Examining rates and patterns of nucleotide substitution in plants

Driven by rapid improvements in affordable computing power and by the even faster accumulation of genomic data, the statistical analysis of molecular sequence data has become an active area of interdisciplinary research. Maximum likelihood methods have become mainstream because of their desirable properties and, more importantly, their potential for providing statistically sound solutions in complex data analysis settings. In this chapter, a review of recent literature focusing on rates and patterns of nucleotide substitution rates in the nuclear, chloroplast, and mitochondrial genomes of plants demonstrates the power and flexibility of these new methods. The emerging picture of the nucleotide substitution process in plants is a complex one. Evolutionary rates are seen to be quite variable, both among genes and among plant lineages. However, there are hints, particularly in the chloroplast, that individual factors can have important effects on many genes simultaneously.     

Intralineage variation in the pattern ofrbcL nucleotide substitution

Variation in chloroplastrbcL sequences was studied in representative species of four different lineages: the tribeRubieae (Rubiaceae), and the generaDrosera (Droseraceae),Nothofagus (Nothofagaceae) andIlex (Aquifoliaceae). Each lineage has its particular non-overlapping set ofrbcL polymorphic sites, indicating that common unconstrainedrbcL sites are not shared. Large differences in the rate and pattern of nucleotide substitution are observed among the four lineages. The genusIlex has the lowest rate of substitution, the lowest transition/transversion ratio, the lowest synonymous/replacement ratio and the lowest number of substitutions at the third codon position. An apparent relationship of these measures to the age of the lineages is observed. The A + T content and codon use among the four lineages are very similar and, apparently, cannot account for the observed differences in patterns of nucleotide substitution. However, the A + T content of the two bases immediately flanking the polymorphic sites is higher inIlex than in the other lineages. This could be correlated with the transversion/transition bias observed inIlex. The particularly low synonymous/replacement ratio found inIlex could also be explained by the small population sizes of species in this genus.     

Estimating a nucleotide substitution rate for maize from polymorphism at a major domestication locus

To estimate a rate for single nucleotide substitutions for maize (Zea mays ssp. mays), we have taken advantage of data from genetic and archaeological studies of the domestication of maize from its wild ancestor, teosinte (Z. mays ssp. parviglumis). Genetic studies have shown that the teosinte branched1 (tb1) gene was a major target of human selection during maize domestication, and sequence diversity in the intergenic region 5' to the tb1-coding sequence is extraordinarily low. We show that polymorphism in this region is consistent with new mutation following fixation for a small number of tb1 haplotypes during domestication. Archeological studies suggest that maize was domesticated approximately 6,250-10,000 years ago and subsequently the size of the maize population is thought to have expanded rapidly. Using the observed number of mutations within the region of selection at tb1, the approximate age of maize domestication, and approximations for the maize genealogy, we have derived estimates for the nucleotide substitution rate for the tb1 intergenic region. Using two approaches, one of which is a coalescent approach, we obtain rate estimates of approximately 2.9 x 10(-8) and 3.3 x 10(-8) substitutions per site per year. We also show that the pattern of polymorphism in the tb1 intergenic region appears to have been strongly affected by the mutagenic effect of DNA methylation. Excluding target sites of symmetric DNA methylation (CG and CNG sites) from analysis, the mutation rate estimates are reduced by approximately 50%-60%, while the rates for CG and CNG sites are nearly an order of magnitude higher. We use rate estimates from the tb1 region to estimate the timing of expansion of transposable elements in the maize genome and suggest that this expansion occurred primarily within the last million years.     

The phylogenetic history of Selaginellaceae based on DNA sequences from the plastid and nucleus: extreme substitution rates and rate heterogeneity

Molecular phylogenetic research on Selaginellaceae has focused on the plastid gene rbcL, which in this family has unusually high substitution rates. Here we develop a molecular data set from the nuclear 26S ribosomal DNA gene with the aim of evaluating and extending the results of previous phylogenetic research. The 26S rDNA and the rbcL regions were sequenced for a sample of 23 species, which represent the main elements of species diversity in the family. The data were analysed independently and in combination using both maximum parsimony and Bayesian inference. Although several between genome differences were found, the general pattern of relationships uncovered by all analyses was very similar. Results corroborate the previous study supporting new groupings not previously recognised on morphological grounds. Substitution rates in the 26S rDNA were also found to be high (26% informative) for the region analysed, but lower than for rbcL (37% informative). These data indicate that high substitution rates might be widespread in all three genomes (i.e., plastid, mitochondrion, and nucleus).     

High rates of nucleotide substitution in nuclear small-subunit (18S) rDNA from holoparasitic flowering plants

Relative rate tests, using Gnetum as a reference taxon, were conducted on nuclear 18S rRNA sequences from 10 angiosperms including autotrophic nonparasites (Arabidopsis, Asarum, Glycine, Malpighia, and Zea), a chlorophyllous hemiparasite (Arceuthobium—Viscaceae), and achlorophyllous holoparasites (Balanophora—Balanophoraceae, Prosopanche—Hydnoraceae, and Rafflesia and Rhizanthes—Rafflesiaceae). Compared with Glycine, the mean number of substitutions per site (K) for five autotrophic angiosperms is 0.036 whereas for the holoparasites K = 0.126, i.e., 3.5 times higher. Comparisons of autotrophic species with short and long generation times showed no differences in K; hence, divergent rRNA sequences in the holoparasites are likely attributable to other mechanisms. These might include genetic bottlenecks, effective population size, and/or molecular drive. High substitution rates appear to be associated only with those parasitic angiosperms that have developed a highly modified haustorial system and extreme nutritional dependence upon the host. At present, high substitution rates in these parasites confound attempts to determine their phylogenetic position relative to other angiosperms. Correspondence to: D.L. Nickrent     

Molecular and morphological characterization of Leveillula (Ascomycota: Erysiphales) on monocotyledonous plants

Leveillula on monocotyledonous plants have been recorded as L. taurica by several authors, whereas the fungus on Allium has been described as an independent species, namely L. allii, by some authors. We sequenced ca 600 bp of the rDNA ITS region for two Leveillula specimens from Allium and Polianthes (both from monocotyledons) and compared them with several already published sequences from Leveillula isolates from dicotyledons. Pair-wise percentages of sequence divergences were calculated for all Leveillula isolates. The ITS sequence of the Polianthes isolate was identical to L. taurica on Helianthus and Vicia. The sequence of the Allium isolate was 99.5 % identical to L. taurica on Euphorbia, Haplophylum, Peganum, etc. These results suggest close relationships between monocot and dicot pathogenic Leveillula species. The identity between two monocot isolates was 98.4 %. Phylogenetic analysis revealed that the two monocot isolates do not group into a clade together. This result suggests that Leveillula acquired parasitism to monocots at least twice independently.     

Analysis of nucleotide substitutions and amino acid conservation in theDrosophila Adh genomic region

The homologous genomic region that contains two paralogous genes,Adh andAdh-dup, was compared in severalDrosophila species. Sequences were analyzed as follows: a) At the nucleotide level, Ka and Ks values were determined for each pair of species. Ka-Adh and Ka-Adh-dup are not significantly different. However, Ks-Adh values are significantly lower than Ks-Adh-dup, which are more variable. In agreement with other reports, lower Ks values forAdh correlate with a high level of gene expression and relatively high percentage of G+C content in the third codon position, while the opposite applies toAdh-dup. b) At the protein level, amino acid comparisons reveal conserved regions shared by ADH and ADH-DUP, which have been assigned to known functional domains. Key residues for dehydrogenasic function are also found in ADH-DUP, thus pointing to a dehydrogenase activity for ADH-DUP, albeit very different from that of ADH.     

Distinct changes of genomic biases in nucleotide substitution at the time of Mammalian radiation

Differences in the regional substitution patterns in the human genome created patterns of large-scale variation of base composition known as genomic isochores. To gain insight into the origin of the genomic isochores, we develop a maximum-likelihood approach to determine the history of substitution patterns in the human genome. This approach utilizes the vast amount of repetitive sequence deposited in the human genome over the past approximately 250 Myr. Using this approach, we estimate the frequencies of seven types of substitutions: the four transversions, two transitions, and the methyl-assisted transition of cytosine in CpG. Comparing substitutional patterns in repetitive elements of various ages, we reconstruct the history of the base-substitutional process in the different isochores for the past 250 Myr. At around 90 MYA (around the time of the mammalian radiation), we find an abrupt fourfold to eightfold increase of the cytosine transition rate in CpG pairs compared with that of the reptilian ancestor. Further analysis of nucleotide substitutions in regions with different GC content reveals concurrent changes in the substitutional patterns. Although the substitutional pattern was dependent on the regional GC content in such ways that it preserved the regional GC content before the mammalian radiation, it lost this dependence afterward. The substitutional pattern changed from an isochore-preserving to an isochore-degrading one. We conclude that isochores have been established before the radiation of the eutherian mammals and have been subject to the process of homogenization since then.     

Evolutionary change of restriction sites under unequal rates of nucleotide substitution among the three positions of codons

Summary Under the assumption of unequal rates of nucleotide substitution among the three positions of codons, mathematical formulas are derived for the probability that a restriction site observed at time 0 in a DNA sequence will be present at time t, the probability that a new restriction site will emerge at a particular place in two descendant sequences, and the proportion of identical restriction sites between two such sequences. All three quantitites are shown to be larger for the case of unequal rates than for the case of equal rates. As a consequence, estimates of nucleotide divergence (2t) between two sequences based on restriction-site data tend to be lower than the actual values of the assumption of equal rates is made but actually does not hold. However, the degree of underestimation is slight if 2t is 0.10 or smaller. The underestimation can be serious when 2t becomes 0.20 or larger, particularly if the substitution rates at the three codon positions are very different or if transitional substitution occurs more frequently than transversional substitution. The underestimation is more serious for four-base enzymes than for sixbase enzymes.     

Contrasting rates of nucleotide substitution in the X-Linked and Y-Linked zinc finger genes

We have sequenced the entire exon (1,180 bp) encoding the zinc finger domain of the X-linked and Y-linked zinc finger genes (ZFX and ZFY, respectively) in the orangutan, the baboon, the squirrel monkey, and the rat; a total of 9,442 by were sequenced. The ratio of the rates of synonymous substitution in the ZFY and ZFX genes is estimated to be 2.1 in primates. This is close to the ratio of 2.3 estimated from primate ZFY and ZFX intron sequences and supports the view that the male-to-female ratio of mutation rate in humans is considerably higher than 1 but not extremely large. The ratio of synonymous substitution rates in ZFY and ZFX is estimated to be 1.3 in the rat lineage but 4.2 in the mouse lineage. The former is close to the estimate (1.4) from introns. The much higher ratio in the mouse lineage (not statistically significant) might have arisen from relaxation of selective constraints. The synonymous divergence between mouse and rat ZFX is considerably lower than that between mouse and rat autosomal genes, agreeing with previous observations and providing some evidence for stronger selective constraints on synonymous changes in X-linked genes than in autosomal genes. At the protein level ZFX has been highly conserved in all placental mammals studied while ZFY has been well conserved in primates and foxes but has evolved rapidly in mice and rats, possibly due to relaxation of functional constraints as a result of the development of X-inactivation of ZFX in rodents. The long persistence of the ZFY-ZFX gene pair in mammals provides some insight into the process of degeneration of Y-linked genes.Correspondence to: W.-H. Li     

Phylogeny of gymnosperms inferred fromrbcL gene sequences

Partial nucleotide sequences of the large subunit of ribulose-1,5-bisphosphate carboxylase (rubisco) gene (1333 base pairs: about 90% of the gene) from several seed plants were determined. Phylogenetic trees based on amino acid sequences were inferred by using the neighbor joining and maximum likelihood methods. The results indicate (1) monophyly of gnetum group (Ephedra, Gnetum, Welwitschia), (2) monophyly of extant gymnosperms containing gnetum group, which contradicts the results of morphological data.     

Stochastic models of molecular evolution and the estimation of phylogeny and rates of nucleotide substitution in the hominoid primates

We have estimated phylogenetic patterns and rates of nucleotide substitution in the hominoid primates using two different probabilistic models of molecular evolution as applied to three different data sets of nucleic acid sequences. The orang-utan was found to be the out-group of the other hominoids examined. Within the African apes and human clade the sister-group relationship of chimpanzee and human was found to be statistically the best, although the magnitude of the error estimates (a reflection of random statistical fluctuations) makes this conclusion tentative. The ψν-globin data sets were found to be statistically the most consistent and gave estimates of the times of divergence of chimpanzee and human from gorilla and of chimpanzee from human as 7·7 ± 1·5 Ma (Millions of years ago) and 7·4 ± 1·5 Ma respectively, although the speculative nature of these estimates is emphasized. In all cases the calibration point was the assumed divergence of the orang-utan from the remaining hominoids at 14·5 Ma. There was no statistically significant evidence of a slowdown in nucleotide substitution rate for the human lineage, or among the hominoids as a whole with respect to the Old and New World monkeys. We advocate the continued use and development of stochastic models of molecular evolution as a basis for phylogenetic estimation. On this basis one can choose between competing hypotheses of relationship in a statistical manner and can provide estimates of the errors involved in such estimations. The assumptions of all stochastic models are open to test and future refinement.     

Phylogenetic relationships of the enigmatic fern families Hymenophyllopsidaceae and Lophosoriaceae: Evidence fromrbcL nucleotide sequences

Nucleotide sequences fromrbcL were used to infer relationships of Lophosoriaceae and Hymenophyllopsidaceae. The phylogenetic positions of these two monotypic fern families have been debated, and neither group had been included in recent molecular systematic studies of ferns. Maximum parsimony analysis of our data supported a sister relationship betweenLophosoria andDicksonia, and also betweenHymenophyllopsis andCyathea. Thus, both newly-examined families appear to be part of a previously characterized and well-supported clade of tree ferns. The inferred relationships ofLophosoria are consistent with most (but not all) recent treatments. However,Hymenophyllopsis includes only small delicate plants superficially similar to filmy ferns (Hymenophyllaceae), very different from the large arborescent taxa. Nevertheless, some synapomorphic characteristics are shared with the tree fern clade. Further studies on gametophytes ofHymenophyllopsis are needed to test these hypotheses of relationship.     

Slow algae, fast fungi: exceptionally high nucleotide substitution rate differences between lichenized fungi Omphalina and their symbiotic green algae Coccomyxa

Omphalina basidiolichens are obligate mutualistic associations of a fungus of the genus Omphalina (the exhabitant) and a unicellular green alga of the genus Coccomyxa (the inhabitant). It has been suggested that symbiotic inhabitants have a lower rate of genetic change compared to exhabitants because the latter are more exposed to abiotic environmental variation and competition from other organisms. In order to test this hypothesis we compared substitution rates in the nuclear ribosomal internal transcribed spacer region (ITS1, 5.8S, ITS2) among fungal species with rates among their respective algal symbionts. To ensure valid comparisons, only taxon pairs (12) with a common evolutionary history were used. On average, substitution rates in the ITS1 portion of Omphalina pairs were 27.5 times higher than rates in the corresponding pairs of Coccomyxa since divergence from their respective ancestor at the base of the Omphalina/Coccomyxa lineage. Substitution rates in the 5.8S and the ITS2 portions were 2.4 and 18.0 times higher, respectively. The highest rate difference (43.0) was found in the ITS1 region. These are, to our knowledge, the highest differences of substitution rates reported for symbiotic organisms. We conclude that the Omphalina model system conforms to the proposed hypothesis of lower substitution rates in the inhabitant, but that the mode of transmission of the inhabitant (vertical versus horizontal) could be a prevailing factor in the regulation of unequal rates of nucleotide substitution between co-evolving symbionts. Our phylogenetic study of Coccomyxa revealed three main lineages within this genus, corresponding to free-living Coccomyxa, individuals isolated from basidiolichens Omphalina and Coccomyxa isolated from ascolichens belonging to the Peltigerales.     

Cophylogeny and disparate rates of evolution in sympatric lineages of chewing lice on pocket gophers

Although molecular-based phylogenetic studies of hosts and parasites are increasingly common in the literature, no study to date has examined two congeneric lineages of parasites that live in sympatry on the same lineage of hosts. This study examines phylogenetic relationships among chewing lice (Phthiraptera: Trichodectidae) of the Geomydoecus coronadoi and Geomydoecus mexicanus species complexes and compares these to phylogenetic patterns in their hosts (pocket gophers of the rodent family Geomyidae). Sympatry of congeneric lice provides a natural experiment to test the hypothesis that closely related lineages of parasites will respond similarly to the same host. Sequence data from the mitochondrial COI and the nuclear EF-1alpha genes confirm that the two louse complexes are reciprocally monophyletic and that individual clades within each species complex parasitize a different species of pocket gopher. Phylogenetic comparisons reveal that both louse complexes show a significant pattern of cophylogeny with their hosts. Comparisons of rates of nucleotide substitution at 4-fold degenerate sites in the COI gene indicate that both groups of lice have significantly higher basal mutation rates than their hosts. The two groups of lice have similar basal rates of mutation, but lice of the G. coronadoi complex show significantly elevated rates of nucleotide substitution at all sites. These rate differences are hypothesized to result from population-level phenomena, such as effective population size, founder effects, and drift, that influence rates of nucleotide substitution.     

Absence in monocotyledonous plants of the diffusible plant factors inducing T-DNA circularization and vir gene expression in Agrobacterium

Summary T-DNA circularization is one of the molecular events specifically induced in agrobacterial cells upon their infection of dicotyledonous plant cells. We developed a seedling co-cultivation procedure to determine whether or not monocotyledonous plants have the ability to induce T-DNA circularization and vir gene expression. Co-cultivation of Agrobacterium tumefaciens with seedlings of dicotyledonous plants showed that the circularization event takes place efficiently. The exudates and extracts of the seedlings also effectively induced T-DNA circularization and vir gene expression, indicating that dicotyledonous seedlings contain diffusible factors capable of inducing these molecular events. In contrast, neither T-DNA circularization nor vir gene expression was detectable when Agrobacterium was incubated with seedlings of monocotyledonous plants. Supplementing with acetosyringone, a known inducer of vir gene expression and T-DNA circularization, resulted in the induction of circularization during co-cultivation with monocotyledonous seedlings. These results indicate that the seedlings of monocotyledonous plants have no detectable amounts of diffusible inducers, unlike dicotyledonous seedlings. Therefore, it is unlikely that the vir genes are expressed in Agrobacterium inoculated in monocotyledonous plants. This may be one of the blocks in tumorigenesis of monocotyledonous plants by Agrobacterium.     

Effects of N addition rates on the productivity ofPicea Sitchensis,Thuja plicata,andTsuga heterophylla seedlings

Seedlings ofPicea sitchensis, Thuja plicata andTsuga heterophylla were supplied N hydroponically at one of four exponentially increasing rates of addition (0.09, 0.07, 0.05, or 0.025 gN^-1 day^-1) for up to 3 months in a naturally illuminated glasshouse. Relative growth rates (RGR) were analyzed as a function of N uptake, the allocation of assimilated N to foliage (LNFR), foliar N concentrations (N_la) and met assimilation rates (NAR), which were combined to estimate N productivity (RGR per unit whole-plant N concentration). Nitrogen accumulation, biomass and N partitioning and RGR and its components varied with species in response to the different N regimes.T. heterophylla had the lowest maximum wholeplant N concentrations (wpN) and specific absorption rates for N and exhibited the least plasticity in root: shoot ratios as wpN increased from 11–21 mg g^-1. In all species, RGR increased linearly with wpN, while LNFR increased curvilinearly. Foliar N (N_la) increased linearly with wpN and NAR increased linearly with N_la. The RGRs ofT. heterophylla were highest at wpNs up to 18 mg g^-1, a result of higher foliar N use efficiencies (NAR/N_la). However, RGR increased more with wpN inT. plicata andP. sitchensis. Although LNFR increased with wpN in all species, foliar N use efficiency declined, possibly due to an increased partitioning of foliar soluble N to non-photosynthetic compounds. Thus, in each species, N productivity did not increase above intermediate levels of wpN: 14 mg g^-1 inT. heterophylla, 16 mg g^-1 inP. sitchensis and 17 mg g^-1 inT. plicata.