BotanicalJoumal of the Lannean Sockg (1997), 123: 353-362. With 4 figures
Infiaspecific variation of leaf anatomy in
Origanum vulgare grown wild in Greece
ARTEMIOS M. BOSABALIDIS
Department of Botay, School
of Biology, Aristotle Universip, Eiessaloniki 54006, Greece
AND
STELLA KOKKINI
Luboratoy of Systematic Botany and Pbtogeography, School 0sBiology,Aristoth Uniuersip,
lhessaloniki 54006, Greece
Receiued Septmber 1996; acceptedf o r publication Noumber I996
The histological components of the leaf were studied in dried herbarium material of the three Organum
uukare subspecies (subsp. hzrtum, subsp. uiria'ulum and subsp. uukare) grown wild in Greece. These three,
geographically distinct, taxa showed remarkable differences in their leaves. The leaves of subsp. hirtum
grown in the Mediterranean climatic zone of Greece are characterized by thick cutinized outer walls of
the epidermal cells and a thick mesophyll with highly developed chlorophyllous tissues. Peltate glandular
trichomes and stomata are numerous on both leaf surfaces. The thickness of the mesophyll decreases
in the other two subspecies grown in the northern part of the country, where a Continental type of
climate occurs. The number of glandular trichomes and stomata also decreases. Besides these differences,
a noticeable reduction in the size of the essential oil-accumulating subcuticular chamber of the glandular
trichomes and in the number of the peribasal cells, has also been recorded in the plants of subsp. uukagare
and uiridulum.
0 1997 The Linnean Society of London
ADDITIONAL KEY WORDS:--essential oils - glandular trichomes - stomata.
CONTENTS
Introduction . . . . . . . .
Material and methods . . . . .
Results . . . . . . . . . .
Cross sections of leaves . . .
Paradermal sections of leaves .
Discussion . . . . . . . . .
References . . . . . . . . .
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353
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355
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36 1
362
INTRODUCTION
Olzganum uukare L. is the most widely distributed species of the genus Olzganum in
Greece. In a taxonomic study of the Greek populations, Kokkini, Vokou & Karousou
0024-4074/97/040353
+ 10 $25.00/0/bt960089
353
0 1997 The Linnean Society of London
354
A. M. BOSABALIDIS AND S. KOKKINI
(1 99 1) reported the occurrence of three, geographically distinct, subspecies in the
country, viz. 0. vulgare subsp. hirtum (Link) Ietswaart [O. hirtum Link, 0. heracleoticum
sensu Halacsy, Consp. Fl. Graer. 2: 255 (1902); Hayek, Prodr. Fl. Penins. Balc. 2:
334 (1931);Rechinger, Fl. Aegaea 532 (1943)& Bot.Jahrb. 80: 395 (1961);Fernades
& Heywood in Tutin et al., F1. Eur. 3: 171 (1972); non L. Sp. PI.: 589 (1753)],
subsp. vulgare and subsp. uiridulum (Martrin-Donos) Nyman [O. viridulum MartrinDonos, 0. herackoticum L., 0. mkare L. var. uinde Boiss., 0. uiride (Boiss.) Halacsy, 0.
uulgare L. subsp. viride (Boiss.) Hayek]. Subspecies hirtum is mainly found on the
islands and southern mainland, whereas towards the north it is mostly confined to
the lowland, coastal areas. Its range in Greece is limited by the prevalence of a
Continental climate, in the northern and central parts of the mainland KotiniZambaka, 1983). Subspecies vulgare is mainly grown in the north-east and northcentral mountainous regions of Greece, and subsp. viridulum in the north-west part
of the mainland (Kokkini et al., 1991).
One of the key characters used in both recent and earlier taxonomic approaches
of 0. vulgare infraspecific taxa is the appearance of sessile glands, i.e. peltate glandular
trichomes (conspicuous or inconspicuous) on the leaves (Boissier, 1879; Halacsy,
1902; Hayek, 1931; Rechinger, 1943; Ietswaart, 1980; Baden, 1991; Kokkini et al.,
1991). Furthermore, a number of studies suggest that the essential oil content and
composition can also be used for the discrimination of the three 0. uukan subspecies
(Kokkini & Vokou, 1989; Kokkini et al., 1991; Vokou, Kokkini & Bessiere, 1993;
Kokkini, Karousou & Vokou, 1994).
The present report, part of a wider project on the morphological and chemical
variation of 0. vulgare plants grown wild in Greece, is focused on the histological/
morphometrical study of the leaves and particularly of the epidermal glandular
trichomes producing essential oil.
MATERLiL i\iw METHODS
Developed middle cauline leaves of 0. vu&are herbarium specimens from fullyflowered plants were used. The specimens collected from the islands of Crete (west
Crete: near the village of Lakki, alt. 550 m, Kokkini 1O.vii. 1984)and Euboea (central
Euboea: Mt Dirfys, above the village of Steni, alt. 950 m, Kokkini 3O.vii. 1985)
corresponded to the subspecies hirtum, those from Mt Menikio (north-east Greece:
above the village of Mikropolis, alt. 750 m, Kokkini 1O.vii. 1986) to subsp. vuZgure,
and the ones from Mt Mitsikeli (north-west Greece: near the village of Anthrakitis,
alt. 850 m, Kokkini 16.viii.1987) to subsp. viridulum (Fig. 1). The specimens are
deposited in thc Herbarium of the Institute of Systematic Botany and Phytogeography
of the Aristotle University of Thessaloniki (TAU).
In order to obtain a clear and reliable picture of the leaf anatomy of the dried
herbarium specimens, we attempted to apply a modified technique of fixation of
fresh leaf material used in electron microscopy (Robinson et al., 1985). Three
leaves from each taxon were used which were softened overnight between waterimpregnated filter papers. For each leaf, one sample was taken from either side of
the middle part of the midvein. The samples were prefixed with a mixture of 2.5%
glutaraldehyde and 2% paraformaldehyde in 0.05 M phosphate buffer and then
postfixed with 1% osmium tetroxide in the same buffer @H 7.0). After dehydration
LEAF ANATOMY OF OHGA..MLM WLGARE
355
Figure I , Localities in Greece where O n g a n u m vdguz specimens used in the present study were collected. (0)subsp.
hirtum; (m) subsp. uulgare; (A)subsp. viridulum.
in an alcohol series, the leaves were embedded in Spurr’s (1969) resin. Semithin
sections (1-2 pm thick) were obtained in a Reichert O m U, ultramicrotome, stained
with 1% toluidine blue 0 in borax and observed in a Zeiss 111 photomicroscope.
The above chemical treatment is to our knowledge applied for the first time to
dried herbarium material. The clear anatomical picture with resolution to the
chloroplast level (see Fig. 2) would be useful to further studies.
The number of stomata and glandular trichomes per leaf surface unit area was
assessed in leaf paradermal sections. Because of the irregular surface of the leaf
blade, paradermal sections were serially cut and the positions of stomata and glandular
trichomes in the successive sections were recorded on a common transparency.
RESULTS
Cross sections of leaves
The three Oeanum uukaw subspecies hirtum, uukaw and viridulum exhibit in leaf
cross sections obvious differences in the thickness of the lamina (Fig. 2). In the leaves
of subsp. hirtum, particularly those from Euboea, the lamina is thicker due to an
3.56
ii hf. BOSAB.UIDIS XYD S. KOKKIKI
h p r e 2.C:omparativr anatomy of 07~qammm@re lea\.es from differenr regions of &xxe. ‘4, subsp. hirtum from
<:rere. B. subsp. h b ? m from Eubora. C . subsp. ru[qaw. D, subsp. i~iridulum.Note tbc differences in the thickness 01
thc leaf blade, thc size of the epidermal and mcsophyll rclls. the presence of glandular trichomes and the thicknes!
of rhr outer periclinal walls of the epidmnal cells. d glandular trichomc, fip palisade parerrchynia, U P tipper
epidermal cells, /e lower epidermal cells, .~pspongy parenrhyna. Scale bar= 60 pm.
LEAF ANATOMY OF ORZGAJCLM WLGARE
357
TABLE
1. Thickness (in p)of the epidermis and the mesophyll tissues of the leaves of Origunum vulp-are
(mean SD)
~
Taxon
subsp. hirtum"
subsp. hirtumb
subsp. mlgare
subsp. utridulum
Upper
epidermis
Palisade
parenchyma
SPonU
parenchyma
27.10f2.07
26.66k3.40
31.80f 10.59
22.96f2.50
64.37k3.39
70.71 f8.96
70.00f9.03
29.58k4.64
72.92f5.42
148.64f7.10
63.53f6.31
27.95f4.27
~~
Lower
epidermis
Entire
leaf
24.80f2.74
21.85k3.44
21.96f3.43
16.79k3.39
189.19*
267.86
185.09
97.28
from Crete
'from Euboea
* sum of the means of the values of the partial leaf components
a
extensive increase of the amount of the spongy mesophyll tissue (Fig. ZB, Table 1).
In subsp. vulgare, the leaf blade has about the same thickness as that of subsp. hirtum
from Crete, but the thickness of the partial histological components of the blade
(upper and lower epidermes, palisade and spongy parenchyma) varies (Fig. 2C,
Table 1).
The leaves of subsp. viridulum were found to have the thinnest lamina (Fig. 2D,
Table l), c. 49% and 64% thinner than that of subsp. hirtum from Crete and Euboea,
respectively. It is also c. 47% thinner than the lamina of subsp. vulgare. The cells of
the palisade parenchyma are not elongate as in the typical dicot leaf, but they are
short, leaving between them large intercellular spaces. An aerenchyrna also exists
in the spongy tissue, the cells of which are very small compared with those of the
leaves from Crete, Menikio and particularly from Euboea.
The leaves of subsp. hirtum, particularly those from Crete, show in cross section
the presence on both surfaces of numerous local depressions bearing glandular
trichomes (Fig. 2A, B). In the adaxial and abaxial epidermal cells, the outer cutinized
periclinal walls are very thick (Fig. ZA, B), a fact associated with a reduced flexibility
of the lamina.
Paradermal sections of leaves
Examination of leaf paradermal sections from plants of all four regions studied,
showed that stomata occur on both leaf surfaces, the lower one having a greater
number (Figs 3, 4; Table 2). Inamdar & Bhatt (1972), who studied 33 species in 17
genera of the Labiatae (the genus Organum was not included), found that in the
majority of species, stomata occurred exclusively on the lower leaf surface (the only
species with stomata on both leaf surfaces were Leucas aspera Spreng., L. zqlanica R.
Br., and Ocimum basilkum L.). However, Cantino (1990) in an extensive survey of
stomatal position in 127 genera of Labiatae (Oeanum was again not included),
mentioned that both hypostomatic and amphistomatic leaves are found in the
members of the family, the presence of the latter type of leaves being slightly more
frequent.
The upper leaf surface is further observed to possess more peltate glandular
trichomes than the lower one (Figs 3, 4; Table 2). An increase in glandular trichome
density on the upper leaf surface has also been noticed in other plants producing
essential oils (Bory & Clair-Maczulajtys, 1981; Ascensao & Pais, 1987; Bosabalidis
A 51 BOS.1R.U.IDIS .lND S KOKKINI
LEAF ANATOMY OF ORIGANUM WLGARE
359
Figure 4. Leaf paradermal sections selected from respective series to show the density of stomata and glandular
richomes, the size of glandular trichomes and the number of epidermal cells surrounding the trichomes. A & B,
ipper and lower leaf surfaces from Organum uulgure subsp. vulgare, C & D, upper and lower leaf surfaces from subsp.
iridulum. Scale bar = 125 pm.
A. M. BOSABALIDIS AND S. KOKKINI
360
TXBLE
2. Number of glandular trichomes and stomata per mm2 of leaf surface area in O@unum vukure
(mean _+ SD)
Lower leaf surface
Upper leaf surface
Taxon
subsp. brtzim*
subsp. hirtum'
subsp. vdgaa
subsp. uiridulum
Glandular ~ c h o m e s
11.30+0.92
7.63 k0.57
4.26k0.29
1.48 f 0.03
Stomata
Glandular trichomes
Stomata
97.67 k8.99
103.08k 12.59
43.3 I f 5.64
5.56k0.63
10.09f 1.77
5.54k0.76
3.19 k0.50
1.16 f0.04
206.32 f24.85
355.26 k52.5 1
283.08 k 38.24
214.21 k33.07
from Crete
Euboea
" from
TABLE
3. Horizontal diameter (pm) of the essential oil-accumulating subcuticular chamber and number
of peribasal epidermal cells in each glandular trichome (surface view of Origunum vulgure leaves)
(mean +_ SD)
Upper leaf surface
Taxon
Lower leaf surface
Subcuticular
chamber
Peribasal cells
Subcuticular
chamber
Perihasal cells
103.07f 3.59
80.43 f 3.78
76.67 k2.09
70.27 2 2.58
17.3Of 1.57
12.86f0.77
14.00 0.82
12.57 f0.53
92.73f3.37
71 .OO f4.7 1
68.27t3.19
63.07 5 2.68
18.565 1.75
_
I
_
subsp. krtum'
subsp. hirbm''
subsp. o&aa
suhsp. iidulurn
a
I'
13.555 0.82
13.50t0.53
14.57k0.85
from Crete
from Euboea
& Exarchou, 1995). The glandular trichomes of the upper epidermis were found to
have larger essential oil-accumulating subcuticular spaces (Table 3). The number
of epidermal cells surrounding the foot cells of the trichomes radially does not
appear to vary remarkably in the populations of the upper and lower leaf epidermes
(Table 3).
Comparative observations made on the density and size of glandular trichomes
and/or stomata per mm2 of both leaf surfaces disclosed that further north the plants
extend the smaller are the number of glandular trichomes and stomata per leaf
surface unit area. The same pattern also holds true for the size of the subcuticular
space and/or the number of the peribasal cells of the trichomes (Table 3).
The leaves of subsp. hirturn from Euboea bear on both surfaces fewer glandular
trichomes and more stomata than the leaves from Crete (Table 2). The subcuticular
space of the trichomes is smaller in the leaves from Euboea and so is the number
of the trichome peribasal cells (Table 3). In subsp. uulgare, most of the parameters
mentioned in Tables 2 and 3 have undergone a reduction in their values compared
with those of subsp. hirtum. The lowest values, however, are estimated in the leaves
of subsp. uiridulurn, in which stomata have almost disappeared from the upper
epidermis (reduction by about 94% compared with subsp. hirtum from Crete) (Fig.
4C, Tables 2, 3). Analogous is also the reduction (by 87%) in the number of
glandular trichomes per mm2 of leaf surface and (by 32%) in the size of the essential
oil-bearing subcuticular chamber. The number of epidermal cells radially surrounding
the foot cell of the glandular trichomes similarly decreases.
LEAF ANATOMY OF ORIGANUM WLGARE
36 1
DISCUSSION
The above findings confirm the observations made by taxonomists using an
ordinary lens or stereoscope, who had noticed the visible differences between the
three subspecies in respect of their glandular trichomes (Boissier, 1879; Halacsy,
1902; Hayek, 1931; Rechinger, 1943; Ietswaart, 1980; Baden, 1991; Kokkini et al.,
1991).
The variability of 0. vukare within Greece, in the leaf parameters investigated in
the present study, can probably be ascribed to the climatic conditions prevailing in
the respective regions. The island of Crete, where the southernmost of the two
subsp. hirtum populations grows, belongs to the Real Mediterranean climatic zone
of Greece, whereas the island of Euboea is found in the Main Transitional climatic
zone between the Real Mediterranean and the Continental Mediterranean zone.
Thus on Crete, the total annual sunshine is increased and the summer precipitation
is decreased, in comparisonwith Euboea. The populations of the other two subspecies,
viridulum and vukare found in Mts Mitsikeli and Menikio, respectively, fall within the
Continental Mediterranean climatic zone of Greece. This zone is characterized by
lower winter temperatures, less sunshine, and higher total precipitation than the
other two zones (Kotini-Zambaka, 1983).
The differences found between the two populations of subsp. hirtum as concerns
the increased number of glandular trichomes and the greater size of the essential
oil-accumulating chamber in each trichome, suggest respective differences in the
essential oil content of the plants. Thus, subsp. hirtum plants collected from Crete
have an essential oil content of 4.8 ml/ 100 g-' dry wt, whereas those from Euboea
have an oil content of 3.2rnl/lOOg-' dry wt (Kokkini & Vokou, 1989; Vokou et
al., 1993). Distillation procedures applied to a high number of 0. vukare samples
from scattered localities all over Greece, have shown that subsp. hirtum, though a
very variable taxon, is always rich in essential oil (its oil content ranges from 1.1 to
8.2 m1/100 g-' dry wt). The other two subspecies have a much lower essential oil
content, with traces up to 0.8 m1/100 g-' dry wt (Kokkini et al., 1991). It should be
mentioned, furthermore, that differences between the subspecies exist not only in
the content of the essential oil, but also in its composition. The essential oil of subsp.
hirtum is characterized by a high phenolic content (up to 94% of the total oil),
whereas that of subspecies uiridulum and vukare has a much lower one (Kokkini et
al., 1991; Vokou et al., 1993; Kokkini et al., 1994).
Regarding the ecological significance of the glandular trichomes and their essential
oils, it has been suggested that they may play a defensive role against microbes and
herbivores. The dense leaf glandular pubescence may also have an effect on
leaf temperature and water loss through increased light reflectance (Levin, 1973;
Ehleringer, 1984; Kelsey, Reynolds & Rodriguez, 1984; Ehleringer & Clark, 1988).
The great number of stomata on the upper leaf side in the plants from the islands
of Crete and Euboea is puzzling. One would expect that in such warm and dry
regions like the Aegean islands, the number of stomata on the upper leaf side should
be limited, since that side is directly exposed to the sun's heat. It might be that in
this case stomata on the upper leaf side are concerned more with the conduction
of C 0 2 rather than of water vapour (Farquhar & Sharkey, 1982).
The anatomical differences found in the leaves of the three 0. vukare subspecies
grown wild in Greece, support their morphological and chemical (essential oil)
differences (Kokkini& Vokou, 1989; Kokkini et al., 1991; Vokou et aL, 1993; Kokkini
362
A. M. BOSABALIDIS AND S. KOKKINI
al., 1994). The overall detected differences should be considered as compensation
responses of 0. vulgare plants to the various climatic conditions prevailing in each
area of the country.
et
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