Flora 233 (2017) 1221

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Flora
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Salvia uliginosa Benth.: Glandular trichomes as bio-factories of

volatiles and essential oil
Claudia Giuliani a,b, , Roberta Ascrizzi c , Corrado Tani d , Martina Bottoni a,b ,
Laura Maleci Bini d , Guido Flamini c , Gelsomina Fico a,b
a
Department of Pharmaceutical Sciences, University of Milan, Via Mangiagalli 25, I-20133 Milan, Italy
b
Ghirardi Botanic Garden, Department of Pharmaceutical Sciences, University of Milan, Via Religione 25, I-25088 Toscolano Maderno, Brescia, Italy
c
Department of Pharmacy, University of Pisa, Via Bonanno 6, I-56126 Pisa, Italy
d
Department of Biology, University of Florence, Via La Pira 4, I-50121 Florence, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Micromorphological, histochemical and ultrastructural investigations on the indumentum of vegetative
Received 8 March 2017 and reproductive organs were carried out for Salvia uliginosa Benth. (Lamiaceae). Besides non-glandular
Received in revised form 5 May 2017 hairs, two types of glandular trichomes (peltate and short-stalked capitate) are described and compared
Accepted 5 May 2017
with trichomes in other Lamiaceae species. The histochemical ndings evidence a similar, complex chem-
Edited by Alessio Papini
ical nature of the secretory products for both types of trichomes: lipids, terpenes, phenols and avonoids.
Available online 10 May 2017
TEM investigation allows to conrm the preliminary histochemical results.
In addition, we report for the rst time the phytochemical characterization of the leaf and ower
Keywords:
Salvia uliginosa benth
volatile emission proles and the analysis of the essential oil obtained from the aerial parts.
indumentum Leaf volatile prole resulted more complex, presenting a total number of constituents almost double
Micro-morphology compared to that found in the owers (50 vs 27 compounds, respectively). Leaf VOC prole is char-
Hs-spme acterized by 30 exclusive compounds, dominated by 1,8-cineole (3.84%) and trans--cadinene. Flower
Gc/ms VOC prole Exhibits 7 exclusive compounds, among which germacrene D (8.65%) is the most abun-
VOC prole dant one. Sesquiterpene hydrocarbons represent the major chemical class in both proles (90.13% in
Essential oil leaves and 92.18% in owers) and 20 common compounds have been detected: among them -elemene,
-caryophyllene, -humulene and bicyclogermacrene are the dominant constituents, with relative per-
centages in the range of about 5.0017.00% for the leaves and of about 9.00-31.00% for the owers.
49 different compounds were identied in the essential oil obtained from the aerial parts. The extraction
yield is 0,02%. Sesquiterpenes represent the most abundant terpene fraction (89.56%). The main com-
pound is bicyclogermacrene (16.30%) followed by germacrene D (14.81%), -caryophyllene (8.57%) and
-cadinene (8.47%), among the hydrocarbons. Spathulenol (12.66%) dominates the oxygenated sesquiter-
penes.
2017 Elsevier GmbH. All rights reserved.

1. Introduction In the Lamiaceae family, glandular hairs represent the primary

The Salvia genus (family Lamiaceae, subfamily Nepetoideae,
tribe Menthae) includes about 9501000 species (Dizkirici et al.,
2015), some of which are of commercial value. The glandular hairs
and the essential oil proles of various species have been charac-
terized and investigated also in relation to their biological activities
(Kintzios, 2000; Fu et al., 2013; Ali et al., 2015).
Corresponding author at: Department of Pharmaceutical Sciences, University of
Milan, Via Mangiagalli 25, I-20133 Milan, Italy.
E-mail address: claudia.giuliani@unimi.it (C. Giuliani).
sites for the synthesis of bioactive volatile compounds, which play a
crucial role in the seduction towards pollinators or seed dispersers
or in the repulsion strategies against phytophagous insects or pests
(Maffei, 2010). In addition, this specialized structures secrete large
quantities of exudates, forming a continuous layer on the plant
surface, which may regulate the interactions with the microbiota
inhabiting the phyllosphere and the anthosphere (Werker, 2000).
As part of a wide-spectrum project on Salvia spp., aimed at
exploring the close relationship between the productivity in sec-
ondary metabolites, their bio-ecological signicance and their
potential importance to humans, in this work we carried out a
micro-morphological and phytochemical survey of the indumen-

http://dx.doi.org/10.1016/j.ora.2017.05.002
0367-2530/ 2017 Elsevier GmbH. All rights reserved.
 C. Giuliani et al. / Flora 233 (2017) 1221
tum of mature leaves and owers of Salvia uliginosa Benth. (section
Uliginosae).
Salvia uliginosa, known as bog sage, is a perennial subshrub
native to South America (Brazil, north-eastern Argentina, Uruguay)
and was rst introduced as ornamental at the Royal Botanic Gar-
dens of Kew in 1913 (Turrill, 1914), because of its beautiful sky-blue
owers, with white beelines on the upper and lower lips. Nowa-
days it is widely cultivated in the gardens of Europe, also in virtue
of its high adaptability to different soil types, sun exposures, and
drainage regimes. Indeed, although both the common name and
the species epithet refer to the boggy conditions in which the plant
grows in the wild in its home range, it may well tolerate dry con-
ditions.
Besides the presence of medium-sized blue owers, this species,
as all the members of section Uliginosae, displays a unique staminal
structure. This synapomorphy consist in an appendage that is a
forward-facing or assurgent tooth located on the ventral side of
the posterior connective branches (Turner, 2009). As most Salvia
species, this plant is bee-pollinated (Wester and Claen-Bockhoff,
2007).
In its native range, S. uliginosa (syn. S. lanceolata Larranaga) was
employed in the folk medicine for its carminative, tonic and stim-
ulant properties (Pinto, 2012).
The glandular indumentum of S. uliginosa has never been
previously investigated. With regard to the phytochemical state-
of-the-art, literature data on the characterization of VOCs lack.
Only one contribution focusing on the essential oil prole of a
population from Brazil is known (Pinto, 2012): hexadecanoid acid,
n-nonadecane and nonadecanal resulted as the main compounds;
the biological activity of the essential oil against Staphylococcus
aureus has been also proved (Pinto, 2012).
The principal goal of this work was to comprehensively char-
acterize the glandular trichomes of both the vegetative and the
reproductive organs by light, scanning electron and transmission
electron microscopy; histochemical procedures were also used to
localize the secreted substances within and on the surfaces of the
hairs. In addition, we combined the micro-morphology of the secre-
tory structures with their productivity in secondary metabolites
through the phytochemical characterization of the vegetative and
oral volatile bouquets and essential oil analysis.
The overall set of such information, and in particular the chemi-
cal nature of the volatile substances emitted, may nally contribute
to shed light on the biotic interactions established by the examined
species, thus constituting the basis for future insights on the ecolog-
ical roles of the secondary metabolites, mainly in relation to insect
pollinators.
2. Materials and methods
2.1. Plant material
Salvia uliginosa was cultivated at the Ghirardi Botanic Garden
(Toscolano Maderno, Bs) of the Department of Pharmaceutical Sci-
ences of the University of Milan. Voucher specimens was deposited
in the Herbarium of the Ghirardi Botanic Garden, University of
Milan, Italy under the accession codes UNIMI 0030/15.
The sampling for the micro-morphological analysis were carried
out on plants in full-bloom in June 2016. Collection of plant material
for the phytochemical investigation (VOCs and essential oil) was
performed simultaneously.
2.2. Micromorphological analysis
The glandular indumentum was examined on both vegetative
and reproductive organs (stems, leaves, bracts, oral buds and
13
full-open owers) by means of light microscopy, as well as both
scanning electron, and transmission electron microscopy.
Five replicates, similar for size, position and developmental
stage, were selected from different individuals for each plant part,
in order to verify the consistency of the trichome morphotypes,
distribution pattern, histochemistry and ultrastructure.
2.2.1. Light microscopy (LM)
Histochemical investigation was performed to evidence the
main chemical classes of metabolites in the trichome secretory
products. For each replicate of all the fresh plant parts, hand-made
sections (4050 m thick) and semi-thin sections (2025 m thick)
obtained by means of a cryostat, were stained with the follow-
ing dyes: Sudan III, IV (Johansen, 1940), Fluoral Yellow (Brundrett
et al., 1991), Nile Red for neutral lipids (Greenspan et al., 1985),
Ruthenium Red for polysaccharides other than cellulose (Jensen,
1962) and Alcian Blue for acidic polysaccharides (Beccari and Mazzi,
1966), Nadi reagent for terpenes (David and Carde, 1964), ferric
trichloride for polyphenols (Gahan, 1984), aluminium trichloride
for avonoids (Gurin et al., 1971), Mercuric Bromophenol Blue
for total proteins (Beccari and Mazzi, 1966). Matchings for all of
the histochemical stains were performed with control procedures.
Autouorescence of the trichome secretory products were also
evaluated. Observations were performed under a Leitz DM-RB Fluo
Optic microscope equipped with a digital camera Nikon DS-L1.
2.2.2. Scanning electron microscopy (SEM)
For each replicate, small segments with a surface of 2530 mm2
were xed in FAA (formaldehyde: glacial acetic acid: 70% ethanol
5:5:90 by volume) at room temperature for 7 days. Then the sam-
ples were dehydrated in ascending ethanol series up to absolute
with 20-min intervals, transferred to acetone, critical point-dried
in a Balzer Union CPD 020 apparatus and sputter coated with gold.
Observations were made under a Philips scanning electron micro-
scope at an accelerating voltage of 20 kV.
2.2.3. Transmission electron microscopy (TEM)
For each replicate of leaves, calices and petals small segments
(510 mm2 ) were xed in 2,5% glutaraldehyde in phosphate buffer
0.1 M pH 7.2 overnight at room temperature. Subsequently they
were rinsed twice in the same buffer, post-xed in 2% osmium
tetroxide for 2 h and washed in distilled water two times for 5 min.
Then, the material was dehydrated in ascending ethanol series up
to absolute and embedded in Spurr resin. The samples were cut in
ultrathin sections (3 m thick) by means of an ultramicrotome and
stained with uranyle acetate and lead citrate. Observations were
made under a Philips EM-300 transmission electron microscope.
2.3. Phytochemical investigation
2.3.1. Volatile organic compounds (VOCs)
Three leaves and three full-opened owers were cut and imme-
diately inserted in two separate glass vials of suitable volume for
the sampling.
HS-SPME Sample analysis Supelco SPME (Solid Phase Micro-
Extraction) devices coated with polydimethylsiloxane (PDMS,
100 m) were used to sampling the headspace. SPME sampling
was performed using the same new bre, preconditioned accord-
ing to the manufacturer instructions, for all the analyses. Sampling
was accomplished in an air-conditioned room (22 1 C) to guar-
antee a stable temperature. The sampling temperature was chosen
to avoid the thermal damage of the supercial glandular hairs
of the leaves and, thus, any articial-induced volatiles release.
After 30 min of equilibration time, the bre was exposed to the
headspace for 30 min. The equilibration time was chosen as the
ideal one after several trials at different intervals. The sampling
14 C. Giuliani et al. / Flora 233 (2017) 1221
time was experimentally determined to obtain an optimal adsorp-
tion of the volatiles, to avoid both under- and over-saturation of the
bre and of the mass spectrometer ion trap. Once sampling was n-
ished, the bre was withdrawn into the needle and transferred to
the injection port of the GCMS system. All the SPME sampling and
desorption conditions were identical for all the samples. Further-
more, blanks were performed before each rst SPME extraction and
randomly repeated during each series. Quantitative comparisons of
relative peaks areas were performed between the same chemicals
in the different samples.
2.3.2. Essential oils (EOs)
Plant aerial parts at blooming were dried at room temperature
and in the dark and stored under the same conditions until the
hydrodistillation process.
The essential oil hydrodistillation was performed in a standard
Clevenger apparatus for 2 h.
GC/MS Analysis The GC/EI-MS analyses were performed with
a Varian CP-3800 apparatus equipped with a DB-5 capillary col-
umn (30 m X 0.25 mm i.d., lm thickness 0.25 m) and a Varian
Saturn 2000 ion-trap mass detector. The oven temperature was
programmed rising from 60 C to 240 C at 3 C/min; injector tem-
perature, 220 C; transfer-line temperature, 240 C; carrier gas, He
(1 ml/min).
Volatiles analysis The essential oils were injected after dilution
in n-hexane HPLC grade at 5%. The identication of the constituents
was based on the comparison of their retention times (tR ) with
those of pure reference samples and their linear retention indices
(LRIs) determined relatively to the tR of a series of n-alkanes. The
mass spectra were compared with those listed in the commercial
libraries NIST 2014 and ADAMS and in a home-made mass-spectral
library, built up from pure substances and components of known
oils, and MS literature data (Stenhagen et al., 1974; Masada, 1976;
Rdel et al., 1982; Davies, 1990).
3. Results
3.1. Micromorphological investigation
3.1.1. Trichomes morphotypes and distribution pattern
SEM analysis revealed that the glandular indumentum of S. uligi-
nosa consist of peltate and capitate hairs (Fig. 1ab). Both hair types
are distributed over both vegetative and reproductive organs and
co-occur with non-glandular trichomes (Fig. 2an).
Peltate trichomes are formed by one basal epidermal cell, a stalk
cell and a broad head of four secretory cells arranged in a single disc
(Fig. 1a). They are endowed with a large apical subcuticular space
in which the secretion is stored.
Peltate trichomes display a distribution pattern that includes
stems, leaves, oral peduncles, calyces and corollas (Table 1). On
stems and peduncles their occurrence and actual density can not
be adequately assessed because of the general pubescence due to
the abundant non-glandular trichomes completely covering the
epidermal surface. They occur on the interveinal regions of leaf
and sepal abaxial surfaces, whereas they lack on the adaxial ones
(Fig. 2af, i). On petals their distribution is limited to the abaxial
side, both over the corolla tube and at the upper and lower lips
(Fig. 2jl).
The capitate hairs are short-stalked trichomes and consist of
one basal epidermal cell, one neck-stalk cell and a round head of
two broad cells surrounded by a medium-wide subcuticular space
(Fig. 1b). They are widespread on the whole plant surface, with
the exception of the stem and the calyx adaxial side (Fig. 2al).
They are mainly located along the veinal system, especially on the
abaxial side of leaves and calyces (Fig. 2e, i). On the leaf adaxial side
they are distributed on the whole lamina and appeared particularly
abundant at the edges (Fig. 2c).
In both types of glandular trichomes, the apical region of the
immature hairs appear wrinkled or slightly smooth, indicative of
the close adhesion of the cuticle to the outer periclinal wall of the
secretory cells. At maturity, the cuticle surface becomes smoother
due to the accumulation of the secretion products within the devel-
oping storing chamber formed by detachment of the cuticle.
Non-branched, uniseriate non-glandular trichomes were
observed on the whole plant surface (Fig. 2aj, l). Two main types
can be distinguished. Type I hairs are multicellular, sharply pointed,
with a basal wider cell and a gradually minor cell diameter toward
the apical region (see Fig. 2b,c); they present distinct articulations
between cells and display different length and a variable number
of cells, ranging from one to ve-six. Their cuticle is smooth or
slightly papillate; an evident crown of swollen epidermal cells
encircles the basal cell of type I hairs (Fig. 2c). These trichomes
can be perpendicular to the plant surface, but generally they tend
to lean toward the apex of the organ bearing them. Longer type
I hairs are situated on stems, leaves (both along the margins and
over midribs and major veins), oral peduncles and on the veinal
system of calyces. Shorter type I hairs occur on the interveinal
regions of leaves and calyces and over the corolla adaxial surface.
Type II hairs, exclusively observed on the corolla abaxial side,
consist of two-four cells with an equal diameter and present
a rounded apex; they are erect and possess a wrinkled cuticle
(Fig. 2k).
3.1.2. Histochemistry of the glandular trichomes
Various histochemical dyes were used to detect the produc-
tion of major chemical compound classes in glandular trichomes
(Table 2, Fig. 3ah). Staining with Ruthenium Red showed that the
cell walls of the secretory heads of peltate and capitate trichomes
contained unesteried pectins (Fig. 3a). Acidic polysaccharides,
detected by Alcian Blue, were present in the cytoplasm of the secre-
tory cells of both type of glandular trichomes, as well as in the
neck cells; the secretory material resulted not stained by the above
mentioned hydrophilic procedures (Table 2).
Total proteins were present in all cells of both types of glandular
trichome (Table 2), whereas the response of the secretory material
stored in the subcuticular spaces to Mercuric Bromophenol Blue
was invariably negative.
The secretory head of peltate and capitate trichomes stained
yellow-greenish with the Fluoral Yellow-088 test, indicating the
presence of total lipids (Fig. 3b, f). NADI reaction resulted in an
intense dark-violet staining of the secretory product within sub-
cuticular space, typically in the form of droplets, as in the peltate
trichomes (Fig. 3c), thus conrming the presence of terpenes in all
trichome types (Table 2).
The head and the secretory products of both types of glandu-
lar trichome contained also phenolic compounds, as indicated by
the greenish-brown staining with Ferric trichloride (Fig. 3g). In par-
ticular, the intense response to Aluminium trichloride, proved by
the brilliant blue staining, indicate the production and release of
avonoidic compounds (Fig. 3d, h).
3.1.3. Ultrastructure of the glandular trichomes
TEM analysis on both glandular trichome types at the active
secretory phase allows to evidence the major cellular compart-
ments involved in the secretion process (Fig. 4).
In mature peltate trichomes (Fig. 4a), the most striking ultra-
structural features of the secreting cells is the presence of several
electron-dense plastids occasionally containing starch granules.
Generally they display large globular intraplastidial bodies and
scarce tubular membrane elements (Fig. 4b, c). Abundant dilated
cisternae originated from the smooth endoplasmic reticulum (SER)
 C. Giuliani et al. / Flora 233 (2017) 1221 15

Fig. 1. ab. Glandular trichomes morphotypes in Salvia uliginosa, LM: a. peltate trichome. b. short capitate trichome.

Table 1
Distribution pattern of the indumentum in Salvia uliginosa.

Trichome type Stem Leaves Calyx Corolla

adaxial abaxial adaxial abaxial adaxial abaxial

non-glandular type I +++ +++ +++ +++ ++

non-glandular type II ++
peltate + + + +
short capitate ++ + + + +

Table 2
Results of the histochemical investigation of the glandular trichomes of Salvia uliginosa.

Staining procedure Target compounds Observed colour Trichome types

peltate short capitate

Fluoral Yellow-088 Total lipids Yellow to orange + ++

Nile Red Neutral lipids Golden-yellow + ++
Nadi reagent Terpenes Violet-blue + +
FeCl3 Polyphenols Emerald-green + +
AlCl3 Flavonoids Blue-green + +
Ruthenium red Polysaccharides/unesteried pectins Pinkish to red +a +a
Alcian blue Acid polysaccharides Pale-blue +a +a
Hg Bromophenol Blue Total proteins Dark Blue +a +a

Results: () absent; () scarce, (+) intense, and (++) very intense.

Symbols indicate the staining response of the secretory products stored in the subcuticular space
a
Refers exclusively to the staining response of the cytoplasm of the glandular cells.

are also present, generally in close association with plastids (Fig. 4c,
d). Golgi stacks and rough endoplasmic reticulum (RER) occur occa-
sionally. Numerous plasmodesmata are present at the tangential
walls of all the trichome cells, especially between the neck cell and
the secreting cells (Fig. 4e), indicating that also the basal cells and
the neck cells take part in the secretion process. The cytoplasm of
neck cell displays large lipidic droplets (Fig. 4e).
With the aging of trichomes plastids become ameboid or
cup-shaped (Fig. 4e) and several autophagic vacuoles containing
myelin-like gures are observed.
In full-active short capitate trichomes, the cytoplasm of the
glandular cells is dense and is characterized by several l vacuoles,
mithocondria, multishaped plastids surrounded by SER cisternae,
and lipidic droplets (Fig. 4). Golgi stacks and RER cisternae generally
lack.
In both peltate and short-capitate trichomes the storing cham-
ber is enclosed by a thick cuticle layer and by the external portion of
the pectic-cellulosic layer of the outer periclinal walls of the glan-
dular cells (Fig. 4f, h). The cuticle remains rmly adherent to the cell
wall at the basal part of the secretory cell, with cuticle detachment
occurring only at the apical region (Fig. 4a, g). In the subcuticular
space the secretion products present a heterogeneous appearance,
however generally globose electron-dense inclusions immersed in
an electron-opaque granular matrix are observed (Fig. 4d).
No pre-arranged openings for the released of the secretion were
observed; the secretion products are presumably extruded through
the intact outer wall or after cuticle breakage, copiously owing out
on the head, along the stalk and on the epidermis. Materials with
various electron-density degrees are occasionally observed across
the intact outer periclinal cell walls (Fig. 4i).
3.2. Phytochemical investigation
3.2.1. VOCs emission
The spontaneous emission prole of S. uliginosa revealed a total
of 57 different compounds: 50 were detected in the leaf samples
and 27 in the ower ones (Table 3).
The leaf samples are dominated by sesquiterpene hydrocarbons,
reaching 90.13%; oxygenated monoterpenes and monoterpene
hydrocarbons followed in comparable relative percentages, 3.84%
16 C. Giuliani et al. / Flora 233 (2017) 1221

Fig. 2. al. Trichomes distribution pattern in Salvia uliginosa, SEM: a. General view of the leaf adaxial surface. bc. Particulars of leaf adaxial surface showing short capitate
trichomes (arrow) and non-glandular trichomes of type I: middle region (b) and leaf margin (c). d. General view of the leaf abaxial surface. e-f. Particulars of the leaf abaxial
surface showing peltate (asterisk), short capitate trichomes (arrow) and non-glandular trichomes of type I: veinal system (e), interveinal region (f). g. General view of a oral
bud. h. Particular of the oral pedicel with abundant non-glandular trichomes of type I. i. Calyx abaxial surface with non-glandular type I, peltate (asterisk) and short capitate
trichomes (arrow). j. General view of the mature corolla. k. Particular of the abaxial surface of the upper lip with non-glandular type II and peltate trichomes (asterisk). l.
Particular of the adaxial surface of the lower lip with non-glandular type I and short capitate trichomes (arrow).

and 3.68%, respectively. The principal compounds in the whole
headspace is -muurolene (see Table 3, 40, 31.43%), followed
by other sesquiterpene hydrocarbons: bicyclogermacrene (43,
16.91%), -caryophyllene (30, 12.91%), -humulene (35, 5.93%) and
-elemene (20, 5.09%). Monoterpene hydrocarbons are only repre-
sented by 1,8-cineole (9, 3.84%).
The ower headspace is mainly rich in terpene hydrocar-
bons, of which sesquiterpene ones account for 92.18%, followed
by monoterpenes (6.42%). The most abundant compound in this
sample is bicyclogermacrene (43), accounting for 31.26%. -
caryophyllene (30, 25.66%), -elemene (20, 13.98%), -humulene
(35, 9.26%) and germacrene D (41, 8.65%) are other relevant
sesquiterpene hydrocarbons. The two most abundant monoterpene
hydrocarbons in the ower samples are (E)--ocimene (11) and
(Z)--ocimene (10), which represent 2.21% and 1.72%, respectively.
20 compounds are common to both leaf and ower headspaces.
As a whole, the minor common compounds occurred in similar
relative percentages; the major ones, corresponding to the domi-
 C. Giuliani et al. / Flora 233 (2017) 1221
Fig. 3. ah. Histochemistry of the glandular trichomes of Salvia uliginosa, LM. ad. Peltate trichome: Alcian Blue (a); Fluoral Yello-088 (b); Nadi reagent (c); AlCl3 (d). eh.
Short capitate trichome: Alcian Blue (e); Fluoral Yello-088 (f); FeCl3 (g); AlCl3 (h).
nant constituents of leaf and ower bouquets, occurred in different
relative abundances that vary in the range of 515% points. -
muurolene (40) is the principal compound of the leaf headspace
(31.43%), where it shows a noteworthy increment in terms of rela-
tive abundance in comparison to the ower sample (0.31%).
30 and 7 exclusive compounds were found in leaf and ower
volatile prole, respectively. The compounds exclusively found in
leaves are in general present at traces levels or in amounts lower
than 1%; the most represented ones are 1,8-cineole (9, 3.84%) and
trans--cadinene (48, 1.86%). In the ower volatile prole the char-
acteristic compounds are represented with relative abundances
lower that 0.6%, with the exception of germacrene D (41, 8.65%).
3.2.2. Essential oil prole
Salvia uliginosa essential oil composition is reported in Table 4:
a total of 49 different compounds were identied. The extraction
yield is 0,02%.
Sesquiterpenes represent the most abundant terpene fraction
(89.56% of the total terpene relative abundance): hydrocarbons
account for 69.39% and oxygenated ones reach 20.17%. The most
abundant compound is bicyclogermacrene (see Table 4, 31, 16.3%),
followed by germacrene D (29, 14.81%), -caryophyllene (22, 8.57%)
and -cadinene (35, 8.47%) among the hydrocarbons. Spathulenol
(39), accounting for 12.66%, dominates among the oxygenated
sesquiterpenes. Monoterpene hydrocarbons represent 6.62% of the
total oil and the main compounds are limonene (9), (Z)--ocimene
(10) and (E)--ocimene (12), with similar relative abundances,
slightly lower than 2%.
17
The non-terpene derivatives (apocarotenoids included) account
for about 0.87% of the essential oil.
4. Discussion
The glandular trichomes observed on the leaves and owers
of Salvia uliginosa correspond to the two main trichome types
observed in the family Lamiaceae: peltate and capitate (Werker,
2000).
In S. uliginosa, the peltate hairs, occurring on the vegetative and
reproductive organs, are formed by one epidermal cell, and by one
stalk-neck cell supporting a wide four-celled head. The presence
of a four-celled head is a character observed in other members
of Salvia (Eiji and Salmaki, 2016); however, within the genus, the
most common head cell disposition consists in six up to eight cells
arranged in a single circle (Eiji and Salmaki, 2016). In most Lami-
aceae a different pattern prevails: four central cells with a variable
number of peripheral cells ranging from six up to 16 (Werker, 1993,
2000). This last pattern has been also documented in Salvia aurea
(Serrato-Valenti et al., 1997).
The micromorphological investigation proved evidence that S.
uliginosa bears only one type of capitate trichome, whereas differ-
ent morphotypes of capitate hairs generally co-occur in the same
plant, also over the same organ (Giuliani and Maleci Bini, 2008).
This evidence is not consistent with the greater number of capi-
tate morphotypes reported by Gupta and Bhambie (1980) for eight
Salvia species, by Werker et al. (1985a) for S. sclarea and S. dominica,
and by Werker et al. (1985b) for S. ofcinalis and S. fruticosa. The co-
18 C. Giuliani et al. / Flora 233 (2017) 1221

Fig. 4. ai. Ultrastructure of the glandular trichomes of Salvia uliginosa, TEM. a-f. Peltate trichome. a. General view of a mature peltate trichomes at the active secreting stage.
b. Glandular cell cytoplasm of a full active peltate trichome. c. Particular of a glandular cell cytoplasm with electron-dense plastids and well-developed SER. d. Particular of
SER and of the subcuticular space containing lipidic droplets immersed in a granular matrix. e. Cytoplasm of the neck cells of a mature peltate trichome, note the numerous
plasmodesmata (arrows). f. Particular of the outer periclinal wall of the glandular cells, note the detachment of the cuticular layer delimiting the subcuticular space. g-i.
Short capitate trichome. g. Secreting cell cytoplasm of short capitate trichomes at the active secreting stage. h, i. Particulars of the cytoplasm of the secreting cell, note the
osmiophilic secreted materials (asterisks) close to the outer periclinal cell wall.
Symbols: cl, cuticular layer; ld, lipidic droplets; p, plastids; pl, pectic-cellulosic layer; m, mithocondria; Ser, SER; ss, subcuticular space; v, vacuoles.

occurrence of different capitate morphotypes is also conrmed in
the detailed analysis of the glandular indumentum of 46 different
species of Salvia of Iranian origin (Eiji and Salmaki, 2016).
Salvia uliginosa short capitate trichomes, widespread on the
whole plant, consist of two secretory head cells supported by one-
celled stalk, and of a basal epidermal cell. This kind of capitate hair
has been already found in Salvia species (Eiji and Salmaki, 2016) and
correspond to type II described by Werker et al. (1985a). In addition,
these trichomes match the type described for several representa-
tives of the genus Stachys (Giuliani and Maleci Bini, 2008).
The histochemical analysis showed that the secretion prod-
ucts of both types of glandular trichomes are characterized by
a similar, complex composition positive to Fluoral Yellow-088,
Nadi reagent, FeCl3 and AlCl3 . Therefore, secretion is constituted of
lipophilic substances, in particular terpenes, phenolic compounds
and avonoids.
Although peltate trichomes were generally considered typical
essential oil producers in Lamiaceae, containing the bulk of the
terpenes produced (Hallahan, 2000; Werker, 2000), capitate tri-
chomes of S. uliginosa seemed to produce great amounts of terpenic
substances as well. Moreover, a copious production of phenolic
and avonoidic substances is demonstrated in both peltate and
short capitate hairs. Chemical complexity of this nature has previ-
ously been reported for Salvia sclarea and S. dominica (Werker et al.,
 C. Giuliani et al. / Flora 233 (2017) 1221 19

Table 3 Table 4
HS-SPME proles of the leaves and owers of Salvia uliginosa. Compositions of the essential oils obtained from the aerial parts of Salvia uliginosa.

l.r.i. Compounds Relative Abundance (%) l.r.i.a Compounds Relative

Abundance (%)
Leaves Flowers
1 931 -thujene tr
1 931 -thujene tr 2 941 -pinene 0.13
2 941 -pinene tr 3 976 sabinene 0.12
3 976 sabinene tr 4 980 1-octen-3-ol tr
4 982 -pinene tr 1.23 5 982 -pinene 0.34
5 987 3-octanone tr 6 993 myrcene 0.33
6 993 myrcene 0.2 0.54 7 1005 -phellandrene 0.13
7 1005 -phellandrene 0.13 0.12 8 1027 p-cymene tr
8 1032 limonene tr 9 1032 limonene 1.69
9 1034 1,8-cineole (=eucalyptol) 3.84 10 1042 (Z)--ocimene 1.95
10 1042 (Z)--ocimene 1.73 1.72 11 1045 phenyl acetaldehyde tr
11 1052 (E)--ocimene 1.32 2.21 12 1052 (E)--ocimene 1.79
12 1090 cis-linalool oxide (furanoid) tr 13 1062 -terpinene tr
13 1101 linalool 0.32 14 1101 linalool 0.13
14 1129 allo-ocimene 0.3 0.6 15 1133 (E)-2,6-dimethyl-1,3,5,7- 0.14
15 1140 nopinone tr octatetraene
16 1143 camphor tr 16 1351 -cubebene 0.81
17 1189 -terpineol tr 17 1372 -ylangene 0.24
18 1204 decanal tr 18 1376 -copaene 0.82
19 1205 verbenone tr 19 1384 -bourbonene 0.51
20 1244 carvacrol methyl ether 0.36 20 1390 -cubebene 0.49
21 1340 -elemene 5.09 13.98 21 1410 -gurjunene tr
22 1351 -cubebene 0.43 22 1420 -caryophyllene 8.57
23 1372 -ylangene 0.32 23 1429 -copaene 1.15
24 1376 -copaene 1.04 0.15 24 1441 aromadendrene 1.68
25 1384 -bourbonene 0.37 0.15 25 1456 -humulene 4.58
26 1390 -cubebene 0.61 0.11 26 1462 cis-muurola-4(14),5-diene 0.27
27 1392 -elemene 0.6 0.27 27 1470 trans-cadina-1(6),4-diene 0.22
28 1395 sativene 0.18 28 1477 -muurolene 4.63
29 1410 -gurjunene 0.16 0.11 29 1481 germacrene D 14.81
30 1414 -ylangene 0.14 30 1485 -selinene 0.19
31 1420 -caryophyllene 12.91 25.64 31 1495 bicyclogermacrene 16.3
32 1429 -copaene 2.24 0.42 32 1498 -muurolene 0.38
33 1441 aromadendrene 1.37 0.71 33 1507 (E,E)--farnesene 0.63
34 1447 cis-muurola-3,5-diene 0.16 34 1511 cis--cadinene 3.88
35 1456 -humulene 5.93 9.26 35 1524 -cadinene 8.47
36 1461 alloaromadendrene 0.29 36 1534 cadina-1,4-diene 0.41
37 1461 alloaromadendrene tr 37 1538 -cadinene 0.35
38 1462 cis-muurola-4(14),5-diene 1.08 38 1575 germacrene D-4-ol 0.19
39 1470 trans-cadina-1(6),4-diene 0.18 39 1576 spathulenol 12.66
40 1477 -muurolene 31.43 0.31 40 1583 globulol 0.13
41 1481 germacrene D 8.65 41 1590 viridiorol 0.27
42 1485 -selinene tr 42 1606 humulene epoxide II 0.26
43 1492 valencene tr 43 1615 humulane-1,6-dien-3-ol 0.36
44 1495 bicyclogermacrene 16.91 31.26 44 1628 1-epi-cubenol 0.23
45 1498 -muurolene 0.4 45 1640 epi--cadinol 1.74
46 1505 -amorphene 0.53 46 1642 epi--muurolol 2.47
47 1507 (E,E)--farnesene 1.05 0.63 47 1661 cis-calamenen-10-ol 0.11
48 1513 trans--cadinene 1.86 48 1682 (Z)--santalol 1.75
49 1515 cycloisolongifol-5-ol 0.59 49 1845 hexahydrofarnesyl acetone 0.87
50 1524 -cadinene 4.46 0.13
51 1534 cadina-1,4-diene 0.3 Monoterpene 6.62
52 1538 -cadinene 0.38 hydrocarbons
53 1556 germacrene B 0.11 Oxygenated 0.13
54 1575 germacrene D-4-ol 0.77 monoterpenes
55 1576 spathulenol tr Sesquiterpene 69.39
56 1581 caryophyllene oxide 0.14 hydrocarbons
57 1815 2-ethylhexyl salicylate 0.12 Oxygenated 20.17
sesquiterpenes
Monoterpene hydrocarbons 3.68 6.42 Apcarotenoids 0.87
Oxygenated monoterpenes 3.84 0.68 Non-terpene derivatives tr
Sesquiterpene hydrocarbons 90.13 92.18
Oxygenated sesquiterpenes 0.91 0.59 Total 97.18
Non-terpene derivatives tr 0.12 a
Linear retention indices on a DB5 column.
Total 98.56 99.99

1985a), S. aurea (Serrato-Valenti et al., 1997) and S. blepharophylla
(Bisio et al., 1999).
In both trichomes types, the secreted material gave invariably
negative responses to the hydrophilic stainings, indicating the lack
or the scarce production of polysaccharides. On the contrary, abun-
dant non-cellulosic and acidic polysaccharides have been reported
in the secreted material of capitate hairs in various Lamiaceae
(Werker, 1993; Giuliani and Maleci Bini, 2008), whereas only traces
of pectic polysaccharidic substances are generally found in the
secreted material of peltate hairs (Werker, 1993; Giuliani and
Maleci Bini, 2008). In S. uliginosa only the wall and the cytoplasm of
the head cells stained positively to Alcian Blue and Ruthenium Red:
in the protoplast, polysaccharides can act on water retention, con-
tributing to the maintenance of the water potential in the tissues
(Werker, 2000).
20 C. Giuliani et al. / Flora 233 (2017) 1221
In the examined species, the trichomes responsible for both
the terpene and avonoid production and storage, as evidenced
with the histochemical stainings and conrmed by TEM analysis,
are both peltate and capitate trichomes, widespread on the whole
plant epidermis. The ultrastructural observations on the cytoplasm
of the secreting cells showed large electron-dense plastids and a
well-developed periplastidial SER, which are considered cellular
compartment responsible for the synthesis and transfer of terpenic
substances (Fahn, 2000; Hallahan, 2000). Moreover, SER elements
are responsible for the intracellular transport of polyphenols (Fahn,
2000).
The limited production of polysaccharidic compounds is sup-
ported by the occasional occurrence of Golgi stacks and RER,
responsible for the synthesis and transport of such substances
(Schnepf, 1974; Ascensao et al., 1995; Fahn, 2000).
The secreted substances copiously accumulate in the subcuticu-
lar space. In both hair types, ultrastructural observations conrmed
that the secretory material is characterized by a heterogeneous
composition: indeed, materials with different electro-density
degrees and appearance are observed in the subcuticular space,
thus indicating the presence of different chemical phases. An
emulsion-like appearance has been described for the secretion
products of peltate trichomes in other Lamiaceae species (Giuliani
and Maleci Bini, 2008).
In these types of trichomes, two potential mechanisms for the
extrusion of the secretory products have been hypothesized: (i) via
pores in the intact cuticular structure, generally described in liter-
ature only in capitate trichomes (Werker, 1993); (ii) by breakage of
the cuticle, common to members of the Lamiaceae (Werker, 1993,
2000).
The volatile components may pass through minute pores of the
intact cuticle. Also avonoids may cross the cell walls and the cuti-
cle, crystallizing on the surface of trichomes and on the epidermis
of the organs bearing them (Wollenweber, 1994). However it can
be argued that the most part of the secretion is extruded after the
rupture of the cuticle, as it generally occur (Giuliani and Maleci Bini,
2008).
With regard to non-glandular trichomes, two distinct morpho-
types were described: type I, widespread on the whole plant, and
type II, occurring exclusively on petals. The presence and dis-
tribution of non-glandular hairs among Salvia species is widely
documented and both simple and branched trichomes have been
described (Eiji and Salmaki, 2016). Our results showed that only
simple trichomes are present in S. uliginosa, as documented
in almost all the New World species investigated so far, since
branched trichomes have been mainly observed in taxa of European
or Asian origin (Eiji and Salmaki, 2016).
About the phytochemical investigation on the vegetative and
ower bouquets, this work represents the rst contribution on the
characterization of the VOC proles in S. uliginosa.
The obtained volatile proles show high level of variability.
Indeed, leaf prole is more complex, presenting a total number of
constituents almost double compared to that found in the ow-
ers. In addition the leaf headspace is characterized by 30 exclusive
compounds, dominated by 1,8-cineole (9) and trans--cadinene
(48). The ower headspace Exhibits 7 exclusive compounds, among
which germacrene D (41) resulted the most abundant one.
However, sesquiterpene hydrocarbons represent the major
chemical class in both proles and 20 common compounds have
been detected. Among them, -elemene (21), -caryophyllene
(30), -humulene (35) and bicyclogermacrene (43) are the dom-
inant compounds detected in comparable relative percentages.
-muurolene (40), principal compound of the leaf prole, is present
in signicantly higher relative abundance (higher by about 30%
points) in comparison to the ower prole.
As most of species included in Salvia genus, S. uliginosa is bee-
pollinated (Wester and Claen-Bockhoff, 2007). At the Ghirardi
Botanical Garden S. uliginosa plants were exclusively visited by
honeybees (Giuliani and Fico, personal observations).
Besides visual signalling, bees are also able to perceive chemi-
cal cues in a multimodal pattern related to their ability to recover
food resources (Wright and Schiestl, 2009). Floral scents are blends
of many different chemical compounds (Dobson 2006), present at
diverse concentrations (Maffei et al., 2011). Honeybees, in particu-
lar, are able to detect subtle differences in the overall intensity or
concentration of single compounds in a scent (Wright et al., 2005).
Previous studies have demonstrated that some of the volatiles
produced by S. uliginosa owers are involved in the attraction of
honeybees, e.g. -caryophyllene (Blight et al., 1997) and germar-
crene D (Lusebrink et al., 2015), the latter exclusively emitted from
S. uliginosa owers. Therefore, a possible role of this volatile in
the seduction strategies towards honeybees may be hypothesized.
Moreover, since -caryophyllene was simultaneously detected in
the leaf headspace, we can suggest a synergistic action leaf-ower
in attracting potential pollinators (Vitalini et al., 2011).
This report is the rst contribution on the composition of the
essential oil of S. uliginosa out of its native range.
The genus Salvia, as most of the aromatic plants of the Lamiaceae
family, are included within the subfamily Nepetoideae (El-Gazzar
and Watson, 1970). However, several studies highlighted that in
Salvia there are also oil-poor species (Malencic et al., 2004).
Indeed, S. uliginosa presents a very low yield of essential oil,
0.02%, as documented in other members of the genus (Malencic
et al., 2004). This result may be related to the fact the glandular tri-
chomes synthesized compounds with low volatility which cannot
be recovered by hydrodistillation (Bicchi et al., 1985).
Noteworthy differences resulted from the comparison of liter-
ature data (Pinto, 2012). In fact, this author reported an essential
oil particularly rich in non-terpene compounds, mainly aliphatic
and carbonyl derivatives, such as hexadecanoic acid (30.1%),
n-nonadecane (17.5%), nonadecanal (4.5%). The only two sesquiter-
penes detected at percentages higher than 1% were spathulenol
(1.9%) and -muurolol (1.1%). Notably, sesquiterpene hydrocar-
bons, which represented the main chemicals of the essential oil
reported in our study, are completely absent in Pinto (2012)
investigation. This behaviour may be related to the very different
environmental and geographical conditions of growth of the plants.
In addition, the analysis reported by Pinto (2012) is incomplete,
since only 64% of the total oil has been identied, therefore this
evidence could furtherly explain the high levels of diversity in the
oil composition with our samples.
The results obtained in the present study agree with previ-
ous reports that oil-poor species of Lamiaceae possess oil rich in
sesquiterpene compounds (Lawrence, 1992; Senatore et al., 1997).
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