shown conclusively at least for Cold Bokkeveld (Anders, 1962c). Here, 
the content of a nonvolatile cosmogenic nuclide, Al^*^, is consistent with the Ne'-^ 

Table 5 
Ages of Carbonaceous Chondrites 



Meteorite 


Group 


K-Ar age 


Cosmic ray exposure 
age 






AE 


m.y. 


Cold Bokkeveld* 


c 


1.2 


0.2 


Felixt 


D 


4.5 


56 


Felix* 


D 


4.1 


48 


Ivunaf 


A 


1.4 


1.6 


Lancet 


D 


<3.9 


5 


Migheit 


C 


4.3 




Mighei* 


C 


2.4 


2.4 


Mokoiaf 


E 


3.4 


13 


Murrayt 


C 


2.5 


4 


Murray* 


C 


1.6 


4 


Orgueil* 


A 


1.3 


3 



*Zahringer (1962). 

tStauffer (1961). 

J Gerling and Rik (1955). 

Table 6 
Carbon Isotopic Composition in Carbonaceous Chondrites (Boato, 1954) 



Meteorite 


Class 


C 


«C" 






% 


%c 


Ivuna 


A 


3.3 


-6.6 


Orgueil 


A 


2.8 


-11.4 


Cold Bokkeveld (London) 


C 


1.55 


-9.4 


Cold Bokkeveld (Paris) 


C 


1.6 


-5.2 


Mighei 


C 


2.6 


-9.9 


Murray 


C 


1.9 


-3.9 


Lance 


D 


0.34 


-15.7 


Mokoia 


E 


0.84 


-17.4 


Forest City 


Ordinary Ch. 


0.08 


-24.3 


Richardton 


Ordinary Ch. 


0.02 


-24.6 



content, so that diffusion losses of the latter seem to be ruled out. The short 
exposure age (0.1 to 0.2 m.y.) would seem to suggest a lunar origin, as proposed 
by Urey (1962), but this hypothesis has its difficulties (Anders, 1962f). 

Other isotope measurements exist that have a bearing on the origin of car- 
bonaceous chondrites. Boato (1954) has measured the carbon isotopic compo- 
sition in these meteorites (table 6). The C^'/O^ ratio is variable from meteor- 
ite to meteorite, and even within the same meteorite (Cold Bokkeveld). It 
is known that living organisms have a preference for C^', so that biogenic ma- 



Anders: Origin of Carbonaceous Chondrites 



527 



terials are generally depleted in C^^ relative to the source material: atmospheric 
CO2 or oceanic bicarbonate (Craig, 1953). This effect is quite pronounced if the 
biogenic carbon comprises only a small fraction of the total available carbon 
(figure 8). 



o 



-36 
-34 
-32 
-30 
-28 
-26 
-24- 
-22- 
-20 
-18 



16 



fO 
O -14 

<:50 

-12 

-10 
-8 
-6 
-4 
-2 

+ 2 
+ 4 
+ 6 
+ 8 
+ 10 



UJ 

< 

Z 

o 

CD 

a: 
< 



- o 



o 
■ o 

' o 

••0 



CO 
UJ 

< 

cr 

CD 
UJ 

h- 

cc 

UJ 

> 

z 



UJ 

z 

CC 
< 

2 






A 



if) 

I- 

z 
< 



UJ 

Z 

< 

2 



UJ 

_J LJ 
Q. 

Quj 
Z UJ 
< q: 

_i I- 

• 

5: 



S 



Q 

o 
o 



Z 

UJ 

u 

UJ 

cc 



Q 

o 
o 



CO 

O 



dip 



_i 
< 
o 
u 






UJ CO 
ZUJ 

h- *< 

COO 





o 



I 

U 



OJ 
O 
U 



H 



c/) 

I- 
Z 

UJ 

Q 
UJ 






UJ 

_l 

O 
CC 

f- 

UJ 
Q. 



UJ 

H 

X 
Q. 






t 




o 

q: 

C/) 

D 
O 

UJ 

Z 

o 






o 
o 

o 
cr 

UJ 
X 
Q. 
CO 

o 



if) 

Q 



< 



Figure 8. Isotopic composition of carbon from various sources. Processes involving a 
partial loss of carbon in the form of volatile compounds {e.g., the formation of petroleum from 
the remains of organisms) result in the depletion of C. Such a depletion is also observed in 
the case of the meteorites (table 6) in which the C" content declines with decreasing total 
carbon content. [Reproduced from Craig (1953), with permission of the editor.] 



528 



Annals New York Academy of Sciences 



Unfortunately, Boato measured only the total combustible carbon, and not 
the fractionation among the several forms of carbon in the meteorite. There 
seems to be a correlation between decreasing C'^ content and decreasing total 
carbon in the meteorite. Boato suggested that this imphed preferential loss 
of C" during partial volatilization, and pointed out that a similar depletion 
had been observed in terrestrial processes that were accompanied by a loss of 
volatiles, e.g., the conversion of dead organisms to petroleum. For the purpose 
of the present discussion, it is immaterial whether this last process is abiotic or 
biotic; any low-temperature process will lead to qualitatively similar fractiona- 
tions. 

Urey (1962) suggested that sulfur metabolizing organisms might be responsi- 
ble for the oxidized sulfur compounds (S and MgS04) in the carbonaceous 
chondrites. However, as seen in table 7, the elemental sulfur in Orgueil is 
enriched in S^^ relative to the sulfate (Thode and DuFresne, 1961), whereas 
sulfur bacteria as well as inorganic processes occurring under equilibrium condi- 



Table 7 
Sulfur Isotopic Composition in Carbonaceous Chondrites 





SSHfoc) 


Object 


S04= 


S° 


Orgueil* 

Gulf Coast salt domes (11 samples) f 
Sulfur Lake, Cj-renaica, N. Africaf 


-1.30 

+41.4 
+ 15.8 


+3.04 

+2.5 
-15.3 



* DuFresne & Thode (1961). 

t Thode, Wanless & VVallouch (1954). 

tions tend to produce just the opposite fractionation, depleting elemental S in 
S''^ (Thode et al., 1954). The equilibrium constant for the reaction 

s^-'Or + H Ss^^ ^ S3404= + H Ss'^ 

is 1.071 at 25° C. (Tudge and Thode, 1950), so that the sulfur and sulfate in 
Orgueil are clearly out of equilibrium. Perhaps the origin of the higher oxida- 
tion states of sulfur will be clarified by further isotope measurements on the 
troilite in Orgueil (Thode and Anders, 1962). 

Boato (1954) also measured the hydrogen isotopic composition of the hy- 
drated silicates in carbonaceous chondrites. His results (table 8) show that 
the D/H ratio in Ivuna, Orgueil, and Mokoia was considerably higher than that 
in terrestrial waters. This fractionation may have been caused by kinetic 
isotope effects during formation of the hydrated silicates (Clayton, 1961) or 
by extensive evaporation of the water in the meteorite parent body. 

Hydrocarhous. Finally, a few words should be said about the hydrocarbons 
(Nagy el al., 1961). This matter has been discussed in greater detail elsewhere 
(Meinschein, 1961; Anders, 1961, 1962a; Nagy el al., 1962a; Meinschein et al, 
1962). For the present discussion, only three of the most salient points will be 
restated. 

Meinschein and his associates certainly deserve great credit for determining 



Anders: Origin of Carbonaceous Chondrites 



529 



the mass spectrum of the hydrocarbons in the meteorite, and for drawing atten- 
tion to its possible resemblance to the mass spectra of biogenic hydrocarbons. 
One point on which we disagree, however, is the extent of such resemblance. 
FIGURES 9 and 10, plotted from their data, show the worst and the best cases, 
respectively. If the comparison is extended to the entire mass spectrum, and 
to a larger variety of biogenic reference materials, certain additional resem- 
blances, but also certain differences appear. It seems very difficult to decide, 
on purely objective grounds, whether these resemblances are strong enough to 
prove a biological origin. 

There is also a question to what extent the peak height at a given mass num- 
ber may be taken as a measure of the amount of parent hydrocarbon of this 
mass. This is a good assumption for the [CnHiH+o]"^ ions derived from the 
CnH.2n+2 paraffius. But as one goes to compounds progressively poorer in 
hydrogen, the ambiguity increases. The [CnH-in-e]"*" ions are derived not only 
from the C„H-2„_6 ( = tetracycloalkane) series, but also from the CuH2u+2 , 

Table 8 
Hydrogen Isotopic Composition in Carbonaceous Chondrites (Boato, 1954) 



Meteorite 


Class 


H2O 


6D 






% 


% 


Ivuna 


A 


7.0 


-1-35.8 


Orgueil 


A 


7.3 


-1-29.0 


Cold Bokkeveld (London) 


C 


7.8 


-13.0 


Cold Bokkeveld (Paris) 


C 


8.0 


-5.8 


Mighei 


C 


8.6 


-6.4 


Murray 


C 


6.8 


+9.6 


Lance 


D 


0.9 


-7.7 


Mokoia 


E 


0.8 


+25.9 


Terrestrial waters 






-15 to +5 



CnHon , CnH2n-2 , aud CnH2n-4 families, with possible additional contributions 
from nitrogen and oxygen compounds. Thus, it seems fair to attribute most 
ob the observed peak height in the C„H2n+2 series to paraffins. However, just 
in the case of this series, the resemblance is rather poor (figure 9), and the 
great difference between the spectra of the original Orgueil distillate (Nagy 
et al., 1961) and the chromatographically separated hydrocarbon fraction 
(Meinschein, 1961) shows that even in this favorable case, some 70 to 90 per 
cent of the originally observed peak height came from compounds other than 
saturated hydrocarbons.* In figure 10, the resemblance is very good, and the 
changes have been moderate, but as pointed out, the peaks in this series contain 
substantial contributions from so many different sources, that it seems unsafe 
to infer a similarity in parent hydrocarbon distribution from a similarity in 
peak heights. 

Finally, one must not overlook the possibility that the observed hydrocarbon 

* This sample is not strictly comparable to the original distillate, collected in the range 
250° to 400° C, because it also contains the 400° to 500° C. fraction. But differences of the 
same order are found between the original distillate and a chromatographically separated 
hydrocarbon fraction of a solvent extract of the whole meteorite (Meinschein et al., 1962). 



530 



Annals New York Academy of Sciences 



distribution was made abiotically by Miller-Urey type reactions in the solar 
nebula. Such reactions are known to produce carbon chains of varying length, 
presumably by free radical reactions. The hydrocarbons in comets and the 
organic material in cosmic dust (Parkin et al., 1%2) may have been produced 
in this way. Meinschein (1961) has argued that such reactions would be 
highly nonselective, showing little preference among the billions of possible 



I.OOOr 



100- 



X 

o 
<v 

a. 



10- 



- 1 1 1 1 1 1 1 1 1 


- 


: C,H2,,2 Series 


- 


A 

/ 1 ,. _^ 


- 


^^,,^1 _,.^ 




■-——-'' — 7 j*^^" ' / 




^•'~"-— — J-"' \ \. 1 




y'' ' * ^V / 




/ * ^X ^ / 




~ / \ \/' '\^ J/\ 


~ 


I / ', / \^ *^ // ^^ 


- 


/ ' ' ^"^"^..J ''^ 


- 


"">. 


- 


-y N^-A 


- 


V^ 


~ 


\ / — 


~ 


\ / 




\ / 


— 


'-- \ / 


- 


» / 


- 


\' 


- 


_ OrgueiKdistillate ) V 


- 


Orguell (saturated HC fraction ) 




~ Butter 




Recent sediments 

1 1 1 1 1 1 1 1 1 





15 



20 
Carbon Number 



25 



Figure 9. Mass spectrum of meteoritic hydrocarbons and 2 Ijiogenic reference materials 
(Nagy el al., 1961). The observed peak heights in the C„Ho„+.. series are probably due, 
mainly, to parent ions of saturated hydrocarbons, although fragment ions of other substances 
also contribute. The difference between the original Orgueil distillate and the chemically 
separated, saturated hydrocarbon fraction indicates that large amounts of other substances 
were present in the distillate. 



isomers. But it is essential not to equate the concepts "abiotic" and "non- 
selective." Industrial chemical syntheses, from polyethylene to medicinals, 
are highly selective, favoring one or a few products over the multitude of others. 
Even Miller-Urey type reactions can be quite selective, as shown by Wilson 
(1960). He obtained products mainly in the mass ranges Ci to C5 and C20 
and up. Although the product distribution in that particular experiment 
(and in the industrial Fischer-Tropsch synthesis of hydrocarbons) may not be 
an accurate match of the Orgueil hydrocarbon distribution, one must remember 
that only an intinitesimal fraction of the possible combinations of conditions 



Anders: Origin of Carbonaceous Chondrites 



531 



(composition, temperature, pressure, time, energy input, catalysts, availability 
of surfaces, etc.) has been explored. 

Some chemical evidence has become available on the Orgueil hydrocarbons 
(Yang and Tsong, 1962). A cyclohexane extract of the meteorite shows nothing 
but C — H groups in its infrared spectrum, indicating that it consists mainly of 
hydrocarbons. The ultraviolet absorption spectrum shows a broad band near 
270 m/i, but virtually no absorption above 300 m/x. Hence, aromatic ring 
systems larger than naphthalene or biphenyl seem to be ruled out. Pre- 
sumably, 1- and 2-ring aromatic systems with aliphatic side chains are present. 
Chromatographic separation on silica gel resolved in the material into 5 spots, 2 
of which fluoresced weakly under ultraviolet light. The material possessed a 
strong, terpene-hke odor. More complex materials, including polynuclear 

1,000 



5 100 
o 

Q. 



20 



z — 1 r- "T 1 1 1 1 1 1 1 1 1 1 


- 


^ ^^^^^^^ ^>— -.'1 /^*.i*ll oio. \ 


^ 


Orgueil (distillate) 


- /\ Orgueil (saturated HC fraction) 


— 


"/'^^ Butter 




jr _^ ^^^ Recent sediments 




^v \ '^5v /" - — — ■-' ^ 


y 


^^^C^^^^''*^--.. "/^ ^ 


v~ 


- C^Hg^.g Series ~~— -' 

1 1 1 1 1 1 1 1 1 1 1 1 1 


\ 



15 



20 
Carbon Number 



25 



Figure 10. Mass spectrum of meteorilic hydrocarbons and 2 biogenic reference materials 
(Nagy et al., 1961). In the CnHsn.e series, the meteorilic and terrestrial mass spectra show a 
strong resemblance to each other, but because the contribution of fragment ions to the peaks 
is quite large in this series, the similarity in peak heights does not necessarily imply a simi- 
larity in hydrocarbon distribution. 



hydrocarbons of higher molecular weight, bearing polar substituents, were 
extracted from the meteorite with more polar solvents, but it seems that none 
of these higher polynuclear hydrocarbons were present in the free state. This 
relative simplicity of the aromatic hydrocarbon fraction was already noted by 
Meinschein el al. (1962), on the basis of mass spectrometric analysis. 

Perhaps the hydrocarbons in Orgueil are of biogenic origin. But in our 
opinion, the present evidence is not suthcient to justify this conclusion. 

Summary 

The carbonaceous chondrites seem to have been produced by the action of 
liquid water on a more primitive source material. Their mineralogy implies 
that this exposure to water occurred at temperatures near 300° K., a pH of 6-10, 
a reduction potential of <— 0.2 volts, and that it lasted for at least 10^ years 
(DuFresne and Anders, 1962). Their high content of chalcophile trace ele- 



532 Annals New York Academy of Sciences 

imiits and primordial nobk- gases suggests a source material more primitive 
than ordinary chondrites; yet the presence of high-temperature minerals 
implies that this source material j)assed through at least one high-temperature 
stage. These conditions would be satisfied by material expelled from the sun 
in a gaseous state (Wood, 1958), and accreted to soUd bodies after condensation 
and cooling (Urey, 1952). 

The exposure to liquid water could have occurred in subsurface regions of an 
asteroid heated by extinct radioactivityor anotherinternal energy source. Sun- 
light for photosynthesis would not reach these regions, but an appreciable 
amount of free energy would be available from the conversion of olivine to 
hydrated silicates. Although this source of energy is finite, it may have 
served as the basis for the evolution of a nonphotosynthetic life form. 
None of the isotopic data suggest the presence of life, however. The fractiona- 
tion between sulfur and sulfate in Orgueil is in the opposite direction from that 
observed for terrestrial sulfur bacteria. The carbon data are inconclusive, hav- 
ing been determined on the total combustible carbon only, rather than on 
individual compounds or fractions. The hydrocarbon data are also not con- 
clusive, since the degree of resemblance to biogenic hydrocarbons and the ability 
of nature to produce such a hydrocarbon distribution by purely abiotic (Miller- 
Urey) reactions are still open to dispute. 

A cknowledgments 

I am greatly indebted to E. R. DuFresne, whose work provided many of the 
basic data cited in this paper. I also want to express my gratitude to N. C. 
Yang and Maria Tsong, who made available their unpublished data on the 
organic matter in Orgueil, and to Frank W. Fitch, who contributed many valu- 
able criticisms. 

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Anders: Origin of Carbonaceous Chondrites 533 

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Zahringer, J. 1962. Z. Naturforsch. 17A. In press. 



AQUEOUS, LOW TEMPERATURE ENVIRONMENT OF THE 
ORGUEIL METEORITE PARENT BODY 

Bartholomew Nagy 
Department of Chemistry, Fordliam University, New York 58, N. Y. 

Warren G. Meinschein 
Esso Research and Engineering Co., Linden, N.J. 

Douglas J. Hennessy 

Department of Chemistry, Fordliam University, New York 58, N.Y. 

Sources of knowledge of the composition of the universe have been limited to 
(1) information which can be deduced from radiated energy and nuclear parti- 
cles, and to (2) the results of the studies of meteorites. The presence of hydrous 
silicates, iron oxide, water soluble salts, and organic matter in the carbonaceous 
chondrites makes the study of this rare group of meteorites especially intriguing. 

There are 19 known carbonaceous chondrites (table 1). All 19 meteorites 
were observed to fall. They all show a few millimeters thick fusion crust. 
Various investigators have found, however, that below the crust the stones are 
unaltered. Carbonaceous chondrites usually have loose textures and many of 
them have a mineralogical composition indicating that they were never sub- 
jected to temperatures higher than 300 to 400° C. 

The Orgueil meteorite, the principal object of the present investigation, fell 
at 8 P.M. on May 14, 1864. Fragments were collected in and about the villages 
of Orgueil, Nohic, and Campas in southern France. The appearance of the 
luminous meteor, the subsequent detonations and the fall were observed by the 
local residents. It is reported that the sound of the detonations was heard 
within an area of approximately 75-miles radius. The combined weight of the 
fragments which are now in various museum collections is approximately 11.5 
kg. Based upon present knowledge of the attrition a stony meteorite under- 
goes when it enters the atmosphere and the loss of fragments scattered by the 
explosions that accompany most of these falls, it is probably safe to assume 
that the Orgueil stone weighed several tons before it fell to earth. 

Carbonaceous chondrites are characterized by the few per cent of carbona- 
ceous matter that they contain, by their water content, and as Urey and Craig^ 
pointed out, by the highly oxidized state of their iron content. Mere traces of 
carbon and water, however, have been found in a large number of stony and in a 
few metallic meteorites, all of which had high temperature histories. Only 4 
of the 19 carbonaceous chondrites have been subjected to organic analysis. 
As recently as 1956, Wiik- observed that". . . the organic compounds are the 
least well known substances in the carbonaceous chondrites." This lack of 
information is probably caused by the fact that only small quantities of organic 
matter can be extracted from these chondrites and that this organic substance 
is difficult to analyze. There are a number of early investigations of varying 
reliability described in the literature; most of these vaguely refer to "bitumi- 
nous" substances, specifying odor, color, etc. 

534 



Nagy et al. : Environment of Orgueil Meteorite Parent Body 535 

Organic Analyses 

Meteorite organic analyses may be divided into two types: (1) the classical 
type analyses (which may involve the combustion of the organic matter and 
the subsequent gravimetric determination of CO2 , the reacting of the extracts 
with acids or alkalies); and (2) the analyses which were based upon spectro- 
scopical (infrared, ultraviolet, mass spectrometry) and chromatographic tech- 
niciues. Many of the former type analyses are either incompletely recorded 
or seem to be unreliable for other reasons. Consequently, only 4 of these 
analyses will be discussed briefly. These are: Berzelius' analysis^ of Alais in 
1834; Wohler's analysis-* of Kaba, in 1858; Berthelot's analysis^" of Orgueil, 
in 1868; and Mueller's analysis^ of Cold Bokkeveld, in 1953. 

Berzelius was the first to ascertain the presence of organic matter in a stony 
meteorite.* He suggested that the Alais organic matter resembled humic acids 



Table 1 
List of Known Carbonaceous Chondrites 



Locality of fall 


Date of fall 


Locality of fall 


Date of fall 


Alais, France 


1806 


Mighei, U.S.S.R. 


1889 


Cold Bokkeveld, South Africa 


1838 


Mokoia, New Zealand 


1908 


Crescent, United States 


1936 


Murray, United States 


1950 


Felix, United States 


1900 


Nawapali, India 


1890 


Haripura, India 


1921 


Nogoya, Argentina 


1879 


Indarch, U.S.S.R. 


1891 


Orgueil, France 


1864 


Ivuna, Tanganyika 


1938 


Santa Cruz, Mexico 


1939 


Kaba, Hungary 


1857 


Simonod, France 


1835 


Lance, France 


1872 


Staroye Boriskino, U.S.S.R. 


1930 






Tonk, India 


1911 



or similar organic materials and observed that the meteorite disintegrated in 
water. 

Within approximately 1 year after its fall, Wohler obtained what was appar- 
ently an uncontaminated sample of the Kaba meteorite. He suggested that the 
meteorite may contain remnants of humic matter. One year later, in a shorter 
note,* Wohler reported that he had identified bituminous material resembling 
ozocerite in Kaba, and stated that this matter "has undoubtedly organic 
origin." This rather important statement came 31 years after this same in- 
vestigator had first discovered that a biochemical (urea) could be synthesized 
from inorganic matter. One must keep in mind, of course, that facilities for a 
comprehensive evaluation of organic compositions were somewhat limited in 
1858. 

Berthelot was the first investigator who obtained hydrocarbons from Orgueil. 

* Berzelius' comments are of interest: ... "Es leidet folglich keinen Zweifel, dass der 
untersuchte Stein, ungeachtet aller seiner Verschiedenheiten im Aeussern, ein Meteorstein 
ist, welcher, aller Wahrscheinlichkeit nach, aus der gewohnlichen Heimath der Meteorsteine 
herstammt." and "Giebt diess moglicherweise einen Wink iiber die Gegenwart organischer 
Gebilde auf anderen VVeltkorpern?" 



536 Annals New York Academy of Sciences 

Gaseous, liquid, and solid hydrocarbons were found to be present after treat- 
ment with hydriodic acid.* 

In 1953, Mueller reanalyzed the Cold Bokkeveld stone. (This meteorite 
had already been studied by Wohler.) Mueller extracted a soft resinous sub- 
stance. Reactions of the organic matter with alkalies suggested that the 
extract consisted basically of complex organic acids, containing some nitrogen, 
sulfur, and halogen. It must be pointed out, however, that the high organic 
halogen content has not yet been confirmed by other investigators. Mueller 
searched for but could not detect graphite in the organic substance; on the other 
hand, he observed 10 to 12 per cent crystalline sulfur. The Cold Bokkeveld 
meteorite was found to contain water; rehydration experiments demonstrated 
that this water was not a terrestrial contamination. This author also suggested 
that the extract resembled humic acid. The author was able to reject the 
carbide theory of hydrocarbon synthesis in meteorites on various experimental 
grounds. An alternate theory was proposed by Mueller, namely, that carbona- 
ceous chondrites are fragmental aggregates, and that the organic matter is the 
result of low temperature condensation from the atmosphere of the meteorite 
parent body. He concluded that the temperature of the meteorite never 
exceeded 200 to 350° C. 

There are few analyses of the second type. In 1959, Sisler^ ran infrared 
spectra on an extract of the Murray meteorite and recorded carbon-hydrogen 
and the carbonyl absorptions. Calvin^" obtained water extracts from Murray 
and Orgueil. The extracts probably contained some hydrocarbons and hetero- 
cyclic bases. It was reported that the ultraviolet absorption curves of the 
extracts, taken at different pH values, showed that there was a pH sensitive 
absorption at the wave length corresponding to the cytosine absorption. 
Amino acid analysis led to negative results; on the other hand, mass spectro- 
scopical data showed what may have been hydrocarbons containing up to 12 
carbon atoms. 

Boato's study" of the distribution of the hydrogen and carbon isotopes in 
carbonaceous chondrites is of considerable interest. This author found that 
vvater, which was distilled from Orgueil, Murray, Ivuna, and Mokoia in vacuo 
and above 180° C. temperature, showed hydrogen isotope ratios that were 
definitely outside the terrestrial range. On the other hand the water which 
was distilled below 180° C. seemed to be a terrestrial contamination. Carbon 
isotope ratios were found to be similar to those on earth. Although Boato 
thought that the carbon compounds could not be derived from living things, 
the C^^ depletion in the Orgueil meteorite which he observed is typical of the 
depletions found in some marine organisms. The author pointed out that 
carbonaceous chondrites are heterogeneous bodies, and suggested that the 
meteorite organic matter is indigenous. 

In a recent pubUcation^^ tj^g results of an analysis of organic matter in the 
Orgueil meteorite were reported. Saturated hydrocarbon groups were identi- 
fied, some of which contained up to 29 carbon atoms per molecule. The 

* Berthelot's original statement reads: J'ai applique la meme methode a la matiere char- 
bonneuse de la meteorite d'Orgueil. J'ai reproduit, en effet, quoique plus peniblemenl qu'ayec 
la houille, une proportion notable de carbures formeniques, C2"H-"+2, comparables aux huiles 
de petrole." 



Nagy et al. : Environment of Orgueil Meteorite Parent Body 537 

preliminary observation was made that the type of molecular species present in 
the meteorite hydrocarbon mixture and the molecular weight range of the 
mixture resembled in many important aspects the hydrocarbons in the products 
of organisms and in sediments on earth. Studies in progress are to extend the 
preliminary investigation.^- The purpose of the present study is to determine 
whether the physical-chemical conditions on the meteorite parent body may 
have been suitable to sustain a form of life. 

Inorganic Analyses 

Carbonaceous chondrites contain only a small percentage of organic matter, 
the remainder consists of inorganic minerals. Their history can be determined 
most clearly only if one has a satisfactory understanding of both their organic 
and inorganic composition. Most stony meteorites, have been subjected to 
inorganic analyses. As early as 1878 Nordenskiold'^ noted the pronounced 
uniformity in the chemical compositions of chondrites. In 1953, Urey and 
Craig^ reviewed some 350 chemical analyses, selected the reliable ones, and 
came to the conclusion that chondrites fell into two distinct groups, a high and 
a low group as far as their total iron content and the oxidation state of their 
iron was concerned. They suggested that the cause of this phenomenon was 
related to the genesis of meteorites. The parent asteroids went through a low 
temperature accumulation process, a high temperature melting and evaporation 
process, a stage of collision with smaller objects and finally a collision of 2 
asteroidal sized bodies. These authors, and later Wiik,- observed that the 
carbonaceous chondrites belonged to the high iron group. Urey and Craig 
suggested that the material forming the carbonaceous chondrites had been 
infiltrated on the parent body by water, carbon compounds, and hydrogen 
sulfide. It is generally agreed that more information is a necessary prereq- 
uisite to a satisfactory understanding of the genesis of these meteorites. 

Wiik- has shown that there are three types of carbonaceous chondrites. The 
first type (Orgueil, Ivuna, Tonk) contains approximately 20 per cent water, 
approximately 22 per cent Si02 and 15 to 18 per cent "FeS." All forms of 
sulfur, including elementary sulfur, were hypothetically combined with the 
iron in the "FeS" reported, but x-ray diffraction data on Orgueil does not show 
any FeS. The second type (Cold Bokkeveld, Murray, Mighei, Staroye 
Boriskino) contains approximately 13 per cent water, 27.5 per cent Si02 , and 
9 per cent "FeS." Neither the first nor the second group contains any metalUc 
iron, nickel, or cobalt. The third type (Lance, Mokoia) contains approximately 
33 to 34 per cent Si02 , less than 1 per cent water and between 5 and 6 per cent 
"FeS." Metallic nickel and iron are present in the third group. Edwards,^* 
using an analytical method developed by Edwards and Urey,^^ found that the 
sodium and potassium distributions in carbonaceous chondrites agreed with 
Wiik's classifications. They noted that there was one exception, the Murray 
meteorite, which gave abnormally low alkali metal values. 

Inorganic analyses of meteorites point out certain important relationships, 
which can serve to supplement mineralogical data. Structural and synthetic 
mineralogy, an active field of study during the preceding 15 years, has been 
repeatedly applied with success to investigations concerned with determining 
the physical-chemical environment during rock and mineral genesis. The 



538 Annals New York Academy of Sciences 

identification of the mineral content of noncarbonaceous chondrites is usually a 
rather straightforward process. Mineral analysis in carbonaceous chondrites 
is more complicated. 

In 1864, Pisani/*'' who was one of the first analysts of Orgueil, noted the 
presence of magnetite and a "serpentine-like" mineral. More recently, Kvasha^^ 
reported finding chlorites in Staroye Boriskino. Stulov^*^ concluded that 
Orgueil, Cold Bokkeveld, and Staroye Boriskino contained chlorite-serpentine 
type minerals. Mason^^ suggested that all carbonaceous chondrites may con- 
tain chlorites. Calvin'" found that the water soluble salts in Orgueil and 
Murray were magnesium sulfate and calcium sulfate, respectively. Sztrokay, 
Tolnay and Foldvary-Vogl''" performed ore microscopical studies and chemical 
analyses on the Kaba meteorite. They suggested that carbonaceous meteor- 
ites may represent an arrested phase of meteorite development. 

Layer lattice silicates (such as chlorite and serpentine) lose structural water 
at elevated temperatures; Mueller'' and Boato^' came to the conclusion that 
water lost at high temperature was not a terrestrial contamination. Conse- 
quently, it is probably safe to conclude that the layer lattice silicates are prod- 
ucts of the meteorite parent body. 

Experimental Studies 

The experiments were designed to examine the mineral composition and, 
through this, the parent environment. Six stony meteorites were studied: 
Orgueil, Murray, Ivuna, Holbrook, St. Marks, and Bruderheim. The last 3 
are not carbonaceous chondrites; they were used as controls. There were 3 
different samples of the Orgueil meteorite. One sample (A) was obtained from 
the collection ot The American A^Iuseum of Natural History, New York. Sam- 
ple (A) has only recently been acquired by this museum; previously it formed 
part of an academic collection in the United States. The hydrocarbon analysis 
reported in the preliminary publication'- was performed on sample (A). 
The second sample (B) was broken off from meteorite specimen No. 519 of 
The American Museum of Natural History. Sample (B) has been in the 
museum collection for several years. The third sample (C) was obtained from 
the U.S. National Museum, Washington, D.C. It was Usted as part of meteor- 
ite specimen No. 234 and it was noted that the museum originally obtained it 
from S. Meunier. The samples of the Ivuna, Holbrook, and St. Marks meteor- 
ites were obtained from The American Museum of Natural History. The 
Murray sample was received from the Institute of Meteoritics, The University 
of New Mexico, Albuquerque, New Mexico, where it had been labeled as I. 
O. M. No. CRi-102. The Bruderheim meteorite was obtained from the Depart- 
ment of Geology, University of Alberta, Edmonton, Alberta, Canada; it had 
been part of specimen B-79. The chemical analyses of the 3 carbonaceous 
chondrites are Usted in table 2. The analysis of 1 of the noncarbonaceous 
chondrites (Holbrook) is included in the table for comparison. The samples 
were examined for visible impurities with a microscope or by visual examination, 
or both. 

Trace Element Analysis 

The origin of carbonaceous chondrites has been discussed repeatedly since 
Berzelius' research in 1834. Recently, Bernal" proposed that the Orgueil 



Nagy et at. : Environment of Orgueil Meteorite Parent Body 539 

meteorite may be part of the primitive earth ''shot off some hundreds of milUons 
of years ago and again united to its parent body." It was, therefore, deemed 
necessary to determine whether Orgueil is really a meteorite of extraterrestrial 
origin. 

Chondrites, as well as sedimentary and igneous rocks on earth, have char- 
acteristic trace element distribution patterns. Fifteen trace elements were 

Table 2 
Chemical Analyses of 4 Meteorites 





Carbonaceous chondrites 


Noncarbonaceous 
chondrite 




Orgueil* 


Ivuna* 


Murray* 


Holbrookf 


Fe 











7.18 


Ni 


— 


— 


— 


1.09 


Co 


• — - 


— 


— 


0.052 


FeSt 


15.07 


18.38 


7.67 


7.94 


SiO. 


22.56 


22.71 


28.69 


40.11 


TiO. 


0.07 


0.07 


0.09 


0.14 


AI2O3 


1.65 


1.62 


2.19 


1.90 


MnO 


0.19 


0.23 


0.21 


0.37 


FeO 


11.39 


9.45 


21.08 


12.01 


MgO 


15.81 


16.10 


19.77 


25.18 


CaO 


1.22 


1.89 


1.92 


1.74 


Na.>0 


0.74 


0.75 


0.22 


0.93 


K2O 


0.07 


0.07 


0.04 


0.10 


P2O5 


0.28 


0.41 


0.32 


0.40 


H,0+ 
HoO- 


19.89 


18.68 


9.98 
2.44 


0.27 


CroOs 


0.36 


0.33 


0.44 


0.45 


NiO 


1.23 


1.34 


1.50 





CoO 


0.06 


0.06 


0.08 





C 


3.10 


4.83 


2.78 





Loss on ignition (or- 


6.96 


4.10 


0.62 





ganic matter) 










Sum 


100.65 


101.02 


100.64 


99.98 



* After Wiik.6 

t After Mason and Wiik.^' 

t Includes ail forms of sulfur, including elementarj^ sulfur. There is no X-ray diffraction 
evidence that FeS, as such, occurs in Orgueil. 
Note. 

H2O, C, and S have been reduced from the value of the loss on ignition. The oxidation 
of FeO, Fe, Ni, and Co have been taken into consideration. The ignition loss as given, is 
an approximate estimate of the amount of organic matter. 

H20~ refers to water removed below 110° C. temperature, H2O+ to water obtained above 
that temperature. 



determined in Orgueil sample (A) by emission spectroscopy. Another ele- 
ment, phosphorus, was determined spectrophotometrically by the molybdenum 
blue method. A Jarrell-Ash, 3.4 m. spectrograph (15,000 lines per inch grating) 
was used for the trace element analysis. All determinations were made in 
duplicate. Germanium was used as internal standard for cobalt, chromium, 
copper, manganese, nickel, and vanadium. No internal standard was used for 
barium, gallium, lithium, strontium, zirconium, scandium, cesium, and rubid- 
ium because these elements were below the limits of detection. No internal 



540 



Annals New York Academy of Sciences 



standard was used for titanium. Kodak SA No. 1 (2200-4650A) and Kodak 
I-N (6700-9500A) plates were used to record the spectrum. The following 
wave lengths ranges were covered (1; 2200-3500A (for Co, Cr, Cu, Ga, Mn, 
Ni, Ti, V, Zr, and Sc) ; (2) 3500-4650A (for Ba and Sr) ; (3) 6700-9500A (for 
Li, Cs, and Rb). 

The trace element data in Orgueil are consistent with the average abundances 
of trace elements in chondrites. Note particularly the Ni, Cr, Co, Ti, Ba, Sr, 
and Rb values. Table 3 also shows that the Orgueil analysis does not agree 
with the average abundances of trace elements in shales and in igneous rocks 
(the latter is commonly referred to as the crustal abundance).* The trace 



Table 3 
Trace Element Abundances in the Orgueil Meteorite, in Chondrites, and in 

Igneous Rocks and Shales 





Meteorites 


Terrestrial rocks 


Element 












Orgueil carbon- 
aceous chondrite 


Chondrites* 


Shalest 


Igneous rockst 


Ba 


<10 


8 


570 


1,000 


Co 


400 


800 


18 


20 


Cr 


2,600 


2,200 


no 


100 


Cs 


10 


0.13 


5 


5 


Cu 


200 


90 


18 


55 


Ga 


<10 


5.3 


13 


19 


Li 


<3 


2.7 


55 


32 


Mn 


1,900 


1,900 


620 


1,000 


Ni 


11,000 


13,400 


64 


35 


Rb 


<10 


3.7 


140 


115 


Sc 


<10 


9.4 


14 


20 


Sr 


<10 


10 


300 


450 


Ti 


200 


790 


4,920 


4,400 


V 


30 


39 


120 


110 


Zr 


<20 


33 


160 


156 


P 


790 









Concentrations in parts per million. 

* After Goldschmidt.2' 

t After Shaw;--* supplemented by Taylor and Sachs" with recent data from the literature. 
{ After Ahrens and Taylor.-^ 

element abundances support the view that the Orgueil sample had an extra- 
terrestrial or precrustal origin. 

Electron Microscopy 

Most of the mineral particles in Orgueil were too small to be visible under the 
polarizing microscope. Therefore, 2 of the samples, (A) and (B), were examined 
with North American Phillips EM-IOOB electron microscopes. Detailed 
measurements were made on sample (A) after a survey has shown that both (A) 
and (B) contained particles which had identical crystal habits. Specimens 
were prepared by dusting with a Q-tip on I'\)rmvar film. Specimens were given 

* It should l)e noted, that it is difficult to establish average values for trace elements in 
shales; this was pointed out by Shaw.-'' 



J 
i 



Nagy et al. : Environment of Orgueil Meteorite Parent Body 541 

a light coating of carbon evaporated under vacuum for stabilization and for 
improved heat conductance under the electron beam. Some specimens were 
shadowed with platinum. 




Figure 1. Electron micrograph of the Orgueil meteorite. A, micaceous particle. Ao, 
micaceous particle with one edge rolled up; B, aggregate of micaceous particles; C", opaque, 
equidimensional particle (probably magnetite). 



In FIGURE 1 is shown 1 of the electron micrographs. It was found that the 
Orgueil meteorite consisted mainly of thin, sheethke particles, and of the 
aggregates of such particles. The flaky crystals had irregular shapes. Their 
average particle size was appro.ximately 0.1 to 0.2 fx. The thickness of the 
flakes was not estimated; it appeared that they were quite thin. Some of the 
flakes showed a tendency of rolling up along one or more edges under the elec- 
tron beam. The flaky particles resembled layer lattice silicates, and particu- 
larly the thin, irregular, and fluffy flakes of montmorillonite clay. In addition 



542 Annals New York Academy of Sciences 

to the flaky mineral, a few opaque and equidimensional particles were also 
visible. These were probably octahedral or dodecahedral crystals of magnetite. 
Their average diameters were 0.2 to 0.5 fi. 

X-ray and Electrott Diffraction Studies 

X-ray and electron diffraction techniques were used to identify the mineral 
matrix of the Orgueil meteorite. The x-ray data were obtained from diffrac- 
tometer patterns, from manual, step-scanning counts, as well as from flat film 
and Debye-Scherrer photographs. 

The 6 meteorites, (including 2 Orgueil samples, A and C), were x-rayed. In 
addition, x-ray patterns were obtained from a sample of salt, extracted with 
water from Orgueil, from 5 samples of Orgueil heated with water in sealed glass 
tubes for a period of several days at 105°, 240°, 350°, and 400° C, respectively, 
and, from samples of Orgueil, Murray, and Holbrook, after being subjected to 
rapid heating in air to 980° C. temperature. The results were compared with 
published data and with the diffraction patterns of the following standards: 
chlorite (clinochlore) from Brinton Quarry, West Chester, Pa.; magnetite from 
Mineville, Adirondack Mts., N.Y. (both were obtained from the Mineral 
Collection of the Department of Geology, Columbia University); serpentine 
(mainly antigorite) from Havana, Cuba (from the Genth Collection, The 
Pennsylvania State University), and iron (metal) powder, C.P. grade. 

The carbonaceous chondrites gave poor diffraction patterns. Apparently, 
this was caused by small particle size and by a strong fluorescence of the sample, 
when subjected to CuKa radiation. Magnetite lines appeared on all Orgueil 
patterns; many of the silicate lines were made visible on photographic film by 
reducing the exposure of the diffuse background with another strip film, put in 
front of the one that was to be used for the diffraction record. Manual, step- 
scanning in the low angle region established a diffuse band related to the char- 
acteristic basal reflections of layer lattice silicates. The counting pattern, 
however, did not show the same resolution as the photographs, where at least 
one of the 001 reflections stood out as a very weak but as a still shghtly notice- 
able line. The hydrothermal treatment of Orgueil failed to improve the 
quahty of the diffraction effects. 

Diffraction data from the Orgueil sihcates are shown in table 4, with some 
layer lattice silicates containing magnesium. Low angle counts obtained from 
oriented slides are shown in figure 2. The oriented samples were prepared by 
subjecting the powdered meteorite to shearing stress with a pestle on abraded 
glass slides. This produced a thin, glossy film in which the mineral flakes 
appear to have been aligned parallel to the glass, as prescribed by earlier experi- 
ments and theory.-^ Each 0.2 degree 26 increment was counted in the 2.0°- 
16.0° 2d range for a period of 134 seconds, with CuKa radiation and a scale 
factor of 256 on a Norelco X-ray diffractometer unit. The statistical probable 
error in the counts, under such experimental conditions, is 0.4 per cent. 

Because of the uncertainty in the position of the 001 reflections positive 
identification was not possible. Chlorite and/or montmorillonite may be 
present; the former is a likelier constituent, considering that chlorites rich in 
iron give weak 1st and 3rd order basal reflections. Reflections extending above 
7 A preclude serpentine. 



i 



Nagy et al. : Environment of Orgueil Meteorite Parent Body 543 



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w 
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h-l 

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2.89 
2.62 

2.32 
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1.74 
1.55 

1.34 








V 

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2.19 
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1.38 




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4.49 
2.64 
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1.74 
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1.32 





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544 



Annals New York Academy of Sciences 



7.6A 



10. OA 



14. OA 



130 



120- 



110 



100 



90 



g 80 

o 
o 

LU 
(/) 

S 70 

Q. 

to 



8 60 




ORGUEIL, (C), ORIENTED SAMPLE 
(U.S. NATIONAL MUSEUM, 
WASHINGTON, D.O" ■ 



ORGUEIL, (A), ORIENTED 
SAMPLE, (THE AMERICAN 
MUSEUM OF NATURAL 
HISTORY, NEWYORK, N.Y. 




26 

Figure 2. Record of manual, step-scanning counts in the low angle region. Norelco 
X-ray diffractomeler, CuKa radiation, fixed time basis. Each point on the graph was counted 
for 134 seconds, and the scale factor was 256. 






Nagy et al. : Environment of Orgueil Meteorite Parent Body 545 

Magnetite (Fe304) is a component in Orgueil;* the magnetite lines were 
sharp enough to ensure that they were not caused by chromite (FeCr204), 
which gives a similar pattern. The 2.97 A, 2.53 A, 1.71 A, and 1.62 A magnetite 
hnes were recorded. Chromite peaks at 4.83 A and 1.91 A were not observed; 
the strongest diffraction effect was sharp and always appeared at 2.53 A. The 
x-ray diffraction patterns showed no evidence for hematite (a-Fe203), pyrite 
(FeS2), troilite (FeS), pyrrhotite (FeySs), metallic iron and nickel, fayalite 
(Fe2Si04), forsterite (Mg2Si04), enstatite (MgSiOs), or gibbsite (Alo03-3H20) 
in Orgueil. The X-ray data indicated that the mineral composition of the 
sample was heterogeneous to some extent. 

The water soluble salt was obtained by heating the sample in water in sealed 
glass tubes at 104° C. for a period of 2 days, after which the supernatant liquid 
was poured off, filtered, and evaporated. The crystalline product was MgS04- 
6H2O; there were a few minor peaks which have not been identified. The 
diffraction patterns showed that subjecting the Orgueil sample to rapid heating 
(6.5° C. per minute) to 980° C. temperature in air, led to the formation of a 
limited cjuantity of olivine (forsterite) and hematite. When the chlorite and 
the serpentine standards were subjected to the identical heat treatment they 
seem to have fully recrystallized into the high temperature minerals. 

The diffraction pattern of Ivuna was almost identical to Orgueil, but Murray 
showed signs of containing olivine. The diffraction patterns of the noncarbona- 
ceous chondrites were sharp and distinct; the results were in agreement with 
published data. 

Electron diffraction studies were conducted in an attempt to confirm the 
x-ray data. Specimens were prepared by dusting with a Q-tip because it was 
thought that this method would lead to a random orientation of the flakes. 
Patterns were taken in selected areas and in manipulator positions. A "beam- 
stop" was used for some patterns; the centers of the patterns were reduced with 
Farmer's reducer. Measurements were made both on plates and on enlarge- 
ments. 

The electron diffraction diagrams showed a series of concentric rings, with a 
hexagonal (or pseudohexagonal) array of spots overimposed on most rings. 
There were also 2 diffuse bands present. 001 reflections were not recorded. 

The electron and X-ray diffraction data were in good agreement (d-values in 
TABLE 4 are based on both). There were only two differences. Electron 
diffraction diagrams did not show magnetite lines (probably because of the 
scarcity of magnetite in the fields that were examined). Furthermore, electron 
diffraction diagrams were always sharp and distinct. The hexagonal pattern 
of spots was related apparently to diffractions from the basic hexagonal building 
units of layer silicate structures. 

Thermogravimetric A nalysis 

In addition to the X-ray and electron diffraction methods, there are 2 thermal 
methods of layer lattice silicate analysis: differential thermal analysis and 
thermogravimetric analysis. Faust^- obtained differential thermal curves on 
Orgueil and Mighei but was unable to interpret the data because of the inter- 

* Part of the magnetite may contain Ni, as NiFe204 . 



546 



Annals New York Academy of Sciences 



ference of a wide range of exothermic effects (caused probably by the combus- 
tion of organic matter). Thermogravimetric analysis was selected because it 
was thought that the temperature-weight curves of untreated meteorite samples 
could be meaningfully interpreted even if there was organic matter present. 
Experiments were performed with the 6 meteorites and with mineral standards, 
as well as with mineral-organic mixtures. A "Stanton Thermo-recording" 
instrument was used; the samples were heated in platinum crucibles. The 
instrument was calibrated for both temperature and weight effects. In addi- 




100 200 300 400 500 600 700 800 900 1000 
TEMPERATURE, "C. 



100 200 300 400 500 600 700 800 900 1000 
TEMPERATURE, °C. 



Figure 3. Thermalbalance curves of meteorites (left) and of mineral standards, and 
of a mineral-organic mixture (right). Heating rate 6.5° C. per minute; each sample weighed 
0.302 gm. 



tion, standard kaolinite (API-No. 17) and montmorillonite (API-No. 25) 
samples were run for the purpose of calibration. Each sample was run at a 
6.5° C. per minute heating rate; sample weights were held identical: 0.302 g. 
The thermobalance curves are shown in figure 3 (left and right). 

The Orgueil curve shows a gradual decrease in weight to the inflection point 
(Ai) at approximately 600° C. temperature. At approximately 900° C. there 
is a second inflection point (Bi). The distillation residue of Orgueil sample 
(A), heated in an initial vacuum of lO"'^ mm. Hg at 510° C. for a period of 2 
hours, showed only the effect at Bi . The weight gain in the 400 to 500° C. 
temperature range may have been caused by the oxidation of magnetite made 
apparent by the removal of volatile organic matter and part of the water. 



Nagy et al. : Environment of Orgueil Meteorite Parent Body 547 

The initial weight loss of the distillation residue may have been caused by the 
loss of rehydrated water. The Ivuna thermobalance curve was very similar 
to that of Orgueil, although the position of point Ai was less well defined. 
Murray lost less weight than either Orgueil or Ivuna. The 3 noncarbonaceous 
chondrites gained weight during heating, caused probably by the oxidation of 
metal. Note that the Holbrook and Bruderheim curves were actually identi- 
cal. The cause of the small inflection at approximately 870° C. (Ci) is not 
known. 

One may compare the meteorite curves with published data and with those 
in Figure 3 {right) in an attempt to evaluate the meteorite compositions. The 
thermogravimetric patterns and the differential thermal curves of chlorites are 
characterized by two high temperature dehydration effects; see Mielenz, 
Schieltz and King,-''^ and Nutting.-^* Serpentine (antigorite), seems to show 
only one principal, dehydration reaction at high temperatures.^^ Certain 
chlorites, containing 2 polymorphic (14A and 7 A) units, had been observed to 
exhibit 3 high temperature, dehydration effects.^^ Montmorillonite may give 
complicated patterns; in most montmorillonites, however, the first dehydration 
reaction occurs at temperatures lower than Ai . Some montmorillonite- 
organic complexes show endothermic reactions in the 850 to 950° C. temperature 
range.^" Talc^^ seems to have only 1 principal, dehydration effect, which occurs 
close to 1000° C. Gibbsites seem to lose the majority of their water below 
425° C. temperature.^^ 

The Orgueil curve (untreated) has 2 inflection points, Ai and Bi , similar to 
chlorite, A2 and B2 . The thermobalance curve of a synthetic mixture con- 
sisting of 68 weight per cent chlorite, 14 per cent magnetite, 8 per cent Mg2S04- 
6H2O, 4 per cent elementary sulfur, and 6 per cent humic acid,* was in part 
similar to the Orgueil curve. Other mixtures, containing either bituminous 
petroleum, asphaltene, graphite, and/or serpentine, were less similar. The 
thermogravimetric pattern of a chlorite sample, ground in and saturated with 
piperidine, has shown that point Bi shifted to higher temperatures. Thermo- 
gravimetric analysis of mineral-organic mixtures suggests that the gradual 
decrease in weight below 600° C. temperature is caused by the volatilization of 
complex organic matter. 

The following samples yielded curves which were dissimilar to Orgueil: 
Recent marine sediment (from the Eastern Atlantic Ocean, 35°57'N, 07°30'W, 
from a depth of 1350 feet, and 575 cm. below the sea bottom); top soil (from an 
oak forest in Hartsdale, N.Y.) ; and a low temperature silicate reaction product. 
The latter sample was prepared by mixing, in stoichiometric proportions, 
sodium silicate and magnesium chloride solutions and allowing them to stand 
for several days at room temperature. It has been claimed in the literature^^'^^ 
that such a low temperature process might yield a product resembling serpen- 
tine. X-ray diffraction patterns of the product did not show serpentine lines 
and most of the weight loss occurred below 300° C. temperature on the thermo- 
balance curve. The soil sample also lost most of its weight at low temperatures. 
The Recent sediment was indicative of clay minerals other than chlorites, and 
also probably other than montmorillonite. 

* The humic acid was prepared by I. A. Breger of the U.S. Geological Survey from Minne- 
sota peat by low temperature alkali extraction, followed by acid precipitation and dialysis. 



548 Annals New York Academy of Sciences 

A Discussion of Conditions on the Parent Body 

Experimental data establish that the Orgueil meteorite consists chiefly of the 
following substances (Hsted in an approximate order of decreasing abundance). 
(1) Hydrous layer lattice sihcate mineral(s), (probably chlorite or, less likely, 
montmorillonite); (2) magnetite; (3) magnesium sulfate; (4) organic matter; 
and (5) elementary sulfur. 

The terrestrial occurrence of the minerals (1, 2, 3, 5) must be briefly con- 
sidered before one attempts to evaluate the environment of the Orgueil parent 
body. As an initial consideration, one may note that hydrous, layer lattice 
silicate minerals can form only in the presence of water (licjuid or vapor). 
Clearly, the parent body must have contained water. 

The chlorite minerals occur in crystalline, metamorphic schists (which had 
high temperature histories), in altered, basic igneous rocks as well as in soils 
and sediments (low temperature history). Serpentine and talc have either 
hydrothermal origins or they are alteration products of igneous rocks. The 
montmorillonite minerals are known to occur both in soils and sediments and 
in rocks altered by hot hydrothermal solutions. Layer sihcate minerals occur 
under a rather wide range of temperatures. 

Magnetite is present in many igneous rocks (which crystallized from molten 
silicates), and in sediments. Epsomite is known to crystallize from (low 
temperature) mineral water; it is often found in limestone caves. Sulphur 
may be the result of either volcanic activity, of the decomposition of H2S in 
thermal springs or of bacterial action in rocks and Recent sediments. 

On the other hand, phase equilibria studies,^^"'*' have demonstrated that 
certain, characteristic high temperature minerals, absent in Orgueil, beg'n to 
form above 450 to 500° C. temperature. This then may be safely assigned as 
the upper limit of the Orgueil temperature history. As to the lower limit of 
the parent body, one must resort to speculation. It is difficult to visualize 
how a great mass of crystalline siUcates could have formed through solid state 
reactions, at temperatures below the freezing point of water. 

Other considerations may narrow down the temperature range. The fact 
that some sulfur and hydrocarbons can be liberated from the stone at tempera- 
tures as low as 150 to 200° C, at slightly reduced pressure, suggests that the 
upper limit of the temperature range could not have been much higher than 
200° C* Furthermore, the composition of the organic matter seems to have 
been altered when the meteorite was heated with water in sealed glass tubes at 
temperatures substantially higher than 200° C. DuFresne and Anders"*- noted 
recently that some strained glass fragments found in the Mighei carbonaceous 
chondrite indicated that the meteorite could not have been subjected to a 
temperature of 180° C. for a period longer than a few weeks. The authors 
claimed that Mighei temperatures could not have exceeded 300° C. It was 
also suggested that the magnesium sulfate veins in Orgueil were produced by 
liquid water. 

There are 2 other useful indications of environment : the oxidation-reduction 
potential (Eh) and the pH. It is known from Pourbaix's*^ fundamental work 

* Gas chromatographic and mass spectrometric analyses indicate that hydrocarbons as 
small as C9 are present in Orgueil. The boiling point of H-nonane is 150° C. at atmospheric 
pressure. 



Nagy et al. : Environment of Orgueil Meteorite Parent Body 549 

on the thermodynamics of dilute aqueous solutions that /ih-pH relationships 
may be used to detine mineral stability. £h-pH diagrams had been used to 
deduce environmental conditions from low temperature mineral paragenesis 
and from sedimentation data; Carrels'^ presented a comprehensive treatise on 
the geological aspects of £h-pH relationships. 
Because of a lack of information, an evaluation of the parent environment in 



(UPPER LIMITED OF WATER 
.STABILITY) 



^^0^ 



Fe203 
(S04--) 




0.0 

-0.2 
-0.4 
-0.6 

-0.8- 

-1.0 




(LOWER LIMIT OF 
WATER STABILITY) 



8 



10 



12 14 



pH 



Figure 4. Stability relationships of iron sulfides and oxides in water at 25° C. and 1 atmos 
total pressure and total dissolved sulfur activity of 10~^ After Garrels.^^ 

terms of £h-pH relations must be basically speculative in nature. Speculation 
is possible, however, if it can be assumed, that: (1) the temperature range was 
approximately 0° to 200° C; (2) organic matter and other minor components 
do not substantially affect known £h-pH relationships; and (3) minerals of the 
Orgueil suite are gentically related. It is known^* from the stability of iron 
oxides in water that pressure has only a limited affect on /?h-pH relationships. 
A change of the temperature from 25° to 100° C. causes a shift in the stability 
fields of solids relative to the £h-pH axes but it does not affect the shape and 
size of the fields. 



550 



Annals New York Academy of Sciences 



In FIGURE 4 is illustrated a common geological phenomenon, i.e., the inter- 
relations between iron oxides and iron sulfides in water at different Eh and 
pH values. The stability relationships on the diagram were calculated by 
Garrels^ for 25° C. temperature and for 1 atmos. pressure at a total dissolved 
sulfur activity of 10~^ Under such circumstances, magnetite is stable under 
mildly reducing conditions and at a pH higher than 7. The SO^T ion is stable 



UJ 



+1.0 
+0.8 

+0.6 
+0.4 
+0.2 

0.0 
-0.2 

-0.4 
-0.6 

-0.8 
-1.0 



OXIDIZING 
VANADIUM 



^^DE POSITS 



% 



V. 



^a 



% 



%, 



MINE '3" 

WATERS, 



'A/ /^ OXIDIZING \ ' 

'/3 DEPOSITS 

RAIN, "^C 



V. 



> 



% 



STREAM '^/V- 

WATER, ^ /■/ 

NORMAL V/C^ 
OCEAN ^ 
WATER 



-^/^ 



H 



Os, 



%. 



V. 



^^. 



^e 



% 



% 



\ 



BOG 



\ 



^T/J/^^WATERS 



GROUND 
'6'/ WATER 

r^^ WATER- '^/•^ 
<0, \ LOGGED ^O f^ 






%,"\"^SALINE "%/), 
^^/>.^ ^ WATERS //^ 






% 



N» 



_L 



_L 



4 6 8 10 12 14 

pH 

Figure 5. Approximate position of some natural environments as characterized by Eh. 
and pH. After Garrels.^^ 



in the magnetite field, but the stability field of elementary sulfur extends 
toward a more oxidizing environment and an acidic pH. The missing mineral 
phases in a given suite are also indicative of environment. Note that py- 
rite forms at the same pH as magnetite but under a more reducing environment 
than magnetite; whereas, hematite forms under a more oxidizing environment. 
Neither pyrite nor hematite has crystallized out in Orgueil, although their 
elementary components are present. Layer lattice silicates are known to occur 
under conditions similar to the magnetite environment in figure 4. 



Nagy et al. : Environment of Orgueil Meteorite Parent Body 55 1 

The relationship shown in figure 4 may be appUcable, in general, to the 
Orgueil meteorite parent body. From this relationship one may then speculate 
that the Orgueil parent body had an aqueous, low temperature, slightly alkaline 
and slightly reducing environment. It seems that sulfur was formed by some 
unrelated process. 

The approximate positions^ of some terrestrial environments as characterized 
by Eh and pK are shown in figure 5. It is interesting that the proposed 
Orgueil environment resembles those terrestrial environments which are isolated 
from the earth's atmosphere (organic rich saline waters).* 

Summary 

The Orgueil meteorite has long been known to contain bound-water, organic 
matter, and sulfur, in addition to silicate, iron oxide, and magnesium sulfate. 
Trace element data in Orgueil, obtained during the present study, were found 
to be consistent with the average abundances of trace elements in chondritic 
meteorites but they appeared dissimilar to average abundances in terrestrial 
shales and igneous rocks. Electron microscopy showed that the meteorite 
consists mainly of micaceous minerals. X-ray, electron diffraction studies and 
thermogravimetric analysis confirmed the occurrence of hydrous layer lattice 
silicates and of magnetite, in addition to some magnesium sulfate. The 
mineral suite prescribes an aqueous environment of the parent body. The 
parent body temperature seems to have been low to moderate and one may 
speculate that the environment was slightly reducing and that the pH was 
slightly alkaline. 

Acknowledgments 

A part of these studies was supported by the National Science Foundation; 
this support is gratefully acknowledged. The authors wish to thank Vincent 
Modzeleski of Fordham University for his able assistance in several experi- 
ments. The authors would like to thank Brian Mason, R. F. Folinsbee, 
Lincoln LaPaz, and E. P. Henderson for providing the meteorite samples and 
Ralph J. Holmes for providing the mineral standards. The recent marine 
sediment sample was received from the Lamont Geological Observatory of 
Columbia University. The trace element analysis was performed by Ledoux 
and Company, Teaneck, N.J.; the electron micrographs and electron diffraction 
patterns were taken by the Ladd Research Industries, Burlington, Vt. The 
authors would like to thank I. Fankuchen of the Polytechnic Institute of Brook- 
lyn for his helpful suggestions regarding the x-ray diffraction analysis and for 
permitting the use of his laboratory for making certain measurements. The 
authors would like to thank Harold C. Urey of the University of California, 
Brian Mason of The American Museum of Natural History, Robert M. Garrels 
of Harvard University, I. Fankuchen of the Polytechnic Institute of Brooklyn, 

* Sagan^*" suggested that indigenous organic matter may exist buried under the surface 
of the moon. He observed that "organisms shielded from solar illumination, perhaps in 
congealed dust matrix interstices, might survive cosmic radiation for 10" years or more; lunar 
subsurface temperatures are too low to impede survival." The Orgueil meteorite may repre- 
sent the remnant of such an undergrounfl habitat, but the experimental data gathered in this 
study do not preclude the possibilitj- that the parent body was of sufficient size to hold an 
atmosphere and thus, bodies of water. 



552 Annals New York Academy of Sciences 

Lincoln LaPaz of The University of New Mexico and J. D. Bernal of Birkbeck 
College, University of London, for reading the manuscript. 

References 

1. Urey, H. C. & H. Craig. 1953. Geochim. et Cosmochim. Acta. 4: 36. 

2. WiiK, H. B. 1956. Geochim. et Cosmochim. .\cta. 9: 279. 

3. Berzelius, J. J. 1834. Ann. Phys. Chem. 33: 113. 

4. WoHLER, M. F. 1858. Sitzher. Akad. Wiss. Wien, Malh-naturw. Kl. 33: 205. 

5. Berthelot, M. 1868. Compt. rend. 67: 849. 

6. Berthelot, M. 1869. J. prakt. Chem. 106: 254. 

7. Mueller, G. 1953. Geochim. et Cosmochim. Acta. 4: 1. 

8. WoHLER, M. F. 1859. Sitzlier. Akad. Wiss. Wien, Math-naturw. Kl. 34: 7. 

9. SiSLER, F. 1959. UnpubUshed work. 

10. Calvin, M. 1961. Chem. Eng. News. 39(21): 96. 

11. BoATO, G. 1954. Geochim. et Cosmochim. Acta. 6: 209. 

12. Nagy, B., W. G. Meinschein & D. J. Hennessy. 1961. Ann. N.Y. Acad. Sci. 93: 25. 

13. NoRDENSKioLD, A. E. 1878. Geol. Foren. i Stockholm Forh. 4: 45. 

14. Edwards, G. 1955. Geochim. et Cosmochim. Acta. 8: 285. 

15. Edwards, G. & H. C. Urey. 1955. Geochim. et Cosmochim. Acta. 7: 154. 

16. PiSANi, F. 1864. Compt. rend. 59: 132. 

17. KvASHA, L. G. 1948. Meteoritika Acad. Sci. U.S.S.R. 4: 83. 

18. Stulov, N. N. 1960. Meteoritika Acad. Sci. U.S.S.R. 19: 81. 

19. Mason, B. 1960. Nature. 186: 230. 

20. Sztrokay, K. I., V. Tolnay & M. Foldvary -Vogl. 1961. Acta Geol. Acad. Sci. Hung. 

7: 57. 

21. Mason, B. & H. B. Wiik. 1961. Geochim. et Cosmochim. Acta. 21: 276. 

22. Bernal, J. D. 1961. Nature. 190: 129. 

23. Goldschmidt, V. M. 1954. Geochemistry. O.xford University Press. London. 

24. Shaw, D. M. 1954. Bull. Geol. Soc. Am.' 65: 1151. 

25. Taylor, S. R. & M. Sachs. 1960. Nature. 188: 387. 

26. Ahrens, L. H. & S. R. Taylor. 1960. Spectrochemical Analysis. Ed. 2. Addison- 

Weslev Publishing Co. Reading, Mass. 

27. Buessem, W. R. & B. Nagy. 1954. Proc. 2nd Nat. Conf. on Clays. : 480. 

28. MacEwan, D. M. C. 1951. X-ray Identification and Structure of the Clay Minerals. 

Mineral Soc. Gr. Brit. Monograph. : 86. 

29. Gruner, J. W. 1934. Z. Krist. 88: 412. 

30. Selfridge, G. C. 1937. Am. Mineralogist. 22: 97. 

31. Brindley, G. W. & K. Robinson. 1951. X-ray Identification and Structure of the 

Clay Minerals. Mineral Soc. Gr. Brit. Monograjjh. : 173. 

32. Faust, G. T. Unpublished work. 

33. Mielenz, R. C, N. C. Schieltz & M. E. King. 1954. Proc. 2nd Nat. Conf. on Clays. 

: 285. 

34. Nutting, P. G. 1943. U.S. Geol. Survey. Bull. 197.E: 197. 

35. Nelson, B. C. & R. Roy. 1954. Proc. 2nd Nat. Conf. on Clays. 335. 

36. Byrne, P. J. S. 1954. Proc. 2nd Nat. Conf. on Clays. : 241. 

37. Strese, H. & U. Hofmann. 1941. Z. anorg. u. allgem. Chem. 247: 65. 

38. Epprecht, W. 1941. Schweiz. mineral. Petrog. Mitt. 27: 1. 

39. BowEN, N. L. & 0. F. Tuttle. 1949. Bull. Geol. Soc. Am. 60: 439. 

40. Yoder, H. S. 1952. Am. J. Sci., Bowen. 569. 

41. TuRNOCK, A. C. 1960. Ann. Rept. Director Geophys. Lab. 98. 

42. DuFresne, E. R. & E. Anders. 1961. Geochim. et Cosmochim. Acta. 23: 200. 

43. PouRBAix, M. J. N. 1949. Thermodynamics of Dilute Aqueous Solutions. Edward 

Arnold & Co. London. 

44. Garrels, R. M. 1960. Mineral Equilibria. Harper and Bros. N.Y. 

45. Sagan, C. 1960. Proc. Natl. Acad. Sci., U.S. 46: 393. 

46. Sagan, C. 1960. Proc. Natl. Acad. Sci., U.S. 46: 396. 



EVIDENCE IN METEORITES OF FORMER LIFE: THE ORGANIC 

COMPOUNDS IN CARBONACEOUS CHONDRITES ARE 

SIMILAR TO THOSE FOUND IN MARINE SEDIMENTS 

W. G. Meinschein 
Esso Research and Engineeriufi Co., Linden, X.J. 

Bartholomew Nagy, Douglas J. Hennessy 

Fordham I'nhersity, New York, N.Y. 

Recently, the composition of the hydrocarbons in the Orgueil carbonaceous 
chondrite has been proposed as evidence for biological activity in the parent 
body.' This apparently novel use of hydrocarbons has created great interest 
and is the subject of appreciable controversy. 

The proposal that certain meteorites were once a part of an extraterrestrial 
biosphere is not new. Analyses of carbonaceous substances in chondrites 
were published first more than 120 years ago. Berzelius,"' in 1834, speculated 
about and decided against the possibility that the humic acid type substances 
in the Alais meteorite were biological products. Wohler,'*-^ however, thought 
that the ozocerite type constituents in the Kaba chondrite, which he investi- 
gated, in 1858, were "undoubtedly of organic origin;" but Berthelot did not 
share Wohler's belief that a resemblance to terrestrial organic matter was 
proof of a biological origin. Berthelot'''' hypothesized a reaction of metallic 
carbides and water to explain the presence of "petroleum-like" hydrocarbons 
in the Orgueil stone. Although Mueller'^ noted experimental evidence against 
the carbide theory of Berthelot, the "chlorobitumens" which were reportedly 
isolated from the Cold Bokkevelt by Mueller^ were not suggested as organic 
products. All of these observational and elemental analyses were far less 
definitive than the analyses that have been made possible by modern technicjues 
and instruments and recent accjuisition of paleobiological reference data. 
Investigations of terrestrial biotic matter and modern analytical methods, 
now, provide a basis for speculations about extraterrestrial life. 

Spectrometric, chromatographic, and new microscopic methods were not 
utilized in the study of the carbonaceous substances in meteorites before 1954. 
Boato"* nieasured the abundances of stable carbon and hydrogen isotopes in 
14 meteorites. He noted that "the carbonaceous material is, of course, not 
derived from living sources, but it is noteworthy that the range of variation 
in C'-^ in the meteorites is of the order of the depletion observed in a terrestrial 
process involving loss of volatile compounds."^ Recent investigations, how- 
ever, do not support the view that a loss of volatile compounds leads to a 
depletion of C''* in sedimental organic matter. Silverman and Epstein'^ and 
Park and Epstein" have found that ecology exerts the principal control on 
terrestrial, organic C'-^ contents. Lipids, which are the major volatile con- 
stituents of plants and animals, have lower C'^ contents than other organic 
compounds." 

Boato's deuterium determinations provided evidence of the extraterrestrial 
origin of meteorites. He found much greater concentrations of deuterium in 
the combined water which were removed from the Orgueil stone at temperatures 

553 



554 Annals New York Academy of Sciences 

exceeding 180° C. than have been found in natural waters on earth. Neither 
deuterium** nor trace nietaP- abundances in the Orgueil meteorite support 
Bernal's''^ contention that this carbonaceous chondrite "may be a part of the 
primitive earth shot ofT hundreds of milHons of years ago and again united to 
its parent body." 

Calvin'^ obtained the ultraviolet absorption spectra of water extracts of the 
Murray and Orgueil meteorites in acidic and basic solutions, and he concluded 
that the absorption wavelengths and the variations in these lengths induced 
by the addition of acids were suggestive of cytosine, a building block of nucleic 
acids which are essential components of all living cells. Briggs'''' reported 
evidence of purines and imidazoles in the Orgueil, Murray, and Mokoia meteor- 
ites. 

Detailed analyses of the amino acids, amino sugars, and sugars in the Bruder- 
heim chondrite and the Murray carbonaceous chondrite have been run by 
Degens and Bajor."'' They determined the quantities of 20 amino acids, 3 
sugars, and 2 amino sugars as well as the presence of cytosine, uracil, and/or 
hypoxanthine in the 2 samples.^'' "After complete extraction of all hydrolyz- 
able matter in the meteorite," the investigators^^ found that "amino acids and 
sugars could be generated within 3 weeks' exposure to open air in c|uantities 
about 20 per cent of the original values." Because of the regeneration, com- 
position, and stabilities of the amino, sugar, and nucleic acid constituents in 
the Bruderheim and Murray, Degens and Bajor"' proposed that the "organic 
constituents analyzed are with great probability terrestrial in origin rather 
than fossil remnants of extraterrestrial life." 

Claus and Nagy have reported "organized elements" in the Orgueil and 
Ivuna'^ and have observed similar elements in the Alais and Tonk carbonaceous 
chondrites.'^ These "organized elements" are dissimilar to any known mineral 
forms but resemble, yet are not identical to certain species of algae. "'^ Recog- 
nizable, well-preserved terrestrial type organisms, also, were seen in the 4 
carbonaceous chondrites, but the latter species were present in much smaller 
numbers than the microfossil-like elements. ''' The terrestrial type organisms 
were assumed to be contaminants acquired on earth, and they represented the 
only specimens resembling microorganisms that were found in the Bruderheim 
and Holbrook chondrite.'^ 

Palynological treatments have provided additional information about some 
of the "organized elements" in meteorites. Staplin'^ added: (1) hydrochloric 
acid to remove the carbonates; (2) hydrofluoric acid to remove the silicates; 
and (3) Schulz solution (nitric acid and potassium chlorate) to bleach the 
residue that he had obtained by these acid treatments of an Orgueil fragment. 
The residue contained "recent (organic) contaminants, a very few well-pre- 
served Cretaceous microfossils, and relatively numerous less well-preserved 
microfossils of unknown age or affinities. The unidentified microfossils, mostly 
in the 10 to 100 n size range, superficially resemble certain of the unicellular 
algae if size, texture, and the presence of an acid resistant pellicle are con- 
sidered." 

Fox^" has made abiotically double walled carbonaceous particles which he 
believes may be formed in shapes and sizes that resemble the "organized ele- 
ments" in meteorites. Fitch et al.,^^ noted that the "organized elements" in 



I 



Meinschein et al. : Evidence in Meteorites of Former Life 555 

carbonaceous chondrites may be minerals or sulfur droplets; whereas Briggs 
and Kitto" conclude that the "complex organic microstructures" in the Mokoia 
meteorite may be either of biogenic or abiogenic origin. It remains to be 
demonstrated, however, that these abiotic products can duplicate the fluores- 
cence, size, and numerical distributions, structural details, biological stain 
acceptance, and behavior during palynological treatment which have been 
reported'^ '^ for the microfossil-like "organized elements" in meteorites. In 
the opinion of Nagy el al.,~^ no organic particles have yet been prepared that 
possess all the properties of "organized elements" or cell remnants. Bernal-^ 
states, "the question of whether the objects admittedly composed of sulfur or 
mineral fragments are or are not identical with the 'organized elements' . . . 
clearly requires for its resolution careful comparisons by a panel of impartial 
experts." Urey^^ feels that "although the present evidence is not conclusive, 
there are good reasons for exploring possible origins of lifelike forms in the 
carbonaceous chondrites other than contamination after their arrival on earth." 

Biological Indicators 

Living things may be grossly regarded as unique assemblages of parts or 
molecules that possess efficient means of synthesizing, using highly select 
arrays of complex molecules, and of reproducing their specie. Plants can con- 
vert several per cents of the solar energy that they receive into molecular 
energy or food. Compounds which form a major portion of the constituent 
parts of organisms comprise an exceedingly small fraction of the compounds 
which theoretically can be made by abiotic reactions. Sagan'''^ reports "the 
most optimistic extrapolation from existing laboratory ultraviolet experimental 
data" for the quantum yield of organic molecules by Miller-Urey-*^ type syn- 
theses is 1 part in 1()(),()()0 parts, and the products of these syntheses are neither 
solely nor entirely the compounds made by living things. Organisms are 
apparently in excess of a thousand times more efficient than abiotic reactions 
which may have occurred in a primordial environment.-^ 

Although some nonbiological process under some presently undelinable 
conditions may duplicate the productive capacities of living cells, available 
data support the view that detectable concentrations of complex molecular 
mixtures composed of compounds resembling those in living cells are products 
of life.'-^ 

Because organisms are efficient and apparently unique producers of certain 
arrays of molecules, plant and animal matter has probably exerted a major 
control on the compositions of many carbonaceous substances in terrestrial 
sediments for the last 2 or more billion years.-^ Either the preserved or the 
altered biosynthetic products in Earth's sediments may provide a valuable, 
legible record of prehistorical life and its evolution. Analyses of extracts of 
terrestrial sediments indicate that ancient plants and animals have left evidence 
of their existence and that some extractable substances of natural samples 
may be used as biological indicators. 

In this investigation, the compositions of the benzene extracts of soils and 
marine sediments froni various regions on earth have been used as references. 
It is postulated that the terrestrial extracts retain evidence of biological activity, 
and it is assumed that similarities between terrestrial and meteoric extracts 



556 Annals New York Academy of Sciences 

constitute evidence that the meteorites were either contaminated while on 
Earth or a part of a parent body which supported hfe. Careful consideration 
will be given to the compositions of the extracts of the sediments and meteorites 
so as to determine, as well as these compositions permit, whether the extractable 
fractions of carbonaceous chondrites are indigenous or contaminants. 

Experimental Procedure 

Solvents and glassware. Reagent Cirade solvents were used exclusively. 
Before use, solvents were distilled through 6 plate glass helices columns, and 
100 gm. aliquots of each solvent batch were blown to constant weight in the 
sample recovery system. Solvents accepted for use contained less than 0.1 mg. 
residue per 100 gm. of solvent and these residues did not absorb detectably in 
either the 2 to 15 yu or 220 to 400 m/i regions. All glassware and porcelain 
used in preparing and analyzing meteorite samples were cleaned with acid 
and carefully rinsed with the accepted solvents. 

Blanks. A blank, which omitted only the meteorite sample, was run on 
each step of sample preparation and analysis. 

Extractions. Consolidated fragments of the l-Orgueil(B) (1.7 gm.), 2- 
Orgueil(C) (14.5 gm.), 1-Murray (1.9 gm.), 2-Murray (10.2 gm.), and Hol- 
brook (1.8 gm.) meteorites were placed individually on glass wool plugs in 5 
glass funnels.-'' Each fragment was rinsed separately with several portions of 
a 1 volume methanol to 9 volumes benzene (9:1 benzene-methanol) solvent. 
Meteorite samples smaller than 2 gm. were rinsed with 25 ml. of solvent, and 
the 2-Orgueil and 2-Murray fragments were rinsed with 80 and 50 ml. of solvent 
respectively. The rinses from each meteorite were analyzed separately. 

The rinsed fragments were crushed to 20 to 40 mesh size, placed separately 
on a glass wool plug above a sintered glass partition between a boiling tiask 
and a water-cooled condenser in an all glass, single piece, Soxhlet-type extractor. 

Small (<2 gm.) and large (>10 gm.) fragments were extracted by slightly 
different procedures. A 25-ml. aliquot of 9:1 benzene-niethanol was added 
to each of the extractors containing the crushed small fragments. After 6 
hours at reflux, the extracts were withdrawn and a second 25 ml. of the solvent 
was added to each unit. The extractions were continued an additional 14 
hours. In this manner, a rinse, a 6-hour extract, and a 6- to 20-hour extract 
of each small fragment was obtained. The large 2-Murray and 2-Orgueil frag- 
ments were extracted for 20 hours with 50 ml. of solvent, so that only a rinse 
and a 20-hour extract of each of these samples was recovered. 

Sample recovery. Solvents were evaporated from rinses, extracts, and from 
the eluates of colloidal copper and silica gel columns. Sample bottles con- 
taining organic solutions of meteorite rinses, extracts, or eluates were placed in 
receptacles or aluminum cups in a constant temperature bath maintained at 
40 ± 1° C. Nitrogen filtered through silica gel was blown over the organic 
solutions for 4 to 6 hours. This recovery procedure removes the solvents and 
most organic compounds from the meteorites that have vapor pressures greater 
than Ci:i w-paraffins. Thus, the hydrocarbons recovered in the meteorite 
samples consisted primarily of Ch and larger molecules. 

In TABLE 1 are presented the weights of the extracts recovered from the 
sulfur removal step. 



I 



Meinschein et al. : Evidence in Meteorites of Former Life 557 

Although a semimicro balance was used in all weighings, 1 or more removals 
of solvent and 2 or more weighings were required to obtain a residue weight. 
Therefore, the weights listed are probably accurate to only ±0.2 mg. 

Sulfur removal. Elemental sulfur was removed from all meteorite rinses and 
extracts by means of colloidal copper columns.^" 

Chromatograpluc separations. The 6-hour extracts of 1-Orgueil and 1- 
Murray, and 20-hour extracts of 2-Murray and 2-Orgueil fragments were 
fractionated on 9-gm. silica gel columns. ^^ The Holbrook extract was too 
small to fractionate. This method of chromatographic separation on silica 
gel has been previously investigated. Thousands of crude oils and organic 
extracts of sediments and organisms have been fractionated by this chromato- 
graphic procedure. Infrared, ultraviolet, and mass spectrometric analyses, 
elemental analyses, and aluminum chromatographic analyses^^''^** of numerous 
fractions of these silica gel eluates have established: (1) the M-heptane eluates 



Table 1 

Organic Residues 

(Weights in milHgrams) 



* A portion of sample lost when solvent "bumped." 

t Not determined. 

X Nonvolatile residue — inorganic salts. 

§ Contained visible traces of colloidal copjjer from sulfur removal step. 

\ Estimated from mass spectra peak heights. 



Sample 


Rinses 


6-Hour e.xtracts 


6 to 20-Hour 
extracts 


1-Orgueil (1.7 gm.)* 
2-Orgueil (14.5 gm.) 
1 -Murray (1.9 gm.) 
2-Murray (10.2 gm.) 
Holbrook (1.8 gm.) 
Blank 


0.1 
0.3 
0.1 

t 
0.2t 

o.i§ 


6.0 

75.0 (20-hour) 
1.1 

6.7 (20-hour) 
0.4§ (O.OH) 

o.m 


0.6 

t 
0.1 

t 
0.1 
0.0 



are composed primarily of saturated hydrocarbons; (2) carbon tetrachloride 
fractions contain saturated hydrocarbons, olefins, traces of some nonpolar 
organic nitrogen and sulfur compounds, and alkyl- and cycloalkylbenzenes; 
(3) benzene eluates contain most of the aromatic hydrocarbons, some organic 
esters, alcohols, and other organic nonhydrocarbons;^i and (4) methanol eluates 
are composed predominately of organic nonhydrocarbons. 

Weights of the chromatographic fractions of 1-Orgueil, 1-Murray, 2-Murray, 
and blanks, which were in most ca.ses too small to be determined accurately, 
are presented in table 2. The 2-Orgueil extract was of sufficient size for a 
triplicate analysis, and the chromatographic data on the 3 alic|uots of this 
sample are given in table 3. 

Infrared spectroscopy. All blanks, rinses, extracts, and the methanol eluates 
of 1-Orgueil and 2-Murray were scanned in the 2 to 15 m region. Infrared 
spectra were obtained of the //-heptane, carbon tetrachloride, benzene, and 
methanol fractions of 2-Orgueil. Scans were run on a Baird Associates Model 
4-55 spectrometer with a sodium chloride prism. Sample and blank cells 
had 0.1-mm. cell lengths and were equipped with sodium chloride windows. 



558 



Annals New York Academy of Sciences 



Blanks, rinses, Holbrook extracts, and 1-Murray 6- to 20-hour extract did not 
absorb significantly in the 2 to 15 ^ infrared region. The infrared spectra of 
the total blanks, and total Holbrook, l-()rgueil, and 2-Murray organic extracts 
are presented in figure 1. In figure 2 are shown the spectra of the individual 
chromatographic fractions of the 2-Orgueil extract. 

Ultraviolet and visual spectroscopy. All blanks, extracts, rinses, and chro- 
matographic fractions were scanned in 220 to 400 ni/u region. Scans were run 
on a Gary model 14 spectrometer with matched cells of 1 cm. in length and 
methanol as solvent. Visual spectra were run on the benzene and methanol 
chromatographic fractions of the 1-Orgueil, and 1-Murray. Absorption 
decreased continuously from 400 to 800 m^ in all of these fractions. The 



Table 2 

Silica Gel Chromatographic Fractions 

(Weights in milligrams) 



1-Orgueil (6.0) 
1 -Murray (1.1) 
2-Murra'v (6.7) 
Blank 



w-Heptane 



0.1 (0.3*) 
0.0 (0.1*) 
0.4 
0.1 (0.0*) 



Carbon 
tetrachloride 



0.2 
0.1 
0.1 
0.0 



Benzene 



0.6 

0.0 (0.1*- 

0.4 

0.0 



t) 



Methanol 



4.4 
0.7 
4.3 
0.1 (O.Ot) 



* Estimated from mass spectra peak heights. 
t Estimated from ultraviolet absorption. 

Table 3 

Silica Gel Chromatographic Fractions of 2-Orgueil 

(Weights in Milligrams) 



I 



2-Orgueil aliquots 



Awt. 24.50 
B wt. 25.09 
C wt. 24.19 





/(Heptane 


2 
2 
2 


32 (9.5%,) 
45 (9.8%,) 
56 (10.6%,) 



Carbon 
tetrachloride 



1.46 (6.0%) 
1.32 (5.3%,) 
1.17 (4.8%,) 



Benzene 



0.76 (3.\%) 
0.82 (3.3'^) 
0.76 (3.1%) 





Methanol 


15 
15 
15 


00 
92 
62 


(61.2%,) 
(63.4%) 
(64.6%) 



Left on column 

4.96 (20.2%) 
4.58 (18.3%) 
4.08 (16.9%) 



Orgueil extracts ab.sorbed the strongest in the visual range, but the visual 
spectra of all extracts lacked any suggestion of a specific absorption at a particu- 
lar wavelength. 

The blanks did not absorb and the chromatographic fractions of the Holbrook 
absorbed only slightly in the 220 to 400 niyu range. The ultraviolet spectra of 
the 1-Orgueil and 2-Murray chromatographic fractions are presented in figures 
3 and 4, respectively. In figure 5 are given the ultraviolet spectra of the 
total Holbrook extract and the total procedure blank. The Murray and 
Orgueil extracts fluoresced in ultraviolet light. 

Mass spectroscopy. Blanks and the rinse; 6-hour and 6- to 20-hour extracts 
of Holbrook; and carbon tetrachloride, benzene, and methanol eluates of the 
Orgueil distillate did not produce measurable peaks at masses greater than 150 
in the mass spectrometer. Measurable mass spectra were obtained of the 4 
individual chromatographic fractions of the 6-hour extract of 1-Orgueil and 



1 



Meinschein et al. : Evidence in Meteorites of Former Life 559 

of the 2-Murray extract; of the composited //-heptane, carbon tetrachloride, 
and benzene eluates and the methanol eluate of the 6-hour extract of l-j\Iurray; 
the rinses and 6- to 20-hour extracts of 1-Orgueil and 1 -Murray and of the 
w-heptane eluate of the Orgueil distillate. Additional fractionations and 
analyses are being run on the 2-Orgueil fractions. All spectra except those 
of the 2-Murray eluates were obtained on a Consolidated 21-103C mass spec- 



3000 2500 2000 



1500 



WAVENUMBERS IN CM"I 
1300 1200 1100 1000 



600 



700 



625 




6 9 10 II 

WAVELENGTH IN MICRONS 



Figure 1. Infrared spectra of total Ijenzene-methanol extracts (free sulfur removed) and 
blanks. Only minor absori)tions appear in the spectra of the blank and Holbrook extract. 
Infrared absorption bands in the 2-Murray and 1 -Orgueil extracts are similar to the bands 
otjserved in the benzene extracts of some terrestrial sediments. 



5000 



3000 2500 2000 



WAVENUMBERS IN CM~' 
1300 1200 MOO 1000 



625 




8 9 10 

WAVELENGTH IN MICRONS 

Figure 2. Infrared spectra of silica gel chromatographic fractions of the 2-Orgueil ben- 
zene-methanol extracts. That «-he]itane and carbon tetrachloride eluates absorb only at 
wavelengths that may be attributed to C — C and C — H bonds. The benzene eluate has a 
small carbonyl absorption near .S.8 fi and absorption bands at 9.7, 12.3, and 13.4 n. These 
latter bands appear in the infrared spectra of the benzene eluates of ancient sediment extracts, 
crude oils, and many recent marine sediment extracts. Absorption Imnds in the methanol 
eluate are typical of the bands found in the spectra of the methanol eluates of many terrestrial 
sediment extracts. 



trometer which is modified for the analysis of high molecular weight organic 
compounds. Operating conditions for the Consolidated instrument were: 
ionization potential, 70 volts (benzene and methanol eluates of 1 -Orgueil were 
run also at 12 volts); ionization current, 45 yuamp.; magnet current, 1.30 amp.; 
scan rate, accelerating potential 3300 to 4000 volts in 20 minutes; temperature, 
ionization chamber, 250° C, and inlet system, 300° C. 

The mass spectra of the 2-Murray eluates were obtained by the Analytical 
Research Division of Esso Research and Engineering Company with a General 



560 



Annals New York Academy of Sciences 



Electric 12-in. mass spectrometer that is modified for high mass analyses. 
Operating conditions of the General Electric instrument were: ionization 
potential, 31 volts (benzene and methanol eluates of 2-Murray were run also 
at 12 volts); ionization current, 50 yuamp.; accelerating potential, 2500 volts; 
scan rate, magnet current 50 to 500 mamp. in 20 minutes; temperatures, sample 
evaporator 255° to 262° C, volume 233° to 240° C, inlet lines 213° to 217° C, 
ionization chamber 174° to 177° C. 



o 
< 

OD 

tr 
o 
to 

CD 
< 



, \.\ I -•■n-HEPTANE 

\ 1/ ll \ • • -CARBON-TET. 

I l|i V- -BENZENE 

' \) ' I— METHANOL 

*O.OImg/ml lo.025 mg/ml \ 

\ I Vl 

I 10.2 mg/ml 

, U-V SOLVENT \ 
\ -METHANOL \ 

\ \ 




"200 240 280 320 

MILLIMICRONS 



360 



400 



Figure 3. Ultraviolet spectra of the silica gel chromatographic fractions of 1-Orgueil 
6-hour extract. Saturated hydrocarbons do not absori) in the ultraviolet range. Aromatic 
hydrocarbons do absorb. These spectra indicate that w-heptane eluate is composed primarily 
of saturated hydrocarbons and that the aromatics in the 1-Orgueil extract are concentrated 
in the benzene eluate. 



< 

GD 

q: 
O 
en 

CD 

< 




200 



240 



280 320 

MILLIMICRONS 



360 



400 



Figure 4. Ultraviolet spectra of the silica gel chromatographic fractions of the 2-Murray 
20-hour extract. Chromatographic fractions of the 2-Murray extract are similar in composi- 
tion to those of 1-Orgueil extract, but the relative concentrations of the aromatic hydrocarbons 
in these extracts vary in much the same manner as do the aromatic contents of different 
terrestrial sediments. 



Meinschein et al. : Evidence in Meteorites of Former Life 561 

The Consolidated and G.E. instruments can be used for accurate analyses 
of mixtures of known hydrocarbons. Concentrations of individual compounds 
can normally be determined within ±2 per cent of the true concentrations. 

Preparation of mass spectral data sheets. The mass spectra were obtained as 
photographic records of the galvanometer deflections produced when the 
ions of each particular mass are brought sequentially into focus. The deflec- 
tions were measured and the heights of the individual peaks were recorded. 
These measured peak heights were corrected by a computer for ions containing 
C'^ and H- (corrections made on the basis of terrestrial abundances of C^'^ and 
H-). The computer totaled and normalized the corrected peak heights to a 
value of 30(),(K)() and printed these heights as a 14-column array. 



UJ 

o 
< 

CD 

cr 
o 

03 

< 



1.0 

0.9 

Q8 

07 

06 

05 

0.4 

03 

02 

Ol 

O.Q 



— TOTAL EXTRACT 

--BLANK (REAGENTS. 
ANALYTICAL PRO- 
CEDURES) 




^<.00lmg 

PHENANTHRENE/ml 

il/2 TOTAL EXTRACT IN 
3 ml METHANOL 



200 240 280 320 

MILLIMICRONS 



360 



400 



Figure 5. Ultraviolet spectra of total Holbrook 6-hour extract and total procedures 
blank. The blank does not absorb significantly in the ultraviolet. This indicates that no 
aromatic contaminants were added to the meteoritic extracts during their analyses. Absorp- 
tion of the total Holbrook extract is very small. The amount of aromatics acquired by the 
Holbrook fragment in almost 50 years of storage could not have exceeded a few micrograms. 

Descriptions of the 14-column array have been published.'*' ••^■''••^'' The com- 
puter arbitrarily labels the 14 columns from left to right with values of .f 
which range as integers from —11 to +2. Use of 14 columns results in the 
placement of homologous ions {i.e., ions of the same structural type which 
differ by CH2 or 14 mass unit groups) of a particular type in a single column. 
The integers heading the columns may represent .v values in the general hydro- 
carbon formula C„H2„+j . Each horizontal row of peak heights is marked 
with a value of n or C f^ which may indicate the number of carbon atoms in 
the ions forming the various peaks. 

The C i^ and x values assigned to the rows and columns of the spectra in 
TABLES 4, 5, and 7 are correct in most cases because saturated hydrocarbons 
in nature yield ions with .v numbers that are predominantly in the x = —11 to 
+ 2 range. However, the x values and thus the C ^ shown in tables 6 and 8 
are subject to a different interpretation. Aromatic molecules in the meteoritic 
extracts have .v values ranging chiefly from .v = —25 to —12, and the columns 
in T.'VBLE 6 and 8 would be more accurately labeled by .v values that are 14 less 



562 



Annals New York Academy of Sciences 



and C # values that are one greater than the values shown. These apparent 
discrepancies in x values and C )^ values in the mass spectral data sheets of 
aromatic fractions are commonly accepted because they simplify the problem of 

Table 4 

Mass Spectrum of the w-Heptane Eluate from Silica Gel of the 1-Orgueil 6-Houe 
Extract. Saturated Hydrocarbon Fraction 

Total ionization = 58022.28 



Normalized isotope corrected peak heights (300,000) 





-11 


-10 


-9 


-8 


-7 


-6 


-5 


-4 


-3 


-2 


-1 





+ 1 


+2 




















-0 


-0 


-0 


-0 


-0 


3 


4 


-0 











-0 


-0 


-0 


-0 


-0 


-0 


-0 


-0 


-0 


-0 


4 


5 


-0 





-0 


-0 


-0 


-0 


-0 


-0 


9389 


3350 


19430 


5997 


16219 


74 


5 


6 


175 


265 


223 


221 


2025 


573 


3593 


1234 


10154 


4045 


13601 


2908 


10411 


67 


6 


7 


263 


86 


285 


229 


5338 


2511 


3087 


1175 


9135 


3219 


11029 


2118 


2317 


70 


/ 


8 


110 


112 


267 


290 


1885 


436 


2621 


1208 


5861 


1812 


6230 


1651 


1639 


47 


8 


9 


479 


183 


603 


258 


1418 


405 


1880 


854 


3944 


1173 


3360 


1079 


1338 


376 


9 


10 


440 


175 


617 


245 


872 


361 


1706 


707 


2465 


734 


1418 


813 


1192 


176 


10 


11 


354 


161 


566 


171 


687 


312 


1682 


572 


1768 


642 


921 


713 


985 


131 


11 


12 


270 


137 


433 


141 


581 


266 


1354 


462 


1409 


479 


698 


650 


867 


114 


12 


13 


208 


101 


286 


142 


544 


280 


1178 


482 


1112 


433 


555 


580 


751 


103 


13 


14 


234 


74 


263 


127 


585 


263 


1166 


373 


982 


410 


461 


511 


668 


59 


14 


15 


172 


139 


252 


265 


1099 


335 


962 


252 


626 


324 


344 


422 


585 


29 


15 


16 


120 


64 


233 


116 


574 


293 


594 


296 


515 


284 


296 


385 


519 


44 


16 


17 


96 


62 


248 


105 


429 


215 


472 


220 


429 


254 


248 


350 


532 


10 


17 


18 


106 


59 


222 


87 


344 


176 


427 


178 


394 


215 


237 


318 


478 


67 


18 


19 


192 


49 


252 


88 


382 


158 


386 


156 


360 


208 


194 


319 


459 


44 


19 


20 


108 


60 


168 


73 


262 


114 


318 


152 


303 


196 


195 


265 


436 


39 


20 


21 


180 


53 


162 


72 


292 


83 


312 


124 


312 


185 


215 


294 


477 


60 


21 


22 


173 


41 


174 


63 


262 


103 


286 


121 


265 


203 


209 


292 


385 


200 


22 


23 


118 


44 


163 


79 


273 


133 


255 


176 


219 


262 


193 


312 


294 


274 


23 


24 


78 


56 


155 


84 


229 


218 


201 


221 


164 


287 


101 


260 


177 


189 


24 


25 


85 


81 


142 


126 


170 


210 


171 


211 


108 


265 


59 


214 


132 


129 


25 


26 


65 


82 


121 


140 


180 


188 


107 


192 


72 


171 


69 


152 


85 


96 


26 


27 


49 


150 


85 


167 


114 


261 


80 


169 


36 


114 


44 


88 


57 


59 


27 


28 


29 


69 


73 


106 


59 


129 


32 


80 


33 


77 


81 


67 


45 


42 


28 


29 


24 


66 


32 


98 


37 


105 


8 


57 





73 





477 


25 


157 


29 


30 





31 


13 


38 


5 





1 





-0 


-0 


-0 


-0 


-0 


-0 


30 



Humble paraffin-naphthene type analyses 



in percentage 




Paraffins 


17.57 


Noncondensed naphthenes 


28.86 


2-Ring naphthenes 


17.16 


3-Ring naphthenes 


11.83 


4-Ring naphthenes 


11.25 


5-Ring naphthenes 


6.41 


6-Ring naphthenes 


6.91 



Total 



100.00 



programming the computer. No problem other than a simple arithmetic 
calculation enters the interpretation of these data sheets. All peaks in tables 6 
and 8 are recorded at their proper mass positions, and the masses and sizes of 
these peaks supply in conjunction with the ultraviolet spectra of the meteoritic 
aromatics all of the information subsequently discussed. 



Meinschein et al. : Evidence in Meteorites of Former Life 563 

Mass spectral data. Mass spectra of complex saturated hydrocarbon mix- 
tures suggest the structures and molecular weight distributions of certain of 
the compounds and compound types that comprise the mixtures. Spectra 

Table 5 

Mass Spectrum of the Carbon Tetrachloride Eluate from Silica Gel of the 

1-Orgueil 6-Hour Extract. Saturated, Olefinic, plus Minor Concentrations 

OF Aromatic Hydrocarbons 

Total ionization = 18452.57 



Normalized isotope corrected peak heights (300,000) 





-11 


-10 


-9 


-8 


-7 


-6 


-5 


-4 


-3 


-2 


-1 





+1 


+2 
-0 























1 


-0 


-0 


-0 


-0 


3 


4 


-0 











-0 


-0 


-0 


-0 


-0 


-0 


-0 


-0 


-0 


-0 


4 


5 


-0 





-0 


-0 


-0 


-0 


-0 


-0 


5456 


2229 


15614 


5938 


13054 


169 


5 


6 


325 


395 


244 


253 


2155 


795 


3026 


849 


7492 


3040 


11285 


2830 


9047 


66 


6 


7 


297 





271 


70 


4296 


981 


2525 


999 


6527 


2071 


7929 


2087 


2119 


66 


7 


8 


226 


169 


466 


586 


3859 


661 


2426 


899 


4490 


1235 


4827 


1999 


1766 


187 


8 


9 


1434 


535 


1203 


653 


4590 


626 


2041 


649 


2911 


871 


2267 


933 


1483 


1157 


9 


10 


1366 


505 


1670 


604 


1966 


538 


1923 


591 


1836 


600 


1250 


729 


2035 


754 


10 


11 


1150 


419 


1658 


294 


1280 


356 


1860 


350 


1206 


836 


973 


812 


1708 


592 


11 


12 


917 


307 


1132 


231 


1043 


309 


1082 


286 


1361 


477 


862 


671 


1344 


377 


12 


13 


615 


211 


705 


167 


792 


236 


1448 


628 


995 


315 


881 


578 


1056 


290 


13 


14 


916 


142 


595 


176 


789 


308 


1156 


412 


892 


370 


706 


513 


1060 


310 


14 


15 


486 


137 


534 


505 


2732 


380 


3586 


237 


655 


286 


457 


368 


690 


150 


15 


16 


366 


122 


519 


160 


478 


427 


626 


900 


475 


204 


421 


354 


558 


165 


16 


17 


313 


160 


413 


157 


672 


222 


444 


247 


443 


214 


433 


347 


701 


66 


17 


18 


522 


51 


331 


56 


285 


130 


320 


90 


311 


206 


223 


384 


611 


140 


18 


19 


279 


208 


334 


130 


349 


177 


306 


201 


367 


172 


258 


325 


439 


154 


19 


20 


244 


163 


260 


143 


297 


249 


61 


136 


287 


235 


276 


235 


422 


154 


20 


21 


307 


64 


230 


52 


219 


136 


232 


98 


306 


179 


252 


238 


500 


130 


21 


22 


242 


44 


187 


141 


165 


177 


222 


80 


276 


200 


213 


163 


402 


85 


22 


23 


205 


36 


140 


54 


154 


82 


276 


99 


252 


120 


182 


186 


362 


73 


23 


24 


239 


57 


133 


167 


188 


35 


205 


84 


240 


202 


207 


142 


241 


54 


24 


25 


100 


93 


61 


70 


69 


167 


58 


231 


138 


158 


155 


302 


98 


84 


25 


26 


79 


32 





79 


53 


57 


56 


56 


154 


44 


153 


126 


178 


50 


26 


27 


85 


79 


80 





114 


74 


161 


39 


72 


35 


126 


68 


163 


55 


27 


28 


67 


35 


109 


23 


79 


98 


106 


21 


128 


82 


92 


74 


96 


25 


28 


29 


95 


26 


94 


124 


111 


227 


109 


515 


97 


358 


114 


279 


75 


150 


29 


30 


21 


92 





121 


51 


100 


87 


105 


100 


117 


46 


101 





6 


30 



Humble parafRn-naphthene type analysis in percentage 

Paraffins 16.78 

Noncondensed naphthenes 22.52 

2-Ring naphthenes 9.86 

3-Ring naphthenes 15.87 

4-Ring naphthenes 17.00 

5-Ring naphthenes 7. 10 

6-Ring naphthenes 10.87 



Total 



100.00 



shown in tables 4, 5, and 7 indicate the relative numbers of ions of various 
masses that were formed when gaseous, predominantly saturated hydrocarbons 
were bombarded by 70-volt electrons. The ions measured consisted chiefly 
of fragment and "parent" ions. Fragment ions are made by the rupture of 
carbon-to-carbon and/or carbon-to-hydrogen bonds of hydrocarbon molecules. 



564 



Annals New York Academy of Sciences 



"Parent" ion.s are formed when a single electron is removed from a molecule. 
Only the positively charged ions with masses greater than 66 are recorded in 
TABLES 4 through 8. 

A saturated hydrocarbon may yield a variety of ions. Any compound can 
form a greater number of small than of large fragments, and large molecules 
can produce more fragments than can small molecules. It is for these reasons 
that the values (peak heights) in tables 4, 5, and 7 generally decrease from the 



Table 6 

Mass Spectrum of the Benzene Eluate from Silica Gel of the 1-Orgueil 

6-HouR Extract. Aromatic Hydrocarbon Fraction 



Total ionization = 3748.80 



Ionizing potential = 12 volts 











Normalized 


isotope 


corrected peak heights (300,000) 












-11 


-10 


-9 


-8 


-7 


-6 


-5 


-4 


-3 


-2 


-1 





+1 
-0 


+2 















3 


-0 


-0 


-0 


-0 


3 


4 


-0 











-0 


-0 


-0 


-0 


-0 


-0 


-0 


-0 


-0 


-0 


4 


5 


-0 





-0 


-0 


-0 


-0 


-0 


-0 


56 


1493 


1254 











5 


6 











3i 








628 


1375 


1565 


11. SO 


1017 











6 


7 











86 





329 


672 


1204 


2602 


2412 


503 











7 


8 

















11 


136 


364 


504 


2171 


982 


121 








8 


9 














391 








538 


563 


558 


1598 





452 





9 


10 











1105 


815 


200 


589 


150 


196 





620 





463 





10 


11 





6 





379 


90 


123 


120 





300 


3141 


551 


447 


477 


315 


11 


12 











137 


358 


8 


52 





1180 


12303 


890 


1918 


1150 


526 


12 


13 





215 


185 





62 








38880 


706 


9265 


379 


871 


83 


515 


13 


14 



















815 


2653 


36 


2021 


461 


442 


221 


336 


14 


15 





51 


134 


1074 


917 


130 


1062 


438 





295 


/ 


211 


341 





15 


16 








182 


428 





308 


243 


969 


121 

















16 


17 





1128 





416 





308 


560 


1546 





141 


271 


83 








17 


18 





244 





129 





446 


614 


4647 











2460 


284 


593 


18 


19 


89 


506 





970 


16 


673 





494 











599 


96 


664 


19 


20 





256 





378 











1074 





133 





313 


157 


895 


20 


21 





167 





226 





702 





320 





686 





555 


18 


1799 


21 


22 





496 





954 





65 





639 


953 


1487 





954 


707 


4257 


22 


23 





585 





403 





1376 





947 


1498 


8527 





1308 


341 


5767 


23 


24 





239 





1275 





2294 


29 


3413 


101 


14557 


167 


2635 


187 


6071 


24 


25 





533 





4600 





2176 





695 V 


44 


6348 





3832 





5896 


25 


26 





758 





2588 





1820 


98 


5077 


22 


4726 





3537 





6088 


26 


27 





3057 





1957 





1185 





2664 





1676 





3262 





1772 


27 


28 





1077 





525 





710 





722 





722 





1628 





1788 


28 


29 





847 





1036 





1069 





49 





-0 


-0 


-0 


-0 


-0 


29 



Humhie aromatics type analysis in i)ercentage 



Alk}l l)cnzenes 
Najjhthalenes 




5.11 
16.56 


Acena])iithenes 




17.75 


Acenaphthyienes 
Phenanthrenes 




11.10 
20.95 


Diacenaphthylenes 

Pyrenes 

Chrysenes 




4.01 
1.43 
0.55 


Acejjyrenes 

Benzpyrene and other organic 


compounds 


1.09 
21.45 



Total 



100.00 



Meinschein et al. : Evidence in Meteorites of Former Life 565 

top toward the bottom of each x column. However, there are exceptions. 
Certain ions have peak heights which exceed or approach in size peaks imme- 
diately above them on the data sheets. These anomalously large peaks provide 
structural and distributional information about some of the compounds in 
mixtures of saturated hydrocarbons. 

Table 7 

Mass Spectrum of w Heptane Eluate from Silica Gel of the 2-Murray 20-Hour 
Extract. Saturated Hydrocarbon Fraction 

Total ionization = 127759.59 











Normalized isotope corrected peak heights (300,000' 












-n 


-10 


-9 


-8 


-7 


-6 


-5 


-4 


-3 


-2 


-1 





+1 


+2 






















-0 


-0 


-0 


-0 


-0 


3 


4 


-0 











-0 


-0 


-0 


-0 


-0 


-0 


-0 


-0 


-0 


-0 


4 


5 


-0 





-0 


-0 


-0 


-0 


-0 


-0 


2678 


1227 


8838 


3342 


12892 


186 


5 


6 


110 


105 


52 


46 


189 


96 


719 


763 


5669 


3153 


9558 


2430 


9621 


151 


6 


7 


101 


48 


38 


20 


475 


177 


1155 


984 


6975 


3298 


10467 


1765 


3703 


87 


7 


8 


73 


38 


53 


70 


581 


233 


1361 


950 


5738 


2329 


7673 


1671 


2742 


81 


8 


9 


98 


51 


164 


99 


624 


295 


1645 


911 


4587 


1738 


4935 


1409 


2134 


85 


9 


10 


127 


58 


256 


108 


616 


326 


1941 


963 


3490 


1243 


2556 


1104 


1702 


79 


10 


11 


139 


67 


296 


114 


595 


326 


2120 


826 


2654 


939 


1700 


913 


1385 


77 


11 


12 


147 


71 


281 


108 


615 


345 


1803 


710 


2252 


773 


1302 


835 


1175 


68 


12 


13 


131 


60 


274 


122 


670 


342 


1622 


659 


1849 


664 


1049 


793 


1108 


64 


13 


14 


135 


49 


282 


118 


761 


348 


1657 


613 


1578 


629 


815 


697 


932 


48 


14 


15 


134 


66 


283 


159 


773 


325 


1138 


483 


1135 


511 


633 


613 


793 


45 


15 


16 


146 


65 


329 


147 


845 


400 


990 


396 


899 


468 


523 


550 


707 


49 


16 


17 


142 


64 


324 


154 


698 


340 


845 


360 


787 


439 


432 


549 


706 


45 


17 


18 


155 


64 


346 


146 


624 


295 


765 


337 


731 


394 


367 


510 


661 


38 


18 


19 


179 


88 


360 


162 


651 


261 


725 


304 


646 


366 


343 


499 


648 


59 


19 


20 


187 


76 


347 


128 


565 


233 


611 


283 


582 


345 


363 


450 


600 


53 


20 


21 


189 


78 


360 


142 


589 


227 


563 


280 


511 


359 


318 


449 


581 


169 


21 


22 


196 


111 


345 


159 


543 


269 


522 


312 


455 


409 


285 


476 


518 


260 


22 


23 


207 


131 


335 


197 


486 


339 


478 


378 


414 


438 


250 


471 


444 


317 


23 


24 


192 


147 


311 


224 


425 


381 


426 


389 


332 


437 


209 


424 


365 


247 


24 


25 


183 


132 


275 


226 


364 


327 


358 


250 


288 


380 


164 


343 


294 


205 


25 


26 


156 


141 


260 


212 


346 


274 


306 


299 


222 


303 


137 


292 


230 


160 


26 


27 


147 


129 


236 


226 


290 


318 


243 


257 


174 


250 


109 


235 


177 


140 


27 


28 


135 


116 


204 


175 


250 


251 


191 


191 


132 


184 


86 


175 


138 


106 


28 


29 


110 


98 


176 


213 


181 


250 


156 


155 


105 


152 


57 


138 


112 


109 


29 


30 


90 


91 


122 


175 


137 


182 


114 


123 


76 


117 


43 


122 


83 


73 


30 


31 


74 


66 


95 


116 


88 


117 


81 


85 


52 


84 


24 


71 


60 


64 


31 


32 


54 


57 


77 


81 


79 


89 


62 


78 


34 


63 


17 


55 


44 


37 


32 


33 


47 


51 


56 


65 


54 


60 


37 


48 


22 


52 


19 


36 


32 


33 


ii 


34 


26 


42 


36 


47 


36 


46 


24 


41 


13 


30 


11 


34 


17 


26 


34 


35 


17 


34 


21 


32 


21 


32 


16 


22 


7 


21 





24 


4 


15 


35 


36 


8 


19 


17 


13 


17 


16 


9 


19 





2 





-0 


-0 


-0 


36 



Humble paraffin-naphthene type analysis in percentage 

Paraffins 16.49 

Noncondensed naphthenes 17.28 

2-Ring naphthenes 18.06 

3-Ring naphthenes 13.87 

4-Ring naphthenes 13.18 

5-Ring naphthenes 10. 12 

6-Ring naphthenes 10.99 



Total 



100.00 



566 



Annals New York Academy of Sciences 



Table 8 

Mass Spectrum of Benzene Eluate from Silica Gel of the 2-Murray 20-Hour Extract. 

Aromatic Hydrocarbon Fraction 



Total ionization = 38025.84 



Ionizing potential = 12 volts 











Normalized isotope correc 


ted peak heights (300,000) 












-11 


-10 


-9 


-8 


-7 


-6 


-5 


-4 


-3 


-2 


-1 





+1 
-0 


+2 




















-0 


-0 


-0 


-0 


3 


4 


-0 











-0 


-0 


-0 


-0 


-0 


-0 


-0 


-0 


-0 


-0 


4 


5 


-0 





-0 


-0 


-0 


-0 


-0 


-0 


121 


114 


272 


334 


433 


57 


5 


6 


149 


64 











36 


47 


9 


249 


286 


417 


290 


416 


53 


6 


7 


85 


4 








93 


137 


78 


122 


356 


274 


540 


259 


304 


57 


7 


8 


21 








130 


315 


172 


112 


221 


424 


288 


512 


264 


237 


59 


8 


9 


75 


129 


344 


252 


489 


292 


176 


244 


419 


236 


435 


179 


219 


82 


9 


10 


175 


235 


402 


241 


447 


267 


270 


200 


373 


228 


298 


141 


245 


235 


10 


11 


148 


307 


453 


214 


368 


224 


343 


188 


332 


276 


511 


340 


289 


280 


11 


12 


274 


280 


452 


201 


296 


214 


216 


161 


326 


257 


1180 


277 


350 


276 


12 


13 


246 


257 


389 


228 


289 


203 


239 


439 


363 


364 


814 


244 


314 


222 


13 


14 


306 


213 


384 


162 


312 


283 


444 


325 


428 


342 


749 


237 


286 


191 


14 


15 


308 


273 


379 


20919 


891 


675 


545 


1124 


447 


1317 


629 


322 


326 


335 


15 


16 


296 


316 


881 


7558 


966 


1345 


604 


1996 


522 


637 


622 


426 


374 


2359 


16 


17 


444 


5966 


1060 


5834 


715 


1750 


524 


3305 


561 


843 


612 


748 


410 


1584 


17 


18 


476 


2587 


741 


4114 


556 


1731 


518 


1972 


489 


956 


518 


10436 


665 


2274 


18 


19 


437 


1837 


552 


4092 


510 


1718 


398 


1475 


438 


1180 


484 


2912 


409 


1534 


19 


20 


375 


1401 


384 


2298 


375 


1365 


374 


4454 


487 


1659 


460 


2195 


337 


1808 


20 


21 


362 


1319 


363 


1761 


237 


1219 


270 


1619 


413 


1449 


416 


1610 


315 


1243 


21 


22 


284 


1107 


291 


2101 


270 


1233 


281 


1357 


360 


1594 


312 


1434 


286 


1074 


22 


23 


265 


1095 


211 


1310 


204 


1013 


271 


1147 


322 


1169 


267 


1119 


233 


940 


23 


24 


193 


915 


203 


1004 


199 


830 


246 


941 


230 


934 


201 


894 


157 


734 


24 


25 


193 


791 


153 


877 


154 


708 


200 


780 


195 


794 


174 


752 


147 


635 


25 


26 


173 


638 


141 


724 


117 


649 


124 


663 


155 


695 


140 


627 


144 


258 


26 


27 


148 


838 


82 


588 


104 


497 


131 


509 


142 


544 


91 


487 


142 


419 


27 


28 


121 


522 


102 


577 


108 


422 


132 


477 


83 


443 


24 


407 


54 


375 


28 


29 


46 


379 


34 


406 


58 


365 


69 


379 


80 


343 


67 


357 


32 


344 


29 


30 


53 


322 


57 


329 


54 


329 


46 


284 


61 


290 


35 


251 


41 


219 


30 


31 


28 


241 


43 


226 


47 


217 


65 


243 


48 


224 


23 


178 


41 


183 


31 


32 


23 


220 


16 


196 


57 


143 


49 


165 


19 


167 


28 


148 


42 


152 


32 


33 


31 


155 


45 


157 





159 





121 


12 


147 





106 


4 


97 


33 


34 





134 





123 


27 


110 


5 


95 


13 


85 





99 





102 


34 


35 


3 


104 


8 


110 





108 





90 





102 





27 





89 


35 


36 





92 





94 





94 





56 





45 





36 





31 


36 


37 





69 





54 





37 





39 





71 





61 





44 


37 


38 





3 





-0 


-0 


-0 


-0 


-0 


-0 


-0 


-0 


-0 


-0 


-0 


38 



Humble aromatic type analysis in percentage 

Alky] benzenes 4.73 

Naphthalenes 3 . 65 

Acenaphthenes 7.72 

Acenaphthylenes 2.81 

Phenanthrenes 6.36 

Diacenaphthylenes 4.75 

Pyrenes 13.14 

Chrysenes 7.57 

Acepyrenes 2 . 93 

Benzpyrenes & other organic compounds 46.34 



Total 



100.00 



Meinschein ct al. : Evidence in Meteorites of Former Life 567 

Large peaks may appear at masses equal to those of molecules, fragments 
containing stable ring systems, or fragments of different structure and common 
mass. w-Parafiftns and polycycloalkanes form more molecular or "parent" ions 
than do branched chain parat^ins or monocycloalkanes. FVagments containing 
the stable, partially hydrogenated phenanthrene ring system frequently give 
large peaks. Isoprenoids usually have a number of methyl branches or substit- 
uents, and several different fragments of the same mass can be formed when 
an isoprenoid loses a methyl group. Common mass ions of isoprenoids, also, 
yield large peaks in the mass spectra of naturally occurring saturated hydro- 
carbons. 

"Parent," stable ring, or common mass ions can be identitied in most cases. 
For convenience, the whole number atomic masses of C = 12 and H = 1 are 
used in calculations of ion mass numbers, and fragment ions generally have odd 
mass numbers. Therefore, stable ring and common mass ions are found 
predominantly in odd numbered .v columns. All "parent" ions of hydrocarbons 
have even mass numbers, and these ions appear in even numbered .v columns. 
Common mass ions normally appear a row above and one column to the left of 
"parent" ions; whereas most stable ring fragment ions have masses 50 or more 
units less than the masses of the molecular ions. Thus, stable ring ions appear 
chietly in odd .v columns several rows above the "parent" ion region. 

Some stable ring ions, however, are formed by breaking 2 bonds. These 
ions have even mass numbers. An example of an even mass stable ring ion is 
the -v = —6 and C^ = 16, mass 218, ion in the cholestane'*^ mass spectrum. 
The even mass numbers of some fragment ions lead to ambiguity in the identi- 
fication of "parent" ions. This ambiguity can be removed in many cases by 
additional fractionations or by information gathered from the mass spectra 
of reference compounds. Studies of thousands of mass spectra of saturated 
hydrocarbon mixtures from plant and animal lipids, sedimental extracts, and 
crude oils have shown that certain fragment and "parent" ions commonly 
yield large peaks at particular masses. Some of these large peaks may be 
absent in the mass spectra of highly paraffinic fractions, but further separations 
usually yield cycloalkyl concentrates in which these large peaks appear as they 
do in most biological and sedimental hydrocarbons. 

The peaks that are normally large in the spectra of naturally occurring 
saturated hydrocarbon mixtures have been mentioned in previous publica- 
tions,^^"^^ and many of these peaks will be indicated, again, in the subsequent 
interpretation of the meteoritic hydrocarbon spectra. But, it is noteworthy 
in the spectra of cholestane^^ that the .v = — 7, C ^ = 26 (common mass ion) 
and 17 (stable ring ion); x = —6, C ;i^ = 27 ("parent" ion) and 16 (stable 
ring ion) ; and .r = — 5, C ^ = 11 (stable ring ion) peaks are large. These same 
mass peaks have been observed to be large in either total or refined saturated 
hydrocarbon fractions which have been isolated from extracts of terrestrial 
sediments. Peaks at x = —6, C# = 28, 29, and/or 30, which may be 
''parent" ions of compounds structurally related to parent sterol hydrocarbons 
other than cholestane, are also prominent in most naturally occurring alkane 
fractions. 

Mass spectra of the meteoritic hydrocarbons presented in tables 6 and 8 



568 Annals New York Academy of Sciences 

were obtained at an ionization potential of 12 volts. At this low potential 
aromatic hydrocarbons yield chiefly "parent" ions. This technique cannot 
be used in the analyses of saturated hydrocarbons because the energies required 
to break bonds and remove electrons are approximately equal in alkanes. 

Nearly all of the large peaks in table 6 are produced by molecular ions, but 
apparently the wide range of electron energies in the ion source of the General 
Electric instrument caused appreciable fragmentation. In table 8 the data on 
many large fragments as well as "parent" ions are presented. Ultraviolet 
spectra provide a valuable assistance in the interpretation of the mass spectra 
of aromatic hydrocarbons. Many of the aromatic hydrocarbons in naturally 
occurring aromatic mixtures can be identified by the combined use of mass and 
ultraviolet spectroscopy. 

Blanks and the Holbrook meteorite. Because limited amounts of carbonaceous 
chondrites are available, minimal sample sizes were used in these investigations. 
To ensure that laboratory contaminants did not significantly affect the results of 
the analyses obtained on the minimal sized samples, an elaborate system of 
blanks was used. In addition, a high temperature meteorite, the Holbrook, 
which should not have contained significant amounts of indigenous organic 
matter served as an indicator of the type of contaminants a meteorite might 
acquire during its fall to earth, contact with earth, and storage in a museum. 
None of the blanks contained organic matter that could be detected by in- 
frared, ultraviolet, or mass spectrometric analyses; and the Holbrook extracts 
showed very small infrared and ultraviolet absorptions as shown in figures 1 
and 5. These controls indicate: (1) that laboratory contaminants did not 
measurably alter the meteorite analyses; (2) that the amount of organic matter 
acquired by meteorites (Holbrook fell in 1912) may be negligible. 

Rinses and extracts. The process of first rinsing and of then extracting the 
meteorites was used to detect contaminants. It was assumed that surface and 
near surface contaminants could be rinsed from the surfaces of the stones. 
Analysis of the rinse fractions (all of which were relatively small) and the 
extracts did not show any marked changes in concentrations of hydrocarbons 
between the exterior and interior portions of the meteorites. These analyses 
suggest that the surfaces of the meteorites had not been contacted or con- 
taminated by significant quantities of extractable organic matter during 
storage. 

Authenticity of meteorite fragments. A complementary publication^- lists 
the reliable sources of the fragments studied in these investigations. This 
complements^ also reviews and presents data that indicate that meteorites 
are of extraterrestrial origin and that the samples used in this study are authen- 
tic meteorites. 

Records of Life on Earth 

Terrestrial organisms or their remnants represent the only established 
references for detection of biological materials. Variations in the appearances, 
behaviors, and compositions of organisms make it apparent that qualitative, 
rather than precise, quantitative measurements or observations provide the 
best means of recognizing previously unseen or unanalyzed forms or remnants 
of life. Presently the extensive data on the fossil remains and organic matter 



Meinschein et al. : Evidence in Meteorites of Former Life 569 

in the sediments on earth are the most acceptable standards for the identifica- 
tion of former life in meteorites. 

Numerous soils and marine sediments have been analyzed by methods 
analogous to those used in this investigation of the Murray and Orgueil carbona- 
ceous chondrites. Additional analyses have been run on saturated hydro- 
carbons from sediments, plants, and animals. A review of these investigations 
of terrestrial samples will serve as a basis for evaluating the analyses of the 
meteoritic hydrocarbons. 

Smith^" found that geologically young hydrocarbons (C* ages 9000 to 14000 
years) isolated from recent sediments have optical activities, infrared spectra, 
elemental and type compositions, and chromatographic properties equivalent 
to hydrocarbons in ancient crude oils. Oakwood^' plotted optical activities 
versus distillation temperatures for hydrocarbons from kelp (a seaweed) and 
crude oil, and he observed that these similar plots peaked in the same tempera- 
ture region.*-' Saturated hydrocarbons in mixtures of plant and animal lipids, 
recent and ancient sediments, and crude oils seem to have equivalent chromato- 
graphic properties and infrared spectra, and similar mass spectrometric cracking 
patterns.^'* C^^ ages^"'*'* and //-paraffin distributions^^ •^**-''^'^^-*^-"^ provide the 
only reported means of distinguishing between the Cu and larger saturated 
hydrocarbons in recent sediments and those in crude oils. 

Wax-esters, closely re.sembling beeswax, have been found in a variety of 
types of soils from arid and humid areas of tropical and temperate regions of 
the world.^^ Blumer^*^ has identified in soils the same aromatic hydrocarbons 
that have been identified in marine sediments.^^ Hunf*^"*^ and Brenneman^" 
have reported similarities between hydrocarbons dispersed in ancient sediments 
and concentrated in crude oils. Bray^' has observed that the aromatic frac- 
tions of all crude oils absorb near 12.35 and lvS.45 m in the infrared.*^ He has 
referred to these absorptions as "oil bands." 

2,6,10,14-Tetramethylpentadecane, pristane, is a norisoprenoid hydro- 
carbon constituent of fish oils.^-'^'' Pristane forms 0.2 and 0.5 per cent, respec- 
tively, of the two crude oils in which it was measured. ^^ 2,6,10,14-Tetra- 
methylhexadecane, phytane, is a diterpenoid or isoprenoid which is, also, a 
common component of fish and crude oils.^^ The concentrations of phytane 
in 10 Iranian oils is to be reported. ^^ It is of interest that the first Cu, or 
larger, branched paraffins, pristane and phytane, isolated from crude oils are 
of an isoprenoid type.'^^ 

When the possible complexity of petroleum is considered, the relatively 
high concentrations of pristane and the measurable quantities of phytane in 
crude oils is noteworthy. There are in excess of 100,000 possible isomers of 
Ci9 and 366,319 possible isomers of Con paraffins.^'' Because crude oils contain 
hydrocarbons composed of more than 50 carbon atoms, the number of paraffins, 
cycloalkanes, and aromatic compounds that might form petroleum is astronomi- 
cally large and of the order of 10'** different hydrocarbons. Because of the 
diversity of compound types and the large range of carbon atom contents of 
the hydrocarbons in petroleum, the abundances of pristane and phytane in 
crude oils suggest a highly selective synthesis of these compounds. The com- 
mon presence of the precisely structured pristane and phytane in fish and 
crude oils may be more than fortuitous. Possibly certain of the stable saturated 



570 Annals New York Academy of Sciences 

hydrocarbons from prehistoric Hfe have been preserved for geological periods 
of time in nature, and these compounds may be used to define and study the 
existence of ancient organisms. 

It has been suggested that saturated isoprenoid type hydrocarbons synthe- 
sized either by living things*-'^* or from sterol and isoprenol remnants of 
organisms^'* •^^•^^•"■^'^ are major sources of naturally occurring alkane hydro- 
carbons. Disagreement on the origin of terrestrial alkanes centers about the 
issue of whether living things*""^^'^^ or chemical reactions acting on plant and 
animal remains'*^ ■^''•^''•^'^''^"'®^ make most of the saturated hydrocarbons in 
nature. Either of these sources would yield biosynthetically controlled prod- 
ucts which could serve as reliable biological indicators. 

Similarities and differences in the benzene and analogous extracts of terrestrial 
sediments may be generally summarized and partially explained. An average 
sediment contains between 20 and 80 parts per million of Cm and larger hydro- 
carbons.^' ■■*" •'*^ ■** These hydrocarbons usually comprise between 10 and 30 
per cent of most extracts, and the hydrocarbons can be separated chromato- 
graphically from the organic oxygen, nitrogen, and sulfur containing molecules 
which make up 70 to 90 per cent of the extract.^' ■^"•^^ Hydrocarbons are 
eluted primarily in the w-heptane, carbon tetrachloride, and benzene eluates; 
whereas the organic nonhydrocarbons appear chiefly in the methanol eluates 
from silica gel chromatographic cokmns. 

The extractable organic nonhydrocarbon fractions from sediments normally 
show hydroxyl or amino (2.9 to 3.0 fx), carbon-hydrogen (3.3 to 3.5 m), carbonyl 
(5.7 to 5.9 fx) and broad absorptions in the 7 to 15 /jl regions of the infrared. 
Saturated and aromatic hydrocarbons from sedimental extracts show the 
usual carbon-hydrogen absorptions in the 3.3 to 3.5 n and 7.2 to 7.8 m regions. 
In addition, the saturated hydrocarbons in most cases absorb at the carbon 
chain frequency near 13.9 n which is indicative of //-paraffins, and aromatics 
generally absorb at the "oil band" frequencies of 12.4 and 13.4 fx. 

Nonlinear polyring aromatics are dominant in sedimental hydrocarbon 
mixtures. Phenanthrenes, chrysenes, pyrenes, and perylenes appear with and 
without alkyl and cycloalkyl substituents in many soils and marine sediments. 
Anthracenes, naphthacenes, and larger linear polyring aromatics have not been 
identified in extracts of soils or marine sediments. Carruthers^'' has isolated 
some alkylanthracenes from crude oils; but in petroleum also, phenanthrenes 
are much more abundant than anthracenes.^^ 

Sedimental extracts can be divided into two broad classes: (1) soil or aerobic, 
and (2) marine sediment or anaerobic. Overlaps do exist. Anaerobes and 
aerobes both live in soils and marine sediments, but aerobes appear dominant 
in most soils. Soil extracts normally contain more wax-esters and less free 
sulfur and aromatic hydrocarbons than do extracts of marine sediments.^' •''^ 
Although the same molecular structures appear to be present^^'^^'*^ in sedi- 
mental hydrocarbons, the ratio of saturated to aromatic hydrocarbons is 
usually greater in soils than in marine sediments.'*' •^'*'^"''*^ 

Apparently, the compositions of sedimental extracts can be grossly explained 
on the basis of the stabilities of plant and animal constituents in different 
natural environments. Chemically and/or biochemically active carbohy- 
drates, proteins, fats, oils, and porphyrins (hemin, etc.) comprise all but a 



Meinschein et al. : Evidence in Meteorites of Former Life 571 

small fraction of the substances in most living cells. Yet, only traces of these 
substances are found in some sediments. i^'®*"*^* Stable saturated hydrocarbons, 
extremely minor constituents of the lipid fractions of most organisms*^'^^'^^'^^- 
^^''^* are present in measurable concentrations in essentially all sediments. 
Likewise, wax-esters, which form thin protective coatings of plants and in- 
sects,^^'''* are apparently stable in aerobic environments and are found com- 
monly in soils.^^ 

Differences in the compositions of the extractable fractions of soils and 
marine sediments may be traceable to the anaerobic activity in sea bottoms. 
Organic acids and alcohols combine to form wax-esters. Anaerobes utilize 
these acids and alcohols and sulfate ions.^^ Conversions of olefinic steroid and 
isoprenoid acids and alcohols into stable aromatic molecules result in large 
energy releases. Anaerobes need energy. Most common foods are almost 
completely consumed by aerobes in surface sediments. To survive, anaerobes 
must frequently use unusual energy sources and reduce sulfates to hydrogen 
sulfide which is oxidized to free sulfur.''* Marine sediments may contain more 
free sulfur and aromatic hydrocarbons and less wax-esters than soils,^^ merely, 
because marine environments are normally more anaerobic^* than nonmarine 
environments. Thus, analyses of sedimental extracts may serve as environ- 
mental as well as biological indicators. 

Meteoritic Extracts 

Because minimal sample sizes were used in this investigation, most meteorite 
extracts were too small to provide reliable data by all of the analytical methods 
used. Only the 2-Orgueil extract was of sufficient size to permit accurate 
chromatographic analysis and to supply ;/-heptane, carbon tetrachloride, and 
benzene fractions which absorbed significantly in the 2 to 15 micron regions 
(table 3, FIGURE 2). However, because the ultraviolet and mass spectro- 
metric data (figures 3 and 4 and tables 4, 5, 6, 7, and 8) indicate that the 
Murray and Orgueil extracts are related much as are terrestrial sediment 
extracts, analyses of the 2-Orgueil sample may be approximately representative 
of other extracts of carbonaceous chondrites. 

The 2-Orgueil chromatographic data in table 3 fall in the terrestrial sedi- 
ment range.^i ■'*'''** Infrared spectra of the total meteorite extracts in figure 1 
and of the chromatographic fractions of the 2-Orgueil in figure 2 indicate 
that all of the major absorption bands may be traceable to hydroxyl or amino 
(2.9 to 3.1 m), carbon to hydrogen {i.i to 3.6 and 7.1 to 7.9 /x), carbonyl (5.6 to 
6.0 and 8.0 to 9.0 pi), aromatic or olefinic (10 to 14.5 m), and carbon-to-carbon 
chain (13.8 to 13.9 /x) groups. The carbonyl absorptions in the Murray and 
Orgueil extracts suggest a complex mixture of carbonyl compounds and the 
mass spectra (tables 6 and 8) show that these samples do not contain apprecia- 
ble concentrations of either fatty acids or beeswax-like esters (absence of 
large peaks at odd carbon numbers in x = — 10 column). Ultraviolet (figures 
3 and 4) and mass (tables 6 and 8) spectra show that the Murray and Orgueil 
extracts contain significant quantities of aromatic hydrocarbons. The Murray 
extracts contained small and the Orgueil extracts contained copious amounts of 
free sulfur. The preceding cursory examination of chromatographic and 
spectrometric data indicate that the Murray and Orgueil extracts resemble 



572 Annals New York Academy of Sciences 

terrestrial marine sediment extracts. Like most marine sediments and unlike 
most soils, the Murray and Orgueil meteorites have benzene extractable frac- 
tions which contain complex mixtures of carbonyl compounds (which are 
not wax-esters) and significant concentrations of aromatic hydrocarbons and 
free sulfur. 

Further consideration of the spectrometric data provides evidence of addi- 
tional similarities and some dissimilarities between meteoritic and terrestrial 
sedimental extracts. In figure 2, the 12.3 and 13.4 m absorption bands of the 
benzene eluate of 2-()rgueil are similar to the "oil bands''^' found in the in- 
frared spectra of the aromatic fractions of all crude oils,'*^'^' but the ultraviolet 
(figure 5) and mass spectra (tables 6 and 8) show that the meteorite aro- 
matic fractions are unusually simple. Aromatic mixtures in crude oils and 
ancient sediments^^'"'-^^'^^ greatly exceed in complexity these meteoritic frac- 
tions. Apparently, certain recent sediments are the only terrestrial samples^^' 
39,45,46 containing naturally formed aromatic mixtures which even approach 
in simplicity the aromatic hydrocarbons from these meteorites. 

Structural types of the major aromatic species in the Orgueil and Murray 
fractions can be deduced from the ultraviolet (figures 3 and 4) and mass 
(tables 6 and 8) data. The ultraviolet spectra indicate the possible aromatic 
nuclei, and the mass data permits the elimination of some possible nuclei 
which do not yield large parent ions in the mass spectra. Based upon the 
ultraviolet and mass data the principal aromatic nuclei are in order of decreas- 
ing abundance: (1) Phenanthrenes, pyrenes, and chrysenes in the Orgueil extract. 
Most abundant aromatic hydrocarbon is phenanthrene. See 178 mass peak 
in X = —4 column at C^ =13 in table 6. (2) Pyrenes, chrysenes, benz(j)- 
fluoranthenes (indicated but not completely identified) and phenanthrenes in 
the Murray extract. Most abundant aromatic hydrocarbon is pyrene. See 
202 mass peak in x = —8 column at C j^ = 15 in table 8. 

Large "parent" ions appear in the 22 to 29 carbon number range of the even 
.r columns in table 6. These ions are made from complex aromatic molecules 
many of which differ in carbon and hydrogen content from aromatics reported 
in the literature. It has been proposed^'* '^^ that naturally occurring aromatics 
may be products of transformations of isoprenoids and steroids, and this 
proposal has recently been supported by the identification of 21 aromatic 
compounds in petroleum. Mair and Martinez-Pico'^'^ note that "most of the 
('21 aromatic') compounds are related to steroids. The results . . . give strong 
support to the theory that steroids and other natural products related to 
phenanthrene are petroleum precursors." Conversions of olefinic steroids 
and terpenes to aromatics necessitate the loss of some alkyl, usually methyl 
substituents, from the highly substituted isoprenoid ring systems. Conse- 
(juently, aromatics formed from terpenes and steroids would contain a lower 
number of carbon atoms than their precursors. The high concentrations of 
largely unreported C2.S through C28 phenanthrenes, chrysenes, and pyrenes 
indicated by the large peaks in table 6 suggest that C27 through C^o steroids 
and triterpenoids may have been a source of the aromatics in the Orgueil 
meteorite. This suggestion is amplified by the common prevalence of the 
same types of nonlinear polyring aromatics in meteoritic and sedimental 



Meinschein et al. : Evidence in Meteorites of Former Life 573 

hydrocarbon mixtures, as is further indicated by the "oil band" absorptions of 
the benzene eluate in figure 2. 

C23 through C28 aromatics are less abundant in the Murray (table 8) 
than in the Orgueil. The lower abundance may indicate that the Murray 
was subjected to a high temperature, more severe environment than the 
Orgueil and the polyalkyl substituted aromatics may have been partly de- 
graded in the Murray. Olivine, a high temperature mineral, is a constituent 
of the Murray meteorite. The simplicity of the aromatic fractions in these 
meteorites may be explained by assuming that more restricted varieties of 
organisms existed in the meteorites than are normally found in recent terrestrial 
sediments. 

/i-Paraffin^'*'^'''^^'*^'*^ and polycycloalkane distributions have been cited as 
evidence of the biologic origin of some of these compounds.-^ •^^•^^■^^ Most 
sediments and organisms contain greater abundances of: (1) odd- than even- 
carbon number //-paraffins in the C21 to C35 range. (2) C24, C27, C28, C29 
and/or C30 than of other C17 and larger tetra-, penta-, and higher polycyclo- 
alkanes. Although alterations, which change some organic molecular struc- 
tures and distributions, decrease the features characterizing biologically 
derived hydrocarbons, these features apparently persist even in the hydro- 
carbons from ancient sediments.^'* ■^^•'*^'^'* 

Many of the features noted previously for terrestrial hydrocarbons appear 
in the mass spectra of the meteoritic hydrocarbons. In the x = -\-2 columns, 
the 23 and 29 carbon number peaks in tables 4 and 7 and the C25, C27, and 
C29 peaks in table 5 are larger than the peaks immediately above and below 
them in the x = -\-2 column. Peaks in the .v = -1-2 are produced by ions 
that have masses equal to paraffins or heptacycloalkanes. Branched paraffins 
do not produce "parent ions" to a measurable degree in the mass spec- 
trometer,^^ '^* and most heptacycloalkanes would contain more than 25 carbon 
atoms in their ring nuclei. Therefore, the above designated peaks probably 
contain w-paraffin "parent peaks," and the "peakings" at odd carbon numbers 
in the C23 to C29 range in these x = -\-2 columns of tables 4, 5, and 7 are similar 
to "peakings" observed in the mass spectra of saturated hydrocarbons from 
various ancient terrestrial sediments.^^ 

Other "peakings" and "inflections" (anomalously small differences in sizes 
between successive peaks in an x column) in tables 4, 5, and 7 are note- 
worthy. These are: C20 through C30 peaks in the odd numbered x columns of 
tables 4, 5, and 7 are approximately as large as the even numbered x peaks 
in this carbon number range. These large odd x peaks and certain of the 
"peakings" in the odd x columns are indicative of poly alkyl-substituted or 
branched chain alkanes such as isoprenoids. Saturated hydrocarbons, made 
by hydrogenating products of the abiotic Fischer-Tropsch synthesis, do not 
yield as large odd .v peaks as do meteoritic and terrestrial alkanes. A com- 
parison of abiotic and meteoritic hydrocarbons will be presented in a subsequent 
publication. 

Because of the chromatographic properties of the fractions and the odd 
mass numbers of the ions the large peaks at x = — 7, C^ = 15 in tables 4 
and 5 and at .v = ~5, C^ = 13 and 15 in table 5 are produced apparently 



574 Annals New York Academy of Sciences 

by nonbasic cyclic nitrogen compounds. These compounds are slightly more 
polar than alkanes and are more concentrated in the carbon tetrachloride 
eluate (table 5) than in the »-heptane eluate (table 4). In table 4, the 
peaks at .v = —6 and — 7, C^ = 16 are larger than those of homologous ions 
at C^ 13, this suggests that the nitrogen ions may have obscured the "peak- 
ings" at X = —6 and — 7, Ci^ = 16 which are generally observed in the mass 
spectra of the terrestrial alkanes. "Peakings" at x = +1, C^ = 21 in 
tables 4 and 5 have an odd mass number and also are suggestive of nitrogen 
compounds. "Peakings" in the 19 to 25 carbon number ranges of the x = 0, 
— 2, and —4 columns which appear in tables 4, 5, or 7 are uncommon in 
sedimental hydrocarbon spectra but are present in the mass spectrum of the 
saturated hydrocarbons from oysters.'^^ Neither crude oil nor sedimental 
organic contaminants are probable sources of the mono-, di-, and tricyclo- 
alkanes which form the ions producing the latter peakings. Nevertheless, 
differences in the carbon numbers at which the "peakings" maxima occur in 
the various columns of tables 4, 5, and 7 as well as the alternate high or low 
values of odd and even C ^ "parent ions" in table 5 may be more suggestive 
of a biological product than of an abiotically formed mixture that is thermo- 
dynamically at equilibrium. 

Contamination 

Carbonaceous chondrites are friable, seemingly porous stones. Olivine, a 
mineral that forms at temperatures above 400° C. is present in the Murray 
but apparently not in the Orgueil stone. Associated minerals in the Orgueil 
suggest that it may have formed in an environment resembling an organic rich 
saline environment on Earth,'- and the compositions of the extractable car- 
bonaceous fractions of the Murray and Orgueil, also, are indicative of marine 
type sedimentary deposits. Although the compositions and the intimate 
associations of the mineral and carbonaceous materials in carbonaceous 
chondrites are not incongruous with a marine ecology, meteorites are likely to 
be contaminated by terrestrial substances. It is important to consider the 
most probable contaminants and the effect that they may have upon the 
composition of the carbon constituents of meteorites. 

All meteorites accepted as carbonaceous chondrites were observed during 
their fall to Earth. Many of the carbonaceous chondrite fragments that have 
been collected are partially coated with a heat altered layer. Charred crusts 
were apparently formed on the lead surfaces of the meteorites when these 
areas were heated to incandescence on entry into Earth's atmosphere. 

In transit to Earth carbonaceous chondrites break-up. Boato's- measure- 
ments show that meteoritic waters released at temperatures above 180° C. 
apparently have not been exchanged with terrestrial waters. Carbonaceous 
chondrites give off sizeable quantities of water at temperatures in excess of 
180° C.,* and additional evidence has been presented'- that these meteorites 
did retain substances in space which normally boil below 180° C. The volatile 
constituents of carbonaceous chondrites suggest that they have restricted the 
egress of gases to the vacuum of space. Perhaps, the interiors of these frag- 
ments are less accessible than their porous structures may indicate, but re- 
gardless of the permeabilities of carbonaceous chondrites, their fall was over 



Meinschein et al. : Evidence in Meteorites of Former Life 575 

quickl}^ and, in the thin molten surface layers of the falling meteorites, tem- 
peratures far in excess of 180° C. were reached. Even from these thin melts, 
sizeable volumes of gases may have been expelled. During their plummet to 
Earth, carbonaceous chondrites probably lost more volatile carbonaceous 
substances then they received from the atmosphere. 

When the meteorites struck Earth, their hot surfaces may have distilled or 
decomposed organic matter, and cool portions of the chondrites may have 
condensed and collected the vapors. But, the burned crusts of recovered 
fragments of carbonaceous chondrites cover only a fraction of the stones. 
These crusts are very thin, and the fragments are friable. It is doubtful that 
the heat or impact energies of these stones could have vaporized more than a 
trace of terrestrial organic matter. If carbonaceous chondrites are con- 
taminated appreciably, they probably acquired most of the contaminants 
after the fragments were collected. 

Analyses of the Holbrook chondrite show that this stone was not greatly 
contaminated either during its fall or almost 50 years of storage on Earth. 
Nevertheless, the carbonaceous matter in the Murray and Orgueil meteorites 
may have been defiled. Carbonaceous chondrites are more porous than 
ordinary chondrites, and the Murray and Orgueil contain higher concentrations 
of carbon than the Holbrook. Organic materials may be strongly adsorbed 
on carbonaceous substances. Thus, the Murray and Orgueil fragments were 
probably more susceptible to contamination than the Holbrook. Notwith- 
standing, the high concentrations of benzene extractable materials in the 
Murray and Orgueil cannot be easily explained by natural contaminants. 
Why in 10 years should the Murray, or in 100 years should the Orgueil stones 
accumulate much more extractable carbonaceous substances than an average 
soil collects in thousands of years? It seems likely that most of the carbon 
compounds in the Murray and Orgueil fragments were either indigenously 
formed in the parent body or carelessly added by man. 

Meteorites are handled, and some are marked for identification. Oily 
hands, paints, wax pencils, polishes on display cases, plasticizers in plastic 
storage cases, microorganisms, pyrolysis products of fossil fuels in urban 
atmospheres, lacquer coatings, and other carbonaceous substances may have 
contacted and contaminated the Murray and Orgueil fragments. Contamina- 
tion has been considered a major problem throughout this investigation, and 
appreciable attention has been paid to this problem. 

Fragments of the Orgueil meteorite were obtained from two museums and 
the Murray fragments came from another collection. Contaminants from 
these various locations should have been quite different, but none of the 
variations in the compositions of the meteoritic extracts suggested significant 
organic contamination. All of the fragments were carefully inspected and no 
evidence of markings or coatings were observed. Microscopical examina- 
tions'^"^ show that recent terrestrial type organisms are present in the Murray^'' 
and Orgueil in numbers that are two or more orders of magnitude less than in 
the average terrestrial sediment. Microorganisms usually contain from 1 to 3 
parts per thousand by weight of hydrocarbons. These concentrations are only 
slightly greater than those of the hydrocarbons in the Orgueil (table 2). 
Terrestrial organisms which have existed in the meteorites, seem to have been 



576 Annals New York Academy of Sciences 

too small in number to have supplied more than a trace of the meteoritic 
hydrocarbons. 

Analytical data provide additional evidence against significant contamina- 
tion of the Orgueil and Murray fragments. Benzpyrenes are common pyrolysis 
products. The air in urban areas contains from 1.5 to 25.5 parts per trillion 
by weight of benzpyrenes.^^ Pyrolysis products of wood include methyl 
alcohol, ketones, organic acids, Cg and smaller alkanes, as well as olefinic 
hydrocarbons. Colored markings usually contain pigments that absorb 
sharply in the visual range. Wax pencils are frequently composed of wax- 
esters or petroleum waxes. Drying oils in paints and lacquers are mixtures of 
olefinic compounds which have carbonyl functional groups. Crude oil dis- 
tillates and polishing oils have limited boiling point ranges and contain chiefly 
C20 and smaller compounds. The aromatic fractions in petroleum are more 
complex than the aromatics in recent sediments or the meteorites.^* ■^^•''^•^^ 

Analyses of the Murray and Orgueil extracts show that they contain: 
(1) negligible concentrations of olefins (and in the Orgueil extract of benz- 
pyrenes); (2) no substances which absorb sharply in the visual region; (3) 
alkane and aromatic hydrocarbons which are distributed as they are in ter- 
restrial marine sediments; (4) hydrocarbons and benzene extractable nonhy- 
drocarbons in the same relative abundances that they are found in sedimental 
extracts. Because the Murray contains about 5 times and the Orgueil more 
than 50 times the amount of benzene extractable materials that is found in an 
average sediment, it is unlikely that these extracts could have been obtained 
from terrestrial sediments which are the only previously reported sources of 
extracts of these compositions. The low recent organism counts'^ '^^ make it 
improbable that terrestrial organisms were a source of the extracts. Qualita- 
tive and quantitative considerations support the view that the Murray and 
Orgueil carbonaceous extracts were predominantly indigenous. 

Nonbiological Sources of Hydrocarbons 

Anders^' suggests that hydrocarbons resembling those in terrestrial organ- 
isms may have been made abiotically in the solar nebula and incorporated 
later in the bodies of the solar system. When life evolved on Earth, he believes 
that these hydrocarbons favored the evolution and survival of organisms which 
could utilize and synthesize hydrocarbons of the types which are now present 
in most solar bodies.^^ Innumerable other speculative sources of hydro- 
carbons may be proposed. Many of these proposals are neither clearly 
supported nor directly denied by experimental data. 

Although the extensive literature on organic reactions define what many 
reactants will form under various conditions, our imaginations may specify 
reactants and conditions that are either untried or unobtainable on Earth. 
Nevertheless, organic chemistry is, in part, a record of the means that have 
been devised by intensive study and extensive research to synthesize biotic 
type products. This record clearly attests that it is extremely difficult by the 
use of abiotic reactions to duplicate most of the individual biological com- 
pounds that have carbon numbers in the range covered by the compounds in 
the benzene extracts of carbonaceous chondrites. After failing to synthesize 
a pristane reference, Bendoraitis et al.,^* isolated this hydrocarbon, which is a 



Meinschein et al. : Evidence in Meteorites of Former Life 577 

minor constituent of fish oils. To avoid the problems of total synthesis, Dean 
and Whitehead-^^ used phytol, a biological product, and exchanged a single 
hydroxyl group for a hydrogen atom to make phytane. Thermodynamically, 
it is, also, difficult to deiine feasible conditions under which hydrocarbons in 
terrestrial sediments may have formed abiotically. Amossow and Wasso- 
jewitsch-"'' have observed that the ecjuilibrium temperatures calculated from 
the abundances of various hydrocarbons in crude oils range between 0° and 
225° C. for a Nebit-Dagh oil, 90° and 1075° C. for a Kara-Tschuchura oil, and 
— 70° and 225° C. for a Kostschage oil. These temperature ranges are in 
great disagreement with the temperatures that are believed to have existed in 
sedimentary basins during petroleum formation. 

Research efforts carried out over a 100-year period have failed to provide 
any evidence that abiotic, radiological, or chemical reactions were a significant 
source of hydrocarbons in terrestrial sediments. A summary of the API 
research on the origin of hydrocarbons notes the inability of radioactive 
induced reactions to make products similar to those found in nature.^* 

C()iicl!isio)is 

Aromatic hydrocarbons have been identified as common constituents of 
meteoritic and terrestrial sedimental extracts. Saturated hydrocarbons 
isolated from the Murray and Orgueil carbonaceous chondrites have infrared 
spectra, molecular weight ranges, and cracking patterns in the mass spec- 
trometer that resemble those of sedimental saturated hydrocarbons. The 
relative amounts of hydrocarbons and nonhydrocarbons, the infrared spectra 
of the nonhydrocarbons, and the free sulfur contents of the benzene extracts 
of the Orgueil and terrestrial marine sediments are similar. Except for the 
relative simplicity of the aromatic fraction from the Orgueil fragment, analyses 
of both the Orgueil and Murray extracts fall within the range of compositional 
variations observed in terrestrial sediment extracts of plant and animal 
hydrocarbons. 

Although further research may provide an alternative explanation for the 
amounts and overall compositions of the benzene extracts of the Murray and 
Orgueil carbonaceous chondrites, many similarities of these extracts to the 
extracts of terrestrial marine sediments have been demonstrated. Lacking 
another experimentally established explanation, we propose that the amounts 
and compositions of the benzene extracts of the Murray and Orgueil are evi- 
dence for biological activity in the parent body of these meteorites. Because 
of the apparent stabilities of certain hydrocarbons in natural environments, 
these compounds may provide a means of tracing the evolution of life in 
primordial times. 

Acknowledgments 

The authors thank Brian Mason, R. F. Folinsbee, Lincoln LaPlaz, and E. P. 
Henderson for providing the meteorite samples. T. C. Menzel, G. G. Wanless, 
and J. J. Waters of Esso Research and Engineering Company ably assisted in 
the analyses of some meteorite extracts. The authors thank the various 
individuals who critically read the manuscript. 



578 Annals New York Academy of Sciences 

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59. Colombo, U. & G. Svioni. 1961. Geochim. et Cosmochim. Acta. 25: 24. 

60. Erdman, J. G. 1961. Geochim. et Cosmochim. Acta. 22: 16. 

61. Breger, I. A. 1959. Preprints General Petroleum Symposium. : 79. Fordham Univ. 

Chemistry Department. New York. 

62. Carruthers, W. 1956. J. Chem. Soc. 1956: 603. 

63. Smith, H. M., H. N. Dunning, H. T. Rall & J. S. Ball. 1959. Proc. Am. Petroleum 

Inst. HI. Refining. 39: 443. 

64. Abelson, p. H. 1959. Geochemistry of Organic Sustances. John Wiley and Sons. 

New York. 

65. Degens, E. T. & M. Bajor. 1960. Gluckauf. 24: 1525. 

66. Valletyne, J. R. 1957. J. Fisheries Research Board Can. 14: i3. 

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Geochim. et Cosmochim. Acta. 19: 272. 

68. Bajor, M. 1960. Braunkohle. 10: 472. 

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United States. Vols. XXV and XXVI. : 1. 

70. Waldron, J. D., D. S. Gowers, A. C. Chibnall & S. H. Piper. 1961. Biochem. J. 78: 

435. 

71. Carruthers, W. & R. A. W. Johnstone. 1959. Nature. 184: 1131. 

72. Wanless, G. G., W. H. King Jr. & J. J. Ritter. 1955. Biochem. J. 59: 684. 

73. Chibnall, A. C. & S. H. Piper. 1934. Biochem. J. 28: 2008. 

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Academic Press. New York. 

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Baltimore. (See other annual volumes, 1943-1952.) 



FURTHER OBSERVATIONS ON THE PROPERTIES OF THE 
"ORGANIZED ELEMENTS" IN CARBONACEOUS 

CHONDRITES 

George Claus 

New York University, Medical Center, Neii< York 16, N.Y. 

Bartholomew Nagy 
Department oj Chemistry, Fordham University, New York 58, N.Y. 

Dominic L. Europa 

Department of Pathology, BeUeviie Hospital, New York 16, N.Y. 

Independent studies, conducted at various laboratories, indicate that the 
"organized elements" do not seem to be terrestrial contaminants. This 
evaluation is based mainly upon a consideration of fine morphological criteria. 
New experiments with biological stains revealed that the organic microstruc- 
tures in carbonaceous meteorites may selectively take stains in the presence 
of mineral matter. 

Claus and Nagy (1961, 1962) and Nagy el al. (1962), described organic 
microstructures that were found embedded in some of the indigenous minerals 
of certain carbonaceous meteorites. These findings have been confirmed, or 
partially confirmed by independent investigators. Reimer (1961), Staplin 
(1962), Palik (1962, 1963), Cholnoky (1962)," and Skuja (1962) examined the 
same meteorite sample as did Claus and Nagy (1961, 1962). Briggs and Kitto 
(1962) described what they thought to be indigenous, stainable microstructures 
in the Mokoia meteorite. However, the last two authors were unable to reach 
a conclusion regarding the origin of these particles. Ross (1962) examined 
another sample of the Orgueil meteorite, from the collection of the British 
Museum, and found microstructures which he believed were of biological origin 
and which were most likely indigenous to the meteorite. Recently, Engels 
(1962) isolated HF-resistant pellicles from another sample of the Orgueil me- 
teorite. Timofeev (1962) found fossilized and indigenous microflora in the 
Mighei carbonaceous chondrite. The microscopical preparations of Claus 
and Nagy have been examined by approximately 100 microbiologists including 
Erdtman, Bourrelly, Papp, Deflandre, Palmer, Durham, Dienes, and Gregory. 
Tentative but divergent identifications were offered by some of these inves- 
tigators. 

Fitch and Anders (1963) argued that the organized elements were silicate 
mineral grains, opaque mineral particles, hydrocarbon globules, coacervates, 
Fox (1961) spheroids, sulfur droplets, pollens, and starch grains or spores, or 
other unknown terrestrial contaminants. Deflandre (1962) stated that the 
organized elements are unspecified terrestrial contaminants or artifacts (except 
those which are embedded in minerals). Urey (1962a) reviewed the available 
information and suggested that the organized elements may indicate, but they 
cannot yet be regarded as conclusive proof for the existence of extraterrestrial 
life. 

Other experimental information which may suggest the presence of extra- 

580 



J 



Claus et al.: "Organized Elements" in Chondrites 581 

terrestrial biological processes includes the finding of what seems to be biochemi- 
cal compounds in carbonaceous meteorites. Nagy et al. (1961a), and Mein- 
schein et al. (1963), reported isolation of complex, saturated, and aromatic 
hydrocarbons, respectively. Anders (1962) and Krejci-Graf (1962) criticized 
these findings and the interpretations. However, a rebuttal has been offered 
(Nagy et al., 1962). Calvin (1961) and Briggs (1961) reported the finding of 
compounds which might be cytosine or purines, respectively. It should be 
noted that bituminous organic matter was isolated from the Orgueil meteorite 
by Cloez (1864), only a few weeks after the meteorite fell. This fact suggests 
that a sizeable portion of the meteoritic organic matter is likely to be of extra- 
terrestrial origin. 

Mineralogical and petrographical studies have shown that the parent body 
(ies) of carbonaceous chondrites was capable of supporting a form of life. It 
seems that liquid water was present and that this low-to-moderate temperature, 
aqueous environment was slightly alkaline and somewhat reducing. Extrap- 
olation of the parent body environment from the known mineral assemblage 
in terms of phase equilibria data in />H-redox systems has been published by 
Nagy et al. (19616), and in more detail (1963). Similar conclusions, arrived 
at by independent studies, have been advanced by DuFresne and Anders (1962). 
Petrographical observations by Nagy and Claus (1963) showed that the meteor- 
ite parent body had been subjected at one time to mechanical stresses that 
produced fractures which were later filled with magnesium sulfate. The 
textural patterns of the Orgueil and Ivuna carbonaceous chondrites resemble 
certain terrestrial rocks, such as pyroclastic rocks, deposited in water from 
fragmental volcanic debris. They also resemble silicate rocks altered by 
hydrothermal solutions. These petrographical studies have also shown that 
the interior of the meteorites does not contain evidence for high temperature 
effects acquired during the fall through the Earth's atmosphere. Conse- 
quently, organic microstructures and unorganized biochemical type compounds 
could have arrived in the meteorites without destruction by heat. The study 
of petrographical thin sections also suggests that the average pore size of the 
Orgueil and Ivuna meteorites is too small (<1 n) to permit the entrance of 
most airborne terrestrial contaminants. 

The micropaleontological examinations, the biochemical analyses, and the 
mineralogical and petrographical measurements strongly suggest that biologi- 
cal activity was active at one time on the meteorite parent body. A full 
evaluation of the origin of the organized elements must involve a consideration 
of fine morphological structures, the applicability of biological stainings and 
other microchemical methods. 

Two suggestions had been advanced to explain the origin of the organized 
elements, provided that they will prove to be indigenous microfossils in the 
carbonaceous chondrites. Bernal (1962) suggested that life may have evolved 
along similar lines at various places in the Universe. Urey (19626) suggested 
that the organized elements are terrestrial forms that contaminated the moon 
from Earth during early geological times. According to Urey's concept, bio- 
logical matter and water may have been transferred to the moon, which was 
at that time closer to earth, by the impact of meteorites into terrestrial bodies 
of water. The carbonaceous meteorites are thought to come from the moon. 



582 Annals New York Academy of Sciences 

The Usefulness of ike Microscopical Evaluation of Morphological Criteria 

Organisms consist of highly organized organic mailer. Consequently, 
they have a characteristic and speciiic chemical composition which reveals 
itself in specific morphology. Most morphological features serve the specific 
life functions of the organisms. 

Morphological features develop through 2 basic processes: hereditary proc- 
esses transmitted through genes from parent to offspring, and environmental 
influences affecting the individual. The first process results in genotypical 
morphological features; the second leads to phenotypical morphologies. Geno- 
typical features are constant within a narrow limit (Dobshansky, 1951), 
whereas the phenotypical features are apt to show wide variations. Identifi- 
cations based upon morphology must be restricted to genotypical features 
(Cholnoky, 1960). This means, of course, that a particular species cannot be 
identified through the examination of a single individual. A series of specimens 
must be examined to define the limits of phenotypical variation. The geno- 
typical and the phenotypical morphological features are functional. However, 
genotypical morphology reflects hereditary needs (phylogenetic adaptation), 
whereas phenotypical features represent individual needs bearing on environ- 
ment (ontogenetic adaptation; Goldschmidt, 1940). 

The relationship between function and morphology is apparent among 
plants of higher and lower orders. For example, two species of the flowering 
plant genus Ambrosia, A. elatior and A. artemisiaefoUa, live in habitats which 
are exposed to different degrees of sunshine and contain different amounts of 
moisture. The latter species, A . artemisiaefoUa, lives in a semidesert environ- 
ment. Consequently, the size of the foliage is smaller than that of the former 
species, A. elatior, and scleral elements are abundant in the leaves to provide 
mechanical support during periods of severe loss of turgor. 

The Ambrosia pollen, i.e., ragweed pollen, shows a characteristic genotypical 
morphology, a solid, spinose exo-exine. Clearly, the spines represent a geno- 
typical feature because they must develop from the tapetal layer of the pollen 
sack. The pollen grains, during their ontogenesis, are not directly exposed to 
environmental influences. The spines are formed by apposition. For the 
same reason the spines of the ragweed pollen are solid rather than hollow. 
The solid intine can be penetrated by 3 pores only. The spines may facilitate 
the transportation of the pollen grains. The characteristic tricolpate structure 
is always observable upon proper focusing of the microscope (Erdtman, 
1952, 1957; Faegri and Iversen, 1950; Jonas, 1952; Hyde and Adams, 1958). 
See FIGURE 1, pollen grain of Ambrosia trijida; figure 2 the same in optical 
section; figure 3, Hystrichosphaeridium sp. from the Upper Cambrian; fig- 
ure 4, pollen grain of Dahlia pinnata; figure 5, Hystrichosphaeridium from 
the Upper Cambrian. (Figures 1, 2, and 4 were taken from Wodehouse, 
1942, and correspond to his numbers; 118, 119, and 115, respectively; figures 
3 and 5 were taken from Timofeev, 1956, and correspond to his numbers 20 
and 19, respectively.) 

There are similar looking species of unicellular, aquatic plants. For ex- 
ample, Hystrichosphaeridium Deflandre is covered with spines. These spines, 
however, serve a different function, develop through different embryological 



Claus et al.: "Organized Elements" in Chondrites 583 

processes and are constructed differently (Deflandre, 1936; Timofeev, 1956; 
Evitt, 1961a, 1962). The spines of real Hystrichospherids grow out from the 
outer layer of the cell wall through intussusception. These hollow spines help 
the organisms to float in water (Schiller, 1933-37). Both pollen and Hystri- 
chosphere spines help to protect the species. On casual observation ragweed 
pollen grains and certain Hyslricliosp/ieres may look alike (cf. figures 1 and 3, 















10 /u, 



Figures 1 to 5. 1, 2, and 4, pollen grains; 3 and 5, Hyslrichospheres. 

and 4 and 5). Very careful microscopical examination is required by experi- 
enced microscopists to distinguish the hollow Hystrichospheridmm spines from 
the solid ragweed pollen spines. This example may emphasize that detailed 
and careful observations are necessary for the identification of all microscopical 
plants and parts of plants. 

Fitch and Anders (1963) questioned the validity of using fine morphological 
criteria in the identification of microorganisms. They claimed that structural 
features less than 1 /x in size are difficult to observe and they suggested that 
the resultant identification must be subjective. Yet the science of systematic 



584 Annals New York Academy of Sciences 

microbiology and micropaleontology provides numerous examples of the 
successful use of fine morphological characteristics (involving features less 
than 1 /x in size) in the identification of protobionta. 

The blue-green algal genus Oscillatoria has 160 morphologically distinct 
taxa, 28 of which are less than 1 ^t in diameter but they are still amenable to 
morphological characterization (Hollerbach et al., 1953). As early as 1871, 
optical microscopy was sufficiently advanced to allow Pfitzer to establish two 
new genera Neidiuni and Anomoeoneis, that were formerly included in Navicnla, 
by observing morphological features less than 0.3 ju in size. The recent work 
of Hay flick (1962) established that primary, atypical pneumonia in humans 
is caused by a pleura pnetimouia-Uke organism (PPLO), less than 0.3 n in size. 
The same detailed morphological observation is also used in characterization of 
microfossils. Recently, Evitt (19616 and 1962) has shown that the group 
of Hyslrichospheres (Precambrain to Recent) consists of polyphyletic members. 
This finding was again based upon the observation of fine morphology, which 
involved the examination of the number of processes, spines, and the plate 
structures. Most biologists agree that the microscopical examination of fine 
morphological features (1 /z or less in size) is not only possible but it is a common 
practice in systematic zoology and botany. 

Modern biological microscopes, if properly used by experienced investi- 
gators, can resolve objects as small as 0.2 /x in diameter. The theoretical 
limit of resolution is 0.10 ^t. Clearly, it is quite possible to observe morpho- 
logical features in the 0.3 to 0.5 n range. Fitch and Anders argued that one 
of the organized elements embedded in mineral matter in one of the thin sec- 
tions of Nagy et al. (1962) cannot be characterized because the spines are 
approximately 0.3 ji long and the resolution of a microscope which they be- 
lieved to be equipped with a regular, high dry objective is 0.3 ju- This argu- 
ment is in error because the spines on this organized element were observed 
with a X/O oil immersion objective, with a numerical aperture of 1.15, which 
gave a lower limit of resolution of 0.22 ^i. The transparency of the embedding 
mineral (magnesium sulfate), its lack of color in the thin section and the lack of 
significant differences in refractive indices in this portion of the microscopical 
preparation prevented any serious interference from attaining the necessary 
resolution. 

Fitch and Anders (1963) proposed a set of criteria to establish that certain 
objects are microfossils. Their criteria are essentially the customary defini- 
tion of life. They suggested that to be able to prove that the organized ele- 
ments are indigenous and extraterrestrial microfossils one must show (1) that 
they have characteristic morphologies, (2) show some evidence of propagation, 
and (3) show signs of metaboHc processes. 

Their first point needs no further discussion. One may reply to their second 
requirement by noting that adjoining organized elements have been observed 
embedded in minerals. This raises the possibility of either copulation or 
division (figurp: 6c). Similar objects (but solitarily) were often found in the 
mineral matrix (figure db). A less direct indication may be derived from the 
possible presence of deoxyribonucleic acid- (DNA)-type material, which will 
be discussed in another chapter. Finally, the presence of what may be bio- 



J 



Claus et al.: "Organized Elements" in Chondrites 585 

genie compounds, such as certain hydrocarbons, cytosine and purines, should 
have already provided an answer to their last postulated requirement. 

The Applicahilily of Biological Stains to the Investigation 
of Meteorite Samples 

The use of biological stains for demonstrating cellular structures is indispen- 
sable in microscopical biology. During the preceding 2 decades a better under- 
standing of the chemical nature of dyes and the chemical reactions involved, 
has made it possible to use the color developed as a specific indicator for the 
presence of a certain compound. With the development of color indices and 
standardization of the marketed dyes (Conn, 1953) many of the unpredictable 
results or uncertainties originating from the varied staining procedures have 
been eliminated. At present there are still some major gaps in the knowledge 
of the reaction mechanisms of several dyes and their exact specificity in several 
cases is not known; however, one is able to use them with a certain degree of 
confidence if one fulfills the following three criteria. (1) Use a whole array 
of structurally different dyes on the same substrate. (2) Use adequate amounts 
of controls. (3) Rely more on stains which chemically react with the sub- 
strate (or before application are in colorless form) than on those which are 
merely adsorptive in nature. 

SUdes were prepared essentially in 2 ways. (1) The sample was either 
crushed or dispersed on the slide in double distilled water, was covered, and 
the aqueous staining solution was, during the period of observation, constantly 
sucked through the preparation. (2) The meteorite and soil samples were 
crushed between 2 slides the surfaces of which were coated with fresh egg 
albumin. The other materials like pollen or starch grains, etc. were dusted 
over the albumin covered slides with a fine brush. The slides prepared in this 
way were then subjected to the staining procedures, washed with tridistilled 
water, dehydrated, mounted in balsam and coverslipped. In cases of the 
eosin and hemato.xylin-eosin staining instead of eggwhite, collodium was used 
for adhesion. The staining with ninhydrin was performed in small test tubes, 
and the material was transferred to glycerin and examined in it. During the 
staining procedures special care was taken not to contaminate or crosscontami- 
nate the preparations, therefore the stains were freshly made up with tri- 
distilled water and the different specimens were stained in separate sets of 
copUn jars. 

As can be seen from table 1, 19 widely differing biological stains were used, 
of which only 1, i.e., safranin, is considered to be a true adsorptive stain (the 
nature of dyeing with the eosin stains, like Azure II, and Dienes PPLO blue 
stain is still debated). Of the 19 stains only 1, Sudan IV, gave negative re- 
sults with the organized elements. All of the other stains were found to be 
positive. One has to emphasize, however, that only a portion of the organized 
elements stained with the different stains and the proportion of the stained to 
the unstained particles varied from stain to stain. One has also to admit that 
several of the dyes used stained not only the organized elements but also a 
portion of the mineral material. However, one could easily distinguish by 



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Claus el al.: "Organized Elements" in Chondrites 589 

the intensity or shade of the color the organized elements from the minerals. 
The staining of the soil samples gave similar results. One may make the fol- 
lowing comments regarding the stains. 

The use of chlor-zinc-iodide in the taxonomy of the OsciUaloriaceae (Cyano- 
phyta) is mandatory. It is one of the best cellulose reagents and the system- 
atics of the sheathed genera of the above mentioned family is based upon the 
positivity or negativity of this reaction, i.e., whether the sheath turns blue or 
does not stain at all. The 2 other color reactions ascribed to this stain are, 
however, somewhat less specitic. It is accepted that chitinous substances 
turn brown, whereas pectic compounds show a yellowish brown coloration. 
The presence of proteinaceous moiety disturbs this reaction, as proteins will 
also assume a yellowish brown color. There is, however, some difference be- 
tween the color given by pectins and that produced by proteins. To differ- 
entiate between these 2 colors requires either color charts or materials for com- 
parison. In the case of the organized elements, chlor-zinc-iodide invariably 
gave a yellowish brown color in the walls, characteristic for pectic substances. 
Although it would be premature to conclude on the basis of this color reaction 
that the organized elements possess walls made up by pectins, one is able to 
rule out the possibility that they are recent pollen or spore contaminants 
because then they should either become blue (cellulose) or dark brown 
(chitin) in their walls. Types 1, 2, 3, and 4 have been seen reacting with this 
stain. 

The blue stain of Dienes was developed for the dyeing of pleuropneumonia- 
Uke organisms (PPLO) as a substitute for the more complicated Giemsa staining 
(Dienes, 1939). It is an alcoholic solution of methylene blue and Azure II. 
Viable PPLO or bacteria will stain deep blue with the stain but will later be- 
come faint due to decolorization of the methylene blue, whereas dying or dead 
bacteria stain pink or do not stain at all. Fungus hyphae or spores usually 
stain very dark blue; cellulose elements, however, stain lilac. This stain was 
selected not so much to study its effect on the organized elements as to enable 
us to recognize terrestrial contaminants. However, in the samples under 
study, no viable bacteria were seen; fungus hyphae were absent and only a 
single gonotokont was observed. The organized elements turned either bluish 
or lilac by the stain but the majority did not stain at all. Types 1, 2, and 3 
have been seen taking up the stain. The mineral matrix in the Orgueil or 
Ivuna meteorites and in the soil samples turned light bluish. 

One of the most surprising results was obtained with the Feulgen stain. 
This staining technique was developed for the selective staining of nuclei and 
chromosomes. It involves the use of the Schiff reagent (leuko-basic-fuchsin) 
and its reaction with the aldehydes obtained by the acid hydrolysis (HCl) of 
deoxyribonucleic acids. The staining is considered to be extremely sensitive 
and very specitic. Since its first description, in 1924, by Feulgen, there has 
been published voluminous literature dealing with the questions of sensitivity 
and of specificity of the technique (Pearse, 1960). Several modifications were 
proposed and, at present, the Feulgen staining has become one of the most 
reliable and one of the most widely used techniques for the demonstration of 
DNA in cells, and for the study of nuclear movements during cell division. 
There are other substances besides DNA that are, however, known to give a 



590 Annals New York Academy of Sciences 

positive Feulgen reaction. These are the plasmalogens (acetal phosphatides), 
(Schubert, personal communication, N.Y.U.). These latter compounds, how- 
ever, are not very likely to occur in either the meteorites or in the soils as they 
are quite unstable and are known to be present only in the central nervous 
system and in the muscles of animals. Another, as yet unidentified substance 
yielding a positive Feulgen reaction is the binding material among the cells of 
some of the species of the green algal genus Oedogonium (Woes-Tschermak, 
personal communication, Vienna, Austria). This material according to our 
observations seems to possess a faint pinkish color even in the unstained, 
living algae, if viewed in the microscope in dimmed oblique light and it may be 
a compound similar to that described by Palik (1928) in Hydrodictyon (Chloro- 
phyta) and named as erythropectin because of its pink color. In the case of the 
Oedogonia the acid hydrolysis seems only to strengthen the pink color already 
present and in reality we may not be dealing with a positive Feulgen reaction. 
This question, however, deserves further investigation. 

By using the Feulgen technique on our samples it was found that a con- 
siderable number of the organized elements of type 1 and type 2, stained 
homogeneously pink with this stain, whereas the mineral material remained 
unstained or took a faint greenish color. It was most instructive to see the 
results of the staining on the different controls; organisms present in the soil 
or dust samples did not stain, except for their nuclei, which turned red. The 
mineral and possible organic material in these samples, similarly to the material 
in the Orgueil and Ivuna meteorites, either remained unstained or took a pale 
greenish color. The same color developed also in the walls of the pollen grains, 
whereas their nuclei stained pink (see figure 7, a io e). Pollen grains as a 
whole never turned pink after the use of this staining method. In 1937, Shuita 
investigated the nucleic divisions of pollen grains and found the Feulgen stain- 
ing (by virtue of its complete noninterference with any other cellular element 
of the grains except their chromatins), was the most suitable stain for such 
type of studies. 

Another interesting observation was made regarding the starches. Because 
we could not find any literature data dealing with the effect of Feulgen stain- 
ing on starch grains it seems to be worthwhile to deal with this problem at 
some length. Starch (amylum) occurs in the phylogeny of plants compara- 
tively early. It is present in the green algae and it remains characteristic for 
the main line of plant evolution up to the Monocotyledoneae. Starch is always 
an intracellular product and in most of the green algae and in the leaves of 
higher plants it is formed in the chloroplasts. In the green algae usually a 
separate organell, the pyrenoid, is in the center of the grains, other grains may, 
however, directly be deposited in the stroma of the plastids or even in the cyto- 
plasm (Czurda, 1928). Fritsch writes (1949, p. 67): "The grains . . . appear 
to grow by apposition of layers on all sides, and their polyhedral form (giving 
the entire group the shape of a shell) is a result of the fact that free deposition 
can take place on the external surface." In cases of grains directly develop- 
ing in the stroma or in the few cases in which they arise in the cytoplasm their 
shape becomes more or less spherical and the layering takes a concentric shape. 
Similarly, in any other starch producing plant the grains consist of an inner. 




Figure 6a Petrographic thin section of the Figure 6b Petrographic thin section of the 
Orgucil meteorite showing two, adjoining Orgueil meteorite showing a singular object, 
organized elements. Magnification 590 x . similar to those in Fig. 6 a. Magnification 590 x . 




Figure 7a Type 1 organized element after Figure 7b A type 2 organized element after 
Feulgen staining. Magnification 1000 x. Feulgen staining. Magnification llQx. 




Figure 7c Cassava starch grain after Feulgen 
staining. Magnification 590 x , 



Figure 7 d Cassava starch grain after periodic 
acid Schiff / PAS / staining. Note that Feulgen 
staining, when administered according to stan- 
dardized techniques, does not stain starch 
whereas PAS does. Magnification 590 x . 




Figure 7e Elm pollen grain after Feulgen 
staining. Note that only the nuclei are stained. 
Magnification 1000 x. 



Figure 8 a A type 2 organized clement in 
optical cross section from a powdered pre- 
paration of the Orgueil meteorite. Magnifi- 
cation 1000 X. 




{■im- 



^<^V ^' 



Figure Sb The same organized element pho- 
tographed by focusing on its top. Note that 
the protrusions have dark centers indicating 
hollow tubes. Magnification 770 x . 



Figure Sc Cross section of an organized ele- 
ment similar to that shown in Figs. 8 a and 
8 b. This object occurs in a petrographic thin 
section of the Ivuna meteorite. Magnification 
770 X . 




Figure 8d Ambrosia pollen grain after Grid- 
ley staining in optical cross section showing 
solid protrusions. Magnification 590 x . 




Figure 8e The same preparation showing the' 
characteristic tricolpate, triporate structure of 
the Ambrosia pollen grain. Magnification 
590 X. 



w 



•"•iC 




• •* 



fclKgy 



Figure 9a Organized element resembling a 
Dinoflagellate cyst focused to show the ridges 
and canals of its surface. Magnification 770 x . 




Figure 9b Same object focused on the sur- 
face appendages. Magnification 770 x . 




Figure 10a A type 2 organized element from 
the Orgueil meteorite. Magnification 590 x . 



Figure 10b A similar object from the Ivuna 
meteorite shown in UV light. Ribs appear in 
the interior of the organized element. Mag- 
nification 400 X . 




Figure 10c Petrographic thin section of the 
Ivuna meteorite seen in UV light, showing an 
organized element in a magnesium sulfate 
vein. Magnification 400 x . 



Figure 10d The same organized element in 
UV light after the blueish colored fluores- 
cense of the mineral material had been elimi- 
nated by an appropriate set of filters. Mag- 
nification 400 X . 




Figure 11a Two type 1 organized elements 
and mineral grains in a powdered sample of 
the Orgueil meteorite. Magnification 590 x . 



Figure 1 1 b An opaque, magnetic particle from 
the Orgueil meteorite. Magnification 590 x. 



$■••* 




• 4.' 

Figure 12a An organized element resembl- 
ing the features of a Thecamoeba, focused on 
the sculptured top of the object. Magnifi- 
cation 770 X . 





Figure 12b Same object focused on the spines 
of its lower surface. Note that in the hollow 
interior a bubble of / possibly / air occurs. 
Magnification 770 x . 




Figure 13a A type 5 organized element after 
Gridley staining. Focused to show the three 
tubular protrusions of the body. Magnifi- 
cation 590 x . 



Figure 13b Same object focused on two of 
the protrusions only. Magnification 590 x . 



Claus et al.\ "Organized Elements" in Chondrites 591 

variedly shaped "nucleus" or hilum and the layered starch is around it. The 
mcoposition of the "hilum" is unknown. It seems that it is proteinaceous in 
nature. The sole exception from this is represented by Rhodophyta, in which 
the so-called Floridean starch develops always in the cytoplasms, is unlayered 
and is lacking a "nucleus." This compound, however, is closer to glycogen in 
its composition as it stains brown instead of blue with iodine. The shape of 
starch grains, their mode of layering and the form of their "nuclei" is constant 
and species specific, thus, it is a true genotypical feature which gives a good 
basis to establish relationships between different plants. The pictures of the 
starch grains in the endospermia of varied Gramineae, the so-called amylo- 
grams, were successfuUy used in the ehicidation of several important micro- 
systematic problems (Soo, 1953). 

None of the 6 starches showed a positive reaction after Feulgen staining. 
Actually, the grains became so translucent that it took considerable time to 
recognize them in regular transmitted light. There were several dark bhie 
dots on the slides, however, which in phase contrast were found to be the 
"nuclei" of the grains; they were invariably surrounded with a translucent 
envelope, showing concentric rings and corresponding in size and shape to the 
grains before treatment. In the case of the Cassava starch (grains of Manihot 
utilissima) the envelope occasionally turned faint blue on exposure to the 
Feulgen stain. It seems that the starch grains are inhomogeneous in their 
chemical composition and the circular striations are caused by a very thin 
layer of a matter different from amylum. The envelope covering the grains 
must be of the same material as those that cause the circles. That we were 
dealing with true structural elements that upon the action of P'eulgen staining 
occasionally turned faint blue could be determined by investigating ruptured 
or broken grains. In these cases fringes of the envelope and of the circles 
could be observed on the edges of the ruptures. No other staining and not 
even the unstained materials gave similar results. The possibility exists that 
the starch was at least partially hydrolyzed by the HCl and that this procedure 
made conditions favorable for the detection of the envelope. The staining 
pattern of the starch "hila" and that of the envelope after the Feulgen stain 
do not allow us, as yet, to conclude anything about their nature. We may, 
however, conjecture, per analogiam, to the green algae that the starch "nuclei" 
may be proteinaceous in nature. 

It is clear from these experimental results that the claim of Fitch and Anders' 
(1963) that one of the type 2 organized elements (stained with Feulgen staining 
and showing a diffuse pink coloration) is a starch grain is unfounded. These 
authors demonstrate that the Schiff reagent is a nonspecific carbohydrate stain, 
which is even capable of reacting with several inorganic materials, including 
clay minerals. They show pictures of deep magenta colored starch grains, 
dyed with the Schiff reagent and conclude that as Feulgen staining uses the 
Schiff reagent, it is nonspecific, hence the organized element in question is most 
probably a starch grain. This conclusion is in error as shown by figures 
la-Id. Figure la shows a type 1 ; figure lb a type 2 organized element after 
Feulgen staining. In figure 7c is shown the Cassava starch with Feulgen and 
in figure Id with PAS staining. 



592 Annals New York Academy of Sciences 

Similarly, the assumption of Gregory (1962) that the organized element in 
question may either be a "Hulle" cell of Aspergillus nidiilans or the pollen 
of a Juiiiperus sp. or Taxus baccata can be rejected on the basis of the stain- 
ing alone, even without taking into account the basic morphological differences. 
It has already been mentioned that with Feulgen staining only the nuclei of the 
pollen grains stain and not the body as a whole. 

Interpreting the results of the diffuse Feulgen staining of the type 1 and 
type 2 organized elements presents a different cjuestion. Bacteria and blue- 
green algae are known to contain chromidial apparatus instead of a compact 
nucleus. It is customary to refer to the central body or centroplasm of the 
Cyanophyta as nuclear equivalents, containing diffused nucleic material. In- 
deed, Feulgen staining in such organisms will result in a diffuse pink coloration 
of the protoplasm. One may thus think that some of the organized elements 
indeed contain diffused nucleic material in their interior. The present evidence, 
however, is not sufficient to substantiate such a supposition. We can only 
state that, according to one of the most specific biological staining techniques 
developed, there is an indication that nucleic acids are present in the interiors 
of some of the type 1 and type 2 organized elements. It is, however, equally 
possible that an as yet unknown material is present in these organized elements, 
which will react with the Feulgen reagent to produce the pink color. 

The Gridley staining was primarily developed (195vS) as "A Stain for Fungi 
in Tissue Sections." It is the combination of Gomori's aldehyde-fuchsin stain 
and the Hotchkiss-McManus technique. As Gridley states (p. 303): "The 
problem of positive tissue elements with the periodic acid-Schiff reaction was 
eliminated by hydrolyzing in chromic acid instead of periodic acid." For 
counter staining Metanil yellow is used. Fungal conidia and hyphae are 
stained deep blue to purple by this method while other tissue elements, mostly 
the proteins, stain yellow with the counter stain. The results of this technique 
on our samples were interesting for 3 reasons. (1) Some of the organized ele- 
ments of type 1, type 2, and type 3, stained lilac with the stain, whereas the 
mineral matrix of the Orgueil and Ivuna meteorite in many instances took a 
dirty purple color. A similar staining pattern of the minerals was observed 
in the soil samples, in which, however, some algae stained rose and fungi be- 
came blue. It was, therefore, surprising to see that the minerals of the Murray 
meteorite did not stain with this stain. The starch grains took a vivid magenta 
color. (2) Type 5 organized elements stained orange with the Gridley stain- 
ing and the surrounding halo turned yellow from the counter stain. The 
meaning of this reaction is obscure. (3) The forms described by Staplin (1962) 
as Coelesliles sexaugulalus from the Orgueil meteorite stained yellow, probably 
from the Metanil yellow. These forms have originally a yellowish-orange 
shade, however, after the exposure to the Gridley staining the change in their 
color was striking. On the slides prepared from the meteorites not a single 
spore or fungus hypha could be detected. In the preparation from Holbrook 
1 specimen of Nitzschia acicularis (a Diatom) was seen. 

A surprising result was obtained by the application of a watery solution of 
Janus green B. Many of the type 1 and a few of type 2 organized elements 
developed a blue-stained reticulate structure in their inside. This stain is a 



Claus et al.: "Organized Elements" in Chondrites 593 

vital or supravital stain generally used for the demonstration of mitochondria. 
It is considered to be more or less specific for ribonucleic acids. The reticulum 
that developed in the organized elements was similar to that obtained after 
their treatment with 6 N HCl. The meaning of this staining pattern is ob- 
scure. 

Metanil yellow alone, in a watery solution was applied to rule out the possi- 
biUty that the observed yellow coloration of Staplin's form after Gridley stain- 
ing was due either to the hydrolysis with chromic acid or to the Schiff reagent. 
As was the case in the Gridley stained preparations, after treatment with the 
simple watery preparations, Staplin's Coelestites turned into a striking yellow. 
A few of type 1 organized elements also became yellow. With our present 
knowledge, we cannot satisfactorily interpret these results. 

Neutral red, usually used in biology for its nontoxic character as a vital or 
supravital stain, stained some of type 1, 2, and 3 organized elements a homo- 
geneous red. 

Ninhydrin, this most sensitive amino-acid or protein reagent, stained lilac 
several type 1 and 2 organized elements; however, it gave a purple coating 
to the mineral debris in the carbonaceous meteorite and soil samples. The 
ordinary stony meteorites gave negative results. 

Periodic acid-Schifif (PAS) reagent seemed to be the least adequate for 
differential staining. Being a general carbohydrate stain, it dyed magenta 
color the pollen and starch grains and algal cells. Several of the organized ele- 
ments of types 1, 2, and 3 also took this stain and developed a color similar to 
those of the algal cells or starch or pollen grains. The minerals of the Orgueil 
and Ivuna meteorites and those of the soil samples also took up the stain. 
There was a significant difference between the shades of the organized elements 
and the starch or pollen grains on one hand and that of the minerals on the 
other; the latter having a more "dirty" magenta color. Furthermore, some 
type 1 organized elements remained totally unstained among well stained 
mineral aggregates. It was interesting to see that the minerals of the Murray 
meteorite did not, or only very occasionally, stain with PAS and that the 
staining of both the Holbrook and Bruderheim meteorites was negative. The 
quality and intensity of the color of the stained organized elements resembled 
closely the color of the controls. One may speculate that some kind of chemical 
similarity may exist between the organized elements and the controls that 
contain carbohydrates. Because of the nonspecificity of this reaction, how- 
ever, it seems advisable not to attempt to reach any premature conclusion in 
this matter. 

It was previously mentioned that Sudan IV was the only stain which left 
unaffected the organized elements, although it stained vivid red the oil drop- 
lets of the terricole Diatoms. 

Toluidine blue gave a blue or pink color with some types 1, 2, and 3 organized 
elements. In several cases the minerals of the carbonaceous meteorites and 
of the soil samples also turned blue. Metachromasia, however, was not ob- 
served with the mineral particles. Samples of Orgueil, after being treated 
with boiling HF for 10 minutes, left acid resistant peUicles of the types 1, and 
2 organized elements, which after staining with toluidine blue showed signs of 



594 Annals New York Academy of Sciences 

metachromasia. It would, however, be somewhat premature to conclude from 
this staining pattern that there are acidic or basic polysaccharides in the acid 
resistant pellicles of the organized elements. 

One may draw the following conclusions from the staining experiments. 

(1) The specificity of some of these stains is not known. However, it seems 
unhkely, that 18 of the 19 stains used, gave positive results by chance. To 
evaluate the meaning of a single staining reaction often seems to be impossible. 
One cannot argue that a sample is of biogenic origin on the basis of a single 
staining. However, if a whole array of different stains are applied, which are 
widely differing in their chemical composition and in their specificity, one can 
point out biogenic material. 

(2) The use of a great variety of stains (some of them specifically developed 
for the scanning of certain microorganisms, like Dienes blue-stain for PPLO 
or the Gridley staining for fungi) facilitated the recognition and, thus, the 
elimination of earthly contaminants in the meteorite samples. As only small 
meteorite fragments or powdered material could be used for these studies, the 
question of contamination could be settled only on the basis of elimination. 
But by the use of the numerous stains and the relatively great number of con- 
trols (including soil from the impact area, and dust from the museum) one 
could recognize and exclude the common contaminants. 

(3) An examination of the soil and dust samples has shown that micro- 
organisms stained differently from the mineral constituents, i.e., the latter 
did not stain at all or took a different color. These and the starch and pollen 
controls have confirmed the specificity of the Feulgen reaction. 

(4) It has been pointed out that not all of the organized elements stained. 
A gradation in the staining was observed with almost every stain (with the 
possible exception of safranine). One reasonable explanation for this phenom- 
enon may be that different degrees of mineralization are present in the or- 
ganized elements. In terrestrial bitumens microfossils are often differen- 
tially mineralized (Andreanszky, 1954). 

Physical and Chemical Observations on the Organized Elements 

Fluorescence in ultraviolet light. When preparations of the 4 carbonaceous 
meteorites were examined with a fluorescent microscope a number of particles 
became readily visible. Most of these particles fluoresced with a greenish- 
yellow light when excited by ultraviolet radiation and when Corming 7-59 + 
Wratten 2B filters were used. A Zeiss fluorescent microscope was applied 
in these studies. Less frequently, particles fluoresced with a green, pink or 
red color upon excitation by ultraviolet light. An examination of the thin 
sections and of the crushed samples has shown that some of the mineral con- 
stituents (possibly those that were coated with bituminous matter) seem to 
have fluoresced with a bluish- white color. The fluorescence of the organized 
elements, in our opinion, can be readily distinguished from the fluorescence of 
the mineral matter. It is, of course, possible to select the right combination 
of filters to exclude the bluish-white fluorescence from the yellowish-green or 
pink fluorescent light. Several of the type 1 organized elements were found to 
fluoresce with greenish-yellow light, contrary to the argument of Fitch and 
Anders (1963). Fitch and Anders also claimed that pink colored fluorescence 



Claus et al.: "Organized Elements" in Chondrites 595 

of organized elements is not a true fluorescence. They suggested that bire- 
fringent particles may appear red when viewed with ultraviolet light in the 
fluorescent microscope. The reason for this could be that the usual filters 
transmit a portion of the red part of the spectrum. However, the particles 
which were described previously were not birefringent when examined in a 
polarizing microscope and they were photographed with an additional set of 
filters (Corming 7-59 + Wratten 2B + Zeiss 064 + 061) that permitted mainly 
blue light to enter the microscope. Consequently, the argument of Fitch and 
Anders is not valid in this case. 

The microscopical assembly used for the fluorescence studies enabled one to 
view objects in UV darkfield illumination, as weU as in regular transmitted 
light. In this way it was possible to select organized elements of distinct 
morphologies for the fluorescence studies. It is true that a few irregularly 
shaped particles also fluoresced in greenish-yellow light. These particles 
could be easily fragments of organized elements, broken during the crushing 
of the meteorite samples. On the other hand, the majority of the irregularly 
shaped particles fluoresced with a different color than the organized elements; 
they emitted bluish-white light and they were probably mineral particles (or 
particles coated with bituminous matter). 

Fluorescent microscopy is also useful to demonstrate certain morphological 
features which are not readily visible in transmitted light. Mitochondriatic 
granules become visible in the UV microscope when they emit fluorescent light 
with or without fluorochromation (Drawert and Metzner, 1956). Similarly, 
fluorescent microscopy was found to be useful to visualize certain morphological 
features that were not visible when the organized elements were examined in 
regular, transmitted fight or with phase contrast microscopy. Figure 106 
shows an organized element that fluoresced with greenish-yeUow light. 
Centripetal ribs were found to be present around the walls. This morphological 
feature is rather unusual, because only a few Diatom species are known to show 
centripetal ribs (Hustedt, 1930). This feature is not identical with the internal 
septae of Coccolilhoplwrideae, Silicoflagellatae, Foramiiiiferae, and certain Di- 
atoms such as Naviculaceae etc., the septae of which extend much farther into 
the cell. The general habit, as seen in transmitted fight, figure 10a, resembles 
a Trachelomonas, a genus of aquatic protophyta, except of the location of the 
pore. However, a Trachelomonas does not have centripetal ribs. The presence 
of the centripetal ribs are of particular interest regarding the argument of 
Fitch and Anders about pollen grains. The exo-exine of pollen grains show 
centrifugal thickenings but they never show centripetal ribs. Fames and 
MacDaniels (1947) state on p. 49 in their "Introduction to Plant Anatomy" 
that: "The external wall layers and surface projections of spores and pollen 
grains are formed in part by tapetal fluid or mother cell cytoplasm." (There- 
fore, only centrifugal thickenings can occur on walls of pollen grains.) It 
seems unlikely, that the particles shown in figure 10, a and 6, are pollen grains 
or spores. It is known that spores of fungi do not fluoresce. Hofler and 
Pecksieder write (1942, p. 117): "Angesichts der weiten Verbreitung primar 
Fluoreszenz im Gewebe der Pilzkorper beriihrte uns die Beobachtung urn so 
auffalliger, dass die Sporen der Hulpilze im UV-Lichl nicht fluoreszierten, viel- 
mehr meisl vdllig unsichtbar waren." 



596 Annals New York Academy of Sciences 

The abundance of the organized elements. The number of the organized 
elements (per milligram) in the Orgueil, Alais, Ivuna, and Tonk meteorites has 
been reported previously (Nagy et al., 1962). The numbers were arrived at 
by counting all types of organized elements or such fragments thereof, which 
appeared to be larger than 50 per cent of 1 particle. The type 1 organized 
elements of Claus and Nagy (1961) are the most abundant; they comprise 
approximately 80 to 90 per cent of all microstructures. This type has also the 
simplest morphology. Consequently, certain investigators were unable to 
distinguish this type of organized elements from mineral particles or were of 
the opinion that the morphological criteria are not sufficient to distinguish 
them from mineral particles. If one chooses to exclude the type 1 organized 
element one will arrive at a count that is substantially lower than that given 
by Claus and Nagy (1961). It should be noted, however, that some biologists, 
on critical examination, were inclined to include this type 1 particle among 
the organized elements (Papp, 1963; Cholnoky, 1963; Skuja, 1962; Palik, 
1963). Also, as Urey (1962o) pointed out, one only needs to have some bio- 
genic and indigenous microstructures in a meteorite to ascertain the existence 
of extraterrestrial life. The total count of organized elements, including type 
1 (1300 to 1700 per mg.) shows good agreement with counts of microplanktons 
in fossil marine populations (1200 per mg.), as reported by Kolbe (1952) and 
with the counts of stainable organic microstructures in the Mokoia meteorite 
(1000 to 1700 per mg.) described by Briggs and Kitto (1962). 

Solubility in acids. The effect of acids and organic solvents on the organized 
elements has been reported previously (Nagy et al., 1962). It is necessary, 
however, to comment again on this subject because Fitch and Anders (1963) 
claimed to have dissolved 97 per cent of an Orgueil meteorite sample by heating 
it for 17 hours at 60° C. in HF and for 18 hours in 6 N HCl at 25° C. The 
remaining residue was reported to be an aggregation of "finely granular, black 
to brown material virtually devoid of any structure." Milder treatments in 
concentrated HF (Urey, 1962o; Staplin, 1962; and Nagy et al., 1962) and 
6 N HCl resulted in a residue which contained several transparent and acid 
resistant pellicles. Organized elements, including type 1, retained their 
characteristic morphologies upon exposure to 6 A^ HCl at room temperature 
for varying periods of time. The type 1 organized elements were not de- 
stroyed when boiled in concentrated HF for 15 minutes. The statement 
made by Fitch and Anders ". . . since they disappear after treatment with HF, 
we believe they are most likely grains of silicate minerals although they are 
classified as organized elements by Nagy and coworkers" seems to be in error. 
In spite of the rather severe treatment they used, Fitch and Anders were still 
able to find some transparent and highly organized structures of undoubtedly 
biogenic nature. 

Problems of contamination. Several claims were made in the literature to 
the effect that the organized elements are terrestrial contaminants (Fitch and 
Anders, 1963; Deflandre, 1962; Gregory, 1962; and Pearson, 1962). Contami- 
nation is, of course, a serious problem and it cannot be fully excluded at the 
present time. However, it should be borne in mind that no trained micro- 
biologist or micropaleontologist who has actually worked with an Orgueil sam- 



Claus et al.: "Organized Elements" in Chondrites 597 

pie, for any length of time, has yet (at the time of this writing) positively 
identified the organized elements as known terrestrial species. Certain com- 
ments, that to us seem to be somewhat vague, such as Deflandre's (1962) 
statement: "Positive identifications in this case are unnecessary and super- 
fluous" cannot be taken too seriously. Deflandre, to our knowledge, has 
never examined a carbonaceous meteorite. Similarly, the criticisms of Gregory 
and Pearson cannot be accepted as strong evidence against the extraterrestrial 
nature of the organized elements because these authors made their identi- 
fications from a set of photographs and sketches that were reproduced rather 
poorly in a scientific journal. (Ciregory saw some of the microscopical prep- 
arations after he submitted his paper to the press.) Gregory and Pearson 
identified the same organized element as 2 different terrestrial contaminants. 
On one occasion a single organized element, when it was briefly shown in a 
microscope to 18 microbiologists, was "identified" as 18 different species of 
protobionta or organic artifacts. 

Fitch and Anders (1963) claim that only a few of their particles, which ac- 
cording to them are mere terrestrial contaminants, survive the combined HF, 
HCl treatment. We found that these particles, when we examined their 
preparations under the microscope, showed morphological features that were 
dissimilar to common airborne contaminants (Wodehouse, 1942, 1945; Gregory, 
1961). They appeared to us identical to some of the forms that we found in 
our preparations. As a matter of fact, we found one of these forms ourselves 
in Fitch and Anders' preparations, in their presence, during their visit to our 
laboratories. 

Fitch and Anders claim that some of these particles are ragweed pollen. 
However, accorchng to their own measurements, given in their report, some of 
those particles seem to be too small to be Ambrosia pollen. It is clear that one 
must both critically evaluate the fine morphology and make accurate measure- 
ments of size to establish a particle as a known terrestrial contaminant. Fur- 
thermore, Fitch's and Anders' contention that some of the organized elements 
in our preparation are ragweed pollen is also untenable because it is based upon 
the comparison of photographs of rather low resolution which do not permit 
the evaluation of fine morphological criteria. In figure 8g are shown some 
ragweed pollen grains, to demonstrate the solid spines of theexo-exine. Figure 
M is an optical cross-section of the same. In figure 86 is shown the type 2 
organized element (note the hollow protrusions). In figure 8c is .shown a 
similar object embedded in minerals. The identification of another organized 
element (figure lb) as either a starch grain or a recent Juniperus pollen is also 
in error because the structure of starch grains shows concentrical layering. 
Juniper pollens are much larger than the object in question, they do not have 
papillae and have rugate exo-exines (Erdtman, 1957). 

Additional sources of possible terrestrial contaminations have been exam- 
ined recently. Soil samples and outcrop samples have been collected in the 
vicinity of the villages of Orgueil and Nohic in Southern France near the lo- 
cation where the meteorite fell. It has been suggested (Bourrelly, 1962) that 
soil and rock samples from the impact area be examined to evaluate the degree 
of contamination from the local environment. Bourrelly noted that the culti- 



598 



Annals New York Academy of Sciences 



Montouban 12 Km 
iJ 



. . /' I.Perllfrot 


^ 2 Lescure 
^_Compsas 


T 




^ 


"""^-^^^C^^ River 


=^^ 




^^^^r^^ Orgueil 


J 





\ 



OI<ind2 Locotions of falls of 
the two Orgueil meteorite 
samples, received from 
the Montouban Museum 



I Locations of the 
soil somples 



1=50,000 



Figure 14. Map of the Orgueil and Campsas area. 



CROSS SECTION ALONG THE ORGUEIL METEORITE TRAJECTORY BETWEEN 
THE VILLAGES OF LAPEYRIERE AND ORGUEIL. 



Lopeyriere 



Campsas 




Ofgueil 



m mmmfi . 



GEOLOGICAL COLUMN AT ORGUEIL 



1 : recent alluvium of the Tarn. 
2;ancient alluvium of the middle 

terrace of the Tarn. 
3:ancient alluvium of the lower 

terrace of the Tarn, 
/♦ifluvlal terraces of the Garonne^ 
5:molasse (Stamplen-Ol Igocene) 

Tert lary 



soils about 30 cm thick 

alluvium (clay), about 60-90 cm thick 

o; o 

O.'o sand and pebbles of the terrace, 
about 5 ni- thick 



molasse (Stampien), clay and sand, 
at least 200 m. thick 




Figure 15. Geological cross-section in the vicinity of Orgueil, in France. 



vation of land in this part of France is basically the same today as it was at 
the time of the fall of the Orgueil meteorite. One may expect, therefore, that 
a microbial population similar to that of 1864 is present in the soil. In figure 
14 are shown the locations of the falls (on May 14, 1864) of the stones and the 
locations where soil samples were collected on March 29, 1^62. In figure 15 
a geological cross-section of the area is shown. The sedimentary strata that 
underlies the alluvium in the Orgueil-Nohic area consists of Tertiary formations 
extending to a depth of at least 600 feet. Staplin (1962) suggested that a few 
Cretaceous microfossil contaminants might have been included in the Orgueil 
meteorite from the soil in the impact area. Our studies, based upon the recent 
field work of Henri Coustau, revealed no Cretaceous outcrops near Orgueil and 



Claus et al.: "Organized Elements" in Chondrites 599 

Nohic. Staplin (1962) was undecided about the Cretaceous identity of the 
forms in cjuestion. It seems that these forms are not Cretaceous contaminants, 
after all. 

Microbiological and micropaleontological examination of the soil and rock 
outcrop samples revealed no forms that were morphologically identical to the 
organized element in the Orgueil meteorite. The species of microorganisms 
that had been identitied from the Orgueil soil samples are listed in table 2. 
These samples still contained a considerable amount of their original water 
content when they arrived at our laboratories, thus several forms could be 
studied while still alive. The soil and rock samples were treated identically 
to the meteorite samples, including the biological staining techniques. We 
concluded that the organized elements in the Orgueil meteorite are not identical 
with the organisms and microfossils that were collected on March 29, 1962, 
by Henri Coustau, in the soils and rocks of the impact area. 

Another source of contamination may be the microorganisms in the air. 
When a meteorite enters the earth's atmosphere it "breathes in" air because of 
the reduced pressure in its interior. It is conceivable that some organisms 
may be sucked in at such time (although the average pore size of the Orgueil 
meteorite is estimated to be less than 1 /x). In order to gather some informa- 
tion about this possibility, particles collected in the atmosphere have been ex- 
amined. The airborne particle samples were received through the courtesy of 
C. W. Phillips, U.S. Army Chemical Corps, Fort Detrick, Md. They were col- 
lected on precleaned microscope slides at the elevation of the collection. It is 
known (Proctor and Parker, 1942) that at the height of between 10 and 30,000 
feet mainly bacteria exist. An examination of the slides revealed no organisms 
that were morphologically identical to the organized elements in the carbon- 
aceous meteorites. There are a number of reports in the literature (Hyde and 
Adams, 1958) on airborne pollen grains and spores; and a few reports on algae in 
the air (Schlichting, 1961). It seems that the organized elements of the meteor- 
ites do not correspond to known airborne contaminants. 

Other possible sources of contaminations, such as chemicals used, including 
the water, have been evaluated previously (Nagy et al., 1962). 

Finally, 2 samples of the Orgueil meteorite were recently obtained from the 
Montauban Museum (through the courtesy of A. Cavaille). These samples 
have been in Montauban, France, which is near Orgueil, continuously since 
approximately 2 weeks after the fall of the meteorite. The samples were kept 
under glass jars; however, they were not stored in a sterile environment. 

A microscopical examination of the Montauban samples revealed identical 
organized elements (except type 5) to those from other museums. It is very 
difhcult to believe that 6 samples of the Orgueil meteorite (from the American 
Museum of Natural History in New York, the U.S. National Museum in 
Washington, D.C., from the British Museum, from the Musee d'Histoire 
Naturelle, Paris and the 2 from Montauban) would have been contaminated 
by identical microorganisms in storage. The organized element that has been 
claimed to be a ragweed pollen by Fitch and Anders, was also found to be pres- 
ent in the Montauban sample. Ragweed (Ambrosia) is a native American 
plant. It was introduced to Europe only in the early part of the twentieth 
century and it is still not a common plant there (Soo, 1953). 



Table 2 

Biological Material Found in Soil and Sedimentary Rock Samples Near the 

Village of Orgueil 



Name of species 


Soil sample A, 
surface 


Soil sample B, 
from 40 cm. depth 


Rock sample from 
quarry 


Achnanllws cf. micrccephala 


+ 


+ 




Actinomyceles 2 different spp. 


+ 


+ 




Adelomycetes spore 5 dilYerent spp. 


+ 


+ 




Amoeba 3 different spp. 


+ 






AmpJiora oval is 




+ 




A nkistrodesm us Jalcatiis 


+ 






Arcella sp. 


+ 






Aspergillus sp. 


+ 






BotryococcHS sp. 


+ 


+ 




Chlorella vulgaris 


+ 






Chloi-ococcuin h umicolum 


+ 


+ 


+ 


Chroococcus tiirgidus 


+ 






Chrysophyta cysta, 2 types 




+ 


+ 


Ciliata 4 different spp. 


+ 


+ 




Closlerium ehrenbergii 


+ 






Cocconeis spp. 2 t>'pes 




+ 


+ 


Cosmarium sp. 




+ 




Cosmarium granulatunt 


+ 






Cymhella ventricosa 


+ 






Cymhella prostrala 




+ 




Difflugia cf. pyriformis 


+ 






Dinohryon serkdaria 






+ 


Euglena deses 




+ 




Euglena sp. cysta 




+ 




Fragilaria pinnala 


+ 


+ 




Gomphonema olivaceuni 


+ 






Kephyrion sp. 






+ 


Melosira varians 




+ 




Navictda rhincliocepl/ala 


+ 






Navicula hungarica var. capitata 


+ 






Navicula pupula 




+ 




Nematoda 2 different s|)p. 


+ 


+ 




Nitzschia acicularis 


+ 


+ 




Nilzschia fruslulum 






+ 


Nitzschia sigmoidea 






+ 


Nostoc fusiforme 


+ 






Oocystis pusilla 




+ 




Oscillaloria animalis 


+ 


+ 




Oscillatoria teyebrijormis 


+ 






Oscillatoria tenuis 


+ 






Pediastrum boryanum var. gramdatum 


+ 






Peridinium sp. (fragment) 






+ 


Phormidium autumnale 


+ 


+ 




Phortnidium foveolarum 


+ 






Pinnularia viridis 


+ 






Pleurococcus naegelii 


+ 






Pollen grains of 








Abies sp. 




+ 




Achillea cf. millefoHum 


+ 






Agrostis cf. vulgaris s. alba 


+ 


+ 




Alnus sp. 






+ 


Bettda sp. 


+ 




+ 


Cirsium sp. 


+ 






Hordeum cf. vulgare 


+ 


+ 




Knautia arvensis 


+ 






Pinus sp. 


+ 




+ 


Polygonum cf. convolvulus 




+ 




Prunus s. Crategus sp. 


+ 






Quercus cf. puhescens 






+ 


Sa gill aria sagiltifolia 


+ 






Salix cf. cinerea s. caprea 


+ 






Senecio cf. vulgaris s. viscosus 


+ 


+ 




Trifolium cf. arvense 


+ 


+ 




Triticnm aestivum 


+ 


+ 




Tussilago farfara 


+ 







i 



( 



tinn 



Claus et al.: "Organized Elements" in Chondrites 

Table 2 — Continued 



601 





Soil sample A, 


Soil sample B, 


Rock sample from 


Name of species 


surface 


from 40 cm. depth 


quarry 


Scenedesmiis aciiminalus 


+ 






Scenedesmus bihtgatiis 


+ 






Scenedesmus obliquns 




+ 




Scenedesmus quadricauda var. longispina 


+ 


+ . 




Steril ni\celia 3 types 


+ 


+ 


+ 


Stephanodiscus lianlzsckii 




+ 




SlicltococcHS minor 


+ 






Strombomonas sp. 






+ 


Surirellii ovata 




+ 




SynechococcHS elongatus 


+ 






Synedra nimpens 


+ 


+ 




Synedra ulna var. oxyrliynchus 




+ 




Tetraedron muHcum 


+ 






Ulollirix syi. 


+ 






Unidentitiahle arthrospores (Noslocalesf) 


+ 


+ 




3 types 








Unidentifiable conidiospores 4 types 


+ 


+ 


+ 


Unidentifiable green algal zygotes or 


+ 


+ 


+ 


zygospores (Oedogoniaceaef Conjii- 








galesff) 5 types 








Unidentifiable moss-protonema 


+ 


+ 




Vampyiella sp. 


+ 






Windier id cf. sessilis 


+ 






Xanthidium sp. 


+ 







On occasion fungi are known to grow on mineral specimens in museums. 
In the growth process the hyphae get attached to or penetrate into the samples. 
Microscopical examination of the thin section of such samples reveals the spores 
and hyphae. No hyphae or remnants of hyphae were yet seen in carbonaceous 
meteorites. This renders unlikely the possibiUty that the organized elements 
are spore contaminants from fungi that grew on the samples in the museums. 
Such a fungal growth would be rather unusual and could occur only in the 
presence of adequate moisture. Some of the mineral components of the Orgueil 
meteorite point out that the samples were kept in dry museum storage. 

Terrestrial contaminations should have been able to enter the pores if the 
interiors of the meteorite are contaminated. Organized elements are embedded 
in minerals and in the mineral aggregates in meteorites, as was reported pre- 
viously. A petrographical study of the thin sections (Nagy and Claus, 1963) 
led to the estimation of the average pore sizes as less than 1 n in diameter. 
The size of the organized elements varies between 3 to 60 m- Although there 
may be a few wider fractures going through the samples it is thought to be 
impossible for organisms to penetrate the dense and unfractured areas of the 
mineral matrix. 

Microscopical preparations of the carbonaceous meteorites have now been 
prepared from time to time, over a period of 1 year (before, during, and after 
the pollination time of ragweed and other flowering plants). No correlation 
has yet been found between the types and numbers of the organized elements 
and the time of preparation of the slides. This suggests that the organized 
elements were not introduced into the sample when the microscopical prepara- 
tions were made. Identical organized elements, such as the particle that is 



602 Annals New York Academy of Sciences 

claimed to be ragweed pollen by Fitch and Anders, have also been found by 
different investigators at different laboratories at different times. 

In our opinion the probability of terrestrial contamination is a most serious 
problem. However, the control experiments described in this report and 
previously (Claus and Nagy, 1961; Nagy el al., 1962) strongly indicate that 
the organized elements (or most of them) are not terrestrial contaminants. Yet 
it must always be borne in mind that even unusual contaminants may become 
included easily in a sample. Microscopical preparations of an Orgueil meteor- 
ite sample provided through the courtesy of Fitch and Anders for us to study, 
contained fragments of a Compsopogou filament (a not too common species 
of Rhodophyta), individuals of Chlorella, a rare species of Ndgeliella, antennae 
of Cladocerae, sqamae of Tilia leaves, and emergentia of unknown origin. 
Although the organized elements were clearly visible, the presence of the 
aquatic contaminants suggested more of a sample of a Recent sediment than 
that of a carbonaceous meteorite. 

The Diverse Morphology of Organized El erne ills 

An examination of approximately 400 microscopical preparations of car- 
bonaceous meteorites, and related material, has as of now revealed 30 distinct 
morphological types of organized elements. Other investigators (Staplin, 
1962; Palik, 1962, 1963; Ross, 1963) found several other types. None of these 
organized elements seems to be identical to known terrestrial species, although 
they resemble them. 

Organisms can be classified into four symmetry groups. The simplest 
symmetry group is the sphere, and the most advanced one is the bilateral type. 
(Asymmetrical categories can be derived from each of the four groups.) Trig- 
onal symmetry is the least common among terrestrial organisms. Organized 
elements, however, often fall into this class. Organized elements contain 
examples of each of the symmetry categories (see figures 6, a and b; 1, a and 
b; 8, a, b, and c; 9, a and b; 10, a, b, c, and d; 11a; 12, a and b; and 13, a and b). 

Conclusions 

Consideration of the fine morphology, physical and chemical tests, staining 
with biological stains, and further evaluation of contaminations suggest that 
the Orgueil, Ivuna, Tonk, and Alais carbonaceous meteorites contain indige- 
nous, organic microstructures which seem to be of biogenic origin. Full proof of 
the indigenous and biological nature of the organized elements is still not 
available but the indications seem to be strong. 

It has been shown that fine morphological criteria are of diagnostic value. 
As a matter of fact, microbiologists and morphologists are using such criteria 
every day in a variety of problems. It has been shown, as it is known to many 
investigators, that morphological features of 0.3 ^ size can be observed and 
identified by optical microscopy. The value of morphological criteria was 
noted by Fournier (1962) when he stated at the First International Conference 
on Palynology, that a worker in biology . . . "classifies his pollen based on 
morphological features alone, a fact that has proven no detriment to his work." 

The criticisms of Fitch and Anders have been considered and found to be 
unacceptable. A critical, systematic and objective evaluation of the organized 



Claus et al.: "Organized Elements" in Chondrites 603 

elements is essential if an accurate identification* of these particles is to be 
achieved. 

A ck nowledgments 

We wish to thank the microbiologists who examined the microscopic prepara- 
tions. Special acknowledgments are made to A. Cavaille of the Montauban 
Museum for providing our new, Orgueil meteorite samples; to Henri Coustau 
of SNPA in Pau for collecting the soil and rock samples and C. W. Phillips of 
the U.S. Army Chemical Corps, Fort Detrick, Aid. for providing the airborne 
dust samples. The pollen samples were obtained from B. Siegel of the Brook- 
lyn Jewish Hospital in New York. We wish to thank Professor Harold C. 
Urey of the University of California for his encouragement and continued in- 
terest. 

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* Because of the obviously great significance of the organized elements, one must, of course, 
be unusually careful in evaluating their true identity. Our objections to the publishing of 
photographs of our yet unreported organized elements, taken of our preparations at our in- 
vitation but published without our permission (.\nders and Fitch, Science, 1962, 138, 1392) 
is based also on the fact that publishing photographs of organized elements adjacent to Recent 
microbiological objects does not seem to facilitate identification because both types of these 
particles are three dimensional objects. Furthermore, in a scientific report, critical or other- 
wise, one is obliged to quote not only criticisms but also confirmations. It is important that 
the organized elements be treated seriously and be evaluated by investigators with suthcient 
experience in observing morphological features because, after all, the organized elements 
could be the first tangible evidence for the existence of extraterrestrial life. 



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PANEL DISCUSSION 

The Identity of the "Organized Elements" 

H. C. Urey (Moderator; University of California, LaJolla, Calif.): In re- 
viewing the events that led to this symposium, the moderator recalled that 
approximately one year ago at The New York Academy of Sciences a presenta- 
tion was made by Nagy el al. regarding the finding of what might be biogenic 
hydrocarbons in the Orgueil meteorite. The moderator stated that although 
he viewed their finding with skepticism at that time, he later looked over the 
mass spectrometric data collected by the investigators and was sufficiently im- 
pressed to suggest that additional analyses be run, such as infrared and ultra- 
violet spectra. Once these suggestions had been carried out, the moderator 
viewed microscopic preparations obtained from the Orgueil and Ivuna meteor- 
ites that reminded him of biological matter. The moderator said he then posed 
the following question to himself: Suppose these were living things, how did 
they become imbedded in dolomite? In a subsequent published article, the 
moderator suggested that these "organized elements" might be earthly forms 
that had somehow been transferred from the earth to the moon in early geo- 
logical times and later had returned to earth in carbonaceous meteorites. 
Reactions among scientists to this theory varied; doubts were expressed, and 
the moderator himself was (and still is) unsure of it. The moderator believes, 
however, that the study of carbonaeceous meteroties for Ufe-like forms is not 
an unreasonable pursuit, particularly when one considers that the United 
States plans to spend some 25 billion dollars to put a man on the moon. 

The moderator acknowledged, on the other hand, that he was also impressed 
by the arguments of Fitch et al. that the "organized elements" might be merely 
terrestrial contaminations, such as ragweed pollen. He noted, however, that 
investagators Nagy, Claus, Meinschein, and Hennessy have been willing to 
show their sample preparations freely and to solicit the opinions of others. He 
noted, also, that they are enthusiastic; and while it is true that enthusiasm 
may lead to errors, it is also true that lack of enthusiasm is not an especially 
strong motivation for further work. 

J. D. Bernal {Department of Physics, Birkbeck College, University of London, 
London, England) : This discussant suggested that the problem be defined in 
terms of the question, "What is it we are looking at?"; also that the problem be 
approached in terms of all related subjects and that carbonaceous meteorites 
be considered only as related to other meteorites. He raised the question of 
whether carbonaceous meteorites represent the beginning or the end of the 
development of meteorite bodies; the origin of these objects, he pointed out, is 
an extremely important question. In his opinion, the "organized elements" 
could be contaminations, "jokes of nature," or remnants of organisms. One 
approach to the contamination problem is to determine whether anything could 
have gotten into the samples since the meteorite fell on earth; another is to 
ascertain whether the "organized elements" resemble any known biological 
forms. If they are not contaminations, the burden of proof lies with Nagy 
et al., and it is the biologists who must evaluate such proof. As for being "jokes 
of nature," the "organized elements" might, for example, be mineral concretions 

606 



Panel Discussion: Identity of "Organized Elements" 607 

that give the appearance of microfossils. Some of the Precambrian micro- 
fossils shown earUer in the sessions might also be such particles. Bernal stated 
that if one could prove that the Precambrian forms were indeed fossils, it 
would perhaps aid in the identification of the "organized elements." Finally, 
if the "organized elements" are neither contaminations nor "jokes of nature," 
one might then wonder whether anyone had "faked" them. Again the burden 
of proof would lie with those who say that the "organized elements" are truly 
indigenous fossils in the meteorites. If they are indigenous microfossils, what 
would this mean? One must consider where these objects might have origi- 
nated, whether life was brought to earth on meteorites, and where hfe might 
first have arisen, on earth or on another body. 

Among other considerations mentioned by Bernal was that the carbonaceous 
meteorites might contain between six and eight per cent of organic matter, most 
of which is definitely not terrestrial contamination. According to Meinschein 
and Nagy, the meteorite hydrocarbons are products of life; but can we really 
say this, or might they be abiotic matter from which life originated? Some 
scientists even question whether petroleum hydrocarbons are of biological 
origin. It was recalled that in an earlier paper in this symposium the syntheses 
of biochemicals from abiotic sources was described; however, no mention was 
made of lipids. Meinschein's and Nagy's evidence depends on Hpids, but 
one does not known whether lipids can be produced abiotically. 

Next, according to Bernal, there is the question of the mineral composition 
of the meteorites. Water must have been present when these minerals were 
synthesized. Were the serpentine minerals the decomposition products of 
other silicates? The discussant pointed out that one fact is known: some of the 
serpentine particles were euhedral. Mason postulated that the primitive 
meteorite aggregated from dust particles, but the carbonaceous meteorites do 
not fit into this picture. Sztrokay found veins filled with bituminous material 
in the Kaba carbonaceous meteorite, which would suggest an elaborate chemical 
history. All of this was very puzzling according to Bernal. One might ask 
why it is necessary to bother with all this when in ten years time someone will 
land on an asteroid and settle the question of extraterrestrial life; however one 
must remember that the solution to this problem depends on many people, 
specialists in their various fields, who must get together and coordinate their 
efforts since no one man can settle this problem alone. 

H. C. Urey: The moderator stated that the subject of extraterrestrial life 
was of such great importance that it might affect scientific thinking about the 
moon and, in fact, about the entire solar system. He did not, however, agree 
with Bernal regarding the origin of meteorites. Moreover, to understand 
better the "organized elements," one must also question what primitive life 
forms would be like. Would they survive as long as present life forms do? 
What would be the biochemical composition of primitive life forms? Would it 
be the same as it is today? Would the porportions of amino acids be the same 
as they are today? These, he felt, were all questions which would yet have to 
be answered. 

B. ]. Cholnoky (National Institute jar Water Research, Pretoria, Republic of 
South Africa) : Cholnoky stated that the only important consideration is whether 
there are or are not fife forms in carbonaceous meteorites. It matters not which 



608 Annals New York Academy of Sciences 

scientist is right or wrong. The problem is basically biological. To the biolo- 
gist it should be of no importance, as far as identitication goes, whether the 
"organized elements" came from meteorites, from outer space, or from some- 
where else; all that should matter is whether the "organized elements" can be 
identified as remnants of once living matter. Cholnoky emphasized that he is 
a microbiologist, who has spent 51 years studying microorganisms. He is not 
particularly interested, he stated, in fossils in meteorites, as such, but only in 
life forms in general. 

There were then two main points which he wanted to make: First, he ex- 
pressed his surprise that physicists and chemists seemed willing to offer critical 
evaluations regarding the biogenicity of organic microstructures. As a biolo- 
gist, he would never think himself competent to comment on purely chemical 
and physical problems. He suggested that physicists and chemists adopt a 
similar attitude regarding biological problems. Consecjuently, he believed 
that the suggestion put forth in an earlier paper by investigators from Florida 
i.e., that protenoid coacervates may resemble living cells in appearance, must be 
rejected. The methods of identification of these must be judged as insufficient; 
any identification of strains of coacervates must be submitted to experts. 
Although experiments with coacervates go back to the work of dejong, and were 
designed to investigate vacuole formations, dejong never said anything about 
cell walls. 

Secondly, Cholnoky commented on claims that the "organized elements" in 
carbonaceous meteorites were only grains of starch or pollen contaminations. 
He said he has seen starch grains under microscopes on innumerable occasions 
and could not identify the "organized elements" as starch grains. To argue a 
point at meetings with photographic evidence was not satisfactory, since micro- 
organisms are three dimensional and their morphology cannot be adequately 
represented in two dimensional photographs. 

Sidney W. Fox {Institute for Space Biosciences, Florida State University, 
Tallahassee, Florida) : The discussant stated that he had heard the word Florida 
mentioned, so he assumed Cholnoky's first point was in reference to his work. 
He wondered if Cholnoky had made correct distinctions. The Florida group 
works with microspheres, which can be separated by centrifugation; these 
microspheres are more stable than the Oparin coacervate droplets. He wanted 
to make another point, which he had forgotten to mention at the earlier session 
that day. Should the micropaleontologists and meteorite investigators con- 
clude that the "organized elements" were not fossils of micro-organisms, but 
preprotobionata, i.e., a type of abiotic microspheres, then this would be an even 
more significant finding, because it would indicate the discovery of precursor 
organic particles from which life forms could have later evolved. 

Robert Ross {Department of Botany, British Museum of Natural History, 
London, England) : This discussant reported on his own studies of the Orgueil 
meteorite: 

The Orgueil meteorite fall consisted of about 20 stones. Two of the speci- 
mens at the British Museum (Natural History) arrived there as complete 
stones. He had studied one of these, which had not yet been examined for 
"organized elements" by other workers. In straight crushed preparations, he 



Panel Discussion: Identity of "Organized Elements" 609 

did not find the large number of particles that he had expected to find after 
reading the report by Claus and Nagy. Nevertheless, he did find a small 
number of particles which, if found in terrestrial samples, he would have said 
to be of biological origin. Two colleagues at the British Museum agreed that 
these particular objects looked as if they might be of biological origin. 

Plans have been made to conduct more refined experiments on these organic 
particles. Electron microprobe analysis. X-ray microanalysis, and electron 
microscopy are being contemplated. Certain additional experiments have 
already been performed, however. In addition to the examination of straight 
crushed preparations, density separations were carried out with aqueous cad- 
mium borotungstate solutions; this liquid was used instead of the organic 
liquids used by Nagy et al. It was thought that the use of inorganic liquids 
would eliminate some of the criticisms raised against these investigators, i.e.^ 
that the "organized elements" were mere droplets of bituminous matter, dis- 
solved and then precipitated from organic liquids. 

A fragment of the Orgueil meteorite was used, and its surface was scraped off 
with sterilized instruments. The sample in water was then subjected to re- 
peated and prolonged freezing and thawing in an attempt to break up the 
mineral matrix and to disintegrate the stone. This process was partially suc- 
cessful. The disintegrated material was then suspended in cadmium boro- 
tungstate solution and centrifuged. Four fractions were obtained, one of 
which sank in liquids of 2.4 density. In the three light fractions, representing 
densities of below 1.6, equal to 1.6, and between 1.6 and 2.4, a number of the 
Type I "organized elements" of Claus and Nagy were found. In these density 
range fractions, furthermore, there were also other objects, which resembled 
collapsed spore membranes. Finally, two unusual forms were found in the 
lightest fraction. Each of these objects consisted of a hollow tube, approxi- 
mately 25/i long and X^in wide. The tubes contained an infilling •'^4m wide, 
that had a refractive index different from the tube walls and quite different 
from the Canada balsam in which these objects were mounted. The fillings 
were probably air bubbles. One end of each of these tubes blended smoothly 
into what looked like a torn piece of membrane, approximately lO^i wide. The 
overall appearance of these forms, the tubes and the torn membranes together, 
approximated a mushroom shape. These forms had been associated with the 
meteorite matrix; the sterile procedures used suggested that they were not con- 
taminations acquired during the study, but were part of the Orgueil meteorite. 
The forms reminded Ross somewhat of the fossil hystrichospheres that Papp 
had described earlier during the sessions. They might be parts torn from such 
an organism. He concluded they were of biogenical origin. Claus and Nagy 
had shown that the objects they found take up biological stains and resist 
acids. These crude tests suggested that they consisted of carbon compounds. 
All this evidence, he believed, adds up to a strong indication, but not proof, 
that there are indigenous remains of living organisms in the Orgueil meteorite. 

H. C. Urey: He expressed the opinion that Ross's findings were quite im- 
pressive. He thought, however, that one might still wonder about what hap- 
pens to living matter when it is fossilized for four and a half billion years; also, 
what would the very earliest forms of life look like? He stated that it would be 



610 Annals New York Academy of Sciences 

significant if one could find many objects with only narrow variations in their 
morphology, since the same types of organisms should not vary widely in 
morphology. 

George Claus {Department of Microbiology, New York University Medical 
Center, New York, N.Y.): Claus stated that Type I of the "organized elements" 
is by far the most common. Morphological variations, as well as size distribu- 
tion of organisms, follow a Gausian distribution curve. "Organized elements" 
follow the same pattern. 

F. W. Fitch (Department of Pathology, University of Chicago, Chicago, III.) : 
According to Fitch, many different kinds of particles described in Orgueil me- 
teorite preparations have been called "organized elements." In his opinion, 
one deals with a heterogeneous population of objects which can be divided into 
two general classes — particles having a simple appearance and particles having 
highly structured morphology. The rather featureless objects are numerous 
but seem to have no specific properties indicating biological origin. Particles 
having complex morphology are quite rare and some may have a biological 
origin. However, there is no proof that they are not terrestrial contaminants. 
Fitch wondered what would be adequate criteria for identifying the "organized 
elements" as extraterrestrial forms having biological origin. He did not be- 
lieve that morphology alone was adequate evidence. There are at least 250,000 
plant species on the earth. It is impossible for any individual to be familiar 
with more than a fraction of these and to identify isolated plant fragments. 
There were no experts specializing in the study of pollen and of fungi at this 
meeting, according to Fitch; therefore evaluation of the objects at this meeting 
must necessarily be incomplete. 

Bartholomew Nagy (Department of Chemistry, Ford ham University, New 
York, N. Y.) : Nagy stated that because there were no experts on pollen at the 
meeting, he and Claus took their microscopic preparations to a meeting they 
attended during the previous week of the First International Congress of 
Palynology at Tucson, Arizona. At this meeting there were approximately 
300 experts on pollen from 22 different countries. The slides were exhibited in 
public, and anyone who wished to examine them under the microscope could 
do so. Approximately 80 specialists did so and to his knowledge, no one 
definitely identified the "organized elements" as recent pollen contaminations. 
Since, however, Anders et al. have argued that the "organized elements" 
were ragweed pollen grains and starch grains, he thought it might be interesting 
to recall Erdtman's comments on the "organized elements." Erdtman is a 
Swedish pollen expert. His first impression was that the "organized elements" 
were indeed pollens; however, after more careful examination, he concluded that 
this was incorrect because they were similar to hystrichospheres, a pelagic form 
of protobionta. 

H. C. Urey: Urey noted that enthusiastic people can make mistakes, but a 
mistake should not stop anyone. If one cannot identify these objects, one 
should consult others. Photographs of objects projected on the screen do not 
settle the question. 

Rainer Berger (Lockheed California Company, Bjtrbank, Calif.) : Berger 
agreed that undoubtedly more experimentation is needed. As Cholnoky 
pointed out, biochemical tests could be inconclusive when applied to fossils. 



Panel Discussion: Identity of "'Organized Elements" 611 

For example, people buried in Pompeii in ash from the eruption of Vesuvius 
apparently have no carbon left, because the tissues have been fully replaced by 
mineral matter. Nobody doubts that they are remnants of people, yet bio- 
chemical tests on them would give negative results. 

Berger went on to say that one does not know what happens to meteorites 
during their passage through the atmosphere. There is a question as to 
whether air or air-borne pollen is sucked in. He wondered if it might be pos- 
sible that the pollen could become imbedded in the meteorite and become 
fossilized during museum storage. He also wondered how long it takes to 
fossilize organisms. 

Warren Meinschein (Esso Research and Engineering Company, Linden, 
N. J.) : Meinschein's opinion was that it requires a long time to fossilize organ- 
isms and it certainly requires water. 

R. Berger: Berger wondered if there was enough water for this to occur in 
the museum. 

D. J. Hennessy {Department of Chemistry, Fordham University, Xew York, 
N. Y.) : According to Hennessy, the issue at the present time was whether these 
"organized elements" were terrestrial or extraterrestrial. Since Orcel in the 
Paris Museum has large, single pieces of the Orgueil meteorite, perhaps he could 
be persuaded to permit drilling into one with a sterile drill to obtain a sample 
from the interior. 

Edward Anders {The Enrico Fermi Institute for Nuclear Studies, University 
of Chicago, Chicago, III.): Anders suggested that it was utterly misleading to 
speak of "organized elements" as if they were a single, well-defined family of 
particles with certain generic properties. Instead, it appeared that the or- 
ganized elements fell into two sharply distinct classes. Particles of the first 
class have a striking morphology, and most of them are probably biogenic. 
However, they are quite rare, even in Nagy's samples, and they have not been 
seen in the Orgueil samples studied at Chicago. Most of them show a strong 
resemblance to common airborne contaminants, such as pollen grains, fly ash, 
etc., and it seems hkely that most of them are in fact terrestrial contaminants. 
Particles of the second class are probably indigenous to the meteorite. But 
they seem to lack all other properties suggestive of a biological origin: their 
morphology is nondescript, and resembles that of mineral grains; they do not 
take biological stains, or take them atypically; they do not fluoresce in ultra- 
violet light; they dissolve in acids; and they have nearly the same density as 
the mineral grains in the meteorite. In view of these findings, Fitch and 
Anders believed that two questions needed to be settled before all others. For 
the particles of the first class, what is the evidence that they are not terrestrial 
contaminants? And for the particles of the second class, what is the evidence 
that they are not in fact mineral grains? 

[Note added by the discussant in proof. Most of the evidence obtained since 
the meeting has favored the view that the majority of the organized elements 
are either contaminants or mineral grains. The spiny Type II elements 
("hystrichospherids"), alleged by us to be ragweed pollen grains, have in fact 
been identified as ragweed pollen by several pollen experts. The Type V ele- 
ment ("dinoflagellate"), discovered by Claus and Nagy on a Gridley-stained 
slide of Orgueil was shown to resemble Gridley-stained ragweed pollen (Fitch 



612 Annals New York Academy of Sciences 

and Anders, Science, 1%.^, in press). The particles of simple morphology, 
which we said resembled mineral grains, do indeed have the chemical composi- 
tion of limonite (hydrated ferric oxide), according to electron microprobe 
analyses by Nagy, Fredriksson, Claus, Anderson, Urey, and Percy (Nature, 
1963, in press), and they dissolve in acids without leaving a structured organic 
residue (Anders and Fitch, Science, 1962, 138: 1392). The case for their bio- 
logical origin now rests entirely on their featureless morphology.] 

H. C. Urey: Ross's findings impressed the moderator. Ross had worked 
with a complete stone which had probably been heated during passage through 
the atmosphere. The moderator wondered whether there had been any signs 
of contamination on Ross's sample, and whether it had been marked with 
paint or mounted on wax. 

R. Ross: The samples of the Orgueil meteorite in the British Museum were 
kept in a box with a glass cover on it. There were no precautions taken during 
the years to keep them sterile in storage. He had used, however, sterilized 
instruments to work with the samples. He had also scraped away exposed 
surfaces of the meteorite with sterile instruments before taking a sample, and 
before placing the samples into water and subjecting them to freezing and 
thawing. 

P. Morrison {Department of Physics, Cornell University, Ithaca, N. Y.): 
Morrison stated that he would like to see a count of the relative distribution 
of the "organized elements." 

G. Claus: According to Claus, "organized elements" were present in Orgueil 
on the average of 1700 per milligram. Type I was by far the most common and 
on one side Type II represented approximately five per cent of the total count. 
Claus noted, regarding the staining, that there are many "organized elements" 
which do not take stains. 

H. C. Urey: This was a puzzle to Urey, and he wondered if the simpler ones 
could be artifacts and the more complicated ones, contaminations. 

Paul Tasch {Department of Geology, University of Wichita, Wichita, Kansas) : 
Tasch observed that, exclusive of magnetic particles, the "organized objects" 
found in some carbonaceous chondrites fall into three classes: (1) terrestrial 
contaminants in addition to those already cited by Claus and Nagy; (2) 
proteinoid microspheres of Fox or organic-chemical analogues of Morrison; and 
(3) indigenous microfossils. He believed that allowance for (1) and (2) had 
been made, but (3) still remained to be explained. The discussant had observed 
three distinct objects in Claus and Nagy's thin sections. Two of these were on 
display at the International Palynological Conference at Tucson, Arizona, and 
one was presented by Claus in his talk. The discussant's first impression of 
one of these objects embedded in salt was that it resembled a chrysophyte; 
another object, named Daidaphore Berzelii, had a hystrichospherid-like organi- 
zation. A third object appeared to have antapical horns and a girdle, thus 
suggesting a dinoflagellate. 

Ross, according to Tasch, had reported how he isolated a distinct object from 
a carbonaceous meteorite, and had indicated his conviction that it was not a 
contaminant, but resembled a process of hystrichosphere. 

Tasch also pointed out that F. L. Staplin of Imperial Oil, Ltd. processed a 
sample of the Orgueil meteorite and wrote a report, soon to be published, which 



Panel Discussion: Identity of "Organized Elements" 613 

the discussant had ah-eady seen in a preprint. Staplin found, after palynologi- 
cal processing of the sample, a group of micro-objects in the fmal residue. In 
his judgment, these were the closest in affinities to a hystrichosphere-leiosphere 
assemblage. Among the objects, he also found some chrysophytes. 

Now, continued Tasch, these three observations, in addition to those of 
Claus, all seemed to be consistent. 

Tasch suggested that to advance this discussion the problem of contamina- 
tions be bypassed and the objects found in the meteorites be accepted as in- 
digenous. What would then follow? Pelagic protists closely resembling 
terrestrial types must have lived on the parent body; once this is admitted, 
then it follows that there were water bodies in which they lived. In addition, 
there must also have been a supply of phosphorous, nitrogen, and other nutrient 
substances. That, according to Tasch, would be as far as paleobiology can 
take it. It would then be necessary for Urey, Bernal, and others to explain 
where it is possible for such water bodies to exist. 

A. Papp {Department of Paleontology, University of Vienna, Vienna, Austria) : 
Papp emphasized that the basic rule of the natural sciences is that an experi- 
ment must be repeatable before one can accept the tindings as valid. Research 
on the "organized elements" started only a short while ago, yet the experiments 
had already been successfully repeated, and independently, by Ross in England, 
Staplin in Canada, and Skuja in Sweden. Now the Anders group came up with 
negative results. A most important cjuestion would be whether any one else 
has found the "organized elements" besides Nagy et al; the answer to this is 
yes. They were found in England, in Canada, in Sweden and, according to 
Papp, one would venture to say they were also found in Chicago. 

There is then the question as to the differences in yield; the answer to this 
would be that the greatest number of objects were found by those who did 
most of the work. Papp illustrated this by pointing out that in two kilograms 
of sedimentary rock one would find more microfossils than in only one gram of 
rock. It would seem that the problem of differences regarding the number of 
"organized elements" would be related to the relative amounts of time spent on 
the problem by the different investigators. 

With respect to attempts to identify and to classify these forms, Papp con- 
cluded that it was unimportant whether they look somewhat like dinoflagel- 
lates or something else. They are something different, and it would be impos- 
sible to include them in terrestrial systems. One could only compare them with 
terrestrial forms and state that they resemble certain terrestrial species, and 
even with this, one would be saying very much. 

In summary, Papp beUeved that the "organized elements" had been proven 
to be organic and that their organized nature had been confirmed independently 
in four countries. He was impressed that "organized elements" were not 
identical to, but only resembled terrestrial organisms and, therefore, he con- 
sidered that the question of their origin was closed. 

H. C. Urey: He and his associates in Lajolla could not find anything in their 
sample of Orgueil. They then sent their samples to Nagy and the "organized 
elements" were encircled on the slides and returned to them. They still could 
not find one. Finally, however, a technician did find an "organized element." 

A. Papp: Six months previously one of Papp's colleagues, a mineralogist in 



614 Annals New York Academy of wSciences 

Vienna, had said that the "organized elements" were mineral spherolites. 
Papp stated that this only showed that one needs extensive trainmg in micro- 
biology to recognize these forms. 

H. C. Urey: Urey mentioned that Volcani, the microbiologist at Lajolla, 
was not discouraged. 

C. M. Palmer {Division of Water Supply and Pollution Control, Robert A. 
Taft Sanitary Engineering Center, Cincinnati, Ohio) : He stated that it should be 
mentioned that Claus was capable of observing and finding structural details 
that other people often overlooked; i.e., details on filamentous algae which had 
never been seen, although the forms had been known for more than 100 years. 

Pierre Bourrelly (Department of Cryptogamic Botany, Miisee d'Histoire 
Naturelle, Paris, France) : Bourrelly saw the microscopic preparations. He 
believed that the "organized elements" were definitely organisms. They did 
not look much like hystrichospheres, and they did not seem to be contaminants. 
He was puzzled because they resembled terrestrial forms; he thought they should 
exhibit greater differences. 

R. Berger: He recalled that Papp had implied that the terrestrial evolu- 
tionary sequence might have occurred elsewhere. There might be an equiva- 
lent biochemistry which would lead to similar organisms. 

J. D. Bernal: He expressed the opinion that the carbonaceous meteorites 
could not be of terrestrial origin because of their unusual mineral content. 
If they were not terrestrial, he questioned what the origin might be. Only 
Earth and Mars are capable of holding water; therefore, if the "organized 
elements" arose elsewhere than on earth, one would be forced to choose between 
two possibilities: that the same biochemistry is prescribed for every origin of 
life and, therefore, life always follows the same trend; or that all life forms 
originated from the same ultimate life source. In other words, one is faced 
with these questions: did life originate in several places as a result of the same 
biochemical mechanism, or did Ufe evolve only once and then spread to different 
places? He added that four billion years might not be a sufficient amount of 
time for biochemical evolution on earth. 

H. C. Urey: According to Dr. Urey, life might have transferred to the moon 
from earth. 

P. Tasch: He was one who considered the moon transfer theory a serious 
possibility, though a difficult one to accept. 

H. DoMBROWSKi {Department of Balneology, Justus-lJebig University 
Giessen, Germany): Dombrowski stated that there is an analogy between the 
problem of "organized elements" and his work obtaining living bacteria from 
salt deposits. Chemists have known for a long time that salts contained less 
than 0.01 per cent of nitrogen. The origin of this nitrogen could not be ex- 
plained until biologists started to work on salt samples. Now it is known that 
this nitrogen content is associated with the bacteria embedded in the salt. 

Brian Mason {Department of Mineralogy, The American Musuem of Natural 
History, New York, N. Y.) : Mason thought that it would be very difficult not 
to contaminate a meteorite in a museum. The American Museum of Natural 
History acquired its Orgueil sample in 1901, but it is not known what happened 
to it before this date. The sample was kept in an open box. It should be 



Panel Discussion: Identity of "Organized Elements" 615 

mentioned that magnesium sulfate in the meteorite in New York is MgS04- 
4H2O; in Chicago it is MgS04- 7H2O. This would only show how the environ- 
ment can effect the "organized elements." 

J. D. Bernal: He mentioned that the Orgueil sample in the Bombay Mu- 
seum in India fell to a fine dust because of the humidity. 

W. G. Meinschein: The hydrocarbon distribution in Orgueil resembled 
biogenic hydrocarbons in Recent marine sediments. Mass spectra showed 
that these hydrocarbons could not have been contaminated by accident, much 
less by intent. The hydrocarbon concentrations were above Recent sediment 
concentration levels. 

B. Mason: This discussant was of the opinion that the organic matter in 
Orgueil was clearly indigenous, but that one must prove that it was not formed 
by inorganic processes. 

W. G. Meinschein: It was emphasized by Meinschein that 23 saturated 
hydrocarbon compounds had been identified. The parent sterol hydrocarbons 
were present and the aromatic hydrocarbons were those which are found in 
Recent sediments. Clearly, he believed, they were of biogenic origin. 



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