Characterizing each step of pollination in Phlox drummondii reveals that a single butterfly species predominates in the pollinator assemblage.

PREMISE: A central goal of pollination biology is to connect plants with the identity of their pollinator(s). While predictions based on floral syndrome traits are extremely useful, direct observation can reveal further details of a species' pollination biology. The wildflower Phlox drummondii has a floral syndrome consistent with pollination by Lepidoptera. We tested this prediction using empirical data. METHODS: We observed each step of pollination in P. drummondii. First, we observed 55.5 h of floral visitation across the species range. We used temporal pollinator exclusion to determine the contribution of diurnal and nocturnal pollination to reproductive output. We then quantified P. drummondii pollen transfer by the dominant floral visitor, Battus philenor. Finally, we tested the effect of B. philenor visitation on P. drummondii reproduction by quantifying fruit set following single pollinator visits. RESULTS: Battus philenor is the primary pollinator of P. drummondii. Pollination is largely diurnal, and we observed a variety of lepidopteran visitors during the diurnal period. However, B. philenor was the most frequent visitor, representing 88.5% of all observed visits. Our results show that B. philenor is an extremely common visitor and also an effective pollinator by demonstrating that individuals transfer pollen between flowers and that a single visit can elicit fruit set. CONCLUSIONS: Our data are consistent with the prediction of lepidopteran pollination and further reveal a single butterfly species, B. philenor, as the primary pollinator. Our study demonstrates the importance of empirical pollinator observations, adds to our understanding of pollination mechanics, and offers a specific case study of butterfly pollination.

Genetic and spatial variation in vegetative and floral traits across a hybrid zone.

PREMISE: Genetic variation influences the potential for evolution to rescue populations from impacts of environmental change. Most studies of genetic variation in fitness-related traits focus on either vegetative or floral traits, with few on floral scent. How vegetative and floral traits compare in potential for adaptive evolution is poorly understood. METHODS: We measured variation across source populations, planting sites, and genetic families for vegetative and floral traits in a hybrid zone. Seeds from families of Ipomopsis aggregata, I. tenuituba, and F1 and F2 hybrids of the two species were planted into three common gardens. Measured traits included specific leaf area (SLA), trichomes, water-use efficiency (WUE), floral morphology, petal color, nectar, and floral volatiles. RESULTS: Vegetative traits SLA and WUE varied greatly among planting sites, while showing weak or no genetic variation among source populations. Specific leaf area and trichomes responded plastically to snowmelt date, and SLA exhibited within-population genetic variation. All aspects of floral morphology varied genetically among source populations, and corolla length, corolla width, and sepal width varied genetically within populations. Heritability was not detected for volatiles due to high environmental variation, although one terpene had high evolvability, and high emission of two terpenes, a class of compounds emitted more strongly from the calyx than the corolla, correlated genetically with sepal width. Environmental variation across sites was weak for floral morphology and stronger for volatiles and vegetative traits. The inheritance of three of four volatiles departed from additive. CONCLUSIONS: Results indicate stronger genetic potential for evolutionary responses to selection in floral morphology compared with scent and vegetative traits and suggest potentially adaptive plasticity in some vegetative traits.

Reference genome of the color polymorphic desert annual plant sandblossoms, Linanthus parryae.

Sandblossoms, Linanthus parryae is a widespread annual plant species found in washes and sandy open habitats across the Mojave Desert and Eastern Sierra Nevada of California. Studies in this species have played a central role in evolutionary biology, serving as the first test cases of the shifting balance theory of evolution, models of isolation by distance, and metrics to describe the genetic structure of natural populations. Despite the importance of L. parryae in the development of landscape genetics and phylogeography, there are no genomic resources available for the species. Through the California Conservation Genomics Project, we assembled the first genome in the genus Linanthus. Using PacBio HiFi long reads and Hi-C chromatin conformation capture, we assembled 123 scaffolds spanning 1.51 Gb of the 1.96 Gb estimated genome, with a contig N50 of 18.7 Mb and a scaffold N50 of 124.8 Mb. This assembly, with a BUSCO completeness score of 88.7%, will allow us to revisit foundational ideas central to our understanding of how evolutionary forces operate in a geographic landscape. In addition, it will be a new resource to uncover adaptations to arid environments in the fragile desert habitat threatened by urban and solar farm development, climate change, and off-road vehicles.

Feeding friend and foe: ample pollen mitigates the effects of pollen theft for a gynodioecious plant, Polemonium foliosissimum (Polemoniaceae).

BACKGROUND AND AIMS: Most angiosperms rely on pollinators to transport pollen and effect fertilization. While some floral visitors are effective pollinators, others act as thieves, consuming pollen but effecting little pollination in return. The importance of pollen theft in male and female reproductive success has received little attention. Here, we examined if pollen consumption by flies altered pollen receipt and exacerbated pollen limitation for a bumblebee-pollinated plant, Polemonium foliosissimum (Polemoniaceae). METHODS: To examine the effect of pollen-thieving flies, we took a three-pronged approach. First, we used single-visit observations to quantify pollen removal and pollen deposition by flies and bumblebees. Second, we manipulated pollen in the neighbourhood around focal plants in two years to test whether pollen reduction reduced pollen receipt. Third, we combined pollen reduction with hand-pollination to test whether pollen thieving exacerbated pollen limitation. Polemonium foliosissimum is gynodioecious in most populations in the Elk Mountains of central Colorado, USA. Thus, we also tested whether pollen theft affected hermaphrodites and females differently. RESULTS: Flies removed significantly more pollen and deposited less pollen per visit than did bumblebees. Reduction of pollen in the neighbourhood around focal plants reduced pollen receipt in both years but only nearly significantly so in 2015. In 2016, plants were significantly pollen-limited; hand-pollination significantly increased seeds per fruit for both hermaphrodites and females. However, the reduction of pollen around focal plants did not exacerbate pollen limitation for either hermaphrodites or females. CONCLUSIONS: Our results suggest that plants tolerate significant consumption of pollen by thieves and pollinators by producing ample pollen to feed both and fertilize available ovules. Our results demonstrate that pollen limitation in P. foliosissimum is driven by lack of effective pollinators rather than lack of pollen. Teasing out these effects elucidates the relative importance of drivers of reproductive success and thus the expected response to selection by different floral visitors.

The role of polyploidy in shaping morphological diversity in natural populations of Phlox amabilis.

PREMISE OF THE STUDY: Studies of natural populations of polyploids increasingly highlight complex patterns of variation in ploidy and geographic distribution of cytotypes. As our understanding of the complexity of polyploidy grows, our understanding of the morphological correlates of polyploidy should expand as well. Here we examine in what ways, and to what degree, polyploidy affects the overall phenotype of a species across its distribution when there are three ploidies and geographic complexity in cytotype distribution. METHODS: We measured 31 morphological traits from stems, leaves, and flowers from up to 25 individuals from 11 sites across the distribution of Phlox amabilis. Chromosome counts and flow cytometry confirmed and expanded upon earlier research documenting diploid, tetraploid, and hexaploid populations, and also identified a site with two ploidies. Univariate and multivariate statistics were used to characterize the morphological effects of polyploidy. KEY RESULTS: We detected significant associations between morphology and ploidy in 11 traits spread across vegetative and reproductive structures. Generally, diploid individuals differed from polyploid individuals to a greater extent, and in different ways, than tetraploid and hexaploid plants differed from each other. Multivariate morphometrics demonstrated that the two primary axes of overall variation are driven by morphological traits associated with polyploidy, and individuals of different ploidies can be discriminated with 95% success. CONCLUSIONS: Polyploidy plays a major role in shaping overall morphological diversity in natural populations of P. amabilis.

Fitness consequences of short- and long-distance pollinations in Phlox hirsuta, an endangered species.

PREMISE OF THE STUDY: The persistence of rare and endangered plant species may depend on the distance pollinators travel when dispersing pollen. Pollinations between adjacent plants, which are often genetically similar, can decrease seed set, germination, and/or progeny vigor due to shared S-alleles or inbreeding depression. Interpopulation pollen dispersal is often suggested as a management tool to increase genetic diversity; however, long-distance pollinations also have the potential to decrease fitness. METHODS: We performed experimental hand pollinations in the field and germination experiments in a growth chamber to determine the effect of intrapopulation pollination distance (1 m, 10 m, and 100 m) on seed set, seed germination, progeny growth, and progeny reproduction in Phlox hirsuta. In addition, we included interpopulation pollinations (6740 m) to determine whether artificial gene flow is a viable management option for this endangered species. KEY RESULTS: Although pollination distance did not affect the number of healthy seeds produced or the likelihood of radicle emergence, it did significantly affect the ability of germinating seeds to successfully produce cotyledons. Outbreeding depression was observed during seed germination and early seedling development. Seedlings resulting from interpopulation pollinations developed more slowly and were less likely to survive to produce cotyledons than seedlings resulting from all three intrapopulation pollination distances. CONCLUSIONS: Our results suggest that the success of P. hirsuta does not depend on the distance pollinators travel within populations and that conservation strategies that involve transporting genes between populations can be counterproductive.

Floral scent in natural hybrids of Ipomopsis (Polemoniaceae) and their parental species.

BACKGROUND AND AIMS: Floral traits, such as floral volatiles, can contribute to pre-zygotic reproductive isolation by promoting species-specific pollinator foraging. When hybrid zones form, floral traits could also influence post-zygotic isolation. This study examined floral volatiles in parental species and natural hybrids in order to explore potential scent mediation of pre-zygotic and post-zygotic isolation. METHODS: Floral bouquets were analysed for the sister species Ipomopsis aggregata and I. tenuituba and their natural hybrids at two contact sites differing in both hybridization rate and temporal foraging pattern of hawkmoth pollinators. Floral volatiles were quantified in diurnal and nocturnal scent samples using gas chromatography-mass spectrometry. KEY RESULTS: The bouquets of parental species and hybrids showed qualitative overlap. All flowers emitted similar sets of monoterpenoid, sesquiterpenoid, aliphatic and benzenoid compounds, but separated into groups defined by multivariate analysis of quantitative emissions. The parental species differed most strikingly in the nitrogenous compound indole, which was found almost exclusively in nocturnal bouquets of I. tenuituba. Natural hybrid bouquets were highly variable, and showed emission rates of several compounds that appeared transgressive. However, indole emission rates were intermediate in the hybrids compared with rates in the parents. Volatile bouquets at the contact site with lower hybridization did not show greater species specificity in overall scent emission, but I. tenuituba presented a stronger indole signal during peak hawkmoth activity at that site. CONCLUSIONS: The two species of Ipomopsis differed in patterns of floral bouquets, with indole emitted in nocturnal I. tenuituba, but not in I. aggregata. Natural hybrid bouquets were not consistently intermediate between the parents, although hybrids were intermediate in indole emission. The indole signal could potentially serve as a hawkmoth attractant that mediates reproductive isolation both before and after hybrid formation.

QTL analysis of divergent floral morphology traits between Gilia yorkii and G. capitata.

Speciation is a complex process typically accompanied by significant genetic and morphological differences between sister populations. In plants, divergent floral morphologies and pollinator differences can result in reproductive isolation between populations. Here, we explore floral trait differences between two recently diverged species, Gilia yorkii and G. capitata. The distributions of floral traits in parental, F1, and F2 populations are compared, and groups of correlated traits are identified. We describe the genetic architecture of floral traits through a quantitative trait locus analysis using an F2 population of 187 individuals. While all identified quantitative trait locus were of moderate (10-25%) effect, interestingly, most quantitative trait locus intervals were non-overlapping, suggesting that, in general, traits do not share a common genetic basis. Our results provide a framework for future identification of genes involved in the evolution of floral morphology.

Interactions of hybridization and mating systems: a case study in Leptosiphon (Polemoniaceae).

PREMISE OF THE STUDY: The roles of hybridization and mating systems in the evolution of angiosperms have been well studied, but less work has focused on their interactions. Self-incompatible and self-compatible species often show asymmetry in heterospecific pollen rejection. Self-fertilization can preempt ovules before opportunities for hybridization. In turn, hybridization might affect mating system evolution through selection for selfing to avoid production of low fitness hybrids. • METHODS: AFLP and morphological analyses were used to test for hybrids in a contact zone between species with contrasting breeding systems. Crossing experiments examined the relative contributions to reproductive isolation of pollen-pistil interactions, timing of self-fertilization, and F1 viability and fertility. A diallel cross of siblings tested for an association between heterospecific incompatibility and S-genotype in the self-incompatible species. • KEY RESULTS: A low frequency of hybrids was detected in the contact zone. Pollen-pistil interactions were partially consistent with the SI × SC rule; some individuals of the self-incompatible species rejected heterospecific pollen, whereas the self-compatible species was fully receptive to it. In the selfing species, individuals with early selfing produced fewer hybrid progeny than did those with delayed self-compatibility when heterospecific pollen was applied after self-pollen. Viability of F1s was high but fertility was low. Variability in heterospecific pollen rejection was not related to S-genotype. • CONCLUSIONS: Both self-fertilization and self-incompatibility are associated with limits to hybridization at this site. The strong effect of timing of selfing on production of low fitness F1s suggests that hybridization might select for early selfing in this population.

Altered precipitation affects plant hybrids differently than their parental species.

PREMISE OF THE STUDY: Future changes in environmental conditions may alter evolutionary processes, including hybridization in nature. Frequency of hybrids could be altered via range shifts by the parental species or by changes in prezygotic or postzygotic reproductive isolation. We examined the potential for range shifts and change in postzygotic isolation by determining effects of increasing and decreasing precipitation on leaf physiology and fitness components in the subalpine herbs Ipomopsis aggregata (Polemoniaceae), I. tenuituba¸ and their natural hybrids in a common garden in the habitat of I. aggregata. METHODS: Summer precipitation was experimentally doubled or halved over 3 yr in comparison with ambient conditions. We measured relative growth rate, specific leaf area, intrinsic water-use efficiency, survival to reproduction, biomass, number of flowers produced, and floral morphology. KEY RESULTS: Ipomopsis tenuituba increased relative growth rate with higher precipitation more so than did I. aggregata during the first summer, but this response did not result in changes across treatments in relative survival or final reproductive success of the two species. When precipitation was reduced, the relative success of hybrids was greater than that of the home species, I. aggregata. In dry conditions, hybrids increased water-use efficiency and fitness as indexed by number of flowers more so than the other plant types did. CONCLUSIONS: Increased reproduction in hybrids in the reduced precipitation regime indicates that postzygotic reproductive isolation may breakdown under imposition of dry conditions. These results suggest the potential for frequency of hybrids to increase if severe droughts become more common.

Complex interactions underlie the correlated evolution of floral traits and their association with pollinators in a clade with diverse pollination systems.

Natural selection by pollinators is an important factor in the morphological diversity and adaptive radiation of flowering plants. Selection by similar pollinators in unrelated plants leads to convergence in floral morphology, or "floral syndromes." Previous investigations into floral syndromes have mostly studied relatively small and/or simple systems, emphasizing vertebrate pollination. Despite the importance of multiple floral traits in plant-pollinator interactions, these studies have examined few quantitative traits, so their co-variation and phenotypic integration have been underexplored. To gain better insights into pollinator-trait dynamics, we investigate the model system of the phlox family (Polemoniaceae), a clade of ∼400 species pollinated by a diversity of vectors. Using a comprehensive phylogeny and large dataset of traits and observations of pollinators, we reconstruct ancestral pollination system, accounting for the temporal history of pollinators. We conduct phylogenetically controlled analyses of trait co-variation and association with pollinators, integrating many analyses over phylogenetic uncertainty. Pollinator shifts are more heterogeneous than previously hypothesized. The evolution of floral traits is partially constrained by phylogenetic history and trait co-variation, but traits are convergent and differences are associated with different pollinators. Trait shifts are usually gradual, rather than rapid, suggesting complex genetic and ecological interactions of flowers at macroevolutionary scales.

The first complete chloroplast genome sequence from Polemonium chinense (Polemoniaceae).

In this study, the complete chloroplast genome of Polemonium chinense was determined by next-generation sequencing technology. The plastome is 155,578 bp in length, with a large single-copy region (LSC) of 85,864 bp and a small single-copy region (SSC) of 18,246 bp, separated by a pair of inverted repeat regions (IRs) of 25,734 bp. It contains 135 genes, including 89 protein-coding genes, 37 tRNA genes, 8 rRNA genes, and 1 pseudogene. The overall GC content is 37.1%, while the corresponding values in the LSC, SSC and IR region are 35.1, 30.3, and 42.9%, respectively. The phylogenetic tree shows that Polemoniaceae is sister to Fouquieriaceae.

The Phenotypic and Genetic Underpinnings of Flower Size in Polemoniaceae.

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.

MULTIPLE ORIGINS OF SELF-COMPATIBILITY IN LINANTHUS SECTION LEPTOSIPHON (POLEMONIACEAE): PHYLOGENETIC EVIDENCE FROM INTERNAL-TRANSCRIBED-SPACER SEQUENCE DATA.

Phylogenetic reconstruction based on sequence variation in the internal transcribed spacer (ITS) region of rDNA was used to investigate the evolutionary dynamics of homomorphic self-incompatibility in Linanthus section Leptosiphon (Polemoniaceae), a group of annual plant species. Hand-pollination experiments revealed that five species were self-incompatible and four were self-compatible. Optimization of breeding systems onto the tree resulting from maximum-likelihood analysis, with no assumptions made about the ancestral condition, indicated that self-incompatibility has been lost four times in this section. An alternative tree rearrangement conforming to the hypothesis of three losses of self-incompatibility did not have a significantly lower likelihood than the maximum-likelihood tree as determined by a paired-sites test, but all rearrangements resulting in fewer than three losses were statistically rejected. Linanthus bicolor, a selfing species, was found to be polyphyletic, with populations from different geographic regions occurring in three well-supported clades. Morphological similarity in these distinct lineages is likely to have resulted from convergent evolution of traits associated with self-fertilization. Selection for reproductive assurance is hypothesized to have played an important role in the recurrent transformations from self-incompatibility to selfing in this group of annual species.