Institute of Cell and Molecular Biology, University of Edinburgh
Correspondence: E-mail: andrew.hudson{at}ed.ac.uk.
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Abstract |
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Key Words: CYCLOIDEA DICHOTOMA TCP genes Antirrhinum Antirrhineae
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Introduction |
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Other functionally characterized CYC-related genes include teosinte branched1 (tb1), which represses growth of axillary shoots and floral organs in maize, and the PCF1 and PCF2 genes of rice (Doebley, Stec, and Hubbard 1997; Kosugi and Ohashi 1997). All these belong to a larger gene family named TCP (from tb1, CYC, and PCF). This family comprises 25 members in A. thaliana, is specific to plants, and encodes noncanonical basic Helix-Loop-Helix (bHLH) transcription factors (Cubas et al. 1999; Riechmann et al. 2000). All CYC-like genes share the TCP box, encoding a 5559 amino acid bHLH motif that is implicated in protein dimerization and DNA binding and an R box encoding a more C-terminal motif of 18 amino acids. Outside these boxes, protein coding sequences (which in CYC, DICH, and tb1 are uninterrupted by introns) show low sequence conservation, except in a region encoding a C-terminal domain of unknown function that has been termed the "end box" (Cubas et al. 1999; Möller et al. 1999; Citerne, Möller, and Cronk 2000). Because some TCP genes have known roles in floral asymmetry, and because of the potential importance of other members of the family in morphological evolution, they have been the subject of phylogenetic analysis in other genera with zygomorphic flowers (e.g., Gesneriaceae: Möller et al. 1999; Citerne, Möller, and Cronk 2000; and grasses: Lukens and Doebley 2001).
A previous study in Antirrhineae claimed to have identified at least five CYC-like loci in Antirrhinum and Misopates species, mainly on the basis of sequence phylogenies (Vieira, Vieira, and Charlesworth 1999). Paralogs and each putative ortholog appeared to be very highly conserved within the genus Antirrhinum, between genera within the Antirrhineae (Antirrhinum, Misopates, Linaria, and Cymbalaria), and with Digitalis purpurea, a more distant member of the Veronicaceae (Olmstead et al. 2001). This finding suggested that CYC-like sequences have evolved at an unusually slow rate in Veronicaceae.
To investigate further the evolutionary dynamics of CYC-like genes, we obtained CYC sequences from 17 Antirrhinum species and sequences of CYC and DICH loci from relatives of Antirrhinum. In contrast to previous studies, we found that CYC represents a single locus in Antirrhinum, that DICH is its closest paralog, and that the two loci have resulted from a gene duplication in the Veronicaceae in the lineage leading to the tribe Antirrhineae. Moreover we show that both CYC and DICH genes have evolved quickly within Veronicaceae, mainly as a result of nucleotide insertions and deletions in regions outside the functionally important TCP and R boxes. Antirrhinum species, with one exception, share similar CYC alleles, suggesting that the genus has undergone a recent radiation within the last 5 myr.
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Materials and Methods |
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For Southern hybridizations, 3 µg of genomic DNA was digested with Hind III, fractionated in 0.7% agarose gels, and blotted onto a nylon membrane. Sequences were detected by hybridization to 32P-labeled probes consisting of either the CYC or DICH sequences from A. majus amplified with primers cycF-cycR or dichF-dichR that had been cloned into pGEM-T. Hybridization was carried out in 2x SSC at 65°C (high stringency), 60°C (medium stringency), or 55°C (low stringency) for 18 hours. Filters were then washed in 0.2x SSC at the equivalent temperature and exposed to X-ray film for 3 days. Between hybridizations, filters were washed in 0.1% SDS at 100°C until no residual signal could be detected with a 3-day exposure to film.
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Results |
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To compare the evolutionary rate of CYC with another nuclear locus, an A. majus EST collection was searched for homologs of D. purpurea nuclear loci present in GenBank. Two Antirrhinum ESTs were identified as homologs of the D. purpurea aldose reductase gene (gi:13160396). Aligning the Antirrhinum and Digitalis sequences revealed no indels and K2P distances of 0.23 and 0.24 between the Digitalis sequence and each of the Antirrhinum ESTs. In comparison, the K2P distance between Antirrhinum CYC and Digitalis CYC-like was 0.28 with 15 indels (the number of indels depending on the alignment chosen), and between Antirrhinum DICH and Digitalis CYC-like the distance was 0.30 with 22 indels. In comparison, the K2P distance between CYC and DICH loci in Antirrhinum was 0.23 with 19 indels. These results suggest that CYC and DICH loci have evolved faster than the aldose reductase locus, largely as a result of indels outside the TCP and R boxes.
The ratio of nonsynonymous to synonymous substitution rates (Ka/Ks) within the TCP and R boxes ranged from 0.1 to 0.4. Outside these regions values were higher (between 0.1 and 0.6), but comparable to those obtained for TCP genes in grasses (Lukens and Doebley 2001). These Ka/Ks values did not provide evidence for positive selection and suggested that the variation within CYC and DICH sequences reflected relaxed evolutionary constraints.
The relationships within the CYC and DICH clades (fig. 3) differed slightly from each other and from the species phylogenies based on chloroplast DNA (fig. 2). Most notably, the clade containing Antirrhinum DICH was placed closer to the Cymbalaria sequence than to Linaria, whereas the Antirrhinum CYC and trnL + F sequences appeared closer to Cymbalaria than to Linaria. Possible explanations for these differences is that they resulted from later gene duplications that gave rise to paralogs within each clade and that the sequences did not represent orthologous genes. Evidence of a later duplication was provided by the two CYC-like sequences obtained from Ch. origanifolium. These were sufficiently different from each other (K2P distance = 0.087) to represent distinct loci rather than alleles. They were, however, more similar to each other than to the sequence from any other taxon, suggesting that they had arisen from a recent duplication in the lineage leading to Chaenorhinum.
The number of CYC and DICH loci was tested further by Southern hybridization of genomic DNA. High stringency hybridization with the A. majus DICH sequence as a probe detected strongly hybridizing bands (of 1.5 kb) in A. majus and M. orontium, but not in their more distant relatives, Cy. muralis and D. purpurea (fig. 4). This suggested that a single DICH locus was present in Antirrhinum and Misopates and that the sequences from these species were more similar to each other than to the DICH-like genes of other taxa. This relationship was consistent with the phylogeny of DICH sequences obtained from these species, except that S. nuttallianus produced a much weaker band than M. orontium in Southern hybridization, even though the DICH sequences from the two species were equally similar to the A. majus probe. One explanation for this discrepancy is that S. nuttallianus has a larger genome than M. orontium, so fewer DICH target sequences were present in equivalent amounts of DNA on Southern blots. Similarly, high stringency hybridization with the A. majus CYC probe detected strong bands in A. majus and M. orontium and weaker bands in S. nuttallianus and Ch. origanifolium. As for DICH, the relative strength of hybridization signals was consistent with the relationship between CYC sequences in these taxa, except that S. nuttallianus and L. vulgaris were weaker than expected. This discrepancy might again reflect relative differences in genome size.
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At low stringency both CYC and DICH probes detected multiple bands in all species, consistent with the presence of multiple members of the TCP gene family.
To investigate further the variation of CYC alleles within the genus Antirrhinum, sequences were obtained from 17 Antirrhinum species accessions representing the full morphological and geographical range of the genus. Phylogenetic analysis gave clear support for monophyly of Antirrhinum CYC in relation to the outgroups M. orontium and S. nuttallianus (fig. 5). The CYC alleles of Antirrhinum species showed low substitution divergence (average P = 0.004; maximum P = 0.017), suggesting that they had diverged relatively recently. Several groups of species were found to share the same or very closely related sequences including (1) A. graniticum and A. barellieri; (2) A. mollissimum and A. hispanicum; (3) A. cirrigherum, A. latifolium, and A. australe; and (4) A. lopesianum, A. meonanthum, and A. braun-blanquetii. However, no bootstrap support was obtained for the distinction of groups (2), (3), or (4) using ML or MP.
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Discussion |
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Our findings differ from those of Vieira, Vieira, and Charlesworth (1999), who proposed the existence of five CYC-like loci in Antirrhineae, including A. majus. Two of their proposed locicyc1a and cyc1bcorrespond to Antirrhinum CYC alleles in our study, and their cyc2, cyc3, and cyc4 loci correspond to Misopates CYC, Misopates DICH, and Antirrhinum DICH, respectively. The previous study is therefore likely to have classified alleles as paralogous genes. In addition, genera as distantly related to Antirrhinum as Linaria, Cymbalaria, and Digitalis were proposed to carry sequences identical, or nearly identical, to those found in Antirrhinum (Vieira, Vieira, and Charlesworth 1999). We found no evidence for sequences so similar to Antirrhinum CYC alleles in more distantly related species, and the sequences that we did obtain from these taxa were not represented in the previous study. Possible explanations for these discrepancies is that the previous PCR primers were able to amplify CYC and DICH loci only from Antirrhinum and Misopates and that samples from more distantly related taxa had been contaminated with these sequences.
We provided further support for the existence of only one CYC and one DICH locus in Antirrhineae by direct sequencing and restriction digestion of PCR products. With the exception of Chaenorhinum, each individual carried only one or two CYC sequences, as expected of alleles. Moreover, the PCR products from A. majus and A. molle, distinguished by a restriction site polymorphism, segregated as alleles of the same locus in the F2 of an A. majus x A. molle hybrid and were used to map the locus to the same position as the cyc mutation (Schwarz-Sommer et al. 2003). Southern hybridization also suggested that DICH was the locus most similar to CYC in Antirrhinum, Sairocarpus, and Misopates, and it failed to provide evidence for sequences identical to Antirrhinum CYC in the more distantly related Linaria, Cymbalaria, and Digitalis.
The sequence phylogeny is consistent with the CYC and DICH loci having arisen from a gene duplication in the Veronicaceae (after the divergence of Digitalis) in an ancestor of the Antirrhineae with asymmetric flowers (Reeves and Olmstead 1998).
Although the relationship between DICH or CYC sequences from Antirrhinum, Sairocarpus, and Misopates is not well resolved, the phylogenies of both loci place Antirrhinum basal to Sairocarpus and Misopates (fig. 3). This contrasts with the phylogeny of the chloroplast trnF + L sequences in which Antirrhinum is basal. Such discrepancies between phylogenies of organelle and nuclear sequences are not uncommon in plants at lower taxonomic levels, and they can be explained by reticulation (e.g., sorting of alleles present in a common ancestral population) or by hybridization (e.g., Schwarzbach and Rieseberg 2002).
At a higher level, the DICH sequences from Linaria were placed basal to DICH from other Antirrhineae, whereas the Linaria CYC and trnL + F sequences were nested within the Antirrhineae. This might be explained by an additional duplication of DICH followed by loss of different paralogs from Linaria and related taxa. Although the presence of two CYC sequences in Chaenorhinum suggested that a more recent duplication had occurred, it appeared likely to have been confined to the lineage leading to Chaenorhinum. Likewise, additional CYC-like or DICH-like genes in all other taxa could not be detected by PCR amplification or Southern hybridization. An alternative explanation for the anomalous position of Linaria DICH sequences is that they had been subjected to different selectional pressures (discussed below).
These comparisons suggested a slightly higher rate of nucleotide substitution in CYC-like genes compared to aldose reductase genes However, they revealed a much higher turnover of indels in CYC-like sequences. Almost all indels are present in regions outside those encoding the functionally important TCP and R boxes. Although the rapid divergence of these regions is likely to represent relaxed evolutionary constraints, adaptive changes cannot be ruled out in the absence of further functional studies.
Rapid sequence evolution can theoretically follow divergence in the expression of duplicate genes, which allows proteins to assume different specialized functions (a case of subfunctionalization; Lynch and Force 2000). Although CYC and DICH partially overlap in function in the floral meristem of A. majus, their expression domains differ, and each also has a unique role, consistent with subfunctionalization following duplication (Carpenter and Coen 1990; Luo et al. 1996, 1999). In L. vulgaris, loss of CYC activity can have the same phenotypic effect as loss of both CYC and DICH in A. majus (Cubas, Vincent, and Coen 1999). This greater dependence on CYC activity in floral asymmetry is unlikely to reflect absence of the DICH locus from Linaria, because we detected DICH sequences in both L. vulgaris and L. maroccana. It might therefore reflect a greater degree of divergence between CYC and DICH functions in Linaria, relative to Antirrhinum. Our finding that the two Linaria DICH sequences are placed basally within the DICH clade (whereas Linaria CYC sequences cluster with Chaenorhinum CYC sequences) is consistent with the Linaria DICH locus having experienced different constraints following divergence of function. An alternative explanation is that CYC and DICH have very similar functions in Linaria and that DICH activity was already absent from the L. vulgaris material in which CYC function was tested experimentally (e.g., because of a recent mutation or epimutation). Floral asymmetry in this case would appear dependent on CYC activity alone.
Rapid sequence evolution has been recorded for several duplicated transcription factor genes in plants, including CYC-like genes of Gesneriaceae and CONSTANS LIKE genes of Brassicaceae (Citerne, Möller, and Cronk 2000; Lagercrantz and Axelsson 2000). No asymmetry in the rate of molecular evolution was detected for CYC and DICH, as has been found for other duplicated transcription factors (e.g., Stauber, Jäckle, and Schmidt-Ott 2002; Stauber, Prell, and Schmidt-Ott 2002).
Only a few nucleotide substitutions were found for CYC alleles within the genus Antirrhinum. One explanation for the similarity of CYC alleles is that speciation in Antirrhinum occurred relatively recentlya view supported by the ability of Antirrhinum species to form fertile hybrids and the low degree of sequence divergence of other nuclear and chloroplast sequences within the genus (our unpublished results). Dating the divergence between the teosinte-branched (tb1) gene in maize and its closest homolog in Oryza sativa to 65 myr before present (Purugganan 1997), provides a conservative divergence estimate of 3.2 to 4.5 x 103 substitutions per million years for CYC-like genes. Assuming that the rates of sequence evolution in CYC and DICH are comparable to their monocot homologs, then the divergence of Antirrhinum species can be estimated to have begun less than 5.3 to 3.7 MYA, and most Antirrhinum species to have arisen within the last million years. These divergence-time estimates for Antirrhinum species are consistent with estimates from other nuclear and chloroplast sequences (our unpublished results).
Several weakly supported clades were detected within CYC alleles from different Antirrhinum species. Species groupings largely corresponded to shared morphological characters and similar geographical distributions (Sutton 1988) and to similarities of other DNA sequences (our unpublished results), suggesting that the CYC locus provides a weak phylogenetic signal at this taxonomic level. This trend, however, is disrupted by A. siculum and A. charidemi, which appear geographically and phylogenetically distinct from the other sampled species but have CYC alleles that group among them.
CYC is linked to the self-incompatibility (S) locus of Antirrhinum by less than 1 cM (Schwarz-Sommer et al. 2003). Antirrhinum species carry functionally equivalent S alleles (e.g., Sherman 1939), which are presumed to be ancient and to have been maintained by balancing selection (Vekemans and Slatkin 1994), as in Solanaceae (Ioerger et al. 1990). Where tested, Antirrhinum S allele sequences show the expected deep divergences (Xu et al. 1996) and provide little species-phylogenetic signal. Although CYC is closely linked to S, it appears not to have been subjected to the same selection, suggesting that recombination has been sufficient to uncouple the fate of the S and CYC loci. However, one exceptional CYC sequence, differing by four trinucleotide insertions, was detected in A. molle. Although this is placed basally to other Antirrhinum CYC sequences, as expected of an old allele, it differs from other Antirrhinum sequences by more trinucleotide insertions than substitutions, and it shares none of its insertions with the outgroup sequences from Misopates or Sairocarpus. The CYC allele in A. molle is therefore likely to have accumulated its insertions very recently. Because the trinucleotide insertions cause amino acid insertions, rapid evolution of the CYC allele in A. molle might have been driven by positive selection. Alternatively, it might reflect an increased rate of insertion mutations or a decreased constraint in the A. molle lineage. Further sampling of sequences within A. molle populations will be necessary to address this question.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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