* Department of Biology, Georgetown University
Biological Dynamics of Forest Fragments Project, National Institute for Research in the Amazon, Manaus, Brazil
Department of Biology, Loyola University, Chicago
Correspondence: E-mail: hamiltm1{at}georgetown.edu.
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Abstract |
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Key Words: homoplasy chloroplast genome indel intergenic relative rate generation time effect
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Introduction |
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Chloroplast DNA has blossomed as an intraspecific genetic marker for at least two practical reasons. First, complete genome sequences and the development of conserved oligonucleotide primers for both coding and noncoding cpDNA regions (e.g., Taberlet et al. 1991; Demesure, Sodzi, and Petit 1995; Dumolin-Lapegue, Pemonge, and Petit 1997; Hamilton 1999a) have provided a ready means to assay cpDNA sequence variation. Second, cpDNA intergenic regions have exhibited substantial intraspecific insertion/deletion (indel) polymorphism within and among plant populations (e.g., McCauley 1994, 1998; Powell et al. 1995; Dumolin-Lapégue, Pemonge, and Petit 1998; Hamilton 1999b; Caron et al. 2000; Desplanque et al. 2000; Dutech, Maggia, and Joly 2000; Muloko-Ntoutoume et al. 2000; Oddou-Muratorio et al. 2001). Although the traditional view that plant cpDNA changes very slowly in nucleotide sequence still holds to some extent (Palmer 1990; Provan, Powell, and Hollingsworth 2001), indels appear to evolve more rapidly and are therefore useful for studies both at the intraspecific and shallow interspecific levels. Despite the availability of intraspecific cpDNA indel variation, employing polymorphic indel haplotypes to make evolutionary inferences about population divergence and gene flow faces several challenges.
Indels clearly do not occur at random locations within organelle genomes, but they are often associated with specific features of DNA sequences. Regions containing repeats that lead to slipped-strand mispairing (e.g., mononucleotide and microsatellite repeats), stem-loop secondary structure, and intramolecular recombination are thought to cause the majority of insertion/deletion mutations (reviewed by Kelchner 2000). Thus, recurrent mutations ("multiple hits") may occur at the same sites and generate homoplasy for organelle indel haplotypes (Clegg et al. 1994). Haplotypes defined by indels then have increased probabilities of being identical in state without being identical by descent. Such homoplastic characters cause character state reversals in phylogenetic trees and violate the basic assumptions of measures of population subdivision (e.g., FST or GST). Indel homoplasy will tend to reduce inferred population subdivision, because populations can share haplotypes as a result of both gene flow and recurrent mutation. This problem has been widely recognized for nuclear microsatellite loci (e.g., Jarne and Lagoda 1996; Goldstein and Pollock 1997; Hedrick 1999) and for the use of indel polymorphism in phylogenetic reconstruction (e.g., Golenberg et al. 1993; Graham et al. 2000; Kelchner 2000). Fewer studies have evaluated the extent of cpDNA homoplasy among recently diverged species (e.g., Doyle et al. 1998) or among populations of a single species (e.g., Desplanque, et al. 2000).
Both indels and nucleotides exhibit homoplasy. Many models of nucleotide substitution are available to estimate actual sequence divergence from observed sequence changes (reviewed in Swofford et al. 1996). At present, there are no generally employed methods to adjust observed numbers of indel changes for multiple unobserved evolutionary events at an indel site, although a few indel mutation models do exist that could form the basis of a correction method (e.g., Tajima and Nei 1984; Thorne, Kishinmo, and Felsenstein 1992; McGuire, Denham, and Balding 2001). Without estimates of rates of indel mutational homoplasy, it is potentially difficult to distinguish the action of evolutionary processes that homogenize populations (e.g., gene flow, selection) from convergence because of saturation of changes as divergence among populations increases.
Nucleotide substitution rate estimates are available for coding regions of the chloroplast genome compared among relatively deeply diverged taxa (reviewed by Clegg et al. 1994; Muse 2000) and more recently for intergenic regions compared at shallow levels of divergence such as within families (e.g., Gaut et al. 1997; reviewed in Kelchner 2000). Because indels found in multiple regions of the cpDNA genome may exhibit absolute rate differences, estimates of rate variation among cpDNA intergenic regions would be helpful when selecting regions for population studies because genetic markers sought for a specific hypothesis must be selected based on the level of lineage resolution required (Avise 1994; Parker et al. 1998). Furthermore, estimates of indel evolutionary rates would be valuable for interpreting the spatial patterns of indel haplotypes observed in population structure studies, providing context on the relative degree of divergence (e.g., numbers of indel changes among populations compared to among closely related species).
Divergence estimates can help identify cpDNA regions exhibiting disproportionately slow or fast rates of evolution, a result consistent with some types of selection (see animal mtDNA review by Ballard and Kreitman 1995). Indel mutations may directly alter fitness and therefore be subject to natural selection. For example, deletion mutations could be favored if smaller genomes have higher fitness in a "race for replication" (reviewed by Rand 1993), causing the rate of indel evolution to differ from a neutral rate. The action of selection on indel haplotypes could be diagnosed as rate heterogeneity among lineages (Sarich and Wilson 1973). Chloroplast regions where mutational changes do not approximate a molecular clock have the potential to greatly complicate the interpretation of spatial patterns of haplotypes observed in population structure studies. Direct selection on haplotypes can result in patterns of population subdivision that are the result of local selection pressures and not historical gene flowfor example, locally adapted haplotypes where the fitness of a given haplotype varies among populations. Direct selection on haplotypes can also result in reduced population subdivision of haplotypes as a result of uniform selection pressures among populations, even though gene flow may be limited (e.g., Stephan et al. 1998).
In this study we examined patterns of inter- and intraspecific sequence variation at six chloroplast intergenic sites for 16 individuals from eight species in the Brazil nut tree family (Lecythidaceae). Our analyses were designed to examine the evolution of indels used previously to estimate population structure in the tropical tree Corythophora alta (Hamilton 1999a). That study found cpDNA indel variation partitioned almost entirely among populations. The pattern is consistent with either very limited seed dispersal (if indels are neutral with low levels of homoplasy), or with regional selection pressures that have influenced the spatial pattern of cpDNA haplotypes. Our goals here were to describe the evolutionary history of cpDNA variation in close relatives of C. alta and to test the assumptions of the population structure study to the extent possible with a phylogenetic analysis. We employed a comparative analysis of cpDNA variation using nucleotide changes as a standard, because they are well understood and thoroughly modeled, to examine the evolution of indel changes, which are much less well understood or modeled. We compared the relative evolutionary rates of cpDNA indel and nucleotide changes to determine if indel homoplasy occurs frequently enough to confound substantially the effects of gene flow with the effects of recurrent mutation. We also sought to determine if the relative rate of cpDNA evolutionary change in C. alta is excessively fast or slow compared to other closely related species. Such relative rate variation could be consistent with homoplasy or with the action of selection on cpDNA haplotypes and would indicate that the distribution of cpDNA variation among populations may not be due exclusively to drift and gene flow. We also sought to determine if there was evidence of rate heterogeneity among the six cpDNA regions examined.
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Materials and Methods |
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Genomic DNA was extracted by grinding frozen leaf tissue in liquid nitrogen and using a DNeasy plant kit (QiaGen, Valencia, Calif.) according to the manufacturer's instructions. The PCR reactions contained 2 µL of DNA template (DNA concentration was not determined), 2 µL of 10x Thermopol buffer (containing 20 mM MgSO4), 0.2 mM each dNTP, 0.4 µm of each primer and 0.4 units of Vent exo- polymerase (New England Biolabs, Cambridge, Mass.) in a total volume of 50 µL. The thermal cycling profiles were 5 min at 96°C followed by 3040 cycles of 96°C for 45 s, annealing temperature for 1 min and 72°C extension for 30 s (see Hamilton 1999b). For trnH-trnL we used 30 cycles and a 55°C annealing temperature. For atpß-rbcL we used 5 min at 94°C followed by 35 cycles of 94°, 55° and 72°C for 1 min each. The PCR products were purified with QiaQuick spin columns (QiaGen), and both strands were sequenced in reactions containing 4.6 µL water, 2 µL template, 2.4 µL primer (1 µM) and 6 µL dRhodamine or Big Dye version 2 terminator reaction ready mix (Applied Biosystems, Foster City, Calif.). Sequence reactions were purified with Centrisep spin columns (Princeton Separations, Adelphia, N.J.) and electrophoresed on a model 377 sequencer (Applied Biosystems, Foster City, Calif.). In a few cases the PCR product repeatedly yielded poor sequence data for one strand, and the PCR product was cloned with the Zero Blunt TOPO PCR Cloning Kit Sequencing Version H (Invitrogen, Carlsbad, Calif.). With cloned PCR products, sequencing reactions used T7 and T3 vector primers instead of region-specific cpDNA primers.
The resulting sequences for each cpDNA region were aligned into contigs for each individual and edited using Sequencher 3.1.1 and 4.1.2 (GeneCodes, Ann Arbor, Mich.). Multiple sequence alignments for each cpDNA region were made manually with the aid of Sequencher from the consensus sequences from each individual. Sequences were trimmed to exclude terminal coding regions and the psbN gene within the psbB-psbH region in all analyses. Gaps in the multiple sequence alignments were positioned to minimize the number of nucleotide differences among sequences where possible. Insertion/deletion characters and states were scored following the "complex" coding method of Simmons and Ochoterena (2000), including the use of ordered step matrices to encode the minimum number of events that separate indel character states where necessary. Indel character states and step matrices were coded using MacClade 4.03. Indel character states were based only on length and did not include nucleotide differences within regions exhibiting sequence length variation. This conservative approach was taken because such nucleotide changes may not be independent of indel events. Single base indels and indels associated with mononucleotide runs were all subject to an additional check against original sequence chromatograms after multiple sequence alignment to verify that they were not a result of base calling error. Sequences were deposited in GenBank individually (accession numbers AY172691 to AY172799) and as multiple sequence alignments for each region or PopSets.
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Statistical Analysis |
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To choose the best supported model of nucleotide evolution for the aligned sequences with hierarchical likelihood ratio tests, we employed the program Modeltest 3.06 (Posada and Crandall 1998). The best-fitting model and the resulting parameter estimates were then used in PAUP* to estimate maximum likelihood pairwise distances between sequences for nucleotide changes only (not indels). When distances were estimated by maximum likelihood for cpDNA regions separately, base frequencies were estimated by maximum likelihood for each region independently.
A likelihood ratio test of the molecular clock for the full five- and six-region data sets as well as for each data set while excluding one region was conducted along the lines of Posada and Crandall (2001). Employing the substitution model selected with Modeltest, we obtained in PAUP* likelihood scores and topologies of phylogenies not assuming a molecular clock. This topology was then loaded as a constraint, and likelihood scores were obtained for phylogenies assuming a molecular clock. The log likelihood scores were used to compute the statistic delta = 2(lnL1 lnL0), which is distributed as a 2 with the number of taxa minus 2 degrees of freedom (see Huelsenbeck and Crandall 1997).
We used Tajima's (1993) 1D and 2D nonparametric tests for deviations from the constant rate expectation that the number of unique changes along one lineage equals that along its sister lineage. Because these tests do not depend on a model of sequence change, they can be applied directly to indel characters as well as to nucleotide substitutions. We used only nucleotide changes that occurred outside of indel sites, consistent with our scoring of indel haplotypes explained above. The 1D was conducted with either nucleotide or indel changes separately using a 2 distribution with 1 degree of freedom. The 2D was applied to nucleotide substitutions and indels to test rate constancy using the joint outcome of the two types of changes using a
2 distribution with 2 degrees of freedom. To obtain the results of the 1D and 2D tests, a program called T1Dand2D (version 6.4) was written in C and used in conjunction with an Excel spreadsheet.
Not all pairs of sequences had sufficient numbers of nucleotide or indel changes to apply the 2 approximation for the 1D, which requires that each lineage have at least six observed changes (Tajima 1993; Nei and Kumar 2000). Thus, for 1D tests where at least one lineage had five or fewer changes we applied an exact binomial test to calculate the probability of observing an outcome as likely or less likely than the one observed under the null hypothesis that 50% of the changes occur on each lineage (using a binomial calculator at http://home.clara.net/sisa/binomial.htm).
We observed that the 2 approximation has a high type I error (null rejected when it is actually true) when at least one lineage had five or fewer changes. The
2 approximation is especially poor for small numbers of changes on both lineages. For example, we observed cases in the 1D test of four changes on one lineage and zero changes on the other lineage. Such an outcome has a probability of 1/16 or 0.0625 when the null hypothesis of equal rates is true, but a probability of 0.0455 under the
2 approximation. Because the 2D test uses the sum of the
2 values for each class of change to determine the overall probability of having observed the joint outcome of changes under the null hypothesis, the exact binomial test cannot be employed. Thus, the 2D tests are not adjusted for the high type I error rate of the
2 approximation and likely overstate the occurrence of deviations from rate constancy when numbers of changes are small for one or both categories of changes. The results indicate cases of the 2D test that have 5 or fewer changes on one lineage for one or both categories of mutational change.
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Results and Discussion |
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Figure 1 shows the frequency distribution of the lengths of indels, which ranged in length from one to 68 base pairs. Shorter indels were more common, with only 11 of 95 (11.6%) indels longer than 20 base pairs. The distribution has a modal indel length of five base pairs. The indel length distribution observed here is somewhat less skewed to the left than a distribution observed for a broad comparison of angiosperms, which had a modal length of one and average length of almost five base pairs (Graham et al. 2000).
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The bootstrap consensus parsimony trees based only on nucleotide data from the five and six region data sets are shown in figure 2. C. cainito was specified as the outgroup for the five region data (fig. 2A), and B. excelsa was used as the outgroup for the six region data (fig. 2B) based on its position in the five region tree. For each region there was a single most parsimonious tree with topology identical to the bootstrap consensus, maximum likelihood, and Neighbor-Joining trees (results not shown). Each branch of these trees shows the inferred nucleotide and indel character state changes used for regression analyses. Increased character state reversals (decreased consistency indices) for trees with combined nucleotide and gap data suggested the possibility of modest homoplasy for either nucleotide or indel data. Consistency indices were higher for phylogenies estimated with nucleotide data alone (0.954 and 0.988) than when estimated using combined data constrained to nucleotide-estimated topology (0.869 and 0.849) for the five and six region data, respectively.
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The quadratic regression terms also provided insight into indel homoplasy, because multiple hits are expected to lead to saturation of changes and a rate of apparent change that declines as divergence increases. There was evidence for weak saturation of apparent changes for the five region data because the quadratic term was significantly negative. It is important to note that the degree of curvature in the regression was slight and the estimate was heavily influenced by one branch with the most changes. The quadratic regression term for the six region data provided no evidence for indel saturation, perhaps because of more recent divergence among taxa and a smaller sample size of branch lengths.
For both the five and six region data sets, Modeltest selected the K81uf + G (also called K3P) model of nucleotide substitution (Kimura 1981). Selection of this model indicated it was most likely that nucleotide frequencies were unequal (estimated frequencies of A = 0.3339, C = 0.1578, G = 0.1455, T = 0.3627 were essentially identical for the two data sets), rates of transition and transversion were unequal, rates varied among sites, and no sites were invariant. The substitution models for the five and six region data sets did differ in their rate matrices (1.0000 1.6091 0.2494 0.2494 1.6091 and 1.0000 1.0954 0.3555 0.3555 1.0954) and gamma parameters (1.1457 and 0.2094). Substitution models for individual regions were K81uf (atpB-rbcL, trnL-trnH and trnS-trnG), HKY (psbB-psbH) or F81 (trnH-psbA and rpl20-5'rps12). The former two models both indicate unequal transition and transversion rates, but the K81uf has two different rates for transversions. The F81 indicates no difference in rates of transition and transversion. The K81uf + G models served as an average model for combined-region data and were used to estimate pairwise distances between sequences (tables 3 and 4). Distances in intraspecific comparisons (within C. alta and L. zabucajo) were 0.4% divergence or less, whereas intergeneric divergences within the Lecythidaceae ranged from above 3% down to those observed at the intraspecific level. Divergences between the Lecythidaceae and the Sapotaceae outgroup were around 7% in all cases. We note that distances estimated under the K81uf + G model, a region-specific model (e.g., K81uf) and uncorrected distances ("p" distances) were very similar, differing by a few percent at most in comparisons among the most diverged lineages (results not shown).
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For the five region data, the two Tajima 1D/2D tests did not in general support deviations from a molecular clock for indel or nucleotide changes (table 5). Both the 1D for nucleotide substitutions alone and the 2D showed that only the E. romeucardosoi lineage gave consistently significant deviations from rate constancy. E. romeucardosoi generally showed relatively fewer nucleotide changes per site and relatively fewer indel changes, suggesting a rate slowdown in that lineage (see table 3 and figure 2A). Most of the remaining lineages did not indicate significant deviations from rate constancy using indel changes alone, nucleotide changes alone, or both classes of changes in the 2D test.
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Differences in the rates of evolutionary change at the intergenic regions examined would cause variation in the levels of divergence among cpDNA regions. To test the hypothesis of regional rate heterogeneity, we estimated pairwise nucleotide distances within chloroplast intergenic regions in the five and six region data sets and then plotted these distances (fig. 4). These calculations show that the range of distances varies with cpDNA region. In the five region data set, trnS-trnG showed the greatest divergences (up to about 12%) as well as the widest variability, whereas atpß-rbcL showed the smallest divergences and variability. In the six region data where fewer, more closely related taxa were compared, trnH-psbA showed divergences over 15% with a broad range of values, and the other five regions showed modest divergence (up to about 4%) and low variability. For trnH-psbA, intraspecific comparisons were 1% to 3% divergence, whereas intergeneric divergence was frequently above 6%. The loci other than trnH-psbA showed fairly uniform divergence in the six region data set. Nucleotide divergence within the trnH-psbA region was slightly greater than that observed for the trnS-trnG region, even though the former was estimated from a set of more closely related taxa. Thus, the trnH-psbA region showed the highest evolutionary rate among the regions examined. These results were consistent with our inability to produce a multiple sequence alignment for all taxa for the trnH-psbA region, as well as with previous observations that the trnH-psbA region evolves rapidly (e.g., Aldrich et al. 1988).
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The Tajima's 1D/2D tests were also used to determine if individual cpDNA regions were associated with any relative rate deviations observed. This test was conducted by applying the Tajima 1D/2D tests to both the five and six region data sets where one region had been deleted (analogous to a delete-one-region jackknife). The general patterns of deviations from the null hypothesis observed in the full data set (tables 5 and 6) were also observed in the delete-one-region data sets (results not shown). The same results were obtained with likelihood ratio tests of a molecular clock conducted with delete-one-region data sets (results not shown). This outcome suggests that neither the trnS-trnG nor the trnH-psbA region was individually causing the relative rate heterogeneity observed in E. romeucardosoi and L. zabucajo despite having larger genetic distances among taxa than other regions (see fig. 4). These results are consistent with deviations from the molecular clock not caused by processes specific to any cpDNA region such as selection, but rather caused by phenomena affecting the entire chloroplast genome such as a generation time effect within specific lineages (Gaut 1998; but see Whittle and Johnston 2003). This finding is consistent with previous evidence that synonymous substitutions in the chloroplast genome routinely demonstrate a generation time effect (Muse and Gaut 1997), suggesting that noncoding regions and synonymous changes may evolve under similar evolutionary processes. A generation time effect would not affect the employment of cpDNA regions in intraspecific population studies because the spatial patterns of haplotypes observed should still reflect gene flow events. A generation time effect may, however, cause some taxa to have more or less divergence among haplotypes to employ as genetic markers compared to related taxa.
Several statistical issues in our employment of Tajima's 1D/2D deserve further comment. Tables 5 and 6 contain a large number of non-independent pairwise comparisons of taxa. Thus, the Tajima 1D/2D results should be interpreted with the qualification that some of the significant results may be spurious because of the large number of tests conducted. For example, in table 5 a total of 150 tests are presented (50 indel 1D, 50 nucleotide 1D, and 50 2D). At a significance level of 0.05 we would expect 5 out of 100 tests to reject the null hypothesis (rate constancy) even when it is true if each comparison is independent (Rice 1989). Therefore, we expect at least 7 or 8 of the tests in table 5 to falsely reject the null hypothesis of rate constancy. Thus instead of placing a large amount of weight on particular significant cells in the table, we considered repeated rejection of the null hypothesis with data from particular lineages to be biologically significant. This is indicated by many significant cells along a row or down a column (e.g., see E. romeucardosoi [Ero] in table 5). A large number of comparisons was not as much an issue in table 6 because it had only 30 tests.
We find it unlikely that the statistical power of the Tajima 1D/2D was low, leading to an insensitive test of rate heterogeneity. Our use of a binomial exact test allowed inclusion of comparisons with few nucleotide or indel changes to achieve additional power. Tajima (1993) and Bromham et al. (2000) both looked at sequence length's influence on power, finding that longer sequences were critical in detection of rate variation. Our sequences were quite long, and therefore we expect high power, with the qualification that power obviously drops as regions are excluded to test for among-region rate variation. Both studies also noted that a distant outgroup lineage can reduce power. Because our C. cainito outgroup showed about 7% nucleotide divergence from the Lecythidaceae, we also tested alternative outgroups with lower levels of nucleotide divergence. As described above, Tajima 1D/2D results were qualitatively identical regardless of outgroup. Although our tests suggest that these chloroplast indel and nucleotide changes are evolving in a neutral fashion, the lack of power might give false confidence that indels meet assumptions of population structure analyses. Yet the critical issue is expressed in the following question: is statistical power too low to detect rate variation which would bias estimates of population structure such as FST? In this case, the data that are available are not affected by non-neutral processes to a great enough extent to bias estimates of population structure. This view parallels the approach of Bromham et al. (2000), who focused specifically on the magnitude of error which would be introduced into the estimation of the date of a common ancestor by assuming the molecular clock when rates vary.
Our data were also collected to test the possibility that natural selection acts directly on observed indels (or nucleotide substitutions). We do not find strong evidence that rates of divergence for chloroplast regions among the Lecythidaceae sampled are heterogeneous with the exception of the E. romeucardosoi and L. zabucajo lineages. This is consistent with no net effect of selection on the rate of chloroplast indel or nucleotide evolution. However, these data cannot test the possibility that selection acting at linked sites in the genome influences the levels or geographic distribution of chloroplast polymorphism. This is so because selective sweeps are not expected to alter the absolute degree of neutral divergence between populations or species because even complete linkage to advantageous or deleterious mutations does not affect the neutral substitution rate (Birky and Walsh 1988; Charlesworth 1998). Testing for the influence of selection at linked sites on levels of indel or nucleotide polymorphism within C. alta will require additional haplotype polymorphism data.
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Conclusions |
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Supplementary Materials |
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Acknowledgements |
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Footnotes |
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