Evidence for a Slowed Rate of Molecular Evolution in the Order Acipenseriformes

Jeannette Krieger and Paul A. Fuerst

*Department of Molecular Genetics
{dagger}Department of Evolution, Ecology and Organismal Biology, The Ohio State University


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
A test of the hypothesis that the members of the order Acipenseriformes (sturgeons and paddlefishes) possess a slowed rate of molecular evolution was carried out by conducting relative-rate comparisons with representatives of four groups of teleost fishes (Cypriniformes, Elopomorpha, Salmonidae, and Percomorpha) using 21 nuclear or mitochondrial protein loci and the nuclear and mitochondrial small subunit rRNA genes, obtained from the literature or our own research. In 70 out of 81 comparisons between individual taxa (86%), acipenseriform sequences showed slower rates of change than the homologous teleost loci examined. When teleost sequences are considered together, 21 of the 23 loci show slower rates of substitution in the acipenseriform lineage. Teleost proteins show 1.85 times as many unique amino acid differences as acipenseriform proteins, when both are compared with outlier sequences. These results support a hypothesis of slowed molecular evolutionary rate in the Acipenseriformes.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Sturgeon and paddlefish are considered "living fossils" (Gardiner 1984Citation ) because although the order Acipenseriformes first appeared in the fossil record approximately 200 MYA, it appears that they have not undergone much morphological change since the origin of the group. In addition, various karyotypic and genetic studies have found limited amounts of change in chromosomes and DNA sequences when different species of acipenseriforms are compared. For example, Kedrova, Wladytchenskaya, and Antonov (1980)Citation found that almost all genome fractions of the four species of sturgeon they examined (Huso huso, Acipenser ruthenus, A. stellatus, and A. gueldenstaedti) were homologous. Birstein and Vasiliev (1987)Citation concluded from their karyotypic study of three sturgeon species containing ~120 chromosomes (H. huso, A. ruthenus, and A. stellatus) and the results of other karyotypic studies of two species (Polyodon spathula—Dingerkus and Howell 1976Citation and Scaphirhynchus platorynchus—Ohno et al. 1969Citation ) containing ~120 chromosomes that the similarity of the karyotypes among these species indicates a slow rate of karyological evolution. In their study of six different species, De la Herrán et al. (2001)Citation found low rates of mutation and homogenization for the HindIII satellite DNA family in sturgeon, which they note is not typical of most other satellite DNAs. Examination of partial and whole nuclear 18S rRNA genes in the Acipenseriformes by Birstein, Hanner, and DeSalle (1997)Citation and J. Krieger and P. A. Fuerst (unpublished data) indicates a very small amount of difference in sequence between functional sequence variants of different species. The study by J. Krieger and P. A. Fuerst (unpublished data) detected only one base substitution in this gene between the functional (expressed) sequences from lake sturgeon (A. fulvescens) and North American paddlefish (P. spathula), which are members of two different families within the order. Examination of mitochondrial DNA (mtDNA) sequences, which are generally considered to be relatively quickly evolving in vertebrates, has also indicated low amounts of sequence divergence among different acipenseriform species relative to the age of the group as a whole (Brown et al. 1996Citation ; Birstein, Hanner, and DeSalle 1997Citation ; Birstein and DeSalle 1998Citation ; Krieger, Fuerst, and Cavender 2000Citation ). The small amount of divergence in various mtDNA sequences (D-loop and cytochrome b, 16S rRNA, 12S rRNA, cytochrome c oxidase subunit II, tRNAPhe and tRNAAsp genes) seen among species of this group could, in part, be because of relatively recent divergence times for the species in this group. The idea of recent divergence times was suggested by Choudhury and Dick (1998)Citation in their study of the historical biogeography of sturgeon. The authors concluded: "It appears that although the acipenserids are a geologically old group, the historical biogeography of surviving lineages is best explained by more recent geological and climatic changes." However, this probably does not completely explain the low levels of divergence among mtDNA sequences from acipenseriform species. Brown et al. (1996)Citation carried out analyses of RFLP patterns and D-loop sequences in four species of North American sturgeon (A. oxyrinchus, A. fulvescens, A. transmontanus, and A. medirostris). They determined that to remain in accordance with the Miocene speciation events postulated to have separated eastern (A. oxyrinchus and A. fulvescens) and western (A. transmontanus and A. medirostris) sturgeon species in North America 7–12 MYA (Cavender 1986Citation ), the mutation rates of mtDNA in sturgeon must be two- to fourfold lower than that of mammalian mtDNA (Brown et al. 1996Citation ).

On the basis of the results of studies like these, it has been suggested (Birstein and Vasiliev 1987Citation ; Brown et al. 1996Citation ) 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. 1992Citation ; Martin, Naylor, and Palumbi 1992Citation ; Martin and Palumbi 1993Citation ; Martin 1999Citation ). 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. 1994Citation ). 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.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Gene and Protein Sequence Selection
The literature was searched in an attempt to identify genes or proteins for which an acipenseriform sequence, an outgroup (shark—Chondrichthyes, lungfish—Dipnoi, lamprey—Petromyzontiformes, or rat—Rodentia) sequence, and sequences from two or more of four teleost ingroups (one representative each from the Salmonidae, Cypriniformes, Percomorpha, and Elopomorpha) were available. Twenty-three genes or proteins were identified that fit these criteria, although sequences from all four teleost ingroups utilized in this study were not available for all the genes or proteins examined. Twenty of these sequences are nuclear: the 18S ribosomal RNA gene (18S rRNA gene), the beta-2 microglobulin protein, the CXC chemokine receptor 4 protein, the follicle-stimulating hormone protein, the glucagon protein, the glycoprotein hormone alpha subunit, the growth hormone protein, the heat shock protein 70, the insulin protein, the prolactin protein, the proopiomelanocortin protein, the proteolipid protein, the recombinase activating protein-1, the recombinase activating protein-2, the rhodopsin protein, the somatolactin protein, the thyroid-stimulating hormone protein, the triosephosphate isomerase protein, and the vitellogenin protein. The other three loci are mitochondrial: the 12S ribosomal RNA gene (12S rRNA gene), the cytochrome c oxidase subunit II protein, and the cytochrome b protein. The amino acid sequences of protein-coding genes were used instead of the nucleotide sequences because the distant relationships between the groups used in this study has likely resulted in the saturation of the nucleotide sequences with mutations, making them less informative for relative-rate analyses. The use of a closely related outgroup for relative-rate tests makes the statistical test of rate constancy more effective in rejecting the null hypothesis of equal rates of molecular change between the ingroup taxa. The outgroup most widely believed to be most closely related to acipenseriforms and teleosts is the order Polypteriformes. They have been suggested to be the sister group of Acipenseriformes + Neopterygii (gars, bowfin, and teleosts) by both morphological and molecular phylogenetic studies (Patterson 1982Citation ; Lauder and Liem 1983Citation ; Lê Lecointre, and Perasso 1993Citation ; Bemis, Findeis, and Grande 1997Citation ; Venkatesh, Ning, and Brenner 1999Citation ). Ideally, sequences from species belonging to the Polypteriformes should therefore be used to represent the outgroup. Unfortunately, relatively few polypteriform gene or protein sequences are available in the literature. Therefore, shark sequences were used when available, then lamprey, then lungfish, and if a fish outgroup sequence was not available, a mammal sequence (rat) was used. When there was more than one sequence reported from a species for a gene or protein in the literature, all available sequences were used together to represent that species. If both sturgeon and paddlefish sequences were available for a particular gene or protein, they were used together to represent the Acipenseriformes. This would compensate for possible rate differences within the Acipenseriformes when comparisons were made with the evolutionary rates in the teleosts (although, as discussed, there is little evidence for large amounts of DNA sequence divergence among acipenseriform species). One of the programs used for the analysis, PHYLTEST, allows multiple sequences to be used in representing a particular lineage (Takezaki, Razhetsky, and Nei 1995Citation ). A list of the species used in the relative-rate tests for each gene or protein, along with the GenBank accession numbers for their sequences, is given in the supplementary materials section.

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 1989Citation ) with the aid of secondary structures created for the 18S and 12S rRNAs of P. spathula based on currently accepted models (Gutell 1994Citation ). 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 1989Citation ). 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. 1997Citation ), 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 microglobulin—122 amino acids, cytochrome b—382 amino acids, cytochrome c oxidase subunit II—230 amino acids, CXC chemokine receptor 4—360 amino acids, follicle-stimulating hormone—129 amino acids, glucagon—36 amino acids, glycoprotein hormone alpha subunit—120 amino acids, growth hormone—193 amino acids, heat shock protein 70—46 amino acids, insulin—53 amino acids, lutenizing hormone—144 amino acids, prolactin—226 amino acids, proopiomelanocortin—97 amino acids, proteolipid protein—153 amino acids, recombinase activating protein-1—326 amino acids, recombinase activating protein-2—259 amino acids, rhodopsin—288 amino acids, somatolactin protein—236 amino acids, thyroid-stimulating hormone—146 amino acids, triosephosphate isomerase—232 amino acids, and vitellogenin—1,102 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 1996Citation ) 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 1980Citation ) 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 1996Citation ) was also used to conduct a two-tailed normal deviate test (Takezaki, Razhetsky, and Nei 1995Citation ) 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.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 1.—Relative-rate test diagram and formulas illustrating the two-cluster test method (Takezaki, Razhetsky, and Nei 1995Citation ) used in the computer program PHYLTEST (Kumar 1996Citation ), which was used in this study

 
We have also utilized the nonparametric relative-rate test of Tajima (1993)Citation to assess the significance of sequence differences in a three taxa situation. This avoids statistical complications that can arise when assuming specific patterns of amino acid or nucleotide substitution. Calculation of relative-rate tests with Tajima's nonparametric relative-rate test was performed using MEGA Version 2.1 (Kumar et al. 2001)Citation . Levels of significance observed in the nonparametric test were compared with those obtained using PHYLTEST. Further, the nonparametric test allows the identification of the number of unique amino acid changes that have occurred in each of the derived lineages (i.e., acipenseriform or teleost) and allows a quantification of the deficiency (or excess) of amino acid changes in the acipenseriform lineage relative to the teleost lineage.

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.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Table 1 shows a summary of the results of the two-cluster relative-rate tests of Takezaki, Razhetsky, and Nei (1995) comparing acipenseriform sequences with those of a cypriniform, a salmonid, a percomorph, an elopomorph, or all available teleost groups combined. Seventy of 81 comparisons (86%) between individual taxonomic groups showed the acipenseriform sequences evolving more slowly than those of the teleosts. When the four teleost groups are combined for analysis, 21 of the 23 loci show acipenseriform sequences evolving more slowly. The consistency of the results when using different outgroups to compare different gene-protein sequences indicates that the use of different taxa to represent the outgroup apparently did not affect the results. When different outgroup sequences were available for a locus, no difference was seen in the patterns of lineage differences or in the significance of the results. The results of 39 of the 104 separate locus comparisons were statistically significant at the 5% level by the two-tailed normal deviate test conducted in PHYLTEST (Kumar 1996Citation ), as indicated by asterisks in Table 1 . Results from Tajima's test agree closely. The sign of all tests is seen to be the same as determined by PHYLTEST. The significance levels are very similar as well. Two comparisons that are significant in table 1 did not show significance using Tajima's test (beta-2 microglobulin and somatolactin, both involving percomorph comparisons), whereas four that are not significant in table 1 were found to show significantly slower changes in acipenseriforms using Tajima's test (insulin comparing percomorphs and elopomorphs, rhodopsin comparing cypriniforms, and cytochrome c oxidase subunit II involving percomorphs). For both tests, all the statistically significant comparisons showed the acipenseriform sequences to be evolving more slowly than the sequences of teleosts. Only one gene and four protein sequences were, in some cases, found to be evolving more quickly in acipenseriforms than in teleosts. Only two loci show a consistent pattern of more rapid evolution in the acipenseriform taxa. Glucagon was evolving more quickly in the acipenseriform in all five comparisons with teleosts, and acipenseriform lutenizing hormone was evolving more quickly in all comparisons, except in that with the percomorph.


View this table:
[in this window]
[in a new window]
 
Table 1 Results of Two-Cluster Relative-Rate Tests (Takezaki, Razhetsky, and Nei 1995) Carried Out with the Computer Program PHYLTEST (Kumar 1996) for Acipenseriforms and Teleosts

 
When considering the overall pattern of relative rates, using the sign test on the direction of change noted in table 1 , results showed a significant pattern of slower change when acipenseriform taxa were compared with each of the four teleost taxon groups (P < 0.01 for comparisons with cypriniform, salmonid, or percomorph taxa, and P < 0.05 when compared with Elopomorpha).

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.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Our results have shown a significantly slower rate of molecular change in the lineage that gave rise to the modern sturgeon and paddlefish, compared with the primary fish lineage which gave rise to the teleost fishes. This is particularly interesting because a previous study that examined perciform and cypriniform cytochrome b gene sequences determined that teleosts have a slower rate of molecular evolution than mammals (Cantatore et al. 1994Citation ). Our results indicate that the molecular evolutionary rate in the Acipenseriformes is likely even further reduced than that in teleosts. About 86% of pairwise relative-rate comparisons between acipenseriforms and teleosts showed slower rates in the Acipenseriformes. An examination of the average pattern of amino acid substitution suggests that the rate of change may be almost twice as fast in the teleost line. In addition, the reduction in evolutionary rate seems to be common to both nuclear and mitochondrial sequences.

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 1970Citation ; Li, Tanimura, and Sharp 1987Citation ; Ohta 1993Citation ; Mooers and Harvey 1994Citation ; Li et al. 1996Citation ) 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. 1994Citation ; Li et al. 1996Citation ). The metabolic rate hypothesis (Martin and Palumbi 1993Citation ; Martin 1999Citation ) 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 5–23 years depending on sex because females generally take longer than males) (Birstein 1993Citation 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)Citation noted that the rate of oxygen use in a salmonid, as determined by Brett (1972)Citation , was 5.5 times higher than that in a white sturgeon (A. transmontanus) of similar weight, as determined by Burggren (1978)Citation . In a study of the metabolism of lake sturgeon, (A. fulvescens), Singer, Mahadevappa, and Ballantyne (1990)Citation 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)Citation 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 1978Citation ; Burggren and Randall 1978Citation ) found that sturgeons have a relatively low resting metabolic rate that does not increase much (2–3 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 1979Citation ; Singer, Mahadevappa, and Ballantyne 1990Citation ).

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. 1996Citation ; Birstein, Hanner, and DeSalle 1997Citation ; Birstein and DeSalle 1998Citation ; Krieger, Fuerst, and Cavender 2000Citation ). 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 1973Citation ; Bromham, Rambaut, and Harvey 1996Citation ; Gissi et al. 2000Citation ). 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. 1996Citation ; Birstein, Hanner, and DeSalle 1997Citation ; Birstein and DeSalle 1998Citation ; Krieger, Fuerst, and Cavender 2000Citation ) 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).


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was partially supported by a Theodore Roosevelt Memorial Fund Grant from the American Museum of Natural History and a Dissertation Improvement Award from the National Science Foundation.


    Footnotes
 
Dan Graur, Reviewing Editor

Keywords: Acipenseriformes evolutionary rates relative-rate test sturgeon teleost Back

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 . Back


    References
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

    Avise J. C., B. W. Bowen, T. Lamb, A. B. Meylan, E. Bermingham, 1992 Mitochondrial DNA evolution at a turtle's pace: evidence for low genetic variability and reduced microevolutionary rate in the Testudines Mol. Biol. Evol 9:457-473[Abstract]

    Bemis W. E., E. K. Findeis, L. Grande, 1997 An overview of Acipenseriformes Environ. Biol. Fish 48:25-71[ISI]

    Birstein V. J., 1993 Sturgeons and paddlefishes: threatened fishes in need of conservation Conserv. Biol 7:773-787[ISI]

    Birstein V. J., R. DeSalle, 1998 Molecular phylogeny of Acipenserinae Mol. Phylogenet. Evol 9:141-155[ISI][Medline]

    Birstein V. J., R. Hanner, R. DeSalle, 1997 Phylogeny of the Acipenseriformes: cytogenetic and molecular approaches Environ. Biol. Fish 48:127-155[ISI]

    Birstein V. J., V. P. Vasiliev, 1987 Tetraploid-octoploid relationships and karyological evolution in the order Acipenseriformes (Pisces): karyotypes, nucleoli, and nucleolus-organizer regions in four acipenserid species Genetica 72:3-12[ISI]

    Brett J. R., 1972 The metabolic demand for oxygen in fish, particularly salmonids, and a comparison with other vertebrates Respir. Physiol 14:151-170[ISI][Medline]

    Bromham L., A. Rambaut, P. H. Harvey, 1996 Determinants of rate variation in mammalian DNA sequence evolution J. Mol. Evol 43:610-621[ISI][Medline]

    Brown J. R., K. Beckenbach, A. T. Beckenbach, M. J. Smith, 1996 Length variation, heteroplasmy and sequence divergence in the mitochondrial DNA of four species of sturgeon (Acipenser) Genetics 142:525-535[Abstract/Free Full Text]

    Burggren W. W., 1978 Gill ventilation in the sturgeon, Acipenser transmontanus: unusual adaptations for bottom dwelling Respir. Physiol 34:153-170[ISI][Medline]

    Burggren W. W., J. Dunn, K. Barnard, 1979 Branchial circulation and gill morphometrics in the sturgeon Acipenser transmontanus, and ancient Chondrosteian fish Can. J. Zool 57:2160-2170[ISI]

    Burggren W. W., D. J. Randall, 1978 Oxygen uptake and transport during hypoxic exposure in the sturgeon Acipenser transmontanus Respir. Physiol 34:171-183[ISI][Medline]

    Cabot E. L., A. T. Beckenbach, 1989 Simultaneous editing of multiple nucleic acid and protein sequences with ESEE Comput. Appl. Biosci 5:233-234[Medline]

    Cantatore P., M. Roberti, G. Pesole, A. Ludovico, F. Milella, M. N. Gadaleta, C. Saccone, 1994 Evolutionary analysis of cytochrome b sequences in some Perciformes: evidence for a slower rate of evolution than in mammals J. Mol. Evol 39:589-597[ISI][Medline]

    Cavender T. M., 1986 Review of the fossil history of North American freshwater fishes Pp. 699–724 in C. H. Hocutt and E. O. Wiley, eds. The zoogeography of North American freshwater fishes. John Wiley & Sons, New York

    Chang B. H.-J., L. C. Shimmin, S.-K. Shyue, D. Hewett-Emmett, W.-H. Li, 1994 Weak male-driven molecular evolution in rodents Proc. Natl. Acad. Sci. USA 91:827-831[Free Full Text]

    Choudhury A., T. A. Dick, 1998 The historical biogeography of sturgeons (Osteichthyes: Acipenseridae): a synthesis of phylogenetics, palaeontology and palaeogeography J. Biogeogr 25:623-640[ISI]

    De la Herrán R., F. Fontana, M. Lanfredi, L. Congiu, M. Leis, R. Rossi, C. Ruiz Rejón, M. Ruis Rejón, M. A. Garrido-Ramos, 2001 Slow rates of evolution and sequence homogenization in an ancient satellite DNA family of sturgeons Mol. Biol. Evol 18:432-436[Free Full Text]

    Dingerkus G., W. M. Howell, 1976 Karyotypic analysis and evidence of tetraploidy in the North American paddlefish, Polyodon spathula Science 194:842-843[ISI][Medline]

    Ewart H. S., W. R. Driedzic, 1987 Enzymes of energy metabolism in salmonid hearts: spongy versus cortical myocardia Can. J. Zool 65:623-627[ISI]

    Gardiner B. G., 1984 Sturgeons as living fossils Pp. 148–152 in N. Eldredge and S. M. Stanley, eds. Living fossils. Springer-Verlag, New York

    Gissi C., A. Reyes, G. Pesole, C. Saccone, 2000 Lineage-specific evolutionary rate in mammalian mtDNA Mol. Biol. Evol 17:1022-1031[Abstract/Free Full Text]

    Gutell R. R., 1994 Collection of small subunit (16S- and 16S-like) ribosomal RNA structures: 1994 Nucleic Acids Res 22:3502-3507[Abstract]

    Kedrova O. S., N. S. Wladytchenskaya, A. S. Antonov, 1980 Single copy and repeated sequence divergency in the fish genomes Mol. Biol. (Russ.) 14:1001-1012

    Kimura M., 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]

    Kohne D. E., 1970 Evolution of higher-organism DNA Q. Rev. Biophys 33:327-375

    Krieger J., P. A. Fuerst, T. M. Cavender, 2000 Phylogenetic relationships of the North American sturgeons (order Acipenseriformes) based on mitochondrial DNA sequences Mol. Phylogenet. Evol 16:64-72[ISI][Medline]

    Kumar S., 1996 PHYLTEST: a program for testing phylogenetic hypothesis Version 2.0. Institute of Molecular and Evolutionary Genetics and Department of Biology, The Pennsylvania State University, University Park, Pennsylvania

    Kumar S., K. Tamura, I. B. Jakobsen, M. Nei, 2001 MEGA2: molecular evolutionary genetics analysis software Arizona State University, Tempe, Ariz

    Lauder G. V., K. F. Liem, 1983 The evolution and interrelationships of the Actinopterygian fishes Bull. Harv. Mus. Comp. Zool 150:95-197

    Lê H. L. V., G. Lecointre, R. Perasso, 1993 A 28S rRNA based phylogeny of the gnathostomes: first steps in the analysis of conflict and congruence with morphologically based cladograms Mol. Phylogenet. Evol 2:31-51[Medline]

    Li W.-H., D. L. Ellesworth, J. Krushkal, B. H.-J. Chang, D. Hewett-Emmett, 1996 Rates of nucleotide substitution in primates and rodents and the generation–time effect hypothesis Mol. Phylogenet. Evol 5:182-187[ISI][Medline]

    Li W.-H., M. Tanimura, P. M. Sharp, 1987 An evaluation of the molecular clock hypothesis using mammalian DNA sequences J. Mol. Evol 25:330-342[ISI][Medline]

    Martin A. P., 1999 Substitution rates of organelle and nuclear genes in sharks: implicating metabolic rate (again) Mol. Biol. Evol 16:996-1002[Abstract]

    Martin A. P., G. J. P. Naylor, S. R. Palumbi, 1992 Rates of mitochondrial DNA evolution in sharks are slow compared to mammals Nature 357:153-155[ISI][Medline]

    Martin A. P., S. R. Palumbi, 1993 Body size, metabolic rate, generation time, and the molecular clock Proc. Natl. Acad. Sci. USA 90:4087-4091[Abstract]

    Mooers A. Ø., P. H. Harvey, 1994 Metabolic rate, generation time and the rate of molecular evolution in birds Mol. Phylogenet. Evol 3:344-350[Medline]

    Ohno S., J. Muramoto, C. Stenius, L. Christian, W. A. Kittrel, N. B. Atkin, 1969 Microchromosomes in holocephalian, chondrostean, and holostean fishes Chromosoma 26:35-40[ISI][Medline]

    Ohta T., 1993 An examination of the generation time effect on molecular evolution Proc. Natl. Acad. Sci. USA 90:10676-10680[Abstract]

    Patterson C., 1982 Morphology and interrelationships of primitive Actinopterygian fishes Am. Zool 22:241-259[ISI]

    Sarich V. M., A. C. Wilson, 1973 Generation time and genomic evolution in primates Science 179:1144-1147[ISI][Medline]

    Singer T. D., V. G. Mahadevappa, J. S. Ballantyne, 1990 Aspects of the energy metabolism of lake sturgeon, Acipenser fulvescens, with special emphasis on lipid and ketone body metabolism Can. J. Fish. Aquat. Sci 47:873-881[ISI]

    Tajima F., 1993 Simple methods for testing molecular clock hypothesis Genetics 135:599-607[Abstract/Free Full Text]

    Takezaki N., A. Razhetsky, M. Nei, 1995 Phylogenetic test of the molecular clock and linearized trees Mol. Biol. Evol 12:823-833[Abstract]

    Thompson J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, D. G. Higgins, 1997 The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools Nucleic Acids Res 24:4876-4882

    Venkatesh B., Y. Ning, S. Brenner, 1999 Late changes in spliceosomal introns define clades in vertebrate evolution Proc. Natl. Acad. Sci. USA 96:10267-10271[Abstract/Free Full Text]

Accepted for publication February 1, 2002.