Transmitting tissue-specific proteins (TTS proteins) are abundant in the extracellular matrix of Nicotiana pistils, and vital for optimal pollen tube growth and seed set. We have identified orthologs from several species in the Solanaceae, including Petunia axillaris axillaris and Petunia integrifolia. We refer to TTS proteins and their orthologs as histidine domain-arabinogalactan proteins (HD-APGs). HD-AGPs have distinctive domains, including a small histidine-rich region and a C-terminal PAC domain. Pairwise comparisons between HD-AGPs of 15 species belonging to Petunia, Nicotiana, and Solanum show that the his-domain and PAC domain are under purifying selection. In contrast, a proline-rich domain (HV2) is conserved among cross-hybridizing species, but variant in species-pairs that are reproductively isolated by post-pollination pre-fertilization reproductive barriers. In particular, variation in a tetrapeptide motif (XKPP) is systematically correlated with the presence of an interspecific reproductive barrier. Ka/Ks ratios are not informative at the infrageneric level, but the ratios reveal a clear signature of positive selection on two hypervariable domains (HV1 and HV2) when HD-AGPs from five solanaceous genera are compared. We propose that sequence divergence in the hypervariable domains of HD-AGPs reinforces sympatric speciation in incipient species that may have first diverged as a consequence of pollinator preferences or other ecological factors.

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