*Department of Plant and Microbial Biology, University of CaliforniaBerkeley;
Celera Diagnostics, Alameda, California
In the entire fungal kingdom, only DNA substitution rates in the SSU rRNA gene (Berbee and Taylor 1993
, 2001
) and amino acid substitution rates (Heckman et al. 2001
) have been estimated and used to date fungal divergences. However, these molecules are not sufficiently variable to date events at or below the genus level. DNA sequences of protein-coding genes and the internal transcribed spacer (ITS) region are sufficiently variable, but their substitution rates are not known. In this article, we investigated the DNA substitution rates in the protein-coding genes and the ITS region by comparison with the SSU rDNA divergence in a representative fungal lineage, Eurotiomycetes (=plectomycetes) (Eriksson and Winka 1998
). The Eurotiomycete lineage is a monophyletic class of Ascomycota (Berbee and Taylor 1992
) and includes many economically important fungi, such as Penicillium chrysogenum (antibiotic production) and Aspergillus oryzae (soy sauce production), as well as many human pathogens such as Coccidioides immitis (lung disease), Histoplasma capsulatum (lung disease), and Trichophyton rubrum (athlete's foot). Estimating DNA substitution rates in quickly evolving molecules such as the ITS and protein-coding genes will not only provide a means to estimate the divergence times between lineages at and below the genus level, but in conjunction with coalescent theory, it may provide information for estimating epidemiological parameters such as effective population size (Watterson 1975
) and recombination rates (Hey and Wakeley 1997
).
With the aid of published Eurotiomycete phylogenies based on SSU rDNA (Ogawa, Yoshimura, and Sugiyama 1997
; Sugiyama, Ohara, and Mikawa 1999
), we searched for pairs of closely related species which had not only SSU rDNA sequences but also sequences of homologous protein-coding genes. Next, we compared the extent of differentiation at synonymous sites of protein-coding genes between sister taxa. It was found that saturation at synonymous sites of protein-coding loci is attained before divergence of the SSU rDNA sequences reaches 1%2%. A 1%2% divergence usually corresponds to divergences seen within a genus or among closely related genera. Therefore, the protein genes should be well suited to dating divergences at and below the genus level.
In this paragraph we briefly outline our methodology. The first step of our approach was to estimate the divergence times between sister species from the SSU rDNA phylogenetic tree (Ochman and Wilson 1987
; Ochman, Elwyn, and Moran 1999
). Twenty-seven Eurotiomycete species and two out-group taxa, i.e., the Sordariomycete Neurospora crassa and the loculoascomycete Capronia pilosella, were subjected to distance analysis. The regularity of nucleotide substitution rates at the SSU rDNA gene was evaluated by the two-cluster test and the branch length test (Takezaki, Rzhetsky, and Nei 1995
; Nei and Kumar 2000
). Rates were not found to be constant for six species and four nodes using the branch length test and the two-cluster test, respectively (P < 0.05, data not shown). Apparently, rate constancy does not hold throughout this system. Because rates may not be constant, we also considered a model in which molecular evolutionary rates vary across lineages instead of remaining constant. We compared the Langley Fitch (LF) algorithm, which assumes rate constancy, with the nonparametric rate-smoothing (NPRS) algorithm, which does not (Langley and Fitch 1974
; Sanderson 1997
). All methods of phylogenetic inference depend heavily on their underlying models. For this reason we used a hierarchical likelihood ratio test to search for the DNA substitution model that best fit our SSU rDNA data set (Posada and Crandall 1998
). Phylogenetic relationships were then inferred using the selected evolutionary model and the heuristic search option for maximum likelihood (ML) implemented in PAUP 4.0b8a (Swofford 2001
). The ML tree obtained was then used to estimate the divergence times between closely related taxa using the NPRS and LF methods. A simple and widely used Kimura two-parameter model was also used to construct a neighbor-joining (NJ) tree; the NJ tree was also subjected to the NPRS and LF methods as a comparison. Both NPRS and LF methods require at least one calibration point to fit branch lengths to the geological time scale. We used the divergence of Eurotiomycetes (=plectomycetes) and Sordariomycetes (=pyrenomycetes) for calibration. Initially, the divergence was set to 100 arbitrary units, and the relative divergence times between taxa were estimated (fig. 1
; details are available in Supplementary Material at http://www.molbiolevol.org/). For all pairs of taxa, the NPRS algorithm gave 15.2% to 87.5% larger values for divergence times than did the LF algorithm. For both NPRS and LF algorithms, most of the divergence values calculated from the NJ tree with a Kimura two-parameter algorithm were larger than those from the ML tree; NJ values were up to 48.6% larger than ML values. A simulation study indicated that divergence times are estimated more accurately using NPRS rather than LF when (1) there are enough data, (2) evolution is not clock-like, and (3) levels of rate autocorrelation are moderate to high (Sanderson 1997
). Unfortunately, although we believe we have sufficient data (1,631 bp), we have no way of assessing the other parameters, so we have used the divergence times of both the NPRS and the LF methods, hoping to account for the ambiguity of the past.
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Our goal was to estimate DNA substitution rates at six independent protein-coding loci and the ITS of the rDNA repeat unit. To obtain substitution rates, we required estimates for divergence times and genetic distances between sister species. Divergence times between sister species of Eurotiomycete fungi were estimated using NPRS and LF algorithms based on three calibration times corresponding to the three estimates of ES divergence; hence, a total of six rate estimates were calculated for each of the species pairs (table 1
). For class 1 chitin synthase (CHS1), class 2 chitin synthase (CHS2), zinc finger protein (creA), and orotidine-5'-phosphate decarboxylase (pyrG), four different measures of genetic distance were calculated: DNA substitutions in synonymous sites (KS), nonsynonymous sites (KA), the third bases of codons (K3rd), and exons. Introns were not included in these data sets. The modified Nei-Gojobori method implemented in MEGA2.0 (Kumar et al. 2000
) was used to estimate KS and KA. The Kimura two-parameter model was used for all the other distance measures. Large parts of the data sets for ADP-ribosylation factor (arf) and alpha tubulin (tub1) are intron sequences, so for these loci four measures of genetic distance were calculated: DNA substitutions in exons, introns, K3rd, and KS. There were no nonsynonymous substitutions in the arf or tub1 loci.
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Only one pair of sister species could be used for substitution rate estimation in the remaining protein-coding loci, CHS2, creA, pyrG, arf, and tub1. KS values for these genes are also in the range of 10-910-8. The KS values for the four species pairs at the CHS1 and one value for each of the CHS2, creA, pyrG, arf, and tub1 loci gave the average of 6.3 ± 5.4 x 10-9 (n = 9) when the ES divergence of 400 Myr and NPRS were used. On average, in CHS1, CHS2, creA, and pyrG, synonymous substitutions were found to accumulate about 22 times faster (KS/KA = 22.2 ± 8.4, n = 7) than nonsynonymous substitutions. This value is higher than the average ratio of Drosophila genes (KS/KA = 8.2) but is not an extreme value (Li 1997
). The KS/KA ratio is positively influenced by both the intensity of negative selection and the population size.
The arf and tub1 data sets consist of exons and introns, for which rates were estimated separately. At arf and tub1 loci, introns mutate 2.2 and 1.6 times faster than synonymous sites and 8.6 and 4.7 times faster than all sites in exons, respectively.
The ITS in the rDNA repeat unit have been widely used for phylogenetic studies (e.g., Berbee et al. 1995
; Cullings, Szaro, and Bruns 1996
) and population studies (e.g., O'Donnell 1992
; Kasuga et al. 1993
). DNA substitution rates were estimated using six pairs of sister species. Unlike with protein-coding genes such as CHS1 (Bowen et al. 1992
) and RPB2 (Liu, Whelen, and Hall 1999
), multiple alignment of DNA sequences from species belonging to different genera was impossible due to the frequent occurrence of indels. The length at the spacer regions (ITS1 + ITS2) varied greatly between the species used in this study, ranging from 302 to 372 bp. DNA substitution rates at the ITS region (ITS1, ITS2 + 5.8S rDNA) vary extensively between lineages, and the average was 1.4 ± 1.3 x 10-9 (n = 5) when the NPRS and an ES divergence of 400 Myr were used. Despite the large functional difference between the ITS region and the protein-coding genes, DNA substitution rates are found to be comparable. In a wide range of plants, substitution rates at the ITS region fall between 1.72 x 10-9 and 7.83 x 10-9 (Richardson et al. 2001
). Therefore, estimates for Eurotiomycetes are comparable to the lower values for plants.
Neutral Mutation Rates Across Kingdoms
Overall, neutral mutation rates in protein-coding genes, which were measured as synonymous substitution rates or approximated as substitution rates at the third bases of codons, ranged from 0.9 x 10-9 to 16.7 x 10-9 substitutions per site per year (NPRS and ES = 400 Myr). These values are in the range of DNA substitution rates in most of the protein-coding genes in plants (Gaut et al. 1996
), animals (Li 1997
), and bacteria (Ochman, Elwyn, and Moran 1999
). For example, synonymous substitution rates in cereals are in the range of 5.1 x 10-9 to 7.1 x 10-9 (Wolfe, Sharp, and Li 1989
). In mammals, rates were between 1.6 x 10-9 and 6.4 x 10-9, and in Drosophila, rates were between 3.7 x 10-9 and 30.0 x 10-9 (Li 1997
; Moriyama and Powell 1997
). In Escherichia coli and Salmonella typhimurium, substitution rates varied from 0.14 x 10-9 to 5.6 x 10-9 (Ochman and Wilson 1987
). Although neutral mutation rates vary across loci, their range is surprisingly constant among the four major clades plants, animals, bacteria, and fungi in spite of the enormous differences in cellular organization, body size, generation time, and ecology of these organisms.
Acknowledgements
The authors are grateful to Michael Sanderson at the University of California at Davis, Chen Su and Helen Piontkivska at Pennsylvania State University, and Naoko Takezaki at the Max Planck Institute for helping with data analysis; Masato Sugiyama at Mitsubishi Chemical for sharing unpublished data with us; and Anne Pringle for comments on the manuscript. Financial support for this work was provided by the National Institutes of Health (grant A1 43491 to J.W.T.).
Footnotes
Julian Adams, Reviewing Editor
Keywords: synonymous substitution rates
molecular clock
plectomycete
fungi
evolution
ITS
Address for correspondence and reprints: Takao Kasuga, Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, California 94720. E-mail: kasugat{at}uclink4.berkeley.edu
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