Polyploidization is common among angiosperms and might induce typically allogamous plants to become autogamous (self-compatible, relying on sexual self-fertilization) or apomictic (achieving asexual reproduction through seeds). This work aimed to determine whether neopolyploidy leads to the breakdown of the self-incompatibility system in the hexaploid non-apomictic species Handroanthus serratifolius (Vahl) S. Grose, through analyses of its floral biology, pollination biology and breeding system. Although anthesis lasted for three days, increasing the overall floral display, receptivity decreased as of the second day. Centridini and Euglossini bees were the main pollinators, and low nectar availability (1.95 ± 1.91 μl/flower) might have obliged them to visit multiple flowers. We observed low reproductive efficacy. That might be explained by self-sterility and by the great number of flowers per individual, which could increase the frequency of geitonogamy. Ovule penetration by the pollen tubes in self-pollinated pistils with posterior abscission indicated late-acting self-incompatibility in H. serratifolius, as observed in other diploid Bignoniaceae species, although inbreeding depression cannot be excluded. The self-sterility found in the monoembryonic, hexaploid individuals studied here contrasts with the results for other neopolyploid Handroanthus and Anemopaegma species, which are often autogamous and apomictic. Our results suggest that neopolyploidy is not the main factor leading to self-fertility in Handroanthus.Key words: apomixis, late-acting self-incompatibility, monoembryony, polyploidy, Tabebuia serratifolia Acta Botanica Brasilica 27(4): 714-722. 2013. Self-sterility in the hexaploid Handroanthus serratifolius(Bignoniaceae), the national flower of Brazil

Neopolyploidy has been associated with gametophytic apomixis and breakdown of gametophytic self-incompatibility. Nevertheless, Bignoniaceae presents agamic polyploid complexes with neopolyploidy associated to sporophytic apomixis. Apomictic populations are commonly polyploid, polyembryonic and self-fertile, while diploids are mostly late-acting self-incompatible (LSI) and monoembryonic. Contrastingly, Handroanthus serratifolius shows hexaploid monoembryonic and polyembryonic populations, although breeding system has been studied only for monoembryonic individuals, which are LSI. Our aim here was to investigate breeding system and early embryology in polyembryonic individuals of H. serratifolius to define if they form an agamic polyploid complex. Experimental pollinations and histological analyzes of ovules and young seeds were carried out. Megasporogenesis and megagametogenesis occurred as in other sexual species of Bignoniaceae. The polyembryonic individuals were self-fertile and double fertilization was observed both after self and cross-pollinated pistils. Adventitious embryos originated from the hypostasis and integument of the ovule, indicating sporophytic apomixis. Adventitious embryo precursor cells occurred in all pistils, including unpollinated ones. But unpollinated pistils aborted possibly due to absence of endosperm, and pollination was required for fruit-set (pseudogamy). It is possible that the self-fertility in polyembryonic individuals ensues as the initial endosperm of self-fertilized ovules supply early adventitious embryos development, and these embryos would later prevent the abortion of selfed pistils. The sporophytic apomixis in polyembryonic populations and the occurrence of sexual monoembryonic populations of H. serratifolius allows us to consider the species part of an agamic polyploid complex. But in contrast with other Handroanthus agamic complexes, both apomictic and sexual LSI plants were hexaploid.

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Mixed cross and self-pollen load on the stigma (mixed pollination) of species with late-acting self-incompatibility system (LSI) can lead to self-fertilized seed production. This "cryptic self-fertility" may allow selfed seedling development in species otherwise largely self-sterile. Our aims were to check if mixed pollinations would lead to fruit set in LSI Adenocalymma peregrinum, and test for evidence of early-acting inbreeding depression in putative selfed seeds from mixed pollinations. Experimental pollinations were carried out in a natural population. Fruit and seed set from self-, cross and mixed pollinations were analysed. Further germination tests were carried out for the seeds obtained from treatments. Our results confirm self-incompatibility, and fruit set from cross-pollinations was three-fold that from mixed pollinations. This low fruit set in mixed pollinations is most likely due to a greater number of self- than cross-fertilized ovules, which promotes LSI action and pistil abortion. Likewise, higher percentage of empty seeds in surviving fruits from mixed pollinations compared with cross-pollinations is probably due to ovule discounting caused by self-fertilization. Moreover, germinability of seeds with developed embryos was lower in fruits from mixed than from cross-pollinations, and the non-viable seeds from mixed pollinations showed one-third of the mass of those from cross-pollinations. The great number of empty seeds, lower germinability, lower mass of non-viable seeds, and higher variation in seed mass distribution in mixed pollinations, strongly suggests early-acing inbreeding depression in putative selfed seeds. In this sense, LSI and inbreeding depression acting together probably constrain self-fertilized seedling establishment in A. peregrinum.

Long-lived flowers increase pollen transfer rates, but these entail high water and carbon maintenance costs. The retention of pollinated and reward-free old flowers enhances pollinator visitation to young receptive flowers by increasing floral display size. This mechanism is associated with acropetal inflorescences or changes in flower colour and openness, but the retention of unchanging solitary flowers remains overlooked. We examined pollination-dependent variation in floral longevity and determined stigmatic receptivity, pollen viability and pollen removal rates among flower ages in Kielmeyera regalis, a Neotropical savanna shrub. We also evaluated the effects of floral display size on pollinator visitation rates. Lastly, we determined whether old flowers are unvisited and exclusively increase pollinator attraction to young flowers through flower removal experiments. Regardless of pollination treatment, flowers lasted fully open with no detectable physical changes for 3 days. Over time, stigmas remained receptive but >95% of pollen was removed. Pollinator visitation significantly increased with floral display size and intermediate percentages (15-30%) of newly opened flowers. Accordingly, the retention of reward-free and unvisited old flowers increased young flower-pollinator interaction. Our results reveal the importance of a prolonged floral longevity in increasing pollinator attraction toward newly opened receptive flowers without changes in flower colour and form. We conclude that the retention of pollinated, reward-free and unvisited colour-unchanged old flowers in K. regalis is a strategy that counteracts the water use costs associated with the maintenance of large flowers with increased mate opportunities in a pollen-limited scenario.