Final Report Summary - MULTISELF (Multiple levels of selection in Fungi)
General background
The research context of the project is the interaction between selection levels, in connection with the broader issue of the emergence of multicellularity. In multicellular organisms, selection can act at the higher level of the whole organism, but also at lower levels, like genes, nuclei, or cells, provided they are genetically heterogeneous. Intra-organismal genetic heterogeneity exists in individuals of a wide range of taxa, and may lead to genetic conflicts between selection levels. The best-known example is cancer, where genetic heterogeneity of cells can leads to the disruption of the whole organism. However, focusing on cheating cell lineages, like cancerous cells, may have lead to overlook that different levels of integration may also act in cooperation rather than generate genetic conflicts.
Fungi are ideal models to test for these issues of multilevel selection because of the heterokaryotic phase in their life cycle, i.e. a phase where genetically divergent nuclei coexist in the mycelium. We investigated if selection at the nuclear level is acting in cooperation, or conflict, with the organismal level. We have combined experimental approaches with genomic approaches to investigate these issues. Our model species, Neurospora tetrasperma, has undergone a recent shift towards permanent heterokaryosis [1]. Nuclei of opposite mating types, mat A and mat a, are packaged into sexual spores (the ascospores) and do not fuse in the mycelium, except at the very onset of meiosis [2]. Occasionally, spores of single mating-type may be formed, restoring an haploid state for the mycelium (homokaryon) until mating takes place again [3]. Heterokaryosis in N. tetrasperma leads to a substantial within-organism variation. Nuclear divergence amounts up to 3.2% on the mating-type chromosome (i.e. the chromosome carrying the mat locus), where over 1500 genes are linked to mat [4-6], whereas the other six chromosomes show more similarity [4]. The genetic divergence also translates into expression divergence between nuclei of different mating type [7], thus likely into phenotypic divergence.
Project purposes and main findings
The first axis of our research focused on nuclear ratios between mating-type nuclei in N. tetrasperma. We developed a qPCR method to be able to follow nuclear ratios in different lineages of our model species, under different growth conditions. This experimental approach allowed us to investigate how different heterokaryosis is from diploidy: we verified that the ratio is homogeneous within the mycelium, but that it can deviate from the diploid-like 1:1-condition. We also showed that different mating type nuclei might have differing or constrained rates of replication in the mycelium, depending on the lineage. Medium had also an effect on the nuclear ratio.
We also measured fitness traits of the mycelium and its component nuclear genotypes. In combining results of both genetic and fitness studies, we found indications of both advantages and disadvantages of heterokaryosis in our study system. First, we found that nuclear types have different fitness optima during the life cycle, indicating division of labor under the heterokaryotic condition. The phenotype of the mycelium is reflecting the nuclear ratio in an additive manner. Thus, our data indicate that within-organism variation results in phenotypic flexibility. Second, in one of the studied strains, we observed that nuclei of one of the mating type enjoyed better transmission than its sister nuclei in asexual spores, and higher replication in the mycelium; at the same time fitness studies showed that the successful nuclear type was being detrimental to the organism: this is a clear case of the existence of selfish nuclear types in our system.
Heterokaryotic fungi thus show a state of multicellularity where transfer of fitness to the higher level of the organism seems, at most, incomplete.
The second axis of this project is a genomic approach investigating more functional aspects of heterokaryosis, specifically, how gene expression is regulated under heterokaryosis. We set up an RNAseq analysis on heterokaryons and homokaryons of N. tetrasperma, using the same lineages as in axis 1 above. We aim at comparing gene expression in homokaryons and heterokaryons differing in their nuclear content, ie mating type nuclear ratio, grown under vegetative or sexual phase of the life cycle.
We will investigate if gene expression in heterokaryons is purely additive between mat A or mat a levels of expression in homokaryons, or if it is non-additive. Non-additive gene expression changes in heterokaryons would reveal an impact of heterokaryosis on the evolution of gene expression, by analogy to hybrids where novel interactions among alleles can result in non-additive gene expression compared to parents [8]. Mating type specific gene expression will also be compared between heterokaryons of different nuclear ratio in N. tetrasperma to understand if gene expression is collinear to DNA content, or if compensation of dosage can occur, indicating a higher level of coordination between nuclear types. Specialization of function of either mating type over the life cycle, as found in axis 1, suggests mating-type specific differential expression depending on the life cycle phase. To be able to compare permanent heterokaryosis to the ancestral state, we included in the design homokaryons of N. tetrasperma sister species N. sitophila and N.crassa which are both predominantly haploid. We expect to observe mating-type specific expression in N.tetrasperma only, as a hallmark of increased complexity in the multicellular state.
Finally, the third axis of the project reuses part of the RNAseq data to investigate the consequences of suppression of recombination on gene expression. A suppression of recombination has occurred along the mating-type chromosome in N. tetrasperma, ensuring the efficiency of mating under permanent heterokaryosis. The non-recombining part encompasses around thousand genes that diverged subsequently between mat A and mat a. The suppression of recombination in the mating-type chromosome renders this chromosome very similar to sex chromosomes in animals and plants. When recombination is suppressed, selection is relaxed and genes degenerate, accumulating deleterious mutations. Molecular degeneration has been shown in N.tetrasperma [9, 10], despite the recent origin of recombination arrest –about 1MA - [1]. How is then expression affected? We’ll compare RNA expression on the mating type chromosome between species with recombining (Neurospora sitophila and N. crassa) and non-recombining (different lines of N.tetrasperma) mating-type chromosomes.
For axes 2 and 3, data is currently under analysis: a pipeline to investigate allele specific expression (here mating type specific expression) is under development. Results and their dissemination are expected under the next year.
Impact
Our results led to a better understanding of heterokaryosis, a common, yet still understudied genetic system, leading to new insights into the evolution of multicellularity. This project has potential expected outputs in cancer research, as cancer being a typical case of a detrimental consequence of conflicts between levels of selection, leading to deleterious effects at the individual level. Our results in the consequences of genetic heterogeneity will be of importance to an evolutionary view of cancer [11]. The ultimate goal is to consider new forms of therapy, in particular therapies that would not select for mutant cells even more selfishly aggressive and replicative.
References
1. Corcoran, P., et al., A global multilocus analysis of the model fungus Neurospora reveals a single recent origin of a novel genetic system. Molecular Phylogenetics and Evolution, 2014. 78: p. 136-147.
2. Raju, N.B. and D.D. Perkins, Diverse programs of ascus development in pseudohomothallic species of Neurospora, Gelasinospora, and Podospora. Developmental Genetics, 1994. 15(1): p. 104-118.
3. Raju, N.B. Functional heterothallism resulting from homokaryotic conidia and ascospores in Neurospora tetrasperma. Mycological Research, 1992. 96: p. 103-116.
4. Corcoran, P., et al., Introgression maintains the genetic integrity of the mating-type determining chromosome of the fungus Neurospora tetrasperma. Genome research, 2016. 26(4): p. 486-98.
5. Ellison, C.E. et al., Massive Changes in Genome Architecture Accompany the Transition to Self-Fertility in the Filamentous Fungus Neurospora tetrasperma. Genetics, 2011. 189(1): p. 55-U652.
6. Merino, S.T. et al., Pseudohomothallism and evolution of the mating-type chromosome in Neurospora tetrasperma. Genetics, 1996. 143(2): p. 789-799.
7. Samils, N., et al., Sex-linked transcriptional divergence in the hermaphrodite fungus Neurospora tetrasperma. Proceedings of the Royal Society B-Biological Sciences, 2013. 280(1764).
8. Bell, G.D.M. et al., RNA-Seq Analysis of Allele-Specific Expression, Hybrid Effects, and Regulatory Divergence in Hybrids Compared with Their Parents from Natural Populations. Genome Biology and Evolution, 2013. 5(7): p. 1309-1323.
9. Whittle, C.A. Y. Sun, and H. Johannesson, Evolution of Synonymous Codon Usage in Neurospora tetrasperma and Neurospora discreta. Genome Biology and Evolution, 2011. 3: p. 332-343.
10. Whittle, C.A. Y. Sun, and H. Johannesson, Degeneration in Codon Usage within the Region of Suppressed Recombination in the Mating-Type Chromosomes of Neurospora tetrasperma. Eukaryotic Cell, 2011. 10(4): p. 594-603.
11. Aktipis, A., Principles of cooperation across systems: from human sharing to multicellularity and cancer. Evolutionary Applications, 2016. 9(1): p. 17-36.
The research context of the project is the interaction between selection levels, in connection with the broader issue of the emergence of multicellularity. In multicellular organisms, selection can act at the higher level of the whole organism, but also at lower levels, like genes, nuclei, or cells, provided they are genetically heterogeneous. Intra-organismal genetic heterogeneity exists in individuals of a wide range of taxa, and may lead to genetic conflicts between selection levels. The best-known example is cancer, where genetic heterogeneity of cells can leads to the disruption of the whole organism. However, focusing on cheating cell lineages, like cancerous cells, may have lead to overlook that different levels of integration may also act in cooperation rather than generate genetic conflicts.
Fungi are ideal models to test for these issues of multilevel selection because of the heterokaryotic phase in their life cycle, i.e. a phase where genetically divergent nuclei coexist in the mycelium. We investigated if selection at the nuclear level is acting in cooperation, or conflict, with the organismal level. We have combined experimental approaches with genomic approaches to investigate these issues. Our model species, Neurospora tetrasperma, has undergone a recent shift towards permanent heterokaryosis [1]. Nuclei of opposite mating types, mat A and mat a, are packaged into sexual spores (the ascospores) and do not fuse in the mycelium, except at the very onset of meiosis [2]. Occasionally, spores of single mating-type may be formed, restoring an haploid state for the mycelium (homokaryon) until mating takes place again [3]. Heterokaryosis in N. tetrasperma leads to a substantial within-organism variation. Nuclear divergence amounts up to 3.2% on the mating-type chromosome (i.e. the chromosome carrying the mat locus), where over 1500 genes are linked to mat [4-6], whereas the other six chromosomes show more similarity [4]. The genetic divergence also translates into expression divergence between nuclei of different mating type [7], thus likely into phenotypic divergence.
Project purposes and main findings
The first axis of our research focused on nuclear ratios between mating-type nuclei in N. tetrasperma. We developed a qPCR method to be able to follow nuclear ratios in different lineages of our model species, under different growth conditions. This experimental approach allowed us to investigate how different heterokaryosis is from diploidy: we verified that the ratio is homogeneous within the mycelium, but that it can deviate from the diploid-like 1:1-condition. We also showed that different mating type nuclei might have differing or constrained rates of replication in the mycelium, depending on the lineage. Medium had also an effect on the nuclear ratio.
We also measured fitness traits of the mycelium and its component nuclear genotypes. In combining results of both genetic and fitness studies, we found indications of both advantages and disadvantages of heterokaryosis in our study system. First, we found that nuclear types have different fitness optima during the life cycle, indicating division of labor under the heterokaryotic condition. The phenotype of the mycelium is reflecting the nuclear ratio in an additive manner. Thus, our data indicate that within-organism variation results in phenotypic flexibility. Second, in one of the studied strains, we observed that nuclei of one of the mating type enjoyed better transmission than its sister nuclei in asexual spores, and higher replication in the mycelium; at the same time fitness studies showed that the successful nuclear type was being detrimental to the organism: this is a clear case of the existence of selfish nuclear types in our system.
Heterokaryotic fungi thus show a state of multicellularity where transfer of fitness to the higher level of the organism seems, at most, incomplete.
The second axis of this project is a genomic approach investigating more functional aspects of heterokaryosis, specifically, how gene expression is regulated under heterokaryosis. We set up an RNAseq analysis on heterokaryons and homokaryons of N. tetrasperma, using the same lineages as in axis 1 above. We aim at comparing gene expression in homokaryons and heterokaryons differing in their nuclear content, ie mating type nuclear ratio, grown under vegetative or sexual phase of the life cycle.
We will investigate if gene expression in heterokaryons is purely additive between mat A or mat a levels of expression in homokaryons, or if it is non-additive. Non-additive gene expression changes in heterokaryons would reveal an impact of heterokaryosis on the evolution of gene expression, by analogy to hybrids where novel interactions among alleles can result in non-additive gene expression compared to parents [8]. Mating type specific gene expression will also be compared between heterokaryons of different nuclear ratio in N. tetrasperma to understand if gene expression is collinear to DNA content, or if compensation of dosage can occur, indicating a higher level of coordination between nuclear types. Specialization of function of either mating type over the life cycle, as found in axis 1, suggests mating-type specific differential expression depending on the life cycle phase. To be able to compare permanent heterokaryosis to the ancestral state, we included in the design homokaryons of N. tetrasperma sister species N. sitophila and N.crassa which are both predominantly haploid. We expect to observe mating-type specific expression in N.tetrasperma only, as a hallmark of increased complexity in the multicellular state.
Finally, the third axis of the project reuses part of the RNAseq data to investigate the consequences of suppression of recombination on gene expression. A suppression of recombination has occurred along the mating-type chromosome in N. tetrasperma, ensuring the efficiency of mating under permanent heterokaryosis. The non-recombining part encompasses around thousand genes that diverged subsequently between mat A and mat a. The suppression of recombination in the mating-type chromosome renders this chromosome very similar to sex chromosomes in animals and plants. When recombination is suppressed, selection is relaxed and genes degenerate, accumulating deleterious mutations. Molecular degeneration has been shown in N.tetrasperma [9, 10], despite the recent origin of recombination arrest –about 1MA - [1]. How is then expression affected? We’ll compare RNA expression on the mating type chromosome between species with recombining (Neurospora sitophila and N. crassa) and non-recombining (different lines of N.tetrasperma) mating-type chromosomes.
For axes 2 and 3, data is currently under analysis: a pipeline to investigate allele specific expression (here mating type specific expression) is under development. Results and their dissemination are expected under the next year.
Impact
Our results led to a better understanding of heterokaryosis, a common, yet still understudied genetic system, leading to new insights into the evolution of multicellularity. This project has potential expected outputs in cancer research, as cancer being a typical case of a detrimental consequence of conflicts between levels of selection, leading to deleterious effects at the individual level. Our results in the consequences of genetic heterogeneity will be of importance to an evolutionary view of cancer [11]. The ultimate goal is to consider new forms of therapy, in particular therapies that would not select for mutant cells even more selfishly aggressive and replicative.
References
1. Corcoran, P., et al., A global multilocus analysis of the model fungus Neurospora reveals a single recent origin of a novel genetic system. Molecular Phylogenetics and Evolution, 2014. 78: p. 136-147.
2. Raju, N.B. and D.D. Perkins, Diverse programs of ascus development in pseudohomothallic species of Neurospora, Gelasinospora, and Podospora. Developmental Genetics, 1994. 15(1): p. 104-118.
3. Raju, N.B. Functional heterothallism resulting from homokaryotic conidia and ascospores in Neurospora tetrasperma. Mycological Research, 1992. 96: p. 103-116.
4. Corcoran, P., et al., Introgression maintains the genetic integrity of the mating-type determining chromosome of the fungus Neurospora tetrasperma. Genome research, 2016. 26(4): p. 486-98.
5. Ellison, C.E. et al., Massive Changes in Genome Architecture Accompany the Transition to Self-Fertility in the Filamentous Fungus Neurospora tetrasperma. Genetics, 2011. 189(1): p. 55-U652.
6. Merino, S.T. et al., Pseudohomothallism and evolution of the mating-type chromosome in Neurospora tetrasperma. Genetics, 1996. 143(2): p. 789-799.
7. Samils, N., et al., Sex-linked transcriptional divergence in the hermaphrodite fungus Neurospora tetrasperma. Proceedings of the Royal Society B-Biological Sciences, 2013. 280(1764).
8. Bell, G.D.M. et al., RNA-Seq Analysis of Allele-Specific Expression, Hybrid Effects, and Regulatory Divergence in Hybrids Compared with Their Parents from Natural Populations. Genome Biology and Evolution, 2013. 5(7): p. 1309-1323.
9. Whittle, C.A. Y. Sun, and H. Johannesson, Evolution of Synonymous Codon Usage in Neurospora tetrasperma and Neurospora discreta. Genome Biology and Evolution, 2011. 3: p. 332-343.
10. Whittle, C.A. Y. Sun, and H. Johannesson, Degeneration in Codon Usage within the Region of Suppressed Recombination in the Mating-Type Chromosomes of Neurospora tetrasperma. Eukaryotic Cell, 2011. 10(4): p. 594-603.
11. Aktipis, A., Principles of cooperation across systems: from human sharing to multicellularity and cancer. Evolutionary Applications, 2016. 9(1): p. 17-36.