Department of Biological Sciences, State University of New York at Buffalo, Buffalo
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Abstract |
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Introduction |
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Although LBA is commonly invoked as a source of inconsistency, it remains unclear how common the theoretical conditions that lead to LBA are in nature (Huelsenbeck 1998
; Sanderson et al. 2000
; Weins and Hollingsworth 2000
). Part of the problem is the lack of a consistent reconstruction method for real data that might remedy the bias. The initial assumption that ML is a more consistent method than MP has been challenged in several studies (Sanderson and Kim 2000
). Even when potential biases are clearly identified in real data, as in the well-studied Strepsiptera-Diptera problem, it is difficult to determine if the inferred phylogeny results from a bias or from shared evolutionary history (Huelsenbeck 1997
; Whiting 1998
; Steel, Huson, and Lockhart 2000
). Independent phylogenetic evidence is necessary to rule out shared evolutionary history as a source of a given phylogenetic relationship (Weins and Hollingsworth 2000
). Also, in many cases multiple biases interact and are difficult to tease apart (Steel, Huson, and Lockhart 2000
).
One nuclear gene that exhibits complex molecular evolution and a demonstrated potential for biased phylogenetic results is the nuclear large subunit (LSU) rRNA gene. Among-taxon base composition and substitution rate differences are commonly observed in this gene. For example, insects exhibit a nonstationarity pattern because dipterans possess LSU stem regions with 10% greater adenine-thymine content than most nondipteran relatives (Friedrich and Tautz 1997
). Also, the dipteran stem lineage possesses a 20-fold greater substitution rate than other holometabolous insects. The overall mosaic of slowly and rapidly evolving regions in LSU rDNA, with variable domains evolving faster (610 times faster in plants) than conserved domains, creates strong among-site rate heterogeneity (Hillis and Dixon 1991
; Kuzoff et al. 1998
). This mosaic pattern also predisposes the gene to differing distributions of variable sites among taxa that possess different overall rates of substitution.
In addition to potential bias, some initial studies of the LSU reported several unique sources of phylogenetic error. These include sequence coevolution, within-individual gene family variation, frequent indel mutations, and cryptically simple repeats (Tautz, Trick, and Dover 1986
; Hancock and Dover 1988
; Bult, Sweere, and Zimmer 1995
; Nunn et al. 1996
). Nevertheless, detailed studies of the entire LSU gene have shown that these sources of error are negligible in some eukaryotes and that this gene yields strong phylogenetic information. For example, Kuzoff et al. (1998)
examined complete LSU sequences from fifteen plant taxa and found significant phylogenetic concordance with 18S rDNA and rbcL gene phylogenies, greater phylogenetic information than other genes, and few apparent sources of phylogenetic error. Likewise, Mallat and Sullivan (1998)
used the entire LSU sequence of 10 chordates to test the hypothesis of cyclostome monophyly. The results indicated strong phylogenetic signal for this question and were concordant with phylogenies based on several nuclear protein-coding genes (Kuraku et al. 1999
) as well as whole mtDNA sequence phylogenies (Delarbre et al. 2000
). Finally, Mugridge et al. (2000)
found that complete sequences recovered the expected topology of sarcocystid protozoans, but the use of shorter LSU segments compromised the phylogeny. Thus, accurate phylogenetic information is present in the few existing studies of the entire gene despite multiple potential sources of error.
Here, we present the first exploration of phylogenetic utility, bias, and inconsistency in nearly complete nuclear LSU ribosomal RNA gene sequences from nonchordate animals. Our data set consists of 12 new sequences from daphniid crustaceans for which there are several existing robust associations based on independent mtDNA sequence, heat shock protein (HSP) 90, and morphological information (Lehman et al. 1995
; Colbourne and Hebert 1996
; Taylor, Hebert, and Colbourne 1996
; Taylor, Finston, and Hebert 1998
; unpublished data). Although these groups (fig. 1
) are approximations, we explore a disagreement that involves the breaking up of a strongly supported cryptic species clade, Daphnia laevis and D. dentifera. These species are so similar in morphology that they have often been incorrectly synonymized as D. longispina (see Brooks 1957
).
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Materials and Methods |
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Sequence Assembly and Alignment
Sequences were assembled and edited with Sequencher 4.0 (Gene Codes Corporation, Ann Arbor, Mich.) and then aligned with Clustal X using the default parameters (Thompson, Higgins, and Gibson 1994
; Thompson et al. 1997
). The alignment was manually adjusted with BioEdit 4.7.1 (Hall 1999
) according to core region rRNA secondary structure (De Rijk et al. 2000
). The alignment length was 4,661 base pairs, but 1,038 sites could not be aligned unambiguously and were excluded from the phylogenetic analysis (the alignment is available at http://www.herbaria.harvard.edu/treebase, study accession number S657, or from A.R.O. upon request). Variable domain boundaries were based on the proposal of De Rijk et al. (2000)
. Sequences were deposited into GenBank and accession numbers are listed in table 1
.
Phylogenetic Analyses
All phylogenetic analyses were conducted in PAUP* 4.0 (Swofford 2000
). Base compositions were calculated for entire LSU sequences, conserved cores, variable domains, and parsimony-informative sites. In order to assess phylogenetic signal in the sequences, the g1 skewness statistic (Hillis and Huelsenbeck 1992
) was calculated from 10,000 random tree length distributions. One taxon of each strongly supported clade was then removed to determine if phylogenetic signal was present in the deeper branches.
Maximum parsimony analysis was used with a branch and bound search algorithm, all characters weighted equally, and gaps treated as characters and as missing data. Nonparametric bootstrapping was performed with 1,000 bootstrap replicates and the MP search settings just listed. Nonparametric bootstrap resampling with MP was carried out with increasing numbers of resampled bases.
Fifty-six ML models were assessed by a series of likelihood ratio tests with the program Modeltest 3.0 (Posada and Crandall 1998
). Hierarchical model fitting indicated that the Tamura-Nei model (TrN) with invariable sites and the gamma parameter (Tamura and Nei 1993
) had the best fit to the data (TrN + I +
; table 3 ). This is a special case of the general time-reversible model with among-site rate variation and the following parameters being estimated from the data: three types of base substitutions, the proportion of invariable sites, and the gamma estimate of among-site rate variation with four rate categories. In order to find the best tree under the ML criterion, we used this model with a heuristic search, tree bisection-reconnection branch swapping, and 10 random sequence taxon additions. To test the hypothesis that the observed tree was more likely than the expected tree, we used parametric bootstrapping (Swofford-Olsen-Waddell-Hillis-test; partial optimization under HA; direct estimation of P-value) (Huelsenbeck and Crandall 1997
; Shimodaira and Hasegawa 1999
; Goldman, Anderson, and Rodrigo 2000
).
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The effect of differing distributions of invariant sites on tree structure was examined by phylogenetic analysis of covariotide data partitions (Lockhart et al. 1998
). The data were partitioned into five categories for two groupslong-branched taxa (D. laevis and D. occidentalis) and other taxa. The categories were: type 1, sites that are invariant across all taxa; type 2, sites that are invariant within D. laevis/D. occidentalis, and within the remaining taxa, but different between these groups (e.g., a site with the bases AA/GGGGGGGGG, where the first two taxa are D. laevis and D. occidentalis); type 3, sites that are invariant across all taxa but vary in D. laevis/D. occidentalis; type 4, sites that are invariant in D. laevis/D. occidentalis but vary in the other taxa; and type 5, sites that vary in both groups. Type 3 and 4 sites are generally considered to be the covariotide sites (Lockhart et al. 1998
). Nevertheless, if the putative LBA group has just two taxa, and a positively misleading shared character occurs at a site that is invariable in other taxa, then a type 2 site will result. Removing these type 2 sites from the analysis should then eliminate or markedly reduce the covariotide bias.
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Results |
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The MP trees generally shared the topology of the expected concordance tree (tree length = 388, consistency index [CI] = 0.789, retention index [RI] = 0.539; with gapped sites, two best trees were found of 465 steps; CI = 0.794, RI = 0.549; fig. 2 ). The only differences in the MP bootstrap consensus tree and the reference tree (fig. 1 ) were the disruption of the D. laevis/D. dentifera clade in favor of a D. laevis/D. occidentalis clade and the movement of D. ephemeralis to a basal position in the ingroup. This position for D. ephemeralis had weak support, but the branch leading to the D. laevis/D. occidentalis clade had strong bootstrap support when gapped sites were included as a fifth character (87%) and moderate support (75%) when gapped sites were scored as missing. The observed tree was eight steps shorter (465 steps) than the best tree found from an analysis constrained to have the expected D. laevis/D. dentifera clade (473 steps). When the number of sites available for resampling was varied (and gapped sites were excluded), the bootstrap value for the D. laevis/D. occidentalis clade increased with the number of sites used, reaching a maximum value of 77% at 3,623 sites (fig. 3 ).
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LBA and Covariotide Analyses
The unexpected D. laevis/D. occidentalis grouping involved the two branches that are the longest in the ingroup. Indeed, at an ML length of 0.042 (fig. 2
), the branch leading to D. laevis is the longest branch among all taxa and greater than five times longer than the other ingroup branches. Simulations designed to determine if the branches are long enough to be misleading were carried out and supported an LBA scenario that involved the long branches leading to D. laevis and D. occidentalis. Even when the simulated data were parameterized with a D. laevis/D. dentifera clade, parsimony recovered the long-branched D. laevis/D. occidentalis clade in a majority of replicates (62%; fig. 4A
) and strict consensus trees of replicates (52%). The true tree was recovered in only 32% of the replicates. The opposite pattern was recovered by ML analysis as the correct D. laevis/D. dentifera clade was recovered in 65% of the replicates, whereas the long-branched clade was recovered in only 14% of replicates. When the true tree was changed to the inferred tree (which contains a D. laevis/D. occidentalis clade), both MP and ML showed strong recovery of the correct tree (90%96%; fig. 4B
).
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Discussion |
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What is the source of the bias? Studies of bias have generally involved deeper phylogenies, often above the ordinal level, where the source of inconsistency is often blurred by the joint action of multiple biases or by taxon-sampling artifacts. The Diptera-Strepsiptera and eukaryotic phyla studies are examples where base compositional and covariotide biases may be acting (Steel, Huson, and Lockhart 2000
). In contrast, the LSU data in daphniids involves an inconsistency at the species level that seems to lack these biases. Among-taxon base compositional bias, for instance, is not significant for the entire dataset, and the covariotide analysis indicates that although D. occidentalis and D. laevis have differing distributions of sites that are free to vary from the rest of the taxa, support for the inconsistency comes from sites that are free to vary in the nonlong-branch taxa. Therefore, base compositional and covariotide biases fail to explain the inconsistency. Also, because the simulated data sets in the parametric bootstrapping lacked compositional and covariotide bias, but still recovered the same inconsistent topology as the observed data, additional biases must be acting.
The analyses are consistent with an LBA bias that results from an accelerated rate of evolution in the variable sites of the D. laevis/D. dubia group. The inconsistency clearly involves the longest branch in the tree and the second longest branch in the ingroup (D. occidentalis), a prediction of an LBA scenario. The correct grouping of D. laevis when no other long-branch attractor is present (i.e., when D. occidentalis is removed) is also consistent with LBA. Finally, even when the simulations contain a D. laevis/D. dentifera tree parameter, MP recovered the incorrect D. laevis/D. occidentalis clade in the majority of replicates. The evidence for long-branch repulsion is weak as both ML and MP recovered the correct long-branched clade in a separate simulation. These findings strongly suggest that an accelerated rate of evolution in the LSU of the lineage leading to D. laevis contributes to an LBA bias.
Long-branch attraction is only one of several explanations for a positively misleading association between D. laevis and D. occidentalis. Other plausible explanations include alignment artifacts, paralogous gene comparisons, cryptic sequence simplicity, erroneous concordance trees, and suboptimal models of evolution. The alignment that we used was conservative with most of the rapidly evolving expansion segments and length-variable regions removed from the analysis. Adding ambiguously aligned sites to the analysis still results in the D. laevis/D. occidentalis clade (not shown). Therefore, alignment is unlikely to be the sole cause of inconsistency. It is possible that a rogue paralogous rDNA copy was sequenced in D. laevis. However, this seems unlikely for two reasons. First, within-individual variation was undetected in the sequenced PCR product of D. laevis in the sites used for phylogenetic analysis. Secondly, many of the unique substitutions in D. laevis were also observed in the sister species D. dubia (fig. 6A ), suggesting that the changes in this lineage are shared and derived rather than artifacts.
Could it be that the LSU tree is correct and the concordance tree incorrect? More genetic characters are needed for a definitive answer to this question. Both morphology and gene sequence (particularly 16S rDNA and HSP90) support this clade. Daphnia laevis and D. dentifera are nearly morphologically indistinguishable, and for many decades they were considered to be the same species. Furthermore, the independent reference trees lack the long branches (i.e., they do not violate the molecular clock assumption) that apparently mislead the nuclear LSU analysis (e.g., Taylor, Finston, and Hebert 1998
). The available independent data indicate that the D. laevis/D. dentifera clade is robust.
Complex ML models have been proposed as a remedy for LBA, but this approach recovered the same LBA clade as that recovered by the MP analysis for daphniid LSU data. Nevertheless, parametric bootstrapping of the data revealed that ML did recover the correct tree for the majority of replicates, whereas MP recovered the incorrect LBA clade for the majority of replicates. This finding supports the notion that ML is much less sensitive to LBA than MP (Huelsenbeck 1997
). However, the differing results of ML under parametric and nonparametric bootstrapping suggest that the model used in the nonparametric analysis is inadequate or that too few characters have been sampled. Because the nuclear LSU has a very complex mode of evolution, the TrN + I +
model may be inappropriate even though model testing determined that it had the best fit to the data. For example, the known aspects of rRNA secondary structure coevolution are unaccounted for in the TrN + I +
model. The Akaike information criterion (Hasegawa 1990
), an alternative way of comparing different models of DNA sequence evolution, indicated that a more complex GTR + I +
model was optimal, but the tree reconstructed with the use of this model also recovered the D. laevis/D. occidentalis clade (not shown).
The use of a conserved gene and the addition of taxa also failed in this case to stave off LBA. It seems that the acceleration in LSU rates occurred before the radiation of the group that can act as a source of additional taxa (such as D. dubia). We know of no other extant species whose addition might break up the long D. laevis/D. dubia or D. laevis/D. occidentalis branch. Inspection of the phylogram (fig. 5C ) reveals that further taxon addition will most likely fail to break up the D. laevis branch. The long branch leading to the D. laevis clade is so long that other Daphnia species are more distant from D. laevis than from the outgroup genera Simocephalus and Ceriodaphnia.
Often a rogue lineage is associated with some aspect of unusual habitat, life history, or molecular evolution that might increase the substitution rate (Nunn et al. 1996
; Stiller and Hall 1999
). Nunn et al. (1996)
, for example, found that the large size of the LSU V3 domain in isopod crustaceans is associated with terrestrial habitats. They proposed that the rapid evolution and large size in terrestrial isopods might be a response to the increased thermal stress of terrestrial life. At present, we can identify nothing exceptional about the biology of the D. laevis lineage compared with that of the other daphniid taxa examined. The habitat of D. laevis is temperate freshwater ponds and lakesless extreme than the snowmelt ponds of the coldwater stenotherm D. ephemeralis, or the Australian saline habitat of D. truncata. There is nothing exceptional about the life history of D. laevis compared with that of D. pulex (Banta et al. 1939). Also, there are no signatures of base compositional bias that might affect substitution rates as in the dipterans. A positive association of gene size and rate of evolution in rDNA genes that appears to occur in protists (Stiller and Hall 1999
) does not exist in daphniidsD. laevis has an unexceptional gene size (table 5
). The factors that lead to substitutional rate acceleration in the D. laevis group are elusive.
Although D. laevis has an unexceptional LSU gene size for daphniids, it is clear that daphniids themselves possess unusually large nuclear LSU rDNA genes. At approximately 4.5 kb (about 84 bp are missing from our reported sequence), the D. pulicaria LSU sequence is smaller than the largest-reported LSU gene, 5.2 kb in the hagfish, but larger than the LSU of most animals and plants (Kuzoff et al. 1998
; Mallat and Sullivan 1998
). Crease and Taylor (1998)
found that the size of the V2 (also known as D2) expansion segment from the LSU and the small subunit (SSU) are highly correlated in branchiopod crustaceans. When combined with the SSU, intergenic, and internal transcribed spacer data (Crease 1993
; Crease and Taylor 1998
; unpublished data), our LSU results make it clear that each gene region in the daphniid nuclear rDNA gene family is exceptionally large in length when compared with that of other animals. As daphniids have the smallest genomes of all the crustaceans examined (Lecher, Defaye, and Noel 1995
), our results bolster the hypothesis that rDNA variable regions are uncoupled from the factors that regulate genome size evolution (Crease and Taylor 1998
).
Slippage and gene familywide base composition biases are consistent with variable region coevolution but fail to explain the existence of rogue variable regions that are markedly expanded compared with neighboring variable regions in an array. Our results have identified the putative hidden break region or V6 (table 4
) as a rogue expansion segment in daphniids. In an extensive comparison of this region with 29 taxa from 12 phyla, Chenuil, Solignac, and Bernard (1997)
found very little size variation in the helix that is expanded in daphniids. In their study, Drosophila had the longest stem at 44 bp. A recent study of vertebrate LSUs reported one chordate taxon with an expanded V6, Branchiostoma floridae (lancelet), which had a size of 180 bp (Mallat and Sullivan 1998
). So, with a broad range of 90351 bp, daphniids possess the largest-reported V6 regions and show more size variation in this region than is found throughout the rest of the reported metazoan phyla. In some animals, the V6 region, which contains the recognition site for the L25 protein (Chenuil, Solignac, and Bernard 1997
), is partially or entirely deleted by processing (Ware, Renkawitz, and Gerbi 1985
). There is no evidence that the largest V6 regions (from D. pulicaria) undergo any size reduction during processing (Taylor, Omilian, and Swain, unpublished data). Because daphniid V6 regions show a broad range of size from just above average size to the largest yet recorded, their study could provide insights into the evolution of rogue rDNA expansion segments. Such studies are important in the light of proposals to code rDNA size variation and other correlated expansion segment features for phylogenetic studies (Billoud et al. 2000
).
Despite several sources of error and potential bias, the nuclear LSU rDNA is useful for reconstructing evolutionary relationships among daphniids that lack strong among-lineage rate heterogeneity. In the present case, rapidly evolving taxa misled the analysis in two ways: LBA involving D. laevis and D. occidentalis, and a reduction in alignable informative sites that affected the position of D. ephemeralis. With these rapidly evolving taxa removed, daphniid phylogenies constructed from nuclear LSU sequence data are well resolved, well supported, and concordant with phylogenies constructed from independent data. Of particular note is a strongly supported and paraphyletic genus Daphnia. Also, there is strong support for the following clades: (D. pulex/D. ambigua), (Daphniopsis/Ctenodaphnia), (D. magna/D. longicephala), and the traditional subgenus Daphnia. Although many of the expansion segments proved unreliable in the alignment, they may contain further phylogenetically informative secondary structural information.
Our first comparison of nearly complete LSU sequences from nonchordate animals has revealed a misleading association involving morphologically cryptic species and a conserved gene. Each optimality criterion is misled by this pattern of evolution, but ML is markedly less sensitive to the observed LBA bias than MP. Unlike most existing case studies, this inconsistency seems caused by substitutional rate acceleration at variable sites rather than base compositional, covariotide, or taxonomic sampling biases. This case may provide a simple empirical case to study LBA remedies because there has been insufficient time for multiple interacting biases to evolve. Alternatively, further taxonomic sampling will reveal additional rogue lineages that possess differing or multiple biases in daphniid LSU sequences. The results suggest that current models of evolution are inadequate for some rDNA genes and that rogue taxa are difficult to predict on the basis of morphological divergence, genome type, and gene sequence conservation. Given this situation, the best guard against inconsistency seems to be the comparison of numerous independent genes.
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Acknowledgements |
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Footnotes |
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Keywords: Daphnia,
long-branch attraction
nuclear large subunit
rDNA
rate acceleration
cryptic species
Abbreviations: CI, consistency index; HSP, heat shock protein; LBA, long-branch attraction; LSU, large subunit; ML, maximum likelihood; MP, maximum parsimony; PCR, polymerase chain reaction; RI, retention index; SSU, small subunit.
Address for correspondence and reprints: Angela R. Omilian, Department of Biology, Jordan Hall, 1001 E. Third Street, Indiana University, Bloomington, Indiana 47405. aomilian{at}indiana.edu
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