Flowering biology and structure of floral nectaries in Galanthus nivalis L.

Abstract

In Poland Galanthus nivalis L. is partially protected. The flowers of this species are one of the first sources of nectar and pollen for insects from February to April. The aim of this study was to present the flowering biology as well as the topography, anatomical, and ultrastructural features of the floral nectary. The flower lifespan, the breeding system, and the mass of pollen and nectar produced by the flowers were determined. Examination of the nectary structure was performed using light, fluorescence, scanning and transmission electron microscopy. The flower of G. nivalis lives for about 30 days. The stamens and pistils mature simultaneously and during this time nectar is secreted. The anthers of one flower produced the large amount of pollen (4 mg). The breeding system of G. nivalis was found to be characterized by partial self-compatibility, outcrossing, and xenogamy. The nectary is located at the top of the inferior ovary. The nectary epidermal cells are characterized by striated cuticular ornamentation. Initially, the secreted nectar formed vesicle-like protuberances under the cuticle. The epidermal and parenchymal cells contain numerous plastids, mitochondria, dictyosomes, ER cisterns, and vesicles fused with the plasmalemma, which indicates granulocrine nectar secretion.

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1 of 20Published by Polish Botanical Society
Acta Societatis Botanicorum Poloniae
ORIGINAL RESEARCH PAPER
Flowering biology and structure of oral
nectaries in Galanthus nivalis L.
Elżbieta Weryszko-Chmielewska*, Mirosława Chwil
Department of Botany, University of Life Sciences in Lublin, Akademicka 15, 20-950 Lublin,
Poland
* Corresponding author. Email: elzbieta.weryszko@up.lublin.pl
Abstract
In Poland Galanthus nivalis L. is partially protected. e owers of this species are
one of the rst sources of nectar and pollen for insects from February to April. e
aim of this study was to present the owering biology as well as the topography,
anatomical, and ultrastructural features of the oral nectary. e ower lifespan,
the breeding system, and the mass of pollen and nectar produced by the owers
were determined. Examination of the nectary structure was performed using light,
uorescence, scanning and transmission electron microscopy. e ower of G. ni-
valis lives for about 30 days. e stamens and pistils mature simultaneously and
during this time nectar is secreted. e anthers of one ower produced the large
amount of pollen (4 mg). e breeding system of G. nivalis was found to be char-
acterized by partial self-compatibility, outcrossing, and xenogamy. e nectary is
located at the top of the inferior ovary. e nectary epidermal cells are charac-
terized by striated cuticular ornamentation. Initially, the secreted nectar formed
vesicle-like protuberances under the cuticle. e epidermal and parenchymal cells
contain numerous plastids, mitochondria, dictyosomes, ER cisterns, and vesicles
fused with the plasmalemma, which indicates granulocrine nectar secretion.
Keywords
Galanthus nivalis; ower; breeding system; pollen mass; nectar production; struc-
ture of nectary
Introduction
e area of occurrence of Galanthus nivalis L. includes Western, Central, and Eastern
Europe, the mountains of the Crimea and the Caucasus as well as Minor Asia. G.
nivalis is a species characteristic of Southern European mixed deciduous forests, par-
ticularly dry-ground forests of the association Carpinion betuli illyricum found in the
Balkan Peninsula [1]. is species is considered to be an indicator plant in phenologi-
cal studies in many European countries: Austria, Bosnia and Herzegovina, Croatia,
the Czech Republic, Germany, Lithuania, the Netherlands, Slovakia, and the UK, in
which the “rst owers open” stage is taken into consideration [2].
In Poland G. nivalis is found in the south, in lower mountainous locations, in the
central part of the country as well as in the Lublin region (Lubelszczyzna) and in the
Wielkopolska region. It is worth stressing that the northern limit of the range of G.
nivalis runs through Poland where it is a species characteristic of mesophilic decidu-
ous forests of the order Fragetalia sylvaticae [3]. Galanthus nivalis was fully protected
in Poland during the period 1946–2014 as a rarely encountered species. Since 2014 it
has been partially protected [4].
DOI: 10.5586/asbp.3486
Publication history
Received: 2015-08-07
Accepted: 2016-01-25
Published: 2016-03-18
Handling editor
Elżbieta Bednarska-Kozakiewicz,
Faculty of Biology and
Environmental Protection,
Nicolaus Copernicus University
in Toruń, Poland
Authors’ contributions
EWC: research designing; EWC,
MC: microscopic analysis,
writing the manuscript,
observation of owering,
collection and analysis of nectar;
photographs: MC
Funding
This research was supported by
the Polish Ministry of Science
and Higher Education as part of
the activities of the University of
Life Sciences in Lublin.
Competing interests
No competing interests have
been declared.
Copyright notice
© The Author(s) 2016. This is an
Open Access article distributed
under the terms of the Creative
Commons Attribution License,
which permits redistribution,
commercial and non-
commercial, provided that the
article is properly cited.
Citation
Weryszko-Chmielewska E,
Chwil M. Flowering biology and
structure of oral nectaries in
Galanthus nivalis L. Acta Soc
Bot Pol. 2016;85(1):3486. http://
dx.doi.org/10.5586/asbp.3486
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2 of 20© The Author(s) 2016 Published by Po lish Botanical Societ y Acta Soc Bot Pol 85(1):3486
Weryszko -Chmielewska an d Chwil / Flowering and nec taries in Galanthus nivalis
In Poland the owering period of G. nivalis lasts from February to April [3]. Aer
winter, the owers of G. nivalis are one of the rst sources of nectar and pollen for
insects. e bell-shaped owers of this species exhibit structural adaptations to the
thermal conditions prevailing in early spring [5,6]. e colored markings in the form
of green spots and stripes on the tepals as well as the scent and nectar produced by the
owers are adaptations to entomogamy [6,7].
e owers of monocot species are highly diverse in terms of the location and
structure of nectaries [8–10]. Some representatives of the Amaryllidaceae family, e.g.,
Narcissus [11], have septal nectaries, while in others the nectaries are located on the
tepals [10,12]. In the literature, no detailed data can be found about the structure of
the G. nivalis nectary. Szafer and Wojtusiakowa [13] reported that in this species nec-
tar is secreted via a nectary disk located on the surface of the inferior ovary. Daumann
[8] found two types of nectaries in this ower: one on the tepals and the other one,
which is disk-shaped, at the base of the ower. Kugler [7] noticed that the thin-walled
tissues of the nectary of G. nivalis were oen chewed by insects visiting its owers.
In our previous study, we found that the oral nectary in G. nivalis was located at
the top of the inferior ovary between the base of the tepals and the style and formed
a bright layer contrasting with the green receptacle fused with the ovary. is work
is a continuation of our earlier study on the micromorphology and anatomy of some
oral structures of this species [6].
Since G. nivalis is a species that attracts interest from the point of view of phyto-
phenology, whose owering is studied in the aspect of its response to climate warm-
ing [14,15], we undertook a study on the ecology of owering of this species in the
conditions of central-eastern Poland. e aim of the research presented in this paper
was to determine the lifespan of the G. nivalis ower, stigma receptivity, the period
of pollen and nectar presentation as well as the mass of pollen and nectar, and also to
evaluate the breeding system of this species. A large part of this paper is devoted to the
results of the investigations of the topography, micromorphology, and the anatomical
and ultrastructural features of the nectary. is study was undertaken to explain the
divergent data found in the literature as regards the location of the nectary in the G.
nivalis ower and to ll the gap in the information concerning the structure of the
nectary gland in this species.
Material and methods
The ower stages in the study
Galanthus nivalis L. plants were supplied by the Botanical Garden of the Maria Curie-
Skłodowska University in Lublin. We selected 10 buds on each of the experimental
plants planted in three districts of the city in order to investigate the ower lifes-
pan and stigma receptivity. e bud swell stage was considered to be the beginning
of owering. We conducted a morphometric analysis of the individual parts of the
ower. We determined the nectary size and stigma receptivity at dierent ower de-
velopment stages: closed bud (I), bud swell (II), bud burst (III), partially open ower
(IV), and fully open ower (V; Fig. 1d–h). At the individual stages, we measured the
perianth length as well as the nectary diameter and height (N= 16).
Stigma receptivity and breeding system
We examined stigma receptivity using the Peroxtesmo Ko test which causes blue or
bluish green coloration within two minutes aer a reaction with peroxidase present in
the tissues of a receptive stigma [16]. e characteristics of the breeding systems and
the outcrossing level of G. nivalis were determined using the method recommended
by Cruden, as described by Dafni [16], aer the determination of the number of pol-
len grains (P; N = 6) and ovules (O) produced by the ower (N = 20).

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Weryszko -Chmielewska an d Chwil / Flowering and nec taries in Galanthus nivalis
Study of nectar and pollen grains
e mass of nectar was determined at bud burst (III) and fully open ower (V) stages
using the method by Jabłoński [17]. For the analysis, we collected 6 nectar samples
from 8–16 owers at either stage. e nectar sugar content was measured using a
refractometer.
e mass of pollen produced by a stamen was measured using the method by
Warakomska [18]. Samples (N = 10) included 50 mature stamens collected at bud
swell stage (II) before the onset of pollen shed.
Nectary structure
To examine the nectary structure, owers were collected at the stages II and V. To
perform preliminary analysis by light microscopy, hand-made longitudinal sections
of fragments of the ovary with a nectary were prepared and stained with Lugol’s solu-
tion and Sudan III in order to detect starch and lipids [19].
Permanent microscope slides
Nectaries sampled from fresh owers were xed in 4% glutaraldehyde at a tempera-
ture of 21°C for 6 hours and in 0.1 M phosphate buer, pH 7.0, at 4°C for 24 hours.
e samples were washed with phosphate buer and treated with 1% OsO4 at 0°C for
2 hours. Nectary fragments were stained with a 0.5% water solution of uranyl acetate.
Next, the plant material was dehydrated in ethanol, embedded in Spurr Low Viscosity
resin, and polymerized at 60°C.
Light microscopy (LM)
For observation of the nectary cells, semi-thin longitudinal 0.8–1-µm thick sections
were stained with toluidine blue (0.25%) [19] and periodic acid-Schi ’s reagent (PAS)
staining was also performed [20]. e slides prepared in this way were analyzed under
a Nikon Eclipse 400 light microscope.
Fluorescence microscopy (FM)
Aer addition of uorochrome (a 0.01% solution of auramine O) [21], hand-made
sections from fresh nectary fragments and semi-thin sections from the xed material
were embedded in a 50% glycerol solution. e observations were carried out under a
NIKON Eclipse 90i uorescence microscope equipped with a uorescein isothiocya-
nate – FITC lter (excitation light 465–495 nm), a DAPI lter (365–461 nm), and a
barrier lter (wavelength 515–555 nm).
Transmission electron microscopy (TEM)
Ultrathin 70-nm thick sections were cut with a Reichert Ultracut S microtome. ey
were stained with an 8% uranyl acetate solution in 0.5% acetic acid for 40 minutes;
the sections were washed twice with distilled water (10 minutes) and contrasted with
the Reynolds reagent (15 minutes) [22]. e slides were dried aer rinsing with water.
e nectary cell ultrastructure in the pre-secretory phase and at full nectar secretion
was observed under a FEI Tecnai G2 Spirit transmission electron microscope.

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Weryszko -Chmielewska an d Chwil / Flowering and nec taries in Galanthus nivalis
Scanning electron microscopy (SEM)
Aer dehydration in increasing concentrations of acetone, i.e., 15, 30, 50, 70, 90, and
99.5% (anhydrous acetone was used twice) for 15 minutes in each concentration, the
xed plant samples were critical-point dried in liquid CO2 in an Emitech K850 dryer
and sputter-coated with gold using an EMITECH K550X sputter coater. e analysis
of the surface of the nectary epidermis was performed under a Tescan Vega II LMU
scanning electron microscope.
Results
Flowering period
In 2015 the owering of G. nivalis lasted from 18 February to 23 March. Observations
carried out simultaneously in three parts of the city produced similar results. e av-
erage ower lifespan was 31 days (Fig. 2). On the rst day of owering, when swollen
buds were observed (Fig. 1e), the mean daily temperature was −0.8°C. e beginning
of senescence of the inner tepals, which took place at a temperature of +5.3°C, was
considered to be the end of owering. During the time of owering, the temperature
increased to +8.5°C and on most days it remained in the range of 4–7°C (Fig. 2).
We found stigmatic receptivity, using the Proxtesmo Ko test (Fig. 1j,k), already at
the closed bud stage (Fig. 1d,e) and it continued until the time of senescence of the
outer tepals (25 days). Over the same time, we observed anther dehiscence and the
presentation of pollen; we also found scent emission by the tepals (sensory method)
and nectar secretion (Fig. 2). Senescence of the outer tepals occurred 5 days earlier
than in the case of the inner tepals, and the scent emitted by the inner tepals was
clearly detectable even when the outer tepals had already lost their attractive appear-
ance. Senescence of the tepals began on 15 March and in some owers the whole
perianth with the stamens fell o already aer 8 days, while in some other ones it
remained desiccated for 27 days (until 10 April). During this period, the temperature
maintained at about 13°C for several days and dierent sized fruit primordia formed
from the ovary of the pistil.
Floral morphology and pollen production
e bell-shaped owers of G. nivalis were composed of 3 white boat-shaped outer
tepals with a length of about 20 millimeters and three inner tepals about 10 mm long
(Tab. 1). e inner tepals were marked with V-shaped green marks on the abaxial
surface and green stripes on the adaxial surface. On cloudy weather the tepals most
frequently remained folded, whereas on sunny days we could observe substantial
opening of the perianth, in particular the outer tepals (Fig. 1a–c). e androecium
consisted of six stamens with an average length of 7 mm, arranged in two whorls.
e large anthers accounted for 80% of the stamen length (Tab. 1). e pistil had an
inferior ovary with an average height of 4.34 mm and a style that reached a length of
8 mm and protruded above the anthers (Fig. 1i). e distance between the stigma and
the apical part of the anthers was 1.5–2 mm. We found dierent numbers of ovules
(33–49) in the ovaries.
e mass of pollen per stamen was 0.66 mg, while the average mass of pollen pro-
duced by one ower was 4 mg (Tab. 1). We determined the average number of pollen
grains produced per stamen, which was 62 850. At an air temperature of 7–8°C, we
observed bees collecting nectar and pollen from the G. nivalis owers (Fig. 1c). Pollen
loads formed from the pollen of this species were orange.

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Weryszko -Chmielewska an d Chwil / Flowering and nec taries in Galanthus nivalis
Fig. 1 Flowers and fragments of the oral parts of Galanthus nivalis (i–k LM; m FM; l,n,o SEM). a–c
Campanulate owers with visible green marks on the tepals. d–h Dierent ower development stages:
closed bud (stage I; d); bud swell (stage II; e); bud burst (stage III; f); beginning of owering (stage IV;
g); ower open (stage V; h). i Flower at bud swell aer cutting tepals (stage II); the nectary is visible on
the surface of the inferior ovary located between the base of the stamens and the base of the pistil’s style
(arrow). j Pistil’s style; visible a receptive stigma aer using Peroxtesmo Ko test (two arrows) and pollen
grains (arrowheads) on the style surface (stage II). k Papillae on the stigma (asterisks); Peroxtesmo Ko
test. l Papillae on the abaxial surface of the epidermis of the inner tepals; visible longitudinal cuticular
striae; SEM. m Cross sectional view of the inner tepal. n Characteristic ribs and numerous stomata
(double-headed arrow) in the adaxial epidermis of the inner tepals. o Stoma and cuticular ornamenta-
tion consisting of twisted striae on the surface of other epidermal cells (inner tepals).

6 of 20© The Author(s) 2016 Published by Po lish Botanical Societ y Acta Soc Bot Pol 85(1):3486
Weryszko -Chmielewska an d Chwil / Flowering and nec taries in Galanthus nivalis
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Fig. 2 Dierent stages of anthesis (31 days) of Galanthus nivalis owers.
Tab. 1 Morphological characters of Galanthus nivalis owers and pollen weight
per ower.
Investigated character Min.–max. Mean SD
Outer tepal length mm 17.0–24.0 20.91 2.07
Inner tepal length 8.5–12.0 10.34 1.77
Anther length 4.0–6.5 5.53 0.62
Stamen length 6.0–7.5 6.97 0.53
Style length 6.5–9.5 7.98 0.76
Ovary height 3.0–6.0 4.34 0.99
Pollen mass per stamen mg 0.61–0.73 0.66 0.06
Pollen mass per ower 3.66–4.38 3.96 0.37

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Weryszko -Chmielewska an d Chwil / Flowering and nec taries in Galanthus nivalis
Nectary size and nectar production
We examined the size of the nectaries
at dierent ower development stages
(Fig. 1d–h). We found that at the suc-
cessive stages (I–V) the nectary size
increased with the elongation of the
perianth. e perianth length (13.1
mm) at closed bud stage (I) was 60%
of the perianth length at stage V (21.8
mm). On the other hand, the nectary
diameter at stage I reached as much as
91% of the nectary diameter at fully-
open ower stage (1.96 mm). Slightly
larger dierences in the nectary height
were found at dierent ower opening
stages. At stage I, the nectary height was
80% of its height at stage V (484 µm; Tab. 2). e nectar produced in the ower is
visible already in a closed bud (stage II) and accumulates in the space between the
staminal laments and the pistil’s style, seeping up to the height of the anthers where
it comes into contact with their adaxial surface (Fig. 1i). Nectar secretion occurred for
about 2/3 of the owering period (20 days).
We measured the mass of nectar secreted by the owers bagged to prevent insect
visitation at ower development stages III and V. We found much more nectar at
stage III (2.66 mg/ower) than at stage V (1.4 mg/ower). At stage III, the nectar was
characterized by lower sugar content (16.2%) than at stage V (22.9%), which may be
attributable to the gradual loss of water by the nectar with the tepals open. However,
due to the larger amount of nectar secreted the mass of sugars oered to insects by
the ower at bud burst stage (III) was higher than the mass of sugars in the nectar in
open owers (V; Ta b. 3 ).
Scent emission
e abaxial (outer) surface of the outer and inner tepals emitting a delicate scent was
covered with dierent sized papillae with characteristic surface striation (Fig. 1l). e
adaxial surface of the inner tepals, covered with green striae (Fig. 1b), gave o a much
stronger scent. In the cross-sectional view of the inner tepals, ribs were visible on
the adaxial surface at the place of occurrence of the green stripes (Fig. 1m). Chlo-
roplasts occurred in the subepidermal layers of parenchyma, while the central part
of the tepals, beneath the ribs, was lled with air canals. SEM examination revealed
that the epidermal cells covering the adaxial surface were mostly isodiametric and
arranged in regular rows. Numerous stomata characterized by a wide opening of the
outer cuticular ledges were distributed among them (Fig. 1n,o). e cuticle found on
the surface of the epidermal cells formed a characteristic pattern composed of twisted
striae (Fig. 1o). e occurrence of numerous stomata is a trait characteristic of tissues
emitting a strong odor (osmophores).
Tab. 2 Bud or outer tepal length and nectary size at dierent development
stages of Galanthus nivalis owers; N = 16.
Nectary develop-
ment stages
Bud / outer tepal
length (mm)
Nectary diam-
eter (mm)
Nectary height
(µm)
Mean ±SD
I 13.1 ±1.8 1.79 ±0.20 390.5 ±4.0
II 16.2 ±1.4 1.86 ±0.31 438.3 ±59.5
III 16.9 ±0.9 1.92 ±0.20 441.0 ±70.6
IV 18.8 ±1.7 1.95 ±0.26 448.4 ±37.4
V 21.8 ±1.8 1.96 ±0.21 484.1 ±71.2
Tab. 3 Nectar production rate in Galanthus nivalis owers.
Flower devel-
opment stages
Nectar mass mg/ower
Nectar sugar percentage
(%)
Nectar sugar mass mg/
ower
min.–max. mean ±SD min.–max. mean ±SD min.–max. mean ±SD
III 1.75–3.67 2.66 ±0.62 15.0–17.5 16.2 ±0.9 0.28–0.59 0.43 ±0.10
V 1.00–2.14 1.40 ±0.32 20.5–28.0 22.9 ±1.8 0.23–0.50 0.32 ±0.08

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Weryszko -Chmielewska an d Chwil / Flowering and nec taries in Galanthus nivalis
Breeding system
In the biology of owering and pollination of plants, functional oral morphology is
of great importance, since it allows their sexual systems to be assessed. Tab. 4 presents
the characteristics of the owers of G. nivalis that enable the assessment of the breed-
ing system of this species. We calculated the outcrossing index (OCI) based on the
ower diameter which was 15–35 mm, depending on perianth opening, and based
on the estimation of the temporal and spatial separation of the stigma and anthers.
e value of OCI = 4 shows that in G. nivalis there is outcrossing and demand for
pollinators, but these owers are partially self-compatible. We also determined the
outcrossing level based on the P/O ratio (pollen grains : ovules). e number of pollen
grains produced per ower reached 377 100, whereas the average number of ovules
per ower was 40. us, the P/O ratio is 9427, which indicates xenogamy.
Nectary micromorphology
e nectary of the Galanthus nivalis owers (Fig. 3a) was located at the top of the in-
ferior ovary between the tepals and the style. e green whitish nectary layer was situ-
ated above the green tissues of the ovary (Fig. 3a,b). Nectary tissues formed conical
convexities between the laments and style (Fig. 3e, Fig. 4a) and circular concavities
lled with nectar at their bases (Fig. 3a,b).
e outer walls of the nectary epidermal cells viewed in SEM were characterized by
striated cuticular ornamentation, which was well visible already in the pre-secretory
phase. e undulating, parallel, densely arranged striae in adjacent cells oen exhib-
ited varied orientation. ey were more loosely arranged at the cell borders (Fig. 4b).
In the initial phase of nectar secretion, small vesicle-like convexities appeared among
the striae (Fig. 4c). eir number and size increased with the increasing secretory
activity of the nectary. e largest vesicles were formed at the borders of adjacent
epidermal cells (Fig. 4d,e). e nectar that accumulated under the cuticle led to the
formation of convexities and vesicles; the diameter of the latter typically reached 4–15
μm. Locally, the secretion residues formed an irregular layer on the epidermis surface
(Fig. 4e). No stomata were observed in the nectary epidermis (Fig. 4a). We also de-
tected nectar secretion within the epidermal cells of the basal part of the laments,
which may imply secretory activity of these oral structures (Fig. 4f).
Nectary anatomy
Prior to nectar secretion, the nectary epidermis exhibited light green epiuorescence
aer the treatment with auramine O and the use of the FITC lter (Fig. 3d), or bright
blue epiuorescence when the DAPI lter was used (Fig. 3c). As shown by uores-
cence microscopy, the layer of the nectary glandular cells was distinguished from
the ovary and receptacle tissues by distinct uorescence (Fig. 3e). e outer walls of
the epidermal cells exhibited the presence of a cutinized layer aer the application
Tab. 4 Outcrossing index and breeding system of Galanthus nivalis.
Flower characteristics Results Value s
1. Flower diameter >6 mm 3
2. Temporal separation of anther dehiscence and stigma receptivity homogamy 0
3. Spatial positioning of the stigma and anthers spatially separated 1
OCI (outcrossing index) 4
Breeding system partially self-compatible, outcrossing, demand for pollinators
e eects given in the table were calculated according to Dafni [16] tests.

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of Sudan III (Fig. 3f, Fig. 5c). Nectar drops were visible in the cross sections of fresh
nectary tissues (Fig. 5c), which corresponds with our SEM observations. e nectary
gland in the owers of G. nivalis is composed of one layer of epidermal cells, 7–10
layers of glandular parenchymal cells, and several layers of sub-nectary parenchyma
(Fig. 5a). ese layers form a 414–616 μm thick nectary ring (mean 484 μm).
Epidermal cells
e height of the nectary epidermis was 13–21 μm. e outer cell wall was substan-
tially thicker than the anticlinal walls and the periclinal inner wall (Fig. 5a–g, Fig. 6a).
e part of the wall in contact with the environment was characterized by intense
Fig. 3 Location of the nectary in G. nivalis owers (a,b,f LM; c–e FM). a Nectary located at the apex of
the inferior ovary between the bases of the tepals and the style; visible projections of whitish nectarifer-
ous tissue between the laments. b Nectary disc with visible shimmering nectar (arrow) accumulated
in the concavities surrounding the laments and the style. c,d Longitudinal section of the oral nectary
at the initial phase of nectar secretion; visible outer layers of the gland, blue (c) and green (d) uores-
cence of the gland layers (stage II); FM. e More intense uorescence of the nectary layer in an older
ower (stage IV); FM. f Protrusion of the nectary tissues between the style base and the lament; visible
orange-stained cutinized layers. c–e Aer application of the DAPI lter (c), aer addition of auramine
O and using the FITC lter (d), autouorescence (e), aer treatment with Sudan III (f). f – lament;
n – nectary; o – ovary; s – style; t – tepal.

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Fig. 4 Surface of the oral nectary (n) and a lament of Galanthus nivalis (SEM). a Protrusion of the nectary tissues (n)
between the style base (asterisk) and the lament (double asterisk). b Surface of nectary epidermal cells with substan-
tially dense cuticular striae (stage I). c,d Fragments of the outer walls of nectary epidermal cells with striated cuticular
ornamentation and dierent-sized nectar-containing vesicles (arrows; stage III). e Surface of nectary epidermal cells
with vesicles (arrows) and secretion residues (double arrow). f Surface of lament epidermal cells with nectar-containing
vesicles (arrow; stage III).

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Fig. 5 Longitudinal sections of the Galanthus nivalis nectary (a–e LM; f–h FM). a Nectary located by the
style; visible small epidermal cells, large parenchymal cells (p) and small subglandular parenchymal cells.
b Outer layers of the nectary tissues; epidermal cells with a thick outer cell wall and undulating cuticle and
three layers of strongly vacuolated nectary parenchyma. c A part of nectary epidermal cells from fresh plant
material; visible orange-stained cutinized layer of the outer cell wall with a drop of nectary on the surface
(double arrow). d,e Nectary epidermal and parenchymal cells; visible pits in the walls (arrows) and secretion
accumulated beneath the cuticle and in the intercellular spaces (asterisk). f Nectary epidermal cells, visible
strongly thickened outer cell walls of the epidermis, large nuclei in the central part of the protoplasts. g Nectary
epidermal cells (stage III), visible uorescence of the nectar accumulated beneath the cuticule (arrowheads),
along the radial epidermal walls (double headed arrow) and in the subepidermal intercellular spaces (two ar-
rows). h Nectary parenchymal cells, visible strong vacuolation, large nuclei and pits in the cell walls (arrows).
Aer treatment: toluidine blue (a,b), Sudan III (c), PAS reaction (d,e), auramine O, and using the FITC lter
(f–h). e – epidermal cells; p – nectary parenchyma cells; s – style.

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undulation (Fig. 5d,e). In preparations from fresh plant material, a few light green
plastids were observed in the epidermal cells. e cell nuclei in this tissue were char-
acterized by large sizes and a lobular shape. ey were located in the central part of the
cell or close to the center (Fig. 5a,b,d,e, Fig. 6a). e cytoplasm was rich in granulari-
ties (Fig. 5b–e) which emitted distinct uorescence (Fig. 5f,g).
Nectary parenchyma
e nectary parenchyma was characterized by large, highly vacuolated cells, whose
cytoplasm and nuclei were most frequently located parietally (Fig. 5a,b,d). e pa-
renchymal cell walls exhibited numerous simple pits (Fig. 5d,e,h). In many regions of
the tissue, we observed large and very large intercellular spaces (Fig. 5a,b,e, Fig. 7a).
Fig. 6 Galanthus nivalis nectary epidermal cells (stage II); TEM. a Cells with a thick outer cell wall, dier-
ent-sized vacuoles, lobular cell nuclei, small plastids, and numerous mitochondria. b Fragment of the outer
cell wall and parietal cytoplasm; a secretion-containing vesicle incorporated into the plasmalemma (arrow),
mitochondrion, rough endoplasmic reticulum, subcuticular spaces (asterisks). c Fragment of the outer cell
wall with a cuticle layer on the surface, micro-channels in the cuticle (two arrows). d Parietal cytoplasm with
rough endoplasmic reticulum, Golgi apparatus, and numerous secretory vesicles (double headed arrows).
c – cuticle; cw – cell wall; G – Golgi apparatus; m – mitochondria; n – nucleus; p – plastid; RER – rough
endoplasmic reticulum.

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Fig. 7 Galanthus nivalis nectary parenchymal cells in dierent stages (TEM). a,b Stage II; d–f Stage V. a Cell
from the subepidermal layer with pleomorphic plastids, small vacuoles, numerous mitochondria, lobular cell
nucleus, dierent-sized vacuoles, numerous endoplasmic reticulum proles, and probably secretions in the
intercellular space (asterisk; stage II). b Fragment of the cytoplasm with numerous Golgi apparatuses, secre-
tory vesicles (arrow head), lipid bodies, rough endoplasmic reticulum, and vesicular structures (arrows) in
the vacuoles (stage II). c Mitochondria arranged in groups close to the cell wall, the plastid, and the nucleus
(stage V). d,e Plastids with electron-dense content, distinct thylakoids without the developed granal part and
with small plastoglobules (double-headed arrow); many plastids exhibit bright regions emerging through
thylakoid stratication (two arrows) or a convexity in the outer region of the plastid, two plastids in close
contact (e), pits (arrow) in the walls (d; stage V). f Pits (arrow) in the cell walls (cw) and invaginations of the
plasmalemma (double arrows; stage V). cw – cell wall; ER – endoplasmic reticulum; G – Golgi apparatus;
l – lipid body; m – mitochondria; n – nucleus; p – plastid; RER – rough endoplasmic reticulum; v – vacuole.

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e parenchyma cells contained small plastids. e PAS staining did not reveal starch
grains before and during nectar secretion.
Using a variety of microscopic techniques, we observed the presence of nectar
in the intercellular spaces, beneath the cuticle layer in the outer epidermal walls
(Fig. 5d,e). In turn, induction of tissue uorescence revealed secretion of signicant
amounts through the nectary epidermis radial walls (Fig. 5g).
Nectary ultrastructure
e nectary epidermis. e epidermal cells had a 4–5 µm-thick outer wall with a
distinct, relatively thin, undulating cuticle layer reaching a thickness of ca. 0.47 µm
(Fig. 6a). e height of the cuticular striae (cuticle convexities) was 1.1–1.3 µm
(Fig. 6c). A very thin cuticular layer raised by nectar secreted was visible on the outer
wall surface (Fig. 6b). e protoplasts of the epidermal cells were characterized by
dense cytoplasm as well as numerous mitochondria and plastids. e lobular cell
nuclei contained dark heterochromatin (Fig. 6a). e plastids had irregular shapes
and oen exhibited elongation. e mitochondria were located singly or in groups
of several. eir internal structure exhibited distinct cristae (Fig. 6b). Numerous
RER proles and secretory vesicles were visible in various regions of the cytoplasm.
Frequently, the RER systems were located near the cell walls (Fig. 6b,d). e Golgi
apparatuses were accompanied by large dictyosomal vesicles (Fig. 6d). Local invagina-
tion of the plasmalemma gave rise to periplasmic spaces. Additionally, the fusion of
secretory vesicles with the plasmalemma, accompanied by the release of nectar to the
aforementioned space was also observed (Fig. 6b).
e nectary parenchyma cells. e walls of the glandular parenchyma cells were
characterized by varied thickness (Fig. 7a). ey contained numerous pits with
plasmodesmata (Fig. 7d,f). Plasmalemma undulations formed periplasmic spaces
(Fig. 7f). e intercellular spaces contained probably secretion (Fig. 7a). e electron-
dense cytoplasm had multiple plastids and mitochondria. Irregular-shaped cell nuclei
contained electron-dense chromatin and an osmophilic nucleolus (Fig. 7a,c). e mi-
tochondria were arranged singly or in groups and had well-developed cristae. Numer-
ous Golgi apparatuses and secretory vesicles were present in the cytoplasm (Fig. 7b).
e plastids contained in the glandular parenchyma cells were usually elongated. ey
were characterized by a system of irregularly arranged thylakoids interspersed with
transparent zones (Fig. 7c–e). A dark homogeneous stroma was visible in the plastids
with a lesser degree of elongation (Fig. 7d). Some plastids contained dark plastoglob-
ules associated with thylakoids. e closely located plastids were linked via convexi-
ties of the envelope portions and external parts of the plastids (Fig. 7e). We observed
mitochondria connected with the plastid too (Fig. 7c). Additionally, lipid bodies were
detected in the cytoplasm (Fig. 7b).
Release of nectar onto the epidermis surface
e initial phase of nectar secretion was signaled by the appearance of tiny vesicles
on the surface of the outer epidermal cell wall (Fig. 4c). Intense nectar secretion
caused bulging (Fig. 4d) and detachment of the cuticle from the cell wall fragments
located underneath and the formation of nectar-lled subcuticular spaces (Fig. 5d,e).
e largest nectar-lled protuberances were observed in the areas of contact of two
or more epidermal cells (Fig. 5e,g). Locally, the cuticle was strongly dilated and the
cuticular striae were stretched (Fig. 5e). e release of nectar onto the surface of epi-
dermal cells proceeded without cuticle rupture, as suggested by observation of nec-
tar droplets above the cuticle layer (Fig. 5c). Signicant amounts of nectar lling the
spaces between the epidermis layer and glandular parenchyma were also visible aer
application of the PAS staining protocol (Fig. 5d,e), and by uorescence microscopy.
e nectar was also accumulated between the anticlinal walls of the epidermal cells,
which were partially detached under the cuticle (Fig. 5g).

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Discussion
Flowering
In Central Europe the onset of owering of G. nivalis marks the beginning of the rst
phenological season [14] termed as early spring [1]. e time of blooming in early
spring is greatly aected by the thermal conditions prevailing in the previous months
[23]. In 2015 we recorded the beginning of G. nivalis owering in Lublin on 18 Febru-
ary, while the end of owering on 23 March (34 days). In 2012, on the other hand,
according to Żuraw et al. [24] the owering of this species in the same city lasted from
6 to 23 March (18 days). Maak and von Starch [14] demonstrated large dierences in
the time of blooming of G. nivalis in northern Germany at 74 study sites over the pe-
riod 1971–1990. In their research, half of the data obtained showed that the beginning
of G. nivalis owering occurred before 28 February, whereas at other study sites it was
recorded between the 38th and 85th day of the year. Budnikov and Kricsfalusy [25]
report that in the East Carpathians the rst G. nivalis owers bloom from the end of
February to the third 10 days of March, depending on the altitude (130–1320 m). e
data given by the above-mentioned authors are in agreement with the G. nivalis ower-
ing period reported for dierent regions of Poland (February–April) [3]. On the other
hand, the beginning of owering of this species is observed much earlier in the UK, as
in the previous years it occurred already in January, on average 25 January [15].
Breeding system
Our study shows that the lifespan of the G. nivalis ower was 31 days in 2015. It should
be noted that it is an exceptionally long owering duration for a ower. Chudzik and
Śnieżko [26] found that the lifespan of unpollinated owers of this species was 20–30
days, whereas pollinated owers bloomed much shorter (3–15 days). In the case of
the owers observed in our study, no such relationship can be demonstrated as fruit
formed from the ovary in all the owers aer the perianth had dropped o.
e owers of G. nivalis can be considered to be homogamous, since we recorded
stigma receptivity and pollen shed at the same time. is character indicates the pos-
sibility that the G. nivalis ower can be pollinated with its own pollen, which may also
be conrmed by the very long owering period and the long persistence of stigma
receptivity as well as by the fact that fruit was set in all owers. e outcrossing index
(OCI), calculated according to the method given by Dafni [16], also shows that the
owers of G. nivalis are partially self-compatible.
Nevertheless, the characters clearly manifested in the owers which indicate ento-
mophily (a large perianth, scent, colored nectar guides, nectar) were also conrmed
by the outcrossing index (outcrossing, demand for pollinators) and based on the de-
termination of the outcrossing level (xenogamy). Chudzik et al. [27] revealed that
when the owers of G. nivalis were pollinated with their own pollen the number of
seed-setting was half lower than in the case of pollination with foreign pollen.
Apicultural value
Pollen and nectar production by plants that bloom earliest in the growing season is of
great importance to bees and other insects seeking food already during early spring
in order to replenish stores used during the winter [28]. e owers of G. nivalis with
their large stamens [6] produce signicant amounts of pollen (4 mg), comparable to
the mass of pollen in some species of the genus Rosa that produce an androecium of
numerous stamens, e.g., R. canina [28]. e nectar contained in these owers (2.66
mg), even though its amount is not evidence of abundant nectar production by this
species, can be a very valuable source of early spring nectar for insects if these owers
occur in great numbers per unit of area.

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Location of the nectary
Our investigations show that the oral nectaries of G. nivalis are located at the top of
the inferior ovary between the tepals and the style base. is location corresponds to
the type of “gynoecial nectaries” and the group of “ovarian nectaries”, according to the
data provided by Bernardello [10].
e literature data indicate that oral nectaries in monocotyledons represent two
main types, i.e, septal and perigonal. Septal nectaries, e.g., present in many represen-
tatives of Asparagales and Liliales [10,12,29], are common in this class of plants [9].
e perigonal type comprises staminal and tepalar nectaries [30,31].
In plants of the family Amaryllidaceae, which produce an inferior ovary, septal
nectaries are the prevalent type [11,12,29,31]. However, Rudall [29] reports that some
species of this family are devoid of septal nectaries (e.g., Cyrtanthus). Bernardello [10]
regards tepal nectaries as the characteristic type in Amaryllidaceae. In turn, Daumann
[8] as well as Dafni and Werker [32] found in Sternbergia (Amaryllidaceae) three
types of nectaries: tepal and staminal nectaries, apart from septal nectaries. In other
representatives of Amaryllidaceae, various authors also report a dual location of the
nectary in the owers. Daumann [8] found that in species of the genus Galanthus two
types of nectary occur: one on the inner tepals, while the other one, which is disk-
shaped, at the base of the ower. Dahlgren et al. [9] also report that in the owers of
Galantheae tribus nectar is secreted in the distal part of the inner tepals and in the
disk at the ower base. In our previous investigations focused on the micromorphol-
ogy of elements of the G. nivalis perianth, we did not however nd nectaries in the
inner tepals [6]. Our study presented in this paper showed that the nectary gland in
the owers of the above-mentioned species is only located at the tip of the ovary, at
the base of the stamens. erefore, our results are only partially in agreement with the
data contained in the papers of other authors concerning the location of the nectary
in the owers of Galanthus nivalis.
Scent emission
Additionally, we drew attention to the more intense scent produced by the inner te-
pals than that of the outer tepals. We found that the structure of the inner tepals had
the characteristics of the organs of odor-producing tissues (osmophores). We showed
that their adaxial epidermis had numerous stomata and the upper surface was in-
creased by the folds of the outer tissues, which facilitates the emission of an odorous
secretion. Vogel [33] demonstrated that in some plants at the time of active emission
of odorous compounds intense transpiration occurs during which stomata open wide.
In G. nivalis the stomata in the epidermis of the tepals were mostly open.
Numerous stomata were earlier observed on the folded surface of the osmophores
in Amorphophallus rivieri [34]. Kugler [7] also drew attention to the release of odorous
compounds by the inner tepals of Galanthus nivalis. Numerous stomata occurred only
on the adaxial ribbed surface that emitted a strong odor, while the abaxial surface was
covered with dierent sized papillae. e results of our study concerning the presence
of stomata in the adaxial epidermis of the inner tepals of G. nivalis are contradictory
to the ndings of Aschan and Pfanz [35] who report that stomata are lacking in the
tepals of this species in both epidermal layers. However, the photosynthetic activity
of the green parts of the tepals which emit a scent, as shown by the above-mentioned
authors, even though it accounted for one fourth of the fully developed leaves, can
be important for the supply of major substrates necessary in the metabolic pathway
leading to the production of odorous substances.
Nectary structure
e outer wall of the epidermal cells of the G. nivalis nectary was covered by an in-
tensely undulating layer of the cuticle. Our observations carried out during the nectary
secretory phase demonstrated that the cuticle in this species was strongly stretched by
the secreted nectar; however, it did not rupture. In G. nivalis Kugler [7] also observed

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Weryszko -Chmielewska an d Chwil / Flowering and nec taries in Galanthus nivalis
stretching of the cuticle on the nectary surface by the accumulating nectar and did
not nd the cuticle to rupture, either. We observed microchannels in the cuticle. is
may suggest that this layer in the nectaries is permeable to nectar. e occurrence of
microchannels in the nectary cuticle has also been observed in other species by Nepi
[36]. is author reported that in a majority of nectaries the cuticle was continuous
and usually cracked in septal nectaries.
Numerous analyses of the anatomy of the nectary have demonstrated that the pa-
renchyma of this gland is composed of small isodiametric cells [36–39]. In contrast,
the G. nivalis nectary was found to consist of large parenchymal cells which were
highly vacuolated during the nectar secretion phase. e tissue was characterized by
the presence of large intercellular spaces. e fact of the occurrence of collenchyma in
the subepidermal layer of the nectary in the owers of the orchid Maxillaria coccinea
can also indicate great diversity in the structure of nectariferous tissues [41].
e G. nivalis nectary was green whitish. In the plastids of the epidermal and pa-
renchymal cells, we did not nd granal thylakoids typical of chloroplasts. Our obser-
vations are in agreement with the data provided by Nepi [36] who claims that most
typically small amounts of light reach the nectaries and, although they are green, their
thylakoids and grana are underdeveloped. Lüttge [42] also found that green plastids of
the nectaries oen lacked granal thylakoids in which photosystem II is located.
e plastids in the G. nivalis nectaries observed in our study were characterized by
the presence of electron-dense stroma, numerous unfused thylakoids and plastoglob-
ules located in their close vicinity. is type of structure is similar to the structure of
tubular chromoplasts, distinguished by various authors [43,44]. e plastids observed
by us in the nectary cells are similar to some extent to chromoplasts occurring in the
white parts of the tepals of this species investigated by Ščepánková and Hudak [5].
However, the unfused thylakoids in the plastids of the nectaries had a more regular
arrangement than that described for the plastids in the tepals.
In the plastids of the G. nivalis nectaries, there were local protrusions that were in
contact with the adjacent plastids. We also observed numerous mitochondria located
in close vicinity of the plastids. Interconnections between plastids have been found in
the cells of many plant species [44]. In cells of tomato, plastid complexes were found
and they comprised the main parts of the plastid body and its stromules [45,46]. It has
been shown that there is an exchange of protein molecules between plastids of higher
plants [47]. e studies of other authors reveal that plastids and mitochondria located
in close vicinity to one another can exchange metabolites [48]. e above-cited au-
thors suggest that plastids and mitochondria may have a synergistic eect on plant
growth and development.
e protrusions found in the plastids of the G. nivalis nectaries can also be ad-
aptations to lower temperature conditions. Giełwanowska et al. [49] as well as
Giełwanowska and Szczuka [50] considered dierent deformations (pockets, lacunae)
in plastids of the leaves of Antarctic plants to be adaptations to extreme conditions.
We observed spherical osmophilic inclusions in the parenchymal cells of the nectar-
ies. Similar inclusions of various sizes were observed in Antarctic plants by the above-
mentioned authors.
We observed numerous plastids, mitochondria, dictyosomes, ER cisterns, and
abundant vesicles in the epidermal and parenchymal cells of the G. nivalis nectary. In
many sites, the vesicles were fused with the plasmalemma, which may indicate nectar
secretion in the granulocrine system. Granulocrine nectar secretion has been demon-
strated in many other monocotyledons species of the genera Asphodelus, Brassavola,
Musa, Platanthera, and Strelitzia [51–53].
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