Printer Friendly


Byline: M. Ibrahim N. Nazar M. Ilyas and T. Mahmood


The importance of genetic variation for sustaining biological diversity and evolutionary processes is widely accepted. The present study aims to investigate the phylogenetic relationships among five selected species of Apocynaceae collected from various regions of Pakistan using chloroplast gene (rps11) which encode ribosomal protein of smaller subunit11. For this purpose rps11 gene was amplified purified sequenced from each species and analyzed with MEGA5. Phylogenetic analysis revealed an overall narrow genetic background (0.5 %) among selected species. Nerium oleander Rhazya stricta and Vinca major were the most similar with a bootstrap value of 24-44 % while Catharanthus roseus and Nerium oleander showed distant relationship. Divergence value was observed within a range of 0.023-9.994 on the basis of pair wise distance calculation. The study further confirmed the use of rps11 gene as a potential candidate for phylogenetic analysis.

Key words: Apocynaceae rps11 Genetic Variation.


Apocynaceae is considered as one of the largest family of angiosperms containing 375 genera and over 5100 species around the globe (Endress et al. 2007). Most species include lianas herbs shrubs trees frequently found in rainforests tropics and subtropics but some can grow in tropical dry and xeric environments (Endress and Bruyns 2000). Modern classification of Endress et al. (2007) divided the family into five subfamilies i.e Asclepiadoideae Secamonoideae and Periplocoideae are from the former Asclepiadaceae and Apocynoideae Rauvolfioideae are from Apocynaceae. Pakistan is hosting 19 genera and 26 species (Nazimuddin et al. 1983) mostly found in North Punjab Azad Kashmir Hazara Rawalpindi Attock Swat Bajaur Waziristan and Salt range area (Ali 1983).

The family carries considerable importance in the field of medicine because of its extensive use in Cancer Chemotherapy skin diseases Diabetes Diarrhea and Malaria (Middleton 2007). Besides its importance in economy and construction the family serve as a potential source for vast amount of chemicals like Alkaloids Steroids Triterpenoids Phenolics from Alstonia spp. (Singh and Singh 2003) and Terpenoids Indole Alkaloids (TIA) Vincristine Vinblastine Catharanthine from Catharanthus roseus species (Gueritte and Fashy 2005). Similarly reports of Shazly et al. (2005) confirmed the presence of Cardiotonic glycoside Neeriifolin' which has insecticidal property and Thevetin that work as heart stimulant. Preservation and successful management of natural flora is deeply associated with the accurate assessment of genetic diversity which in turn is of paramount importance for possible adaptation and consequently for prolong survival of a species (Hanski and Ovaskainnen 2000). A series of technique and genetic marker system has been developed for estimation and analysis of genetic variability at inter and intraspecific level. A wide range of molecular approaches has been used by numerous research groups to find the natural relationship within various groups of Apocynaceae (Potgieter and Albert 2001; Rapini et al. 2003; Rapini et al. 2006; Livshultz et al. 2007; Simoes et al. 2007; Livshultz 2010). A recent review by Nazar et al. (2013) has significantly emphasized on the systematics and taxonomy of Apocynaceae.

Chloroplast DNA can serve as a main source of phylogenetic analysis (Small et al. 2005) at different systematic level in various genera of plant kingdom (Kallersjo et al. 1998). In past chloroplast genome has been assessed for phylogenetic study of Apocynaceae in genus Asclepias (Mark et al. 2011) Hoya (Livia et al. 2011) Diplolepis (Hechem et al. 2011) and Pachypodium (Burg et al. 2013) subfamily Secamonoideae plus Asclepiadoideae (Tatyana et al. 2010) sub tribe Metastelmatinae (Silva et al. 2012) and Rauvolfioideae (Mahadani et al. 2013) by various research groups. The present study aims to investigate the genetic variability within selected species of Apocynaceae using rps11 gene and to use this information for better management and phylogenetic analysis. MATERIALS AND METHODS

Plant Collection: Five species of Apocynaceae were collected from different regions of Pakistan (Table 1) and were identified in the National Herbarium of Pakistan Department of Plant Sciences Quaid-i-Azam University Islamabad.

DNA Extraction: DNA was extracted using CTAB (Cetyltrimethyl ammonium bromide) method (Richards 1997) with little alteration (Mahmood et al. 2011a) and its quality was checked on 1% agarose gel via gel documentation system (Dolphin-Doc Plus Wealtech).

Primer Designing and PCR: A set of primers and PCR conditions were used as given by Mahmood et al. (2011a) with slight modification. Annealing temperature was 56 C0 with 25 ul of reaction mixture which contain 12.5 l PCR Master Mix (2X) (MBI Fermentas) 1 l (25 pmol) of forward and reverse primer each 1 l template and 9.5 l of nanopure water. PCR (Multigene Labnet) was used for amplification and products were checked on 1.5 % agarose gel.

Sequencing: PCR products were purified by JET quick PCR Product Purification Spin Kit (Genomed) resolved on 2% agarose gel and sequenced by Macrogen (Korea).

Phylogram Construction: Sequences were analyzed using Molecular Evolutionary Genetics Analysis (MEGA5) and dendrogram was constructed by neighbour-joining (NJ) method to check the phylogeny and genetic variability. Pair wise distance analysis was conducted to calculate evolutionary divergence using MEGA5.


Phylogenetic Analysis: Phylogenetic Analysis divided the species into two clusters. Cluster 1 consists of three species showing overall bootstrap value from 24-44 % while cluster 2 includes only one species. Values of evolutionary divergence ranged from 0.023-9.994 among selected species (Table 2). Lowest divergence value was observed between Alstonia scholaris and Rhazya stricta while highest divergence value was observed between Catharanthus roseus and Nerium oleander. Catharanthus roseus emerged as the most primitive species forming an out group (Fig. 1). Previously genetic variability was assessed among various species of Apocynaceae using RAPD markers CAPS technique and sequences of rps11 region (Mahmood et al. 2010; 2011 a b c). According to our knowledge no earlier work has been reported on selected species using rps11 as a marker for estimation of genetic variability. Overall the results are an indicator of change in the chloroplast DNA during the course of evolution.

The present study showed narrow genetic background among selected species with 0.5% genetic diversity which is lower than already reported results of 15% 34% and 42% (Mahmood et al. 2011a b c) which is due to variation in marker selection and species number. Dendrogram divided the species into two clusters which is accordance to the result already reported by Mahmood et al. (2011a) but contradictory to the reported results of Mahmood et al. (2011b) which divided the species into three clusters which may be attributed to different marker system (RAPD) used for phylogenetic studies. Earlier work of Mahmood et al. (2011b) placed Catharanthus roseus and Alstonia scholaris in separate cluster while in this study they appear as an out group to the remaining species and are considered as the most primitive and common ancestor. Nerium Oleander (Apocynoideae) revealed maximum similarity (44%) with Rhazya Stricta

(Rauvolfioideae) which is contradictory to the reported results of Endress an d Bruyns (2000) based on morphological data such results indicated a common lineage history for both species. In future the close relationship between Nerium oleander and Rhazya stricta and distant relationship of Catharanthus roseus require further confirmation not only at molecular but also at morphological and biochemical level using enormous number of species and markers.

Table 1. List of selected species along with their subfamily order longitude latitude and area of collection

S.No###Species Name###Subfamily###Order###Site name###Longitude and latitude

1###Nerium oleander###Apocynoideae###Gentianales###Islamabad###33 42' N and 73 10' E

2###Catharanthus roseus###Rauvolfioideae###Gentianales###Islamabad###33 42' N and 73 10' E

3###Alstonia scholaris###Rauvolfioideae###Gentianales###Islamabad###33 42' N and 73 10' E

4###Vinca major###Rauvolfioideae###Gentianales###Swat###34 51' N and 72 33' E

5###Rhazya stricta###Rauvolfioideae###Gentianales###Nowshehra###34 0' N and 71 58' E

Table 2. Estimation of evolutionary divergence among rps 11 gene sequences for five species of family








Conclusion: The study confirmed the use of rps11 as a source for assessing genetic variation in Apocynaceae showing distant relationship of Catharanthus roseus (Rauvolfioideae) with Nerium oleander (Apocynoideae) and close genetic relationship between Nerium oleander and Rhazya stricta.

Acknowledgement: We are thankful to Higher Education Commission Islamabad Pakistan for providing financial assistance.


Ali S. I. (1983). Asclepiadaceae. Flora Pak. 150: 1-65. Burge D.O. K. Mugford A.P. Hastings and A.A. Agrawal (2013). Phylogeny of the plant genus Pachypodium (Apocynaceae). Peer J1:e70http ://

Endress M. E. and P. V. Bruyns (2000). A revised classification of the Apocynaceae. Botanic. Rev. 66: 1-56.

Endress M.E. R.W. Van der Ham S. Nilsson L. Civeyrel M.W. Chase B. Sennblad K. Potgieter J. Joseph M. Powell D. Lorence Y.M. Zimmerman and V.A. Albert (2007). Aphylogenetic analysis of Alyxieae (Apocynaceae) based on rbcL matK trnLintron trnL-Fspacer sequences and morphological characters. Ann. Missouri. Bot. Gard. 94: 1-35.

Gueritte F. and J. Fahy (2005). The Vinca alkaloids. In: Cragg G.M. Kingston L. Newman D.J. (Eds.) Anticancer agents from natural product. Tayler and Francis Boca Raton Florida pp: 123-136.

Hanski I. and O. Ovaskainen (2000). The metapopulation capacity of a fragmented landscape. Nature. 404: 755-758.

Kallersjo M. J. S. Farris M. W. Chase B. Bremer M. F. Fay C. J. Humphries G. Petersen O. Seberg and K. Bremer (1998). Simultaneous parsimony jackknife analysis of 2538 rbcL DNA sequences reveals support for major clades of green plants land plants seed plant and flowering plants. Plant. Syst. Evol. 213: 259-287.

Livia W. G. K. Muellner and N. Alexandra (2011). Revisiting the wax plants (Hoya Marsdenieae Apocynaceae): Phylogenetic tree using the matK gene and psbA-trnH intergenic spacer. Taxon. 60(1): 4-14.

Livshultz T. (2010). The phylogenetic position of milkweeds (Apocynaceae subfamilies Secamonoideae and Asclepiadoideae): evidence from the nucleus and chloroplast. Taxon. 59: 10161030.

Livshultz T. D. J. Middleton M. E Endress and J. Williams (2007). Phylogeny of Apocynoideae and the APSA clade. Ann. Missouri Bot. Gard. 94: 323-361.

Mahadani P. G. D. Sharma and S. K. Ghosh (2013). Identification of ethnomedicinal plants (Rauvolfioideae: Apocynaceae) through DNA barcoding from northeast India. Pharmacogn. Mag. 9(35): 255-263.

Mahmood T. A. Iqbal N. Nazar I. Naveed B.H. Abbasi and S.M.S. Naqvi (2011b). Assessment of genetic variability among selected species of Apocynaceae. Biologia. (66)1: 64-67.

Mahmood T. F. Meer F. Munir N. Nazar and I. Naveed (2011a). Genetic diversity of selected Apocynaceae species based on chloroplast gene rps11. J. Medic. Plants Res. 5(17): 4382-4387.

Mahmood T. S. Muhammad and Z. K. Shinwari (2010). Molecular and morphological characterization of Caralluma species. Pakistan J. Bot. 42(2): 1163- 1171.

Mahmood T. A. Tariq N. Nazar B.H. Abbasi and S.M.S. Naqvi (2011c). Comparative assessment of genetic variability in Cryptolepis buchananii Tylophora hirsute and Wattakaka volubilis. Pakistan J. Bot. 43(5): 2295-2300.

Mark F. C. David E. Chris M. Gamer J. Roberta and P. Steven (2011). Phylogenetic Relationships of Asclepias (Apocynaceae) inferred from Non- coding Chloroplast DNA Sequences. Syst. Bot. 36(4): 1008-1023.

Middleton D.J. (2007). Apocynaceae (subfamilies Rauvolfioideae and Apocynoideae) Flora Malesiana ser.I seed plants. The National Herbarium of the Netherlands Leiden TheNetherlands.18: 474.

Nazar N. D. J. Goyder J. J. Clarkson T. Mahmood and M.W. Chase (2013). The taxonomy and systematics of Apocynaceae: where we stand in 2012. Bot. J. Linn. Soc. 171(3): 482-490. Nazimuddin S. M. Qaiser E. Nasir and S. I. Ali (1983). Flora of Pakistan Shamim Printing Press Karachi Pakistan: 84: 95-101.

Potgieter K. and V. A. Albert (2001). Phylogenetic relationships within Apocynaceae based on trnL intron and trnL-F spacer sequences and propagule characters. Ann. Missouri. Bot. Gard. 88: 523-549.

Rapini A. M. W. Chase and T. U. P. Konno (2006). Phylogenetics of South American Asclepiadoideae (Apocynaceae). Taxon 55: 119- 124. Rapini A. M. W. Chase D. J. Goyder and J. Griffiths (2003). Asclepiadeae classification: evaluating the phylogenetic relationships of New World Asclepiadoideae (Apocynaceae). Taxon 52: 33- 50.

Richards E. J. (1997). Preparation of plant DNA using CTAB. In: Ausubel FM. Brent F. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. (Eds.) Short protocol in molecular Biology. John Wiley and Sons New York pp: 210-211.

Simoes A. O. T. Livshultz T. Conti and E. Endress (2007). Phylogeny and systematics of the Rauvolfioideae (Apocynaceae) based on molecular and morphological evidence. Ann. Missouri. Bot. Gard. 94: 268297.

Singh S. K. and A. Sinhg (2003). Molluscicidal and anticholinesterase activity of Alstonia schlaris plant against fresh water snail Lymnaea acuminate. Pakistan J. Bio. Sci. 6(16): 1442- 1446.

Shazly M. M. E. M. Zayat and W. HermersdOrfer (2005). Insecticidal activity mammalian cytotoxicity and mutagenicity of an ethanolic extract from Nerium oleander (Apocynaceae). Ann. Appl. Biol. 136(2): 153157.

Small R. L. E. B. Lickey J. Shaw and W. D. Hauk (2005). Amplification of noncoding chloroplast DNA for phylogenetic studies in lycophytes and monilophytes with a comparative example of relative phylogenetic utility from Ophioglossaceae. Mol. Phylogen. Evol.36: 509- 522.

Tatyana L. (2010). The phylogenetic position of milkweeds (Apocynaceae subfamilies Secamonoideae and Asclepiadoideae): Evidence from the nucleus and chloroplast. Taxon 59(4): 1016-1030.

Silva U. C. Soares R. Alessandro; L.S. R.P. Luz and V.B. Cassio (2012). Taxonomic Considerations on Metastelmatinae (Apocynaceae) Based on Plastid and Nuclear DNA. Taxon 37(3): 795- 806.

Hechem V. C. CalviAo and E. Cecilia (2011). Molecular phylogeny of Diplolepis (Apocynaceae-Asclepiadoideae) and allied genera and taxonomic implications. Taxon. 60(3): 638-648.
COPYRIGHT 2014 Asianet-Pakistan
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2014 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Publication:Journal of Animal and Plant Sciences
Geographic Code:9PAKI
Date:Aug 31, 2014

Terms of use | Privacy policy | Copyright © 2018 Farlex, Inc. | Feedback | For webmasters