Cleistogamous Research Articles

Identification of microRNAs and their target genes associated with chasmogamous and cleistogamous flower development in Viola prionantha.

Differentially expressed microRNAs were found associated with the development of chasmogamous and cleistogamous flowers in Viola prionantha, revealing potential roles of microRNAs in the developmental evolution of dimorphic flowers. In Viola prionantha, chasmogamous (CH) flowers are induced by short daylight, while cleistogamous (CL) flowers are triggered by long daylight. How environmental factors and microRNAs (miRNAs) affect dimorphic flower formation remains unknown. In this study, small RNA sequencing was performed on CH and CL floral buds at different developmental stages in V. prionantha, differentially expressed miRNAs (DEmiRNAs) were identified, and their target genes were predicted. In CL flowers, Viola prionantha miR393 (vpr-miR393a/b) and vpr-miRN3366 were highly expressed, while in CH flowers, vpr-miRN2005, vpr-miR172e-2, vpr-miR166m-3, vpr-miR396f-2, and vpr-miR482d-2 were highly expressed. In the auxin-activated signaling pathway, vpr-miR393a/b and vpr-miRN2005 could target Vpr-TIR1/AFB and Vpr-ARF2, respectively, and other DEmiRNAs could target genes involved in the regulation of transcription, e.g., Vpr-AP2-7. Moreover, Vpr-UFO and Vpr-YAB5, the main regulators in petal and stamen development, were co-expressed with Vpr-TIR1/AFB and Vpr-ARF2 and showed lower expression in CL flowers than in CH flowers. Some V. prionantha genes relating to the stress/defense responses were co-expressed with Vpr-TIR1/AFB, Vpr-ARF2, and Vpr-AP2-7 and highly expressed in CL flowers. Therefore, in V. prionantha, CH-CL flower development may be regulated by the identified DEmiRNAs and their target genes, thus providing the first insight into the formation of dimorphic flowers in Viola.

A novel type of neighbour perception elicits reproductive plasticity in an annual plant with a mixed mating system.

Plants display various forms of phenotypic plasticity in anticipation of changing conditions, many of which are influenced by information obtained from neighbouring plants. Here, we tested the hypothesis that cleistogamic Lamium amplexicaule plants can adaptively modify production of chasmogamous (CH) and cleistogamous (CL) flowers based on the perception of conspecific neighbours. The production and proportion of CH and CL flowers was examined in individual L. amplexicaule grown at varying densities or treated with root leachates from plants grown at different densities. When growing at high density or treated with root leachates from high-density pots, L. amplexicaule increased production of more expensive, potentially outcrossing CH flowers. In contrast, single plants or plants treated with root leachates from empty pots or single-source plants predominantly developed cheaper, self-pollinated CL flowers. The results demonstrate a novel root-based neighbour-perception modality that enables plants to adaptively adjust production of CH and CL flowers in response to the presence of potential reproductive partners. Further research is needed to explore the broader ecological implications of this novel interplant cueing on reproductive bet-hedging and plasticity in natural settings, as well as to identify the involved cues and their mode of operation.

Spatiotemporal variation of chasmogamy and cleistogamy in a native perennial grass: fecundity, reproductive allocation and allometry.

It is difficult to assess the relative variability or stability of chasmogamous (CH) and cleistogamous (CL) reproduction in perennial herbs with mixed mating because long-term data in natural populations are unavailable. Here, the aim was to quantify and compare spatial (between-habitat) and temporal (among-year) variation in CH and CL reproduction over 5 years in two subpopulations of the native perennial grass Danthonia compressa. This species produces CH spikelets on terminal panicles in early summer, while axillary CL spikelets, including a basal cleistogene, mature into the autumn. Flowering tillers were collected from a sunny woodland edge and an adjacent shady interior habitat for 5 consecutive years (2017-21). Seed set, fecundity, seed mass and biomass allocation were recorded for the two floral types along with tiller vegetative mass. Bivariate line fitting was used for allometric analysis of CH and CL fecundity. Seed set, fecundity, mass per seed and allocation to seeds differed between floral types and showed significant variation between habitats and among years. Seed set and fecundity in CH panicles were greater than that of axillary CL panicles in most years. Tiller mass positively affected axillary CL seed production and mass of the basal cleistogene. Fecundity and allocation among years were more variable for CH compared to CL reproduction. High seed set and fecundity of CH spikelets suggest that pollination does not limit reproduction via chasmogamy. Late maturation of axillary CL spikelets provides additional fecundity, especially in larger plants along sunny woodland edges. The heavy cleistogene at the tiller base could be important to population persistence, analogous to the axillary bud bank of other perennial grasses that are not cleistogamous. The spatiotemporal stability of CL reproduction underscores the ecological significance of cleistogamy to reproductive fitness.

Issue Information

Molecular EcologyVolume 32, Issue 6 p. i-ii ISSUE INFORMATIONFree Access Issue Information First published: 07 March 2023 https://doi.org/10.1111/mec.16512AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract Cover Illustrations: (Left to Right): (Image 1): The Blue-faced Honeyeater (Entomyzon cyanotis) is a widespread honeyeater in Australia and exhibits classic patterns of biogeography and genetic differentiation. On p. 1248-1270, Burley et al. use short- and long-read whole genome sequence data to characterize the phylogeography of this species and report on the discovery of a neo-sex chromosome, a first for the honeyeater family (Meliphagidae); (Image 2): A golden takin dancing on the rocks; (Image 3): The hot-spring snake (Thermophis baileyi) is endemic to the Qinghai-Tibet Plateau at elevations above 3600 m; (Image 4): Ancient Daphnia pulicaria resurrected from the South Center Lake (MN, USA ) from an ephippia originally laid down circa 1418-1301 A.D.; (Image 5): Inflorescence of the mangrove species Lumitzera racemosa, a woody plant that has adapted to the extreme intertidal environments; (Image 6): Eelpout Ophthalmolycus amberensis captured in the Gerlache Strait; (Image 7): A breeding pair of Amphilophus xiloensis, one of species that make up the Midas cichlid adaptive radiation. In the picture the female (smaller) is gold colocea. The genetic basis of both troits were uncovered through long- read sequencing. The genetic cause of sex determination is reported in this issue; (Image 8): Morphology of the chasmogamous (CH) and cleistogamous (CL) flowers and petals. Scale bars, 1 cm; (Image 9): Lycaeides butterfly from the Dubois hybrid zone perched on its host plant, alfalfa; (Image 10): Male Xylocopa sonorina on a flower; (Image 11): piRNA clusters are evolving rapidly in Drosophila; (Image 12): Dotplot of the self-alignment of an array of satellite DNA; (Image 13): Haplotype blocks span through time and genomic space; (Image 14): Adults of two sympatric species of Lake Whitefish, Coregonus clupeaformis spp. Normal & Dwarf from Cliff Lake, Maine, USA. Photo Credit: (Left to Right): (Image 1): Angus Emmott; (Image 2): Naxun Zhao; (Image 3): Yu-Fan Wang; (Image 4): Dagmar Frisch; (Image 5): Cairong Zhong; (Image 6): Thomas Desvignes; (Image 7): Ad konings; (Image 8): Congcong Dong; (Image 9): Lauren K. Lucas; (Image 10): Kathy Keatley Garvey; (Image 11): Filip Wierzbicki, Robert Kofler, Sarah Signor; (Image 12): Valentina Peona; (Image 13): N/A; (Image 14): Illustration by Paul Vecsei for Louis Bernatchez. Volume32, Issue6Special Issue: ECOLOGICAL AND EVOLUTIONARY INFERENCES FROM LONG-READ SEQUENCINGMarch 2023Pages i-ii RelatedInformation

Comparative ecology of chasmogamous and cleistogamous reproduction inDanthonia compressa: effects of floral type and maternal environment

The significance of maternal and progeny environment to chasmogamy (CH) and cleistogamy (CL) in perennial plants is not well known. The perennial grass Danthonia compressa produces CH spikelets capable of outcrossing on terminal panicles in summer followed by selfing axillary CL spikelets in autumn. This study compared CH and CL seed mass, germination, seedling size, tillering, and reproduction in a sunny woodland edge and shaded interior habitat in the Catskill Mountains of New York State. CL seeds were significantly heavier than CH seeds. Numbers and mass of both seed types were greater in the edge. CL seeds showed much greater germination than CH seeds in both environments during spring, 2018; CL seedlings were larger than CH seedlings in the sunnier habitat. Survival was much greater in the edge than interior and higher for CL seedlings. After 27 months, maternal effects were mostly not detected and there was no significant effect of plant type (CH vs CL) on tiller production, final biomass, or reproduction via CH and CL. For this native grass, non-dispersed CL seeds may be more critical to seedling establishment and population persistence due to greater mass and germinability, and better plant survival, relative to smaller, dispersible CH seeds.

The potential for floral evolution in response to competing selection pressures following the loss of hawkmoth pollination in Ruellia humilis.

In the absence of hawkmoth pollinators, chasmogamous (CH) flowers of Ruellia humilis self-pollinate by two secondary mechanisms. Other floral visitors might exert selection on CH floral traits to restore outcrossing, but at the same time preferential predation of CH seeds generates selection to increase the allocation of resources to cleistogamous (CL) flowers. To assess the potential for an evolutionary response to these competing selection pressures, we estimated additive genetic variances ( ) and covariances for 14 reproductive traits and three fitness components in a Missouri population lacking hawkmoth pollinators. We found significant for all 11 floral traits and two measures of resource allocation to CL flowers, indicating the potential for a short-term response to selection on most reproductive traits. Selection generated by seed predators is predicted to increase the percentage of CL flowers by 0.24% per generation, and mean stigma-anther separation is predicted to decrease as a correlated response, increasing the fraction of plants that engage in prior selfing. However, the initial response to this selection is opposed by strong directional dominance. The predicted evolutionary decrease in the number of CH flowers available for potential outcrossing, combined with the apparent preclusion of potential diurnal pollinators by the pollen-harvesting activities of sweat bees, suggest that 100% cleistogamy is the likely outcome of evolution in the absence of hawkmoths. However, rare mutations with large effects, such as delaying budbreak until after sunrise, could provide pathways for the restoration of outcrossing that are not reachable by gradual quantitative-genetic evolution.

Climate change is associated with increased allocation to potential outcrossing in a common mixed mating species.

PremiseAlthough the balance between cross‐ and self‐fertilization is driven by the environment, no long‐term study has documented whether anthropogenic climate change is affecting reproductive strategy allocation in species with mixed mating systems. Here, we test whether the common blue violet (Viola sororia; Violaceae) has altered relative allocation to the production of potentially outcrossing flowers as the climate has changed throughout the 20th century.MethodsUsing herbarium records spanning from 1875 to 2015 from the central United States, we quantified production of obligately selfing cleistogamous (CL) flowers and potentially outcrossing chasmogamous (CH) flowers by V. sororia, coupled these records with historic temperature and precipitation data, and tested whether changes to the proportion of CL flowers correlate with temporal climate trends.ResultsWe find that V. sororia progressively produced lower proportions of CL flowers across the past century and in environments with lower mean annual temperature and higher total annual precipitation. We also find that both CL and CH flower phenology has advanced across this time period.ConclusionsOur results suggest that V. sororia has responded to lower temperatures and greater water availability by shifting reproductive strategy allocation away from selfing and toward potential outcrossing. This provides the first long‐term study of how climate change may affect relative allocation to potential outcrossing in species with mixed mating systems. By revealing that CL flowering is associated with low water availability and high temperature, our results suggest the production of obligately selfing flowers is favored in water limited environments.

Cleistogamous spike and chasmogamous spike carbon remobilization improve the seed potential yield of Cleistogenes songorica under water stress

AbstractDevelopmental signals and environmental stresses regulate carbon distribution in the vegetative and reproductive organs of plants and affect seed yield. Cleistogenes songorica is a xerophytic grass with great potential application value in ecological restoration. However, how carbohydrate transport and distribution during grain filling affect the seed yield of C. songorica under water stress is not clear. The present study showed that the soluble sugar and starch contents of cleistogamous (CL) spikes and chasmogamous (CH) spikes were significantly higher at the milk stage, which was attributed to a significantly higher seed number and seed yield per spike under water stress conditions than under well-watered conditions (P < 0.01). RNA-seq data revealed a total of 54,525 differentially expressed genes (DEGs) under water stress conditions, but only 3744 DEGs were shared among all comparison groups. Weighted gene co-expression network analysis showed that the transport and distribution of carbohydrates were regulated by ABA-responsive genes (CsABA8OX1_1, CsABA8OX1_2, CsABA8OX2_1, CsABA8OX2_2, CsNCED3, CsNCED1_1, CsNCED1_2 and CsNCED4_1) and sugar transport and starch synthesis genes (CsSUS1, CsSUS2, CsSUS3, CsAGP1, CsAGP4, CsAGP5, CsSSS1 and CsSBE5) under water stress conditions. These genes jointly regulated carbohydrate remobilization in sources (stems, leaves and sheaths) to promote grain filling and improve seed yield. The present study helped to clarify the phenotypic, metabolic and transcriptional response mechanisms of vegetative organs, such as stems and leaves, and reproductive organs, such as CL spikes and CH spikes, to promote carbohydrate redistribution under water stress, and it provides theoretical guidance for improving seed yields.

Selective forces on the maintenance of outcrossing in an almost exclusively cleistogamous violet species.

Cleistogamous species constitute interesting study systems to resolve the longstanding question of how outcrossing is maintained given that seed production is ensured through selfing. In this work, we investigate the selective forces that allow the persistence of producing self-pollinated cleistogamous (CL) and chasmogamous (CH) flowers in Viola jaubertiana Marès & Vigin. We monitored three populations at different elevation for two years, and studied the flowering phenology and the relative contribution of each flower morph to parental fitness. We tested whether allocation to CH and CL flowers differed across populations and if it covaried with herbivory and water stress conditions. We also performed hand-pollination and bagging experiments in CH flowers to estimate inbreeding depression and heterosis. The CH flowers open in winter under unfavorable conditions for pollination, show high pollen limitation and no-delayed selfing, and thus produce a low amount of seeds. Conversely, CL flowers appear in early spring, are physiologically cheaper to produce (i.e., dry weight is 3.4 times lower than that of CH flowers), and yield approximately 100 times more seeds than CH flowers. The CH flowers were favored under water stress and low herbivory. Crosses between populations showed up to 25% greater fitness than those within populations. Despite the great pollen limitation in CH flowers, we suggest that the interaction among different environmental determinants and heterosis are probably sufficient forces to maintain chasmogamy in this long-lived species, reducing deleterious fixed mutations in the selfed lines.

Seasonal and Simultaneous Cleistogamy in Rostrate Violets (Viola, subsect. Rostratae, Violaceae).

The special mixed reproductive system, i.e., the ability of an individual plant to develop both open, chasmogamous (CH) flowers adapted to cross-pollination and closed, cleistogamous (CL) flowers with obligate self-pollinating, is a common phenomenon in Viola L. In most sections of Northern Hemisphere violets, cleistogamy is seasonal, and CH and CL flowers develop sequentially in the season. Non-seasonal cleistogamy (simultaneous) is a rare phenomenon in rostrate violets. In the current study, we focused on modification of the CH/CL mating system in V. caspia by environmental conditions, resulting in a gradual switch from temporal cleistogamy, occurring in nature, to simultaneous cleistogamy under greenhouse conditions. V. reichenbachiana with seasonal cleistogamy was the control for V. caspia with the labile seasonal/simultaneous cleistogamy system. In simultaneous cleistogamy, the CH and CL flowers, fruits and seeds developed on an individual plant at the same time on the same branch. The typical difference between CH and CL flowers’ pistils is a straight style ending with a head-like stigma in CH and a curved style in CL adapted to self-pollination. This trait persists in the fruit and seed stages, allowing for easy recognition of fruit of CL and CH flowers in simultaneous cleistogamy. Floral meristems of CH flowers of V. reichenbachiana developed on the rhizome at the end of the growing season under short-day conditions and remained dormant until the following season. The CL floral meristems formed under long-day conditions on elongating lateral branches in the upper leaf axils. The daily temperature influenced the variable CH/CL ratio of V. caspia in nature and greenhouse conditions. Regulation of the CL/CH flower ratio by modifying environmental factors is important for basic research on genetic/epigenetic regulation of cleistogamy and for practical use to produce genetically stable lines of economically important species via CL seeds.

DOES CLEISTOGAMOUS SELF-FERTILIZATION PROVIDE REPRODUCTIVE ASSURANCE AGAINST SEED PREDATION IN RUELLIA HUMILIS?

Mixed mating is thought to be adaptive in many species of flowering plants because self-fertilization provides reproductive assurance when pollinator densities are low. In species with dimorphic flowers, automatic self-fertilization by small cleistogamous (CL) flowers might also provide reproductive assurance by avoiding floral herbivory. We tested this hypothesis by quantifying flower production, seed production, and seed predation across an entire reproductive season within a Missouri population of Ruellia humilis, a species with mixed mating enforced by dimorphic flowers. The production of CL flowers was delayed relative to the production of open-pollinated chasmogamous (CH) flowers, consistent with the hypothesis of reproductive assurance. The rate of seed predation by larvae of the Noctuid moth Tripudia rectangula was much higher for fruits produced by CH flowers (69%) than by CL flowers (18%). Thus, although CH flowers self-pollinate in the absence of outcross pollen, CL flowers are a much more economical source of selfed seeds, not only because they require fewer resources but also because they experience much lower levels of seed predation. Seed predation by Tripudia is common in many other cleistogamous species of Ruellia native to the southern United States and Mexico and might have played a significant role in the evolution of CL flowers in this large genus.

Gibberellins are required for dimorphic flower development in Viola philippica

Photoperiod is a major determinant of chasmogamous (CH)–cleistogamous (CL) dimorphic flower development in Viola philippica, and only long-day (LD) conditions induce CL flowers. In this study, it was found that the active gibberellin (GA) content in CL floral buds was higher than in CH floral buds formed under short-day (SD) conditions, suggesting that the biosynthesis of active GAs is enhanced by a longer photoperiod and may be associated with dimorphic flower development. Thus, the next step was to molecularly characterize the key V. philippica GA synthesis genes GA 20-oxidase (VpGA20ox) and GA 3-oxidase (VpGA3ox). In terms of the expression of VpGA20ox and VpGA3ox, it was found that the active GAs could be upregulated in developing pistils under both LD and SD conditions to develop functional pistils, and GAs could also accumulate in the stamens under SD conditions. The anthers and the adjacent petals were well developed under SD conditions. In contrast, the above-mentioned floral organs displayed low GA contents under LD conditions and were poorly developed. Although the application of paclobutrazol, an inhibitor of GA synthesis, did not reverse CL development under LD conditions, exogenous GAs could partially trigger the transition from CH to CL flowers under relative SD conditions (≤12 h daylight). This was coupled with the downregulation of B-class MADS-box genes, thereby restraining stamen and petal development. Both VpGA20ox and VpGA3ox exhibited similar expression profiles with B-class MADS-box genes in the development of the stamens and petals. Therefore, in response to photoperiod, GA signaling could affect the expression of B-class homeotic genes and regulate dimorphic flower development in Viola. As a compensation for poorly-developed nectaries, anthers, and petals, filament elongation, style shortness, and inward bending could ensure self-pollination in CL flowers. This work provides new insights into the regulation of CH–CL floral development and the evolutionary significance of the formation of dimorphic flowers.

Pollination of the Orchid Habenaria rhodocheila by the Swallowtail Butterfly Papilio helenus in Subtropical Evergreen Broad-Leaved Forests in Southern China

Habenaria rhodocheila is different from other species in the genus as it has bright yellow-orange, orange-red or red floral colors and grows in subtropical evergreen broad-leaved forests. It is listed as a level II protected plant in China. This study focused on details of the pollination mechanism, including floral structure, the pollinator and its behavior, the pollination process, and fruit set in its natural environment compared to controlled artificial pollination. Our results showed that the flowers of H. rhodocheila are self-compatible, but rely on a pollinating agent due to the lack of an automatic self-pollination mechanism. At three sites the flowers of H. rhodocheila were frequently pollinated by a species of the swallowtail butterfly Papilio helenus L. During pollination, the butterfly inserts its proboscis into the spur aperture. The base of the proboscis then contacts the viscidia and the pollinarium sticks to the insect, followed by contact between pollinia and stigmas. The length of the proboscis of P. helenus is comparable to, but slightly shorter than the length of the floral spur. The pollination activity generally occurs between 9:30 AM to 1:30 PM daily and is especially frequent between 10:30 AM and 11:30 AM. These results suggest that the similarity between the length of the butterfly's proboscis and the length of the orchid's spur is a major factor contributing to the efficient pollination of H. rhodocheila in its natural habitat. Most species in Habenaria are moth-pollinated, whereas our research has shown that H. rhodocheila in subtropical evergreen broad-leaved forests in China is specifically adapted to butterfly pollination by P. helenus.

Genome-Wide Analysis of the Role of NAC Family in Flower Development and Abiotic Stress Responses in Cleistogenes songorica.

Plant-specific NAC (NAM, ATAF, CUC) transcription factor (TF) family plays important roles in biological processes such as plant growth and response to stress. Nevertheless, no information is known about NAC TFs in Cleistogenes songorica, a prominent xerophyte desert grass in northwestern China. In this study, 162 NAC genes were found from the Cleistogenes songorica genome, among which 156 C. songorica NAC (CsNAC) genes (96.3%) were mapped onto 20 chromosomes. The phylogenetic tree constructed by CsNAC and rice NAC TFs can be separated into 14 subfamilies. Syntenic and Ka/Ks analyses showed that CsNACs were primarily expanded by genomewide replication events, and purifying selection was the primary force driving the evolution of CsNAC family genes. The CsNAC gene expression profiles showed that 36 CsNAC genes showed differential expression between cleistogamous (CL) and chasmogamous (CH) flowers. One hundred and two CsNAC genes showed differential expression under heat, cold, drought, salt and ABA treatment. Twenty-three CsNAC genes were commonly differentially expressed both under stress responses and during dimorphic floret development. Gene Ontology (GO) annotation, coexpression network and qRT-PCR tests revealed that these CsNAC genes may simultaneously regulate dimorphic floret development and the response to stress. Our results may help to characterize the NAC transcription factors in C. songorica and provide new insights into the functional research and application of the NAC family in crop improvement, especially in dimorphic floret plants.

Analysis of Six Transcription Factor Families Explores Transcript Divergence of Cleistogamous and Chasmogamous Flowers in Cleistogenes songorica.

Cleistogenes songorica is a cultivated turfgrass species that employs a mixed breeding system. To determine the morphological differences and molecular mechanisms of the chasmogamous (CH) and cleistogamous (CL) flowers of this species, we evaluated seed traits and analyzed six transcription factor (TF) families related to floral development. The seed traits from the CH and CL flowers were significantly different. In total, 12 CsAP2, 13 CsSPL, 9 CsGRF, 21 CsMYB, 15 CsMADS, and 1 CsLFY differentially expressed genes were identified from the transcriptome of the C. songorica flowers, which was further supported by evolutionary relationships and conserved motifs. All collinear gene pairs had a Ka/Ks ratio <1. Analysis of the promoters and miRNAs of the TFs revealed that the members of the six TF families may coregulate the divergence of CH and CL flowers during evolution. Two CsAP2, 8 CsSPL, 6 CsGRF, 3 CsMYB (targeted by miR172s, miR156s, miR396a/b and miR159a/b, respectively), and 15 CsMADS genes as well as 1 CsLFY gene may be involved in the development of CH and CL flowers. This study is the first to analyze the differences between CH and CL flowers at the TF-family level, which will help in the understanding of dimorphic turfgrasses.

Comprehensive analysis of bZIP transcription factors uncovers their roles during dimorphic floret differentiation and stress response in Cleistogenes songorica

BackgroundTranscription factors act as important regulators of transcription networks. Basic leucine zipper (bZIP) transcription factors have been shown to be involved in multiple biological processes in plants. However, no information is available for the bZIP family in Cleistogenes songorica, which is an important xerophytic and allotetraploid grass in desert grasslands.ResultsIn this study, 86 CsbZIPs were identified in the allotetraploid C. songorica genome. For location analysis, CsbZIPs were distributed evenly across two subgenomes of C. songorica. Phylogenetic tree analysis among three species indicated that CsbZIPs were evolutionarily more closely related to OsbZIPs than AtbZIPs. Syntenic and phylogenetic analyses confirmed that the CsbZIPs were mainly expanded by whole-genome duplication events. Furthermore, it was determined that rice and C. songorica might have undergone purified selection during their long evolutionary history by calculating the Ks values and Ka/Ks ratios of orthologous gene pairs. By analysing the expression patterns of CsbZIPs in different tissues and under abiotic stresses, 21 CsbZIP genes were differentially expressed between chasmogamous (CH) and cleistogamous (CL) flowers, including two FLOWERING LOCUS D (FD) genes. In shoots and roots, 79.1 and 87.2% of the CsbZIP genes, respectively, displayed transcript changes under at least one stress treatment, such as heat, cold, drought and salt. Strikingly, 17 common CsbZIP genes showed differential expression under stress response and during CL flowering. Co-expression network, GO annotation and real-time quantitative reverse transcription PCR (qRT-PCR) analyses revealed a close relationship between CL flowering-associated genes and abiotic stress-related genes.ConclusionsBZIP TFs were comprehensively analysed and identified in allotetraploid C. songorica. Our results provide insights into the evolutionary history of the bZIP family in C. songorica and provide abiotic stress-responsive and CL-associated candidate CsbZIP genes for potential applications in the genetic improvement of plants.

Bees may drive the reproduction of four sympatric cacti in a vanishing coastal mediterranean-type ecosystem.

BackgroundSympatric congeneric plants might share pollinators, or each species might avoid competition by evolving specialized traits that generate partitions in pollinator assemblages. In both cases, pollen limitation (a decrease in the quality and quantity of compatible reproductive pollen) can occur, driving the plant mating system to autogamy as a mechanism of reproductive assurance. We assessed the relationships between pollinator assemblages and mating systems in a group of sympatric congeneric plants. We attempted to answer the following questions: (i) How similar are pollinator assemblages among sympatric cactus species? (ii) Which mating systems do sympatric cactus species use?MethodsWe studied sympatric Eriosyce taxa that inhabit a threatened coastal strip in a mediterranean-type ecosystem in central Chile. We performed field observations on four taxa and characterized pollinators during the years 2016 and 2017. We estimated differences in the pollinator assemblages using the Bray–Curtis index. To elucidate the mating systems, we conducted hand-pollination experiments using three treatments: manual cross-pollination, automatic self-pollination, and control (unmanipulated individuals). We tested differences in seed production for statistical significance using Kruskal–Wallis analysis.ResultsEriosyce subgibbosa showed a distinctive pollinator assemblage among the sympatric species that we studied (similarity ranged from 0% to 8%); it was visited by small bees and was the only species that was visited by the giant hummingbird Patagona gigas. Pollinator assemblages were similar between E. chilensis (year 2016 = 4 species; 2017 = 8) and E. chilensis var. albidiflora (2016 = 7; 2017 = 4); however, those of E. curvispina var. mutabilis (2016 = 7; 2017 = 6) were less similar to those of the aforementioned species. E. curvispina var. mutabilis showed the highest interannual variation in its pollinator assemblage (18% similarity). Reproduction in E. subgibbosa largely depends on pollinators, although it showed some degree of autogamy. Autonomous pollination was unfeasible in E. chilensis, which depended on flower visitors for its reproductive success. Both E. chilensis var. albidiflora and E. curvispina var. mutabilis showed some degree of autogamy.DiscussionWe observed differences in pollinator assemblages between E. subgibbosa and the remaining Eriosyce taxa, which depend on hymenopterans for pollen transfer. Pollinator assemblages showed considerable interannual variation, especially those of E. subgibbosa (ornithophilous syndrome) and E. curvispina var. mutabilis (melitophilous syndrome). Autogamous reproduction in these taxa may act as a reproductive assurance mechanism when pollinator availability is unpredictable. Our study contributes to improving our understanding of the reproductive systems of ecological interactions between threatened species in a Chilean mediterranean-type ecosystem.

Low levels of inbreeding depression and enhanced fitness in cleistogamous progeny in the annual plantTriodanis perfoliata

The maintenance of outcrossing in cleistogamous plants that produce both open, facultatively outcrossing chasmogamous (CH), and closed, obligate selfing cleistogamous (CL) flowers is puzzling because CL reproduction is thought to be more reliable and less costly. A possible explanation for the maintenance of CH flowers is the avoidance of inbreeding depression. However, inbreeding depression for cleistogamous species has rarely been quantified. In this study, we estimate levels of inbreeding depression in plants from three populations of Triodanis perfoliata (L.) Nieuwl., a dimorphic cleistogamous annual, under greenhouse conditions. Estimates of inbreeding depression at multiple life stages in all three populations were low and often not different from zero. Inbreeding depression at specific life stages varied, with two populations showing later-acting inbreeding depression, which is also found in other selfing species. In two of the study populations, selfed CL progeny outperformed selfed CH progeny, indicating a flower-type effect. The low levels of inbreeding depression and the superior fitness of CL compared with selfed CH flowers that we observed make the maintenance of CH flowers in this system surprising, and suggest that other advantages of outcrossing CH flowers are likely responsible for maintaining mixed mating in this species.

The role of phenotypic plasticity and pollination environment in the cleistogamous, mixed mating breeding system of Triodanis perfoliata.

The role of variable pollination environments in maintaining mixed mating systems is an active area of research. Dimorphic cleistogamy, in which a plant reproduces by both open, facultative outcrossing chasmogamous (CH) flowers and closed, cleistogamous (CL) flowers presents an excellent opportunity to study mixed mating. For example, plastic responses in allocation to an optimal floral type could serve as an adaptive strategy that maintains mixed mating under variable pollination environments. We tested for pollen limitation and plastic responses in allocation to different floral types under manipulated pollination conditions in the dimorphic cleistogamous, mixed mating annual, Triodanis perfoliata. Using a field population, we quantified pollen limitation, auto-fertility and plastic responses in the breeding system by measuring allocation to flower number and seed set of floral types. We found no evidence for pollen limitation for CH flowers, and CH flowers had low efficacy of autonomous selfing. Importantly, we found that T. perfoliata alters floral number following changes in pollination conditions, with pollen-supplemented plants having lower relative CH flower number than non-supplemented plants. Breeding system plasticity may allow for benefits from outcrossing through CH flowers, but also increased overall fitness through relatively cheap CL reproduction. After CH flowers receive pollen, subsequent production of CH flowers was reduced, which may be due to resource limitation. Our findings did not support a theoretical model predicting increased CH flowers with high pollination levels. These results increase our understanding of the role of pollination services and resource allocation in the maintenance of mixed mating systems, which also warrants further investigation.

Analysis of microRNA reveals cleistogamous and chasmogamous floret divergence in dimorphic plant

Cleistogenes songorica, a grass species that exhibits two spatially different type of inflorescence, chastogamy (CH), flowers localized at the top, and cleistogamy (CL) flowers embedded in leaf sheath. This study aimed at dissecting reasons underlying these distinct floral development patterns at morphological and microRNA level. Phenotyping for CH and CL was conducted and four small RNA libraries were constructed from the CH and CL flowers for high-throughput sequencing to identify the differentiated miRNAs. As results, spikelet, stigma, anther, lemma and lodicule length of CH flowers were found larger than that of CL, and so was seed setting. Also, 17 flower-related differential expression miRNAs were identified which were associated with floral organ development and morphogenesis, and the flower development. Further results showed that miR159a.1-CL3996.Contig2 pair was related to anther development, miR156a-5p-CL1954.Contig2 was linked to response to high light intensity, miR408-3p/miR408d-Unigene429 was related to pollination and Unigene429 positively regulated flower development. To our knowledge, this is the first study on differential miRNA accumulation between CH and CL flowers and our study serves as a foundation to the future elucidation of regulatory mechanisms of miRNAs in the divergent development of CL and CH flowers in a single plant.