Department of Biological Sciences, Texas Tech University
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Domain length variation was examined by comparing the unaligned sequence lengths for each individual. These values were compared with all individuals of a given species to the ETAS domain, ranging in length from 251 to 276 bp. This domain contained the 24-bp duplication of C. rufocanus. Excluding this duplication, the size range for this domain was 251252 bp. Clethrionomys gapperi was the only species with length variation in this domain (251252 bp). The central domain ranged in length from 311 to 312 bp. Only two species examined (C. gapperi and C. californicus) had length variation in this domain. The third domain, CSB, ranged in length from 379 to 384 bp. The CSB domain was variable in length in all but one species (C. californicus). However, this lack of length variation in C. californicus was probably due to the small number of individuals (n = 2) included in the analysis. The length variation in the CSB domain was caused by several single-nucleotide indels which were not present in all individuals.
Base Composition Heterogeneity
Base composition varied significantly among domains of the Clethrionomys control region (table 3
). All taxa showed similar composition biases of A = T > C > G in the ETAS domain, T > C > A > G in the central domain, and A > T > C > G in the CSB domain. Clethrionomys follows the general mammalian mtDNA control region pattern of (A+T) > (C+G) in all domains (Sbisà et al. 1997
).
|
To determine the type and pattern of mutations, comparisons were made within species and populations. The restriction of this method to intraspecific variation was necessary to reduce the number of assumptions resulting from the extensive variation in this molecule. At many nucleotide positions, there were three or more character states present. Therefore, we were unable to specifically characterize each position for interspecific comparisons. Variation in the control region was classified as pyrimidine or purine transitions (ts-pyr, ts-pur), transversions (tv), or insertions/deletions (indels). Pyrimidine : purine transition (ts-pyr : ts-pur) and transition : transversion (ts : tv) ratios were calculated for all species examined (table 4 ). Domains differed significantly in frequency of variable nucleotide positions; however, substitution ratios (i.e., ts : tv : indel or ts-pyr : ts-pur) did not differ significantly between domains.
|
Intraspecific Genetic Distance
Genetic distance (Tamura and Nei 1993
) ranges were calculated for the three species of Clethrionomys that were represented by more than two individuals. Values presented represent the genetic distances between the two most closely related individuals and the two most distantly related individuals within a species. Clethrionomys glareolus had a range of distance values of 0.0020.014. Within our sample, the genetic distance of C. rutilus ranged from 0.004 to 0.032, and that of C. rufocanus ranged from 0.009 to 0.018. Clethrionomys gapperi had the largest genetic distance values, ranging from 0.013 to 0.078. It was not possible to determine a range of distance values for C. californicus because of the small sample size (n = 2). The genetic distance for C. californicus was 0.008.
Conservation of Structural Elements
There were two ETAS elements in the control region. ETAS1 was located approximately 2550 bp from the tRNAPro gene at the 5' end of the control region. This element was highly conserved within Clethrionomys. Only 7 of the 57 (12%) nucleotide positions were variable within the ETAS1 element, which was significantly lower than the 24.7% average variable nucleotide frequency for the remaining ETAS domain (2, P = 0.030). In addition, the consensus sequence for ETAS1 in Clethrionomys differed from Mus (Sbisà et al. 1997
) at only four sites. Unlike ETAS1, ETAS2 was highly variable in Clethrionomys, with approximately 25 of the 54 (47%) nucleotide positions being variable. This frequency was significantly higher than that of the remaining ETAS domain, which had only 16% variable nucleotide positions (
2, P < 0.0001).
There were three CSB elements found in the control region of Clethrionomys. The first was CSB1, which was moderately conserved. Within this element, 10 of the 25 (40%) nucleotide positions were variable. This level of conservation (60%) was the same as that found in the remaining CSB domain (60%). CSB2 and CSB3 were the other two CSB elements found in Clethrionomys; however, CSB2 was not well conserved. CSB2 had only seven conserved nucleotide positions out of its 19-bp length (60% variable), which made it significantly more variable than the remaining CSB domain (2, P = 0.032). CSB3 was the most conserved of all of the structural elements found in the control region. The CSB3 element was significantly more conserved than the remaining CSB domain (
2, P = 0.009). Only 2 of the 18 (11%) nucleotide positions found within this element were variable. In fact, we found no intraspecific variation in the CSB3 element. In addition to the previously documented CSB elements, we also identified a "CSB1-like" element. However, this element was not in the CSB domain. The CSB1-like element was found 14 bp downstream of the ETAS2 element in the ETAS domain. The consensus sequence of this element for Clethrionomys was 5'-AAMYATTAATGYTYDAHAGACATA. When all specimens were included in a single alignment, only 6 of the 24 (25%) nucleotide positions were variable. When compared with the CSB1 element of Mus musculus (Sbisà et al. 1997
), the consensus sequence of this CSB1-like element differed at three nucleotide positions and was polymorphic at five additional positions. Although the level of conservation in this CSB1-like element was higher than that found in the CSB1 element in the CSB domain, this level of conservation was not higher than the 22% average for the remaining ETAS domain (55 of 252 positions variable;
2, P = 0.704).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There are differences in base composition between all three domains. Base composition heterogeneity has previously been documented in several other mammalian taxa by Sbisà et al. (1997)
. In addition, there are differences in the frequencies of variable nucleotide positions within the three domains (table 3 and fig. 2
). When all species of Clethrionomys are included, 159 out of the 395 (40%) nucleotide positions within the CSB domain are variable. In the ETAS domain, 61 of the 276 (22%) nucleotide positions are variable. However, in the central domain, only 37 of the 313 (11.3%) nucleotide positions are variable. This conserved central domain has levels of interspecific variation similar to those of mitochondrial rRNA and tRNA genes, as well as nonsynonymous sites of protein-coding genes in several mammalian groups (Pesole et al. 1999
), whereas the ETAS and CSB domains have much faster evolutionary rates. This interspecific pattern of polymorphic nucleotide position heterogeneity varies slightly across taxonomic levels. When the frequency of variable sites within a species is calculated, the percentages of variable nucleotide positions in the CSB, ETAS, and central domains were 5.9%, 4.6%, and 1.0%, respectively (table 3
). Finally, the distribution of tandem repeats and short indels is heterogeneous among domains. The only tandem repeat is found in the ETAS domain of C. rufocanus. Only one species (C. gapperi) has an indel in the ETAS domain, two species have indels in the central domain (C. gapperi and C. californicus), and four of the five species included have indels in the CSB domain. The CSB domain contains significantly more indels than the other two domains (
2, P = 0.009). In fact, 14 of the 19 intraspecific indels occurred in the CSB domain.
The amounts of variation found in some of the structural elements were higher than expected based on the level of variation found within the same domain. The fact that some of these elements are conserved across different orders (Brown et al. 1986
; Saccone, Attimonelli, and Sbisà 1987
; Sbisà et al. 1997
) suggests that they should be highly conserved within a single genus. The CSB3 and ETAS1 elements were highly conserved within Clethrionomys (>87%), providing additional support for their existence and functionality. The remaining elements were at least as variable as the domains in which they were found, thus raising doubts about the functionality of those elements. Functions have been proposed for both CSB and ETAS elements. Saccone, Attimonelli, and Sbisà (1987)
suggest that CSB and ETAS elements are associated with start and stop sites, respectively, for D-loop strand synthesis. Doda, Wright, and Clayton (1981)
suggest that displacement-loop strands terminated near specific sequences within the ETAS domain of humans and mice. The fact that many of these elements are not well conserved in the generic analysis suggests that these elements have one or more of the following characteristics. First, the elements may not have a crucial role in replication. Second, only one of the CSB elements and one of the ETAS elements may be needed for mitochondrial DNA replication to function properly. If only one of each type of element is necessary, any additional elements could be variable without detrimental effects. Third, the elements may be able to function properly even with multiple variable nucleotide combinations. If these elements function primarily via secondary structure, high levels of variation may not significantly affect function.
Sbisà et al. (1997)
suggest that even though CSB1 was the least conserved sequence block in their study, it is functionally the most important element. They base their conclusion on the observation that CSB1 has been identified in all mammals examined, while CSB2 and CSB3 are sometimes absent. In the current study, CSB3 was the most conserved sequence block, followed by CSB1 and, finally, CSB2, which was the least conserved. A CSB1-like element identified in the ETAS domain of Clethrionomys was highly conserved, with only three fixed differences when compared with the CSB1 element of Mus.
Functions have also been attributed to both ETAS elements. ETAS1 may contain a recognition signal for termination of the nascent DNA chain. ETAS2 may contain binding sites for termination factors (Sbisà et al. 1997
). If ETAS2 has a critical role in mtDNA replication, its function must not be compromised by multiple nucleotide combinations. However, the high level of conservation found within the ETAS1 element supports the hypothesis that this element may be conserved to function in mtDNA replication.
The single tandem repeat found in C. rufocanus is the only documented tandem repeat present in the ETAS domain of rodents. This likely dates the origin of this unique feature as subsequent to the divergence of C. rufocanus from the remaining species of Clethrionomys. We used the proposed time of the Microtus/Clethrionomys divergence (78 MYA; Martin et al. 2000)
to estimate an overall control region rate of evolution for arvicoline rodents (3.4%3.9%/Myr), although this rate varies among domains, as expected (ETAS, 3.6%4.2%; central, 1.5%1.7%; CSB, 5.8%6.6%). We then used this overall rate to estimate the date of the divergence of C. rufocanus from the remaining Clethrionomys species. It appears that C. rufocanus diverged approximately 4.24.8 MYA. If these dates are accurate, then C. rufocanus probably evolved this repeat in the last 4.8 Myr. However, it is also possible that other species of Clethrionomys had this tandem repeat or several repeats at one time, but the repeats were subsequently lost in all of the species except C. rufocanus.
Species-level analyses revealed a nucleotide bias within all species examined. A bias towards transitions over both transversions and indels was apparent. Ts : tv ratios were as high as 9.5 to 1 (C. rutilus). These results provide additional support for the conclusions of Brown et al. (1986)
, who noted a similar bias when comparing different species of Rattus. In addition, we also documented a significant bias toward pyrimidine (C
T) transitions over purine (A
G) transitions in four of the five species (table 4
). For example, C. californicus exhibited a pyrimidine : purine transition ratio of 5 to 1. This pyrimidine transition bias differs from the results of Brown et al. (1986)
, who were unable to show any pyrimidine transitional bias when comparing two different species of Rattus. Although we were able to document a bias in nucleotide substitution type within the control region, we were unable to document any substantial differences in transition substitution bias between domains.
Concerning the selection of an appropriate section of the control region for maximum variability, the ETAS and CSB domains provide the greatest variability for population analyses. The ETAS has been shown to be effective for population analysis (Hoelzel et al. 1993
; Goldberg and Ruvolo 1997
; Ishibashi et al. 1997
; Stacy et al. 1997
; Bickham et al. 1998
; Slade et al. 1998
; Rosel et al. 1999
; Ehrich et al. 2000
; Kerth, Mayer, and Konig 2000
; Matson et al. 2000
). If small indels are a concern, the CSB region should be avoided. However, if indels are of interest for the analysis, the CSB domain is an ideal region for examination. The central domain might prove useful in interspecific, intergeneric, or even family-level studies. For most interspecific analyses, the ETAS and central domains combined provide adequate resolution. The mtDNA control region can provide an appropriate region for examination for a number of different types of studies. However, the use of a single domain may provide all of the resolution necessary for a specific analysis, thus reducing the cost of a research project by reducing the required amount of DNA sequencing.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
1 Present address: Department of Wildlife and Fisheries Sciences, Texas A&M University.
1 Keywords: control region
D-loop
Clethrionomys
evolutionary rate
mitochondrial DNA
2 Address for correspondence and reprints: Robert J. Baker, Department of Biological Sciences, Texas Tech University, Lubbock, Texas 79409-3131. rjbaker{at}ttu.edu
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arctander P., C. Johansen, M.-A. Coutellec-Vreto, 1999 Phylogeography of three closely related African bovids (Tribe Alcelaphini) Mol. Biol. Evol 16:1724-1739
Arnason U., A. Gullberg, E. Johnsson, C. Ledje, 1993 The nucleotide sequence of the mitochondrial DNA molecule of the grey seal, Halichoerus grypus, and a comparison with mitochondrial sequences of other true seals J. Mol. Evol 37:323-330[ISI][Medline]
Arnason U., E. Johnsson, 1992 The complete mitochondrial DNA sequence of the harbor seal, Phoca vitulina J. Mol. Evol 34:493-505[ISI][Medline]
Bickham J. W., T. R. Loughlin, D. G. Calkins, J. K. Wickliffe, J. C. Patton, 1998 Genetic variability and population decline in Steller sea lions from the Gulf of Alaska J. Mammal 79:1390-1395[ISI]
Biju-Duval C., H. Ennafaa, N. Dennebouy, M. Monnerot, F. Mignotte, R. C. Soriguer, A. El Gaaïed, A. El Hili, J.-C. Mounolou, 1991 Mitochondrial DNA evolution in lagomorphs: origin of systematic heteroplasmy and organization of diversity in European rabbits J. Mol. Evol 33:92-102[ISI]
Brown G., G. Gadaleta, G. Pepe, C. Saccone, E. Sbis, 1986 Structural conservation and variation in the D-loop-containing region of vertebrate mitochondrial DNA J. Mol. Biol 192:503-511[ISI][Medline]
Castro-Campillo A., H. R. Roberts, D. J. Schmidly, R. D. Bradley, 1999 Systematic status of Peromyscus boylii ambiguus based on morphologic and molecular data J. Mammal 80:1214-1231[ISI]
Chang D. D., W. W. Hauswirth, D. A. Clayton, 1985 Replication priming and transcription initiate from precisely the same site in mouse mitochondrial DNA EMBO J 4:1559-1567[Abstract]
Doda J. N., C. T. Wright, D. A. Clayton, 1981 Elongation of displacement-loop strands in human and mouse mitochondrial DNA is arrested near specific template sequences Proc. Natl. Acad. Sci. USA 78:6116-6120[Abstract]
Ehrich D., V. B. Federov, N. C. Stenseth, C. J. Krebs, A. Kenney, 2000 Phylogeography and mitochondrial DNA (mtDNA) diversity in North American collared lemmings (Dicrostonyx groenlandicus) Mol. Ecol 9:329-337[ISI][Medline]
Excoffier L., Z. Yang, 1999 Substitution rate variation among sites in mitochondrial hypervariable region I of humans and chimpanzees Mol. Biol. Evol 16:1357-1368[Abstract]
Fallon B., P. Barrett, O. Carroll, P. Henderson, J. Donlon, J. Fairley, 1997 Hepatic phenylalanine hydroxylase of bank voles Clethrionomys glareolus Schreber as a biomarker of pollution Proc. R. Irish Acad. B 97:115-119
Fumagalli L., P. Taberlet, L. Favre, J. Hausser, 1996 Origin and evolution of homologous repeated sequences in the mitochondrial DNA control region of shrews Mol. Biol. Evol 13:31-46[Abstract]
Ghivizzani S. C., S. L. D. Mackay, C. S. Madsen, P. J. Laipis, W. W. Hauswirth, 1993 Transcribed heteroplasmic repeated sequences in the porcine mitochondrial DNA D-loop region J. Mol. Evol 37:36-47[ISI][Medline]
Ghivizzani S. C., C. S. Madsen, M. R. Nelen, C. V. Ammini, W. W. Hauswirth, 1994 In organello footprint analysis of human mitochondrial DNA: human mitochondrial transcription at the origin of replication Mol. Cell. Biol 14:7717-7730[Abstract]
Goldberg T. L., M. Ruvolo, 1997 The geographic apportionment of mitochondrial genetic diversity in east African chimpanzees, Pan troglodytes schweinfurthii Mol. Biol. Evol 14:976-984[Abstract]
Hoelzel A. R., J. Halley, S. J. O'Brian, C. Campagna, T. Arnbom, B. Le Boeuf, K. Ralls, G. A. Dover, 1993 Elephant seal genetic variation and the use of simulation models to investigate historical population bottlenecks J. Hered 84:443-449[ISI][Medline]
Hoelzel A. R., J. M. Hancock, G. A. Dover, 1993 Generation of VNTRs and heteroplasmy by sequence turnover in the mitochondrial control region of two elephant seal species J. Mol. Evol 37:190-197[ISI][Medline]
Hoelzel A. R., J. V. Lopez, G. A. Dover, S. J. O'Brian, 1994 Rapid evolution of a heteroplasmic repetitive sequence in the mitochondrial DNA control region of carnivores J. Mol. Evol 39:191-199[ISI][Medline]
Horling J., A. Lundkvist, M. Jaarola, A. Plyusnin, H. Tegelstrom, K. Persson, H. Lehvaslaiho, B. Hornfeldt, A. Vaheri, B. Niklasson, 1996 Distribution and genetic heterogeneity of Puumala virus in Sweden J. Gen. Virol 77:2555-2562[Abstract]
Ishibashi Y., T. Saitoh, S. Abe, M. C. Yoshida, 1997 Sex-related spatial kin structure in a spring population of grey-sided voles Clethrionomys rufocanus as revealed by mitochondrial and microsatellite DNA analyses Mol. Ecol 6:63-71[ISI][Medline]
Kerth G., F. Mayer, B. Konig, 2000 Mitochondrial DNA (mtDNA) reveals that female Bechstein's bats live in closed societies Mol. Ecol 9:793-800[ISI][Medline]
Kocher T. D., W. K. Thomas, A. Meyer, S. V. Edwards, S. Pääbo, F. X. Villablanca, A. C. Wilson, 1989 Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers Proc. Natl. Acad. Sci. USA 86:6196-6200[Abstract]
Koh H. S., W. J. Lee, T. D. Kocher, 2000 The genetic relationships of two subspecies of striped field mice, Apodemus agrarius coreae and Apodemus agrarius chejuensis Heredity 85:30-36[ISI][Medline]
Labuda M., O. Kozuch, E. Zuffova, E. Eleckova, R. S. Hails, P. A. Nuttall, 1997 Tick-borne virus transmission between ticks cofeeding on specific immune natural rodent hosts Virology 235:138-143[ISI][Medline]
Longmire J. L., M. Maltbie, R. J. Baker, 1997 Use of "lysis buffer" in DNA isolation and its implication for museum collections Occas. Pap. Mus. Tex. Tech Univ 163:1-3
Lundkvist A., Y. Cheng, K. B. Sjolander, B. Niklasson, A. Vaheri, A. Plyusnin, 1997 Cell culture adaptation of Puumala hantavirus changes the infectivity for its natural reservoir, Clethrionomys glareolus, and leads to accumulation of mutants with altered genomic RNA S segment J. Virol 71:9515-9523[Abstract]
Lutz S., H. Witting, H.-J. Weisser, et al. (11 co-authors) 2000 Is it possible to differentiate mtDNA by means of HVIII in samples that cannot be distinguished by sequencing the HVI and HVII regions? Forensic Sci. Int 113:97-101[ISI][Medline]
Martin Y., G. Gerlach, C. Schlotterer, A. Meyer, 2000 Molecular phylogeny of European muroid rodents based on complete cytochrome b sequences Mol. Phylogenet. Evol 16:37-47[ISI][Medline]
Matson C. W., B. E. Rodgers, R. K. Chesser, R. J. Baker, 2000 Genetic diversity of Clethrionomys glareolus populations from highly contaminated sites in the Chornobyl region Environ. Toxicol. Chem 19:2130-2135[ISI]
Mignotte F., M. Gueride, A.-M. Champagne, J.-C. Mounolou, 1990 Direct repeats in the non-coding region of rabbit mitochondrial DNA Eur. J. Biochem 194:561-571[Abstract]
Nordyke K. A., S. W. Buskirk, 1991 Southern red-backed vole, Clethrionomys gapperi, populations in relation to stand succession and old-growth character in the central Rocky Mountains Can. Field Nat 105:330-334[ISI]
Pesole G., C. Gissi, A. De Chirico, C. Saccone, 1999 Nucleotide substitution rate of mammalian mitochondrial genomes J. Mol. Evol 48:427-434[ISI][Medline]
Plyusnin A., O. Vapalahti, K. Ulfves, H. Lehvaslaiho, N. Apekina, I. Gavrilovskaya, V. Blinov, A. Vaheri, 1994 Sequences of wild Puumala virus genes show a correlation of genetic variation with geographic origin of the strains J. Gen. Virol 75:405-409[Abstract]
Rosel P. E., S. C. France, J. Y. Wang, T. D. Kocher, 1999 Genetic structure of harbour porpoise Phocoena phocoena populations in the northwest Atlantic based on mitochondrial and nuclear markers Mol. Ecol 8:S41-S54[ISI][Medline]
Saccone C., M. Attimonelli, E. Sbis, 1987 Structural elements highly preserved during the evolution of the D-loop-containing region in vertebrate mitochondrial DNA J. Mol. Evol 26:205-211[ISI][Medline]
Sbis E., F. Tanzariello, A. Reyes, G. Pesole, C. Saccone, 1997 Mammalian mitochondrial D-loop region structural analysis: identification of new conserved sequences and their functional and evolutionary implications Gene 205:125-140[ISI][Medline]
Shadel G. S., D. A. Clayton, 1997 Mitochondrial DNA maintenance in vertebrates Annu. Rev. Biochem 66:409-435[ISI][Medline]
Slade R. W., C. Moritz, A. R. Hoelzel, H. R. Burton, 1998 Molecular population genetics of the southern elephant seal Mirounga leonina Genetics 149:1945-1957
Stacy J. E., P. E. Jorde, H. Steen, R. A. Ims, A. Purvis, K. S. Jakobsen, 1997 Lack of concordance between mtDNA gene flow and population density fluctuations in the bank vole Mol. Ecol 6:751-759[ISI][Medline]
Stewart D. T., A. J. Baker, 1994a. Patterns of sequence variation in the mitochondrial D-loop region of shrews Mol. Biol. Evol 11:9-21[Abstract]
. 1994b. Evolution of mtDNA D-loop sequences and their use in phylogenetic studies of shrews in the subgenus Otisorex (Sorex: Soricidae: Insectivora) Mol. Phylogenet. Evol 3:38-46[Medline]
Swofford D. L., 1998 PAUP* Phylogenetic analysis using parsimony (*and other methods). Version 4. Sinauer, Sunderland, Mass
Tamura K., M. Nei, 1993 Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees Mol. Biol. Evol 10:512-526[Abstract]
Tryland M., T. Sandvik, R. Mehl, M. Bennett, T. Traavik, O. Olsvik, 1998 Serosurvey for orthopoxviruses in rodents and shrews from Norway J. Wildl. Dis 34:240-250[Abstract]
Vigilant L., M. Stoneking, H. Harpending, K. Hawkes, A. C. Wilson, 1991 African populations and the evolution of human mitochondrial DNA Science 253:1503-1507[ISI][Medline]
Walberg M. W., D. A. Clayton, 1981 Sequence and properties of the human KB cell and mouse L cell D-loop regions of mitochondrial DNA Nucleic Acids Res 9:5411-5421[Abstract]
Wilkinson G. S., A. M. Chapman, 1991 Length and sequence variation in evening bat d-loop mtDNA Genetics 128:607-617
Wilkinson G. S., F. Mayer, G. Kerth, B. Petri, 1997 Evolution of repeated sequence arrays in the D-loop region of bat mitochondrial DNA Genetics 146:1035-1048