*Department of Biology, Colgate University, New York;
Department of Biology and Institute of Molecular Evolutionary Genetics, Penn State University
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Abstract |
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Introduction |
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For protein-coding genes that have evolved under purifying selection, the rate of nonsynonymous substitutions and dN/dS are expected to increase when a gene is converted to a pseudogene or when changes in the environment, life history, or developmental program render the gene's function obsolete. Obsolete genes may persist as open reading frames for long periods of time because of chance alone (Marshall, Raff EC, and Raff RA 1994
). However, before the full-length open reading frame is interrupted by a stop codon, the cessation of purifying selection is expected to become evident in the pattern of nucleotide substitutions. Genes or pseudogenes that are not under the influence of purifying selection should on an average have dN/dS rate ratios (=
) equal to unity. This a priori expectation provides a basis to test for the loss of selectively maintained gene function (Crandall and Hillis 1997
; Nei, Zhang, and Yokoyama 1997
).
The null hypothesis for such comparative tests is that is the same in hypothetically constrained and unconstrained lineages. As in any formal hypothesis test, simply failing to reject the null hypothesis does not prove its veracity. The test may not have sufficient power to detect a change in
. Given the narrowly defined alternative hypothesis in tests for the loss of selective constraint (
= dN/dS = 1), it is relatively easy to simulate sequence evolution according to the alternative hypothesis and estimate the type-II error rate, ß. The type-II error rate is the probability of failing to reject the null hypothesis when the alternative is true. In cases where ß is very small, and yet the null hypothesis is not rejected, one must seriously consider the possibility that the null hypothesis is correct. In the context presented here, when a change in the environment, life history, or developmental program that has rendered known gene function obsolete is not associated with a change in
, selective maintenance of some unknown gene function may be implicated.
Here, we provide a strategy for performing such a power analysis and apply it to the analyses of a photosynthetic gene, rbcL, in nonphotosynthetic parasitic plants (Wolfe and dePamphilis 1997
, 1998
) and a photoreceptor gene coding for an opsin in cave-dwelling crayfish (Crandall and Hillis 1997
). In both examples the failure to detect a change in the selective constraint has raised the question of an alternative function for these genes.
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Materials and Methods |
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Sequence Data
Three plastid genes from eight photosynthetic and seven nonphotosynthetic plant species were included in the rbcL analysis. The rbcL genes sampled from the nonphotosynthetic species were intact, with open reading frames and no apparent amino acid substitutions, relative to photosynthetic seed plants in the region coding for the active site (Kellogg and Juliano 1997
; Wolfe and dePamphilis 1998
). A combined phylogenetic analysis of plastid-encoded intron maturase (matK) and ribosomal protein (rps2) was performed to establish the topological relationships of the species included in the rbcL analysis. Neither of these genes is involved in photosynthesis. Most of the matK (Young, Steiner, and dePamphilis 1999
), rps2 (dePamphilis, Young, and Wolfe 1997
), and rbcL (Wolfe and dePamphilis 1997
, 1998
) sequences were collected for previous studies (table 1
). Additional matK sequences were collected using a Beckman CEQ2000 automated sequencer following the manufacture's protocols as modified by Barkman et al. (2000)
. One-quarter volume sequencing reactions and postsequencing cleanup were performed in 96-well microtiter plates. The methodological details for PCR amplification were as reported by Young, Steiner, and dePamphilis (1999)
. PCR products were sequenced directly following Qiagen PCR column cleanup. All sequences are available on GenBank (table 1
), and the sequence alignments for all three genes have been placed in TreeBASE (http://www.treebase.org/treebase/). The lengths of aligned sequences were 1,320 bp (440 codons) for rbcL, 1,632 bp for matK, and 616 bp for rps2.
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The resulting ML phylogeny was used to identify five pairs of photosynthetic and nonphotosynthetic lineages and an out-group for each pair (figs. 2 and 3 ). Separate tests for change in constraint on rbcL genes in the nonphotosynthetic lineage were performed for each of the five subsets.
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0 if j is a stop codon
0 if i and j differ at more than a single position
j for synonymous transversions
j for synonymous transitions
j for nonsynonymous transversions
j for nonsynonymous transitions.
For each of the five comparisons, the estimated parameters included the transition-transversion rate ratio (), the per site nonsynonymous-synonymous rate ratio,
, and the expected equilibrium frequencies for all 61 amino acidcoding codons,
j. Codon frequencies were estimated from the observed nucleotide frequencies at each of the three codon positions. Ancestral sequences were also estimated in CODEML following the procedure described by Yang, Kumar, and Nei (1995)
. The probability of stop codons arising, pstop, in the absence of selective maintenance was approximated by allowing qij
0 when j is a stop codon and then summing pij(t) across the inferred ancestral sequence for all three stop codons. Note that this approximation assumes that ancestral sequences were estimated without error. The probability of stop codons arising in nonphotosynthetic clades, pstop(clade), for Harveya and Orobanche was approximated as:
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For each of the five analyses, a null distribution for the likelihood ratio was generated using separate Monte-Carlo simulations. The sequence-generating simulation program is available upon request. For each analysis, 100 data sets were simulated using the basal ancestral sequence, model parameters, and branch lengths estimated from the original data sets under the single-rate ratio model (fig. 1
). Following the procedures of Goldman (1993
, Huelsenbeck and Ranala 1997)
, the likelihood ratio was calculated for each simulated data set. P-values were estimated from the simulated null distributions and compared with P-values obtained by assuming that two times the nested likelihood ratio approximates a chi-square distribution for long sequences evolving under the null model (Yang and Bielawski 2000
).
In order to assess the power of each test, 100 additional data sets were simulated according to the two-rate ratio model (fig. 1 ). The likelihood ratio statistic, , was calculated for each simulated data set, and the distribution of the resulting
's was compared with the null distribution. The probability of failing to reject a false null hypothesis, ß, was estimated as the proportion of the power distribution with
less than the upper 5% of the null distribution.
Opsin Analysis
Others have taken alternative approaches for assessing variation among evolutionary lineages in the strength of purifying selection acting on protein-coding genes (Crandall and Hillis 1997
; Nei, Zhang, and Yokoyama 1997
). Here, we use one example to demonstrate how the Monte-Carlo simulation approach described for the likelihood ratio test can be used for any loss-of-constraint test performed on protein-coding genes.
Opsins are photosensitive pigments that play a central role in color vision for vertebrates (Yokoyama S and Yokoyama R 1996
) and invertebrates (Briscoe and Chittka 2001
). Opsins have also been implicated as playing a role in the entrainment of circadian rhythms in vertebrates, including subterranean blind mole rats (Spalax ehrenbergi) (Argamaso et al. 1995
; David-Gray et al. 1999
; Janssen et al. 2000
). Crandall and Hillis (1997)
tested for loss of selective constraint on opsin genes sampled from three independent cave-dwelling crayfish species: Cambarus hubrichti, Orconectes australis, and Procambarus orcinus. Separate comparative analyses were performed to assess rhodopsin gene evolution in each cave-dwelling lineage relative to a surface-dwelling sister species. Analogous to our rbcL investigation, three-taxon data sets included a cave-dwelling species, a surface-dwelling congener, and an out-group taxon (Crandall and Hillis 1997
). Ancestral nucleotide sequences were estimated using CODEML (Yang 1999
), and nonsynonymous and synonymous substitutions were inferred for each pair of related cave- and surface-dwelling crayfish lineages. A 2 x 2 contingency table analysis was then used to test for an association between habitat and the type of inferred nucleotide substitution (nonsynonymous vs. synonymous). When the contingency table analyses and Kolmogorov-Smirnov tests for differences in nucleotide and amino acid substitution matrices all failed to detect a change in the cave-dwelling lineages, Crandall and Hillis (1997)
hypothesized that rhodopsin may have a function other than photoreception in lightless cave environments.
In order to assess the power of the contingency table analysis performed by Crandall and Hillis, we simulated opsin gene evolution taking the same approach described for the rbcL power analysis. For each three-taxon analysis specified in the original study, sequences were simulated with the per site nonsynonymous-synonymous rate ratio set at cave = 1.0 for the cave-dwelling species. Branch lengths, rate ratios for branches leading to the surface-dwelling crayfish,
surface, and ancestral sequence were estimated in CODEML (Yang 1999
).
Following the procedure of Crandall and Hillis, the number of nonsynonymous and synonymous substitutions along branches leading to the cave- and surface-dwelling species were inferred and used to test for independence between habitat and substitution type for all the 100 simulated data sets. The distribution of P-values obtained for Fisher's exact test was calculated for each analysis. Whereas the original paper reported the probability of the inferred contingency table values for nonsynonymouns and synonymous substitutions in cave- and surface-dwelling crayfish under the null hypothesis, we calculated P-values for the more appropriate one-tailed test. The P-values for the one-tailed test are influenced not only by the probability of the observed contingency table values but also by the probabilities of all other contingency tables that would suggest a stronger association while maintaining equal row and column totals. The power of each analysis, ß, was estimated in two ways. First, ß was estimated as the frequency of simulated data sets, where Pone-tailed > 0.05 (fig. 1
). As a comparison, ß was also estimated using a more standard power analysis for Fisher's exact test (Cassagrande, Pike, and Smith 1978
) implemented in DSTPLAN (Brown et al. 2000
).
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Results and Discussion |
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For two of the five subsets, the likelihood ratio test resulted in the rejection of the null hypothesis that was equal in the nonphotosynthetic and photosynthetic lineages (table 2
). Note that for all the likelihood ratio tests, the P-values derived from our simulationbased null distribution were very similar to those based on the chi-square null distribution (table 2 ). The rate ratios differed significantly between Alectra orobanchoides, a holoparasite, and A. sessiliflora, a photosynthetic hemiparasite (table 2
). In this example, the power of the likelihood ratio test was suggested by the significant difference detected in the dN/dS rate ratio along a branch with only five inferred nucleotide substitutions (fig. 3
). However, the probability of failing to reject the null hypothesis when
n = 1.0 on a phylogeny with similar ancestral sequence, branch lengths, and transition bias was quite high (ß = 0.77, fig. 4A
).
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In the Harveya analysis, the tests for change in with loss of photosynthesis was more powerful when photosynthesis was assumed to be lost early in the history of the holoparasitic clades (table 2
, fig. 5
). This makes sense because there is more time for change in constraint to affect the number of observed synonymous and nonsynonymous substitutions. The branches leading to both Orobanche species were quite long (fig. 3E
), and there was no overlap observed between
distributions estimated from data simulated under the null hypothesis (
p =
n) and the alternate hypothesis (
n = 1) (fig. 5
).
Despite the power of the likelihood ratio tests in the Harveya and Orobanche analyses, no change in was detected when the rbcL genes from holoparasitic sister species were assumed to be evolving under the same degree of constraint. Even under the null model, however, the inferred ratio of amino acid to nucleotide substitutions was much higher for H. capensis relative to H. purpurea, and for O. corymbosa relative to O. fasciculata (fig. 3
). On the basis of this observation, we estimated the likelihood ratios for change in
along the branches leading to H. capensis and O. corymbosa (table 2
). For each of these analyses, the P-values reported in table 2
would lead us to reject the null hypothesis if we had proposed a priori that the rbcL genes in H. capensis and O. corymbosa were evolving differently from the rbcL genes in their respective sister species. However, because we posed this hypothesis only after inspecting the inferred substitution patterns, the reported P-values are inflated, and should be interpreted with caution.
With this caveat in mind, the combined results of the likelihood ratio tests and power analyses clearly demonstrate that rbcL evolution has been constrained in the lineages leading to the holoparasitic species S. gesnerioides, H. purpurea, and O. fasciculata. These species are achlorophyllous and thought to be totally dependent on their hosts for carbohydrates. Although weak rbcL gene expression has been detected in L. clandestina (Bricaud, Thalouarn, and Renaudin 1986
; Delavault, Sakanyan, and Thalouarn 1995
; Lusson, Delavault, and Thalouarn 1998
), there are no rbcL gene expression data for S. gesnerioides, H. purpurea, and O. fasciculata. Intact rbcL transcription promoters have been sequenced from both O. fasciculata and O. corymbosa (Wolfe and dePamphilis 1997
). Interestingly, the stem-loop structure deduced for the 3' untranscribed portion of the rbcL gene in photosynthetic plants, thought to be important in termination, is conserved in O. corymbosa but degenerate in O. fasciculate (Wolfe and dePamphilis 1997
).
Although the rbcL genes in some of the holoparasitic lineages included in this investigation are apparently evolving under relaxed selective constraint, it is not surprising that the rbcL reading frames are still intact. Given the inferred ancestral sequences and the relatively short branches along which we are assuming loss of gene function, the probability of stop codons arising in the holoparasitic lineages was inferred to be quite low in most cases (table 2
). As others have pointed out, unexpressed or obsolete genes may persist as open reading frames for some time even in the absence of selective maintenance (Marshall, Raff EC, and Raff RA 1994
). It is also worth noting that the probability of the origin of a stop codon is not perfectly proportional to branch length. The probability is strongly influenced by the frequency of codons in the ancestral gene that are within one nucleotide substitution of a stop codon. As a consequence, the stop codon probabilities reported in table 2
may be inaccurate if the inferred ancestral sequences differ from the true ancestral sequences.
Because length mutations were not considered, the probability that an open reading frame would be truncated in the absence of selective constraint is even greater than the reported stop codon probabilities (table 2
). Analyses of the noncoding regions of the plastid genome suggest that length mutations, especially those involving nucleotide repeats, may be as common as base changes (e.g., Golenberg et al. 1993
). With this in mind, intact functional rbcL sequences can be taken as additional circumstantial evidence that the large subunit of RUBISCO has been selectively maintained in some holoparasitic lineages.
Crayfish Opsin Analysis
Although the contingency test for change in dN/dS used by Crandall and Hillis in their analysis of opsin genes in cave-dwelling crayfish (Crandall and Hillis 1997
) differed from the likelihood ratio test described previously, the power of the test was also assessed with data sets simulated under the alternative hypothesis that hypothetically unconstrained sequences were evolving with dN equal to dS. For all the three contingency tests performed in the original article, ß was found to be greater than 65% (fig. 6
). The more standard power analysis of Cassagrande, Pike, and Smith gave very similar estimates of ß (fig. 6 ). The results of the contingency analyses, therefore, are not suggestive of selectively maintained function for opsin in cave-dwelling crayfish.
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The analyses of protein-coding genes described here are obviously simplistic. A single-rate ratio parameter, , is estimated and modeled for the entire gene. Studies of rbcL evolution have clearly demonstrated variation in amino acid substitution rates among structural domains (Kellogg and Juliano 1997
; Wolfe and dePamphilis 1998
). Recent advances in the modeling of amino acid substitution patterns among the structural domains exhibited in globular proteins (Goldman, Thorne, and Jones 1998
) and transmembrane proteins (Liò and Goldman 1999
) suggest that the estimation of variation in evolutionary rates among gene sequences and phylogenies would be enhanced by including information on the secondary structure. Even in instances when protein structure is not known, analyses allowing for among site variation in
could help elucidate the function of particular sequence domains (Yang et al. 2000
; Yang and Bielawski 2000). The likelihood ratio tests available in PAML for assessing variation in
across a protein-coding gene (Yang et al. 2000
) have been shown to be quite powerful (Anisimova, Bielawski, and Yang 2001
). The number of parameters estimated in such an analysis, however, could be quite large in tests of variation in
across a coding gene and among branches on a phylogeny. Failure to detect lineage-specific adaptive or diversifying selection in such analyses would be difficult to interpret without an accompanying power analysis.
Tests for variation in among domains and branches may be especially weak when branch lengths are short, the variation in selection among domains is subtle, or domain size is limited to a few codons. For example, most of the variation in the spectral sensitivity of vertebrate opsins can be explained by the amino acid composition at just five sites (Yokoyama and Radlwimmer 2001
). Moreover, adaptive substitutions at one or two of these sites have resulted in spectral tuning that has maximized light sensitivity in extreme environments (Yokoyama 1997
). The two functional opsin genes known from the Comoran coelacanth (Latimeria chalumnae) have evolved to produce pigments with maximal sensitivity (
max) in the very narrow range of the light spectrum that reaches its deep-sea habitat (Yokoyama et al. 1999
). Functional cone (David-Gray et al. 1999
) and rod (Janssen et al. 2000
) opsin genes have been identified for the subterranean blind mole rat S. ehrenbergi. As with the coelacanth opsins,
max for the cone opsin shows an adaptive shift to the red portion of the spectrum relative to
max for homologous opsins in other mammals. Although experiments and anatomical studies have shown that S. ehrenbergi is indeed blind, multiple lines of evidence suggest that the reduced, subcutaneous eye and its photosensitive pigments have been coopted to aid in light-mediated entrainment of circadian rhythms (Argamaso et al. 1995
; David-Gray et al. 1999
; Janssen et al. 2000
). In these examples, adaptive evolution has been demonstrated by linking change in amino acid sequence with change in protein function (Yokoyama S and Yokoyama R 1996
; David-Gray et al. 1999
; Yokoyama et al. 1999
). A change in the ratio of nonsynonymous to synonymous nucleotide substitutions has not been demonstrated in these cases.
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Conclusions |
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Testing the fit of gene sequence data to explicit evolutionary models can certainly increase our understanding of gene function. The results of such tests, however, must be interpreted with caution. In some instances power analysis can guide the interpretation of statistical tests that have failed to detect change or reject a null hypothesis. In earlier articles, Wolfe and dePamphilis (1997
, 1998
) found no evidence of reduced evolutionary constraint on rbcL genes of the nonphotosynthetic plants included in their analysis. Three hypotheses were offered to explain their surprising result: (1) the plants are photosynthetic for at least a portion of their life, (2) the carboxylase or oxygenase activity of the peptide encoded by rbcL serves a critical function in some nonphotosynthetic plants, or (3) the open reading frame for rbcL is simply persisting by chance, and it will eventually be interrupted by a stop codon or length mutation. The power analyses performed here suggest that we can reject the third hypothesis for the persistence of rbcL in S. gesnerioides, Harveya purpurea, and Orobanche fasciculata. Physiological and gene expression studies are necessary to test the remaining two hypotheses.
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Acknowledgements |
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Footnotes |
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Keywords: molecular evolution
statistical power
likelihood ratio test
rbcL
opsin
rhodopsin
crayfish
Orobanchaceae
Scrophulariaceae
Address for correspondence and reprints: Jim Leebens-Mack, Department of Biology and Life Sciences Consortium, 518 Mueller Laboratory, Penn State University, University Park, Pennsylvania 16802. jleebensmack{at}psu.edu
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