Pl. Syst. Evol. 261: 245–256 (2006) DOI 10.1007/s00606-006-0445-6 A duplication of gcyc predates divergence within tribe Coronanthereae (Gesneriaceae): Phylogenetic analysis and evolution J. F. Smith1, M. M. Funke1, and V. L. Woo2 1Department of Biology, Boise State University, Boise, Idaho USA 2School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand Received February 8, 2006; accepted March 10, 2006 Published online: September 19, 2006 Ó Springer-Verlag 2006 Abstract. Recent investigations in Gesneriaceae genes (Luo et al. 1996, Almeida et al. 1997, have indicated that the cycloidea homolog, gcyc, Reeves and Olmstead 1998). By studying remains functional at the DNA level and rates of mutant plants of Antirrhinum majus L. (snap- sequence divergence in this gene are not statistically dragon) that lack the wild type floral bilateral different across all taxa regardless of floral symme- symmetry, Luo et al. (1996, 1999) identified two cyc try. A duplication of g has been detected within loci that are essential for the development of Coronanthereae, a tribe that has phylogenetic bilaterally symmetrical flowers. These are cy- affinities to subfamily Gesnerioideae and includes two genera with radially symmetrical corollas. cloidea (cyc) and dichotoma (dich). Both genes Duplication of gcyc has been detected in all are essential for full bilateral symmetry in Coronanthereae except Sarmienta. All paralogs Antirrhinum but the role of cyc is more impor- appear functional at the DNA level. Likewise, tant and acts early in floral development. The there is no increased sequence divergence between developmental aspects of these flower symme- the two copies, nor between species with radially try genes in Antirrhinum have stimulated inter- symmetrical flowers to those with bilateral symme- est in the evolution of plant groups with both try. The duplication of gcyc in Coronanthereae is bilateral and radial floral symmetry (Coen and most likely a result of polyploidy since Coronan- Nugent 1994; Coen et al. 1995; Coen 1996; thereae have the highest chromosome counts of all Cronk and Mo¨ ller 1997; Endress 1997; Run- Gesneriaceae. ning 1997; Donoghue et al. 1998; Baum 1998; Moller et al. 1999; Citerne et al. 2000; Theißen Key words: Gesneriaceae, Coronanthereae, paralogs, ¨ gcyc, floral symmetry, phylogeny, polyploidy. 2000; Cronk 2002; Cubas 2002; Gillies et al. 2002; Hileman and Baum 2003; Hileman et al. 2003; Smith et al. 2004a, b). One family of Asteridae that manifests Floral symmetry is a trait of systematic and shifts in floral symmetry is Gesneriaceae ecological importance in Asteridae (Cronk (Cronk and Mo¨ ller 1997). Gesneriaceae and and Mo¨ ller 1997, Donoghue et al. 1998) and nearly all close relatives of the family (placed aspects of symmetry in Asteridae are thought to in Lamiales sensu APGII 2003) have bilater- be dependent on a small set of floral symmetry ally symmetrical flowers. Additionally, peloric 246 J. F. Smith et al.: Duplication of gcyc in Coronanthereae mutants of Saintpaulia Wendl., Sinningia and another within tribe Coronanthereae speciosa, and Sinningia cardinalis (Lehm.) H. (Wang et al. 2004). E. Moore indicate that shifts from bilateral Tribe Coronanthereae has been poorly to radial symmetry can occur within a species studied within Gesneriaceae (Weber 2004). as it has been seen for Antirrhinum (Luo et All species placed in this tribe have long been al. 1996, 1999) and Linaria Mill. (Cubas et al. considered a monophyletic group and are 1999). Phylogenetic analyses (Smith 1996, unique among all Gesneriaceae in having 2000; Smith et al. 1997) indicate that the nectaries embedded in the ovaries (Wiehler ancestral condition for Gesneriaceae is bilat- 1983, Weber 2004). Additionally, chromosome eral symmetry and that radially symmetrical counts for these species are among the highest flowers have been derived several times from for all Gesneriaceae with counts ranging from within otherwise bilaterally symmetrical flow- estimates of n=37 to n = +/) 45 (Wiehler ered clades (Cronk and Mo¨ ller 1997, Mo¨ ller 1983, Skog 1984, Weber 2004). These counts et al. 1999, Smith 2000). Mo¨ ller et al. (1999) are relative to n = 8–14 and 16 for other identified a homolog to cyc in Gesneriaceae, Gesnerioideae (Wiehler 1983, Skog 1984). gcyc. Investigations into the function of gcyc Seedlings are isocotylous (Weber 2004), indi- in taxa with radially symmetrical taxa have cating that there may be a closer link to not revealed any mutations that may be Gesnerioideae since members of Cyrtandroi- directly responsible for the shift in floral deae are distinguished by anisocotylous seed- symmetry (Mo¨ ller et al. 1999; Citerne et al. lings (Burtt 1963). 2000; Smith et al. 2004a, b; Wang et al. Early phylogenetic analysis of Gesneria- 2004). Likewise, these same studies did not ceae indicated that Coronanthereae were sister discover a statistically significant increased to the remainder of subfamily Gesnerioideae rate of sequence divergence in taxa with based either on morphological (Smith 1996) or radially symmetrical flowers in comparison to molecular characters (Smith et al. 1997). their bilaterally symmetrical flowered sister Subsequent analyses at the family level have, taxa, indicating that gcyc is still under 1) supported this position (Smith 2000), 2) positive selection and may have functions shown an unresolved polytomy among the beyond determining floral symmetry in Ges- Coronanthereae, Napeantheae, Beslerieae, and neriaceae. the remainder of Gesnerioideae (Mayer et al. For most Gesneriaceae, gcyc exists as a 2003), 3) shown that Coronanthereae are a single copy, at least based on the means that part of Gesnerioideae, but the relationships have been used to detect multiple copies such between Coronanthereae, Beslerieae and Na- as PCR and cloning (Mo¨ ller et al. 1999; peantheae are poorly supported (Smith et al. Citerne et al. 2000; Smith et al. 2004a, b; 2004a). Wang et al. 2004). However, duplications have The tribe comprises nine genera distrib- been detected within Gesneriaceae, and based uted either in the South Pacific (Negria, on current phylogenetic understanding, imply Coronanthera, Depanthus, Rhabdothamnus), at least four separate duplication events, one Australia (Lenbrassia, Fieldia) or Southern during the early divergence of the family giving South America (Sarmienta, Asteranthera, Mit- rise to gcyc1 and gcyc2 that may have occurred raria). This tribe often has been treated as a prior to the divergence between Gesnerioideae separate subfamily of Gesneriaceae, Coro- and Cyrtandroideae or may only be ancestral nantheroideae (Wiehler 1983, Burtt and to Cyrtandroideae. Another duplication has Wiehler 1995) since its distribution bridges been detected within Saintpaulia and Strepto- that of the two other subfamilies, Gesnerioi- carpus (Citerne et al. 2000), and the remaining deae with a primarily New World distribu- two consist of a duplication that preceded the tion, and Cyrtandroideae that is nearly divergence of Loxostigma and Didymocarpus exclusively Old World. J. F. Smith et al.: Duplication of gcyc in Coronanthereae 247 With the exception of Coronanthera which copy number nuclear gene, gcyc followed the comprises 11 species and Depanthus with two protocols of Smith et al. (2004a). Up to 20 clones species, all other genera are monotypic. Burtt per species were sequenced in order to detect both (1998) has hinted that Depanthus may be paralogs. Sequences were obtained from both congeneric with Coronanthera. Both genera strands of all gene regions to be examined via are known from New Caledonia and Depan- PCR amplification and sequencing with a Li-Cor LongreadIR 4200 automated sequencer. The thus is separated from Coronanthera on the pGEM-T vector system kit (Promega) was used basis of having a radially symmetrical corolla to clone gcyc. Wang et al. (2004) detected paralogs with five fertile stamens as opposed to the of gcyc in Fieldia in their analysis. To determine bilabiate corolla with four fertile coherent which of each of the paralogs in Coronanthereae stamens found in Coronanthera. Thus the matched these sequences, both Fieldia gcyc1E species of Depanthus represent a shift from (AY423151) and gcyc1F (AY423152) were in- bilateral to full radial symmetry. Likewise, cluded in one phylogenetic analysis to resolve Lenbrassia has been suggested as being synon- which of the two paralogs from within Fieldia ymous with Fieldia (Burtt 1999). The species matched gcyc1E and gcyc1F. The gcyc1E of are similar morphologically, but differ in habit, Mitraria (AY423153) from Wang et al. (2004) Lenbrassia is a small tree and Fieldia is an was also included. epiphytic subshrub with a nearly regular The data were analyzed using PAUP* 4.0b10 (Swofford 2000) utilizing both maximum parsi- corolla (Weber 2004), potentially again repre- mony (MP) and maximum likelihood (ML). Indels senting a shift from bilateral to radial symme- were either treated as missing data, or rescored try, at least in the corolla. Both species are following the methods of Simmons and Ochoterena from Australia. (2000). Additionally, since gcyc lacks introns, The duplication of gcyc within Coronan- indels were rescored as multi-state characters using thereae allows for a further investigation on the amino acid translation as a template and the potential functionality of this gene within scoring characters as groups of three base pairs Gesneriaceae. This study proposes to examine corresponding to codons (Baum et al. 1994). For sequences of gcyc in Coronanthereae