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Department of Ecology and Evolutionary Biology, Princeton University, Princeton, New Jersey 08544
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ABSTRACT |
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Cellular slime molds are soil organisms, yet since they were discovered in 1869 they have been studied on agar surfaces. Here the behavior of a number of species is examined and it is evident that they have different responses to directional light and they all thrive in the presence of soil. While phototaxis clearly plays a significant role in their ability to come to the soil surface for dispersal, even more important are gradients in the soil: both temperature gradients known from earlier studies, and as we show here gas gradients, presumably ammonia as a repellent and oxygen as an attractant. There are numerous differences in both morphology and behavior among slime mold species, some of which are likely to be the result of natural selection to particular habitats, while others could be explained more easily by neutral phenotypic variation.
Key words: adaptation, gas-taxis, phototaxis, soil ecology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONDISCUSSIONLITERATURE CITED |
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). In the beginning of the 20th century only a handful of species were known, but today about 100 species have been identified ( James Cavender pers comm). This slow realization of their richness in nature is no doubt because they are so small and inconspicuous in the soil and those working with them have had to rely entirely on bringing them out in enrichment cultures in the laboratory. There has been hardly any work with them directly in the soil: Since 1869 cultivating them on agar in Petri plates has been the norm. It might be helpful to remind the reader that the life cycle of cellular slime molds is unusual in that feeding occurs first by the separate vegetative amoebae and when the bacterial food has been consumed, starvation is followed by the aggregation of the amoebae to form a multicellular organism that ultimately becomes a fruiting body, a stalk made up of vacuolated, dead cells and a terminal mass of unicellular spores, or sorus.
Behavior in the soil.— –
It has been known for a long time that the large species of slime molds are capable of migration and tend to feed below in the soil and migrate upward to fruit at the surface, a location that is optimal for spore dispersal because of the traffic of invertebrates that pick up the adhesive spores and carry them to fresh patches of bacterial food. This upward movement needs an explanation because it is known that the migration stage is affected by gravity, and in the absence of all other cues (light, temperature and gas gradients) they will migrate downward (Häder and Hansel 1991; see also Bonner et al 1988, where, in the presence of ammonia, all tactic responses are blocked: slugs and fruiting bodies hang downward from an inverted agar surface).
It is well known that there are a number of taxes that could be responsible for the upward movement. The migrating cell masses are highly phototactic, which certainly would play a significant role in their upward movement in the daytime. They also are extremely sensitive to temperature gradients, and Whitaker and Poff (1980) showed that they have a sophisticated mechanism letting them, in the absence of light, to move upward in both night and day. During the day they move upward toward the warmth, but in the cool of the night they become negatively thermotactic and continue to move upward, in this case to the now cooler surface.
What we find in this study is that gas gradients also play a key role. In the absence of directional light and temperature gradients the migrating cell masses (now commonly called slugs, although more formally they are known as pseudoplasmodia) nevertheless will migrate upward to the surface and, as we shall see, there is good evidence they are guided by gas gradients.
The other major object of this study is to survey some of the common large species of cellular slime molds found in the soil, to examine the significance of the differences in their morphologies and the differences in their behaviors. In the case of the former we briefly shall review the life history and morphology of eight species and this will be followed by an examination of their different behaviors, with special reference to phototaxis and gas orientation. With this background the slime molds then will be examined in soil, to do a comparative study in an environment that more closely resembles their natural one.
Slime mold morphology.— –
There are a number of major differences in the morphology of cellular slime mold fruiting bodies. (For details and references see the monograph of Raper 1984.) All species have a slender stalk supporting a mass of spores or sorus. (There may be multiple sori.) Of these some are small, generally 1 mm or less in height. The number of amoebae that enter an aggregate determines size in cellular slime molds; feeding and growth occur first and is followed by the development of a multicellular fruiting body. Many more species are longer than 1 mm, some as long as 10 mm. Because only the larger species migrate there is a fundamental difference between large and small species in that the smaller ones fruit where they have fed, while the larger ones can move away to a remote spot for spore dispersal. Those that do migrate can either do so forming a long stalk as they move, while others have a stalkless migration such as that found in the familiar species, Dictyostelium discoideum.
Migration with a stalk is found in D. mucoroides and D. purpureum. There is a variant of D. mucoroides that is of special interest: it is D. mucoroides var. stoloniferum that has secondary fruiting bodies. As is well known for other species the spore masses or sori produce a germination inhibitor. However in this variant there is none, so when a sorus collides with the substratum the spores immediately hatch amoebae that aggregate without feeding and produce a second generation of small fruiting bodies (Cavender and Raper 1968).
There is also variation in the placement of the spores on the stalk. A common form is a single terminal sorus, as is found in many species, including D. discoideum. However in species of Polysphondylium, an equally abundant genus, there are numerous sori on one stalk, radiating outward in regular whorls, each held out by a delicate ministalk. In the case of D. rosarium, instead of whorls the sori are left behind at fairly regular intervals-like beads on a rosary-along the solitary stalk.
Finally D. polycephalum is unusual in that it has a long, thin slug that migrates considerable distances, and when it fruits 2–6 papilla appear in a globular cell mass, each of which forms a small fruiting body, the stalks of which cling together for three quarters of their height, and then they separate to form a whorl of sori.
These eight larger species with their different morphologies are drawn diagrammatically (FIG. 1). Also indicated is their geographic distribution, based on the work of Raper (review 1984) and Cavender and his associates (Swanson et al 1999).
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, Häder and Burkart 1983). It is also known that low concentrations of ammonia gas inhibit phototaxis (Bonner et al 1988).
For this aspect of the study, five of the eight species were used. A Petri dish containing nonnutrient agar was inoculated in the center with a loopful of Escherichia coli B/r and slime mold spores were added. The plate was placed in a darkroom approximately 60 cm from a 4 watt incandescent bulb (resulting in a temperature of 26 C). Once migration had run its course the tracks were marked on the back of the plate with a glass marking pen and photocopied. (FIG. 2 shows such tracings for four species.) Note that there is considerable variation in the angle of the migration paths. This was measured by drawing a line from the inoculation spot to the terminal slug and with a protractor measuring the angle deviating a straight line toward the light.
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.)
For each species, in addition to the angle, the width of the slug was measured as an indication of its size. In the first three species the angle variation did not correlate with size, but it clearly did so in the case of D. discoideum: the larger the slug the poorer it was in orientating (FIG. 3). D. discoideum is the only species of the four that has stalkless migration; the other three species always have a stalk right up to the sensitive tip as they crawl along. Because orientation involves the lens effect, the light intensity at the dark side of the slug is affected by the slug thickness in D. discoideum. Perhaps the optical properties of the stalk play a role in the different behavior of the other species.
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:334). However they both clearly show gas orientation, as do all the other species tested. The evidence for this is the simple observation that when they are inoculated in one spot on the agar, all the slugs move out from one another like spokes in a wheel.
Gas-taxis.— –
The matter of gas orientation is of special significance when considering the existence of slime molds in the soil. It has long been known that slugs and rising fruiting bodies emit ammonia gas that causes them to move away from one another; indeed it is the cause of the fruiting bodies rising at right angles from the substratum by equalizing the concentration of the gas on all sides (Bonner and Dodd 1962). The best evidence for gas orientation is demonstrated by placing charcoal near a slug or a rising fruiting body: The cell masses will orient into the charcoal because the charcoal adsorbs ammonia.
It was discovered in this study that slime molds also will orient toward soil; clearly, like charcoal, it also removes ammonia, because it too has a large surface for the adsorption of the gas. However, soil is less effective than charcoal; the orientation toward a small heap of soil is less dramatic than to a comparable heap of charcoal but nevertheless is clearly evident (FIG. 4).
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). Soil experiments.— – To study slime molds in the soil, the following method was devised. Nonnutrient agar about 4 mm deep was placed on the bottom of an 8 cm diam, 5 cm high glass-crystallizing dish and inoculated with a loopful of E. coli B/r and slime mold spores, sometimes in more than one spot. These samples were covered with soil from a deciduous forest. The soil was unsterilized and slightly damp. The dishes were covered with the top of a Petri dish and placed by a north window in the laboratory.
Initially the agar was covered with soil about 7–10 mm deep, although the surface of the soil was so uneven it was difficult to determine an exact figure. It was immediately obvious in the case of some species that the slugs and the fruiting bodies that emerged were exceptionally large, more so than the controls on the surface of plain agar. Clearly these species thrived under these conditions.
P. pallidum.. The most dramatic improvement in fruiting body size occurred with this species. Normally, on agar, P. pallidum is considered a relatively delicate species and if grown on agar the largest fruiting bodies will have about 12 whorls. Those that emerged were distinctly larger, occasionally reaching stalks with 20 whorls. This would imply that the soil was acting directly on aggregation, permitting the aggregates to be larger.
The other striking feature of their fruiting bodies was that they only rarely emerged vertically, as they do more commonly on agar, but more often migrate horizontally along the soil surface. Perhaps this is because, as mentioned earlier, the soil attracts the elongating tips of the developing fruiting bodies, although the effect of gravity on their greater weight cannot be ruled out.
P. violaceum.. This species was similar to P. pallidum in its appearance emerging from the soil. The difference came on the agar controls: In the absence of soil, as is well known, it develops more robustly than P. pallidum. Therefore the salubrious effect of soil was less evident. Here also the fruiting bodies are often horizontal on the soil surface.
D. mucoroides.. The fruiting bodies of this species appeared healthy as they emerged from the soil, but they did almost as well on agar alone. Mature specimens tended to surface more vertically than Polysphondylium.
D. mucoroides var. stoloniferum.. On agar this form spreads out more and fruits in a larger area than any of the other species. This tendency is especially marked on the soil surface where it clearly thrives. The initial stalks not only radiate from the point of inoculation, but small secondary fruiting bodies are evident at the periphery.
D. purpureum.. This species, both with and without soil, closely resembled D. mucoroides.
D. discoideum.. This species similarly seemed to thrive on the soil surface, as it did on agar.
D. rosarium.. The development of this species was enhanced greatly by the presence of soil, as opposed to agar, in a manner comparable with its effect on P. pallidum. On agar 5–25 sori beaded along the fruiting body stalk compared to 30–50 in soil. On soil more stalks emerged horizontally in contrast to their vertical emergence on an agar surface.
D. polycephalum.. While this species formed abundant long, thin slugs on agar, it was reluctant to fruit. However, in soil it produces masses of fruiting bodies.
It is known from earlier work that cellular slime molds could be isolated in soil at a depth of 20 cm, although they are far more abundant near the surface (review: Raper, 1984:43). This raises the questions: Can deep amoebae aggregate and migrate to fruit at the soil surface? Or have their spores or amoebae simply been washed away by rain? Or consumed by earthworms, as Huss (1989) demonstrated? The fact that they are sparse at greater depths would seem to indicate that aggregation well below the surface is unlikely.
To find the limits of vertical migration the loopsful of E. coli and P. pallidum or D. discoideum spores were covered with soil of different depths of approximately 1.5, 2 and 4.5 cm. Under these conditions the soil was too deep to allow the fruiting bodies to emerge; the upper limit was below 1.5 cm.
Another unknown was whether the size of the soil particles made a difference. Some of the forest soil was dried and sieved (mesh sizes were 10, 60 and 230). The first consisted of dirt particles about 1 mm diam; the second one was like sand; the third was fine silt. Water was added to each and they were placed, at a depth of 5–10 mm, over agar inoculated with separate clumps of E. coli B/r containing the spores of P. pallidum, D. discoideum or D. rosarium. The slime molds emerged through all three porosities with equal success; clearly migration occurred through a wide range of soil particle sizes. This was further supported by placing a layer of charcoal, or fine Darco, or diatomaceous earth, or purified sand over the developing slime molds; in each case they emerged to produce similar healthy fruiting bodies.
Further experiments were run on dishes with and without soil, in the light and the dark. As has been known for many years slime molds raised in the dark on agar will produce larger fruiting bodies than in the light (Potts 1902, Harper 1932). Therefore it was not surprising to find that the cultures on agar in the dark were almost as large as those emerging from soil. The dishes with soil in the dark were indistinguishable from those raised in soil in the light of the north window. Because the number of amoebae that aggregate to a center determines size, perhaps the aggregations are equally large when covered with soil because they too are in the dark.
Gas-taxis in the soil.— – Does gas-taxis play a role in the emergence of slime molds in soil? The value of surface fruiting to spore dispersal was demonstrated when unsterilized forest soil in the crystallizing dish contained a small spider, an insect larva or a mite. In no time at all the fruiting bodies and sori disappeared; the roaming invertebrate had spread them.
One can show that gas-taxis is necessary in those situations where all the other taxes can be ruled out. For instance, as mentioned earlier, we know that temperature gradients can aid slug migration in soil, but in our experiments in dishes at room temperature with less that 1 cm of soil, it was unlikely that temperature gradients existed. Also if phototaxis is ruled out, either by doing the experiment in the dark or using those species incapable of orienting to directional light, it is clear that there must be another orienting mechanism responsible for the upward movement.
Orientation by gas gradients is the obvious answer. Two gasses can orient migrating slugs: ammonia, as we have just discussed, and oxygen. It was shown by Sternfeld and David (1981) that slugs respond to oxygen gradients: they move toward higher concentrations of oxygen (review: Bonner 2003). In soil both of these gasses could be involved in the upward movement of slugs: Ammonia would be more concentrated in the soil than in the air above it, and the reverse would be true for oxygen; therefore both gases could be involved in emergence.
To test these possibilities, an experiment was run in which there was no temperature gradient or directional light and a slice of soil was suspended so that there were two soil-air interfaces close to one another, but pointing in opposite directions. Soil was placed between two nylon screens (pore size: 0.5 mm) 12 mm apart, and small chunks of nonnutrient agar inoculated with E. coli B/r and P. pallidum were lodged in the middle, halfway between the two surfaces (FIG. 5). In this way the gas gradients would exist at both surfaces. The soil slice was placed in the middle of a plastic bottle that was kept suitably humid at both ends. After 4–5 d, fruiting bodies appeared on both sides; the slugs moved both upward and downward, and this was true in the dark as well as in uniform light. The obvious explanation is the two gas gradients of ammonia and oxygen on each side of the soil slice oriented the slugs to both surfaces.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONDISCUSSIONLITERATURE CITED |
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First it should be pointed out that cellular slime molds are especially healthy when raised in the soil. That they are well adapted to their natural environment was demonstrated in an interesting experiment by Ponte et al (1998) who showed that a mutant in which a knockout of a gene resulted in the absence of a cell-adhesion molecule nevertheless grew well on an agar surface, yet thrived poorly in the soil. The molecule is adaptive and needed in its normal soil environment but is of no importance when grown on an artificial agar surface.
The larger species of cellular slime molds that are capable of migration thrive within the top 1 cm of soil. As a rule they move to the surface for fruiting-body formation and sporulation, an optimal position for spore dispersal by passing invertebrates. They have many physiological mechanisms to guide them upward: phototaxis in the daytime, thermotaxis that operates both day and night, as do gas-taxis in gradients of ammonia and oxygen.
Why are three mechanisms needed to achieve the same goal? The most likely answer is that there is significant selection pressure for dispersing the spores at the surface and that any mechanism that arose through variation and mutation would be retained to assure the reliability of the upward movement. The dominant system seems to be gas-taxis because this is the only system that is present in all species and is independent of such environmental conditions as are phototaxis, and to a lesser degree thermotaxis.
The key evolutionary question is why is there so much variation in the morphology of different species, and similarly why is there variation in their physiology, such as their different responses to directional light? What are the adaptive advantages of these various morphologies and behaviors? After all, if we think of larger animals and plants we can readily see the connection between their shapes and how they manage in their ecological niches: Tigers have great jaws and teeth to obtain their diet of raw meat; trees have leaf patterns so that they can be optimally effective in catching sunlight in their particular environment. All that is obvious to us, but we have no such ready explanations for the different shapes or behaviors of cellular slime molds.
The only cases where we have some clues are in a few species that have a restricted geographic distribution. Of the species examined here D. mucoroides var. stoloniferum is found only in moist tropical rain forests, and D. rosarium in arid areas. The fact the D. mucoroides var. stoloniferum has lost its germination inhibitor and as we have seen is capable of spreading horizontally large distances on the surface of the soil, we could imagine it is ideally suited to tropical conditions where the relative humidity remains high at all times. The chances of desiccation are minimal and therefore spreading both primary and secondary sori as they radiate outward is safe and offers the maximal numbers of spore masses for distribution by passing invertebrates.
The case of D. rosarium is harder to rationalize. They do not respond to directional light yet they clearly manage to rise to the surface; presumably they rely on gas-taxis and possibly on thermotaxis. What is not known—and this is the key issue—is how they develop and fruit under relatively dry conditions.
The most straightforward example of adaptation to a particular environment comes from Cavender’s (1978) discovery of a new species, D. septentrionalis, in the cool environment of Alaska. It grows optimally at 16 C and fails to develop above 20 C, far below the temperature range of any other known species.
These are exceptions because most species are cosmopolitan in their distribution. They not only are found globally but also are found side-by-side in both temperate and tropical localities (Swanson et al 1999). This is true of species of both Dictyostelium and Polysphondylium. There is no clue as to why some have multiple sori, either in whorls or in a row on the stalk, and others have a single terminal sorus, and why some species have stalkless migration and the others not. And they frequently exist together in the same plot of soil.
As is so often the case with such questions there are two possible answers. It could be that selection is operating, but we have no clue as to what conditions favor one kind of fruiting body over the other. The alternative hypothesis is that their differences have arisen by chance mutation and that there are no advantages of one form over the other in their soil environment; they are selectively neutral.
The fact that the species differ both morphologically and in their phototactic behavior yet thrive as neighbors in the same environment strongly points to the second hypothesis. This is by no means a unique situation: Consider the great variety in morphology of different species of radiolaria and foraminfera. Here too each form appears to be as successful as the other; there are no evident advantages to a particular shape as D’Arcy Thompson had argued (1961:196ff, Bonner 1996:17ff). Each appears to be equally adapted to their common environment. This is a neutral-phenotype theory of evolution, where a variety of morphologies are equally successful in a particular environment, in contrast to the neutral-gene theory of Kimura (1983). In the former natural selection fails to discriminate among phenotypes, each of which has a separate genotype; in the latter selection fails to discriminate among genotypes and they all have the same phenotype.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: jtbonner{at}princeton.edu
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONDISCUSSIONLITERATURE CITED |
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———. 2003. The evolution of development in the cellular slime molds. Evolution and Development 5:305–313.[CrossRef]
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