The molecular basis of phenotypic evolution: beyond the usual suspects.

It has been well documented that mutations in coding DNA or cis-regulatory elements underlie natural phenotypic variation in many organisms. However, the development of sophisticated functional tools in recent years in a wide range of traditionally non-model systems have revealed many 'unusual suspects' in the molecular bases of phenotypic evolution, including upstream open reading frames (uORFs), cryptic splice sites, and small RNAs. Furthermore, large-scale genome sequencing, especially long-read sequencing, has identified a cornucopia of structural variation underlying phenotypic divergence and elucidated the composition of supergenes that control complex multi-trait polymorphisms. In this review article we highlight recent studies that demonstrate this great diversity of molecular mechanisms producing adaptive genetic variation and the panoply of evolutionary paths leading to the 'grandeur of life'.

Genetic basis of nectar guide trichome variation between bumblebee- and self-pollinated monkeyflowers (Mimulus): role of the MIXTA-like gene GUIDELESS.

Nectar guide trichomes play crucial ecological roles in bee-pollinated flowers, as they serve as footholds and guides for foraging bees to access the floral rewards. However, the genetic basis of natural variation in nectar guide trichomes among species remains poorly understood. In this study, we performed genetic analysis of nectar guide trichome variation between two closely related monkeyflower (Mimulus) species, the bumblebee-pollinated Mimulus lewisii and self-pollinated M. parishii. We demonstrate that a MIXTA-like R2R3-MYB gene, GUIDELESS, is a major contributor to the nectar guide trichome length variation between the two species. The short-haired M. parishii carries a recessive allele due to non-synonymous substitutions in a highly conserved motif among MIXTA-like MYB proteins. Furthermore, our results suggest that besides GUIDELESS, additional loci encoding repressors of trichome elongation also contribute to the transition from bumblebee-pollination to selfing. Taken together, these results suggest that during a pollination syndrome switch, changes in seemingly complex traits such as nectar guide trichomes could have a relatively simple genetic basis, involving just a few genes of large effects.

Ancient and recent introgression shape the evolutionary history of pollinator adaptation and speciation in a model monkeyflower radiation (Mimulus section Erythranthe).

Inferences about past processes of adaptation and speciation require a gene-scale and genome-wide understanding of the evolutionary history of diverging taxa. In this study, we use genome-wide capture of nuclear gene sequences, plus skimming of organellar sequences, to investigate the phylogenomics of monkeyflowers in Mimulus section Erythranthe (27 accessions from seven species). Taxa within Erythranthe, particularly the parapatric and putatively sister species M. lewisii (bee-pollinated) and M. cardinalis (hummingbird-pollinated), have been a model system for investigating the ecological genetics of speciation and adaptation for over five decades. Across >8000 nuclear loci, multiple methods resolve a predominant species tree in which M. cardinalis groups with other hummingbird-pollinated taxa (37% of gene trees), rather than being sister to M. lewisii (32% of gene trees). We independently corroborate a single evolution of hummingbird pollination syndrome in Erythranthe by demonstrating functional redundancy in genetic complementation tests of floral traits in hybrids; together, these analyses overturn a textbook case of pollination-syndrome convergence. Strong asymmetries in allele sharing (Patterson's D-statistic and related tests) indicate that gene tree discordance reflects ancient and recent introgression rather than incomplete lineage sorting. Consistent with abundant introgression blurring the history of divergence, low-recombination and adaptation-associated regions support the new species tree, while high-recombination regions generate phylogenetic evidence for sister status for M. lewisii and M. cardinalis. Population-level sampling of core taxa also revealed two instances of chloroplast capture, with Sierran M. lewisii and Southern Californian M. parishii each carrying organelle genomes nested within respective sympatric M. cardinalis clades. A recent organellar transfer from M. cardinalis, an outcrosser where selfish cytonuclear dynamics are more likely, may account for the unexpected cytoplasmic male sterility effects of selfer M. parishii organelles in hybrids with M. lewisii. Overall, our phylogenomic results reveal extensive reticulation throughout the evolutionary history of a classic monkeyflower radiation, suggesting that natural selection (re-)assembles and maintains species-diagnostic traits and barriers in the face of gene flow. Our findings further underline the challenges, even in reproductively isolated species, in distinguishing re-use of adaptive alleles from true convergence and emphasize the value of a phylogenomic framework for reconstructing the evolutionary genetics of adaptation and speciation.

To stripe or not to stripe: the origin of a novel foliar pigmentation pattern in monkeyflowers (Mimulus).

The origin of phenotypic novelty is one of the most challenging problems in evolutionary biology. Although genetic regulatory network rewiring or co-option has been widely recognised as a major contributor, in most cases how such genetic rewiring/co-option happens is completely unknown. We have studied a novel foliar pigmentation pattern that evolved recently in the monkeyflower species Mimulus verbenaceus. Through genome-wide association tests using wild-collected samples, experimental crosses of laboratory inbred lines, gene expression analyses, and functional assays, we identified an anthocyanin-activating R2R3-MYB gene, STRIPY, as the causal gene triggering the emergence of the discrete, mediolateral anthocyanin stripe in the M. verbenaceus leaf. Chemical mutagenesis revealed the existence of upstream activators and repressors that form a 'hidden' prepattern along the leaf proximodistal axis, potentiating the unique expression pattern of STRIPY. Population genomics analyses did not reveal signatures of positive selection, indicating that nonadaptive processes may be responsible for the establishment of this novel trait in the wild. This study demonstrates that the origin of phenotypic novelty requires at least two separate phases, potentiation and actualisation. The foliar stripe pattern of M. verbenaceus provides an excellent platform to probe the molecular details of both processes in future studies.

A Tetratricopeptide Repeat Protein Regulates Carotenoid Biosynthesis and Chromoplast Development in Monkeyflowers (Mimulus).

Little is known about the factors regulating carotenoid biosynthesis in flowers. Here, we characterized the REDUCED CAROTENOID PIGMENTATION2 (RCP2) locus from two monkeyflower (Mimulus) species, the bumblebee-pollinated species Mimulus lewisii and the hummingbird-pollinated species Mimulus verbenaceus We show that loss-of-function mutations of RCP2 cause drastic down-regulation of the entire carotenoid biosynthetic pathway. The causal gene underlying RCP2 encodes a tetratricopeptide repeat protein that is closely related to the Arabidopsis (Arabidopsis thaliana) REDUCED CHLOROPLAST COVERAGE proteins. RCP2 appears to regulate carotenoid biosynthesis independently of RCP1, a previously identified R2R3-MYB master regulator of carotenoid biosynthesis. We show that RCP2 is necessary and sufficient for chromoplast development and carotenoid accumulation in floral tissues. Simultaneous down-regulation of RCP2 and two closely related paralogs, RCP2-L1 and RCP2-L2, yielded plants with pale leaves deficient in chlorophyll and carotenoids and with reduced chloroplast compartment size. Finally, we demonstrate that M. verbenaceus is just as amenable to chemical mutagenesis and in planta transformation as the more extensively studied M. lewisii, making these two species an excellent platform for comparative developmental genetics studies of closely related species with dramatic phenotypic divergence.

Developmental Genetics of Corolla Tube Formation: Role of the tasiRNA-ARF Pathway and a Conceptual Model.

Over 80,000 angiosperm species produce flowers with petals fused into a corolla tube. The corolla tube contributes to the tremendous diversity of flower morphology and plays a critical role in plant reproduction, yet it remains one of the least understood plant structures from a developmental genetics perspective. Through mutant analyses and transgenic experiments, we show that the tasiRNA-ARF pathway is required for corolla tube formation in the monkeyflower species Mimulus lewisii Loss-of-function mutations in the M. lewisii orthologs of ARGONAUTE7 and SUPPRESSOR OF GENE SILENCING3 cause a dramatic decrease in abundance of TAS3-derived small RNAs and a moderate upregulation of AUXIN RESPONSE FACTOR3 (ARF3) and ARF4, which lead to inhibition of lateral expansion of the bases of petal primordia and complete arrest of the upward growth of the interprimordial regions, resulting in unfused corollas. Using the DR5 auxin-responsive promoter, we discovered that auxin signaling is continuous along the petal primordium base and the interprimordial region during the critical stage of corolla tube formation in the wild type, similar to the spatial pattern of MlARF4 expression. Auxin response is much weaker and more restricted in the mutant. Furthermore, exogenous application of a polar auxin transport inhibitor to wild-type floral apices disrupted petal fusion. Together, these results suggest a new conceptual model highlighting the central role of auxin-directed synchronized growth of the petal primordium base and the interprimordial region in corolla tube formation.

Comparative analysis of corolla tube development across three closely related Mimulus species with different pollination syndromes.

Fusion of petals to form a corolla tube is considered a key innovation contributing to the diversification of many flowering plant lineages. Corolla tube length often varies dramatically among species and is a major determinant of pollinator preference. However, our understanding of the developmental dynamics underlying corolla tube length variation is very limited. Here we examined corolla tube growth in the Mimulus lewisii species complex, an emerging model system for studying the developmental genetics and evo-devo of pollinator-associated floral traits. We compared developmental and cellular processes associated with corolla tube length variation among the bee-pollinated M. lewisii, the hummingbird-pollinated Mimulus verbenaceus, and the self-pollinated Mimulus parishii. We found that in all three species, cell size is non-uniformly distributed along the mature tube, with the longest cells just distal to the stamen insertion site. Differences in corolla tube length among the three species are not associated with processes of organogenesis or early development but are associated with variation in multiple processes occurring later in development, including the location and duration of cell division and cell elongation. The tube growth curves of the small-flowered M. parishii and large-flowered M. lewisii are essentially indistinguishable, except that M. parishii tubes stop growing earlier at a smaller size, suggesting a critical role of heterochrony in the shift from outcrossing to selfing. These results not only highlight the developmental process associated with corolla tube variation among species but also provide a baseline reference for future developmental genetic analyses of mutants or transgenic plants with altered corolla tube morphology in this emerging model system.

Repressors of anthocyanin biosynthesis.

Anthocyanins play a variety of adaptive roles in both vegetative tissues and reproductive organs of plants. The broad functionality of these compounds requires sophisticated regulation of the anthocyanin biosynthesis pathway to allow proper localization, timing, and optimal intensity of pigment deposition. While it is well-established that the committed steps of anthocyanin biosynthesis are activated by a highly conserved MYB-bHLH-WDR (MBW) protein complex in virtually all flowering plants, anthocyanin repression seems to be achieved by a wide variety of protein and small RNA families that function in different tissue types and in response to different developmental, environmental, and hormonal cues. In this review, we survey recent progress in the identification of anthocyanin repressors and the characterization of their molecular mechanisms. We find that these seemingly very different repression modules act through a remarkably similar logic, the so-called 'double-negative logic'. Much of the double-negative regulation of anthocyanin production involves signal-induced degradation or sequestration of the repressors from the MBW protein complex. We discuss the functional and evolutionary advantages of this logic design compared with simple or sequential positive regulation. These advantages provide a plausible explanation as to why plants have evolved so many anthocyanin repressors.

The leaf polarity factors SGS3 and YABBYs regulate style elongation through auxin signaling in Mimulus lewisii.

Style length is a major determinant of breeding strategies in flowering plants and can vary dramatically between and within species. However, little is known about the genetic and developmental control of style elongation. We characterized the role of two classes of leaf adaxial-abaxial polarity factors, SUPPRESSOR OF GENE SILENCING3 (SGS3) and the YABBY family transcription factors, in the regulation of style elongation in Mimulus lewisii. We also examined the spatiotemporal patterns of auxin response during style development. Loss of SGS3 function led to reduced style length via limiting cell division, and downregulation of YABBY genes by RNA interference resulted in shorter styles by decreasing both cell division and cell elongation. We discovered an auxin response minimum between the stigma and ovary during the early stages of pistil development that marks style differentiation. Subsequent redistribution of auxin response to this region was correlated with style elongation. Auxin response was substantially altered when both SGS3 and YABBY functions were disrupted. We suggest that auxin signaling plays a central role in style elongation and that the way in which auxin signaling controls the different cell division and elongation patterns underpinning natural style length variation is a major question for future research.

A Novel R2R3-MYB Transcription Factor Contributes to Petal Blotch Formation by Regulating Organ-Specific Expression of PsCHS in Tree Peony (Paeonia suffruticosa).

Flower color patterns play critical roles in plant-pollinator interactions and represent one of the most common adaptations during angiosperm evolution. However, the molecular mechanisms underlying flower color pattern formation are less understood in non-model organisms. The aim of this study was to identify genes involved in the formation of petal blotches in tree peony (Paeonia suffruticosa) through transcriptome profiling and functional experiments. We identified an R2R3-MYB gene, PsMYB12, representing a distinct R2R3-MYB subgroup, with a spatiotemporal expression pattern tightly associated with petal blotch development. We further demonstrated that PsMYB12 interacts with a basic helix-loop-helix (bHLH) and a WD40 protein in a regulatory complex that directly activates PsCHS expression, which is also specific to the petal blotches. Together, these findings advance our understanding of the molecular mechanisms of pigment pattern formation beyond model plants. They also benefit molecular breeding of tree peony cultivars with novel color patterns and promote germplasm innovation.