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A Phylogenetic Analysis of the Cucurbitaceae: Evidences From International Journal of Bioinformatics Research, ISSN: 0975–3087, Volume 1, Issue 2, 2009, pp-47-53 A phylogenetic analysis of the Cucurbitaceae : evidences from mat k nucleotide sequences Uma Reddy B. Department of Bioinformatics, Gulbarga University, Gulbarga, India, E. mail: [email protected], Ph: +91-9449329266 Abstract- MaturaseK gene ( mat K) of chloroplast is highly conserved in plant systematics which is involved in Group-II intron splicing. The size of the gene is approximately 1500 base pairs in length, located with in the intron of trn K. In the present study, mat K gene from Cucurbitaceae was taken for the analysis of variants, parsimony site, patterns, transition/ transversion rate and phylogeny. The family Cucurbitaceae comprises over 120 genera and 825 species with medicinal values. The mat K gene sequences have been withdrawn from GenBank and used for the analysis. The multiple sequence alignments were performed by Genebee-ClustalW. The phylogenetic analyses were carried out by MEGA (Molecular Evolutionary Genetics Analysis) software package based on Unweighted Pair Group Method with Arithmetic Mean (UPGMA) and Maximum Parsimony (MP) methods. The results indicated that the Trichosanthes, Austrobryonia, Momordica, Neoalsomitra, Gomphogyne, Sicyos genera showed polyphyletic condition. The overall invariants between the 88 taxa are 28.5%, average composition of nucleotides among taxa are found to be approximately, A-0.30, T-0.37, G-0.15, C-0.17 and the ratio of transition and transversion is 0.824. Phylogenetic tree was designed to identify the ideal regions that could be used for defining the inter and intra-generic relationships. From this study, it could be concluded that, the chloroplast mat K gene sequences can unambiguously resolve the relationships, as well as provided a good indication of major supra-generic groupings among the selected members of the family Cucurbitaceae . Key Words: mat K gene, polyphyletic, Cucurbitaceae , Phylogenetic relationships, MEGA INTRODUCTION METHODOLOGY Advancement in molecular biology and DNA Data collection: The entire coding region of sequencing techniques has enabled to mat K sequences of 88 different species characterize the genomes of various organisms belonging to 54 genera of Cucurbitaceae are rapidly. Analyses of the DNA sequences of presented in the table-1. Generic and species various species are providing valuable information were obtained from taxonomy information about their taxonomy, gene makeup database of National Centre for Biotechnology and utilizations. The mat K gene is one of the Information (NCBI) ( http://www.ncbi.nlm.nih.gov/Genbank/index.html ) . most rapidly evolving plastid-coding regions [1]. It is approximately 1550 base pairs (bp) long and Sequence analysis: The data analysis was encodes a maturase involved in splicing type –II done for the 88 different species belonging to 54 introns from RNA transcripts [2]. The mat K gene genera of Cucurbitaceae for which the sequence has been widely employed as a sequences are available in GenBank to find the powerful tool in examining inter- and intragenous interspecific and intergeneric relationship among phylogeny due to its high substitution rates [3 -4]. the selected genera. Multiple sequence Recent studies have shown the usefulness of this alignment was performed by using Genebee- gene for resolving intergeneric or interspecific ClustalW service ( http://www.genebee.msu.su/services/phtree_reduced.html ) to relationships among flowering plants. For findout the basic sequence statistics including example, Malphigiaceae [5], Poaceae [6], nucleotide frequencies, transition/ transversion Cornaceae [7], Orchidaceae [8–9]. (Ts/Tv) ratio and variability in different regions of Cucurbitaceae is one of the largest families of sequences. flowering plants, comprising over 120 genera and 825 species [10]. The family is economically very Phylogenetic analysis: The phylogenetic tree important being the major source of food and was constructed on the basis of mat K gene forage and its great diversity (ranked as fifth sequences ranging between (1051 to 1393) of 88 largest family in flowering plants) has also taxa of 54 genera belong to the family attracted much interest in ecological as well as Cucurbitaceae , based on UPGMA and MP systematic studies [11]. The main objective to methods using MEGA software [12]. Tree elucidate the systematic positions of members of statistics included the consistency index (CI) [13], the family Cucurbitaceae also aimed to generate retention index (RC) [14] and rescaled parallel data sets for representatives of all genera consistency index (RCI) for all sites [14]. Branch to strengthen the morphology based lengths (ACCTRAN optimization with equal classification. weights) and the level of support for branches of the phylogenetic trees was evaluated with the bootstrap analysis [15] to verify the strength of the branches based on 100 replicates, using Bioinfo Publications, International Journal of Bioinformatics Research, ISSN: 0975–3087, Volume 1, Issue 2, 2009 A phylogenetic analysis of the Cucurbitaceae : evidences from matk nucleotide sequences branch and bound search. Bootstrap percentages and striate pollen grains, tendrils distally bifid, are described as high (85-100%), moderate (75 – seeds mostly winged. 84%) or low (50 – 74%) [16]. The number of In case of clade-II, only Gomphogyne cissiformis nucleotide substitutions per site was estimated by belongs to the tribe Zanoniae showing the typical Kimura’s two parameter method [17] with the character is tendrils distally bifid, seeds mostly DNADIST program. winged. Where as, remaining two taxa, Sicyos hillebrandii and S. baderoa belongs to tribe RESULTS AND DISCUSSION Sicyeae sharing common features like The study presented here is a preliminary trichomatous nectary, 4 to 10 – colporate pollen attempt to employ plastid DNA sequence of a few grains, ovules solitary, pendulous, pollen selective genera in Cucurbitaceae to address spinulose, polyporate. They are closely related interfamilial relationships; it is evidenced from the and this relationship is strongly supported by high Fig-1. The results of the multiple sequence bootstrap values (98%). alignment studies demonstrated that, 695 The clade-IV is also divided into 2 subclades, the variable sites and 257 parsimony sites. The subclade-II consists of total four taxa, of which overall invariants between the 88 taxa are 28.5%, Luffa graveolens and L. echinata belongs to tribe average nucleotide composition in 88 taxa found Benincaseae, sharing the common features like to be approximately, A-0.30, T-0.37, G-0.15, C- ovules many, horizontal, pollen reticulate, 0.17 and transition and transversion ratio is triporate and their association is strongly 0.824. Phylogenetic tree was designed to identify supported by very high bootstrap, values, 97%. the ideal regions that could be used for defining Where as, Trichosanthes schlechteri and the inter and intra-generic relationships. Gymnopetalum chinense belongs to subtribe Phylogenetic tree of family Cucurbitaceae of 88 Trichosanthinae of tribe Trichosantheae, are taxa were inferred using UPGMA (MP) method. sisterly originated with the Luffa species, and the The length of the tree was found to be 18473. relationship among these two tribal members is The consistency index is 0.161641, the retention supported by very low bootstrap values i.e., 44%. index is 0.657458 and the rescaled consistency These Trichosantheae tribal members sharing index for all sites is 0.106272. All positions commonly pollen striate, smooth or verrucose, containing gaps and missing data were flower longer stamens united with triplicate eliminated from the dataset. A total of 1000 thecae characters. residues were used from the sequences of 88 In case of clade-V, the Tricyclandra leandril and selected taxa to construct a phylogenetic tree, Trochomeria macrocarpa belongs to tribe out of which 257 are invariants and potentially Trichosantheae and Melothrieae respectively, informative. The family tree obtained from the sharing some of the common features like, styles UPGMA method was split into fourteen well united into a single column, receptacle tube supported clades and also noticed that, the few relatively longer, alike in male and female selected genera namely, Trichosanthes, flowers. Other four members of the same clade Austrobryonia, Momordica, Neoalsomitra, namely, Austrobryonia centralis, Polyclathra Gomphogyne, Sicyos showed polyphyletic cucumerina, Scopellaria marginata, condition, indicating that the members of these Schizocarpum palmeri belongs to the subfamily genera are evolved several times during Cucurbitoideae, exhibiting common features are evolutionary processes. styles united into a single column, ovules many, The UPGMA method of phylogenetic analysis horizontal or 1 to few ascending pollen, spinose strongly suggests that, the Clade – I consists of pentaporate. two subclades, which are sisterly origin and In clade-VI, out of three species, Sicyosperma majority of the taxa present in this clade consists gracile and Rytidostylis ciliate are belongs to of 1146 nucleotide residues. Subclade-I consists subtribe Cyclantherinae of tribe Sicyeae and of six species, of which, five belongs to tribe exhibiting strictly some common characters are Trichosantheae, namely, Trichosanthes ovigera, trichomatous nectary, 4 to 10 colporate pollen T. cucumerina, T. bracteata, T. subvelutina, grains, ovules solitary, pendulous, pollen Hodgsonia heteroclite and are
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