Ziziphora L. comprises annual and perennial herbs in Iran. These herbaceous or sub shrubby plants have verticil lasters which are scattered in leaf axils or crowded in a terminal capitulum. This genus by about 25 to 30 species in Africa, Asia and Europe has only 4 species in Iran. The only perennial Ziziphora in Iran is Z. clinopodioides Lam. This species contains some subspecies according to Flora iranica (Rechinger, 1982). These subspecies are mainly distinguished from each other by means of calyx characters.
Lamiaceae family has been used for centuries as flavoring agent or spice in different foods and also in traditional medicines for treatment of digestive and viral diseases. Utilization of Ziziphora clinopodioides in yoghurt in many parts of Iran is an example of its daily medicinal usage. This species is used with yogurt, salad and many dishes. Some research has considered this species as a medicinal herb in Iran. Khodaparast et al. (2007) studied the interaction between these compounds and yoghurt to evaluate the effect of Ziziphora clinopodioides on growth of yoghurt starter culture.
Ziziphora in Iran is represented by four species. These are Z. capitata L., Z. clinopodioides Lam, Z. persica Bunge. and Z. tenuior L. All these species are aromatic and montane taxa of Iran. From these taxa Z. clinopodioides is distributed in Caucasia, North of Iraq, Iran, Afghanistan and Central Asia. These species are often a mat-forming perennial with prostrate to erect stems mainly much-branched at base. Their leaves show a high variation in size and shape. In Iran it is a plant of rocky slopes and scree. Subdivision of this polymorphic, basically Irano-Turanian, species into formal infra-specific taxa does not seem to be practical, since the variation of different characters is far from homologous and is poorly correlated with variation within the species (Sensu lato). Various morphological groups of this species in Turkey have also been recognized on the basis of their habit, leaf-shape and indumentums according to Hedge and Lamond (1982).
MATERIALS AND METHODS
Populations of Z. clinipodioides were collected from 8 different localities
of Iran from 2007 till 2008 (Table 1). In each sampling site
10 individuals were gathered. Macro and micro morphological characters were
studied (Table 2, 3). In Macro-morphological
studies 43 features were studied from vegetative and reproductive parts of individuals.
Micro-morphological studies considered epidermis and pollen grains. Dorsal epidermis
was studied by tissue removal using methylene green.
Pollen for examination by LM was prepared according to the Woodehouse method
(Woodehouse, 1935). Pollen grains of the 4 recognized subspecies taxa of Zizizohora
clinopodioides were mostly taken from herbarium material deposited at
the herbarium of Alzahra University (Tehran, Iran). The polar axis (P), equatorial
axis (E), corpus length (Clg) and width (Clt), apocolpium diameter and mesocolpium,
exine and intine thickness were measured on the pollen grains. Measurements
and light micrographs were taken with an Olympus DP12 microscope. The measured
pollen diameters were based on at least 20 samples and other characters on approximately
10 samples under the light microscope. For SEM, pollen grains were transferred
directly to a stub and micrographs were obtained using an XL-30 ESEM-FEG/PHILIPS
microscope. Pollen grains were treated by Physical vapor deposition method under
sputter coater. In general, the pollen terminology follows Punt et al.
||Address of Z. clinopodioides populations
|| Qualitative studied characters in Z. clinopodioides
|| Quantitative characters studied in Ziziphora clinopodioides
Pollens were studied by SEM without any treatment. Anatomical features of leaf were studied by hand made cross sections which were double colored by Carmen and Methylene green.
In order to detect significant differences in studied characters among populations,
Analysis of Variance (ANOVA) followed by the Least Significant Differences (LSD)
tests were performed. To reveal taxa relationships we used cluster analysis
and Principal Component Analysis (PCA). For multivariate analysis the mean of
quantitative characters were used while qualitative characters were coded as
binary/multi-state characters. Standardized variables were used for multivariate
statistical analysis. The average taxonomic distances and squared euclidean
distances were used as dissimilarity coefficient in cluster analysis of morphological
data. In order to determine the most variable morphological characters among
the species studied, factor analysis based on principal components analysis
was performed. SPSS ver. 15 was used for statistical analysis.
Anatomical observations: In leaf transactions of different populations
some differences were observed. In subsp. rigida (Boiss) Rech. f., hair
frequency was more in dorsal leaf surface, both surface had cuticle, a single
epidermis layer and there are fibers in both adaxial and abaxial leaf surface
but adaxial fibers are larger. Sclerenchyma are present between xylem and phloem
in mid rib (Hour population) or absent (Alvares population), both leaf surfaces
are smooth (Fig. 1A-I). In elburzensis
Rech. f. subsp. hair frequency is same in both leaf surface (28 km to Germi
population) or dorsal is more (Neor and 5 km to Germi populations). Mid rib
surface is smooth, cuticle thickness is same in both leaf surface and fibers
are present in both side of leaf (Neor and 5 km to Germi populations) or only
in adaxial surface (28 km to Germi) (Fig. 2A-F).
Subsp. szowitsii Rech. f., showed more hair frequency in leaf abaxial
surface, mid rib surface is smooth, both leaf surfaces had cuticle. There were
some sclerenchyma layers between xylem and phloem and some fiber layers in dorsal
leaf surface (Fig. 1). In subsp. pseudodasyanta Rech.
f., leaf dorsal surface had more hairs and there were observed some glandular
hairs. Midrib surface was smooth. There was no sclerenchyma between xylem and
phloem (Fig. 1).
||Leaf blade anatomical variations in different Z. clinopodioides
subspecies in Iran. First row (A, B and C Z. clinopodioides subsp.
pseudodasyantha), middle row (D, E and F, Z. clinopodioides subsp.
rigida) and lower row (G, H and I, Z. clinopodioides subsp.
szowitsii). (A: Simple hair, B: Glandular hair, C: Upper collenchyma,
D: Upper parenchyma, E: Phloem, F: Xylem, G: Lower fibers, H: Lower collenchyma,
I: Lower epidermis, J: Palisade parenchyma, K: Spongy parenchyma, L: Upper
fibers, S: Sclerenchyma, P: Upper cuticle, R: Lower cuticle, N: Secondary
veins, M: Orbicular sclerenchyma, X: Xylem along M, Y: Phloem along M)
||Leaf blade anatomical variations in different populations
of Z. clinopodioides subsp. elburzensis in Iran (A, D and
E, Neor Lake), (B, 28 km to Germi) and (C and F, 5 km to Germi). Abbreviations
as Fig. 1
|| Stomata types of Ziziphora clinopodioides subspecies
Studying epidermis characters showed that in different subspecies of Z.
clinopodioides there are some differences in cell wall shape and cell shape.
The epidermal cells are variously shaped with sinuous walls in all studied subspecies.
The epidermal cells have wavy walls. The cells exhibit a somewhat quadrangular
shape and wavy cell walls in elburzensis subspecies. In subsp. rigida
some how arc shape cell walls without much sinuate cell walls were observed
(Fig. 3). In all subspecies the stomata are superficial. Although,
other stomata types have been reported from the Lamiaceae only anomocytic stomata
is recorded in the Z. clinopodioides subspecies here.
||SEM and LM micrographs of Z. clinopodioides
subspecies pollen grains. Upper row: Equatorial view, Middle row; Surface
detail, Lower row; LM at polar view. (A) elburzensis, (B) rigida
and (C) szowitzii
Palynological comparisons: The pollen grains are monads, six-zonocolpate, prolate, prolate-spheroidal to spheroid as pollen grains did not show particular differences in LM observations, SEM micrographs were used. Polar axis was of 25.9-26.9 μ and an equatorial diameter of 17.1-19.7 μ of the examined populations, the largest pollen grains, on average, were observed in Mazandaran population (Z. clinopodioides subsp. szowitsii) and the smallest pollen grains, were observed in Ghozlou population (subspecies rigida). The longest corpus was observed in subsp. rigida and the shortest was in subsp. elburzensis. The exine sculpture is bireticulate. In the bireticulate ornamentations number of perforations and mesh size are not identical. The average diameter of the small perforations is less than 1.50 μ. Present results are a confirmation of the genus position in Mentheae and Nepetoideae. Pollen grains are illustrated in LM and SEM (Fig. 4). The ectocolpi are distributed symmetrically, elongated, usually shallow and narrowing at the poles.
Morphological observations: Although, there are many thing in common in studied populations but there are fine visible modifications. Some of these differences are related to calyx features. In elburzensis subspecies, calyx length is 3-4.02 mm, plants are glabrous to hairy with hairs up to 0.25 mm length. Hair are very infrequent and sparsely distributed. These hair are straight and white colored. The rigida subspecies have calyx with 3.3-4.7 mm length. Calyx is pedicled and its base is hairy. Hair length is 0.37 to 0.81 mm. Hair are white colored and with medium frequency. In szowitsii calyx is 5.13 mm and calyx hair are about 0.4 mm length. Hair are whitish and dense. Ovate shape leaves are observed in pseudodasyatha subspecies which is mainly covered with dense hairs, 0.31 to 0.597 mm length. Leaves width is more than their length. A comparison of hair at dorsal surface of calyx is given in Fig. 5.
The cluster analysis of Z. clinopodioides subspecies of Iran, based
on both quantitative and qualitative characters presented two major clusters
(Fig. 6). At first step three subspecies is separated from
Z. clinopodioides subsp. szowitsii. There are sub clusters, one
is containing populations belonging to Z. clinopodioides subsp. rigida
but Z. clinopodioides subsp. elburzensis are inserted in different
clusters (one population near rigida, another near pseudodasyantha
and one population makes a separate cluster itself). As it is evident Z.
clinopodioides subsp. szowitsii, pseudodasyantha and subsp.
rigida are clearly separated but it seems that subsp. elburzensis
is not clearly limited by the studied set of characters (Fig.
|| Calyx dorsal surface in different subspecies of Z.
clinopodioides in Iran
||WARD clustering of Z. clinopodioides subspecies based
on mean of quantitative and qualitative morphological characters in Iran
In order to determine the most variable characters among the studied species,
Factor analysis based on PCA was performed revealing that the first two factors
comprise about 58% of total variation. In the first factor with about 32% of
total variation, characters such as inflorescence leaf base and hair and calyx
hair type possessed the highest positive correlation (≥0.7). In the second
factor with about 26% of total variation, characters as inflorescence leaf shape,
calyx hair density and leaf hair type possessed the highest positive correlations.
In the third factor with about 14% of total variation, characters as stem hair
density and corolla length cause high variation. These are the most variable
morphological characters among Z. clinopodioides subspecies in Iran (Table
||Factor analysis results based on morphological characters
for Z. clinopodioides subspecies of Iran
As no one has studied Ziziphora pollen grains before, observations could
not be compared with others findings. The studied subspecies are considered
to be most closely related to Lycopus L. and Mentha L. in the
tribe Mentheae (Briquet, 1896). According to the present results, the pollen
morphology in Ziziphora clinopodioides may be helpful in elucidating
the infra specific relationships. Studies so far in the Ziziphora indicate
that additional sources of potentially very useful characters are available
from the pollen morphology. As with any morphological study, the more complete
the data, the more convincing the subsequent analyses regarding phylogeny and
|| PCA ordination of the Z. clinopodioides subspecies
based on morphological characters
In this study all leaf micro-characters of epidermis were shown to be of great importance in the taxonomy of the Z. clinopodioides subspecies. They allowed in many instances clear separation between various taxa (Fig. 7). The rigida subspecies seems to be clearly separated (red circle in Fig. 7). Other clearly separated subspecies are pseudodasyantha (green circle in Fig. 7) and szowotsii (Dark blue circle). As in Fig. 7 it is evident the elburzensis subspecies has not been clearly separated and is distributed between other subspecies and its limitation needs further considerations. Due to the studied micro and macro-morphological characters an identification key to separate four Z. clinopodioides subspecies has been provided.
||Inflorescence leaf wider than other leaves
||Inflorescence leaf not wider than other leaves
|| Calyx hair frequent and dense
||Calyx hair sparse
||Leaf hair dense
|| Leaf hair sparse
The use of micro morphological characters as complement of macro-characters for taxonomic purposes is highly recommended to study the specific or infra-specific limits of Lamiaceae. These subspecies were not studied before and this project was the first step in Iran to study these subspecies. The separation of Z. clinopodioides subsp. elburzensis should be revised due to the differences in soil types and habitats occupied by this subspecies.