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Abstract PremiseDetecting clear tissue‐ and organ‐specific patterns of gene expression is key to understanding the genetic mechanisms that control plant development. In situ hybridization (ISH) of mRNA is one of the most precise, yet most challenging approaches to gene expression assays. Methods and ResultsDetection of histone H4 expression in reproductive tissues ofMimulus lewisii, a model angiosperm, was optimized using the RNAscope ISH assay. The optimized protocol was used to detect histone H4 expression in reproductive tissues of two gymnosperm species,Taxodium distichumandJuniperus virginiana, without further need for species‐specific optimization. Additionally, the optimized protocol was used to detect expression ofCYCLOIDEAtranscription factors inM. lewisiireproductive tissues without further optimization and with results similar to those previously reported. ConclusionsThe RNAscope assay can quickly and sensitively generate high‐quality ISH results in reproductive tissues across a breadth of plant species. 

In the formation of species, adaptation by natural selection generates distinct combinations of traits that function well together. The maintenance of adaptive trait combinations in the face of gene flow depends on the strength and nature of selection acting on the underlying genetic loci. Floral pollination syndromes exemplify the evolution of trait combinations adaptive for particular pollinators. The North American wildflower genusPenstemondisplays remarkable floral syndrome convergence, with at least 20 separate lineages that have evolved from ancestral bee pollination syndrome (wide blue-purple flowers that present a landing platform for bees and small amounts of nectar) to hummingbird pollination syndrome (bright red narrowly tubular flowers offering copious nectar). Related taxa that differ in floral syndrome offer an attractive opportunity to examine the genomic basis of complex trait divergence. In this study, we characterized genomic divergence among 229 individuals from aPenstemonspecies complex that includes both bee and hummingbird floral syndromes. Field plants are easily classified into species based on phenotypic differences and hybrids displaying intermediate floral syndromes are rare. Despite unambiguous phenotypic differences, genome-wide differentiation between species is minimal. Hummingbird-adapted populations are more genetically similar to nearby bee-adapted populations than to geographically distant hummingbird-adapted populations, in terms of genome-wided_XY. However, a small number of genetic loci are strongly differentiated between species. These approximately 20 “species-diagnostic loci,” which appear to have nearly fixed differences between pollination syndromes, are sprinkled throughout the genome in high recombination regions. Several map closely to previously established floral trait quantitative trait loci (QTLs). The striking difference between the diagnostic loci and the genome as whole suggests strong selection to maintain distinct combinations of traits, but with sufficient gene flow to homogenize the genomic background. A surprisingly small number of alleles confer phenotypic differences that form the basis of species identity in this species complex.