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Keywords:

  • Environment;
  • molecular evolutionary rate;
  • plants;
  • pollen : ovule ratio;
  • seed bank persistence;
  • 18S ITS

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supplementary material
  9. Supporting Information

One of the main goals of molecular evolutionary biology is to determine the factors that influence the evolutionary rate of selectively neutral DNA, but much remains unknown, especially for plants. Key factors that could alter the mutation rate include environmental tolerances (because they reflect a plants vulnerability to changes in habitat), the pollen : ovule ratio (as it is associated with the number of mitotic divisions) and seed longevity (because this influences the number of generations per unit time in plants). This is the first study to demonstrate that seed bank persistence and drought tolerance are positively associated with molecular evolutionary rates in plants and that pollen : ovule ratio, shade tolerance and salinity tolerance have no detectable relationship. The implications of the findings to our understanding of the impact of environmental agents, the number of cell divisions and cell aging on neutral DNA sequence evolution are discussed.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supplementary material
  9. Supporting Information

Data on the influence of the environment on the evolutionary rate of selectively neutral DNA, including introns, intergenic spacers and silent sites of genes, has been very limited to date (e.g. Bleiweiss, 1998; Hebert et al., 2002), especially in plants. Given that the rate of molecular evolutionary rate of selectively neutral DNA (henceforth referred to as molecular evolutionary rates) equals the point mutation rate (Kimura, 1983), the environment could have a significant impact on DNA sequence evolution. First, the environment might directly induce mutations (Friedberg et al., 1995; Britt, 1999). Second, it might give rise to long-term physiological/genetic level changes that impact either the point mutation rate per cell division (e.g. efficiency of replication or repair machinery) or the number of cell divisions per generation. The lack of data on the factors that affect molecular evolutionary rates is surprising given that, due to their sessile nature and lack of sequestered germ-line, plants are particularly vulnerable to their environment and thus to incurring environmentally related mutations (Whitham & Slobodchikoff, 1981; Gill & Halverson, 1984; Sutherland & Watkinson, 1986; Klekowski, 1988). Thus, the environment could play a key role in molecular evolutionary variation in plants and warrants further investigation.

A primary challenge in assessing the relationship between environmental factors and molecular evolutionary rates is obtaining reliable information about taxon-specific habitat characteristics. One might be interested in examining UV, for example, as it has been shown to be mutagenic (Friedberg et al., 1995; Britt, 1999) and thus could influence molecular evolutionary rates. Determining the historical UV exposure for a wide range of taxa is a formidable task, however and thus other, fewer complexes, approaches need to be considered. One plausible method is to compare molecular evolutionary rates among plants with different levels of shade tolerance. Shade tolerance is an effective gauge for UV exposure as taxa with higher shade tolerance are likely to have a history of lower exposure to UV than shade intolerant taxa. Similarly, tolerance information regarding salinity (i.e. salinity tolerance) and low-water availability (i.e. drought tolerance), each of which is believed to detrimentally alter the DNA (Tesi et al., 1994; Lerner et al., 1994; Albinsky et al., 1999; Hammermann et al., 2000; Wang & Zhang, 2001; Tuteja et al., 2001; Hebert et al., 2002; Hall et al., 2003), can be used to assess whether these environmental parameters influence molecular evolutionary rates in plants.

Given that the molecular evolutionary rate could be affected either by a change in the mutation rate per cell division or by a change in the number of cell divisions per generation, an important issue to consider when assessing factors underlying evolutionary rates is the role of replication errors (i.e. cell division rates). Evidence from animals has lead to the suggestion that many base substitution mutations are replication errors occurring during the synthesis-stage of the cell cycle. This opinion has largely been based on the findings that the molecular evolutionary rate tends to be higher for DNA carried by the male than the female germ-line in humans and numerous other taxa where the male undergoes more cell divisions (Shimmin et al., 1993; Chang et al., 1996; Ellegren & Fridolfsson, 1997; Makova & Li, 2002). In addition, taxa with shorter generation times have been shown to have higher molecular evolutionary rates, a trend consistent with the differences in the number of germ-line DNA replications per unit time (i.e. more cell divisions in taxa with shorter generation times, Wu & Li, 1985; Ohta, 1993). In plants, the role of replication errors is largely unknown. Although male-biased evolutionary rates have been detected in certain plant taxa (Whittle & Johnston, 2002) and generation-time effects have been observed in some but not all plant groups (e.g. Gaut et al., 1996; Laroche & Bousquet, 1999; Andreasen & Baldwin, 2001; Whittle & Johnston, 2003), it is not known whether replication errors underlie these trends. The main challenge in assessing the role of DNA replication on the molecular evolutionary rate in plants has been obtaining precise estimates of the number of cell divisions per generation (and thus per unit time). Approximate estimates, however, could provide valuable information. For example, one could approximate the number of cell divisions during sperm vs. egg production by calculating the ratio of the number of pollen to ovules per individual. A positive relationship between the pollen : ovule ratio and molecular evolutionary rates would suggest that many mutations are caused by replication errors. In terms of generation time, it is possible that the lack of an effect that has reported in some studies (Whittle & Johnston, 2003) is caused by the presence of seed banks, which act to increase generation time and reduce DNA replications (cell divisions) per unit time, potentially masking any influence of cell division rates on molecular evolutionary rates. Accordingly, an inverse relationship between seed bank persistence and molecular evolutionary rates would be consistent with the notion that most mutations are replication errors and with the generation time theory.

In order to assess the relationship between a particular factor and the molecular evolutionary rate, it is imperative to eliminate phylogenetic bias (Felsenstein, 1985). The use of phylogenetically independent comparisons of closely related species pairs with contrasting values for the feature of interest (e.g. shade tolerant vs. intolerant) provides an effective means to obtain a series of data points that can be utilized in statistical analysis (Bromham et al., 1996; Ackerly, 2000; Whittle & Johnston, 2003). The statistical test that best accounts for recent switches in the characteristics under study and for variation in the relatedness among taxon pairs is the sign test (Sokal & Rohlf, 1995; Ackerly, 2000; Whittle & Johnston, 2003). This approach has the advantages that it is easy to apply and is conservative.

The objective of the present study is to use phylogenetically independent comparisons combined with sign tests to assess whether the molecular evolutionary rates of the nuclear 18S internal transcribed spacer (ITS) region (i.e. ITS1 plus ITS2) in plants are associated with shade tolerance, salinity tolerance, drought tolerance, the pollen : ovule ratio, or the persistence of seed banks. The predictions are as follows. Based solely on their short-term impact on DNA, molecular evolutionary rates are predicted to be positively correlated with salinity and drought tolerance and inversely related to shade tolerance (increased tolerance, reduced UV exposure). Based on the assumption that there is an association between cell division and mutation rates, it is predicted that neutral molecular evolutionary rates will be positively correlated with the pollen : ovule ratio and inversely related to seed bank persistence.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supplementary material
  9. Supporting Information

A separate dataset was gathered and analysed for each factor under investigation including shade-tolerance, salinity tolerance, drought tolerance and the pollen : ovule ratio and seed bank persistence. Taxon pairs were chosen based on the following criteria: (1) the complete DNA sequences of 18S ITS region was available from Genbank, (2) the two taxa represent the same genus and (3) the two taxa had contrasting values for the factor of interest (based on the appropriate literature, see http://www.blackwellpublishing.com/products/journals/suppmat/JEB/JEB977/JEB977sm.htm). To ensure independence of pairs, no more than one taxon pair was chosen per genus. When more than two possible taxon pairs were identified within a genus, the two taxa with the greatest difference in trait value for the factor of interest were selected. Once the final group of taxon pairs was chosen, each taxon per pair was placed into one of two contrasting categories [i.e. shade tolerance (tolerant vs. intolerant), saline tolerance (tolerant vs. intolerant), drought tolerance (tolerant vs. intolerant), the pollen : ovule ratio (high vs. low) and seed bank persistence (transient vs. persistent), see Supplementary material]. It should be noted that the information for shade, saline and drought tolerance, gathered mostly from USDA NRCS (2004), was based on data collected from the scientific literature, grey literature, agency documents and from the knowledge of plant specialists (USDA NRCS, 2004). A reference taxon for each pair-wise comparison was chosen, wherever possible, from a closely related genus in the same family and used to measure branch lengths to each taxon of the contrasted pair from their common ancestor. Phylogenies in the literature were examined to ensure that the reference taxon is outside the clade containing the two compared taxa. Although there are multiple copies of the ITS region in many plant taxa, the paralogues are nearly uniform and thus this has little/no impact for analyses based on between species comparisons (Baldwin et al., 1995).

For each set of three taxa (the two compared taxa and the reference taxon), DNA sequences were aligned for the ITS region using Clustal X (Jeanmougin et al., 1998) and gaps were removed. The phylogenetic branch length (PBL) to each taxon following their divergence from the common ancestor (i.e. the number of nucleotide substitutions per nucleotide site) was inferred based on the maximum likelihood model described by Tamura & Nei (1993) with gamma variation and three rate classes using the software HYPHY (Muse & Pond, 2000). Branch lengths were also determined using Kimura's (1980) two-parameter substitution model, which yielded similar results (data not shown). A maximum likelihood relative rate test was also conducted using HYPHY for each taxon pair. Aligned sequences ranged from 294 to 470 bp.

Statistical analysis

A sign test was conducted with respect to each trait (shade, saline and drought tolerance, pollen : ovule ratio and seed bank persistence) at the ITS region. The main analysis thus comprises five separate sign tests. As a supplemental analysis, parallel sign tests were conducted for each of the five factors under study using only the most genetically divergent taxon pairs (sum of the PBLs to the common ancestor > 0.10) and using only the least divergent pairs (sum of PBL < 0.1). In order to assess whether the overall data affirms/refutes the null hypothesis that molecular evolutionary rates are independent of the five parameters, it was determined whether the proportion of sign tests having statistically significant P-values from the main analysis was greater than that expected by chance according to the Binomial distribution (i.e. whether the totality of the data is inconsistent with null hypothesis). In order to determine which specific traits are statistically significant after accounting for multiple comparisons with five traits would require an approach such as the Bonferroni correction. In this study, no single result in the main analysis remains statistically significant after the Bonferroni correction. This correction, however, is known to be highly conservative (Sokal & Rohlf, 1995). In addition, the sign test is conservative (Sokal & Rohlf, 1995; Ackerly, 2000), the approach of using phylogenetically independent contrasts is conservative (Ackerly, 2000) and the datasets are modest in size (using available data). Consequently, although the overall data indicate that one or more of the traits affect molecular evolutionary rates (Table 1), the results for the individual traits represent preliminary findings that must be confirmed as more data becomes available.

Table 1.  Sign tests based on phylogenetic branch lengths (PBL) at the 18S internal transcribed spacer region (ITS1 plus ITS2) for taxon pairs with different values for shade tolerance, saline tolerance, drought tolerance, the pollen : ovule ratio and seed bank persistence.
 Sign tests for the ITS region
Pairs with sum of PBL <0.1Pairs with sum of PBL > 0.1Across all pairs
  1. Bold indicates statistical significance (P < 0.05). Detailed data is provided in Supplementary material.

Shade tolerance
 Proportion of positive signs (tolerant vs. intolerant)6/164/510/21
 Proportion of negative signs (tolerant vs. intolerant)10/161/511/21
 P-value of sign test0.4540.3751
Saline tolerance
 Proportion of positive signs (tolerant vs. intolerant)9/163/712/23
 Proportion of negative signs (tolerant vs. intolerant)7/164/711/23
 P-value of sign test0.80311
Drought tolerance
 Proportion of positive signs (tolerant vs. intolerant)17/211/418/25
 Proportion of negative signs (tolerant vs. intolerant)4/213/47/25
 P-value of sign test0.0070.6250.043
Pollen : Ovule ratio
 Proportion of positive signs (high vs. low P : O ratio)4/93/77/16
 Proportion of negative signs (high vs. low P : O ratio)5/94/79/16
 P-value of sign test110.803
Seed bank persistence
 Proportion of positive signs (transient vs. persistent)1/92/63/15
 Proportion of negative signs (transient vs. persistent)8/94/612/15
 P-value of sign test0.0400.6880.035
The probability of obtaining statistical significance in two or more of five sign tests for the analysis across all taxon pairs according to the Binomial distribution0.023

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supplementary material
  9. Supporting Information

Shade and salt tolerance

No statistically significant differences were detected between shade tolerant and shade intolerant species or between salt tolerant and salt intolerant species, based on an analysis of 21 and 23 taxon pairs, respectively. The absence of a relationship between shade and saline tolerance and molecular evolutionary rates is inconsistent with the predictions for these parameters and is surprising given that UV and salt, respectively, can markedly alter the integrity of DNA and thus possibly lead to mutations (Friedberg et al., 1995). One explanation for this finding is that plants have the capability to induce physiological changes (adaptive or not) that protect the DNA and/or DNA processing machinery when exposed to these particular environments. Another possibility is that multiple historical switches in shade or in salt tolerances diluted any effect on molecular evolutionary rates. Further study will be needed to determine whether a relationship can be detected for a broader range of plant taxa and DNA regions.

Drought tolerance

The PBL was longer for the relatively drought tolerant than the intolerant taxon for 18 of 25 taxon pairs (P < 0.05, Table 1). This trend is further supported by the fact that the branch length was longer for a larger fraction of drought tolerant taxa (17 of 21, P < 0.01) when examining only the least divergent taxon pairs (sum of PBL < 0.1). The influence of drought tolerance is surprising as one might expect that drought and salinity tolerance would have the same impact on evolutionary rates given that reduced water availability leads to increased salinity levels both within the soil and the plant (Xu et al., 2002). It would also be expected that all three environmental factors, including shade, saline and drought tolerance would have a similar effect on evolutionary rates if a stress-mediated effect on the mutation rate played a major role in these plants. That an effect was only detected for drought tolerance suggests that the elevated evolutionary rate may be caused by factors specific to low-water conditions (e.g. drought-specific physiological changes that alter gene expression; altering the level of DNA damage, repair and/or accuracy of DNA replication). This is consistent with evidence showing that water stress is correlated to changes in DNA content of certain plant species such as Morus L. (Barathi et al., 2001) and to the level of DNA damage and the accuracy of DNA repair in species of Phragmites (Wang & Zhang, 2001). Another factor that could contribute to these results is that taxa in arid regions tend to produce seeds that are subjected to very dry conditions for extended time periods, a factor known to lead to high levels of DNA damage in the embryo (Osborne, 2000). Overall, the association between molecular evolutionary rates and drought tolerance is consistent with the predicted results based on the detrimental impact of low-water conditions on DNA. Alternatively, drought tolerant species might undergo more cell divisions per unit time.

The pollen : ovule ratio

Based on an assessment of a total of 16 taxon pairs, no statistically significant differences were detected in molecular evolutionary rates between taxa with higher and lower pollen : ovule ratios. These results suggest either that cell divisions during reproduction have no/little impact on mutation rates, that the effect is minimal relative to the number of mutations that occur prior to the formation of the flowers and/or that replication-independent events play a greater role in determining the mutation rate among plants. The results also suggest that male-biased evolutionary rates in certain plants (Whittle & Johnston, 2002) may not be related to differences in cell division number during flowering and thus, other factors are likely responsible. The assessment of the pollen : ovule ratio thus contradicts the predicted results.

Seed bank persistence

The PBL was longer for the taxon with a persistent rather than a transient seed bank in 12 of 15 comparisons (P < 0.05). When including only the least divergent pairs, the taxon with a persistent seed bank had a longer branch length for eight of nine taxon pairs (P < 0.05). The results indicate that more mutations occur per unit time in taxa whose seeds undergo longer periods before germination. Although the role of seed aging in plant molecular evolutionary biology has received little attention to date, there is substantial evidence to suggest it affects the heritable mutation rate. For example, studies of variation in AFLPs and other genetic markers indicate that naturally aged rye (Secale cereale) seeds contain higher levels of point mutations than unaged seeds and that these mutations are transmitted to the progeny for at least three generations (Chwedorzewska et al., 2002a,b). It has also been shown that plants produced from older seeds contain higher levels of heritable mutations in Crepis (Gerassimova, 1935) and in Zea mays (Peto, 1933). In addition, pollen abortions (Cartledge & Blakeslee, 1934) and recessive visible mutations have been reported to be substantially higher in Datura plants produced from older seeds (e.g. pollen abortion increases from one to more than eight percent over 10 years, Cartledge & Blakeslee, 1934). These mutations in older seeds are likely caused by the inaccurate repair of DNA damage, such as strand breaks, which accumulate in a time-dependent manner in seeds (Cheah & Osborne, 1978) and/or by impairment of the DNA replication machinery over time. It has been shown that older seeds have a lower RNA and protein content (suggesting substantial degradation, Begnami & Cortelazzo, 1996; Reuzeau & Cavalie, 1997), reduced ability to translate RNA (Reuzeau & Cavalie, 1997) and lowered activity of enzymes (Basavarajappa et al., 1991) such as RNA poly (A) polymerase (Grilli et al., 1995; Reuzeau & Cavalie, 1997), each of which might negatively influence the level and/or activity of molecules involved in DNA replication and repair. The presence of a higher molecular evolutionary rate in taxa with extended seed longevity in the present results combined with the tendency of many heritable mutations to occur during seed aging suggests that seed bank longevity could play an important role in increasing the rate of molecular evolution in plants.

These findings contradict the initial hypothesis that taxa with persistent seed banks would have a lower evolutionary rate according to the generation-time theory. Instead it appears that, on average, more heritable mutations occur per unit time during seed aging than during the lifetime of the plant (i.e. from seed germination to seed maturation). Given that most embryonic cells in seeds are in the resting stage during aging (G0/G1 stage, Georgieva et al., 1994; Vazquez-Ramos & Sanchez, 2003), the finding of higher evolutionary rates in taxa with persistent seed banks suggests that many mutations arise from replication-independent events in plants.

Caveats

There are two caveats to the present findings. First, given that this is the first such study in plants, it should be emphasized that the results represent trends specific to these relatively modest datasets. Although the findings are strengthened by the conservative nature of the sign tests and the phylogenetically independent contrasts as well as by the fact that two of the five factors under study were statistically significant, more than that expected by chance (P < 0.023, Table 1), further investigation will be needed to support or refute the generality of these relationships among plants. Secondly, although the 18S ITS region is an intergenic spacer and thus its sequence evolution is likely largely determined by mutation rates, it is transcribed and thus small regions could be subjected to some level of selection in certain plant species (Baldwin et al., 1995), potentially influencing some of the results. Altogether, further information from different and larger datasets and DNA regions will help further elucidate the factors underlying molecular evolutionary rate variation in plants.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supplementary material
  9. Supporting Information

The author would like to thank Dr S.P. Otto for valuable discussions on this topic and for ongoing feedback on the manuscript as well as Dr. M.O. Johnston for providing helpful comments. Thanks also to the anonymous reviewers. This work was supported by an NSERC PDF to C.-A.W. and an NSERC discovery grant to S.P. Otto.

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  2. Abstract
  3. Introduction
  4. Methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supplementary material
  9. Supporting Information
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Supplementary material

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supplementary material
  9. Supporting Information

Table S1 Taxon pairs for comparisons of shade tolerance and their phylogenetic branch lengths (PBL).

Table S2 Taxon pairs for comparisons of saline tolerance and their phylogenetic branch lengths (PBL).

Table S3 Taxon pairs for comparisons of drought tolerance and their phylogenetic branch lengths (PBL).

Table S4 Taxon pairs for taxa with different pollen :ovule ratios and their phylogenetic branch lengths (PBL).

Table S5 Pairs of taxa with transient vs. persistent seed banks, their annual/perennial life history and phylogenetic branch lengths (PBL).

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supplementary material
  9. Supporting Information

Table 1. Taxon pairs for comparisons of shade tolerance and their phylogenetic branch lengths (PBL).

Table 2. Taxon pairs for comparisons of saline-tolerance and their phylogenetic branch lengths (PBL).

Table 3. Taxon pairs for comparisons of drought tolerance and their phylogenetic branch lengths (PBL).

Table 4. Taxon pairs for taxa with different pollen : ovule ratios and their phylogenetic branch lengths (PBL).

Table 5. Pairs of taxa with transient versus persistent seed banks, their annual/perennial life history and phylogenetic branch lengths (PBL).

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