Do we truly understand pollination syndromes in Petunia as much as we suppose?

Petunia is endemic to South America grasslands; member of this genus exhibit variation in flower colour and shape, attracting bees, hawkmoths or hummingbirds. This group of plants is thus an excellent model system for evolutionary studies of diversification associated with pollinator shifts. Our aims were to identify the legitimate pollinator of Petunia secreta, a rare and endemic species, and to assess the importance of floral traits in pollinator attraction in this Petunia species. To determine the legitimate pollinator, field observations were conducted, and all floral visitors were recorded and evaluated. We also measured the nectar volume and sugar concentration. To characterize morphological cues for pollinators, we assessed the ultraviolet (UV)-light response in detached flowers, and characterized the floral pigments and pollen volatile scents for four different Petunia species that present different pollination syndromes. Petunia secreta shares the most recent ancestor with a white hawkmoth-pollinated species, P. axillaris, but presents flavonols and anthocyanin pigments responsible for the pink corolla colour and UV-light responses that are common to bee-pollinated Petunia species. Our study showed that a solitary bee in the genus Pseudagapostemon was the most frequent pollinator of P. secreta, and these bees collect only pollen as a reward. Despite being mainly bee-pollinated, different functional groups of pollinators visit P. secreta. Nectar volume, sugar concentration per flower, morphology and components of pollen scent would appear to be attractive to several different pollinator groups. Notably, the corolla includes a narrow tube with nectar at its base that cannot be reached by Pseudagapostemon, and flowers of P. secreta appear to follow an evolutionary transition, with traits attractive to several functional groups of pollinators. Additionally, the present study shows that differences in the volatiles of pollen scent are relevant for plant mutualistic and antagonist interactions in Petunia species and that pollen scent profile plays a key role in characterizing pollination syndromes.

Nuclear and plastid markers reveal the persistence of genetic identity: a new perspective on the evolutionary history of Petunia exserta.

Recently divergent species that can hybridize are ideal models for investigating the genetic exchanges that can occur while preserving the species boundaries. Petunia exserta is an endemic species from a very limited and specific area that grows exclusively in rocky shelters. These shaded spots are an inhospitable habitat for all other Petunia species, including the closely related and widely distributed species P. axillaris. Individuals with intermediate morphologic characteristics have been found near the rocky shelters and were believed to be putative hybrids between P. exserta and P. axillaris, suggesting a situation where Petunia exserta is losing its genetic identity. In the current study, we analyzed the plastid intergenic spacers trnS/trnG and trnH/psbA and six nuclear CAPS markers in a large sampling design of both species to understand the evolutionary process occurring in this biological system. Bayesian clustering methods, cpDNA haplotype networks, genetic diversity statistics, and coalescence-based analyses support a scenario where hybridization occurs while two genetic clusters corresponding to two species are maintained. Our results reinforce the importance of coupling differentially inherited markers with an extensive geographic sample to assess the evolutionary dynamics of recently diverged species that can hybridize.

Diversification in the South American Pampas: the genetic and morphological variation of the widespread Petunia axillaris complex (Solanaceae).

Understanding the spatiotemporal distribution of genetic variation and the ways in which this distribution is connected to the ecological context of natural populations is fundamental for understanding the nature and mode of intraspecific and, ultimately, interspecific differentiation. The Petunia axillaris complex is endemic to the grasslands of southern South America and includes three subspecies: P. a. axillaris, P. a. parodii and P. a. subandina. These subspecies are traditionally delimited based on both geography and floral morphology, although the latter is highly variable. Here, we determined the patterns of genetic (nuclear and cpDNA), morphological and ecological (bioclimatic) variation of a large number of P. axillaris populations and found that they are mostly coincident with subspecies delimitation. The nuclear data suggest that the subspecies are likely independent evolutionary units, and their morphological differences may be associated with local adaptations to diverse climatic and/or edaphic conditions and population isolation. The demographic dynamics over time estimated by skyline plot analyses showed different patterns for each subspecies in the last 100 000 years, which is compatible with a divergence time between 35 000 and 107 000 years ago between P. a. axillaris and P. a. parodii, as estimated with the IMa program. Coalescent simulation tests using Approximate Bayesian Computation do not support previous suggestions of extensive gene flow between P. a. axillaris and P. a. parodii in their contact zone.

Asymmetric effects of loss and gain of a floral trait on pollinator preference.

Shifts in pollination syndromes involve coordinated changes in multiple floral traits. This raises the question of how plants can cope with rapid changes in pollinator availability by the slow process of accumulation of mutations in multiple genes. Here we study the transition from bee to hawkmoth pollination in the genus Petunia. Interspecific crosses followed by single locus introgressions were used to recreate putative intermediate evolutionary stages in the evolution of moth pollination. The effect of the loss/gain of petal color was asymmetric: it had no influence on the established pollinator but enhanced visitation by the new pollinator. Therefore, shifts in pollination syndromes may proceed through intermediate stages of reduced specialization and consequently enhanced reproductive assurance. The loss of petal color in moth-pollinated Petunia involves null mutations in a single regulatory gene, An2. Such simple genetic changes may be sufficiently rapid and frequent to ensure survival during pollinator failure.

Isolation barriers between petunia axillaris and Petunia integrifolia (Solanaceae).

The isolation barriers restricting gene flow between populations or species are of crucial interest for understanding how biological species arise and how they are maintained. Few studies have examined the entire range of possible isolation barriers from geographic isolation to next generation hybrid viability. Here, we present a detailed analysis of isolation barriers between two flowering plant species of the genus Petunia (Solanaceae). Petunia integrifolia and P. axillaris feature divergent pollination syndromes but can produce fertile hybrids when crossed in the laboratory. Both Petunia species are primarily isolated in space but appear not to hybridize in sympatry. Our experiments demonstrate that pollinator isolation is very high but not strong enough to explain the absence of hybrids in nature. However, pollinator isolation in conjunction with male gametic isolation (i.e., pollen-pistil interaction) can explain the lack of natural hybridization, while postzygotic isolation barriers are low or nonexistent. Our study supports the notion that reproductive isolation in flowering plants is mainly caused by pre- rather than postzygotic isolation mechanisms.