Rapid Evolution of a Cyclin A Inhibitor Gene, roughex, in Drosophila

Sergei N. Avedisov, Igor B. Rogozin, Eugene V. Koonin and Barbara J. Thomas

Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Interspecific Comparison of rux...
 Conclusions
 Acknowledgements
 References
 
The recent sequencing of the complete genome of the fruit fly Drosophila melanogaster has yielded about 30% of the predicted genes with no obvious counterparts in other organisms. These rapidly evolving genes remain largely unexplored. Here, we present evidence for a striking variability in an important Drosophila cell cycle regulator encoded by the gene roughex (rux) in closely related fly species. The unusual level of Rux protein variability indicates that there are very low overall constraints on amino acid substitutions. Despite the lack of sequence similarity, certain common features, including the presence of a C-terminal nuclear localization signal and a functionally important N-terminal RXL cyclin-binding motif, exist between Rux and cyclin-dependent kinase inhibitors of the Cip/Kip family. These results indicate that even some genes involved in key regulatory processes in eukaryotes evolve at extremely high rates.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Interspecific Comparison of rux...
 Conclusions
 Acknowledgements
 References
 
The cell cycle is one of the most conserved cellular processes in eukaryotes, integrating multiple regulatory pathways involved in both proliferation and differentiation. Progression through the cell cycle in eukaryotes is strictly regulated by the orderly action of a specific class of protein kinases (cyclin-dependent kinases [CDKs]) and their partner regulatory proteins, cyclins (Pines 1998Citation ; Sheaff and Roberts 1998Citation ). The activity of cyclin-CDK complexes is regulated by differential phosphorylation of the CDK subunit and by interaction with specific regulatory proteins, cyclin-dependent kinase inhibitors (CKIs).

The key role that cell cycle regulation plays in developing organisms might imply that its participating components will be highly conserved among diverse eukaryotic taxa. Indeed, cyclins and CDKs are well conserved and easily recognizable from yeast to humans (Pines 1994Citation ; Jeffrey et al. 1995Citation ; Liu and Kipreos 2000Citation ). Recent analysis of the Drosophila genome sequence supports previous suggestions of strong parallels between many fly and vertebrate cell cycle regulators (Rubin et al. 2000)Citation . In addition, there are numerous reports demonstrating proper function of cell cycle regulators in heterologous systems (e.g., Lehner and O'Farrell 1990Citation ; Lew, Dulic, and Reed 1991Citation ; Geng et al. 1999Citation ). Significantly, Xenopus cyclin A (CycA) can bind to and activate the yeast Cdc28 kinase in vivo (Funakoshi et al. 1997Citation ). Conversely, several genes that have been previously shown to contribute directly to different stages of cell cycle regulation in flies do not as yet have homologs in other genomes analyzed (e.g., thr [D'Andrea et al. 1993Citation ], rux [Thomas et al. 1994Citation ], pim [Stratmann and Lehner 1996Citation ], rca1 [Dong et al. 1997Citation ]). This observation challenges the concept of the conservation of the cell cycle machinery and raises questions about the evolutionary origin of these genes. One gene that has no apparent homolog in other eukaryotes is the CycA inhibitor rux.

Rux is an essential cell cycle regulator in Drosophila, and viability in loss-of-function rux alleles is reduced to roughly 10% of wild type (Gonczy, Thomas, and DiNardo 1994Citation ; Thomas et al. 1994Citation ). It has been demonstrated that rux acts to down-regulate CycA-dependent activity during the G1 phase and is responsible for a temporary G1 arrest during several stages of Drosophila development (Sprenger, Yakubovich, and O'Farrell 1997Citation ; Thomas et al. 1997Citation ). In addition, Rux regulates the second meiotic division during Drosophila spermatogenesis (Gonczy, Thomas, and DiNardo 1994Citation ; Thomas et al. 1994Citation ) and functions during embryogenesis in the exit from mitosis (Foley and Sprenger 2001)Citation . Recent studies (Foley, O'Farrell, and Sprenger 1999Citation ; Avedisov et al. 2000Citation ) revealed different levels of regulation of CycA by Rux and suggested a mechanism by which Rux mediates cell cycle arrest. Specifically, we defined two small but functionally important structural motifs within the Rux protein (Avedisov et al. 2000)Citation . An N-terminal Leu-31 has been found to be critical for the association between Rux and CycA and for the inhibition by Rux of CycA-dependent kinase activity. This residue is a part of a canonical RXL sequence which mediates, in part, the binding of human CKIs p21, p27, and p57 to CycA-Cdk2 and CycE-Cdk2 (Adams et al. 1996Citation ; Chen et al. 1996Citation ). The second motif is a C-terminal bipartite nuclear localization signal (NLS) which is responsible for targeting Rux to the nucleus. Overexpression of wild-type Rux protein in the developing eye disc also results in translocation of CycA to the nucleus, where CycA protein is destroyed, and this activity of Rux is also dependent on the C-terminal NLS. Thus, Rux may influence the intracellular distribution of CycA and promote its destruction (Avedisov et al. 2000)Citation .

The rux gene is present in a single copy at cytological position 5C on the X chromosome of Drosophila melanogaster. The gene encodes a 335-amino-acid protein (fig. 1 ) with no homologs detectable in current protein databases. In this study, we performed a phylogenetic analysis of Rux in eight species of the D. melanogaster subgroup and showed that the protein evolves unexpectedly rapidly. This report represents one of the first studies of a hypervariable gene with a known essential function in closely related Drosophila species.



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Fig. 1.—Scheme of the organization of the rux gene in Drosophila melanogaster. An intron is indicated by a line; exons are indicated by boxes. Filled-in portions of exons are coding portions. CBS = CycA-binding sequence; NLS = nuclear localization signal; rp = six-amino-acid repeats. Under the gene structure, the shaded bars indicate two previously characterized functional regions of the protein (Avedisov et al. 2000)Citation . Four potential CDK phosphorylation sites are represented by arrowheads; three RXL sequences are shown above the diagram

 

    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Interspecific Comparison of rux...
 Conclusions
 Acknowledgements
 References
 
Drosophila Stocks
Drosophila simulans (sim; stock number 14021-0251.165), Drosophila mauritiana (mau; stock number 14021-0241.41), Drosophila sechellia (sec; stock number 14021-0248.1), Drosophila yakuba (yak; stock number 14021-0261.0), Drosophila erecta (ere; stock number 14021-0224.0), and Drosophila orena (ore; stock number 14021-0245.0) were obtained from the National Drosophila Species Resource Center at Bowling Green State University. Drosophila teissieri (tei; stock number S160) flies were obtained from the UMEA Drosophila Stock Center (Sweden).

DNA Extraction, PCR Amplification, and Sequencing
Genomic DNA was extracted from 50 adult flies from each strain as described (Chia et al. 1985Citation ). Different combinations of degenerate primers were used for PCR amplification of regions of the rux gene from eight Drosophila species. Standard PCR amplification conditions were 30 cycles of denaturation at 95°C for 30 s, primer annealing at 50°C or 55°C for 30 s, and primer extension at 70°C for 3 min. Oligonucleotide sequence information and details of PCR strategy are available on request.

The amplified fragments were cloned into the pCR2.1 vector (Invitrogen) and sequenced. Sequences were verified by sequencing directly from the amplified genomic DNA. Sequencing was done on an automated sequencer (Applied Biosystems) using the ABI PRISM dye-terminator cycle-sequencing kit (Perkin-Elmer). The D. simulans, D. mauritiana, D. sechellia, D. teissieri, D. yakuba, D. erecta, and D. orena rux sequences have been deposited in the GenBank database under accession numbers AF327884AF327890.

Data Analysis
The nonredundant protein sequence database at the National Center for Biotechnology Information (NCBI, National Institutes of Health, Bethesda, Md.) was searched using the gapped BLAST program (Altschul et al. 1997Citation ). Protein sequences were compared with domain-specific sequence profiles using the SMART system (Schultz et al. 1998Citation ) and the Conserved Domain (CD) search at the NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Multiple protein and nucleotide sequence alignments were constructed using the CLUSTAL W (version 1.8) program (Baylor College of Medicine software package) (Thompson, Higgins, and Gibson 1994Citation ) and then adjusted by hand. Protein secondary-structure prediction was performed using the PHD program with a multiple-protein-sequence alignment supplied as the query (Rost and Sander 1994Citation ). Sequence-structure threading was performed using the hybrid fold prediction method (Fischer 2000)Citation .

The Pamilo-Bianchi-Li (PBL) method (Li 1993Citation ; Pamilo and Bianchi 1993Citation ) and the K-Estimator (Comeron 1999Citation ), MEGA2 (Kumar, Tamura, and Nei 1994Citation ), and YN00 (Yang and Nielsen 2000)Citation programs were used to estimate the numbers of synonymous (Ks) and nonsynonymous (Ka) substitutions per site. Phylogenetic trees based on multiple alignments of nucleotide sequences were constructed using the neighbor-joining (Saitou and Nei 1987Citation ) and maximum-parsimony (Fitch 1971Citation ) methods as implemented in the PAUP* program (Swofford 1998Citation ) with default parameters.


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Interspecific Comparison of rux...
 Conclusions
 Acknowledgements
 References
 
rux Belongs to a Hypervariable Fraction of the Drosophila Genome
Since Rux has no detectable homologs in existing databases, we cloned sequences homologous to rux from various fruit fly species. We first tested Drosophila virilis, a species of the subgenus Drosophila, which has been estimated to have diverged from melanogaster (subgenus Sophophora) ~40–60 MYA (Beverley and Wilson 1984Citation ; Russo, Takezaki, and Nei 1995Citation ) and which is commonly used for comparisons with melanogaster. However, both low-stringency Southern blot analysis and PCR with degenerate oligonucleotide primers from different regions of the rux gene failed to detect sequences homologous to rux in D. virilis (data not shown). We similarly tested several other species from both the Sophophora and the Drosophila subgenera, including six closely related species of the melanogaster group, Drosophila ananassae, Drosophila auraria, Drosophila lucipennis, Drosophila pseudotakahashii, Drosophila elegans, and Drosophila eugracilis (estimated to have diverged from the melanogaster subgroup 17–20 MYA; Lachaise et al. 1988Citation ). Even in these more closely related species, we were unable to detect a rux homolog. We conclude that rux belongs to the subset of Drosophila genes with extremely high evolutionary divergence rates (Schmid and Tautz 1997Citation ).

As a next step in identifying Rux homologs, we tested the eight known species from the melanogaster subgroup. According to the most recent estimates for individual species from this subgroup, the D. melanogaster lineage split from D. yakuba approximately 5.1 ± 0.8 MYA, from D. mauritiana 2.7 ± 0.4 MYA, and from D. simulans 2.3 ± 0.3 MYA (Li, Satta, and Takahata 1999Citation ). Positive results were obtained in cross-hybridization experiments, and the corresponding genomic sequences were recovered by PCR. The alignment of the inferred polypeptides is shown in figure 2 . The most surprising result is the unusually low level of similarity between Rux proteins from these closely related species, in which orthologous proteins typically show negligible amino acid sequence difference (table 1 ). Thus, Rux seems to be one of the most diverged Drosophila proteins described to date (see table 1 and below).



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Fig. 2.—Alignment of Rux protein sequences. The amino acid sequences deduced from the nucleotide sequence of a Drosophila melanogaster Rux cDNA (mel; Thomas et al. 1994Citation ) and Drosophila simulans (sim), Drosophila mauritiana (mau), Drosophila sechellia (sec), Drosophila teissieri (tei), Drosophila yakuba (yak), Drosophila erecta (ere), and Drosophila orena (ore) Rux genomic DNA are aligned. Amino acids are numbered consecutively for each species and indicated at the right margin. A dot in the alignment indicates an identical amino acid in a species and D. melanogaster, whereas a dash represents a gap. The RXL sites are underlined, the putative CDK phosphorylation sites are in boldface, and the basic clusters of the bipartite NLS are in italics. Secondary-structure prediction (2s) is shown at the top of the alignment; e indicates extended conformation (ß-strand), and h indicates {alpha}-helix

 

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Table 1 Comparison of Amino Acid Replacements for Different Characterized Proteins

 
The unrooted trees derived from the alignment are shown in figure 3 ; as could be expected for a single-copy nuclear gene, the phylogeny of rux is completely consistent with that of the flies themselves and includes three major clusters that matched three species complexes, i.e., the melanogaster complex, the yakuba complex, and the erecta/orena complex (Lachaise et al. 1988Citation ). A significant difference (P < 0.001) in substitution rates (Tajima 1993Citation ) was found between D. orena and D. erecta rux genes.



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Fig. 3.—Phylogenetic trees of rux nucleotide sequences. A, Unrooted neighbor-joining tree. The scale is based on the number of nucleotide substitutions per site between the two sequences calculated by the Kimura (1980) two-parameter method. The numbers for the interior branches refer to the bootstrap values with 1,000 pseudoreplicates. B, A maximum-parsimony tree constructed using PAUP*

 

    Interspecific Comparison of rux Within the melanogaster Subgroup
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Interspecific Comparison of rux...
 Conclusions
 Acknowledgements
 References
 
The sequence comparison of Rux proteins reveals a surprisingly large number of amino acid replacements, as well as insertions and deletions. The overall level of similarity is substantially lower than that for other functionally characterized orthologous genes compared between these closely related Drosophila species (table 1 ) and is comparable to the average level of protein similarity between orthologs from the distantly related D. virilis and D. melanogaster (see, e.g., Hart et al. 1993Citation ; Newfeld et al. 1997Citation and references therein). Moreover, these comparisons underestimate the variability of the Rux protein because they do not take into account insertions and deletions. Comparisons with uncharacterized fast-evolving genes (including partially sequenced open reading frames) identified recently in the Drosophila genome (Schmid and Tautz 1997Citation ) indicate that the rate of Rux divergence is comparable to or higher than those of the two most rapidly diverging genes: for the D. melanogaster/D. yakuba species pair, Ka values calculated by the K-Estimator program (Comeron 1999Citation ) were 0.157, 0.168, 0.116, and 0.108 for Rux, 1G5, 1E9, and 2D9, respectively. These rates were significantly higher than those of several other fast-evolving genes (e.g., Ka values for 2A5 and 2C9 were 0.020 and 0.018, respectively).

By aligning the sequences from all eight species, evolutionarily conserved regions and variations in selective constraints along the Rux protein sequence were identified. The predicted size of the Rux protein differed for each species (fig. 2 ). These differences in length were largely due to different copy numbers of a six-amino-acid repeat unit in the C-terminal region of the protein. Interestingly, expansion of this sequence has occurred only in the species of the melanogaster complex, while both the yakuba and the erecta/orena lineages retain a single unit. While the function of these repeats is unclear, the observed length divergence among the species of the melanogaster complex may reflect the evolution of an as yet unknown regulatory pattern.

The distribution of replacements along the Rux protein sequence is nonuniform. To reveal patterns that may be specific to previously described functional domains of this protein (Avedisov et al. 2000)Citation , the coding region was subdivided into two partially overlapping regions and further characterized (table 2 ). The region that is important for interaction with CycA resides within the N-terminal two thirds of the protein (amino acids 1–217), whereas the C-terminal portion (amino acids 188–335) has been shown to be responsible for the proper intracellular localization of wild-type Rux and contains the NLS (Avedisov et al. 2000)Citation . Most of the amino acid substitutions have occurred in the C-terminal third of the protein. This region also harbors seven of the 10 insertions/deletions detected in the alignment. This suggests that the C-terminal part of the protein is under noticeably reduced selective constraint, although most of the residues that compose the bipartite NLS consensus are conserved between the eight species. In some of the species, the first arginine residue in the distal basic cluster of the NLS consensus is replaced by proline (fig. 2 ). To examine the possibility that this point substitution changes the subcellular localization of Rux protein in these species, we expressed in Drosophila cultured cells a chimeric protein in which the N-terminal 39 amino acids were from D. melanogaster Rux and the remaining 301 amino acids were from D. erecta Rux. The C-terminal portion derived from the D. erecta protein contained the entire region required for nuclear localization (Avedisov et al. 2000)Citation , including an NLS of the structure RKR-(X)10-PKR. Similar to the D. melanogaster Rux, this chimeric protein localized predominantly to the nucleus (data not shown), indicating that the subcellular localization of Rux is conserved in different fly species despite the deviation of the NLS sequence from the consensus in some of those species.


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Table 2 Comparison of Amino acid Replacements for Different Functional Regions of Roughex

 
As expected, the three previously identified RXL sequences were invariant in all eight species. A relatively high level of amino acid conservation was also observed in the regions surrounding the RXL sequences. The N-terminal RXL sequence has been shown to be critical for the CycA-binding and inhibitory function of Rux, whereas the other two motifs have been suggested to modulate the CycA/Rux interaction (Avedisov et al. 2000)Citation . An additional amino acid stretch of high conservation is located between amino acids 122 and 137, suggesting an important role for this region of the protein in rux function.

Synonymous and Nonsynonymous Substitutions in the rux Gene
We calculated the numbers of synonymous (Ks) and nonsynonymous (Ka) substitutions per site between rux gene sequences from different species (table 3 ). The Ka/Ks ratio is normally used to estimate the mode and the strength of selection acting on a coding sequence (for a recent review, see Kreitman and Comeron 1999Citation ). For Rux, each pairwise comparison produces Ks values exceeding Ka; i.e., there is no clear evidence of positive selection. However, the Ka/Ks ratios for this protein are among the highest reported for Drosophila species (Schmid and Tautz 1997Citation ; Schmid et al. 1999Citation ), which suggests relaxed purifying selection. Table 3 shows the data produced by the PBL (Li 1993Citation ; Pamilo and Bianchi 1993Citation ) and maximum-likelihood (Yang and Nielsen 2000)Citation methods; several other methods for Ks and Ka calculation implemented in the MEGA and K-Estimator packages yielded very similar results (data not shown). These observations confirm that there are low overall constraints on nonsynonymous substitutions in the rux gene.


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Table 3 Numbers of Synonymous (Ks) and Nonsynonymous (Ka) Substitutions per Site in the rux Coding Region

 
Judged by the Ka/Ks values, the upstream two thirds of the protein is under stronger purifying selection than the downstream third (table 4 ). Interestingly, the substitution rates in the intron sequences of the gene are lower than the Ks values in the coding region (table 5 ). One possible explanation is that the intron in rux is relatively short (90–105 bp, depending on the species), and conserved splicing signals may represent a significant proportion of the intron sequences. Therefore, these sequences may be under stronger evolutionary constraints than synonymous sites in the coding region.


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Table 4 Ka/Ks for Different Functional Regions of Roughex

 

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Table 5 Numbers of Substitutions per Site in the rux Intron Sequences

 
In cases of rapid evolution, some regions of a gene can be expected to evolve under positive selection even when Ka < Ks for the entire coding region (Kreitman and Comeron 1999Citation ). However, our searches using a sliding-window technique (window length varied from 60 to 360 bp) in pairwise alignments (Endo, Ikeo, and Gojobori 1996Citation ) did not reveal strong indications that positive selection operates in the rux gene. Interestingly, Ka/Ks values exceeded 1 for some pairwise comparisons (table 4 ), but various statistical tests of positive selection implemented in the K-Estimator and MEGA2 programs did not confirm that the observed excess was significant. We cannot reject the hypothesis that some sites in the rux gene experience positive selection; a rigorous test of this hypothesis requires a larger data set (Suzuki and Gojobori 1999Citation ).


    Conclusions
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Interspecific Comparison of rux...
 Conclusions
 Acknowledgements
 References
 
In many organisms, cell cycle inhibitors are surprisingly dissimilar at the amino acid level compared with their highly conserved substrates, cyclins and CDKs. For example, in the yeast Saccharomyces cerevisiae, no apparent homologs were found for metazoan CKIs, although a functional analog, Sic1 (Schwob et al. 1994Citation ), exists. The identification of two regions important for Rux function (Avedisov et al. 2000)Citation prompted us to compare the overall structures and organizations of several cell cycle inhibitors and Rux (table 6 ). Although Rux does not have any detectable sequence similarity to members of the Cip/Kip family of CKIs, certain structural features seem to be shared by these proteins. Like Rux, the Cip/Kip proteins are relatively small and contain a bipartite NLS near their C-termini; the importance of this motif for nuclear targeting has recently been demonstrated for p21 and p27 (Zeng et al. 2000)Citation . Furthermore, a consensus RXL cyclin-binding motif is located at the N-terminus of both groups of inhibitors. Finally, the amino-terminal region responsible for the inhibitory effect is more conserved than the C-terminal region both in the p21 subfamily of mammalian CKIs (Sherr and Roberts 1995Citation ) and among the Rux homologs from different Drosophila species (this report). Threading analysis failed to detect structural similarity between Rux and p27Kip, whose crystal structure has been determined (Russo et al. 1996Citation ). However, multiple-alignment–based secondary-structure prediction for Rux indicated a structural organization with some general similarities to p27Kip, with a predicted ß-sheet surrounded by extended {alpha}-helices (fig. 2 ). The origin of the Rux gene remains unknown. In principle, it cannot be ruled out that this gene has evolved from a common ancestor with the Cip/Kip family, but the sequences have diverged to the extent that no similarity is detectable even by the most sensitive sequence comparison methods or by threading. Determination of the three-dimensional structure of Rux will help to solve the issue of a possible structural and evolutionary relationship between Rux and the Cip/Kip family of CKIs.


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Table 6 Structural Features that Rux Shares with Cyclin-Dependent Kinase Inhibitors of the Cip/Kip Family

 
Very high divergence rates have recently been reported for a substantial fraction of D. melanogaster genes (Schmid and Tautz 1997Citation ). These rapidly evolving genes were identified by differential screening of cDNAs between D. yakuba and D. virilis. Several clones identified in this study were surprisingly diverged (Schmid et al. 1999Citation ), but none of these cDNAs has been characterized at the functional level. In contrast, many functionally characterized genes tend to diverge at significantly slower rates (e.g., Schmid and Tautz 1997Citation ). These observations might suggest that the fast-evolving genes are responsible for fly-specific functions and, as a result, have no detectable sequence homologs in distantly related taxa. However, we show here that the most rapidly evolving fraction may include critical genes whose function is conserved in general terms in all eukaryotes. Conceivably, Rux homologs are not restricted in their phyletic distribution to just one subgroup of the genus Drosophila, but homologs from more distant species so far could not be identified because of their rapid divergence.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Interspecific Comparison of rux...
 Conclusions
 Acknowledgements
 References
 
We thank Josep Comeron for help with data analysis, and Karl Schmid, Yurii Ilyin, Mark Mortin, and Michael Lichten for critical comments on the manuscript.


    Footnotes
 
Claudia Kappen, Reviewing Editor

1 Present address: Engelhardt Institute of Molecular Biology, Moscow, Russia. Back

2 Present address: National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland. Back

1 Keywords: roughex cell cycle inhibitor cyclin A evolution Drosophila Back

2 Address for correspondence and reprints: Sergei N. Avedisov, Engelhardt Institute of Molecular Biology, 32 Vavilov Street, Moscow 117984, Russia. ave{at}genome.eimb.relarn.ru . Back


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 Results and Discussion
 Interspecific Comparison of rux...
 Conclusions
 Acknowledgements
 References
 

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Accepted for publication July 25, 2001.