Abstract: Electrophoretic seed protein patterns of a number of accessions of Salicornia europaea L. sl., S. prostrata Palas, S. fragilis P.W. Ball and Tutin, Sarcocornia fruticosa (L.) A. J. Scott, Sarcocornia perennis (Miller.) A. J. Scott, Arthrocnemum glaucum (Del.) Ung.-Sternb., Microcnemum coralloides (Loscos and Pardo) subsp. anatolicum Wagenitz and Halocnemum strobilaceum (Pall.) Bieb. were electrophoretically analysed on SDS-PAGE. In total 48 different bands were identified. The obtained data have been treated numerically using the cluster analysis method of unweighted pair group (UPGMA). Finally it was determined that all species separated according to seed protein profiles. And the cladogram obtained studied taxa have been given.
INTRODUCTION
Salicornia L. (Chenopodiaceae) is a genus of annual, apparently leafless halophytic herbs that have articulated, succulent stems. World wide there are 13 species with innumerable variants (Scott, 1977). The number of European species varies between seven (Scott, 1977) and eight (Ball and Akeroyd, 1993). There is no revision for Asian Salicornia, most of the floras cited only Salicornia europaea L. sensu lato (Iljin, 1936; Grubov, 2000; Hedge, 1997; Bolous, 1996). Freitag et al. (2001) recorded S. perennans Wild. From northern Caspian and Akhani (2003) described S. persica Akhani from central Iran. There are three Salicornia species known in Turkey S. europaea L., S. prostrata Pallas, S. fragilis P.W. Ball and Tutin, each are known from other parts of Europe (Ball, 1967a, b). In Salicornia, a combination of inbreeding, which allows the development of locally differentiated populations, considerable phenotypic plasticity, a much reduced morphology of the plant and the inadequacy of herbarium material in representing the succulent growth form has created great taxonomic difficulties (Ball, 1960, 1964; Davy et al., 2001).
Arthrocnemum is a genus of succulent halophytic subshurbs or shrubs closely related to Salicornia. There are three species known from Europe, Middle East and Turkey; A. perenne (Mill.) Moss., A. fruticosum (L.) Moq. and Arthrocnemum glaucum (Del.) Ung.-Sternb. (Ball, 1967a, b, 1993). The limits of the genera Salicornia, Arthrocnemum and Sarcocornia are not clear. Arthrocnemum perenne and A. fruticosum are assigned to three different genera (Salicornia, Arthrocnemum and Sarcocornia) by different authors (Ball, 1993, 1967a, b; Scott, 1977; Freitag, 2000). The distinctness of Arthrocnemum perenne and A. fruticosum is not clear (Freitag, 2000).
It is therefore difficult to find convincing species delimitations on the basis of morphological characters only. Molecular studies have shown that even morphologically hardly distinguishable species are genetically distinct (Wolff and Jefferies, 1987; Jefferies and Gottlieb, 1982). The high stability of seed proteins makes them a powerful tool in elucidation of the origin, evolution and relationship of taxa (El Naggar, 2001; Bhargava et al., 2005; Vladova et al., 2000). Therefore, we tried to distinguish the Turkish Salicornia L. species and allied taxa with the aid of seed protein markers in this study. Salicornia species have seed dimorphism except S. pusilla J. Woods (König, 1960; Dalby, 1962; Ungar, 1979). Central seeds were used for the study. The obtained data were analyzed by numerical analysis (cluster analysis) based on Jaccard’s coefficient (Sneath and Sokal, 1973).
MATERIALS AND METHODS
First specimens to be identified have been collected from the cited localities (Table 1) in flowering time in 70% alcohol (Ball, 1960). After identification of the specimens, seed specimens have been collected from the same localities. Five individual plants with seed per populations have been sampled for Salicornia, one per others. Voucher specimens of studied taxa are deposited in Herbarium ANK.
| Table 1: | Localities of the studied taxa |
| Fig. 1: | The photograph of electhrophoretic banding patterns of the studied taxa. M is Molecular Weight Marker, Numbers are representing taxa in same order in Table 1 |
Seeds of each individual plant were ground separately to fine flour in a prechilled mortar and pestle. Several extraction methods have been tried but the best results could be obtained as follows; Proteins were extracted (0.3 g seed flour to 150 μL extract) in the 0.5 M Tris/HCI buffer, pH 6.8. The extract was centrifuged at 13000 rpm for 3 min and the 75 μL supernatant was decanted and 25 μL concentrated sample buffer (4X) added and boiled for 5 min and aliquots used in PAGE.
Five individual per Salicornia sp. populations and one individual per each of other taxa have been examined electrophoretically. Electrophoresis was carried out in the modified discontinuous SDS-PAGE system of Laemmli (1970) using 10% acrylamide resolving gel (pH 8.6) and 4% stacking gel (pH 6.8). Gels were stained by 0.1% Coomassie Brilant Blue R-250 and destained in destaining solution (25% isopropyl alcohol, 10% glacial acetic acid and 65% distilled water). After decolorization, gels were photographed. The electhrophoretic banding patterns of the studied taxa are shown in Fig. 1 and the drawing of it is in Fig. 2.
| Fig. 2: | The drawing of electhrophoretic banding patterns of the studied taxa. M is Molecular Weight Marker, Numbers are representing taxa in the same order as in Table 1 |
In total, 48 different bands were identified. Forty eight protein characters scored for each of the 15 Operational Taxonomic Units (OTU’s). Each character was classified as presence (1) and absence (0). The Jaccard’s coefficient Sj = a/(a+b+c), (where a is the number of characters shared by a pair of samples, b is the number of characters found in one of a pair only and c is the number of characters found only in the other one of a pair) was used a measure of similarity of pattern (Sneath and Sokal, 1973). The matrix of Jacard’s coefficients was used in a pair-wise cluster analysis using the unweighted pair group method (UPGMA) using arithmetic average to produce a phenogram of similarities.
RESULTS AND DISCUSSION
Two different morph of S. europaea sl. has been observed in Çorum, Sungurlu. Both morph included to study and treated as different taxon. In total, 48 different bands were identified (Fig. 1 and 2). Seed protein variation in population level has been observed only in the Tarsus population of S. europaea sl and Samsun population of S. prostrata. Each variant have been run in final gel and treated as different OTUs for statistical analysis (Fig. 3).
Cladogram has distinguished Halocnemum strobilaceum in the other taxa, Papini et al. (2004) also found similar results for Halocnemum in their ITS based study. The cladogram showed that genus Sarcocornia and Arthrocnemum are sister taxa. In ITS based cladogram of Papini et al. (2004) genus Sarcocornia is not distinguished in genus Arthrocnemum and they advocate threating them in genus Arthrocnemum. Kadereit et al. (2005), Davy et al. (2006) support distinctions between Arthrocnemum and Sarcocornia in their DNA based studies. Genus Sarcocornia was closer to Arthrocnemum than Salicornia in the cladogram, Papini et al. (2004) also found similar results while Kadereit et al. (2005) found Sarcocornia and Salicornia closer. Actually, morphologically Salicornia and Sarcocornia are closer except former is annual and the latter is perennial.
| Table 2: | Matrix of similarity between studied taxa. Numbers are representing taxa in the same order as in Table 1 |
| Fig. 3: | Dendrogram shows the relationships between the taxa based on protein characters |
Microcnemum corraloides subsp. anatolicum is closer to Arthrocnemum and Sarcocornia than Salicornia in the cladogram.
Cladogram has distinguished the known species of Salicornia, S. europaea, S. prostrata, S. fragilis from each other. On the other hand it showed that S. europaea clade is so variable in seed proteins. S. europaea sl. is also so variable morphologically therefore it needs to be investigated in details.
ACKNOWLEDGMENTS
The authors are grateful to Prof. Dr. Cumhur Çökmüş, Ankara University, Turkey for his kind helps in laboratory analyses.