*Department of Biology, Faculty of Sciences, Kyushu University;
Bio-resources Technology Division, Forestry and Forest Product Research Institute, Kukizaki, Ibaraki, Japan
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Abstract |
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Introduction |
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One group of species that has not been well studied for molecular evolution is conifer trees. Conifers belong to gymnosperms, and there are currently about 550 species in the group. They have large genome sizes and long life expectancy and have undergone only a few chromosome duplications in their evolution. In addition, they are wind-pollinated and probably for this reason are less differentiated within species (Hamrick and Godt 1990
). These characteristics make this group an interesting target of a molecular evolutionary study.
In this study, we surveyed the molecular evolutionary characteristics of nuclear genes in 10 species of Cupressaceae sensu lato (s.l.), one group of conifers. Their phylogenetic relationships have been inferred using plastid DNA (Brunsfeld et al. 1994
; Tsumura et al. 1995
; Gadek et al. 2000
; Kusumi et al. 2000
), rDNA (Stefanovic et al. 1998
), and immunological analysis (Price and Lowenstein 1989
). These analyses provided well-resolved phylogenetic relationships within Cupressaceae s.l. Gadek et al. (2000)
combined nonmolecular and molecular data and proposed a new infrafamilial classification of Cupressaceae s.l., in which seven subfamilies were recognized.
Meanwhile, in collaboration with other researchers, we have started a genome project of Cryptomeria japonica, which belongs to the Taxodioideae of Cupressaceae s.l. (based on the new classification, Gadek et al. 2000
). Cryptomeria japonica is one of the most important timbers in Japan because of its excellent growth and wood quality. Tsumura et al. (1997)
constructed sequenced-taggedsite (STS) markers, and Iwata et al. (2001)
constructed cleaved amplified polymorphic sequence (CAPS) markers in C. japonica, using libraries from 3-day imbibed embryos and inner bark, adding more markers to a linkage map based on RFLP, RAPD, and isozyme and morphological loci (Mukai et al. 1995
). Furthermore, expressed sequence tags (ESTs) analysis was also carried out (Ujino-Ihara et al. 2000
), and more than 2,000 partial sequences of C. japonica were obtained from the cDNA clones isolated from a library derived from the inner bark tissues. These genetic information and the phylogenetic analyses provide us an opportunity to study the molecular evolution of C. japonica and its related species. Here, we study the molecular evolution of 11 nuclear genes from cDNA clones, whose functions were inferred by homology.
We study the 11 genes from 10 species, including species from three subfamilies of Cupressaceae s.l., Taxodioideae, Cupressoideae, and Sequoioideae. The Taxodioideae include three genera, Cryptomeria, Taxodium, and Glyptostrobus. The inferred relationships among these genera are well supported, and this clade is sister to the traditional Cupressaceae sensu stricto (s.s.), which is subdivided into the Cupressoideae and the Callitroideae according to the new classification (Gadek et al. 2000
). In contrast with the Taxodioideae, the Cupressoideae include 10 genera comprising more than 100 species. Many of the modern genera of these subfamilies originated before the end of the Mesozoic, and the Cupressoideae-Callitroideae clade and Taxodioideae likely diverged roughly 100 MYA (Miller 1977, 1988
). Thus, there has been enough time for these species, potentially, to accumulate sequence variation to study the evolutionary dynamics of genes. Because the Sequoioideae is sister to those two subfamilies, this subfamily was chosen as an outgroup. We characterize the molecular evolution of each of the 11 genes from species of these conifer subfamilies by estimating the synonymous (silent, dS) and nonsynonymous (amino acid replacing, dN) substitution rates in protein-coding regions and the nucleotide substitution rate (K) in noncoding regions.
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Materials and Methods |
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Sequence Analyses
Sequence alignments were first performed by Clustal X (Thompson et al. 1997
) and then refined manually. In order to know the relationships among sequences, we first constructed neighbor-joining trees (Saitou and Nei 1987
) of the 11 genes, using these alignments of DNA sequences. The numbers of synonymous and nonsynonymous substitutions per site (dS and dN) and numbers of nucleotide substitutions per site (K) in noncoding regions were estimated by maximum likelihood (ML), using the CODEML and BASEML programs of the PAML package (Yang 2000
). For the estimation of dS and dN, we used models allowing transition-transversion rate bias and unequal codon frequencies, which were determined using the empirical nucleotide frequencies at the three positions of the codon (F3 x 4 model; Yang and Nielsen 1998
). K was estimated under Kimura's two-parameter model (Kimura 1980
) that also accounts for transition-transversion rate bias. The parameters, dS, dN, and K, were first estimated pairwise by ML for each gene, and the average numbers of substitutions between subfamilies, with their variances, were obtained using the method of Nei and Jin (1989)
. The one-degree-of-freedom (1D) relative rate test of Tajima (1993)
was used to compare the accumulation of site differences between Taxodioideae and Cupressoideae species.
Lineage-specific estimates of dS and dN were obtained by ML using the sequences from three species, Metasequoia glyptostroboides (or Sequoia sempervirens), C. japonica, and Thujopsis dolabrata. In the latter two species, sequences of all 11 nuclear genes could be determined, and so these species were used as representatives of the respective subfamilies. We used two models of the dN/dS () ratio. The first model assumed the same ratio for all branches of the M. glyptostroboides, C. japonica, and T. dolabrata, whereas the second model allowed independent
ratios for the three branches.
G+C content at third codon positions synonymous site (GC3s) and codon usage bias, measured using the effective number of codons (ENC; Wright 1990
), were computed in all sequences using the program CODON (Lloyd and Sharp 1992
).
To test the presence of codon (amino acid) sites with = dN/dS > 1 which can be considered as candidate sites undergoing diversifying selection and to identify them, ML models with variable
ratios among sites were used to analyze each of the 11 nuclear gene data (Nielsen and Yang 1998
). We use the following six models for the
distribution (table 4
), implemented in CODEML program (Yang 2000
). Model 0 (M0) assumes one
ratio (
0) for all codon sites. The neutral model (M1) assumes conserved sites with
0 = 0 and neutral sites with
1 = 1 with a proportion p0 and a proportion p1 = 1 - p0. The selection model (M2) adds an additional
class with frequency p2 = 1 - p0 - p1, with
2 estimated from the data. The discrete model (M3) uses a general discrete distribution with three site classes, with the proportions (p0, p1, and p2) and the
ratios (
0,
1, and
2) estimated from the data. The beta model (M7) assumes that the
ratio varies according to a beta distribution B(p, q), whose domain is bounded within the interval (0, 1). Thus, this model does not allow for codon sites with
> 1. The beta &
model (M8) adds a discrete
class to the beta (M7) model to account for codons with
> 1. Sites with
drawn from the beta distribution B(p, q) occur in a proportion p0, and the rest belong to a discrete
class (
1) and occur in proportion p1 = 1 - p0. We compared each of four pairs of models (M0 vs. M3, M1 vs. M2, M1 vs. M3, and M7 vs. M8) by likelihood-ratio tests (LRTs) to examine the statistical significance of the fit of the model (Yang et al. 2000
).
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Results |
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Because it is important to compare orthologous, not paralogous, genes for estimating substitution rates, we first examine this issue. The highest synonymous substitution rate is that of the Chs, and the lowest is that of the HemA (see table 2
). The former is more than eight times higher than the latter, and this high estimate merits special concern because an overestimation of the substitution rate is expected if we compare paralogous genes. We examined the phylogenetic relationship of the eight Chs sequences using a pine Chs gene obtained from a public database as an outgroup. Topological relationships should not reflect species relationships if we compare paralogous genes, but we obtained exactly the same topological relationship as that obtained from plastid genes by Kusumi et al. (2000)
(data not shown). In addition, Chs was directly sequenced from PCR products, and no sequence heterogeneity was observed. Therefore, we tentatively conclude that the eight Chs sequences are orthologous and that Chs has high synonymous substitution rates, although there is a small possibility that Chs genes are paralogs that retain phylogenetic relationships like the plastid genes. In other genes, we examined the topological relationships without taking outgroup genes (fig. 1
). In all genes, the topological relationships are essentially the same as those of the plastid genes, though gene duplications after the divergence of Cupressoideae and Taxodioideae were found in a few genes. We therefore believe that we are measuring substitution rates of orthologous genes.
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For noncoding regions, only three data sets of introns and two of 3'-untranslated regions (UTRs) were obtained. In three out of five cases, because sequences were short or include extensive indels (or both), lengths of aligned segments became less than 200 bp. Introns of the GapC and Rabggtb genes were relatively long (1,094 and 617 bp each), and estimates from these regions were considered reliable. For GapC and Rabggtb, average nucleotide substitution rates (K) are higher in the Cupressoideae lineage.
In the previous study, we applied Tajima's 1D relative rate test to plastid genes (chlL, matK, and rbcL) and the 28S rRNA gene to test the homogeneity of substitution rates among the lineages. The results indicated that changes in the nucleotide substitution rate have occurred across multiple loci. We also applied the analysis to the 11 nuclear genes. Among 114 tests, only 10 comparisons were significant at the 5% level, and 9 out of 10 significant results indicated higher rates of accumulation of nucleotide substitutions in the Cupressoideae lineage. Although most comparisons of pairs were not significant, in 69% of the comparisons, we observed larger numbers of different sites (Tajima's m in the method) in the Cupressoideae lineage.
The values of the (=dN/dS) ratio estimated using the average synonymous and nonsynonymous substitution rates are also shown in table 2
. As in the previous study, two plastid genes show higher values of the
ratio between Sequoioideae and Cupressoideae. However, 8 out of 11 nuclear genes show higher value of the
ratio between Sequoioideae and Taxodioideae. The mean value of
ratios among the nuclear genes between Sequoioideae and Taxodioideae is 0.153, and it is higher than that between Sequoioideae and Cupressoideae, 0.129.
To test whether the variation of the ratio between the two lineages is significant, we carried out the LRT under two models, one assuming the same
ratio for all branches (lineages) of the tree and another assuming different
ratios among branches. For these three branch analyses, we used sequences from three species, M. glyptostroboides (or S. sempervirens), C. japonica, and T. dolabrata, as representatives of respective subfamilies. The latter two species were chosen because all 11 genes could be sequenced from them. If genes are duplicated (e.g., F3h, Rabggtb), we used copy A (see fig. 1
). The sequences used in analyses are marked by an * in figure 1
. The same choice was made for the following analyses using these three species. First, we assumed a star phylogeny for the estimation. By the LRT, only one gene, the Pat gene, shows a significant difference of the
ratio in the three lineages (P < 0.05). The same analysis was performed using the rooted tree (the outgroup is M. glyptostroboides or S. sempervirens), and the result was similar to that using the star phylogeny. To sum up, heterogeneity of nucleotide substitution rates in the two lineages has occurred in both the nuclear and plastid genomes, but these lineage effects did not significantly change the
ratios of the respective lineages.
From the above ML estimation, the numbers of synonymous and nonsynonymous substitutions for the three branches, M. glyptostroboides (or S. sempervirens), C. japonica, and T. dolabrata, were also obtained. To examine variability of the numbers of synonymous and nonsynonymous substitutions among lineages, we calculated the dispersion index (R) with and without the weighting factor of Gillespie (1989)
. The weighting factors are proportional to the total number of substitutions across all 11 genes along the lineage in the respective categories (synonymous and nonsynonymous). They are 1.453, 0.665, and 0.883 for M. glyptostroboides, C. japonica, and T. dolabrata lineages, respectively, for nonsynonymous substitutions, and 1.319, 0.700, and 0.981, respectively, for synonymous substitutions. We used the dN and dS estimates from the ML method under the model of different
ratios among branches. The results are shown in table 3
. In both categories of substitutions, adjustments for lineage effects by the weighting factors did lower the average values of R. For the synonymous substitutions, without weights (equally weighted) R ranges from 0.281 to 18.612, with an average value of 4.106. When weights are used, R ranges from 0.092 to 5.734, with the average value being reduced to 2.104. For the nonsynonymous substitutions, equally weighted R ranges from 13.229 to 0.000, with an average value of 2.500, but weighted R ranges from 2.772 to 0.136, with a reduced average value of 0.968. This last estimate is surprisingly close to 1, which is expected under a simple Poisson process with the same rate among lineages (Kimura 1983, p. 69
). Therefore, the lineage effects seem to be a significant factor of variation in synonymous and especially in nonsynonymous numbers of substitutions.
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We subsequently examined whether GC3s, ENC, and dN were related to dS variation among the genes, using the data from M. glyptostroboides (or S. sempervirens), C. japonica, and T. dolabrata. We estimated lineage-specific dS and dN under the model with different ratios among branches. Before examining the relationships of dS wth GC3s, ENC, and dN, we checked whether estimates of dS (dN) in the C. japonica and T. dolabrata lineages are correlated across loci or not. The coefficient of determination (r2) of dS estimates is 0.708 (P = 0.005) and that of dN estimates is 0.696 (P = 0.0007) between these two lineages. Subsequently, the relationships of synonymous substitution rate with nonsynonymous substitution rate, GC content, and codon bias were evaluated by linear regression, using the lineage-specific dS and dN, GC3s, and ENC values.
In both lineages, the correlation between dS and dN did not differ significantly from zero (fig. 2a and b,
dotted line). However, these plots have an outlier gene (Chs). This gene has a high dS and a low ratio. Because the number of nonsynonymous differences of the Chs genes between these two species was one, a large variance of the estimates of dS was expected. Thus, we also carried out regression excluding the Chs genes. When the Chs gene was removed, estimates of dS were positively correlated with dN in both lineages (C. japonica, r2 = 0.476, P = 0.0249; T. dolabrata, r2 = 0.793, P = 0.0002) (fig. 2a and b,
solid line). GC3s is also positively correlated with dS, and the correlation coefficient was significant in both lineages (C. japonica, r2 = 0.786, P < 0.0001; T. dolabrata, r2 = 0.403, P = 0.0341) (fig. 2c and d
). On the other hand, dS was not correlated with ENC in either lineage (C. japonica, r2 = 0.22, P = 0.1499; T. dolabrata, r2 = 0.061, P = 0.4766) (fig. 2e and f
).
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Discussion |
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Rate Variation Among Genes
As shown in other organisms, synonymous substitution rates of the nuclear genes vary among loci in conifers. We showed that the synonymous substitution rate was positively correlated with the nonsynonymous substitution rate and GC3s but not with ENC (fig. 2
). This pattern is similar to those reported in mammals (Bielawski, Dunn, and Yang 2000
; Hurst and Williams 2000
). It is well known that mammalian genomes are made up of large regions of distinct base composition, the so-called isochores (Bernardi et al. 1985
), and that the GC content at third codon positions of a gene is correlated with the GC content of the regions in which that gene resides (Ikemura 1985
). Recently, Matassi, Sharp, and Gautier (1999)
investigated synonymous substitution rates and GC content at silent sites among genes lying within 1 cM of each other in mouse and human. They found regional similarity both in the synonymous substitution rate and GC content, but did not find a significant correlation between them. However, other analyses using an ML method indicated a positive correlation between synonymous substitution rate and GC3s (Bielawski, Dunn, and Yang 2000
; Hurst and Williams 2000)
. In addition, Francino and Ochman (1999)
reported that interspecific variation in two globin pseudogenes that reside in different isochores was consistent with the effects of differential GC mutation pressure. These results suggest that variation in synonymous substitution rate among genes at least partially reflects region-specific synonymous mutation rates (Wolfe, Sharp, and Li 1989
), and such regional differences are related to GC content (Ticher and Graur 1989
). Because compositional analyses of genomes and genes have not been carried out in the conifers studied here, we could not conclude whether the positive correlation of the synonymous substitution rate with GC3s observed both in mammal and conifers is caused by a similar evolutionary process or not. Nonetheless, it is interesting that organisms that have different lifecycles and habitats demonstrated similar patterns of nucleotide substitutions.
The nuclear genomes of angiosperms are characterized by compositional compartmentalization (Salinas et al. 1988
), and differences of compositional patterns among genomes have been observed. The genomes of Gramineae are GC rich, and their coding sequences cover a broad compositional range, whereas the genomes of dicots, Arabidopsis, soybean, pea, tobacco, tomato, and potato are GC poor, and their coding sequences cover a narrow GC range (Salinas et al. 1988
; Matassi et al. 1989
; Carels et al. 1998
). However, studies on the relationship between the synonymous substitution rate and base composition are limited. In the Gramineae, Alvarez-Valin et al. (1999)
reported a negative correlation between the synonymous rate and GC3s, and this result is contrary to that observed in conifers. Furthermore, we did not find a correlation between codon usage and synonymous substitution rates (fig. 2e and f), but Fennoy and Bailey-Serres (1993)
suggested that codon usage in maize might reflect both regional bias on nucleotide composition and selection on the third position. What factors make the opposite signs of the correlation between the synonymous rate and GC3s observed in conifers and Gramineae is currently unknown.
A significant positive correlation between synonymous (dS) and nonsynonymous (dN) substitution rates was also found in Drosophila (Dunn, Bielawski, and Yang 2001)
, but it was weak, if existent, in mammals (Bielawski, Dunn, and Yang 2000
; Hurst and Williams 2000
). A positive correlation is expected if both types of substitutions are mutation-driven. Mutation-driven models of molecular evolution include the neutral (Kimura 1968
; see Ohta and Ina 1995
, for a theoretical treatment of the correlation), nearly neutral (Ohta 1973, 1992
) and advantageous mutation models. If environmental fluctuations drive amino acid substitutions as in the SAS-CFF model of Gillespie (1978)
, a positive correlation between dS and dN is not expected unless synonymous substitutions are also driven by the same force. Because we found a positive correlation between synonymous and nonsynonymous substitution rates when the Chs gene was excluded, substitution data in conifers are consistent with the hypothesis of mutation-driven evolution. The exclusion of Chs is justified because the number of nonsynonymous substitutions between C. japonica and T. dolabrata is very small.
The mutation-driven hypothesis of nonsynonymous substitutions is also consistent with the observation of the low dispersion indices, R, for nonsynonymous substitutions. Except in strong interaction models such as the house-of-cards model (see Iwasa 1993
; Gillespie 1994
), the dispersion index is expected to be close to 1, under various mutation-driven models, unless underlying changes of parameters (e.g., population size) are very slow (Araki and Tachida 1997
; Cutler 2000
). The dispersion index is large in mammals (Gillespie 1989
; Ohta 1995
) but not large in Drosophila (Zeng et al. 1998
). Therefore, we suggest that nonsynonymous substitutions in conifers are mainly driven by mutation, not by diversifying selection as suggested by Gillespie (1989)
for mammalian genes. Because the number of sample genes is small, more genes need to be examined to generalize these findings.
Possibility for the Adaptive Selection at the Protein Level
ML analyses based on the M3 model identified three sites and one site with > 1 in the Ferr and F3h genes, respectively, suggesting that they are candidate sites under positive selection. The M8 model also detected the same candidate sites, but the test results were not significant. Because the number of sequences (s = 6) was small and sequence length (n = 115) was short in the Ferr genes in our study, the failure of obtaining a significance between M8 and M7 may be just a lack of power in the LRT. Because the neutrality of synonymous substitutions is not yet known in conifers, the fact that
> 1 in some sites does not automatically mean that those sites are under positive selection, but at least they are good candidates for further research. The ferredoxin donates electrons to several proteins, which are important components of the photosynthesis apparatus and the nitrate reduction in plants. It is notable that such a basic gene has candidate sites for positive selection.
Molecular Evolution in Conifers
In summary, synonymous substitution rates for conifer nuclear genes are higher in Cupressoideae than in Taxodioideae, are variable among loci, and correlate with GC content and nonsynonymous rates but not with ENC. The dispersion indices in nonsynonymous substitutions are close to 1. Some characteristics are similar to those of mammals but others are not. These features of nucleotide substitutions in conifers are considered to reflect various factors that affected the evolution of those species and their ancestors. In addition to increasing the number of genes to examine as to what extent the general features found in this study hold, it is necessary to identify those evolutionary factors and evaluate their effects by accumulating more information on conifers that have been attracting less attention than other taxa thus far.
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Acknowledgements |
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Footnotes |
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Keywords: Cupressaceae
lineage effect
substitution rate
dispersion index
the neutral theory
Address for correspondence and reprints: Hidenori Tachida, Department of Biology, Faculty of Sciences, Kyushu University, Ropponmatsu, Fukuoka 810-8560, Japan. htachscb{at}mbox.nc.kyushu-u.ac.jp
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akashi H., 1995 Inferring weak selection from pattern of polymorphism and divergence at silent sites in Drosophila DNA Genetics 139:1067-1076
Alvarez-Valin F., K. Jabbari, N. Carels, G. Bernardi, 1999 Synonymous and nonsynonymous substitution in genes from Gramineae: intragenic correlations J. Mol. Evol 49:330-342[ISI][Medline]
Araki H., H. Tachida, 1997 Bottleneck effect on evolutionary rate in the nearly neutral mutation model Genetics 147:907-914
Bernardi G., 2000 The compositional evolution of vertebrate genomes Gene 259: (Special issue) 31-43.[ISI][Medline]
Bernardi G., B. Oloffson, J. Flipski, M. Zerial, J. Salinas, G. Cuny, M. Meunierrotival, F. Rodier, 1985 The mosaic genome of warm-blooded vertebrates Science 228:953-958[ISI][Medline]
Bielawski J. P., K. A. Dunn, Z. Yang, 2000 Rates of nucleotide substitution and mammalian nuclear gene evolution: approximate and maximum-likelihood methods leads to different conclusions Genetics 156:1299-1308
Brunsfeld S. J., P. S. Soltis, D. E. Soltis, P. A. Gadek, C. J. Quinn, D. D. Strenge, T. A. Ranker, 1994 Phylogenetic relationships among the genera of Taxodiaceae and Cupressaceae: evidence from rbcL sequences Syst. Bot 19:253-262[ISI]
Carels N., P. Hatey, K. Jabbari, G. Bernardi, 1998 Compositional properties of homologous coding sequence from plants J. Mol. Evol 46:45-53[ISI][Medline]
Cutler D. J., 2000 Understanding the overdispersed molecular clock Genetics 154:1403-1417
Dunn K. A., J. P. Bielawski, Z. Yang, 2001 Substitution rates in Drosophila nuclear genes: implications for translational selection Genetics 157:295-305
Eyre-Walker A., B. S. Gaut, 1997 Correlated rates of synonymous site evolution among plant genomes Mol. Biol. Evol 14:455-460[Abstract]
Fennoy S. L., J. Bailey-Serres, 1993 Synonymous codon usage in Zea mays L. nuclear genes is varied by levels of C and G-ending codons Nucleic Acids Res 21:5294-5300[Abstract]
Francino M. P., H. Ochman, 1999 Isochores results from mutation not selection Nature 400:30-31[ISI][Medline]
Gadek P. A., D. L. Alpers, M. M. Heslewood, C. J. Quinn, 2000 Relationships within Cupressaceae sensu lato: a combined morphological and molecular approach Am. J. Bot 87:1044-1057
Gaut B. S., 1998 Molecular clocks and nucleotide substitution rates in higher plants Evol. Biol 30:93-120[ISI]
Gaut B. S., B. R. Morton, B. M. McCaig, M. T. Clegg, 1996 Substitution rate comparisons between grasses and palms: synonymous rate differences at the nuclear gene Adh parallel rate differences at the plastid gene rbcL Proc. Natl. Acad. Sci. USA 93:10274-10279
Gillespie J. H., 1978 A general model to account for enzyme variation in natural populations. V. The SAS-CFF model Theor. Popul. Biol 13:1-45[ISI][Medline]
. 1989 Lineage effects and the index of dispersion of molecular evolution Mol. Biol. Evol 6:636-647[Abstract]
. 1991 The causes of molecular evolution Oxford University Press, Oxford, U.K
. 1994 Substitution processes in molecular evolution. III Deleterious alleles. Genetics 138:943-952
Hamrick J. L., M. J. Godt, 1990 Allozyme diversity in plant species Pp. 4363 in A. H. D. Brown, M. T. Clegg, A. L. Kahler, and B. S. Weir, eds. Plant population genetics, breeding, and genetic resources. Sinauer, Sunderland, Mass
Hurst L. D., E. J. B. Williams, 2000 Covariation of GC content and the silent site substitution rate in rodents: implications for methodology and for the evolution of isochores Gene 261:107-114[ISI][Medline]
Ikemura T., 1985 Codon usage and tRNA content in unicellular and multicellular organisms Mol. Biol. Evol 2:13-34[Abstract]
Iwasa Y., 1993 Overdispersed molecular evolution in constant environments J. Theor. Biol 164:373-393[ISI][Medline]
Iwata H., T. Ihara-Ujino, K. Yoshimura, K. Nagasaka, Y. Tsumura, 2001 Cleaved amplified polymorphic sequence markers in Sugi, Cryptomeria japonica Theor. Appl. Genet 103:881-895[ISI]
Kimura M., 1968 Evolutionary rate at the molecular level Nature 217:624-626[ISI][Medline]
. 1980 A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences J. Mol. Evol 16:111-120[ISI][Medline]
. 1983 The neutral theory of molecular evolution Cambridge University Press, Cambridge, U.K
Kusumi J., Y. Tsumura, H. Yoshimaru, H. Tachida, 2000 Phylogenetic relationships in Taxodiaceae and Cupressaceae sensu stricto based on matK gene, chlL gene, trnL-trnF IGS region, and trnL intron sequences Am. J. Bot 87:1480-1488
Li W.-H., 1997 Molecular evolution Sinauer, Sunderland, Mass
Lloyd A. T., P. M. Sharp, 1992 CODONS: a microcomputer program for codon usage analysis J. Hered 83:239-240[ISI][Medline]
Matassi G., L. M. Montero, J. Salinas, G. Bernardi, 1989 The isochores organization and compositional distribution of homologous coding sequences in nuclear genomes of plants Nucleic Acids Res 17:5273-5290[Abstract]
Matassi G., P. M. Sharp, C. Gautier, 1999 Chromosomal location effects on gene sequence evolution in mammals Curr. Biol 9:786-791[ISI][Medline]
Miller C. N., 1977 Mesozoic conifers Bot. Rev 43:217-280[ISI]
. 1988 The origin of modern conifer families Pp. 448486 in C. B. Beck, ed. Origin and evolution of gymnosperms. Columbia University Press, New York
Mukai Y., Y. Suyama, Y. Tsumuta, T. Kawahara, H. Yoshimaru, T. Kondo, N. Tomaru, N. Kuramoto, M. Murai, 1995 A linkage map for sugi (Cryptomeria japonica) based on RFLP, RAPD, and isozyme loci Theor. Appl. Genet 90:835-840[ISI]
Nei M., L. Jin, 1989 Variances of the average number of nucleotide substitutions within and between populations Mol. Biol. Evol 6:290-300[Abstract]
Nickrent D. L., R. J. Duff, A. E. Colwell, A. D. Wolfe, N. D. Young, K. E. Steiner, R. J. Duff, 1998 Molecular phylogenetic and evolutionary studies of parasitic plants Pp. 211241 in D. E. Soltis, P. S. Soltis, and J. J. Doyle, eds. Molecular systematics of plants, 2nd edition. Chapman & Hall, New York
Nielsen R., Z. Yang, 1998 Likelihood models for detecting positively selected amino-acid sites and applications to the HIV-1 envelope gene Genetics 148:929-936
Ohta T., 1973 Slightly deleterious mutant substitutions in evolution Nature 246:96-98[ISI][Medline]
. 1992 The nearly neutral theory of molecular evolution Annu. Rev. Syst. Ecol 23:263-286[ISI]
. 1995 Synonymous and nonsynonymous substitutions in mammalian genes and the nearly neutral theory J. Mol. Evol 40:56-63[ISI][Medline]
Ohta T., Y. Ina, 1995 Variation in synonymous substitution rates among mammalian genes and the correlation between synonymous and nonsynonymous divergences J. Mol. Evol 41:717-720[ISI][Medline]
Price R. A., J. M. Lowenstein, 1989 An immunological comparison of the Sciadopityaceae, Taxodiaceae, and Cupressaceae Syst. Bot 14:141-149[ISI]
Saitou N., M. Nei, 1987 The neighbor-joining method: a new method for reconstructing phylogenetic trees Mol. Biol. Evol 4:406-425[Abstract]
Salinas J., G. Mattasi, L. M. Montero, G. Bernardi, 1988 Compositional compartmentalization and compositional patterns in the nuclear genomes of plants Nucleic Acids Res 16:4269-4285[Abstract]
Stefanovic S., M. Jager, J. Deutsch, J. Broutin, M. Masselot, 1998 Phylogenetic relationships of conifers inferred from partial 28S rRNA gene sequences Am. J. Bot 85:688-697[Abstract]
Tajima F., 1993 Simple methods for testing the molecular evolutionary clock hypothesis Genetics 135:599-607
Thompson J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, D. G. Higgins, 1997 The Clustal-X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools Nucleic Acid Res 25:4876-4882
Ticher A., D. Graur, 1989 Nucleic acid composition, codon usage, and the rate of synonymous substitution in protein-coding genes J. Mol. Evol 28:286-298[ISI][Medline]
Tsumura Y., Y. Suyama, K. Yoshimura, N. Shirato, Y. Mukai, 1997 Sequence-tagged-sites (STSs) of cDNA clones in Cryptomeria japonica and their evaluation as molecular markers in conifers Theor. Appl. Genet 94:764-772[ISI]
Tsumura Y., K. Yoshimura, N. Tomaru, K. Ohba, 1995 Molecular phylogeny of conifers using RFLP analysis of PCR-amplified specific chloroplast genes Theor. Appl. Genet 91:1222-1236[ISI]
Ujino-Ihara T., K. Yoshimura, Y. Ugawa, H. Yoshimaru, K. Nagasaka, Y. Tsumura, 2000 Expression analysis of ESTs derived from the inner bark of Cryptomeria japonica Plant Mol. Biol 43:451-457[ISI][Medline]
Wolfe K. H., P. M. Sharp, W.-H. Li, 1989 Mutation rates differ among regions of the mammalian genome Nature 337:283-285[ISI][Medline]
Wright F., 1990 The effective number of codons' used in a gene Gene 87:23-29[ISI][Medline]
Yang Z., 2000 PAML: phylogenetic analysis by maximum likelihood. Version 3.0 University College London, U.K
Yang Z., R. Nielsen, 1998 Synonymous and nonsynonymous rate variation in nuclear genes of mammals J. Mol. Evol 46:409-418[ISI][Medline]
Yang Z., R. Nielsen, N. Goldman, A.-M. K. Pedersen, 2000 Codon-substitution models for heterogeneous selection pressure at amino acid sites Genetics 155:431-449
Zeng L.-W., J. M. Comeron, B. Chen, M. Kreitman, 1998 The molecular clock revisited: the rate of synonymous vs replacement change in Drosophila. Genetica 102/103:369382