Abstract: Some natural Lathyrus aphaca L. varieties have been investigated from chemotaxonomic point of view. Seed fatty acid composition of four L. aphaca varieties (affinis, biflorus, pseudoaphaca and modestus) of genus Lathyrus was determined and their taxonomic relationships were also evaluated using chemical and numerical taxonomic techniques. The Lathyrus aphaca varieties seed contained profitable composition of poly-unsaturated fatty acids, linoleic and palmitic acid. The lowest fatty acid component was margaric, pentadecanoic and myristic acid. It is determined that the seed lipids contained a relatively large proportion of un-saturated fatty acids with linoleic acid as predominant. The results showed that intraspecific variation of fatty acid patterns was low in L. aphaca varieties with the exception of modestus, which was found partly different by fatty acid composition from the others.
Introduction
The fact that, species producing unusual fatty acids may become extinct before their genetic potential to produce technically interesting raw material has been fully recognized and investigated. The rate at which progress in new techniques, e.g. gene transfer from wild plant species into agronomically developed crops as rapeseed, can be achieved is in direct competition with the rate at which plant species, and their genetical potential, are disappearing from the globe (Aitzetmüller & Tsevegsüren, 1994). Thus, the study of plant chemicals in terms of technical and pharmaceutical importance is increased all over the world. Kupicha (1983) lists 152 species in the Lathyrus genus (Fabaceae) which are distributed from sub-arctic to sub-tropics. It includes a range of grain, forage, pasture and ornamental crops (Enneking, 1998). The genus comprises of 67-70 taxa in the level of species, subspecies and variety (Davis, 1970; Maxted et al., 1988; Ertekin & Saya, 1996) and it is represented by 10 sections in Flora of Turkey. The Aphaca (Adans) Reichb. (Guss.) Arc. section is represented by 2 species, L. aphaca (L.) and L. stenolobus (Boiss), according to Davis (1970). L. aphaca is comprised of 6 varieties in Flora of Turkey and its complex reaches its maximum variability in Anatolia. Several of the varieties have ecogeographical preferences and intermediates are relatively uncommon in Turkey. The classification accommodates most of the variation found in turkey, but varietal misidentification is easy in the absence of notes on flower colour. Seed - coat pattern is probably not a reliable diagnostic character.
L. aphaca var. biflorus Post. is widespread except in N. east Anatolia. L. aphaca var. pseudoaphaca (Boiss.) Davis usually has a narrow standard than others. Some morphological differences were reported among the varieties, especially flowers colour, peduncle and the stipule characters. However, Lathyrus genus is very important plant as a cultivar and crop in Turkey and other parts of the world. Doğan et al. (1992) divided Lathyrus genus into Orobus and Lathyrus sub-genera. Then they reducted the section number of Lathyrus from 10 to 9.
Many wild species of plants are capable of synthesizing unusual and technically interesting fatty acid structures in their seed lipids (Smith, 1970; Badami & Patil, 1981; Aitzetmüller & Tsevegsüren, 1994). Unusual FAs accumulated in the seeds of higher plants can be of chemotaxonomical interest and their presence or absence may indicate closer or more distant relationships between the considered species (Aitzetmuller, 1993) as it has already been demonstrated (Hegnauer, 1989; Harborne & Turner, 1984; Jmbs and Pham, 1996; Valesco and Goffman, 1999). There is some evidence that the rare fatty acids, may be harmful to animals eating the seeds. Erucic acid, a characteristic acid of the Cruciferae, has been described as having toxic effects in mammals, if ingested in sufficient amounts(Harborne & Turner, 1984).
It is aimed in this research to indicate the fatty acid composition of L. aphaca varieties and chemical relationships among these taxa and also compare them chemotaxonomically. In this way it was studied whether the fatty acid made a positive contribution to the taxonomy of these plants or not. The study is very important in view of infraspesific chemical variation in the Lathyrus taxa. On the other hand, it is very useful to determine these chemicals in plant, in field of chemosystematics, pharmacology and agronomy.
Materials and Methods
Plant material: Seeds of four L. aphaca varieties were collected from natural habitats, shown in Table 1.
Lipid Extraction: Seeds (2g) of each sample were homogenized with isopropanol. The latter procedure - extraction and purification - was made according to Kates (1986) method. Methylation was carried out according to Christie (1984).
Gas Chromatography: Analysis of fatty acid methyl esters was carried out and the resultant mixture of fatty acid methyl ester was injected onto a UNICAM – 610 GC. FID detector, carrier gas (N, ml /min) 2.5 capillary column (15 m X 0.32 mm) packed with 70% BPX –70 were used for analysis. The temperature of column was 185 0C. One μL was introduced onto the column.The quantity of fatty acid was calculated as μg/ g. Identification of fatty acid components was carried out with the help of authentic standard and retention time of components used in fatty acid analysis. For all samples the procedure was performed in triplicate and the mean values were stated.
Cluster analysis technique – Euclidean distance – in SPSS 9.05 computer program was applied on the chemical data.
Results and Discussion
The data obtained from the analysis is presented in Tables 2a,b. Despite the mentioned morphological differences (Davis, 1970: Şahin et al., 2000) that exist among the L. aphaca varieties they had similar fatty acid patterns.
| Table 1: | Locations of studied Lathyrus aphaca varieties. |
| Table 2a: | Fatty acid composition of seed lipids of Lathyrus aphaca varieties (μg/g, %) |
| Fatty acids: 13:0 (Tridecanoic acid ), 14:0 (Myristic acid), 15:0 (Pentadecanoic acid), 16:0 (Palmitic acid), 17:0 (Margaric acid), 18:0 (Stearic acid), 18:1 (Oleic acid), 18:2 (Linoleic acid), 18:3 (Linolenic acid). | |
| Table 2b: | SFA (Saturated fatty acids) and USFA (Unsaturated fatty acid) amount of Lathyrus aphaca varieties studied (μg/g). |
The major FAs encountered were 18:2, 16:0, 18:3, 18:1 and 18:0 respectively.
Comparison of fatty acid composition of L. aphaca varieties revealed specific features in FA composition, characteristic of these varieties that would have a taxonomic value. Four L. aphaca varieties were found different in fatty acid composition quantitatively.
In investigation, 9 fatty acid components were determined in each taxa, 12:0, 19:0 and 20:0 fatty acid components were not found throughout. The amount of saturated fatty acids (SFA- 13:0, 14:0, 15:0, 16:0:17:0,18:0) and also unsaturated fatty acid (USFA - 18:1 + 18:2 + 18:3) are shown in Table 2b. It can be said that Lathyrus taxa studied were not rich in point of saturated fatty acid component and amount (Table 2a and b).
The lowest fatty acid component was Margaric acid (17:0) in all of the taxa, this is very rare fatty acid component in the plant seed lipids. Pentadecanoic and myristic acid also were the second lowest components. The highest fatty acid component was the Linoleic acid in all of the Lathyrus taxa. While Palmitic acid was the second abundant fatty acid component in all varieties except pseudoaphaca. It is reported that γ- linolenic acid had great interest for the pharmaceutical industry and is now produced from oilseeds, where it is present at 5-10% levels. Some of unusual fatty acids were present in small amounts only in the seed oils and they were chemotaxonomically significant becuse of their constant presence in all the species of one genus or a few genera, combined with their constant absence throughout the species of other genera (Aitzetmüller & Tsevegsüren, 1994).
The ratio of 18:3 and 18:2 (18:3/18:2) fatty acid components were found 0.16 in var. pseudoaphaca, 0.11 in var. affinis, 0.14 in var. biflorus and 0.04 in var. modestus. It is reported that the fatty acid composition and the ratio of (18:3/18:2)can serve as taxonomic markers in some family member (Marin et al., 1991). The differences among the taxa by 18:3/18:2 ratio, showed that first three varieties were very similar and var. modestus was found highly different from others.
| Fig. 1: | Affinities relationships between samples of Lathyrus aphaca varieties, based on the distribution of seed fatty (1. L. aphaca var. psudoaphaca, 2. L. aphaca var. affinis, 3. L. aphaca var. biflorus, 4. L. aphaca var. modestus). |
It is reported that some morphological confusions and controversies were determined among the Lathyrus species and varieties. L. aphaca var. affinis (Guss.) Arc.- was reported as depauperate plant and easily mistaken for var. pseudoaphaca (Davis, 1970).
It can be said that fatty acid amount of Lathyrus sp. studied in general were similar in point of both SFA and USFA. In the same way, oleic, linoleic and palmitic acid were the main components in seed oil. This phenomenon was reported by other researchers (Daulatabad, 1987; Kwieicinska & Matyka, 1986; Bağcı & Şahin, unpublished).
The results of the Euclidean distance method of cluster analysis for the specimen data set are summarized in Fig. 1. In the dendrogram, L. aphaca varieties studied connected with each other at top level. These varieties were found very similar in view of fatty acid composition as shown in dendrogram. But L. aphaca var. modestus was determined as a single taxon. They formed two groups, first three varieties – var. pseudoaphaca, var. affinis, var. biflorus- and the last, that stayed as single specie in dendrogram. L. aphaca varieties affinis and biflorus were found the closest two taxon in the dendrogram and var. pseudoaphaca was the third closest taxon with the both.
Chowdury & Banerjii (1995) analyzed the seeds from 3 plant species belonging to Crotalaria for fat and fatty acids and found that all the seed fats resembled the simple linoleic-oleic-palmitic type. The results obtained in present study confirmed that the linoleic – palmitic and linolenic type fatty acid composition was dominant in the Lathyrus taxa, particularly comparing with the other plant species. Nine samples of the legume seeds including L. sativus were analyzed for fatty acids and tocopherol contents. In the result, they reported that the legume seeds had profitable poly unsaturated fatty acids composition especially, from linoleic and linolenic acid point of view (Grela & Günter, 1999).
In morphologic and karyologic analysis of some Lathyrus sp., L. aphaca, var. modestus (Şahin, 1993) was found to be different from others (Lathyrus rotindifolius Willd., subsp. miniatus (Bieb.ex.stev) Davis, L. cassius Boiss., L. cicera L. ) in terms of chromosomal karyology. Şahin et al. (2000) studied, chromosome number of Lathyrus taxa and L. aphaca varieties - modestus, affinis and pseudoaphaca, these were found same (2n = 14). In the dendrogram of karyologic analysis L. aphaca var. affinis and var. pseudoaphaca formed a small duplet group and var. modestus was found similar with the other big Lathyrus group containing var. modestus, L. sativus, L. stenophyllus and the others (Şahin et al., 2000). The dendrogram result in this study was supported with this finding.
In the Lamarque and Guzma’ n (1997) study on the Prosopis chilensis stated that fatty acids had no significant correlation with geographic parameters analyzed, some unsaturated fatty acids correlated significantly with altitude and the variability observed might indicate that there is sufficient intraspecific difference to permit improvement by selection and breeding. The results obtained also were very important by showing infraspecific variation determined in the Lathyrus taxa. It is required to enlarge the study with different parameters especially inter and intraspecific variation.Velasco & Goffman, (2000) reported that the fatty acid composition of the seed oil has been used as a criterion in searching for a ratio of infrageneric classification of Linum L.
Previous studies have revealed the chemotaxonomic value of fatty acids in several plant families and species (Senatore, & Basso, 1994: Agnase et al., 1998) which is confirmed for the Lathyrus aphaca varieties in the present study. In conclusion fatty acid composition of Lathyrus sp. studied can be used as taxonomic markers, but it will require to make population studies by means of biosystematics in detail. A more detailed investigation could afford a proper evaluation of polymorphisms based on fatty acid distributions in Lathyrus genus. It is reported that, especially in the interspecific hybridization studies in genus Lathyrus are essential for clarifying the genetic relationships among species of this important genus (Yamamoto, 1984). Unusual and technically interesting fatty acids and their sporadic occurence in seed fats is genetically determined and they are highly significant indicators of phylogenetic relationships (Aitzetmuller, 1995).