Comparative genome biology has unveiled the polyploid origin of all angiosperms and the role of recurrent polyploidization in the amplification of gene families and the structuring of genomes. Which species share certain ancient polyploidy events, and which do not, is ill defined because of the limited number of sequenced genomes and transcriptomes and their uneven phylogenetic distribution. Previously, it has been suggested that most, but probably not all, of the eudicots have shared an ancient hexaploidy event, referred to as the gamma triplication. In this study, detailed phylogenies of subfamilies of MADS-box genes suggest that the gamma triplication has occurred before the divergence of Gunnerales but after the divergence of Buxales and Trochodendrales. Large-scale phylogenetic and K(S)-based approaches on the inflorescence transcriptomes of Gunnera manicata (Gunnerales) and Pachysandra terminalis (Buxales) provide further support for this placement, enabling us to position the gamma triplication in the stem lineage of the core eudicots. This triplication likely initiated the functional diversification of key regulators of reproductive development in the core eudicots, comprising 75% of flowering plants. Although it is possible that the gamma event triggered early core eudicot diversification, our dating estimates suggest that the event occurred early in the stem lineage, well before the rapid speciation of the earliest core eudicot lineages. The evolutionary significance of this paleopolyploidy event may thus rather lie in establishing a species lineage that was resilient to extinction, but with the genomic potential for later diversification. We consider that the traits generated from this potential characterize extant core eudicots both chemically and morphologically.

A key question regarding protein evolution is how proteins adapt to the dynamic environment in which they function and how in turn their evolution shapes the protein interaction network. We used extant and resurrected ancestral plant MADS-domain transcription factors to understand how SEPALLATA3, a protein with hub and glue properties, evolved and takes part in network organization. Although the density of dimeric interactions was saturated in the network, many new interactions became mediated by SEPALLATA3 after a whole genome triplication event. By swapping SEPALLATA3 and its ancestors between dimeric networks of different ages, we found that the protein lost the capacity of promiscuous interaction and acquired specificity in evolution. This was accompanied with constraints on conformations through proline residue accumulation, which made the protein less flexible. SHORT VEGETATIVE PHASE on the other hand (non-hub) was able to gain protein-protein interactions due to a C-terminal domain insertion, allowing for a larger interaction interface. These findings illustrate that protein interaction evolution occurs at the level of conformational dynamics, when the binding mechanism concerns an induced fit or conformational selection. Proteins can evolve towards increased specificity with reduced flexibility when the complexity of the protein interaction network requires specificity.

Evolution of morphology includes evolutionary shifts of developmental processes in space or in time. Heterochronic evolution is defined as a temporal shift. The concept of heterochrony has been very rewarding to investigators of both animal and plant developmental evolution, because it has strong explanatory power when trying to understand morphological diversity. While for animals, extensive literature on heterochrony developed along with the field of evolution of development, in plants the concept has been applied less often and is less elaborately developed. Yet novel genetic findings highlight heterochrony as a developmental and evolutionary process in plants. Similar to what has been found for the worm Caenorhabditis, a heterochronic gene pathway controlling developmental timing has been elucidated in flowering plants. Two antagonistic microRNA’s miR156 and miR172 target two gene families of transcription factors, SQUAMOSA PROMOTOR BINDING PROTEIN-LIKE and APETALA2-like, respectively. Here, we propose that this finding now allows the molecular investigation of cases of heterochronic evolution in plants. We illustrate this point by examining microRNA expression patterns in the Antirrhinum majus incomposita and choripetala heterochronic mutants. Some of the more beautiful putative cases of heterochronic evolution can be found outside flowering plants, but little is known about the extent of conservation of this flowering plant pathway in other land plants. We show that the expression of an APETALA2-like gene decreases with age in a fern species. This contributes to the idea that ferns share some heterochronic gene functions with flowering plants.

The evolution of plants is characterized by several rounds of ancient whole genome duplication, sometimes closely associated with the origin of large groups of species. A good example is the γ triplication at the origin of core eudicots. Core eudicots comprise about 75% of flowering plants and are characterized by the canalization of reproductive development. To better understand the impact of this genomic event, we studied the protein interaction network of MADS-domain transcription factors, which are key regulators of reproductive development. We accurately inferred, resurrected and tested the interactions of ancestral proteins before and after the triplication and directly compared these ancestral networks to the networks of Arabidopsis and tomato. We find that the γ triplication generated a dramatically innovated network that strongly rewired through the addition of many new interactions. Many of these interactions were established between paralogous proteins and a new interaction partner, establishing new redundancy. Simulations show that both node and edge addition through the triplication were important to maintain modularity in the network. In addition to generating insights into the impact of whole genome duplication and elementary processes involved in network evolution, our data provide a resource for comparative developmental biology in flowering plants.

The evolution of plants is characterized by whole-genome duplications, sometimes closely associated with the origin of large groups of species. The gamma (γ) genome triplication occurred at the origin of the core eudicots, which comprise ∼75% of flowering plants. To better understand the impact of whole-genome duplication, we studied the protein interaction network of MADS domain transcription factors, which are key regulators of reproductive development. We reconstructed, synthesized, and tested the interactions of ancestral proteins immediately before and closely after the triplication and directly compared these ancestral networks to the extant networks of Arabidopsis thaliana and tomato (Solanum lycopersicum). We found that gamma expanded the MADS domain interaction network more strongly than subsequent genomic events. This event strongly rewired MADS domain interactions and allowed for the evolution of new functions and installed robustness through new redundancy. Despite extensive rewiring, the organization of the network was maintained through gamma. New interactions and protein retention compensated for its potentially destructive impact on network organization. Post gamma, the network evolved from an organization around the single hub SEP3 to a network organized around multiple hubs and well-connected proteins lost, rather than gained, interactions. The data provide a resource for comparative developmental biology in flowering plants.