Department of Genetics, North Carolina State University
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Abstract |
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Introduction |
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The Hawaiian silversword alliance (Asteraceae-Madiinae) is an outstanding example of plant adaptive radiation (Raven, Evert, and Eichhorn 1999
, pp. 250251; Schluter 2000
) and serves as a key system for understanding the origin of plant morphological diversity (Robichaux et al. 1990
). The 30 extant species in the Hawaiian Madiinae putatively arose from a herbaceous allopolyploid North American tarweed ancestor
5 MYA (Baldwin et al. 1991
; Baldwin and Sanderson 1998
; Barrier et al. 1999
) and have subsequently evolved spectacular differences in vegetative and reproductive morphology, as well as in physiology and habitat (Carr 1985
; Robichaux et al. 1990
). Shoot elongation in the basal rosette plants of the genus Argyroxiphium is largely limited to reproductive stems, whereas the other two genera (Wilkesia and Dubautia) have elongated woody vegetative stems. The Dubautia spp., moreover, show tremendous variation in the rate and extent of stem elongation and vary from diminutive shrubs and cushion plants (e.g., D. scabra, D. ciliolata, D. waialealeae) to trees up to 6 m tall (D. reticulata, D. knudsenii, and D. arborea) (Carr 1985
; Robichaux et al. 1990
). Some of the most spectacular morphological differences occur between closely related taxa. For example, D. arborea and D. ciliolata are essentially indistinguishable by microsatellite and AFLP polymorphisms (unpublished data; E. Friar, personal communication) but exhibit striking differences in height, leaf size and morphology, and floral and capitulescence characteristics (Carr 1985
; Robichaux et al. 1990
).
Genes involved in transcriptional regulation could be especially important in the evolution of major phenotypic differences because they tend to control specific developmental pathways (Doebley and Lukens 1998
; Purugganan 2000
). Consistent with this prediction, Barrier, Robichaux, and Purugganan (2001)
found accelerated rates of evolution in ASAP1 and ASAP3/TM6, orthologues of the floral homeotic genes APETALA1 and APETALA3, in Hawaiian Madiinae lineages relative to the North American lineages. Significantly less rate acceleration was found in orthologues of the structural gene CAB9. These results suggest that positive selection, and not merely relaxation of selection because of gene duplication, has contributed to the accelerated evolutionary rates in ASAP1 and ASAP3. It is not clear, however, whether these results apply to transcriptional regulators in general or only to a small subset of regulatory loci.
Regulatory genes known to play key roles in plant growth regulation would seem especially likely to be under selection in the silversword alliance. The Arabidopsis genes GA INSENSITIVE (GAI) and RGA (Peng et al. 1997
; Silverstone, Ciampaglio, and Sun 1998
) and related genes of the DELLA subfamily encode growth regulators (Peng et al. 1999
) and have been implicated in quantitative variation in developmental traits (Thornsberry et al. 2001
). The DELLA genes, which also include the three RGL genes in Arabidopsis (Dill and Sun 2001
; Wen and Chang 2002
), Rht-1 in wheat, d8 in maize (Peng et al. 1999
), and SLR1 in rice (Ogawa et al. 2000
; Ikeda et al. 2001
), are a subset of the GRAS family of plant transcriptional regulatory genes (Pysh et al. 1999
) whose products modulate gibberellin (GA) responses (Peng et al. 1999
). Semidominant mutations in GAI, RGA, RGL1, Rht-1, d8, and SLR1, all of which involve deletions in or truncations of the N-terminal DELLA region for which the gene subfamily is named, have been shown to confer a GA-unresponsive dwarf phenotype (Peng et al. 1997
; Peng et al. 1999
; Dill and Sun 2001
; Ikeda et al. 2001
; Wen and Chang 2002
). Mutants are viable and have become important constituents of green revolution grain varieties (Peng et al. 1999
). Different d8 and Rht-1 mutants show varying degrees of dwarfism, suggesting that alterations within the DELLA region may have broad importance for the modulation of gibberellin responses (Peng et al. 1999
). A broader spectrum of developmental variation in maize, including time to flowering, has been shown to be strongly associated with polymorphisms in d8 outside the DELLA region and with sites in the promoter (Thornsberry et al. 2001
). DELLA genes differ somewhat from one another in expression patterns and effects of GA on expression levels, suggesting some degree of variation in gene regulation (Silverstone, Ciampaglio, and Sun 1998
; Ogawa et al. 2000
; Wen and Chang 2002
). The finding that the wild-type DELLA proteins quantitatively affect GA-induced growth responses in a dose-dependent manner (Dill and Sun 2001
; Fu et al. 2001
; King, Moritz, and Harberd 2001
; Wen and Chang 2002
) and the association of sites in the d8 promoter with flowering time (Thornsberry et al. 2001
) also suggest that transcript levels might be involved in phenotypic variation. Posttranslational regulation is also important, at least in RGA and SLR1 proteins, which undergo rapid degradation in response to the GA treatment (Silverstone et al. 2001
; Itoh et al. 2002
).
Our objective in this study was to test whether the acceleration in protein evolution observed in ASAP1 and ASAP3/TM6 (Barrier, Robichaux, and Purugganan 2001
) can be generalized to other regulatory genes in the Hawaiian silversword alliance. We tested whether the rates of evolution were accelerated in Hawaiian relative to North American lineages of DaGAI-A and DaGAI-B, two DELLA genes in the silversword alliance, and whether some codons showed evidence of positive selection. Furthermore, to test the prediction that cis-regulatory regions of transcriptional regulators are the most likely locations for important variation (Doebley and Lukens 1998
), we evaluated portions of the DaGAI upstream flanking regions as well as the coding regions. Finally, the results would also provide some evidence of whether DaGAI might have played a major role in at least some of the episodes of morphological evolution that gave rise to the 30 existing Hawaiian Madiinae. The molecular signature of such a role could include mutations in key conserved residues and domains, such as the DELLA region important in GA responsiveness, or accelerated evolutionary rates in putative promoter sequences.
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Materials and Methods |
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Isolation and Sequencing of GAI Homologues
Short genomic fragments (130140 bp) of a GAI homologue (designated DaGAI) were isolated by PCR from D. arborea, using degenerate primers designed from conserved domains in the N-terminal regions of the published sequences. An initial round of amplification was performed using primers gaiU103deg (5'-TACAAGGTNMGNTCNTCNGANATG-3') and gaiL278deg (5'-GGAGGRTTNARNTCNGWNARCAT-3'), followed by seminested amplifications using gaiU103deg and gaiL234deg (5'-CTCCGCNGGRTTRTARTGNACNGT-3') or gaiU136deg (5'-GCTCAGAARYTNGARCARYTNGA-3') and gaiL278deg.
Nested forward primers for 3' rapid amplification of cDNA ends (3' RACE) (Frohman, Dush, and Martin 1988
) were designed from degenerate PCR products. First-strand cDNA synthesis and nested amplification of DaGAI transcripts were done with a 3' RACE kit (Life Technologies), using total RNA from D. arborea shoot tips and leaves as the template. Nested reverse primers designed from 3' RACE products were used to obtain genomic sequences from D. arborea corresponding to the 5'-end of the DaGAI-coding region and approximately 900 bp of the upstream flanking sequences, using the Universal GenomeWalker kit (Clontech) to perform anchored PCR (Siebert et al. 1995
). Further verification of DaGAI expression in elongating shoot tips was obtained with RT-PCR using the Access RT-PCR kit (Promega) and gene-specific primers. Reactions without reverse transcriptase were used as negative controls for genomic DNA contamination.
Specific PCR primers designed from the D. arborea sequences or subsequently obtained sequence data were used to amplify DaGAI from nine Hawaiian Madiinae (D. arborea, D. ciliolata, D. menziesii, D. knudsenii, D. raillardioides, D. microcephala, A. sandwicense, A. kauense, and W. gymnoxiphium) and four North American Madiinae (A. madioides, C. muirii, M. sativa, and C. multiglandulosa). PCR amplification was performed using the proofreading Pwo polymerase (Roche) to minimize the amount of sequence error introduced during PCR. The DaGAI region was amplified as two overlapping fragments; one fragment covered virtually the entire putative coding region, and the second fragment encompassed 6001,000 bp of the upstream flanking sequence plus
400 bp of the 5'-end of the coding region. Overlapping regions were used to determine which sequences from the two sets of products were allelic. Further GenomeWalker library construction and amplification were required to isolate the upstream region from C. multiglandulosa and the second DaGAI copy in D. arborea.
All PCR products were cloned into TOPO TA or Zero Blunt vectors (Invitrogen) for sequencing. Cycle sequencing reactions were performed using BigDye terminator-polymerase mixes (Applied Biosystems), and sequences were resolved on ABI 377 automated sequencers or ABI 3700 DNA analyzers. PHRED (Ewing and Green 1998
; Ewing et al. 1998
) was used to call bases and assign quality scores. Most, but not all, regions were covered by overlapping sequence reads. Discrepancies and unique sequence changes at sites with single coverage and low-quality scores were visually rechecked against chromatograms. All sequences are deposited in GenBank (accession numbers AF492562AF492590).
Sequence Alignment and Phylogeny Reconstruction
The two DaGAI sequences from D. arborea were visually aligned with d8 from maize, SLR1 from rice, and the five known GAI family members in Arabidopsis thaliana: GAI, RGA, and the three "RGA-like" sequences that have been designated as RGL1, RGL2, and RGL3 (Dill and Sun 2001
). Phylogenies were evaluated with PAUP* Version 4.0b6 (Swofford 1998
). Only sites that could be aligned confidently in the in-group taxa were included, and third codon positions were excluded. The 3' portion of the GRAS family SCARECROW gene from A. thaliana, which can be aligned with the DELLA subfamily sequences, was included as an out-group. Neighbor-joining tree construction used the HKY85 substitution model with gamma rate variation, and shape parameter
= 0.5. Maximum parsimony trees were constructed using a branch-and-bound search to find the single minimum-length tree. Gaps were treated as missing data. Branch support for both neighbor-joining and maximum parsimony trees was evaluated using 500 bootstrap replicates. A maximum likelihood tree was constructed using the HKY85 substitution model with gamma rate variation, with the shape parameter estimated from the data. A heuristic search was performed using four random addition replicates followed by tree bisection/reconnection (TBR) branch swapping.
The alignment of sequences from the thirteen Hawaiian and North American Madiinae taxa included both copies from D. arborea, D. menziesii, D. raillardioides, A. sandwicense, and A. kauense for a total of 18 sequences. Calycadenia multiglandulosa was chosen as an out-group for phylogenetic analyses, on the basis of previous phylogenetic investigations in the Madiinae (Baldwin and Wessa 2000
; Barrier, Robichaux, and Purugganan 2001
). Maximum parsimony trees were generated in PAUP* using a branch-and-bound search. Branch support was evaluated using 500 bootstrap replicates. Maximum likelihood analysis used the HKY85 substitution model with the gamma shape parameter estimated from the data. A maximum likelihood tree was generated heuristically using five random addition replicates followed by TBR branch swapping.
Molecular Evolution Analyses
Maximum likelihood analyses of molecular evolution in DaGAI were carried out in HY-PHY version 0.901 beta (peppercat.stat.ncsu.edu/hyphy) using the maximum likelihood tree topology generated in PAUP*. Nucleotide-based relative rate tests used the HKY85 substitution model with global or local transition-transversion ratios. Codon-based analyses of replacement-synonymous substitution ratios (KA/KS, or
) and relative rates used the Muse-Gaut 3 x 4 codon substitution model (Muse and Gaut 1994
), in which the separate nucleotide frequencies at each of the three codon positions are used to calculate the codon substitution matrix. Nested models with different numbers of constrained parameters were used to conduct likelihood ratio hypothesis tests. Models included (1) a global model in which a single
ratio was applied to the entire tree; (2) a 3-
model in which three separate
ratios were applied to branches descending from the most recent common ancestor (MRCA) of the Hawaiian A copy, those descending from the MRCA of the Hawaiian B copy, and to North American branches, respectively; (3) 2-
models in which two of the three categories in the 3-
model were combined; and (4) an unrestricted local model in which separate KA and KS values were determined for each branch of the tree. In the 2-
and 3-
models, the branches leading to the common ancestors of the Hawaiian A and B copies were considered to be North American, even though some of the evolution along these branches may have occurred after Hawaiian introduction. The null distribution of the likelihood ratio test statistic was assumed to follow a
2 distribution with degrees of freedom equal to the difference in the number of unconstrained parameters between the two hypotheses. Analyses were performed for the DaGAI-coding region as a whole and for sliding windows of 300 bp with 150-bp offsets between successive windows. To test the sensitivity of the results to the tree topology, all analyses were also run using an alternate topology, with slight differences in both Hawaiian gene lineages, generated in HY-PHY by stepwise addition without branch swapping.
Tests of variable selection among codons used two different null models. In the first (neutral) model, all codons were assumed to belong either to an invariant ( = 0) or a neutral (
= 1) class. This model was tested against an alternative (selection) model containing a third class of sites with an
ratio estimated from the data. In the second null model (beta model),
was assumed to follow a beta distribution with 0
1, which was discretized into three classes of equal frequency for the purposes of estimation. This model was tested against an alternative (beta+
) model containing an additional class of positively selected sites, in which
was constrained to be greater than 1.0 and was estimated from the data. The sensitivity of this test to model assumptions was tested by reducing the number of discretized ß distribution categories to two and by changing the codon substitution model to the Goldman-Yang 3 x 4 model (Goldman and Yang 1994
).
Analyses of the upstream flanking sequences were done in HY-PHY using the HKY85 nucleotide substitution model. The upstream sequence was divided into three regions (proximal, intermediate, and distal) on the basis of the changes in nucleotide composition and the frequency of indels. Constraint and branch length heterogeneity were evaluated by comparing each region separately with the third codon position sites in the coding region. Three nested models were used for each comparison: (1) a model in which both the upstream region and third position sites were constrained to have identical branch lengths; (2) a model in which branch length ratios were constrained to be identical but could vary proportionately between the two regions; and (3) a model in which all branch lengths were estimated independently for the two sections. The likelihood ratio test of model 3 against model 2 (the relative ratio test) evaluates branch ratio heterogeneity between the two sections, and the test of model 2 against model 1 evaluates differences in overall substitution rates between the sections. A sliding window analysis was also performed, using windows of 200 bp and offsets of 50 bp between successive windows.
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Results and Discussion |
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A comprehensive alignment of the DaGAI sequences from the Madiinae encompassed 0.61.0 kb of the upstream flanking sequence plus the entire coding region, except for 65 bases at the 3'-end. Pairwise nucleotide distances for the DaGAI coding region sequences show divergences of 1.9% or less within both DaGAI-A and DaGAI-B in the Hawaiian taxa. Distances between A and B copies range from 5.6% to 6.5%. The C. multiglandulosa sequence was by far the most divergent, with distances of 16.7%17.4% from the DaGAI-A sequences, 17.6%18.1% from the DaGAI-B sequences, and 17.7%18.3% from the other North American DaGAI sequences. Calycadenia multiglandulosa was included as an out-group on the basis of previous studies that have shown it to belong to a separate tarweed lineage, whereas the other three North American species were included as close relatives of the silversword alliance ancestors (Baldwin and Wessa 2000
; Barrier, Robichaux, and Purugganan 2001
). The distances are thus consistent with a common origin for all the DaGAI sequences within a single lineage of the Madiinae.
Phylogenetic trees were constructed from the entire aligned coding regions. Two minimum-length trees were found using maximum parsimony. Two likelihood peak "islands" were found when maximum likelihood methods were used, and the tree corresponding to the higher peak was identical in topology to one of the two minimum-length parsimony trees (fig. 1
). The Hawaiian A and B copy clades were strongly supported in maximum parsimony bootstrap analyses. Anisocarpus madioides was strongly supported as sister to the Hawaiian A copy, consistent with the findings for the A copy of ASAP3/TM6 relative to A. scabridus, the sister species to A. madioides (Barrier et al. 1999
; Barrier, Robichaux, and Purugganan 2001
). The B copy clade was strongly supported as sister to all the other ingroup taxa, in contrast with the sister relationship to C. muirii shown by ASAP3/TM6 (Barrier et al. 1999
; Barrier, Robichaux, and Purugganan 2001
). Support was much weaker for clades within the Hawaiian species for both the A and B copies, consistent with a rapid adaptive radiation of the silversword alliance after introduction to Hawaii. All within-copy clades with at least moderate support were consistent with chloroplast DNA and rDNA ITS phylogenies (Baldwin et al. 1991
; Baldwin and Sanderson 1998
). Phylogenetic trees constructed from upstream flanking sequences produced similar topologies with respect to the major clades (data not shown).
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Common Ancestry of Monocot and Dicot DELLA Genes
We aligned the sequences of DaGAI-A and DaGAI-B from D. arborea with publicly available sequences of GAI, RGA, and three other RGA-like sequences (RGL1, RGL2, and RGL3) from A. thaliana, d8 from maize, and SLR1 from rice. We constructed phylogenetic trees from the nine genes using conserved regions of the A. thaliana SCARECROW gene as an out-group. The neighbor joining, maximum parsimony, and maximum likelihood methods produced identical topologies (fig. 2
). The two DaGAI copies formed a strongly supported clade, as did the Arabidopsis GAI-RGA and RGL2-RGL3 gene pairs. The GAI-RGA and RGL2-RGL3 sister relationships are also supported by genomic evidence; GAI and RGA are located in duplicated Arabidopsis chromosomal blocks (10b and 10a) identified by Vision, Brown, and Tanksley (2000)
, as are RGL2 and RGL3 (blocks 71a and 71b). The two DaGAI sequences grouped with the three Arabidopsis RGL sequences rather than to GAI and RGA, but bootstrap support for this branch was weak (54% with maximum parsimony and <50% with neighbor-joining). The two monocot sequences form a strongly supported sister clade to the dicot sequences, which provides phylogenetic support for previous assumptions of orthology between the monocot sequences and GAI (Peng et al. 1999
).
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The ambiguous relationships between DaGAI and the Arabidopsis DELLA genes may reflect duplication dates for the GAI-RGA and RGL gene ancestors close to the time of the rosid-asterid divergence 112156 MYA (Yang et al. 1999
). In a comparison of a tomato genomic region with the Arabidopsis genome sequence, Ku et al. (2000)
found evidence of two genomic duplications in the Arabidopsis lineage, the older of which may have occurred very near the time of the rosid-asterid divergence. If the GAI-RGA and RGL lineages diverged before the rosid-asterid divergence and DaGAI were orthologous to the latter, which is weakly suggested by our data, another, as yet undetected, set of DELLA loci orthologous to GAI and RGA may be present in the Madiinae. Genes in both the GAI-RGA and RGL lineages in Arabidopsis, however, have now been shown to function in regulation of gibberellin growth responses (Wen and Chang 2002
), so the precise phylogenetic relationships between DaGAI and the Arabidopsis genes lack any obvious functional implications.
Patterns of Sequence Conservation and Divergence in DaGAI
The DaGAI sequences shared conserved features that have been previously reported for the DELLA subfamily (Peng et al. 1999
), including (1) a conserved N-terminal DELLA domain, (2) regions of leucine-heptad repeats, (3) two valine-rich regions, (4) key conserved residues in an SH2-like domain, and (5) a conserved tyrosine near the C-terminus that is a putative phosphorylation site in signal transducer and activator of transcription (STAT) proteins (see fig. 3
). The putative start of translation is identified by the amino acid sequence MKR a short distance upstream from the start of the DELLA domain, as in the other GAI homologues. The only substantial difference is that conserved arginine residues have been changed (to alanine and glutamine, respectively) in both sections of the putative bipartite nuclear localization signal.
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Rates of Neutral Molecular Evolution are Similar Between A and B Copies of DaGAI
We conducted relative rate tests on the DaGAI-A and DaGAI-B lineages to evaluate whether rates of evolution differed between copies. We applied maximum likelihood codon and nucleotide substitution models to the D. arborea A and B copies, using C. multiglandulosa as the out-group. Both synonymous and replacement substitution rates were higher for the A copy than for the B copy in the codon models, but the differences were not significant. Similarly, relative rates did not differ significantly with the nucleotide models applied to codon positions 12 only or position 3 only. Elevated substitution rates for the A copy were marginally significant only when maximum likelihood nucleotide substitution models were applied to the entire coding region (P = 0.045 using a global transition-transversion rate). The branch lengths of the maximum likelihood tree (fig. 1
) appear to show that faster rates of evolution in the A copy lineage relative to the B copy lineage are confined to the more basal, North American branches, or are related to the placement of the root. Thus, there is no suggestion of differences in neutral evolutionary rates between the two DaGAI copies in the Hawaiian lineages.
Selective Constraints Differ Between the Hawaiian and North American DaGAI Lineages
To evaluate the heterogeneity in the selective constraint in DaGAI, we analyzed the rates of synonymous (KA) and nonsynonymous (KS) substitutions along each branch of the maximum likelihood tree using codon-based maximum likelihood models. We excluded C. multiglandulosa from these analyses so that the results would not be unduly influenced by evolution along the out-group branch. We imposed different sets of constraints on KA/KS (or ) ratios along various tree branches (table 1
), which allowed us to conduct a number of nested hypothesis tests of constraint and selection (table 2
). We also tested the robustness of the maximum likelihood models to alterations in the tree topology and obtained very similar results when an alternative topology was used (data not shown).
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The test results suggest both a general relaxation of the selective constraint in the Hawaiian copies and a relaxation of constraint specific to the Hawaiian A copy. Nevertheless, even the A copy branches remained under substantial selective constraint ( = 0.37). These results stand in sharp contrast to results from other regulatory genes (Barrier, Robichaux, and Purugganan 2001
), with average
ratios of 0.79 and 0.98 for ASAP3/TM6 and ASAP1, respectively. The mild degree of relaxed selective constraint in the two Hawaiian copies of DaGAI corresponds more closely to that for the structural gene ASCAB9 (Barrier, Robichaux, and Purugganan 2001
).
The North American and two Hawaiian DaGAI branches showed similar patterns of selective constraint and maintained the same relative order of ratios across nearly the entire coding region. We used a sliding window analysis to evaluate the patterns of constraint in the three sets of DaGAI branches (fig. 4
and table 3
). The patterns of constraint in all three sets of branches closely mirrored the degree of conservation observed in the broader DELLA subfamily. Only two nonsynonymous substitutions were observed within either the Hawaiian A or B copies over the entire DELLA region. The section encompassing the valine-rich regions and the C-terminal region beginning with the SH2-like domain also showed especially strong constraint. The variable region downstream of the DELLA region and, to a lesser extent, the region just upstream of the SH2-like domain showed relaxed levels of constraint.
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It is important to note that the evolutionary rate analyses require only that both DaGAI-A and DaGAI-B be monophyletic, that their common ancestor would have lived after the divergence from C. multiglandulosa, and that the MRCAs of the Hawaiian A and B copies would have lived near the time of the Hawaiian introduction. The relative percentages of sequence divergence and the gene tree (fig. 1 ) support these assumptions. Neither hybrid introgression that conjoined the DaGAI-A and DaGAI-B ancestors before allotetraploidy nor duplication within the A or B copy lineages after introduction to Hawaii would affect any of the aforementioned analyses. The possible presence of other DELLA genes in the Madiinae, whether the result of recent or older duplications, likewise would not affect the validity of our analyses. The strength and patterns of selective constraint we have found suggest that the DaGAI genes have important functions in spite of whatever additional duplications may have occurred.
No Evidence for Coding Region Sites Under Positive Selection
We tested the presence of individual codons in DaGAI that may be under positive selection. Even though DaGAI-A and DaGAI-B are under strong overall selective constraint, individual codons representing key functional sites in the proteins could be experiencing positive selection and contributing to adaptive variation. If this were the case, we would expect to find a class of sites with ratios greater than 1. A model incorporating a class of sites under selection was significant when tested against the neutral model (table 4
). The estimated
ratio for the additional rate class, however, was far less than 1.0, indicating purifying rather than positive selection. Likewise, addition of a category of sites under positive selection into a beta model (beta+
model) did not produce a significant increase in likelihood, thus providing no evidence for positive selection. This test was somewhat sensitive to the particular assumptions used and became significant when the number of discrete categories used to approximate the beta distribution was reduced from three to two (data not shown). In all model variants, however, the estimated
value was not much greater than 1.0 and never exceeded 1.40. The likelihood improvements with the beta+
model thus appear to be because of a better fit of the constraint distribution and not the presence of sites under positive selection. These results suggest that a subset of codons in DaGAI is evolving much faster than the gene as a whole but under approximate neutrality rather than under positive selection.
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The Hawaiian B copy had an elevated number of indels but not substitutions, relative to the A copy for the upstream flanking sequence as a whole. The differences in numbers of substitutions versus indels between the D. arborea and A. sandwicense A and B copies were significant in a contingency test (P = 0.041), indicating heterogeneity between the indel and the substitution rates.
Relatively few published studies have examined upstream flanking sequences in a phylogenetic context, so there is limited data that can be used to evaluate whether the patterns we observed are typical. Some recent studies (although involving enzyme-encoding rather than regulatory genes) have found higher rates of sequence polymorphism and divergence in functionally important promoter segments than in adjacent regions, and adaptive explanations have been proposed (Crawford, Segal, and Barnett 1999
; Miyashita 2001
). Such patterns, however, could also be caused by coevolved sequence differences that preserve existing functions rather than selection for novel functions (Ludwig et al. 2000
). The lack of correlation between indel and substitution rates in the upstream regions is not readily explained. One possibility is that the substitutions that modify the transcription factorbinding sites are more deleterious than the indels that change the spacing between sites. Testing this hypothesis, however, would require detailed functional studies.
The elevated substitution rates in the middle upstream region, the branch length heterogeneity between the proximal upstream region and the coding region, and the decoupled rates of indels and substitutions are all possible indicators of positive selection in the DaGAI upstream flanking region. None of these observations, however, demonstrate positive selection to the exclusion of other possibilities. Further studies that include within-species sampling of alleles would allow the use of more sensitive tests to distinguish between relaxed selective constraint and positive selection (e.g., the HKA test, Hudson, Kreitman, and Aguade 1987
). Ambiguous alignments in parts of the middle region could have influenced some of the results, but the high variability in this region cannot be explained as an artifact of alignment. Elevated rates of substitution and numerous indels are apparent even within the A and B lineages, where the alignments are straightforward.
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Summary |
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Lukens and Doebley (2001)
also found a lack of evidence for positive selection among tb1-like sequences in grasses of the tribe Andropogoneae. Both the tb1-like and DELLA genes are putative transcription factors with effects on plant morphology and have had important roles in domesticated crops. One possible explanation is that plant growth and morphology may be affected by large numbers of genes. Individual genes may thus be under very weak selection or else beneficial mutations with large effects may have occurred by chance in other genes. It is also possible that other DELLA genes in the Madiinae, especially those that may have arisen from earlier genomic duplications, might show very different evolutionary patterns.
We were also interested in the more specific question of whether DaGAI itself might have had a major role in the adaptive radiation of the silversword alliance, similar to that of DELLA genes in domesticated crops. In this regard, the absence of relaxation in the selective constraint in the DELLA region is especially noteworthy. The dwarf phenotypes of the "green revolution" wheat varieties, mediated by DELLA truncations in Rht-1 genes with consequent reductions in gibberellin responsiveness, are of great advantage because of reduced wind and rain damage and less expenditure on vegetative biomass production (Peng et al. 1999
). We had predicted that similar, but possibly less drastic, DELLA mutations might have been important in adaptive evolution in the silversword alliance as well. The region around the SH2-like domain, in which maize indels are strongly associated with flowering time (Thornsberry et al. 2001
), likewise shows no evidence of relaxed selection. One possible explanation for our failure to obtain such results is that maintenance of normal gibberellin responsiveness may be important for long-term fitness in natural environments, even those that promote strong adaptive divergence in growth habit, in contrast with some crop environments. This explanation assumes that DaGAI has a function similar to that of other DELLA genes, which seems reasonable, given their functional conservation in taxa as diverse as A. thaliana and various grasses (Peng et al. 1997
, 1999
; Fu et al. 2001
; Ikeda et al. 2001
; Wen and Chang 2002
).
The possibility of divergent selection on the upstream DaGAI regions may warrant further investigation. In particular, some of the larger indels in the upstream region co-occur with major changes in the growth form within the Hawaiian Madiinae. Indels in the upstream region of d8 show strong association with the developmental phenotypes in maize, suggesting a phenotypic role for regulatory variation in DELLA genes (Thornsberry et al. 2001
). The recently demonstrated importance of dosage level in growth response modulation by DELLA genes in Arabidopsis and transgenic rice (Dill and Sun 2001
; Fu et al. 2001
; King, Moritz, and Harberd 2001
; Wen and Chang 2002
) suggests that changes in gene copy number could also have profound phenotypic effects. Study of DaGAI expression levels and molecular population genetics would be warranted in pairs of closely related taxa with large differences in growth form.
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Acknowledgements |
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Footnotes |
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1 Present address: Department of Biology, University of North Carolina at Greensboro
Abbreviations: GAI, GA INSENSITIVE; MITE, miniature inverted-repeat transposable element; MRCA, most recent common ancestor.
Keywords: Madiinae
silversword alliance
GAI
adaptive radiation
selection
regulatory genes
Address for correspondence and reprints: David L. Remington, Department of Biology, University of North Carolina at Greensboro, P.O. Box 26174, Greensboro, NC 27402. E-mail: dlreming{at}uncg.edu
.
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References |
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