Flower color polymorphism of a wild Iris on the Qinghai-Tibet plateau.

BACKGROUND: Flower color plays a crucial role in attracting pollinators and facilitating environmental adaptation. Investigating the causes of flower color polymorphism and understanding their potential effects on both ecology and genetics can enhance our understanding of flower color polymorphism in wild plant. RESULTS: In this study, we examined the differences of potential male and female fitness between purple- and yellow- flower individuals in Iris potaninii on the Qinghai-Tibet Plateau, and screened key genes and positively selective genes involved in flower color change. Our results showed that yellow flower exhibited a higher pollen-to-ovule ratio. Yellow flowers were derived from purple flowers due to the loss of anthocyanins, and F3H could be an essential gene affecting flower color variation though expression regulation and sequence polymorphism in this species. Furthermore, our findings suggest that genes positively selected in yellow-flowered I. potaninii might be involved in nucleotide excision repair and plant-pathogen interactions. CONCLUSIONS: These results suggest that F3H induces the flower color variation of Iris potaninii, and the subsequent ecological and additive positive selection on yellow flowers may further enhance plant adaptations to alpine environments.

A Novel R2R3-MYB Transcription Factor Contributes to Petal Blotch Formation by Regulating Organ-Specific Expression of PsCHS in Tree Peony (Paeonia suffruticosa).

Flower color patterns play critical roles in plant-pollinator interactions and represent one of the most common adaptations during angiosperm evolution. However, the molecular mechanisms underlying flower color pattern formation are less understood in non-model organisms. The aim of this study was to identify genes involved in the formation of petal blotches in tree peony (Paeonia suffruticosa) through transcriptome profiling and functional experiments. We identified an R2R3-MYB gene, PsMYB12, representing a distinct R2R3-MYB subgroup, with a spatiotemporal expression pattern tightly associated with petal blotch development. We further demonstrated that PsMYB12 interacts with a basic helix-loop-helix (bHLH) and a WD40 protein in a regulatory complex that directly activates PsCHS expression, which is also specific to the petal blotches. Together, these findings advance our understanding of the molecular mechanisms of pigment pattern formation beyond model plants. They also benefit molecular breeding of tree peony cultivars with novel color patterns and promote germplasm innovation.

A century of pollination success revealed by herbarium specimens of seed pods.

A widely observed pollinator decline around the world has led to the prediction that terrestrial ecosystems could be disrupted as plant pollination suffers, but declining pollination success has not been tested rigorously in wild plants, and it still remains unclear how pollination success of plant species responds differently in the context of pollinator decline. By viewing the number of seeds per pod as a quantitative measure of successful pollination, we examined seed pods in 4637 herbarium specimens of 109 obligately outcrossing legumes collected over the past century. We found that only 13 species showed significant temporal change with nine of those as an increase. None of the three subfamilies of legumes showed a consistent trend, and the subfamily Papilionoideae with the most specialized flowers, had increasing seed number per pod more often than decreasing. We conclude that legume pollination in China shows no sign of disruption and the effects of plant-pollinator disruption may be more complicated than simplistic predictions have allowed.

Mixed effects of honey bees on pollination function in the Tibetan alpine grasslands.

The global expansion of domesticated plant and animal species has profoundly impacted biodiversity and ecosystem functions. However, the spillover effect of non-native honey bees from mass-flowering crops into adjacent natural vegetation on pollination function within plant communities remains unclear. To address this, we conduct field experiments to investigate the ecological impacts of honey bees (Apis mellifera) and a mass-flowering crop (Brassica rapa var. oleifera) on pollinator communities, plant-pollinator interactions, and reproductive performance of wild plants in 48 pollinator-limited alpine grasslands. Our findings indicate that the transition of dominant pollinators from flies to honey bees enhances visitation fidelity of pollinator species and reconfigures pollination interactions due to an increase in competition between honey bees and native pollinator species. Additionally, honey bees increase, decrease or do not alter plant reproductive success, depending on the plant species. Here, we report the mixed effects of honey bees on pollination function in pollinator-limited alpine grasslands.

The effects of changes in flowering plant composition caused by nitrogen and phosphorus enrichment on plant-pollinator interactions in a Tibetan alpine grassland.

Soil eutrophication from atmospheric deposition and fertilization threatens biodiversity and the functioning of terrestrial ecosystems worldwide. Increases in soil nitrogen (N) and phosphorus (P) content can alter the biomass and structure of plant communities in grassland ecosystems; however, the impact of these changes on plant-pollinator interactions is not yet clear. In this study, we tested how changes in flowering plant diversity and composition due to N and P enrichment affected pollinator communities and pollination interactions. Our experiments, conducted in a Tibetan alpine grassland, included four fertilization treatments: N (10 g N m-2 year-1), P (5 g P m-2 year-1), a combination of N and P (N + P), and control. We found that changes in flowering plant composition and diversity under the N and P treatments did not alter the pollinator richness or abundance. The N and P treatments also had limited effects on the plant-pollinator interactions, including the interaction numbers, visit numbers, plant and pollinator species dissimilarity, plant-pollinator interaction dissimilarity, average number of pollinator species attracted by each plant species (vulnerability), and average number of plant species visited by each pollinator species (generality). However, the N + P treatment increased the species and interaction dissimilarity in flowering plant and pollinator communities and decreased the generality in plant-pollinator interactions. These data highlight that changes in flowering plants caused by N + P enrichment alter pollination interactions between flowering plants and pollinators. Owing to changes in flowering plant communities, the plant-pollinator interactions could be sensitive to the changing environment in alpine regions.

Multiple lines of evidence supports the two varieties of Halenia elliptica (Gentianaceae) as two species.

Delimiting species requires multiple sources of evidence. Here, we delimited two varieties of Halenia elliptica (Gentianaceae) using several lines of evidence, including morphological traits and mating system in a sympatric population, phylogenetic relationships based on nrITS and cpDNA (rpl16) data, and complete chloroplast genome sequences. Comparative analysis of 21 morphological traits clearly separates the two varieties of H. elliptica. Examination of the flowering process and pollination treatments indicate that H. elliptica var. grandiflora produces seeds via outcrossing, whereas H. elliptica var. elliptica produces seeds via mixed mating. Furthermore, hand-pollinated hybridization of the two varieties produced no seeds. Observations of pollinators showed that when bees began a pollination bout on H. elliptica var. grandiflora they preferred to continue pollinating this variety; however, when they began a pollination bout on H. elliptica var. elliptica, they showed no preference for either variety. Phylogenetic analysis confirmed the monophyly of H. elliptica, which was further divided into two monophyletic clades corresponding to the two varieties. A large number of variants from the chloroplast genomes reflected remarkable genetic dissimilarities between the two varieties of H. elliptica. We recommend that the two varieties of H. elliptica should be revised as two species (H. elliptica and H. grandiflora). Our findings indicate that H. elliptica varieties may have split into two separate species due to a shift in mating system, changes in flowering phenology and/or post-pollination reproductive isolation.

Selective seed abortion induced by nectar robbing in the selfing plant Comastoma pulmonarium.

• Self-pollination often provides plants with the benefit of reproductive assurance; thus, it is generally assumed that species' interactions that alter floral attractiveness or rewards, such as nectar robbing, will have little effect on the seed production of selfing species. We challenge this view with experimental data from Comastoma pulmonarium, a selfing annual experiencing a high ratio of nectar robbing in the Qinghai-Tibet Plateau. • We manipulated robbing (robbed or netted) and pollination mode (hand-selfed or hand-outcrossed) in a factorial design and measured the number of developing ovules and mature seeds, together with seed weight and seed germination, in each treatment. • Robbing decreased the number of mature seeds, but not the number of developing ovules, suggesting a negative influence of robbers through indirect effects via selective seed abortion. We found no evidence for early-acting inbreeding depression, but found later-acting inbreeding depression. Our data also suggested that later-acting inbreeding depression of progeny from robbed flowers could be reduced in comparison with that from unrobbed flowers. • We suggest that nectar robbing can have both negative and positive effects on the quantity and quality, respectively, of progeny produced in selfing plants, and challenge the view that robbing has no effect on selfing species.

Pollinator individual-based networks reveal the specialized plant-pollinator mutualism in two biodiverse communities.

Generalization of pollination systems is widely accepted by ecologists in the studies of plant-pollinator interaction networks at the community level, but the degree of generalization of pollination networks remains largely unknown at the individual pollinator level. Using potential legitimate pollinators that were constantly visiting flowers in two alpine meadow communities, we analyzed the differences in the pollination network structure between the pollinator individual level and species level. The results showed that compared to the pollinator species-based networks, the linkage density, interaction diversity, interaction evenness, the average plant linkage level, and interaction diversity increased, but connectance, degree of nestedness, the average of pollinator linkage level, and interaction diversity decreased in the pollinator individual-based networks, indicating that pollinator individuals had a narrower food niche than their counterpart species. Pollination networks at the pollinator individual level were more specialized at the network level (H'2) and the plant species node level (d') than at the pollinator species-level networks, reducing the chance of underestimating levels of specialization in pollination systems. The results emphasize that research into pollinator individual-based pollination networks will improve our understanding of the pollination networks at the pollinator species level and the coevolution of flowering plants and pollinators.

Sexual conflict in protandrous flowers and the evolution of gynodioecy.

Sexual interference between male and female function in hermaphrodite plants is reduced by protandry. In environments with insufficient pollinator service, prolongation of male function owing to limited pollen removal could restrict the duration of female function and lower seed production. We provide evidence that this form of sexual conflict has played a role in the spread of females in gynodioecious populations of Cyananthus delavayi in the pollen-limited environments in which this subalpine species occurs. Using field experiments involving artificial pollen removal from the strongly protandrous flowers of hermaphrodites, we demonstrated a trade-off between male- and female-phase duration with no influence on overall floral longevity. Pollen removal at the beginning of anthesis resulted in hermaphrodite seed production matching that of females. In contrast, restricted pollen removal increased the duration of male function at the expense of female function lowering maternal fertility compared to females. This pattern was evident in five populations with females experiencing a twofold average seed fertility advantage compared to hermaphrodites. Gynodioecy often appears to evolve from protandrous ancestors and pollen limitation is widespread in flowering plants suggesting that sexual conflict may play an unappreciated role in the evolution of this form of sexual dimorphism.

Delayed selfing in an alpine biennial Gentianopsis paludosa (Gentianaceae) in the Qinghai-Tibetan plateau.

Delayed selfing could provide ovules with an opportunity to be fertilized as a means of "pollination assurance" before the flowers wilt. It could, thus, be regarded as an adaptation to unpredictable pollinator environments. Within the alpine biennial Gentianopsis paludosa, the showy flowers and herkogamy at the early stage of a flower's life cycle may favor outcrossing. As the flower ages, anthers contact the central stigma due to the elongation of all filaments, resulting in autonomous selfing. Flower visitors are extremely rare in a high altitude population; and examination of the mating system indicates that G. paludosa is self-pollinated under natural conditions in this population. While at the lower altitude, the bumblebee visitation rate is relatively high but possibly unreliable. Stigma receptivity is the highest on the third day of anthesis, and decreases thereafter. Pollen viability is the highest when flowers open, and gradually decreases later. Self-pollination of G. paludosa occurs at the late stage of a flower's lifecycle when stigma receptivity and pollen viability have both decreased, suggesting delayed selfing and assurance of seed production. This delayed selfing could assure seed production under the constraints of pollinator scarcity, but ensure outcrossing when pollinators were available. Such a flexible pollination mechanism is highly adaptive in the alpine environment of the Qinghai-Tibetan Plateau.

Development of Microsatellite Markers for a Dioecious Herpetospermum pedunculosum (Cucurbitaceae).

Understanding the evolution of flower diversity is a central topic in plant evolutionary ecology, and natural selection on floral traits via male fitness could be estimated quantitatively using microsatellites. Here, based on RNA sequencing, we developed simple sequence repeat primers and verified polymorphisms in 2 wild populations of Herpetospermum pedunculosum (Cucurbitaceae), a dioecious annual plants native to the Himalaya Mountains. A total of 131 paired primers were designed; 15 paired primers were found to be polymorphic, with the expected heterozygosity varying between 0.280 and 0.767. We also identified 58 genotypes in 20 plants from the 2 populations. Conclusively, these primers could be effective in examining male fitness and population genetic structure of H pedunculosum in future studies.

Cell number explains the intraspecific spur-length variation in an Aquilegia species.

Variations of nectar spur length allow pollinators to utilize resources in novel ways, leading to the different selective pressures on spurs and allowing taxa to diversify. However, the mechanisms underlying spur length variation remain unclear. Interspecific comparisons of spur length suggest that both cell division and anisotropic expansion could explain the changes of spur length, and that hormone-related genes contribute to the process of spur formation. In contrast, little is known about intraspecific spur length variation. In Aquilegia rockii, spur length varies strikingly, ranging from 1 mm to 18 mm. To examine the potential mechanisms underlying spur length variation in A. rockii, we observed cell morphology and analyzed RNA-seq of short- and long-spurred flowers. Scanning electron microscopy revealed that at two positions on spurs there were no differences in either cell density or cell anisotropy between short- and long-spurred flowers, suggesting that in A. rockii changes in cell number may explain variations in spur length. In addition, we screened transcriptomes of short- and long-spurred flowers for differentially expressed genes; this screen identified several genes linked to cell division (e.g., F-box, CDKB2-2, and LST8), a finding which is consistent with our analysis of the cellular morphology of spurs. However, we did not find any highly expressed genes involved in the hormone pathway in long-spurred flowers. In contrast to previous hypotheses that anisotropic cell expansion leads to interspecific spur variation in Aquilegia, our results suggest that cell number changes and related genes are mainly responsible for spur length variations of A. rockii. Furthermore, the underlying mechanisms of similar floral traits in morphology may be quite different, enriching our understanding of the mechanisms of flower diversity in angiosperms.

The coexistence of hermaphroditic and dioecious plants is associated with polyploidy and gender dimorphism in Dasiphora fruticosa.

Dasiphora fruticosa comprises male, female and hermaphrodite plants, which are distributed sympatrically in some populations on the Qinghai-Tibet Plateau. To explore what governs the coexistence of these three sexual phenotypes, we investigated the DNA contents, pollen and ovule production, pollen deposition, and performed hand-pollination in both hermaphroditic and dioecious individuals of D. fruticosa. Flow cytometry confirmed that the DNA content of males and females were almost twice as much as that of the hermaphrodites. Male and female flowers produced more pollen grains and ovules than hermaphroditic flowers. Hand-pollinated treatments showed that unisexual flowers were sterile in one sexual function and bisexual flowers were fertile for both functions, but no sterile seeds were produced between unisexual and bisexual flowers. Our findings imply that polyploidy is related to gender dimorphism, and both are likely to play a strong role in the coexistence of two cryptic biological species of D. fruticosa (low ploidy hermaphroditic species and high ploidy dioecious species) in the Qinghai-Tibet Plateau.

Equipped for Migrations Across High Latitude Regions? Reduced Spur Length and Outcrossing Rate in a Biennial Halenia elliptica (Gentianaceae) With Mixed Mating System Along a Latitude Gradient.

Halenia (Gentianaceae) originated from the mountain regions of East Asia, and diversified in America following long migrations via Beringia. While Halenia elliptica, one species of the genus in China, migrated toward high latitudes in China. Spur length of H. elliptica is highly variable. We examined the relationship between spur length and mating pattern along a latitude gradient. Field experiments were performed in two populations of H. elliptica, and we found that this species could produce seeds via both autonomous selfing and the aid of pollinators, suggesting a mixed mating system. In seven populations of H. elliptica along a latitudinal gradient, we found a trend of decrease in spur length with the increase of latitude. Based on molecular data from 11 microsatellite loci, we found that multilocus outcrossing rate decreased with the increase of latitude while the estimated inbreeding depression increased significantly, indicating that a high degree of inbreeding depression might have prevented evolution toward complete selfing in the high latitude populations with short spur length, and thus maintained mixed mating system of H. elliptica. Our results suggest that the mixed mating system of this species might be helpful in overcoming pollinator scarcity in newly colonized populations toward high latitudes after its origination in the mountain regions of China, and the decrease of spur length in the high latitude populations could result from reduced resource allocation to pollinator associated traits.

Differentiation in drought tolerance mirrors the geographic distributions of alpine plants on the Qinghai-Tibet Plateau and adjacent highlands.

Climatic tolerance, especially drought tolerance, is one of the major factors shaping the geographic distributions of plant species. Thus, the general decline in rainfall from the Himalaya-Hengduan Mountains (HHM) to the inner Qinghai-Tibet Plateau (QTP) might account for the significant differences in species distributions and richness between the two regions. To test this hypothesis, we conducted a water stress experiment using four Anisodus species (A. tanguticus, A. luridus, A. carniolicoides and A. acutangulus), which were treated with different levels of water stress in a glasshouse, and examined their differences in physiological responses. The results suggest that A. tanguticus, which inhabits the inner QTP, generally has higher fitness under severe water stress than the other species based on its high root:shoot ratio, long-term water use efficiency and photosynthetic rate, indicating that it possesses a genetically based drought tolerance mechanism. Our results suggest that plant species inhabiting the inner QTP may be more drought tolerant than those inhabiting the HHM regions. This provides a new example supporting the hypothesis that climatic tolerance plays a major role in shaping plant distributions on the QTP and its adjacent highlands and presents new insights into the patterns of geographic distribution and diversity of the plants inhabiting these areas.

Evaluation of pollinator effectiveness based on pollen deposition and seed production in a gynodieocious alpine plant, Cyananthus delavayi.

Examining variations in pollinator effectiveness can enhance our understanding of how pollinators and plants interact. Pollen deposition and seed production after a single visit by a pollinator are often used to estimate pollinator effectiveness. However, seed production is not always directly related to pollen deposition because not all pollen grains that are deposited on a stigma are compatible or conspecific. In the field, we tested pollinator effectiveness based on pollen deposition and the resulting seed production after single visits by different pollinator groups in a gynodieocious alpine plant Cyananthus delavayi (Campanulaceae). Our results showed that mean pollen deposition was generally inconsistent with mean seed production when comparisons were performed among different pollinator groups and sexes. In general, the correlations were not significant between pollen deposition and seed production in both perfect and female flowers after single visits by halictid bees, bumble bees, and hoverflies. We suggest seed set of virgin flowers after single visits is a more reliable indicator of pollinator effectiveness than pollen deposition and would be a better indicator of pollinator effectiveness for future studies.

Intensified wind pollination mediated by pollen dimorphism after range expansion in an ambophilous biennial Aconitum gymnandrum.

Pollination systems and associated floral traits generally differ between core and marginal populations of a species. However, such differences are rarely examined in plants with a mixed wind- and bumblebee-pollination system, and the role of wind pollination during range expansion in ambophilous plants remains unclear. We compared floral traits and the contributions of bumblebee and wind pollination in refugium and marginal populations of the ambophilous plant Aconitum gymnandrum. We found that most floral traits differed between the two populations, and those traits associated with the shift to wind pollination were pronounced in the marginal population. Bumblebee visitation rates varied significantly, but were generally low in the marginal population. Wind pollination occurred in both populations, and the efficiency was lower than that of bumblebee pollination. Two types of pollen grains, namely round and fusiform pollen, were transported to a stigma by bumblebees and wind, but fusiform pollen contributed to wind pollination to a larger degree, especially in the marginal population. Our results suggest that wind pollination was enhanced by pollen dimorphism in the marginal population of A. gymnandrum, and wind pollination may provide reproductive assurance when bumblebee activity is unpredictable during range expansion, indicating that ambophily is stable in this species and shift in pollination system could be common when plants colonize new habitats.

Divergence in Eco-Physiological Responses to Drought Mirrors the Distinct Distribution of Chamerion angustifolium Cytotypes in the Himalaya-Hengduan Mountains Region.

Polyploid species generally occupy harsher habitats (characterized by cold, drought and/or high altitude) than diploids, but the converse was observed for Chamerion angustifolium, in which diploid plants generally inhabit higher altitudes than their polyploid derivatives. Plants at high altitudes may experience cold-induced water stress, and we therefore examined the physiological responses of diploid and hexaploid C. angustifolium to water stress to better understand the ecological differentiation of plants with different ploidy levels. We conducted a common garden experiment by subjecting seedlings of different ploidy levels to low, moderate, and severe water stress. Fourteen indicators of physiological fitness were measured, and the anatomical characteristics of the leaves of each cytotype were determined. Both cytotypes were influenced by drought, and diploids exhibited higher fitness in terms of constant root:shoot ratio (R:S ratio) and maximum quantum yield of PS II (Fv/Fm ), less reduced maximal photosynthetic rate (A max), transpiration rate (E), intercellular CO2 concentration (C i) and stomatal conductance (g s), and higher long-term water use efficiency (WUEL) under severe water stress than did hexaploids. Analysis of leaf anatomy revealed morphological adjustments for tolerating water deficiency in diploids, in the form of closely packed mesophyll cells and small conduits in the midvein. Our results indicate that diploid C. angustifolium is more tolerant of drought than hexaploid plants, ensuring the successful survival of the diploid at high altitudes. This eco-physiological divergence may facilitate the species with different cytotypes to colonize new and large geographic ranges with heterogeneous environmental conditions.

Changes of flowering phenology and flower size in rosaceous plants from a biodiversity hotspot in the past century.

Responses of plant traits to climate changes are complex, which could be mirrored by the investigations of herbarium specimens. By examining specimens of Rosa and Cotoneaster species collected since 1920s in Hengduan Mountains, we analyzed the changes of flowering phenology and flower size in the past century when climate changes were considered to be intensified. We found that flowering phenology of Rosa showed no significant change, but flowering phenology of Cotoneaster was delayed in recent years. Flower size of Rosa species showed a marginally significant decrease over the past century. The results suggested that responses of flowering time to global changes and pollinator mediated selection on floral traits might be more complex than what were expected. Our results indicated that future researches based on investigations of herbarium specimens should be carried out on multiple plant species with different flower structures and life histories to better understand the effects of climate changes on plant traits.