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Differences in plant annual/perennial habit are hypothesized to cause a generation time effect on divergence rates. Previous studies that compared rates of divergence for internal transcribed spacer (ITS1 and ITS2) sequences of nuclear ribosomal DNA (nrDNA) in angiosperms have reached contradictory conclusions about whether differences in generation times (or other life history features) are associated with divergence rate heterogeneity. We compared annual/perennial ITS divergence rates using published sequence data, employing sampling criteria to control for possible artifacts that might obscure any actual rate variation caused by annual/perennial differences.
Relative rate tests employing ITS sequences from 16 phylogenetically-independent annual/perennial species pairs rejected rate homogeneity in only a few comparisons, with annuals more frequently exhibiting faster substitution rates. Treating branch length differences categorically (annual faster or perennial faster regardless of magnitude) with a sign test often indicated an excess of annuals with faster substitution rates. Annuals showed an approximately 1.6-fold rate acceleration in nucleotide substitution models for ITS. Relative rates of three nuclear loci and two chloroplast regions for the annual Arabidopsis thaliana compared with two closely related Arabidopsis perennials indicated that divergence was faster for the annual. In contrast, A. thaliana ITS divergence rates were sometimes faster and sometimes slower than the perennial. In simulations, divergence rate differences of at least 3.5-fold were required to reject rate constancy in > 80 % of replicates using a nucleotide substitution model observed for the combination of ITS1 and ITS2. Simulations also showed that categorical treatment of branch length differences detected rate heterogeneity > 80% of the time with a 1.5-fold or greater rate difference.
Although rate homogeneity was not rejected in many comparisons, in cases of significant rate heterogeneity annuals frequently exhibited faster substitution rates. Our results suggest that annual taxa may exhibit a less than 2-fold rate acceleration at ITS. Since the rate difference is small and ITS lacks statistical power to reject rate homogeneity, further studies with greater power will be required to adequately test the hypothesis that annual and perennial plants have heterogeneous substitution rates. Arabidopsis sequence data suggest that relative rate tests based on multiple loci may be able to distinguish a weak acceleration in annual plants. The failure to detect rate heterogeneity with ITS in past studies may be largely a product of low statistical power.
Comparative studies of molecular substitution rates between lineages provide insights into the mechanisms that cause evolution of DNA sequences. Under the neutral theory [1,2] rates of nucleotide substitutions are expected to be equal to rates of mutation, thus a constant rate of nucleotide substitution in homologous DNA sequences should be observed among lineages that share mutation rates. Neutral theory assumes that genetic drift is the primary evolutionary mechanism causing molecular evolution and predicts that rates of sequence change would be both constant over time and independent of the effective population size. Heterogeneity in substitution rates can be explained under neutral theory by either unevenness of mutation rates at individual loci (manifested as locus effects) or correlated mutation rates across all loci within species (manifested as lineage effects). Alternatively, natural selection may cause rate heterogeneity among loci and lineages via purifying selection that reduces the probability of substitution due to functional constraint or through the increased probability of substitution associated with positive natural selection [3-6]. Identifying causes of rate heterogeneity as well as specific variables that affect underlying mutation and substitution rates is fundamental to understanding the mechanisms that cause evolution of DNA sequences (reviewed in [7]).
Differences in generation time could affect substitution rates, causing lineage effects on substitution rates if organisms with shorter generation times experience more mutations per unit of chronological time than organisms with longer generation times. This neutral explanation for rate heterogeneity among lineages is commonly called the generation time hypothesis. Under the generation time hypothesis, lineage-specific heterogeneity in rates of divergence can be explained by differences in the number of germ line cell divisions per unit time among lineages that otherwise share constant mutation rates. Therefore, under the generation time hypothesis substitution rates are expected to be negatively correlated with generation time [5,8,9]. Generation time effects on synonymous substitution rates have been widely observed at multiple loci for several mammalian species [2,3,9-15]. Generation-time-like effects have also been tested for in organism such as RNA viruses where faster substitution rates were correlated with higher frequencies of replication [16] and in spore-forming bacteria where rates of divergence were not related to spore dormancy [17].
In angiosperms, expected generation time impacts on rates of molecular evolution are not as clear as in animals since plants lack distinct germ and somatic cell lines. Plant cells are totipotent and the number of cell divisions between germination and gamete production can vary from individual to individual and even among parts of a single individual. The generation time hypothesis modified for plants assumes that variation in the frequency of cell replication is correlated with differences in annual/perennial habit. Since annuals have shorter minimum time to first flowering than perennials, it has been assumed that annuals would also experience a higher frequency of cell replication per chronological time and thereby a faster rate of divergence when compared to perennials [18]. The generation time hypothesis has been invoked to explain why annual species exhibited higher rates of molecular evolution than perennial species for several nuclear, mitochondrial and chloroplast loci (e.g. [19-23]). However, results from studies that support a generation time effect in plants have two primary limitations [24]. First, some studies used multiple non-independent comparisons in their analyses that may lead to statistical difficulties as well as potential phylogenetic bias. Second, the taxa compared were highly divergent so that other evolved differences in addition to generation time could also have caused the rate variation observed. Comparing divergence rates in phylogenetically-independent sets of annual/perennial pairs that are recently diverged can correct for these two pitfalls when testing for a generation time effect in angiosperms [24].
Loci that can be used to estimate divergence rates are limited in the vast majority of angiosperms, which restricts comparisons of substitution rates in multiple independent sets of recently diverged plant taxa. For example, the plant mitochondrial genome exhibits a fast pace of structural evolution but the lowest rate of nucleotide substitutions of all three plant genomes making it especially difficult to obtain sequences in multiple plant lineages with sufficient divergence [18,25-28]. Universal primers are available for multiple chloroplast regions but, like mitochondrial regions, the utility of these regions is often limited by low sequence divergence at shallow phylogenetic relatedness. Nuclear loci are not widely available in multiple plant lineages since nuclear genomes have variable architecture, abundant multigene families with rapid duplication and loss complicating the identification of orthologous loci [18,27]. There is also a wide range of substitution rates among nuclear DNA sequences in plants [29], requiring multiple loci in comparative studies to average rates over independent loci.
The internal transcribed spacers (ITS1 and ITS2) of nuclear ribosomal DNA (nrDNA) are the only nuclear DNA markers currently available for comparative tests of the generation time hypothesis in a broad range of recently diverged plant taxa for several reasons. First, ITS regions are universally amplifiable in plants and many plant taxa have been sequenced. Second, ITS regions are highly variable at the nucleotide level. Third, it is commonly believed that ITS multicopy arrays are homogenized by concerted evolution so that intraspecific polymorphism does not complicate estimates of divergence [30,31]. Moreover, ITS regions have been used extensively in molecular evolution studies of plants such as demonstrating that rates of ITS nucleotide substitution are associated with species diversity [32], reproductive isolation and life history [33], and environmental variables ([33]; but see [34]).
Two recent studies compared rates of ITS1 and ITS2 divergence using phylogenetically independent sets of angiosperms differing in life history but reached opposite conclusions about whether differences in life history affect rates of divergence. In the first study, Whittle and Johnston [24] did not find an association between relative rates of nucleotide substitution and annual/perennial life history in 22 species pairs, leading them to conclude that the generation time hypothesis does not apply to angiosperms. In another recent study, clades with a predominantly herbaceous life history exhibited an almost twice-faster average rate of divergence than predominantly long-lived woody clades using 28 independently calibrated absolute rates of ITS nucleotide substitution [35]. Both studies consistently did not reject the null hypothesis of constant divergence rates when comparing life histories. Since low statistical power of rate tests was suspected, both papers also treated substitution rate differences qualitatively or categorically (e.g. annual is faster or perennial is faster regardless of the magnitude of the rate difference). These conflicting results mandate further research into whether differences in generation times are correlated with substitution rates in angiosperms.
Given that ITS1 and ITS2 are currently among the only sequences available to test for rate heterogeneity among a wide sampling of plant taxa, it is essential to assess the statistical power of rate heterogeneity tests based on ITS sequences. It is critical to determine the magnitude of rate heterogeneity required to reliably reject the null hypothesis of rate constancy when evaluating whether differences in annual/perennial habit have heterogeneous substitution rates. Low statistical power will result in type II errors (incorrectly failing to reject the null hypothesis of rate constancy) that could lead to an erroneous conclusion that annual/perennial habit is not associated with divergence rates. One main cause of low statistical power is a small number of nucleotide substitutions available to estimate divergence, a common situation when recently diverged species are being compared. Simulations have shown that the power of Tajima's relative rate test [36], distance-based relative rate tests [9], and the maximum-likelihood relative ratio test [6] are all dependent on sequence lengths, the relatedness of the outgroup taxa, and the employment of an appropriate model of nucleotide substitution [37,38]. The alternative approach of categorical treatment of substitution rate differences in annual/perennial comparisons is based on the assumption that the direction of rate differences would accurately test rate heterogeneity. However, this approach has not yet been subjected to a rigorous power analysis.
In this article, we test whether annual/perennial habit affects rates of divergence by comparing both relative rates of molecular evolution and categorical branch length differences in 16 independent annual/perennial species pairs. ITS1 and ITS2 sequences were obtained from GenBank under strict sampling criteria designed to control for artifacts contributing additional variation in divergence rates that could obscure any rate variation caused by differences in life history. The criteria were that each annual/perennial pair was recently diverged, had at least eight nucleotide changes between taxa, had ITS sequences for two outgroup taxa available, and the ITS sequences were originally obtained from a single PCR amplicon. The power of maximum likelihood relative rate tests was investigated by determining the degree of rate heterogeneity required to reliably reject rate constancy for DNA sequences simulated under average nucleotide substitution parameters of ITS sequences. We also used simulations to assess whether categorical treatment of branch length differences is an appropriate method to test for rate heterogeneity when a relative rate test does not reject rate constancy. In addition, we utilized sequences of three nuclear loci, two chloroplast regions and multiple intra-specific nrDNA ribotypes for the annual Arabidopsis thaliana and two closely related perennials (Arabidopsis lyrata subsp. lyrata and A. lyrata subsp. petrea) to test whether substitution rate differences at the ITS regions were correlated across multiple loci as expected under the generation time hypothesis.
ITS annual/perennial substitution rates
The edited sequences had between 210–267 sites for ITS1, between 183–257 sites for ITS2 and between 420–505 sites for the combined ITS region. Results from the maximum-likelihood relative rate test and the categorical treatment of branch length differences, as well as the estimated rate differences in 16 independent annual/perennial comparisons for the ITS1, ITS2 and combined ITS data using two outgroup taxa, are summarized in Table 1. The maximum-likelihood relative rate test rejected the null hypothesis of rate constancy in 12 out of 48 comparisons with less divergent outgroups and in 13 out of 48 comparisons with more divergent outgroups. In most tests where the null was rejected, annual species exhibited faster rates of nucleotide substitution than perennial species (10 annuals versus 2 perennials with less divergent outgroups and 8 annuals versus 5 perennials with more divergent outgroups). Similar results for relative rate tests were obtained with Tajima's 1D relative rate test (results not shown). After treating branch length differences categorically, a sign test showed a significant excess of faster substitution rates in annual taxa for all ITS sequence regions with both more and less diverged outgroups with the exception of ITS1 using more diverged outgroups (Table 1). In summary, the results indicated rate homogeneity for many comparisons but annuals more frequently exhibited significantly faster rates of nucleotide substitution when there was significant rate heterogeneity.
Table 1. Relative branch lengths for ITS1, ITS2 and combined ITS sequences between comparisons of recently diverged annual (before the slash) and perennial (after the slash) species when two different phylogenetically related outgroup taxa are used.
Using two outgroup taxa with different levels of divergence in each annual/perennial comparison showed that substitution rates varied slightly but did not change the general conclusion that annuals exhibited faster rates of substitution than perennials. For ITS1 using less divergent outgroups, relative rate tests rejected rate constancy in three of 16 comparisons, indicating that three annual species exhibited a significantly faster rate of substitution. In the same way, annual taxa exhibited longer branch lengths in 12 of the 16 categorical comparisons (sign test, p = 0.038). When the same annual/perennial species pairs where compared using more divergent outgroups, two annuals and one perennial showed significantly faster rates by relative rate tests while 11 of 16 qualitative comparisons (sign test, p = 0.105) exhibited longer branch lengths for annual taxa. For the ITS2 data, four cases (three annuals and one perennial) rejected rate constancy when less divergent outgroups were employed and in 13 of 16 qualitative comparisons (sign test, p = 0.011) annuals showed longer branch lengths. If more divergent outgroups were used, five cases (three annuals and two perennials) rejected rate constancy and 12 of 16 qualitative comparisons (sign test, p = 0.038) exhibited longer branch lengths for annual taxa. For the combined ITS sequence data with less diverged outgroups, four annuals and one perennial species exhibited significantly faster substitution rates by relative rate tests and 13 of 16 qualitative comparisons (sign test, p = 0.011) exhibited longer branch lengths for the annual taxa. If more divergent outgroups were used with combined ITS sequence data, three annuals and two perennials rejected rate constancy and 12 of 16 qualitative comparisons (sign test, p = 0.038) exhibited longer branch lengths for annual taxa.
Power simulations
The phylogeny used in the simulations is shown in Figure 1 and the nucleotide substitution parameter sets implemented in simulations are given in Table 2. The Kimura 2 parameter nucleotide substitution model (K80 or K2P), which assumes equal base frequencies and variable transition and transversion frequencies [39], was the one most frequently estimated for each of the 16 annual/perennial species pair comparisons for all six ITS sequence datasets (results not shown, substitution models available from the authors). Overall, the simulations indicated that a maximum likelihood relative rate test had an increasing chance to detect rate heterogeneity for ITS-like sequences as the rate difference between ingroup taxa increased (Figure 2). Relative rate tests rejected the null hypothesis in no more than 80% of replicate simulations for a given set or parameters for both ITS1-like and ITS2-like sequences, independently of the level of divergence of the outgroup used (see solid lines in Figure 2A, B, D and 2E). With a 3-fold or less rate difference between ingroup taxa, ITS1-like and ITS2-like simulated sequences rejected rate constancy for no more than 50% of replicates. For combined ITS-like sequences, the power of the test increased and rate constancy was rejected in about 80% of replicates with a 3.5-fold or greater rate difference between ingroup taxa (see solid lines in Figure 2C and 2F).
Table 2. Nucleotide substitution parameters estimated from ITS sequence data of 16 annual/perennial pairs used to simulate DNA sequences with Seq-Gen.
Figure 1. Schematic of the tree topology used when simulating DNA sequence triplets for power analyses. The program used to simulate DNA sequences (Seq-Gen) continues to add nucleotide changes until threshold divergence values have been reached. These threshold divergence values were obtained by averaging the estimated sets of branch lengths from actual ITS sequences for 16 annual/perennial/outgroup comparisons (Table 1). These averaged values are given in Phylip format in Table 2. The threshold divergence values for the perennial-like taxon (DIA-perennial) and the outgroup-like taxon (DR-outgroup, DR-IA) were kept constant for each outgroup (closer and further) and each set of nucleotide substitution parameters (ITS1-like, ITS2-like and Combined-ITS-like). To model rate heterogeneity, the threshold divergence value of the annual-like taxon (DIA-annual) for each set of replicate simulations was determined by multiplying the perennial-like taxon divergence threshold (DIA-perennial) by1.5 to 5 in steps of 0.5.
Figure 2. The frequency of significant rate heterogeneity by maximum likelihood relative rate tests along with the frequency of rate heterogeneity indicated by categorical rate comparisons in simulated data. Each data point represents the proportion of 1000 replicate simulated ITS-like sequence triplets with one of the ingroup taxon evolving with a substitution rate parameter between 1.5 and 5 times faster than the other ingroup taxon. In addition, the graphs show the proportion of 1000 replicates which correctly indicated that the taxon with the faster substitution rate parameter exhibited a higher substitution rate using qualitative substitution rate comparisons regardless of whether the maximum likelihood relative rate test rejected rate constancy. Simulated sequences were obtained with mean divergence and nucleotide substitution parameter sets estimated from actual ITS sequences (see Table 2).
The proportion of replicates where the faster evolving taxon had a qualitatively higher substitution rate was at least 70% even with a rate difference as low as 1.5-fold. Categorical rate comparisons identified the annual-like taxon as faster in 100% of replicates when the rate difference was 3-fold or greater for all three ITS-like sequences. The proportion of replicates with significant rate heterogeneity for each of the ITS-like sequences was similar between the more and less divergent outgroups.
The distributions of estimated rate differences between ingroup taxa among replicate sequence simulations for all six different nucleotide substitution model parameter sets (see Table 2) are shown in Figure 3. Estimated rate differences within a nucleotide substitution parameter exhibited high variation and extremely long tails for all six nucleotide substitution parameter sets. The mean and coefficient of variation were not appropriate to summarize the distributions because of non-normality (20 or so very extreme values at each tail lead to a large difference between the mean and mode). Medians and 95% confidence intervals (CI) of estimated substitution rate differences for replicate simulations were: median = 1.74, CI = -2.2 – 14.6 for ITS1-like sequences; median = 1.52, CI = -2.9 – 10.1 for ITS2-like sequences; median = 1.77, CI = -1.4 – 5.8 for combined-ITS-like sequences with a less diverged outgroup. Using a more diverged outgroup, replicate simulations exhibited rate difference distributions of median = 1.56, CI = -3.2 – 25.1 for ITS1-like sequences; median = 1.17, CI = -4.9 – 8.4 for ITS2-like sequences; median = 1.42, CI = -2.1 – 4.2 for combined-ITS-like sequences. The median value of the estimated rate differences was nearly identical in each case to the rate difference parameter in Table 2 and 95% CIs of estimated rate differences overlapped extensively.
Figure 3. Histograms of estimated substitution rate differences between annual-like and perennial-like species pairs in 1000 independent replicates that were simulated under the average nucleotide substitution parameter sets estimated from actual ITS sequences (see Table 2). The value axis gives the ratio of the faster substitution rate over the slower substitution rate. The rate difference ratio was assigned a negative value when the perennial-like taxon had a faster estimated substitution rate and was positive when the annual-like taxon had a faster estimated substitution rate.
Arabidopsis annual/perennial substitution rates
Results of relative rate tests, estimates of substitution rates and estimated substitution rate differences between the annual A. thaliana and the perennials A. lyrata and A. petraea for various nrDNA ribotypes, the three nuclear loci and two chloroplast regions are summarized in Table 3. Rate constancy was rejected in a single instance, where the chloroplast region had a significantly faster substitution rate for the annual A. thaliana. Qualitatively, substitution rate differences estimated at each of the three nuclear loci were similar and uniformly indicated a faster rate for the annual species. The average substitution rate difference for all nuclear loci was 1.35-fold between A. thaliana and A. lyrata and 1.32-fold between A. thaliana and A. petraea.
Table 3. Estimated branch lengths and substitution rate differences (Rate Δ) for comparisons between the annual Arabidopsis thaliana and the two perennials Arabidopsis lyrata subspecies lyrata and Arabidopsis lyrata subspecies petraea using five nuclear loci (ITS1, ITS2, Chs, Adh, PgiC), two chloroplast regions (rbcL and matK) and Crucihimalaya himalaica as the outgroup.
When the three nuclear loci were concatenated into a single sequence, a Tamura-Nei nucleotide substitution model with a gamma parameter was obtained (results not shown). When the entire concatenated nuclear sequence was used in the likelihood relative rate test, A. thaliana showed significantly faster divergence rates when compared to both A. lyrata (p = 0.017) and A. petraea (p = 0.025; results not shown). For the concatenated nuclear sequences, the average substitution rate difference was 1.35-fold between A. thaliana and A. lyrata and 1.33-fold between A. thaliana and A. petraea.
When the two chloroplast regions were concatenated into a single sequence, a Hasegawa-Kishino-Yano nucleotide substitution model best fit the data (results not shown). In contrast with the results from the concatenated nuclear sequences, relative rate tests did not reject the null hypothesis of rate constancy for the concatenated chloroplast sequences between A. thaliana and both A. lyrata (p = 0.093) and A. petraea (p = 0.092; results not shown). Categorical analyses for the concatenated chloroplast sequences indicated a faster divergence rate for A. thaliana when compared to A. lyrata (1.7-fold difference) and A. petraea (1.8-fold difference).
No relative rate tests rejected rate constancy for any comparison of Arabidopsis ITS sequences. For all ITS sequences in Arabidopsis, categorical treatment of substitution rates as well as estimated rate differences between annual and perennial taxa showed a roughly equal number of cases where the annual and the perennial exhibited a faster substitution rate. Sometimes the taxon with the faster rate for ITS1 had the slower rate for ITS2, such as when A. petraea (R1 ribotype) had a qualitatively higher substitution rate at ITS1 but qualitatively lower substitution rate at ITS2. In another case, using the alternative ribotype R2 for A. petraea both ITS1 and ITS2 exhibited qualitatively higher substitution rates in the annual taxon. When additional outgroups and nrDNA ribotypes were used in the annual/perennial/outgroup comparisons for ITS (data not shown), both A. thaliana and the perennial taxa had qualitatively faster substitution rates with about equal frequency. The alternating pattern of either the annual or perennial taxon exhibiting a faster estimated substitution rate for ITS sequences was in contrast to the consistent pattern of faster estimated substitution rates for the annual A. thaliana at the three nuclear loci and the two chloroplast regions.
Overall, the null hypothesis of rate constancy for ITS sequences was not rejected in the majority of annual/perennial comparisons based on both maximum-likelihood and Tajima's 1D relative rate tests. When rate constancy was rejected, annuals exhibited higher rates of nucleotide substitution in most cases. Categorical treatment of branch length differences indicated that an excess number of annual species had higher rates of nucleotide substitution. Because these two patterns are expected under the generation time hypothesis for plants, these results support the hypothesis that differences in the annual/perennial habit are associated with rates of molecular evolution in angiosperms.
The simulations reported in this paper supply several insights. First, the simulations showed how often relative rate tests based on ITS-like sequences reject the null hypothesis of rate constancy when rates are in fact unequal. Second, the simulations showed how frequently a categorical comparison of branch lengths detects faster substitution rates even when relative rate tests do not reject the null hypothesis. Third, the simulations provide context for observations of ITS substitution rate homogeneity or heterogeneity reported in earlier studies, in particular, why substitution rates may not have been associated with differences in annual/perennial habit [24,35]. Because ITS sequences are short and have few diverged sites when compared between recently diverged taxa, relatively low power to reject rate constancy seemed possible. Indeed, the simulations showed that the statistical power to detect rate heterogeneity using ITS sequences is generally low for rate differences less than 3-fold. For simulations based on ITS1-like and ITS2-like nucleotide substitution parameter sets, the relative rate test only achieved an 80% probability of rejecting rate homogeneity with 4.5 or 5-fold rate differences. These power analyses for ITS-like sequences agree with a more general previous study that demonstrated a high type II error for Tajima's 1D test when the DNA sequences compared are short and have few diverged sites [38]. The simulations further suggest that categorical treatment of branch length differences is a more powerful indicator of rate heterogeneity at low to moderate substitution rate differences compared to relative rate tests, at least for ITS-like sequences. However, conclusions about the statistical power of the categorical rate comparisons only apply to the average nucleotide substitution model and divergence parameters used in the simulations and may not be a general phenomenon.
The best-documented case of a generation time effect is the 2- to 3-fold faster substitution rate in rodents compared to hominids [12,7]. This well studied example provides some perspective on the magnitude of rate differences we might expect to observe in plants if a generation time effect actually operates. In plants, the magnitude of rate differences between annual and perennial taxa was 2-fold at synonymous sites in the mitochondrial coxI gene [40], 4-fold at both synonymous and non-synonymous sites in the chloroplast rbcL gene [20,21,27], and 2.5-fold at synonymous sites in nuclear Adh loci [20] (Eyre-Walker and Gaut 1997). All of these studies were limited to comparisons of highly divergent annual and perennial species and therefore confounding factors other than differences in generation time might have lead to an overestimation of the impact of annual/perennial habit on substitution rates. In a study focused on phylogenetically independent comparisons, Kay and collaborators [35] found that clades with a predominantly herbaceous life history exhibited divergence rates for ITS sequences almost two times faster than clades with a predominantly long-lived woody life history in 28 phylogenies representing 21 different angiosperm families. The overall substitution rate differences estimated for the annual/perennial species comparisons in this paper were of similar magnitude to the ITS rate differences observed by Kay et al. [35]. Here, annuals evolved on average 1.6 times faster rate than perennials when a less divergent outgroup was used, while a slightly lower 1.4-fold average acceleration of annuals was observed with more divergent outgroup taxa. Categorical comparisons for ITS sequences also indicated that faster rates of substitution were correlated with annual habit. Therefore, the ITS results in this paper support a weak substitution rate acceleration for annuals consistent with a generation time effect in plants. We also believe that our sampling methods controlled for rate heterogeneity caused by variables other than annual/perennial habit and helped to better detect a weak generation time effect. This explains in part why our results are distinct from those of Whittle and Johnston [24], even though both studies were based on some of the same ITS data and used similar relative rate tests.
The Arabidopsis sequence data also support a weak annual acceleration in substitution rates. The annual A. thaliana had a significantly faster substitution rate for the chloroplast rbcL locus when compared with A. petraea. In addition, categorical rate comparisons consistently showed a faster substitution rates for A. thaliana for all the nuclear loci and chloroplast regions, even though rate constancy was not rejected by relative rate tests. When the three nuclear loci were combined, A. thaliana had a significantly faster substitution rate than either perennial. The only exception to the pattern of faster divergence rates for A. thaliana were at ITS sequences. The lack of any relative rate tests rejecting rate homogeneity and about half of qualitative comparisons indicating annuals were faster, all suggest that the ITS sequences showed no evidence of a faster substitution rate for A. thaliana. However, the simulation results showed that ITS-like sequences have little power to reject rate constancy when substitution rates are less than 2-fold different. So the pattern of about half of the qualitative rate comparisons showing a faster substitution rate for annuals is consistent with random variation about a mean rate difference of zero. Interestingly, similar results indicating a consistently higher number of synonymous substitutions (but rate constancy was not rejected by Tajima's relative rate test) in A. thaliana than in A. lyrata were observed in five out of six loci using the closely related outgroup species Capsella rubella and Arabidopsis graba [41].
Recent divergence of annual/perennial taxa is an advantage when attempting to infer the possible causes of rate heterogeneity because it reduces the number of evolutionary changes that distinguish the taxa in addition to annual/perennial habit. Unfortunately, that advantage may come at the cost of statistical power to detect potential rate heterogeneity. Recent divergence also means that few substitutions have occurred in the two taxa being compared so that the number of nucleotide changes will be small. The Arabidopsis data further suggest that statistical power to compare annual/perennial substitution rates is limiting. The individual Arabidopsis nuclear loci did not show significant rate heterogeneity. However, the larger sample of changes in the three loci combined showed the approximately 1.4-fold rate difference between annual and perennial was significantly faster for the annual. Since we did not distinguish among synonymous and nonsynonymous sites in the Arabidopsis sequences, the significant rate difference is an average across all types of nucleotide sites and reflects the net substitution rate of neutral sites and any sites influenced by positive or negative selection.
In addition to the low power of ITS sequences to test relative rate hypotheses, ITS sequences may have other limitations that further hamper their ability to detect rate heterogeneity. There is the possibility that the ITS1 and ITS2 sequences might be subject to different selective pressures [42] resulting in region-specific rates of ITS substitution when natural selection is stronger than genetic drift. The ITS sequence data in this paper indicated a pattern of annual taxa exhibiting faster substitution rates that was consistent between both ITS1 and ITS2 regions. Such a pattern is not expected if ITS1 and ITS2 regions experience locus-specific selection pressures. In addition, it has been hypothesized that incomplete concerted evolution could independently affect rates of molecular evolution at either ITS1 or ITS2 [30]. Reports of multiple nrDNA haplotypes within individuals are becoming increasingly common [e.g. [43-51]] suggesting that complete concerted evolution should not always be assumed for ITS sequences. The different intraspecific nrDNA ribotypes used in the Arabidopsis annual/perennial comparisons did in fact change the perception of substitution rates between ITS1 and ITS2 regions. Either ITS1 or ITS2 was observed to have the faster substitution rate for annuals depending on the nrDNA ribotypes used in the Arabidopsis annual/perennial comparison. Thus, the Arabidopsis ITS data suggest that estimates of substitution rates may depend on the nrDNA ribotype employed in comparisons. If selection pressures or polymorphism dynamics have a greater impact on estimates of substitution rates than does a weak generation time effect, any acceleration in the substitution rate of annuals will be difficult to detect with ITS.
The underlying biological mechanisms that might cause an acceleration of substitution rates in annuals are still unclear [18,35], although life history features that influence the number of rounds of DNA replication per unit of calendar time are capable of altering the relative substitution rate when mutation rates are constant. Identifying the underlying cause or causes of rate heterogeneity is difficult because variables such as the combined effects of organism size and temperature on metabolic rate [52,53], the influx of environmental energy that may lead to mutation [54], and mating system [55] are potentially confounded with differences in generation time. Annual or perennial habit may itself have a variable relationship to the generation time pertinent to substitution rates. For example, many perennials are able to flower in their first year like annuals while other perennial species may require many years until first flower. The total range of possible generation times in plants is very large since some woody perennials may live for thousands of years. The species in this study all fall at the short generation time end of this range since they are either annuals or short-lived perennials. Therefore, the suggestion of a weak annual/perennial substitution rate difference in our study may not apply if plant groups containing perennials with longer lives and greater time to first flower were compared.
ITS substitution rates in 16 phylogenetically-independent comparisons of annual and perennial taxa and from the combination of nuclear loci and chloroplast genome regions in annual and perennial Arabidopsis suggest a modest rate acceleration of less than 2-fold in annuals. These results support an association between rates of nucleotide substitution and annual/perennial habit in plants as expected under the generation time hypothesis. Separately, simulations showed that relative rate tests employing ITS-like sequences are not expected to be powerful enough to reject rate homogeneity when substitution rate differences are small. Given that the power of ITS sequences to test for generation time effects is very low, the conclusion by Whittle and Johnston [24] that no annual/perennial effect on substitution rates exits seems unwarranted. The small substitution rate differences observed here and in other studies points out that testing the generation time hypothesis among closely related plant species will require multiple loci to achieve sufficient power, as was the case in the now classic examples of animal generation time effects. While their availability in many plant taxa facilitates phylogenetically-independent comparisons, ITS sequences by themselves are not likely to be a powerful tool to test hypotheses involving substitution rate heterogeneity. Further studies with greater statistical power have to be carried out before drawing a definitive conclusion about patterns of relative substitution rate heterogeneity in annual and perennial plants and its possible causes.
DNA sequences
Partial nrDNA sequences from 64 different species representing 13 angiosperm plant families were retrieved from GenBank (Table 4). Because rate heterogeneity at nrDNA sequences may have multiple causes, we established three criteria designed to control for artifacts possibly contributing to rate heterogeneity that may obscure any rate variation caused by differences in annual/perennial life history.
Table 4. Genbank accession numbers for annual/perennial species pair and outgroup sequences, where the first taxon is the annual and the second taxon is the perennial for each annual/perennial pair, and for outgroups the first taxon listed is less diverged and the second is more diverged.
The first criterion was that only ITS1 and ITS2 sequences obtained from the same PCR amplicon were sampled to distinguish between functional and non-functional copies. A functional copy is expected to be under strong selective constraints limiting its substitution rate while a non-functional copy (pseudogene) is expected to exhibit a higher rate of nucleotide substitution when compared to a functionally constrained copy. Nuclear ribosomal DNA regions are usually located in chromosomal regions within nucleolus organizer regions (NORs) in the form of tandemly repeated arrays. Each nrDNA copy is organized into less constrained ITS1 and ITS2 regions separated by the 163–164 base pair 5.8S region, which is highly conserved when functional copies are compared within genera or between recently diverged genera [42,56]. A rigorous method to detect pseudogenes is to compare estimated divergence at conserved sequence regions with estimated divergence at unconstrained sequence regions [57]. Thus, we compared nucleotide divergence for 5.8S, ITS1 and ITS2 regions for each set of nrDNA sequences from the annual/perennial/outgroup comparison. We excluded from further analyses nrDNA sequences that exhibited either 1) a high divergence at the 5.8S region relative to the others 5.8S regions, or 2) divergence of the 5.8S region that was approximately equal to divergence at the ITS1 and ITS2 regions within a species, because these are patterns consistent with a lack of 5.8S functional constraint. This is a conservative sampling approach to prevent inadvertently combining functional and non-functional nrDNA copies in comparisons of annual and perennial taxa that could hamper our ability to detect possible associations between divergence rates and life history. The criterion of using nrDNA sequences from the same PCR amplicon was restrictive in that it caused us to exclude numerous possible ITS sequences available in GenBank.
The second criterion was that pairs of annual/perennial taxa sampled had nrDNA sequences from two outgroup taxa that were relatively closely related within the same family. This permitted examination of the impact of outgroup divergence on relative rate comparisons between annual and perennial taxa and prevented our relative rate estimates and hypothesis tests from being contingent on the peculiarities of a single outgroup.
The third criterion was that each of the ITS1 and ITS2 sequences were required to have at least eight nucleotide changes between annual and perennial species. Complete ITS sequences (ITS1, 5.8S and ITS2) were approximately 600 base pairs long on average so that eight nucleotide changes in 600 base pairs is equal to 1.3% divergence. Since the power of relative rate tests depends on the number of substitutions, this criterion prevented sampling of sequences likely to have little statistical power to reject the null hypothesis of rate homogeneity.
The annual Arabidopsis thaliana and the perennials A. lyrata subsp. lyrata (A. lyrata) and A. lyrata subsp. petraea (A. petraea) met the three sampling criteria for nrDNA and also offered three additional nuclear loci (Chs, Adh and PgiC) and the rbcL and matK chloroplast regions (see accession numbers in Table 5). The Arabidopsis taxa also offer intra-specific variation in nrDNA ribotypes and consequently polymorphic ITS1 and ITS2 sequences that permitted us to test whether relative rate comparisons were influenced by nrDNA polymorphism.
Table 5. GenBank accession numbers for sequences sampled of the annual Arabidopsis thaliana, the two perennials Arabidopsis lyrata subspecies lyrata and Arabidopsis lyrata subspecies petraea as well as the outgroup Crucihimalaya himalaica.
Alignment and phylogenetic analyses
Sequences were aligned into contigs for each comparison of an annual, perennial and an outgroup taxon and edited using Sequencher 4.5 (Gene Codes, Ann Arbor, Mich.). Gapped positions were pruned from alignments before analyses. Modeltest 3.5 [58] was used to determine the most likely nucleotide substitution model and the associated parameters for each triplet annual/perennial/outgroup sequence comparison. Branch lengths for each annual/perennial comparison were determined for ITS1, ITS2 and for the combination of both ITS1 and ITS2 regions (the combined ITS region) using PAUP* v.4.0b10 [59] and HyPhy [60] under the parameters of the substitution model determined in Modeltest. To express differences in nucleotide substitution rates between annual/perennial pairs, the substitution rate of the taxon exhibiting longer branch length was divided by the substitution rate of the taxon with the shorter branch length. In addition, the generation time hypothesis has been tested via categorizing branch length differences (e.g. annual faster than perennial or perennial faster than annual) even when rate homogeneity is not rejected by a relative rate test [24]. Thus, annual/perennial branch length differences were also summarized by a categorical variable to indicate whether the annual or perennial species exhibited the higher rate of substitution. Following Whittle and Johnston [24], we performed sign tests of the null hypothesis that annuals and perennials exhibited a longer branch length with equal frequency. In contrast to Whittle and Johnston [24], we employed one-tailed versions of the test because under the alternative hypothesis of rate heterogeneity annuals are expected to have a faster substitution rate than perennials.
Relative rate tests
To test for differences in substitution rates among taxa, both maximum-likelihood [61] and Tajima's 1D [36] relative rates tests were applied to the ITS sequences for each independent annual/perennial species pair. The relative rate test compares the number of nucleotide substitutions that occurred in one of the ingroup species to the number of substitutions that occurred in the other ingroup species utilizing the outgroup to identify those substitutions that can be unambiguously assigned to one of the ingroup taxa [62,63]. Under the null hypothesis of equal substitution rates in each lineage, the number of nucleotide changes is expected to be equal for the two taxa. The maximum-likelihood relative rate test is considered one of the most powerful and flexible tests for rate heterogeneity, but it requires knowledge of the nucleotide substitution pattern, any substitution rate variation among sites in addition to the phylogenetic relationship among sequences [27]. The maximum-likelihood relative rate tests were implemented in HyPhy [60] and used the nucleotide substitution models from Modeltest.
An alternative relative rate test which does not require an explicit nucleotide substitution model is Tajima's 1D test [36]. Although Tajima's 1D test cannot correct for saturation, apparent divergences are not expected to be gross under-estimates of true divergences for recently diverged taxa. The null hypothesis of rate constancy can be tested with Tajima's 1D using a chi-square with one degree of freedom as implemented in the T1Dand2D v4OS program [64]. Because sites with gaps or ambiguous base calls can be considered as an additional change by the Tajima's 1D program, they were excluded from the analyses.
Computer simulations
We investigated the power of maximum likelihood relative rate tests for each of the ITS1, ITS2 and combined ITS regions by computer simulation by utilizing empirically estimated sequence substitution parameters. Simulation parameters were based on nucleotoide substitution parameters estimated from ITS sequences and were divided into two groups based on divergence of the outgroup within each annual/perennial pair (Table 2). Transition/transversion ratio, sequence length and substitution rate difference between annuals and perennials were averaged over all independent annual/perennial pairs. Because the nucleotide substitution models for annual/perennial/outgroup triplets were somewhat variable (see Results), the most frequently obtained nucleotide substitution model was employed in the simulations.
The combination of SG Runner (T. Wilcox; homepage.mac.com/tpwilcox/) and Seq-Gen [65] were used to model each of the ITS regions. Seq-Gen simulates nucleotide substitution within lineages until a given threshold of divergence between the ingroup taxa has been reached. This threshold divergence value was obtained by averaging the estimated divergences (or branch lengths) from all annual/perennial/outgroup triplets (see Figure 1). Each ITS-like data consisted of 1000 DNA sequence triplets simulated with one of the ingroup taxon having a substitution rate between 1.5 and 5 times faster than the other ingroup taxon. The threshold branch length values of the taxon with the slower substitution rate parameter sets (also denoted as the perennial-like taxon) and the outgroup taxa were kept constant. The threshold branch length value of the taxon with the faster substitution rate parameter sets (also denoted as the annual-like taxon) was simulated with a rate difference of 1.5 to 5 times increasing in steps of 0.5 times the threshold branch length value of the perennial-like taxon.
Each set of triplet sequences was analyzed in PAUP* to calculate the relative branch lengths and the maximum likelihood values of each constrained and unconstrained tree. Then, a likelihood ratio test (LRT) was carried out for each of the 1000 replicates using an Excel spreadsheet and used to calculate the proportion of replicates where the null hypothesis of rate constancy was rejected. The percent of cases where the LRT rejected rate constancy was divided into instances where either the faster evolving annual-like taxon or the slower evolving perennial-like taxon had the longer estimated branch length. In addition, branch length differences in each replicate simulation were categorized into qualitative outcomes of annual-like taxon faster or perennial-like taxon faster, independent of whether or not the relative rate test rejected rate constancy. This provided an estimate of the proportion of replicates where the categorical comparison of rates detected rate heterogeneity. In order to evaluate the variation in estimates of annual/perennial rate differences in sequences most similar to actual ITS data, one set of 1000 replicate simulations were carried out using the nucleotide substitution model parameters and average rate differences estimated from the ITS sequences of the 16 annual/perennial pairs (see bottom rows of Table 2). The distribution of the estimated rate differences between the ingroup taxa in 1000 replicate triplet sequences was plotted in histograms for the six combinations of three ITS nucleotide substitution models and more and less diverged outgroups.
Authors' contributions
The four authors jointly conceived and designed the study. DSH and OFP assembled the data and performed the analyses. JMB and MBH coordinated the study. MBH and DSH wrote the manuscript. All authors read, edited and approved the final manuscript.
We thank P. Armbruster, M. Cummings, C. Drummond, C. Lund and W. Hahn for discussion and comments. B. Johnson provided statistical advice on the sign test. Two anonymous reviewers provided helpful comments that improved the manuscript. This work was supported by a doctoral fellowship to D. F. Soria-Hernanz from the Spanish Ministerio de Educacion y Ciencia, graduate support from Georgetown University and the Department of Biology, the Cosmos Foundation, and a National Science Foundation grant to M.B.H. (DEB9983014). Publication charges supported by the Department of Biology, Georgetown University.
Kimura M: Evolutionary rate at the molecular level.
Nature 1968, 217:624-626. PubMed Abstract | Publisher Full Text
Kimura M: The neutral theory of molecular evolution. New York: Cambridge University Press; 1983.
Gillespie JH: The causes of molecular evolution. New York: Oxford University Press; 1991.
Ohta T: The nearly neutral theory of molecular evolution.
Annu Rev Syst Ecol 1992, 23:263-286. Publisher Full Text
Ohta T, Gillespie JH: Development of Neutral and Nearly Neutral Theories.
Theor Popul Biol 1996, 49:128-142. PubMed Abstract | Publisher Full Text
Muse SV, Gaut BS: Comparing patterns of nucleotide substitution rates among chloroplast loci using the relative ratio test.
Genetics 1997, 146:393-399. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Bromham L, Penny D: The moderm molecular clock.
Nature Reviews Genetics 2003, 4:216-224. PubMed Abstract | Publisher Full Text
Laird CD, McConaughy BL, McCarthy BJ: Rate of Fixation of Nucleotide Substitutions in Evolution.
Nature 1969, 224:149-154. PubMed Abstract | Publisher Full Text
Wu ML, Li WH: Evidence for higher rates of nucleotide substitution in rodents than in man.
Proceedings of the National Academy of Sciences USA 1985, 82:1741-1745. Publisher Full Text
Ohta T: An examination of the generation-time effect on molecular evolution.
Proceedings of the National Academy of Sciences USA 1993, 90:10676-10680. Publisher Full Text
Ohta T: Synonymous and nonsynonymous substitutions in mammalian genes and the nearly neutral theory.
Journal of Molecular Evolution 1995, 40:56-63. PubMed Abstract | Publisher Full Text
Li W, Ellsworth DL, Krushkal J, Chang B, Hewett-Emmett D: Rates of nucleotide substitution in primates and rodents and the generation-time effect hypothesis.
Molecular Phylogenetics and Evolution 1996, 5:182-187. Publisher Full Text
Pesole G, Gissi C, De Chirico A: Nucleotide substitution rate of mammalian mitochondrial genomes.
Journal of Molecular Evolution 1999, 48:427-434. PubMed Abstract | Publisher Full Text
Gissi C, Reyes A, Pesole G, Saccone C: Lineage-Specific Evolutionary Rate in Mammalian mtDNA.
Mol Biol Evol 2000, 17(7):1022-1031. PubMed Abstract | Publisher Full Text
Kumar S, Subramanian S: Mutation rates in mammalian genomes.
Proceedings of the National Academy of Sciences 2002, 99:803-808. Publisher Full Text
Hanada K, Suzuki Y, Gojobori T: A large variation in the rates of synonymous substitution for RNA viruses and its relationship to a diversity of viral infection and transmission modes.
Molecular Biology Evolution 2004, 21(6):1074-1080. Publisher Full Text
Maughan H: Rates of molecular evolution in bacteria are relatively constant despite spore dormancy.
Evolution 2007, 61(2):280-288. PubMed Abstract | Publisher Full Text
Gaut BS: Molecular clocks and nucleotide substitution rates in higher plants.
Gaut BS, Muse SV, Clark WD, Clegg MT: Relative rates of nucleotide substitution at the rbcL locus of the monocotydelonous plants.
Journal of Molecular Evolution 1992, 35:292-303. PubMed Abstract | Publisher Full Text
Eyre-Walker A, Gaut BS: Correlated rates of synonymous site evolution across plant genomes.
Mol Biol Evol 1997, 14(4):455-460. PubMed Abstract | Publisher Full Text
Gaut BS, Clark LG, Wendel JF, Muse SV: Comparisons of the molecular evolutionary/process at rbcL and ndhF in the grass family (Poaceae).
Mol Biol Evol 1997, 14(7):769-777. PubMed Abstract | Publisher Full Text
Laroche J, Bousquet J: Evolution of the mitochondrial rps3 intron in perennial and annual angiosperms and homology to nad5 intron 1.
Mol Biol Evol 1999, 16(4):441-452. PubMed Abstract | Publisher Full Text
Andreasen K, Baldwin BG: Unequal evolutionary rates between annual and perennial lineages of checker mallows (Sidalcea, Malvaceae): Evidence from 18S–26S rDNA internal and external transcribed spacers.
Mol Biol Evol 2001, 18(6):936-944. PubMed Abstract | Publisher Full Text
Whittle G, Johnston MO: Broad-scale analysis contradicts the theory that generation time affects molecular evolutionary rates in plants.
Journal of Molecular Evolution 2003, 56:223-233. PubMed Abstract | Publisher Full Text
Wolfe KH, Li W-H, Sharp P: Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast, and nuclear DNAs.
Proceedings of the National Academy of Sciences USA 1987, 84:9054-9058. Publisher Full Text
Palmer JD, Herbon L: Plant mitochondrial DNA evolves rapidly in structure, but slowly in sequence.
Journal of Molecular Evolution 1988, 28:87-97. PubMed Abstract | Publisher Full Text
Muse SV: Examining rates and patterns of nucleotide substitution in plants.
Plant Molecular Biology 2000, 42:25-43. PubMed Abstract | Publisher Full Text
Palmer JD, Adams KL, Cho Y, Parkinson CL, Qiu Y-L, Song K: Dynamic evolution of plant mitochondrial genomes: mobile genes and introns, and highly variable mutation rates.
Proceedings of the National Academy of Sciences USA 2000, 97:6960-6966. Publisher Full Text
Small RL, Cronn RC, Wendel JF: L.A.S. Johnson Review No. 2. Use of nuclear genes for phylogeny reconstruction in plants.
Australian Systematic Botany 2004, 17:145-170. Publisher Full Text
Alvarez I, Wendel JF: Ribosomal ITS sequences and plant phylogenetic inference.
Molecular Phylogenetics and Evolution 2003, 29:417-434. Publisher Full Text
Nei M, Rooney A: Concerted and birth-and-death evolution of multigene families.
Annual Review of Genetics 2005, 22:121-152. Publisher Full Text
Xiang Q, Zhang W, Ricklefs R, Qian H, Chen Z, Wen J, Li JC: Regional differences in rates of plant speciation and molecular evolution: a comparison between Eastern Asia and Eastern North America.
Evolution 2004, 58:2175-2184. PubMed Abstract
Archibald J, Mort M, Crawford D, Kelly J: Life history affects the evolution of reproductive isolation among species of Coreopsis (Asteraceae).
Evolution 2005, 59:2362-2369. PubMed Abstract
Brown JM, Pauly GB: Increased rates of molecular evolution in an equatorial plant clade: an effect of environment or phylogenetic nonindependence?
Evolution 2005, 59:238-242. PubMed Abstract
Kay KM, Whittall JB, Hodges SA: A survey of nuclear ribosomal internal transcribed spacer substitution rates across angiosperms: an approximate molecular clock with life history effects.
BMC Evolutionary Biology 2006., 6 PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Tajima F: Simple methods for testing the molecular evolutionary clock hypothesis.
Genetics 1993, 135:599-607. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Rambaut A, Bromham L: Estimating divergence dates from molecular sequences.
Mol Biol Evol 1998, 15(4):442-448. PubMed Abstract | Publisher Full Text
Bromham L, Penny D, Rambaut A, Hendy MD: The power of relative rates tests depends on the data.
Journal of Molecular Evolution 2000, 50:296-301. PubMed Abstract | Publisher Full Text
Kimura M: A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences.
Journal of Molecular Evolution 1980, 16:111-120. PubMed Abstract | Publisher Full Text
Laroche J, Li P, Maggia L, Bousquet J: Molecular evolution of angiosperm mitochondrial introns and exons.
Proceedings of the National Academy of Sciences USA 1997, 94(11):5722-5727. Publisher Full Text
Wright SI, Lauga B, Charlesworth D: Rates and patterns of molecular evolution in inbred and outbred Arabidopsis.
Mol Biol Evol 2002, 19(9):1407-1420. PubMed Abstract | Publisher Full Text
Baldwin BG, Sanderson MJ, Porter JM, Wojciechowski MF, Campbell CS, Donoghue MJ: The ITS region of nuclear ribosomal DNA: A valuable source of evidence on angiosperm phylogeny.
Annals of the Missouri Botanical Garden 1995, 82:247-277. Publisher Full Text
Buckler ES, Ippolito A, Holtsford TP: The Evolution of Ribosomal DNA: Divergent Paralogues and Phylogenetic Implications.
Genetics 1997, 145:821-832. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Hughes C, Bailey C, Harris S: Divergent and reticulate species relationships in Leucaena (Fabaceae) inferred from multiple data sources: insights into polyploid origins and nrDNA polymorphism.
Andreasen K, Baldwin BG: Nuclear ribosomal DNA sequence polymorphism and hybridization in checker mallows (Sidalcea, Malvaceae).
Molecular Phylogenetics and Evolution 2003, 29:563-581. Publisher Full Text
Koch MA, Dobe C, Mitchell-Olds T: Multiple Hybrid Formation in Natural Populations: Concerted Evolution of the Internal Transcribed Spacer of Nuclear Ribosomal DNA (ITS) in North American Arabis divaricarpa (Brassicaceae).
Molecular Biology Evolution 2003, 20:338-350. Publisher Full Text
Nieto Feliner G, Gutiérrez Larena B, Fuertes Aguilar J: Fine-scale geographical structure, intra-individual polymorphism and recombination in nuclear internal transcribed spacers in Armenia (Plumbaginaceae).
Annals of Botany 2004, 93:189-200. PubMed Abstract | Publisher Full Text
Razafimandimbison SG, EA Kellogg, Bremer B: Recent origin and phylogenetic utility of divergent ITS putative pseudogenes: A case study from Naucleeae (Rubiaceae).
Systematic Biology 2004, 53:177-192. PubMed Abstract | Publisher Full Text
Okuyama Y, Fujii N, Wakabayashi M, Kawakita A, Ito M, Watanabe M, Murakami N, Kato M: Nonuniform concerted evolution and chloroplast capture: Heterogeneity of observed introgression patterns in three molecular data partition phylogenies of Asian mitella (Saxifragaceae).
Molecular Biology Evolution 2005, 22:285-296. Publisher Full Text
Won H, Renner SS: The internal transcribed spacer of nuclear ribosomal DNA in the gymnosperm Gnetum.
Molecular Phylogenetics and Evolution 2005, 36:581-597. Publisher Full Text
Noyes RD: Intraspecific nuclear ribosomal DNA divergence and reticulation in sexual diploid Erigeron strigosus (Asteraceae).
American Journal of Botany 2006, 93:470-479. Publisher Full Text
Gillooly J, Allen A, West G, Brown J: The rate of DNA evolution: Effects of body size and temperature on the molecular clock.
Proceedings of the National Academy of Sciences USA 2005, 102:140-145. Publisher Full Text
Gillooly J, Brown J, West G, Savage V, Charnov E: Effects of size and temperature on metabolic rate.
Science 2001, 293:2248-2251. PubMed Abstract | Publisher Full Text
Davies T, Savolainen V, Chase M, Moat J, Barraclough TG: Environmental energy and evolutionary rates in flowering plants.
Proc R Soc Lond B 2004, 271:2195-2200. Publisher Full Text
Charlesworth D, Wright S: Breeding systems and genome evolution.
Curr Opin Genet Dev 2001, 11(6):685-690. PubMed Abstract | Publisher Full Text
Hershkovitz MA, Zimmer EA, Hahn WJ: Ribosomal DNA and angiosperm systematics.
In Molecular Systematics and Plant Evolution Edited by R. B. a. Hollingsworth RGP. 1999, 268-326.
Bailey CD, Carr T, Harris S, Hughes C: Characterization of angiosperm nrDNA polymorphim, paralogy, and pseudogenes.
Molecular Phylogenetics and Evolution 2003, 29:435-455. Publisher Full Text
Posada D, Crandall KA: Modeltest: testing the model of DNA substitution.
Bioinformatics 1998, 14:817-818. PubMed Abstract | Publisher Full Text
Swofford DL: PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4. Sinauer Associates, Sunderland Mass; 2002. PubMed Abstract | Publisher Full Text
Kosakovsky Pond SL, Frost SDW, Muse SV: Hypothesis testing using phylogenetics (HyPhy).
Bioinformatics 2005, 21:676-679.
5.PubMed Abstract | Publisher Full Text
Muse SV, Weir BS: Testing for equality of evolutionary rates.
Genetics 1992, 132:269-276. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Sarich VM, Wilson AC: Immunological time scale for hominid evolution.
Science 1967, 158:1200-1203. PubMed Abstract | Publisher Full Text
Nei M, Kumar S: Molecular Evolution and Phylogenetic. Oxford University Press, Oxford; 2000.
Hamilton MB, Braverman JM, Soria-Hernanz DF: Patterns and relative rates of nucleotide and insertion/deletion evolution at six chloroplast intergenic regions in New World species of Lecythidaceae.
Molecular Biology Evolution 2003, 20:1710-1721. Publisher Full Text
Rambaut A, Grassly NC: Seq-Gen: An application for the Monte Carlo simulation of DNA sequence evolution along phylogenetic trees.
Comput Appl Biosci 1997, 13:235-238. PubMed Abstract
Bell CD, Patterson RW: Molecular phylogeny and biogeography of Linanthus (Polemoniaceae).
American Journal of Botany 2000, 87:1857-1870. PubMed Abstract | Publisher Full Text


text:
In many ways Lucy Li is a typical 10-year-old. Li likes food — Chinese food, french fries and ice cream are favorites. Her music preferences run to rock and hip-hop. Her favorite song is "It's Time" by Imagine Dragons. She likes to read. She's an A student who enjoys history.
WHEN: Aug. 5-11
WHERE: Country Club of Charleston
WHAT: An international field of 156 golfers play 36 holes of stroke play Monday and Tuesday, with the top 64 advancing to the match play portion of the tournament.
Tee times: Monday and Tuesday begin at 7:30 a.m. and continue until 2:30 p.m.
ADMISSION: Free
PARKING: No parking at the Country Club. Public parking is available at the vacant space located at Folly Road and Albemarle Road, with shuttles running every 15 minutes from 5:15 a.m. until 9 p.m.
TV: The Golf Channel (3-5 p.m. Wednesday; 4-6 p.m. Thursday-Sunday)
On Monday, Li will earn a place in golf history when the youngster from Redwood, Calif., hits her opening tee shot at 7:40 a.m. in the U.S. Women's Amateur Championship at the Country Club of Charleston. At 10 years, 10 months and 4 days, she will be the youngest participant ever in the event that dates to 1895. The previous youngest golfer was Latanna Stone, who competed in the 2012 Women's Amateur at the age of 10 years, 11 months and 2 days.
This isn't Li's first historic step into the U.S. Golf Association history book. In June, Li (10 years, 8 months, 16 days) qualified for the Women's Amateur Public Links Championship and made it into the match play portion of the event, breaking a record previously held by Michelle Wie (10 years, 8 months, 23 days).
Li said she didn't realize she was making history "until I saw the articles." But there was no free ride into the Public Links or the Women's Amateur.
Li earned her way into this week's tournament with birdies on two of her last three holes en route to a 75 in a qualifying event in her home state.
There's an athletic bounce in her step as the 4-11, 80-pound golf prodigy walks down the fairways, accompanied by her mother and aunt. She swings her arms from side to side, snapping her fingers because "I just like snapping my fingers." She averages about 225 yards off the tee, good for a 10-year-old but yards short of her older competition. To make up for the lack of distance, she owns an outstanding short game.
For the past 2½ years, Li has been under the tutelage of noted golf instructor Jim McLean, who has golf schools in Florida, Texas and Arizona.
"She is still learning to play. She makes great contact almost every time and she has improved her accuracy," McLean said. "It's fun to watch her. I'm very proud of Lucy, the person she is, how mature, how smart and how nice she is. She's a very nice girl."
Li's parents sought out numerous activities for her as she grew up. She took diving lessons at nearby Stanford and dove from the 10-meter platform at age 4 even though she couldn't swim. She also tried gymnastics and music. Li was 7 when her parents discovered her aptitude for golf. Li's brother Luke, who will be a freshman at Princeton this fall, was the captain of his high school golf team. While the family was watching him on the practice range, someone asked Lucy to take a swing.
McLean recalled his first meeting with Li's parents, who asked him to become her instructor. McLean, who works with numerous PGA and LPGA professionals, told Li's parents that he could put her with talented teachers, but Li's parents were insistent that they wanted McLean. Reluctantly, he agreed to work with her once every couple of weeks. McLean soon discovered that Li had talent, drive and desire, traits that have allowed her to make giant strides at a young age.
Li said working with McLean has been fun, an attitude she tries to carry with her to the golf course.
"He's very funny," Li said of McLean. "He wants everything to be natural."
Through McLean, Li has made the acquaintance of Rory McIlroy (her favorite golfer "because he's so nice"), Phil Mickelson, Dustin Johnson, Justin Rose, Jason Day, Keegan Bradley and others.
"This summer has been very exciting. There's been a lot of traveling and sightseeing," Li said. "My goal is to try to play as well as I can. The golf course is pretty long, especially the par-4s. Other than that, it's pretty straightforward. The greens can be tricky."
McLean said he would love to see Li make history but has a previous obligation.
"I try to make tournaments a fun experience for her," McLean said. "Obviously, at 10 years old to compete in the U.S. Women's is a huge win. There's no downside whatsoever."
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subset: commoncrawl


text:
Below is the poem entitled heard which was written by poet
Jonas. Please feel free to comment on this poem. However, please remember, PoetrySoup is a place of encouragement and growth.
Read Poems by
Bart Jonas
I have heard the light
Her radiant but sad muse
Wavering across the spectrum LIFE
These ears are tuned to the melody
that continues to play throughout our
passages in this stratosphere Today
You might think Me a strange
to remember every frequency that
keeps ringing across my melancholy face
as Myself,
Sensitive to the words
Verses that are unique and distinctively different
From any sound that reverberates around this Mighty World
Does it not surprise that my Ears are the same as these eyes
Made for listening as well for Peering
I am not such a predator that uses it for leering
For attuning,
into & outer space of this Earth,
Her Vineyard of Wine that keeps expanding all of her taste
Not such a Waste,mind you
If you heard what I have..
You Would Listen with Love as well
I can Tell!!
A comment has not been posted for this poem. Encourage a poet by being the first to comment.


text:
Details about Body Lift
A well-known form of cosmetic surgery to correct any excess skin which may be loose or sagging and improve the shape and tone of the underlying tissue. A body lift is mostly performed after massive weight loss has left the excess skin lose and flabby.
The incisions are based on the area of the body, which is being treated, patient preference can also come into call here.
Many of the most common body lift procedures include other surgeries such as abdominoplasty, thigh lift, arm lift and a breast lift.
Video about the procedure Body Lift
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text:
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Full Disclosure: These are my personal opinions. No company is silly enough to claim them. I am a mobility and SMAC analyst, consultant and writer. I work with and have worked with many of the companies mentioned in my articles.
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@ThingsExpo Stories
SYS-CON Events announced today that HPM Networks will exhibit at the 17th International Cloud Expo®, which will take place on November 3–5, 2015, at the Santa Clara Convention Center in Santa Clara, CA. For 20 years, HPM Networks has been integrating technology solutions that solve complex business challenges. HPM Networks has designed solutions for both SMB and enterprise customers throughout the San Francisco Bay Area.
Aug. 3, 2015 06:45 PM EDT Reads: 561
For IoT to grow as quickly as analyst firms' project, a lot is going to fall on developers to quickly bring applications to market. But the lack of a standard development platform threatens to slow growth and make application development more time consuming and costly, much like we've seen in the mobile space. In his session at @ThingsExpo, Mike Weiner, Product Manager of the Omega DevCloud with KORE Telematics Inc., discussed the evolving requirements for developers as IoT matures and conducted a live demonstration of how quickly application development can happen when the need to comply wit...
Aug. 2, 2015 11:15 AM EDT Reads: 374
The Internet of Everything (IoE) brings together people, process, data and things to make networked connections more relevant and valuable than ever before – transforming information into knowledge and knowledge into wisdom. IoE creates new capabilities, richer experiences, and unprecedented opportunities to improve business and government operations, decision making and mission support capabilities.
Aug. 1, 2015 10:00 AM EDT Reads: 351
Explosive growth in connected devices. Enormous amounts of data for collection and analysis. Critical use of data for split-second decision making and actionable information. All three are factors in making the Internet of Things a reality. Yet, any one factor would have an IT organization pondering its infrastructure strategy. How should your organization enhance its IT framework to enable an Internet of Things implementation? In his session at @ThingsExpo, James Kirkland, Red Hat's Chief Architect for the Internet of Things and Intelligent Systems, described how to revolutionize your archit...
Jul. 30, 2015 07:30 PM EDT Reads: 1,438
MuleSoft has announced the findings of its 2015 Connectivity Benchmark Report on the adoption and business impact of APIs. The findings suggest traditional businesses are quickly evolving into "composable enterprises" built out of hundreds of connected software services, applications and devices. Most are embracing the Internet of Things (IoT) and microservices technologies like Docker. A majority are integrating wearables, like smart watches, and more than half plan to generate revenue with APIs within the next year.
Jul. 30, 2015 02:30 PM EDT Reads: 164
Growth hacking is common for startups to make unheard-of progress in building their business. Career Hacks can help Geek Girls and those who support them (yes, that's you too, Dad!) to excel in this typically male-dominated world. Get ready to learn the facts: Is there a bias against women in the tech / developer communities? Why are women 50% of the workforce, but hold only 24% of the STEM or IT positions? Some beginnings of what to do about it! In her Opening Keynote at 16th Cloud Expo, Sandy Carter, IBM General Manager Cloud Ecosystem and Developers, and a Social Business Evangelist, d...
Jul. 30, 2015 12:00 PM EDT Reads: 2,096
In his keynote at 16th Cloud Expo, Rodney Rogers, CEO of Virtustream, discussed the evolution of the company from inception to its recent acquisition by EMC – including personal insights, lessons learned (and some WTF moments) along the way. Learn how Virtustream's unique approach of combining the economics and elasticity of the consumer cloud model with proper performance, application automation and security into a platform became a breakout success with enterprise customers and a natural fit for the EMC Federation.
Jul. 30, 2015 09:00 AM EDT Reads: 2,183
The Internet of Things is not only adding billions of sensors and billions of terabytes to the Internet. It is also forcing a fundamental change in the way we envision Information Technology. For the first time, more data is being created by devices at the edge of the Internet rather than from centralized systems. What does this mean for today's IT professional? In this Power Panel at @ThingsExpo, moderated by Conference Chair Roger Strukhoff, panelists addressed this very serious issue of profound change in the industry.
Jul. 29, 2015 03:00 PM EDT Reads: 1,311
Discussions about cloud computing are evolving into discussions about enterprise IT in general. As enterprises increasingly migrate toward their own unique clouds, new issues such as the use of containers and microservices emerge to keep things interesting. In this Power Panel at 16th Cloud Expo, moderated by Conference Chair Roger Strukhoff, panelists addressed the state of cloud computing today, and what enterprise IT professionals need to know about how the latest topics and trends affect their organization.
Jul. 29, 2015 02:00 PM EDT Reads: 1,220
It is one thing to build single industrial IoT applications, but what will it take to build the Smart Cities and truly society-changing applications of the future? The technology won't be the problem, it will be the number of parties that need to work together and be aligned in their motivation to succeed. In his session at @ThingsExpo, Jason Mondanaro, Director, Product Management at Metanga, discussed how you can plan to cooperate, partner, and form lasting all-star teams to change the world and it starts with business models and monetization strategies.
Jul. 28, 2015 04:30 PM EDT Reads: 1,783
Converging digital disruptions is creating a major sea change - Cisco calls this the Internet of Everything (IoE). IoE is the network connection of People, Process, Data and Things, fueled by Cloud, Mobile, Social, Analytics and Security, and it represents a $19Trillion value-at-stake over the next 10 years. In her keynote at @ThingsExpo, Manjula Talreja, VP of Cisco Consulting Services, discussed IoE and the enormous opportunities it provides to public and private firms alike. She will share what businesses must do to thrive in the IoE economy, citing examples from several industry sectors.
Jul. 28, 2015 11:00 AM EDT Reads: 2,057
There will be 150 billion connected devices by 2020. New digital businesses have already disrupted value chains across every industry. APIs are at the center of the digital business. You need to understand what assets you have that can be exposed digitally, what their digital value chain is, and how to create an effective business model around that value chain to compete in this economy. No enterprise can be complacent and not engage in the digital economy. Learn how to be the disruptor and not the disruptee.
Jul. 27, 2015 10:00 AM EDT Reads: 2,057
Akana has released Envision, an enhanced API analytics platform that helps enterprises mine critical insights across their digital eco-systems, understand their customers and partners and offer value-added personalized services. "In today's digital economy, data-driven insights are proving to be a key differentiator for businesses. Understanding the data that is being tunneled through their APIs and how it can be used to optimize their business and operations is of paramount importance," said Alistair Farquharson, CTO of Akana.
Jul. 27, 2015 09:00 AM EDT Reads: 340
Business as usual for IT is evolving into a "Make or Buy" decision on a service-by-service conversation with input from the LOBs. How does your organization move forward with cloud? In his general session at 16th Cloud Expo, Paul Maravei, Regional Sales Manager, Hybrid Cloud and Managed Services at Cisco, discusses how Cisco and its partners offer a market-leading portfolio and ecosystem of cloud infrastructure and application services that allow you to uniquely and securely combine cloud business applications and services across multiple cloud delivery models.
Jul. 27, 2015 08:00 AM EDT Reads: 1,918
The enterprise market will drive IoT device adoption over the next five years. In his session at @ThingsExpo, John Greenough, an analyst at BI Intelligence, division of Business Insider, analyzed how companies will adopt IoT products and the associated cost of adopting those products. John Greenough is the lead analyst covering the Internet of Things for BI Intelligence- Business Insider's paid research service. Numerous IoT companies have cited his analysis of the IoT. Prior to joining BI Intelligence, he worked analyzing bank technology for Corporate Insight and The Clearing House Payment...
Jul. 26, 2015 09:00 PM EDT Reads: 1,600
"Optimal Design is a technology integration and product development firm that specializes in connecting devices to the cloud," stated Joe Wascow, Co-Founder & CMO of Optimal Design, in this SYS-CON.tv interview at @ThingsExpo, held June 9-11, 2015, at the Javits Center in New York City.
Jul. 25, 2015 02:00 PM EDT Reads: 408
SYS-CON Events announced today that CommVault has been named "Bronze Sponsor" of SYS-CON's 17th International Cloud Expo®, which will take place on November 3–5, 2015, at the Santa Clara Convention Center in Santa Clara, CA. A singular vision – a belief in a better way to address current and future data management needs – guides CommVault in the development of Singular Information Management® solutions for high-performance data protection, universal availability and simplified management of data on complex storage networks. CommVault's exclusive single-platform architecture gives companies unp...
Jul. 25, 2015 01:00 PM EDT Reads: 1,984
Electric Cloud and Arynga have announced a product integration partnership that will bring Continuous Delivery solutions to the automotive Internet-of-Things (IoT) market. The joint solution will help automotive manufacturers, OEMs and system integrators adopt DevOps automation and Continuous Delivery practices that reduce software build and release cycle times within the complex and specific parameters of embedded and IoT software systems.
Jul. 25, 2015 12:15 PM EDT Reads: 490
"ciqada is a combined platform of hardware modules and server products that lets people take their existing devices or new devices and lets them be accessible over the Internet for their users," noted Geoff Engelstein of ciqada, a division of Mars International, in this SYS-CON.tv interview at @ThingsExpo, held June 9-11, 2015, at the Javits Center in New York City.
Jul. 25, 2015 12:00 PM EDT Reads: 1,568
Internet of Things is moving from being a hype to a reality. Experts estimate that internet connected cars will grow to 152 million, while over 100 million internet connected wireless light bulbs and lamps will be operational by 2020. These and many other intriguing statistics highlight the importance of Internet powered devices and how market penetration is going to multiply many times over in the next few years.
Jul. 25, 2015 09:00 AM EDT Reads: 1,510


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Well, sure, but what about in Cataclysm? With all those stat changes you guys talked about at BlizzCon, there has to be some surprises on the way when we see our first glimpse of Cataclysm gear."They're just numbers. We don't think it starts getting scary until the human mind literally has trouble processing the digits. If your new staff has 152,420 spell power, well, yeah that's a mouthful. But if your new staff has 4000 spell power, the only thing that can really be freaking you out is that 4000 is just a really big number compared to the 100 spell power you might be used to. Automobiles used to cost hundreds of dollars instead of tens of thousands of dollars too.... :)"
Not too surprising, really; with the removal of spell power, we're gonna see a whole bunch of Int on items, for example, so the first time we see that Sunken City of Vashj'ir green staff, we're probably gonna have a bit of an "AaaaAAAH!" moment."Gear inflation is just part of MMOs, or at least the kind of MMO that we make. Players are going to freak when they see the Cataclysm gear (because believe me, we did), but it's also exciting too. :)"
There's also a bit of insight into exactly what happened with Wrath's gear scaling. Blame Ulduar.
Editor's Note: Car? What about a Yacht?
Patch 3.3 is the last major patch of Wrath of the Lich King. With the new Icecrown Citadel 5-man dungeons and 10/25-man raid arriving soon, patch 3.3 will deal the final blow to Arthas. WoW.com's Guide to Patch 3.3 will keep you updated with all the latest patch news.


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By Army Staff Sgt. Garrett Ralston3rd Armored Cavalry Regiment
BABIL PROVINCE, Iraq, Nov. 18, 2010 A Humvee sits outside the laundry facility on Contingency Operating Site Kalsu here as other vehicles pass by.
As the truck's motor idles, a young soldier sitting in the rear seat peruses a magazine. He is alone as the vehicle's other occupants, including its driver, are retrieving their laundry.
"Where's the driver for this vehicle?" said a voice from outside the truck.
"They're all inside getting their laundry, Pete," answered the soldier inside the truck.
Peter Higgins then sternly tells the soldier, "Someone needs to be in the driver's seat if the truck is running. You guys know the rules."
As Higgins walks on, the truck's driver appears.
"Who was that?" asked the driver.
"Just 'Safety Pete' getting on me about the truck running with nobody in the driver's seat," said the soldier.
The soldiers correct the problem and get moving for the day's mission. They don't question Higgins, they say, because he's simply doing his job. That's why they call him "Safety Pete."
Higgins, a safety and occupational health specialist, has for the past three years worked to ensure that 3rd Armored Cavalry Regiment soldiers operate their equipment in a safe manner.
Higgins said he retired from active-Army service in 1999 as a first sergeant and then went to work as a teacher in Killeen, Texas. He said he didn't care much for teaching, so he later went to work as a parole officer for the state of Texas.
"Both were good jobs, but I found myself missing the Army and especially working with soldiers," Higgins said.
After 9/11, Higgins decided he wanted to work for the military as a civilian. Shortly thereafter, the previous 3rd ACR commander contacted him and Higgins went to work for the regiment as its safety officer.
"I spend a lot of my time walking or driving around looking for hazards and unsafe actions or practices the soldiers may be involved in," Higgins said. "When I see something, I typically grab another soldier and let them correct the problem so that others don't make the same mistakes. It comes down to taking care of the soldier, period."
Higgins recently detected a problem with the fire suppression system mounted on a series of trucks in the regiment's inventory. A missed step in the system's installation, coupled with unclear directions for inspection, posed a serious danger to the soldiers.
"I never realized when I reported it how far up it would actually go," he said. "The issue has gone all the way to the Department of Defense, and corrections are now being made to the vehicle's manual."
Higgins said he endeavors "to ensure the safety of the soldiers who are out here doing the missions every day."
"Accidents will happen," he acknowledged, "but they can be prevented if the right information is passed along."
Article is closed to new comments.
The opinions expressed in the following comments do not necessarily reflect those of the U.S. Department of Defense.
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text: I didn't say she was the best player... No, seriously. She took a blow to the head by her own player when coming out to gather a through pass, which you will see in the highlights because the collision led to the 4-4 equaliser, and she looked injured after that, lying on the ground for a while and pressing an ice bag to her cheek during the break before extra time. Yet she went on to play the full game, fearlessly charging out of the goal to gather through passes on more than one occasion, and she made some good saves throughout the match. At one point she made a great reflex save that turned a shot I was sure would go in out for a corner. On a different note: Germany 0, North Korea 1 HT. Great curling strike into the top corner.


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This tour focuses on the Disneyland known personally by Walt Disney. As you walk around the park your tour guide will share with you the lesser known details of his plans and hopes for various parts of the park. The guide will also share stories from the park's early days. While the tour does not spend much time showing you the behind-the-scenes working of the park highlights do include a visit to the lobby of Club 33 and some extra time with The Enchanted Tiki Room seeing pieces up close.
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