ORIGINAL ARTICLE Phylogenetic utility of MORE AXILLARY GROWTH4 (MAX4)-like genes: a case study in Digitalis/Isoplexis (Plantaginaceae) L. J. Kelly Æ A. Culham Received: 4 June 2007 / Accepted: 21 December 2007 / Published online: 10 May 2008 Ó Springer-Verlag 2008 Abstract We present the first assessment of phylogenetic utility of a potential novel low-copy nuclear gene region in flowering plants. A fragmentof the MORE AXILLARY GROWTH 4 gene (MAX4, also known as RAMOSUS1 and DECREASED APICAL DOMINANCE1), predicted to span two introns,was isolated from members of Digitalis/ Isoplexis.Phylogenetic analyses, underboth maximum parsimony and Bayesian inference, were performed and revealed evidence of putative MAX4-like paralogues. The MAX4-like trees were compared with those obtained for Digitalis/Isoplexis using ITS and trnL-F, revealing a high degreeof incongruence betweenthesedifferentDNA regions.Network analyses indicate complex patterns of evolution between the MAX4 sequences, which cannot be adequately represented on bifurcating trees. The incidence of paralogy restricts the use ofMAX4 in phylogenetic inference within the study group, although MAX4 could potentially be used in combination with other DNA regions for resolving species relationships in caseswhere para- logues can be clearly identified. Keywords Digitalis Isoplexis Low-copy nuclear gene region MAX4/RMS1/DAD1 Molecular phylogeny Network Paralogy Introduction One of the greatest challenges facing plant molecular phy logenetics remains the development of appropriate DNA regions to act as sources of characters. It is widely unders that in order to reconstruct species relationships accurate data from multiple independently evolving DNA regions need to be incorporated into phylogenetic analyses (Pamil and Nei 1988; Doyle 1992; Strand et al. 1997; Soltis and Soltis 2000; Cronn et al. 2002a; Rokas et al. 2003; Rokas a Carroll 2005). Plastid DNA has been the most widely used source ofcharacters for phylogenetic inference in plants (Small et al. 2004). The internal transcribed spacer region (ITS) of nuclear ribosomal DNA (nrDNA) is one of the most common sources of characters for use in low-level phylo- genetic investigations (A ´ lvarez and Wendel 2003; Hughes et al. 2006). Whilst congruence between plastid DNA and nrDNA trees can be used to provide confidence that the relationships inferred arean accurate reconstruction of organismal history, in some cases it is necessary to seek additional loci to allow resolution of conflict between com- peting hypotheses (Cronn et al. 2002a). It has been sugge that nuclear genes represent a virtually unlimited source o molecular characters for use in phylogenetic inference of plants (Sang 2002; A ´ lvarez and Wendel 2003; Small et al. 2004), and may offer a variety of other beneficial features (for reviews see Sang 2002; Mort and Crawford 2004; Sma et al. 2004). Consequently there has been a steady increa the number of low-copy nuclear gene regions being devel- oped for use in phylogenetic investigations of plants (e.g. Mathews and Sharrock 1996; Mason-Gamer et al. 1998; Tank and Sang 2001; Martins and Barkman 2005; Syring et al. 2005; Whittall et al. 2006). Nevertheless, there are s relatively few regions that have been tested in a range of lineages(Mortand Crawford 2004; Smallet al. 2004). L. J. Kelly A. Culham Centre for Plant Diversity and Systematics, School of Biological Sciences, University of Reading, Whiteknights, Reading RG6 6AS, UK L. J. Kelly (&) Jodrell Laboratories, Royal Botanic Gardens, Kew,Richmond, Surrey TW9 3DS, UK e-mail: l.kelly@rbgkew.org.uk 123 Plant Syst Evol (2008) 273:133–149 DOI 10.1007/s00606-008-0008-0