Aagaard, J., Willis, J., and Phillips, P. 2006

J Mol Evol (2006) xx:1–13 DOI: 10.1007/s00239-005-0306-x Relaxed Selection Among Duplicate Floral Regulatory Genes in Lamiales Jan E. Aagaard,1,3 John H. Willis,2 Patrick C. Phillips1 1 Center for Ecology and Evolutionary Biology, University of Oregon, Eugene, OR 97403, USA 2 Department of Biology, Duke University, Durham, NC 27708, USA 3 Department of Genome Sciences, University of Washington, Box 357730, Seattle, WA 98195, USA Received: 19 December 2005 / Accepted: 12 June 2006 [Reviewing Editor: Dr. Yves van de Peer] Abstract. Polyploidization is a prevalent mode of responsible for preservation of co-orthologs of FLO genome diversification within plants. Most gene and DEF in Lamiales, including interactions among duplicates arising from polyploidization (paralogs) the genes of this regulatory pathway. are typically lost, although a subset may be maintained under selection due to dosage, partitioning of Key words: Gene duplication — Floral regulatory gene function, or acquisition of novel functions. Be- genes cause they experience selection in the presence of other duplicate loci across the genome, interactions among genes may also play a significant role in the maintenance of paralogs resulting from polyploidization. Previously, we identified duplicates of the Introduction genes LFY/FLO and AP3/DEF that directly interact in a floral regulatory pathway and are thought to be Polyploidization occurs frequently in flowering the result of ancient polyploidization in the Lamiales plants. Estimates of the frequency of polyploidization (> 50 mya). Although duplicates of MADS box based on cytological studies vary widely but sug- genes including AP3/DEF are common throughout gest up to 70% of angiosperms may be polyploids the angiosperm lineage, LFY/FLO duplicates in (Wendel 2000; Otto and Whitton 2000). Analysis of Lamiales are the first reported outside of tetraploid cDNA and genomic sequences similarly shows that taxa. In order to explore hypotheses for the joint both recent and ancient polyploidization among crop preservation of these interacting floral regulatory and model plant taxa are common (Blanc and Wolfe genes including novel LFY/FLO paralogs, here we 2004a). The relative importance of polyploidization clone FLO and DEF duplicates from additional to the evolution and diversification of flowering Lamiales taxa and apply codon substitution models plants (angiosperms) has been extensively debated to test how selection acts on both genes following (e.g., Stebbins 1971; Levin 1983) and is reviewed duplication. We find acceleration in the ratio of elsewhere (Otto and Whitton 2000). It is clear, how- nonsynonymous-to-synonymous nucleotide substitu- ever, that polyploidization contributes significantly tions for one (FLO) or both (DEF) paralogs that to the long-term genomic content of angiosperm appears to be due to relaxed purifying selection as taxa. For example, more than half of all genes from opposed to positive selection and shows a different Arabidopsis thaliana are thought to be the result of pattern among functional domains of these genes. ancient polyploidization (Maere et al. 2005), with Several mechanisms are discussed that might be approximately 20% of predicted genes resulting from a whole-genome duplication event that occurred Correspondence to: Jan E. Aagaard; email: jaagaard@gs. 24–40 million years ago (mya; Blanc et al. 2003; Blanc washington.edu and Wolfe 2004b). 2 Because the contribution of polyploidization to have broad impact on duplicate gene preservation plant evolution is ultimately a function of the across the polyploid genome through concerted duplicate genes that are maintained within ge- divergence of the interacting members of genetic nomes, it is important to understand both the pathways and gene networks (Blanc and Wolfe patterns and mechanisms of duplicate gene preser- 2004b), essentially a multilocus extension of sub- vation. The majority of duplicates (paralogs) arising functionalization models. from polyploidization in plants are silenced. In the Previously, we identified duplicates of several case of A.thaliana , nearly 70% of paralog pairs regulatory genes important in floral development from the last polyploid event have lost one member within the order of tricolpate plants Lamiales (Aag- (Blanc et al. 2003). Although there is some sug- aard et al. 2005). These include co-orthologs of the gestion that paralogs resulting from polyploidiza- floral meristem identity gene FLO (Coen et al. 1990) tion may persist longer (Otto and Whitton 2000; and floral homeotic MADS box gene DEF (Sommer Moore and Purugganan 2003; Lynch and Conery et al. 1990) from the model Lamiales species Antir- 2001), this is generally consistent with patterns for rhinum majus. Orthologs of FLO and DEF from A. smaller-scale duplication events (e.g., tandem or thaliana (LFY and AP3, respectively [Weigel et al. segmental duplications) which suggest that silencing 1992; Jack et al. 1992]) carry out similar functions in due to loss of function mutations predominates the floral regulatory pathway as in A.majus , where (Walsh 1995), resulting in a half-life for fully LFY/FLO positively regulates the expression of the redundant paralogs which is generally less than 10 downstream gene AP3/DEF (Ingram et al. 1997; million years (Lynch and Conery 2000). However, Weigel and Meyerowitz 1993). Duplication of the A. the probability that one member of a paralog pair majus genes FLO and DEF in Lamiales is thought to is lost appears to differ markedly among functional be the result of a whole-genome duplication (poly- classes of genes depending on the mechanism of ploidization) which occurred after the split between gene duplication. For example, in A.thaliana par- the ancestor of A.majus (Veronicaceae) and the alogs of regulatory genes involved in transcription lineage leading to many of the other families of and signal transduction are retained at a higher Lamiales including Verbenaceae, Paulowniaceae, rate if duplicates are the result of polyploidization Phrymaceae, and Orobanchaceae (Aagaard et al. rather than smaller-scale gene duplication events 2005). Co-orthologs of both genes (FLOA and (Maere et al. 2005). FLOB, DEFA and DEFB) are expressed and appear Paralogs that escape the typical fate of silencing functional in multiple taxa from these four families, and are maintained within genomes under selection and have been maintained within multiple lineages may be preserved via a number of mechanisms. that diverged 30–50 mya (Wickstrom et al. 2001). Theory suggests that preservation of paralogs is Significantly, duplicates of AP3/DEF from several primarily due to parsing of the ancestral single- points along the angiosperm phylogeny are known copy geneÕs functions among duplicates (subfunc- (Theissen et al. 2000; Kramer et al. 1998, 2003), al- tionalization) rather than the acquisition of novel though Lamiales represents an as yet novel case of gene function driven by positive selection, based in ancient LFY/FLO paralogs (Cronk 2001). large part on the predominance of degenerate ra- The purpose of the present study is to explore ther than adaptive mutations (e.g., Force et al. hypotheses regarding the evolutionary forces acting 1999). Subfunctionalization can result from com- among these paralogs that may be responsible for the plementary degenerate mutations restricted to reg- maintenance of duplicate copies. Here we clone ulatory regions as in the model of Force et al. additional co-orthologs of the floral regulatory genes (1999) or involve functional domains within protein FLO and DEF from four taxa within the Lamiales coding regions, resulting in an altered pattern of family Phrymaceae (sensu Beardsley and Olmstead amino acid substitution between duplicates (Der- 2002) to supplement our earlier work (Aagaard et al. mitzakis and Clark 2001). Because paralogs arising 2005) in order to have sufficient power for likelihood- from polyploidization experience selection along based statistical tests employing codon substitution with other duplicate loci across the genome, inter- models (Aagaard and Phillips 2005). We then test for actions among loci may also contribute significantly rate heterogeneity and the role of selection in diver- to their maintenance. Such interactions could result gence among FLO and DEF paralogs following from simple dosage effects, which should predomi- duplication in Lamiales using several different codon nate among genes functioning within metabolic substitution models. Based on these results, we ex- pathways and regulatory genes involved in signal plore several mechanisms to explain the maintenance transduction and transcription (Birchler et al. of the joint preservation of paralogs from multiple 2001). Alternately, divergence in the timing or genes that are interacting members of the floral reg- expression domains among pairs of paralogs could ulatory pathway. 3 spanning intron 2, 5¢-RACE was used to extend sequence to the 5¢- termini of coding regions. In contrast, AP3/DEF-like homologs were cloned directly from 3¢-RACE cDNA pools. Briefly, a single degenerate primer at the 5¢ terminus of the DEF coding region (5¢-ATGGCTCGTGGGAAGATHCARAT-3¢) was used in 3¢- RACE, after which PCR products were directly cloned and sequenced (at least 12clones sequenced per 3 ¢RACE PCR). Nucleotide alignments of the newly cloned LFY/FLO- and AP3/DEF-like genes from Phrymaceae as well as Solanales and Lamiales homologs (Supplementary Table S1) were carried out initially based on the translated nucleotide (protein) sequence using the ClustalX algorithm

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