Impaginato 39 1. Introduction Berberis microphylla G. Forst. is a Patagonian native shrub commonly named “calafate”, with a large distribution from Neuquén (37° SL) to Tierra del Fuego (54° 8´ SL) (Orsi, 1984). This species has a growing economic potential due to the production of fruits as a non-timber forest product (Tacón Clavaín, 2004). In fact, its dark blue berries are consumed fresh, as jams and preserves, and are used for the production of soft drinks and ice cream. Moreover, the fruits have a high content of carbohydrates, phe- nols and antioxidants (Arena and Curvetto, 2008; Arena et al., 2011, 2012, 2013 b). The genetic and morphological analysis of spontaneous accessions in natural populations of B. microphylla grown on Tierra del Fuego (Giordani et al., 2016), as well as the changes in form and leaf anatomy due to weather conditions (Radice and Arena, 2015) were recently studied. The study of flower anatomy related to blooming is an important step for programs of genetic resource conservation and improvement, complementing basic studies of floral biology (de Castro Nunes et al., 2012; Wetzstein et al., 2014). Flower structure and floral biology was well described by Arena et al. (2011), like the phenological stages (Arena et al., 2013 a) and flower bud differentiation (Arena and Radice, 2014). More recently, a comprehensive study of pollen grain was published (Radice and Arena, 2016 a). Nevertheless, pollination was not clear until now. Fertilization of Patagonian Berberis has been classi- fied as cross-pollination by Orsi (1984). However, Hegi (1958) and Romeo et al. (2005) referred the Berberis species as autogamous due to the absence of visiting insects together with extreme climatic con- d i t i o n s p r e v a l e n t i n m o s t a r e a s o f P a t a g o n i a . However, during flowering, the activity of different Syrphidae was observed (Radice et al., 2016). On the other hand, floral movements have been appointed as mechanisms to facilitate self-pollination (Darwin, 1862). Stamen movement has been documented in a few plant families, among them Berberidaceae (Lechowski and Bialczyk, 1992). However, results of controlled treatments of self- and cross-pollination compared with those of open-pollination performed during three different periods in B. microphylla do Adv. Hort. Sci., 2017 31(1): 39-44 DOI: 10.13128/ahs-20724 Flower anatomy related to blooming development of Berberis microphylla G. Forst (Berberidaceae) S. Radice, M. Arena Department of Plant Physiology, Facultad de Agronomía y Ciencias Agroalimentarias UM-CONICET, Machado 914, Lab. 501, B1708EOH, Morón. Prov. de Buenos Aires, Argentina. Key words: anthesis, barberry, nectar, Patagonia, pollen grain, stigma. Abstract: Berberis microphylla G. Forst. is a Patagonian native shrub commonly named “calafate”, which has a growing economic potential due to its dark blue berries that are consumed fresh, as jams and preserves, and are used for the pro- duction of soft drinks and ice cream. Moreover, the fruits have a high content of carbohydrates, phenols and antioxi- dants. The objective of this work was to show the changes observed in the flower from the emergence in relation to the floral phases and the importance that they have on pollination and fertilization. During the anthesis, the nectar is excret- ed inside and outside of the petal through the epidermis of the secretory tissue. The epidermis of the stigma is papillae with cells of greater length in the periphery of this structure simulating an additional ring. Secretory tissue is also present on the area of the fusion carpel. During anthesis, the epidermis glands of the stigma showed active secretion and these conditions favor pollen grain germination. Germinated pollen grains were observed after 12 hours of pollination and ten days later the pollen tube reached the ovule area. Pollen tube grew surrounded the ovules and probably some of them already accomplished the fertilization. (*) Corresponding author: siradice@yahoo.com Received for publication 19 November 2016 Accepted for publication 15 February 2017 Copyright: © 2017 Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2017 31(1): 39-44 40 not support this hypothesis (Radice and Arena, 2016 b). Thus, self-pollination resulted only in pollen ger- mination on the stigmas but the pollen tubes were not able to reach the ovules. According to these antecedents, anatomical studies are necessary for a better understanding of the physiological processes during floral biology and pollination that interact with Syrphidae activity. The objective of this work was to show the changes observed in the flower from its emergence in relation to the floral phases and the importance that they have on pollination and fertil- ization. 2. Materials and Methods Plant material Flowers of Berberis microphylla G. Forst. (n = 20) on growth stages ranging from 53 to 68 on the BBCH scale proposed for B. buxifolia (Arena et al., 2013 a) were collected on plants grown near Ushuaia city, Tierra del Fuego (54° 48’ SL, 68° 19’ WL and 30 m asl), and were fixed in FAA (formaldehyde, 100 mL; ethyl alcohol, 500 mL; acetic acid, 50 mL; distilled water, 350 mL). Light microscopy Button flowers were dehydrated in an ethanol series and embedded in Spurr’s resin. Thin sections (75-90 nm thick) were stained with uranyl acetate and lead citrate. Fluorescent microscopy Flowers fixed in FAA were washed with distilled water and softened with NaOH (8N) as described by Martin (1959). Then, they were stained with aniline blue to study pollen tube growth. Squash material was observed by a Leica microscope (DM 2500) with DAPI filter. Scanning electron microscopy (SEM) Button flowers fixed in FAA were dehydrated in an ethanol series and critical point drying technique was employed. Samples were sputter coated with 20 nm gold and observed with a Philips XL 30 SEM. Ovules and seeds relation The number of ovules on button flowers (n= 130) and the number of seeds on formed fruits (n=100) were counted. 3. Results Pistil of B. microphylla is similar to a bottle (Fig. 1A). Flowers before anthesis (growth stage 54) showed anthers with microspores at an advanced stage of development and underdeveloped ovules (Figs. 1A-D). In effect, microsporangia contain tapetal cells metabolized, i.e. a thick portion was deposited on the wall of the microspore and inside it is possible to observe vegetative and generative cells that are surrounded by a thin delicate wall (Fig. 1D). On the other hand, ovules are rudimentary with an active cellular proliferation on the nucellus and integu- ments (Fig. 1C). The epidermis of the stigma is covered by secreto- ry cells (Figs. 2 A, C, E) and the short style is also recovered by glands (Fig. 2B). Petals have two thick nectaries on the basal position (Fig. 3A). Both petal epidermises are also formed by glandular cells (Figs. 3B, D, E, F). On a later flower phase (growth stage 59), the epidermis and nectariferous cells of the nec- tar have dense cytoplasm and conspicuous nucleus (Figs. 3C). Nectar is abundant in the intercellular spaces (Fig. 3C). Flowers on the anthesis phase (growth stage 60) showed nectariferous cells with a gradual diminution of staining density (Fig. 3G). Total production of nectar per flower is poor; it is secreted through the gland cells that are present on the epi- dermis of the nectaries (Figs. 3C, G) and two epider- Fig. 1 - Bottom flower of B. microphylla G. Forst (light micro- graph). A, longitudinal section of a flower on stage 54. B, anther with mature pollen grains; C, rudimentary ovule with nucellus (n) and integuments (i) in development; D, detail of pollen grain with exine (e), Vacuole (v) and gener- ative cell (gc) contained into the vegetative cell; n= nucle- us. Bars: A = 200 µm; B, D = 10 µm; C = 50 µm. Radice and Arena - Flower anatomy related to blooming development of Berberi microphylla G. Forst 41 mal layers of petals (Figs. 3E,F). Nectar is exuded through the cuticle with rupture of its outer layer. Greater presence of vacuoles is observed in both nectar tissues (Fig. 3G). Flowers on growth stage 59 showed a mono- carpellary pistil with a clavate shaped stigma (Fig. 4A). Epidermis of the stigma is papillae (Figs. 4B, C, E) with cells of greater length in the periphery of this structure simulating an additional ring (Fig. 4E). These cells secrete a sticky substance that keeps always the stigma hydrated during the anthesis phase (Fig. 2F). Secretory tissue is also present on the area of the fusion carpel (Figs. 4B, D). On the other hand, stigmas on growth stage 59 are receptive, i.e. they can promote pollen germination while stigma on less developed growth phases is not receptive (Fig. 2E). Flowers on anthesis phase (growth stage 60) Fig. 2 - SEM micrograph of the pistil of B. microphylla G. Forst. A, stigma of a flower on stage 59; B, detail of box in the pic- ture A, periphery of the upper end of the ovary with glands; C, stigma front view shown in A; D, stigma front view shown of a flower on stage 60; E, detail of box in the picture D, long hairs peripheral stigma with pollen grains attached; F, detail of circle in the picture C, epidermal cells of stigma; G, detail of circle in the picture D, active secre- tions glands; H, mixture of pollen grains wrapped in stig- matic mucilage, arrow points to a grain of foreign pollen. Bars: A-E =100 µm; F-H= 50 µm. Fig. 3 - Nectary of B. microphylla G. Forst. A-D SEM micrograph. B- C, E-G light micrograph. A, nectary view at the base of petal; B, section of a petal and a nectariferous area; C, detail of tissue of nectariferous area of a flower on stage 59; intercellular space with nectar (arrows); D, detail of circle in the picture A, epidermis with glands, arrows show secretory glands. E, detail of circle in the picture B, petal outer epidermis; F, detail of circle in the picture B, petal inner epidermis; G, detail of tissue of nectariferous area of a flower on stage 60, arrow indicates an epidermal cell in active secretion. Bars: A-B= 100 µm; C= 10 µm; D= 50 µm; E-G= 10 µm. Fig. 4 - Light micrograph of the pistil of B. microphylla G. Forst. A, longitudinal section of a flower on stage 59.B, view of stig- ma and the upper end of the ovary; C, glandular cells of the flat part of stigma; D, detail of glandular cells of fusion area of the carpel; E, detail of hairs surrounding the stig- ma. Bars: A= 100 µm; B= 200 µm; C-E= 50 µm. Adv. Hort. Sci., 2017 31(1): 39-44 42 showed mature pollen grain with a well-formed external wall and the cytoplasm of the vegetative cell rich of starch (Fig. 5E). In this phase anthers are dehiscent. On the contrary, ovules are externally coated by the integuments and attached to the ovary by the funiculus on basal placentation (Fig. 5D). Internally, the ovules present some delay in compari- son to the pollen grain development. Pollinated flow- ers show ovules with the embryo sac with egg cell, synergists, antipodes and polar nuclei cells developed (Figs. 5 A-C); nevertheless, no pollinated pistils show ovules with megaspore mother cells without devel- opment. Pollen is deposited on the stigma mainly between the secretory cells that surround this structure (Figs. 2D, G). Germinated pollen grains were observed after 12 hours from pollination (Fig. 6B). After 24 hours pollen tube crosses the stigma (Fig. 6A) and ten days later the pollen tube reached the ovule area. Pollen tube grew surrounded the ovules and probably some of them accomplished the fertilization (Figs. 7A, B). Subsequently seed growth is observed (Fig. 7C) but this process is not given in all the ovules. In effect, on a selected natural population the ovules and seeds were counted, and an average of 8.95 (ranged from 6.9 to 10.0) and 5.28 (ranged from 3.3 to 7.2), respectively, were registered, i.e. 40.97% of all ovules produced aborted. 4. Discussion and Conclusions Flowering plants are associated with a broad spec- trum of animal pollinators, among these bees consti- tute an important but not exclusive one (Dötterl and Vereecken, 2010). In effect, it has already been demonstrated that B. microphylla is not self-fertile so it depends on insect pollination (Radice and Arena, 2016 b). Tierra del Fuego (Argentina) offers an extreme climatic situation where the bees cannot prosper; accordingly calafate flowers are visited by different syrphids (Radice et al., 2016). Pollination by insects, including flies, is commonly a mutualistic interaction, in which both the plant and the insect benefit; thus, anatomical organization and pollina- tion strategies developed on the flowers must be adapted to the environmental conditions. Calafate shrubs bloom with abundant yellow flow- ers that produce aromatic nectar (Radice et al., 2016), that exudates inside the flower as well as the outside through the petals mainly in their insertion Fig. 5 - Details of the ovary and pollen grains of a flower on anthesis stage of B. G. Forst. A-C, embryo sac with egg cell (A, arrow), synergids (B, arrow) and antipodes (C, arrow) and polar nuclei (C, arrows head); D, SEM micrograph of ovules on the basal insertion of ovary; E, mature pollen grains with cytoplasm rich in starch grains. Bars: A-C= 200 µm; D= 100 µm; E= 10 µm. Fig. 6 - Fluorescent light micrograph of pollen tube germinated on pistils of B. microphylla G. Forst. A, view of stigma with pollen grain germinated and pollen tubes inserted in the ovary; B, pollen tubes on the first stage of growth; C, pollen tube penetrating the ovule. Bars: A-C= 100 µm. Radice and Arena - Flower anatomy related to blooming development of Berberi microphylla G. Forst 43 area. This particularity is very important because the fluorescent emission of nectar attracts pollinating insects. B. microphylla have perigonial nectaries type “1a” according to the topographic classification of Fahn (1982). Unlike what was found on the nectar tissue of Berberis corymbosa by Bernardello et al. (2000), no stomata were found on this species either on histological sections or through SEM observations. In effect, nectar is exuded through the epidermis as cited by Bernardello (2007). Berberis produce small amounts of nectar per flower; effectively it was regis- tered less than 1 µl on B. corymbosa (Bernardello et al., 2000) and 1.57 µl on B. microphylla (Radice al., 2016). Nectar concentration was considered as inter- mediate for B. buxifolia (31.2±14.8%) (Chalcoff et al., 2006). This result is in coincidence with Radice et al. (2016) who measured 36.28 °Brix in nectar of the Berberis population studied. Stigma epidermis is covered by hairs that secrete a stigmatic fluid to promote pollen germination. Surface hairs on the stigma can be seen in others species like Papaver rhoeas, or Lupinus luteus (Fahn, 1982). Once overcome the stigma, the pollen tubes grow through the carpel wall. In effect the margin of the carpel is covered by glands that nourish the ger- minated pollen. The Berberidaceae are generally con- sidered to have originated in some part of the rana- lian complex (Chapman, 1936), i.e., it is belonging to the group of the oldest dicotyledons which is con- firmed by its structure devoid of carpelar style (Fahn, 1982). There are three important elements to attract the pollinating insect on B. microphylla such as color, scent and nectar. It has long been known that bees utilize not only visual but also olfactory flower cues f o r f i n d i n g s u i t a b l e h o s t p l a n t s ( D ö t t e r l a n d Vereecken, 2010). This pollination strategies present in calafate could be useful in other growth areas of the species. On the other hand, young flies in the presence of generic floral scent respond more strongly to a uniformly yellow cue than to any other uniform color cue (green, white, black, blue, red) except for ultraviolet (Brodie et al., 2015). Nectar is the most commonly sought reward by flower-visiting flies (Woodcock et al., 2014) because carbohydrates contained in the nectar provide short-term energy supply. So the abundant number of yellow flowers plus the fluorescent emitted cue by nectar must con- stitute a strong attraction for syrphids. Acknowledgements Authors acknowledge to the Prefectura Naval Argentina, the technical assistance of Isabel Farías. 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