Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(1): 55-81, 2019 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/cayologia-251 Citation: N. Nazar, J.J. Clarkson, D. Goyder, E. Kaky, T. Mahmood, M.W. Chase (2019) Phylogenetic relation- ships in Apocynaceae based on nucle- ar 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, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant 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 2 Department 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 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, inter- generic 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 Peri- plocoideae are nested within Apocynoideae. The APSA clade (Apocynoideae, Peri- plocoideae, Secamonoideae and Asclepiadoideae) is strongly supported here, but the crown clade of Apocynaceae (comprised of subfamilies Asclepiadoideae, Secamonoide- ae, Periplocoideae and Echiteae, Mesechiteae, Odontadenieae and Apocyneae of Apo- cynoideae) 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, Phy- tochrome A. 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 vari- ous 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). Subfami- lies Rauvolfioideae and Apocynoideae have been recov- ered 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: Tabernae- montaneae, Alstonieae, Alyxieae, Carisseae, Hunterieae, Melodineae, Plumerieae, Vinceae, Willughbeieae, Aspi- dospermeae and Amsonieae. Monophyly of most tribes in the subfamily has always remained suspect (Potgi- eter and Albert 2001; Sennblad and Bremer 2002); how- ever, 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 rec- ognized by Endress and Bruyns (2000): Wrightieae, Malouetieae, Apocyneae, Echiteae and Mesechiteae. Since this publication, five more tribes, Nerieae (Senn- blad and Bremer 2002), Odontadenieae (Endress et al. 2007a), Baisseeae (Endress et al. 2007a) and Rhabdade- nieae (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 (Apo- cynoideae, Periplocoideae, Secamonoideae, Asclepia- doideae) clade (Judd et al. 1994; Sennblad and Bremer 1996, 2002; Civeyrel et al. 1998; Potgieter and Albert 2001). Periplocoideae were recognized until the last dec- ades 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 Ascle- piadoideae five tribes have been recognized: Asclepiade- ae, Ceropegieae, Marsdenieae, Fockeeae and Eustegieae (Endress et al. 2007a; Endress et al., 2014)). Endress and Bruyns (2000) delimited the tribes of Asclepiadoide- ae on the basis of the orientation of pollinia in pollen sacs: upwardly directed in Ceropegieae-Marsdenieae and pendulous in Asclepiadeae. Erect pollinia are con- sidered a primitive character and also found in Seca- monoideae and Fockeeae (Kunze 1993). Recognition of Fockeeae as a tribe separate from Marsdenieae in Ascle- piadoideae 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 analy- ses (Potgieter and Albert, 2001; Rapini et al., 2003; Ver- hoeven 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 Glos- sonematinae) 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 (contain- ing Malagasy Cynanchum species), but New World sec- tions of the subtribe are polyphyletic (Liede and Taüber 2002; Rapini et al. 2006). Furthermore, cladistic analy- ses of Goyder et al. (2007) and Fishbein et al. (2011) have emphasized that generic delimitation of subtribe Ascle- piadineae is problematic. These studies concluded that Asclepiadoideae still needs further investigation to iden- tify monophyletic groups and find morphological char- acters 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 com- bination with morphological datasets. Livshultz (2010) presented a study using the low- copy 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. How- ever, there are still many other areas within the fam- ily 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 resolu- tion within the subfamily Rauvolfioideae. 57Phylogenetic relationships in Apocynaceae 2. MATERIALS AND METHODS 2.1. DNA extraction and amplification Taxa of Apocynaceae used here were either collect- ed from the field in Pakistan or sampled from the DNA Bank at The Royal Botanic Gardens, Kew (https://dna- bank.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 con- taining plastic beads (using a Genogrinder 2010, SPEX CertiPrep Ltd, Harrow, Middlesex, UK) and then fol- lowing 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/eth- idium bromide. Samples were then purified using a den- sity 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 Livs- hultz (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 degrad- ed DNA, especially from herbarium samples, Platinum® taq DNA polymerase (Invitrogen) was used. The reac- tion 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 vol- ume with nuclease free water. The following PCR pro- gram 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 car- ried out at 72 °C for 7 min. Higher annealing tempera- tures 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 poly- morphisms. 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, Michi- gan, 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 fol- lowing 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 topolo- gies as the basis for assessing incongruence (which we did not observe here). For the Bayesian results, we considered posterior probabilities (PP) > 0.95 as well- supported; < 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 incon- gruent clades, so we produced combined analyses of trnL-F and PHYA. The combined dataset (trnL-F and PHYA) com- prises 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 parsi- mony 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 opti- mization 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 poten- tially 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 sam- pling 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 collec- tion are given. Taxa Voucher detail Country Regions sequenced Asclepiadoideae – Asclepiadeae: Metastelmatinae Blepharodon lineare (Decne.) Decne. Forzza et al. 2027 Argentina trnL-F and PHYA Asclepiadoideae – Asclepiadeae: Oxypetalinae Araujia sericifera Brot. Forster 7656 Australia PHYA Funastrum clausum (Jacq.) Schltr. Mello- Silva et al. 1919 Argentina PHYA Oxypetalum capitatum Mart. Mello- Silva et al. 1924 Argentina PHYA Philibertia discolor (Schltr.) Goyder Mello- Silva et al. 1887 Argentina PHYA Philibertia lysimachioides (Wedd.) T. Mey. Mello- Silva et al. 1886 Argentina PHYA Asclepiadoideae – Asclepiadeae: Gonolobinae Matelea pseudobarbata (Pitter) Woodson M. Endress 97-08 Costa Rica PHYA Asclepiadoideae – Asclepiadeae: Asclepiadinae Calotropis procera (Aiton) W. T. Aiton Naz001* Pakistan trnL-F and PHYA Kanahia laniflora (Forssk.) R. Br. Goyder et al. 3931 Tanzania PHYA Pergularia daemia (Forssk.) Chiov. Naz024* Pakistan PHYA Pergularia tomentosa L. Naz012* Pakistan trnL-F and PHYA Stenostelma corniculatum (E. Mey.) Bullock Balkwill 10908 South Africa PHYA Xysmalobium parviflorum Harv. ex Scott-Elliot Killick & Vahrmeijer 3658 South Africa PHYA Asclepiadoideae – Asclepiadeae: Cynanchinae Cynanchum viminale (L.) Bassi Chase 731 ** PHYA Cynanchum jacquemontianum Decne. Naz010* Pakistan trnL-F and PHYA Cynanchum obtusifolium L.f. P. Bruyns Vch South Africa PHYA Asclepiadoideae – Asclepiadeae: Tylophorinae Tylophora hirsuta (Wall.) Wight Naz014* Pakistan trnL-F and PHYA Unplace genus Oxystelma esculentum (L. f.) Sm. Naz020* Pakistan trnL-F and PHYA Asclepiadoideae – Asclepiadeae: Astephaninae Eustegia minuta (L. F.) N. E. Br. P. Bruyns 4357 South Africa PHYA Oncinema lineare (L. F.) Bullock P. Bruyns Vch? South Africa PHYA Schubertia grandiflora Mart. Irwin et al. 31285 Brazil PHYA Asclepiadoideae – Marsdenieae Dischidia lanceolata Decne. Chase 734 Indonesia PHYA Dregea abyssinica K.Schum. Goyder et al. 3918 Tanzania PHYA Gymnema sylvestre (Retz.) Schultz. Chase 3902 India trnL-F and PHYA Hoya finalasonii Wight Chase 17138 India PHYA Hoya manipurensis Deb. Chase 733 Thailand PHYA Marsdenia carvalhoi Morillo & Carnevali Chase 3904 Brazil trnL-F and PHYA Rhyssolobium dumosum E. Mey. P. V. Bruyns 3948 South Africa PHYA Staphanotis floribunda Brongn. Chase 732 Senegal trnL-F and PHYA Wattakaka volubilis (Linn.f.) Stapf. Naz006* Pakistan trnL-F and PHYA Asclepiadoideae – Ceropegieae Caralluma tuberculata N.E. Br. Naz019* Pakistan trnL-F and PHYA Ceropegia sandersonii Decne.ex Hook. Chase 17507 ** PHYA Duvalia polita N. E. Br. Kew ** PHYA Boucerosia indica Dalzell Chase 2861 India PHYA Leptadenia pyrotechnica Naz018* Pakistan trnL-F and PHYA Neoschumannia kamerunensis Chase 3903 Cameroon PHYA Quaqua incarnata (L. f.) Bruyns Chase 9818 South Africa PHYA Heterostemma acuminatum Decne. Forster 5090 ** PHYA 59Phylogenetic relationships in Apocynaceae 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 fre- quencies of all observed bi-partitions (i.e. the posterior probabilities for each node). Taxa Voucher detail Country Regions sequenced Secamonoideae Secamone alpini Schult. P. Bruyns Vch South Africa trnL-F and PHYA Periplocoideae Cryptolepis buchananii Roemer & Schult. Naz002* Pakistan trnL-F and PHYA Cryptolepis decidua (Planch. ex Benth.) N. E. Br. P. V. Bruyns s.n. (east of Fish R.) Namibia trnL-F and PHYA Hemidesmus indicus (L.) R.Br. ex Schult. Chase 725 Tamil Nadu PHYA Periploca aphylla Decne. Naz004* Pakistan trnL-F and PHYA Raphionacme hirsuta (E.Mey.sec.N.E.Brown) R.A.Dyer CFR 15 South Africa PHYA Schlechterella abyssinicum (Chiov.) Venter & R. L. Verh. Chase 720 Ethopia trnL-F and PHYA Apocynoideae - Malouetieae Kibatalia gitingensis (Elmer) Woodson Liede 3268 ** trnL-F and PHYA Pachypodium leallii Welw. Chase 735 South Africa trnL-F and PHYA Apocynoideae - Nerieae Adenium obesum (Forssk.) Roem. & Schult. Chase 727 Somalia trnL-F and PHYA Nerium oleander L. Naz015* Pakistan trnL-F and PHYA Apocynoideae - Apocyneae Beaumontia grandiflora (Roxb.) Wall. Naz008* Pakistan trnL-F and PHYA Trachelospermum jasminoides (Lindl.) Lem. Naz022* Pakistan trnL-F and PHYA Apocynoideae - Echiteae Fernaldia pandurata (A.DC.) Woodson M Endress, Zurich ** PHYA Rauvolfioideae - Wrightieae Pleioceras barteri Baill. Endress, P. 99-10 Ivory Coast PHYA Rauvolfioideae - Carisseae Carissa spinarum L. Naz017* Pakistan trnL-F and PHYA Rauvolfioideae - Plumerieae Anechites nerium Urb. Bremer et al. 3386 UPS ** PHYA Skytanthus acutus Meyen M. Endress, Zurich ** PHYA Thevetia peruviana (Pers.) K. Schum. Naz013* Pakistan trnL-F and PHYA Rauvolfioideae - Vinceae Amsonia hurbritchii Woodson Chase 19252 USA trnL-F and PHYA Petchia ceylanica (Wight) Livera R. Olmor s. n Germany trnL-F and PHYA Rauvolfia serpentina (L.) Benth. Naz003* Pakistan trnL-F and PHYA Rhazya orientalis A.DC. M. Endress s.n. Zurich trnL-F and PHYA Vinca major L. Naz025* Pakistan trnL-F Rauvolfioideae - Tabernaemontaneae Tabernaemonta divericata (L.) R. Br. exRoem.Schult Chase 5571 Bangladesh PHYA Rauvolfioideae - Hunterieae Gonioma kamassi E.Mey. Chase 5806 South Africa trnL-F and PHYA Rauvolfioideae - Alyxieae Alyxia buxifolia R. Br. Smith, R.J. (RJS202) Australia PHYA Rauvolfioideae - Alstonieae Alstonia scholaris (L.) R. Br. 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. 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 pub- lished in Rapini et al. (2003), Sennblad and Bremer (1998) and Livshultz (2010) with updated nomenclature (Endress et al., 2014). Species Name PHYA trnL-F Subtribe Tribe Subfamily Adenium obesum (Forssk.) Roem. & Schult. LT972249 HE805526 Nerieae Apocynoideae Aganosma wallichii G. Don. GU901319 EF456127 Ichnocarpinae Apocyneae Apocynoideae Alstonia scholaris (L.) R. Br. LR027092 HE805532 Alstonieae Rauvolfioideae Alyxia buxifolia R. Br. LT972244 AF214152 Alyxieae Rauvolfioideae Amsonia hurbritchii Woodson LR027376 Amsonieae Rauvolfioideae Anechites nerium Urb. LT972245 AM295087 Thevetiinae Plumerieae Rauvolfioideae Angadenia berteroi (A.DC.) Miers GU901358 EF456246 Echiteae Apocynoideae Anodendron paniculatum A. DC. GU901327 EF456194 Papuechitinae Apocyneae Apocynoideae Apocynum androsaemifolium L. GU901328 AF214308 Apocyinae Apocyneae Apocynoideae Araujia sericifera Brot. LT972246 AJ704332 Oxypetalinae Asclepiadeae Asclepiadoideae Artia balansae (Baillon) Pichon ex Guillaumin GU901372 EF456142 Echiteae Apocynoideae Baissea multiflora A. DC. GU901330 EF456199 Baisseeae Apocynoideae Beaumontia grandiflora (Roxb.) Wall. LR027094 HE805527 Beaumontiinae Apocyneae Apocynoideae Blepharodon linere (Decne.) Decne. LR026999 AY163668 Metastelmatinae Asclepiadeae Asclepiadoideae Boucerosia indica Dalzell HF969013 AF214202 Ceropegieae Asclepiadoideae Calotropis procera (Aiton) W. T. Aiton LT972247 HE805509 Asclepiadinae Asclepiadeae Asclepiadoideae Caralluma tuberculata N.E. Br. LT972248 HE805510 Ceropegieae Asclepiadoideae Carissa spinarum L. LR027375 HE805533 Carisseae Rauvolfioideae Ceropegia sandersonii Decne.ex Hook. HF969012 AF214179 Ceropegieae Asclepiadoideae Chonemorpha fragrans (Moon) Alston GU901332 EF456132 Chonemorphinae Apocyneae Apocynoideae Cleghornia malaccensis (Hook. f.) King & Gamble GU901333 EF456241 Apocyinae Apocyneae Apocynoideae Cryptolepis buchananii Roemer & Schult. HG004619 HE805522 Periplocoideae Cryptolepis decidua (Planch. ex Benth.) N. E. Br. HG004618 HE805523 Periplocoideae Cycladenia humilis Bentham GU901367 EF456140 Odontadenieae Apocynoideae Cynanchum jacquemontianum Decne. LR027368 HE805511 Cynanchinae Asclepiadeae Asclepiadoideae Cynanchum obtusifolium L.f. HF969010 AJ428692 Cynanchinae Asclepiadeae Asclepiadoideae Cynanchum viminale (L.) Bassi HG004632 AJ290912 Cynanchinae Asclepiadeae Asclepiadoideae Dischidia lanceolata Decne. LR028004 Marsdenieae Asclepiadoideae Dregea abyssinica K.Schum. HG004620 Marsdenieae Asclepiadoideae Duvalia polita N. E. Br. HF969009 AJ488374 Ceropegieae Asclepiadoideae Echites umbellatus Jacq. GU901387 EF456186 Echiteae Apocynoideae Elytropus chilensis Müll. Arg. GU901398 EF456171 Odontadenieae Apocynoideae Epigynum cochinchinense (Pierre) D.J. Middleton GU901340 EF456147 Ichnocarpinae Apocyneae Apocynoideae Eustegia minuta (L.f.) N.E.Br. LR027089 AJ410207 Eustegieae Asclepiadeae Asclepiadoideae Fernaldia pandurata (A.DC.) Woodson GU901329 EF456209 Echiteae Apocynoideae Finlaysonia insularum (King & Gamble) Venter GU901341 EF456105 Periplocoideae Fockea edulis K. Schum. LR027374 AF214199 Fockeeae Asclepiadoideae Forsteronia guyanensis Müll.Arg. GU901359 EF456153 Mesechiteae Apocynoideae Funastrum clausum (Jacq.) Schltr. HG004645 AJ428794 Oxypetalinae Asclepiadeae Asclepiadoideae Gonioma kamassi E.Mey. HG004623 HE805535 Hunterieae Rauvolfioideae Gymnanthera oblonga (Burm. f.) P.S. Green GU901348 EF456106 Periplocoideae Gymnema sylvestre (Retz.) Schultz. HG004637 HE805512 Marsdenieae Asclepiadoideae Hemidesmus indicus (L.) R.Br. ex Schult. HG004617 DQ916877 Periplocoideae Heterostemma acuminatum Decne. AJ574827 Ceropegieae Asclepiadoideae Hoya finalasonii Wight HG004636 Marsdenieae Asclepiadoideae Hoya manipurensis Deb. LR027373 AF214227 Marsdenieae Asclepiadoideae Ichnocarpus frutescens R. Br. GU901356 EF456136 Ichnocarpinae Apocyneae Apocynoideae Kanahia laniflora (Forssk.) R. Br. HG004642 AY163695 Asclepiadinae Asclepiadeae Asclepiadoideae Kibatalia gitingensis (Elmer) Woodson HG004629 HE805528 Malouetieae Apocynoideae 61Phylogenetic relationships in Apocynaceae Species Name PHYA trnL-F Subtribe Tribe Subfamily Laubertia contorta (Mart.& Galeotti) Woodson GU901375 EF456180 Echiteae Apocynoideae Leptadenia pyrotechnica HG004614 HE805513 Ceropegieae Asclepiadoideae Mandevilla boliviensis Decne. GU901343 EF456134 Mesechiteae Apocynoideae Marsdenia carvalhoi Morillo & Carnevali LR027091 DQ334521 Marsdenieae Asclepiadoideae Marsdenia glabra Costantin LR027370 EF456114 Marsdenieae Asclepiadoideae Matelea pseudobarbata (Pitter) Woodson HG004621 Gonolobinae Asclepiadeae Asclepiadoideae Microloma tenuifolium (L.) Kuntze LR027371 AJ410230 Astephaninae Asclepiadeae Asclepiadoideae Motandra guineensis A. DC. GU901361 EF456210 Baisseeae Apocynoideae Neoschumannia kamerunensis Schltr. HG004613 AJ410054 Ceropegieae Asclepiadoideae Nerium oleander L LR027093 HE805529 Nerieae Apocynoideae Odontadenia perrotteti (A. DC.) Woodson GU901335 EF456211 Odontadenieae Apocynoideae Oncinema lineare (L. F.) Bullock LR027090 AJ428827 Astephaninae Asclepiadeae Asclepiadoideae Oncinotis tenuiloba Stapf GU901368 EF456141 Baisseeae Apocynoideae Orthanthera jasminiflora Schinz AJ574827 Ceropegieae Oxypetalum capitatum Mart. HG004644 AY163710 Oxypetalinae Asclepiadeae Asclepiadoideae Oxystelma esculentum (L. f.) Sm. HF969014 AJ290887 Asclepiadeae Asclepiadoideae Pachypodium leallii Welw. HG004628 HE805530 Malouetieae Apocynoideae Papuechites aambe Markgr. GU901370 EF456189 Papuechitinae Apocyneae Apocynoideae Parameria laevigata (Juss.) Mold. GU901371 EF456197 Urceolinae Apocyneae Apocynoideae Parsonsia eucalyptophylla F. Muell. GU901380 EF456215 Echiteae Apocynoideae Peltastes isthmicus Woodson GU901324 EF456129 Echiteae Apocynoideae Pentalinon luteum (L.) B.F. Hansen & R.P. Wunderlin HG004631 EF456191 Echiteae Apocynoideae Pergularia daemia (Forssk.) Chiov. HG004641 JN205300 Asclepiadinae Asclepiadeae Asclepiadoideae Pergularia tomentosa L. HG004640 HE805514 Asclepiadinae Asclepiadeae Asclepiadoideae Periploca aphylla Decne. HG004616 HE805524 Periplocoideae Petchia ceylanica (Wight) Livera HG004624 AM295093 Catharanthinae Vinceae Rauvolfioideae Petopentia natalensis (Schltr.) Bullock GU901376 EF456107 Periplocoideae Philibertia discolor (Schltr.) Goyder LR027369 AY163700 Oxypetalinae Asclepiadeae Asclepiadoideae Philibertia lysimachioides (Wedd.) T. Mey. HG004643 AJ290900 Oxypetalinae Asclepiadeae Asclepiadoideae Phyllanthera grayi (P.I. Forst.) Venter GU901377 EF456103 Periplocoideae Pinochia corymbosa (Jacq.) M.E. Endress & B.F. Hansen GU901378 EF456167 Odontadenieae Apocynoideae Pleioceras barteri Baill. LR027096 EF456251 Wrightieae Apocynoideae Prestonia lagoensis (Müll. Arg.) Woodson GU901337 EF456237 Echiteae Apocynoideae Quaqua incarnata (L. f.) Bruyns HG004612 AJ488455 Ceropegieae Asclepiadoideae Raphionacme hirsuta (E.Mey.sec.N.E.Brown) R.A.Dyer HG004615 AJ581825 Periplocoideae Rauvolfia serpentina (L.) Benth. HG004625 HE805539 Rauvolfiinae Vinceae Rauvolfioideae Rhabdadenia biflora Müll.Arg. LR028003 Rhabdadenieae Apocynoideae Rhazya orientalis A.DC. AM295095 Vinceae Rauvolfioideae Rhodocalyx rotundifolius Müll. Arg. GU901396 EF456238 Echiteae Apocynoideae Rhyssolobium dumosum E. Mey. HG004635 AM233378 Marsdenieae Asclepiadoideae Schlechterella abyssinicum (Chiov.) Venter & R. L. Verh. HG004611 HE805525 Periplocoideae Schubertia grandiflora Mart. HG004622 AJ428827 Astephaninae Asclepiadeae Asclepiadoideae Secamone alpini Schult. LR027095 HE805519 Secamonoideae Secamone elliptica R. Br. GU901389 EF456116 Secamonoideae Secondatia densiflora A. DC. GU901339 EF456228 Odontadenieae Apocynoideae Sindechites chinensis Oliv. & Tsiang: GU901393 EF456244 Amphineuriinae Apocyneae Apocynoideae Skytanthus acutus Meyen HG004627 AF214269 Thevetiinae Plumerieae Rauvolfioideae Staphanotis floribunda Brongn. HG004634 HE805517 Marsdenieae Asclepiadoideae Stenostelma corniculatum (E. Mey.) Bullock HG004639 AY163722 Asclepiadinae Asclepiadeae Asclepiadoideae Stipecoma peltigera Müll. Arg. GU901394 EF456193 Echiteae Apocynoideae Tabernaemonta divericata (L.) R. Br. exRoem.Schult AF214399 Tabernaemontaneae Rauvolfioideae 62 Nazia Nazar et al. 3. RESULTS 3.1. Incongruence As mentioned above, well-supported clades incon- gruent 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 par- simony informative. Analysis produced 13960 equally most-parsimonious trees with 3284 steps and a consist- ency index of 0.49 and retention index of 0.71. Mr Mod- eltest 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 hetero- geneity 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). Rauvolfioide- ae and Apocynoideae are non-monophyletic, but the subfamilies of the traditional Asclepiadaceae are strong- 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 analy- ses Tabernaemontana falls with Vinceae, whereas Hunt- erieae 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). Rhabdad- enia, 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 Rhabdad- enia 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 sup- ported 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 con- firmed (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 sup- port 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). Species Name PHYA trnL-F Subtribe Tribe Subfamily Temnadenia odorifera (Vell.) J.F. Morales: GU901373 EF456179 Echiteae Apocynoideae Thevetia peruviana (Pers.) K. Schum. LR027097 Thevetiinae Plumerieae Rauvolfioideae Thyrsanthella difformis (Walter) Pichon GU901391 EF456177 Odontadenieae Apocynoideae Toxocarpus villosus(Blume) Decne GU901399 EF456117 Secamonoideae Trachelospermum jasminoides (Lindl.) Lem. HG004630 HE805531 Chonemorphinae Apocyneae Apocynoideae Tylophora hirsuta (Wall.) Wight HE805515 Tylophorinae Asclepiadeae Asclepiadoideae Urceola lucida Benth. & Hook. f. GU901400 EF456226 Urceolinae Apocyneae Apocynoideae Vallaris solanacea (Roth) O. Kuntze GU901401 EF456162 Beaumontiinae Apocyneae Apocynoideae Vinca major L. LR028005 HE805541 Vincinae Vinceae Rauvolfioideae Wattakaka volubilis (Linn.f.) Stapf. HF969011 HE805516 Marsdenieae Asclepiadoideae Xysmalobium parviflorum Harv. ex Scott-Elliot HG004638 AM295674 Asclepiadinae Asclepiadeae Asclepiadoideae Zygostelma benthamii Baill GU901404 EF456109 Periplocoideae 63Phylogenetic relationships in Apocynaceae Eustegia of the new tribe Eustegieae is recovered as sis- ter 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. Sub- tribe Astephaninae (PP 1.0) is sister to rest of Asclepia- deae. The informally named ACT clade is not recovered here due to the position of Cynanchineae (Figures 1, 2). 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 jacquemon- tianum. 64 Nazia Nazar et al. 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 sup- port 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). Fig. 1. (Continued). 65Phylogenetic relationships in Apocynaceae 4. DISCUSSION In the separate Bayesian and MP analyses, well- supported 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 previ- ously 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 Fig 2. Bayesian analysis of Apocynaceae using combined datasets (PHYA and trnL-F). Posterior probabilities are shown along branches. Cynanchum (P) = Cynanchum jacquemontianum. 66 Nazia Nazar et al. 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 Fig. 2. (Continued). 67Phylogenetic 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 phy- logenetic analyses of Apocynaceae (Sennblad and Brem- er 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 sup- ported (PP 0.91) sister-group relationship of Malouetie- ae 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, Trachelosper- mum, 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 sub- tribe 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 Ichnocarpi- nae of the tribe Apocyneae described in updated classi- fication 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 Apocynoide- ae are similar to those observed in the studies of Livs- hultz (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 sub- family Periplocoideae. This relationship was originally suggested by Macfarlane (1933) on the basis of their geography (Livshultz et al. 2007). In previous phylo- genetic analyses, this relationship has frequently been noted, but with weak support (Sennblad et al. 1998; Pot- gieter 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 sup- port in the Bayesian analysis (PP 1.0) and comparatively weak bootstrap support (BP 78). In contrast, tetrad bear- ing Periplocoideae are most closely related to pollinium- bearing 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 classi- fication of Endress and Bruyns (2000), Baissea and Mot- andra are grouped with Prestonia and Cycladenia on the basis of corona characters (particularly finger-like pro- jections above the stamens). In molecular phylogenetic analyses Prestonia forms a group with the ‘core Echiteae’, and Baissea and Mot- andra 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 Neri- um (Nerieae). Therefore, morphologically, the African 68 Nazia Nazar et al. clade still needs additional characters to justify its sepa- rate tribal identity as the sister group of the milkweeds. 4.3 Periplocoideae In both these analyses (Bayesian and MP), the posi- tion 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 morpholo- gy (Table 3), the subfamily is regarded as an intermedi- ate stage in a transition series between characters typi- cal 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 trans- lators in some Periplocoideae and then further aggrega- tion leading to a pollinia in some Periplocoideae (Nils- son et al. 1993; Verhoeven and Venter 1998; Livshultz et al. 2007). Therefore, Periplocoideae as sister to the milk- weeds 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 sup- ported in the Bayesian tree (PP 1.0) and receives rela- tively less support in the MP analysis (BP 70). These results show Periploca (the type genus of subfamily Peri- plocoideae) 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 Peri- plocoideae. 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 Reference Rauvolfioideae Corolla with sinistrorse aestivation in bud; anthers free from style head; staminal filaments free; sclerified anther wings absent; pollen granular; stylar head secretions not differentiated; fruit a berry drupe or follicle; seeds lacking a coma Sennblad (1997); Endress and Bruyns (2000) Apocynoideae Corolla with dextrorse aestivation in bud; anthers adnate to style head; staminal filaments free; sclerified anther wings absent; pollen granular; stylar head secretions not differentiated; fruit a follicle; seeds comose Endress et al. (1996); Endress and Bruyns (2000) Periplocoideae Corolla with dextrorse to valvate aestivation in bud; anthers adnate to style head; staminal filaments free; sclerified anther wings absent; pollen in tetrads, sometimes clumped into pollinia lacking waxy coating; stylar head secretions forming spoonlike translators with sticky basal viscidium; pollinia if present 4 per translator; fruit a follicle; seeds comose Verhoeven and Venter (1998); Endress and Bruyns (2000); Goyder et al. (2012) Secamonoideae Corolla with dextrorse or sinistrorse to valvate aestivation in bud; anthers and style head fused to form gynostegium; staminal filaments fused into a tube; sclerified anther wings present; pollen in tetrads clumped into pollinia lacking 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 Civeyral (1996); Verhoeven and Venter (1998); Endress and Bruyns (2000); Goyder et al. (2012) Asclepiadoideae Corolla with dextrorse to valvate aestivation in bud; anthers and style head fused to form gynostegium; staminal filaments fused into a tube; sclerified anther wings present; pollen in tetrads clumped into pollinia encased in waxy coating; 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 Klackenberg (1995b); Civeyral (1996); Endress and Bruyns (2000); Goyder et al. (2012) 69Phylogenetic 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 sup- port (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 Apoc- ynaceae, comprises ~3000 species distributed world- wide (Goyder, 2006). Currently, five tribes are rec- ognized 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: Anisoto- minae, Heterostemminae, Leptadeniinae and Stapelii- nae. 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 fea- tures (Meve 1995; Meve and Liede 2001a, 2001b and 2004). The Hoya/Dischidia group forms a well-support- ed 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 Potgi- eter 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, Stepha- notis and Wattakaka. However, the position of Rhysso- lobium 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 mem- bers of Marsdenieae; in both analyses, the position of this genus is poorly supported. This result is congru- ent with Meve and Liede (2004) and Wanntorp et al. (2006a). Monophyly of Ceropegieae-Marsdenieae (which pos- sess erect pollinia, regarded as a primitive condition in Asclepiadoideae; Kunz, 1993) is well supported in Bayes- ian 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 Mars- denieae 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 unit- ed 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 Asclepiadoide- ae (Albers and Meve, 2001). 4.6 Asclepiadeae Asclepiadeae, the largest tribe of Asclepiadoideae having pendent pollinia (Table 3) and reduced chro- mosome number (x=10, x=9) from basic number (x=11) (Albers and Meve, 2001), are recovered here as mono- phyletic. 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 analy- sis as compared to parsimony. In a broad overview of Apocynaceae conducted by Rapini et al. (2003), three main clades were defined — Astephaninae compris- ing of only three genera Astephanus, Microloma and Oncinema sensu Liede (2001), ACTG (Asclepiadinae, Cynanchinae, Tylophorinae and Glossonematinae) and MOG (Metastelmatinae, Oxypetalinae and Gonolobi- nae). 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 Nazia Nazar et al. 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 posi- tion sister to the rest of the AT clade, as also observed here; however, in previous molecular phylogenetic analy- ses using plastid loci this close relationship was not well- supported. In the updated classification of Apocynaceae by Endress et al. (2014), Oxystelma was placed in sub- tribe Asclepiadineae. Cynanchineae here comprised of only Old World taxa (Cynanchum viminale, C. jacque- montianum 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 con- trasted 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; Liede- Schumann 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 sis- ter to Metastelmatinae and Oxypetalinae, respectively. According to Liede (1997) Funastrum clausum was pre- viously included in Metastelmatinae on the basis of morphological characters, but in the most recent classi- fication (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, Oxy- petalum 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 Philber- tia 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 Apo- cynaceae when compared to previous studies, but the relationships recovered are not in particular markedly divergent from those obtained previously with just plas- tid data. The present analyses concluded that Rauvolf- ioideae, Apocynoideae and the traditional Asclepiadace- ae 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 Peri- plocoideae are nested within Apocynoideae, in a posi- tion comparable to that in Livshultz et al. (2007). Peri- plocoideae 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. Old World Cynanchineae forms a well-supported group within the New World MOG clade. In the present study support for clades are com- paratively better than in studies where plastid regions alone were sequenced. In the future, there is a need to sequence greater numbers of taxa of Apocynaceae to further refine the relationships in the family. There is also a need to be increased field collection of material so that high-quality DNA can be recovered from a wider range of Apocynaceae taxa. 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