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loral biology of the dioecious species Laurus nobilis L. (Lauraceae)

ttore Pacini ∗, Nicola Sciannandrone, Massimo Nepiepartment of Life Sciences, Siena University, via P.A. Mattioli 4, 53100 Siena, Italy

r t i c l e i n f o

rticle history:eceived 29 August 2013ccepted 10 February 2014dited by R. Löschvailable online xxx

eywords:ntherollen presentationeversible anther openingollen water content and viabilityectaries and nectarollination in a dioecious species

a b s t r a c t

The present study examines the cytological, physiological, chemical and ecological characteristics ofpollen and nectar offered by male and female flowers of the dioecious plant Laurus nobilis. The variousphases of floral phenology and the insect pollinators were observed. We used cytological methods todetermine anther, pollen and nectary structure. Nectar sugar composition was evaluated by HPLC. Pollenviability in time was compared with cytoplasmic and intine water content. Pollen presentation was foundto be reversible by opening and closing of anther valves, determined by hydration of the mechanicallayer of the anther. Pollen, covered by pollenkitt, was presented for dispersal for 3 consecutive days andduring this time the intine and cytoplasm lost water and pollen viability diminished. At germination exineruptured together with the outermost layer of the intine. Nectaries of male flowers were observed on theanther filament and on staminodes of female flowers. The nectar consisted almost entirely of sucrose andwas more concentrated in male flowers. Secreted through stomata, nectar was presented in a thin layer.In the study area, the main pollinators (about half the total number of all visits) were hymenopterans.

ntroduction

Angiosperms may have hermaphrodite or male and female flow-rs, which may be on the same or different plants. The latter case isermed dioecy and characterises about 6% of angiosperms (Rennernd Ricklefs, 1995). In dioecious plants, male and female flowers areormally morphologically quite similar if pollination is zoophilous,ut may be very different, due to flower adaptations facilitatingollen release and capture, if the dispersing agent is wind or water.

Pollen is a generalised reward because its primary function iso transport male gametes. In some species it is also a reward forollinators. Pollen maturation is accompanied by anther changes

n the following order: formation of a mechanical layer, tapetalegeneration, disappearance of locular fluid, and dehydration of theechanical layer and other anther parts (Borg et al., 2009). Pollen

lso undergoes partial dehydration with consequent changes in

Please cite this article in press as: Pacini, E., et al., Floral biology of

http://dx.doi.org/10.1016/j.flora.2014.02.001

olume and form (Franchi et al., 2011; Pacini, 1994). The pro-rammed dehydration of anther and pollen inactivates or reducesollen biochemical mechanisms (Borg et al., 2009). This occurs

∗ Corresponding author. Tel.: +39 0577232963; fax: +39 0577232860.E-mail address: ettore.pacini@unisi.it (E. Pacini).

usually during the period prior to seed quiescence (Franchi et al.,2011). It also enables the anther to open and present pollen fordispersal (Keijzer, 1987). The substance produced by tapetal degen-eration, especially in entomophilous plants, becomes deposited onthe outer surface of pollen during the final stages of dehydration(Weber, 1991), constituting the pollenkitt, which has various tasksaccording to the pollination mode. Its most common functions areto keep pollen grains together and attached to the anther, and tofavour adhesion to bodies of visiting animals (Pacini and Hesse,2005).

Pollen may be presented to dispersing agents in the open anther(primary presentation) or in other flower parts (secondary pre-sentation). This is made possible by pollenkitt (Pacini and Hesse,2005). As early as in the late 19th century, the Austrian naturalistKerner von Marilaun (1895/1896) observed that the anthers of Lau-rus nobilis and many other Lauraceae do not open in the usual waybut by upward inversion of valves, exposing pollen at the valve tip.

Pollen water content at anther opening, and during presentationand dispersal, varies from species to species in relation to environ-

the dioecious species Laurus nobilis L. (Lauraceae). Flora (2014),

mental relative humidity. It is generally in the range of 10–20%but in many plants it may exceed 30% (Franchi et al., 2011; Nepiet al., 2001). In ripe angiosperm pollen the vegetative cell alwayscontains polysaccharide reserves, such as starch and/or soluble or

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nsoluble cytoplasmic carbohydrates (Pacini, 1996). Honeybees andther pollinators prefer pollen of species with soluble and insolubleytoplasmic carbohydrates to pollen of species with amyloplastsFranchi et al., 1996).

In the case of dioecious and monoecious entomophilous species,ectar may be a reward for pollinators. Unlike pollen, nectar is apecialised reward because it is only useful to collectors (pollina-ors and predators). In plants with flowers of a single sex (dioeciousnd monoecious), nectaries may be found in flowers of one sex onlyr in both sexes, though they may be disposed differently (Pacini,010). In the case of Osyris alba (Santalaceae) only male flowersave nectaries and female flowers are visited by insects attractedy the deception of false anthers (Aronne et al., 1993). Nectar pro-uction and secretion are processes having high energy costs, and

ndeed uncollected nectar may be reabsorbed, especially in flowershat produce it in abundance (Pacini and Nepi, 2007).

The quantity of nectar secreted by a flower varies widely andepends on the main type of insect pollinator, if any, and on theumber of ovules to be fertilised (Pacini et al., 2003). Nectar secre-ion and the life of a flower may last a few hours or many days, asn the case of extrafloral nectaries. In hermaphrodite and unisexualowers, the volume of nectar and its composition differs in relationo sexual expression and type of pollinator (Pacini and Nepi, 2007).ugars are the main components of nectar, together with a com-lex mixture of other substances, such as amino acids, enzymes,ineral salts and secondary metabolites, the relative proportions

f which have proven highly variable in the species so far studiedNepi et al., 2012; Nicolson and Thonburg, 2007).

In this paper we examined some aspects of pollen biology andollination in bay laurel, Laurus nobilis L. (Lauraceae), a dioeciousediterranean plant, often growing as an ornamental tree, that

ffers different rewards for pollinators in male and female flow-rs. In particular the aims of the present study are: (i) to describehe mechanism of anther opening and how it is influenced by rel-tive humidity, (ii) to determine if the thick intine of pollen act as

water reservoir during pollen presentation, (iii) to know morebout nectaries and nectar in male and female individuals of thisioecious species.

aterials and methods

tudy site and materials

The research was conducted in a small garden close to Sienaentre (43◦19′07′′ N, 11◦19′50′′ E) with a male and a female Laurusobilis L. growing about 20 m apart and without any co-floweringlant. The trees were 10–15 years old. Male and female flowers onranches of similar age and exposure were used. The study wasonducted in the first half of April 2010 and 2011.

lower development and duration – male and femaleeceptivity

Flower development was tracked in 30 marked male and femaleowers. We monitored floral stages, counting the days between onetage and another until the end of blooming. Female receptivity waseasured in ten female flowers at the above mentioned stages byeans of the stigma peroxidase test, using Peroxtesmo KOTM paper,

s described by Dafni and Motte Maués (1998).

ariations in volume, water content and viability of pollen

Pollen was collected from five flowers with closed (bud) andpen anthers (at different intervals since opening). Viability wasetermined by the fluorochromatic reaction (Heslop-Harrison

PRESSx (2014) xxx–xxx

et al., 1984). Beginning at time 0 (anther opening) we took samplesevery 3–4 h for 48 h and every 7–9 h until 72 h.

To study changes in pollen volume in the anther and duringpresentation, we prepared microscope slides. Pollen was obtainedfrom closed anthers by crushing and from open anthers using a nee-dle, and placed in immersion oil. Since the pollen was spherical withuniform wall thickness, we measured pollen and cytoplasmic diam-eter under the microscope. We then calculated pollen grain andcytoplasmic volume by the formula V = 4/3 �r3, where r is radius.Wall volume was obtained by subtracting cytoplasmic from grainvolume. We then looked for correlations between pollen grain vol-ume, cytoplasmic volume and wall volume at the following times:0 h (time 0, pollen exposure), 7 h, 14 h, 24 h, 36 h and 55 h of expo-sure. Pollen viability and variation of volume were recorded for atotal of 300 grains (3 × 100). Correlations were analysed by the non-parametric Spearman rank test, using the programme Statistica(release 6.2, alpha = 0.05).

The presence or absence of locular fluid was detected by mak-ing transverse sections of maturing anthers by hand and observingthem in immersion oil on cover slips under the microscope.

Water content of ripe pollen was determined on the first dayof presentation and for three consecutive days with differentweather conditions. Water content was calculated from the differ-ence between fresh weight of pollen from newly opened anthersand dry weight after dehydration at 105 ◦C until constant weight.

Pollen tube emission

The onset of pollen germination was followed in vivo and invitro. Newly opened female flowers were pollinated at 9 am withpollen collected from male flowers under the stereomicroscopeusing an eyelash mounted on a toothpick, and deposited on thestigma. Pollen germination was checked as described by Shivanna(2003).

For germination in vitro, pollen was collected in the same wayfrom newly opened anthers and deposited in a drop of culturemedium supplemented with 30% sucrose (Brewebaker and Kwack,1963). Germination was checked by light microscope observation.The onset of pollen tube emission was recorded for 300 grains(3 × 100) at different times.

Histochemistry

Mature anthers were fixed and embedded as described by Federand O’Brien (1968). Sections (1–2 �m) were tested for: (1) totalpolysaccharides with periodic acid Schiff (PAS) (Jensen, 1961);(2) pectins with ruthenium red (Jensen, 1961); (3) nucleic acidswith DAPI (Coleman and Goff, 1985); (4) total proteins with mer-curic bromophenol blue (Barka and Anderson, 1963); (5) exineand cuticle with auramine O (Heslop-Harrison, 1977); (6) callosewith Sirofluor (Evans and Hoynes, 1982). Nectary parenchyma cellswere tested for autofluorescence of chlorophyll in chloroplasts byfluorescence microscopy. To test for starch in chloroplasts of nec-tary parenchyma at different times of day we used Lugol reagent(Jensen, 1961). The same reagent was used for starch in freshpollen. The localisation of nectar-producing cells was determinedby the method described in Dafni (1992). Pollenkitt on pollen grainswas detected staining them with scarlet R in ethanol (Heslop-Harrison and Heslop-Harrison, 1985) and by the method describedby Teppner (2009).

Nectar sampling and sugar analysis

To study nectar we used the protected crop system: budswere bagged to prevent pollinators from collecting nectar. Thewhole inflorescence was bagged with tulle (1 mm mesh). Since the

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Table 1Sequence of nectar presence/absence, stigma receptivity and pollen presentation in bagged male and female flowers of Laurus nobilis. Stigma receptivity increases at days 4and 5.

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6–7

♀ Nectar secretion − + + + + −Nectaries fall

Stigma receptivity − + + ++ ++ Stigma-stylefall♂ Nectar secretion + + + + −

Nectariesbecomebrownish

−Nectaries fall

Pollen presentation + + + + + Anthersbecomeadpressed andbrownish, thenthey fall

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Fig. 1. Scheme of male (♂) and female (♀) flowers. Male flowers have two seriesof anthers, the inner ones bearing nectaries. Receptivity is centripetal and nectar is

plant produced more flowers per branch (of the same length) thanthe female plant and the inflorescences were wider because thestamens pushed the petals outward (Fig. 1). The mean life of maleflowers was shorter than that of female flowers (Table 2). Stigma

Table 2Main differences between male and female flowers of Laurus nobilis (mean ± SD,n = 50). Nectar sugar concentration is indicated in arbitrary units corresponding tothe chromatographic peaks area.

Features ♂ ♀Petal length (mm) 3.1 ± 0.05 3.3 ± 0.08Flower diameter (mm) 7.7 ± 0.11 5.5 ± 0.20

uantity of nectar was small and it was highly viscous, we deter-ined its presence by observing the thickness of nectaries in

mmersion oil under the microscope.We determined the quantity of total sugars per male and female

ower. It proved impossible to measure other characteristics, suchs volume of nectar secreted, concentration and daily production,ecause the quantity was too small. Nectar was sampled with wicksf filter paper (Whatman no. 1) from about 150 flowers of eachex. Nectar of ten flowers was absorbed on each wick triangle.he surface of the nectary was touched with the tip of the papericks, which was then checked under the microscope for nectar

nd pollen. If pollen was found, the sample was discarded. Samplesere frozen at −20 ◦C until analysis. Starting this latter, the sam-les were thawed to ambient temperature, and nectar was thenecovered from the filter paper by static elution with 100 �l dis-illed water for 2–4 min, followed by a centrifugation for 5 min at1,000 × g.

Analysis of the sugar profile of the supernatant was conductedy HPLC using a chromatograph having an injection loop with aominal volume of 20 �l, a Waters Sugar-Pack I ion exchange col-mn (6.5–300 mm) and a Waters 2410 refractive index detector.he column was run under isocratic conditions with MilliQ water asobile phase, at a flow of 0.5 ml min−1 at 90 ◦C. The instrument was

alibrated with solutions having known and increasing concentra-ions of sugars common in nectar, i.e. sucrose, glucose and fructoseNicolson and Thonburg, 2007), made up using certified materialsnd MilliQ water. A single analysis was performed for each sample.he chromatographic data were analysed using Data Apex Clarityite 3.0 software for qualitative determination of sugars.

ollinators

Insects visiting male and female flowers were monitored on 5thnd 6th April 2010. The number of visits was counted for flowers ofach sex on two branches each carrying about 50 open flowers. Thebservations were made by the same person (NS) during the follow-ng time intervals: 10–11 am, 11–12 am, 3–4 pm and 4–5 pm. Thebserver moved from one plant to the other after an hour. Weatheras similar on the two days, fair with moderate wind.

esults

orphology of male and female flowers

Five to six flowers are united in umbel inflorescences in male and

emale inflorescences of L. nobilis. Each flower has a perianth withour actinomorphic petals. Female flowers are pale yellow, withuperior ovary and a single pistil with one ovule and 4 staminodes,bout 4–5 mm long, containing nectaries (Figs. 1 and 2A). During

already present in the innermost series when the outermost anthers expose theirpollen. Female flowers have a single ovule in the ovary (ov) and a central stigma (st).The ovary is surrounded by four staminodes bearing nectaries. Scale bar = 0.2 cm.

receptivity, an abscission zone in the style was evident between thebase and the ovary (Fig. 2A). The style-stigma complex was shedtwo days after pollination. Male flowers are pale yellow (Fig. 2B)and consist of 8–14 stamens, all about 6–7 mm long. Only centralstamens have nectaries (Figs. 1 and 2C).

Male and female receptivity

Male and female flowers opened at the same time (8–9 am). Fourstages of receptivity were observed: young flower (day 1 of bloom-ing), mature flower (days 2 and 3), almost senescent flower (days4 and 5) and senescent flower (days 6 and 7): Table 1. The male

Inflorescence diameter (mm) 14.0 ± 0.19 12.0 ± 0.31Number of flowers/inflorescences 5.7 ± 0.12 5.1 ± 0.13Flower lifespan 4 d 5–6 dNectar sugar concentration ++ +

4 E. Pacini et al. / Flora xxx (2014) xxx–xxx

Fig. 2. Male and female flowers of Laurus nobilis. (A) Detail of female flower showing staminodes (S) and nectaries (N). The ovary (ov) bears the style and stigma (ST). Atthe base of the style, the site where abscission tissue will form after pollination and fertilisation is already evident (arrow). (B) Male flower at the onset of floral anthesis.T er witc ed colo

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he stamens diverge from the receptacle to which they are attached. (C) Male flowovered by nectar. (D) Male flower at the end of receptivity. The anthers have chang

eceptivity of female flowers began on day 2 and increased up toay 5; this was shown by stigma colour that became more intenseevealing increasing peroxidase activity. The stigma was shed onay 6–7.

ollen presentationOpening of the anthers, which are bilocular, occurred in a cen-

ripetal manner: first the anthers of external stamens withoutectaries, followed by those of inner stamens bearing nectariesFig. 1). On anthesis of male flowers (Fig. 2B) the anthers divergedith respect to each other, inclining at 45◦ to the flower axis

Fig. 2C). The anthers are extrorse, i.e. they are opening outwards.xternal anthers opened after 9 am and the time taken for openinganged from 15 min to 1 h, depending on the weather conditions.nternal anthers opened 1–2 h later, depending on the weather.udden environmental changes such as rain slowed or prevented

nther opening, whereas temperature increase and low relativeumidity accelerated anthesis.

Pollen was presented on the internal surface of the valves, twoer anther, which inverted upwards (Fig. 3A and B). Inversion of the

h dehiscent anthers presenting pollen. The nectaries of the innermost anthers areur and all pollen has been removed. A: scale bar = 0.5 mm; B, C, D: scale bar = 1 mm.

valves is due to disappearance of locular fluid, separation of valvecells from the rest of the anther (Fig. 3A), drying of the mechan-ical layer (Figs. 3C, D and 4), and drying of other anther cells atthe end of receptivity (Fig. 2D and 4), in that order. Detachmentof valve profile cells from the rest of the anther depended on: (a)separation of small cells along the valve edge separating the valveas a distinct unit from the rest of the anther (Fig. 3C); (b) pres-ence of lignified cell thickenings in the anther mechanical layerlimited to the valves (Fig. 3C and D). The mechanical layer con-sists of a single layer of cells located only under the epidermal cellsof the valves. The cells of the valve mechanical layer have ligni-fied thickenings, especially in the inner circumferential and radialwalls, and little cytoplasmic residues (Fig. 3D). Tension in cells of thevalve mechanical layer increases with drying, causing progressivedetachment of the valve from the edge. With drying, mechanicallayer cells change from being prismatic when the valve is closed

to truncated pyramidal when the valve is open. The flat, outward-facing surface increases in area by distension with respect to thesituation when the valve os closed. Complete upward inversion ofthe valve (Figs. 2C, 3B and 4) took 15–60 min., depending on air

Fig. 3. Anthers and anthesis. (A) An anther begins to open. (B) The anther valve begins to open. (C) Transverse section of an anther loculus before the first haploid mitosis.Note areas where the valve will detach (arrows) and the lignified thickenings of the mechanical layer of innermost cells. D. Section of the previous image at the same stage,observed by fluorescence microscopy with auramine O. Note fluorescent lignified thickenings of the mechanical layer, the exine on the pollen grains and the peritapetalm rest ob

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embrane that contains elements of sporopollenin. The cuticle of the valve and thear = 60 �m.

elative humidity (Fig. 4). On inversion of the valve, pollen retainedy pollenkitt becomes exposed in the upper part of the valveFigs. 2C and 4). Dry air conditions after 9 am caused inversionf the valves, while nocturnal humidity and moisture from rainaused lowering of valves and protection of pollen (Fig. 4). Almostll the pollen in each locule remained attached to the invertedalve margin. Very few pollen grains (not quantified but evidenty stereomicroscopic observation) remained on the locular cavityurface.

In the absence of insects that actively collected pollen or became

usted with it while collecting nectar, the pollen remained at thealve tip for about five days, when the live cells of all stamens ofhe flower withered, turned brown and collapsed together intohe flower (Fig. 2D). Although we did not study anemophilous

f the anther is thin and only weakly fluorescent. A, B: scale bar = 100 �m; C, D: scale

dispersal of pollen, this way of pollen transfer cannot be excludedwhen wind is sufficiently strong to detach pollen from thevalves.

Pollen cytology and germination

No cytological differences were found between pollen ofexternal anthers without nectaries and internal anthers bearingnectaries. Pollen is binucleate at maturity (Fig. 5A) and its sporo-derm is composed of very thin exine bearing conical spines and

a very thick intine (about one tenth of pollen diameter) withoutapertures. The intine consists of three layers (Fig. 5B): a thick cen-tral pectin layer and two external and internal fine PAS-positivelayers were observed under the exine (Fig. 5B). When exposed for

Please cite this article in press as: Pacini, E., et al., Floral biology of the dioecious species Laurus nobilis L. (Lauraceae). Flora (2014),http://dx.doi.org/10.1016/j.flora.2014.02.001

Fig. 4. Diagram showing manner of pollen presentation and its various stages. Only anther valve cells have a mechanical layer under the epidermis. At maturity, the layerconsists of dead cells with lignified thickenings orientated so as to open the valve (1–3) on detachment of the valve from the rest of the anther. Opening occurs after locularfluid (pale blue, resp. light-grey) disappearance and by dehydration of mechanical layer cells, which change from prismatic with rectangular bases to prismatic with trapezoidbases. This causes the valve to invert upwards, taking the pollen retained by pollenkitt (yellow) with it (3). When the valve cells rehydrate due to high relative humidity orrain, they change shape and the valve closes, protecting the pollen (3′). Pollen is presented for dispersal for 5 days (3–4); on day 6 the rest of the anther also dehydrates andthe valve folds towards the centre of the flower. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Ripe pollen structure. (A) Pollen observed by flurorescence microscopy using DAPI stain (DNA stains blue). DNA of the generative cell (gn) is condensed and is thereforemore fluorescent than that of the vegetative cell (vn). The exine is autofluorescent and bears spines on a thin basal layer. (B) Pollen before hydrolysis of starch (PAS). Theintine consists of three layers: the external two are more PAS-positive than the thicker inner layer. Scale bars = 10 �m.

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Fig. 7. Variations in volume of pollen grain, intine and protoplast. Total volume,

suggests that it accumulated by gravity.It was impossible to measure the nectar volume, but no great

differences seemed to exist between the male and female flowers.However, it is worth noting that male flowers have an average of

ig. 6. Decrease in total volume of pollen grain, intine and cytoplasm in the varioustages before and at anther opening. dba = days before anthesis (n = 300).

ispersal, pollen grains were held together and attached to thenther by a coating of pollenkitt.

The cytoplasm of the vegetative cell stores starch in the initialinucleate stage (Fig. 5B). The starch is hydrolysed before dehydra-ion of the anther, developing an intensely PAS-positive cytoplasm.

At the start of anthesis, pollen had a water content of 34–41%,epending on the day of sampling. General pollen morphologyid not change during development and presentation, the pollenemaining spherical. Only the total volume varied with changes inydration and this was due to variations in intine thickness andytoplasm diameter.

Our observations of variations in cytoplasm, intine and pollenolume before anther opening showed changes due to disappear-nce of locular fluid and subsequent slight water loss in the earlytages of presentation (Fig. 6). In general we observed a direct corre-ation between total volume, cytoplasm volume of the vegetativeell and wall volume during presentation (Fig. 7). At time 0 and

h, total volume was significantly correlated with cytoplasm vol-me (R = 0.95, p < 0.05); wall volume was also correlated with totalolume (R = 0.77, p < 0.05). After 14 h there was only a direct signif-cant correlation between total and cytoplasmic volume (R = 0.96,

< 0.05), while wall volume was not correlated. At 24 h and 55 h, correlation was found between cytoplasmic volume and wallolume (R = 0.63, p < 0.05; R = 0.62, p < 0.05 respectively). At 36 h aignificant correlation was found between total and wall volumeR = 0.88, p < 0.05).

The viability of pollen remaining in the anther decreased withime, dropping sharply beyond 14 h after anthesis (Fig. 8). Pollenegan to germinate about 60 min after pollination on the stigmand after about 40 min in vitro, when the position where the pollenube would form was indicated by a callose dome after 30 minFig. 9A). The increase in volume of the cytoplasm and intine rup-ured the external intine layer and the overlying exine sculptureFig. 9B), after which the pollen cytoplasm and the inner intine layerppeared through the external intine (Fig. 9B) and the pollen tubeegan to grow (Fig. 9C).

ectaries and nectar

The nectaries of male and female flowers are both kidney-haped; they are situated half way along staminodes in femaleowers (Fig. 10A) and below the anthers in male flowers (Fig. 10B).

n both, the nectary consists of an epidermis with stomata locatedn shallow grooves (Fig. 10C and D), an underlying parenchymaFig. 10C) and a branching vascular bundle including phloem andylem. The nectar-producing parenchyma is most probably able

o conduct photosynthesis as the cells contained autofluorescenthloroplasts (Fig. 10C). Small starch grains were visible in chloro-lasts of nectaries observed in the daytime and were absent inlastids of nectaries observed at 8 am the next morning.

intine volume and cytoplasmic volume decrease sharply beyond 7 h of exposure(n = 300).

The nectar is secreted through open stomata (nectarostomata).At the onset of secretion, the nectar is visible as drops at thesestomata (Fig. 10A). The nectar is viscous and spreads more or lessuniformly over the nectary surface as secretion proceeds. In thelast days of receptivity of bagged male and female flowers, the nec-tar layer was more abundant in the lower part of nectaries, which

Fig. 8. Pollen viability during floral anthesis. Viability remains constant for about14 h, and then falls sharply (n = 300). dba = days before anthesis.

F ittedi isibleb ). Scale

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ig. 9. In vitro pollen tube emission. (A) The area where the pollen tube will be emntine wall open under pressure of the underlying tube; developing tube becomes vreaks through the exine and the outer intine layer after about 45 min (bright field

ve pairs of nectaries, whereas female flowers only have four. Inoth flowers, the nectar was presented for four days, staggered by aay according to the respective receptivities (Table 1). The nectaries

f anthers and staminodes turned brown when secretion ceasednd were shed the next day (Table 1).

ig. 10. Nectaries of male and female flowers. (A) Nectary of a male flower at the start oar = 0.4 mm). (B) Stamen of the inner circle with nectaries stained with Ruthenium red toar = 0.6 mm). (C) Section of a nectary just before the start of secretion. The stoma (arrowower with two nectaries at the beginning of secretion. Arrow indicates a drop of nectar

is visible as a callose dome after about 15 min (Sirofluor). (B) Exine and the outer after about 30 min (auramine O). (C) The pollen grain with developing pollen tube

bars = 10 �m.

Sugar determinations showed only sucrose, which differedin quantity between male and female flowers. In male flowers,the sucrose concentration of the nectar, as extracted from the

paper triangles averaged 0.91 ± 0.16 mg ml−1 and in female flowers0.43 ± 0.6 mg ml−1.

f secretion. Arrow indicates a stoma which has already exuded nectar (SEM; scale reveal nectar secretion. Note intense staining of nectaries and anther valves (scale) is not yet open (autofluorescence) (scale bar = 50 �m). (D) Staminode of a female(scale bar = 0.6 mm).

Table 3Number of visits by different groups of insects to male and female flowers of Laurus nobilis at different times of day in the observation period. Hymenopterans were theprinciple pollinators.

Male flower Female flower

April 5th hour 10–11 15–16 Total 10–11 15–16 TotalApril 6th hour 11–12 16–17 11–12 16–17

Hymenoptera 6 9 8 6 29 5 7 6 6 24Lepidopetra 3 3 4 3 13 2 4 2 3 11Coleoptera 3 2 1 3 9 3 4 2 1 10

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Diptera 1 0 2 2

Total 13 14 15 14

ollinators

Male and female flowers of L. nobilis were visited by differ-nt orders of insects (Table 3), which collected pollen and nectar.ymenopterans (Apis mellifera, Bombus lucorum, Xylocopa violacea)ccounted for almost 50% of the visits observed. Other insects werehe lepidopteran Gonepteryx rhamni, ∼25% of visits, the coleopter-ns Meligethes sp. and Oxythyrea funesta with ∼20%, and sporadicisits by the dipteran Musca domestica and aphids (Table 3). Theuration of visits was not timed, but hymenopterans stayed longern male flowers. The number of visits to male and female flowersas similar (Table 3).

iscussion

ioecy and reproduction in Laurus nobilis

Laurus nobilis is not considered to be a native species of EuropeTutin et al., 1964–1980), though it was certainly introduced a longime ago from a moist, subtropical climate (Rodriguez-Sanchezt al., 2009). Its reproductive cycle is nevertheless complete in cen-ral and southern Italy and female plants produce fruits in autumn,he seeds of which germinate. Being a dioecious entomophilouslant, (a) sexual reproduction always involves wide genetic reas-ortment, (b) sexual reproduction may only occur if pollinatingnsects are present during flower receptivity, and (c) these insects

ust be sensitive to attraction by male and female flowers even ifot with an equal rate. Flamini et al. (2002) found that male and

emale flowers emitted chemically different perfumes originatingrom different parts of the flowers. Further, (d) male and femalelants must grow sufficiently close to each other to be within theange of pollinating insects, (e) the pollen must reach the stigma in

viable state, and (f) there must not be other plants in the vicinityhat are more attractive to pollinators. The present research wasonducted in a garden in a period when any other plants were notn bloom, so that bay laurel flowers were visited by different typesf insects, rewards were collected, pollination occurred, and theruits ripened.

In plants with separate male and female flowers (monoeciousnd dioecious) and entomophilous pollination, the flowers mustppear sufficiently similar to insect eyes to prevent discrimination.n the case of bay laurel, the male and female flowers were visited

ore or less equally.

nthers and pollen presentation

The anthers of bay laurel flowers are disposed in inner and outerircles, the latter being without nectaries. Anther opening by theechanism of inversion of valves is uncommon, being typical of

auraceae and few other families (Castro, 1981; D’Arcy and Keating,996; Hufford, 1996). Anthers always open through a well definedequence of events: (i) locular fluid, which nourishes the pollen,vaporates or is reabsorbed by other parts of the flower (Pacini,

5 4 1 2 2 96 14 16 12 12 54

1994); (ii) water is lost by cells of the epidermis and the mechanicallayer, and in certain species also by other anther parts; (iii) the smallcells around the stomium detach by lysis from the middle lamella(Hufford, 1996) – the stomium is the area where the anther opensand may be a longitudinal or differently profiled slit; (iv) the partsof the anther bearing the mechanical layer invert outwards; (v)pollen engulfed by pollenkitt remains on the internal surface of theanther, (whereas in other species pollen without pollenkitt leavesthe anther as soon as the latter opens: Bianchini and Pacini, 1996;Carrizo et al., 2006; Castro, 1981; Keijzer, 1999; Pacini, 1994).

Bay laurel anthers have two U-shaped stomia; usually,angiosperm anthers have four linear stomia, other Lauraceae, suchas Umbellularia californica, have four curved stomia (Hufford, 1996).

Valve opening is triggered by the disposition of the anthermechanical layer cells, which are dead and dehydrated at matu-rity. Differences in thickness and the arrangement of lignified cellthickenings determines the manner of anther opening (Manning,1996). After detachment of the cells on three sides of its border, thevalve inverts upwards, exposing pollen, retained by pollenkitt onthe valve inner surface. Since valve opening is slow, very few grainsof pollen are lost; indeed, only a few grains seem to remain in thelocular cavity.

In the case of bay laurel, evaporation of water from the valveepidermis is possible because the cuticle is thin and discontinuous,especially between cells. The mechanical layer dries by evapora-tion, because when it rains or at times of high relative humidity,the valve cells rehydrate and increase in volume. Consequently,the valve hinges downwards in this case, returning to its positionprior to presentation, and protects the pollen. According to Kernervon Marilaun (1895/1896) and Endress (1994), this mechanism ofpresentation and protection of pollen is typical of tropical plants,which grow in humid conditions, and many of them bloom at night.Inversion of the valve, and its return to initial disposition under highrelative humidity, is possible because of the flexibility of the hingebetween valve and the rest of the anther.

The mechanism enabling inversion of the valve and pollen pre-sentation is a biological mechanism involving coordinated aspectsof anther physiology, such as detachment of small cells on thevalve edges and drying of the mechanical layer. On the other hand,return of the valve to its initial position due to rain or high humid-ity is a passive physical phenomenon due solely to rehydration ofdead valve cells that return to their original prismatic form, whichcauses lowering of the valve. Changes in position of reproductivestructures with changes in environmental relative humidity arecommon in all land plants, especially angiosperms, where anthersoften close temporarily, or mechanisms exist to avoid early rehy-dration of pollen (Edwards and Jordan, 1992; Li et al., 2012).

According to Hufford (1996), this type of pollen presentationis a specialisation with respect to common pollen presentation by

longitudinal opening of the anther and indicates a selective advan-tage in pollen presentation. According to Endress (1994), on theother hand, it is a primitive character from which the more commonlongitudinal stomium aperture was derived.

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ollen structure and viability in time

In most plants, pollen has two walls, often interrupted by aper-ures for pollen tube emission. However, particularly in plantsrowing in environments with high relative humidity at pollina-ion, the exine may be very thin, discontinuous and even absent, asn aquatic dicots with under-water pollination and various preva-ently tropical species (Kress, 1986; Pacini and Hesse, 2011). In L.obilis, the exine is thin and covered with tiny spines, and therere no pores – which means that there is no predetermined sitef pollen tube emission; rather, the site is determined by adhesiono the stigma. In vitro it is not clear, how the site of pollen tubemission becomes determined. The intine is very thick and con-ists prevalently of pectin. It accumulates much water and containsvaginations of the plasma membrane, sealed by formation of a fur-her intine layer (Kubitzki, 1980). A similar pollen structure is alsoound in various monocots of tropical environments (Kress, 1986)nd in some gymnosperms with taxoid pollen (Pacini et al., 1999).his similarity in intine structure is also seen at germination, whichs preceded by release of the exine and part of the intine (Chichiriccònd Pacini, 2007). However, in gymnosperms the intine may be aoor water reserve for the cytoplasm during dispersal, since it is noturgid as in bay laurel and the pollen has a water content below 20%Nepi et al., 2001).

The degree of dehydration of pollen and its rehydration on thetigma is regulated by the elasticity of the exine, intine and cyto-hysiological features of the vegetative cell. In the case of bay laurelnd all plants with thin, discontinuous exine, this process is regu-ated prevalently by the intine and the cytoplasm.

It is plausible to deduce that in the present study, bay pollennderwent a decrease in volume due to loss of water, largely fromhe cytoplasm (the wall loses much less), up to 14 h after exposure.rom 24 h onward, the decrease in total pollen volume was due toater loss by the wall, because the cytoplasm had by now reached

ts dehydration limit. Viability data showed a close link with volumeecrease due to water loss, as observed also in pumpkin, anotherpecies with recalcitrant pollen (Nepi and Pacini, 1993). The termrecalcitrant’ was coined in analogy to seeds (Franchi et al., 2011),o indicate pollen presented for dispersal with a water content thatxceeds 30%. Pollen and seeds that are highly dehydrated at dis-ersal are known as ‘orthodox’ and have a water content of lesshan 30%. The term ‘orthodox’ indicates that this pattern is com-

on to most pollen and seeds (Franchi et al., 2011). All this meanshat according to the level of dehydration of pollen and seeds,lants may be classified in four categories: orthodox pollen andeeds, orthodox pollen and recalcitrant seeds, recalcitrant pollennd seeds, and recalcitrant pollen with orthodox seeds. Bay laurel isoubly recalcitrant, since both pollen and seeds are dispersed with

water content of more than 30% (Franchi et al., 2011). The presentesearch demonstrates that Laurus nobilis pollen maintains its via-ility for less than a day, like other recalcitrant pollen (Nepi et al.,001). However, unlike certain other recalcitrant pollen, it does notave a callosic wall at dispersal, as do have pollen of Cucurbita pepond certain grasses, and/or cytoplasmic reserves of callose (Heslop-arrison, 1987; Nepi et al., 1995). Moreover, its germination time

n vivo and in vitro is slightly longer than that of other pollen inhis category. Usually, recalcitrant pollen germinates on the stigman 5–30 min, whereas orthodox pollen always takes longer than0 min. Pollen tube emission in bay laurel depends on the initialater content and on pollen volume, and naturally also on that of

he counterpart tissue that provides the water for rehydration.Orthodoxy and recalcitrance have advantages and disadvan-

ages. Orthodox pollen and seeds may be dispersed over greateristances and resist water stress better, however they take longero rehydrate and germinate. Recalcitrant pollen and seeds are notrogrammed to be dispersed over great distances; they lose water

PRESSx (2014) xxx–xxx

readily and their viability is therefore shorter, though they germi-nate more rapidly. This means that orthodoxy and recalcitrance areadapted to different climates. Recalcitrant pollen and seeds performbetter in the tropics and under particular conditions, such as pollendispersal at night or during humid periods (Franchi et al., 2011).

Nectaries and nectar

Male and female flowers of L. nobilis have morphologically andstructurally similar nectaries on stamens and staminodes, respec-tively, a characteristic of many Laurales (Bernardello, 2007). Laurelnectaries consist of an epidermis, an underlying photosynthesis-ing parenchyma and a vascular bundle consisting of phloem andxylem. Like in most species with permanently open stomata (nec-tarostomata), which are situated in the centre of slight depressionsin the nectary surface, the nectar exudes through these stomata(Nepi, 2007). The presence of starch in chloroplasts during the dayand its absence the next morning suggests that the starch producedby photosynthesis is immediately hydrolysed to produce some ofthe nectar soluble carbohydrates.

The term “nectar presentation” indicates the manner in whichnectar is exposed for collection. It may be on the nectary surface ordistant from it, for example in spurs, as they occur in certain Ranun-culaceae, Scrophulariaceae and Orchidaceae (Pacini et al., 2003).In the present species, nectar is presented on the nectary surface,and because of its high viscosity, it accumulates in a layer which isthicker in the lower part of the nectary. Only nectar carbohydrateswere found in the analysis we did. They were mainly sucrose in bothmale and female flowers, though the sucrose quantity in nectar offemale flowers was about half that of male flowers.

Nectar viscosity in male and female flowers would depend notonly on sugars but also on substances, such as amino acids and lowmolecular weight proteins, always present in nectar (Nepi et al.,2012; Nicolson and Thonburg, 2007) but not investigated in thepresent study. Research currently underway by the present authorsshows that the nectar of certain species, especially if exposed,contains low molecular weight pectins that could contribute toviscosity and impede evaporation during the warmer hours of theday.

The small quantity of nectar produced per nectary in bay laurelmay be related to the presence of a single ovule in the ovary. Thismeans that few pollen grains are needed on the stigma to ensurefertilisation, and thus less frequent pollinator visits and lower nec-tar volume as reward to these visitors may suffice.

Pollen is protected from increased humidity and rain by lower-ing of the valves, but the nectar is not protected. However, sincethe nectary is most probably able to conduct photosynthesis andis supposedly active for several days, the nectar removed by sur-plus ambient moisture may be replaced. Male flowers invest morein pollination because they have more nectaries and more concen-trated nectar, as well as pollen. However, the investment is limitedto the pollination period, whereas in female flowers it continuesthrough pollination to fertilisation and ripening of the fruit.

In the study site, nectar was mainly collected by honeybees andconsumed immediately and more or less completely by the respec-tive bee colony (Ricciardelli D’Albore and Persano Oddo, 1978) asit is the case with nectar of many species that flower between win-ter and early spring, and is not being used to produce honey. Beesstayed on male flowers for slightly longer time than on female flow-ers and visited them slightly more frequently, presumably becausemale flowers offer pollen and nectar, and the nectar is more concen-trated. Ricciardelli D’Albore and Torini D’Ambrosio (1982) studied

pollinators of bay laurel at Perugia (Central Italy) and Turin (NorthItaly), observing that dipterans were the most constant visitorsand honeybees played the leading role, in the absence of moreattractive flower alternatives. This means that in a plant with wide

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eographical distribution, such as bay laurel, the spectrum of pol-inators may be different in relatively close sites, such as Siena anderugia (about 50 km), and may also depend on the availability ofther flowers.

onclusions

The study of Laurus nobilis pollination biology presented in thisaper lead to some interesting results. Laurel anthers open by aalve and pollen grains are exposed on it, the valve can reverse andlose the anther under high humidity or rain. The pollen exine ishin and the thick intine functions as a reserve of water, keepinghe cytoplasm wet, so that pollen viability is kept high for 14 h,fterwards it declines rapidly. The composition of nectar is sub-tantially the same in both sexes, but concentration is higher inales. Notwithstanding the differences in the rewards offered byale and female flowers, the number of visits by insects is very

imilar.

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