Dying of thirst: Osmoregulation by a hawkmoth pollinator in response to variability in ambient humidity and nectar availability.

Climate-induced shifts in flowering phenology can disrupt pollinator-floral resource synchrony, especially in desert ecosystems where rainfall dictates both. However, baseline metrics to gauge pollinator health in the wild amidst rapid climate change are lacking. Our laboratory-based study establishes a baseline for pollinator physiological state by exploring how osmotic conditions influence survivorship in a desert hawkmoth pollinator, Manduca sexta. We sampled hemolymph osmolality from over 1000 lab-grown moths at 20 %, 50 %, and 80 % ambient humidity levels. Starved moths maintained healthy osmolality of 350-400 mmol/kg for 1-3 days after eclosion regardless of ambient humidity, but it sharply rose to 550 mmol/kg after 4-5 days in low and moderate humidity, and after 5 days in high humidity. Starved moths in low humidity conditions perished within 5 days, while those in high humidity survived twice as long. Moths fed synthetic Datura wrightii nectar, synthetic Agave palmeri nectar, or water, maintained osmolality within a healthy range of 350-400mmol/kg. The same was true for moths fed authentic floral nectars from Datura and Agave plants, although moths consumed more synthetic than authentic nectars, possibly due to non-sugar constituents. Simulating a 4-day mismatch between pollinator emergence and nectar availability, a single nectar meal osmotically rescued moths under dry ambient conditions. Our findings highlight hemolymph osmolality as a rapid and accurate biomarker distinguishing dehydrated from hydrated states in insect pollinators.

Co-flowering richness has variable effects on pollen quantity and quality limitation in four Clarkia species.

BACKGROUND AND AIMS: Pollination failure occurs from insufficient pollen quantity or quality. However, the relative contributions of pollen quantity vs quality to overall pollen limitation, and how this is affected by the co-flowering context, remain unknown for most plant populations. Here, we studied patterns of pollen deposition and pollen tube formation across populations of four predominately outcrossing species in the genus Clarkia to evaluate how richness of co-flowering congeners affects the contribution of pollen quantity and quality to pollen limitation. METHODS: We partition variation in pollen deposition and pollen tube production across individuals, populations and species to identify the main sources of variation in components of reproductive success. We further quantify the relative contribution of pollen quantity and quality limitation to the reproductive success of the four Clarkia species using piecewise regression analyses. Finally, we evaluate how variation in the number of co-flowering Clarkia species in the community affects the strength of pollen quality and quality limitation. RESULTS: Across all contexts, pollen deposition and the proportion of pollen tubes produced varied greatly among individuals, populations, and species, and these were not always correlated. For instance, C. xantiana received the smallest pollen loads yet produced the highest proportion of pollen tubes, while C. speciosa exhibited the opposite pattern. Yet, co-flowering richness had variable effects on the strength of pollen quantity and quality limitation among populations. Specifically, breakpoint values, which are an indicator of overall pollen limitation, were two times larger in the four-species community compared with one and two-species communities for two Clarkia species, suggesting that pollen limitation can increase with increasing richness of co-flowering congeners. CONCLUSIONS: Our results reveal a complex interplay between quantity and quality of pollen limitation and co-flowering context that may have different evolutionary outcomes across species and populations.

Organ-specific volatiles from Sonoran desert Krameria flowers as potential signals for oil-collecting bees.

The evolution of flowers that offer oils as rewards and are pollinated by specialized bees represents a distinctive theme in plant-pollinator co-diversification. Some plants that offer acetylated glycerols as floral oils emit diacetin, a volatile by-product of oil metabolism, which is utilized by oil-collecting bees as an index signal for the presence of floral oil. However, floral oils in the genus Krameria (Krameriaceae) contain ß-acetoxy-substituted fatty acids instead of acetylated glycerols, making them unlikely to emit diacetin as an oil-bee attractant. We analyzed floral headspace composition from K. bicolor and K. erecta, native to the Sonoran Desert of southwestern North America, in search of alternative candidates for volatile index signals. Using solid-phase microextraction, combined with gas chromatography-mass spectrometry, we identified 26 and 45 floral volatiles, respectively, from whole flowers and dissected flower parts of these two Krameria species. As expected, diacetin was not detected. Instead, ß-ionone emerged as a strong candidate for an index signal, as it was uniquely present in dissected oil-producing floral tissues (elaiophores) of K. bicolor, as well as the larval cells and provisions from its oil-bee pollinator, Centris cockerelli. This finding suggests that the floral oil of K. bicolor is perfused with ß-ionone in its tissue of origin and retains the distinctive raspberry-like scent of this volatile after being harvested by C. cockerelli bees. In contrast, the elaiophores of K. erecta, which are not thought to be pollinated by C. cockerelli, produced a blend of anise-related oxygenated aromatics not found in the elaiophores of K. bicolor. Our findings suggest that ß-ionone has the potential to impact oil-foraging by C. cockerelli bees through several potential mechanisms, including larval imprinting on scented provisions or innate or learned preferences by foraging adults.

A phylogeny of the evening primrose family (Onagraceae) using a target enrichment approach with 303 nuclear loci.

BACKGROUND: The evening primrose family (Onagraceae) includes 664 species (803 taxa) with a center of diversity in the Americas, especially western North America. Ongoing research in Onagraceae includes exploring striking variation in floral morphology, scent composition, and breeding system, as well as the role of these traits in driving diversity among plants and their interacting pollinators and herbivores. However, these efforts are limited by the lack of a comprehensive, well-resolved phylogeny. Previous phylogenetic studies based on a few loci strongly support the monophyly of the family and the sister relationship of the two largest tribes but fail to resolve several key relationships. RESULTS: We used a target enrichment approach to reconstruct the phylogeny of Onagraceae using 303 highly conserved, low-copy nuclear loci. We present a phylogeny for Onagraceae with 169 individuals representing 152 taxa sampled across the family, including extensive sampling within the largest tribe, Onagreae. Deep splits within the family are strongly supported, whereas relationships among closely related genera and species are characterized by extensive conflict among individual gene trees. CONCLUSIONS: This phylogenetic resource will augment current research projects focused throughout the family in genomics, ecology, coevolutionary dynamics, biogeography, and the evolution of characters driving diversification in the family.

Reproductive strategies and their consequences for divergence, gene flow, and genetic diversity in three taxa of Clarkia.

Differences in reproductive strategies can have important implications for macro- and micro-evolutionary processes. We used a comparative approach through a population genetics lens to evaluate how three distinct reproductive strategies shape patterns of divergence among as well as gene flow and genetic diversity within three closely related taxa in the genus Clarkia. One taxon is a predominantly autonomous self-fertilizer and the other two taxa are predominantly outcrossing but vary in the primary pollinator they attract. In genotyping populations using genotyping-by-sequencing and comparing loci shared across taxa, our results suggest that differences in reproductive strategies in part promote evolutionary divergence among these closely related taxa. Contrary to expectations, we found that the selfing taxon had the highest levels of heterozygosity but a low rate of polymorphism. The high levels of fixed heterozygosity for a subset of loci suggests this pattern is driven by the presence of structural rearrangements in chromosomes common in other Clarkia taxa. In evaluating patterns within taxa, we found a complex interplay between reproductive strategy and geographic distribution. Differences in the mobility of primary pollinators did not translate to a difference in rates of genetic diversity and gene flow within taxa - a pattern likely due to one taxon having a patchier distribution and a less temporally and spatially reliable pollinator. Taken together, this work advances our understanding of the factors that shape gene flow and the distribution of genetic diversity within and among closely related taxa.

Hidden worlds within flowers.

There is a growing realization that ecological interactions take place at many scales, from acorns to forests, and that formerly overlooked community members, particularly microbes, can play outsized ecological roles. Beyond their primary function as the reproductive organs of angiosperms, flowers constitute resource-rich, ephemeral habitats teeming with flower-loving symbionts, or 'anthophiles'. The physical, chemical, and structural properties of flowers combine to create a habitat filter, selectively determining which anthophiles can reside there, and how, and when they interact. The microhabitats within flowers can provide shelter from predators or inclement weather, places to eat, sleep, thermoregulate, hunt, mate or reproduce. In turn, floral microhabitats contain the full range of mutualists, antagonists and apparent commensals, whose complex interactions impact how flowers look and smell, how profitable they are to foraging pollinators, and how selection feeds back upon the traits shaping those interactions. Recent studies suggest coevolutionary paths by which floral symbionts might be co-opted as mutualists and provide compelling examples in which ambush predators or florivores are recruited as floral allies. Unbiased studies that include the full roster of floral symbionts are likely to reveal novel links and additional nuance in the rich ecological communities hidden within flowers.

Context and the functional use of information in insect sensory ecology.

Context-specific behaviors emerge from the interaction between an animal's internal state and its external environment. Although the importance of context is acknowledged in the field of insect sensory ecology, there is a lack of synthesis on this topic stemming from challenges in conceptualizing 'context'. We address this challenge by gleaning over the recent findings on the sensory ecology of mosquitoes and other insect pollinators. We discuss internal states and their temporal dynamics, from those lasting minutes to hours (host-seeking) to those lasting days to weeks (diapause, migration). Of the many patterns reviewed, at least three were common to all taxa studied. First, different sensory cues gain prominence depending on the insect's internal state. Second, similar sensory circuits between related species can result in different behavioral outcomes. And third, ambient conditions can dramatically alter internal states and behaviors.

Cone humidity is a strong attractant in an obligate cycad pollination system.

Studies of pollination biology often focus on visual and olfactory aspects of attraction, with few studies addressing behavioral responses and morphological adaptation to primary metabolic attributes. As part of an in-depth study of obligate nursery pollination of cycads, we find that Rhopalotria furfuracea weevils show a strong physiological response and behavioral orientation to the cone humidity of the host plant Zamia furfuracea in an equally sensitive manner to their responses to Z. furfuracea-produced cone volatiles. Our results demonstrate that weevils can perceive fine-scale differences in relative humidity (RH) and that individuals exhibit a strong behavioral preference for higher RH in binary choice assays. Host plant Z. furfuracea produces a localized cloud of higher than ambient humidity around both pollen and ovulate cones, and R. furfuracea weevils preferentially land at the zone of maximum humidity on ovulate cones, i.e., the cracks between rows of megasporophylls that provide access to the ovules. Moreover, R. furfuracea weevils exhibit striking antennal morphological traits associated with RH perception, suggesting the importance of humidity sensing in the evolution of this insect lineage. Results from this study suggest that humidity functions in a signal-like fashion in this highly specialized pollination system and help to characterize a key pollination-mediating trait in an ancient plant lineage.

Target Enrichment and Extensive Population Sampling Help Untangle the Recent, Rapid Radiation of Oenothera Sect. Calylophus.

Oenothera sect. Calylophus is a North American group of 13 recognized taxa in the evening primrose family (Onagraceae) with an evolutionary history that may include independent origins of bee pollination, edaphic endemism, and permanent translocation heterozygosity. Like other groups that radiated relatively recently and rapidly, taxon boundaries within Oenothera sect. Calylophus have remained challenging to circumscribe. In this study, we used target enrichment, flanking noncoding regions, gene tree/species tree methods, tests for gene flow modified for target-enrichment data, and morphometric analysis to reconstruct phylogenetic hypotheses, evaluate current taxon circumscriptions, and examine character evolution in Oenothera sect. Calylophus. Because sect. Calylophus comprises a clade with a relatively restricted geographic range, we were able to extensively sample across the range of geographic, edaphic, and morphological diversity in the group. We found that the combination of exons and flanking noncoding regions led to improved support for species relationships. We reconstructed potential hybrid origins of some accessions and note that if processes such as hybridization are not taken into account, the number of inferred evolutionary transitions may be artificially inflated. We recovered strong evidence for multiple evolutionary origins of bee pollination from ancestral hawkmoth pollination, edaphic specialization on gypsum, and permanent translocation heterozygosity. This study applies newly emerging techniques alongside dense infraspecific sampling and morphological analyses to effectively reconstruct the recalcitrant history of a rapid radiation. [Gypsum endemism; Oenothera sect. Calylophus; Onagraceae; phylogenomics; pollinator shift; recent radiation; target enrichment.].