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Widely documented in animals, behavioural thermoregulation mitigates negative impacts of climate change. Plants experience especially strong thermal variability but evidence for plant behavioural thermoregulation is limited. Along a montane elevation gradient, Argentina anserina flowers warm more in alpine populations than at lower elevation. We linked floral temperature with phenotypes to identify warming mechanisms and documented petal movement and pollinator visitation using time-lapse cameras. High elevation flowers were more cupped, focused light deeper within flowers and were more responsive to air temperature than low; cupping when cold and flattening when warm. At high elevation, a 20° increase in petal angle resulted in a 0.46°C increase in warming. Warming increased pollinator visitation, especially under cooler high elevation temperatures. A plasticity study revealed constitutive elevational differences in petal cupping and stronger temperature-induced floral plasticity in high elevation populations. Thus, plant populations have evolved different behavioural responses to temperature driving differences in thermoregulatory capacity.

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PREMISE: Agriculture expansion, livestock, and global change have transformed biological communities and altered, through aerosols and direct deposition, N:P balance in soils of inter-Andean valleys, potentially affecting flowering phenology of many species and thereby flowering synchrony and plant reproduction. METHODS: We evaluated the influence of variation in temperature and moisture along the local elevational gradient and treatments with the addition of N and P and grazing on flowering synchrony and reproduction of Croton, a dominant shrub of the inter-Andean dry scrub. Along the elevational gradient (300 m difference between the lowest and highest site), we set up plots with and without grazing nested with four nutrient treatments: control and addition of N or P alone or combined N + P. We recorded the number of female and male flowers in bloom monthly from September 2017 to August 2019 to calculate flowering synchrony. We assessed fruiting, seed mass, and pre-dispersal seed predation. RESULTS: Higher growing-season soil temperatures, which were negatively associated with local elevation and higher nitrogen availability promoted flowering synchrony of Croton, particularly among larger plants. Greater flowering synchrony, high soil temperatures, and addition of N + P resulted in production of more fruits of Croton, but also intensified pre-dispersal seed predation. CONCLUSIONS: Temperature, availability of moisture throughout the elevational gradient, and nutrient manipulation affected flowering synchrony, which subsequently affected production of fruits in Croton. These results emphasize the critical role of current anthropogenic changes in climate and nutrient availability on flowering synchrony and reproduction of Croton, a dominant plant of the inter-Andean scrub.

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Despite the abundant evidence of impairments to plant performance and survival under hotter-drought conditions, little is known about the vulnerability of reproductive organs to climate extremes. Here, by conducting a comparative analysis between flowers and leaves, we investigated how variations in key morphophysiological traits related to carbon and water economics can explain the differential vulnerabilities to heat and drought among these functionally diverse organs. Due to their lower construction costs, despite having a higher water storage capacity, flowers were more prone to turgor loss (higher turgor loss point; ΨTLP) than leaves, thus evidencing a trade-off between carbon investment and drought tolerance in reproductive organs. Importantly, the higher ΨTLP of flowers also resulted in narrow turgor safety margins (TSM). Moreover, compared to leaves, the cuticle of flowers had an overall higher thermal vulnerability, which also resulted in low leakage safety margins (LSM). As a result, the combination of low TSMs and LSMs may have negative impacts on reproduction success since they strongly influenced the time to turgor loss under simulated hotter-drought conditions. Overall, our results improve the knowledge of unexplored aspects of flower structure and function and highlight likely threats to successful plant reproduction in a warmer and drier world.

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Introduction: In Eragrostis curvula, commonly known as weeping lovegrass, a synthetic diploidization event of the facultative apomictic tetraploid Tanganyika INTA cv. originated from the sexual diploid Victoria cv. Apomixis is an asexual reproduction by seeds in which the progeny is genetically identical to the maternal plant. Methods: To assess the genomic changes related to ploidy and to the reproductive mode occurring during diploidization, a mapping approach was followed to obtain the first E. curvula pangenome assembly. In this way, gDNA of Tanganyika INTA was extracted and sequenced in 2x250 Illumina pair-end reads and mapped against the Victoria genome assembly. The unmapped reads were used for variant calling, while the mapped reads were assembled using Masurca software. Results: The length of the assembly was 28,982,419 bp distributed in 18,032 contigs, and the variable genes annotated in these contigs rendered 3,952 gene models. Functional annotation of the genes showed that the reproductive pathway was differentially enriched. PCR amplification in gDNA and cDNA of Tanganyika INTA and Victoria was conducted to validate the presence/absence variation in five genes related to reproduction and ploidy. The polyploid nature of the Tanganyika INTA genome was also evaluated through the variant calling analysis showing the single nucleotide polymorphism (SNP) coverage and allele frequency distribution with a segmental allotetraploid pairing behavior. Discussion: The results presented here suggest that the genes were lost in Tanganyika INTA during the diploidization process that was conducted to suppress the apomictic pathway, affecting severely the fertility of Victoria cv.

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Brazil is one of the centers of diversity of Cactaceae, yet studies addressing both pollination biology and the breeding system in Brazilian cacti are scarce. We herein present a detailed analysis of two native species with economic relevance: Cereus hildmannianus and Pereskia aculeata. The first species produce edible, sweet, spineless fruits and the second species produces leaves with high protein content. Pollination studies were undertaken through fieldwork observations in three localities of Rio Grande do Sul, Brazil, over two flowering seasons, totaling over 130 observation hours. Breeding systems were elucidated utilizing controlled pollinations. Cereus hildmannianus is solely pollinated by nectar-gathering species of Sphingidae hawk moths. In contrast, the flowers of P. aculeata are pollinated by predominantly native Hymenoptera but also by Coleoptera and Diptera, which gather pollen and/or nectar. Both cacti species are pollinator-dependent; neither intact nor emasculated flowers turn into fruit, yet whereas C. hildmannianus is self-incompatible, P. aculeata is fully self-compatible. In sum, C. hildmannianus is more restrictive and specialized regarding its pollination and breeding system, whereas P. aculeata is more generalist. Understanding the pollination needs of these species is a necessary starting point towards their conservation but also for their proper management and eventual domestication.

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Successful plant reproduction and fruit formation depend on adequate pollen and pistil development, and pollen-pistil interactions. In Nicotiana tabacum, pollen tubes grow through the intercellular spaces of pistil-specialized tissues, stigmatic secretory zone, and stylar transmitting tissue (STT). These intercellular spaces are supposed to be formed by the modulation of cell wall pectin esterification. Previously we have identified a gene preferentially expressed in pistils encoding a putative pectin acetylesterase (PAE), named NtPAE1. Here, we characterized the NtPAE1 gene and performed genome-wide and phylogenetic analyses of PAEs. We identified 30 PAE sequences in the N. tabacum genome, distributed in four clades. The expression of NtPAE1 was assessed by RT-qPCR and in situ hybridization. We confirmed NtPAE1 preferential expression in stigmas/styles and ovaries and demonstrated its high expression in the STT. Structural predictions and comparisons between NtPAE1 and functional enzymes validated its identity as a PAE. Transgenic plants were produced, overexpressing and silencing the NtPAE1 gene. Overexpressed plants displayed smaller flowers while silencing plants exhibited collapsed pollen grains, which hardly germinate. NtPAE1 silencing plants do not produce fruits, due to impaired pollen tube growth in their STTs. Thus, NtPAE1 is an essential enzyme regulating pectin modifications in flowers and, ultimately, in plant reproduction.

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The flowers of the species of Malpighiaceae in the Neotropical Region are relatively uniform in their morphology due to their dependence on oil-collecting bees as their main pollinators. However, many species of the genus Galphimia seem to have acquired a different floral syndrome, lacking markedly zygomorphic flowers and developed elaiophores in the calyx. Likewise, these species present anthers with great development, probably in response to the selection of pollinators that collect pollen. Galphimia australis incorporated some of these traits but also retained some residual characteristics typical of species pollinated by oil bees. This leads to many questions on how these flowers ensure their pollination. Inquiring about the reduction or modification of these characteristics allows us to understand how G. australis achieves a different pollination syndrome. In this research, we carry out a detailed morphological and anatomical study of the flowers and pollen grain devolvement of G. australis and floral visitors were observed and captured. Results were analyzed in order to determine how this species changed from the oil-floral syndrome, typical of neotropical Malpighiaceae, to one syndrome with pollen as the main reward.

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Floral traits mediate the roles of distinct animals as effective pollinators along a generalization/specialization continuum. Many plant species are visited by different pollinator functional groups and the specific contribution of each group is expected to reflect the set of floral characteristics defined by pollination syndromes. Although considered a highly specialized nectarivorous group, hummingbirds frequently visit flowers lacking apparent specialization to bird pollination. How they contribute to the reproduction of these plants, however, has not been evaluated through field experiments considering multiple non-related plant species simultaneously. Here, we investigated hummingbirds' contributions to the pollination of ten plant species comprising a gradient of adaptation to bird pollination in the Brazilian rupestrian grasslands. We excluded hummingbirds from flowers and evaluated their relative contribution in comparison to insects (mainly bees) on conspecific/heterospecific pollen deposition and fruit set. Floral traits that are typically associated with bird pollination were associated with increased pollen deposition, but not with fruit set in the presence of hummingbirds. With hummingbirds, conspecific and heterospecific pollen deposition increased in most species, while fruit set increased in four plant species with varying degrees of fit to ornithophily. Our results show that assessing the relative contribution of specific pollinator groups may depend on when this contribution is measured, i.e. pollen deposition or fruit set. Considering fruit set, our results indicate that hummingbirds contributed to plant reproduction independently of the fit to bird pollination syndrome. This emphasizes their importance as under-appreciated generalized pollinators in some communities.

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Mitochondrial adrenodoxins (ADXs) are small iron-sulfur proteins with electron transfer properties. In animals, ADXs transfer electrons between an adrenodoxin reductase (ADXR) and mitochondrial P450s, which is crucial for steroidogenesis. Here we show that a plant mitochondrial steroidogenic pathway, dependent on an ADXR-ADX-P450 shuttle, is essential for female gametogenesis and early embryogenesis through a maternal effect. The steroid profile of maternal and gametophytic tissues of wild-type (WT) and adxr ovules revealed that homocastasterone is the main steroid present in WT gametophytes and that its levels are reduced in the mutant ovules. The application of exogenous homocastasterone partially rescued adxr and P450 mutant phenotypes, indicating that gametophytic homocastasterone biosynthesis is affected in the mutants and that a deficiency of this hormone causes the phenotypic alterations observed. These findings also suggest not only a remarkable similarity between steroid biosynthetic pathways in plants and animals but also a common function during sexual reproduction.

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Pollination and reproduction are important processes for understanding plant community dynamics. Information regarding pollination and reproduction is urgent for threatened ecosystems, such as tropical montane ecosystems. In tropical mountains, pollination patterns are expected to conform to the reproductive assurance theory (due to low pollinator activity) and old, climatically buffered and infertile landscapes (OCBIL) theory (due to restricted plant range size). For 82 plant species of the Itatiaia National Park (including endemic and endangered species), we evaluated at least one of the following features: pollinator identity, flower color and size, flowering phenology, and pollinator dependence. Most plant species (ca. 60%) were pollinated by two or more functional groups of pollinators (generalized pollination), with high importance of flies as pollinators. There was low pollinator activity overall (less than one visit per flower per hour). Notably, the invasive honeybee Apis mellifera L. performed half of the visits to this entire plant community, suggesting an impact on the native pollinator fauna and consequently on the native flora. Most endemic plants were generalized with white and small flowers, while endangered species were pollination-specialized with colorful and large flowers. Thus, endangered species are susceptible to changes in pollinator fauna. Flowering seasonality reflected the importance of climatic constraints in this environment. One-third of the plant species were autogamous. Our data suggest that pollinator scarcity may have promoted reproductive assurance strategies such as generalization and pollinator independence. Our community-level study highlighted consistent pollination patterns for tropical mountains and emphasized threats for specialized endangered species.

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Nutrient addition experiments indicate that nitrogen and phosphorus limit plant processes in many tropical forests. However, the long-term consequences for forest structure and species composition remain unexplored. We are positioned to evaluate potential long-term consequences of nutrient addition in central Panama where we have maintained a factorial nitrogen-phosphorus-potassium fertilization experiment for 21 yr and an independent study quantified the species-specific nutrient requirements of 550 local tree species. Here, we ask whether nutrients limit reproduction at the species and community levels. We also ask whether species-specific reproductive responses to nutrient addition are stronger among species associated with naturally fertile soils, which could contribute to a shift in species composition. We quantified species-level reproductive responses for 38 focal species in the 21st year of the experiment and community-level reproductive litter production for the first 20 yr. Species-level reproductive responses to nitrogen and potassium addition were weak, inconsistent across species, and insignificant across the 38 focal species. In contrast, species-level responses to phosphorus addition were consistently and significantly positive across the 38 focal species but were unrelated to species-specific phosphorus requirements documented independently for the same species. Community-level reproductive litter production was unaffected by nutrient addition, possibly because spatial and temporal variation is large. We conclude that phosphorus limits reproduction by trees in our experiment but find no evidence that reproductive responses to phosphorus addition favor species associated with naturally phosphorus-rich soils.

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Domestication is an evolutionary process with an impact on plant reproduction. Many domesticated plants are self-compatible (i.e., they lack mechanisms to reject their own pollen), but few domesticated plants are fully or partially self-incompatible. We used the husk tomato, Physalis philadelphica, as a study model to investigate changes in the reproductive strategy of an annual partially self-incompatible plant during the process of domestication. Wild and cultivated populations of this species coexist in close proximity. These different populations present a high level of morphological and genetic variation associated with different degrees of domestication. We hypothesized that artificial selection favors self-compatibility in cultivated plants through changes in their reproductive strategy and some reproductive parameters associated with domestication. To test this hypothesis, we characterized the floral morphology and some reproductive parameters of weedy plants (wild plants), landraces (semi-domesticated plant), and commercial plants (domesticated plants). We conducted an artificial crossing experiment, germinated the seeds, and recorded seedling growth. Commercial plants had the largest flowers and the highest number of ovules. Yet, they did not differ in other reproductive parameters (e.g., herkogamy, size of pollen grains, stigmatic area, and pollen:ovule ratio) from landraces and weedy plants. Physalis philadelphica produced fruits by autonomous autogamy in the artificial crossing experiment. These fruits were the smallest and lightest fruits at all degrees of domestication; however, fruit set of autonomous autogamy was higher in weedy plants. In addition, fruit production was higher when weedy plants donated pollen to commercial plants. Although seeds produced by autonomous autogamy of weedy plants had a low germination percentage, their cotyledons and the embryonic foliage leaves appeared earlier than in landraces and commercial plants. In conclusion, the domestication syndrome in this plant was manifested as increments in flower size and ovule production. Contrary to expectations, there was higher fruit production by autonomous autogamy in weedy plants than in cultivated plants. It seems that artificial selection in P. philadelphica favors self-incompatibility in cultivated plants. Nonetheless, spontaneous self-pollination seems to be advantageous in weedy populations because they produced viable seeds from which cotyledons and the embryonic foliage leaves emerged earlier than in cultivated plants.

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BACKGROUND: Apomixis, an asexual mode of plant reproduction, is a genetically heritable trait evolutionarily related to sexuality, which enables the fixation of heterozygous genetic combinations through the development of maternal seeds. Recently, reference floral transcriptomes were generated from sexual and apomictic biotypes of Paspalum notatum, one of the most well-known plant models for the study of apomixis. However, the transcriptome dynamics, the occurrence of apomixis vs. sexual expression heterochronicity across consecutive developmental steps and the orientation of transcription (sense/antisense) remain unexplored. RESULTS: We produced 24 Illumina TruSeq®/ Hiseq 1500 sense/antisense floral transcriptome libraries covering four developmental stages (premeiosis, meiosis, postmeiosis, and anthesis) in biological triplicates, from an obligate apomictic and a full sexual genotype. De novo assemblies with Trinity yielded 103,699 and 100,114 transcripts for the apomictic and sexual samples respectively. A global comparative analysis involving reads from all developmental stages revealed 19,352 differentially expressed sense transcripts, of which 13,205 (68%) and 6147 (32%) were up- and down-regulated in apomictic samples with respect to the sexual ones. Interestingly, 100 differentially expressed antisense transcripts were detected, 55 (55%) of them up- and 45 (45%) down-regulated in apomictic libraries. A stage-by-stage comparative analysis showed a higher number of differentially expressed candidates due to heterochronicity discrimination: the highest number of differential sense transcripts was detected at premeiosis (23,651), followed by meiosis (22,830), postmeiosis (19,100), and anthesis (17,962), while the highest number of differential antisense transcripts were detected at anthesis (495), followed by postmeiosis (164), meiosis (120) and premeiosis (115). Members of the AP2, ARF, MYB and WRKY transcription factor families, as well as the auxin, jasmonate and cytokinin plant hormone families appeared broadly deregulated. Moreover, the chronological expression profile of several well-characterized apomixis controllers was examined in detail. CONCLUSIONS: This work provides a quantitative sense/antisense gene expression catalogue covering several subsequent reproductive developmental stages from premeiosis to anthesis for apomictic and sexual P. notatum, with potential to reveal heterochronic expression between reproductive types and discover sense/antisense mediated regulation. We detected a contrasting transcriptional and hormonal control in apomixis and sexuality as well as specific sense/antisense modulation occurring at the onset of parthenogenesis.

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One of the most common evolutionary transitions in angiosperms is the reproductive change from outcrossing to selfing, commonly associated with changes in floral biology and genetic diversity. Here, we aim to test whether self-compatibility leads to a reduction of floral traits and genetic diversity. For this, we experimentally estimate levels of self-compatibility, measure three floral traits and estimate four genetic diversity parameters using nine microsatellites in nine Calceolaria species. Our analysis indicated that four of the study species were self-incompatible. In addition, we found that self-compatible species did not show a reduction in floral traits size, but rather displayed larger corolla and elaiophore areas. Our analyses of genetic diversity identified larger allele number and observed heterozygosity in selfers than in outcrossers, but did not find larger inbreeding in the self-compatible species. Even though our results contradict our expectations, in the case of Calceolaria, their high dependence on only two genera of oil-bees puts the genus in a vulnerable reproductive position, probably facilitating the evolution of reproductive assurance mechanisms in the absence of pollinators. As a result, plants maintain their pollinator attraction traits while evolving the ability to self, possibly in a delayed way.

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In the past decades, the grasses of the Paspalum genus have emerged as a versatile model allowing evolutionary, genetic, molecular, and developmental studies on apomixis as well as successful breeding applications. The rise of such an archetypal system progressed through integrative phases, which were essential to draw conclusions based on solid standards. Here, we review the steps adopted in Paspalum to establish the current body of knowledge on apomixis and provide model breeding programs for other agronomically important apomictic crops. In particular, we discuss the need for previous detailed cytoembryological and cytogenetic germplasm characterization; the establishment of sexual and apomictic materials of identical ploidy level; the development of segregating populations useful for inheritance analysis, positional mapping, and epigenetic control studies; the development of omics data resources; the identification of key molecular pathways via comparative gene expression studies; the accurate molecular characterization of genomic loci governing apomixis; the in-depth functional analysis of selected candidate genes in apomictic and model species; the successful building of a sexual/apomictic combined breeding scheme.

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MAIN CONCLUSION: In Brachiaria brizantha BbrizSERK1, BbrizSERK2 and BbrizSERK3 were identified. SERK expression marks somatic embryogenesis, sexual MMC, and sexual and apomictic PMC. BbrizSERK3 might have a regulatory role in reproductive development. Somatic embryogenesis receptor-like kinase (SERK) consists of plasma membrane receptor genes that have been characterized in various species, associated with several aspects of plant development, including reproduction. SERK genes are involved in anther development and in early embryo development in sexual and asexual seed formation. To comprehend the complexity of the SERK genes and their function in Brachiaria reproduction, we performed a homology-based search in a genomic database of a sexual B. brizantha and identified sequences of three SERK genes, BbrizSERK1, BbrizSERK2, and BbrizSERK3. RNASeq data showed equivalent abundance of BbrizSERK1 and BbrizSERK2 transcripts in ovaries at early megasporogenesis of sexuals and apomicts, while BbrizSERK3 transcripts were more abundant in ovaries of sexuals than in apomicts. BbrizSERK3 results in three coding sequences due to alternative splicing, among them Variant 1 results in a protein with all the predicted domains of a SERK. BbrizSERK transcripts were detected in male reproductive tissues of both sexual and apomictic plants, suggesting a role in controlling anther development. BbrizSERK transcripts were detected early in ovule development, in the integuments, and in the megaspore mother cell of the sexual plant, but not in the cells that give rise to apomictic embryo sacs, suggesting a role in female reproductive development of sexuals. This paper provides evidences that SERK genes plays a role in the onset and establishment of somatic embryogenesis and in the reproductive development of B. brizantha and suggests a distinct role of BbrizSERK in apomixis initiation.

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Genetic diversity is a key factor for population survival and evolution. However, anthropogenic habitat disturbance can erode it, making populations more prone to extinction. Aiming to assess the global effects of habitat disturbance on plant genetic variation, we conducted a meta-analysis based on 92 case studies obtained from published literature. We compared the effects of habitat fragmentation and degradation on plant allelic richness and gene diversity (equivalent to expected heterozygosity) and tested whether such changes are sensitive to different life-forms, life spans, mating systems, and commonness. Anthropogenic disturbance had a negative effect on allelic richness, but not on gene diversity. Habitat fragmentation had a negative effect on genetic variation, whereas habitat degradation had no effect. When we examined the individual effects in fragmented habitats, allelic richness and gene diversity decreased, but this decrease was strongly dependent on certain plant traits. Specifically, common long-lived trees and self-incompatible species were more susceptible to allelic richness loss. Conversely, gene diversity decreased in common short-lived species (herbs) with self-compatible reproduction. In a wider geographical context, tropical plant communities were more sensitive to allelic richness loss, whereas temperate plant communities were more sensitive to gene diversity loss. Our synthesis showed complex responses to habitat disturbance among plant species. In many cases, the absence of effects could be the result of the time elapsed since the disturbance event or reproductive systems favoring self-pollination, but attention must be paid to those plant species that are more susceptible to losing genetic diversity, and appropriate conservation should be actions taken.

Meta-Análisis de los Efectos Diferenciales de la Fragmentación y Degradación del Hábitat sobre la Diversidad Genética de las Plantas Resumen La diversidad genética es un factor clave para la supervivencia y evolución de las poblaciones. Sin embargo, la perturbación antropogénica de los hábitats puede dañar esta diversidad, volviendo a las poblaciones más susceptibles a la extinción. Con el objetivo de evaluar los efectos globales de la perturbación del hábitat sobre la variación genética de las plantas, realizamos un meta-análisis basado en 92 estudios de caso obtenidos de la literatura publicada. Comparamos los efectos de la degradación y fragmentación del hábitat sobre la riqueza de alelos y la diversidad de genes (equivalente a la heterocigosidad esperada) de las plantas y probamos si dichos cambios son sensibles para diferentes formas de vida, tiempos de vida, sistemas de apareamiento y preponderancia. La perturbación antropogénica tuvo un efecto negativo sobre la riqueza de alelos, pero no sobre la diversidad genética. La fragmentación del hábitat tuvo un efecto negativo sobre la variación genética, mientras que la degradación del hábitat no tuvo efecto. Cuando examinamos los efectos individuales en los hábitats fragmentados, la riqueza de alelos y la diversidad genética disminuyeron, pero esta disminución estuvo vinculada fuertemente con ciertas características de las plantas. Específicamente, los árboles de larga vida y las especies auto-incompatibles fueron más susceptibles a la pérdida de la riqueza de alelos. De manera contraria, la diversidad genética disminuyó en especies comunes de vida corta (hierbas) con reproducción auto-compatibles. En un contexto geográfico más amplio, las comunidades de plantas tropicales fueron más sensibles a la pérdida de la riqueza de alelos, mientras que las comunidades de plantas de zonas templadas fueron más sensibles a la pérdida de diversidad de genes. Nuestra síntesis mostró respuestas complejas a la perturbación del hábitat entre las especies de plantas. En muchos casos, la ausencia de efectos podría ser el resultado del tiempo transcurrido desde el evento de perturbación o los sistemas reproductivos que favorecen la auto-polinización, pero se le debe prestar atención a aquellas especies de plantas que son más susceptibles a la pérdida de la diversidad genética, para así poder realizar las acciones de conservación apropiadas.

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Aposporous apomictic plants form clonal maternal seeds by inducing the emergence of non-reduced (2n) embryo sacs in the ovule nucellus and the development of embryos by parthenogenesis. In previous work, we reported a plant-specific TRIMETHYLGUANOSINE SYNTHASE 1 (TGS1) gene (PN_TGS1-like) showing expression levels positively correlated with sexuality rates in facultative apomictic Paspalum notatum. PN_ TGS1-like displayed contrasting in situ hybridization patterns in apomictic and sexual plant ovules from premeiosis to anthesis. Here we transformed sexual P. notatum with a TGS1-like antisense construction under a constitutive promoter, in order to produce lines with reduced transcript representation. Antisense plants developed prominent trichomes on the adaxial leaf surface, a trait absent from control genotypes. Reproductive development analysis revealed occasional formation of twin ovules. While control individuals typically displayed a single meiotic embryo sac per ovule, antisense lines showed 12.93-15.79% of ovules bearing extra nuclei, which can be assigned to aposporous-like embryo sacs (AES-like) or, alternatively, to gametophytes with a misguided cell fate development. Moreover, around 8.42-9.52% of ovules showed what looked like a combination of meiotic and aposporous-like sacs. Besides, 32.5% of ovules at early developmental stages displayed nucellar cells with prominent nuclei resembling apospory initials (AIs), which surrounded the megaspore mother cell (MMC) or the MMC-derived meiotic products. Two or more concurrent meiosis events were never detected, which suggest a non-reduced nature for the extra nuclei observed in the mature ovules, unless they were generated by proliferation and misguided differentiation of the legitimate meiotic products. The antisense lines produced a similar amount of viable even-sized pollen with respect to control genotypes, and formed an equivalent full seed set (∼9% of total seeds) after self-pollination. Flow cytometry analyses of caryopses derived from antisense lines revealed that all full seeds had originated from meiotic embryo sacs (i.e. by sexuality). A reduction of 25.55% in the germination percentage was detected when comparing antisense lines with controls. Our results indicate that PN_ TGS1-like influences ovule, gametophyte and possibly embryo development.

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BACKGROUND: Apomixis is considered an evolutionary deviation of the sexual reproductive pathway leading to the generation of clonal maternal progenies by seeds. Recent evidence from model and non-model species suggested that this trait could be modulated by epigenetic mechanisms involving small RNAs (sRNAs). Here we profiled floral sRNAs originated from apomictic and sexual Paspalum notatum genotypes in order to identify molecular pathways under epigenetic control that might be involved in the transition from sexuality to agamospermy. RESULTS: The mining of genes participating in sRNA-directed pathways from floral Paspalum transcriptomic resources showed these routes are functional during reproductive development, with several members differentially expressed in apomictic and sexual plants. Triplicate floral sRNA libraries derived from apomictic and a sexual genotypes were characterized by using high-throughput sequencing technology. EdgeR was apply to compare the number of sRNA reads between sexual and apomictic libraries that map over all Paspalum floral transcripts. A total of 1525 transcripts showed differential sRNA representation, including genes related to meiosis, plant hormone signaling, biomolecules transport, transcription control and cell cycle. Survey for miRNA precursors on transcriptome and genome references allowed the discovery of 124 entities, including 40 conserved and 8 novel ones. Fifty-six clusters were differentially represented in apomictic and sexual plants. All differentially expressed miRNAs were up-regulated in apomictic libraries but miR2275, which showed different family members with opposed representation. Examination of predicted miRNAs targets detected 374 potential candidates. Considering sRNA, miRNAs and target surveys together, 14 genes previously described as related with auxin metabolism, transport and signaling were detected, including AMINO ACID/AUXIN PERMEASE 15, IAA-AMIDO SYNTHETASE GH3-8, IAA30, miR160, miR167, miR164, miR319, ARF2, ARF8, ARF10, ARF12, AFB2, PROLIFERATING CELL FACTOR 6 and NITRATE TRANSPORTER 1.1. CONCLUSIONS: This work provides a comprehensive survey of the sRNA differential representation in flowers of sexual and apomictic Paspalum notatum plants. An integration of the small RNA profiling data presented here and previous transcriptomic information suggests that sRNA-mediated regulation of auxin pathways is pivotal in promoting apomixis. These results will underlie future functional characterization of the molecular components mediating the switch from sexuality to apomixis.

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Most of the world's land surface is currently under human use and natural habitats remain as fragmented samples of the original landscapes. Measuring the quality of plant progeny sired in these pervasive environments represents a fundamental endeavour for predicting the evolutionary potential of plant populations remaining in fragmented habitats and thus their ability to adapt to changing environments. By means of hierarchical and phylogenetically independent meta-analyses we reviewed habitat fragmentation effects on the genetic and biological characteristics of progenies across 179 plant species. Progeny sired in fragmented habitats showed overall genetic erosion in contrast with progeny sired in continuous habitats, with the exception of plants pollinated by vertebrates. Similarly, plant progeny in fragmented habitats showed reduced germination, survival and growth. Habitat fragmentation had stronger negative effects on the progeny vigour of outcrossing- than mixed-mating plant species, except for vertebrate-pollinated species. Finally, we observed that increased inbreeding coefficients due to fragmentation correlated negatively with progeny vigour. Our findings reveal a gloomy future for angiosperms remaining in fragmented habitats as fewer sired progeny of lower quality may decrease recruitment of plant populations, thereby increasing their probability of extinction.

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