Firenze University Press www.fupress.com/caryologia Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: N. Nazar, J.J. Clarkson, D. Goyder, E. Kaky, T. Mahmood, M.W. Chase (2019) Phylogenetic relationships in Apocynaceae based on nuclear PHYA and plastid trnL-F sequences, with a focus on tribal relationships. Caryologia 72(1): 55-81. doi: 10.13128/ cayologia-251 Received: 24th July 2018 Accepted: 18th October 2018 Published: 10th May 2019 Copyright: © 2019 N. Nazar, J.J. Clarkson, D. Goyder, E. Kaky, T. Mahmood, M.W. Chase. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Phylogenetic relationships in Apocynaceae based on nuclear PHYA and plastid trnL-F sequences, with a focus on tribal relationships Nazia Nazar1,2,3,*, James J. Clarkson3, David Goyder3, Emad Kaky4,5, Tariq Mahmood2, Mark W. Chase3 1 Department of Plant Sciences, University of Nottingham, LR12 5RD, United Kingdom of Plant Science, Quaid-i-Azam University Islamabad, Pakistan 3 Royal Botanic Gardens Kew, Richmond, Surrey, TW9 3AE, United Kingdom 4 Kalar Technical Institute, Sulaimani Polytechnic University, Iraq 5 Life Science School, University of Nottingham, NG7 2RD, United Kingdom * Corresponding author, E-mail: Nazia.Nazar1@nottingham.ac.uk 2 Department Abstract. To date, most molecular phylogenetic studies of Apocynaceae have been based on plastid DNA regions or nuclear ribosomal DNA. In this study, we used part of the PHYA (phytochrome A) exon, a low-copy nuclear gene, and combined it with the trnL-F region (intron and spacer) to investigate placement of Periplocoideae, intergeneric relationships of Asclepiadoideae and relationships within Rauvolfioideae. We included 112 taxa representing most major clades of Apocynaceae. The study confirms that both subfamilies Apocynoideae and Rauvolfioideae are paraphyletic and that Periplocoideae are nested within Apocynoideae. The APSA clade (Apocynoideae, Periplocoideae, Secamonoideae and Asclepiadoideae) is strongly supported here, but the crown clade of Apocynaceae (comprised of subfamilies Asclepiadoideae, Secamonoideae, Periplocoideae and Echiteae, Mesechiteae, Odontadenieae and Apocyneae of Apocynoideae) has only moderate support. The present study places Periplocoideae as part of the sister group to the rest of the crown clade. This contrasts with results from the previous only PHYA and plastid marker–based studies in which periplocoids appeared as sister to a clade comprising Baisseeae (Apocynoideae) plus Secamonoideae and Asclepiadoideae. Old World Cynanchinae form a well-supported clade with the New World MOG (Metastelmatinae, Oxypetalinae and Gonolobinae) tribes rather than with the largely Old World. Asclepiadinae and Tylophorinae, as suggested by earlier studies. In our combined analyses, resolution among most groups is improved as compared to previous plastid-only analyses. Keywords. Apocynaceae, Asclepiadeae, Baisseeae, Periplocoideae, Phylogeny, Phytochrome A. Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(1): 55-81, 2019 ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/cayologia-251 56 Nazia Nazar et al. 1. INTRODUCTION Since Endress and Bruyns (2000), Apocynaceae sensu lato have been investigated with molecular data, mostly plastid, to evaluate relationships among various groups proposed in their classification (Potgieter and Albert 2001; Rapini et al. 2003 and 2006; Livshultz et al. 2007; Simões et al. 2007; Livshultz 2010). Subfamilies Rauvolfioideae and Apocynoideae have been recovered as non-monophyletic (e.g., Sennblad et al. 1998; Potgieter and Albert 2001; Livshultz et al. 2007; Simões et al. 2004). In more recent classifications (Endress et al. 2007a; Simões et al. 2007; Endress et al., 2014)), ten tribes have been proposed in Rauvolfioideae: Tabernaemontaneae, Alstonieae, Alyxieae, Carisseae, Hunterieae, Melodineae, Plumerieae, Vinceae, Willughbeieae, Aspidospermeae and Amsonieae. Monophyly of most tribes in the subfamily has always remained suspect (Potgieter and Albert 2001; Sennblad and Bremer 2002); however, in the recent phylogenetic analysis by Simões et al. (2007), six clades (out of nine tribes sensu Endress and Bruyns 2000) were identified in Rauvolfioideae, which could be referred to Willughbeieae, Tabernaemontaneae, Hunterieae, Alyxieae, Plumerieae, and Carisseae, while Melodineae, Alstonieae and Vinceae were polyphyletic Similarly, in Apocynoideae, five tribes were recognized by Endress and Bruyns (2000): Wrightieae, Malouetieae, Apocyneae, Echiteae and Mesechiteae. Since this publication, five more tribes, Nerieae (Sennblad and Bremer 2002), Odontadenieae (Endress et al. 2007a), Baisseeae (Endress et al. 2007a) and Rhabdadenieae (Endress et al., 2014) have been recognized in this subfamily. Baisseeae are considered a sister group of the milkweeds (Asclepiadoideae-Secamonoideae) rather than subfamily Periplocoideae on the basis of various phylogenetic studies (Sennblad and Bremer 1996, 2000 and 2002; Potgieter and Albert 2001; Lahaye et al. 2005; Livshultz et al. 2007). Also phylogenetic analyses firmly support placement of Periplocoideae in the APSA (Apocynoideae, Periplocoideae, Secamonoideae, Asclepiadoideae) clade (Judd et al. 1994; Sennblad and Bremer 1996, 2002; Civeyrel et al. 1998; Potgieter and Albert 2001). Periplocoideae were recognized until the last decades of the 20th century as members of Asclepiadaceae (Kunze 1990 and 1993; Venter et al. 1990; Dave and Kuriachen 1991; Liede and Kunze 1993; Nilsson et al. 1993; Swarupanandan et al. 1996). In subfamily Asclepiadoideae five tribes have been recognized: Asclepiadeae, Ceropegieae, Marsdenieae, Fockeeae and Eustegieae (Endress et al. 2007a; Endress et al., 2014)). Endress and Bruyns (2000) delimited the tribes of Asclepiadoideae on the basis of the orientation of pollinia in pollen sacs: upwardly directed in Ceropegieae-Marsdenieae and pendulous in Asclepiadeae. Erect pollinia are considered a primitive character and also found in Secamonoideae and Fockeeae (Kunze 1993). Recognition of Fockeeae as a tribe separate from Marsdenieae in Asclepiadoideae by Kunze et al. (1994) is disputed by Endress and Bruyns (2000) due to insufficient taxon sampling in Marsdenieae. The isolated basal position of Fockeeae has been confirmed by subsequent phylogenetic analyses (Potgieter and Albert, 2001; Rapini et al., 2003; Verhoeven et al., 2003). Rapini et al. (2003) identified three main clades in Asclepiadeae that could be referred to as subtribes: Astephaninae and two multiple subtribe clades, ACTG (Asclepiadinae, Cynanchinae, Tylophorinae and Glossonematinae) and MOG. Subtribe Glossonematinae was later dissolved by Liede et al. (2002), Glossonema and Odontanthera were included in Cynanchineae and a third genus of the tribe Solenostemma belongs to none of the subtribes presently recognized in the Asclepiadeae (Endress et al. 2007a). Cynanchinae within the ACT clade are divided into a monophyletic Old World succulent group (containing Malagasy Cynanchum species), but New World sections of the subtribe are polyphyletic (Liede and Taüber 2002; Rapini et al. 2006). Furthermore, cladistic analyses of Goyder et al. (2007) and Fishbein et al. (2011) have emphasized that generic delimitation of subtribe Asclepiadineae is problematic. These studies concluded that Asclepiadoideae still needs further investigation to identify monophyletic groups and find morphological characters by which to recognize them. To date, almost all broader molecular phylogenetic studies of Apocynaceae have been based on plastid DNA, either alone or in combination with morphological datasets. Livshultz (2010) presented a study using the lowcopy nuclear gene, PHYA (phytochrome A, exon 1) for a number of Apocynaceae groupings. Her approach proved useful in describing the status of tribe Baisseeae as the sister group of the milkweeds (i.e. Asclepiadoideae and Secamonoideae) rather than Periplocoideae. However, there are still many other areas within the family where resolution/support is low. For this study, we sequenced the same region of PHYA 1 (first exon) as in Livshultz (2010) for a broader dataset sampled across the family and combined these data with the widely sampled plastid trnL-F (intron/spacer) region. Our main goals are to: 1) further improve resolution in the primary clades of Asclepiadoideae (one of the groups from the crown clade defined by Livshultz 2010), 2) examine the position of Periplocoideae in Apocynaceae and 3) improve resolution within the subfamily Rauvolfioideae. 57 Phylogenetic relationships in Apocynaceae 2. MATERIALS AND METHODS 2.1. DNA extraction and amplification Taxa of Apocynaceae used here were either collected from the field in Pakistan or sampled from the DNA Bank at The Royal Botanic Gardens, Kew (https://dnabank.science.kew.org/). A complete list of taxa including voucher details, taxonomic treatment and provenance are provided in Table 1. Total genomic DNA was extracted from silica-dried field collections following the 2 ×CTAB protocol of Richard (1997) with modifications described by Nazar and Mahmood (2010). DNA from herbarium specimens was isolated by pulverising dry material in tubes containing plastic beads (using a Genogrinder 2010, SPEX CertiPrep Ltd, Harrow, Middlesex, UK) and then following a modified Doyle and Doyle (1987) 2 ×CTAB method. To isolate DNA from these samples, we used precipitation in chilled ethanol (-20 °C) for at least 24 hr and then resuspended in 1.55 g/ml caesium chloride/ethidium bromide. Samples were then purified using a density gradient, followed by removal of the ethidium and caesium chloride with butanol/dialysis and storage in Tris EDTA. Primers (PHYA 2059F, 2745F, 2971R, 3560R) used to amplify the first exon of PHYA are those of Livshultz (2010). The region was amplified using ReddyMix PCR Mastermix (Thermo Scientific, Epsom, Surrey, UK) in a 25 μl reaction volume. Degraded DNA (and/ or possibly impure DNA), in some samples caused problems for amplification using the ReddyMix PCR Mastermix. To amplify the target regions from degraded DNA, especially from herbarium samples, Platinum® taq DNA polymerase (Invitrogen) was used. The reaction mix (25 μl total volume) consisted of 2.5 μl 10 ×PCR buffer, 2 μl MgCl2 (50 mM/ml), 1 μl of BSA (50 mg/ml), 0.6 μl of each primer (0.1 ng/μl), 0.2 μl of 5 U/ μl of Platinum taq DNA polymerase, made up to volume with nuclease free water. The following PCR program was used for amplification: initial denaturation at 94 °C for 2 min, followed by 35 cycles of denaturation at 94 °C for 20 sec, annealing at 50 °C for 30 sec and extension at 72 °C for 2 min. A final extension was carried out at 72 °C for 7 min. Higher annealing temperatures reduced yields, and we found that using 50 °C did not cause amplification of more than one region (i.e. the sequencing reactions were free from obvious polymorphisms. PCR products were cleaned using NucleoSpin® Extract II mini-columns (Macherey-Nagel, Duren, Germany) following the manufacturer’s protocols. For cleaning of cycle sequencing products, we used precipita- tion in ethanol (using EDTA). Samples were sequenced on an ABI 3730 automated sequencer according to the manufacturer’s protocols (Applied Biosystems, Inc.). Electropherograms were edited and assembled using Sequencher version 4.5 (Gene Codes, Ann Arbor, Michigan, USA); these sequences were easily aligned by eye in PAUP following the suggestions of Kelchner (2000). 2.2. Data analysis Incongruence between trnL-F and PHYA results was assessed by looking for contradictory clades in both PHYA and trnL-F Bayesian and parsimony trees by following the same criteria regarding bootstrap support used by Livshultz (2010). Several studies have shown that the incongruence length test (ILD) proposed by Ferris et al. (1994) is too sensitive and unreliable for detection of incongruence (Darlu and Lecointre, 2002), so we did not use it here and have instead relied on inspection for well supported but different tree topologies as the basis for assessing incongruence (which we did not observe here). For the Bayesian results, we considered posterior probabilities (PP) > 0.95 as wellsupported; < PP 0.95 is considered weakly supported and not indicative of incongruence. For the parsimony results, we considered bootstrap percentages (BP) of 80 as the cut-off for assessing incongruence. The separate analyses did not produce any clear evidence for incongruent clades, so we produced combined analyses of trnL-F and PHYA. The combined dataset (trnL-F and PHYA) comprises of 112 sequences — 47 sequences from study of Livshultz (2010) are included (Table 2). Phylogenetic analyses were performed using maximum parsimony (MP; PAUP version 4.0b10, Swofford 2002) and Bayesian methods (Mr. Bayes ver.3.1, Huelsenbeck and Ronquist, 2001). Gaps were treated as missing data. For the parsimony analyses, the combined data matrix was analysed using tree bisection-reconnection (TBR) swapping and 1000 replicates of random taxon-addition, holding 10 trees at each step to reduced time searching islands of equally parsimonious trees. DELTRAN character optimization was used to illustrate branch lengths (due to reported errors with ACCTRAN optimization in PAUP version 4.0b). For Bayesian analysis, a HKY85 model was specified in which all transitions and transversions have potentially different rates. More complex models were also tested, but these yielded the same tree with similar PP. The analysis was performed with 500,000 generations of Markov chain Monte Carlo with equal rates and a sampling frequency of 10. Microsoft excel was used to plot 58 Nazia Nazar et al. Table 1. A list of the samples from Pakistan and The Royal Botanic Gardens Kew, London with vouchers information and place of collection are given. Taxa Asclepiadoideae – Asclepiadeae: Metastelmatinae Blepharodon lineare (Decne.) Decne. Asclepiadoideae – Asclepiadeae: Oxypetalinae Araujia sericifera Brot. Funastrum clausum (Jacq.) Schltr. Oxypetalum capitatum Mart. Philibertia discolor (Schltr.) Goyder Philibertia lysimachioides (Wedd.) T. Mey. Asclepiadoideae – Asclepiadeae: Gonolobinae Matelea pseudobarbata (Pitter) Woodson Asclepiadoideae – Asclepiadeae: Asclepiadinae Calotropis procera (Aiton) W. T. Aiton Kanahia laniflora (Forssk.) R. Br. Pergularia daemia (Forssk.) Chiov. Pergularia tomentosa L. Stenostelma corniculatum (E. Mey.) Bullock Xysmalobium parviflorum Harv. ex Scott-Elliot Asclepiadoideae – Asclepiadeae: Cynanchinae Cynanchum viminale (L.) Bassi Cynanchum jacquemontianum Decne. Cynanchum obtusifolium L.f. Asclepiadoideae – Asclepiadeae: Tylophorinae Tylophora hirsuta (Wall.) Wight Unplace genus Oxystelma esculentum (L. f.) Sm. Asclepiadoideae – Asclepiadeae: Astephaninae Eustegia minuta (L. F.) N. E. Br. Oncinema lineare (L. F.) Bullock Schubertia grandiflora Mart. Asclepiadoideae – Marsdenieae Dischidia lanceolata Decne. Dregea abyssinica K.Schum. Gymnema sylvestre (Retz.) Schultz. Hoya finalasonii Wight Hoya manipurensis Deb. Marsdenia carvalhoi Morillo & Carnevali Rhyssolobium dumosum E. Mey. Staphanotis floribunda Brongn. Wattakaka volubilis (Linn.f.) Stapf. Asclepiadoideae – Ceropegieae Caralluma tuberculata N.E. Br. Ceropegia sandersonii Decne.ex Hook. Duvalia polita N. E. Br. Boucerosia indica Dalzell Leptadenia pyrotechnica Neoschumannia kamerunensis Quaqua incarnata (L. f.) Bruyns Heterostemma acuminatum Decne. Voucher detail Country Regions sequenced Forzza et al. 2027 Argentina trnL-F and PHYA Forster 7656 Mello- Silva et al. 1919 Mello- Silva et al. 1924 Mello- Silva et al. 1887 Mello- Silva et al. 1886 Australia Argentina Argentina Argentina Argentina PHYA PHYA PHYA PHYA PHYA M. Endress 97-08 Costa Rica PHYA Naz001* Goyder et al. 3931 Naz024* Naz012* Balkwill 10908 Killick & Vahrmeijer 3658 Pakistan Tanzania Pakistan Pakistan South Africa South Africa trnL-F and PHYA PHYA PHYA trnL-F and PHYA PHYA PHYA Chase 731 Naz010* P. Bruyns Vch ** Pakistan South Africa PHYA trnL-F and PHYA PHYA Naz014* Pakistan trnL-F and PHYA Naz020* Pakistan trnL-F and PHYA P. Bruyns 4357 P. Bruyns Vch? Irwin et al. 31285 South Africa South Africa Brazil PHYA PHYA PHYA Chase 734 Goyder et al. 3918 Chase 3902 Chase 17138 Chase 733 Chase 3904 P. V. Bruyns 3948 Chase 732 Naz006* Indonesia Tanzania India India Thailand Brazil South Africa Senegal Pakistan PHYA PHYA trnL-F and PHYA PHYA PHYA trnL-F and PHYA PHYA trnL-F and PHYA trnL-F and PHYA Naz019* Chase 17507 Kew Chase 2861 Naz018* Chase 3903 Chase 9818 Forster 5090 Pakistan ** ** India Pakistan Cameroon South Africa ** trnL-F and PHYA PHYA PHYA PHYA trnL-F and PHYA PHYA PHYA PHYA 59 Phylogenetic relationships in Apocynaceae Taxa Secamonoideae Secamone alpini Schult. Periplocoideae Cryptolepis buchananii Roemer & Schult. Cryptolepis decidua (Planch. ex Benth.) N. E. Br. Hemidesmus indicus (L.) R.Br. ex Schult. Periploca aphylla Decne. Raphionacme hirsuta (E.Mey.sec.N.E.Brown) R.A.Dyer Schlechterella abyssinicum (Chiov.) Venter & R. L. Verh. Apocynoideae - Malouetieae Kibatalia gitingensis (Elmer) Woodson Pachypodium leallii Welw. Apocynoideae - Nerieae Adenium obesum (Forssk.) Roem. & Schult. Nerium oleander L. Apocynoideae - Apocyneae Beaumontia grandiflora (Roxb.) Wall. Trachelospermum jasminoides (Lindl.) Lem. Apocynoideae - Echiteae Fernaldia pandurata (A.DC.) Woodson Rauvolfioideae - Wrightieae Pleioceras barteri Baill. Rauvolfioideae - Carisseae Carissa spinarum L. Rauvolfioideae - Plumerieae Anechites nerium Urb. Skytanthus acutus Meyen Thevetia peruviana (Pers.) K. Schum. Rauvolfioideae - Vinceae Amsonia hurbritchii Woodson Petchia ceylanica (Wight) Livera Rauvolfia serpentina (L.) Benth. Rhazya orientalis A.DC. Vinca major L. Rauvolfioideae - Tabernaemontaneae Tabernaemonta divericata (L.) R. Br. exRoem.Schult Rauvolfioideae - Hunterieae Gonioma kamassi E.Mey. Rauvolfioideae - Alyxieae Alyxia buxifolia R. Br. Rauvolfioideae - Alstonieae Alstonia scholaris (L.) R. Br. Voucher detail Country Regions sequenced P. Bruyns Vch South Africa trnL-F and PHYA Naz002* P. V. Bruyns s.n. (east of Fish R.) Chase 725 Naz004* CFR 15 Chase 720 Pakistan trnL-F and PHYA Namibia trnL-F and PHYA Tamil Nadu Pakistan South Africa Ethopia PHYA trnL-F and PHYA PHYA trnL-F and PHYA Liede 3268 Chase 735 ** South Africa trnL-F and PHYA trnL-F and PHYA Chase 727 Naz015* Somalia Pakistan trnL-F and PHYA trnL-F and PHYA Naz008* Naz022* Pakistan Pakistan trnL-F and PHYA trnL-F and PHYA M Endress, Zurich ** PHYA Endress, P. 99-10 Ivory Coast PHYA Naz017* Pakistan trnL-F and PHYA Bremer et al. 3386 UPS M. Endress, Zurich Naz013* ** ** Pakistan PHYA PHYA trnL-F and PHYA Chase 19252 R. Olmor s. n Naz003* M. Endress s.n. Naz025* USA Germany Pakistan Zurich Pakistan trnL-F and PHYA trnL-F and PHYA trnL-F and PHYA trnL-F and PHYA trnL-F Chase 5571 Bangladesh PHYA Chase 5806 South Africa trnL-F and PHYA Smith, R.J. (RJS202) Australia PHYA Naz007* Pakistan trnL-F and PHYA *Vouchers specimens are preserved in the Plant Biochemistry and Molecular Biology Laboratory of Quaid-i-sAzam University, Islamabad, Pakistan. ** Information not present in Kew’s databases. generation number against InL to find the ‘burn in’. Trees of low PP were deleted, and all remaining trees were imported into PAUP 4.0b10. A Bayesian tree (i.e., a majority-rule consensus tree) was produced showing frequencies of all observed bi-partitions (i.e. the posterior probabilities for each node). 60 Nazia Nazar et al. Table 2. A list of taxa with GenBank accession numbers used in trnL-F and PHYA analyses, sequenced in present study, previously published in Rapini et al. (2003), Sennblad and Bremer (1998) and Livshultz (2010) with updated nomenclature (Endress et al., 2014). Species Name Adenium obesum (Forssk.) Roem. & Schult. Aganosma wallichii G. Don. Alstonia scholaris (L.) R. Br. Alyxia buxifolia R. Br. Amsonia hurbritchii Woodson Anechites nerium Urb. Angadenia berteroi (A.DC.) Miers Anodendron paniculatum A. DC. Apocynum androsaemifolium L. Araujia sericifera Brot. Artia balansae (Baillon) Pichon ex Guillaumin Baissea multiflora A. DC. Beaumontia grandiflora (Roxb.) Wall. Blepharodon linere (Decne.) Decne. Boucerosia indica Dalzell Calotropis procera (Aiton) W. T. Aiton Caralluma tuberculata N.E. Br. Carissa spinarum L. Ceropegia sandersonii Decne.ex Hook. Chonemorpha fragrans (Moon) Alston Cleghornia malaccensis (Hook. f.) King & Gamble Cryptolepis buchananii Roemer & Schult. Cryptolepis decidua (Planch. ex Benth.) N. E. Br. Cycladenia humilis Bentham Cynanchum jacquemontianum Decne. Cynanchum obtusifolium L.f. Cynanchum viminale (L.) Bassi Dischidia lanceolata Decne. Dregea abyssinica K.Schum. Duvalia polita N. E. Br. Echites umbellatus Jacq. Elytropus chilensis Müll. Arg. Epigynum cochinchinense (Pierre) D.J. Middleton Eustegia minuta (L.f.) N.E.Br. Fernaldia pandurata (A.DC.) Woodson Finlaysonia insularum (King & Gamble) Venter Fockea edulis K. Schum. Forsteronia guyanensis Müll.Arg. Funastrum clausum (Jacq.) Schltr. Gonioma kamassi E.Mey. Gymnanthera oblonga (Burm. f.) P.S. Green Gymnema sylvestre (Retz.) Schultz. Hemidesmus indicus (L.) R.Br. ex Schult. Heterostemma acuminatum Decne. Hoya finalasonii Wight Hoya manipurensis Deb. Ichnocarpus frutescens R. Br. Kanahia laniflora (Forssk.) R. Br. Kibatalia gitingensis (Elmer) Woodson PHYA trnL-F LT972249 GU901319 LR027092 LT972244 LR027376 LT972245 GU901358 GU901327 GU901328 LT972246 GU901372 GU901330 LR027094 LR026999 HF969013 LT972247 LT972248 LR027375 HF969012 GU901332 GU901333 HG004619 HG004618 GU901367 LR027368 HF969010 HG004632 LR028004 HG004620 HF969009 GU901387 GU901398 GU901340 LR027089 GU901329 GU901341 LR027374 GU901359 HG004645 HG004623 GU901348 HG004637 HG004617 HE805526 EF456127 HE805532 AF214152 HG004636 LR027373 GU901356 HG004642 HG004629 Subtribe Ichnocarpinae AM295087 Thevetiinae EF456246 EF456194 Papuechitinae AF214308 Apocyinae AJ704332 Oxypetalinae EF456142 EF456199 HE805527 Beaumontiinae AY163668 Metastelmatinae AF214202 HE805509 Asclepiadinae HE805510 HE805533 AF214179 EF456132 Chonemorphinae EF456241 Apocyinae HE805522 HE805523 EF456140 HE805511 Cynanchinae AJ428692 Cynanchinae AJ290912 Cynanchinae AJ488374 EF456186 EF456171 EF456147 AJ410207 EF456209 EF456105 AF214199 EF456153 AJ428794 HE805535 EF456106 HE805512 DQ916877 AJ574827 AF214227 EF456136 AY163695 HE805528 Ichnocarpinae Eustegieae Oxypetalinae Tribe Subfamily Nerieae Apocyneae Alstonieae Alyxieae Amsonieae Plumerieae Echiteae Apocyneae Apocyneae Asclepiadeae Echiteae Baisseeae Apocyneae Asclepiadeae Ceropegieae Asclepiadeae Ceropegieae Carisseae Ceropegieae Apocyneae Apocyneae Apocynoideae Apocynoideae Rauvolfioideae Rauvolfioideae Rauvolfioideae Rauvolfioideae Apocynoideae Apocynoideae Apocynoideae Asclepiadoideae Apocynoideae Apocynoideae Apocynoideae Asclepiadoideae Asclepiadoideae Asclepiadoideae Asclepiadoideae Rauvolfioideae Asclepiadoideae Apocynoideae Apocynoideae Periplocoideae Periplocoideae Apocynoideae Asclepiadoideae Asclepiadoideae Asclepiadoideae Asclepiadoideae Asclepiadoideae Asclepiadoideae Apocynoideae Apocynoideae Apocynoideae Asclepiadoideae Apocynoideae Periplocoideae Asclepiadoideae Apocynoideae Asclepiadoideae Rauvolfioideae Periplocoideae Asclepiadoideae Periplocoideae Asclepiadoideae Asclepiadoideae Asclepiadoideae Apocynoideae Asclepiadoideae Apocynoideae Odontadenieae Asclepiadeae Asclepiadeae Asclepiadeae Marsdenieae Marsdenieae Ceropegieae Echiteae Odontadenieae Apocyneae Asclepiadeae Echiteae Fockeeae Mesechiteae Asclepiadeae Hunterieae Marsdenieae Ichnocarpinae Asclepiadinae Ceropegieae Marsdenieae Marsdenieae Apocyneae Asclepiadeae Malouetieae 61 Phylogenetic relationships in Apocynaceae Species Name PHYA trnL-F Laubertia contorta (Mart.& Galeotti) Woodson Leptadenia pyrotechnica Mandevilla boliviensis Decne. Marsdenia carvalhoi Morillo & Carnevali Marsdenia glabra Costantin Matelea pseudobarbata (Pitter) Woodson Microloma tenuifolium (L.) Kuntze Motandra guineensis A. DC. Neoschumannia kamerunensis Schltr. Nerium oleander L Odontadenia perrotteti (A. DC.) Woodson Oncinema lineare (L. F.) Bullock Oncinotis tenuiloba Stapf Orthanthera jasminiflora Schinz Oxypetalum capitatum Mart. Oxystelma esculentum (L. f.) Sm. Pachypodium leallii Welw. Papuechites aambe Markgr. Parameria laevigata (Juss.) Mold. Parsonsia eucalyptophylla F. Muell. Peltastes isthmicus Woodson Pentalinon luteum (L.) B.F. Hansen & R.P. Wunderlin Pergularia daemia (Forssk.) Chiov. Pergularia tomentosa L. Periploca aphylla Decne. Petchia ceylanica (Wight) Livera Petopentia natalensis (Schltr.) Bullock Philibertia discolor (Schltr.) Goyder Philibertia lysimachioides (Wedd.) T. Mey. Phyllanthera grayi (P.I. Forst.) Venter Pinochia corymbosa (Jacq.) M.E. Endress & B.F. Hansen Pleioceras barteri Baill. Prestonia lagoensis (Müll. Arg.) Woodson Quaqua incarnata (L. f.) Bruyns Raphionacme hirsuta (E.Mey.sec.N.E.Brown) R.A.Dyer Rauvolfia serpentina (L.) Benth. Rhabdadenia biflora Müll.Arg. Rhazya orientalis A.DC. Rhodocalyx rotundifolius Müll. Arg. Rhyssolobium dumosum E. Mey. Schlechterella abyssinicum (Chiov.) Venter & R. L. Verh. Schubertia grandiflora Mart. Secamone alpini Schult. Secamone elliptica R. Br. Secondatia densiflora A. DC. Sindechites chinensis Oliv. & Tsiang: Skytanthus acutus Meyen Staphanotis floribunda Brongn. Stenostelma corniculatum (E. Mey.) Bullock Stipecoma peltigera Müll. Arg. Tabernaemonta divericata (L.) R. Br. exRoem.Schult GU901375 HG004614 GU901343 LR027091 LR027370 HG004621 LR027371 GU901361 HG004613 LR027093 GU901335 LR027090 GU901368 EF456180 HE805513 EF456134 DQ334521 EF456114 HG004644 HF969014 HG004628 GU901370 GU901371 GU901380 GU901324 HG004631 HG004641 HG004640 HG004616 HG004624 GU901376 LR027369 HG004643 GU901377 GU901378 LR027096 GU901337 HG004612 HG004615 HG004625 LR028003 GU901396 HG004635 HG004611 HG004622 LR027095 GU901389 GU901339 GU901393 HG004627 HG004634 HG004639 GU901394 AJ410230 EF456210 AJ410054 HE805529 EF456211 AJ428827 EF456141 AJ574827 AY163710 AJ290887 HE805530 EF456189 EF456197 EF456215 EF456129 EF456191 JN205300 HE805514 HE805524 AM295093 EF456107 AY163700 AJ290900 EF456103 EF456167 EF456251 EF456237 AJ488455 AJ581825 HE805539 Subtribe Gonolobinae Astephaninae Astephaninae Oxypetalinae Papuechitinae Urceolinae Asclepiadinae Asclepiadinae Catharanthinae Oxypetalinae Oxypetalinae Rauvolfiinae AM295095 EF456238 AM233378 HE805525 AJ428827 Astephaninae HE805519 EF456116 EF456228 EF456244 Amphineuriinae AF214269 Thevetiinae HE805517 AY163722 Asclepiadinae EF456193 AF214399 Tribe Subfamily Echiteae Ceropegieae Mesechiteae Marsdenieae Marsdenieae Asclepiadeae Asclepiadeae Baisseeae Ceropegieae Nerieae Odontadenieae Asclepiadeae Baisseeae Ceropegieae Asclepiadeae Asclepiadeae Malouetieae Apocyneae Apocyneae Echiteae Echiteae Echiteae Asclepiadeae Asclepiadeae Apocynoideae Asclepiadoideae Apocynoideae Asclepiadoideae Asclepiadoideae Asclepiadoideae Asclepiadoideae Apocynoideae Asclepiadoideae Apocynoideae Apocynoideae Asclepiadoideae Apocynoideae Asclepiadoideae Asclepiadoideae Apocynoideae Apocynoideae Apocynoideae Apocynoideae Apocynoideae Apocynoideae Asclepiadoideae Asclepiadoideae Periplocoideae Vinceae Rauvolfioideae Periplocoideae Asclepiadeae Asclepiadoideae Asclepiadeae Asclepiadoideae Periplocoideae Odontadenieae Apocynoideae Wrightieae Apocynoideae Echiteae Apocynoideae Ceropegieae Asclepiadoideae Periplocoideae Vinceae Rauvolfioideae Rhabdadenieae Apocynoideae Vinceae Rauvolfioideae Echiteae Apocynoideae Marsdenieae Asclepiadoideae Periplocoideae Asclepiadeae Asclepiadoideae Secamonoideae Secamonoideae Odontadenieae Apocynoideae Apocyneae Apocynoideae Plumerieae Rauvolfioideae Marsdenieae Asclepiadoideae Asclepiadeae Asclepiadoideae Echiteae Apocynoideae Tabernaemontaneae Rauvolfioideae 62 Nazia Nazar et al. Species Name Temnadenia odorifera (Vell.) J.F. Morales: Thevetia peruviana (Pers.) K. Schum. Thyrsanthella difformis (Walter) Pichon Toxocarpus villosus(Blume) Decne Trachelospermum jasminoides (Lindl.) Lem. Tylophora hirsuta (Wall.) Wight Urceola lucida Benth. & Hook. f. Vallaris solanacea (Roth) O. Kuntze Vinca major L. Wattakaka volubilis (Linn.f.) Stapf. Xysmalobium parviflorum Harv. ex Scott-Elliot Zygostelma benthamii Baill PHYA trnL-F GU901373 LR027097 GU901391 GU901399 HG004630 EF456179 GU901400 GU901401 LR028005 HF969011 HG004638 GU901404 3. RESULTS 3.1. Incongruence As mentioned above, well-supported clades incongruent between the Bayesian and parsimony results were not observed. In some cases, trnL-F provided higher support for certain clades than did PHYA, but in other cases the reverse was true. Overall resolution produced by trnL-F for both Bayesian and parsimony analyses was lower than for PHYA. We will not describe the results of the separate analyses (they are highly similar), but we do include figures here for comparison (Supplementary data (Figures 1a, 1b, 2a, 2b)); we confine our discussion to only the combined results because the individual gene trees are congruent and the combined results are better resolved and have higher support. 3.2. Combined trnL-F and PHYA analyses The dataset comprises 112 taxa and 2325 characters, of which 1400 are contributed by PHYA and 975 from trnL-F. In the parsimony analysis, 701 characters (479 from PHYA and 222 from trnL-F) proved to be parsimony informative. Analysis produced 13960 equally most-parsimonious trees with 3284 steps and a consistency index of 0.49 and retention index of 0.71. Mr Modeltest indicated that the best fit model was a general time reversible model with an alpha parameter for the shape of the gamma distribution to account for rate heterogeneity among sites (GTR+G+I). A burn in period of 2 106 generations per run was removed. The Bayesian tree (Figure 2) generally depicts more resolved groups as compared to the parsimony tree (Figure 1). Rauvolfioideae and Apocynoideae are non-monophyletic, but the subfamilies of the traditional Asclepiadaceae are strong- Subtribe Tribe Subfamily Thevetiinae Echiteae Plumerieae Odontadenieae Apocynoideae Rauvolfioideae Apocynoideae Secamonoideae Apocynoideae Asclepiadoideae Apocynoideae Apocynoideae Rauvolfioideae Asclepiadoideae Asclepiadoideae Periplocoideae EF456177 EF456117 HE805531 Chonemorphinae HE805515 Tylophorinae EF456226 Urceolinae EF456162 Beaumontiinae HE805541 Vincinae HE805516 AM295674 Asclepiadinae EF456109 Apocyneae Asclepiadeae Apocyneae Apocyneae Vinceae Marsdenieae Asclepiadeae ly supported. The APSA clade receives high support (BP 99; PP 1.0), and Wrightieae emerge as sister to the rest of the clade. In Rauvolfioideae, resolution of groups is low in both analyses. Monophyly of Plumerieae receives low support in both analyses (BP 59; PP 0.93), whereas Vinceae are paraphyletic in the MP analysis and poorly supported in the Bayesian tree (PP 0.83). In both analyses Tabernaemontana falls with Vinceae, whereas Hunterieae cluster with the Amsonia-Rhazya clade (BP 100; PP 1.0), but this relationship is not well supported. The position of Carisseae is found here as sister to the APSA clade with weak support (BP 59; PP 0.91). APSA is well supported (BP 100; PP 1.0). Rhabdadenia, the only genus of Rhabdadenieae, forms a weakly supported clade with members of Malouetieae in the MP analysis (Figure 1), whereas in the Bayesian tree Rhabdadenia appears elsewhere (Figure 2). Periplocoideae receive good support in both analyses (BP 100; PP 1.0), but they are embedded in Apocynoideae. The clade comprising Odontadenieae, Mesechiteae, Echiteae and Apocyneae (all Apocynoideae) is well supported (BP 99; PP 1.0). Baisseeae (sensu Endress et al. 2007a) are supported (BP 99; PP1.0) with Dewevrella as their sister. This clade forms a strongly supported sister to the milkweeds in the Bayesian tree (PP 1.0) and is relatively less well supported in the parsimony tree (BP 78). The Secamonoideae-Asclepiadoideae clade receives strong support in both analyses (BP 100; PP 1.0), and the position of Fockeeae as sister to the rest is confirmed (BP 100; PP1.0). Members of Ceropegieae form a well-supported clade (BP 99; PP 1.0) in Asclepiadoideae. The monophyly of Marsdenieae receives strong support only in the Bayesian analysis (PP 0.99; BP 78). The close relationship between these tribes receives strong support also only in the Bayesian tree (PP 1.0; BP 53). Phylogenetic relationships in Apocynaceae 63 Fig. 1. One of the most parsimonious trees for Apocynaceae based on sequences of the combined dataset (PHYA and trnL-F). Bootstrap percentages > 50 and consistent with the strict consensus tree are indicated below branches. Cynanchum (P) = Cynanchum jacquemontianum. Eustegia of the new tribe Eustegieae is recovered as sister to the combined Marsdenieae-Ceropegieae clade (BP 53; PP 1.0). The major clades in Asclepiadeae receive strong support in the Bayesian analysis, whereas reso- lution is relatively poor in the parsimony analysis. Subtribe Astephaninae (PP 1.0) is sister to rest of Asclepiadeae. The informally named ACT clade is not recovered here due to the position of Cynanchineae (Figures 1, 2). 64 Nazia Nazar et al. Fig. 1. (Continued). Asclepiadineae and Tylophorinae form a well-supported clade (the AT clade) with Oxystelma as their sister (PP 1.0). Cynanchineae here comprise just the Old World genus Cynanchum, which forms a strongly supported clade sister to the MOG clade (BP 84; PP 1.0). The MOG clade (all New World) receives strong support only in the Bayesian tree (PP 1.0). Within MOG, Gonolobineae are monophyletic (PP 1.0). Blepharodon (Metastelmatinae) is weakly supported (PP 0.61) as sister to Funastrum (Oxypetalinae), resulting in Oxypetalinae not being monophyletic. Within Oxypetalinae, Araujia, Philibertia and Oxypetalum form a well-supported clade (PP 1.0). Phylogenetic relationships in Apocynaceae 65 Fig 2. Bayesian analysis of Apocynaceae using combined datasets (PHYA and trnL-F). Posterior probabilities are shown along branches. Cynanchum (P) = Cynanchum jacquemontianum. 4. DISCUSSION In the separate Bayesian and MP analyses, wellsupported incongruent nodes are not observed, which was also reported by Livshultz (2010). Relationships are better supported in the combined results (Figures 1, 2) compared to the separate trnL-F and PHYA trees. Here, we confine our discussion of results to the combined analyses. These results are broadly congruent with previously published phylogenetic studies of Apocynaceae (Livshultz et al. 2007; Simões et al. 2007; Endress et al. 2007a), and like these both subfamilies of Apocynaceae sensu stricto are not resolved as monophyletic. On the 66 Nazia Nazar et al. Fig. 2. (Continued). basis of evidence from previous studies (Sennblad and Bremer 2002; Simões et al. 2004 and 2007), members of Alstonieae are sister to the rest of Apocynaceae. In our study, Alstonia was designated as the outgroup (Figures 1, 2). The separate position of Amsonia and Rhazya from the rest of Vinceae is in agreement with earlier DNA studies (Potgieter and Albert 2001; Endress et al. 2007b; Simões et al. 2007). However, a floral study conducted by Endress et al. (2007b) on Amsonia and Rhazya suggested that these two genera are more similar to Catharanthus 67 Phylogenetic relationships in Apocynaceae and Vinca, but our results place the former pair with Hunterieae. Endress and Bruyns (2000) treated Rhazya as a synonym of Amsonia on the basis of similar fruits, seeds and f loral morphology (Pichon 1949; Nilsson 1986), and this relationship is also strongly supported in our study (BP 100; PP 1.0). As in Simões et al. (2007), monophyly of Plumerieae did not receive strong support in our analyses. Carisseae emerge as a sister group to the APSA clade, corresponding with the results of Civeyrel et al. (1998), Potgieter and Albert (2001), Simões et al. (2007) and Livshultz et al. (2007). 4.1 APSA clade Wrightieae of subfamily Apocynoideae is sister to the rest of the APSA clade as was the case in other phylogenetic analyses of Apocynaceae (Sennblad and Bremer 1996 and 2002; Sennblad et al. 1998; Potgieter and Albert 2001; Livshultz et al. 2007; Livshultz 2010). A strongly supported clade termed as the ‘crown clade’ by Livshultz et al. (2007) received less support in our Bayesian tree (PP 0.92) and is weakly supported in our MP analysis as compared to Livshultz et al. (2007) and Livshultz (2010). However the moderately supported (PP 0.91) sister-group relationship of Malouetieae with the crown clade, as illustrated in recent studies (Livshultz et al. 2007; Livshultz 2010), is also confirmed in our analyses. Pachypodium has traditionally been included in Echiteae (Pichon 1950), but Endress and Bruyns (2000) transferred this genus into Malouetieae, and this change was supported by Livshultz et al. (2007) and our study. Old World Apocyneae form a well-supported clade with the New World tribes (Odontadenieae, Echiteae and Mesechiteae) of Apocynoideae in both analyses. In a recent phylogenetic analysis (Livshultz 2010), this clade received less support: BP 68 compared to BP 100/ PP 1.0 here. Monophyly of Apocyneae is not supported by the MP analysis as compared to 100 BP in Livshultz (2010), but in the Bayesian tree they receive low support (PP 0.84). Our sampling of more taxa may be responsible for the shift in support observed in our results relative to those of Livshultz (2010). The topology in Apocyneae is somewhat inconsistent with that in Livshultz (2010), only by adding Trachelospermum, a basal clade (PP 0.72) emerges comprising of Beaumontia, Trachelospermum, Vallaris, Sindechites, Papuechites and Anodendron. In previous phylogenetic studies (Potgieter and Albert 2001; Sennblad and Bremer 2002; Simões et al. 2004 and 2007) Beaumontia and Trachelospermum form a clade with Chonemorpha, but here Chonemorpha from subtribe Chonemorphinae is sister to Urceola from subtribe Urceolinae (BP 100; PP 1.0; Figures 1, 2). In the present study, only two subtribes Apocyinae and Ichnocarpinae of the tribe Apocyneae described in updated classification of Apocynaceae (Endress et al., 2014) appeared monophyletic. New World Apocynoideae (Echiteae, Mesechiteae and Odontadenieae) do not form a well-supported clade in our analyses as observed by Livshultz et al. (2007) and Livshultz (2010). In our analyses we did not add more taxa to the New World Apocynoideae group. Therefore intergeneric relationships of New World Apocynoideae are similar to those observed in the studies of Livshultz (2010) (Figures 1, 2). Endress et al. (2014) recently described subtribe Rhabdadenieae, which is sister to the crown clade (as a separate clade in the Bayesian analysis and with members of Malouetieae in MP) as observed by Livshultz (2010). 4.2 Baisseeae (African clade) Endress et al. (2007a) defined a new tribe Baisseeae comprising three African genera – Baissea, Oncinotis and Motandra – and Livshultz et al. (2007) stated that Baisseeae are sister to the milkweeds rather than subfamily Periplocoideae. This relationship was originally suggested by Macfarlane (1933) on the basis of their geography (Livshultz et al. 2007). In previous phylogenetic analyses, this relationship has frequently been noted, but with weak support (Sennblad et al. 1998; Potgieter and Albert 2001; Sennblad and Bremer 2002) and more recently with stronger support (Lahaye et al. 2007; Livshultz et al. 2007; Simões et al. 2007). In our analysis, this sister relationship of Baisseeae receives strong support in the Bayesian analysis (PP 1.0) and comparatively weak bootstrap support (BP 78). In contrast, tetrad bearing Periplocoideae are most closely related to polliniumbearing milkweeds, and Baisseae in the present study and previous molecular studies (Sennblad and Bremer, 2000; Livshultz et al. 2007) received strong support as sister to the pollinium-bearing milkweeds. In the classification of Endress and Bruyns (2000), Baissea and Motandra are grouped with Prestonia and Cycladenia on the basis of corona characters (particularly finger-like projections above the stamens). In molecular phylogenetic analyses Prestonia forms a group with the ‘core Echiteae’, and Baissea and Motandra form a separate clade (Baisseeae; Livshultz 2010). Recently, Livshultz et al. (2007) identified these genera as having colleters on the adaxial surface of their petiole (rarely extending onto the base). However, this character is shared by Farquharia (Malouetieae), Isonema and Nerium (Nerieae). Therefore, morphologically, the African 68 Nazia Nazar et al. clade still needs additional characters to justify its separate tribal identity as the sister group of the milkweeds. 4.3 Periplocoideae In both these analyses (Bayesian and MP), the position of Periplocoideae in Apocynoideae differs from the analyses of Livshultz (2010). However, this result has been observed in other previous studies (Sennblad and Bremer 2000; Potgieter and Albert 2001; Livshultz et al. 2007; Livshultz 2010). On the basis of floral morphology (Table 3), the subfamily is regarded as an intermediate stage in a transition series between characters typical of Apocynoideae and those of milkweeds (Demeter 1922; Safwat 1962; Cronquist 1981; Rosatti 1989; Endress 1994, 2001 and 2004; Endress and Bruyns 2000; Wyatt et al. 2000). Apocynum has pollen in tetrads with simple translators, which is frequently considered to be the first stage in this series (Demeter 1922; Safwat 1962; Nilsson et al. 1993 and also cited by Livshultz et al. 2007). This is followed by pollen in tetrads with spoon-shaped translators in some Periplocoideae and then further aggregation leading to a pollinia in some Periplocoideae (Nilsson et al. 1993; Verhoeven and Venter 1998; Livshultz et al. 2007). Therefore, Periplocoideae as sister to the milkweeds is a common concept in the literature, but results of phylogenetic analyses have shown that Periplocoideae are more closely related to Apocynaceae sensu stricto; instead, Baisseeae are the sister of the milkweeds (Kunze 1996; Judd et al. 1994; Struwe et al. 1994; Sennblad and Bremer 1996; Endress 1997; Sennblad 1997; Potgieter and Albert 2001; Sennblad and Bremer 2002; Livshultz et al. 2007). Pollen in tetrads and pollinia have evolved in parallel in the APSA clade (Livshultz et al. 2007). In this analysis, Periplocoideae are well supported (BP 100; PP 1.0) as observed in Livshultz et al. (2007) and Livshultz (2010). The grooved translator clade described by Ionta and Judd (2007) is also well supported in the Bayesian tree (PP 1.0) and receives relatively less support in the MP analysis (BP 70). These results show Periploca (the type genus of subfamily Periplocoideae) is sister to the rest of the subfamily, which can be contrasted with the findings of Ionta and Judd (2007), in which Phyllanthera is sister to the rest of Periplocoideae. Note that Phyllanthera is sister to Petopentia (BP 92; PP 1.0) with these data. 4.4 Asclepiadoideae-Secamonoideae (milkweed clade) Secamonoideae have commonly been observed as sister of Asclepiadoideae (Sennblad and Bremer 1996, 2000 and 2002; Civeyrel et al. 1998; Civeyrel and Rowe Table 3. Key morphological characters in subfamilies of family Apocynaceae. Subfamily Key Characters Rauvolfioideae Corolla with sinistrorse aestivation in bud; anthers free from style head; staminal Sennblad (1997); Endress and Bruyns filaments free; sclerified anther wings absent; pollen granular; stylar head (2000) secretions not differentiated; fruit a berry drupe or follicle; seeds lacking a coma Corolla with dextrorse aestivation in bud; anthers adnate to style head; staminal Endress et al. (1996); Endress and filaments free; sclerified anther wings absent; pollen granular; stylar head Bruyns (2000) secretions not differentiated; fruit a follicle; seeds comose Corolla with dextrorse to valvate aestivation in bud; anthers adnate to style Verhoeven and Venter (1998); Endress head; staminal filaments free; sclerified anther wings absent; pollen in tetrads, and Bruyns (2000); Goyder et al. sometimes clumped into pollinia lacking waxy coating; stylar head secretions (2012) forming spoonlike translators with sticky basal viscidium; pollinia if present 4 per translator; fruit a follicle; seeds comose Civeyral (1996); Verhoeven and Venter Corolla with dextrorse or sinistrorse to valvate aestivation in bud; anthers and (1998); Endress and Bruyns (2000); style head fused to form gynostegium; staminal filaments fused into a tube; sclerified anther wings present; pollen in tetrads clumped into pollinia lacking Goyder et al. (2012) waxy coating; stylar head secretions differentiated into pale soft translator lacking clearly structured translator arms (pollinia fused directly to corpusculum or on short stalks); pollinarium with 4(-5) pollinia; fruit a follicle; seeds comose Corolla with dextrorse to valvate aestivation in bud; anthers and style head fused Klackenberg (1995b); Civeyral (1996); to form gynostegium; staminal filaments fused into a tube; sclerified anther Endress and Bruyns (2000); Goyder et wings present; pollen in tetrads clumped into pollinia encased in waxy coating; al. (2012) stylar head secretions differentiated into dark hard translator with translator arms (pollinia (mostly) linked to corpusculum via variously structured translator arms); pollinarium with 2 pollinia; fruit a follicle; seeds comose Apocynoideae Periplocoideae Secamonoideae Asclepiadoideae Reference 69 Phylogenetic relationships in Apocynaceae 2001; Fishbein 2001; Potgieter and Albert 2001; Lahaye et al. 2005 and 2007; Livshultz et al. 2007). In our study, this clade receives strong support (BP 100; PP 1.0). Although not broadly sampled here, the included taxa confirm monophyly of Secamonoideae with high support (BP 99; PP 1.0) Secamone is not recovered here as monophyletic, which is congruent with the results of Lahaye et al. (2007). Asclepiadoideae, the largest subfamily of Apocynaceae, comprises ~3000 species distributed worldwide (Goyder, 2006). Currently, five tribes are recognized in the subfamily: Fockeeae, Ceropegieae, Marsdenieae,Asclepiadeae (Endress et al., 2007a) and Eustegieae (Endress et al., 2014). The position here for Fockeeae is consistent with previous analyses (Civeyrel et al. 1998; Fishbein 2001; Potgieter and Albert 2001; Rapini et al. 2003; Livshultz et al. 2007; Livshultz 2010). Eustegia is a monotypic genus with pendent pollinia, placed in to separate tribe of Asclepiadoideae (Goyder 2006), but phylogenetic studies based on plastid markers (Liede 2001; Rapini et al. 2003; Goyder et al. 2007) have placed Eustegia sister to the Marsdenieae-Ceropegieae clade, a result confirmed by our results 4.5 Ceropegieae-Marsdenieae clade Meve and Liede (2004) recognized four subtribes in Ceropegieae based on anatomical characters: Anisotominae, Heterostemminae, Leptadeniinae and Stapeliinae. In our study Leptadeniinae are sister to the rest of Ceropegieae. Stapeliinae receive strong support (BP 100; PP 1.0), and Anisotominae are sister to Stapeliinae with strong support in the Bayesian analysis (PP 1.0) and moderate support in the parsimony analysis (BP 88). Both subtribes have overlapping morphological features (Meve 1995; Meve and Liede 2001a, 2001b and 2004). The Hoya/Dischidia group forms a well-supported subclade in both analyses (BP 100; PP 1.0) and along with members of the genus Marsdenia they receive strong support in the Bayesian analysis (PP 1.0) and moderate MP support (BP 88). The association of Hoya and Dischidia has previously been supported by Potgieter and Albert (2001), Livshultz (2002 and 2003), Rapini et al. (2003), Meve and Liede (2004) and Wanntorp et al. (2006a and 2006b). There is little molecular phylogenetic data available for Marsdenieae; however, recently a few studies have focused on Hoya (Wanntorp and Forster 2007; Wanntorp and Kunz 2009; Wanntrop et al. 2011). Another well-supported subclade (BP 97; PP 1.0) in Marsdenieae is comprised of Dregea, Gymnema, Stephanotis and Wattakaka. However, the position of Rhyssolobium seems unclear in both analyses. In the Bayesian analysis this genus is sister to the subclade that is sister to the rest, whereas with MP it is sister to other members of Marsdenieae; in both analyses, the position of this genus is poorly supported. This result is congruent with Meve and Liede (2004) and Wanntorp et al. (2006a). Monophyly of Ceropegieae-Marsdenieae (which possess erect pollinia, regarded as a primitive condition in Asclepiadoideae; Kunz, 1993) is well supported in Bayesian analysis (PP 1.0). In earlier studies (Orbigny, 1843; Decaisne, 1844) Ceropegieae and Marsdenieae sensu Endress and Bruyns (2000) were considered a single entity. However Endress and Bruyns (2000) treated Marsdenieae and Ceropegieae as two tribes, due to the lack of hyaline insertion crest on outer surface of pollinium and absence of an outer corona and milky latex in former (Bruyns and Forster 1991; Omlor 1998; Meve and Liede 2004). However, Swarupanandan et al. (1996) again united these two tribes, and this idea was later supported by molecular phylogenetic analyses (Potgieter and Albert 2001; Rapini et al. 2003; Meve and Liede 2004). Both tribes have also been observed to have the lowest level of polyploidy compared to other member of Asclepiadoideae (Albers and Meve, 2001). 4.6 Asclepiadeae Asclepiadeae, the largest tribe of Asclepiadoideae having pendent pollinia (Table 3) and reduced chromosome number (x=10, x=9) from basic number (x=11) (Albers and Meve, 2001), are recovered here as monophyletic. The African genus Eustegia appearing as sister to the Ceropegieae-Marsdenieae clade is now recognized as separate tribe Eustegieae in Asclepiadoideae (Endress et al., 2014). Higher levels of intergeneric resolution in Asclepiadeae are recovered in the Bayesian analysis as compared to parsimony. In a broad overview of Apocynaceae conducted by Rapini et al. (2003), three main clades were defined — Astephaninae comprising of only three genera Astephanus, Microloma and Oncinema sensu Liede (2001), ACTG (Asclepiadinae, Cynanchinae, Tylophorinae and Glossonematinae) and MOG (Metastelmatinae, Oxypetalinae and Gonolobinae). In the present study, Oncinema and Microloma of Astephaninae are well supported as sister to the rest of Asclepiadeae, a result similar to previous molecular studies (Liede 2001; Rapini et al. 2003; Figures 1, 2). Of the other two clades recovered by Rapini et al. (2003), the MOG clade is resolved as monophyletic, whereas the ACT clade remains non-monophyletic with these data. Oxystelma is recovered here as sister to the Asclepiadine- 70 ae-Tylophorinae clade (AT clade) with strong support in the Bayesian analysis (PP 1.0). Oxystelma was among the incertae sedis of Asclepiadoideae (Liede and Taüber 2000; Endress et al. 2007a), and previously Liede (1997) included it in Metastelmatinae. In subsequent molecular phylogenetic analyses (e.g., Potgieter and Albert 2001; Liede and Taüber 2002; Liede et al. 2002; Rapini et al. 2003) the genus failed to a form a clade with members of Metastelmatinae. Instead, this genus occupied a position sister to the rest of the AT clade, as also observed here; however, in previous molecular phylogenetic analyses using plastid loci this close relationship was not wellsupported. In the updated classification of Apocynaceae by Endress et al. (2014), Oxystelma was placed in subtribe Asclepiadineae. Cynanchineae here comprised of only Old World taxa (Cynanchum viminale, C. jacquemontianum and C. obtusifolium) appear as sister of the MOG clade (PP 1.0), which is comprised of members from the New World. However, these results can be contrasted with Rapini et al. (2003) where Cynanchinae are embedded in the ACT clade (but without support). The MOG clade (New World) is recovered here with high support (PP 1.0) as observed in previous studies (Liede and Taüber 2000, 2002; Rapini et al. 2003; LiedeSchumann et al. 2005; Rapini et al. 2006). Blepharodon lineare and Funastrum clausum were resolved taxa in the study of Rapini et al. (2006) and appeared as sister to Metastelmatinae and Oxypetalinae, respectively. According to Liede (1997) Funastrum clausum was previously included in Metastelmatinae on the basis of morphological characters, but in the most recent classification (Endress et al. 2007a; Endress et al., 2014) and also various molecular studies (Rapini et al. 2006) it is placed in Oxypetalinae. However here in the Bayesian analysis the relationship between Blepharodon lineare and Funastrum clausum is unclear, but their sister-group position to the rest of Oxypetalinae is well supported (PP 1.0; Figure 2). The MP analysis fails to produce good resolution in the MOG clade. In the present study, Oxypetalum is sister to Araujia-Philbertia, similar to the result of Rapini et al. (2006). However, in earlier studies (with fewer data) a close relationship between Philbertia and Blepharodon (Liede and Taüber 2000) or Philbertia and Funastrum (Rapini et al. 2003) was observed. Gonolobineae receive strong support with these data (BP 97; PP 1.0). Our study included a low-copy nuclear region and shows better resolution within some key clades in Apocynaceae when compared to previous studies, but the relationships recovered are not in particular markedly divergent from those obtained previously with just plastid data. The present analyses concluded that Rauvolf- Nazia Nazar et al. ioideae, Apocynoideae and the traditional Asclepiadaceae are all non-monophyletic groups and that, in contrast, the APSA clade is well supported. The crown clade of Livshultz et al. (2007) and Livshultz (2010) received only moderate support here. Our studies confirm that Periplocoideae are nested within Apocynoideae, in a position comparable to that in Livshultz et al. (2007). Periplocoideae should be placed in Apocynoideae rather than thought of as the sister group of the milkweeds. The sister group relationship between Baisseeae and the milkweeds is also confirmed by our analyses. The ACT clade was not monophyletic, whereas the MOG clade was. 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Bootstrap percentages > 50 and consistent with the strict consensus tree are indicated below branches. Cynanchum (P) = Cynanchum jacquemontianum. Phylogenetic relationships in Apocynaceae Fig. 1a. (Continued). 75 76 Nazia Nazar et al. Fig. 1b. Bayesian tree based on only PHYA sequences. PP values are indicated above the branches. Values > 0.95 are considered as strong support. Cynanchum (P) = Cynanchum jacquemontianum. Phylogenetic relationships in Apocynaceae Fig. 1b. (Continued). 77 78 Nazia Nazar et al. Fig. 2a. Parsimony analysis of trnL-F sequences of taxa present in combined analyses. Bootstrap percentages > 50 are indicated below branches. Cynanchum (P) = Cynanchum jacquemontianum. Phylogenetic relationships in Apocynaceae Fig. 2a. (Continued). 79 80 Nazia Nazar et al. Fig. 2b. Bayesian analysis of trnL-F sequences of taxa included in combined analyses. Values > 0.95 are considered as strong support. 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