Intra-specific variations in Silene : Morphometry and micromorphometry analyses

The Silene species are annual, biennial or perennial herbs mainly distributed in the northern hemisphere, Europe, Asia and northern Africa. The section, Inflatae Boiss., is the smallest section of the genus containing three species and four subspecies in Iran, out of which one subspecies is endemic with very restricted distribution in the western and northern regions. Studies on seed morphology with scanning electron microscope (SEM) have revealed taxonomically useful microcharacters. The present study was performed to study morphological and micromorphological features in seven Silene species and subspecies in the sect Auriculatae and Inflatae growing in Iran, to study their intraspecific variations and to find out if such data can be used in taxonomy of the genus. The grouping obtained by unweighted pair group method with arithmetic mean (UPGMA) dendrogram separated the species of the section Inflatae from the species of the section Auriculatae. Siahbishe population of Silene vulgaris differed in morphological characters and was placed some distance from the other studied populations. It also had the highest values of total haploid chromosome length (50.51 μM), size of the longest chromosome, (5.42 μM), size of the shortest chromosome (2.88 μM) and the mean chromosome length (4.21 μM). Therefore morphological differences of this population may be related to its higher total chromosome length (genome size), compared to those of other populations studied. We also suggested that the Siahbishe population to be considered as a new variety of S. vulgaris, based on both morphological and cytological grounds. The seed type in both section are symmetrical reniform, asymmetric reniform and rounded-reniform. In the sect. Auriculate, the length of seed ranged from 1.22 to 2.23 mm, while the size of seed width ranged from 0.86 to 1.38 mm. In the sect. Inflatae, the length of seed ranged from 1.30 to 1.86 mm and the seed width ranged from 0.80 to 1.42 mm. These two sections differed in some other seed characteristics which are discussed.


INTRODUCTION
The genus Silene L. (Caryophyllaceae) consists of about 700 species mainly distributed in the northern hemisphere, Europe, Asia and Northern Africa (Bari, 1973;Greuter, 1995).Based on morphological characters, Chowdhuri (1957) classified the Silene species into 22 sections, which was not supported by molecular studies *Corresponding author.E-mail: msheidai@yahoo.com.
Abbreviations: UPGMA, Unweighted pair group method with arithmetic mean; SEM, scanning electron microscope.
Thus, x = 9, 10, 12 and 23 are the known basic chromosome numbers in Silene.
The section Inflatae Boiss. is the smallest section of the genus containing three species and four subspecies in Iran, out of which one subspecies is endemic with very restricted distribution in western and northern regions (Melzheimer, 1980).The members of this section are perennial with unifloral or dichasial inflorescence, vosiculate-planned calyx with 10 to 20 fibrous and the fruit capsules which are often scattered.On the other hand, the section Auriculatae (Boiss.)Schischkin, is the largest section of the genus, containing about 35 species in Iran, out of which 21 species are endemic with very restricted distribution in mountainous areas such as Elburz, Zagros and Azarbayejan (Melzheimer, 1988).The members of this section are caespitose plants with large flowers placed at the end of short stems.Their inflorescence is unifloral or dichasial.Calyx is cylindricalclavate, pubscent or glandular-pubescent.The petals have conspicuous auricule at the end of claws.
Seed coat morphology is surprisingly slightly affected by environmental conditions under which the plant grows (Barthlott, 1984;Özcan, 2002;Gontcharova et al., 2009;Abdel Khalik, 2006;Wada and Reed, 2008;Karihaloo and Malik, 1996;Gandhi et al., 2011;Plazal et al., 2004;Hong et al., 1999).Studies on seed morphology with scanning electron microscopy (SEM) have revealed taxonomically-important microcharacters, such as cell shape (Canne, 1979;Hong et al., 1999;Gandhi et al., 2011, Plazal et al,. 2004), cellular arrangements (Clark and Jernstedt, 1978;Hong et al., 1999), and the protrusions and patterns of the radial walls (Carolin, 1980, Hong et al., 1999).In Caryophyllaceae, seeds are relatively small (usually less than 3 mm long), and the testa is often variously sculptured (Bittrich, 1993).A few SEM studies have been performed on seed morphology in Caryophyllaceae, including Arenaria L. (Wofford, 1981;Wyatt, 1984), Sagina L. (Crow, 1979) a few local taxa in Silene ) Melzheimer, 1977), and Melandrium Roehl.(Chung and Lee, 1988;Wada and Reed, 2008) The seeds of eight species of Silene in Korea were examined with a SEM to evaluate the systematic significance of seed coat microstructure (Hong et al., 1999).SEM technique was used to investigate the macro and micromorphology of seed in 17 species (12 genera) of Caryophyllaceae in Turkey and some important character were identified, including hylar zone, seed surface type, tubercle shape, surface granulation, suture outline and seed type (Yıldız, 2002).The present study was performed to investigate morphological and micromorphological features in seven Silene species and subspecies in the sect Auriculatae L. and Inflatae growing in Iran to illustrate intraspecific variations and to find out if such data is of taxonomic value in the genus Silene.

MATERIALS AND METHODS
Morphological and micro-morphological studies were performed in seven Silene species and subspecies from the sect.Auriculatae L. Tabaripour et al. 5209 and Inflatae growing in Iran (Table 1).

Morphometry
In total, 40 morphological characters were used for morphometry, including quantitative (Table 2) and qualitative characters (Table 3) taken from published materials on Silene (Oxelman, 1996) and personal observation in the field.Quantitative morphological characters were randomly measured in at least five plants and the means were used in phonetic analysis.Qualitative characters were coded binary or multistate characters accordingly.Grouping of the species based on morphology characteristics was performed using different clustering methods including unweighted paired group with arithmetic average (UPGMA) and neighbor-Joining (NJ) methods, as well as principal coordinate analysis (PCoA) (Podani, 2000).

Micromorphometry
In total, 17 seed characteristics were used for micromorphometry (Table 4).Seed characters were taken from the studies made by Stearn (1996), Prentice (1978), Hong et al. (1999) and our personal observation in the laboratory.SEM micrographs of seeds were taken by Cam Scan MV 2300 SEM at an accelerating voltage 25.0 kV.Fully mature and undamaged seeds were selected.The seeds were thoroughly vacuum-coated with gold to provide conductivity for the SEM images at Tehran University (Tehran, Iran) Electron Microscopy Unit and examined in three different positions including, surface, ventral and dorsal views using different magnifications.
Seed quantitative morphological characters were randomly measured in at least three plants and the means were used in analysis.Qualitative characters were coded as binary or multistate characters and grouping of the species based on micromorphological characteristics was performed by using PCoA method (Podani, 2000).Statistical analyses were performed using the SPSS ver. 10 (1999) software and NTSYS ver. 2. (1998).

Morphometry
The grouping obtained by UPGMA dendrogram (Figure 1), produced four major clusters.The species of the sect.Inflatae are placed in the first three major clusters, separated from the sect.Auriculatae which comprised the fourth cluster.The members of these two sections differed in characters like habit form, the basal leaf shape, leaf and stem indumentum as well as capsule form.The populations of S. vulgaris form the first major cluster in which Haraz-Panjab, Semnan-Shahdej and Firooz-kooh populations are placed close to each other showing morphological similarities.They differ only in the length of corona in cauline leaf.
Chaloos to Siahbishe population joins the other S. vulgaris populations with some distance, differing in   SL, Seed length (cm); SW, seed width (cm); R, length/width ratio; TL, testa cell length (µM); TW, testa cell width (µM); TR, ratio of testa cell length/ width; HL, hilum length (µM); HW, hilum width (µM); HR, ratio of hilum length/width; TB, Tubercul height (µM).some distance from the other S. vulgaris populations, also has the highest values of total haploid chromosome length (50.51 µm), size of the longest chromosome, (5.42 µM), size of the shortest chromosome (2.88 µM) and the mean chromosome length (4.21 µM).Therefore, morphological differences of this population may be related to its higher total chromosome length (genome size) compared to those of other populations studied.Hence, we suggest that the Siahbishe population be considered as a new variety of S. vulgaris, based on both morphological and cytological grounds.
The second major cluster is formed by populations of S. odontopetala.Binalud population joins the other S. odontopetala populations with some distance.According to the subspecies description provided in Flora Iranica, this population belongs to the S. odontopetala sub.odontopetala.The other populations show similarity and form a group together.According to the subspecies description provided in Flora Iranica, Touchal population belongs to the S. odontopetala subsp.physocalyx.Therefore, the populations showing morphological similarities to it are considered to be S. odontopetala subsp.physocalyx.The third major cluster is formed by populations of S. pungens in which Anhar to Marmisho road popu-lation is placed far from the other three populations studied due to difference in characters like calyx length and alar pedicel length.Unfortunately, these populations have not been studied from karyotype point of view and we are not able to compare morphological differences with those characters.Separation of the species studied in the sect.Inflatae from the sect.Auriculatae is well supported in morphological tree.Furthermore, inclusion of different popula-tions of each species in the sect.Inflatae indicates proper identification of these three species and possible lack of introgression or any other pollen exchange among them as no overlap was observed in morphological characteristics in these species.
The forth major cluster is formed by the populations of S. aucheriana, S. sisianica, Silene pseudaucheriana and S. gynodioica from the sect.Auriculatae.S. sisianica shows morphological similarity to S. gynodioica and are placed close to each other.Affinity between these species has been suggested in Flora Iranica too.Two populations of Mishodagh and Marand in S. sisianica show some morphological differences and join each other with some distance.Moreover, the populations of S. aucheriana show close affinity in morphological characters and are placed close to each other.They mainly differ in some of their mor-phological quantitative characters.

Micromorphometry
SEM images of the mature seeds in the studied species are presented in Figures 2A to W The seed type in both section are symmetrical reniform, asymmetric reniform and rounded-reniform (Table 4).In the sect.Auriculate, the length of seed ranged from 1.22 mm in the Ghoshchi population of S. sisianica to 2.23 mm in Oshtorankuh population of S. pseudaucheriana, while the size of seed width, ranged from 0.86 mm in Ghoshchi population of S. sisianica to 1.38 mm in Oshtorankuh population of S. pseudaucheriana.In the sect.Inflatae, the length of seed ranged from 1.30 mm in the Tochal population of S. odontopetala to 1.86 mm in Siahbishe population of S. vulgaris, while the seed size of width ranged from 0.80 mm in Dizin population of S. odontopetala to 1.42 mm in Siahbishe population of S. vulgaris.Furthermore, in the section, the seed length/width ratio ranged from 1.21 in Tochal population of S. odontopetala to 1.71 in Dizin population of S. odontopetala while, in the sect.Auriculate, this ratio ranged from 1.42 in Oshtorankuh population of S. aucheriana to 1.77 in Ghoshchi The t-test analysis performed (data not shown) for quantitative seed characters between members of two sections showed significant difference for characters like ratio of length/width, length and width of seed surface, the ratio of these characters, length and width of hilar zone.Moreover, analysis of variance (ANOVA) test performed (data not shown) for quantitative seed characters showed significant difference for the same characters among three species of S. aucheriana, S. odontopetala and S. vulgaris.PCoA grouping of the species based on micromorphometry data (Figure 3) also separated the species of the sections Inflatae and Auriculatae into almost distinct groups showing the use of micromorphometry in taxonomy of the group.
In general, the present findings reveal intraspecific

Figure 2 Figure 3 .
Figure 2 Contd.Representative SEM of seeds in Silene species studied.P. Surface view in Shahdej population of S. vulgaris.Q. Dorsal view in Shahdej population of S. vulgaris.R. Surface view in Firuzkuh population of S. vulgaris.S. Ventral view in Firuzkuh population of S. vulgaris.T. Dorsal view in Firuzkuh population of S. vulgaris.U. Surface view in Chakolbozakuh population of S. odontopetala.V. Ventral view in Chakolbozakuh population of S. odontopetala.W. Dorsal view in Chakolbozakuh population of S. odontopetala.The scale for all photos except for P and Q is 500 µM.For P and Q it is 1 mm.

Table 1 .
Silene species studied and their localities.

Table 2 .
Quantitative morphological characters in Silene species studied.
(Sheidai et al., 2011)e shape and basal leaf length and width, length of stamen filament linkage to corona, length/width seed and pedicel length.According to Flora Iranica, these populations with ovate-oblanceolate leaves are from the subsp.vulgaris.Our previous cytological study in the same populations(Sheidai et al., 2011)showed uniform karyotype features in these populations including somatic chromosome number and polyploidy level (2n = 2x = 24), karyotype formulae (12 m) and karyotype symmetry (1A class of Stebbins , classification).However, they differed somewhat in total chromosome length and the mean chromosome length.Siahbishe population, which differed in morphological characters and is placed with

Table 3 .
Qualitative morphological characters in Silene species.

Table 4 .
Seed quantitative characters in Silene species.