Arielle M. Cooley

Biologists have long been interested in understanding genetic constraints on the evolution of development. For example, noncoding changes in a gene might be favored over coding changes if they are less constrained by pleiotropic effects. Here, we evaluate the importance of coding‐sequence changes to the recent evolution of a novel anthocyanin pigmentation trait in the monkeyflower genus Mimulus. The magenta‐flowered Mimulus luteus var. variegatus recently gained petal lobe anthocyanin pigmentation via a single‐locus Mendelian difference from its sister taxon, the yellow‐flowered M. l. luteus. Previous work showed that the differentially expressed transcription factor gene MYB5a/NEGAN is the single causal gene. However, it was not clear whether MYB5a coding‐sequence evolution (in addition to the observed patterns of differential expression) might also have contributed to increased anthocyanin production in M. l. variegatus. Quantitative image analysis of tobacco leaves, transfected with MYB5a coding sequence from each taxon, revealed robust anthocyanin production driven by both alleles. Counter to expectations, significantly higher anthocyanin production was driven by the allele from the low‐anthocyanin M. l. luteus, a result that was confirmed through both a replication of the initial study and analysis by an alternative method of spectrophotometry on extracted leaf anthocyanins. Together with previously published expression studies, our findings support the hypothesis that petal pigment in M. l. variegatus was not gained by protein‐coding changes, but instead solely via noncoding cis‐regulatory evolution. Finally, while constructing the transgenes needed for this experiment, we unexpectedly discovered two sites in MYB5a that appear to be post‐transcriptionally edited—a phenomenon that has been rarely reported, and even less often explored, for nuclear‐encoded plant mRNAs.

The origin of phenotypic novelty is a perennial question of genetics and evolution. To date, few studies of biological pattern formation specifically address multi-generational aspects of inheritance and phenotypic novelty. For quantitative traits influenced by many segregating alleles, offspring phenotypes are often intermediate to parental values. In other cases, offspring phenotypes can be transgressive to parental values. For example, in the model organism Mimulus (monkeyflower), the offspring of parents with solid-colored petals exhibit novel spotted petal phenotypes. These patterns are controlled by an activator-inhibitor gene regulatory network with a small number of loci. Here we develop and analyze a model of hybridization and pattern formation that accounts for the inheritance of a diploid gene regulatory network composed of either homozygous or heterozygous alleles. We find that the resulting model of multi-generational Turing-type pattern formation can reproduce transgressive petal phenotypes similar to those observed in Mimulus. The model gives insight into how non-patterned parent phenotypes can yield phenotypically transgressive, patterned offspring, aiding in the development of empirically testable hypotheses.

The origin of phenotypic novelty is a perennial question of genetics and evolution. To date, few studies of biological pattern formation specifically address multi-generational aspects of inheritance and phenotypic novelty. For quantitative traits influenced by many segregating alleles, offspring phenotypes are often intermediate to parental values. In other cases, offspring phenotypes can be transgressive to parental values. For example, in the model organism Mimulus (monkeyflower), the offspring of parents with solid-colored petals exhibit novel spotted petal phenotypes. These patterns are controlled by an activator-inhibitor gene regulatory network with a small number of loci. Here we develop and analyze a model of hybridization and pattern formation that accounts for inheritance in diploid gene regulatory networks composed of either homozygous or heterozygous alleles. We find that the resulting model of multi-generational Turing-type pattern formation can reproduce the transgressive petal phenotypes observed in Mimulus . The model gives insight into how non-patterned parent phenotypes can yield phenotypically transgressive, patterned offspring, aiding in the development of empirically testable hypotheses.

Phenotypic transitions in related taxa often share a common genetic basis, which suggests that there are constraints that shape the process of evolution at the genetic level. For example, noncoding changes in a gene might be favored relative to coding changes due to being less constrained by pleiotropic effects. Here we evaluate the importance of coding-sequence changes to the recent evolution of a novel anthocyanin pigmentation trait in the monkeyflower genus Mimulus . The magenta-flowered Mimulus luteus var. variegatus recently gained petal lobe anthocyanin pigmentation via a single-locus Mendelian difference from its sister taxon, the yellow-flowered M. l. luteus . Previous work showed that the differentially expressed transcription factor gene MYB5a/NEGAN is the single causal gene. However, it was not clear whether MYB5a coding-sequence evolution (in addition to the observed patterns of differential expression) might also have contributed to increased anthocyanin production in M. l. variegatus . Quantitative image analysis of tobacco leaves, transfected with highly expressed MYB5a coding sequence from each taxon, revealed robust anthocyanin production driven by both alleles compared to a negative control. Counter to expectations, significantly higher anthocyanin production was driven by the coding sequence from the low-anthocyanin taxon M. l. luteus . Together with previously-published expression studies, this supports the hypothesis that petal pigment in M. l. variegatus was not gained by protein-coding changes, but instead via non- coding cis -regulatory evolution. Finally, while constructing the transgenes needed for this experiment, we unexpectedly discovered two sites in MYB5a that appear to be post- transcriptionally edited – a phenomenon that has been rarely reported, and even less often explored, for nuclear-encoded plant mRNAs.

Hybridization generates inter-genomic interactions, which may result in unique traits not seen in either parent species. Here we explore the genetic basis of both carotenoid and anthocyanin floral pigmentation in hybrids between monkeyflower species Mimulus cupreus and M. luteus var. variegatus. Mimulus cupreus has abundant yellow carotenoid pigmentation in its petal lobes, while M. l. variegatus has a derived reduction in carotenoid intensity. Thus, as expected, carotenoid intensity segregates in an F2 hybrid population. More surprisingly, both species appear to have petal lobes solidly and identically covered in magenta anthocyanin pigment (which, when overlaid on the bright yellow carotenoid background, leads to an orange color in M. cupreus ), yet F1 and F2 hybrids exhibit novel and complex spatial patterns of anthocyanin spotting. A rare yellow morph of M. cupreus , which lacks petal anthocyanins, also generates spatially patterned offspring when hybridized with M. l. variegatus . We use this cross, together with newly developed high-quality genome assembly of M. l. luteus and image analysis tools, to investigate the genetic architecture of color and pattern variation in an F2 hybrid population. We report a single QTL, containing the Beta-carotene hydroxylase ( BCH ) gene, associated with the non-patterned carotenoid reduction in M. l. variegatus . HPLC shows that relative beta-carotene abundance differs between dark yellow and light yellow petals, supporting a causal role for BCH . The presence versus absence of petal lobe anthocyanin segregates in a 3:1 ratio, and we report (as expected) an associated QTL encompassing the anthocyanin activator MYB5a/NEGAN which has previously been shown to be both necessary and sufficient to activate petal lobe anthocyanins in M. l. variegatus . Anthocyanin patterning was more complex, with seven QTLs associated with five quantitative patterning traits on the upper petals; 11 on the lower petals; and three qualitative whole-flower patterning traits. Although power was too limited to effectively test for epistatic interactions in this cross, the QTLs provide candidate genomic regions for further investigating the molecular mechanisms of spatially complex floral color patterning, and multiple candidate genes are identified including anthocyanin activators and an anthocyanin repressor.

The Plant Cell, Volume 33, Issue 7, koab117, https://doi.org/10.1093/plcell/koab117

Environmental adaptation and species divergence often involve suites of co‐evolving traits. Pigmentation in insects presents a variable, adaptive, and well‐characterized class of phenotypes for which correlations with multiple other traits have been demonstrated. In Drosophila, the pigmentation genes ebony and tan have pleiotropic effects on flies' response to light, creating the potential for correlated evolution of pigmentation and vision. Here, we investigate differences in light preference within and between two sister species, Drosophila americana and D. novamexicana, which differ in pigmentation in part because of evolution at ebony and tan and occupy environments that differ in many variables including solar radiation. We hypothesized that lighter pigmentation would be correlated with a greater preference for environmental light and tested this hypothesis using a habitat choice experiment. In a first set of experiments, using males of D. novamexicana line N14 and D. americana line A00, the light‐bodied D. novamexicana was found slightly but significantly more often than D. americana in the light habitat. A second experiment, which included additional lines and females as well as males, failed to find any significant difference between D. novamexicana‐N14 and D. americana‐A00. Additionally, the other dark line of D. americana (A04) was found in the light habitat more often than the light‐bodied D. novamexicana‐N14, in contrast to our predictions. However, the lightest line of D. americana, A01, was found substantially and significantly more often in the light habitat than the two darker lines of D. americana, thus providing partial support for our hypothesis. Finally, across all four lines, females were found more often in the light habitat than their more darkly pigmented male counterparts. Additional replication is needed to corroborate these findings and evaluate conflicting results, with the consistent effect of sex within and between species providing an especially intriguing avenue for further research.

Endosperm is an angiosperm innovation central to their reproduction whose development, and thus seed viability, is controlled by genomic imprinting, where expression from certain genes is parent-specific. Unsuccessful imprinting has been linked to failed inter-specific and inter-ploidy hybridization. Despite their importance in plant speciation, the underlying mechanisms behind these endosperm-based barriers remain poorly understood. Here, we describe one such barrier between diploid Mimulus guttatus and tetraploid Mimulus luteus. The two parents differ in endosperm DNA methylation, expression dynamics, and imprinted genes. Hybrid seeds suffer from underdeveloped endosperm, reducing viability, or arrested endosperm and seed abortion when M. guttatus or M. luteus is seed parent, respectively, and transgressive methylation and expression patterns emerge. The two inherited M. luteus subgenomes, genetically distinct but epigenetically similar, are expressionally dominant over the M. guttatus genome in hybrid embryos and especially their endosperm, where paternal imprints are perturbed. In aborted seeds, de novo methylation is inhibited, potentially owing to incompatible paternal instructions of imbalanced dosage from M. guttatus imprints. We suggest that diverged epigenetic/regulatory landscapes between parental genomes induce epigenetic repatterning and global shifts in expression, which, in endosperm, may uniquely facilitate incompatible interactions between divergent imprinting schemes, potentially driving rapid barriers.

Much of the visual diversity of angiosperms is due to the frequent evolution of novel pigmentation patterns in flowers. The gene network responsible for anthocyanin pigmentation, in particular, has become a model for investigating how genetic changes give rise to phenotypic innovation. In the monkeyflower genus Mimulus, an evolutionarily recent gain of petal lobe anthocyanin pigmentation in M. luteus var. variegatus was previously mapped to genomic region pla2. Here, we use sequence and expression analysis, followed by transgenic manipulation of gene expression, to identify MYB5a—orthologous to the NEGAN transcriptional activator from M. lewisii—as the gene responsible for the transition to anthocyanin-pigmented petals in M. l. variegatus. In other monkeyflower taxa, MYB5a/NEGAN is part of a reaction-diffusion network that produces semi-repeating spotting patterns, such as the array of spots in the nectar guides of both M. lewisii and M. guttatus. Its co-option for the evolution of an apparently non-patterned trait—the solid petal lobe pigmentation of M. l. variegatus—illustrates how reaction-diffusion can contribute to evolutionary novelty in non-obvious ways. Transcriptome sequencing of a MYB5a RNAi line of M. l. variegatus reveals that this genetically simple change, which we hypothesize to be a regulatory mutation in cis to MYB5a, has cascading effects on gene expression, not only on the enzyme-encoding genes traditionally thought of as the targets of MYB5a but also on all of its known partners in the anthocyanin regulatory network.

Much of the visual diversity of angiosperms is due to the frequent evolution of novel pigmentation patterns in flowers. The gene network responsible for anthocyanin pigmentation, in particular, has become a model for investigating how genetic changes give rise to phenotypic innovation. In the monkeyflower genus Mimulus , an evolutionarily recent gain of petal lobe anthocyanin pigmentation in M. luteus var. variegatus was previously mapped to genomic region pla2 . Here, we use DNA sequence analysis and spatiotemporal patterns of gene expression to identify MYB5a - homologous to the NEGAN transcriptional activator from M. lewisii - as a likely candidate gene within the pla2 region. Transgenic manipulation of gene expression confirms that MYB5a is both necessary and sufficient for petal lobe anthocyanin pigmentation. The deployment of MYB5a/NEGAN to the petal lobe stands in contrast to its more restricted role as a nectar guide anthocyanin activator in other Mimulus species. Transcriptome sequencing of a MYB5a RNAi line reveals the degree to which other regulators of the anthocyanin pathway - including R3 MYB repressors and bHLH and WD40 co-activators - are responsive to the level of expression of MYB5a . Overall, this work reveals that a genetically simple change, which we hypothesize to be a regulatory mutation in cis to MYB5a , has cascading effects on gene expression, not only on the genes downstream of MYB5a but also on all of its known partners in the anthocyanin regulatory network. Graphical : Solid black arrows indicate the direction (though not magnitude) of gene expression change, following RNAi knockdown of MYB5a/NEGAN in M. l. variegatus . The number of black arrows corresponds to the number of gene copies identified in the transcriptome. Grey symbols denote positive and negative regulatory interactions. RTO is an R3 MYB protein that inhibits anthocyanin biosynthesis by sequestering bHLH proteins away from the MBW complex.