Plant Genomics & Developmental Biology Consultant / Expert Offering Creative Paradigms for Scientific Advancement in Agriculture and Medicine, with Emphasis on Plant Genetics / Genetic Engineering and Developmental Biology, e.g., Organogenesis. Michael M. Lieber, Ph.D. Berkeley, CA (510) 526-4224

Adaptively Responsive Mutation on the Karyotypic Level Manifested as Pattern Differentiation and Morphogenesis in a Fungus: Further Steps into a New Paradigm, with Implications for Agriculture

 I. Introduction: Environmentally Responsive Mutagenesis on Different Levels of the Genome

  Environmentally Responsive Mutation on the Molecular/Gene Level

Relatively recent investigations of mutations generated in various unicellular or simple, undifferentiated colonial organisms have revealed what can be considered as the beginnings of a paradigm shift in biology. During the past 27 years, adaptively responsive, enhanced mutation has been found in bacteria and yeast, and in 1969, evident within the unicellular green alga, Chlamydomonas, an eukaryote. Though in the case of yeast, there was also a far earlier report (Lindegren, 1966) of adaptively responsive mutation. Such enhanced mutations, linked to stress, enabled the quick adaptation of the single cells of the organism to changed, stressful situations. In some studies, such adaptation to a particular nutritional stress enabled single cells in non-growing bacterial colonies to produce adapted, growing clones or sectors, called papillae. While in many other studies involving other types of nutritional stress, such adaptation enabled the growth of whole colonies from single cells during the stressful conditions. In 1989, 1990, 1998, and 2000, as well as in 2001, the author showed, through his own work on bacteria under nutritional stress, that the occurrence of adaptively responsive mutations resulting in growing colonies is under internal, genetic control or regulation, hence non-random, demonstrating developmental features. As pointed out, this suggested the evolution of an inner, mutator capacity that could have modulated evolution itself. Earlier in 1967, it was also pointed out by the author that the enhanced occurrence of many types of mutation was non-random, being under genetic regulation through internal mutator processes, whose existence in the past could have enhanced the degree of evolution from within. (Also, see references in Lieber, 2011 and references via http://www.googlescholar.com. )

The non-randomness of enhanced mutations, occurring within organisms under non-lethal stress, has become clearly manifested repeatedly in the last 27 years of mutation research. However, during this 27-year period of investigations, the particular, non-random mutations studied were only adaptively responsive mutations to nutritional requirements and to the stress of antibiotics. And, in the case of the green alga, the adaptively responsive, frequent mutations occurring on the culture medium enabled growth of many colonies of joined cells in the presence of a growth inhibitor in the culture medium, another type of chemical stress. These adaptively responsive, enhanced mutations enabled adaptation to stress on the biochemical or molecular level of organization in unicellular organisms, as opposed to higher levels of organization, such as on the level of morphogenesis in a multicellular, differentiated organism, relatively far more complex than bacteria colonies and the colonial algae. (Also, see Addendum I.)
 

     Environmentally Responsive Mutations on the Karyotypic Level

  In unique contrast, very frequent mutations at the chromosomal level due to or in response to physical stress can occur under inner control that lead to adaptive changes in differentiation of pattern and morphology in olive-green, multicellular fungal colonies having a central, crinkled morphology, sparsely populated with conidiophores/conidia, and of reduced growth rate at high temperature. (See Lieber, 1998 for a description and earlier, relevant references. View PDF of Article.) An environmentally responsive, innerly-controlled, greatly enhanced mutation, through an environmentally responsive, complex, dual mutator, on a higher genomic level determining development, was shown to exist many years previously to most of the mutation studies referred to above, with significant implications for evolution. (Investigations, observations and evidence are described in Lieber, 1972, 1976b. Some of these observations and related evidence is summarized in Addendum II.)

Investigation with the multicellular, differentiated eukaryotic fungus, Asperillus nidulans, an ascomycete, once considered a lower plant, revealed that very frequent mutations on the karyotypical or chromosomal level of organization were an adaptive response to high temperature stress. These environmentally responsive, adaptive, karyotypic mutations resulted in the production of many yellow sectors in each sparsely conidiated, olive-green colony. The sectors were composed of yellow, a-sexual reproductive structures, the conidiophores made up of yellow conidia-spores, the means of a-sexual production. The ensuing, enhanced production of such mutant sectors manifested itself phenotypically in response to a higher temperature level as a new type of pattern-differentiation through such sectors and morphological change within fungal colonies (Lieber, 1972, 1976b, 1998.)

                              Criteria for Adaptive Features:

  Such a new pattern of differentiation and change in morphology, based on inner-controlled though environmentally responsive genomic changes, a responsive, inner-controlled hypermutation, were adaptive in the following ways to temperature stress: These genomic changes enabled, under temperature stress, major increases in the production of yellow a-sexual spores within differentiated, mutant yellow sectors. Such genomic changes also enabled the greatly increased growth rate of such, yellow sectors and their normal, flat morphology. Such spores are necessary for a-sexual inheritance, and their flat, high growth-rate morphology is characteristic of Aspergillii surviving well in various environmental niches. In other words, "the improved morphology, growth-rate, and conidial production of such sectors would now suggest [and reflect] an adaptively responsive, inner-directed mutagenesis to temperature stress." (Lieber, 1998) This new pattern of differentiation on the phenotypic level would be an expression of such beneficial , enhanced responsiveness to physical stress through environmentally responsive mutation. (To view colonies displaying such differentiation, the reader is directed to the photographs at the end of this article. Also, go to Figure 8.). This mutator system is in itself an example of a developmental system capable of further evolution through its own inner-controlled, adaptively, responsive, enhanced mutability at the karyotypic level.

II. Details of the Investigation with Aspergillus nidulans, a Plant-Like Organism

The fungus investigated, Aspergillus nidilans, is a normally haploid, eukaryotic ascomycete with eight chromosomes. Its colonies have internally septate hyphae made up of multinucleated cells divided by the septae. Without chromosomal re-arrangements or new chromosomal configurations within the haploid genome, the fungus produces flat, grass green colonies due to green conidiophores emerging vertically from multinucleated hyphae composing the colonies. The colonies display high growth rates at various temperatures (Lieber, 1972, 1976a.). Colonies with a single, new chromosomal configuration in each of their haploid nuclei have a crinkled morphology and a reduced growth rate, especially at high temperature. One new chromosomal configuration, a uni-mutator, responds mutagenically to various temperatures, ensuing in colonies producing, at various mean-frequencies, yellow mutant sectors with improved growth rates (Lieber, 1976a.) Colonies of this genotype were used as controls in the investigations described below. This fungus produces a-sexually reproductive spores. Each spore, a conidium, has a single nucleus.

Other strains of Aspergillus nidulans investigated at various temperatures had two chromosomal, non-uniform configurations in the haploid genome. These configurations have respectively partial duplications in trans of chromosomes I (Dp I) and III (Dp III), the latter of various sizes. (Dp I and Dp III were carried on respective chromosomal translocations.) Aspergillus colonies with these two configurations in the haploid genome are much smaller than normal colonies. They display a crinkled morphology, especially pronounced at high temperature, and produce far less vegetative spores or conidia at higher temperature, e.g. 39.5 degrees centigrade (Lieber, 1972, 1976b.) One of these configurations, referred to as Dp I, contains two genes for conidial or conidiophore color, one green and one for yellow. The two color alleles are heterozygous within the duplication, the green allele being dominant to the yellow allele, hence the green or olive-green color of the colonies, that is colonies having green or olive-green conidia and conidiophores within the crinkled area. In some nuclei, a specific region of Dp I containing the green allele is subject to deletions, resulting in yellow sectors of increased growth rate.

   As earlier investigations have documented with evidence, the frequency of such deletion, and of corresponding yellow-sector production, is influenced greatly by the other duplication, Dp III after it becomes reduced in size and by temperature. Dp III of reduced size responsively controls the degree of specific deletions from Dp I (Lieber, 1972. 1976b). Dp III, before it is reduced due to deletions from it, also affects or enhances the degree of deletions from Dp I, but to a far lesser extent than the reduced III duplication. For example, the dual mutator strain with the reduced Dp III, present from the start of colonial growth, produces far more yellow mutant sectors per colony than colonies having the intact, non-reduced Dp III present from the start of colonial growth. And this difference in mutation frequencies is significantly different at P< 0.01. This clearly indicates that as Dp III becomes reduced in size due to deletions, it can greatly enhance the degree of deletions from Dp I. This also demonstrates that the very instability of this mutator system can lead to its greatly enhanced mutator effects.

    Description and Involvement of the Responsive, Karyotypic Dual Mutator

Modulated by temperature and the age of the conidia from which colonies are obtained, Dp III, depending on its size, controls the degree, timing and pattern of deletion including the green allele on Dp I within colony genomes. (Lieber, 1972, 1976b). After  Dp III becomes reduced in size as a result of deletions having occurred from it, the reduced Dp III enhances the deletion of the genetic region including the green-allele region of Dp I within colony genomes. (Genetic analysis confirmed the occurrence of such deletions.) A deleted segment, a type of transposition element from Dp III, probably inserted near the green allele on Dp I, may have induced under the control of the reduced Dp III such deletion.  It was proposed that induced heterochromatization by Dp III within a region of Dp I, containing the inserted element, was involved. When this occurs under a temperature stress, that is, at a high temperature opposed to a lower temperature, this mutagenic, deletional interaction of the two configurations, via a likely transposition process, is enhanced to an even a far greater degree in colonies having a particular culture history.

  Moreover, this mutagenic enhancement is clearly regulated, since the improved, yellow sectors, as a consequence of the deletions from Dp I in many nuclei, all emerge at the same time during colonial growth. Furthermore, this temporal control of deletion, clearly under the control of the reduced Dp III, becomes far more pronounced or effective at the stressful, higher temperature. (Evidence presented in Lieber, 1972, 1976b.). This would be a controlled hyper-mutagenesis responding or tuned to a temperature stress. This would be through a environmentally responsive, two-part mutator system on the karyotypic level. The severe dampening, epigenetic influence of age-affected conidia on the degree of mutagenetic interaction, in cultures obtained from these conidia, is also suppressed epigenetically through this higher temperature [Lieber, 1972].

  Specifically, at that higher temperature, irrespective of the age-state (or epigenetic state) of the conidia producing the fungal colonies, the adaptive consequences of this very frequent, deletional mutagenesis or instability, possibly during mitosis, on the karyotypic level of organization, are fungal colonies that each symmetrically produce many yellow sectors of increased growth rate, with abundance of conidia or conidiophores, and of a relatively smooth or non-crinkled morphology. Such are consequences that are very much adaptive to the new temperature situation or stress, especially in the long-term, from the standpoint of the evolution of new adaptive strains of Aspergillus in terms of new differentiation patterns. Moreover, the configurationally, partial duplications, controlling such adaptation, are in effect an adaptively responsive, complex, two-part mutator system on the chromosomal or karyotypic level, and a system which exhibits internal regulation, though environmentally sensitive or responsive, and one whose mutagenic behavior is a-sexually inheritable via conidia and sexually transmittable to a F1 generation via ascospores (Lieber, 1972, 1976b.) As can be seen, this dual mutator system derives from genomic re-organizations on the karyotypic level. Many types of mutator systems, environmentally sensitive, derive from past genetic re-organizations (Lieber, 1972, 1976b).

   Karyotypic Dual Mutator Responds to Stress with Beneficial, Phenotypic Effects     

This situation with Aspergillus indicates that inner-controlled, internally-regulated, very frequent karyotypic change can nevertheless be induced or influenced by a physical stress, namely high temperature, in such a manner that such controlled karyotypic changes result in adaptive changes on the differentiation/morphological, phenotypic level. This is shown as being manifested by the  greatly increased production of a-sexual spores, increased growth rate, and a flat morphology characteristic of well adapted Aspergilli in diverse regions. This would be an example of a karyotypic-based, adaptively responsive differentiation/morphological change responding to a physical stress, a situation that has not been demonstrated before. This is highly significant as it now demonstrates that morphological and differentiation patterns can be adaptively responsive to stress through a stress-induced or modulated mutagenesis involving genomic configurations. These are in effect developmental, controlling elements on the karyotypic level, apparently regulating the excision and insertion of smaller transposition elements within the configurations.

The physical stress influences, possibly via cytoplasmic changes and related nuclear-membrane distortions, the inner-controlled mutagenic interaction of the genomic configurations, in such a way, that control becomes enhanced leading responsively (within one generation) to very frequent, karyotypic-based, controlled changes in differentiation and morphogenesis in fungal colonies. As well as involving the controlled insertion and release of small heterochromatic controlling-elements through controlled intrachromosomal recombination, the stress-related mechanism of such control could also involve controlled inaccessibility of genomic regions to possible nuclear-membrane replication sites and consequent controlled deletion of genomic regions, where cytoplasmic induced heterochromization could play a role in the accessibility (Lieber, 1976b).

 Such responsive enhancement on the karyotypic level enables in some manner, communicated dynamically through different levels of organization, effective adaptive changes on the phenotypic level, that is, on the organismal level. The resulting karytotypic alterations or variations have become co-extensive with the many yellow mutant-sectors within each of a large number olive green colonies, and thereby, coextensive with a new, adaptive pattern of differentiation and morphogenesis. This is in effect a responsively induced, new karyotypic analogue of an adaptive differentiation and morphogenesis within a short period. It would be important to know the relationship-translation between karyotypic changes at one level and those adaptive, phenotypic changes at a higher level. Interestingly, from the perspective or level of population genetics, this coextensive karyotypic variation with phenotypic variation on one scale would also be like an induced, phenotype/genetic variation within a population on a macro-scale.

 III. An Adaptive Phenomenon Apparently Unique in the History of Such Investigations

In the history of investigations into adaptively or environmentally responsive mutagenesis, the adaptive phenomenon involving Aspergillus was not previously observed. This was especially the process whereby controlled, very frequent karyotypic change under and through physical stress can be manifested adaptively in a short period, as very frequent, adaptive changes in morphology, growth, and patterns of differentiation within growing, multicellular fungal colonies under stress. It is appreciated, however, that such responsive adaptation via karyotypic-mutator systems, whether or not transposition elements are involved, may not be perfect, as some karyotypic changes could be deleterious. Nevertheless, the type of environmentally responsive mutator systems within Aspergillus could have themselves evolved into more effective mutator systems, with developmental features. Such features would have lead to more effectively adaptive developmental or morphological solutions to various types of environmental and internally-related epigenetic stress.

   In the laboratory, a small number of genetic crosses involving the hybridization of different karyotypes of Aspergillus resulted in the production of  the karyotype-based mutator system. Analogously, during evolution, such  repeated hybridizations within very short periods could have ensued in many organisms having environmentally responsive karyotypic mutator systems affecting differentiation and morphogenesis, and thereby their inner evolution. In this manner, many adaptive changes in the developments of organisms could have been generated repeatedly in very short periods during their evolution.

   Phenotypic Effects of Karyotypic Mutator Similar to Genetic Assimilation

This phenomenon brings to mind the phenomenon of the genetic assimilation of induced morphological  or phenotypic changes involving stress in Drosophila, first discovered and investigated by C. H. Waddington in the 1950s (Waddington, 1953, 1956a, & 1959.) For example, when developing Drosophila embryos are subject to ether vapor stress-treatments or shocks during a certain period in their development, a portion of the Drosophila develop two thoraxes with two pairs of wings in adult flies. During each fly generation exposed to ether stress, developed bithorax flies were inbreed or crossed. When after a relatively small number of generations of such inbreeding under stress, a very large proportion of the subsequent progeny resulting from repeated inbreeding for the new morphology, when not subject to ether vapor shocks during embryogenesis, still developed the bithorax phenotype as adults.

 In response to stress, the new morphogenesis---and the changed pattern of regulatory processes connectedly involved---have become genetically inheritable or assimilated in a relatively short period in some manner. In other experiments, where inbreeding also involved a relatively small number of adult generations, other types of morphological changes, such as changes in wing morphology, eye morphology and in anal excretory papillae, were also inheritably assimilated or canalized when generations of their developing embryos were subject to other types of imposed environmental stresses, such as temperature shocks with regard to wing and eye development and intense salt treatments of embryo food with regard to papilla size.

Though it was not demonstrated that many of such responsive, genetically based (or canalized), environmentally responsive morphological changes were adaptive to the environmental stresses, the genetically assimilated increase in papilla size as a response to salt stress may, however, have allowed adaptation to the increased salinity in a relatively short period and to any future increases, as Waddington pointed out. It was not ascertained whether or not new mutations on the gene level were induced through the imposed environmental stresses during embryogenesis, though this possibility cannot be ruled out, and is worthy of further investigations. (As will be described shortly, a subsequent investigation does in fact suggest genetic changes being involved.) Also, these morphological changes might have enabled the development of less obvious, internal adaptive features in a complementarity with the evident, canalized morphological changes. This possibility would also be well worth investigating. In this connection, see Lieber, 2011 regarding enabling mutations.

More recent, stress-involved assimilation-experiments were performed with a black caterpillar species. Developing progeny of such were subject to heat shocks within each developing, caterpillar generation. As a consequence, green adults developed during each of 13 generations subject to heat shock. Subsequently, developing caterpillars eventually became inheritably green without heat shock after 13 generations through repeated inbreeding of green progeny caterpillars that had developed in each of those 13 generations (Suzuki and Nijhout, 2006.) As the authors point out, it is feasible that such inheritably acquired color via heat stress would be adaptive as an effective camouflage in a environment of green, leafy vegetation during the warm season, and thus evolutionally adaptive in a relatively very short period in the context of evolution.

 It was Waddington who had pointed out the importance of genetic assimilation in morphological and pattern evolution. Such developmental, genetic assimilation of features at the organismal level during evolution could have involved some types of genomic change on the karyotypic level, which they and their effects could have become repeatedly combined through a relatively short period of inbreeding, thereby accounting for an adaptive assimilation during a relatively, very short period, enabling an accelerated evolution. (Relevantly, the adaptive, Aspergillus mutator-system was created through types of inbreeding involving reorganized chromosomes.) Regarding such genetic or inheritable assimilation of environmentally induced characters, a change in genomic organization is clearly suggested (Piaget, 1974.)

 In  a more recent investigation with Drosophila, the  genetic assimilation of the cross veinless phenotype, through heat shocks, was demonstrated again. In that investigation, chromatin changes ultimately led to such genetic assimilation. It was suggested that chromosomal rearrangements in chromatin modifier genes occurred.  This ensued in position effect variegation via long range heterochromatization, and thereby gene silencing, of genes involved in wing development (Nair and Dearden , 2016.) These findings suggest that chromatin changes could also have played a role in various genetic assimilations of phenotypic changes due to environmental stresses.  Such inheritable assimilation of environmentally-induced morphological and regulatory changes, likely based on chromatin changes or rearrangements and less evident, enabled features, could have contributed to the rapid evolution of developmental systems in various organisms.

      Possible Role of Karyotypic Mutators in Genetic Assimilation:

The role of karyotypic mutators in this cannot be ruled out. This becomes especially feasible in view of the following found with the Aspergillus mutator system: one can generate, through an a-sexual selection from an extremely high mutant-sector, colonial producer at high temperature, a group of colonies with a significantly, further-increased mean frequency of yellow, mutant sectors at high temperature (mean frequency of 21.00 mutant sectors per colony) compared to the mean mutant-sector frequency of another group of colonies at high temperature (mean frequency of 13.7 mutant sectors per colony) [Lieber, 1972]. Through recent analysis, these means are significantly different at P < 0.001. This would certainly suggest a genetic assimilation of a further increased karyotypic mutator effect at high temperature, possibly involving the stabilization of an epigenetic change, itself stressful. And the high temperature stress would be mutagenic in the context of the inner-mutator process. In a way, this would be a non-linear, epigenetic extension of the mutator process. Occurring in other situations, this could have affected the rate of morphological evolution itself.

IV. The Evolution of Developmental Systems Due to Responsive Genomic Changes

In this connection, a high, non-linear rate or burst of karyotypic evolution has been correlated with a high, non-linear rate of morphological evolution in mammals and in higher plants (Wilson et al., 1977.) This may have involved karyotypic mutator systems similar to those described in Aspergillus (Cherry et al., 1978). Furthermore, such karyotypic mutator systems might even have been mutagenically responsive to various environmental and internal pressures or stresses, such as extremes in temperature and pre-mature aging. The consequences of such might very well have been corresponding, nearly immediate morphological changes that were adaptive to the new stresses. The inner-directed changes or reorganizations on the karyotypic level of genetic architecture could have resulted in corresponding, sudden reorganizations of regulatory genetic networks, leading to the higher level morphological changes. This may very well have accounted for the high, non-linear rates of morphological evolution of the mammals and higher plants. And there is some evidence that "morphological evolution relies predominately on changes in the architecture of genetic regulatory networks" (Prud'homme et al., 2007). Wilson et al. in 1977 also postulated the necessary involvement of genetic regulatory regions in the morphological evolution of mammals and of higher plants.

 Such changing architecture would have to correspond to or composes the dynamic architecture of morphogenesis, even though the avenues of this will have to be defined.  Again, the problem is how different levels of organization dynamically interrelate and integrate with one another. With regard to the Aspergillus mutator-system, there is clearly a significant correspondence between inner-controlled karyotypic change at one level and adaptive phenotypic change at another, involving differentiation and morphogenesis.

 A karyotypic-regulatory, architectural basis for morphological evolution, responsive to stress, appears to be reflected in plant evolution . Namely, frequent duplications of karyoptype, leading to polyploidy and corresponding morphological changes during plant evolution, have been shown to be associated with periods of environmental stress (Vanneste et al., 2014.) Polyploidy in plants and general karyotypic change have been very adaptive and have greatly contributed to plant speciation. It cannot be ruled out that such changes in ploidy or karyotype have had, or involved, a developmental, mutator effect, determining in a controlled, specific, and refined manner genomic changes on the karyotypic level. Such mutator systems could have had their origin in those very karyoptypic re-organizations. Relevantly, the creation of allotetraploids can create karyotypic re-organizations that lead to or determine further genomic changes: "Recent studies, mostly with plants, suggest that polyploidization can induce a flurry of genetic and epigenetic events that include DNA sequence elimination and gene silencing." (Pikaand, 2001). And following the hybridization of two wheat species, an allopolyploid was created. In such, it was found that "instantaneous genetic and epigenetic changes in the wheat genome [was] caused by allopolyploidization" (M. Feldman and A. Levy, 2009).

 Such internally directed, further genomic change or re-organization could also define the degree of evolution. In various plants and animals, there are additional examples where there are subsequent karyotypic changes, such as karotypic instabilities "in response to changes in ploidy and interspecific hybridizations" (see Shaperio, 2011). The existence of these processes in the past could have accelerated morphological evolution. As long ago as 1940, the geneticist, Richard Goldschmidt, argued that evolution, especially macro-evolution, could have involved the responsive or directed generation of mutation on the karyotypic/chromosomal level of organization, ensuing in the sudden occurrence of organisms with new developmental, primary patterns (Goldschmidt, 1940.)

Karyotypic Mutator Systems Become Part of Developmental Systems While  Enhancing their Evolution:  The Evolvability of Karyotypic Mutators through Their Inner-Determined Mutations Enhances the Evolution of Developmental systems

As also pointed out several years ago by the author, karyotypic mutator systems may have contributed to and may have themselves become part of the evolution of developmental systems in various organisms, and in so doing, determining the very rate or degree of such an evolution (Lieber, 1972, 1975, 1976b, 1998.), thereby accelerating and enhancing the evolution of developmental systems. It is feasible in view of the adaptive Aspergillus system that such developmental systems would have been the result of an adaptively or environmentally responsive and evolving mutator system. Such an evolving system would have been due to its own inner-controlled, responsive instability or hypermutation. This would be its evolvability, which is a genetic-system's capacity to generate, new adaptive variations on different levels of organization. A consequence of this  evolvability would have been the evolution in various organisms of even more effective, mutator-based developmental systems. Wherein, inner-controlled, minute karyotypic changes would have occurred as features of ontogeny. The would constitute the enhanced evolvability of the evolution of adaptive development.

 Specifically, such an evolving and integrative mutator system, involving the architecture of the karyotype, would have determined the very inner-evolvability of the evolution of development in various organisms, including and especially in higher plants. In effect, the responsively evolving karyotypic-mutator-system would be the responsively evolving capacity to evolve adaptive developmental systems, the inner-evolving evolvability of evolution. Selection becomes synonymous with the increasing evolving capacity for an increasing adaptive evolution; where, such evolving or changing capability would have involved environmentally responsive, yet inner directed, changing developmental and responsive mutator systems on all levels across the succeeding generations. Another avenue for evolution involving mutators increasing from within the evolving capacity for adaptive evolution, and hence implicit selection, might have entailed a modern version of pangenesis, proposed by the author in 1967. (See Mutation, Development and Evolution.)

 V. Likely Consequences and Possibilities from the Evolution of Karyotypic Mutator Systems
 

 The Karyotpic, Dual Hypermutator System is a Developmental System Operating Through Responsive Temporal, Coordinate Control

Though originally occurring years ago, and first described in 1972, investigations of the fungus, Aspergillus nidulans, have nevertheless explicitly revealed, through further examination and interpretation (of earlier, referenced, published data), a new type of environmentally responsive, adaptive mutation of high degree on the karyotypic/chromosonal level, manifested phenotypically as adaptive changes in growth, differentiation and morphology. This phenomenon exhibited a temporal, coordinate, global, control through Aspergillus colonies involving many nuclei in a common cytoplasm, possibly mediated through the re-establishment of balanced forces within the colonies. This would have been a control globally responsive to an environmental stress. And such a responsive, global control  would have enabled a quick, adaptive response to a physical stress to and through the fungal colonies.

That is, the responsive, controlled, genomic-involved phenomenon occurs within Aspergillus colonies when a flexible or plastic accommodation to physical stress is necessary. By means of its timing and coordination within Aspergillus colonies, the global phenomenon is adaptively developmental through different levels of organization, from karyotype to pattern differentiation and morphogenesis on the level of the organism. Its coordination through controlled temporality is a key adaptive feature of this responsive, global phenomenon. This would be a coordination, a globalization, that might have possibly involved, through periodic physiological and cytoplasmic changes in growing colonies, global forces of a field achieving a stabilizing equilibrium or uniformity of forces through different levels of organization of the fungus.  Be this as it may, the capacity to generate this adaptively responsive phenomenon and the adaptive, developmental consequences or features are themselves inheritable through sexual crosses involving meiosis (Lieber, 1972, 1976b). See Figure 99 located near the end of this article.

This phenomenon might also be indirectly related to other environmentally responsive changes in development that have temporal, coordinating features and that become inheritable, such as genetic assimilation. Such genetic assimilation, through a type of  developmental feed-back, could also define or influence regulatory, cytoplasmic changes during development. In effect, this karyotypic-mutator system is a responsive regulatory system, to its dynamic internal milieu, producing new karyotypic configuations and an associated hierarchy of genetic and epigenetic regulatory changes that ensue in a new adaptive morphology and pattern. There may be other, unknown types of environmentally responsive mutator-systems yet to be discovered, which have also played a significant role in the developmental evolution of organisms. Nevertheless, it is likely that many developmental and growth-pattern systems in plants and animals have evolved from a basic, known developmental, karyotypic-mutator system, such as the one discovered in Aspergillus.  Such systems could also involve, refined, somatic intra-chromosomal recombination. In fact, the process of deletion and transposition in the Aspegillus mutator system was proposed to the scientific community as involving specific, somatic intra-chromosomal recombination implicating heterochromatin (Lieber, 1972, 1976b). Though not proposed at the time, such could occur through a chromocenter. In various organisms, the heterochromatic regions, the centromeres, of the chromosomes in a genome are joined together in a heterochromatic structure, referred to as a chromocenter, though it is likely that such heterochromatin extends beyond the centromeres to some extent. Such a chromocenter and the extended heterochromatin could provide an avenue for genetic exchange, via recombination, between chromosomes during interphase. In various organisms, including higher plants, such centromeres are composed of repetitive DNA and retro-transposons. Clustered centromeres, the chromocenters, in certain fungi are in contact with the nuclear envelope before mitosis. (See V. Yadav et. al., 2019 for updated research in this regard.)  In general, such contact or attachment to the nuclear membrane might stabilize the genetic exchange via transposons. Other related mechanisms, involving proposed nuclear-envelope replication-sites and heterochromatic regions, could also have played a supplementary role in the inner-controlled mutagenesis (Lieber, 1976b).

    Many Developmental Systems May have Evolved from Karyotpic Mutators

 In various invertebrate animals, controlled karyotypic changes, such as deletions of heterochromatin, do occur within somatic cells as opposed to germ cells, during development (Goday and Estaban, 2001; Beerman, 1966; Waddington, 1956b, p. 352). Such deletions or excisions may occur through intra-chromosomal recombination (Beerman, 1966). And in certain amphibians, development is known to involve the creation of inheritable, irreversible nuclear (or chromosomal) changes within somatic tissue (see Fischberg and Blackler, 1961), these changes possibly being deletions. During lymphocyte differentiation in mammals, there is a regulation of genomic rearrangement events in those cells (Alt et al., 1986). It is well known that very high frequency, genomic changes involving somatic hypermutation/intra-chromosomal recombination in developmental, immunological tissues (B lymphocytes) occur as a controlled, adaptive response to internal environmental stresses, such as bacteria and viruses or other foreign antigens (Teng and Papavasilion, 2007; Ziqiano et al., 2004; Mange and Mange, 1990). The developmental consequence is diverse antibody production, which is adaptive.

In various plants, there are controlled changes in ploidy in different cells during development (Bino et al, 1993; Galbrath et al., 1991). In Nicotiana, controlled deletions of heterochromatin in somatic cells, possibly involving intra-chromosomal recombination, occur frequently during development, which results in color variegation of the flowers (Burns and Gerstel, 1967). In maize, some features of development are based on a transposition-insertion-deletion, controlling-element system, with many variations of such (McClintock, 1951, 1965). Dr. McClintock proposed that many other aspects of maize development could be so based, as well. As in Aspergillus, such a system in maize derived from a chromosomal or karyotypic reorganization Such a system in maize and its variations are temperature and age sensitive.

These developmental systems have characteristics suggesting their evolution from responsive, karyotypic-based mutators. It is likely that other developmental systems having occurred through the evolution of environmentally responsive, changing karyotypes and based on innerlly-controlled, refined genomic changes, controlling genetic expression, will be demonstrated. Such a system of refined, controlled genomic changes could involve the excision of genetic regions, the transposition of such, and their re-insertion into other sections of the genome. It is not difficult to imagine the evolution of such a refined system from a karyotypic-based mutator system, where in such a refined system, genetic material is not lost in most cases, but excised, transposed, and re-inserted, with developmental effects on higher levels of organization. The earlier and interim stages of such an evolution may be exemplified in many current organisms.

Hence, it is predicted that more and various karyotypic-based mutator-systems, responsively generating or leading to frequent adaptive, inheritable changes in differentiation and morphology within short periods, will be detected in various organisms. As with the Aspergillus system, these mutator-systems may form the basis for the future evolution of more complex and refined developmental and growth pattern systems, leading to more adaptive and, in many cases, productive organisms. This would include cultivated and nurtured plants used in agriculture and horticulture, but among the harmful, could include organisms that are pathogenic to such plants, as well. There is the likelihood that the environmentally responsive mutator systems in bacteria, Aspergillus and maize are genetically related through evolution (Lieber, 1998).

   This makes the aforementioned prediction even more feasible. The developmental, Ac-Ds controlling-element system in maize is very similar to the dual mutator system in Aspergillus (Lieber, 1972,1976b). The adaptively responsive phenomenon exhibited by Aspergillus (once classified as a lower plant) strengthens the case for the widespread occurrence of various types of adaptively responsive mutagenesis in various organisms. This gives greater feasibility to the conclusions stemming from those earlier investigations of adaptively responsive mutagenesis. The developmental, adaptive system in Aspergillus makes the following situation even more feasible. Namely,  environmentally responsive, inheritable mutator systems of various types, especially those with developmental features, have played a significant role in a responsively accelerated, adaptive, developmental evolution. This would pertain especially to the  evolution of animals and plants, including the progenitors of cultivated crops and of pathogenic organisms.

                          Predicted Responsive Mutator System

In fact, what appears to be a variation of such predicted situations, as described above, was recently described in April, 2014. When a soil fungus pathogenic to rice was subject in one experiment to increasing copper concentrations, which increases are normally toxic to the fungus, and to temperature shocks in other experiments, significant genomic rearrangements occurred in response to both types of stresses via the agency of transposition elements or TEs (Chadha and Sharma, 2014.) With increasing concentrations of copper in the culture medium, the fungal colonies became resistant, and were able to grow, which was correlated with increased or frequent genomic change through the insertion of certain TEs. Moreover, increased copper resistance was associated with frequent color changes of the colonies from grey to white, the changes appearing as white sectors in photographs, and also judging from the photographs, morphological changes were also generated. As noted by the authors, colonies adapted to the highest copper concentration exhibited dense aerial hyphae. Those colonies were completely white. In earlier investigations by these authors, temperature shocks or stresses affected fungal growth and resulted in morphological transitions such as pigment changes and the production of aerial hyphae (Personal Communication.)

These responsive, frequent genomic changes to stress appeared to have occurred over a short period, as implied by the data. Under field conditions, where there are high concentrations of copper in the soil in which the fungus resides, and the soil is very warm due to a tropical environment, this fungus exhibits a high degree of genetic diversity or genetic rearrangements, "suggesting [according to the authors] that high copper content of soil and temperature stress are among the important environmental factors responsible for the high genetic diversity of the pathogen under field conditions." The further implication is that such adaptive, genetic diversity was responsively induced via TEs over a short period.

They state: "Whereas, extensive research over the last several decades has elucidated numerous molecular responses to stress, it is much less known how these translate into organismal–level responses." They suggest that environmentally responsive TEs reflect such a translation. Does the color and morphological change of the colonies with regard to copper concentration also reflect such a translation? Recall in this connection, that a process involving transposition elements may also have been involved in the adaptively responsive mutator situation in Aspergillus nidulans, where frequent adaptive changes involving color-pattern differentiation, growth and morphology were generated over a short period. In support of such involvement of controlling elements, transposition of genetic elements, thought to be tandem duplications, from chromosome to chromosome in Aspergillus nidulans induced morphological and pigment changes, as brown sector variants, within short periods (Azevedo and Roper, 1970.) These transposing elements responsible for those phenotypic changes in Aspergillus diploids had their source in a duplication derived from Dp I. Whether or not such phenotypic changes, based on such small, mobile, karyotypic segments, were adaptive was, however, not investigated.

Yet, studies by the author showed that intermediate temperatures of 39.5 degrees C and 36 degrees C, as opposed to temperatures of 42 degrees C and 28 degrees C, could significantly increase, within a nine-day period, the frequency of generation of this genetically based phenotype associated with Dp I (Lieber, 1972.). Moreover,  the generation of these brown variants were also associated with Dp III. The generation of these were confined to 42 degrees C, suggesting their enhancement by this high temperature.

Relevantly, in Aspergillus diploids having a partial chromosome III segment in trans in triplicate gave rise repeatedly to haploid derivatives having mutations on chromosomes I and V (Lieber, 1972.) Perhaps, such chromosomal triplications in trans controlled  the induction of such mutations through the insertion of transposition elements into those chromosomes. Perhaps, such elements originated from the triplications themselves. Such configurations could also have played some role in an adaptively responsive mutagenesis.

The adaptive processes as reflected by internally regulated, frequent karyotypic change and environmentally responsive TEs may only be markers or shadows of a deeper, more encompassing adaptive dynamic; the elucidation of which may give better insight into the translation mentioned above. With this in mind, the following questions arise: How and why would the environmentally responsive and innerly-controlled karyotypic changes, mediated by TEs, develop into adaptive phenotypes? What are the underlying connections that translate environmental cues or stresses into adaptive, organismal, developmental responses, from phenome to genome and through genome to phenome? The authors of the 2014 publication regarding the pathogenic fungus do point out that the TEs investigated do behave in different ways and are highly specific; responding differently to different environmental clues or stresses. Again, what is the basis of such specificity of action leading to a correct phenotypic adaptation?

 VI. Conclusion. Towards Strengthening the New Paradigm with Constructive Results

 More Examples of Environmentally Responsive Mutator Systems and What Their Existence Indicates

Though it appears karyotypic-mutator systems, through their own environmentally responsive, inner-controlled instability, could have adaptively evolved into many current developmental systems based upon inner-controlled genomic changes involving transposing genes, thereby having increased through time the implicit selection and evolvability of such systems, it is still not clear in many ways as to how specific adaptive changes on various levels could have been mediated during that evolution. In this regard, could a type of dynamic, epigenetic imprinting due to various stresses, via cytoplasmic states, cellular membranes, the cytoskeleton and nuclear matrix, on chromosomal behavior and architecture, be involved in such specifically responsive adaptations? And could such an imprinting account for a likely accelerated evolution of pathogenic organisms and higher plants, through an epigenetic imprinting process regulating and determining lasting karyotypic mutator influences on the very developmentally-involved epigenesis? Most relevantly, and predictable in this regard, inheritable epigenetic modifications in plants occur due to environmental stresses (Boyko et al., 2011). Such inheritable, adaptive epigenetic modifications, which the authors refer to as epimutations, are associated with an increased frequency of genomic rearrangements, whose generation appears to be non-random.  Moreover, "epigenetic transgenerational inheritance of altered stress responses" in rats is described by D. Crews et al, 2012.

Such a further evolved, environmentally responsive process in plants and animals could be considered as a transgenerational, environmentally responsive developmental system, perhaps a variation of genetic assimilation. It would be one manifesting and occurring through dynamic connections across different levels. As far as elucidating the dynamic underlying such specific connections and interconnected adaptations on various levels of organization, including the environmentally responsive, transgenerational epigenetic-karyotypic level, one must look for more interconnected, holistic and imaginative explanations, based on new assumptions. One such assumption or hypothesis could assert a nexus of external and internal forces imprinting or driving stable-specificity through instability within and between cellular epigenomes or architectures, coordinately and globally shaping such architectures, where a maximum of dynamic uniformity in non-uniformity would prevail, achieving maximum stability and completion through all architectures, and through their force connections to the external environment. (See, Lieber, 1996.) 

Such completion could involve the reduction or resolution of internal stresses, such as non-uniform tensile stresses within various regions and periods, through the generation of new, internal force configurations guiding development. (See Elder, 1990 for a relevant account of the proposed reduction of tensile stresses within singularities in ontogeny through cell movements in morphogenesis.) In another account, the reduction of non-uniform tensile stresses and pressures during frog development occurs through  the cell movements of morphogenesis. Such movements, guided by the non-uniform tensions and pressures, ensue in the establishment of a equilibrium of forces in the regions of the completed morphogenesis (Cherdantsev et al., 1994). Morphogenesis is guided by non-uniform stresses and resolves them. These explanations, pertaining to dynamic completion, could and should be tested by experiment in order to gain a more complete, empirically-based picture and so enable scientists to arrive at a heuristic, universal principle in biology.

 In his 2011 publication, and in related articles, the geneticist, James Shapiro, speaks of "natural genetic engineering systems" in which highly controlled or regulated genomic rearrangements on different scales occur in bacteria, fungi, plants, insects, and mammals, especially in controlled and creative response to various types of stresses. As he illustrates, in the past, these non-randomly generated rearrangements in response to environmental conditions could have been transmitted through subsequent generations, thus defining and enhancing evolution, especially enhancing the creative capacity for organisms to have evolved adaptively in response to environmental stresses. The Aspergillus dual mutator system, through its responsive developmental features, would be a prime example of a natural genomic engineering system. In referring to the dual mutator system in Aspergillus, Dr. Shapiro stated in a personal communication: "I completely agree with you about the environmental responsiveness of genome operators."

Dr. Shapiro demonstrates, importantly, that in various organisms under stress the engineering of genomic change has, in many cases, repeatedly involved the specific, regulated patterns of transposon/retotransposon excisions and insertions into critical genomic regions, modifying genomic/chromosomal organization, at times involving chromosomal rearrangements at higher scales and genomic amplifications, and, in so doing, regulating genetic expression through different organizational levels. as well as determining genomic imprinting. He describes how this could be controlled or directed epigenetically at the cellular level utilizing inclusively various molecular, non-linear signaling networks. If I understand him correctly, such inner-directed epigenetic processes, responsive creatively to environmental stresses by incorporating them, ensue in inheritable, adaptive phenotypes in evolution.

However, as is the case with regard to the relationship between karyotypic change and morphological evolution, it is not clear how these networks are arranged and function hierarchically in organismic architecture through various levels of organismic organization to generate a responsive differentiation and morphogenesis, which is adaptive. Relatedly, it is also unclear how these ordered, molecular signaling networks connect to or involve the capacity to effect adaptive changes at the phenotypic level of organization. Nor does the heritability of the capacity to effect such responsive patterns of changed phenotypic characteristics appear to be addressed clearly through the coordination of such non-linear networks. 

Specifically, how do such networks define and implicate developmental mutator- systems and their phenotypes, which through their own controlled instability at different levels, evolve into the effective developmental systems of various organisms. Also, responsive transposons can induce inheritable changes in morphogenesis in Drosophila, as pointed out by Dr. Shapiro. Yet, how does the conception of molecular networks apply to this type of situation. What is the hierarchal connection between such networks, their environments, and the evolving capability for the evolution of an adaptive, dynamic geometry of morphogenesis. In effect, how do these networks determine and define responsively the dynamic, capacity of organisms to evolve adaptively in time through all geometrical levels.

A responsive transcalar epigenome, hierarchically and dynamically flexible involving specific, structuring/shaping force-configurations as signals, must be involved in some intricate and geometrically unifying way, through and accounting for all levels of organization. These shaping force-configurations or force-fields of biological geometry or manifolds, possibly driving towards dynamic stability and reduced stress through all levels of the organism in its connection to its environment, would be mediated and enabled epigenetically, perhaps at times, more karyotypically than epigenetically. Though with the exception of three publications (P. Lieber, 1969, M. Lieber, 1996, and M. Lieber, 2006), a unifying principle to explain the why and how of this appears to be lacking in current, even holistic, biological thought and one that may be demonstrated in future research. It appears Professor Shapiro sees this in some way, as he writes, "At present, our understanding of basic principles governing this overall control architecture is severely limited, and it certainly deserves to be a prime subject of 21st Century research." (Shapiro, 2011).

Knowing such a principle or principles may enable scientists to counter or reverse the generation and evolution of pathogenic organisms and promote the evolution of pathogenic resistance in crops, as well. Be this as it may, and pointing to aspects of such a principle, environmentally responsive and innerly-controlled, adaptively changing karyotypic-mutator systems, involving transposons, could have provided the inner dynamic and capacity for various, enhanced macro- and micro-evolutions of various organisms and their developmental processes over relatively short periods. Using tissue culture methods, the creation and application of such mutator systems in an epigenetic context, involving transmitted energies and stresses, may even become a significant parameter in a near-future evolution, through genetic engineering, of more productive and age-resistant plant-cultivars with altered, adaptive developmental and growth pattern systems. These would be developmental changes and features analogous to those generated by the mutator-system in Aspergillus.

Evolution as a Responive Stabilzation Process to Stress Through Environmentally  Responsive Mutators Operating on Different Levels of Genomic Organization while Influencing Phenotypic Development
 

The Aspergillus-mutator-system is an early and significant example (effectively in 1972) of an internally regulated hypermutator-system in a multicellular organism enabling, quick adaptive responsiveness, on various levels of organization, to new environmentally-induced conditions in the organism, and thereby innerly and developmentally evolutionary. The Aspegillus-mutator-system can certainly be seen as being within an epigenetic system guiding, and being cyclically influenced by, inner mutator processes, and one most likely prone to inheritable imprinting.

 
This would be a type of mutator-based, multilevel epigenetic system probably forming the evolved basis for many, present day developmental and growth-pattern systems, at least significant features of such, where controlled genomic change through responsively regulated genetic deletion, transposition and re-insertion could be involved in many situations. Of course in many cases, regulated  gene activation and suppression occur as features of development. Yet, such genetic behavior is dependent on chromosomal configurations or states, such as heterochromatin and methylation. And, predictably, these could very well be epigenetically controlled, and controlling, through the environmentally-influenced deletion, re-insertion and expression of genetic factors, such as transposons, a process representing a type of position effect variegation through regulated intra-chromosomal behavior. Modern genetic research has provided supportive evidence of this (Ito et al. 2016), giving further support to the predictions presented in this article.

As shown by Ito et al., an epigenetic system in a higher plant can induce enhanced, inheritable, and adaptive mutation, through transposon insertion, enabling seed germination in response to a chemical stress in the culture medium that inhibits such germination in culture. This is an evolved, mutator-based system controlling development across generations, in which, transposon activity in progeny must also be induced or enabled by heat treatment of the parental generation: Such prior heat treatment of the parent plant, and ensuing transposon activity within the parent, also enables responsive transposon activity to a chemical stress in seed progeny, promoting their germination. Namely, the chemical-stress induction of beneficial mutations enabling seed germination, through the chemical-stress-responsive insertions of transposons into specific genes within the seed-progeny, requires prior heat treatment of the parental generation. 

 Thus, heat stress itself would seemingly be acting or being utilized in a potentating-mutagenic, epigenetically adaptive fashion across generations, necessary for transposon-induced germination of seeds. However, an implicated, controlling methylation of the inserted transposons---where methylation is under the regulation of another genetic region within the system---can inhibit the expression of the adaptive mutations, ensuing in re-sensitivity to the chemical stress, whereby germination again becomes inhibited. Subsequent heat treatment of the seeds reactivates the expression of the beneficial mutations, controlling germination, ensuing in the re-activation of germination, as well as the expression of genes.adjacent to the beneficial ones. This occurs through heat-induced demethylation of the inserted transposons that created the beneficial mutations and heat-induced demethylation of adjacent regions.

The regulated methylation could mask the effect of such mutant genes in vivo when conditions would require plant dormancy. Under such cold conditions, as their research implies, the effect of the mutant genes would be non-adaptive but adaptive under warm conditions or heat. The chemical stress is in fact a plant hormone that induces dormancy under cold conditions. In view of this, the evolved epigenetic control of mutant induction and expression would quickly be able to accommodate plants to changing environmental conditions, allowing for and inhibiting development when respectively necessary, and in a heritable fashion. And as noted, induced karyotypic change can cause genetic deletions and gene silencing in plants (Pikaand, 2001), which could be adaptive. Even though all the adaptive dynamics across different levels of organization have not been clarified in various studies, the predicted systems or processes such as these can nevertheless be seen as also contributing to the beginning stages of a new paradigm for mutation and evolution.

                               A New Paradigm for Biology?

A new paradigm encompassing rapid, non-random mutation and evolution not only becomes creditable but very feasible. As viewed through this paradigm change, environmentally responsive, enhanced genetic mutation on various genomic levels of architecture can occur while defining or structuring levels of biological evolution so guided responsively via epigenesis by that mutation. This would be, through mutator-processes, an inner-regulated, responsively enhanced mutation to stresses. Thereby, this would have been a mutator-defined mutation enabling and controlling the rapid and responsively accelerated evolution of organismal, developmental capabilities and their expression. As the studies of the Aspergillus hypermutator-system indicate, these environmentally responsive, regulatory mutator systems could have themselves evolved in response to stress to become even more effective, responsive sources of beneficial mutations, underlying the increasing capacity for increasingly adaptive developmental evolution. Through such inner-operating mutator systems with developmental, responsive features, the evolvability of evolution would have become greatly enhanced through time, and this will probably continue into the future.

 In an investigation of bacterial evolution, hypermutators were found to generate, through many bacterial generations, increasing frequencies of adaptively beneficial mutations in response to increasing levels of alcohol stress. Some were far more effective and faster in doing so than others. When the stress subsided for the bacteria, that is when the bacteria became adapted, the hypermutators ceased to generate the mutations (Swings et al., 2017). As the data suggests, such mutators could have themselves evolved in response to stress so as to become more effective and quick generators of beneficial mutations, enabling adaptation to severe stress. Even in a lower organism, this shows types of developmental systems, controlling mutation, quickly responsive or "adaptively tuned", as the authors would put it, to changing environmental stresses, some severe. This very capability, to regulate or promote adaptively responsive mutation to stress, itself responsively and adaptively evolves, as could have karyotype-based mutator systems. In their studies, this evolvability of controlled mutation was also reflected on the molecular level. On this level, these regulated mutations occurred through mismatch repair or misrepair of DNA sequences. In 1989, it was proposed that misrepair of DNA sequences due to transposon insertion into the bacterial chromosome was involved in a global, inner-controlled hypermutation in bacteria, also argued to be environmentally responsive (Lieber, 1989).  Responsive mutators, with developmental features, would appear to have been operating at all genomic levels in all types of organisms. This would have a significant bearing on evolution.

Environmentally responsive mutators, in various organisms, operating on different hereditary levels, from the  molecular-genetic to the genomic/karyotypic, or even trans-genomic or epigenomic, could have enabled a hierarchy of evolution. This could have allowed for the rapid and simultaneous creation of new organisms on various taxonomic levels. Goldschmidt in 1940 did argue for a hierarchy of evolution based on changing karyotypic structures or chromosomal patterns that modified development. This view would appear to suggest that such a hierarchical, multi-dimensional evolution could have had an inner parameter, which might be construed as having developmental features. Such would also have had bearing on the evolution of behavior and the capacity for abstract symbolization in humans. As Jablonka and Lamb illustrate (2014), evolution has had four dimensions, namely the genetic, epigenetic, behavioral and that of symbolization. Such dimensions are illustrated as being environmentally responsive and interactive with one another, generating adaptive variation along their respective, mutually influencing avenues. All of these mutually contigent avenues would themselves have had environmentally responsive, developmental features through space-time.

From what we have seen, on a deeper, more inclusive level, the evolution of developmental and growth pattern systems would appear, in fact, to have an inner, ordering, stabilizing dynamic or component capable of quickly accommodating adaptively to environmental and internally-related epigenetic stresses, which tend to destabilize, and which in this context are mutagenic. Such accommodation, however, would involve the stable assimilation  and reduction of those very stresses into the dynamic configurations and architectures of all organisms, and thereby the development of adaptive phenotypes across many generations.

Evolving organisms are adaptively tuned to stresses through inner-regulated, environmentally responsive mutator-systems, enabling the organisms' adaptive stabilization though space-time and across many generations. Thus, evolution itself, on different levels, would appear to be a stabilizing, trans-generational, evolving developmental process, enhancing the very effective capacity of such a process to evolve, countering destabilization via multilevel, mutator-controlled, environmentally responsive mutation, through space-time. Such a developing, inner drive to evolving stabilization or dynamic completion could also be construed as an implicit, inner-driven, evolving selection process.

This would be a selection having a deeper, subtler, and more encompassing meaning than the one used traditionally in connection to discussions about evolution.  It would be a selection manifesting an increasing, inner capacity, enabled by an evolving environmentally responsive, inner directed mutator architecture, for adaptive evolution. This and the  perspective presented in this article would not only have significant implications for agricultural research, such as crop improvement, but could guide medical research, as well. Furthermore, it would give deep insight as to what it means to be human and to what our biology and minds mean for our own future evolution. Do we evolve so as to overcome our own limiting, destabilizing and destructive constraints? What future realities does our greatly enhanced, mutator-based evolvability foresee for us?

Michael M. Lieber, August, 2014, December, 2015. and July, 2017

   Photographs of Aspergillus Colonies with Dual Mutator

 
Colonies of Aspergillus nidulans from a large group of colonies each having produced through a karyotypic, dual mutator system many mutant yellow sectors in response to a temperature stress. The improved morphology, growth-rate, and conidial production of such sectors would indicate an adaptively responsive, inner-directed mutagenesis to temperature stress. This would be an environmentally responsive hypermutation expressed phenotypically as a new pattern of differentiation encompassing morphological change within sectors. Also, note in the bottom photograph the two white sectors of normal morphology and improved growth rate. These arise due to mutations within a gene epistatically controlling the production of colored conidia. Such sectors were only generated, though infrequently, in the strain with the two partial duplications in the genome. Their generation in this situation might have been due to the insertion of a small genetic element deleted from the reduced Dp III into (or very near to) the epistatic gene on chromosome II, resulting in the suppression of pigment production. Such insertion would have been in concurrence with deletions that would have otherwise produced yellow sectors.


 

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                                                  Biography

            
                                            Addendum I

Though technically considered single-cellular organisms, bacteria and yeast grow into colonies of many cells. Shapiro (1988) argues that bacteria should be considered as multicellular organisms. Nevertheless, compared to Aspergillus nidulans, they would still be very simple multicellular, undifferentiated organisms. As noted, non-growing colonies of bacteria subject to a particular, nutritional stress produced mutations in single cells that gave rise to protruding, growing sectors of cell clones adapted to the nutritional stress. These sectors stain red while non-growing colonies from which they emerge are white. The production of such adaptive sectors under stress is very analogous, if not related, to the production of the mutant, adaptive yellow sectors in the colonies of the Aspergillus-mutator strain. This is especially so as the production of such adaptive sectors by the non-growing, stressed bacterial colonies was under internal, genetic control involving the excision of insertion elements from a regulatory gene (Hall, 1988).

This internal, genetic control responsive to environmental stress might reflect a very early form of developmental system displayed by the bacterial colonies. It would be a basic system producing adaptive sector-variegation or differentiation throughout the colonies by means of responsive, controlled genetic change. Each such colony with genetic-based, responsive, adaptive variegation might be considered as an adaptive, developmental whole or unit. This very early developmental system in bacteria might very well have evolved into developmental, karyotypic mutator-systems such as exhibited by the Aspergillus system and possibly in other, higher organisms, such as maize. In this regard, suggesting a developmental process, a two-part mutator system in bacteria, adaptively responsive to environmental, nutritional stress exhibited temporal control of the occurrence of adaptive mutations (Lieber, 1989, 2001).

This temporal process involved transposons and recombination based on a mutant gene. In related studies with bacterial strains having a two-part mutator system involving transposons and recombination based on a mutant gene, directed, programmed mutagenesis in eight different bacterial strains enabled the same high frequency of growing colonies (within a short period) of each strain on respective media lacking the same group of amino acids for which the respective strains were genetically auxotrophic. This was in contrast to the response of auxotrophic, non-mutator strains. In effect (and implicitly), this represented adaptively responsive, directed mutation uniformly connected to overcoming the same multiple nutritional stresses within a short period, where transposition and recombination were involved (Lieber, 1989, e.g., pages 399, 391 and 385; Lieber.1998). Compared to the frequency of spontaneous mutations that would enable growth to the same group of amino acids under non-selective, non stressful conditions, the frequency of mutations, involving transposons, conferring adaptation under the nutritional stresses, would be exceedingly high.

Regarding transposon control, it was proposed in 1989 that this type of adaptively responsive mutagenesis occurred through misrepair of DNA due to the physical distortions of the bacterial DNA chromosome arising from the insertion of transposons into the chromosome (Lieber, 1989). Recent research has shown that adaptively responsive, increasing hypermutation, over many generations, enables many bacteria exposed to increasing alcohol stress, over those generations, to survive. Such beneficial mutations, promoted by the mutators, have occurred quickly in great frequency through the regulatory mutators promoting mismatch repair (or misrepair) of DNA sequences within the bacterial chromosome. (Swings et al., 2017). As the authors also point out, when the bacteria become adapted to the severe alcohol stress, thereby being no longer under stress, the hypermutation ceases. In view of these different investigations, responsive, developmental features were clearly indicated within varying levels of adaptively responsive mutagenesis tuned or connected to various degrees of environmental stresses.

With regard to the earlier research on hypermutagenic colonies, each growing colony would represent an outcome of a subpopulation of stressed cells within a manifold of non-growing cells on the culture medium, which, through hypermutability of their genomes linked to the nutritional stresses, became adaptive to the stresses. These growing colonies would represent variegation within a sea or manifold of stressed, non-growing cells. This manifold with adaptive, growing variegations might be likened to a macro-colony undergoing inner-controlled, adaptive variegation or differentiation. This would make such a macro- colony a developmentally responsive entity, in many ways related to the adaptively responsive Aspergillus colonies through their environmentally responsive variegation/differentiation. This might suggest deep evolutionary, regulatory connections between bacteria and the fungi.

Very simple colonial organisms have, it would appear, inner mutator-systems capable of evolving into complex, environmentally responsive mutator-systems in relatively higher organisms, such as in Aspergillus and maize (See Lieber, 1998). Environmentally responsive mutator-systems with developmental features of various complexities appear to have been pervasive throughout the evolution of life through their own evolution. The evolution of such developmental mutator-systems from earlier, simpler ones to more complex ones would have given a further inner, evolving evolvability to evolution.
 

                                               References


Hall, B. (1988). Adaptive evolution that requires multiple spontaneous mutations. I. Mutations involving an insertion sequence. Genetics 126: 5-16.

Lieber, M. (1989). New developments on the generation of mutations in Escherichia coli lysogens. Acta Microbiologica Hungarica 36(4): 377-413.

Lieber, M. (1998). Environmentally responsive mutator systems: toward a unifying perspective, Riv.Biol./B. Forum 91: 425-458.

Lieber, M. (2001). Temporal control of environmentally responsive hypermutation involving cryptic genes. Mutation Research 473: 255-257.

Shapiro, J. (1988). Bacteria as multicellular organisms. Scientific America. June: 82-89

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Swings, T. et al., 2017. Adaptive tuning of mutation rates allows fast response to lethal stress in Escherichia coli. eLife 2017;6:e22939. DOI: 10.7554/eLife.22939
 
 
  
                                                   Addendum II

Originally the karyotypic, dual mutator system in many green colonies of Aspegullus nidulans, having the reduced Dp III along with Dp I in its genome and designated as strain h, produced a very high frequency of programmed mutations at 36 degrees C involving the gene for green color on Dp I, as exhibited or manifested by the production, at the same time, of many mutant yellow sectors per colony (Lieber, 1976b, 1972). (Controls at this temperature having only Dp I produced a mean of one yellow sector per colony.) Retrospectively considered, this lower temperature could also have been stressful to Aspergillus strain/cultures having the dual mutator system, ensuing in the production of a very high frequency of mutant yellow sectors by the green colonies. The yellow mutant sectors were of increased growth rate relative to the green colonies. (See Figure 7.)  Also, see Figure 81 as well as Figure 88, and Figure 78. (These photos show four colonies from a large group of colonies having produced a high mean number of yellow sectors. Such colonies were termed h colonies or the h strain.). In view of this, an increasingly stressful range of temperatures from 36 degrees C to 39.5 degrees C, enabling adaptive mutation on the karyotypic level, is indicated.

Conidia, which gave rise a-sexually to new colonies, obtained from the above cultures after their storage at very low temperature for four and one-half months, gave rise, however, to green colonies having a very low frequency of mutant yellow sectors per colony at 36 degrees C. This frequency of mutant sector production was the same as that produced at 36 degrees C by colonies only having Dp I, the uni-mutator strain designated as P. (See Figure 3.) This frequency was a mean of one mutant yellow sector per colony.

The first green, dual mutator strain h had originally been derived from the green improved sectors produced by another, newly generated, dual mutator strain, designated as RP-81. RP-81 non-aged, colonies were grown at 36 degrees C. RP-81* has partial duplications of chromosomes I and III in a haploid genome. These green sectors of greatly increased growth rate are indicative of deletions having occurred from the partial III, chromosomal duplication, reducing its size or content, being referred to as the reduced Dp III or the changed III duplication. (This was confirmed through genetic analysis, Lieber, 1972, 1976b.)

From such newly generated green sectors produced by colonies derived from an aged strain of RP-81**  through deletions, newly produced conidia were isolated and used to produce new, green colonies of strain h at 36 degrees C. (Strain h has a reduced III duplication, reduced Dp III, and the partial chromosomal I duplication, Dp I). These new colonies produced on average 3.4 yellow sectors per colony. This frequency was significantly higher at P < 0. 01 than the mean frequency of such produced by strain P, which was a mean of 1.1 sectors per colony. Though significantly higher than the frequency of mutant yellow sectors produced by the uni-mutator strain P, this frequency was nevertheless far lower than the frequency of such, which the original, non-aged, h strain had produced four and half months earlier. Moreover, the  mutant sectors produced by this dual mutator strain four and one-half months later had not emerged at the same time, suggesting the loss of the program control of mutation that was displayed by its progenitor.

One of these h colonies, h123, had produced many yellow sectors. Conidia from such was used to to create another group of h colonioes at 36 degrees C., the group designated as h123 colonies. This group of h123 colonies produced a mean of 5.3 yellow sectors per colony. Figure 1 below shows one of those colonies. This mean was significantly higher at P< 0.01 than the mean mutant frequencies of one yellow sector per colony.

Furthermore, when h colonial cultures from new a-sexual conidia, isolated from newly generated green sectors of newly cultured RP-81, were grown at the higher temperature of 39.5 degrees C, the frequency of mutant yellow sectors greatly increased or enhanced. This was a mean of 13.7 yellow mutant sectors per green colony, significantly higher  at P < 0.01 compared to the mean frequency of mutant sectors produced by the same strain at 36 degrees C (3.4 yellow sectors per colony), and significantly higher at P< 0.01 compared to the frequency of yellow mutant sectors produced by the uni-mutator strain at 39.5 degrees C, which was a mean frequency of 5 yellow mutant sectors per colony. (See Figure 2 below.) Also, at this higher temperature, all mutant sectors emerged at the same time from the h colonies, indicating the re-emergence of the responsive, temporal program guiding the degree of mutation (Lieber, 1972, 1976b). The vertices of the yellow sectors being equidistant from colony centers indicated that such yellow mutant sectors emerged at the same time or nearly so. (See images.)

 As described (Lieber, 1972), some of these h colonies cultured at 39.5 degrees C had produced far more mutant sectors per colony compared to the mean number per colony of the group. From one of such colonies,  h126, conidia were obtained and used to produce new h colonies cultured again at 39.5 degrees C and at 36 degrees C. These were referred to as h126 colonies. P colonies, having the karyotypic, uni-mutator, Dp I, were also grown at 39.5 degrees C and 36 degrees C. The P colony groups at 36 degrees C and 39.5 degrees C respectively produced a mean of 1.2 yellow sectors per colony and  a mean of 5.4 yellow sectors per colony, indicating that the uni-muator is also responsive to high temperature. 

The group of h126 colonies cultured at 36 degrees C produced a mean of  3.8 yellow sector per colony. Those yellow sectors had not emerged at the same time. The group of h126 colonies cultured at 39.5 degrees C had produced a mean number of 21 yellow mutant yellow sectors per colony, which at P < 0.01 was significantly higher from the means 3.8 and 5.4.  And these sectors emerged from each  h126 colony at the same time, again demonstrating the re-emergence of the temporal program governing responsive mutagenesis. (See images) As pointed out then by this author, these results clearly indicated that the dual mutator system was highly responsive to the increase in temperature by generating a high frequency of mutations in a highly programmed manner.

My conclusion at the time, however, was that the mutagenic interaction between the two components of the dual mutator system was not more effective at the higher temperature than at the lower. This conclusion was assessed later to be incorrect, especially as this conclusion was inconsistent with the experimental findings, e.g., the re-emergence of the temporal control of mutation due to growth at that higher temperature. Moreover, as the evidence indicates, the high temperature and newly generated conidia from the newly generated green sectors, and from the newly generated h126 colony that had produced many yellow sectors at 39.5 degrees C, enabled the dual mutator system, h colonies to become very effective as a group in promoting in a regulated manner a very high frequency of mutations from Dp I, and which were exhibited phenotypically as the many mutant yellow sectors produced, at the same time, by the respective h colonies.

 As the evidence indicates here, the stress of high temperature enabled or stimulated the karyotypic, dual mutator system to promote in a programmed or controlled manner a very high degree of mutations, which appeared to be beneficial under those conditions. A stress on the karyotypic, dual mutator genome can also operate, it would appear, through a lower temperature when the physiological effects of age are not operating, resulting also in an environmentally responsive, controlled, enhanced mutation. The particular physiological state occurring through age-affected cultures would appear to have suppressed a programmed, mutagenic response to stress. As the observations indicated, growth at the higher or stressful temperature negated such suppression, enabling the re-establishment of an effective, programmed, mutagenic interaction between the components of the karyotypic, dual mutator system.

Investigations described in Lieber, 1976a, also showed that a green, karyotypic-uni-mutator strain of Aspegillus, carrying only Dp I, can also respond mutagenically to a high temperature, by producing yellow sectors of improved growth rate and morphology at a later culture period. However, there was not clear evidence of the type of programmed mutagenesis that was operating in the dual mutator system. As indicated in Lieber, 1972, 1976b and by the information presented here, the reduced Dp III component of the dual mutator system assumed the role of the regulator of the degree of mutations involving the Dp I component, taking programmed control of the Dp I uni-mutator when physiological/environmental conditions allowed or induced.

As demonstrated in Lieber, 1972 and 1976b, colony size or circumference is not a factor in different mutation frequencies. As can be seen in the photos, only one colony existed per culture plate. Equal numbers of colonies were used in the respective experimental and control groups. The number of colonies in each group was generally 40, though in one experiment 33 colonies were studied in respective experimental and control groups. Colonies were cultured for 10-12 days.

 In another addendum to this article, the author intends to include written sections from Lieber, 1972, which is the author's Ph.D. dissertation. This should provide further support to the account and perspective presented in this article.

*Groups of RP81 non-aged, colonies cultured at 36 degrees C produce mutant yellow sectors at respective means of about three yellow sectors per colony. The colonies were obtained from a newly isolated RP-81 strain. These yellow sectors generally emerge from the green improved sectors produced by the RP81 colonies. In order for colonies, which are derived from such sectors, to respectively produce very high frequencies of yellow sectors, the colonies must arise or generate from germinating conidia in the centers of the culture medium on Petri dishes. This is described fully, with implications, in Lieber, 1972, 1976b.

**These RP81 colonies had come from aged conidia of a stored RP-81 colony.

                                                          Figure 1

A colony of the dual mutator strain h123 from a group of h123 colonies cultured at 36 degrees C for 10 days. Related h colonies of this strain at 39.5 degrees C are presented in the first group of photographs in this article.

                                                            Figure 2

A colony of the uni-mutator, strain P, from a group of colonies grown at 39.5 degrees C for 10 days. The mutant yellow sectors are of increased growth rate and improved morphology. Most  appear to have arisen about the same time during the later culture period. This would suggest that the uni-mutator, Dp I, is also capable of responding to a temperature stress at a certain time when in the P genome. However,  while in the h strain or genome, its response to such stress can be greatly controlled or programmed by the reduced Dp III. The P colonies grown at this elevated temperature were also used as controls for the h colonies grown under temperature stress.

                                                                   Figure 3

A colony of the uni-mutator, strain P, from groups of colonies grown at 36 degrees C for 10 days.

                                                              Figure 78

Cultured at 36 degrees C, a colony of strain 78 (another colony from a group of h colonies), with the dual mutator system in its genome. This lower temperature could also have been stressful. More specifically, the dual mutator system is likely responding in a controlled, mutagenic manner to a epigenetic stress mediated by temperature and the related cytoplasmic/physiological situation. This could be construed as being an internal stress connected to genomic and environmental conditions. After 4.5 months, cultures obtained from this colony (and others) via aged conidia, gave rise to colonies at 36 degrees C in which the dual mutator effect has been lost or suppressed, only to be re-activated in other h colonies through newly regenerated conidia and a stressful, high temperature, creating a new stressful epigenetic condition to which responsive, karyotypic mutator/phenotypic adaptation occurs again.

                                                               Figure 81

 Cultured at 36 degrees C, a colony of strain h81(another colony from a group of h colonies), with the dual mutator system in its genome. This lower temperature could also have been stressful. More specifically, the dual mutator system is likely responding in a controlled, mutagenic manner to a epigenetic stress mediated by temperature and the related cytoplasmic/physiological situation. This could be construed as being an internal stress connected to genomic and environmental conditions. After 4.5 months, cultures obtained from this colony (and others) via aged conidia, gave rise to colonies at 36 degrees C in which the dual mutator effect has been lost or suppressed, only to be re-activated in other h colonies through newly regenerated conidia and a stressful, high temperature, creating a new stressful epigenetic condition to which responsive, karyotypic mutator/phenotypic adaptation occurs again.

                                                           Figure 88

Cultured at 36 degrees C, a colony of strain h88 (another colony from a group of h colonies), with the dual mutator system in its genome. This lower temperature could also have been stressful. More specifically, the dual mutator system is likely responding in a controlled, mutagenic manner to a epigenetic stress mediated by temperature and the related cytoplasmic/physiological situation. This could be construed as being an internal stress connected to genomic and environmental conditions. After 4.5 months, cultures obtained from this colony (and others) via aged conidia, gave rise to colonies at 36 degrees C in which the dual mutator effect has been lost or suppressed, only to be re-activated in other h colonies through newly regenerated conidia and a stressful, high temperature, creating a new stressful epigenetic condition to which responsive, karyotypic mutator/phenotypic adaptation occurs again.

                                                                      Figure 99

A second generation, dual-mutator strain cultured at 39.5 degrees C. This colony is from a group of 40 colonies derived from one colony obtained from the conidia of a h type sector, with a reduced III duplication and the I duplication. The h type sector had been produced by a colony whose second generation genome is similar to that of RP-81. These colonies, cultured for 12 days, produced a mean number of 15.1 mutant yellow sectors per colony.

                                                           References

Lieber, M. (1972). Environmental and genetic factors affecting instability at mitosis in Aspergillus nidulans. Ph.D. Thesis, University of Sheffield.

Lieber  M. (1976a). The effects of temperature on genetic instability in Aspergillus nidulans, Mutation Res. 34: 94-122.

Lieber, M. (1976b). The genetic instability and mutagenic interaction of chromosomal duplications present together in haploid strains of Aspergillus nidulans, Mutation Res. 37: 33-66.

                                                         Addendum III

    As indicated previously, there are many possible mechanisms or avenues whereby the dual mutator  system in Aspergillus operates in response to stress. With regard to the single mutator system in Aspergillus, Dp I, the author proposed in 1968 that the insertion of a gene, uvs, which is deficient in the repair of damaged DNA due to UV exposure into a Dp I genome, could enhance, through its presence, the deletional instability of that duplication strain, as marked by a significant increase in the frequency of yellow sectors produced by green Dp I colonies. This hypothesis was to be tested as part of the author's proposed graduate research at the Department of Genetics, University of Sheffield, England. However, the author did not pursue this research, concentrating instead on investigations pertaining to the dual mutator strain of Aspergillus. Others in that department did eventually pursue that area of research, leading to its publication by Burr et al. in 1971 and 1982. As their research showed, the addition of a given uvs gene, uvsB, deficient in the function of gene repair, into the genome of the Dp I strain of Aspergillus, ensued, through that gene's presence, in a significant increase in the deletion of the genetic region containing the green pigment gene, y+. This was reflected by a significant increase in the frequency of yellow sectors produced by green colonies. Approximate means of 5 yellow sectors (of improved growth rates) per green colony were noted in experimental groups as compared to approximate means of 1 yellow sector (of improved growth rate) per green colony in the control groups, which had the gene for DNA repair, uvs+. Through an additional experiment, the role of the duplication in the stability was also indicated. In view of the mutagenic effect of the uvsB gene, it is seen that this role can be influenced or modified by other genetic factors in the genome, such as genetic repair systems. Culture temperature was 37 degrees C.

In the author's study of the effects of culturing at high temperature, 39.5 degrees C, on deletional instability of the Dp I strain with a normal repair system, approximate means of 5 yellow sectors (of improved growth rates) per green colony were observed in experimental groups as opposed to approximate means of 1 yellow sector (of improved growth rate) per green colony in control groups, the latter being cultured at 36 degrees C. It was concluded that culturing at a increased temperature enhanced deletional instability, namely, an increase in the frequency of deletions including y+ (Lieber, 1976a). The identity between the degree in increased instability due to deficient DNA repair in a Dp I strain because of the presence of a repair-deficient gene, uvsB, in the Dp I genome, with the degree of increased instability of a non-uvs, Dp I-strain, due to an increased temperature, now suggests that culturing at an increased temperature also impaired DNA repair, leading to increased instability. As the increase in yellow sector frequency at 39.5 degrees C occurred within a narrow period during late growth of the green colonies, it is suggested that the impaired genetic repair process was subsumed or coordinated under some type temporal program.

At the time, in 1976, one conjectured that increased temperature increased heterochromatization during a specific or narrow period, and that increased deletion occurred through such. Now, perhaps, one can also conjecture that increased heterochromatization within the genome inactivated genes (uvs+) responsible for genetic repair, leading to increased deletion at the higher temperature. Also, distortion of cytoplasmic membrane structures due to increased temperature, where mRNA translation is anchored, might have also inhibited the production of repair enzymes through a destabilizing of the anchoring.

All of this would be worthy of further investigation, especially a study of the effects of uv-deficient, repair genes on the dual mutator system at various temperatures. It is likely that genetic repair systems underlie, while being subsumed by, in some manner the controlled, adaptively responsive mutagenic behavior of the dual mutator system in Aspergillus, especially with regard to stress. However, it is presently unknown how and why this is the situation. The existence of such repair systems do not explain the programmed, mutagenic behavior of the dual mutator strain in response to temperature stress. Though, it appears that the existence of such genetic repair systems, at some basic molecular level, nevertheless, must enable such responsive, programmed mutator processes to occur at higher levels of organization, while also being subsumed by such programmed mutator processes.

Further research may show that  such systems involved in genetic repair might in fact function as coordinated, misrepair systems, whose coordination through various levels of organization via forces, is enabled through changes in force-fields, due to stress, across different levels of organization.

                                                References

Burr, K., Palmer, H., and Roper, J. A. Mitotic Non-Conformity in Aspergillus nidulans: The Effect of Reduced DNA Repair. Heredity 27, 387

Burr, K. W., Roper, J. A., and Janice Relton. (1982). Modification of chromosome instability in Aspergillus nidulans, Journal of General Microbiology, 128: 2899-2907.

Lieber  M. (1976a). The effects of temperature on genetic instability in Aspergillus nidulans, Mutation Res. 34: 94-122.

                                                Addendum IV

  The original data on which this article is based is presented in the author's Ph.D thesis (Lieber, 1972). One can download this thesis by accessing http://ethos.bl.uk/Home.do  This is e-Thesis Online Service administered by the British Library. Register on this site, and then in the site, type in the title of the thesis: Environmental and Genetic Factors Affecting Instability at Mitosis in Aspergillus nidulans in order to initiate the download. Once downloaded, begin reading the thesis on page 214.

                                                 Addendum V

As documented, there is much evidence for the existence and behavior of the dual mutator system on the karyotypic level. Nevertheless, a person may wonder how one's research peers viewed it. In 1976, the author received many requests for reprints of his publications on the subject. In a personal communication to the author, Barbara McClintock, the future Nobel Laureate, stated that she recognized the importance of those investigations. This gives the author's investigations a valid and credible place in the history of biology.

                                    Letter from Dr. Barbara McClintock