The Phenotypic and Genetic Underpinnings of Flower Size in Polemoniaceae

Abstract

Corolla length is a labile flower feature and has strong implications for pollinator success. However, the phenotypic and genetic bases of corolla elongation are not well known, largely due to a lack of good candidate genes for potential genetic exploration and functional work. We investigate both the cellular phenotypic differences in corolla length, as well as the genetic control of this trait, in Saltugilia (Polemoniaceae). Taxa in this clade exhibit a large range of flower sizes and differ dramatically in pollinator guilds. Flowers of each species were collected from multiple individuals during four stages of flower development to ascertain if cell number or cell size is more important in determining flower size. In Saltugilia, increased flower size during development appears to be driven more by cell size than cell number. Differences in flower size between species are governed by both cell size and cell number, with the large-flowered S. splendens subsp. grantii having nearly twice as many cells as the small-flowered species. Fully mature flowers of all taxa contain jigsaw cells similar to cells seen in sepals and leaves; however, these cells are not typically found in the developing flowers of most species. The proportion of this cell type in mature flowers appears to have substantial implications, comprising 17-68% of the overall flower size. To identify candidate genes responsible for differences in cell area and cell type, transcriptomes were generated for two individuals of the species with the smallest (S. australis) and largest (S. splendens subsp. grantii) flowers across the same four developmental stages visualized with confocal microscopy. Analyses identified genes associated with cell wall formation that are up-regulated in the mature flower stage compared to mid-stage flowers (75% of mature size). This developmental change is associated with the origin of jigsaw cells in the corolla tube of mature flowers. Further comparisons between mature flowers in the two species revealed 354 transcripts that are up-regulated in the large-flowered S. splendens subsp. grantii compared to the small-flowered S. australis. These results are likely broadly applicable to Polemoniaceae, a clade of nearly 400 species, with extensive variation in floral form and shape.

Keywords: Polemoniaceae; Saltugilia; comparative transcriptomics; floral evo-devo; flower epidermal cells; phylogeny; pollinator-mediated selection.

Figure 1

Phylogenetic relationships of the taxa in Saltugilia using a maximum-likelihood framework with bootstrap support values shown. (A) concatenated data set comprising plastid coding regions, (B) complete plastome sequence, and (C) concatenated data set of 90 nuclear loci.

Figure 2

Examples of flowers and cells visualized. (A) The four species of Saltugilia grown in the greenhouses at the University of Florida. From left to right: S. splendens subsp. grantii, S. splendens subsp. splendens, S. caruifolia and S. australis. (B) Flower of Gilia stellata, one of the species used as an outgroup. (C) S. latimeri collected in Joshua Tree National Park, CA, USA. (D) Prepped microscope slide of the small stage of S. australis. (E) Prepped microscope slide of the mature stage of S. splendens subsp. grantii. (F–I) Representatives of the four types of cells observed in flower material: conical, transition, jigsaw and elongated cells respectively. Scale bars in (A–C,E) are 1 cm, and (F–I) are 20 μM.

Figure 3

Representative cell area vs. cell circularity through development of conical, transition, elongated and jigsaw cells. The closer the circularity value is to 1, the more round the cell.

Figure 4

Box plots comparing the area of each cell type in mature flowers of all species. (A) Mature conical cells, (B) mature transition cells, (C) mature jigsaw cells, (D) mature elongated cells.

Figure 5

Proportions of cell types through the four stages of development for all taxa investigated in one plant of each taxon.

Figure 6

Estimated cell counts for each of the four individual plants for each taxon of Saltugilia. Number of cells was estimated by using the mean bounding box width for each cell type through flower development.

Figure 7

OrthoVenn diagrams of pooled reads for (A) each stage of development: small, half, mid and mature for S. australis, (B) each stage of development: small, half, mid and mature for S. splendens subsp. grantii, and (C) S. australis and S. splendens subsp. grantii showing clusters of proteins that are shared and unique to each species.

Figure 8

Differential gene expression analysis among half, mid and mature stages of development for two plants of S. australis. Cutoff for differentially expressed genes was a 4-fold change in expression levels with a p-value of 0.05. Genes colored in yellow are up-regulated and genes colored purple are down-regulated compared to the other stages.

Figure 9

Differential gene expression analysis between mature stages of development for two plants of S. australis and S. splendens subsp. grantii. Cutoff for differentially expressed genes was a 4-fold change in expression levels with a p-value of 0.05. Genes colored in yellow are up-regulated and genes colored purple are down-regulated compared to the other species.

Figure 10

Ancestral-state reconstructions of pollinators (A–D) and size (E,F) under a maximum parsimony (MP) and maximum-likelihood (ML) framework using the two competing topologies. (A) MP of pollinators using the topology from plastid coding genes, (B) ML of pollinators using the topology from plastid coding genes, (C) MP of pollinators using the topology from the complete plastome and nuclear genes, (D) ML of pollinators using the topology from the complete plastome and nuclear genes, (E) continuous reconstruction of flower size using the topology from plastid coding genes, and (F) continuous reconstruction of flower size using the topology from the complete plastome and nuclear genes.