Abstract:
A mechanism of variation and adaptation which is phenotypic rather than genotypic was found with the morel mushroom. Two types of Morchella variation were studied—spore strain variation manifested through an anomaly on petri plates, and differences in ascocarp (fruiting body) tissue thickness as a method of coping with weather. A phenotypic origin for the variations was indicated by functionality and a distribution which was random rather than Mendelian. Visible examples of stable variations in interbreeding populations indicate that this mechanism is widespread in the plant and animal kingdoms.

Introduction:
Phenotypic variation as an adaptation mechanism was not previously known to exist in biology. It is manifested as stable variations which are distributed randomly in an interbreeding population, where a Mendelian distribution and homogenization of variations would otherwise be expected.

Gene exchange homogenizes the gene pool for an interbreeding population. The dangers of inbreeding demonstrate this principle. Specifically, half of the genes of parents are discarded during meiosis. Recombining genes while discarding half each time is a process which homogenizes the gene pool.

Variations then come from outside sources. They are generated through mutations being acted upon by environmental conditions to select for modified genes which are adapted for survival functions. When they enter a population through breeding, they show a mixture of types only until sufficient gene exchange occurs to homogenize the gene pool.

Morchella researchers [1][2][3] found multiple alleles for several genes when using gel electrophoresis to separate allozymes. They looked for patterns as an indication of taxonomic relationships. The results were scrambled and confusing, but some clustering was said to be an indication of phylogenetic relatedness. The high degree of variation was unexpected and interpreted to indicate Mendelian gene exchange.

When a high degree of unexpected functional variation was found in Morchella by this author it pointed to a non-Mendelian mechanism. Variations were studied in anomalous growth on petri plates and tissue types for mushrooms found in the wild.

Materials and Methods:
Cultures were started from single, germinating ascospores derived from dried ascocarp tissue of M. angusticeps from Michigan. Tissue was rehydrated in sdH₂O; spores were pressed out with a glass rod; dilutions were made when needed; and a drop of suspension was plated on thin agar without nutrients. Germinating spores were selected in 16-24 h by observing microscopically through the bottom of the plate and marking the location. About 10 mm² of agar was transferred from the marked area.

For subculturing, the media composition was dextrose 2%, casein hydrolysate 0.5%, arginine HCl 0.5%, K-acetate 0.1%, basal medium pH 7.0 and agar 2%. The basal medium consisted of the mineral elements of the yeast formula [4] without nitrogen. Organic nutrients were filter sterilized separately and concentrated to prevent heat damage. The incubation temperature was 18°C.

Single germinating ascospores from the same mushroom were used for inoculum (two per plate); so the variables on a plate were intrinsic to the mycelium rather than due to cultural conditions.

Results:
The mycelial growth shown in figure 1 is an anomaly. Mushroom mycelium is never pigmented, since color serves no function below the surface of the normal growth medium. The black pigment visible in this case is the pigment of the ascocarp. The anomaly also contains structured layers including tissue similar to that of the ascocarp. The anomaly serves to highlight intrinsic variations.

(expanded)

The differences on each plate are intrinsic to the mycelium, since the inocula were single germinating ascospores with no significant media transferred with them. The inoculating spores were all derived from the same ascocarp. This means that the dramatic differences in appearance were the result of the ascocarp producing spores which were intrinsically different. The sample shown is 4 typical plates out of 12 inoculated.

Figure 2 shows variations in another species, M. esculenta from central South Dakota. Here the differences are in ascocarp tissue thickness. The top row shows extremes which are thin tissued, and the bottom row thick tissued. These examples were selected from about thirty mushrooms randomly picked from the same area. In other words, these extremes are not uncommon, while most of the morels are in-between the extremes.

(expanded)

In this example, thin-tissued morels were light brown, and thick-tissued ones were gray. Morphological differences were visible around the base of the cap and in the density and curvature of the ridges. Tissue mass was often disordered on the cap of thick-tissued morels. Morels are hollow, and therefore the restrictions at the base reduce internal space.

These differences stem from the need for the fleshy ascomycetes to shrink through dehydration as a method of creating a force for ejecting the spores from the tissue. Thin tissued morel ascocarps are needed in years when there is much rain and humidity slowing the rate of dehydration. A process which is too slow will result in deliquescence of tissue before spores are emitted. The deliquescence appears to result from bacterial attack. Thick tissued morels are need for dry years to prevent the tissue from dehydrating before spores are formed.

Discussion:
These variations in spore types and tissue thickness do not follow the Mendelian pattern of inheritance. Instead of being passed down from parent to offspring, they are randomly distributed through the population.

Previous Morchella researchers assumed the variations which they observed through multiple alleles were an indication of Mendelian gene exchange [1][2][3]. If gene exchange were involved, the variations would have to be transitory and originating from an external source. Yet the variations were found in all species studied, which were collected over distances of several different states in the US and several years of time [5] . The variations were obviously not transitory, and they were not assumed to be, since they were said to indicate phylogenetic differences.

The random distribution of the variations in gene pools which should be relatively homogeneous indicates that the differences are phenotypic rather than genotypic. The large number of genes found to have several alleles [1][2][3] indicates that different alleles are turned on or off in a semistable manner during spore formation. This mechanism is certainly not new or radical, as it controls cellular differences during embryonic development. What's new is to find such variations being expressed between individuals within genetically similar populations.

Interpretation of earlier results was confused by the finding that Morchella mycelia originating from different spore types create a line of unusual, high-density growth between them, even when the spores originate from the same ascocarp [6][7]. The unusual growth was assume to be related to mating. But mating never involves such growth with other species; and the cells within the unusual growth were blocky and walled off, which indicates separation.

The fact that the separating line of growth is not found in other species, and the cells are walled off within it, indicate that the function is to prevent nuclear exchange between different phenotypes, which would dilute or neutralize the essential differences. The separating line should therefore be viewed as a barrier.

Phenotypic variations create rapid response to environmental conditions which normally vary in critical ways. This effect can be observed in plant and animal life as stable variations in interbreeding populations. The test for phenotypic variation is random distribution of variations.

For example, a common weed called creeping jenny or field bindweed shows some patches which have purple flowers, some with white flowers and some in-between. Since these variations are stable and random, they would be phenotypic but not genotypic. Another example is elm trees. Even when growing near each other, they vary in how early they form seeds in the spring. Phenotypic variation is their method of coping with the contradictory requirements of getting their soft seeds into the ground early in the spring and the danger of a late frost destroying them on the trees. In the animal world, the variations between individuals in fast muscles and slow muscles is a good candidate for phenotypic variation. To verify this, a statistical study to determine randomness of distribution would be required.

References:
1. Gessner, R. V., Romano, M. A. & Schultz, R. W. (1987). Allelic variation and segregation in Morchella deliciosa and M. esculenta. Mycologia 79, 683-687.

2. Yoon, C., Gessner, R. V. & Romano, M. A. (1990). Population genetics and systematics of the Morchella esculenta complex. Mycologia 82, 227-235.

3. Royse, D. J. & May, B. (1990). Interspecific allozyme variation among Morchella spp. and its inference for systematics within the genus. Biochemical Systematics and Ecology 18, 475-479.

4. Wickerham, L. J. (1951). Taxonomy of yeasts. U.S. Department of Agriculture, Washington, D. C. Technical bulletin 1029.

5. Jung, S. W., Gessner, R. V., Kuedell, K. C. & Romano, M. A. (1993). Systematics of Morchella esculenta complex using enzyme-linked immunosorbent assay. Mycologia 85, 677-684.

6. Hervey, A., Bistis, G. & Leong, I. (1978). Cultural studies of single ascospore isolates of Morchellla esculenta. Mycologia 70, 1269-1274.

7. Volk, T. J. & Leonard, T. J. (1989). Experimental studies on the morel, I. Heterokaryon formation between monoascosporous strains of Morchella. Mycologia 81, 523-531.