International Journal of Bioinformatics Research, ISSN: 0975–3087, Volume 1, Issue 2, 2009, pp-27-36

Molecular phylogeny of angiospermic families using RBC L gene sequences

B. Uma Reddy* Department of Bioinformatics, Gulbarga University, Gulbarga, India E. mail: [email protected], Ph. No: +91-9449329266

Abstract : The present study was undertaken to assess the role of plastid-rbc L (ribulose-1, 5-bisphosphate carboxylase large-subunit) gene sequences in addressing the evolutionary relationships within the angiosperms at inter and intra-familial levels using computational experiment. In order to elucidate the relationships, a set of 92 chloroplast rbc L sequences representing from 90 taxa of 12 genera and 10 angiospermic plant families (dicot and monocots) were withdrawn from the GenBank database. The multiple sequence alignment was performed using Genebee-ClustalW service to findout the regions of conserved or indels among the sequences. The phylogenetic tree was inferred from these sequences by employing Bootstrap method of UPGMA (Unweighted Pair Group Method with Arithmetic Mean) using MEGA (Molecular Evolutionary Genetics Analysis) software. The consistency of these generic-wise groupings was further confirmed by the MP (Maximum-Parsimony), ME (Minimum-Evolution) and NJ (Neighbor-Joining) methods. The analysis of these studies strongly indicates that, out of the 12 selected genera Trichosanthes (Cucurbitaceae) , Phyllanthus (), Austrobryonia (Cucurbitaceae) , Solanum () , Piper (Piperaceae) and Saxifraga (Saxifragaceae) are grouped into separate clusters and exhibiting monophyletic conditions. Where as, () , ( ) , Cassia (Caesalpinioideae, sub-family) , Canna (Cannaceae) , Mentha () , are paraphyletic and the members of the (Lamiaceae) are distributed throughout these hiraeoid clades, confirming the polyphyly of this large . Similar observations were noticed in all four methods. Thus, chloroplast rbc L gene sequences can unambiguously resolve the relationships, as well as provided a good indication of major supra-generic groupings among the selected angiospermic plant families and also gave many clues for future studies. Key Words: rbc L gene, monophyletic, paraphyletic, polyphyletic, Phylogenetic relationship, MEGA

INTRODUCTION Rhamnaceae [9], Araucariaceae [10]. The The use of DNA nucleotide sequence data has present study was undertaken to evaluate the awakened a quickly growing interest in molecular relationships at the intra and inter familial, systematic analyses. Since last decade intergeneric and interspecific levels, and also to molecular systematics in has progressed examine the rates, patterns and types of rapidly with in-vitro DNA amplification and direct nucleotide substitutions within the rbc L gene in all sequencing methods. In angiosperm systematics, the selected taxa of each genera, through a this molecular approach has been effective in computational experiment using 92 chloroplast addressing many phylogenetic questions that had rbc L gene sequences from 90 taxa, 12 genera, not been solved only by using phenotypic 10 families, and discussed the utilization of characters . The gene for the large subunit of the plastid-rbc L gene for molecular phylogeny. ribulose-bisphosphate carboxylase/oxygenase (rbc L), located on the chloroplast genome, is an MATERIALS AND METHODS appropriate choice for inference of phylogenetic Cladistic parsimony analyses of a total 92 rbc L relationships at higher taxonomic levels [1—3]. nucleotide sequence data representing 90 Because of its slow synonymous nucleotide (Spp) and 2 duplicates from substitution rate in comparison with nuclear Austrobryonia micrantha and Mentha longifolia of genes and its functional constraint that reduces 12 genera, 10 angiospermic plant families (both the evolutionary rate of non-synonymous dicots and monocots) are presented here in the substitution [4]. The substitution rate of rbc L table-1, were withdrawn from GenBank appears appropriate for studies involving taxa database). The multiple sequence alignment was that diverged from 10’s of million years [5]. In performed using Genebee-ClustalW service. All recent years, there has been a growing interest in gap characters were scored as missing data using sequences of the gene coding for the large rather than a fifth character. Phylogenetic subunit of ribulose-1, 5-bisphosphate analyses of the sequence data was analyzed with carboxylase/ oxygenase ( rbc L) to estimate the UPGMA using MEGA software. The consistency plant phylogenies [6]. Several research groups of these generic wise groupings was further have shown the usefulness of this gene for confirmed by the MP, NJ and ME methods as solving intergeneric and interspecific implemented in MEGA 4.1 software [11]. Tree relationships among flowering plants example, statistics included the consistency index (CI) [12], Cucurbitaceae [7] , Orchidaceae [8], retention index (RC) [13] and rescaled

Bioinfo Publications, International Journal of Bioinformatics Research, ISSN: 0975–3087, Volume 1, Issue 2, 2009 Molecular phylogeny of angiospermic plant families using RBCL gene sequences

consistency index (RCI) [13]. Branch lengths (ACCTRAN optimization with equal weights) and (ACCTRAN optimization with equal weights) and bootstrap are shown. The composition of adenine the level of support for branches of the (A)-0.27, thymine (T)-0.29, guanine (G)-0.25 and phylogenetic trees was evaluated with the cytosine (C)-0.19 nucleotides were observed as bootstrap analysis [14] to verify the strength of constant in all the test genera. But slight changes the branches based on 100 replicates, using were noticed in the ratio of transition and branch and bound search. Bootstrap percentages transversion substitutions. In case of Saxifraga are described as high (85-100%), moderate (75 – the ratio of transition and transversion is 1.83, 84%) or low (50 – 74%) [15]. The number of Asparagus is 0.7, Cassia is 1.33, Drypetes is nucleotide substitutions per site was estimated by 1.63, Salvia is 2.7, Mentha is 1.8, Trichosanthes Kimura’s two parameter method [16] with the is 0.5, Piper is 1.0, Phyllanthus is 0.7. Perhaps DNADIST program. the members within genera of Austrobryonia, Solanum and Canna are exhibiting highest level RESULTS of similarity, the rate of transition and The 12 genera of angiosperms namely, transversion ratios are almost negligible. In these Trichosanthes (4 Spp), Solanum (2 Spp), selected taxa, there is no significant cluster at the Austrobryonia (4 Spp and 5 sequences), Piper level of subclass was found, particularly, there is (11 Spp), Phyllanthus (9 Spp.), Saxifraga (2 no clear-cut segregation between monocots and Spp.), Drypetes (10 Spp), Asparagus (3 Spp), dicotyledonous plants was observed in the tree Salvia (35 Spp.), Cassia (4 Spp), Canna (3 Spp), topology. The members of the genera Canna and Mentha (3 Spp and 4 sequences) were selected Asparagus belongs to are for the study are highly divergent in several nested within the clades of the dicot plant respects from one another. But the properties, families. The length of the rbc L gene sequence in such as, biochemical, morphological and the members of few genera included in the cytological characteristics of species within each present study possess fixed number of nucleotide genus are exhibiting greater level of similarity. residues, which is evident from the genus This in turn inferred that the molecular makeup of Trichosanthes is 1356, Phyllanthus -1408, these organisms at the generic and family levels Saxifraga – 1346 and 1377, Solanum – 1370, is also highly conserved. The analysis of the Austobryonia – 1355, Piper – 1428. Where as, above studies strongly indicates that, out of the considerable variation in substitution rates and 12 selected genera, the members of length of the sequences were observed in some Trichosanthes, Austrobryonia, Phyllanthus, other selected genera namely, Saxifraga stellaris Solanum, Piper and Saxifraga are nested into a and S. integrifolia– 1346 and 1377, respectively. separate clusters as a monophyletic group, within Similar observations were found with Asparagus the major clades and sub-clades in the tree officinalis, A. cochinchinensis and A. capensis – topology, this is strongly supported by high 1206, 1369 and 1340, respectively, Drypetes – bootstrap replications (100%). Where as, 1398, except D. littoralis and D. roxburghii -1331 Drypetes, Asparagus, Cassia, Canna, Mentha and 1420, respectively. Canna indica and C. are paraphyletic and the members of the Salvia glauca – 1327 but C. tuerckheimii – 1334; are distributed throughout these hiraeoid clades, Mentha rotundifolia, longifolia (2) – 1420 and M. confirming the polyphyletic condition of this large suaveolens, M. longifolia – 1402; Cassia senna, genus. These observations were noticed in all C. didymobotrya – 1368, C. grandis – 1421, C. UPGMA, MP, ME and NJ methods, but only the fistula- 1465. However, the sequence length of positions of the generic-wise clusters were rbc L in 35 members of the genus Salvia is altered into the tree topology in all these ranging between 968 -1474. But the members methods. The members of the Asparagus of this genus located in the subclades-I of Clade- () and Canna (Cannaceae) were V are strictly containing 1323 and subclades-I of nested within the angiospermic dicot plant Clade-I containing 1371 nucleotide residues only, families also exhibiting the paraphyletic and these exhibited highest level of sequence conditions of these genera with selected dicot similarity and originated from one common plant families. The total length of the tree is ancestral forms. Where as, the members of this 14029. Of the 92 sequences ranging between genus located in the sub-clade-IV of clade-I, 968 – 1471 in the data matrix, of which only 877 clade-II, and clade – III are found to be highly nucleotide residues were used to infer the variable. The UPGMA method of phylogenetic phylogenetic relationships among the selected analysis strongly suggests that, the members of taxa and were shown to be invariant and 305 the genus Trichosanthes are evolved first and were variable. These trees were characterized by forms a primitive, and then Phyllanthus, a consistency index (CI) of 0.187540, retention Drypetes, Saxifraga, Solanum, Austrobryonia are index (RI) of 0.733592 and the rescaled evolved in a sequential order. Where as, the consistency index i.e., RCI = 0.137578 (for all members of the genus Piper are found to be late sites). The strict consensus of these weighted evolved and inferred them as highly advanced trees is shown in the Fig 1-4. Branch lengths among the selected genera. Where as, the

28 Bioinfo Publications, International Journal of Bioinformatics Research, ISSN: 0975–3087, Volume 1, Issue 2, 2009 B. Uma Reddy

members of the genus Mentha are evolved twice selected dicot plant families. The present data with the members of Salvia. Mentha suaveolens also reject the views of Burger, 1997; Taylor and and M. longifolia are early evolved, they are Hickey, 1992 [17–18], that Piperaceae are basal found in the cluster-I and forms a basal group. among most of the primitive angiosperms and But the M. rotundifolia and M. longifolia (2) are that, the monocots included in the present study late evolved and nested in a cluster-V indicating were derived from them. In contrast to the Shu - that they are advanced. Among the four species Miaw et al., 1997 [19], that the monocots of the Cassia, only the C. senna and C. included in the present studies are not clustered didymobotrya exhibited high level of similarity, into a separate monophyletic group. The this is supported by high bootstrap values of members of the genera Canna and Asparagus 100% and occupying in the basal group. Where belongs to monocotyledons are nested within the as, Cassia grandis nested with in the members of major clades of the dicot plant families. Salvia in clade-II and Cassia fistula originated Therefore, the plastid-rbc L gene sequence data sisterly with the members of the genus Saxifraga. lend support to Hutchinson’s view that “the single Similarities between these individuals are cotyledon, parallel-veined leaves, absence of strongly supported with bootstrap values as 96%. cambium, dissected stele and the adventitious In case of the genus Canna, only the C. indica root system of monocots are all regarded as and C. glauca are highly similar, supporting with apomorphies within the angiosperms. Moreover bootstrap values of 100% and located in the monocots are regarded as derived from dicots, major clade-I. Another member of the same the point of origin being Ranales [20]. These genus C. tuerckheimii is located in the clade-IV. results show that analyses of the chloroplast Due to high sequence diversity among the three DNA rbc L is a useful approach for inferring selected species of Asparagus are distributed phylogenetic relationships especially at the distantly into the tree topology and indicated that, supra-generic level. this is evolved several times during evolution. A. capensis is found in clade-I and referred as less CONCLUSION evolved or primitive, where as, A. However, the present study, the tree relations cochinchinensis in the cluster III and A. officinalis among the genera are well resolved and is in Clade-V and this A. officinalis is inferred as providing support for the monophyly of large highly evolved among other selected species. clades and subclades for many of the selected Among the 35 species, the members located in plant genera such as Trichosanthes, the subclade-I of clade-I and members in the Austrobryonia, Saxifraga, Solanum, Phyllanthus subclade –I of clade-V are exhibiting greater level and Piper. The polyphyletic condition of the of sequence similarity, members of the same genus Salvia is supported by the higher rate of genus located in the other clades and subclades the transition and transversion ratio is 2.7, 554 are displaying much variable. It is evident that the gaps as well as significant variation in the total members of this large genus evolved several number nucleotide residues in the different times and are distributed into several patches all members of the genus Salvia among 35 species along the tree topology (FIG -1). The similar (i.e., 968 in Salvia nubicola to 1474 in Salvia monophyletic, paraphyletic and polyphyletic divinorum ). The present investigation also conditions of these selected genera were also concludes that, the gene plastid-rbc L alone is not confirmed through other NJ, MP, and ME likely to provide robust estimates of phylogeny. methods, indicating that these conditions are Data from additional molecular (complete nuclear strictly conserved. Only the relative position of and chloroplast genetic systems) and these generic level groupings was altered (FIG - morphological sources and their combined 2-4). analyses are necessary to establish a stable and firm base for a plant systematics (Family DISCUSSION classification) that better reflects phylogenetic The study presented here is an attempt to relationships. employ a set of 92 plastid rbc L gene sequences of 90 taxa belonging to 12 genera of 10 angiospermic plant families to address intra REFERENCES familial relationships. The analyses of rbc L [1] Chase, M. W.; Soltis, D.E.; Olmstead, R.G.; nucleotide sequences presented here provide a Morgan, D.; Les, D. H.; Mishler, B. D.; great deal of support for previous hypotheses of Duvall, M. R.; Price, J. H.; Hillis, H. G.; relationships within the family and generic levels Qiu, Y. L.; Kron, K. A.; Rettig, J. H.; of some angiospermic plants. The members of Conti, E.; Palmer, J. D.; Manhart, J. R.; the genera belongs to Asparagus (Asparagales) Sytsma, K. J.; Michaels, H. J.; Kress, W. and Canna (Cannaceae) belongs to the sub- J.; Karol, K. G.; Clark, W. D.; Hedren, class monocotyledonous were nested within the M.; Gaul, B. S.; Jansen, R. K.; Kim, K. angiospermic dicot plant families also exhibiting J.; Wimpee, C. F.; Smith, J. F.; Furnier, the paraphyletic conditions of these genera with G.R.; Strauss, S. H.; Xiang, Q. Y.;

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Table: List of the rbc L sequences using figure-1 Sl. Accession No of TAXA USED REFFERENCES No Number base FAMILY (GenBank) pairs 01 AY663611 1408 Phyllanthus polyphyllus Phyllanthaceae [21] 02 AY663610 1408 Phyllanthus nutans Phyllanthaceae [21] 03 AY663609 1408 Phyllanthus nummariifolius Phyllanthaceae [21] 04 AY663608 1408 Phyllanthus lokohensis Phyllanthaceae [21] 05 AY663607 1408 Phyllanthus juglandifolius Phyllanthaceae [21] 06 AY663606 1408 Phyllanthus fluitans Phyllanthaceae [21] 07 AY663605 1408 Phyllanthus flagelliformis Phyllanthaceae [21] 08 AY663603 1408 Phyllanthus calycinus Phyllanthaceae [21] 09 AB233921 1331 Phyllanthus flexuosus Phyllanthaceae [22] 10 EF450315 1428 Piper nigrum Piperaceae Yang et al., (Unpublished) 11 EF450314 1428 Piper chinense Piperaceae Yang et al., (Unpublished) 12 EF450313 1428 Piper laetispicum Piperaceae Yang et al., (Unpublished) 13 EF450312 1428 Piper hainanse Piperaceae Yang et al., (Unpublished) 14 EF450310 1428 Piper hancei Piperaceae Yang et al., (Unpublished) 15 EF450309 1428 Piper sarmentosum Piperaceae Yang et al., (Unpublished) 16 EF450311 1428 Piper betle Piperaceae Yang et al., (Unpublished) 17 EF450308 1428 Piper kadsura Piperaceae Yang et al., (Unpublished) 18 EF450307 1428 Piper wallichii Piperaceae Yang et al., (Unpublished) 19 EF450305 1428 Piper austrosinense Piperaceae Yang et al., (Unpublished) 20 EF450304 1428 Piper longum Piperaceae Yang et al., (Unpublished) 21 EU155605 1356 Trichosanthes schlechteri Cucurbitaceae [23] 22 EU155604 1356 Trichosanthes pentaphylla Cucurbitaceae [23] 23 EU155603 1356 Trichosanthes cucumerina Cucurbitaceae [23] 24 EU155602 1356 Trichosanthes bracteata Cucurbitaceae [23] 25 EF487553 1355 Austrobryonia pilbarensis Cucurbitaceae [23] 26 EF487552 1355 Austrobryonia micrantha Cucurbitaceae [23] HS4V 27 EF487551 1355 Austrobryonia micrantha Cucurbitaceae [23] HS411 28 EF487550 1355 Austrobryonia centralis Cucurbitaceae [23] 29 EF487549 1355 Austrobryonia argillicola Cucurbitaceae [23] 30 AF378764 1334 Canna tuerckheimii Cannaceae [24] 31 AF378763 1327 Canna indica Cannaceae [24] 32 AF378774 1327 Canna glauca Cannaceae Prince (Unpublished) 33 AY570390 1371 Mentha longifolia Lamiaceae [25] 34 Z37417 1420 Mentha rotundifolia Lamiaceae [26] 35 Z37415 1420 Mentha longifolia (2) Lamiaceae [26] 36 U28876 1402 Mentha suaveolens Lamiaceae [27] 37 L14407 1474 Salvia divinorum Lamiaceae [28] 38 AB295077 1323 Salvia plebeia Lamiaceae Sudarmono and Okada (Unpublished) 39 AB295076 1323 Salvia glabrescens Lamiaceae Sudarmono and Okada (Unpublished) 40 AB295073 1323 Salvia nipponica Lamiaceae Sudarmono and Okada (Unpublished) 41 AB295070 1323 Salvia arisanensis Lamiaceae Sudarmono and Okada (Unpublished) 42 AB295069 1323 Salvia hayatana Lamiaceae Sudarmono and Okada (Unpublished) 43 AB295068 1323 Salvia pygmea Lamiaceae Sudarmono and Okada (Unpublished) 44 AB295067 1323 Salvia lutescens Lamiaceae Sudarmono and Okada (Unpublished) 45 AY570451 1162 Salvia viscosa Lamiaceae [25] 46 AY570450 1368 Salvia viridis Lamiaceae [25] 47 AY570449 1353 Salvia staminea Lamiaceae [25]

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Molecular phylogeny of angiospermic plant families using RBCL gene sequences

48 AY570448 1187 Salvia tiliifolia Lamiaceae [25] 49 AY570447 1349 Salvia texana Lamiaceae [25] 50 AY570446 1365 Salvia sylvestris Lamiaceae [25] 51 AY570445 1356 Salvia taraxacifolia Lamiaceae [25] 52 AY570444 1371 Salvia summa Lamiaceae [25] 53 AY570443 1350 Salvia subincisa Lamiaceae [25] 54 AY570442 1371 Salvia spathaceae Lamiaceae [25] 55 AY570439 1349 Salvia sclarea Lamiaceae [25] 56 AY570438 1371 Salvia roemeriana Lamiaceae [25] 57 AY570437 1177 Salvia ringens Lamiaceae [25] 58 AY570436 1364 Salvia pratensis Lamiaceae [25] 59 AY570435 1126 Salvia polystachya Lamiaceae [25] 60 AY570434 1371 Salvia pentstemonoides Lamiaceae [25] 61 AY570433 1186 Salvia palaestina Lamiaceae [25] 62 AY570432 1371 Salvia pachyphylla Lamiaceae [25] 63 AY570431 1355 Salvia officinalis Lamiaceae [25] 64 AY570430 968 Salvia nubicola Lamiaceae [25] 65 AY570427 1182 Salvia nilotica Lamiaceae [25] 66 AY570428 1113 Salvia misella Lamiaceae [25] 67 AY570426 1371 Salvia lyrata Lamiaceae [25] 68 AY570425 1357 Salvia lycoides Lamiaceae [25] 69 AY570424 1296 Salvia involucrate Lamiaceae [25] 70 AY570423 1115 Salvia verbenaca Lamiaceae [25] 71 AY570422 1135 Salvia hirsuta Lamiaceae [21] 72 AY663639 1398 Drypetes staudtii Putranjivaceae [21] 73 AY663638 1398 Drypetes lateriflora Putranjivaceae [21] 74 AY663637 1398 Drypetes gilgiana Putranjivaceae [21] 75 AY663636 1398 Drypetes floribunda Putranjivaceae [21] 76 AY663635 1398 Drypetes fallax Putranjivaceae [21] 77 AY663634 1398 Drypetes diversifolia Putranjivaceae [21] 78 AY663633 1398 Putranjivaceae [21] 79 AY663632 1398 Drypetes brownie Putranjivaceae [21] 80 M95757 1420 Drypetes roxburghii Putranjivaceae [29] 81 AB233926 1331 Drypetes littoralis Putranjivaceae [22] 82 AM234244 1421 Cassia grandis Caesalpinioideae [30] 83 U74195 1465 Cassia fistula Caesalpinioideae [31] 84 Z70155 1368 Cassia senna Caesalpinioideae [31] 85 Z70154 1368 Cassia didymobotrya Caesalpinioideae Kaess, (Unpublished). 86 AY149374 1206 Asparagus officinalis Asparagaceae McPherson et al., (Unpublished) 87 AB029849 1369 Asparagus cochinensis Asparagaceae [32] 88 AM234843 1340 Asparagus capsensis Asparagaceae [30] 89 AF374732 1346 Saxifraga stellaris Saxifragaceae [33] 90 L01953 1377 Saxifraga integrifolia Saxifragaceae [33] 91 FJ914175 1370 Solanum chilense Solanceae Dillon et al., (Unpublished) 92 FJ914174 1370 Solanum pennellii Solanceae Dillon et al., (Unpublished)

32 Bioinfo Publications, International Journal of Bioinformatics Research, ISSN: 0975–3087, Volume 1, Issue 2, 2009 B. Uma Reddy

22 Piper nigrum 0.0000 53 0.0000 Piper austrosinense 0.0000 56 0.0006 Piper wallichii 0.0007 0.0000 13 Piper laetispicum 0.0006 0.0001 81 Piper sarmentosum 18 0.0000 0.0013 Piper longum Piper [Piperaceae] Subclade - II 0.0004 0.0000 Piper kadsura 53 33 0.0006 0.0009 62 Piper chinense 0.0000 0.0000 100 0.0006 Piper hancei 0.0000 0.0514 Piper betle 0.0018 100 Piper hainanense CLADE - V 2.9732 0.0019 Asparagus officinalis 0.0500 53 Mentha rotundifolia 0.0033 0.0125 100 Mentha longifolia 0.0375 0.0083 87 Salvia plebeia 0.0042 0.0070 47 100 Salvia glabrescens 7 0.0000 Subclade - I 0.0013 0.0042 Salvia nipponica 0.3018 64 0.0000 0.0029 Salvia arisanensis Salvia [Lamiaceae] 33 0.0040 Salvia lutescens 0.0001 92 0.0011 2 0.0029 92 Salvia hayatana 0.0000 2.9073 0.0011 Salvia pygmaea 0.0000 36 Salvia tiliifolia 2.3685 Salvia [Lamiaceae] 0.6579 Salvia involucrata 2.3685 Salvia lycioides 0 3.3282 3.5798 Salvia misella 4.5465 17 Austrobryonia pilbarensis 0.0000 17 0.0000 Austrobryonia micrantha 0.0000 86 0.0000 Austrobryonia micrantha(2) Austrobryonia [Cucurbitaceae] Subclade - IV 2 100 0.0011 0.0000 Austrobryonia argillicola 1.6889 2.5908 0.0000 14 Austrobryonia centralis 0.0011 0.4800 Salvia officinalis 33 2.2921 0.2998 100 Solanum chilense Subclade - III 0.0000 Solanum [Solanaceae] 4 2.2921 Solanum pennellii 1.4746 0.0000 Salvia hirsuta 2.7221 Canna tuerckheimii 6 2.3779 CLADE - IV 0.3498 100 Saxifraga stellaris 0 0.0093 Saxifraga [Saxifragaceae] 96 0.0263 Saxifraga integrifolia Subclade - II 1.8296 37 0.0134 0.0093 0.3442 Cassia fistula 0.0356 18 Drypetes staudtii 0.0023 100 18 0.0000 Drypetes fallax 0.0023 2.3289 0.0006 28 Drypetes floribunda 0.0011 0.0023 Drypetes gilgiana 100 0.0029 Drypetes [Putanjivaceae] Subclade - I 37 Drypetes lateriflora 0.0451 0.0023 19 0.0009 Drypetes deplanchei 0.0023 0.0008 27 Drypetes diversifolia 0.0029 0.0003 Drypetes brownii 1.3224 0.0029 14 Salvia pratensis 3.0102 4.9248 Asparagus cochinchinensis Out group 0 3.0102 100 Salvia texana 1.8802 0.0000 Salvia [Lamiaceae] CLADE - III 14 2.9688 Salvia sclarea 0.0000 4.9662 47 Salvia subincisa 2.3426 0.6262 Salvia verbenaca 2.3426 26 Salvia viscosa 2.8156 5.4417 Cassia grandis 0 2.8156 3.3875 Salvia palaestina 5 3.1560 Salvia [Lamiaceae] CLADE - II Salvia polystachya 5.1013 14 2.8919 0.2641 100 Salvia staminea 0.0006 2.8913 Salvia ringens 0.0006 100 Canna indica 0.0000 Canna [Cannaceae] Subclade - VII 9.0506 Canna glauca 0.0000 57 Phyllanthus nutans 0.0029 30 0.0009 Phyllanthus juglandifolius 0.0029 34 0.0005 Phyllanthus polyphyllus 26 0.0014 0.0037 Phyllanthus flagelliformis 0.0012 0.0042 32 Phyllanthus fluitans Phyllanthus [Phyllanthaceae] 0.0056 0 0.0009 42 Phyllanthus calycinus Subclade - VI 3.9166 100 0.0058 0.0011 Phyllanthus flexuosus 0.0317 0.0058 100 51 Phyllanthus nummulariifolius 0.0058 2.4614 0.0020 Phyllanthus lokohensis 32 0.0058 2.5010 Drypetes littoralis 0.0395 Salvia sylvestris 2.5009 100 Salvia viridis 1 0.0516 23 4.0488 2.5299 Asparagus capensis Out group Salvia [Lamiaceae] Subclade - V 0.0516 1.3161 Salvia taraxacifolia 2.5815 37 Salvia nubicola 1 2.3166 Salvia [Lamiaceae] Subclade - IV CLADE - I 1.1042 0.9530 Salvia nilotica 2.3166 71 Trichosanthes pentaphylla 0.0006 6 76 0.0009 Trichosanthes cucumerina 0.6282 0.0006 Trichosanthes [Cucurbitaceae] Subclade - III 100 0.0016 Trichosanthes schlechteri 2.6454 0.0014 Trichosanthes bracteata 27 0.0031 Drypetes roxburghii 0.6211 0.0574 100 100 Cassia senna 0.0034 Cassia [Caesalpinoideae] Subclade - II 2.5911 0.0371 Cassia didymobotrya 0.0034 85 Salvia divinorum 0.0168 0.0073 100 Salvia spathacea 100 0.0000 25 0.0058 Salvia pachyphylla 0.0332 0.0000 0.0003 Mentha suaveolens Out group 42 0.0058 0.0012 Mentha longifolia Out group Salvia [Lamiaceae] Subclade - I 72 0.0040 Salvia lyrata 0.0021 74 0.0019 Salvia pentstemonoides 0.0021 59 0.0011 92 Salvia summa 0.0008 0.0000 0.0011 Salvia roemeriana 0.0000 Fig -1 Phylogenetic tree from 92 rbc L sequences representing 90 taxa of family Cucurbitaceae, were inferred using UPGMA method. Bootstrap percentages and branch lengths are indicated above and below the branches, respectively.

International Journal of Bioinformatics Research, ISSN: 0975–3087, Volume 1, Issue 2, 2009 33

Molecular phylogeny of angiospermic plant families using RBCL gene sequences

86 Salvia summa

60 Salvia roemeriana Salvia (Lamiaceae) Salvia pentstemonoides 69 36 Salvia lyrata 59 Mentha longifolia (2) 99 Mentha suaveolens Salvia divinorum

40 39 Salvia spathacea Salvia (Lamiaceae) 94 Salvia pachyphylla 99 Cassia senna Cassia (Caesalpinioideae - sub family) 99 Cassia didymobotrya 1 Drypetes roxburghii Salvia taraxacifolia Trichosanthes bracteata 11 3 Trichosanthes schlechteri 99 Trichosanthes (Cucurbitaceae) 62 Trichosanthes pentaphylla 0 70 Trichosanthes cucumerina Salvia nilotica 2 Salvia nubicola Salvia verbenaca Salvia subincisa 0 Asparagus capensis 5 99 Salvia staminea Salvia (Lamiaceae) 18 Salvia ringens Drypetes littoralis 14 Phyllanthus flagelliformis

9 99 14 Phyllanthus nutans

37 Phyllanthus fluitans 99 Phyllanthus juglandifolius

82 Phyllanthus nummulariifolius Phyllanthus (Phyllanthaceae) 30 Phyllanthus lokohensis

33 Phyllanthus polyphyllus

30 Phyllanthus calycinus 74 Phyllanthus flexuosus Mentha rotundifolia Mentha (Lamiaceae) 3 99 Mentha longifolia

94 Salvia glabrescens 50 Salvia nipponica 33 Salvia plebeia Salvia viridis 57 83 Salvia sylvestris Salvia (Lamiaceae) Salvia lutescens 99 70 Salvia hayatana 72 Salvia pygmaea Salvia arisanensis 99 Asparagus officinalis Piper kadsura 2 18 29 Piper chinense

99 Piper hancei

23 Piper hainanense 28 Piper betle

65 Piper sarmentosum Piper (Piperaceae) 49 Piper longum

5 Piper nigrum Piper austrosinense 66 15 Piper laetispicum 11 Piper wallichii Canna tuerckheimii Asparagus cochinchinensis 0 0 99 Salvia texana Salvia {Lamiaceae) 0 Salvia sclarea

99 Canna indica 3 Canna (Cannaceae) Canna glauca

21 Salvia polystachya Cassia grandis 12 Salvia tiliifolia 3 Salvia viscosa Salvia (Lamiaceae) 0 1 Salvia involucrata 0 1 Salvia palaestina 8 Salvia lycioides Austrobryonia centralis

0 Austrobryonia pilbarensis 99 Austrobryonia micrantha Austrobryonia (Cucurbitaceae) 56 15 Austrobryonia micrantha(2) 13 Austrobryonia argillicola 99 Salvia officinalis Salvia hirsuta Salvia pratensis Salvia misella

0 99 Saxifraga stellaris Saxifraga (Saxifragaceae) 75 Saxifraga integrifolia 10 Cassia fistula Drypetes brownii 99 49 Drypetes lateriflora 13 99 Drypetes deplanchei Drypetes diversifolia Drypetes (Putranjivaceae) 56 Drypetes staudtii Drypetes gilgiana 50 51 Drypetes floribunda 21 Drypetes fallax Solanum chilense Solanum pennellii Fig-2 Phylogenetic tree from 92 rbc L sequences representing 90 taxa of family Cucurbitaceae, were inferred using the Maximum Parsimony method (length = 14029, CI = CI = 0.187540; RI = 0.733592; RCI = 0.137578 (for all sites).

34 Bioinfo Publications, International Journal of Bioinformatics Research, ISSN: 0975–3087, Volume 1, Issue 2, 2009 B. Uma Reddy

21 Salvia polystachya 10 Salvia palaestina 3 Salvia involucrata Salvia (Lamiaceae) 1 Salvia tiliifolia 0 Salvia viscosa 0 Cassia grandis 0 Salvia plebeia 0 Mentha longifolia (2) 0 Salvia pygmaea 0 Salvia hayatana 0 Salvia glabrescens 0 Salvia nipponica 0 Salvia lutescens 0 Salvia arisanensis 0 Mentha rotundifolia 0 Asparagus officinalis 0 Piper hancei 0 Piper kadsura 0 Piper chinense 0 Piper hainanense

0 Piper sarmentosum 14 Piper longum 0 Piper betle 0 Piper laetispicum 0 Piper wallichii 0 Piper austrosinense

0 Piper nigrum Asparagus cochinchinensis Asparagus capensis 0 Salvia viridis 0 90 Salvia staminea Salvia (Lamiaceae) 0 Salvia ringens

6 Drypetes littoralis Phyllanthus flagelliformis 67 Phyllanthus nutans 47 Phyllanthus juglandifolius 27 Phyllanthus calycinus 17 Phyllanthus polyphyllus Phyllanthus (Phyllanthaceae) 13 Phyllanthus lokohensis 5 Phyllanthus flexuosus 12 Phyllanthus nummulariifolius 12 18 Phyllanthus fluitans

17 Austrobryonia pilbarensis

66 Austrobryonia micrantha Austrobryonia micrantha(2) Austrobryonia (Cucurbitaceae) 83 17 Austrobryonia argillicola 13 Austrobryonia centralis

3 Solanum chilense Solanum (Solanaceae) 88 Solanum pennellii Salvia hirsuta 1 Saxifraga stellaris Saxifraga integrifolia 55 Drypetes deplanchei 33 Drypetes lateriflora 29 Drypetes floribunda 18 Drypetes diversifolia 13 Drypetes staudtii Drypetes (Putranjivaceae) 3 Drypetes fallax 6 Drypetes brownii 2 2 Drypetes gilgiana 13 Cassia fistula

5 Canna tuerckheimii 0 Salvia lycioides Salvia (Lamiaceae) Salvia officinalis

7 Salvia sylvestris 0 Salvia pratensis Salvia (Lamiaceae) 0 Salvia verbenaca

92 Salvia texana 12 Salvia sclarea 0 Salvia (Lamiaceae) 5 Salvia subincisa Salvia misella Salvia nilotica 0 Salvia taraxacifolia

0 26 Trichosanthes schlechteri 53 Trichosanthes cucumerina Trichosanthes (Cucurbitaceae) 0 91 Trichosanthes pentaphylla Trichosanthes bracteata

0 Salvia nubicola

97 Canna indica Canna (Cannaceae) 1 Canna glauca

7 Mentha suaveolens Mentha longifolia 85 Salvia roemeriana 9 Salvia summa 3 Salvia pachyphylla 1 Salvia spathacea 4 Salvia lyrata 0 Salvia (Lamiaceae) Salvia pentstemonoides 3 Drypetes roxburghii 0 Salvia divinorum 7 11 Cassia senna Cassia (out group from Salvia) 41 Cassia didymobotrya Fig -3 Phylogenetic tree from 92 rbc L sequences belongs to 90 taxa of 12 genera and 10 families by the Neighbor-Joining method. Numbers above branches indicate percentage of 500 bootstrap replications. Horizontal bar indicates the nucleotide substitution scale

International Journal of Bioinformatics Research, ISSN: 0975–3087, Volume 1, Issue 2, 2009 35

Molecular phylogeny of angiospermic plant families using RBCL gene sequences

7 Piper nigrum 0 Piper wallichii 0 Piper kadsura 0 Piper laetispicum 0 Piper sarmentosum 0 Piper (Piperaceae) Piper longum 0 Piper chinense 0 Piper hancei 0 Piper betle 0 Piper austrosinense 0 Salvia arisanensis 0 Salvia plebeia 0 Asparagus officinalis 0 Salvia hayatana 3 Salvia pygmaea 0 Piper hainanense 2 Salvia nipponica 3 Salvia glabrescens 11 Mentha rotundifolia 65 Salvia lutescens Mentha longifolia

16 Austrobryonia micrantha(2) 0 13 Austrobryonia argillicola 61 Austrobryonia micrantha Austrobrypnia (Cucurbitaceae) 86 Austrobryonia pilbarensis Austrobryonia centralis

2 Salvia hirsuta

87 Solanum chilense Solanum (Solanaceae) 6 Solanum pennellii

8 Saxifraga integrifolia Drypetes floribunda 72 0 Drypetes diversifolia 12 Saxifraga stellaris 1 Cassia fistula 1 Drypetes brownii 5 Drypetes lateriflora 7 0 Drypetes deplanchei 3 Drypetes (Putranjivaceae) Drypetes staudtii 5 7 Drypetes gilgiana 7 Drypetes fallax Asparagus cochinchinensis Canna tuerckheimii

0 Salvia officinalis Salvia (Lamiaceae) 3 Salvia lycioides

5 Salvia viscosa Salvia involucrata

0 Salvia tiliifolia Salvia (Lamiaceae) Cassia grandis 1 8 Salvia polystachya 21 Salvia palaestina

95 Salvia texana 10 Salvia sclarea 3 Salvia subincisa

0 Salvia misella Salvia (Lamiaceae) Salvia verbenaca

1 Salvia sylvestris 6 Salvia pratensis Asparagus capensis 0 Salvia viridis

11 Phyllanthus nummulariifolius 6 Phyllanthus fluitans 8 Phyllanthus flexuosus 0 4 Phyllanthus lokohensis 8 Phyllanthus polyphyllus Phyllanthus (Phyllanthaceae) 8 Phyllanthus calycinus 14 Phyllanthus juglandifolius 0 37 Phyllanthus nutans 79 Phyllanthus flagelliformis 5 Drypetes littoralis Salvia staminea Salvia (Lamiaceae) 89 Salvia ringens

20 Trichosanthes schlechteri 47 Trichosanthes cucumerina Trichosanthes (Cucurbitaceae) 0 90 Trichosanthes pentaphylla 9 Trichosanthes bracteata Salvia taraxacifolia

96 Canna indica Canna (Cannaceae) 0 4 Canna glauca Salvia nubicola

0 Salvia nilotica Mentha suaveolens 16 Salvia pentstemonoides 88 Salvia summa 12 Salvia roemeriana 3 Salvia spathacea 1 Salvia pachyphylla 6 Salvia lyrata 0 Mentha longifolia (2) 5 Drypetes roxburghii 2 Salvia divinorum 7 12 Cassia senna Cassia (Caesalpinioide) 40 Cassia didymobotrya Fig -4 Phylogenetic tree from 92 rbc L sequences belongs to 90 taxa of 12 genera and 10 families using Minimum Evolution method. Numbers above branches indicate percentage of 500 bootstrap replications.

36 Bioinfo Publications, International Journal of Bioinformatics Research, ISSN: 0975–3087, Volume 1, Issue 2, 2009