The genus, Trifolium L., is one of the most important genera of the
Fabaceae (papilionaceae) familym which consists of 52 annual and
perennial forage species in Iran. After Medicago, this species is regarded
as the second valuable forage plant in Iran, of which certain species such as
T. alexandrium and T. repens have a high agronomic value.
L. was investigated in the form of 6 sections, i.e., Vesicaria,
Trifolium, Lotoidea, Chronosemium, Mistyllus, Trichocephalum.
The Lotoidea section (Heller, 1971-72) comprises 5 perennial species, namely T. radicosum, T. hybridum, T. montanum, T. ambiguum and T. repens and 2 annual species, i.e., T. nigrescens and T. glomeratum in Iran. This section is considered as the most complex section of this genus in term of taxonomy (Zohary and Heller, 1984).
Among the perennial species, the two species of T. ambiguum and T. montanum have great similarities in reproductive organs and the calyx characteristics, making their taxonomic identification a hard job. Moreover, there are many similarities between the perennial species of T. repens and the annual species of T. nigrescens, particularly concerning the shapes of calyx, standard, legume, corolla and leaflets, whereby morphological characteristics are not capable of distinguishing these species from one another accurately.
T. radicosum, the endemic species of Iran, has not undergone any biosystematic
study. The geographical dispersion of this endemic species is in the provinces
of Mazandaran and Tehran. With regards to seeds, there are several studies that
analyse different aspects such as chemical compositions (Kakea and Esen, 1993),
observations through fluorescence microscopy techniques or genetic variation
(Smith et al., 1995).On the other hand, to use of SEM allows for the
observation of structures which would be difficult by other means and some authors
have highlighted the importance of this technique for the study of seed-coats
specially for those genus or species in which identification is complicated,
such as the Trifolium (Zohary and Heller, 1984) or Astragalus
(Karamian and Ranjbar, 2005) in Fabaceae family, where features of seed morphology
have been widely used to distinguish the different taxa or to find affinities
between them. The aim of present study was to illustrate the role of seed features
in the identification of species of Trifolium (Lotoidae section)
in Iran and to relate such characters to the systematics of this section. The
size, weight, dimensions and shape of the Trifolium seed are different
in its various species and are therefore of taxonomic value. In fact, most of
the information on seed micromorphology in the genus Trifolium is related
to the works of Zohary and Heller (1984).
Seed shapes in the species of this genus include ovoid, elliptic and oblong-elliptic. Seed surface may be smooth, scabrate, tuberculate or rugulate. In the species relating to three subspecies of the Lotoidea section, including Oxaliodea, Ochreata and Lotoidea, the hilum shape is apical (Zohary and Heller, 1984). In the present study, the seed characteristics of the Lotoidea section in Iran were analyzed for the purpose of studying the phenetic relationship of the species with one another and for using such characteristics in systematics studies.
MATERIALS AND METHODS
Seed morphology was studied in 7 taxa of the Trifolium, Lotoidea section, including T. repens, T. radicosum, T. nigrescens, T. hybridum, T. glomeratum, T. montanum and T. ambiguum from Iran. The study was based on dry herbarium specimens from the Department of Plant Genetics and Genetic Resource, Seed and Plant Improvement Institute of Karaj. To observe the seeds under the Scanning Electron Microscope (SEM) they were covered by a thin layer of platinum. The measurements are based on the SEM data obtained.
The SEM images included those with a magnification of 95x to observe the general view of the seeds and those with a magnification of 5500x to view the seed sculpture. In addition, a stereomicroscope was used to study hilum shape, which was drawn with a magnification of 40x. Qualitative characteristics including length, width and L/W ratio of the seed as well as the qualitative characteristics of seed sculpture and hilum shape were measured and investigated on each of the samples. The terminology of seed-coat surface sculpturing basically follows Steran (1992) and Font Quer (1993).
RESULTS AND DISCUSSION
The qualitative and quantitative characteristics investigated in the present study included length, width and L/W ratio of the seed, seed shape, hilum shape, seed sculpture and seed color (Aghabaighi, 1998). The size, weight, dimensions and shape of the Trifolium seed are different in its various species and are therefore of taxonomic value. There are wide variety shapes (Lenticular, oblong-ovoide, reniform and ovoid). The seed coat is wrinkled tubercle, reticulate or regulate. Hillum shape is circular to square, elliptic or spindle.
The smallest length and width were those of the species T. repens and the largest, T. radicosum. In the two species of T. ambiguum and T. montanum the seed shape is different despite close morphological characteristics. Seed shape in T. ambiguum was lenticular and in T. montanum, oblong-ovoid, which conforms to the results obtained by Zohary and Heller (1984). In T. radicosum, which is morphologically different from the other species of this section, the seed shape was also different and was elliptic. As with T. radicosum, the T. glomeratum species showed significant differences with the other species with regard to certain morphological characteristics, seed shape is different from the other species and is reniform. The ovoid shape is the dominant shape in the species of this section, which was observed in the species of T. repens, T. nigrescens and T. hybridum. Seed color did not play any particular role in differentiating the species of the Lotoidea section. The seeds were mostly brown to light brown in color.
Hilum shape was different in the two species of T. repens and its ancestral
species, i.e., T. nigrescens(Fig. 1). Hilum shape in
T. repens was rounded while in T. nigrescens it was elliptic.
Thus, hilum shape played a decisive role in differentiating these two species
(the results has not shown). Hilum shape in the two species of T. ambiguum
and T. montanum was similar and of the four-sided type The spindle-shaped
hilum of T. radicosum was considered as another differentiating characteristic
of this species. In the two species of T. glomeratum and T. hybridum,
which showed little morphological similarities with respect to leaflet shape,
inflorescence shape, standard shape and absence of tricome in the calyx tube
and tooth, the hilum shape was physical and stood in an elliptic shape.
Figure 2 shows the seed sculptures of the species of this
section. The investigations revealed that seed sculptures in the species of
this section have variations. In the two very close species, i.e., T. nigrescens
and T. repens, the difference in seed sculpture is quite evident
(Fig. 2e and g). In T. repens, the
seed surface reticulate, while in T. nigrescens the sculpture is of
wrinkled tubercle type.
Tubercular sculptures in the two shapes of smooth tubercles and wrinkled tubercles
were seen in the four species of T. nigrescens, T. hybridum, T.
radicosum and T. glomeratum (Fig. 2b-c
and e-f). Seeds with smooth tubercle (star-shaped) were only
seen in T. glomeratum (Fig. 2b).
||Hillum shape of Lotoidae species
Based on the above characteristics, a cluster dendrogram were drawn for the
species of the Lotoidea section through the UPGMA and Ward methods as
shown in the (Fig. 3). The dendrogram was itself divided into
4 main clusters. The first cluster included T. repens only. This result
showed that T. repens can be separated from its highly similar species,
i.e., T. nigrescens based on seed characteristics. Thus, the micromorphological
study of the seed played an important role in differentiating these two species
which are hardly differentiable in terms of such morphological characteristics
as leaflet shape, inflorescence shape, corolla color, standard shape, calyx
shape and legume shape.
The second cluster included three species: the two species of T. nigrescens and T. hybridum under one sub-cluster and T. glomeratum alone under another sub-cluster and situated near T. hybridum with a similarity level of 3.67. An investigation of these three species showed that morphologically they are close to one another and the findings of the seed micromorphologic study also confirmed these results.
There were two species in the third cluster, i.e., T. montanum and T. ambiguum with a similarity level of 4.2. These species are highly similar to each other morphologically and the results of the seed micromorphologic study very well demonstrate such similarity and places them in a separate cluster.
The fourth cluster included T. radicosum alone which showed the greatest difference with the other species. The morphological characteristics of this species, including purple corolla, oblong standard and elliptic seeds, the pollen grain features and the chromosome morphology strengthen the probability that this species is separate from the Lotoidea section.
The cluster dendrogram showed that the results of the seed micromorphologic
study highly confirm to the results of morphological studies of the above species.
||Seed sculpture: a) T. ambiguum b) T. glomeratum c) T. hybridum
d) T. montanum e) T. nigrescens f) T. radicosum g) T. repens
The results obtained confirm the usefulness of seed characters for the identification
of the species and highlight the division of near species in this section. Zohary
and Heller (1984) divided this section into 3 sub section based on seed morphology.
In the species relating to three subspecies of the Lotoidea section,
Ochreata and Lotoidea, the hilum shape
is apical. In the other species of the Lotoidea section and in almost
all the other sections of this genus, the hilum situation of the seeds have
either a marginal or central position.
Watson et al. (2000) by using ITS marker support that sect. Lotoidae
species form the basal grade in the molecular phylogenies. In their cluster
T. montanum and T. ambigum near each other with high parsimony.
Present results correspond with their work. On the other hand, based on morphological,
palynology and chromosome number in sect. Lotoidae in Iran, T. radicosum,
the endemic species in Iran, has different characters in pollen shape, sculpture
and karyotype with other species in this section. Micromorphological studies
support these results.
||Cluster dendrogram of Lotoidea species through UPGMA
and Ward method
The following shows the identification key of the species of the Lotoidea
section based on seed characteristics:
Based on the results, micromorphologic characteristics of the seed in the species
of the Lotoidea section had effective role in determining the phenetic
relationship of these species.