PALEVO 1631-0683 Elsevier S1631-0683(03)00031-9 10.1016/S1631-0683(03)00031-9 Research article Évolution Independent evolution of Cycloidea-like sequences in several angiosperm taxa Damerval Catherine damerval@moulon.inra.fr a b Manuel Michaël c Station de génétique végétale, INRA/UPS/INA-PG/CNRS UMR 8120, La Ferme du Moulon, 91190 Gif-sur-Yvette, France Groupe « Développement et Évolution », université Pierre-et-Marie-Curie/CNRS UMR 7622, 9, quai Saint-Bernard, case 241, 75252 Paris cedex 05, France Service commun de biosystématique, université Pierre-et-Marie-Curie, 9, quai Saint-Bernard, case 241, 75252 Paris cedex 05, France 2 3 S1631-0683(00)X0011-5 241 250 © 2003 Académie des sciences. Published by Elsevier B.V. All rights reserved. 2003 Académie des sciences Full (PDF)

The TCP family of putative transcriptional factors, defined by the founding members Teosinte branched1, Cycloidea and PCF, is characterised by a specific basic helix-loop-helix domain. CYC and PCF subfamilies have been defined on the basis of sequence differences in conserved domains. Twenty-four coding sequences containing a TCP domain were found in the complete genome of Arabidopsis thaliana using BLAST searches. A neighbour-joining analysis of 112 sequences from various angiosperm taxa, belonging to the TCP family, suggests (i) homoplasy for the presence/absence of an R domain, (ii) multiple independent duplications leading to the wide diversity of CYC subfamily sequences in the five plant families sampled.

Évolution indépendante de séquences homologues de Cycloidea dans plusieurs taxons d’angiospermes. La famille de facteurs de transcription «TCP », définie initialement à partir des gènes Teosinte branched1, Cycloidea et PCF, est caractérisée par un domaine hélice-boucle-hélice basique original. Les sous-familles CYC et PCF ont été définies d’après divers domaines conservés. Vingt-quatre séquences codantes comportant un domaine TCP ont été trouvées dans le génome complet d’Arabidopsis thaliana. Une analyse de neighbour-joining sur 112 séquences de la famille TCP suggère (i) l’existence d’homoplasie pour la présence du domaine R, (ii) des événements multiples de duplication de gènes conduisant à une grande diversité de la sous-famille CYC, indépendants dans les cinq familles de plantes étudiées.

TCP family, CYC subfamily, Cycloidea, Arabidopsis, Angiosperm, Molecular phylogeny, Evolution Famille TCP, Sous-famille CYC, Cycloidea, Arabidopsis, Angiospermes, Phylogénie moléculaire, Évolution presented Presented by Philippe Taquet Version française abrégée Introduction

Les gènes Cycloidea (CYC) et Dichotoma (DICH) interviennent dans le contrôle du développement floral chez Antirrhinum majus, où ils sont responsables de la zygomorphie de la fleur adulte [16] and [17]. Ces deux gènes codent des protéines comportant un domaine conservé de type bHLH (hélice-boucle-hélice basique) dénommé domaine TCP (du nom des gènes qui définissent la famille, Teosinte Branched1 (TB1), CYC et PCF), qui caractérise une famille d’activateurs de transcription supposés [6]. Cette famille n’a jusqu’ici été trouvée que chez les végétaux. Les quelques membres dont on a étudié l’expression (CYC, DICH, LCYC-orthologue de CYC chez Linaria vulgaris [5], TCP1, TCP2 et TCP3 chez Arabidopsis [5], [6], [7], [16] and [17]) semblent intervenir dans des processus de croissance et de développement (voir aussi [8], [9] and [12].

Deux sous-familles sont définies sur la base de la séquence du domaine TCP et d’autres régions conservées [6]. La première, la sous-famille CYC, possède un signal de localisation nucléaire en deux parties, altéré dans la seconde, la sous-famille PCF. Des différences existent également dans la composition des hélices et de la boucle qui les sépare, ainsi que dans la longueur de la seconde hélice. Certains membres de la sous-famille CYC, dont CYC, DICH et TB1, comportent en outre un domaine riche en acides aminés polaires, situé à des distances variables en aval du domaine TCP, dit domaine R. Les membres de la sous-famille PCF comportent d’autres domaines conservés adjacents au domaine TCP [6].

La diversité des gènes homologues de CYC a été analysée dans deux familles botaniques de l’ordre des Lamiales sensu APG [2], les Veronicaceae sensu Olmstead et al. [18] et les Gesneriaceae [3] and [20]. Une famille oligogénique de cinq membres au moins a été mise en évidence chez les Veronicaceae, et de deux membres chez les Gesneriaceae. Les duplications/pertes de gènes à l’origine de cette complexité semblent s’être produites de manière indépendante dans les deux taxons [3].

Afin de préciser la complexité de la famille TCP et l’évolution des gènes de la sous-famille CYC, nous avons tout d’abord recensé les séquences codantes comportant un domaine TCP dans le génome complet d’Arabidopsis thaliana (Brassicaceae), puis réalisé une phylogénie de 112 séquences extraites de Genbank, en axant l’analyse sur les gènes de la sous-famille CYC, principalement chez les eudicotylédones.

 Matériel et méthodes

La recherche de séquences codantes comportant un domaine TCP dans le génome complet d’Arabidopsis thaliana a été faite par BLAST (BLASTp et tBLASTn, [1]) à l’aide de séquences complètes d’homologues de CYC et des domaines TCP de CYC et de PCF1.

Les séquences des gènes des sous-familles CYC et PCF ont été obtenues à partir de Genbank [3], [5], [15], [16], [17] and [20]. L’échantillon de gènes TB1 a été volontairement restreint après une analyse préliminaire, afin de focaliser l’étude sur les eudicotylédones.

Au total 112 séquences ont été analysées. L’alignement nucléotidique a été réalisé grâce au logiciel BioEdit (version 4.8.6) [13] et amélioré grâce aux séquences protéiques. La divergence importante des séquences en dehors des domaines TCP et R nous a conduits à ne conserver que ces deux domaines et la région qui les sépare pour les analyses phylogénétiques (834 positions). Pour les séquences dépourvues de domaine R (toutes celles de la sous-famille PCF et certaines de la sous-famille CYC), seule la séquence du domaine TCP a été prise en compte (177 nucléotides), les autres positions étant codées en données manquantes. Les délétions insérées pour maximiser l’alignement ont également été codées en données manquantes. Les analyses phylogénétiques ont été réalisées avec le logiciel PAUP (version 4.06b) [19] sur les alignements nucléotidiques et protéiques. La méthode du neighbour-joining (correction de Jukes et Cantor pour la distance nucléotidique [14]) a été appliquée à la matrice complète, et une analyse de parcimonie a été faite sur un échantillon restreint à 21 séquences.

 Résultats et discussion

Vingt-quatre séquences codantes comportant un domaine TCP ont été trouvées dans le génome complet d’Arabidopsis thaliana (voir aussi [4]). Onze appartiennent à la sous-famille CYC (dont cinq comportent également un domaine R) et 13 à la sous-famille PCF (Tableau 1).

Une analyse par neighbour-joining a été réalisée sur ces 24 séquences et 88 séquences extraites de Genbank, représentant la diversité des séquences TCP connues jusqu’ici principalement chez les eudicotylédones. Les résultats obtenus sur les alignements nucléotidiques (834 positions) et protéiques (278 positions) sont comparables et seul l’arbre issu de l’analyse nucléotidique est présenté (Fig. 1). Comme nous souhaitions focaliser l’analyse sur l’évolution des gènes de la sous-famille CYC, nous avons arbitrairement raciné l’arbre sur la sous-famille PCF.

Les séquences dépourvues de domaine R ne forment pas un groupe monophylétique, mais apparaissent en trois groupes, qui comportent dans certains cas des séquences portant le domaine R. Il semble donc exister de l’homoplasie pour la présence/absence de ce domaine. L’hypothèse la plus parcimonieuse serait qu’il est apparu une fois à la base de la sous-famille CYC, et a été perdu plusieurs fois indépendamment (au minimum trois fois d’après l’arbre obtenu).

Par ailleurs, l’ensemble des séquences de Veronicaceae et de Gesneriaceae constitue un groupe monophylétique (bootstrap 81%). À l’intérieur de ce groupe, des groupes d’orthologie bien soutenus correspondent aux différents membres des familles oligogéniques mises en évidence chez ces deux familles botaniques par les études antérieures [3] and [20]. Il est remarquable qu’aucun panachage de séquences issues des deux taxons n’apparaisse. De même, les duplications à l’origine des séquences de Mimulus (Phrymaceae) ont eu lieu indépendamment de celles des Veronicaceae et Gesneriaceae. Enfin, parmi les cinq séquences d’Arabidopsis possédant à la fois les domaines TCP et R, aucun orthologue, ni du gène CYC d’A. majus, ni d’aucun autre gène de l’analyse, n’apparaît. Nous confirmons, donc sur un échantillonnage taxinomique plus important que celui de Citerne et al. [3], que l’évolution des gènes de la sous-famille CYC s’est faite par des duplications/pertes multiples de gènes, qui ont eu lieu de manière indépendante dans les différents taxons. L’analyse de parcimonie confirme ces résultats (Fig. 2).

Cette évolution indépendante selon les taxons pose la question de la conservation de la fonction et du rôle des gènes de la sous-famille CYC. Cette fonction semble liée à la croissance et au développement ; la symétrie/asymétrie du territoire d’expression de ces gènes pourrait déterminer l’apparition de structures morphologiques symétriques ou non. Un échantillonnage taxinomique plus large ainsi que des études d’expression plus nombreuses sont indispensables pour mieux comprendre l’évolution de la sous-famille CYC, et pour examiner son rôle dans l’élaboration de structures morphologiques symétriques ou non.

 Introduction

The TCP family of putative transcription factors is characterised by an original conserved basic-Helix-Loop-Helix (bHLH) domain that was initially found in Teosinte branched1 (TB1) of Zea mays, Cycloidea (CYC) and Dichotoma (DICH) from Antirrhinum majus, and two Oryza sativa DNA-binding proteins, PCF1 and PCF2. The initials of the founding members (TB1, CYC and PCF) were used to name the family [6]. So far, the TCP family appears to be restricted to plants.

Two subfamilies were defined based on the characteristics of the TCP domain and other conserved regions [6]. The first one (CYC subfamily), including CYC and DICH from A. majus and TB1 from Z. mays, has a bipartite nuclear localisation signal (NLS), while the second one (PCF subfamily), including the PCFs from O. sativa, has only a portion of the bipartite NLS. Moreover, differences in residue composition exist in the hydrophobic faces of the helices and the loop, and helix II is shorter in the PCF subfamily. Some members of the CYC subfamily are characterised by a second domain rich in polar residues, located at various distances downstream to the TCP domain, called the R domain [6]. A third conserved domain, rich in proline and serine residues, has been described as specific to the TB1 protein in Andropogoneae and a few other grasses [15]. Members of the PCF subfamily share conserved regions adjacent to the TCP domain [6].

Genes belonging to this family have been suggested to play a role in plant growth and development. Cycloidea (CYC) and Dichotoma (DICH) genes are involved in the control of floral development in Antirrhinum majus, promoting a zygomorphic adult flower in wild-type genotypes [16] and [17]. Both are expressed asymmetrically, in the dorsal part of developing floral meristems. CYC has been found to repress a D-cyclin gene in the dorsal stamen of Antirrhinum flower [12]. In maize, TB1 is involved in arresting axillary meristem growth, reducing internode elongation in axillary branches and arresting petal and stamen development in female flowers [8] and [9]. PCF1 and PCF2 were isolated on the basis of their ability to bind to promoter elements of a rice gene whose product is involved in DNA replication and cell cycle control. Among new genes discovered in this family [6], expression of TCP1, TCP2 and TCP3 genes of Arabidopsis are observed in developing floral meristems. Expression of TCP2 and TCP3 does not differ between dorsal and ventral part of the primordia [6], while expression of TCP1 is transient in the dorsal part of the primordia [7].

Within the CYC subfamily, the diversity of so-called Cycloidea-like genes has been analysed in two angiosperm families belonging to the order Lamiales sensu APG [2]. In twelve species of Veronicaceae sensu Olmstead et al. [18], an oligogenic family of at least five members, called CYC1A and 1B, CYC2, CYC3 and CYC4, was found. The orthologues of four of the five genes were found in all sampled taxa, and CYC3 was present in Misopates orontium, but not deeply investigated in the other taxa. CYC1A and 1B are very similar and must have originated through a recent duplication [20]. In the family Gesneriaceae, several Cycloidea-like sequences, termed GCYC, were detected from most species studied [3]. Orthologues of GCYC1 occurred in all studied taxa, with a recent duplication in the Streptocarpus/Saintpaulia clade giving birth to GCYC1A and 1B. Orthologues of GCYC2 were detected only in Ramonda, Conandron and Haberlea.

In the Lamiales sensu APG, a zygomorphic flower is predominant and believed to be the ancestral state [11]. Under this assumption, the actinomorphic flowers observed in many species among the Gesneriaceae should be derived. However, no clear relationship appeared between the number of GCYC genes or their sequence and flower actinomorphy or zygomorphy. Based on an average mutation rate estimated for the CYC gene in Veronicaceae (1.5 × 10–9 nucleotide per year), the divergence between (CYC1, CYC2) on the one hand, and (CYC3, CYC4) on the other hand was estimated to occur about 75 Myr ago, which suggests that orthologues should exist in other angiosperm families [20]. However, this molecular datation is strongly questioned by the fact that no orthologues of CYC, DICH and LCYC (an orthologue of CYC in Linaria vulgaris [5]) from Veronicaceae seem to exist among the GCYC sequences of Gesneriaceae [3].

The availability of the full genomic sequence of Arabidopsis thaliana (Brassicaceae) gives the opportunity to count and compare all members of the TCP family in a given genome. We included the 24 Arabidopsis sequences with a TCP domain also found by Cubas [4] in a phylogenetic analysis of CYC-like sequences available in Genbank, with a specific focus on eudicotyledon species. Our objectives were to investigate the evolution of the CYC subfamily, and to search for putative orthologues of CYC, which would give clues on possible involvement of CYC-like genes in floral symmetry in taxa other than Veronicaeae.

 Material and methods Search for TCP homologous coding sequences in the full genomic sequence of <italic>Arabidopsis</italic>

In a first step, translated sequences of Y16313 (Antirrhinum majus), AF208338 (Sreptocarpus holstii), AF208318 (Ramonda myconi), AF208335 (Streptocarpus dunnii), AF146880 and AF146873 (Misopates orontium), AF199465 (DICH from Antirrhinum majus), AF161252 (LCYC from Linaria vulgaris) and AF146862 (Linaria triornithophora) were used to perform BLASTp [1] searches on the non-redundant database with an advanced search restricted to Arabidopsis thaliana (ORGN) and a threshold of 10–4 for the e value. To check that no sequence was missed using this procedure, (i) tBLASTn searches were performed on the Arabidopsis genome, using the nucleotide sequence of the CYC (Y16313) and the PCF1 TCP domains, and (ii) every retrieved sequence was used to perform BLASTp on the full Arabidopsis genome.

 CYC subfamily sequences

Eighty eight sequences were retrieved from Genbank (see Fig. 1): 48 from [20], 26 (the Sinningia peloric mutant sequence being excluded) from [3], four corresponding to two Mimulus species, the LCYC gene of Linaria vulgaris [5], the originally isolated CYC and DICH sequences [16] and [17], a CYCLOIDEA-like sequence from Lycopersicon esculentum, a TCP3 homologous sequence from Oryza sativa, one TB1 sequence from Oryza sativa and one TB1 gene from Populus.

In a first step, all TB1 sequences from the Andropogoneae analysed in [15] were retrieved and included in the neighbour-joining analysis (see below). However, since we wanted to focus on the eudicotyledon sequences, we present our results including only the TB1 sequence from Capillipedium parviflorum, chosen on the basis of its position in the complete analysis.

 Multiple sequence comparison and sequence analysis

DNA alignments were constructed using ClustalW as implemented in BioEdit version 4.8.6 [13], and visually refined on the basis of amino acid sequences. Because the sequences from Veronicaceae, Gesneriaceae, and Mimulus, all belonging to the Lamiales sensu APG, were very divergent outside the conserved domains, we chose to work in a region extending from the TCP to the R domains. The more variable interdomain region (i.e. between TCP and R domains) was kept because assessment of primary homology among the sequences appeared reasonable, at least among groups of related sequences. For TCP sequences devoid of an R domain, only the TCP domain was taken into account (177 nucleotides, 59 amino acid residues) and all other positions were coded as missing data. The total number of nucleic acid positions in the alignment was 834, of which 415 were constant. Phylogenetic analyses were performed using PAUP version 4.06b [19], from both amino acid and nucleotide matrices. The neighbour-joining (NJ) method was used with distance p for amino acid data and correction of Jukes and Cantor [14] for nucleotide data. Bootstrap was performed with 1000 replicates.

The choice of NJ can be criticized, because distance methods are not phylogenetic in their principle, and because NJ is less performing than other available methods, like maximum parsimony or maximum likelihood. However, in practice, NJ analyses efficiently recover the main sequence clusters from datasets such as ours (i.e. with many sequences and few informative positions), provided that significant nodes are distinguished from artefactual ones in the entirely resolved tree produced. Branch length and bootstrap values are the two criteria we used to identify significant nodes in the NJ trees. Maximum parsimony was also performed, but with the complete dataset, the high number of sequences relatively to the number of informative positions resulted in a huge number of equally parsimonious minimal topologies (as a result of the high degree of irresolution) and, for that reason, even the heuristic analysis could not be completed. A common practice in such a case is to show a strict-consensus tree computed from an arbitrary number of minimal trees, after the search has been aborted; but this results in neglecting a number of alternative minimal topologies, thus underestimating the irresolution. We preferred, as an alternative strategy, to reduce the number of sequences, on the basis of the initial NJ topology. A maximum parsimony analysis was thus performed on a subset of 21 sequences with a heuristic search strategy. Characters were unordered and were given equal weight, and gaps were treated as missing data. Random addition sequence of taxa was used with 100 replicates, followed by TBR (tree bisection-reconnection) branch swapping on the best trees. The option ‘MULTREES on’ was used. Bootstrap analyses (1000 replicates) were performed with the search option set to heuristic and random addition of taxa generating 50 starting tree replicates.

 Results and discussion TCP family sequences in <italic>Arabidopsis</italic> genome

The TCP domain and several Cycloidea protein sequences were used for searching sequences showing similarity in the complete genome of Arabidopsis thaliana. Twenty-four distinct sequences were found dispersed on the five chromosomes. It was not possible to establish the correspondence between all the sequences we retrieved and the accession numbers for the 24 sequences reported by Cubas [4]. Our designation of the TCPs is thus partly different from hers (Table 1). Thirteen sequences belong to the PCF subfamily. Among the eleven sequences belonging to the CYC subfamily, five have an R domain that is characteristic of CYC- or TB1-like proteins (TCP1, TCP2, TCP14, TCP15 and TCP18- see Table 1); they are distributed on three different chromosomes.

 Diversity and evolution of sequences belonging to the CYC subfamily

After searches for TCP family sequences in GenBank, a dataset including 112 distinct members was constituted, including the 24 sequences from A. thaliana. A neighbour-joining distance analysis was performed from the 834-nucleotide alignments, including the TCP domain, the interdomain variable sequence, and the R domain. Since we were mostly interested in the evolution of the CYC subfamily in eudicotyledon species, the resulting tree has been arbitrarily rooted with the PCF subfamily as the outgroup (Fig. 1), but it should be recalled in the following that this tree is actually unrooted.

 Evolution of the R domain

The eight genes from the CYC subfamily without an R domain (including the six genes from Arabidopsis) do not constitute a monophyletic group, but rather occur on three separate branches (Fig. 1). Some of them are closer to TCPs with an R domain, suggesting homoplasy for the presence/absence of the domain. Lack of resolution makes it difficult to appreciate the precise number of evolutionary events; furthermore, it is not possible to polarise the changes in the absence of a root. Assuming that the root is somewhere on the branch linking CYC and PCF subfamilies, the most parsimonious scenario would be an acquisition of the R domain in an ancestor of the whole CYC subfamily, followed by at least three independent losses. In any way, the occurrence of homoplasy for the presence/absence of the R domain should be borne in mind when assessing relationships of orthology and possible functional conservation of genes.

 Relations of orthology and multiple independent duplications within the CYC subfamily

All sequences from Veronicaceae and Gesneriaceae constitute a monophyletic group (bootstrap value 81%), but among this clade, sequences from Veronicaceae or Gesneriaceae are not mixed. Instead, four well-supported clades can be recognised: GCYC1 (bootstrap value 95%), GCYC2 (bootstrap value 99%), (CYC1, CYC2) (bootstrap value 98%) and (CYC3, CYC4) (bootstrap value 100%), of which the first two contain only sequences from Gesneriaceae, and the last two contain only sequences from Veronicaceae. The CYC gene isolated for its major effect on floral symmetry in Antirrhinum belongs to the CYC1 group of orthology, as well as the LCYC gene whose mutation is responsible for the Linaria peloric mutant [5]. DICH, also involved in zygomorphy in Antirrhinum, appears as a member of the CYC4 group of orthology. No relation of orthology emerges between genes from Veronicaceae and Gesneriaceae, which indicates that in these two closely related families, gene diversity among the CYC subfamily was acquired independently, as already noticed by Citerne et al. [3]. In the same way, the four Mimulus sequences group in two well-supported clades that do not aggregate with sequences from either Veronicaceae or Gesneriaceae.

The TCP1 gene from A. thaliana was suggested to be the orthologue of the CYCLOIDEA gene from A. majus [4] and [7]. From our analysis, it is clear that TCP1 is not an orthologue of CYC. Indeed, no relation of orthology can be proposed between particular TCP genes from Arabidopsis and Veronicaceae, respectively.

TB1 genes from Oryza sativa and Capillipedium form together a strongly supported clade. When all TB1 sequences from ref [15] were included in the neighbour-joining analysis, orthology relationship could be well established among the genes of the grass species analysed, but no orthologous relationship occurred with genes from other taxa. In order to further examine relationships among the genes belonging to the CYC subfamily in eudicotyledon species, 21 sequences were selected from the main clades present in the neighbour-joining tree, including two PCF subfamily sequences (see Fig. 1) and were analysed using the maximum parsimony method. The dataset contained 264 informative characters. A single most parsimonious tree (1048 steps in length) was obtained (CI = 0.70, HI = 0.30, RI = 0.61, RC = 0.43), which, consistently with the neighbour-joining analysis on the full set of sequences, did not support any orthology relationship between genes from distinct taxonomic groups (Fig. 2).

 Conclusion

The present study encompasses a broader gene sampling than in any previously published analysis of TCP-domain containing genes. The evolution of CYC subfamily genes appears to have occurred through multiple duplications (and possibly gene losses) taking place after the divergence between the various sampled taxonomic groups. The other possibility of multiple ancestral sequences homogenised through gene conversion, then following divergent evolution in the various taxa seems to be dismissed by the mapping of TCP family genes on different chromosomes in Arabidopsis thaliana.

The question of a possible conservation of function and expression across the different gene subgroups and taxa is presently unanswered. Expression data are recorded for six genes (CYC, DICH, LCYC, TCP1, TCP2 and TCP3), and indicate a dorso-ventral asymmetric expression in developing meristem for CYC, DICH, LCYC and TCP1, and a symmetric one for TCP2 and TCP3. The asymmetric expression of CYC, DICH and LCYC is clearly associated with a zygomorphic flower in Anthirrhinum and Linaria [5], [16] and [17]. The transient expression of TCP1 in Arabidopsis may account for both a precocious asymmetry in sepal development [10], and the lack of dorsoventral asymmetry in the fully developed perianth. The primitive function of CYC subfamily genes seems to be related to growth and development of primordia. These genes thus would offer evolution a molecular basis to develop repeatedly asymmetric morphological structures, simply as a consequence of asymmetric expression.

At present, data on expression or function are very scarce from a taxonomic point of view, and sequence data is strongly biased since most data come from Lamiales and A. thaliana. Improved knowledge about the evolution of structure and function of CYC subfamily genes will come from a better sampling of various angiosperm taxa.

 Acknowledgements

We thank Professors H. Le Guyader and J. Deutsch for critical reading of the manuscript. This work was supported by a grant from the ‘Centre national de la recherche scientifique’ (France) allocated to C.D.

 Altschul S.F Madden J.L Schäffer A.A Zhang J Zhang Z Miller W Lipman D.J Gapped BLAST and PSI-BLAST: a new generation of protein database search programs Nucleic Acids Res. 25 1997 3389–3402 Angiosperm phylogeny Group (The) An ordinal classification for the families of flowering plants Ann. Missouri Botanical Garden 85 1998 531–553 Citerne H Möller M Cronk Q.C.B Diversity of Cycloidea-like genes in Gesneriaceae in relation to floral symmetry Ann. Bot. 86 2000 167–176 Cubas P Role of TCP genes in the evolution of morphological characters in Angiosperms Cronk Q.C.B Bateman R.M Hawkins J.A Developmental genetics and plant evolution, The Systematics Assoc. Spec. Vol. No. 65 2002 247–266 Cubas P Vincent C Coen E An epigenetic mutation responsible for natural variation in floral symmetry Nature 401 1999 157–161 Cubas P Lauter N Doebley J Coen E The TCP domain: a motif found in proteins regulating plant growth and development Plant J. 18 1999 215–222 Cubas P Coen E Zapater J.M.M Ancient asymmetries in the evolution of flowers Curr. Biol 11 2001 1050–1052 Doebley J Stec A Gustus C Teosinte branched1 and the origin of maize: evidence for epistasis and the evoution of dominance Genetics 141 1995 333–346 Doebley J Stec A Hubbard L The evolution of apical dominance in maize Nature 386 1997 485–488 Endress P.K Evolution and floral diversity: the phylogenetic surroundings of Arabidopsis and Antirrhinum Int. J. Plant Sci. 153 1992 S106–S122 Endress P.K Antirrhinum and the Asteridae: evolutionary changes of floral symmetry Symp. Soc. Exp. Biol. 51 1997 133–140 Gaudin V Lunness P.A Fobert P.R Towers M Riou-Khamlichi C Murray J.A.H Coen E Doonan J.H The expression of D-Cyclin genes defines distinct developmental zones in snapdragon apical meristems and is locally regulated by the Cycloidea gene Plant Physiol. 122 2000 1137–1148 Hall T.A BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT Nucl. Acids Symp. Ser. 41 1999 95–98 Jukes T.H Cantor C.R Evolution of protein molecules Munro H.N Mammalian protein metabolism 1969 Academic Press New York 21–132 Lukens L Doebley J Molecular evolution of the Teosinte Branched1 gene among maize and related grasses Mol. Biol. Evol. 18 2001 627–638 Luo D Carpenter R Vincent C Copsey L Coen E Origin of floral asymmetry in Antirrhinum Nature 383 1995 794–799 Luo D Carpenter R Copsey L Vincent C Clark J Coen E Control of organ asymmetry in flowers of Antirrhinum Cell 99 1999 367–376 Olmstead R.G DePamphilis C.W Wolfe A.D Young N.D Elisons W.J Reeves P.A Disintegration of the Scrophulariaceae Am. J. Bot. 88 2001 348–361 Swofford D.L PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods), Version 4.06b 2001 Sinauer Associates Massachusetts, Sunderland Vieira C.P Vieira J Charlesworth D Evolution of the Cycloidea gene family in Antirrhinum and Misopates Mol. Biol. Evol. 16 1999 1474–1483

Neighbour-joining tree resulting from the analysis of the 834-nucleotide alignment, including 112 genes, arbitrarily rooted on the PCF subfamily of TCP sequences. Bootstrap values above 60% are indicated on the branches (a few values above 60% on terminal branches are not indicated for clarity reasons). The 24 TCP coding sequences found in the A. thaliana genome are named TCP#, according to Table 1. Among the CYC subfamily, sequences devoid of an R domain appear in grey rectangles. The original Cycloidea gene of A. majus is in bold. Accession numbers are given following species names. Sequences used in the maximum parsimony analysis are indicated by a star.

Fig. 1. Arbre obtenu par la méthode du neighbour-joining sur la matrice nucléotidique de 834 positions et 112 gènes, avec les séquences de la sous-famille PCF en groupe externe. Les valeurs de bootstrap supérieures à 60% sont indiquées (quelques valeurs supérieures à 60% sur les branches terminales n’ont pas été indiquées pour des raisons de clarté). Les 24 séquences TCP d’Arabidopsis sont nommées TCP#, selon le Tableau 1. Les séquences de la sous-famille CYC sans domaine R figurent dans un rectangle gris ; le gène Cycloidea, originellement cloné chez A. majus, est indiqué en gras. Les numéros d’accession suivent les noms d’espèces. Les séquences utilisées dans l’analyse de parcimonie sont indiquées par un astérisque.

Consensus maximum parsimony tree obtained from the sample of 21 sequences, using two PCF subfamily sequences as the outgroup. Bootstrap values above 60% are indicated.

Fig. 2. Arbre consensus issu de l’analyse de l’échantillon de 21 séquences par la méthode de parcimonie. Les deux séquences de la sous-famille PCF ont été utilisées comme groupe externe. Les valeurs de bootstrap supérieures à 60% sont indiquées.

Name, characteristics and chromosomal assignation of TCP homologous coding sequences found in the Arabidopsis genome. The asterisk points to sequences numbered as in Cubas [4].

Tableau 1

Nom, caractéristiques et localisation chromosomique des séquences codantes comportant un domaine TCP, trouvées dans le génome complet d’Arabidopsis. L’astérisque indique les séquences qui ont pu être numérotées de manière identique à celle adoptée par Cubas [4].

Accession number/Protein_id TCP designation Domain types Chromosome AC002130/AAG00255 TCP1 [5] CYC-type TCP, R I AC011914/AAG52043 TCP14 CYC-type TCP, R I AC073506/AAG50562 TCP15 CYC-type TCP, R I AP001303/BAB02213 TCP18* CYC-type TCP, R III AL021710/CAA16719 TCP2 [5] CYC-type TCP, R IV AF072134/ AAC24010 TCP3 [5] CYC-type TCP I AC005311/AAC63845 TCP10* CYC-type TCP II AP000370/BAA97066 TCP22 CYC-type TCP III AC011664/AAF14823 TCP13* CYC-type TCP III AL357612/CAB93708 TCP17* CYC-type TCP V AB008269/BAB10646 TCP5 [5] CYC-type TCP V AC069273/AAG51130 TCP12 PCF-type TCP I AC013289/AAG52540 TCP24 PCF-type TCP I AC079131/AAG50759 TCP8 [5] PCF-type TCP I AC007887/AAF79358 TCP23* PCF-type TCP I AC006922/AAD31585 TCP11* PCF-type TCP II AC003680/AAC06168 TCP21 PCF-type TCP II AL138649/CAB72153 TCP16* PCF-type TCP III AY058874/AAL24261 TCP9 [5] PCF-type TCP III AB026649/BAB01082 TCP20* PCF-type TCP III AB025623/BAA97226 TCP19* PCF-type TCP V AB007648/BAB11183 TCP4 PCF-type TCP V AL392174/CAC08333 TCP7 [5] PCF-type TCP V AB010072/BAB09705 TCP6 [5] PCF-type TCP V