Molecular Phylogenetic Relationship among Closely Related Species of the Clover Genus (Trifolium – Leguminosae) in Palestine / West Bank

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Hassan, Rana
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جامعة النجاح الوطنية
Leguminosae family is the third largest family of angiosperms; which comprises three sub-families: Caesalpinioideae, Mimosoideae and Papilionoideae. Legumes are flowering plants in Leguminosae family that include a large number of species. Trifolium L. (Clover genus, True Clover) is one of the most important genera in legumes with agricultural values. A Mediterranean region contains the highest biological diversity due to the variation in Mediterranean climate. The native distribution of Clover genus is found there. To resolve the conflicts in the morphological characters in some of the closely related species of Trifolium, it was necessary to use molecular markers. Internal Transcribed Spacer (ITS) regions of the nuclear ribosomal DNA (nrDNA) are the most widely used marker in plant research. They have been proved to be useful source of information for phylogenetic studies in many angiosperm families including Leguminosae. Fresh specimens of different species of Trifolium were collected during the flowering period (March – May 2017) from different locations in Palestine/West Bank. Then a representative plant specimen of each species was deposited at An-Najah National University Herbarium. Depending on Flora Palaestina, and using of Stereo microscope, the collected samples were identified and classified based on their morphological characteristics. CTAB extraction protocol was followed for plant genomic DNA extraction. The ITS regions (ITS-1 spacer, 5.8S rDNA and ITS-2 spacer) were amplified using universal primers. Phylogenetic tree was constructed for the 12 classified samples that belong to Clover genus (Trifolium L.) using Neighbor-Joining method and Salvia candidissima was used as the out-group. Kimura's 2-parameter method was used to compute Pair-wise distances. All DNA sequences that were used in the study were deposited at the GeneBank database under accession numbers. Molecular ITS sequence analysis of both T.purpurem and T.dasyurum showed that they are identical species, since they have identical ITS sequences (0% bp differences) and were clustered together in the same clade I/ sub- clade Ia. This coincides with the high resemblance of their morphological characters. T.campestre and T.grandiflorum could be regarded taxonomically as one species, since they also have identical ITS sequence (0% bp differences) and were clustered together in the same clade II/ sub-clade IIb in the constructed phylogenetic tree. Based on this study, it is recommended to identify both T.purpurem and T.dasyurum as same species. Accordingly depending on the nomenclature rules T.dasyurum (1832) can be considered as synonym to T.purpurem (1807), which can be referred as the valid name of that species. Similarly this can be applied to both T.campestre (1804) and T.grandiflorum (1767). The former one could be regarded as synonym to T.grandiflorum which could be taken as the valid name. In addition, the current conducted traditional morphological and molecular taxonomical study is the first of its type that was able to highlight the phylogenetic relationship among closely related species of the genus Trifolium in Palestine/West Bank. This paves the way for future in-depth researches on different more species of the same genus. In addition, using other molecular markers could reveal more clarification of the taxonomy of Trifolium different species.