Annals of Botany 112: 1723–1742, 2013 doi:10.1093/aob/mct222, available online at www.aob.oxfordjournals.org Evolutionary history of the Afro-Madagascan Ixora species (Rubiaceae): species diversification and distribution of key morphological traits inferred from dated molecular phylogenetic trees J. Tosh1,2,*, S. Dessein3, S. Buerki4, I. Groeninckx1, A. Mouly5,6,B.Bremer6,E.F.Smets1,7,8 and P. De Block3 1Laboratory of Plant Systematics, KU Leuven, Kasteelpark Arenberg 31, PO Box 2437, BE-3001 Leuven, Belgium, 2Ashdown House School, Forest Row, East Sussex RH18 5JY, UK, 3National Botanic Garden of Belgium, Domein van Bouchout, BE-1860 Meise, Belgium, 4Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AE, UK, 5Laboratoire Chrono-environnement, UMR CNRS 6249, Universite´ de Franche-Comte´, 16 Route de Gray, F-25030 Besanc¸on cedex, France, 6Bergius Foundation, Royal Swedish Academy of Sciences and Botany Department, Stockholm University, SE-106 91, Stockholm, Sweden, 7National Herbarium 8 of The Netherlands, Leiden University Branch, PO Box 9514, NL-2300 RA Leiden, The Netherlands and Netherlands Centre for Downloaded from Biodiversity Naturalis, PO Box 9517, NL-2300 RA Leiden, The Netherlands * For correspondence. E-mail: [email protected] Received: 9 April 2013 Returned for revision: 20 May 2013 Accepted: 6 August 2013 Published electronically: 18 October 2013 http://aob.oxfordjournals.org/ † Background and Aims Previous workon the pantropical genus Ixora has revealed an Afro-Madagascan clade, but as yet no study has focused in detail on the evolutionary history and morphological trends in this group. Here the evo- lutionaryhistoryof Afro-MadagascanIxora spp.(aclade of approx.80 taxa) is investigated andthe phylogenetic trees compared with several key morphological traits in taxa occurring in Madagascar. † Methods Phylogenetic relationships of Afro-Madagascan Ixora are assessed using sequence data from four plastid regions ( petD, rps16, rpoB-trnC and trnL-trnF) and nuclear ribosomal external transcribed spacer (ETS) and internal transcribed spacer (ITS) regions. The phylogenetic distribution of key morphological characters is assessed. Bayesian inference (implemented in BEAST) is used to estimate the temporal origin of Ixora based on fossil evidence. † Key Results Two separate lineages of Madagascan taxa are recovered, one of which is nested in a group of East at Stockholms Universitet on March 3, 2014 African taxa. Divergence in Ixora is estimated to have commenced during the mid Miocene, with extensive clado- genesis occurring in the Afro-Madagascan clade during the Pliocene onwards. † Conclusions Both lineages of Madagascan Ixora exhibit morphological innovationsthat are rare throughout the rest of the genus, including a trend towards pauciflorous inflorescences and a trend towards extreme corolla tube length, suggesting that the same ecological and selective pressures are acting upon taxa from both Madagascan lineages. Novel ecological opportunities resulting from climate-induced habitat fragmentation and corolla tube length diver- sification are likely to have facilitated species radiation on Madagascar. Key words: Rubiaceae, Ixora, Afro-Madagascan, molecular phylogenetics, molecular dating, biogeography, ETS, ITS, petD, rps16, rpoB-trnC, trnL-trnF. INTRODUCTION 1998). In continental Africa, Ixora mainly occurs in the Guineo-Congolian Regional Centre of Endemism (RCE) (fol- The pantropical genus Ixora is one of the largest genera in lowing White, 1983), but also in the Afromontane archipelago- Rubiaceae, with approx. 530 species of shrubs and small trees like RCE, and extends into the Zambezian RCE, the Swahilian that typically grow in humid rain forest (Davis et al., 2009). RCE and the Swahilian/Maputaland regional transition zone The centre of species diversity for the genus is in South-East (RTZ) (De Block, 1998). In Madagascar, Rubiaceae are most nu- Asia, in particular Borneo (Lorence et al., 2007). Although no merous and species rich in the evergreen humid forests (Davis modern monograph of Ixora exists, there have been a number and Bridson, 2003). Ixora is no exception to this, occurring of revisions focusing on specific geographical regions (e.g. De most frequently in the humid evergreen forest (littoral, lowland Block, 1998, revision of continental African Ixora spp.; De and montane) on the eastern coast of Madagascar, although Block, 2013, revision of Madagascan Ixora spp.). Phylogenetic Ixora spp. also occur in the semi-deciduous forest of studies of Ixora have primarily focused on the tribal placement Madagascar (De Block, 2003, 2013). and circumscription of the genus (Andreasen and Bremer, Ixora is one of the most easily recognizable genera in 1996, 2000; Mouly, 2007; Mouly et al., 2009a). Most recently, Rubiaceae, in part due to the often striking inflorescences and tet- Mouly et al. (2009b) identified some well-supported, geograph- ramerous flowers (Fig. 1). Diagnostic features for the genus ically defined lineages, including an ‘Afro-Madagascan’ clade. (adapted from De Block, 2007) include articulated petioles, nar- There are approx. 80 Afro-Madagascan Ixora spp. distributed rowly tubular tetramerous flowers, bilobed stigmas, bilocular equally between continental Africa and Madagascar (De Block, ovaries and fruits (or, rarely, with more than two locules), # The Author 2013. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] 1724 Tosh et al. — Evolutionary history of the Afro-Madagascan Ixora species (Rubiaceae) A B C D E F Downloaded from G H http://aob.oxfordjournals.org/ at Stockholms Universitet on March 3, 2014 I J K L M F IG. 1. Example of morphological variation in Ixora. (A) Inflorescence of Ixora regalis. (B) Inflorescence of Ixora elliotii. (C) Inflorescence of Ixora emirnensis. (D) Pendulous inflorescence of Ixora mangabensis. (E) Inflorescence of Ixora densithyrsa. (F) Inflorescence of Ixora siphonantha. (G) Front view of a flower of Ixora guillotii. (H) Flowering branch of Ixora rakotonasoloi. (I) Mature fruits of Ixora guillotii. (J) Fruits of Ixora quadrilocularis. (K) Flowering node of Ixora homolleae. (L) Articulate petioles of Ixora finlaysoniana. (M) Articulate petioles of Ixora homolleae. uniovulate locules and seeds with a large adaxial hilar cavity. In flowers (De Block, 1998, 2003). This is particularly the case contrast, identification at the species level is difficult, with for the African representatives of the genus, which De Block species distinguished on the basis of minor and often continuous (1998) described as ‘extremely homogeneous’ in their charac- characters, typically involving features of the inflorescence and ters. On Madagascar, there are several morphological traits Tosh et al. — Evolutionary history of the Afro-Madagascan Ixora species (Rubiaceae) 1725 occurring in Ixora that are absent in the continental African taxa 25 mL, and contained 1 mL of each primer (100 ng mL21), and rare in the genus as a whole. These include: (1) reduction of 0.35 mL of Biotaq DNA polymerase, 2.5 mLof10× NH4 reac- the number of flowers per inflorescence towards solitary flowers; tion buffer, 1.5 mLof50mM MgCl2,2.5 mLof10mM dNTPs, (2) increase from two- to four-locular ovaries; and (3) increase 1 mL of bovine serum albumin (BSA; 0.4 %) and 2 mL of total towards large flowers (corolla tubes .15 cm long) and fruits genomic DNA. Amplification of petD, rps16 and trnL-trnF (De Block, 2007, 2008, 2013). used the following temperature profile: 94 8C for 3 min; 32 In the present study, we further investigate the phylogenetic cycles of 94 8C for 1 min, 50 8C for 1 min, 72 8C for 1.5 min; relationships of Madagascan and continental African Ixora final extension of 72 8C for 7 min. The amplification profile for spp. using molecular sequence data from four plastid regions rpoB-trnC was: 94 8C for 3 min; 32 cycles of 94 8C for 1 min, ( petD, rps16, rpoB-trnC and trnL-trnF) and nuclear ribosomal 53 8C for 1 min, 72 8C for 2 min; final extension of 72 8C for external transcribed spacer (ETS) and internal transcribed 7 min. spacer (ITS) regions. The purpose of this study is to improve PCR mixes for nuclear regions were the same as for plastid taxon sampling of both African and Madagascan species in regions, except that 1 mL of dimethylsulfoxide (DMSO) was order to: (1) test existing hypotheses concerning the evolutionary added per 25 mL. The ITS amplification profile was: 94 8Cfor affinities within and between African and Madagascan species; 3 min; 32 cycles of 94 8C for 1 min, 52 8C for 1 min, 72 8C for (2) assess the distribution of key morphological innovations of 1 min; final extension of 72 8C for 7 min. The ETS amplification Downloaded from the Madagascan species on our molecular phylogenetic trees; profilewas: 97 8C for 1 min; 40 cycles of 97 8C for 10 s, 55 8C for and (3) investigate the age of species diversification and dispersal 30 s, 72 8C for 30 s; final extension of 72 8C for 7 min. All amp- using molecular dating techniques. lification products were purified using Nucleospin purification columns and sent to Macrogen Inc. (Seoul, South Korea) for sequencing. http://aob.oxfordjournals.org/ MATERIALS AND METHODS Taxon sampling and DNA preparation Sequence alignment and phylogenetic analyses Extensive fieldwork was undertaken in eastern and northern Contiguous sequences were assembled and edited using the Madagascar in order to collect herbarium, alcohol and DNA ma- Staden software package (Staden et al., 1998). Sequences were terial of Madagascan Ixora spp. This material was used in the manually aligned in MacClade v. 4.04 (Maddison and molecular and the morphological study. We included 67 Ixora Maddison, 2002) without difficulty due to low levels of sequence accessions, representing approx. 50 species that occur through- variation. All variable nucleotide positions were verified against out the global distribution of the genus (Table 1). In particular, the original electropherograms. Gaps were treated as missing at Stockholms Universitet on March 3, 2014 our taxon sampling is focused on Madagascan and African data; potentially informative indels were coded using the species.