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| INTRODUCTION |
| In spite of
the great economical importance of Vitis vinifera, the systematic
classification of Vitaceae family has been based until today on
morphological data, as the classification of the majority of the
flowering plants families. Only recently the phylogeny of flowering
plants is becoming disclosed in more detail, largely following the
refining of cladistic methods, the introduction of molecular sequence
data and the availability of information technology suitable for
analyzing and comparing large data amounts. The increasing availability
of DNA sequences makes now possible the comparison between the classification
based on morphological characters and the molecular phylogenies
obtained from DNA sequences. These comparisons revealed that classical
systematics are often highly idiosyncratic, grouping flowering plants
(above the family level), in artificial systems (systems that comprise
many non-monophyletic taxa). The recent molecular genetic studies most often concern the phylogenetic relationships within families or even species, but on the other hand the use of highly conserved DNA regions may be advantageously used for phylogeny reconstruction, especially among distantly related taxonomic units. The most widely used sequences for phylogenetic analysis are ribosomal DNAs (rDNAs), foundable in nuclear and organelles genomes, from the bacteria to higher organisms. Chloroplast genomes are highly conserved both for gene size and sequence (Palmer 1990). For example, the mean nucleotide similarity of ten angiosperms' 16S rDNA is 97.6%, demonstrating the highly conserved nature of this gene. We report here an attempt to classify V. vinifera among spermatophytes, by using partial DNA sequences encoding for the chloroplast ribosomes16S and 23S to. Comparisons between V. vinifera 16S and 23S rDNAs sequences and the homologous regions of 25 and 12 other species of spermatophytes respectively, were carried out by using maximum likelihood, maximum parsimony and distance methods to construct phylogenetic trees. Data from both genes clustered V. vinifera with rosids and asterids within the core eudicots, which is in agreement with most recent systematic data. |
| MATERIALS AND METHODS |
| rDNA sequences |
| Vitis vinifera 16S and 23S partial DNA sequences were obtained by sequencing. Sequences are available in GenBank (http://www.ncbi.nlm.nih.gov/) with the respective accession numbers: AF263516, AF263517, AF143264, AF143267. The Vitis sequences used for comparison were AF143264, AF143267 (see Note below).Nucleotide sequence data for the chloroplastic 16S and 23S rDNA of 22 and 11 angiosperm species respectively, plus the equivalent sequences of the gymnosperm Pinus thunbergii were retrieved from GenBank (Table I). |
| Sequence analysis |
| Nucleotide sequences corresponding to a 493 bp fragment of the 16S rDNA and a 425 bp fragment of 23S rDNA were aligned using the ClustalX program (version 1.8, Thompson et al 1997). Two matrices of 26 and 13 aligned sequences were generated with 505 and 439 characters for the 16S and 23S rDNAs respectively. Pinus was selected as the outgroup taxon. Phylogenetic relationships were inferred using maximum parsimony strategy implemented using the branch-and-bound option in MEGA program (version 1.02, Kumar 1993). Nucleotide sequence data were also analyzed by maximum likelihood using DNAML program of PHYLIP suite (version 3.573c, Felsenstein 1994) with Pinus as outgroup and using a transition:transversion ratio of 2.0:1.0. Finally distance matrix analysis of sequences was performed. Distance matrices were calculated with the program DNADIST of PHYLIP suite, by using Kimura 2 parameters model with transition:transversion ratio of 2.0:1.0. Trees based on distance data were inferred by the neighbor-joining method. |
| RESULTS AND DISCUTION |
| In order to examine the phylogenetic relationships of Vitis vinifera to flowering plant species we aligned Vitis 16S and 23S chloroplastic rDNA sequences with the respective sequences available in GenBank. The two sets of aligned sequences were used to construct the trees corresponding to 16S and 23S sequence data using maximum parsimony (Figures 1 and 4), maximum likelihood (Figures 2 and 5) and neighbor joining (Figures 3 and 6) methods. |
| Figure
1. Phylogenetic interrelationships of 25 species of flowering
plants, compiled from a region (493 bp) of chloroplastic 16S rDNA
using maximum likelihood method and Pinus us outgroup. Figure 2. Phylogenetic interrelationships of 25 species of flowering plants, compiled from a region (493 bp) of chloroplastic 16S rDNA using maximum likelihood method and Pinus us outgroup. Figure 3. Phylogenetic interrelationships of 25 species of flowering plants, compiled from a region (493 bp) of chloroplastic 16S rDNA, using neighbor joining method (the distances were calculated with Kimura 2 parameters model) and Pinus as outgroup. Figure 4. Phylogenetic interrelationships of 12 species of flowering plants, compiled from a region (428 bp) of chloroplastic 23S rDNA, using maximum parsimony method and Pinus as outgroup. Figure 5. Phylogenetic interrelationships of 12 species of flowering plants, compiled from a region (428 bp) of chloroplastic 23S rDNA, using maximum likelihood method and Pinus as outgroup. Figure 6. Phylogenetic interrelationships of 12 species of flowering plants, compiled from a region (428 bp) of chloroplastic 23S rDNA, using neighbor joining method (the distances were calculated with Kimura 2 parameters model) and Pinus as outgroup. |
| The six phylogenetic trees produced, broadly depict the evolutionary relationships of flowering plants with some exceptions. Monocots are clustered together the same as rosids and asterids. In some trees the Fabaceae species are outside rosid cluster. Vitis vinifera is clustered with rosids or it is placed alone before rosids and asterids. These results are in accordance with the recent classification schemes that consider Vitaceae family among the isolated and phylogenetically basal families of core eudicots not placed in any order (Bremer et al 1999). |
| Table I. Species used in this study (available chloroplastic 16S and 23S rDNA sequences are marked in the last two columns. |
| NOTE |
| Vitis vinifera
16S ribosomal RNA gene, partial sequence; chloroplast gene for chloroplast
product. Burger JT, Vivier MA and Pretorius IS. GenBank Accession
AF263516 Vitis vinifera 23S ribosomal RNA gene, complete sequence; chloroplast gene for chloroplast product. Burger JT, Vivier MA and Pretorius IS. GenBank Accession AF263517 Vitis vinifera chloroplast 16S rDNA (partial sequence) DNA sequence (493 bp). Lefort F, Primikirios NI and Roubelakis-Angelakis KA. GenBank Accession 143264 Vitis vinifera chloroplast 23s rDNA (partial sequence) DNA sequence (425 bp). Lefort F, Primikirios NI and Roubelakis-Angelakis KA. GenBank Accession 143267 |
| REFERENCES |
| Bremer
K, Bremer B and Thulin M (1999) Introduction to phylogeny and systematics
of flowering plants. Department of Systematic Botany, Evolutionary
Biology Center, Uppsala University. Felsenstein J (1994) PHYLIP (phylogeny inference package). Version 3.5c. Distributed by the author, Department of Genetics, University of Washington, Seattle. Kumar S, Tamura K and Nei M (1993) MEGA: Molecular Evolutionary Genetics Analysis, version 1.0. The Pennsylvania State University, University Park, PA 16802. Palmer JD (1990) Contrasting modes and tempos of genome evolution in land plant organelles. Trends Genet 6: 115-120. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F and Higgins DG (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 24:4876-4882. * Thiswork was financially supported by the Interreg Operational Programme. |