DIPLACUS ANALYSES
Transition map illustrating the shift from Diplacus australis to Diplacus puniceus:
Over the past 70 years, pollinator dynamics within the Diplacus puniceus/Diplacus australis species pair in Southern California have undergone substantial shifts, driven by changing ecological pressures. Hummingbirds—most notably Calypte anna—have increased in abundance and ecological dominance, leading to a marked increase in their visitation to the previously insect-pollinated D. australis. This alteration in pollinator assemblage has exerted strong selective pressure on floral phenotypes, resulting in observable evolutionary changes in flower color and morphology. Central to this phenotypic transition is the R2R3MYB2 transcription factor, which regulates the biosynthetic pathway responsible for the shift from yellow to red floral pigmentation. The transition facilitates adaptation to hummingbird pollination syndromes and underscores the interplay between pollinator behavior and floral evolution. Despite a deceleration of these changes over the past two decades, the evolutionary process remains incomplete. Along the B1–B6 transect, trait values shift continuously, with flower color transitioning from yellow to red. Notably, since 2018, pure yellow-flowered populations have been absent from the easternmost site (B1; see sampling sites), marking a significant milestone in this ongoing evolutionary process.
Overview of the R2R3-MYB2 transcription factor structure in Diplacus aurantiacus:
A) Alignment. The coding sequence for helix two (R2-1) i s interrupted by an intron that contains a short tandem repeat (STR) co-activator, followed by the second segment of helix two (R2-2). The initial portion of helix three (R3-1) is immediately succeeded by intron 2, which also harbors a co-activator. The latter segment of helix three leads to the C-terminal end, which features a splicing motif and the S6B motif.
B) DNA sequences illustrate the concept of "missing heritability." The R-allele (in red) possesses a slightly different promoter sequence compared to the r-allele (in yellow). Additionally, the R-allele contains two long, variable poly-TA STRs, a distinct splicing motif, and a 6B activator motif. Focusing only on the coding region, the two ecotypes appear to differ solely in their C-terminal sequence; however, this perspective overlooks the important contribution of the STRs.
IGV excerpts of Maur_12720:
From left to right, the elements shown are the promoter, first intron, second intron, and S6B motif. From top to bottom, the species presented are Diplacus puniceus, a somatic derivative of D. australis, and Diplacus australis.
Sufficient activation of the MYB transcription factor (TF) requires an appropriate promoter motif, long poly(TA) short tandem repeats (STR), and the S6B motif, as seen in D. puniceus. Without these features, MaMYB expression is down-regulated. Sample 2009_1 (orange) shows a semi-active state, with only some epidermal cells displaying full MYB activation. Many of these cells either lack a regular promoter, contain short poly(TA) STRs, or lack the S6B motif. Epidermal cells show considerable diversity in regulatory motifs due to somatic variation.
STRs are highly polymorphic in transition zones or at their boundaries, displaying marked variation in mutation rates and patterns, and are often expanded in red morphs. As a result, polymorphism levels vary greatly across different STR loci. Consequently, the STR pattern at 2010_3R is unstable, exhibiting exceptionally high variance.
Flavonoid Biosynthesis Pathway and Heat-map:
This figure illustrates the Flavonoid Biosynthesis Pathway in yellow and red variants of Diplacus puniceus. (A) The pathway initiates with the phenylpropanoid pathway (1), advancing through the early (2) and late (3) stages of Anthocyanin Biosynthesis (ABS). Global regulators are positioned adjacent to both ABS stages. (B) The accompanying heat-map displays the activity of key enzymes and transcription factors (TFs) in yellow and red flowers at both the bud and flowering stages.
Arrows in (A) and color-coded panels in (B) highlight differential enzyme and TF activity. Anthocyanin production is significantly higher in red petals, but minimal or absent in yellow petals.
Concluding remarks By Marc. W. Schmid & Rolf Baumberger (09-23-26)
Short tandem repeats (STRs) play a significant functional role within the first and second introns. This discovery sheds light on the functional organization of STRs in the R2R3-MYB2 gene across the coastal Diplacus genus. This group of subshrubs encompasses a range of species and subspecies distributed throughout California, predominantly distinguished by yellow flowers, though some species exhibit red flowers. Notably, all 18 demes possess the same anthocyanin regulatory MYB gene, MaMYB2.
Although the yellow-flowered and red-flowered ecotypes of Diplacus puniceus can interbreed, they are genetically distinct, with 59 genes exhibiting differential expression. A crucial driver of early speciation is the R2R3-MYB2 transcription factor MAUR_1270 (MaMYB2). This factor is highly expressed in red-flowered D. puniceus but is down-regulated in D. australis, the yellow-flowered ecotype of D. puniceus. MAUR_1270 acts as the principal regulator of floral anthocyanin throughout all coastal D. aurantiacus species.
Generally, yellow-flowering species of the Diplacus genus possess low-copy-number polyTA repeats in the introns of MAUR_1270 and lack the S6B motif. Two exceptions to this pattern are D. grandiflorus, which also lacks the S6B motif but contains a long polyTA repeat, and D. aridus, which mirrors D. puniceus except for a very short second polyTA repeat. In contrast, all red-flowered species within the coastal Diplacus genus display varying polyTA repeat lengths in both the first and second introns. Moreover, these red-flowered species consistently feature an S6B repeat at the C-terminal end of the R2R3-MYB2 coding region.
Based on these observations, we propose a model to address the missing heritability of the R2R3-MYB gene in D. aurantiacus by incorporating a second STR in the second intron. Using the Integrated Genomics Viewer (IGV) to compare cultivated Diplacus species with wild-sampled individuals, we observed rapid changes in DNA copy number across several intron polyTA STRs during anthesis or as spontaneous events. These alterations, driven by somatic mutations along the STRs, are associated with variations in floral color. In response to environmental stresses, such as temperature fluctuations and increased hummingbird visitation, a yellow-flowered Diplacus australis individual may transition to red flowers and exhibit other floral trait changes. This transformation takes place after all through internal STR tuning mechanisms, resulting in gradual poly-allelism at STR loci in either direction, and is heritable.
Data source: 436 genomic Diplacus datasets retrieved from the Sequence Read Archive (SRA) at the National Library of Medicine.
Note: The availability of enhanced datasets (such as those generated from long-read PacBio sequencing) and a high-quality, searchable genome assembly for Diplacus puniceus would enable much deeper analysis. Currently, the evolutionary history of these Diplacus species is based more on speculation than on robust scientific evidence. One should have long-read sequences of all varieties of Diplacus that one can encounter.
09-26