American Journal of Botany 96(5): 989–1010. 2009. A PRELIMINARY PHYLOGENY OF THE ‘ DIDYMOCARPOID GESNERIACEAE ’ BASED ON THREE MOLECULAR DATA SETS: I NCONGRUENCE WITH AVAILABLE TRIBAL CLASSIFICATIONS 1 Michael M ö ller, 2,5 Martin Pfosser, 3,6 Chang-Gee Jang, 3,7 Veronika Mayer, 3 Alexandra Clark, 2 Michelle L. Hollingsworth, 2 Michael H. J. Barfuss, 3 Yin-Zheng Wang, 4 Michael Kiehn, 3 and Anton Weber 3 2 Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh EH3 5LR, Scotland, UK; 3 Faculty Centre of Biodiversity, University of Vienna, Rennweg 14, A-1030 Vienna, Austria and 4 Institute of Botany, Chinese Academy of Sciences, Beijing, China The ‘ didymocarpoid Gesneriaceae ’ (traditional subfam. Cyrtandroideae excluding Epithemateae) are the largest group of Old World Gesneriaceae, comprising 85 genera and 1800 species. We attempt to resolve their hitherto poorly understood generic rela- tionships using three molecular markers on 145 species, of which 128 belong to didymocarpoid Gesneriaceae. Our analyses demonstrate that consistent topological relationships can be retrieved from data sets with missing data using subsamples and dif- ferent combinations of gene sequences. We show that all available classifi cations in Old World Gesneriaceae are artifi cial and do not refl ect natural relationships. At the base of the didymocarpoids are grades of clades comprising isolated genera and small groups from Asia and Europe. These are followed by a clade comprising the African and Madagascan genera. The remaining clades represent the advanced Asiatic and Malesian genera. They include a major group with mostly twisted capsules. The much larger group of remaining genera comprises exclusively genera with straight capsules and the huge genus Cyrtandra with indehis- cent fruits. Several genera such as Briggsia , Henckelia , and Chirita are not monophyletic; Chirita is even distributed throughout fi ve clades. This degree of incongruence between molecular phylogenies, traditional classifi cations, and generic delimitations in- dicates the problems with classifi cations based on, sometimes a single, morphological characters. Key words: atp B- rbc L spacer; Bayesian inference analysis; ITS; maximum parsimony; molecular phylogeny; Old World Gesneriaceae; taxonomy; trnL-F intron-spacer. Gesneriaceae is a medium-sized family, comprising between 1983 ) have received much attention by molecular systematists 150 and 160 genera, and over 3200 species ( Weber, 2004 ; We- and apparently approach consolidation regarding a phyloge- ber and Skog, 2007 ). The distribution is mainly tropical and netic and formal classifi cation ( Smith, 1996 , 2000 ; Smith et al., subtropical, both in the Old and the New World, with minor 1997a , b ; Zimmer et al., 2002 ; Perret et al., 2003 ; Roalson et al., outliers to the north (e.g., Pyrenees, Balkan peninsula, central 2005a , b ), the paleotropical Gesneriaceae (subfam. Cyrtandroi- and northern China) and to the south (southeastern Australia, deae, now Didymocarpoideae) lag behind. The last attempt at a New Caledonia, New Zealand, southern Chile). formal (morphological) classifi cation was by Burtt and Wiehler While the neotropical Gesneriaceae (subfam. Gesnerioideae, (1995) , who distinguished fi ve tribes: (1) Cyrtandreae (with in- including Coronanthereae, raised to subfamily rank by Wiehler, dehiscent, berry-like fruits); (2) Trichosporeae (with append- aged seeds); (3) Epithemateae (Klugieae and Loxonieae sensu 1 Manuscript received 26 August 2008; revision accepted 12 December 2008. Burtt (1963) , a group rather diffi cult to characterize, partly The authors thank the horticulturists at the Royal Botanic Garden with peculiar anatomical characters such as secretory canals Edinburgh, in particular, S. Scott, S. Barber, and A. Ansell; and A. Sieder and meduallary vascular bundles); (4) Didymocarpeae (the at the Botanic Garden Vienna, for excellent work in maintaining and many remaining genera arranged alphabetically to express the accurately curating the living Gesneriaceae collections. They are also wide lack of phylogenetic understanding); and Titanotricheae grateful to staff at KIB for assistance with fi eldwork, especially Dr. D. Z. including the sole genus Titanotrichum . Additional tribes Li and Dr. L. M. Gao, and for the provision of samples by Profs. Z. J. Gu (Saintpaulieae, Ramondeae, Rhynchotecheae) have been rec- and C. Q. Zhang (KIB). The work was carried out and is still continued in the frame of a multidisciplinary cooperation project between the ognized by Ivanina (1965a , b , 1967 ) and Wang et al. (1990 , University of Vienna (supported by the Austrian Science Fund (FWF)- 1992 ). proj. no. P-13107-Bio) and the Royal Botanic Garden Edinburgh Detailed morphological investigations (Weber, 1975 – 1988, (RBGE). RBGE is supported by the Scottish Government Rural and as cited in Mayer et al., 2003 ) and, more recently, a molecular Environment Research and Analysis Directorate (RERAD). Fieldwork analysis based on cpDNA sequences ( Mayer et al., 2003 ) of M.M. was supported by the Percy Sladen Memorial Fund, Oleg showed that the Epithemateae form a distinct clade, sister to the Polunin Memorial Fund, Davis Expedition Fund of the University of remaining Old World Gesneriaceae. Both the morphological in- Edinburgh, and FWF. 5 vestigations and the molecular analyses led to a fairly good un- Author for correspondence (e-mail: [email protected] ) derstanding of the phylogenetic relationships for six of seven 6 Present address: Biology Centre Linz, Ober ö sterreichisches Landes- museum, Johann Wilhelm Klein Stra ß e 73, A-4040 Linz, Austria genera of the ‘ epithematoid Gesneriaceae ’ , as they are infor- 7 Present address: Department of Biology Education, College of Educa- mally referred to in Weber (2004) and Weber and Skog (2007) . tion, Kongju National University, Gongju, Korea The present paper deals with the diverse non-epithematoid Old World Gesneriaceae, the ‘ didymocarpoid Gesneriaceae ’ . A doi:10.3732/ajb.0800291 number of genera had already been included in the molecular 989 990 American Journal of Botany [Vol. 96 analysis of Mayer et al. (2003), and preliminary results of the thermal cycler. The 50 µ L reactions contained 5 µ L 10× NH4 reaction buffer current study formed the basis of the informal classifi cation (Bioline, UK), 5 µ L dNTPs (2 mM), 2.5 µ L MgCl 2 (50 mM), 1.5 µ L of each presented by Weber (2004) . In the meantime, the number of primer (10 µ M), 32.25 µ L dH2 O, 1.25 U Biotaq polymerase (Bioline, UK) and 1.0 µ L DNA template DNA. The PCR thermocycle profi le for trnL-F was: ini- samples included in the molecular analysis has considerably in- tial denaturation for 4 min at 94° C; followed by 30 cycles of 45 s at 94° C, 45 s creased, and suffi cient results are available to make some head- at 55° C and 3 min at 72° C; with a fi nal extension step for 10 min at 72 ° C. ITS way in our understanding of relationships in this group, amplifi cations were carried out using the PCR profi le described in White et al. particularly with respect to previous subdivisions into tribes. (1990) or M ö ller and Cronk (1997) . Nonetheless, we abstain from a new formal classifi cation at this PCR products were run on 1% agarose gels to check for amplifi cation suc- point because a signifi cant number of genera are not yet in- cess and quality. Amplifi ed fragments were purifi ed using QIAquick PCR purifi cation kits (Qiagen, Crawley, UK) following the manufacturer ’ s proto- cluded in our analysis. col, and sequenced using the dideoxy chain-termination method. Cycle se- Effectively analyzing large data sets is challenging and com- quencing was performed as in Mayer et al. (2003) , or in 10 µ L reactions plete analyses computationally intractable (e.g., Savolainen and containing 4 µ L DTCS Quickstart mix (Beckman Coulter, High Wycombe, Chase, 2003 ). Furthermore, sample-rich analyses including UK), 1 µ L primer (10 µ M), 3 µ L dH2 O and 2 µ L purifi ed PCR product, under multiple data sets often suffer from missing data or issues of the following PCR conditions: 35 cycles of 96° C for 20 s, 50° C for 20 s and incongruencies between data sets ( Sanderson and Shaffer, 60° C for 4 min. Sequencing primers were identical to those used for PCR with the addition that primer ‘ d ’ ( Taberlet et al., 1991 ) was also used to se- 2002 ). Here we address the issue of missing data by analyzing quence the trnL intron region. Sequencing atpB-rbcL followed Mayer et al. selected subsets of samples with complete data compared to a (2003) . Sequencing PCR products were purifi ed following the manufactur- complete sample set with missing data, and demonstrate that er ’ s instructions, then run and analyzed on a CEQ 8000 Genetic Analysis “ correct ” topologies of phylogenetic trees can be recovered System (Beckman Coulter, UK). Editing and assemblage of contigs was per- from largely incomplete data matrices. formed using the programs CEQuence Investigator (Beckman Coulter, High Wycombe, UK) and Sequencher 4.5 (Gene Codes Corp, Ann Arbor, USA), and for atpB-rbcL as in Mayer et al. (2003) . The cpDNA alignment matrices were assembled manually, based on those of Mayer et al. (2003) . ITS se- MATERIALS AND METHODS quences were initially aligned using the program CLUSTAL W (Larkin et al. 2007) and the alignment then adjusted manually, as described in M ö ller and Plant materials— Material for DNA extraction came in various forms and Cronk (1997) . from diverse sources, including fresh leaves from research collections (E, HBV), silica-dried