Genetic Diversity of Amsonia Orientalis

Biologia 69/6: 742—749, 2014 Section Botany DOI: 10.2478/s11756-014-0368-6

Genetic diversity of Amsonia orientalis

Cem Tolga Gurkanli¨ 1*, Ibrahim˙ Ozkoc¨ ¸2,EmineBanuAydin 2,ArdaAcem˙i3 &FazılOzen¨ 3

1Department of Fisheries Technology Engineering, Fatsa Faculty of Marine Sciences, Ordu University, 52400,Fatsa, Ordu/Turkey; e–mail: [email protected], [email protected] 2Department of Biology, Faculty of Arts and Sciences, Ondokuz Mayıs University, 55139, Atakum, Samsun/Turkey 3Department of Biology, Faculty of Arts and Sciences, Kocaeli University, 41380, Kocaeli/Turkey

Abstract: Amsonia orientalis Decne. (Apocynaceae), is a rare and threatened plant species which is located only in a constricted area in northeast of Greece and northwest of Turkey in the world. Although phylogenetic analysis depending on nucleotide sequences of genes from different sources (nucleus, mitochondria and chloroplast) became a major tool for classification of plant species, there is still a big lack of information about A. orientalis in the international molecular data bases such as NCBI. In the current study, we phylogenetically analyzed three commonly used molecular markers (18S rDNA, 18S-28S rDNA-ITS region and trnL–F intergenic spacer) from A. orientalis samples collected from Turkey to determine the genetic diversity and also to question the systematic position of A. orientalis.Asaresult,A. orientalis samples clearly showed close relation with Alyxieae tribe rather than Vinceae. And this result brings the necessity to reconsider the morphological characters that have used to delimit the tribes of Rauvolfioideae. Key words: Amsonia orientalis; Apocynaceae; 18S rDNA; 18S–28S rDNA-ITS; trnL–F spacer

Introduction Ozen¨ (2006) implemented an ex situ protection since the habitats of the plant were under serious threat. In Genus Amsonia Walter from Apocynaceae (dogbane) addition to the ex situ protection, some in vitro prop- family covers 22 plant species most of that have agation studies were conducted as conservation strate- medicinal importance including antimicrobial and anti– gies as well (Oz¨ et al. 2008; Acemi et al. 2012; Acemi tumour activities besides their horticultural usage. Al- et al. 2013). though majority of these species are distributed in In the last 2 decades phylogenetic approaches Americas, few of them are native to South Europe, Far based on nucleotide sequences of several molecular East and Turkey (Dabine et al. 1986; Rahman & Za- markers including plastid trn intron, rbcL, ndhFetc., man 1988; Rahman et al. 1989; Sauerwein 1991; Darke have been preferred as an alternative method for clas- 2005). Amsonia species are commonly known as “blue sical Linnaean system for identification and classifica- stars” since they have pale blue colored and star shaped tion of plant taxa. So far, several studies have been flowers. As one of these species, Amsonia orientalis published about the molecular diversity and phylo- Decne. [syn. Rhazya orientalis (Decne.) A. DC.] is a genetic systematics of different plant families includ- rare medicinal and ornamental plant which has natu- ing Lamiaceae (Cantino et al. 1997), Ericaceae (Kron ral distribution only in Turkey and Greece in the world 1997) and Scrophulariaceae (Olmstead et al. 2001). (Tutin et al. 1968; Davis 1978). Although this plant In the last decade, molecular diversity of Apocy- is reported from 3 localities in Turkey which includes naceae family has also significantly exposed with sev- Istanbul,˙ Bursa and Balıkesir provinces (Tutin et al. eral papers beginning with the pioneering and com- 1968), recent field studies indicated a more limited dis- prehensive studies of Potgieter & Albert (2001) and tribution area that covers 10 km2 in Balıkesir province Sennblad & Bremer (2002) which deal with the phy- (Akyal¸cin et al. 2006). Ekim et al. (2000) placed A. ori- logenetic analysis of genes such as rbcL, ndhF, trnL entalis to the category of “critically endangered” (CR) and trnL–F intergenic spacer. However, A. orientalis in the “Red Data Book of Turkish Plants”. Further- was ignored in most these studies most probably be- more, the plant has been placed in the list of the plant cause of its limited natural distribution and that is species that must be conserved on European scale by why still there is a big lack of information about The European Council in the Bern Convention (Euro- genetic diversity of this species. The main goal of pean Council, 1979). In this context, to protect the re- this study is to complete this deficiency in the avail- tained A. orientalis populations in Balıkesir province, able literature and also to question the true system-

* Corresponding author

c 2014 Institute of Botany, Slovak Academy of Sciences Genetic diversity of Amsonia orientalis 743

Table. 1 PCR protocols and primers used in this study. ID, initial denaturation; C, PCR Cycle number; D, denaturation; A, annealing; E, extention; FE, ﬁnal extention.

Locus Primer ID C D A E FE

18S rDNA NS1/NS4 (White et al. 1990) 95 ◦C/3 min ×35 94 ◦C/1 min 51 ◦C/1 min 72 ◦C/1 min 72 ◦C/5 min 18S rDNA NS3/NS8 (White et al. 1990) 95 ◦C/3 min ×35 94 ◦C/1 min 60 ◦C/1 min 72 ◦C/2 min 72 ◦C/10 min trnL–F intergenic spacer TrnL(c)/TrnL(f) 95 ◦C/5 min ×35 94 ◦C/1 min 56 ◦C/1 min 72 ◦C/1 min 72 ◦C/5 min (Taberlet et al. 1991) 18S-28S rDNA–ITS ITS1/ITS4 (White et al. 1990) 94 ◦C/3 min ×35 94 ◦C/1 min 49 ◦C/2 min 72 ◦C/3 min 72 ◦C/7 min

atic position of A. orientalis in Apocynaceae fam- haplotypes in data sets were obtained either from the clos- ily. est BLAST hits of our sequences in the study and from the available literatures. Multiple nucleotide sequence align- ments were generated using ClustalX (Thompson et al. Material and methods 1997) and optimized by hand using BioEdit (Hall 1999). To determine the most appropriate DNA substitution model Plant materials for our data sets, the Akaike information criterion (AIC) Amsonia orientalis plant samples were collected from its (Akaika 1974) and Bayesian information criterion (BIC) wild populations located in Adnan Menderes (Ams–1), tests were applied with jModelTest v. 0.1 package pro- ¨ Gaziosmanpa¸sa (Ams–2), Pa¸sa Alanı (Ams–3) and Omerli gram (Guindon & Gascuel 2003; Posada 2008). Genetic dis- (Ams–4) districts of Balıkesir province of Turkey. The tances for data sets were calculated using MEGA version 6 voucher specimens were deposited in the herbarium of (Tamura et al. 2013). Uludag University (BULU, specimen no: 18138). To evaluate the phylogenetic relationships among samples Neighbor-Joining (NJ) (Saitou & Nei 1987), Maximum- DNA extraction Parsimony (MP) and Maximum–Likelihood (ML) algo- DNA was extracted from fresh and young leaves and roots rithms were used. NJ and MP analysis were performed with according to Temizkan & Arda (2004) with some modifica- software program PAUP* v.4.0b10 (Swofford 1998) and ML tions. As first step, 20–50 mg of plant material was grounded was applied with PhyML 3.0 (Guindon & Gascuel 2003). with 500 µL cetyltrimethyl ammonium bromide (CTAB) The heuristic search approach was applied for the MP anal- buffer (100 mM Tris-HCl, pH 8.0, 20 mM EDTA, 2% CTAB, yses using TBR swapping algorithm with 10 random repe- 1.4 M NaCl, and 0.1% b-mercaptoethanol) with a sterilized titions and then strict consensus trees were generated from ◦ mortar and pestle, and heated to 65 C. The solution was ex- equally parsimonious trees. Bootstrap analysis of the MP tracted twice with chloroform : isoamyl alcohol (24 : 1) by trees were conducted with 1.000 replications using 10 ran- centrifugation at maximum speed at room temperature. The dom repetitions for each replication. And bootstrap analy- supernatant was precipitated with 0.6 volumes of ice–cold sis of the NJ and ML trees were conducted with 10.000 and ethanol. The precipitate was then collected by centrifuga- 1.000 replications, respectively. All new sequences obtained tion at maximum speed. The pellet was washed with 70% in this study were deposited in EMBL data bank under ac- ethanol and air dried. The DNA pellet was finally dissolved cession numbers KJ649324–KJ649333 (See figure legends). in 25 µL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and stored at –20 ◦C prior to use. For some samples, DNA was extracted with DNeasy plant mini kit (Qiagen, Results Germany) according to the manufacturer’s instructions. Phylogenetic analysis of our 18S rDNA nucleotide se- PCR amplification quences together with the ones obtained from GenBank The PCR protocols and DNA primers shown in the Table 1 (see the legend of Fig. 1) were carried out with 1,636 were used to amplify 18S rDNA, 18S–28S rDNA-ITS and aligned nucleotides (see Table 2 for details). MP anal- trnL–F intergenic spacer loci with a MWG Primus thermal ysis yielded a single most parsimonious tree with 60 cycler. All amplifications were performed in a 25 µLvolume steps. AIC and BIC tests suggested two different sub- containing, 10 ng genomic DNA as template, 0.4 pmol of each primers, 0.8 mM dNTP mix (Amresco), 1U Taq poly- stitution models (see Table 2). Since the highest boot- merase, 1.5 mM MgCl2, 1X PCR buffer and distilled H2O. strap values were obtained with TPM2+I+G substi- The PCR products were electrophoresed on 1% agarose gel tution model, the NJ tree drawn with this model was (Amresco, Solon, Ohio) prepared in 1X TBE (Tris-Borate- showninFig.1andthebootstrapvaluesobtainedfrom EDTA) buffer. After stained with ethidium bromide, gels ML and MP analyses were also stated on the nodes of were visualized with the GeneGenius Bio imaging system the tree in parenthesis. On the tree, Alstonia scholaris (Syngene, Synoptics Group, Cambridge, UK). from Rauvolfioideae subfamily showed close relation with Apocynum sp. and Nerium oleander from Apoc- Phylogenetic analysis ynoideae subfamily with 99.8% and 99.7% nucleotide Nucleotide sequencings for the loci mentioned above were sequence similarities, respectively. Our Amsonia orien- performed in both directions with the same primers used talis (Rauvolfioideae subfamily) samples (Ams-1, Ams- for the PCR amplifications (see Table 1). Sequencings were made commercially by Macrogen Inc., Korea, using the 2) showed the same 18S rDNA nucleotide sequence and sequencer Roche 454 GS-FLX Titanium. Sequences were appeared as sister to the lineage above. This relation checked and edited using the program SeqMan II module supported with 51% and 53% bootstrap values in MP of the LASERGENE 99 system (Applied Biosystem). The and ML trees, respectively. 18S rDNA nucleotide se- 744 C.T. Gurkanli¨ et al.

Fig. 1. NJ tree showing the phylogenetic relations among 18S rDNA nucleotide sequences obtained in this study (bold on the tree) and the ones obtained from GenBank (regular on the tree). On the tree, only the bootstrap values greater than 50% have been shown and the bootstrap values of MP (Length: 60 steps; CI: 0.917; RI: 0.706; HI: 0.083) and ML trees were stated in parenthesis with this order. Accession numbers and sources of 18S rDNA haplotypes used for phylogeny are as follows – Nucleotide sequences obtained from GenBank: Alstonia scholaris AF107570; Apocynum sp. AF107571; Nerium oleander AF107572 (Albach et al. 2001); Cerbera manghas AY289645 (Shi et al. 2005); Thevetia sp. U42499 (Soltis et al. 1997); Orphium frutescens HQ448763 (Lemaire et al. 2011); Chionophila jamesii U59944 (Nickrent & Duﬀ 1996). Nucleotide sequences obtained in this study: Ams-1 KJ649324; Ams-2 KJ649325. Genetic diversity of Amsonia orientalis 745

Fig. 2. NJ tree showing the phylogenetic relations of 18S-28S rDNA ITS nucleotide sequences obtained in this study (bold on the tree) and the ones obtained from GenBank (regular on the tree). On the tree, only the bootstrap values greater than 50% have been shown and the bootstrap values of MP (Length: 665; CI: 0.638; RI: 0.615; HI: 0.362) and ML trees were stated in parenthesis with this order. Accession numbers and sources of 18S rDNA haplotypes used for phylogeny are as follows – Nucleotide sequences obtained from GenBank: Poacynum pictum DQ451830 (Zhang et al. unpublished); Apocynum cannabinum DQ005966 (Kress et al. 2005); Carisseae carandas HQ386688; Tabernaemontaneae divarica HQ386697; Thevetia neriifolia HQ386699; Val laris solanacea HQ386700 (Sathishkumar et al. unpublished); Nerium oleander HE602380 (Kool et al. 2012); Rauvolﬁa serpentina JQ230964 (Malik & Babbar unpublished); Alstonia scholaris HQ130656; Catharanthus roseus HQ130657; Plumeria alba HQ130658 (Sathishkumar et al. unpublished); Aspidosperma cruentum FJ037792 (Gonzalez et al. 2009); Swetia lurida FJ010795 (Joshi & Li unpublished); Gentianella pilosa AJ580554 (Jang & Greimler unpublished). Nucleotide sequences obtained in this study: Ams-1 KJ649326; Ams-2 KJ649327; Ams-3 KJ649328; Ams-4 KJ649329. 746 C.T. Gurkanli¨ et al.

Table 2. Nucleotide sequence and phylogeny informations of 18S rDNA, 18S–28S rDNA ITS and trnL–F intergenic spacer data sets analyzed in the study.

18S rDNA 18S–28S rDNA–ITS trnL–F intergenic spacer

Sequence Length 1650 650 340 Polymorphic Sites 51 261 21 Parsimony Informative Sites 11 192 12 Model suggested with AIC test GTR+I+G (I: 0.86; G: 0.776) GTR+I+G (I: 0.347; G: 1.247) TVM+G (G: 1.107) Model suggested with BIC tests TPM2+I+G (I: 0.869; G: 0.771) GTR+G (G: 0.404) HKY+G (G: 0.91) quence similarities between A. orientalis and Apoc- Amsonia formed a monophyletic lineage. The trnL– ynoideae members (Apocynum sp. and N. oleander)was F intergenic spacer nucleotide sequence similarities 99.6% and also was 99.5% between A. orientalis and A. amongst Vinceae tribe species mentioned above were scholaris. Two other Rauvolfioideae members Cerbera determined as between 96% and 99.2%. Unexpectedly, manghas and Thevetia sp. appeared as distantly related an Alstonieae tribe species Laxoplumeria baehniana to A. orientalis with 99.3% and 99.4% 18S rDNA nu- also appeared within this lineage and showed nucleotide cleotide sequence similarities, respectively. sequence similarity between 95.7% and 97.8% with the Phylogenetic analysis of our 18S-28S rDNA ITS Vinceae tribe species. Willughbeieae tribe species (Saba nucleotide sequences together with the ones obtained comorensis and Lacmellea aculeata)appearedassister from GenBank (see the legend of Fig. 2) were carried to the Vinceae tribe lineage with 57% and 50% boot- out with 530 aligned nucleotides (see Table 2 for de- strap values in NJ and MP trees, respectively. Interest- tails). MP analysis yielded 6 most parsimonious trees ingly, Amsonia genus which is assumed to be a mem- with 665 steps. In this study, we considered the NJ ber of Vinceae tribe, formed a totally different lineage and ML trees drawn with the GTR+G model which from other Vinceae tribe species and showed close rela- showed the highest bootstrap values (see Table 2 for tion with Pteralyxia kauaiensis which belongs to Alyx- details). On the tree, Rauvolfioideae subfamily species ieae tribe (Fig. 3). The trnL-F intergenic spacer nu- grouped in four main lineages. Vinceae tribe mem- cleotide sequence similarities between Amsonia genus bers Rauvolfia serpentina and Catharanthus roseus and and P. kauaiensis were determined as between 94.6% also Tabernaemontaneae tribe member Tabernaemon- and 93.9% and this relation supported with 54% boot- tana divarica constituted the Lineage-I with nucleotide strap value in MP analysis. All of our A. orientalis sequence similarities between 87.3% and 85.4%. Addi- samples (Ams-1, Ams-2, Ams-3, Ams-4) showed the tionally, the robustness of this group was supported same trnL–F intergenic spacer nucleotide sequence and with bootstrap values more than 80% in NJ, ML and formed a group with another A. orientalis sample MP trees. Unexpectedly, this lineage appeared as sister (AF214262) with 99.6% nucleotide sequence similarity with Apocynoideae subfamily with nucleotide similari- and this relation was supported with 99%, 99% and 98% ties between 85.2% and 78.8%. Our A. orientalis sam- bootstrap values in NJ, MP and ML trees, respectively. ples that showed three different 18S–28S rDNA ITS nu- Another species from Amsonia genus, A. tabernaemon- cleotide sequences, appeared as distantly related with tana, appeared as sister to the A. orientalis group and other Vinceae tribe members (Lineage-I) and formed this relation supported with 99% bootstrap values in the Lineage-II with Carisseae carandas from Carisseae alltrees.ThetrnL–F intergenic spacer nucleotide se- tribe. This relation was supported with 98% and 53% quence similarity between A. orientalis and A. taber- bootstrap values in NJ and MP trees, respectively. naemontana was 97.1%. The overall mean genetic dis- And the nucleotide sequence similarities among these tance among the tribes in Fig. 3 was 0.068 and the two species were between 93% and 92%. Plumeria alba genetic distance between the Vinceae tribe lineage and and Thevetia neriifolia from Plumerieae tribe and also Amsonia genus lineage was 0.073. Alstonia scholaris from Alstoniaeae tribe formed the Lineage-III with nucleotide similarities between 87.7% Discussion and 83.9%. Another Alstonieae tribe member Aspi- dosperma cruentum formed the Lineage–IV by itself. Rauvolfioideae, one of the 5 subfamilies of Apocy- Phylogenetic analysis of our trnL-F intergenic naceae family (order: Gentianales) has comprised of 9 spacer sequences together with the ones obtained from tribes; Alstonieae, Willughbeieae, Tabernaemontaneae, GenBank (see the legend of Fig. 3) were carried out Melodineae, Hunterieae, Plumerieae, Carisseae, Alyx- with 281 aligned nucleotides. MP analysis yielded 12 ieae and Vinceae that mainly delimited by fruit char- most parsimonious trees with 107 steps. In this study, acters. Although this character seems distinctive and we considered the NJ and ML trees created with sufficient for classifying taxa, it has generally accepted HKY+G substitution model since the highest bootstrap that groupings depending on a single character may values were obtained with this model (Fig. 3). be risky especially when the character subjects to high In the tree, all Vinceae tribe species (Ochrosia range of selective pressure during the evolutionary pro- coccinea, Neisosperma nakaiana, Catharanthus roseus, cess as in the case of fruit structures (Endress & Bruyns Petchia ceylanica and Rauvolfia serpentina) except 2000). It is already known that typical fleshy and in- Genetic diversity of Amsonia orientalis 747

Fig. 3. NJ tree showing the phylogenetic relations of trnL–F intergenic spacer nucleotide sequences obtained in this study (bold on the tree) and the ones obtained from GenBank (regular on the tree). On the tree, only the bootstrap values greater than 50% have been shown and the bootstrap values of MP (Length: 107 steps; CI: 0.916; RI: 0.812; HI: 0.084) and ML trees were stated in parenthesis with this order. Accession numbers and sources of 18S rDNA haplotypes used for phylogeny are as follows – Nu- cleotide sequences obtained from GenBank: Amsonia tabernamontana AF214153; Carissa bispinosa AF214172; Catharanthus roseus AF214175; Lacmellea aculeata AF214217; Laxoplumeria baehniana AF214219; Melodinus monogynus AF214226; Neisosperma nakaiana AF214231; Pteralyxia kauaiensis AF214258; Rauvolfia serpentina AF214260; Amsonia orientalis AF214262; Saba comorensis AF214263 (Potgieter & Albert 2001); Hellea rubrostipulata AJ346957 (Razafimandimbison & Bremer 2002); Cerbera manghas EF456094 (Livshultz et al. 2007); Ochrosia coccinea AM295092; Petchia ceylanica AM295093 (Endress et al. 2007). Nucleotide sequences obtained in this study: Ams–1 KJ649330; Ams–2 KJ649331; Ams–3 KJ649332; Ams–4 KJ649333. 748 C.T. Gurkanli¨ et al. dehiscent fruit structure has independently evolved at position of A. orientalis. As a result, congruent with least 3 times in Rauvolfioideae (Endress et al. 1996; the trnL–F intergenic spacer, 18S–28S rDNA ITS phy- Sennblad & Bremer 1996; Sennblad 1997). This means logeny clearly revealed the unrelatedness of A. orien- that, the structure of fruit is not a homologous char- talis with Vinceae tribe. Nevertheless, because of lack- acter for Rauvolfioideae and the existence of taxa that ing of 18S–28S rDNA ITS nucleotide sequences belong- have dehiscent and dry pericarped fruit structure in the ingtoAlyxieaetribemembers in international data family supports the homoplasious nature of the charac- bases we could not test the relationship of A. orien- ter as well (Endress & Bruyns 2000). Congruent with talis and Alyxieae tribe using this molecular marker. this finding, recent phylogenetic studies based on molec- Small sub-unite (SSU) of nuclear ribosomal DNA ular and morphological data revealed some misclassi- (18S rDNA) is an important molecular tool for inferring fications in the tribal concept of Rauvolfioideae and phylogenies among eukaryotic organisms. Although its suggested reclassifications for them (Potgieter & Albert immense importance, still there are not enough 18S 2001; Sennblad & Bremer 2002; Simoes et al. 2007). In rDNA sequences belonging to the members of Apoc- this context, genus Amsonia appeared as one of these ynaceae family in databases to infer an comprehensive conflicting taxa. Due to some morphological data En- phylogeny which may gives tips for true genology of A. dress & Bruyns (2000) placed Amsonia in Vinceae tribe orientalis. which also contains Catharanthus, Kopsia, Ochrosia, As conclusion, our results from trnL–F intergenic Rauvolfia, Vinca and Petchia genera. On the other spacer and 18S–23S rDNA ITS nucleotide sequence hand, some recent publications questioned this classifi- phylogeny together with the results from previous stud- cation. In one of them, Potgieter & Albert (2001) phylo- ies of Potgieter & Albert (2001) and Simoes et al. (2007) genetically analysed the combined data from nucleotide suggests that Amsonia genus is not phylogenetically re- sequences of plastid trnLintronandtrnL–F intergenic lated with Vinceae tribe but can be belonging to Alyx- spacer and also 6 propagule characters of species be- ieae. And because of the obvious incongruences between longing to Apocynaceae s.l. family. As a result, Vinceae the taxonomic relations obtained from molecular phy- tribe genera Ochrosia, Rauvolfia, Vinca, Catharanthus logenetic approach and conventional morphology based and Neisosperma formed a lineage with two genera of concept, the morphological characters that plays role Alstonieae tribe, Laxoplumeria and Tonduzia.Onthe on delimitation of tribes of Rauvolfioideae should be other hand, Amsonia species (A. tabernaemontana and reconsidered. A. orientalis) appeared as distantly related with other Vinceae tribe genera mentioned above and formed a lin- References eage with Thevetia from Plumerieae tribe. And this lineage appeared in the same main lineage with Alyxieae Acemi A., Ozen F. & Kiran R. 2012. Development of an efficient tribe that contains Condylocarpon, Alyxia, Lepiniopsis, callus production protocol for Amsonia orientalis: A critically Plectaneia genera. In a more recent study, Simoes et al. endangered medicinal plant. Eurasia J. Biosci. 6: 105–112. ¨ (2007) had similar results with the combined phyloge- Acemi A., Ozen F. & Kıran R. 2013. In vitro propagation of Am- sonia orientalis Decne. from nodal segments of adult plants. netic analysis of five chloroplast regions (matK, rbcL, Propag. Ornam. Plants. 13: 25–32. rpl16 intron and 3’ trnK intron). In the phylogenetic Akaike H. 1974. A new look at statistical model identification. tree drawn with the Bayesian approach, all Vinceae IEEE T. Automat. Contr. 19: 716–723. tribe genera except for Amsonia formed a lineage which Akyal¸cın H., Ozen¨ F. & Dülger B. 2006. Anatomy, morphology, supported with significant posterior probability values. palynology and antimicrobial activity of Amsonia orientalis Decne. (Apocynaceae) growing in Turkey. Inter. J. Bot. 2: On the other hand, Amsonia species (A. elliptica and 93–99. A. tabernaemontana) formed a distinct lineage with Albach D.C., Soltis P.S., Soltis D.E. & Olmstead R.G. 2001. Alyxieae tribe members (Alyxia reinwardtii, Chilocar- Phylogenetic analysis of Asterids based on sequences of four 88: pus costatus and Condylocarpon isthmicum)andthis genes. Ann. Mo. Bot. Gard. 163–212. > Cantino P.D., Olmstead R.G. & Wagstaff S.J. 1997. A Compari- lineage was supported with significantly high ( 80) son of phylogenetic nomenclature with the current system: A posterior probability values. Our results from trnL-F in- botanical case study. Systematic Biol. 46: 313–331. tergenic spacer nucleotide sequence phylogeny (Fig. 3) Dabine L.E., Turiak G.Y., Nyaradine-Szabady J., Zambo I., perfectly match with the previous findings that indi- Tetenyi P. & Hermecz I. 1986. Determination of secologanin content from shoots of Rhazya orientalis (Decne.) A. DC. cate Amsonia (A. orientalis and A. tabernaemontana) Using the hplc method. Herba. Hungarica 25: 141–150. genus is related with Alyxieae but not with Vinceae Darke R. 2005. Amsonia in cultivation. The Plantsman New Se- tribe. The genetic distance between the Vinceae tribe ries 4: 2. lineage and the Amsonia genus lineage (0.073) which Davis P.H. 1978. Flora of Turkey and East Aegean Islands. 6: is greater than the overall mean distance (0.068) also 161, Univ. Press of Edinburg. Ekim T., Koyuncu M., Vural M., Duman H., Ayta¸cZ.&Adıgüzel supports this suggestion. N. 2000. Türkiye Bitkileri Kırmızı Kitabı. Türkiye Tabiatını Evolutionary comments depending on single gene Koruma Derne˘gi Yayını, Ankara. phylogeny maybe misleading. That is why it is more Endress M.E. & Bruyns P.V. 2000. A revised classification of the reasonable to use as many genes as possible from dif- Apocynaceae s.l. Bot. Rev. 66: 1–56. ferent sources (nuclear, mitochondrial and chloroplast Endress M.E., van der Ham R.W.J.M., Nilsson S., Civeyrel L., Chase M.W., Sennblad B., Potgieter K., Joseph J., Powell M., DNA) to analyze plant taxa. For this reason, we used Lorence D., Zimmerman Y.M. & Albert V.A. 2007. A phy- 18S-28S rDNA ITS region to confirm the phylogenetic logenetic analysis of Alyxieae (Apocynaceae) based on rbcl, Genetic diversity of Amsonia orientalis 749

matK, trnL intron, trnL–F spacer sequences, and morpholog- Razafimandimbison S.G. & Bremer B. 2002. Phylogeny and clas- ical characters. Ann. Mo. Bot. Gard. 94: 1–35. sification of Naucleeae s.l. (Rubiaceae) inferred from molecu- Endress P.K. 1996. Homoplasy in angiosperm flowers, pp. 303- lar (ITS, rBCL, and tRNT–F) and morphological data. Am. 325. In: Sanderson M.J. & Hufford L. (eds), Homoplasy: The J. Bot. 89: 1027–1041. Recurrence of Similarity in Evolution, Academic Pres, San Saitou N. & Nei M. 1987. The neighbor-joining method: A new Diego, USA. method for reconstructing phylogenetic trees. Mol. Biol. Evol. European Council 1979. Convention on the Conservation of Eu- 4: 406–425. ropean Wildlife and Natural Habitats. Appendix I, http:// Sauerwein M., Ishimaru K. & Shimomura K. 1991. Indole alka- conventions.coe.int/Treaty/en/Treaties/Html/104.htm loids of Amsonia elliptica.Phytochem.30: 1153–1155. Gonzalez M.A., Baraloto C., Engel J., Mori S.A., Petronelli P., Sennblad B. & Bremer B. 1996. The familial and subfamilial rela- Riera B., Roger A., Thebaud C. & Chave J. 2009. Identifica- tionships of Apocynaceae and Asclepiadaceae evaluated with tion of Amazonian trees with DNA barcodes. PLoS One 4: rbcL Data. Pl. Syst. Evol. 202: 153–175. E7483. Sennblad B. 1997. Phylogeny of the Apocynaceae s.l. (PhD. Dis- Guindon S. & Gascuel O. 2003. A simple, fast and accurate algo- sertation summary). Acta Universitas Uppsaliensis. Compre- rithm to estimate large phylogenies by maximum-likelihood. hansive Summaries Uppsala Dissertations, Faculty of Science Syst. Biol. 52: 696–704. and Technology, 295, 18 pp, Uppsala. Hall T.A. 1999. BioEdit: a user-friendly biological sequence align- Sennblad B. & Bremer B. 2002. Classification of Apocynaceae ment editor and analysis program for Windows 95/98/NT. s.l. according to a new approach combining Linnaean and Nucl. Acid. S. 41: 95–98. phylogenetic taxonomy. Syst. Biol. 51: 389–409. Kool A., de Boer H.J., Kruger A., Rydberg A., Abbad A., Bjork Shi S., Huang Y., Zeng K., Tan F., He H., Huang J. & Fu Y. L. & Martin G. 2012. Molecular identification of commer- 2005. Molecular phylogenetic analysis of mangroves: indepen- cialized medicinal plants in southern morocco. PLoS One 7: dent evolutionary origins of vivipary and salt secretion. Mol. E39459. Phylogenet. Evol. 34: 159–166. Kress W.J., Wurdack K.J., Zimmer E.A., Weigt L.A. & Janzen Simoes A.O., Livshultz T., Conti E. & Endress M.E. 2007. Phy- D.H. 2005. Use of DNA barcodes to identify flowering plants. logeny and systematics of the Rauvolfioideae (Apocynaceae) Proc. Natl. Acad. Sci. USA 102: 8369-8374. based on molecular and morphological evidence. Ann. Mo. Kron K.A. 1997. Phylogenetic relationships of Rhododendroideae Bot. Gard. 94: 268–297. (Ericaceae). Am. J. Bot. 84: 973–980. Soltis D.E., Soltis P.S., Nickrent D.L., Johnson L.A., Hakn W.J., Lemaire B., Huysmans S., Smets E. & Merckx V. 2011. Rate Hoot S.B., Sweere J.A., Kuzoff R.K., Kron K.A., Chase accelerations in nuclear 18S rDNA of mycoheterotrophic and M.W., Swensen S.M., Zimmer E.A., Chaw S.M., Gillespie parasitic angiosperms. J. Plant. Res. 124: 561–576. L.J., Kress W.K. & Sytsma K.J. 1997. Angiosperm phylogeny Livshultz T., Middleton D.J., Endress M.E. & Williams J.K. inferred from 18S ribosomal DNA sequences. Ann. Mo. Bot. 2007. Phylogeny of Apocynaoideae and the APSA clade Gard. 84: 1–49. (Apocynaceae S.L.). Ann. Mo. Bot. Gard. 94: 324–359. Swofford D.L. 1998. PAUP* Phylogenetic Analysis Using Par- Nickrent D.L. & Duff R.J. 1996. Molecular studies of parasitic simony (*and Other Methods). Version 4 beta 10. Sinauer plants using ribosomal RNA, pp. 28-52. In: Moreno M.T., Associates, Sunderland, Massachusetts. Cubero J.I., Berner D., Joel D., Musselman L.J. & Parker C. Taberlet P., Gielly L., Pautou G. & Bouvet J. 1991. Univer- (eds), Advances in Parasitic Plant Research. sal primers for amplification of three non-coding regions of Olmstead R.G., Depamphilis C.W., Wolfe A.D., Young N.D., chloroplast DNA. Plant Mol. Biol. 17: 1105–1109. Elisons W.J. & Reeves P.A. 2001. Disintegration of the Scro- Tamura K., Stecher G., Peterson D., Filipski A. & Kumar S. phulariaceae. Am. J. Bot. 88: 348–361. 2013. Molecular Evolutionary Genetics Analysis version 6.0. Oz¨ G.C., Yüzba¸sio˘glu E., Erol O. & Uzen¨ E. 2008. In vitro prop- Mol. Biol. Evol. 30: 2725–2729. agation of Amsonia orientalis Decne (Apocynaceae). Afr. J. Temizkan G. & Arda N. 2004. Moleküler Biyolojide Kullanılan Biotechnol. 7: 3638–3641. Yöntemler. Nobel Tıp Kitabevleri, 62, Ýstanbul. Ozen¨ F. 2006. Türkiye’de tükenme tehlikesinde olan bir türün Thompson J.D., Gibson T.J., Plewniak F., Jeanmougin F., Hig- otekolojisi: Amsonia orientalis Decne. (Apocynaceae). BAU¨ gins D.G. 1997. The ClustalX-Windows interface: Flexible Fen. Bil. Enst. Dergisi 8: 1.m strategies for multiple sequence alignment aided by quality Posada D. 2008. jModel test: phylogenetic model averaging. Mol. analysis tools. Nucleic Acids. Res. 25: 4876–4882. Biol. Evol. 25: 1253–1256. Tutin T.G., Heywood V.H., Burges N.A., Moore D.M., Valentine Potgieter K. & Albert V.A. 2001. Phylogenetic relationships D.H., Walters S.M., Webb D.A. 1968. Flora Europaea. Vol. within Apocynaceae S.L. based on trnL intron and trnL–F 3, Cambridge Univ. Press, Cambridge. spacer sequences and propagule characters. Ann. Mo. Bot. White T.J., Burns T., Lee S., Taylor J. 1990. Amplification and Gard. 88: 523–549. direct sequencing of fungal ridosomal RNA gene for phyloge- Rahman A.U. & Zaman K. 1988. Studies on the alkaloids of netics, pp 315-322. In: Innis M.A., Gelfaund D.H., Sninsky Rhazya stricta.Phytochem.27: 1926–1928. J.J. & White T.J. (eds), PCR protocols: a guide to methods Rahman A.U., Qureshi M.M., Zaman K., Malik S. & Ali S.S. and applications, Academic Pres, San Diego, CA, USA. 1989. The alkaloids of Rhazya stricta and R. orientalis-a review. Fitote. 60: 291–322. Received November 1, 2013 Accepted February 14, 2014