Protoplasma (2018) 255:1763–1776

https://doi.org/10.1007/s00709-018-1269-2

ORIGINAL ARTICLE

Micromorphological and histochemical attributes of flowers and floral

reward in Linaria vulgaris (Plantaginaceae)
Jacek Jachuła 1 & Agata Konarska 1 & Bożena Denisow 1

Received: 21 February 2018 / Accepted: 23 May 2018 / Published online: 3 June 2018
# The Author(s) 2018

Abstract
The self-incompatible flowers of Linaria vulgaris have developed a range of mechanisms for attraction of insect visitors/
pollinators and deterrence of ineffective pollinators and herbivores. These adaptive traits include the flower size and symmetry,
the presence of a spur as a Bsecondary nectar presenter,^ olfactory (secondary metabolites) and sensual (scent, flower color, nectar
guide—contrasting palate) signals, and floral rewards, i.e. pollen and nectar. Histochemical tests revealed that the floral glandular
trichomes produced essential oils and flavonoids, and pollen grains contained flavonoids, terpenoids, and steroids, which play a
role of olfactory attractants/repellents. The nectary gland is disc-shaped and located at the base of the ovary. Nectar is secreted
through numerous modified stomata. Nectar secretion began in the bud stage and lasted to the end of anthesis. The amount of
produced nectar depended on the flower age and ranged from 0.21 to 3.95 mg/flower (mean = 1.51 mg). The concentration of
sugars in the nectar reached up to 57.0%. Both the nectar amount and sugar concentration demonstrated a significant year and
population effect. Pollen production was variable between the years of the study. On average, a single flower of L. vulgaris
produced 0.31 mg of pollen. The spectrum of insect visitors in the flowers of L. vulgaris differed significantly between
populations. In the urban site, Bombus terrestris and Apis mellifera were the most common visitors, while a considerable number
of visits of wasps and syrphid flies were noted in the rural site.

Keywords Histochemical tests . Nectar and pollen rewards . Trichomes . Insect visitors

Introduction vegetative reproduction; the main stem is capable of forming

up to 100 secondary shoots and surviving up to 4 years
Linaria vulgaris (L.) Mill., a perennial herb, belongs to the (Newman and Thomson 2005a). Sexual reproduction also oc-
family Scrophulariaceae, according to classic taxonomy sys- curs. L. vulgaris is considered to be an obligate outcrosser and
tems (Cronquist 1981) or Plantaginaceae, according to the evolved several adaptations towards attracting insects, e.g.,
modern phylogeny system APG IV (Angiosperm Phylogeny well-noticeable flowers (Stout et al. 2000). However, the
Group). The species is native to temperate regions of Eurasia. flowers of L. vulgaris are zygomorphic, deeply spurred, with
However, it has also been successfully introduced to North closed access to the corolla throat (Fernández-Mazuecos et al.
America, Australia, New Zealand, and South Africa (CABI 2013). Such flowers are traditionally viewed as highly spe-
datasheet). L. vulgaris is now considered as an invasive spe- cialized (Stebbins 1970). Specialized flowers are however al-
cies in the USA and in Canada (Sing and Peterson 2011). The so visited by insect that do not fit Bpollination syndrome^ and
species occurs both in cultivated and uncultivated areas, but pollinator composition may differ considerably between pop-
grows especially vigorously in disturbed habitats (Ward et al. ulations (even located closely to one another) (Nepi et al.
2009). The spread of L. vulgaris populations is due to efficient 2003; de Merxem et al. 2009). Specific flower visitors are
especially sensitive to changes in pollinating fauna induced
Handling Editor: Hanns H. Kassemeyer
by habitat types, i.e., functional group of insect to plant spe-
cies can differ greatly between urban and suburban environ-
* Bożena Denisow ments compared to semi-natural and agricultural ones (Geslin
bozena.denisow@up.lublin.pl et al. 2013).
Flower specialization is a result of co-evolution between
1
Department of Botany, University of Life Sciences in Lublin, 15 plants and specialized pollinators (Cacho et al. 2010). The
Akademicka St., 20-950 Lublin, Poland

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1764 J. Jachuła et al.

plant-insect interactions are based on diverse signals per- (phenology, anthesis length), (ii) identified the pattern of dis-
ceived by insects, which have to learn to use different sensory tribution of trichomes on the calyx and corolla, (iii) tried to
channels to make the food search effective (Renner 2006). recognize the main chemical classes of metabolites present in
From the plant point of view, the floral shape and size, flower floral parts and pollen grains, and (iv) assessed the quantity of
arrangements, color, and/or odor are important for advertise- floral reward (nectar and pollen). In addition, we tried to eval-
ment of rewarding flowers ready for pollination (Fernández- uate whether the insect visitors are interested in L. vulgaris
Mazuecos et al. 2013; Sulborska et al. 2014; Balamurali et al. flowers; therefore, we made observations of the insect visitor
2015). activity and spectrum. We also examined whether there are
Floral trichomes—hair-like epidermal structures—have differences in insect visitors composition in highly specialized
been considered to play a role in plant-pollinator relations, flowers of L. vulgaris between two populations (rural and
e.g., signaling the pathway to the reward (Owen and urban).
Bradshaw 2011). In many Linaria species, corolla and calyx
are covered with trichomes—glandular and/or non-glandular
(Segarra and Mateu 2001; Saez and Crespo 2005). Petal tri- Material and methods
chomes may form clusters visible as a contrasting palate and
signal the pathway to the reward (e. g., Owen and Bradshaw Study area
2011). Glandular trichomes may produce different classes of
secondary chemicals, which are stored or volatilized at the The field observations of Linaria vulgaris (L.) Mill. were
plant surface to attract pollinators and/or defend against non- carried out in 2013–2014 on Lublin Upland (51° 15′ 44 ′ N,
effective insect visitors or even are important in plant resis- 22° 30′ 48′ E, SE Poland). Two populations (separated by
tance (Glas et al. 2012; Konarska 2017). approx. 10 km) were selected for the experiment. The first
A crucial role in pollinator attraction is attributable to nec- population was grown in a rural area (in Jastków; R-popula-
tar and pollen, which are considered as main constituents in tion), and the second population originated from an urban area
plant/animal interactions. In most angiosperms, nectar is an (in Lublin, U-population). Every year, we used plants from the
aqueous sugar-rich solution composed of three common same self-renewing population of the same experimental
sugars (sucrose, glucose, fructose) that acts as energetic re- patches (approx. 4–6 m2 each). The plants of both populations
ward for pollinators (Antoń and Denisow 2014; Denisow et were grown on loess soil at pH 6–7 in full-sun sites.
al. 2016). The nectar parameters, nectar volume, sugar con-
centration, and relative composition of sugars vary widely
across species (Chalcoff et al. 2017) and have an impact on Study methods
food selection by insect visitors or pollinators (Baker and
Baker 1983; Nicolson 2007; Rodríguez-Riaño et al. 2014). Flowering and insect observations
There is ample evidence that nectar production and the sugar
concentration in nectar is an environmentally and physiolog- The duration of flowering was recorded; the beginning of
ically related issue (e.g., Petanidou and Smets 1996). In L. flowering was defined when 2–5% of flowers were in com-
vulgaris, numerous nectarostomata are involved in the exuda- plete swelling and the end of flowering was identified when
tion process. Nectar of L. vulgaris is composed mainly of almost 90% of individuals finished blooming (=corolla
sucrose, glucose, and fructose; however, trace amounts of wilted). The life span of an individual flower was defined as
rafinose have also been detected (Nepi et al. 2003). As nectar the period between lower lip folding (=the start of palate pre-
is gathered in the deep corolla spur, the nectar guides present sentation) and corolla wilting. The intensity (=number) and
in the corolla are supposed to increase the efficiency of the spectrum of floral insect visitors were noted. Due to the long
pollination process (Stout et al. 2000; Vargas et al. 2010). blooming period of the species, the observations of floral in-
Pollen is the main source of proteins, lipids, sterols, vita- sect visitors were conducted in June, July, August, and
mins, and hormones and is a key component of balanced September. In each period, the survey was performed for
insect pollinator diet necessary to provide the required propor- two to three consecutive days at 1-hour intervals between
tion of nutrients (Filipiak et al. 2017). There is weak evidence 5.00 and 18.00 (GMT + 2 h). Each census of observation
that some pollen traits (odor, protein content) may be attribut- was 5–10 min long. During the observations, the weather
ed to whether the insect visitors/pollinators are inclined to conditions were as follows: daily temperature above 10 °C,
collect pollen or nectar or both (Dobson and Bergström wind speed < 10 km h−1 with no precipitation. In the case of
2000; Pacini and Hesse 2005; Denisow et al. 2018). very strong wind or rain, the observations were ceased and
The aim of this paper was to present floral features of L. completed on a subsequent day. All insect visitors were noted
vulgaris that can be attributable to the interaction with insect in each observation period. The observations of flowering and
visitors. In particular, we (i) examined the flowering biology insect foraging were conducted in each population (rural—R

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Micromorphological and histochemical attributes of flowers and floral reward in Linaria vulgaris... 1765

and urban—U). Due to difficulties in taxonomic identification of glandular trichomes was observed. Images were acquired
of insects in the field, only some insect visitors were identified with a digital camera Nikon Fi1 and NIS – Elements Br 2
to the species level. Insects were divided in several groups: software.
Apis mellifera, Bombus terrestris, other Bombus spp. (includ- Standard control procedures were conducted simultaneous-
ing B. lapidarius, B. hortorum, B. pascuorum, and B. ly for all the histochemical and fluorescence methods used,
sylvarum), Andrena sp., Vespula vulgaris, syrphid flies, other following the recommendations of the respective authors.
dipterans, and Lepidoptera. The identification of Bombus spp.
was based on Pawlikowski and Pawlikowski (2012). Scanning electron microscopy

Microscopic examinations Five samples of all flower parts (calyces, corollas, stamens,
pistils with nectaries) were fixed in a 4% glutaraldehyde so-
In 2014, the floral microstructure was examined in 2–3 day of lution in 0.1 M phosphate buffer (pH 7.0) for 12 h at room
anthesis in light microscopy (LM), fluorescence light micros- temperature. Later, the plant samples were rinsed in the same
copy (FLM), and scanning electron microscopy (SEM). buffer four times and dehydrated in ethanol series (30, 50, 70,
Samples for microscopic investigations were collected from 90, 95%), and subsequently three times in absolute alcohol.
flowers (n = 30) of different individuals (n = 10) from the rural After dehydration, the plant material was transferred to ace-
and urban populations. tone, dried at critical point in liquid CO2 using Bal-Tec CPD
030, and coated with gold using the Polaron SC 7640 sputter
Light microscopy coater. The number of nectarostomata within a 1-cm2 area of
the nectary epidermis and the length and width of ten stomatal
The height (at the highest point) and the external diameter of pores were measured using morphology software combined
fresh nectaries (n = 10) as well as the length of different types with scanning electron microscopy (SEM). The surface of
of trichomes located on the calyx and corolla (n = 20) were calyces, corollas, stamens, pistils, pollen grains, and nectaries
measured. were investigated and depicted at an accelerating voltage of
30 kV using a TESCAN/VEGA LMU scanning electron
Histochemistry microscope.

Fresh hand-made sections of calyces and corollas with tri- Quantity of floral rewards
chomes and pollen grains were tested using the following
histochemical tests: iodine iodide solution for starch and pro- Every year, nectar was sampled with a micropipette and col-
teins, Ruthenium Red for polysaccharides other than cellulose lected on five separate dates of the study period (Jabłoński
(Johansen 1940; Jensen 1962), Nile Blue for neutral and acid- 2002). Nectar production was determined during the peak of
ic lipids (Jensen 1962), Sudan III (Johansen 1940) and Sudan the L. vulgaris flowering period (i.e., in late June or the be-
Red for total lipids (Brundrett et al. 1991), ferric trichloride for ginning of July). Prior to nectar sampling, the flowers devel-
polyphenols (Johansen 1940), potassium dichromate for tan- oping on different inflorescences were randomly selected and
nins (Gabe 1968), Nadi reagent for terpenoids (David and marked. Then, the inflorescences were bagged (n = 26–30 per
Carde 1964), and concentrated sulfuric acid for sesquiterpenes year and study site) with tulle isolators (mesh size < 1 mm).
(Cappelletti et al. 1986). The stained sections were observed Twice a day, in the morning (i.e., 7–9 a.m.) and evening (i.e.,
and photographed with a Nikon Eclipse 400 light microscope. 7–9 p.m.), we monitored the flowers and noted the progress of
flowering. The amount of nectar produced was determined in
Fluorescence microscopy the bud stage, in 1-day (=when the lower lip folded and the
palate started being presented), 2-day, 3-day, and 4-day
Pollen grains and fresh hand-made corolla sections with glan- flowers; each sample contained nectar from 15 to 20 flowers
dular trichomes were analyzed using a fluorescence micro- of different individuals. On the appropriate day of anthesis,
scope equipped with filter sets: Cy5 (EX 590–650; BA 663– flowers were removed from the plants and transported into the
738), TRITC (EX 525–565; BA 555–600), and FITC (EX laboratory (ca within 2 hours). Micropipettes with the collect-
465–495; BA 515–555). Lipophilic substances and essential ed nectar were reweighed (WPS-36 analytical balance
oils were detected by induction of fluorescence with the RADWAG, Poland). The sugar concentration in the nectar
Neutral Red fluorochrome (Conn 1953; Lulai and Morgan (% w/w) was determined in each sample using an Abbe re-
1992), steroids with the antimony trichloride fluorochrome fractometer. The mass of the nectar and secreted sugars (in
(Mace et al. 1974), flavonoids with the aluminum chloride mg) was calculated.
(Guérin et al. 1971), and magnesium acetate (Charrière- Pollen production was monitored in the full blooming
Ladreix 1976) fluorochromes. Autofluorescence of secretion phase. Closed anthers (n = 100) were extracted from the

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1766 J. Jachuła et al.

flowers. The anthers were placed in tarred glass containers in per stem, averaged 34.2 ± 7. In each population, the L.
four replications. The glass containers with collected anthers vulgaris flower life span was longer in September (3.8 ±
were inserted into a dryer (Elcon CL 65) for several days, at a 0.6 days) than in July (3.1 ± 0.5 days). The inflorescence life
temperature ca. 33 °C. After the anthers had burst, the pollen span ranged from 7 to 9 days (mean = 7.8 days).
was rinsed from the anthers with 70% ethanol (4–10 ml). The
accuracy of the pollen rinsing was checked under a stereomi- Flower micromorphology and histochemistry
croscope with a × 5 power. The mass of pollen produced was
calculated per flower (Denisow 2011). Numerous glandular trichomes were identified on both the
pedicel and the calyx tube fused of 5 sepals (Fig. 1c–e). The
Data analysis glandular trichomes with a multicellular basal foot cells rooted
in the epidermis and a 4-celled capitate bulbous (spherical)
Data are presented as mean values ± SD (standard deviation). head were approximately 100 μm long (range of 80–
We used ANOVA (analysis of variance) to evaluate differ- 124 μm). Glandular trichomes produced exudates composed
ences in the mean values of the analyzed features (number of lipids, phenolic compounds, and tannins (Table 2).
of flowers per inflorescence, nectar amount per flower, nectar The flowers of L. vulgaris emitted a pleasant Bhoney^
sugar concentration, sugars mass per flower, pollen mass per scent. The tubular, 2-lipped corolla was brightly yellow due
flower) between the populations and within the populations to the presence of flavonoid pigments in its epidermal cells
between the years of the study. The Tukey HSD test was (Fig. 2a, b). The lower lip of the corolla consisted of a 2-lobed,
incorporated for post hoc comparison of means at α = 0.05. well-developed, orange-colored projection (palate), which
The data were analyzed using STATISTICA 6.0 (Statsoft Inc.) obstructed the opening to the throat of the corolla (Figs. 1a
software. and 2a, b). Numerous papillae (50–98 μm long) covered by a
striated cuticle were found on the upper surface of the palate
(Fig. 2c–e). The papilla cells contained lipids, pectins, tannins,
and phenolic compounds (Fig. 2o–q, Table 2). The lower part
Results of the palate formed a shield protecting the stamens.
Numerous unicellular or bicellular non-glandular trichomes
Flowering containing living cytoplasm and yellow pigments in vacuoles
were identified on this palate surface (Fig. 2b–g). These tri-
The onset of flowering of L. vulgaris was noted during June/ chomes, with a length of approx. 200–609 μm, were covered
July, while the end of blooming was recorded in September/ by regular striated cuticle. Lipids, which exhibited a positive
October. Only slight differences (3–4 days) were found in the reaction with Sudan III, Sudan Red, and Nile blue, phenolic
duration of the blooming period and full bloom phase between compounds, and flavonoids were detected in the cells of the
the rural and urban populations. However, the full bloom pe- non-glandular trichomes (Fig. 2k–n, Table 2).
riod differed between years of study in the rural population During the course of anthesis, a change in the palate color
(Table 1). The flowers, arranged in a monopodial inflores- was observed. In the bud stage and in 1-day flowers, the palate
cence of the raceme type, developed in acropetal succession was yellowish; it was light orange in 2-day flowers and, in 3-
(i.e. the lower flowers were older than the upper ones). The day and 4-day flowers, the palate changed color to intensive
zygomorphic flowers of L. vulgaris are hermaphroditic orange.
(Fig. 1a, b). The number of flowers ranged from 12 to 41 At the base of the palate, at the site where the lower and
upper petal lips were fused forming the apical part of the
corolla throat, conical non-glandular trichomes with an ap-
Table 1 The period of blooming of Linaria vulgaris in 2013–2014, SE
proximate length of 130 μm were observed (Fig. 2h, i).
Poland These non-glandular trichomes were directed downwardly in-
to the corolla throat and towards the spur. These trichomes
Population Year Blooming period (days) Full bloom were covered by a cuticle, whose striae formed a specific
period (days)
Bstitch^ (Fig. 2i, j).
Rural (Jastków) 2013 15 June–10 October (118) 67 Additionally, two upwardly folded lobes of the upper lip
2014 23 June–15 October (115) 50 near the throat opening exhibited many capitate glandular tri-
Mean 116.5 58.5 chomes with a height of approx. 85 μm, composed of a 1- or
Urban (Lublin) 2013 19 June–02 October (106) 54 2-celled base and a 4- or 8-celled, 1-, 2-, or 3-layered secretory
2014 14 June–10 October (119) 57 head, as well as a few papillae with a length similar to that of
Mean 112.5 55.5 the glandular trichomes located on the palate surface (Fig. 3a–
e). Histochemical tests revealed the content of lipids, pectins,

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Micromorphological and histochemical attributes of flowers and floral reward in Linaria vulgaris... 1767

Fig. 1 Micromorphology of
sepals and peduncle of a Linaria
vulgaris flower. a View of the
flower from the side of the lower
lip. b View of the flower from the
side of the upper lip. c Surface of
the flower peduncle with
glandular trichomes (arrows) and
stomata (arrowheads). d
Glandular trichome on the flower
peduncle. e Glandular trichome
from the sepal with visible
secretion (arrows). pe peduncle,
se sepals, ll lower petal lip, ul
upper petal lip, pa palate, lo lobes
of the upper lip, sp spur, hc head
cells, sc stalk cells

phenolic compounds, terpenoids, sesquiterpenes, steroids, and asymmetrical disc with a diameter of approx. 1.6 mm
flavonoids, whereas the papillae reacted positively with Sudan (Fig. 4a–c). The nectary was the highest at the lower lip (av-
III, Sudan Red, and Nile Blue (Fig. 3f–w, Table 2). erage 444 ± 38 μm) and the lowest on the other side (average
Furthermore, the glandular trichomes, mainly the secretory 25 ± 7 μm). Nectar is secreted through numerous modified
head, showed blue autofluorescence under ultraviolet light nectarostomata located mainly at the level of the glandular
(Fig. 3w), whereas the papillae showed weak green-blue au- epidermal cells at the side of the lower lip (Fig. 4d–f). On
tofluorescence (not shown). average, there were 475 nectarostomata per mm2 of the nec-
The green, fleshy nectary gland in the L. vulgaris flowers tary epidermis; they were characterized by a mean length of
was located at the ovary base and had a shape of an 23.5 μm and a width of 17.8 μm.

Table 2 Metabolites identified in pollen grains and trichomes of Linaria vulgaris flowers by histochemical and fluorescence tests

Staining Target compounds Sepal glandular Upper lip lobe Upper Lower lip Lower lip Pollen
trichomes glandular lip lobe non-glandular papillae grains
trichomes papillae trichomes

Sudan III Total lipids + + + + + +

Sudan Red Total lipids + + + + + +
Nile Blue Acid and neutral lipids + + + + + +
Nadi reagent Terpenoids (essential oils) – + – – – +
Concentrated sulfuric acid Sesquiterpenes – + – – – –
Ruthenium Red Polysaccharides other than – + – – + +
cellulose
Ferric trichloride Polyphenols + + – – + –
Potassium dichromate Tannins + + – – – –
Iodine iodide solution Starch – – – – – –
Proteins – – – – – +
Neutral Red under UV Lipids and essentials oils nd + – – + +
Aluminum chloride under UV Flavonoids nd + – + – +
Magnesium acetate under UV Flavonoids nd + – + – +
Antimony trichloride under UV Terpens contain steroids nd + – – – +

– negative, + positive, nd not done

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1768 J. Jachuła et al.

Fig. 2 Micromorphology and

histochemistry of papillae and
non-glandular trichomes from the
lower petal lip palate. a
Longitudinal section through a
flower with a visible spur (arrow)
and palate (stars) with orange
color. b Fragment of the palate
with non-glandular trichomes
(arrows). c Surface of the palate
with papillae (black stars) and
non-glandular trichomes (white
stars). d, e Papillae (arrows) on
the palate surface. f Non-
glandular trichomes with
intensive orange pigments in cell
vacuoles. g Non-glandular
trichomes with a striated cuticle.
h, i Conical papillae on the inner
surface of the corolla throat. j
Striations of the cuticle visible on
the papilla surface. k Non-
glandular trichomes after Sudan
III staining. l After Sudan Red
staining. m After Nile Blue
staining. n After ferric chloride
staining. o Papillae after Nile Blue
staining. p After Ruthenium Red
staining. q After potassium
dichromate staining. pa palate, sp
spur, st stamens

In the cells of the inner epidermis of the spur, where abun- oils, flavonoids, and steroids were evidenced in the pollen
dant nectar was accumulated, there were numerous calcium grains (Fig. 5g–j, Table 2).
oxalate crystals with various shapes, most commonly
appearing as blocks and multifaceted druse crystals Nectar and pollen rewards
(Fig. 4g). In turn, there were many budding yeast cells in the
form of 4–5-armed Bwindmills^ observed on the surface of the The floral nectar was accumulated in the spur. Nectar release
inner epidermis of the spur (Fig. 4h–j). began in the bud stage (approx. 4–6 h before lower lip folding)
The stigmata of the 2-carpelled pistil were formed of nu- and lasted to the end of anthesis (i.e., when the flower began to
merous papillae with a fluffy secretion and germinating pollen wilt). Significant effect of flower age was found for the mass
grains on their surface (Fig. 5a, b). The style epidermis cells of nectar per flower (F4,76 = 22.525, P < 0.001), the concen-
exhibited massive cuticular striae. The pistil was surrounded tration of sugars in nectar (F4,76 = 52.714, P < 0.001), and for
by 2 longer and 2 shorter stamens, whose filaments were cov- the mass of sugars per flower (F4,76 = 24.488, P < 0.001).
ered by numerous non-glandular trichomes at the base. Nectar production and nectar sugars concentration increased
The anther epidermal cells had a polygonal shape and, gradually throughout flower development, peaked in 3-day
likewise the style, were covered by a striated cuticle flowers, and decreased slightly towards the end of flower life
(Fig. 5c, d). The pollen grains of L. vulgaris were tricolpate span (Fig. 6).
with faveolate (reticulate) ornamentation of the exine (Fig. 5e, On average, the flowers of L. vulgaris from the rural pop-
f). Numerous lipid-protein granules were deposited inside the ulation (R) secreted 1.5-fold more nectar (F1,15 = 8.589, P =
mesh of the reticulate exine. Additionally, pectins, essential 0.011) than the flowers of L. vulgaris growing in the urban

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Micromorphological and histochemical attributes of flowers and floral reward in Linaria vulgaris... 1769

Fig. 3 Micromorphology and

histochemistry of glandular
trichomes on the upper petal lip
lobes. a Fragment of the upper lip
lobe with glandular trichomes
(arrows). b Surface of the upper
lip lobe with glandular trichomes
(arrowheads) and papillae
(arrows). c Glandular trichome
and papilla on the lobe surface. d,
e Multicellular heads of glandular
trichomes with secretion visible
on their surface. f–h Different
type of glandular trichomes. f
Trichome with a multicellular
several-layer head. g Trichome
with a four-celled one-layer head.
h Trichome with a multicellular
one-layer head. i–w
Histochemistry of glandular
trichomes. i–l Lipids in trichome
cells after Sudan III (i), Sudan
Red (j, k), and Nile Blue (l)
staining. k Four-celled trichome
head, top view. m Sesquiterpenes
after concentrated sulfuric acid
staining. n Polysaccharides other
than cellulose after Ruthenium
Red staining. o Tannins after
potassium dichromate staining. p
Polyphenols after ferric chloride
staining. q Terpenoids after Nadi
reagent staining. r–t Fluorescence
of flavonoids under UV light with
magnesium acetate (r) and with
aluminum chloride (s, t). u
Fluorescence of steroids under
UV with antimony trichloride. v
Fluorescence of lipids and
essential oils under UV with
Neutral Red. w Secretion
autofluorescence under UV. hc
head cells, sc stalk cells

population (U) (Table 3). The nectar amount demonstrated a flowers, pollen release was observed in the closed bud stage.
significant year effect (F 1,15 = 9.640, P = 0.008). The nectar No significant population effect was found for the pollen pro-
concentration was high and ranged between 36.5 and 57.0%. duction per flower (F 1,15 = 0.067, P = 0.799); however, year-
Significant year and population effects on the nectar sugar to-year disparities were found (F 1,15 = 16.808, P = 0.001). In
concentration were found (for year F 1,15 = 5.285, P = 0.037; 2014, the flowers of L. vulgaris produced 1.5- to 2-fold more
for population F 1,15 = 12.846, P = 0.003). pollen than in 2013 (Table 4).
On average, the total mass of sugar in the nectar was In good weather conditions (sunny, no precipitation), the
1.1 mg/flower. The total mass of sugar in the nectar available flowers of L. vulgaris attracted numerous insect visitors, i.e.,
per flower differed significantly between the populations (F representatives of Hymenoptera, Diptera, and Lepidoptera
1,15 = 10.531, P = 0.006) and the years of the study (F 1,15 = (Fig. 7). The insect foraged throughout the day from ca. 6.00
9.393, P = 0.008). to 19.00 h. The visits of Bombus species (including B.
Dehiscence of L. vulgaris anthers began as soon as the terrestris, B. hortorum, B. pascuorum, B. lapidarius, B.
palate started being presented. In approximately 10% of the sylvarum) and Apis mellifera were distributed quite evenly

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1770 J. Jachuła et al.

Fig. 4 Micromorphology of the

nectary and spur in L. vulgaris
flowers. a, b Nectary (stars) at the
base of the ovary visible from the
side of the lower lip. c Cross-
section of the ovary with the
nectary (arrow). d Fragment of
the nectary surface with
nectarostomata (arrows). e, f
Nectarostomata in nectary
epidermis with visible secretion. g
Crystals of calcium oxalate
(arrows) in the inner epidermis of
the spur. h–j Yeast cells (arrows)
visible on the inner epidermis of
the spur surface; i—side view. j—
top view. ov ovary

throughout the day, while dipterans and hymenopterans for- considered as a highly specialized species with nectar reward
aged on the flowers mainly in evening hours. The spectrum of available only to heavy and long-tongued bumblebees. In our
insect visitors differed between the populations and the years study, both short-tongued (Apis mellifera, Bombus terrestris,
of the study. Both richness and abundance of insect visitors B. lapidarius) and long-tongued (B. hortorum, B. pascuorum,
were higher in the rural site (Jastków) than in the urban site B. sylvarum) bees were noted searching for floral nectar in a
(Lublin). In the rural population, syrphid flies (33.3% of the legitimate way. Moreover, dipterans and solitary bees (e.g.,
visits) and Vespula vulgaris (25.0% of the visits each) were Andrena spp.) were recorded; however, these insects were
most frequently noted in 2013. In the urban population, in only occasionally able to get inside the flower. This is in
both study years, B. terrestris (52.9%, on average) and A. agreement with the study of Stout et al. (2000), who often
mellifera (28.0%, on average) were the main visitors. observed small dipterans and syrphids approaching L.
vulgaris flowers but were not able to collect the nectar. In
our study, nectar robbing was observed. Vespula vulgaris
Discussion and B. terrestris (about 40% of total individuals) were seen
biting holes in spurs (or re-using already bitten holes), which
Insects rely on diverse floral signals (e.g., visual, olfactory) to is in line with the observations made by Corbet et al. (1981). L.
find flowers and make use of nectar and/or pollen floral re- vulgaris develop nototribic flowers and only insects pushing
wards (Lunau 2000). We observed insects foraging for both their heads through the corolla entrance in search for nectar
nectar and pollen, which did not support the classical view that can deposit pollen on the stigma. Nepi et al. (2003) found that
Linaria vulgaris develops Bnectar flowers^ (Knuth et al. only long-tongued B. pascorum and two Lepidoptera repre-
1904). We observed insect visitors (mainly bumblebees) for- sentatives were able to pollinate the flowers in legitimate way,
aging for pollen, which is in line with Newman and Thomson while other insect visitors acted as primary or secondary nec-
(2005b). According to Vargas et al. (2010), L. vulgaris is tar robbers. Flowers of L. vulgaris appear to be

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Micromorphological and histochemical attributes of flowers and floral reward in Linaria vulgaris... 1771

Fig. 5 Micromorphology of the

generative organs of L. vulgaris
flowers. a Fragment of the style
and stigma with visible pollen
grains. b Striated cuticle on the
style surface and papillae on the
stigma with flocculent secretion.
c, d Cells of anther epidermis with
massive cuticular striations. e, f
Tricolpate pollen grains in SEM;
pollenkitt granules visible in the
mesh of the reticulate exine
(arrows). g Proteins in pollen
grains (reaction with the iodine
iodide solution). h Terpenoids
(Nadi reagent staining). i Lipids
(Sudan III staining). j Flavonoids
(magnesium acetate under UV). st
style, sti stigma, pa papillae

Bphenotypically specialized^ and could be expected to have species as Becological generalist^ (Ollerton et al. 2007). We
functionally specialized pollination system, but the spectrum evidenced that the flowers of L. vulgaris attract a wide range
of insect visitors observed in our study suggests to classify the of insects reaching (or at least trying to reach) nectar from the

Fig. 6 Effect of flower age on

nectar mass, nectar sugar
concentration and nectar sugar
mass in Linaria vulgaris. Values
are means calculated across the
years of study and populations.
Vertical bars show 95%
confidence intervals

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1772 J. Jachuła et al.

Table 3 Nectar amount, sugar

concentration, and sugar mass per Population Year Nectar amount/1 flower Sugar concentration Sugar mass /1 flower
flower at the peak of nectar (mg) (%) (mg)
production (=3-day flowers) in
Linaria vulgaris in 2013–2014, Mean SD Mean SD Mean SD
SE Poland
Rural (Jastków) 2013 2.08a 0.32 46.4a 4.48 0.96a 0.14
2014 3.54b 0.27 54.1b 1.98 1.91b 0.16
Mean 2.81B 50.3B 1.44B
Urban (Lublin) 2013 1.54a 0.20 37.9a 2.53 0.58a 0.03
2014 2.11b 0.36 44.1a 3.65 0.94b 0.20
Mean 1.82A 41.0A 0.76A

Means followed by the same small letters are not significantly different between years and values followed by the
same capital letters are not significantly different between study populations at α = 0.05 according to the Tukey
HSD test
SD standard deviation

side of corolla lobes and presumably touching the reproduc- Medicago sativa and Rorippa sylvestris were heavily used by
tive organs. However, the pollinator effectiveness was not bumblebees and honeybees. On the contrary, there was no
measured, which is the limitation of our research. other attractive co-flowering species in the urban site, which
The composition of insect visitors to L. vulgaris flowers forced bees to visit L. vulgaris, i.e., the only flowering species
differed between the urban and rural habitats. This observation available.
indicates that, even in the case of a species with highly spe- In L. vulgaris, the bright orange-colored palate forms a
cialized flowers (zygomorphic, spurred corolla), the spectrum landing platform for pollinators. No doubt, in a flower with
of floral visitors considerably depends on spatial and temporal a closed corolla and a deep nectar spur, the bright colored
variation in the composition and abundance of local insect landing palate provides pollinators with the information
guild, which is consistent with previous reports (e.g., about the route to the nectar. In fact, we observed insects
Kameyama and Kudo 2009; Zych et al. 2013; Antoń and using the palate as a landing platform, crawling inside to
Denisow 2014; Denisow et al. 2014; Ziemiański and Zych reach the nectar, and touching reproductive organs, possibly
2016). transferring pollen. Leonard et al. (2013) found that the nectar
Moreover, the difference in the composition of insect visi- guides exert an impact on insect behavior, e.g., they can re-
tors between the study populations may be explained by the duce nectar robbing, increase the relative frequency of legiti-
presence or absence of alternative flowering plants in the mate visits, and allow insects to save energy while searching
study site during the flowering of L. vulgaris. In the rural site, for sugar-rich nectar. Therefore, the benefits of nectar guides
are potentially shared by both the plant and the pollinator (e.g.,
Leonard et al. 2013). Moreover, floral pigments that produce
Table 4 Pollen production in flowers of Linaria vulgaris in 2013–2014, patterns attractive to pollinators may also deter florivores
SE Poland (Gronquist et al. 2001). In Linaria species, the significant
inter-species variation in the color of both the corolla and the
Population Year Mass of pollen/1 flower (mg)
palate is suggested to have a high taxonomic significance
Min-max Mean SD (Fernández-Mazuecos et al. 2013).
The upper surface of the palate is roofed by non-glandular
Rural (Jastków) 2013 0.14–0.37 0.23a 0.09 trichomes and numerous papillae covered with a striated cuti-
2014 0.33–0.50 0.41b 0.07 cle. These anatomical features of the upper surface of the
Mean 0.32A palate increase friction and provide a perfect surface for in-
Urban (Lublin) 2013 0.16–0.29 0.23a 0.05 sects to hold firmly on the flower. Moreover, the non-
2014 0.26–0.45 0.37b 0.07 glandular trichomes of considerable length present on the pal-
Mean 0.30A ate surface are potentially involved in protection against air-
borne fungal propagules or dust particles (Mayekiso et al.
Means followed by the same small letters are not significantly different
between years and values followed by the same capital letters are not
2008). The papillae and non-glandular trichomes of a palate
significantly different between study populations at α = 0.05 according store tannins and phenolic compounds, which are likely re-
to the Tukey HSD test sponsible for flower protection against herbivores and patho-
SD standard deviation gens. Plant pathogens are often transferred between plant

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Micromorphological and histochemical attributes of flowers and floral reward in Linaria vulgaris... 1773

Fig. 7 Insect visitors spectrum in

Linaria vulgaris in two
populations in rural and urban
landscape in 2013–2014, SE
Poland

individuals by herbivorous insects. The insect repellent activ- insect foragers (Pyper 1998; Odell et al. 1999; Wright et al.
ity together with strong antifungal and antibacterial activities 2005; Gong et al. 2015). Therefore, it is argued that the floral
of tannins and polyphenols confirmed in several studies scent composition could potentially mediate plant-pollinator
(Lattanzio et al. 2006; Montenegro et al. 2013) are considered interactions and can even be useful in prediction of the polli-
important for plant protection. Phenolic compounds are also nation system. Several other biological activities of essential
supposed to be involved in UV filtration and can ameliorate oils are related to phytoalexins, insect antifeedants, phero-
the effect of intense summer solar radiation (Morey et al. mones, defensive agents, allelochemicals, or signaling mole-
2016). cules (Pichersky and Gershenzon 2002). In L. vulgaris, the
The scent of flowers is considered to be a complex olfac- gynoecial ring-like nectary gland with different heights is lo-
tory signal whose function is both to attract pollinators and/or cated at the base of the superior ovary and represents the
to repeal unwelcome insect visitors and deter herbivores to nectaria-persistentia type, characteristic for the representa-
prevent consumption of reproductive plant structures tives of Scrophulariaceae s.l. (Smets 1986; Bernardello
(Schiestl and Ayasse 2002; Raguso 2008). The flowers of L. 2007). Nectar is released via permanently opened
vulgaris produced an expressive Bhoney^ fragrance. nectarostomata. This species-specific nectar secretion was
Presumably, the scent was emitted by numerous glandular previously observed by Gaffal et al. (1998) and Nepi et al.
trichomes located on the upper corolla lip and volatile terpe- (2003).
noids found in the pollenkitt of pollen grains (discussed later). On average, 1.5-fold more nectar was produced in the
We have demonstrated that the exudates of glandular tri- flowers of L. vulgaris in the rural habitat. The variability in
chomes contain flavonoids, terpenoids, steroids, sesquiter- the nectar amount is quite common and can be attributable to
penes, polyphenols, and tannins. In particular, terpenoids diverse environmental factors, i.e., temperature, relative hu-
and sesquiterpenes found in essential oils have long been rec- midity, soil moisture, and soil nutrients (e.g., Petanidou and
ognized as a source of plant derived flavors and fragrances Smets 1996; Denisow et al. 2014). The amount of nectar pro-
(Byers et al. 2014; Hambäck 2016; Lucas-Barbosa et al. duction in L. vulgaris was reported to be linked to the plant
2016). For example, Sutton (1988) and Tekaya-Karoui et al. and flower age and differ significantly between geographical
(2010) recognized a specific pleasant smell similar to violets localizations (Nepi et al. 2003), which is consistent with our
and/or strawberries in certain species of Linaria, and identi- observations. The diversity in the nectar amount can have an
fied 49 volatile components in essential oils of L. heterophylla impact on pollinators’ behavior and their efficiency in the
flowers. Terpene-producing trichomes similar to those ob- process of pollen transfer and donation (e.g., Antoń and
served in Linaria were also found in the leaves of Denisow 2014).
Calceolaria adscendens, a species that is traditionally includ- Regardless of the year of the study and the population, the
ed in the Scrophulariaceae s.l. (Sacchetti et al. 1999). As re- nectar of L. vulgaris was highly concentrated. Such nectar
ported for the representatives of the genera Antirrhinum, offers great energetic reward and, although it can be relatively
Buddleja, and Bartsia (Scrophulariaceae s.l.), the intensity of difficult to collect and transport, it is considered to be pre-
floral scent emissions was associated with a subtle change in ferred by bumblebees, which can maximize their energy in-
the corolla color and was able to influence the activity of take and can effectively pollinate the flower (Harder 1986;

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1774 J. Jachuła et al.

Nicolson et al. 2013). The pollination of L. vulgaris mainly by responsible for attraction of insect visitors/effective pollina-
bumblebees was reported by Stout et al. (2000) and Newman tors and exclusion of ineffective pollinators and/or herbivores
and Thomson (2005b). In our field studies, bumblebees were in L. vulgaris.
important visitors of the L. vulgaris flowers, in particular in
the urban habitat, accounting for ca. 50% of the total insect Funding information This research was supported financially by the
Ministry of Science and Higher Education of Poland as part of the statu-
visitors.
tory activities (projects OKB/DS/2, OKB/DS/8, and OKB/MN/6) of the
In addition to nectar, L. vulgaris offers pollen reward. The Department of Botany, University of Life Sciences in Lublin.
amount of produced pollen was similar in both populations
(mean = 0.31 mg per flower). However, the pollen productiv- Compliance with ethical standards
ity of the flowers differed considerably between the years of
the study. In 2013, the amount of produced pollen was con- Conflict of interest The authors declare that they have no conflict of
siderably lower in both populations than in 2014. interest.
Microsporogenesis and pollen production are highly attribut- Open Access This article is distributed under the terms of the Creative
able to weather conditions, and even empty anthers can devel- Commons Attribution 4.0 International License (http://
op in unfavorable conditions, e.g., in precipitation deficit creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
(Denisow 2011; Khanduri 2011). In the study region, a signif- distribution, and reproduction in any medium, provided you give appro-
priate credit to the original author(s) and the source, provide a link to the
icant shortage of rainfalls was noted in 2013, which can be the
Creative Commons license, and indicate if changes were made.
cause of the decrease in pollen production in L. vulgaris.
During the anther dehiscence, the mature pollen grains of
L. vulgaris were already covered by pollenkitt that formed
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