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Abstract Background Insects detect environmental chemicals via a large and rapidly evolving family of chemosensory receptor proteins. Although our understanding of the molecular genetic basis for Drosophila chemoreception has increased enormously in the last decade, similar understanding in other insects remains limited. The tobacco hornworm, Manduca sexta, has long been an important model for insect chemosensation, particularly from ecological, behavioral, and physiological standpoints. It is also a major agricultural pest on solanaceous crops. However, little sequence information and lack of genetic tools has prevented molecular genetic analysis in this species. The ability to connect molecular genetic mechanisms, including potential lineage-specific changes in chemosensory genes, to ecologically relevant behaviors and specializations in M. sexta would be greatly beneficial. Results Here, we sequenced transcriptomes from adult and larval chemosensory tissues and identified chemosensory genes based on sequence homology. We also used dsRNA feeding as a method to induce RNA interference in larval chemosensory tissues. Conclusions We report identification of new chemosensory receptor genes including 17 novel odorant receptors and one novel gustatory receptor. Further, we demonstrate that systemic RNA interference can be used in larval olfactory neurons to reduce expression of chemosensory receptor transcripts. Together, our results further the development of M. sexta as a model for functional analysis of insect chemosensation.

Abstract Background Many bacterial chromosomes display nucleotide asymmetry, or skew, between the leading and lagging strands of replication. Mutational differences between these strands result in an overall pattern of skew that is centered about the origin of replication. Such a pattern could also arise from selection coupled with a bias for genes coded on the leading strand. The relative contributions of selection and mutation in producing compositional skew are largely unknown. Results We describe a model to quantify the contribution of mutational differences between the leading and lagging strands in producing replication-induced skew. When the origin and terminus of replication are known, the model can be used to estimate the relative accumulation of G over C and of A over T on the leading strand due to replication effects in a chromosome with bidirectional replication arms. The model may also be implemented in a maximum likelihood framework to estimate the locations of origin and terminus. We find that our estimations for the origin and terminus agree very well with the location of genes that are thought to be associated with the replication origin. This indicates that our model provides an accurate, objective method of determining the replication arms and also provides support for the hypothesis that these genes represent an ancestral cluster of origin-associated genes. Conclusion The model has several advantages over other methods of analyzing genome skew. First, it quantifies the role of mutation in generating skew so that its effect on composition, for example codon bias, can be assessed. Second, it provides an objective method for locating origin and terminus, one that is based on chromosome-wide accumulation of leading vs lagging strand nucleotide differences. Finally, the model has the potential to be utilized in a maximum likelihood framework in order to analyze the effect of chromosome rearrangements on nucleotide composition.

Hoxa5 plays numerous roles in development, but its downstream molecular effects are mostly unknown. We applied bulk RNA-seq assays to characterize the transcriptional impact of the loss of Hoxa5 gene function in seven different biological contexts, including developing respiratory and musculoskeletal tissues that present phenotypes in Hoxa5 mouse mutants. This global analysis revealed few common transcriptional changes, suggesting that HOXA5 acts mainly via the regulation of context-specific effectors. However, Hox genes themselves appeared as potentially conserved targets of HOXA5 across tissues. Notably, a trend toward reduced expression of HoxA genes was observed in Hoxa5 null mutants in several tissue contexts. Comparative analysis of epigenetic marks along the HoxA cluster in lung tissue from two different Hoxa5 mutant mouse lines revealed limited effect of either mutation indicating that Hoxa5 gene targeting did not significantly perturb the chromatin landscape of the surrounding HoxA cluster. Combined with the shared impact of the two Hoxa5 mutant alleles on phenotype and Hox expression, these data argue against the contribution of local cis effects to Hoxa5 mutant phenotypes and support the notion that the HOXA5 protein acts in trans in the control of Hox gene expression., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Mutations and subsequent repair processes are known to be strongly context-dependent in the flowering-plant chloroplast genome. At least six flanking bases, three on each side, can have an influence on the relative rates of different types of mutation at any given site. In this analysis, examine context and substitution at noncoding and fourfold degenerate coding sites in gymnosperm DNA. The sequences are analyzed in sets of three, allowing the inference of the substitution direction and the generation of context-dependent rate matrices. The size of the dataset limits the analysis to the tetranucleotide context of the sites, but the evidence shows that there are significant contextual effects, with patterns that are similar to those observed in angiosperms. These effects most likely represent an influence on the underlying mutation/repair dynamics. The data extend the plastome lineages that feature very complex patterns of mutation, which can have significant effects on the evolutionary dynamics of the chloroplast genome.

A common genome composition pattern in eubacteria is an asymmetry between the leading and lagging strands resulting in opposite skew patterns in the two replichores that lie between the origin and terminus of replication. Although this pattern has been reported for a couple of isolated plastid genomes, it is not clear how widespread it is overall in this chromosome. Using a random walk approach, we examine plastid genomes outside of the land plants, which are excluded since they are known not to initiate replication at a single site, for such a pattern of asymmetry. Although it is not a common feature, we find that it is detectable in the plastid genome of species from several diverse lineages. The euglenozoa in particular show a strong skew pattern as do several rhodophytes. There is a weaker pattern in some chlorophytes but it is not apparent in other lineages. The ramifications of this for analyses of plastid evolution are discussed.

Fourfold degenerate sites within coding regions and intergenic sites have both been used as estimates of neutral evolution. In chloroplast DNA, the pattern of substitution at intergenic sites is strongly dependent on the composition of the surrounding hexanucleotide composed of the three base pairs on each side, which suggests that the mutation process is highly context-dependent in this genome. This study examines the context-dependency of substitutions at fourfold degenerate sites in protein-coding regions and compares the pattern to what has been observed at intergenic sites. Overall, there is strong similarity between the two types of sites, but there are some intriguing differences. One of these is that substitutions of G and C are significantly higher at fourfold degenerate sites across a range of contexts. In fact, A → T and T → A substitutions are the only substitution types that occur at a lower rate at fourfold degenerate sites. The data are not consistent with selective constraints being responsible for the difference in substitution patterns between intergenic and fourfold degenerate sites. Rather, it is suggested that the difference may be a result of different epigenetic modifications that result in slightly different mutation patterns in coding and intergenic DNA.

Substitutions between closely related noncoding chloroplast DNA sequences are studied with respect to the composition of the 3 bases on each side of the substitution, that is the hexanucleotide context. There is about 100-fold variation in rate, among the contexts, particularly on substitutions of A and T. Rate heterogeneity of transitions differs from that of transversions, resulting in a more than 200-fold variation in the transitions: transversion bias. The data are consistent with a CpG effect, and it is shown that both the A + T content and the arrangement of purines/pyrimidines along the same DNA strand are correlated with rate variation. Expected equilibrium A + T content ranges from 36.4% to 82.8% across contexts, while G-C skew ranges from -77.4 to 72.2 and A-T skew ranges from -63.9 to 68.2. The predicted equilibria are associated with specific features of the content of the hexanucleotide context, and also show close agreement with the observed context-dependent compositions. Finally, by controlling for the content of nucleotides closer to the substitution site, it is shown that both the third and fourth nucleotide removed on each side of the substitution directly influence substitution dynamics at that site. Overall, the results demonstrate that noncoding sites in different contexts are evolving along very different evolutionary trajectories and that substitution dynamics are far more complex than typically assumed. This has important implications for a number of types of sequence analysis, particularly analyses of natural selection, and the context-dependent substitution matrices developed here can be applied in future analyses., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

The influence of neighboring base composition, or context, on substitution bias at fourfold degenerate coding sites and in intergenic regions in plastid DNA is compared across the angiosperms, gymnosperms, ferns, liverworts, chlorophytes, stramenopiles and rhodophytes. An influence of flanking base G + C content on the relative rates of transitions and transversions is observed in all lineages and extends up to four nucleotides from the site of substitution in some. Despite finding context effects in all lineages, significant differences were observed between lineages. Overall, the data suggest that context is a general factor affecting mutation bias in plastid DNA but that the dynamics of the influence have evolved over time. It is also shown that, although there are similar effects of context on substitution bias at fourfold degenerate coding sites and at sites within intergenic regions, there are also small but significant differences, suggesting that there could be some selection on some of these sites and that there could be some difference in the mutation and/or repair process between coding and noncoding DNA., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Two competing proposals about the degree to which selection affects codon usage of angiosperm chloroplast genes are examined. The first, based on observations that codon usage does not match expectations under the naïve assumption that base composition will be identical at all neutral sites, is that selection plays a significant role. The second is that codon usage is determined almost solely by mutation bias and drift, with selection influencing only one or two highly expressed genes, in particular psbA. First it is shown that, as a result of an influence of neighboring base composition on mutation dynamics, compositional biases are expected to be widely divergent at different sites in the absence of selection. The observed mutation properties are then used to predict expected neutral codon usage biases and to show that observed deviations from the naïve expectations are in fact expected given the context-dependent mutational dynamics. It is also shown that there is a match between the observed and expected codon usage when context effects are taken into consideration, with psbA being a notable exception. Overall, the data support the model that selection is not a widespread factor affecting the codon usage of angiosperm chloroplast genes and highlight the need to have an accurate model of mutational dynamics., (© 2021. The Author(s).)

The codon usage of the Angiosperm psbA gene is atypical for flowering plant chloroplast genes but similar to the codon usage observed in highly expressed plastid genes from some other Plantae, particularly Chlorobionta, lineages. The pattern of codon bias in these genes is suggestive of selection for a set of translationally optimal codons but the degree of bias towards these optimal codons is much weaker in the flowering plant psbA gene than in high expression plastid genes from lineages such as certain green algal groups. Two scenarios have been proposed to explain these observations. One is that the flowering plant psbA gene is currently under weak selective constraints for translation efficiency, the other is that there are no current selective constraints and we are observing the remnants of an ancestral codon adaptation that is decaying under mutational pressure. We test these two models using simulations studies that incorporate the context-dependent mutational properties of plant chloroplast DNA. We first reconstruct ancestral sequences and then simulate their evolution in the absence of selection on codon usage by using mutation dynamics estimated from intergenic regions. The results show that psbA has a significantly higher level of codon adaptation than expected while other chloroplast genes are within the range predicted by the simulations. These results suggest that there have been selective constraints on the codon usage of the flowering plant psbA gene during Angiosperm evolution., Competing Interests: The authors have declared that no competing interests exist.

Metabolites have essential roles in microbial communities, including as mediators of nutrient and energy exchange, cell-to-cell communication, and antibiosis. However, detecting and quantifying metabolites and other chemicals in samples having extremes in salt or mineral content using liquid chromatography-mass spectrometry (LC-MS)-based methods remains a significant challenge. Here, we report a facile method based on in situ chemical derivatization followed by extraction for analysis of metabolites and other chemicals in hypersaline samples, enabling for the first time direct LC-MS-based exometabolomics analysis in sample matrices containing up to 2 M total dissolved salts. The method, MetFish, is applicable to molecules containing amine, carboxylic acid, carbonyl, or hydroxyl functional groups, and it can be integrated into either targeted or untargeted analysis pipelines. In targeted analyses, MetFish provided limits of quantification as low as 1 nM, broad linear dynamic ranges (up to 5 to 6 orders of magnitude) with excellent linearity, and low median interday reproducibility (e.g., 2.6%). MetFish was successfully applied in targeted and untargeted exometabolomics analyses of microbial consortia, quantifying amino acid dynamics in the exometabolome during community succession; in situ in a native prairie soil, whose exometabolome was isolated using a hypersaline extraction; and in input and produced fluids from a hydraulically fractured well, identifying dramatic changes in the exometabolome over time in the well. IMPORTANCE The identification and accurate quantification of metabolites using electrospray ionization-mass spectrometry (ESI-MS) in hypersaline samples is a challenge due to matrix effects. Clean-up and desalting strategies that typically work well for samples with lower salt concentrations are often ineffective in hypersaline samples. To address this gap, we developed and demonstrated a simple yet sensitive and accurate method-MetFish-using chemical derivatization to enable mass spectrometry-based metabolomics in a variety of hypersaline samples from varied ecosystems and containing up to 2 M dissolved salts.

Increasing anthropogenic inputs of fixed nitrogen are leading to greater eutrophication of aquatic environments, but it is unclear how this impacts the flux and fate of carbon in lacustrine and riverine systems. Here, we present evidence that the form of nitrogen governs the partitioning of carbon among members in a genome-sequenced, model phototrophic biofilm of 20 members. Consumption of NO 3 - as the sole nitrogen source unexpectedly resulted in more rapid transfer of carbon to heterotrophs than when NH 4 + was also provided, suggesting alterations in the form of carbon exchanged. The form of nitrogen dramatically impacted net community nitrogen, but not carbon, uptake rates. Furthermore, this alteration in nitrogen form caused very large but focused alterations to community structure, strongly impacting the abundance of only two species within the biofilm and modestly impacting a third member species. Our data suggest that nitrogen metabolism may coordinate coupled carbon-nitrogen biogeochemical cycling in benthic biofilms and, potentially, in phototroph-heterotroph consortia more broadly. It further indicates that the form of nitrogen inputs may significantly impact the contribution of these communities to carbon partitioning across the terrestrial-aquatic interface. IMPORTANCE Anthropogenic inputs of nitrogen into aquatic ecosystems, and especially those of agricultural origin, involve a mix of chemical species. Although it is well-known in general that nitrogen eutrophication markedly influences the metabolism of aquatic phototrophic communities, relatively little is known regarding whether the specific chemical form of nitrogen inputs matter. Our data suggest that the nitrogen form alters the rate of nitrogen uptake significantly, whereas corresponding alterations in carbon uptake were minor. However, differences imposed by uptake of divergent nitrogen forms may result in alterations among phototroph-heterotroph interactions that rewire community metabolism. Furthermore, our data hint that availability of other nutrients (i.e., iron) might mediate the linkage between carbon and nitrogen cycling in these communities. Taken together, our data suggest that different nitrogen forms should be examined for divergent impacts on phototrophic communities in fluvial systems and that these anthropogenic nitrogen inputs may significantly differ in their ultimate biogeochemical impacts., (Copyright © 2020 Anderton et al.)

Codon adaptation is codon usage bias that results from selective pressure to increase the translation efficiency of a gene. Codon adaptation has been studied across a wide range of genomes and some early analyses of plastids have shown evidence for codon adaptation in a limited set of highly expressed plastid genes. Here we study codon usage bias across all fully sequenced plastid genomes which includes representatives of the Rhodophyta, Alveolata, Cryptophyta, Euglenozoa, Glaucocystophyceae, Rhizaria, Stramenopiles and numerous lineages within the Viridiplantae, including Chlorophyta and Embryophyta. We show evidence that codon adaptation occurs in all genomes except for two, Theileria parva and Heicosporidium sp., both of which have highly reduced gene contents and no photosynthesis genes. We also show evidence that selection for codon adaptation increases the representation of the same set of codons, which we refer to as the adaptive codons, across this wide range of taxa, which is probably due to common features descended from the initial endosymbiont. We use various measures to estimate the relative strength of selection in the different lineages and show that it appears to be fairly strong in certain Stramenopiles and Chlorophyta lineages but relatively weak in many members of the Rhodophyta, Euglenozoa and Embryophyta. Given these results we propose that codon adaptation in plastids is widespread and displays the same general features as adaptation in eubacterial genomes.

Previous studies have shown that the pattern of single nucleotide polymorphism (SNP) in Arabidopsis (Arabidopsis thaliana) deviates from the distribution expected under a neutral model. Here, we test whether or not ancestral misinference could explain this deviation. We start by showing that there are significant and complex influences of context on mutation dynamics as inferred from SNP frequency, in Arabidopsis, and compare the results to observations about context dependency that have been made on a previous analysis of a maize (Zea mays) SNP dataset. The data concerning heterogeneity across sites are then used to make corrections for ancestral misinference in a context-dependent manner. Using Arabidopsis lyrata to infer the ancestral state for SNPs, we show that the resulting unfolded site frequency spectrum (SFS) in Arabidopsis is skewed toward sites with high frequency derived nucleotides. Sites are also partitioned into two general functional classes, second codon position and 4-fold degenerate sites. These two classes show different SFS; although both show an overrepresentation of high frequency derived sites, low frequency derived sites are vastly overrepresented at the second codon position, but significantly underrepresented at 4-fold degenerate sites. We find that these results are robust to corrections for ancestral misinference, even when context-dependent variation in mutation properties is taken into consideration. The data suggest that, in addition to purifying selection, complex demographic events and/or linked positive selection need to be invoked to explain the SFS, and they highlight the importance of sequence context in analyses of genome-wide variation.

We assess the similarity of base substitution processes, described by empirically derived 4 x 4 matrices, using chi-square homogeneity tests. Such significance analyses allow us to assess variation in sequence evolution across sites and we apply them to matrices derived from noncoding sites in different contexts in grass chloroplast DNA. We show that there is statistically significant variation in rates and patterns of mutation among noncoding sites in different contexts and then demonstrate a similar and significant influence of context on substitutions at fourfold degenerate sites of coding regions from grass chloroplast DNA. These results show that context has the same general effect on substitution bias in coding and noncoding DNA: the A+T content of flanking bases is correlated with rate of substitution, transition bias, and GC --> AT pressure, while the number of flanking pyrimidines on a single strand is correlated with a mutational bias, or skew, toward pyrimidines. Despite the similarity in general trends, however, when we compare coding and noncoding matrices we find that there is a statistically significant difference between them even when we control for context. Most noticeably, fourfold degenerate sites in coding sequences are undergoing substitution at a higher rate and there are also significant differences in the relationship between pyrimidines skew and the number of flanking pyrimidines. Possible reasons for the differences between coding and noncoding sites are discussed. Furthermore, our analysis illustrates a simple statistical way for comparing substitution processes across sites allowing us to better study variation in evolutionary processes across a genome.

Highly expressed nuclear genes from Arabidopsis thaliana show an increased frequency of codons that match abundant tRNAs, and it has been suggested that this reflects a selective pressure to increase translation efficiency. Here we explore the possibility that the difference in codon usage between highly expressed genes and other Arabidopsis genes is not the result of selection but, rather, arises from mutation biases. Specifically, we explore the possibility that an influence of transcription level on mutational properties coupled with a context dependency of mutations, both of which have been observed in various organisms, contribute to variation in codon-usage bias across genes. Using noncoding sites immediately flanking both high- and low-expression-coding sequences to infer context-dependent composition biases, we analyze codon-usage bias across genes. The data show that mutation bias cannot explain codon usage of high-expression genes in Arabidopsis and, surprisingly, also indicate that even low-expression genes are under selective constraints. In addition, the data indicate that the general preference for certain codons is context dependent; the composition of the 3' nucleotide, that is, the first position of the next codon, is correlated with what codon is found at an increased frequency in highly expressed genes. This context dependency indicates that selective pressure on codon usage is more complex than previously thought. Overall, the study supports previous suggestions that selection plays a significant role in determining codon usage of nuclear genes in A. thaliana.

RNA editing alters the nucleotide sequence of an RNA molecule so that it deviates from the sequence of its DNA template. Different RNA-editing systems are found in the major eukaryotic lineages, and these systems are thought to have evolved independently. In this study, we provide a detailed analysis of data on C-to-U editing sites in land plant chloroplasts and propose a model for the evolution of RNA editing in land plants. First, our data suggest that the limited RNA-editing system of seed plants and the much more extensive systems found in hornworts and ferns are of monophyletic origin. Further, although some eukaryotic editing systems appear to have evolved to regulate gene expression, or at least are now involved in gene regulation, there is no evidence that RNA editing plays a role in gene regulation in land plant chloroplasts. Instead, our results suggest that land plant chloroplast C-to-U RNA editing originated as a mechanism to generate variation at the RNA level, which could complement variation at the DNA level. Under this model, many of the original sites, particularly in seed plants, have been subsequently lost due to mutation at the DNA level, and the function of extant sites is merely to conserve certain codons. This is the first comprehensive model for the evolution of the chloroplast RNA-editing system of land plants and may also be applicable to the evolution of RNA editing in plant mitochondria.

We examine variation in mutation dynamics across a single genome (Zea mays ssp. mays) in relation to regional and flanking base composition using a data set of 10,472 SNPs generated by resequencing 1776 transcribed regions. We report several relationships between flanking base composition and mutation pattern. The A + T content of the two sites immediately flanking the mutation site is correlated with rate, transition bias, and GC --> AT pressure. We also observe a significant CpG effect, or increase in transition rate at CpG sites. At the regional level we find that the strength of the CpG effect is correlated with regional A + T content, ranging from a 1.7-fold increase in transition rate in relatively G + C-rich regions to a 2.6-fold increase in A + T-rich regions. We also observe a relationship between locus A + T content and GC --> AT pressure. This regional effect is in opposition to the influence of the two immediate neighbors in that GC --> AT pressure increases with increasing locus A + T content but decreases with increasing flanking base A + T content and may represent a relationship between genome location and mutation bias. The data indicate multiple context effects on mutations, resulting in significant variation in mutation dynamics across the genome.

The influence of local base composition on mutations in chloroplast DNA (cpDNA) is studied in detail and the resulting, empirically derived, mutation dynamics are used to analyze both base composition and codon usage bias. A 4 x 4 substitution matrix is generated for each of the 16 possible flanking base combinations (contexts) using 17,253 noncoding sites, 1309 of which are variable, from an alignment of three complete grass chloroplast genome sequences. It is shown that substitution bias at these sites is correlated with flanking base composition and that the A+T content of these flanking sites as well as the number of flanking pyrimidines on the same strand appears to have general influences on substitution properties. The context-dependent equilibrium base frequencies predicted from these matrices are then applied to two analyses. The first examines whether or not context dependency of mutations is sufficient to generate average compositional differences between noncoding cpDNA and silent sites of coding sequences. It is found that these two classes of sites exist, on average, in very different contexts and that the observed mutation dynamics are expected to generate significant differences in overall composition bias that are similar to the differences observed in cpDNA. Context dependency, however, cannot account for all of the observed differences: although silent sites in coding regions appear to be at the equilibrium predicted, noncoding cpDNA has a significantly lower A+T content than expected from its own substitution dynamics, possibly due to the influence of indels. The second study examines the codon usage of low-expression chloroplast genes. When context is accounted for, codon usage is very similar to what is predicted by the substitution dynamics of noncoding cpDNA. However, certain codon groups show significant deviation when followed by a purine in a manner suggesting some form of weak selection other than translation efficiency. Overall, the findings indicate that a full understanding of mutational dynamics is critical to understanding the role selection plays in generating composition bias and sequence structure.

A previously employed method that uses the composition of noncoding DNA as the basis of a test for selection between synonymous codons in plastid genes is reevaluated. The test requires the assumption that in the absence of selective differences between synonymous codons the composition of silent sites in coding sequences will match the composition of noncoding sites. It is demonstrated here that this assumption is not necessarily true and, more generally, that using compositional properties to draw inferences about selection on silent changes in coding sequences is much more problematic than commonly assumed. This is so because selection on nonsynonymous changes can influence the composition of synonymous sites (i.e., codon usage) in a complex manner, meaning that the composition biases of different silent sites, including neutral noncoding DNA, are not comparable. These findings also draw into question the commonly utilized method of investigating how selection to increase translation accuracy influences codon usage. The work then focuses on implications for studies that assess codon adaptation, which is selection on codon usage to enhance translation rate, in plastid genes. A new test that does not require the use of noncoding DNA is proposed and applied. The results of this test suggest that far fewer plastid genes display codon adaptation than previously thought.

Highly expressed plastid genes display codon adaptation, which is defined as a bias toward a set of codons which are complementary to abundant tRNAs. This type of adaptation is similar to what is observed in highly expressed Escherichia coli genes and is probably the result of selection to increase translation efficiency. In the current work, the codon adaptation of plastid genes is studied with regard to three specific features that have been observed in E. coli and which may influence translation efficiency. These features are (1) a relatively low codon adaptation at the 5' end of highly expressed genes, (2) an influence of neighboring codons on codon usage at a particular site (codon context), and (3) a correlation between the level of codon adaptation of a gene and its amino acid content. All three features are found in plastid genes. First, highly expressed plastid genes have a noticeable decrease in codon adaptation over the first 10-20 codons. Second, for the twofold degenerate NNY codon groups, highly expressed genes have an overall bias toward the NNC codon, but this is not observed when the 3' neighboring base is a G. At these sites highly expressed genes are biased toward NNT instead of NNC. Third, plastid genes that have higher codon adaptations also tend to have an increased usage of amino acids with a high G + C content at the first two codon positions and GNN codons in particular. The correlation between codon adaptation and amino acid content exists separately for both cytosolic and membrane proteins and is not related to any obvious functional property. It is suggested that at certain sites selection discriminates between nonsynonymous codons based on translational, not functional, differences, with the result that the amino acid sequence of highly expressed proteins is partially influenced by selection for increased translation efficiency.

We investigated the evolutionary dynamics of the Adh gene family within the grasses (Poaceae), with the goal of using molecular evolutionary tools to understand the process of gene family diversification. We analyzed 21 Adh sequences representing a broad array of grasses. Phylogenetic analyses suggested that Adh duplicated into Adh1 and Adh2 before the radiation of the grasses roughly 65 MYA. Gene structure, including intron length, has varied little over this period. Conservation of intron length prompted investigation into the dynamics of intron evolution, particularly the ability of intron sequences to form secondary structures. Intron sequences did not have an extremely high or low minimum free energy of folding relative to permuted sequences, suggesting that individual Adh introns do not evolve under secondary structural constraints. For coding sequences, the diversification of Adh1 and Adh2 was marked by a shift in third-position G + C content. This shift may reflect differential selection for codon use. Diversification between Adh1 and Adh2 was also typified by a shift in nonsynonymous nucleotide substitution rates, but there was no evidence that relatively fast nonsynonymous nucleotide substitution rates in the Adh2 clade were a product of diversifying selection. Gene conversion may have played a role in retarding diversification of Adh1 and Adh2 in rice, but there is no evidence of gene conversion between paralogs in other taxa. Although the reasons for retention of two functional Adh genes remain obscure, we propose that a shift in gene expression was important for the retention of the two Adh gene copies within the grasses.

It is shown that the two strands of the chloroplast genome from Euglena gracilis are asymmetric with regards to nucleotide composition. This asymmetry switches at both the origin of replication and a location that is halfway around the circular genome from the origin. In both halves of the genome the leading strand is G+T-rich, having a bias toward G over C and T over A, and the lagging strand is A+C-rich. This asymmetry is probably the result of a difference in mutation dynamics between the leading and lagging strands. In addition to composition asymmetry, the two strands differ with regards to coding content. In both halves of the genome the vast majority of genes are coded by the leading strand. These two aspects of strand asymmetry are then applied to a statistical test for selection on codon usage. The results indicate that selection on codon usage is limited to genes on the leading strand; no gene on the A+C-rich lagging strand shows evidence for selection, suggesting that highly expressed genes are coded predominantly on the strand of DNA that is the leading strand during replication. On the basis of these observations it is proposed that the coding strand bias is generated by selection to code highly expressed genes on the leading strand to coordinate the direction of replication and transcription, thereby increasing the potential rate of both reactions.

A morphological data set and three sources of data from the chloroplast genome (two genes and a restriction site survey) were used to reconstruct the phylogenetic history of the pickerelweed family Pontederiaceae. The chloroplast data converged towards a single tree, presumably the true chloroplast phylogeny of the family. Unrooted trees estimated from each of the three chloroplast data sets were identical or extremely similar in shape to each other and mostly robustly supported. There was no evidence of significant heterogeneity among the data sets, and the few topological differences seen among unrooted trees from each chloroplast data set are probably artifacts of sampling error on short branches. Despite well-documented differences in rates of evolution for different characters in individual data sets, equally weighted parsimony permits accurate reconstructions of chloroplast relationships in Pontederiaceae. A separate morphology-based data set yielded trees that were very different from the chloroplast trees. Although there was substantial support from the morphological evidence for several major clades supported by chloroplast trees, most of the conflicting phylogenetic structure on the morphology trees was not robust. Nonetheless, several statistical tests of incongruence indicate significant heterogeneity between molecules and morphology. The source of this apparent incongruence appears to be a low ratio of phylogenetic signal to noise in the morphological data.

In the plant chloroplast genome the codon usage of the highly expressed psbA gene is unique and is adapted to the tRNA population, probably due to selection for translation efficiency. In this study the role of selection on codon usage in each of the fully sequenced chloroplast genomes, in addition to Chlamydomonas reinhardtii, is investigated by measuring adaptation to this pattern of codon usage. A method is developed which tests selection on each gene individually by constructing sequences with the same amino acid composition as the gene and randomly assigning codons based on the nucleotide composition of noncoding regions of that genome. The codon bias of the actual gene is then compared to a distribution of random sequences. The data indicate that within the algae selection is strong in Cyanophora paradoxa, affecting a majority of genes, of intermediate intensity in Odontella sinensis, and weaker in Porphyra purpurea and Euglena gracilis. In the plants, selection is found to be quite weak in Pinus thunbergii and the angiosperms but there is evidence that an intermediate level of selection exists in the liverwort Marchantia polymorpha. The role of selection is then further investigated in two comparative studies. It is shown that average relative codon bias is correlated with expression level and that, despite saturation levels of substitution, there is a strong correlation among the algae genomes in the degree of codon bias of homologous genes. All of these data indicate that selection for translation efficiency plays a significant role in determining the codon bias of chloroplast genes but that it acts with different intensities in different lineages. In general it is stronger in the algae than the higher plants, but within the algae Euglena is found to have several unusual features which are noted. The factors that might be responsible for this variation in intensity among the various genomes are discussed.

The psbA gene of the chloroplast genome has a codon usage that is unusual for plant chloroplast genes. In the present study the evolutionary status of this codon usage is tested by reconstructing putative ancestral psbA sequences to determine the pattern of change in codon bias during angiosperm divergence. It is shown that the codon biases of the ancestral genes are much stronger than all extant flowering plant psbA genes. This is related to previous work that demonstrated a significant increase in synonymous substitution in psbA relative to other chloroplast genes. It is suggested, based on the two lines of evidence, that the codon bias of this gene currently is not being maintained by selection. Rather, the atypical codon bias simply may be a remnant of an ancestral codon bias that now is being degraded by the mutation bias of the chloroplast genome, in other words, that the psbA gene is not at equilibrium. A model for the evolution of selective pressure on the codon usage of plant chloroplast genes is discussed.

Substitutions occurring in noncoding sequences of the plant chloroplast genome violate the independence of sites that is assumed by substitution models in molecular evolution. The probability that a substitution at a site is a transversion, as opposed to a transition, increases significantly with increasing A + T content of the two adjacent nucleotides. In the present study, this dependency of substitutions on local context is examined further in a number of noncoding regions from the chloroplast genome of members of the grass family (Poaceae). Two features were examined; the influence of specific neighboring bases, as opposed to the general A + T content, on transversion proportion and an influence on substitutions by nucleotides other than the two immediately adjacent to the site of substitution. In both cases, a significant effect was found. In the case of specific nucleotides, transversion proportion is significantly higher at sites with a pyrimidine immediately 5' on either strand. Substitutions at sites of the type YNR, where N is the site of substitution, have the highest rate of transversion. This specific effect is secondary to the A + T content effect such that, in terms of proportion of substitutions that are transversions, the nucleotides are ranked T > A > C > G as to their effect when they are immediately 5' to the site of substitution. In the case of nucleotides other than the immediate neighbors, a significant influence on substitution dynamics is observed in the case where the two neighboring bases are both A and/or T. Thus, substitutions are primarily, but not exclusively, influenced by the composition of the two nucleotides that are immediately adjacent. These results indicate that the pattern of molecular evolution of the plant chloroplast genome is extremely complex as a result of a variety of inter-site dependencies.

The psbA gene of the flowering plant chloroplast genome has a pattern of codon bias that differs from all other angiosperm chloroplast genes. In psbA, unlike all other chloroplast genes, the third-codon-position composition does not reflect the general genome compositional bias of a high A+T content. Instead, in specific synonymous groups, the codon use of psbA more closely corresponds to the tRNA population available for translation. Since it requires a composition unlike the genome composition bias, this pattern of codon use is likely to be the result of selection. Selective constraints on codon use are expected to result in decreased rates of synonymous substitution, and it has been observed that psbA has the lowest rate of synonymous substitution among plant chloroplast genes. In the present study, this is examined further by testing whether or not those synonymous groups that specifically have an atypical codon use in psbA have correspondingly low rates of silent substitution. An analysis of synonymous substitution rate, performed separately for different degeneracy classes of amino acids, shows that, contrary to the expectation, those sites that are presumed, based on their codon use, to be under selective constraint in psbA do not show low rates of substitution and are not responsible for the overall low rate of synonymous substitution in this gene. Instead, they actually show increased rates of substitution relative to other chloroplast genes. Two hypotheses concerning the role of selection in psbA are advanced to explain the results.

The alcohol dehydrogenase (Adh; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) gene family has two or three loci in a broad array of angiosperm species. The relative stability in the number of Adh loci led Gottlieb [Gottlieb, L. D. (1982) Science 216, 373-380] to propose that the Adh gene family arose from an ancient gene duplication. In this study, the isolation of three loci from the California fan palm (Washingtonia robusta) is reported. The three loci from palm are highly diverged. One palm Adh gene, referred to here as adhB, has been completely sequenced, including 950 nucleotides of the upstream regulatory region. For the second locus, adhA, 81% of the exon sequence is complete. Both show the same basic structure as grass Adh genes in terms of intron number and intron location. The third locus, adhC, for which only a small amount of sequence is available (12% of exon sequence) appears to be more highly diverged. Comparison of the Adh gene families from palms and grasses shows that the adh1 and adh2 genes of grasses, and the adhA and adhB genes of palms, arose by duplication following the divergence of the two families. This finding suggests that the multiple Adh loci in different monocot lineages are not the result of a single ancestral duplication but, rather, of multiple duplication events.

A number of studies have noted that nucleotide substitution rates at the chloroplast-encoded rbcL locus violate the molecular clock principle. Substitution rate variation at this plastid gene is particularly pronounced between palms and grasses; for example, a previous study estimated that substitution rates in rbcL sequences are approximately 5-fold faster in grasses than in palms. To determine whether a proportionate change in substitution rates also occurs in plant nuclear genes, we characterized nucleotide substitution rates in palm and grass sequences for the nuclear gene Adh. In this article, we report that palm sequences evolve at a rate of 2.61 x 10(-9) substitution per synonymous site per year, a rate which is slower than most plant nuclear genes. Grass Adh sequences evolve approximately 2.5-fold faster than palms at synonymous sites. Thus, synonymous rates in nuclear Adh genes show a marked decrease in palms relative to grasses, paralleling the pattern found at the plastid rbcL locus. This shared pattern indicates that synonymous rates are correlated between a nuclear and a plastid gene. Remarkably, nonsynonymous rates do not show this correlation. Nonsynonymous rates vary between two duplicated grass Adh loci, and nonsynonymous rates at the palm Adh locus are not markedly reduced relative to grasses.

Plant chloroplast genes have a codon use that reflects the genome compositional bias of a high A+T content with the single exception of the highly translated psbA gene which codes for the photosystem II D1 protein. The codon usage of plant psbA corresponds more closely to the limited tRNA population of the chloroplast and is very similar to the codon use observed in the chloroplast genes of the green alga Chlamydomonas reinhardtii. This pattern of codon use may be an adaptation for increased translation efficiency. A correspondence between codon use of plant psbA and Chlamydomonas chloroplast genes and the tRNAs coded by the chloroplast genome, however, is not observed in all synonymous codon groups. It is shown here that the degree of correspondence between codon use and tRNA population in different synonymous groups is correlated with the second codon position composition. Synonymous groups with an A or T at the second codon position have a high representation of codons for which a complementary tRNA is coded by the chloroplast genome. Those with a G or C at the second position have an increased representation of codons that bind a chloroplast tRNA by wobble. It is proposed that the difference between synonymous groups in terms of codon adaptation to the tRNA population in plant psbA and Chlamydomonas chloroplast genes may be the result of differences in second position composition.

Nucleotide sequence from a region of the chloroplast genome is presented for 12 species spanning four subfamilies of the grass family. The region contains the coding sequence for the rbcL gene and the intergenic spacer between the gene coding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) and the photosystem I gene psaI. This intergenic spacer contains a pseudogene for rpl23 as well as two noncoding segments with different A+T contents. Using the sequence of rbcL a chloroplast phylogeny of this family was constructed by parsimony. Variable sites of the two noncoding segments were traced onto the phylogeny to study the dynamics of base substitution. This was also performed for the fourfold-degenerate sites of the rbcL gene. A wide variation in transversion/transition is observed between the two noncoding segments and between the noncoding DNA and the fourfold-degenerate sites of rbcL. This variation is correlated with regional A+T content. As regional A+T content decreases, the ratio of transversions to transitions also decreases. Substitutions were then scored in relation to neighboring base composition. The composition of the two bases immediately flanking each substitution is highly correlated with the transversion/transition bias. When both the 5' and 3' flanking bases are an A or a T, transversions are observed 2.2 times as frequently as transitions. When either or both neighbors are a C or a G, the opposite trend is found; transitions are observed 1.5 times more frequently than transversions.

The correspondence between the transversion/transition ratio and the neighboring base composition in chloroplast DNA is examined. For 18 noncoding regions of the chloroplast genome, alignments between rice (Oryza sativa) and maize (Zea mays) were generated by two different methods. Difficulties of aligning noncoding DNA are discussed, and the alignments are analyzed in a manner that reduces alignment artifacts. Sequence divergence is < 10%, so multiple substitutions at a site are assumed to be rare. Observed substitutions were analyzed with respect to the A+T content of the two immediately flanking bases. It is shown that as this content increases, the proportion of transversions also increases. When both the 5'- and 3'-flanking nucleotides are G or C (A+T content of 0), only 25% of the observed substitutions are transversions. However, when both the 5'- and 3'-flanking nucleotides are A or T (A+T content of 2), 57% of the observed substitutions are transversions. Therefore, the influence of flanking base composition on substitutions, previously reported for a single noncoding region, is a general feature of the chloroplast genome.

The chloroplast genome (cpDNA) of plants has been a focus of research in plant molecular evolution and systematics. Several features of this genome have facilitated molecular evolutionary analyses. First, the genome is small and constitutes an abundant component of cellular DNA. Second, the chloroplast genome has been extensively characterized at the molecular level providing the basic information to support comparative evolutionary research. And third, rates of nucleotide substitution are relatively slow and therefore provide the appropriate window of resolution to study plant phylogeny at deep levels of evolution. Despite a conservative rate of evolution and a relatively stable gene content, comparative molecular analyses reveal complex patterns of mutational changes. Non-coding regions of cpDNA diverge through insertion/deletion changes that are sometimes site dependent. Coding genes exhibit different patterns of codon bias that appear to violate the equilibrium assumptions of some evolutionary models. Rates of molecular change often vary among plant families and orders in a manner that violates the assumption of a simple molecular clock. Finally, protein-coding genes exhibit patterns of amino acid change that appear to depend on protein structure, and these patterns may reveal subtle aspects of structure/function relationships. Only comparative studies of molecular sequences have the resolution to reveal this underlying complexity. A complete description of the complexity of molecular change is essential to a full understanding of the mechanisms of evolutionary change and in the formulation of realistic models of mutational processes.

Codon fitnesses for chloroplast genes were estimated using the relative synonymous codon use of psbA, which has a different pattern of codon use than other chloroplast genes and is the major translation product of the chloroplast. These estimates were used to calculate the codon adaptation index (CAI) of chloroplast genes from Marchantia polymorpha, Nicotiana tabacum, and Chlamydomonas reinhardtii. The genes with the highest CAI values in M. polymorpha correspond to those that are expressed at the highest levels. The rate of divergence between M. polymorpha and both C. reinhardtii and N. tabacum is inversely related to the CAI value of the M. polymorpha gene. The data suggest that selection is acting on the synonymous codon use of the highly expressed genes of the M. polymorpha chloroplast genome. The data set is inconclusive about N. tabacum genes, but, as there is a weaker correspondence between CAI value and expression level, it suggests that selection is not operating in this lineage.

Codon use in the three sequenced chloroplast genomes (Marchantia, Oryza, and Nicotiana) is examined. The chloroplast has a bias in that codons NNA and NNT are favored over synonymous NNC and NNG codons. This appears to be a consequence of an overall high A + T content of the genome. This pattern of codon use is not followed by the psb A gene of all three genomes and other psb A sequences examined. In this gene, the codon use favors NNC over NNT for twofold degenerate amino acids. In each case the only tRNA coded by the genome is complementary to the NNC codon. This codon use is similar to the codon use by chloroplast genes examined from Chlamydomonas reinhardtii. Since psb A is the major translation product of the chloroplast, this suggests that selection is acting on the codon use of this gene to adapt codons to tRNA availability, as previously suggested for unicellular organisms.