Gradual pollen presentation in Vaccinium corymbosum 'Bluecrop': an adaptive mechanism to improve pollination efficiency and outcrossing.

Gradual pollen presentation is a plant reproductive mechanism to improve pollination efficiency and accuracy and promote outcrossing. Vaccinium corymbosum 'Bluecrop' has a typical gradual pollen presentation mechanism. 'Bluecrop' exhibits an inverted bell-shaped flower with a white coloration. By investigating the flower syndrome, pollination characteristics, pollination efficiency, and breeding system of 'Bluecrop', this study aims to explore the adaptive significance of these traits. The results showed 'Bluecrop' released pollen gradually through anther poricidal dehiscence. Among different pollinators, Apis mellifera and Bombus can pollinate effectively, and the mechanism of gradual pollen presentation significantly improved the efficiency of pollen transfer. This characteristic limits the amount of pollen removed by the pollinators and prolongs pollen presentation, thus attracting more pollinators and thereby increasing male fitness. The nectar secretion of 'Bluecrop' is gradual, with a large nectar production and a long phase of nectar secretion, enhance visitation frequencies and the chances of successful pollination. At the same time, campanulate corolla can protect pollen as well as nectar from waste due to environmental factors and other effects. The breeding system of 'Bluecrop' relies mainly on outcrossing because of its low affinity for self-fertilization and good interaction with pollinating insects. Thus, the special floral syndrome and the mechanism of secondary pollen presentation are significant in improving pollination efficiency and promoting the reproductive success of 'Bluecrop' by outcrossing. It can provide a certain theoretical basis for the future propagation breeding of 'Bluecrop'.

The AGL6-ELF3-FT circuit controls flowering time in Arabidopsis.

Adjusting the timing of floral transition is essential for reproductive success in plants. A number of flowering regulators integrate internal and external signals to precisely determine the time to flower. We here report that the AGAMOUS-LIKE 6 (AGL6) - EARLY FLOWERING 3 (ELF3) module regulates flowering in the FLOWERING LOCUS T (FT)-dependent pathway in Arabidopsis. The AGL6 transcriptional repressor promotes floral transition by directly suppressing ELF3, which in turn directly represses FT expression that acts as a floral integrator. Indeed, ELF3 is epistatic to AGL6 in the control of floral transition. Overall, our findings propose that the AGL6-ELF3 module contributes to fine-tuning flowering time in plants.

GhTKPR1_8 functions to inhibit anther dehiscence and reduce pollen viability in cotton.

Sporopollenin, as the main component of the pollen exine, is a highly resistant polymer that provides structural integrity under unfavourable environmental conditions. Tetraketone α-pyrone reductase 1 (TKPR1) is essential for sporopollenin formation, catalyzing the reduction of tetraketone carbonyl to hydroxylated α-pyrone. The functional role of TKPR1 in male sterility has been reported in flowering plants such as maize, rice, and Arabidopsis. However, the molecular cloning and functional characterization of TKPR1 in cotton remain unaddressed. In this study, we identified 68 TKPR1s from four cotton species, categorized into three clades. Transcriptomics and RT-qPCR demonstrated that GhTKPR1_8 exhibited typical expression patterns in the tetrad stage of the anther. GhTKPR1_8 was localized to the endoplasmic reticulum. Moreover, ABORTED MICROSPORES (GhAMS) transcriptionally activated GhTKPR1_8 as indicated by luciferase complementation tests. GhTKPR1_8-knockdown inhibited anther dehiscence and reduced pollen viability in cotton. Additionally, overexpression of GhTKPR1_8 in the attkpr1 mutant restored its male sterile phenotype. This study offers novel insights into the investigation of TKPR1 in cotton while providing genetic resources for studying male sterility.

Unravelling some factors affecting sexual reproduction in rock-specialist shrub: Insight from an endemic Daphne arbuscula (Thymelaeaceae).

The role of endemic species in global biodiversity is pivotal, and understanding their biology and ecology is imperative for their fitness and long-term survival, particularly in the face of ongoing climatic oscillations. Our primary goal was to investigate the sexual reproduction level of the endangered Western Carpathian endemic Daphne arbuscula (Thymelaeaceae), which inhabits extreme rocky habitats, and to comprehend the influence of specific factors on its reproductive success. We conducted the research across four populations, varying in size and environmental conditions. Over two years, we monitored flower and fruit production, analyzed genetic variability within and among populations, and studied pollination mechanisms. Daphne arbuscula proved to be strictly self-incompatible, with significant variations in flower and fruit production among populations and seasons. The average fruit production percentage consistently remained below 50% across populations, indicating challenges in sexual reproduction. Cold and harsh weather during the reproductive phase had a substantial negative impact on sexual reproduction efficacy, leading to decreased fruit production. Nevertheless, several individuals in sheltered microhabitats displayed significantly higher fruit production, ranging from 60% to 83%, emphasizing the critical role of microhabitat heterogeneity in sustaining sexual reproduction in this species. We found no pronounced differences in genetic diversity within or among populations, suggesting that genetic factors may not critically influence the reproductive success of this endemic species. The implications of our findings might be of paramount importance for the long-term survival of D. arbuscula and offer valuable insights for the development of effective conservation strategies for this species.

Comprehensive analysis of stress-activated protein kinase genes (OsSAPKs) in rice flowering time.

MAIN CONCLUSION: The rice SnRK2 members SAPK4, SAPK5, SAPK7 and SAPK10 are positive regulators involved in the regulation of rice flowering, while other single mutants exhibited no effect on rice flowering. The rice SnRK2 family, comprising 10 members known as SAPK (SnRK2-Associated Protein Kinase), is pivotal in the abscisic acid (ABA) pathway and crucial for various biological processes, such as drought resistance and salt tolerance. Additionally, these members have been implicated in the regulation of rice heading date, a key trait influencing planting area and yield. In this study, we utilized gene editing technology to create mutants in the Songjing 2 (SJ2) background, enabling a comprehensive analyze the role of each SAPK member in rice flowering. We found that SAPK1, SAPK2, and SAPK3 may not directly participate in the regulatory network of rice heading date, while SAPK4, SAPK5, and SAPK7 play positive roles in rice flowering regulation. Notably, polygene deletion resulted in an additive effect on delaying flowering. Our findings corroborate the previous studies indicating the positive regulatory role of SAPK10 in rice flowering, as evidenced by delayed flowering observed in sapk9/10 double mutants. Moving forward, our future research will focus on analyzing the molecular mechanisms underlying SAPKs involvement in rice flowering regulation, aiming to enhance our understanding of the rice heading date relationship network and lay a theoretical foundation for breeding efforts to alter rice ripening dates.

Unveiling the phenology and associated floral regulatory pathways of Humulus lupulus L. in subtropical conditions.

MAIN CONCLUSION: The hop phenological cycle was described in subtropical condition of Brazil showing that flowering can happen at any time of year and this was related to developmental molecular pathways. Hops are traditionally produced in temperate regions, as it was believed that vernalization was necessary for flowering. Nevertheless, recent studies have revealed the potential for hops to flower in tropical and subtropical climates. In this work, we observed that hops in the subtropical climate of Minas Gerais, Brazil grow and flower multiple times throughout the year, independently of the season, contrasting with what happens in temperate regions. This could be due to the photoperiod consistently being inductive, with daylight hours below the described threshold (16.5 h critical). We observed that when the plants reached 7-9 nodes, the leaves began to transition from heart-shaped to trilobed-shaped, which could be indicative of the juvenile to adult transition. This could be related to the fact that the 5th node (in plants with 10 nodes) had the highest expression of miR156, while two miR172s increased in the 20th node (in plants with 25 nodes). Hop flowers appeared later, in the 25th or 28th nodes, and the expression of HlFT3 and HlFT5 was upregulated in plants between 15 and 20 nodes, while the expression of HlTFL3 was upregulated in plants with 20 nodes. These results indicate the role of axillary meristem age in regulating this process and suggest that the florigenic signal should be maintained until the hop plants bloom. In addition, it is possible that the expression of TFL is not sufficient to inhibit flowering in these conditions and promote branching. These findings suggest that the reproductive transition in hop under inductive photoperiodic conditions could occur in plants between 15 and 20 nodes. Our study sheds light on the intricate molecular mechanisms underlying hop floral development, paving the way for potential advancements in hop production on a global scale.

Signalling cascades choreographing petal cell death: implications for postharvest quality.

Senescence is a multifaceted and dynamic developmental phase pivotal in the plant's lifecycle, exerting significant influence and involving intricate regulatory mechanisms marked by a variety of structural, biochemical and molecular alterations. Biochemical changes, including reactive oxygen species (ROS) generation, membrane deterioration, nucleic acid degradation and protein degradation, characterize flower senescence. The progression of senescence entails a meticulously orchestrated network of interconnected molecular mechanisms and signalling pathways, ensuring its synchronized and efficient execution. Within flowering plants, petal senescence emerges as a crucial aspect significantly impacting flower longevity and postharvest quality, emphasizing the pressing necessity of unravelling the underlying signalling cascades orchestrating this process. Understanding the complex signalling pathways regulating petal senescence holds paramount importance, not only shedding light on the broader phenomenon of plant senescence but also paving the way for the development of targeted strategies to enhance the postharvest longevity of cut flowers. Various signalling pathways participate in petal senescence, encompassing hormone signalling, calcium signalling, protein kinase signalling and ROS signalling. Among these, the ethylene signalling pathway is extensively studied, and the manipulation of genes associated with ethylene biosynthesis or signal transduction has demonstrated the potential to enhance flower longevity. A thorough understanding of these complex pathways is critical for effectively delaying flower senescence, thereby enhancing postharvest quality and ornamental value. Therefore, this review adopts a viewpoint that combines fundamental research into the molecular intricacies of senescence with a practical orientation towards developing strategies for improving the postharvest quality of cut flowers. The innovation of this review is to shed light on the pivotal signalling cascades underpinning flower senescence and offer insights into potential approaches for modulating these pathways to postpone petal senescence in ornamental plants.

Harmful self-pollination drives gynodioecy in European chestnut, a self-incompatible tree.

PREMISE: Gynodioecy is a rare sexual system in which two genders (sensu Lloyd, 1980), cosexuals and females, coexist. To survive, female plants must compensate for their lack of siring capacity and male attractiveness. In European chestnut (Castanea sativa), an outcrossing tree, self-pollination reduces fruit set in cosexual individuals because of late-acting self-incompatibility and early inbreeding depression. Could this negative sexual interaction explain the presence of females in this species? METHODS: We studied gender variation in wild populations of European chestnut. In addition, we compared fruit set (the proportion of flowers giving fruits) and other key female fitness components as well as reproductive allocation between genders. We then performed emasculation experiments in cosexual trees, by removing nectar-producing fertile male inflorescences. We also removed sterile but nectar-producing male inflorescences from female trees, as a control. RESULTS: We found a highly variable proportion of male-sterile individuals in the wild in European chestnut. In the experimental plot, trees from each gender had similar size, flower density, and burr set, but different fruit set. Removing nectar-producing male inflorescences from branches or entire trees increased fruit set in cosexual but not in female trees. CONCLUSIONS: These results show that self-pollination impairs fruit set in cosexual trees. Female trees avoid these problems as they do not produce pollen but continue to attract pollinators thanks to their rewarding male-sterile inflorescences, resulting in a much higher fruit set than in cosexuals. This demonstrates that even outcrossed plants can benefit from the cessation of self-pollination, to the point that unisexuality can evolve.

Island plants with newly discovered reproductive traits have higher capacity for uniparental reproduction, supporting Baker's law.

Uniparental reproduction is advantageous when lack of mates limits outcrossing opportunities in plants. Baker's law predicts an enrichment of uniparental reproduction in habitats colonized via long-distance dispersal, such as volcanic islands. To test it, we analyzed reproductive traits at multiple hierarchical levels and compared seed-set after selfing and crossing experiments in both island and mainland populations of Limonium lobatum, a widespread species that Baker assumed to be self-incompatible because it had been described as pollen-stigma dimorphic, i.e., characterized by floral morphs differing in pollen-surface morphology and stigma-papillae shape that are typically self-incompatible. We discovered new types and combinations of pollen and stigma traits hitherto unknown in the literature on pollen-stigma dimorphism and a lack of correspondence between such combinations and pollen compatibility. Contrary to previous reports, we conclude that Limonium lobatum comprises both self-compatible and self-incompatible plants characterized by both known and previously undescribed combinations of reproductive traits. Most importantly, plants with novel combinations are overrepresented on islands, selfed seed-set is higher in islands than the mainland, and insular plants with novel pollen-stigma trait-combinations disproportionally contribute to uniparental reproduction on islands. Our results thus support Baker's law, connecting research on reproductive and island biology.

Genome-wide identification of the plant homeodomain-finger family in rye and ScPHD5 functions in cold tolerance and flowering time.

KEY MESSAGE: 111 PHD genes were newly identified in rye genome and ScPHD5's role in regulating cold tolerance and flowering time was suggested. Plant homeodomain (PHD)-finger proteins regulate the physical properties of chromatin and control plant development and stress tolerance. Although rye (Secale cereale L.) is a major winter crop, PHD-finger proteins in rye have not been studied. Here, we identified 111 PHD genes in the rye genome that exhibited diverse gene and protein sequence structures. Phylogenetic tree analysis revealed that PHDs were genetically close in monocots and diverged from those in dicots. Duplication and synteny analyses demonstrated that ScPHDs have undergone several duplications during evolution and that high synteny is conserved among the Triticeae species. Tissue-specific and abiotic stress-responsive gene expression analyses indicated that ScPHDs were highly expressed in spikelets and developing seeds and were responsive to cold and drought stress. One of these genes, ScPHD5, was selected for further functional characterization. ScPHD5 was highly expressed in the spike tissues and was localized in the nuclei of rye protoplasts and tobacco leaves. ScPHD5-overexpressing Brachypodium was more tolerant to freezing stress than wild-type (WT), with increased CBF and COR gene expression. Additionally, these transgenic plants displayed an extremely early flowering phenotype that flowered more than two weeks earlier than the WT, and vernalization genes, rather than photoperiod genes, were increased in the WT. RNA-seq analysis revealed that diverse stress response genes, including HSPs, HSFs, LEAs, and MADS-box genes, were also upregulated in transgenic plants. Our study will help elucidate the roles of PHD genes in plant development and abiotic stress tolerance in rye.

Fields of flowers with few strikes: how oligolectic bees manage their foraging behavior on Calibrachoa elegans (Solanaceae).

In specialized plant-pollinator associations, partners may exhibit adaptive traits, which favor the maintenance of the interaction. The association between Calibrachoa elegans (Solanaceae) and its oligolectic bee pollinator, Hexantheda missionica (Colletidae), is mutualistic and forms a narrowly specialized pollination system. Flowers of C. elegans are pollinated exclusively by this bee species, and the bees restrict their pollen resources to this plant species. The pollen presentation schedules of C. elegans were evaluated at the population level to test the hypothesis that H. missionica females adjust their foraging behavior to the resource offering regime of C. elegans plants. For this, the number of new flowers and anthers opened per hour (as a proxy for pollen offering) was determined, and pollen advertisement was correlated with the frequency of flower visits during the day. Preferences of female bees for flowers of different stages were also investigated, and their efficiency as pollinators was evaluated. Pollen offering by C. elegans was found to be partitioned throughout the day through scattered flower openings. Females of H. missionica indeed adjusted their foraging activity to the most profitable periods of pollen availability. The females preferred new, pollen-rich flowers over old ones and gathered pollen and nectar selectively according to flower age. Such behaviors must optimize female bee foraging efficiency on flowers. Female bees set 93% of fruit after a single visit. These findings guarantee their importance as pollinators and the persistence of the specialized plant-pollinator association.

Ecological drivers of flower-leaf sequences: aridity and proxies for pollinator attraction select for flowering-first in the American plums.

Across temperate forests, many tree species produce flowers before their leaves emerge. This flower-leaf phenological sequence, known as hysteranthy, is generally described as an adaptation for wind pollination. However, this explanation does not address why hysteranthy is also common in biotically pollinated taxa. We quantified flower-leaf sequence variation in the American plums (Prunus, subg. Prunus sect. Prunocerasus), a clade of insect-pollinated trees, using herbaria specimens and Bayesian hierarchical modeling. We tested two common, but rarely interrogated hypotheses - that hysteranthy confers aridity tolerance and/or pollinator visibility - by modeling the associations between hysteranthy and related traits. To understand how these phenology-trait associations were sensitive to taxonomic scale and flower-leaf sequence classification, we then extended these analyses to all Prunus species in North America. Our findings across two taxonomic levels support the hypotheses that hysteranthy may help temporally partition hydraulic demand to reduce water stress and increase pollinator visibility - thereby reducing selective pressure on inflorescence size. Our results provide foundational insights into the evolution of flower-leaf sequences in the genus Prunus, with implications for understanding these patterns in biotically pollinated plants in general. Our approach suggests a path to advance these hypotheses to other clades, but teasing out drivers fully will require new experiments.

Quantifying the production of plant pollen at the farm scale.

Plant pollen is rich in protein, sterols and lipids, providing crucial nutrition for many pollinators. However, we know very little about the quantity, quality and timing of pollen availability in real landscapes, limiting our ability to improve food supply for pollinators. We quantify the floral longevity and pollen production of a whole plant community for the first time, enabling us to calculate daily pollen availability. We combine these data with floral abundance and nectar measures from UK farmland to quantify pollen and nectar production at the landscape scale throughout the year. Pollen and nectar production were significantly correlated at the floral unit, and landscape level. The species providing the highest quantity of pollen on farmland were Salix spp. (38%), Filipendula ulmaria (14%), Rubus fruticosus (10%) and Taraxacum officinale (9%). Hedgerows were the most pollen-rich habitats, but permanent pasture provided the majority of pollen at the landscape scale, because of its large area. Pollen and nectar were closely associated in their phenology, with both peaking in late April, before declining steeply in June and remaining low throughout the year. Our data provide a starting point for including pollen in floral resource assessments and ensuring the nutritional requirements of pollinators are met in farmland landscapes.

First evidence of late-acting self-incompatibility in the Aristolochiaceae.

Most Aristolochiaceae species studied so far are from temperate regions, bearing self-compatible protogynous trap flowers. Although self-incompatibility has been suggested for tropical species, the causes of self-sterility in this family remain unknown. To fill this gap, we studied the pollination of the tropical Aristolochia esperanzae, including the physical and physiological anti-selfing mechanisms. Floral visitors trapped inside flowers were collected to determine the pollinators. Protogyny was characterized by observing the temporal expression of sexual phases and stigmatic receptivity tests. The breeding system was investigated using hand-pollination treatments. Pollen tube growth was observed using epifluorescence to identify the self-incompatibility mechanism. Flies were the most frequent visitors found inside A. esperanzae trap flowers, with individuals from the family Ulidiidae being potential pollinators since they carried pollen. The characteristic flower odour and presence of larvae indicate that A. esperanzae deceives flies through oviposition-site mimicry. Although this species showed incomplete protogyny, stigmatic receptivity decreased during the male phase, avoiding self-pollination. Fruits developed only after cross- and open pollination, indicating that the population is non-autonomous, non-apomictic, and self-sterile. This occurred through a delay in the growth of geitonogamous pollen tubes to the ovary and lower ovule penetration, indicating a late-acting self-incompatibility mechanism. Our findings expand the number of families in which late-acting self-incompatibility has been reported, demonstrating that it is more widespread than previously thought, especially when considering less-studied tropical species among the basal angiosperms.

High floral disparity without pollinator shifts in buzz-bee-pollinated Melastomataceae.

Shifts among functional pollinator groups are commonly regarded as sources of floral morphological diversity (disparity) through the formation of distinct pollination syndromes. While pollination syndromes may be used for predicting pollinators, their predictive accuracy remains debated, and they are rarely used to test whether floral disparity is indeed associated with pollinator shifts. We apply classification models trained and validated on 44 functional floral traits across 252 species with empirical pollinator observations and then use the validated models to predict pollinators for 159 species lacking observations. In addition, we employ multivariate statistics and phylogenetic comparative analyses to test whether pollinator shifts are the main source of floral disparity in Melastomataceae. We find strong support for four well-differentiated pollination syndromes ('buzz-bee', 'nectar-foraging vertebrate', 'food-body-foraging vertebrate', 'generalist'). While pollinator shifts add significantly to floral disparity, we find that the most species-rich 'buzz-bee' pollination syndrome is most disparate, indicating that high floral disparity may evolve without pollinator shifts. Also, relatively species-poor clades and geographic areas contributed substantially to total disparity. Finally, our results show that machine-learning approaches are a powerful tool for evaluating the predictive accuracy of the pollination syndrome concept as well as for predicting pollinators where observations are missing.

Self-(in)compatibility in Tunisian apple accessions [Malus domestica. Borkh]: S-genotypes identification and pollen tube growth analysis.

MAIN CONCLUSION: Self-incompatibility studies have revealed a potential use of Tunisian apple resources for crop improvement and modern breeding programs and a likely correlation between the pollen tube growth and flowering period. Apples [Malus domestica. Borkh] exhibit an S-RNase-based gametophytic self-incompatibility (GSI) system. Four primer combinations were used to S-genotype eighteen Tunisian local apple accessions and twelve introduced accessions that served as references. Within the Tunisian local accessions, S2, S3, S7, and S28 S-alleles were the most frequent and were assigned to 14 S-genotypes; among them, S7S28, S3S7, S2S5, and S2S3 were the most abundant. PCA plot showed that population structuring was affected by the S-alleles frequencies and revealed a modern origin of the Tunisian varieties rather than being ancient ones. Nonetheless, the results obtained with 17 SSR markers showed a separate grouping of local Tunisian accessions that calls into question the hypothesis discussed. Pollination experiments showed that the pollen started to germinate within 24 h of pollination but 48 h after pollination in the "El Fessi" accession. The first pollen tubes arrived in the styles within 36 h of pollination in two early flowering accessions known as "Arbi" and "Bokri", and after 72 h of pollination in late flowering "El Fessi" and 48 h after pollination in remaining accessions. The first pollen tube arrests were observed in accessions "Arbi" and "Bokri" within 84 h of pollination, within 108 h of pollination in "El Fessi" and within 108 h of pollination in remaining accessions. In the apple accession called "Boutabgaya," the pollen tubes reached the base of the style within 120 h of pollination without being aborted. Nevertheless, the self-compatible nature of "Boutabgaya" needs more studies to be confirmed. However, our results revealed the malfunction of the female component of the GSI in this accession. To conclude, this work paved the path for further studies to enhance the insight (i) into the relation between the flowering period and the pollen tube growth, (ii) self-compatible nature of "Boutabgaya", and (iii) the origin of the Tunisian apple.

Chinese cherry CpMYB44-CpSPDS2 module regulates spermidine content and florescence in tobacco.

The flower bud differentiation plays a crucial role in cherry yield and quality. In a preliminary study, we revealed the promotion of spermidine (Spd) in bud differentiation and quality. However, the molecular mechanism underlying Spd regulating cherry bud differentiation remains unclear. To address this research gap, we cloned CpSPDS2, a gene that encodes Spd synthase and is highly expressed in whole flowers and pistils of the Chinese cherry (cv. 'Manaohong'). Furthermore, an overexpression vector with this gene was constructed to transform tobacco plants. The findings demonstrated that transgenic lines exhibited higher Spd content, an earlier flowering time by 6 d, and more lateral buds and flowers than wild-type lines. Additionally, yeast one-hybrid assays and two-luciferase experiments confirmed that the R2R3-MYB transcription factor (CpMYB44) directly binds to and activates the CpSPDS2 promoter transcription. It is indicated that CpMYB44 promotes Spd accumulation via regulating CpSPDS2 expression, thus accelerating the flower growth. This research provides a basis for resolving the molecular mechanism of CpSPDS2 involved in cherry bud differentiation.

HD-Zip proteins modify floral structures for self-pollination in tomato.

Cleistogamy is a type of self-pollination that relies on the formation of a stigma-enclosing floral structure. We identify three homeodomain-leucine zipper IV (HD-Zip IV) genes that coordinately promote the formation of interlocking trichomes at the anther margin to unite neighboring anthers, generating a closed anther cone and cleistogamy (flower morphology necessitating strict self-pollination). These HD-Zip IV genes also control style length by regulating the transition from cell division to endoreduplication. The expression of these HD-Zip IV genes and their downstream gene, Style 2.1, was sequentially modified to shape the cleistogamy morphology during tomato evolution and domestication. Our results provide insights into the molecular basis of cleistogamy in modern tomato and suggest targets for improving fruit set and preventing pollen contamination in genetically modified crops.

A Long-Lived Alpine Perennial Advances Flowering under Warmer Conditions but Not Enough to Maintain Reproductive Success.

AbstractAssessing whether phenological shifts in response to climate change confer a fitness advantage requires investigating the relationships among phenology, fitness, and environmental drivers of selection. Despite widely documented advancements in phenology with warming climate, we lack empirical estimates of how selection on phenology varies in response to continuous climate drivers or how phenological shifts in response to warming conditions affect fitness. We leverage an unusual long-term dataset with repeated, individual measurements of phenology and reproduction in a long-lived alpine plant. We analyze phenotypic plasticity in flowering phenology in relation to two climate drivers, snowmelt timing and growing degree days (GDDs). Plants flower earlier with increased GDDs and earlier snowmelt, and directional selection also favors earlier flowering under these conditions. However, reproduction still declines with warming and early snowmelt, even when flowering is early. Furthermore, the steepness of this reproductive decline increases dramatically with warming conditions, resulting in very little fruit production regardless of flowering time once GDDs exceed approximately 225 degree days or snowmelt occurs before May 15. Even though advancing phenology confers a fitness advantage relative to stasis, these shifts are insufficient to maintain reproduction under warming, highlighting limits to the potential benefits of phenological plasticity under climate change.

Is self-incompatibility a reproductive barrier for hybridization in a sympatric species?

PREMISE: Barriers at different reproductive stages contribute to reproductive isolation. Self-incompatibility (SI) systems that prevent self-pollination could also act to control interspecific pollination and contribute to reproductive isolation, preventing hybridization. Here we evaluated whether SI contributes to reproductive isolation among four co-occurring Opuntia species that flower at similar times and may hybridize with each other. METHODS: We assessed whether Opuntia cantabrigiensis, O. robusta, O. streptacantha, and O. tomentosa, were self-compatible and formed hybrid seeds in five manipulation treatments to achieve self-pollination, intraspecific cross-pollination, open pollination (control), interspecific crosses or apomixis, then recorded flowering phenology and synchrony. RESULTS: All species flowered in the spring with a degree of synchrony, so that two pairs of species were predisposed to interspecific pollination (O. cantabrigiensis with O. robusta, O. streptacantha with O. tomentosa). All species had distinct reproductive systems: Opuntia cantabrigiensis is self-incompatible and did not produce hybrid seeds as an interspecific pollen recipient; O. robusta is a dioecious species, which formed a low proportion of hybrid seeds; O. streptacantha and O. tomentosa are self-compatible and produced hybrid seeds. CONCLUSIONS: Opuntia cantabrigiensis had a strong pollen-pistil barrier, likely due to its self-incompatibility. Opuntia robusta, the dioecious species, is an obligate outcrosser and probably partially lost its ability to prevent interspecific pollen germination. Given that the self-compatible species can set hybrid seeds, we conclude that pollen-pistil interaction and high flowering synchrony represent weak barriers; whether reproductive isolation occurs later in their life cycle (e.g., germination or seedling survival) needs to be determined.