*Department of Molecular Genetics
Department of Evolution, Ecology and Organismal Biology, The Ohio State University
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Abstract |
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Introduction |
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On the basis of the results of studies like these, it has been suggested (Birstein and Vasiliev 1987
; Brown et al. 1996
) that the members of the order Acipenseriformes possess reduced rates of evolution. Reduced evolutionary rates (as compared with mammals) have also been found in sharks and turtles, two groups which share some life history characteristics with sturgeon (long generation time, large body size, ectothermy, and low metabolic rate) (Avise et al. 1992
; Martin, Naylor, and Palumbi 1992
; Martin and Palumbi 1993
; Martin 1999
). In addition, reduced evolutionary rates were also discovered in bony fish (as compared with mammals) when the cytochrome b gene sequences of several species of Perciformes and Cypriniformes (Teleostei) were examined (Cantatore et al. 1994
). The evidence presented above prompted us to formally test the hypothesis that the Acipenseriformes possess a reduced rate of molecular evolution. This study utilizes gene and protein sequences available from the literature and our research to carry out relative-rate tests comparing the rates of molecular evolution in the Acipenseriformes and another group of fish, the teleosts.
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Materials and Methods |
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Sequence Alignment and Relative-Rate Analyses
The 18S and 12S rRNA gene sequences were aligned by eye using the computer program ESEE (Cabot and Beckenbach 1989
) with the aid of secondary structures created for the 18S and 12S rRNAs of P. spathula based on currently accepted models (Gutell 1994
). Structural loop regions of the 18S rRNA gene sequences that could not be reliably aligned because of differences in length were omitted from the analyses. The 18S rRNA gene alignment was 1,787 base pairs in length, and the 12S rRNA gene alignment was 997 base pairs in length. The two rRNA gene alignments may be found in the alignment database at the European Bioinformatics Institute (http://www.ebi.ac.uk) (see Supplementary Material for a list of the accession numbers).
The cytochrome b, cytochrome c oxidase subunit II, glucagon, glycoprotein hormone alpha subunit, heat shock protein 70, insulin, recombinase activating protein-1, and recombinase activating protein-2 protein sequences were easily aligned by eye using the computer program ESEE (Cabot and Beckenbach 1989
). The beta-2 microglobulin, CXC chemokine receptor 4, follicle-stimulating hormone, growth hormone, lutenizing hormone, prolactin, proopiomelanocortin, proteolipid protein, rhodopsin, somatolactin, thyroid-stimulating hormone, triosephosphate isomerase, and vitellogenin protein sequences were aligned using the computer sequence alignment program CLUSTALX (Thompson et al. 1997
), and then any necessary adjustments to the alignments were made by hand. The insulin protein alignment includes sequences for both the A and B chains. The proopiomelanocortin protein alignment contains only regions of the molecule that represent corticotropin, alpha-melanotropin, beta-melanotropin, and beta-endorphin, and any intervening regions that were similar in sequence, because these were the only regions that could be reliably identified and aligned for the seven species examined. A short sequence found at the amino end of the growth hormone of four species used in the tests was not available for the other two species used, so this region was omitted from the alignment. Only the amino half of the vitellogenin protein alignment was used for the tests because it was too difficult to accurately align the carboxyl end. Also, only partial follicle-stimulating hormone, lutenizing hormone, proteolipid protein, recombinase activating protein-1, recombinase activating protein-2, rhodopsin protein, and triosephosphate isomerase sequences were available, so just these available regions were used. (The recombinase activating protein-2 alignment is actually a combination of sequences from two different regions of the protein.) The lengths of the final protein sequence alignments were as follows: beta-2 microglobulin122 amino acids, cytochrome b382 amino acids, cytochrome c oxidase subunit II230 amino acids, CXC chemokine receptor 4360 amino acids, follicle-stimulating hormone129 amino acids, glucagon36 amino acids, glycoprotein hormone alpha subunit120 amino acids, growth hormone193 amino acids, heat shock protein 7046 amino acids, insulin53 amino acids, lutenizing hormone144 amino acids, prolactin226 amino acids, proopiomelanocortin97 amino acids, proteolipid protein153 amino acids, recombinase activating protein-1326 amino acids, recombinase activating protein-2259 amino acids, rhodopsin288 amino acids, somatolactin protein236 amino acids, thyroid-stimulating hormone146 amino acids, triosephosphate isomerase232 amino acids, and vitellogenin1,102 amino acids. The alignments for these 21 protein sequences may also be found in the alignment database at the European Bioinformatics Institute (http://www.ebi.ac.uk) (see Supplementary Material for a list of the accession numbers).
The relative-rate test was carried out separately with representatives of all available teleost ingroups utilized in this study in order to determine if the trend in acipenseriform evolutionary rate was consistent when acipenseriforms were compared with different teleost species. In an attempt to compensate for possible rate differences among the teleost species, the rate test for each gene was also carried out using the available teleost sequences included in the study for that gene combined together as the ingroup, allowing the acipenseriform rate to be compared with the overall teleost rate. For each gene or protein examined, the computer program PHYLTEST (Kumar 1996
) was used to carry out the two-cluster relative-rate test of Takezaki, Razhetsky, and Nei (1995), which is illustrated in figure 1
. The test uses, as a reference taxon, an outgroup that diverged from the lineage leading to the two taxa being compared before the latter diverged from each other. The reference taxon allows indirect comparison of the amount of change found in the lineages leading to each of the ingroups since the two ingroups diverged from one another (point X in fig. 1
). This is possible because one can measure the amount of change that has occurred between the outgroup and each ingroup, as well as between the two ingroups by calculating genetic distances. The formulas shown in figure 1 illustrate how these genetic distances can be used to determine the amount of change taking place in the lineages leading to each ingroup since they diverged from one another (AX and BX in fig. 1
). In this way, one can obtain a comparison of the relative rates of evolution in the two ingroups without knowing the exact timing of their divergence from the fossil record. For relative-rate comparisons of protein sequences, the Poisson correction distance was used, whereas the Kimura two-parameter distance (Kimura 1980
) was used for tests involving gene sequences. These corrected distances were chosen because of the distant evolutionary relationships between the taxa compared in the tests. PHYLTEST (Kumar 1996
) was also used to conduct a two-tailed normal deviate test (Takezaki, Razhetsky, and Nei 1995
) for each relative-rate comparison to determine if the differences in evolutionary rates between the acipenseriforms and teleosts were statistically different form zero at the 5% level.
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Because our conclusions are not necessarily based on the significance of any single locus comparison, we have also considered the overall patterns of rates using a nonparametric sign test based on the binomial distribution. Signs of the relative rate in the acipenseriform lineage compared with a specific teleost taxon lineage were examined, and the significance of the overall patterns of rate differences was determined. In addition, using the information obtained from the Tajima test on the number of unique changes, the proportion of unique changes at a locus in paired lineages was examined in the entire protein data set using paired t-tests.
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Results |
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The relative proportion of unique amino acid differences in the data was evaluated (as identified from the alignments using the Tajima's relative-rate test in MEGA). In a comparison of outliers with cypriniform and acipenseriform sequences, 4,463 amino acid sites were aligned. Of these, 517 sites (11.6%) showed cyprinform-specific amino acids, whereas only 290 (6.5%) showed acipenseriform-specific changes. Using a paired t-test to evaluate differences over all protein loci, acipenseriform taxa showed a highly significant deficiency of changes (t = 2.71, df = 19, P < 0.01 for a one-tailed test). For the salmonid-acipenseriform comparison, 4,431 amino acid sites were compared, with 526 (11.9%) salmonid and 278 (6.3%) acipenseriform-specific changes. Differences over all protein loci evaluated by the paired t-test show acipenseriform taxa with a highly significant deficiency of changes (t = 3.30, df = 19, P < 0.01 for a one-tailed test). For the percomorph-acipenseriform comparison, 4,502 amino acid sites were compared, with 550 (12.2%) percomorph and 289 (6.4%) acipenseriform-specific changes. Differences over all protein loci in the paired t-test show acipenseriform taxa with a highly significant deficiency of changes (t = 3.80, df = 19, P < 0.01 for a one-tailed test). Finally, for the elopomorph-acipenseriform comparison, 2,151 amino acid sites were compared, with 208 (9.7%) elopomorph and 115 (5.3%) acipenseriform-specific changes. Differences over all protein loci in the paired t-test show acipenseriform taxa with a significant deficiency of changes (t = 1.80, df = 12, P < 0.05 for a one-tailed test). Overall, the proportion of unique changes in the four teleost lineages averaged 1.85 times that seen in the acipenseriform lineage.
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Discussion |
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A number of hypotheses have been proposed to explain the apparent slowed rates of molecular evolution observed in other taxa. Among these, the hypotheses that seem to be most relevant to a consideration of acipenseriform-teleost contrasts involve the inverse relationships between body size, generation time, or metabolic rate (or all) and rates of molecular evolution. The generation time hypothesis (Kohne 1970
; Li, Tanimura, and Sharp 1987
; Ohta 1993
; Mooers and Harvey 1994
; Li et al. 1996
) is based on the assumption that species with shorter generation times (rapid sexual maturation) possess germ lines that have a larger number of DNA replication events per year, so that they have a greater chance of replication error per unit time. However, germ line DNA replication events may not always be directly correlated to generation time; species may differ in the number of cell divisions per generation (Chang et al. 1994
; Li et al. 1996
). The metabolic rate hypothesis (Martin and Palumbi 1993
; Martin 1999
) proposes that higher metabolic rates produce more DNA-damaging chemicals (particularly free oxygen radicals), which increases the mutation rate relative to species with lower metabolic rates.
Contemporary acipenseriform species possess long generation times and low metabolic rates. Although there are ranges in body size and ages at sexual maturity depending on the species considered, sturgeon and paddlefish are some of the largest freshwater fish species. In nature, these fish take a relatively long time to reach sexual maturity, as compared with other fish species (approximately 523 years depending on sex because females generally take longer than males) (Birstein 1993
and references therein). In addition, the large body size and ectothermy of these fish suggests that they possess a relatively low metabolic rate, which has also been supported by experimental evidence. Singer, Mahadevappa, and Ballantyne (1990)
noted that the rate of oxygen use in a salmonid, as determined by Brett (1972)
, was 5.5 times higher than that in a white sturgeon (A. transmontanus) of similar weight, as determined by Burggren (1978)
. In a study of the metabolism of lake sturgeon, (A. fulvescens), Singer, Mahadevappa, and Ballantyne (1990)
found that the levels of citrate synthase (an enzyme involved in the Krebs cycle and characteristic of oxidative metabolism) in the sturgeon heart were four times lower than those found in the hearts of salmonids (Ewart and Driedzic 1987), again suggesting an overall lower metabolic rate. Burggren, Dunn, and Barnard (1979)
determined the weight-specific gill area of lamellar blood channels in white sturgeon to be among the lowest found in many fish species examined, which is believed to reflect a low activity level and metabolic rate in sturgeon. Additional studies (Burggren 1978
; Burggren and Randall 1978
) found that sturgeons have a relatively low resting metabolic rate that does not increase much (23 times) during maximum swimming speeds and is lower than that of many fish of comparable sizes tested at similar temperatures. The low metabolic rate of sturgeons is probably a consequence of their slow moving, bottom feeding lifestyle (Burggren, Dunn, and Barnard 1979
; Singer, Mahadevappa, and Ballantyne 1990
).
Thus, the long generation time and low metabolic rate (or both) of the Acipenseriformes could be responsible for the slowed rate of molecular evolution observed in sturgeon and paddlefish (see Brown et al. 1996
; Birstein, Hanner, and DeSalle 1997
; Birstein and DeSalle 1998
; Krieger, Fuerst, and Cavender 2000
). It should be noted, however, that some studies on mammalian taxa failed to find correlations between generation time, body size, or metabolic rate (or all) and evolutionary rate (Sarich and Wilson 1973
; Bromham, Rambaut, and Harvey 1996
; Gissi et al. 2000
). Therefore, different factors may be influencing rates of molecular evolution in different groups, possibly even factors that have not yet been considered.
In conclusion, evidence has been presented for a reduced rate of molecular evolution in a sample of 23 nuclear and mitochondrial loci in the Acipenseriformes compared with teleost fishes by conducting relative-rate tests. This phenomenon may be related to the life history and metabolic characters possessed by sturgeon and paddlefish. A slowed molecular evolutionary rate in the Acipenseriformes would explain the observed low levels of genetic divergence observed among species of the group (for example: Brown et al. 1996
; Birstein, Hanner, and DeSalle 1997
; Birstein and DeSalle 1998
; Krieger, Fuerst, and Cavender 2000
) and may also help explain the recent discovery of the existence of intraindividual variation of the 18S rRNA gene in sturgeon (J. Krieger and P. A. Fuerst, unpublished data).
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Acknowledgements |
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Footnotes |
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Keywords: Acipenseriformes
evolutionary rates
relative-rate test
sturgeon
teleost
Address for correspondence and reprints: Jeannette Krieger, Department of Molecular Genetics, The Ohio State University, 484 West Twelfth Avenue, Columbus, Ohio 43210. krieger.15{at}osu.edu
; jkrieg{at}frontiernet.net
.
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