The myxomycetes--some problems and unanswered questions.

CRC Critical Reviews in Microbiology

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The Myxomycetes - Some Problems and Unanswered Questions William D. Gray & Lindsay S. Olive To cite this article: William D. Gray & Lindsay S. Olive (1976) The Myxomycetes - Some Problems and Unanswered Questions, CRC Critical Reviews in Microbiology, 4:3, 225-248 To link to this article: http://dx.doi.org/10.3109/10408417609106943

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Date: 25 October 2015, At: 13:22

THE MYXOMYCETES - SOME PROBLEMS AND UNANSWERED QUESTIONS

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Author: William D. Gray Department of Biological Sciences Northern Illinois University DeKalb, Illinois Referee: Lindsay S. Olive Department of Botany University o f North Carolina Chapel Hill, North Carolina

INTRODUCTION When I first saw a myxomycete in 1931 and almost immediately became interested in this unique and fascinating group of living organisms, the world of most biological scientists was in general a relatively uncomplicated affair. By presentday standards the field of biochemistry was still in its infancy and we had not yet heard the term “molecular biology.” We were beginning to understand that protoplasm was undoubtedly something more than mere blobs of a mysterious living something since Chambers, Seifriz, and others were using crude micromanipulators to dissect tiny cells and their components and others were injecting fine metal particles into amebae which were then placed in a magnetic field, centrifuged, or subjected to some other form of abuse. However, all t h i vitamins had not yet been discovered, the first electron microscope had just been built, most people still believed that the atom could not be split, and fust-year graduate students were not in a position to speak in offhand and knowledgeable terms of the information they had just uncovered through the use of gas chromatography or an amino acid analyzer. The year 1931 was one of the Great Depression

years, and most universities and colleges (plus the inhabitants thereof) were beginning to encounter increasingly severe financial problems. The purchase of new and more sophisticated (for that time) equipment or perhaps even a small amount of some moderately expensive chemical compound often presented considerable difficulties for the scientist and frequently was quite impossible under the existing economic conditions. The u p todate worker who wanted to measure pH more often than not did so by adding a dye to the solution he was testing and comparing the resultant color with different colored liquids in a series of sealed test tubes, the critical one of which was too often missing. The better equipped and more venturesome worker attempted to measure pH using a procedure called the quinhydrone method. To quantitate amino groups the investigator usually relied on one of the most improbable pieces of laboratory equipment ever devised - the Van Slyke Micro Amino Analyzer - and the metabolites of living organisms were painstakingly isolated and purified and then laboriously identified by the standard organic analytical procedures of the times. Faculty and graduate students alike who were conducting research were more often than not March 1976

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hard-pressed to accumulate the equipment and simple materials necessary for the furtherance of their work; “making do” with what was available was more the rule than the exception. It was not uncommon for a research problem to shift direction, not because certain experimentally derived leads had pointed t o the advisability of the investigator making such a shift, but simply because of the unavailability of materials or equipment. As strange and unreal as it may seem to the present generation of more affluent graduate students, this writer has often seen graduate students in plant physiology whose major problems were simply those of accumulating sufficient numbers of flower pots to use for the growth of the plants needed in their research. Similarly, those individuals attempting t o work with microorganisms in pure culture sometimes had difficulty in obtaining enough test tubes, Petri dishes, or small flasks to conduct their work properly. The memories of the several stark tragedies that occurred when inevitably a dozen or more test tubes, were accidently broken are still quite vivid. There were no such items as discardable glassware discardable graduate students perhaps, but not discardable glassware. I well recall one instance when as a graduate student a sufficient number of small staining dishes were not available t o enable me to set up as many moist chamber cultures of Physanim polycephalum as I felt I needed. Not only were enough such dishes not available, but Cunds for the purchase of additional dishes were also not obtainable. My departmental chairman, who of necessity had to be quite resourceful under such distressing conditions, had a marvelous idea for substitutes for the staining dishes. Stacked on an old shelf in a dusty corner of our ancient building were scores and scores of old-fashioned salt cellars - small squares of solid glass (many with fancy patterns pressed into the sides), each with a round depression in the top. These “dishes” served my purpose quite admirably, but even today when I browse in an antique store and see the current prices on such items, I have a twinge of conscience when I recall that I carelessly broke quite a few of them. By that time, however, I was a well-treated graduate student in one of America’s oldest, most reputable, and least financially pressed universities and so I could not help but wonder how the poor people in the less-blessed colleges and universities were doing. Such then were the general circumstances that prevailed when as an undergraduate I was first 226

CRC Crirical Reviews it! .Wicrobiologv

introduced to the myxomycetes. The introduction was a simple one: My professor of mycology handed me a small herbarium box and told me to study the myxomycete specimen that it contained. I t quickly became apparent that identification of the specimen would present no serious problem because the covert unconcealing of the poorly concealed label revealed in the beautiful script of a well-known, turn-of-the-century mycologist the welcome information that this was a specimen of Arcyria incarnata. Also on the label was pertinent information relative t o where and when the specimen had been collected. Thus, early in my first encounter with a myxomycete I was in approximately the same position I had frequently been in in my fourth grade arithmetic class: The answer to every problem was neatly and conveniently located at the back of the book. However, this similarity of positions became even more obvious whe.n it became apparent (as it frequently had in the fourth grade) that possessing the answer does not necessarily imply a knowledge of how to solve the problem properly. There was still work to be done. Now one had to consult some such book as Macbride’s’’ The North American Slime Molds, lister'^'^ A Monograph of the Mycetozoa, or Massee’? * A Monograph of the Myxogastres. It would have been very helpful t o have had access to Macbride and Martin’s79 The Myxomycetes or Martin and Alexopoulos’s’ The Myxomycetes, but the first w3s then only in the process of being published and the latter had not yet even been c o n t e m p l a t e d . Of course, Rostafinski’s’ Sluzowce was said to be available, but consideration of this latter had t o be eliminated immediately for very sound linguistic reasons. Unfortunately, in my sphere of acquaintances at DePauw University in 1931 there was n o one who read Polish. In fact, I have a strong suspicion that most o f us who called ourselves students at that time had only a hazy idea of exactly where Poland was located, and I further suspect that many of us remained in this general state of geographic ignorance until Poland began t o figure strongly in the newspaper headlines at the outset of World War 11. My linguistic incompetence thus forced me to narrow the field of available books down t o three. The illustrations in Macbride’s book were poor and therefore it did not survive the first elimination round. Massee’s book was incredible (it still is!), even to a rank beginner, so it was Lister’s monograph or nothing. This latter appeared to be


a happy choice since it was large and contained many colored illustrations. Having selected a book, the general idea was that YOU took a specimen, examined it carefully, and then traced its identity by means of a key. This all sounds very straightforward and presumably quite simple, but it should be pointed out that at that time most of my experience with keys was limited to using one to gain access to my dormitory room. In spite of my inexperience, I soon grasped the basic idea of how to use a key, although even today I still have trouble when I encounter a key in which one is faced with making a decision as to whether some structure is very dark salmon pink or very light red. However, in spite of the fact that I had now at least partially mastered a new tool, it proved quite impossible for me or my instructor to use the key and trace my specimen to the species incarnata. Anyone can make a mistake, and so assuming that the collector of my specimen had done so, I merely looked for another species in the genus Arcyria whose description more nearly fitted the specimen at hand. Careful observation of this specimen and careful reading of the descriptions of every species in the genus (I had already tacitly assumed that since Lister had written a large book on the subject, the work had to be complete and authoritative) revealed that it did not represent any known species of Arcyria. Obviously, I, a rank amateur, had discovered a long-hidden new species at age 19 - a highly exhilarating and ego-expanding experience. Why not? DeBary had done it in Germany at a tender age, so why shouldn’t one of our bl>ys? However, at this point another minor difficulty now appeared on the scene: My myxomycete not only was not Arcyria incamata, it was not even an Arcyria. To complicate matters even further, it was not even in the family to which Arcyria belonged. I did not discover a new species but eventually found that I was looking at a very typical specimen of the common Hemitrichia vesparium (presently called Metatrichia vesparium), a species that had been discovered, adequately descnled, and named years before I was even born. This finding certainly did nothing to inhate my ego but was most rewarding in that it taught me a very valuable lesson at a very early age: Even people who write books (as had the individual who had misnamed my specimen) can make mistakes. I know this to be even more true today than I knew it then because now I have

written books, and, as my predecessors and mentors before me, 1, too, have made mistakes. Although this first experience with a myxomycete made me painfully aware of the fact that it was highly improbable that I was the person who was going to set the biological world afire through my contributions to myxomycete taxonomy, it was quite valuable to me in that it first led me into the literature of the field. A thorough search of the literature quickly revealed that there was much yet to be learned about this anomalous group of organisms, although the general developmental cycle had been known since the time of DeBary.’ Fortunately, at the time there was no doubt as to where these organisms belonged in the world of living things - differences of opinion, yes, but doubts, no. Botanists said that they were plants and called them myxomycetes. Zoologists said that they were animals and called them mycetozoa. Two workers in England even pre-’ ferred to call them “myxies” because this word rhymed with pyxies. The number of active myxomycete investigators in the world in the early 1930s was quite small - so small, in fact, that b y 1938 I had established correspondence with most of them. All were helpful, all were encouraging, and all were apparently quite tolerant of beginning students. Among these individuals were Macbride, Brandza, Skupienski, Krzemieniewska, Emoto, Miss G. Lister, and others. Dr. G. W. Martin frequently provided me with encouragement personally and by letter, as did Robert Hagelstein and William Seifriz. The interest gained during those exciting times has stayed with me all these years, although admittedly I have not found it possible to retain myxomycete study as a full-time academic career. Fortunately for those who were aware of their inadequacies or lack of interest in the field of myxomycete taxonomy, an event occurred in 1931 that opened new vistas for me and hundreds of investigators who have come on to the scene more recently. Prior to that time most investigators who were seriously interested in the physiology or development of myxomycetes had to rely primarily on being able to collect plasmodia in their natural habitats in the field during the proper season, an extremely inconvenient and restrictive situation since summers were usually spent in the field, and autumn, winter, and spring in the laboratory and classroom. While some slight successes had been achieved y6

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by various investigators in their efforts to cultivate myxomycetes under the artificial conditions of the laboratory, Howard’s’’ development of a relatively easy and reliable technique for culturing Physarum polycephalum marked the first instance in which interested investigators could have living myxomycete plasmodia available to them at all times in virtually unlimited quantities. When I first read Howard’s paper, tried his method, and found that it worked very well, I was highly enthusiastic and remained so for a number of years. Now after 44 years - certainly a sufficient period of time for proper reflection - I have grave doubts because, just as many people are unable to see the forest for the trees, it seems that many people have been unable to see the myxomycetes for Physarum polycephalum. In no way is this meant to be a criticism of Howard or his work and it should not be so construed. Howard’s work on rnyxomycetes was excellent; he accomplished something that no one had accomplished previously and should be commended highly for his resourcefulness and ingenuity. From the standpoint of those primarily interested in the myxomycetes per se, criticism cannot be justly leveled at Howard or his work but rather at many of the works that were made possible by Howard’s discovery. The literature shows quite clearly that Howard himself was interested in the myxomycetes as unique living organisms. His first papers4 dealt with the life history and development of Physarum polycephalum . His second paper,” published later in the same year (1931), described his method of culturing this species, and his third papers6 described nuclear division in Physarum. His fourth and fifth (coauthored by Cunie in 1932) were concerned with the parasitic habits of myxomycete plasmodia. The response to Howard’s development of a reliable method for culturing Physarum polycephalum was immediate and voluminous. This was primarily because he had provided investigators from a wide variety of disciplines with a means of obtaining almost unlimited quantities of protoplasm which could be used in a wide variety of studies, many of which had nothing to do with myxomycetes as unique living entities. This developing situation was soon further enhanced by the simplification of culture techniques for Physarum polycephalum that were introduced by Camp‘ in 1936. Camp’s moist chamber method for the cultivation of this species made even larger quantities of plasmodia easily available. Further228


more, plasmodia did not have to be removed (frequently with much damage) from the surfaces of agar plates, and the necessity for sterilizing glassware and media had been eliminated. Now an investigator who did not even have access to an autoclave could cultivate plasmodia of Physarum polycephalum in quantities limited only by available time, patience, space, quantity of suitable glassware, and quantity of rolled oats, which could be purchased at that time for five cents a box. All that he needed by way of materials was a covered moist chamber, one half of a Petri dish (either top or bottom would serve), some dry rolled oats, a piece of filter paper, and a small amount of tap water. If fiter paper was not available, a piece of porous towel paper would serve just as well. Surely the millenium in rnyxomycete studies had now been reached, except for one small item: Physarum polycephalum is not a “typical” myxomycete and its behavior and activities should iri’no way be considered typical of the group as a whole until several other species are demonstrated to behave in the same manner. Just by way of a very simple example, it should be stated that very few other species can be easily cultured in this manner, and of those few it is a rare culture in which growth is as profuse as that commonly shown by Physarum polycephalum when it is cultured in a moist chamber. More significantly, however, the description by Alexopoulos’ of three quite different and well-defined types of plasmodia has laid to rest the once rather general assumption that the phaneroplasmodium of Physarum polycephalum is typical of the plasmodia of myxomycetes in general. Thus, Howard, and later Camp, provided the means by which large quantities of an easily cultivated atypical rnyxomycete plasmodium may be obtained, and these means have since been pursued with real vengeance. In itself this would probably not be too serious, provided that all aspects of the activities and characteristics of this species, as a myxomycete, had been thoroughly explored. Then, at least, we would know all about an atypical myxomycete. Unfortunately, this is not the situation, and we are now faced with the existence of literally hundreds of published reports on myxomycetes (i.e., Physarum polycephalum), many of which have little if anything to do with myxomycetes, although in fairness it must be admitted that they supply us with a surprising diversity of observations and analyses of this one species. In their book, S i o l o ~of the Myxo-


mycetes, Gray and Alexopou~os4 have included slightly more than 500 references, of which nearly one fourth are papers concerned wholly with work involving Physarum polycephalum - nearly 25% of the work devoted to less than 0.25% of the myxomycete species that have been described. Similarly, in the chapter devoted to myxomycetes in his recent book, TheMycerozoans, Oliveg6 cites approximately 300 references, many of which are concerned only with work on Physarum polycephalum. Perhaps it may seem unfair to cite only one paper in a matter of this type; however, I can assure the interested reader that a cursory examination of the literature will reveal additional examples. In 1938 L e p ~ wpublished ~ ~ a paper based on his work with Physarum polycephalum. It was a sound paper in which the author attempted to describe the protoplasmic reactions that occurred when he treated living plasmodia with various snake venoms and alkaloids. Such work undoubtedly has its value in the fields of toxicology and pharmacology, but it contributes nothing to our knowledge of the myxomycetes as living o r g a n i s m s because, as Gray and Alexopoulos40 have pointed out, t o date no myxomycetes have been reported to have been bitten by venomous snakes. The undue emphasis that has been placed on Physarum polycephalum , primarily because of its availability and ease of culture, has also unquestionably had its effect on biological training at the collegiate level. Thus, an extremely high percentage of several generations of biology students has been exposed to only this one species of myxomycete, hardly a fair or comprehensive representation of the group. At this point a serious question must be raised concerning the integrity of this well-known species which has been so widely used in so many diverse investigations in so many far-flung laboratories following the contributions of Howard and Camp. Once a culture was obtained, plasmodia were cultivated and maintained under a great diversity of conditions depending on the investigators, their available facilities, and the nature of the problems they were exploring. Some stock cultures were maintained in light and some in dark, some where it was relatively warm and some where it was cool, some in constant temperature rooms into which filtered air was introduced and some on tables in chemical laboratories where they were exposed to a great host of different gaseous compounds. It is highly improbable after a few years of such diverse

treatments that everyone who had obtained a start from the original culture now had exactly the same organism at hand. After all, a plasmodium consists largely of relatively unprotected protoplasm (or whatever we choose to call the basic living substance) and in all probability is highly subject to the effects of many mutagenic agents. Thus, by 1939 Gray3' was able to demonstrate that plasmodia of all of the eight strains of Physarum polycephahm that he examined would not fuse, which they should have done had they been identical. Much stronger and more specific evidence of the diversity of cultured strains of this species is provided by an examination of the different chromosome numbers that have been reported by d i f f e r e n t investigators for Physarum polycephalum. Thus, Ross'0s reported a diploid chromosome count of 90 ? 3, Guttes et aL4' reported a diploid number of 20 and a hap1o.B number of 8, while Koevenig and Jackson" reported a diploid number of 56 f 2 but noted that there were some large nuclei in which the chromosomes numbered over 100. It is true that these chromosomes are difficult to count, but such difficulty would scarcely account for reported diploid counts ranging from 20 to 100. It is possible that some or all of these investigators erred in their counts, but it is equally possible (and in my opinion quite probable) that they were working with different genetic strains of the same species - strains that may well have developed in the hands of past investigators who were cultivating plasmodia under widely diverse conditions for widely diverse purposes. The work of ROSS'O 7 suggests that within a single isolate wide ploidy ranges may exist after long periods of laboratory culture, and Kerr" has shown that some ploidy changes result from incomplete nuclear division. Different strains certainly exist and could scarcely be expected to behave uniformly in an identical manner either physiologically, morphogenetically, or biochemically. Therefore, any unusual attribute of a distinct genetic strain of Physarum polycephalum would not necessarily be true of other strains and certainly not of myxomycetes in general. Perhaps I am now about to engage in a bit of wishful thinking. If so, I may as well reveal it. I would like to see more of the time, effort, and research funds now spent on myxomycetes devoted to these organisms as snch and not to Physarum polycephalum as a convenient and easily March 1976

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cultured test organism. There was comparatively little that anyone could tell you about myxomycetes 40 years ago (other than their taxonomy and the rough details of their developmental cycle), but that certainly need not be the situation today. Students trained in the biological sciences today have access to biochemical training that was simply unheard of in the 1930s. Also available to them are many types of sophisticated equipment that were not even on the drawing board four decades ago. With rapid air transport and the frequent convening of international symposia on a wide variety of scientific subjects, communication in the scientific community today is much more rapid, and a slight advance made by one investigator quickly becomes the common property of his colleagues all over the world. Although the various research funding agencies have retrenched somewhat during the past several years, funds for research became increasingly available shortly after World War 11, thus making it easier for the interested investigator to finance his research. With these and other obvious advantages, a bright new generation of investigators could provide us with much new information concerning the myxomycetes primarily as unique living organisms and not as mere sources of easily available living substance for use in a wide variety of investigations that often have little real bearing on the myxomycetes. As a matter of fact, a very large number of well-trained, highly competent investigators already exist. All that is needed to acheve the myxomycetological Utopia I envisage is a slight shift in emphasis. One may well question the importance of thoroughly studying all aspects of some 400 plus species of an anomalous, inconspicuous group of organisms. Such importance can, of course, be defended on academic grounds in general, but can easily be defended on ecological grounds alone. Ecological awareness is becoming more and more a part of the American scene, and it is now beginning to appear that it might be an exercise in good judgment to learn everything we can about the role that each type of organism plays in our biological environment - while we still have one! In passing it might be just briefly noted that at this time (over three centuries after they were first called to our attention) we have only a few vague ideas concerning the possible roles that the myxomYcetes may play in the niches they OCCUPY in their natural habitats. It is with these thoughts and reminiscences in 230


mind that this present paper is written. In spite of the voluminous literature which began to appear in the 1930s and the promises which molecular biology seemed to hold in more recent times, it now seems that many of the problems relative to the activities of myxomycetes that faced me in 1931 are still facing me today. Perhaps the blame is partly mine, but I cannot accept this simple, self-incriminating answer as the complete one. Students of mycology in the 1930s were not biochemically trained and so were not equipped to sohe many of the complex problems which the myxomycetes present. Neither did students of that era have available to them the sophisticated measuring devices and highly developed analytical techniques that are almost routinely available today. Students of today are no longer limited by such handicaps, and so a very fundamental question must be raised: Should students of biology today be encouraged to be primarily interested‘in exploring a great series of what can easily develop into what I would term “fringe problems” or should they be interested in exploring the basic problems relative to a unique group of organisms in such manner that we may glimpse a more complete picture of such organisms? I will not attempt to answer this question, although my own thoughts on the matter probably show through fairly clearly; I will merely point out a few problems and unanswered questions concerning myxomycetes that to my personal knowledge have been facing us for over four decades.

THE PROBLEM OF MYXOMYCETE CULTIVATION In spite of the relatively small number of species involved, the problems of myxomycete taxonomy have most certainly not been simple ones over the years. Although I have already confessed my inadequacies in this area, nonetheless I have had to identify a great many myxomycete specimens and am well aware of the pitfalls that may be encountered. Myxomycete taxonomy has never been easy, and hence my sympathies are wholly with those who have had the courage and interest to attempt to sort out the taxonomic problems of this group. In spite of the many innate difficulties associated with the myxomycetes, the taxonomists have now presented us with a quite workable system for the moment. The earlier larger works, of course, are those of Rostafinski,’ l o Macbride,” Lister,” Macbride


and Martin,” and H a g e l ~ t e i n . ~ These ~ works ultimately culminated in the excellent book of Martin and Alexopoulosao and more recently the broad treatment of the mycetozoans by Olive.Y6 The small list above is not meant to imply that the cited workers made the only contributions. There were a great many other investigators over the years who made significant but smaller contributions that cannot be listed here. Whether a taxonomist’s work culminated in describing a new species, compiling a list of local species, or preparing a large monograph, he and his taxonomic colleagues shared a common difficulty: For the most part they were all working with specimens of myxomycetes collected in their natural habitats in the field. Here I am not referring to any of the discomforts or possible dangers (during my 8 to 9 years of collecting in Indiana I was twice bitten by snakes, which may have served to dampen my enthusiasm somewhat) that conceivably may be associated with collecting myxomycetes. On the contrary, I am referring to the fact that for his studies the taxonomist has had to rely almost completely o n the study of naturally occurring fructifications, which, even in the same species, may vary over wide and presently unknown limits. Comments have already been made concerning an unprotected plasmodium’s susceptibility to the effects of a variety of environmental factors and need not be repeated here. Because of this extreme susceptibility it is obvious that different fructifications of the same species might be expected to exhibit a wide range of variation if they developed under widely different sets of environmental conditions. Taxonomists have long recognized that such variations exist and have attempted to allow for them, but their extent is not known for most species, and taxonomic problems with resultant differences of opinion are most certain to present themselves with regularity until the range of such variations is known for each species. Some such variations are known and a few will be briefly mentioned here by way of example. Brandza’,’ noted early that sunlight had effects on both color and structure of myxomycete fructifications, although whether these effects were due to intense light, elevated temperature, or a combination of both is not known. Gilbert3’ suggested that humidity had a great effect on the development of Didymium iridis, especially with respect to size, and Skupienska’ ” observed that

the nature of the substrate greatly affected the sporangia of Didymium nigripes that developed there. On beer-wort agar this latter species formed large sporangia on short stipes, but on plain agar the sporangia were small and the stipes were long. However, Skupienska also found that substrate composition had effects other than those on size, since the nature of the peridium was also affected. On plain agar there was an abundance of.lime crystals on the peridium, on h o p ’ s agar a lesser amount, and on beer-wort agar there were no crystals a t all. The budding taxonomist or possibly even the experienced systematist could conceivably have some problems when confronted with a specimen of Didymium on which the presumably rather characteristic lime crystals were lacking. Koevenigb9 noted that under some culture conditions the fructifications of Physarum gyrosum may resemble those of Physarum compressum, Physanun cinereum, or even Physarella oblongu. This investigator considered moisture (both in the substrate and in the surrounding air) to be the primary factor in determining sporangium shape, but this point should be further investigated. Similarly, Gray3 noted some effects of humidity on the character of the peridium of Physarum ji’avicomum: Sporangia that developed under dry conditions had limy peridia, while sporangia with limeless peridia were developed under humid conditions. As in the case of Koevenig’s work, this latter work should be repeated under more precisely controlled conditions of humidity. Skupienski’ ” ” found that temperature affected the morphology of the sporangium as well as the color of the stipe and columella of Didymium nigripes. Reaction of the substrate also had its effects o n the fructifications that developed there: Large, more calcareous sporangia were formed at a pH of 7.8, while at pH 5.5 sporangia were small. Solis’ 2 4 studied some effects of the environment and found that the number of sporangial lobes of Physarum nicaraguense vaned markedly with temperature. Thus, the average number o f lobes per sporangium developed at 15°C was 3.72, while the average number of lobes on those developed at 25°C was 6.28, almost a doubling of lobe number with a 10” rise in temperature. From these few examples it is evident that various environmental factors may have a profound effect on developing fructifica51

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Therefore, one of the major problems in myxotions, resulting in marked variations among the mycete studies today is that of finding methods of mature structures. In view of the fact that the bringing greater and greater numbers of species characters of the mature fructifications often play into culture in the laboratory. This is frequently a an important role in establishing the identity of a difficult task, although occasionally it proves to be myxomycete, it is easy to see how environmental unexpectedly easy. For example, Gray3 easily factors may create Some problems in this area. obtained Physarum tenerum in culture from spores However, some characters of some species seem on the first attempt - not a major achievemept in to be quite stable. For example, W ~ l l m a n ‘ ~ ~ any sense but probably merely a reflection of the found that sporangia of the common Comtricha fact that n o other investigator had seriously typhoides that developed in the laboratory on a attempted to culture this species prior to th?t substrate on which there was free water were not time. On the other hand, only repeated failures typical; in fact, they could not even be recognized were achieved in attempts to bring Physarum as this species on the basis of sporangial characters. pulchenfmum into culture. Spores of this latter Nonetheless, most of the spores that developed in species would show almost 100% germination in these aberrant sporangia were quite characteristic 30 min or less in “Schuylkill Punch’’ (a local for the spores of Comahicha typhoides. For euphemism for Philadelphia drinking water), but anyone familiar with the characteristic spores of at no time were zygotes or plasmodia ever obthis species, identification could be accomplished tained. To date, the laboratory cultivation of this without much difficulty, but if the inexperienced latter species has still not been reported. student encountered such aberrant fructifications in a field collection, identification might present a Perhaps one of the most discouraging features frustrating and insoluble problem. Unfortunately, of culturing other species of myxomycetes is that as viewed with the ordinary light microscope, the they never seem to produce such extensive growth spores of most myxomycete species are not so as Physanim polycephalum usually exhibits in characteristic as to enable one to identify them on moist chamber culture. However, this should not the basis of spore characters alone, as is the case of be a deterrent because to answer many of the Comatricha typhoides. However, the scanning questions concerning a myxomycete such luxurielectron microscope may provide some assistance ant growth is not at all necessary, Thus, in the identification of myxomycetes on the basis McManusE4 was able to obtain Clastoderm of spore characteristics. Schoknecht and Small’ debaiyaniun in culture and later was able to study studied spores and capillitia of 51 species by the ultrastructure of nuclear division in the small scanning electron microscopy and found that all of plasmodia of this species (McManus and Roth“). them possessed some type of ornamentation. They Similar accomplishments have been made by other concluded that spore wall morphologies are similar investigators with other species. Bringing a species within taxonomic groups and are the most stable into cultivation for the first time is often a characteristics for taxonomic purposes. time-consuming process frequently accompanied An obvious answer to solving many of the by failure, but even so there is no valid reason for perplexing problems of myxomycete taxonomy not believing that every species of myxomycete would be to cultivate a great many more species can be cultured in the laboratory since at least one under controlled conditions in the laboratory, species of every order of myxomycetes has been subject them to a wide range of environmental cultured (Gray and A l e x o p o ~ l o s ~Sometimes ~). it conditions, and note the resultant range of variais simply a matter of trying several procedures tion. Encouraged by Howard’s successful cultivauntil one that works is chanced upon. Several tion of Physarurn polycephalum in 1931, a considerations must be borne in mind, the first number of subsequent investigators have successbeing that what works with one species may not fully cultured other species. However, less than necessarily work with another. In a general sense it 15% of the total species have been successfully should not be expected to. After all, the fertilizer cultivated in the laboratory from spore t o spore. applied b y a corn farmer is different from that Of those that have been cultured in the laboratory, used by the soybean farmer; obviously the requirenone has been thoroughly investigated from the ments of corn and soybeans are different. Difstandpoint of the effect of a wide variety of ferent species of myxomycetes might well also be environmental factors on the morphological charexpected to have different requirements for acters of the mature fructifications. growth and life cycle completion. 232



Another important consideration that must be borne in mind is that in the spore-to-spore cultivation of a myxomycete, several quite different and distinct processes must occur: spore germination, division of gametes, fusion of gametes, growth of zygote and subsequent plasmodial growth, initiation of the reproductive stage, completion of morphogenesis, and maturation of the fructification. What may be the optimum set of conditions for one of these processes may not necessarily be optimum for a l l or any of the others and conceivably could be such as to prevent the occurrence of one or more of the other processes, The successful cultivation of a species simply involves the establishment of a set of conditions (chemical, physical, and biological) in which all of these different processes may occur. This does not imply that the first successful procedure developed must necessanly be optimum for each of the major processes involved in successful spore-to-spore cultivation. It simply means that conditions must be such that each of the major processes is allowed to proceed to the extent that life cycle completion is not prevented. Thus, if spores germinate but plasmodia never appear, one must attempt to alter conditions in such manner as to favor myxarnoebal division, myxamoebal fusion, and plasmodial growth. With our present state of knowledge, the choice of such alterations must be made quite arbitrarily, but the patient and imaginative will find that there are almost innumerable such choices that may be made. Once fairly satisfactory plasmodial growth of a species is obtained, the refinement and improvement of cultural procedure becomes simpler, although complete success is not always attained at this, stage. Probably every investigator who has worked with the cultivation of myxomycetes has encountered at one time or another a plasmodial culture which would persist and remain active for long periods of time but would never enter the reproductive stage; until. this latter process occurred he could never even be really sure that he was actually culturing the species that he believed he was culturing. Very probably some nutritional factor necessary for the initiation of morphogenesis (Daniel and Rusch' showed that niacin was necessary for the initiation of sporulation in Physamm polycephalum) was missing from the substrate or some other condition of the environment was such that this process could not occur. When a yellow-pigmented plasmodium is first brought into culture, the initiation of the repro?'

ductive phase is usually readily accomplished by the use of a suitable source of visible radiation, provided that nutritional conditions are suitable, but, as will be discussed later, the factors that initiate reproduction in nonpigmented plasmodia are presently unknown. More often than not, the first attempts to bring a myxomycete species into cultivation from Spores results in cultures in which there are many other microorganisms. Sometimes such contaminants can be quite unexpected and disconcerting, as was the case one morning when this writer discovered a small but perfectly formed basidiocarp of the common field mushroom, Agaricus campestris, lifting the lid from the petri plate culture of what was supposed to be Didymium nigripes. This was somewhat unusual since the contaminants are commonly yeast, bacteria, and/or mold-type fungi. However, once a plasmodium is obtained in culture the investigator can devote some effort to the cleaning up of its biological environment. By using a culture medium which contains some antibiotic, other organisms can sometimes be controlled, but a question can justifiably be raised concerning the possible effects of such antibiotics on the myxomycetes themselves. It may be preferable to obtain the first plasmodial culture on a substrate free of such physiologically potent substances as antibiotics, the total effects of which on the various processes of myxomycetes are largely unknown, and then proceed to obtain relatively cleaner cultures by other less drastic means. This frequently can be accomplished by selecting an area where the plasmodium and surrounding agar are relatively free of other microorganism, cutting out a smalI block of agar on which the plasmodium rests, and transferring the block to a fresh, sterile agar plate. Gray3 obtained Physamm didermoides and Physarella oblonga in culture, cleaned the cultures in this manner, and maintained both species in culture continuously for nearly a year, and many other investigators have had similar successful results with other species. The ultimate goal in the culture of myxomycetes now appears to be that of obtaining them in pure culture on sterile, chemically defined medium that is free even of the dead remains of other microorganisms. Plasmodia o f . Physarum polycephalum have been obtained in such pure culture (Daniel and R u s ~ h : ~Daniel et al?j), as have those of Physamm flavicomum (Ross and Sunshine' " ') and Physarum rigidum (Henney and Henney: Henney and LynchS2),but at this time March 1976

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the unquestioned spore-to-spore cultivation of any myxomycete in which all the usual stages of the life cycle occur has not yet been achieved in this manner. The value of plasmodia derived from pure cultures o n chemically defined medium for certain types of biochemical studies involqing the isolation and quantitation of specific compounds is beyond question. In fact, in such types of studies pure cultures are obviously a necessity. However, I do question the necessity for such material in all cases of the study of myxomycetes per se. Having dealt with literally thousands and thousands of pure cultures of microorganisms for many years, one lesson has been firmly driven home: The behavior of a microorganism in pure culture may in very few ways reflect its behavior in its natural habitats. By the very nature of the niches that most saprobic microorganisms occupy in the world of living things, two things must be true: (1) They must be remarkably efficient in their carbon metabolism and (2) they are rarely if ever to be found unassociated with other microorganisms. Thus, those nonphotosynthetic microorganisms which could not compete with other microorganisms and at the same time efficiently convert substrate carbon to tissue carbon quickly disappeared from the evolutionary scene, and hence those forms which did not have these capabilities are no longer with us today. When a microorganism is placed in pure culture, away from the competition of other organisms and often in a nutritive situation which it would rarely if ever encounter under natural conditions, its metabolism may and often does change quite markedly. An example of this “deranged” metabolism that becomes apparent when a microorganism is taken from its natural habitat and placed in pure culture is easily provided by Aspergillus niger. This common black mold is ubiquitous and may be found on such diverse substrates as dead leaves, an old leather shoe, in soil, on the painted concrete wall of a moist basement, and in hundreds of other habitats where available carbon compounds exist in extremely low concentrations. Nevertheless it survives, completes its life cycle, and seems to thrive. What happens when it is brought into pure culture, does not have t o compete with other living organisms, and exists under “luxury” conditions with regard to energy source supply? That depends wholly on the strain used and the environmental conditions that are maintained for its culture. Under some conditions commercial 234


quantities of citric acid are synth.esesized, under others gallic acid is produced, and in still others amylase is synthesized in recoverable qyantities, to mention just a few. Such diverse capabilities of Aspergillus niger are obviously of great monetary value to the businessman and a source of satisfaction to the industrial mycologist but are of little or no value in explaining how this organism usually behaves. In all probability this must also be true for myxomycetes since such organisms are typically t o be found growing on substrates of low nutrient concentration and in association with many other living things - that is how they live and grow. As they usually occur, these organisms are subjected to all of the subtle influences (nutritional and otherwise) of the members of their biological environment. To completely eliminate this part of their environment while attempting t o study many of their activities creates an unreal and atypical situation and one that is rarely if ever encountered by any myxomycete as it normally develops. However, one must be practical since a plasmodium overrun by mycelia of Mucor, Rhizopus, or some other mold is usually of little value to the investigator, although such casualties frequently occur to myxomycetes under natural conditions, as is evidenced by the fact that it is not uncommon to collect or receive a specimen covered with spores that do not even remotely resemble those of any known myxomycete. There must be some middle ground in which the entire biological environment of the myxomycete has not been eliminated but, on the other hand, in which the desired organism is not completely overrun or destroyed by competing microorganisms. The various attempts to culture myxomycetes monoxenically (e.g., Ross’ and Henney”) probably rested on the practical desire to maintain specific organisms in culture but basically were attempts to achieve this middle ground. Although there is no evidence that symbiotic relationships exist between plasmodia and certain bacteria as Pinoy’ and Skupienski’ z o have suggested, the fact still remains that not infrequently a waning plasmodium can be revived by feeding it bacteria or yeast. I have already admitted the absolute necessity of having pure cultures for certain types of investigations and I repeat it here. However, I also repeat that pure cultures are not at all necessary for many kinds of worthy investigations that serve to expand our knowledge of the myxomycetes, and I wonder if sometimes too much time, effort,

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and materials are not involved in attempts to achieve pure cultures. There is an old saying that the horse comes before the cart. However, sometimes it may be expedient to put the cart before the horse for a time, but basically first things should still come first. Perhaps in the myriad attempts to obtain pure cultures we have too often put the cart before the horse. In support of my belief that there is a tremendous amount of information to be gained from cultures that are not axenic, I submit that there is a growing body of information concerning the genetics, fine structure, morphology, and physiology of myxomycetes that has been derived from studies of organisms that were not grown in pure culture. When practical laboratory culture methods have been devised for the majority of myxomycete species (or ideally all of them since species-wise this is a relatively small group), the effects of various environmental factors can then be tested and noted and thus information concerning the range of variation within each species will become increasingly greater. Such information will be of great value to the taxonomist not only in setting the limits of each species but also in the interpretation and accurate identification of many of the specimens taken from their natural habitats. In addition to those obvious advantages to the taxonomist, the potential morphological and physiological “fallout” from such culturing would be tremendous in volume and in the end would help us to a better understanding of this group.

THE QUESTION OF THE PHOTORECEPTOR One of the most remarkable attributes of myxomycetes may be observed in the transformation of a plasmodium, which is the assimilative structure, into reproductive bodies - sporangia, plasmodiocarps, or aethalia. This transformation (at least the readily visible part) is often quite rapid and typically is complete since rarely are the assimilative and reproductive structures of an individual myxomycete found coexisting. This shift from assimilation to reproduction is an inexorable one, and when the “commitment to sporulate” occurs in a plasmodium, the process cannot be reversed. Naturally such an irreversible system offers a splendid opportunity to study morphogenesis, from the morphological as well as the fine structural and biochemical standpoints. Although earlier workers (e.g., Skupienski’ O )

’

had suspected that light might be involved in triggering the onset of the reproductive phase, Gray3 offered experimental evidence that such was the case. Investigation of four yellowpigmented species, ten nonpigmented species, and one “variable plasmodial type” soon revealed that yellow-pigmented plasmodia did not enter the reproductive phase unless they were illuminated. Much of this research was conducted with the now ubiquitous Physarum polycephalum because it was the only species that could be easily cultivated in quantity at that time. However, enough other species with yellow-pigmented plasmodia were observed to permit the investigator to conclude that light initiates the onset of the reproductive stage in plasmodia of this type. Other investigations (e.g., Sobels and Van der Brugge,lZ3 Gehenio and Luyet,j0 Gray,36j37¶39Straub,I3* R a k o c ~ y , ’Daniel ~~ and R ~ s c h : ~K ~ e v e n i g , ~ ’ and Fergus and Schein”) conducted with other species as well as some of those observed by Gray3 served to confirm this general conclusion, and it is commonly accepted today. Next, it became increasingly obvious that if light was essential, there was some type of photoreceptor involved in the triggering action that light exercised in the initiation of reproduction. Since only yellow-pigmented plasmodia were so affected, it seemed logical to theorize that the photoreceptor was in some way associated with the yellow color and might even be one or more yellow pigments. In very simple experiments Straub13’ provided good evidence that a photoreceptor does exist and that the compound(s) formed as a result of the light reaction can, in fact, be transferred in such fashion that plasmodia which would not fruit now do so. Using plasmodia of Didymium nigripes, Straub found that the Iength of the period of illumination required for fruiting of underilluminated plasmodia could be shortened by allowing such plasmodia to feed on bits of properly illuminated ones. More recently Rakoczy’ O 0 has postulated that a compound (Substance A) is synthesized during the assimilative phase of the plasmodium in either light or dark. Thus, the synthesis of Substance A is light independent and occurs in the presence of the proper precursor when suitabie environmental conditions are maintained. She further postulated that when Substance A is illuminated, Substance B is formed and when it accumulates in sufficient quantity, sporulation is triggered. This is a workable hypothesis and is in agreement with many March 1976

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reported observations. For example, sporulation cannot be triggered until a sufficient quantity of Substance B is formed, which is in agreement with the observations of several workers relative to the indirect relationship that exists between intensity of illumination and time required for the onset of sporulation. Certainly Substance B could not be formed in sufficient quantity unless there was an adequate supply of Substance A; this may explain why there must be assimilation over a minimum period of time before reproduction can ensue. If Rakoczy’s postulate is valid, Substance A is the precursor of the photoreceptor and must be synthesized from a previously existing compound. Since Daniel and Rusch26 have shown that Physamm polycephalum requires niacin for sporulation, they concluded that either a distal metabolite of niacin is required for sporulation or that niacin induces changes that cause light sensitivity. Perhaps niacin or one of its metabolites is the precursor of Rakoczy’s “Substance A,” but at this stage we do not know. Of course, there may be an alternative view embodying the concept that some compound(s) present during the early part of the assimilative stage interfered with the onset of reproduction. If this is true, reproduction can occur only when the interfering compound(s) has been destroyed through a photochemical reaction, although the observations of Straub13’ d o not support this view. Several ideas have been advanced concerning the chemical identity of the photoreceptor. Gray3* measured as great as 910% increases in total riboflavin content in 4-day moist chamber cultures and suggested that part of the yellow coloration of the plasmodium of Physarum polycephalum may be due to the presence of this B-vitamin. This idea has gained little attention and no support, probably because the work was not conducted with axenic cultures, and perhaps should be discarded. However, because of the known photolabile nature of riboflavin, it might be well to reexamine the idea in terms of the alternative view expressed above. WolfI4 isolated two pigments from plasmodia of Physarum polycephalum which he designated Component 1 and Component 2 and concluded that only Component 1 had the characteristics of the photoreceptor. Both compounds were identified as pteridines. However, Rakoczy,” in her studies with Physarum nudum, concluded from the action spectrum she obtained that if the pigments of Physarum polycephalum and Phy-

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sarum nudum were the same, it was Wolf’s Component 2 which absorbed radiant energy. Other investigators have expressed different opinions regarding the chemical nature of the pigments. Thus, Dresden’ a concluded that the yellow color of plasmodia of Physamm polycephalum is due to the presence of a peptidelike pigment. Kuraishi et al.72 disagreed with both Wolf and Dresden, denying that the three pigments they obtained from Physarum polycephalum were either peptides or pteridines. They did not identify these pigments (designated A, B, and C) chemically but did state that A could easily be converted to B and B to C. Brewer’ later purified three pigments (which he also designated A, B, and C) from plasmodia of Physamm polycephalwn He reported pigment A to be a water-soluble, nonaromatic hydrochloride compound containing a hexaene chromophore and probably the amide function and one or more strongly basic nitrogen functions. Pigment B was considered an amphoteric compound with a polyene chromophore (a conjugated heptaene); it contained the carboxyl function and was alkali soluble. The absorption maximum and extinction of pigment B were found to be pH dependent reminiscent of what Gray3 had previously found with a crude extract of Physarum polycephalum plasmodium. Pigment C was very similar to pigment B in its properties and was considered to be an isolation artifact of pigment B. Daniel” stated that a carbonyl group is an essential part of the chromophore of a pigment of Physarum polycephalum, and since he found a pigment component that reacted with semicarbazide (which reacts with Schiff bases), he suggested that the strong basic function observed by Brewer might be explained on the basis of a quaternary form of a Schiff base. In contrast to the above investigators, all of whom worked with Physarum polycephalum, Nair and Zabkag extracted the plasmodia1 pigments from this as well as four other species. They reported recovering one pigment from Physarum gyrosum, four from Physarum polycephalum, three from Physarella oblonga, six from Didymium iridis, and, as might be expected, none from the white plasmodium of Didymium squmulosum. Of the 14 pigments they recovered, suggestions were made regarding the chemical identity of only 3. Thus, they reported a flavone in Physarum gyrosum and a phenolic compound in both Physarum polycephalum and Didym-urn iridis. If we ignore Gray’s3’ hint that the photolabile


riboflavin might in some way be involved in the light absorption process that ultimately leads t o reproduction, we are still faced with several choices regarding the chemical identity of the pigment(s) of Physarum polycephalum Is it a pteridine as Wolf'47 suggested, or could it be a peptide, a polyene, or a phenol? None of the suggestions advanced by the investigators noted above has been verified by other investigators using Physarum polycephalum as the test organism. However, same support of Wolfs hypothesis that the pigment may be a pteridine was supplied in 1973 by Rakoczy"' using Physarum nudum This latter worker found that the 12 fractions that she obtained from the plasmodium of this species all had the characteristics of pteridines. Nonetheless, at the present time we must recognize that we d o not possess any indisputable evidence regarding the chemical identity of this reputedly important pigment(s). In 1968 Gray and Alexopoulos4 stated ". . . it is evident that the important pigment component, whose role as a photoreceptor in the initiation of morphogenesis is now established almost beyond question, still remains without positive identification." Part of this statement is correct, but there is a distinct possibility that part may be incorrect. Certainly the statement that the pigment remains without positive identification is valid, but it may be that its role as a photoreceptor in the initiation of morphogenesis has not been established almost beyond question. The only evidence we have to d a t e is purely circumstantial: All yellowpigmented species that have been thoroughly investigated to date have been found to require light for sporulation. Perhaps we have all been misled: I by the data I obtained experimentally, many others by me and the data they derived independently. In spite of the fact that there is strong circumstantial evidence that a yellow pigment(s) is involved in the initiation of the process of morphogenesis, the actual proof is still lacking. In view of the several absorption spectra of the yellow pigment(s) that have been reported by several investigators, there is no question but that it is a photoreceptor. However, whether or not it is the photoreceptor we are seeking to involve in the initiation of morphogenesis yet remains to be established. Therefore, it might be profitable to initiate a search for a photoreceptor thus far ignored. Although the absorption spectrum of the crude plasmodial extract prepared by Gray37 was in

general agreement with the action spectrum of Physarum polycephalum, the possibility still exists that the yellow pigment is not the specific photoreceptor we are looking for. The preponderance of evidence seems to point to the fact that it is, but some room should be left for an alternative. The importance of identifying the photoreceptor (whether it is one of the yellow pigments or some other compound) cannot be overemphasized since the orderly sequence of biochemical reactions preceding observable structural changes can never be elucidated in its entirety until the first step in the sequence is known. The above comments are not meant to imply that no progress has been made in the investigation of morphogenesis - far from it. As a matter of fact, we are now in possession of a very sizeable number of facts; unfortunately, at this time they are largely unconnected. Thus, it has been reported that there is an extrusion of cellular water during sporulation. ( H a r ~ e r , " Daniel"), ~ that there are changes in nucleic acids, proteins, and polysaccharides (Daniel,'8~Z' Zeldin and Ward'49,150), that glucose inhibits sporulation (Daniel and Rusch"), that indole-3-acetic acid is present in spores but not plasmodia of Physarum polycephalum (Still,' 2 8 Still and Ward'"), that plasmodia treated for sporulation (i.e., illuminated under proper conditions) exhibit phenolase activity while untreated plasmodia do not (Daniel' ), that illumination of plasmodia results in changes in ATP level (Daniel'8~19*21),and that there is a shift in oxidises during sporulation (Ward' 8-' '). It has further been reported (Daniel") that light inhibits respiration of both sporulating and growing plasmodia and also the respiration of isolated mitochondria, that the inactivation of -SH groups in the plasmodium may stimulate sporulation (Ward and and that the probat:.: sites of the light reaction are mitochondria (Daniel' ). These and others are all interesting and important pieces of information, but in order t o understand the shift from assimilation to reproduction, they will all have to be linked together in some logical and orderly fashion - a procedure which cannot be accomplished at this time. For a more extensive discussion of the biochemistry of differentiation, the recent should be consulted. review of Sauer' Daniel and RuschZ5 made recommendations for the establishment of conditions favoring the induction of sporulation in axenically grown plasmodia of Physarum polycephalum: (1) optimal growth age, coinciding with nutrient exhaustion, (2) 4 days of

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incubation in the dark at 21.5”C on medium containing only inorganic salts and niacin, (3) exposure to light (wavelength 350 to 500 nm) for 2 hr, and (4) return to darkness. According to Daniel and Rusch, sporulation then occurs in 12 to 16 hr. Rusch”’ later reported that the evidence now indicates that about 2 hr after the period of illumination, plasmodia contain all of the messenger RNA required for sporulation. Thus, when plasmodia are manipulated under precisely controlled conditions, the time at which the “commitment to sporulate” point is reached by the plasmodium now seems established within fairly narrow limits. This knowledge should now make the work of the biochemist searching for the photoreceptor somewhat simpler and conceivably could lead to the identification of this elusive compound. One suggestion might be made. In their studies, Daniel and Rusch’’ understandably attempted to establish a set of conditions under which sporulation would occur as quickly as possible. Perhaps it might provide the biochemist with better opportunity if the process was spread over a longer time period. This could readily be accomplished by proper manipulation of temperature, pH, light intensity, or light quality (Gray34 ’). 13

INITIATION OF REPRODUCTION IN NONPIGMENTED PLASMODIA Although, as has been noted above, we still do not know the chemical nature of the photoreceptor in light-sensitive pigmented plasmodia, at least we have more information concerning the initiation of reproduction in these types than we have concerning this same process in nonpigmented plasmodia. On the basis of the results obtained by many investigators, it is now generally agreed that visible light (or very long-wave ultraviolet) triggers reproduction in yellow-pigmented plasmodia, although from that point forward our information is not complete. In the case of nonpigmented plasmodia, we do not even know what initiates this series of changes. Vouk’ 3 6 observed that nonpigmented plasmodia would sporulate eqdally well in light or darkness, an observation confirmed by Gray33 in his investigation of the plasmodia of ten nonpigmented species, one of which was later found (Naussg4) to be black when mature. However, in

1961 McManus’ reported that the nonpigmented plasmodium of Stemonitis fusca will not sporulate unless it is illuminated, and Straub’ ” stated that Didymium nigripes requires light for fruiting, a report verified by Fergus and Schein,” who emphasized the fact that a white plasmodium required light. These reports of a light dependence of Didymium nigri$es are rather easily explained because, even though the plasmodium of this species may appear white, Lieth74 isolated four pigments from it and one of these had an absorption spectrum similar to that of a crude plasmodial extract from Physarum polycephalum (Gray3 ’). The report of McManus’’ is still unverified and still unexplained. However, four possible explanations seem apparent. First, Stemonitis fusca may be an exceptional nonpigmented type, and hence we would have to discard the general conclusion that nonpigmented plasmodia do not require light for sporulation. Second, the plasmodia may appear nonpigmented to the unaided eye but in reality contain one or more pigments, as in the case ofDidymiumnigripes. Third, Stemonitis fisca conceivably could be another example of the type reported by Koevenig6’ and also by Fergus and S ~ h e i n . ’ ~ Koevenig found that Physarum gyrosum is light dependent for sporulation but that the plasmodium of this species is white. However, the plasmodium requires light and always turns yellow prior to fruiting. There is still another possible explanation: Perhaps at some stage Stemonitis fusca* is pigmented, if only transiently. It is not too difficult to fail to observe a change in plasmodial color. Thus, Gray33 reported that the plasmodium of Metatrichia vesparum was nonpigmented, while Naussg4 reported that it was black (other workers reported other colors). It was not until some years later that Wollman’ 4 8 observed that the plasmodium of this species is white at first and later becomes black. Hence, Gray had obviously failed to observe the black mature stage, and Nauss had failed to observe the early nonpigmented stage. The problem of Stemonitis fusca can be solved only by ( I ) careful observation ofplasmodial color throughout the life cycle, (2) determination of whether or not a pigment may be isolated from the plasmodium, even though it may appear white, and (3) verification of the report by McManus that light is required for sporulation by this species. Thus, aside from the presently unexplained

*Mims (Protoplasma, 77, 35, 1973) has reported that the plasmodium of Stemonitis vuginiensis varies in color from white to light yellow, and Indira (Trans. Br. Mycot. Soc., 56,25 1,1971) made a similar report for the plasmodium of Stemoniris herbaticu. Perhaps the plasmodia1 color of Stemonitis fuscu is similarly variable.

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report of McManus," wadable information indicates that light is not necessary for induction of the reproductive phase in myxomycetes with nonpigmented plasmodia. Nevertheless, such plasmodia obviously do enter 'the reproductive stage, and if a species with such plasmodia is successfully cultured in such manner that fruiting occurs, it often does so with remarkable regularity if a standard set of cultural conditions is maintained. For example, plasmodia of Physarum dideimoides, maintained under uniform conditions, usually fruited in about 21 days in the absence of light (Gray36). Either a factor other than light is responsible for initiation of the reproductive process or nonpigmented plasmodia do possess an innate rhythmical fruiting pattern such as that first reported by CayleyI4 for several species of Didymium and later erroneously for Physarum polycephalum by Seifriz and Russell.' I It seems quite likely that one myxomycete species may spend a greater or lesser period of time in the active assimilative stage than another species, and this may have been what Cayley and later Seifriz and Russell had in mind when they postulated an innate fruiting rhythm. However, it is far more probable that reproduction in .nonpigmented plasmodia is triggered by some factor (or factors) in the environment. Such factors might be chemical, physical, or biological. A fruitful approach might be to start with the assumption that the presence of or the exhaustion of some nutritional factor might be the triggering factor and then proceed with an investigation along these lines. The idea of the possible involvement of a nutritional factor is not wholly without basis since Daniel and Rusch' have shown that even though light is the triggering factor in initiating reproduction in Physamrn polycephalum,it cannot do so unless niacin is present in the substrate.

THE PROBLEM OF SOLUBLE MATERIALS INTAKE It is quite probable that the first serious investigator to patiently observe the behavior of an active myxomycete plasmodium noted its capability of engulfing solid particles. Small particulates such as bacteria, yeast, algae, fungus spores, and other bits of living or dead organic material were engulfed and then completely or partially digested, depending on the nature of the material. Later it was determined experimentally that other materials such as fmely divided particles of an insoluble

nontoxic dye might also be ingested but were later expelled unchanged on to the substrate by means of contractile vacuoles. Thus, the ability of plasmodia to obtain food by the engulfment of small solid particulates was established quite early in myxomycete studies and has been observed many times since by a variety of investigators. However, it has also long been assumed that in addition to the engulfment of particulates, plasmodia can obtain nutrients in solution by the diffusion of dissolved materials through the membrane(s) which bounds the plasmodium. On the basis of the known behavior of many other types of living cells, t h s seems a natural and logical assumption and could probably remain unchallenged had it not been for the observations of Guttes and Guttes4' in 1960. These workers reported that the plasmodium of Physarum polycephalum engulfs small droplets of liquid from the surrounding medium in the same manner that it engulfs small particulates -a process known as pinocytosis. Thus, the observations of Guttes and Guttes" have shown that water (plus any dissolved substances that it might contain) may enter a plasmodium in a way other than simple, direct diffusion through the bounding membrane(s). Thls then serves as a basis for raising the question as to whether water and the substances dissolved in it need ever enter a plasmodium in the manner considered typical for most living cells. The probability is that it does, but the fact still remains that at this time it has never been proved or disproved. This will be a very difficult problem to solve and, in fact, with presently available procedures may not be soluble beyond question. A necessary first approach would be to determine the frequency and extent of pinocytosis -a painstaking and unspectacular undertaking. If it is found that pinocytosis is of infrequent occurrence but that a plasmodium still thrives in a liquid medium in which the principal energy source is in solution, it could then be justifiably concluded that dissolved materials are not obtained principally in this manner and that they probably enter the plasmodium primarily by diffusion through the bounding membrane(s). On the other hand, if pinocytosis should be found to be a regularly and frequently occurring phenomenon, it would seem obvious that much (and perhaps all) of the plasmodium's nutritive and water requirements are obtained through pinocytosis and the engulfment of particulates. We will have to be satisfied with a solution based largely on circumstantial evidence and best inferences, but at this March1976

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time we do not even possess enough circumstantial evidence to make a final judgment. One further point should be made. If the preponderance of evidence should point to the conclusion that a plasmodium may obtain most of its water and materials in solution through the process of pinocytosis, it might be most illuminating to take some closer secondlooks at the nature of the membrane(s) that bounds the plasmodium.


THE PROBLEM OF PROTOPLASMIC STREAMING AND ITS COORDINATION One of the most interesting and easily observed processes in active myxomycete plasmodia is that of protoplasmic streaming, and it rarely fails to fascinate the student who observes it for the first time. A sense of the inexorable power and great vitality of the plasmodium is certainly felt by the observer of this phenomenon. Perhaps this feeling is due in part to the fact that many students, viewing plasmodial protoplasmic streaming for the first time, had at some prior time viewed the cyclic streaming in a cell of Elodea leaf. By comparison this cyclic movement appeared slow and sluggish, as in fact it was since Zurzycki' reported a rate of flow of 6 pm/sec in Elodea densa, while a rate of 1250 pm/sec in V o ~ k reported ' ~ ~ Didymium nigripes, and later Kamiya" reported the even higher rate of 1350 pm/sec for Physamm polycephalum . In addition to differences in rate of flow, protoplasmic streaming in plasmodia also differs from that in Elodea in that instead of appearing cyclic it usually alternates in a rhythmical pattern, flowing first in one direction, slowing to a stop, and then flowing back in the opposite direction. Vouk'36 referred t o this as progressive and regressive streaming - one progressive followed by a regressive streaming constituting one complete rhythm - but Seifriz' applied the term "shuttle streaming" t o describe this alternating direction of flow. Shuttle streaming is more easily observed in a strand of a large phaneroplasmodium, but apparently it begins quite early in the life of a plasmodium since Ross'o8 reported that it begins at about the 20-nucleate stage. observed and timed shuttle streaming Vouk' and reported that progressive streaming (in the direction of plasmodial movement) is always longer than regressive streaming. Since plasmodial movement obviously depends on the transportation of more protoplasm in the direction of movement than in the reverse direction, Vouk's

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view necessitates the assumption that a greater volume of protoplasm is transported during progressive flow than it is during regressive flow and that volume transported is directly related to duration of flow. Kamiya6' used a doublechamber volumeter which enabled him to measure volume of protoplasm moved during a single streaming. He correlated this with duration of flow and found that progressive flow is not always of longer duration since in 9 4 out of 229 complete streaming rhythms, regressive flow was longer than progressive flow. It seems possible that these differences can be explained on the basis of differences in age and vigor of the plasmodia as well as the culture conditions employed by various investigators since Cummins and Rusch' found that in well-fed plasmodia which were not migrating, duration of regressive flow was about the same as that of progressive flow but that in unfed migrating plasmodia duration of progressive flow was generally longer than that of regressive flow. As early as 1943 Seifriz' was able to list 11 different hypotheses that purported to explain the mechanism of protoplasmic streaming, citing 225 references in his review. Sixteen years later, Kamiya,63 reviewing the same subject as Seifriz, cited 428 references. Obviously there has been no dearth of interest in protoplasmic streaming, and, in view of the fact that there is still no agreement regarding the mechanism by which this process is accomplished, such interest will undoubtedly continue. Thus, to Seifriz's list of 11 possible explanations of streaming may be added the diffusion drag hypothesis of Stewart and Stewart,' 2 7 the contraction-hydraulic hypothesis (Jahns9 and Jahn et aL6*), and the suggestion by Rhealo4 that streaming may be due to peristaltic waves of contraction occurring along the plasmodial strands. More recently Park and Robinsong8 have proposed that streaming may be based on internal water distribution since they found that expansion and contraction of vacuoles in the anterior lobes of the plasmodium correlate with protoplasmic streaming. These latter investigators suggest that the viscosity of the outer gel layer increases or decreases as water passes into or out of the vacuolar system and that such changes in viscosity produce the motivating forces for protoplasmic streaming. This hypothesis should receive further attention, but it does seem possible that the expansion and contraction of vacuoles observed by Park and Robinson could conceivably be an effect rather than the cause of streaming. AIlen and Price4 assumed the energy-rich phos-

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phate bond t o be the immediate source of energy for streaming, and Kamiya et al.64 suggested that ATP formed during fermentation might be this source. These latter workers observed a decided increase in motive force about 6 to 7 min after a plasmodium had been treated with 2 X M ATP; addition of AMP did not produce increases comparable with those caused by ATP. Ts'o et al.' 3 4 also observed an increased rate of streaming when they introduced ATP directly into plasmodia by microinjection. Later Hatano and Takeuchi4 found that on the average there was 0.4 pnol of ATP per gram wet weight of plasmodium and there was no significant change in ATP content under conditions of low oxygen tension. While there now seems to be little doubt that ATP is the immediate source of energy for protoplasmic streaming, there have been some differences of opinion regarding how this compound is produced. Clark" and Kitching and Pirenne6' stated that streaming in plasmodia ceases with removal of oxygen. Moore' observed that plasmodia of Physarum polycephalwn did not survive long periods of anaerobiasis and was of the opinion that the energy for streaming was produced by aerobic respiration. However, Seifriz and Urback' found that streaming continued in atmospheres containing as little as 0.3% oxygen and concluded that respiration is primarily anaerobic. L ~ e w yobserved ~ ~ that the plasmodium of Physarum polycephalum disintegrated in pure nitrogen in 20 to 80 min but that the addition of 5% carbon dioxide prevented disintegration and that streaming continued for more than 24 hr. Ohtags found that removal of oxygen had n o effect on motive force generation, and Kamiya6 reported that conditions that suppress oxygen uptake increase the motive force back of streaming, while conditions that block glycolysis decrease the motive force if respiration is not affected. Hence, the more recent evidence seems to favor the concept that the ATP which provides the energy for protoplasmic streaming is formed primarily (perhaps entirely) by anaerobic means. If it is assumed that ATP is the source of energy for streaming, there still remains the question as to how the plasmodium transforms chemical energy into the mechanical work of transporting protoplasm. Camp'' suggested that an explanation of protoplasmic streaming might be related to muscle contraction, and in 1952 isolated from plasmodia of Physarum polycephalum a protein which like muscle actomyosin contracts in the

presence of ATP. Nakajimag' observed that contraction of plasmodia occurs at points where ATP is applied, and Hatano and O ~ s a w s4'a ~ isolsted ~ from Physarum polycephalum an actinlike protein which they were able to cross complex with muscle myosin. Later Oosawa et al.97 isolated a plasmodia1 myosin which they were able to cross complex with muscle actin. In the meantime various workers were searching for fibrils in plasmodia, and on the basis of the proportion of workers who have reported them, the evidence for their presence is overwhelming. Thus, Sponsler and Bath,' Kishimot~,~~ W o h l f a r t h - B o ttermann,14 4 6 Nakajima,' Nakajima and Allen?' Rhea,'03 Nagai and K a m i ~ a , ' ~McManus," U S U ~ , ' ~and ' Takata et al.' " all reported fibrils to be present in plasmodia. However, Stewart and .Stewart' and Terada' searched for fibrils and were unable to find such structures. It should be noted that while McManus" found fibrils in plasmodia of four other species of myxomycetes, she failed to find them in the aphanoplasmodium of Stemonitis ~ U S C Q .On the basis of these many reports there is no doubt that fibrils exist in plasmodia, but it has not yet been definitely established that the fibrils observed in ultrastructural studies consist of contractile protein complexes. However, Nachmias" has reported that the myosin that he separated from plasmodial actomyosin and then precipitated from a KC1 solution consisted of filaments that were similar to those seen in fixed and sectioned material. Once proof is obtained that establishes with certainty the exact mechanism of protoplasmic streaming, a great step forward will have been taken. And yet even at that point the real problem will then just be ready to be attacked. Even incontrovertible proof of the exact nature of the mechanism of protoplasmic streaming will be only a first step in the direction of solving the major problem - elucidation of the biochemical means by which streaming in the many different areas of a plasmodium is regulated and coordinated. In the study of protoplasmic streaming it seems more often than not to have been completely forgotten that the small strand that the investigator is viewing is but a small portion of a much larger living organism that ordinarily behaves as a unit. There is no question but that the potential for streaming resides throughout the plasmodium since Winer and Moore' early demonstrated that this potential exists in relatively small bits of

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protoplasm. However, this does not mean that each small area of plasmodium is acting independently with respect to streaming. In fact, the streaming in each small section is coordinated with the streaming in every other small section in such a manner that the whole plasmodium behaves as a single living unit; otherwise, a plasmodium would at intervals either tear itself apart and form a number of separate plasmodia or else it would flow into one large heap. That this coordinating mechanism occasionally ceases to function is evidenced by the fact that sometimes a single plasmodium in culture will for no observable reason separate into two or more smaller plasmodia, each of which will then behave as a single unit, even to the extent of forming separate groups of spor angia at separate times. There is one tantalizing hint as to the possible nature of the coordinating mechanism in plasmodia. The enzyme acetylcholinesterase has long been known to play a role in nerve impulse transmission, and the presence of this enzyme in plasmodia of Physarum polycephalztm was reported by Hatano and N a k a j i ~ n a . ~On ~ ? the ~~ basis of this finding, the Japanese investigators then suggested that perhaps plasmodia have a primitive neuromotor system which is functional in protoplasmic movement. The presence of acetylcholinesterase and acetylcholine was later reported in another species, Physarella oblonga, by Hoitink and Van Dijk,s3 who found that acetylcholine caused a lengthening in the duration of streaming, whereas adrenaline and noradrenaline caused a shortening. If the presence of acetylcholinesterase can be demonstrated by investigations of plasmodia of a variety of other species, then its general occurrence in plasmodia can be assumed and its possible role in streaming investigated. A possible first approach might be to quantitate acetylcholinesterase in plasmodia of Physamm polycephafwn before and at various intervals after the commitment to sporulate point had been reached in order to determine whether signifrcant differences in amounts could be detected. Similar quantitations could then be made on plasmodia of two species that behave quite differently when they sporulate. Gray and A l e x o p o u l ~ s@. ~ ~ 149) have suggested two such species as Arcyria nutans and -4rcyria cinerea, the first of which forms many closely packed sporangia from a single heap of plasmodium, while the plasmodium of the second breaks into a number of small, scattered masses, each of which forms a 242


sporangium. It seems possible that the coordinating mechanism may cease to function ‘at different points in the life cycles of two such species, and if the chemical components of the mechanism are identified, quantitation at the proper points in time might well serve to throw further light upon this subject.

THE IDENTITY OF THE GROSSER MYXOMYCETE MEMBRANES At several stages in the life cycle of a myxomycete a variety of easily observable membranes (the term membrane is used here in the most broad general sense) are formed. For example, the active plasmodium is completely encased in a slimy, membranous plasmodia1 sheath which is shed on to the substrate at the posterior end as the plasmodium migrates from location to location; presumably this sheath is formed at the anterior end, where it is seen to be thinnest. When the plasmodium enters the reproductive stage, a peridium is formed on the surface of each fruiting body. During the formation of fruiting bodies, spores are delimited and a spore wall is formed around each spore. After a spore has germinated and the germination products (swarm cells * myxamoebae) encounter adverse conditions, they may encyst, and during this process microcysr walls are formed. Similarly, when a plasmodium encounters conditions which favor sclerotization, it divides into discrete spherules (macrocysts), each of which is enclosed by a spherufe wlf. More attention has been directed by a greater number of investigators toward the identification of spore wall material than to the other gross membranes listed above. Thus, Boic7 was of the opinion that they consisted of pure cellulose, but Kiese166 stated that they were composed of “myxoglucosan.” Goodwin3’ was also of the opinion that spore walls were composed of cellulose, but Schuster’ l 4 concluded that they contained chitin in addition to cellulose. Koevenig7O merely stated that spore walls contained a polysaccharide, but McCormick et a1.82 came to the conclusion that they consisted of a galactosamine polymer and a glycoprotein. Such is the present state of our knowledge of the moststudied gross membrane in myxomycetes. Perhaps this is a trivial point, but the question must be raised as to why the term “spore” is still retained for the structure that bears this name in the myxomycetes since it is the immediate forerunner


of gametes, the usual site of meiosis, and is certainly not a spore in the usual mycological sense. No attempts seem to have been made to establish the chemical identity of the microcyst wall - the membrane formed when a myxamoeba encysts. However, it seems altogether possible that it could be similar chemically to sclerotial spherule walls, which McCormick et aL8' reported as consisting of galactosamine and a glycoprotein. The presence of galactose, which occurs rarely in nature, may indicate that myxomycetes are not closely related t o the fungi or euprotists (Oliveg6). The plasmodium is usually. considered a naked protoplasmic body, and it is true that this structure is not enclosed by a rigid wall. On the contrary, it is encased in a slimy, elastic sheath which apparently is continuously formed at the advancing anterior end of the plasmodium and deposited as waste on the substrate at the posterior end, thus leaving the familiar "slime track." m e a l o 3 considers this sheath a type of exoskeleton, and both he and McManussS have reported it to be fibrillar in structure. Jahn et aL6' have shown that this sheath may be expanded but will return to almost normal size even when expanded two or three diameters by forcing air into the plasmasol. Few investigators have directed their attention toward establishing the chemical identity of this sheath material. However, Simon and Henney'" reported that it consists of a glycoprotein, but McCormick et a1." have stated that it is a sulfated galactose polymer containing traces of rhamnose. Concerning the chemical nature of the peridium there is little information. The nature of the peridia of myxomycete fructifications varies with species and to a certain extent with the conditions existing during sporulation. In some species (notably those of the Physurida) there are greater or lesser amounts of limy material deposited in and on the peridia, but in others such as species of Trichiu or Sternonitis, the peridia consist of thin membranes devoid of lime. The lime in peridia is considered to be CaC03, but the chemical nature of nonlimy peridia yet remains to be established. Obviously all these membranous structures require further investigation. Since the position of the myxomycetes in the classification of living things is still not settled, it would be most helpful to know more about the chemical identity of each of these membranes so that more plausible rela-

tionships or lack of relationships with other groups of organisms might be postulated. Unfortunately, as briefly noted above, our information in this area is still quite scanty.

SUMMATION From the above short list of problems it should not be implied that these are the only outstanding questions regarding the myxomycetes. On the contrary, there are many other still unanswered questions; the ones listed above just happen to be major problems that have been with us a great many years. The above account is presented primarily to call attention to these problems and is certainly not meant to imply that little progress has been made in the area of myxomycete studies - far from it. In spite of the preponderance of attention paid to just one species, during the past four decades a very sizeable body of important' and relevant information has accumulated. For example, although a general idea of the developmental cycle had been known for years, in 1931 the complete details of the myxomycete life cycle had not been established; they could not be given with accuracy since the point at which meiosis occurs was generally believed to be at the time of the nuclear divisions that occurred in the sporangium just prior to spore delimitation, although there was some small dissent from this view. Now, because of the discovery of synap tonemal complexes in maturing spores (Aldrich,* Aldrich and M i m ~ ,Haskins ~ et aL4'), it appears that Von S t o s ~ h " ~was correct in his early view that meiosis usually occurs in the developing spores. Any attempt t o conduct research on the genetics of the myxomycetes would have been considered foolhardy and next to impossible in the early 1930s. Now, in view of the researches of such investigators as Collins,' Carlile,' Dee?' and others, we are beginning to gain an understanding of some of the facets of myxomycete gene tics. Rusch and his many associates in Wisconsin have made a tremendous number of contributions t o our knowledge of Physarum polycephuium, and it seems probable that such work may ultimately give us a more or less complete picture of the biochemistry o f differentiation in this muchstudied species. It is hoped that the techniques developed and refmed in connection with the study of this species may then be applied successMarch 1976

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fully to investigations of other species which will then give us a much more well-rounded picture of the entire group. In the taxonomic area we are greatly indebted to C. J. Alexopoulos and the late Professor G. W. Martin for their excellent treatise T&e Myxomycetes, which appeared in 1969. While all taxonomic problems have not been solved, this work has served to bring considerably more order into a complex and often frustrating area of myxomycete studies. However, Olive’ has recently introduced a major change in classification by t r a n s f e r ring t h e e xosporous Ceratiomyxomycetidae, recognized by Martin and Alexopoulos as a subclass of Myxomycites, to the Protostelia, where it now has family status. At the moment, the taxonomy of the myxomycetes is probably in about as satisfactory a state as may be expected until the range of variation of each species is established through laboratory cultivation followed by careful observation of all or most of the species in this group.

Much credit must also be given to Alexopoulos and his students for their varied researches in areas outside the taxonomic one. Whether or not it has been deliberate is not known, but they have obviously directed their attention away from Physarum polycephalum and hence have helped to present us with a more comprehensive view of the myxomycetes as a whole, not only from the standpoint of their laboratory cultivation but also from those of their morphology, physiology, fine structure, and genetics. Although the data are coming forth slowly and from widely different sources, more and more evidence has accumulated that supports the view that myxomycetes are not fungi and certainly not plants. Exactly where they belong in a system of classification of living things cannot be stated with any certainty at this time, but as more and more biochemical evidence accumulates, we will be in a much more sound position t o speculate regarding their affinities. Hopefully this will occur long before the passing of another four decades.

REFERENCES 1. Alexopoulos, C. J., Gross morphology of the plasmodium and its possible significance in the relationships among the myxomycetes, Mycologiu, 52, 1, 1960. 2. Aldrich, H. C., Pre- and postmeiotic events in spores of the myxomycete, Didymium iridis, J. Cell Biol., 47, 4a, 1970. 3. Aldrich, H. C. and Mims, C. W., Synaptonemal complexes and meiosis in myxomycetes, Am. J. Bot., 57,935,1970. 4. m e n , P. J. and Price, W. H.,The reIation between respiration and protoplasmic flow in the s h e mold, Physarum polycephalum, Am. J. Bot., 37, 393, 1950. 5. Bay, A. de, Ueber die Myxomyceten, Bof. Z., 16,357, 1858. 6. Bary, A. de, Die Mycetozoen, Ein Beitrag zur Kenntnis der niedersten Thiere, 2. Wiss. Zool., 10, 88,1859. 7. Boic, D., bber den chemischen Character der Peridie, des Kapillitiums und der Sporenmembranen bei Myxomyzeten,Acfu Bot. Insr. Bot. Univ. Zagreb, 1,44,1925. 8. Brandza, M., Sur l’influence de la chaleur et de I’evaporation rapide sur les Myxomycktes calcaries vivant en plein soleil, C. R. Acad. Sci. (Paris), 182,488, 1926. 9. Brandza, M., Sur la polychromie des Myxomycdtesvivant en plein soleil, C.R. Acad. Sci. (Paris), 182,987, 1926. 10. Brewer, E. N., Culture and chemical composition of the slime mold, Physarum polycephalum, Diss.Absfr., 25, 6190, 1965. 11. Camp, W. G.,A method of cultivating myxomycete plasmodia, Bull. Torrey Bot. Club, 63,205, 1936. 12. Camp, W. G,The structure and activities of myxomycete plasmodia, BUN. Torrey Bot. Club, 64,307, 1937. 13. Carlile, M. J., Cell fusion and somatic incompatibility in myxomycetes, Ber. Dfsch Bot. Ces., 86, 123,177, 1973. 14. Cayley, D. M., Some observations on mycetozoa of the genus Didymium, Trans. Br. Mycol. SOC.,14,227,1929. 15. Clark, J., Ober den Einfluss niederer Sauerstoffpressungen auf die Bewegungen des Protoplasmas. Ber. Dfsch. Bot. Ces., 6, 273, 1888. 16. Collins, 0.R,Experiments on the genetics of a slime mold, Didymium iridis, Am. Biol. Tach., 31, 333969. 17. Cummins, J. E. and Rusch, H.P., Natural synchrony in a slime mold, Endeavour, 27, 124, 1968. 18. Daniel, J. W., Photo induced polysaccharide synthesis during differentiation of a myxomycete, Fed. Proc., 23, 320, 1964. 244


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IV. Purification and observations of some physico-chemical properties, Annu. Rep. Res. Group Biophys. Jap., 4, 25, 1964. 49. Hatano; S. and Takeuchi, I., ATP content in myxomycete plasmodium and its levels in relation t o some external conditions, Protoplusma, 52, 169, 1960. 50. Henney, M. R., The Mating Type System in the Myxomycete Physarurnflavicomum and the Ascomycete Anixiopsis stercorurh, F'h.D. dissertation, University of Texas, Austin, 1966. 51. Henney, H. R. and Henney, M. R, Nutritional requirements for the growth in pure culture of the myxomycete Physurum rigidum and related species, J. Gen. Microbiol.. 53. 333, 1968. 52. Henney, H. R. and Lynch, T., Growth of Physarurn flavicomum and Physarurn rigidurn in chemically defined minimal media, J. Bacteriol., 9 9 , s 31, 1969. 53. Hoitink, A. W. J. H. and Van Dijk, C., The influence of neurohumoral transmitter substances on protoplasmic streaming in the myxomycete Physurelia oblonga,J. CeN. Physiol.. 67, 133, 1967. 54. Howard, F. L,The life history ofPhysurum polycephalum, Am. J. Bot., 18, 116, 1931. 55. Howard, F. L., Laboratory cultivation of myxomycete plasmodia, Am. J. Bot., 18,624, 1931. 56. Howard, F. L., Nuclear division m plasmodia of Physarurn,Ann. Bot., 4 6 , 4 6 1 , 1932. 51. Howard, F. L. and Cunie, M. E., Parasitism of myxomycete plasmodia on the sporophores of hymenomycetes, J. Arnold Arb., 13, 210, 1932.


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58. Howard, F. L. and Currie, M. E., Parasitism of myxomycete plasmodia on fungous mycelia, J. ArnoldArb., 13,438, 1932. 59. Jahn, T. L., Protoplasmic flow in the mycetozoan, Pbysarum. 11. The mechanism of flow; a reevaluation of the contraction-hydraulic theory and of the diffusion drag hypothesis, Biorbeology, 2, 133, 1964. 60. Jahn, T. L., Rinaldi, R. A., and Brown, M., Protoplasmic flow in the mycetozoan, Pbysarum. I. Geometry of the plasmodium and the observable facts of flow, Biorbeology, 2, 123, 1964. 61. Kamiya, N., The protoplasmic flow in the myxomycete plasmodium as revealed by volumetric analysis, Protoplasma, 39,344,1950. 62. Kamiya, N., The rate of the protoplasmic flow in the myxomycete plasmodium I, Cytologia, 15, 194,1950. 63. Kamiya, N., Protoplasmic streaming, Protoplasmologia, 8, Pt. 3a, 195 9. 64. Kamiya, N., Nakajima, H., and Abe, S., Physiology of the motive force of protoplasmic streaming, Protoplasma, 48, 94, 1957. 65. Kerr, S. 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