*Department of Applied Plant Science, The Queen's University of Belfast, Belfast, N. Ireland; and
Institut für Tierzucht und Genetik, Veterinärmedizinsche Universität Wien, Vienna, Austria
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Abstract |
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Introduction |
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The classic example of a multigene family is the DNA coding for the RNA components of the ribosome (rDNA). One interesting aspect of rDNA evolution is that in many species, e.g., Drosophila (Tartof and Dawid 1976
), humans (Worton et al. 1988
), and Allium cepa (Panzera et al. 1996
), more than a single nucleolus organizer region (NOR) has been described. Many studies, covering a wide phylogenetic range, have suggested that concerted evolution is preventing sequence divergence among the rDNA families located at different chromosomal positions (Arnheim et al. 1980
). The most convincing demonstrations of the homogenization process among rDNA families located on different chromosomes come from species with a hybrid origin. Despite diverged rDNA copies in the ancestors, the rDNA in those species was converted to a single sequence variant similar to one of the ancestral rDNA sequences (Hillis et al. 1991
; Wendel, Schnabel, and Seelanan 1995a
). Regardless of small differences being reported between rDNA families located in different chromosomal positions, in a phylogenetic context the rDNA has been treated as a single-copy gene (Hillis and Dixon 1991
).
Insights into the functional importance of concerted evolution come from studies on the expression of rDNA genes in hybrids. Allotetraploid species, such as Brassica napus (Chen and Pikaard 1997
; Frieman et al. 1999
) and Arabidopsis suecica (Chen, Comai, and Pikaard 1998
), still carry rDNA sequences from both progenitors, but only a single rDNA type is expressed. This phenomenon is called nucleolar dominance and has also been observed in hybrid Xenopus and Drosophila individuals (Honjo and Reeder 1973
; Durica and Krider 1977
). While the molecular basis of this phenomenon is not yet fully resolved, DNA methylation and histone deacetylation seem to be important, along with large chromatin structure based mechanisms (Frieman et al. 1999
).
Different selective forces are acting on the rDNA region which result in varying degrees of sequence conservation across single repeat units. Consequently, each part can be employed for specific phylogenetic questions across a broad taxonomic spectrum (Hillis and Dixon 1991
). Recently, particularly for plant systematics, the faster-evolving internal transcribed spacer (ITS) sequences have developed into a ubiquitous tool for phylogenetic reconstruction of closely related species. Very rarely, however, have the underlying assumptions associated with rDNA evolution been tested. We used a particularly difficult evolutionary settingQuercus petraea and Quercus robur, two species which frequently hybridize but still form two discrete genetic entities (Muir, Fleming, and Schlötterer 2000
)to study the molecular evolution of the rDNA. Based on ITS and 5.8S sequences, we show that both oak species, Q. petraea and Q. robur, carry three divergent rDNA clusters, of which only a single one has maintained function.
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Materials and Methods |
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PCR Amplification
Plant-specific primers were designed by hand from a published alignment of 18S and 28S sequences (Schlötterer 1998
). To account for polymorphisms in the alignment, wobbles were incorporated to recognize all plant ribosomal targets. The primer sequences were CCT TMT CAT YTA GAG GAA GGA G for 18S and CCG CTT ATT KAT ATG CTT AAA for 28S. A 40-µl reaction was prepared with 100 ng of genomic DNA, 1.5 mM MgCl2, 200 µM dNTPs, 1 µM of each primer, and 1 U Taq polymerase. The cycling profile consisted of an initial denaturation step of 3 min, followed by 40 cycles of 60 s at 94°C, 60 s at 56°C, and 90 s at 72°C. PCR products were blunt-ended using Klenow polymerase I (GibcoBRL) and subsequently cloned into M13mp19 (Yanisch-Perron, Vieira, and Messing 1985
).
Cloning and Sequencing
The standard 20-µl ligation mix contained 20100 ng of phosphorylated PCR product, 100 ng M13mp19, 1 U T4 ligase (Promega), and 1 x T4 ligation buffer (Promega). Ligation was carried out at 18°C overnight. Clones carrying inserts were identified with blue/white selection. Sequencing templates were prepared from overnight cultures of positive clones using standard protocols. Clones were sequenced using ABI dye terminator chemistry (Perkin Elmer) according to the manufacturer's instructions and run on an ABI 377 automated sequencer.
RT-PCR
Total genomic RNA was extracted from a single Q. robur individual. Fifty milligrams of fresh leaf material was homogenized using liquid nitrogen with a mortar and pestle. The homogenate was subsequently treated with TRIzol (GibcoBRL) according to the manufacturer's instructions. The RNA was treated with 20 U DNase I (Boehringer Mannheim) before first-strand cDNA synthesis of the 5.8S using 200 U Superscript II (GibcoBRL). Primers were designed to amplify the full range of genomic 5.8S rDNA sequences in Q. petraea/Q. robur. Their sequences were CRA CTC TCR GCA ACG GAT A for 5.8Sf and YRT GAC ACC CAG GCA RAC for 5.8Sr. The cDNA was amplified using the same reaction conditions and cycling profile as that given for the 18S and 28S primers. The cDNA was subsequently cloned into M13mp19. To verify the absence of DNA in the RNA extraction, four templates (total genomic DNA, DNase-treated RNA, the cDNA, and a negative control) were amplified with the 5.8S primers using the same PCR mix. The RNA extraction was considered DNA-free, provided that PCR products were present in the correct size range for DNA and cDNA templates, but were absent in the DNase-treated RNA and negative control reactions. As an additional control, the ITS2-spanning primers 5.8f and 28S (mentioned above) were used with the same templates. DNA was considered absent only if the ITS fragment was absent in the cDNA control.
Data Analyses
Phylogeny Reconstruction
Sequences were edited in Sequence Navigator (Perkin Elmer), aligned using CLUSTAL W (Thompson, Higgins, and Gibson 1994
), and manually adjusted. Phylogenetic reconstruction was carried out with the software package PUZZLE, version 4.0 (Strimmer and von Haeseler 1996
), using the HKY (Hasegawa, Kishino, and Yano 1985
) model of sequence evolution with eight categories of rate heterogeneity. The phylogenetic tree was graphically displayed using TREEVIEW (Page 1996
). Two Fagaceae sequences from GenBank (Colombobalanus excelsa and Trigonobalanus verticillata; accession numbers AF098412 and AF098413) were used as an outgroup. Tables of polymorphic sites, including singletons, were produced with the aid of the SITES program (Hey and Wakeley 1997
).
Relative-Rate Test
A two-cluster relative-rate test (Takezaki, Rzhetsky, and Nei 1995
) implemented in the software package PHYLTEST, version 2.0 (Kumar 1996
), was used to examine the evolutionary rate constancy of rDNA genes among the three divergent clusters. A representative of each haplotype was included in the analysis. The haplotypes were grouped on the basis of the phylogenetic analysis and the test was conducted separately for the 5.8S and ITS2 regions. Rate constancy was examined for pairs of linearized trees (Takezaki, Rzhetsky, and Nei 1995
) using Kimura's (1980)
distance with rate heterogeneity. The Gamma distribution shape parameter, alpha (1.41), was estimated from the data set using PUZZLE, version 4.0.
Methylation-Related Substitutions
Deamination-like substitutions (CT and G
A) were examined at cytosine sites on both strands of the 5.8S and ITS2 sequences. Possible sites of methylation (CpG or CpNpG; Gardiner-Garden, Sved, and Frommer 1992
) were determined for the reconstructed ancestral sequence at internal node I (fig. 1
). The ancestral character states were inferred using maximum likelihood in PAUP, version 4.0b3a (Swofford 1999
), using the Fagaceae sequences as the outgroup. Putative methylation sites present in the ancestral sequence were considered for the analysis. To test whether the putative pseudogenes (clades P1 and P2) showed an elevated rate of deamination-like substitutions, we counted for each clade separately the number of sites showing at least one deamination-like substitution. These observations were compared with the remainder of the sequence, which did not contain methylation sites. We counted for each group separately those sites that had a mutation in at least one haplotype. Significance levels were determined using a G-test with Yates' correction in a 2 x 2 contingency table.
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Results |
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Phylogeny Reconstruction
Phylogenetic analysis of the combined 5.8S and ITS2 regions indicated the presence of three highly diverged clades. The clades were supported by quartet puzzling values of 100% (fig. 1 ). Interestingly, mapping the species from which the clone was obtained onto the phylogenetic tree did not indicate any concordance. Sequences from both species were distributed among the three divergent clades. The same pattern was obtained when the phylogenetic reconstruction was based on either the 5.8S or the ITS2 sequences, respectively. Hence, the three distinct clades predate the species split of Q. robur and Q. petraea, which were previously shown to be two distinct taxonomic units (Muir, Fleming, and Schlötterer 2000
).
Polymorphism
Seventy PCR clones, originating from five Q. petraea individuals and two Q. robur individuals, were sequenced for the 5.8S and ITS2 regions. The average pairwise differences () between the sequences were 0.054 for the 5.8S region and 0.092 for the ITS2 region. The higher variability of the ITS region was also reflected in the number of observed haplotypes. Eight distinct haplotypes were observed in the 5.8S region, and 12 were observed in the ITS2 region (fig. 2a and b
).
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Recombination
Two sequences obtained from the same PCR reaction showed evidence for recombination between two clades. As both sequences had the recombination break point at the same position, we regarded them as the outcome of jumping PCR (Pääbo, Irwin, and Wilson 1990
) and excluded them from the analysis. Visual inspection indicated that haplotypes (Q. petraea JL) share some sites with the two other clades, which may indicate recombination (fig 2b
). In contrast to the excluded sequences, the haplotypes were obtained from two different oak trees and carried specific mutations. Thus, we regard them as true recombinants.
Functionality Tests
The presence of three divergent rDNA genes in oaks raised the question of whether all three sequences are functional. To address this question, we conducted four different types of analyses (relative-rate tests, methylation-mutation analysis, evolutionary comparisons, and cDNA analysis) to determine the functional status of these genes.
Relative-Rate Test
Assuming that all three rDNA lineages have maintained their function, they should be exposed to similar evolutionary constraints and thus show similar rates of evolution. If some of them have lost function, then these lineages are expected to show an elevated rate of evolution. To discriminate between these two alternative hypotheses, we conducted a relative-rate test. The results in table 1
show a significantly lower substitution rate for the F lineage (denoted as functional) than for both the P1 and the P2 lineages (denoted as pseudogenes). Rate constancy can be rejected at the 1% level for both the coding 5.8S and the noncoding ITS2 between the functional gene and the two pseudogenes. However, rate constancy cannot be rejected between the two pseudogenes in the spacer region.
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Methylation-mutation analysis
Pseudogenes are often characterized by cytosine mutations at methylation sites (Li, Wu, and Luo 1984
). If P1 and P2 are pseudogenes but F remains active, then the pseudogenes should show an elevated frequency of cytosine mutations at methylation sites. A G-test with Yates' correction was used to investigate whether deamination-like base substitutions were equally distributed among all clades. Table 2
shows a significantly higher frequency of deamination-like base substitutions at methylation sites in the P1 lineage compared with F, but not in the P2 lineage. Most likely, the P2 lineage has also lost function because the same elevated pattern of deamination-like substitutions present in P1 is clearly visible despite not being statistically significant.
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cDNA
Additional evidence for the expression of clade F and for the loss of function in clades P1 and P2 comes from cDNA analysis. Sequence variation in the 5.8S sequences allowed us to discriminate between the three clades, even if only a short fragment of the 5.8S region was amplified. Twelve cDNA clones were sequenced, and all showed 100% sequence identity with the most frequent clone present in clade F. No clones were detected from clade P1 or P2.
Assuming that all three rDNA genes are functional, equally likely to be cloned, and represented in the genome according to the frequencies cloned, the probability of obtaining 12 clones from clade F is (20/70)12, or 2 x 10-7. In other words, the chances of yielding cDNA clones from the same clade are extremely small. Hence, our cDNA analysis also suggests that P1 and P2 are not expressed.
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Discussion |
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The lack of a significant difference between species is surprising, as we detected three divergent rDNA clades in both species. This observation clearly violates the general rule of concerted evolution in rDNA families located on different chromosomes. The observed average divergence between the three rDNA families is too high to be explained by different homogenization rates between homologous and nonhomologous rDNA copies, as previously suggested (Schlötterer and Tautz 1994
; Sang, Crawford, and Stuessy 1995
). Assuming a mutation rate of 5 x 10-9 (Wolfe, Li, and Sharp 1987
) in the ITS2, we estimate that the three rDNA families in Q. petraea and Q. robur have been separated for about 1317 Myr (for clades P1 and P2, respectively).
Some evidence for the presence of more than a single NOR in Q. petraea and Q. robur stems from fluorescence in situ hybridization. Zoldos et al. (1999)
detected two NORs located on different chromosomes. Interestingly, most of the hybridization signal was detected at one NOR. The other NOR hybridized only weakly and could not be detected in some chromosome preparations. This observation contrasts markedly with our cloning of genomic rDNA sequences, as we detected three rDNA families, which were represented at similar frequencies (fig. 1
). This discrepancy may be explained by a strong amplification and cloning bias in our experiments or by difficulties with chromosomal preparations from oaks. An alternative explanation of how the in situ hybridization data could be reconciled with our sequence analysis is provided by a recent study of Arabidopsis thaliana. Cloix et al. (2000) demonstrated that functional and nonfunctional 5S sequences are interspersed in an array and concerted evolution continues despite this arrangement. Accordingly, functional, and nonfunctional rDNA sequences may be interspersed in oaks. Further studies are required to resolve this question.
Intensive studies of the evolutionary dynamics of rDNA gene families in hybrids have revealed three different outcomes regarding the presence of diverged NORs. (1) The rDNA families are converted to a single sequence type. The rDNA evolution in allopolyploid cotton species is a particularly good example of this mechanism (Wendel, Schnabel, and Seelanan 1995a
). (2) Rather than converting the rDNA family to one type, gene conversion and recombination could generate chimeric molecules, which will most likely be homogenized to a single rDNA molecule, with a sequence intermediate between the two ancestral sequences (van Houten, Scarlett, and Bachmann 1993
; Wendel, Schnabel, and Seelanan 1995b
). (3) One of the two rDNA families present in hybrids is silenced, and only one copy remains functional (Chen, Comai, and Pikaard 1998
; Frieman et al. 1999
).
In Q. petraea and Q. robur, we demonstrated a very high level of sequence homogeneity within each rDNA cluster, suggesting ongoing concerted evolution. Nevertheless, between the three rDNA families, extensive sequence divergence was detected, indicative of genetic isolation of those three rDNA families. Reverse transcription implied that only one of the three clades was transcribed, and so the situation resembles the phenomenon of nucleolar dominance, which is well documented for allopolyploid species and hybrids. Previous studies on A. cepa also detected a lack of expression of some of the rDNA clusters, together with some sequence divergence between the rDNA clusters (Panzera et al. 1996
). Therefore, even in a species that is not described as allopolyploid, nucleolar dominance occurs. While the situation for oaks resembles that for A. cepa, such a large sequence divergence between rDNA clusters is unusual (but see Hugall, Stanton, and Moritz 1999
) and raises the question of whether all rDNA copies have retained functionality.
Further insight comes from a relative-rate test performed on the rDNA sequences from the two oak species. Compared with the functional gene family, we detected a significantly elevated rate of sequence change in the two rDNA gene families which are not expressed. More importantly, 5.8S sequences are affected by this increased rate of evolutionary change, suggesting that these 5.8S genes have lost their functional constraints and evolve as pseudogenes. While it is possible that a local increase in mutation rate has caused this effect, three lines of evidence argue in favor of the pseudogene hypothesis. First, the less constrained ITS sequences do not show the effect of rate heterogeneity as strongly as the 5.8S sequences, indicating that the mutational dynamics changed more dramatically for the 5.8S region. Second, the ratio of ITS2 and 5.8S within-clade variability is higher in the functional clade, suggesting higher sequence conservation in the 5.8S region of the functional clade (table 3 ). Finally, sites in the 5.8S sequence that are highly conserved over a wide phylogenetic range are more conserved in the functional clade than in the clades containing the putative pseudogenes. Hence, combining all lines of evidence, we conclude that nucleolar dominance is not suppressing the expression of two of the three rDNA clades in Q. petraea and Q. robur. Rather, these sequences have lost functionality and should be regarded as pseudogenes.
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In this study, we demonstrated that within a single individual, multiple rDNA copies exist which are quite divergent. Direct sequencing of PCR products would probably have resulted in an unreadable sequence. Assuming that the PCR products are cloned and a single clone is sequenced from each species, several different phylogenetic reconstructions could have been obtained (depending on which of the three clades was included in the analysis). Consequently, phylogenetic reconstruction based on ITS sequences may bear some intrinsic risks, but if the evolutionary dynamics of this multigene family are included in the interpretation and acquisition of the data, ITS sequences could still provide a useful tool for phylogenetic reconstruction.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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1 Abbreviations: ITS, internal transcribed spacer; NOR, nucleolus organizer region; rDNA, ribosomal DNA.
2 Keywords: rDNA
Quercus
concerted evolution
NOR
nucleolar dominance
pseudogenes
3 Address for correspondence and reprints: Christian Schlötterer, Institut für Tierzucht und Genetik, Veterinärmedizinsche Universität Wien, Josef Baumann Gasse 1, 1210, Vienna, Austria. E-mail: christian.schloetterer{at}vu-wien.ac.at
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literature cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arnheim, N., M. Krystal, R. Schmickel, G. Wilson, O. Ryder, and E. Zimmer. 1980. Molecular evidence for genetic exchanges among ribosomal genes on nonhomologous chromosomes in man and apes. Proc. Natl. Acad. Sci. USA 77:73237327.
Chen, Z. J., L. Comai, and C. S. Pikaard. 1998. Gene dosage and stochastic effects determine the severity and direction of uniparental ribosomal RNA gene silencing (nucleolar dominance) in Arabidopsis allopolyploids. Proc. Natl. Acad. Sci. USA 95:1489114896.
Chen, Z. J., and C. S. Pikaard. 1997. Transcriptional analysis of nucleolar dominance in polyploid plants: biased expression/silencing of progenitor rRNA genes is developmentally regulated in Brassica. Proc. Natl. Acad. Sci. USA 94:34423447.
Clapham, A. R., T. G. Tutin, and E. F. Warburg. 1981. Excursion flora of the British Isles. Cambridge University Press, Cambridge, England.
Cloix, C., S. Tutois, O. Mathieu, C. Cuvillier, M. C. Espagnol, G. Picard, and S. Tourmente. 2000. Analysis of 5S rDNA arrays in Arabidopsis thaliana: physical mapping and chromosome-specific polymorphisms. Genome Res. 10:679690.
Dover, G. 1982. Molecular drive: a cohesive mode of species evolution. Nature 299:111117.
Durica, D. S., and H. M. Krider. 1977. Studies on the ribosomal RNA cistrons in interspecific Drosophila hybrids. I. Nucleolar dominance. Dev. Biol. 59:6274.[ISI][Medline]
Frieman, M., Z. J. Chen, J. Saez-Vasquez, L. A. Shen, and C. S. Pikaard. 1999. RNA polymerase I transcription in a Brassica interspecific hybrid and its progenitors: tests of transcription factor involvement in nucleolar dominance. Genetics 152:451460.
Gardiner-Garden, M., J. A. Sved, and M. Frommer. 1992. Methylation sites in angiosperm genes. J. Mol. Evol. 34:219230.[ISI]
Hasegawa, M., H. Kishino, and T. A. Yano. 1985. Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. J. Mol. Evol. 22:160174.[ISI][Medline]
Hey, J., and J. Wakeley. 1997. A coalescent estimator of the population recombination rate. Genetics 145:833846.
Hillis, D. M., and M. T. Dixon. 1991. Ribosomal DNA: molecular evolution and phylogenetic inference. Q. Rev. Biol. 66:411446.[Medline]
Hillis, D. M., C. Moritz, C. A. Porter, and R. J. Baker. 1991. Evidence for biased gene conversion in concerted evolution of ribosomal DNA. Science 251:308310.
Honjo, T., and R. H. Reeder. 1973. Preferential transcription of Xenopus laevis ribosomal RNA in interspecies hybrids between Xenopus laevis and Xenopus mulleri. J. Mol. Biol. 80:217228.
Hugall, A., J. Stanton, and C. Moritz. 1999. Reticulate evolution and the origins of ribosomal internal transcribed spacer diversity in apomictic Meloidogyne. Mol. Biol. Evol. 16:157164.[Abstract]
Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16:111120.[ISI][Medline]
Kumar, S. 1996. PHYLTEST: phylogenetic hypothesis testing, version 2.0. Pennsylvania State University, University Park.
Li, W.-H., C.-I. Wu, and C.-C. Luo. 1984. Nonrandomness of point mutations as reflected in nucleotide substitutions in pseudogenes and its evolutionary implications. J. Mol. Evol. 21:5871.[ISI][Medline]
Modrich, P., and R. Lahue. 1996. Mismatch repair in replication fidelity, genetic recombination and cancer biology. Annu. Rev. Biochem. 65:101133.[ISI][Medline]
Muir, G., C. C. Fleming, and C. Schlötterer. 2000. Species status of hybridizing oaks. Nature 405:1016.
Pääbo, S., D. M. Irwin, and A. C. Wilson. 1990. DNA damage promotes jumping between templates during enzymatic amplification. J. Biol. Chem. 265:47184721.
Page, R. D. M. 1996. TREEVIEW: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12:357358.[Medline]
Panzera, F., M. I. Giménez-Abián, J. F. López-Sáez, G. Giménez-MartÍn, A. Cuadrado, P. J. Shaw, A. F. Beven, J. L. Cánovas, and C. De la Torre. 1996. Nucleolar organizer expression in Allium cepa L. chromosomes. Chromosoma 105:1219.
Petit, M. A., J. Dimpfl, M. Radman, and H. Echols. 1991. Control of large chromosomal duplications in Escherichia coli by the mismatch repair system. Genetics 129:327332.
Sang, T., D. J. Crawford, and T. F. Stuessy. 1995. Documentation of reticulate evolution in peonies (Paeonia) using internal transcribed spacer sequences of nuclear ribosomal DNA: implications for biogeography and concerted evolution. Proc. Natl. Acad. Sci. USA 92:68136817.
Schlötterer, C. 1998. Ribosomal DNA probes and primers. Pp. 267276 in A. Karp, P. G. Isaac, and D. S. Ingram, eds. Molecular tools for screening biodiversity: plants and animals. Chapman and Hall, London.
Schlötterer, C., and D. Tautz. 1994. Chromosomal homogeneity of Drosophila ribosomal DNA arrays suggests intrachromosmal exchanges drive concerted evolution. Curr. Biol. 4:777783.[ISI][Medline]
Strimmer, K., and A. von Haeseler. 1996. Quartet puzzling: a quartet maximum-likelihood method for reconstructing tree topologies. Mol. Biol. Evol. 13:964969.
Swofford, D. L. 1999. PAUP. Phylogenetic analysis using parsimony. Version 4.0. Sinauer, Sunderland, Mass.
Takezaki, N., A. Rzhetsky, and M. Nei. 1995. Phylogenetic test of the molecular clock and linearized trees. Mol. Biol. Evol. 12:823833.[Abstract]
Tartof, K. D., and I. B. Dawid. 1976. Similarities and differences in the structure of X and Y chromosome rRNA genes of Drosophila. Nature 263:2730.
Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:46734680.[Abstract]
van Houten, W. H. J., N. Scarlett, and K. Bachmann. 1993. Nuclear DNA markers of the Australian tetraploid Microseris scapigera and its North American diploid relatives. Theor. Appl. Genet. 87:498505.[ISI]
Wendel, J. F., A. Schnabel, and T. Seelanan. 1995a. Bidirectional interlocus concerted evolution following allopolyploid speciation in cotton (Gossypium). Proc. Natl. Acad. Sci. USA 92:280284.
. 1995b. An unusual ribosomal DNA sequence from Gossypium gossypioides reveals ancient, cryptic, intergenomic introgression. Mol. Phylogenet. Evol. 4:298313.
Whittemore, A. T., and B. A. Schaal. 1991. Interspecific gene flow in sympatric oaks. Proc. Natl. Acad. Sci. USA 88:25402544.
Wolfe, K. H., W.-H. Li, and P. M. Sharp. 1987. Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast, and nuclear DNAs. Proc. Natl. Acad. Sci. USA 84:90549058.
Worton, R. G., J. Sutherland, J. E. Sylvester, H. F. Willard, S. Bodrug, I. Dube, C. Duff, V. Kean, P. N. Ray, and R. D. Schmickel. 1988. Human ribosomal RNA genes: orientation of the tandem array and conservation of the 5' end. Science 239:6468.
Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13 mp18 and pUC19 vectors. Gene 33:103119.
Zoldos, V., D. Papes, M. Cerbah, O. Panaud, V. Besendorfer, and S. Siljak-Yakovlev. 1999. Molecular-cytogenetic studies of ribosomal genes and heterochromatin reveal conserved genome organization among 11 Quercus species. Theor. Appl. Genet. 99:969977.[ISI]