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From The Cover
Evolution
Divergent gene copies in the asexual class Bdelloidea (Rotifera) separated before the bdelloid radiation or within bdelloid families
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Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biology Laboratory, Woods Hole, MA 02543; ¶Section of Integrative Biology and Center for Computational Biology and Bioinformatics, University of Texas, Austin, TX 78712; and ||Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138
Contributed by Matthew Meselson, October 15, 2003
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Abstract |
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Rotifers of the asexual class Bdelloidea are unusual in possessing two or more divergent copies of every gene that has been examined. Phylogenetic analysis of the heat-shock gene hsp82 and the TATA-box-binding protein gene tbp in multiple bdelloid species suggested that for each gene, each copy belonged to one of two lineages that began to diverge before the bdelloid radiation. Such gene trees are consistent with the two lineages having descended from former alleles that began to diverge after meiotic segregation ceased or from subgenomes of an alloploid ancestor of the bdelloids. However, the original analyses of bdelloid gene-copy divergence used only a single outgroup species and were based on parsimony and neighbor joining. We have now used maximum likelihood and Bayesian inference methods and, for hsp82, multiple outgroups in an attempt to produce more robust gene trees. Here we report that the available data do not unambiguously discriminate between gene trees that root the origin of hsp82 and tbp copy divergence before the bdelloid radiation and those which indicate that the gene copies began to diverge within bdelloid families. The remarkable presence of multiple diverged gene copies in individual genomes is nevertheless consistent with the loss of sex in an ancient ancestor of bdelloids.
Genomes of rotifers of the asexual class Bdelloidea are unusual in that they possess two or more divergent copies of every gene that has been examined and lack closely similar copies (ref. 1 and D.B.M.W. and M.M., unpublished data). Previously (1), two of us presented gene trees of the heat-shock gene hsp82 and the TATA-box-binding protein gene tbp that were consistent with the separation of the most divergent copies of both genes before the bdelloid radiation and after the separation of bdelloids from their sister taxon, the facultatively sexual monogonont rotifers (for hsp82, see Fig. 1A). Although the possibility was cited that these copies could represent ancient duplications or components of an ancient polyploid, the time of their apparent separation was seen as consistent with their descent from a pair of former alleles.
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The original trees were generated by parsimony and neighbor joining based on 4-fold degenerate codon positions and used a single outgroup to root the bdelloid gene copies. Because multiple outgroups, more sophisticated evolutionary models, and maximum likelihood (ML) and Bayesian inference methods can produce a more robust phylogeny, we conducted new analyses (initiated by D.M.H.). We find that phylogenetic analysis of the available data cannot discriminate between gene trees that differ in the placement of the root of hsp82 and of tbp bdelloid gene divergence, thereby leaving it unclear whether the most divergent gene copies separated before the bdelloid radiation or within bdelloid families.
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Materials and Methods |
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ML Analysis of hsp82. MODELTEST (3) was used to determine appropriate nucleotide-based evolutionary models. Three partitions of the data were considered: all nucleotides, codon first and second positions, and codon third positions. For each partition, the best model by the Akaike Information Criterion was General Time Reversible with a gamma-shape parameter and proportion of invariant sites to estimate rate heterogeneity. The alpha-shape parameter of the gamma distribution and the percent of invariant sites for the three partitions of the data set were 1.27 and 0.35, 0.32 and 0, and 2.51 and 0, respectively. Likelihood ratio tests showed a simpler model (Tajima-Nei) to be sufficient for all nucleotides and for codon third positions; this model produced the same topology with slightly lower bootstrap support (data not shown).
PAUP* 4.0b10 (4) was used to find the best ML tree by using a heuristic search with tree bisection-reconnection and 1,000 random-addition-sequence replications. Bootstrap values were generated in heuristic searches with 1,000 bootstrapped data sets and 10 random-addition-sequence replications for each bootstrap replicate.
For codon-based ML, the codeml program in PAML 3.13A (5) was used to determine the ML score for the tree in Fig. 1B under a variety of models. The most appropriate model, as determined by likelihood ratio tests and Akaike Information Criterion, was codon equilibrium frequencies estimated as free parameters from the existing codon frequencies (60 parameters) and nonsynonymous/synonymous (dN/dS) ratios estimated for each branch of the tree (39 parameters). The transition-transversion frequency was estimated (1 parameter). The ML trees were found by nearest-neighbor interchange using kappa = 1.5, found when exploring models. When comparing hsp82 trees, the parameter estimations did not always converge; therefore, the tree-comparison program was run 10 times. The standard deviations of the average ML scores for the best and second-best trees were 0.005% and 0.076%, respectively. The best model that did not include the 60-parameter estimate of equilibrium codon frequencies was identical to the one above except that codon equilibrium frequencies were assumed to be equal.
ML trees were evaluated with CONSEL (6), which uses the site likelihoods of each position to determine the significance of a tree by the bootstrap and posterior probability criteria as well as Kishino-Hasegawa and Shimodaira-Hasegawa tests.
Bayesian Analysis of hsp82. Bayesian analyses were performed with MRBAYES 3.0B4 (7). The nucleotide-substitution model was General Time Reversible with a gamma-shape parameter and proportion of invariant sites to estimate rate heterogeneity. For analysis of all nucleotide positions, substitution rates and rate-heterogeneity parameters were estimated for codon first and second positions and for codon third positions independently by using the "unlink" option. Similar posterior probability support for the clade of Philodinidae was obtained without this option or by using a site-specific rate model (data not shown). Markovchain Monte Carlo chain length for all analyses was 2 x 106 generations with trees sampled every 100 generations; the first 104 trees (representing 106 generations) were discarded as burn-in. Additional runs with the same conditions produced the same topology with insignificant differences in posterior probability of any node.
Phylogenetic Analysis of tbp. The likelihood ratio tests implemented in MODELTEST evaluated the Hasegawa-Kishino-Yano model (empirical base frequencies, one transition-transversion rate) with gamma-shape parameters of 0.25 and 0.36 and no invariant sites to be a sufficient explanation of the total nucleotide and codon-only data, respectively. The program dnaml in PHYLIP 3.6 was used to find the best ML tree by using the "jumble" option and global rearrangements. Bootstrap values were generated in an identical manner from 1,000 bootstrapped data sets; for the codons-only analysis, the bootstrap replicates were made at the codon rather than nucleotide level by using a perl script written by D.B.M.W. (available on request). Codon-based ML analysis was conducted as for hsp82 except that likelihood ratio tests and Akaike Information Criterion indicated that determining codon equilibrium frequencies from nucleotide frequencies (3 parameters) was sufficient. Because of the small number of sequences, analysis of bootstrap replicates was practical; 200 bootstrap replicates were each used in nearest-neighbor interchange searches for the best ML tree. Bayesian analysis using the Hasegawa-Kishino-Yano model with a gamma-shape parameter was conducted as described for hsp82 except that the data set included an additional unlinked partition containing the two introns. For analyses of codon first and second positions, the simpler model of Jukes-Cantor with a gamma-shape parameter of 0.12 was selected; more complex models produced similar results (data not shown).
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Results and Discussion |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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No new outgroup sequences are available for tbp. Gene trees produced by ML and Bayesian analysis of all nucleotides and by ML using codon models are identical to the tree presented previously (1), as are ML and Bayesian trees when codon third positions and introns are excluded (Fig. 2). However, this tree topology is not well supported and is not significantly better than one in which the outgroup is positioned such that highly divergent copies separate only within bdelloid families. When codon third positions are included but introns, which are not present in the outgroup, are excluded, ML and Bayesian analyses produce several different trees depending on small changes in model parameters, and none of the trees are well supported (data not shown). Under any method of analysis, the very long branch length to the single outgroup probably precludes accurate rooting of the bdelloid gene copies.
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Thus, the available hsp82 and tbp data (alignments of 304 codons for four bdelloid species and of 109 codons plus 136 intron positions for three bdelloid species, respectively) are insufficient to discriminate between gene trees in which the highly divergent copies of hsp82 and of tbp within individual bdelloid genomes separated before the bdelloid radiation or within individual bdelloid families. The existing evidence for the persistence of the descendants of former allele pairs or homeologs in individual bdelloid genomes is therefore equivocal. Nevertheless, the presence in individual bdelloid genomes of divergent copies of each gene examined, the presence of chromosomes without morphological homologs, the lack of long interspersed nuclear element-like and gypsy-like retrotransposons, and the long-standing failure to find meiosis, males, or hermaphrodites in so large and diverse a taxon continues to argue for the ancient asexuality of bdelloid rotifers (1, 7-16).
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Acknowledgements |
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Footnotes |
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Present address: Center for Bioinformatics and Computational Biology, University of Maryland, College Park, MD 20742. 
To whom correspondence should be addressed at: Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543. E-mail: dmarkwelch{at}mbl.edu.
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