Fourfold Degenerate Sites Research Articles

Mutation Rate and Effective Population Size of the Model Cooperative Bacterium Myxococcus xanthus.

Intrinsic rates of genetic mutation have diverged greatly across taxa and exhibit statistical associations with several other parameters and features. These include effective population size (Ne), genome size, and gametic multicellularity, with the latter being associated with both increased mutation rates and decreased effective population sizes. However, data sufficient to test for possible relationships between microbial multicellularity and mutation rate (µ) are lacking. Here, we report estimates of two key population-genetic parameters, Ne and µ, for Myxococcus xanthus, a bacterial model organism for the study of aggregative multicellular development, predation, and social swarming. To estimate µ, we conducted an ∼400-day mutation accumulation experiment with 46 lineages subjected to regular single colony bottlenecks prior to clonal regrowth. Upon conclusion, we sequenced one clonal-isolate genome per lineage. Given collective evolution for 85,323 generations across all lines, we calculate a per base-pair mutation rate of ∼5.5 × 10-10 per site per generation, one of the highest mutation rates among free-living eubacteria. Given our estimate of µ, we derived Ne at ∼107 from neutral diversity at four-fold degenerate sites across two dozen M. xanthus natural isolates. This estimate is below average for eubacteria and strengthens an already clear negative correlation between µ and Ne in prokaryotes. The higher and lower than average mutation rate and Ne for M. xanthus, respectively, amplify the question of whether any features of its multicellular life cycle-such as group-size reduction during fruiting-body development-or its highly structured spatial distribution have significantly influenced how these parameters have evolved.

The genomic signatures of evolutionary stasis.

Evolutionary stasis characterizes lineages that seldom speciate and show little phenotypic change over long stretches of geological time. Although lineages that appear to exhibit evolutionary stasis are often called living fossils, no single mechanism is thought to be responsible for their slow rates of morphological evolution and low species diversity. Some analyses of molecular evolutionary rates in a handful of living fossil lineages have indicated that these clades exhibit slow rates of genomic change. Here, we investigate mechanisms of evolutionary stasis using a dataset of 1,105 exons for 481 vertebrate species. We demonstrate that two ancient clades of ray-finned fishes classically called living fossils, gars and sturgeons, exhibit the lowest rates of molecular substitution in protein-coding genes among all jawed vertebrates. Comparably low rates of evolution are observed at fourfold degenerate sites in gars and sturgeons, implying a mechanism of stasis decoupled from selection that we speculate is linked to a highly effective DNA repair apparatus. We show that two gar species last sharing common ancestry over 100 million years ago produce morphologically intermediate and fertile hybrids in the wild. This makes gars the oldest naturally hybridizing divergence among eukaryotes and supports a theoretical prediction that slow rates of nucleotide substitution across the genome slow the accumulation of genetic incompatibilities, enabling hybridization across deeply divergent lineages and slowing the rate of speciation over geological timescales. Our results help establish molecular stasis as a barrier to speciation and phenotypic innovation and provide a mechanism to explain the low species diversity in living fossil lineages.

Consequences of asymmetric mutational pressure for the dynamic of linear B-cell epitopes repertoire of influenza a virus neuraminidase rearrangement

Full-length nucleotide sequences of avian influenza A virus neuraminidase coding region (20,631 sequences) were analyzed and compared with those isolated from viruses infecting human and swine (63,750 sequences). If in fourfold degenerate sites there is asymmetric A-bias that may be more or less asymmetric depending on the type of neuraminidase and the host, than in twofold degenerate sites from third codon positions there is a strong asymmetric U-bias in coding regions of N4, N5, and N8 isolated from viruses infecting birds, as well as in those of N1 and N2 isolated from viruses infecting human, swine, and birds, while in coding regions of N9 isolated from birds, there is surprisingly strong C-bias, and in sequences of N3, N6, and N7 the usage of C is quite close to the level of U. Revealed stabilization of both U and C in twofold degenerate sites is the evidence of frequent changes in mutational pressure direction. Asymmetric mutational pressure was one of the sources of amino acid replacements that resulted in an equal percentage of sites with appeared and disappeared linear B-cell epitopes in N1, N2, N4, and N5 (33.62–35.33% vs. 32.41–36.45%, respectively), and controlled by the immune pressure it resulted in a stronger tendency to disappear for B-cell epitopes of N3, N6, N7, N8, and N9 of avian viruses (8.74–28.77% vs. 28.96–38.89%). The lack of correlation between nucleotide usages in fourfold and twofold degenerate sites for three nucleotides, except U, is a strong evidence of mutational pressure theory.

DNA repair-related genes are the key for stony coral ancestors to survive under elevated levels of UVR

DNA damage response (DDR) is a complicated network to defend against physical or chemical changes in DNA in all animals. Elevated levels of ultraviolet radiation (UVR) caused DNA damage, which was a reason for the mass extinction that occurred at the Devonian/Carboniferous (D/C) boundary approximately 359 million years ago (Ma). However, the molecular adaptation of the stony coral ancestors that strangely survived the D/C boundary mass extinction is not well understood. In the present study, the molecular clock analysis using fourfold degenerate sites of 1,463 homologous genes of different stony coral species (a representative group of marine organisms with calcareous skeletons) suggested that their common ancestors originated 384.24 Ma, i.e., slightly earlier than the D/C transition. We identified 21 rapidly evolving genes (REGs) and 49 positive selection genes (PSGs) that were significantly enriched in diverse pathways, including the mitotic cell cycle process, intracellular protein transport, and DNA synthesis involved in DNA repair. Interestingly, four REGs and 21 PSGs were significantly enriched in DDR pathways, including the mitotic cell cycle process, DNA synthesis involved in DNA repair, and cellular response to DNA damage stimulus pathways. We hypothesize that enriched DDR genes are likely involved in the enhanced ability of ancient stony corals to detect and repair DNA damage. For example, the DNA polymerase epsilon catalytic subunit (POLE) gene, which encodes the DNA polymerase epsilon catalytic subunit A, mediates interaction with the other three catalytic subunits through its nonenzymatic carboxy-terminal domain. POLE may potentially enhance the binding ability to other subunits to strengthen its function because eight positive selection sites were distributed in the C-terminal of POLE and on the surface of the simulated 3D protein model. Therefore, our results demonstrated for the first time that the precise transfer of DNA information may help stony coral ancestors survive in elevated levels of ultraviolet radiation, suggesting that DDR levels may be critical to the environmental adaptation of calcareous skeletal organisms during climate change.

Silver nanoparticles elevate mutagenesis of eukaryotic genomes.

Metal nanoparticles, especially silver, have been used in various medical scenarios, due to their excellent antimicrobial effects. Recent studies have shown that AgNPs do not exert mutagenic effects on target bacteria, but the degree to which they compromise eukaryotic genomes remains unclear. To study this, we evaluated the mutagenic effects of AgNPs on the fission yeast Schizosaccharomyces pombe ATCC-16979, of which ∼23% genes are homologous to human ones, at single-nucleotide resolution, and whole-genome scale by running 283 mutation accumulation lines for ∼260,000 cell divisions in total. We also explored the action and mutagenesis mechanisms using differential gene-expression analysis based on RNAseq. Upon AgNPs treatment, the genomic base-substitution mutation rate of S. pombe at four-fold degenerate sites increased by 3.46×, and small indels were prone to occur in genomic regions that are not simple sequence repeats. The G:C → T:A transversion rate was also significantly increased, likely mostly from oxidative damage. Thus, in addition to their antimicrobial potency, AgNPs might pose slight genotoxicity threats to eukaryotic and possibly human genomes, though at a low magnitude.

Do Noncoding and Coding Sites in Angiosperm Chloroplast DNA Have Different Mutation Processes?

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.

A Chromosome-Scale Genome Assembly of Mitragyna speciosa (Kratom) and the Assessment of Its Genetic Diversity in Thailand.

Simple SummaryMitragyna speciosa (Kratom) is a narcotic plant indigenous to Southeast Asian countries, including Thailand. Traditionally, M. speciosa has been used as medicine to treat diarrhea and has anti-coughing, analgesic, and fever-reducing properties. Its leaves are commonly chewed by workers during physical labor for their coca-like stimulant effect to increase stamina and endurance. To identify important bioactive alkaloids with potential pharmaceutical uses, we performed a whole genome sequencing of Kratom to obtain information relating to the gene content in its genome, which will facilitate an improved understanding of the biosynthesis pathway and provide resources for assessing the genetic diversity in M. speciosa.Mitragyna speciosa (Kratom) is a tropical narcotic plant native to Southeast Asia with unique pharmacological properties. Here, we report the first chromosome-scale assembly of the M. speciosa genome. We employed PacBio sequencing to obtain a preliminary assembly, which was subsequently scaffolded using the chromatin contact mapping technique (Hi-C) into 22 pseudomolecules. The final assembly was 692 Mb with a scaffold N50 of 26 Mb. We annotated a total of 39,708 protein-coding genes, and our gene predictions recovered 98.4% of the highly conserved orthologs based on the BUSCO analysis. The phylogenetic analysis revealed that M. speciosa diverged from the last common ancestors of Coffea arabica and Coffea canephora approximately 47.6 million years ago. Our analysis of the sequence divergence at fourfold-degenerate sites from orthologous gene pairs provided evidence supporting a genome-wide duplication in M. speciosa, agreeing with the report that members of the genus Mitragyna are tetraploid. The STRUCTURE and principal component analyses demonstrated that the 85 M. speciosa accessions included in this study were an admixture of two subpopulations. The availability of our high-quality chromosome-level genome assembly and the transcriptomic resources will be useful for future studies on the alkaloid biosynthesis pathway, as well as comparative phylogenetic studies in Mitragyna and related species.

Chromosome-level genome assembly of Indian mangrove (Ceriops tagal) revealed a genome-wide duplication event predating the divergence of Rhizophoraceae mangrove species.

Mangrove ecosystems are unique, highly diverse, provide benefits to humans, and aid in coastal protection. The Indian mangrove, or spurred mangrove, [Ceriops tagal (Perr.) C. B. Rob.] is a member of the Rhizophoraceae family and is commonly found along the intertidal zones in tropical regions in Southeast Asia, southern Asia, and Africa. Here, we present the first high-quality reference genome assembly of the Ceriops species. A preliminary draft assembly, generated from the 10× Genomics linked-read library, was scaffolded using the proximity ligation chromatin contact mapping technique (Hi-C) to obtain a chromosome-scale assembly of 231,919,005 bases with an N50 length of 11,408,429 bases. The benchmarking universal single-copy orthologs (BUSCO) analysis revealed that C. tagal gene predictions recovered 95.8% of the highly conserved orthologs. Phylogenetic analyses suggested that C. tagal diverged from the last common ancestor of flat-leaf spurred mangrove [C. decandra (Griff.) Ding Hou] and C. zippeliana Blume ∼10.4 million yr ago (MYA), and the last common ancestor of genera Ceriops, Kandelia, and Rhizophora diverged from that of genus Bruguiera ∼49.4 MYA. In addition, our analysis of the transversion rate at fourfold-degenerate sites from orthologous gene pairs provided evidence supporting a recent whole-genome duplication in C. tagal. The STRUCTURE and principal component analyses illustrated that C. tagal individuals investigated in this study were the admixture of two subpopulations, the genetic background of which was influenced primarily by location. The availability of genomic and transcriptomic resources and biodiversity data reported in this work will be useful for future studies that may shed light on adaptive evolutions of mangrove species.

A positive correlation between GC content and growth temperature in prokaryotes

BackgroundGC pairs are generally more stable than AT pairs; GC-rich genomes were proposed to be more adapted to high temperatures than AT-rich genomes. Previous studies consistently showed positive correlations between growth temperature and the GC contents of structural RNA genes. However, for the whole genome sequences and the silent sites of the codons in protein-coding genes, the relationship between GC content and growth temperature is in a long-lasting debate.ResultsWith a dataset much larger than previous studies (681 bacteria and 155 archaea with completely assembled genomes), our phylogenetic comparative analyses showed positive correlations between optimal growth temperature (Topt) and GC content both in bacterial and archaeal structural RNA genes and in bacterial whole genome sequences, chromosomal sequences, plasmid sequences, core genes, and accessory genes. However, in the 155 archaea, we did not observe a significant positive correlation of Topt with whole-genome GC content (GCw) or GC content at four-fold degenerate sites. We randomly drew 155 samples from the 681 bacteria for 1000 rounds. In most cases (> 95%), the positive correlations between Topt and genomic GC contents became statistically nonsignificant (P > 0.05). This result suggested that the small sample sizes might account for the lack of positive correlations between growth temperature and genomic GC content in the 155 archaea and the bacterial samples of previous studies. Comparing the GC content among four categories (psychrophiles/psychrotrophiles, mesophiles, thermophiles, and hyperthermophiles) also revealed a positive correlation between GCw and growth temperature in bacteria. By including the GCw of incompletely assembled genomes, we expanded the sample size of archaea to 303. Positive correlations between GCw and Topt appear especially after excluding the halophilic archaea whose GC contents might be strongly shaped by intense UV radiation.ConclusionsThis study explains the previous contradictory observations and ends a long debate. Prokaryotes growing in high temperatures have higher GC contents. Thermal adaptation is one possible explanation for the positive association. Meanwhile, we propose that the elevated efficiency of DNA repair in response to heat mutagenesis might have the by-product of increasing GC content like that happens in intracellular symbionts and marine bacterioplankton.

A chromosome-scale reference genome assembly of yellow mangrove (Bruguiera parviflora) reveals a whole genome duplication event associated with the Rhizophoraceae lineage.

Mangrove forest ecosystems support a diverse flora and fauna of marine and terrestrial species and have important direct and indirect economic, ecological and social values to mankind. Yellow mangrove (Bruguiera parviflora) belongs to the Rhizophoraceae family and is widely distributed in the intertidal zones along sheltered coastal areas in tropical latitudes. Here, we present a high-quality, chromosome-level assembly of the B.parviflora genome. We employed the 10x Genomics linked-read technology to obtain a preliminary assembly, which was subsequently scaffolded using the long-range chromatin contact mapping technique (HiC) to obtain a final assembly containing 213,026,782 bases in 10,045 scaffolds with an N50 length of 10,906,948 bases. Our gene prediction recovered 96.5% of the highly conserved orthologues in the Embryophyta lineage based on the Benchmarking Universal Single-Copy Orthologues (BUSCO) analysis. We analysed the transversion rate at fourfold-degenerate sites from orthologous gene pairs and discovered evidence supporting a recent whole-genome duplication event in B.parviflora and other Rhizophoreae members. Comparative studies based on single-copy orthologous genes indicated that B.parviflora and Bruguiera gymnorrhiza diverged approximately 24.1 million years ago. The population structure analysis revealed that 63 B.parviflora accessions from different geographical regions in Thailand were an admixture of two subpopulations. The examination of alternative splicing events in B.parviflora showed that the most prevalent splicing mechanism was intron retention. This high-quality genome assembly together with the genetic diversity information obtained from the germplasm provide useful genomic resources for future studies on comparative phylogenetics and evolution of adaptive traits in mangrove species.

Genomic Characterization of the Barnacle Balanus improvisus Reveals Extreme Nucleotide Diversity in Coding Regions

Barnacles are key marine crustaceans in several habitats, and they constitute a common practical problem by causing biofouling on man-made marine constructions and ships. Despite causing considerable ecological and economic impacts, there is a surprising void of basic genomic knowledge, and a barnacle reference genome is lacking. We here set out to characterize the genome of the bay barnacle Balanus improvisus (= Amphibalanus improvisus) based on short-read whole-genome sequencing and experimental genome size estimation. We show both experimentally (DNA staining and flow cytometry) and computationally (k-mer analysis) that B. improvisus has a haploid genome size of ~ 740 Mbp. A pilot genome assembly rendered a total assembly size of ~ 600 Mbp and was highly fragmented with an N50 of only 2.2 kbp. Further assembly-based and assembly-free analyses revealed that the very limited assembly contiguity is due to the B. improvisus genome having an extremely high nucleotide diversity (π) in coding regions (average π ≈ 5% and average π in fourfold degenerate sites ≈ 20%), and an overall high repeat content (at least 40%). We also report on high variation in the α-octopamine receptor OctA (average π = 3.6%), which might increase the risk that barnacle populations evolve resistance toward antifouling agents. The genomic features described here can help in planning for a future high-quality reference genome, which is urgently needed to properly explore and understand proteins of interest in barnacle biology and marine biotechnology and for developing better antifouling strategies.

De novo assembly of a chromosome-scale reference genome for the northern flicker Colaptes auratus.

The northern flicker, Colaptes auratus, is a widely distributed North American woodpecker and a long-standing focal species for the study of ecology, behavior, phenotypic differentiation, and hybridization. We present here a highly contiguous de novo genome assembly of C. auratus, the first such assembly for the species and the first published chromosome-level assembly for woodpeckers (Picidae). The assembly was generated using a combination of short-read Chromium 10× and long-read PacBio sequencing, and further scaffolded with chromatin conformation capture (Hi-C) reads. The resulting genome assembly is 1.378 Gb in size, with a scaffold N50 of 11 and a scaffold L50 of 43.948 Mb. This assembly contains 87.4–91.7% of genes present across four sets of universal single-copy orthologs found in tetrapods and birds. We annotated the assembly both for genes and repetitive content, identifying 18,745 genes and a prevalence of ∼28.0% repetitive elements. Lastly, we used fourfold degenerate sites from neutrally evolving genes to estimate a mutation rate for C. auratus, which we estimated to be 4.007 × 10−9 substitutions/site/year, about 1.5× times faster than an earlier mutation rate estimate of the family. The highly contiguous assembly and annotations we report will serve as a resource for future studies on the genomics of C. auratus and comparative evolution of woodpeckers.

Translation-Associated Mutational U-Pressure in the First ORF of SARS-CoV-2 and Other Coronaviruses.

Within 4 months of the ongoing COVID-19 pandemic caused by SARS-CoV-2, more than 250 nucleotide mutations have been detected in ORF1ab of the virus isolated from infected persons from different parts of the globe. These observations open up an obvious question about the rate and direction of mutational pressure for further vaccine and therapeutics designing. In this study, we did a comparative analysis of ORF1a and ORF1b by using the first isolate (Wuhan strain) as the parent sequence. We observed that most of the nucleotide mutations are C to U transitions. The rate of synonymous C to U transitions is significantly higher than the rate of non-synonymous ones, indicating negative selection on amino acid substitutions. Further, trends in nucleotide usage bias have been investigated in 49 coronaviruses species. A strong bias in nucleotide usage in fourfold degenerate sites toward uracil residues is seen in ORF1ab of all the studied coronaviruses: both in the ORF1a and in the ORF1b translated thanks to the programmed ribosomal frameshifting that has an efficiency of 14 – 45% in different species. A more substantial mutational U-pressure is observed in ORF1a than in ORF1b perhaps because ORF1a is translated more frequently than ORF1b. Mutational U-pressure is there even in ORFs that are not translated from genomic RNA plus strands, but the bias is weaker than in ORF1ab. Unlike other nucleotide mutations, mutational U-pressure caused by cytosine deamination, mostly occurring during the RNA plus strand replication and also translation, cannot be corrected by the proof-reading machinery of coronaviruses. The knowledge generated on the mutational U-pressure that becomes stronger during translation of viral RNA plus strands has implications for vaccine and nucleoside analog development for treating COVID-19 and other coronavirus infections.

Exonic splice regulation imposes strong selection at synonymous sites.

What proportion of coding sequence nucleotides have roles in splicing, and how strong is the selection that maintains them? Despite a large body of research into exonic splice regulatory signals, these questions have not been answered. This is because, to our knowledge, previous investigations have not explicitly disentangled the frequency of splice regulatory elements from the strength of the evolutionary constraint under which they evolve. Current data are consistent both with a scenario of weak and diffuse constraint, enveloping large swaths of sequence, as well as with well-defined pockets of strong purifying selection. In the former case, natural selection on exonic splice enhancers (ESEs) might primarily act as a slight modifier of codon usage bias. In the latter, mutations that disrupt ESEs are likely to have large fitness and, potentially, clinical effects. To distinguish between these scenarios, we used several different methods to determine the distribution of selection coefficients for new mutations within ESEs. The analyses converged to suggest that ∼15%–20% of fourfold degenerate sites are part of functional ESEs. Most of these sites are under strong evolutionary constraint. Therefore, exonic splice regulation does not simply impose a weak bias that gently nudges coding sequence evolution in a particular direction. Rather, the selection to preserve these motifs is a strong force that severely constrains the evolution of a substantial proportion of coding nucleotides. Thus synonymous mutations that disrupt ESEs should be considered as a potentially common cause of single-locus genetic disorders.

Genomic distribution and estimation of nucleotide diversity in natural populations: perspectives from the collared flycatcher (Ficedula albicollis) genome.

Properly estimating genetic diversity in populations of nonmodel species requires a basic understanding of how diversity is distributed across the genome and among individuals. To this end, we analysed whole-genome resequencing data from 20 collared flycatchers (genome size ≈1.1Gb; 10.13 million single nucleotide polymorphisms detected). Genomewide nucleotide diversity was almost identical among individuals (mean=0.00394, range=0.00384-0.00401), but diversity levels varied extensively across the genome (95% confidence interval for 200-kb windows=0.0013-0.0053). Diversity was related to selective constraint such that in comparison with intergenic DNA, diversity at fourfold degenerate sites was reduced to 85%, 3' UTRs to 82%, 5' UTRs to 70% and nondegenerate sites to 12%. There was a strong positive correlation between diversity and chromosome size, probably driven by a higher density of targets for selection on smaller chromosomes increasing the diversity-reducing effect of linked selection. Simulations exploring the ability of sequence data from a small number of genetic markers to capture the observed diversity clearly demonstrated that diversity estimation from finite sampling of such data is bound to be associated with large confidence intervals. Nevertheless, we show that precision in diversity estimation in large outbred population benefits from increasing the number of loci rather than the number of individuals. Simulations mimicking RAD sequencing showed that this approach gives accurate estimates of genomewide diversity. Based on the patterns of observed diversity and the performed simulations, we provide broad recommendations for how genetic diversity should be estimated in natural populations.

The geographic distribution and complex evolutionary history of the NX-2 trichothecene chemotype from Fusarium graminearum

Fusarium graminearum and 21 related species comprising the F. sambucinum species complex lineage 1 (FSAMSC-1) are the most important Fusarium Head Blight pathogens of cereal crops world-wide. FSAMSC-1 species typically produce type B trichothecenes. However, some F. graminearum strains were recently found to produce a novel type A trichothecene (NX-2) resulting from functional variation in the trichothecene biosynthetic enzyme Tri1. We used a PCR-RFLP assay targeting the TRI1 gene to identify the NX-2 allele among a global collection of 2515 F. graminearum. NX-2 isolates were only found in southern Canada and the northern U.S., where they were observed at low frequency (1.8%), but over a broader geographic range and set of cereal hosts than previously recognized. Phylogenetic analyses of TRI1 and adjacent genes produced gene trees that were incongruent with the history of species divergence within FSAMSC-1, indicating trans-species evolution of ancestral polymorphism. In addition, placement of NX-2 strains in the TRI1 gene tree was influenced by the accumulation of nonsynonymous substitutions associated with the evolution of the NX-2 chemotype, and a significant (P<0.001) change in selection pressure was observed along the NX-2 branch (ω=1.16) in comparison to other branches (ω=0.17) in the TRI1 phylogeny. Parameter estimates were consistent with positive selection for specific amino-acid changes during the evolution of NX-2, but direct tests of positive selection were not significant. Phylogenetic analyses of fourfold degenerate sites and intron sequences in TRI1 indicated the NX-2 chemotype had a single evolutionary origin and evolved recently from a type B ancestor. Our results indicate the NX-2 chemotype may be indigenous, and possibly endemic, to southern Canada and the northern U.S. In addition, we demonstrate that the evolution of TRI1 within FSAMSC-1 has been complex, with evidence of trans-species evolution and chemotype-specific shifts in selective constraint.

Are Synonymous Sites in Primates and Rodents Functionally Constrained?

It has been claimed that synonymous sites in mammals are under selective constraint. Furthermore, in many studies the selective constraint at such sites in primates was claimed to be more stringent than that in rodents. Given the larger effective population sizes in rodents than in primates, the theoretical expectation is that selection in rodents would be more effective than that in primates. To resolve this contradiction between expectations and observations, we used processed pseudogenes as a model for strict neutral evolution, and estimated selective constraint on synonymous sites using the rate of substitution at pseudosynonymous and pseudononsynonymous sites in pseudogenes as the neutral expectation. After controlling for the effects of GC content, our results were similar to those from previous studies, i.e., synonymous sites in primates exhibited evidence for higher selective constraint that those in rodents. Specifically, our results indicated that in primates up to 24% of synonymous sites could be under purifying selection, while in rodents synonymous sites evolved neutrally. To further control for shifts in GC content, we estimated selective constraint at fourfold degenerate sites using a maximum parsimony approach. This allowed us to estimate selective constraint using mutational patterns that cause a shift in GC content (GT ↔ TG, CT ↔ TC, GA ↔ AG, and CA ↔ AC) and ones that do not (AT ↔ TA and CG ↔ GC). Using this approach, we found that synonymous sites evolve neutrally in both primates and rodents. Apparent deviations from neutrality were caused by a higher rate of C → A and C → T mutations in pseudogenes. Such differences are most likely caused by the shift in GC content experienced by pseudogenes. We conclude that previous estimates according to which 20-40% of synonymous sites in primates were under selective constraint were most likely artifacts of the biased pattern of mutation.

Rearrangement and evolution of mitochondrial genomes in parrots

Mitochondrial genome rearrangements that result in control region duplication have been described for a variety of birds, but the mechanisms leading to their appearance and maintenance remain unclear, and their effect on sequence evolution has not been explored. A recent survey of mitochondrial genomes in the Psittaciformes (parrots) found that control region duplications have arisen independently at least six times across the order. We analyzed complete mitochondrial genome sequences from 20 parrot species, including representatives of each lineage with control region duplications, to document the gene order changes and to examine effects of genome rearrangements on patterns of sequence evolution. The gene order previously reported for Amazona parrots was found for four of the six independently derived genome rearrangements, and a previously undescribed gene order was found in Prioniturus luconensis, representing a fifth clade with rearranged genomes; the gene order resulting from the remaining rearrangement event could not be confirmed. In all rearranged genomes, two copies of the control region are present and are very similar at the sequence level, while duplicates of the other genes involved in the rearrangement show signs of degeneration or have been lost altogether. We compared rates of sequence evolution in genomes with and without control region duplications and did not find a consistent acceleration or deceleration associated with the duplications. This could be due to the fact that most of the genome rearrangement events in parrots are ancient, and additionally, to an effect of body size on evolutionary rate that we found for mitochondrial but not nuclear sequences. Base composition analyses found that relative to other birds, parrots have unusually strong compositional asymmetry (AT- and GC-skew) in their coding sequences, especially at fourfold degenerate sites. Furthermore, we found higher AT skew in species with control region duplications. One potential cause for this compositional asymmetry is that parrots have unusually slow mtDNA replication. If this is the case, then any replicative advantage provided by having a second control region could result in selection for maintenance of both control regions once duplicated.

The complete nucleotide sequence of the mitochondrial genome of Bactrocera minax (Diptera: Tephritidae).

The complete 16,043 bp mitochondrial genome (mitogenome) of Bactrocera minax (Diptera: Tephritidae) has been sequenced. The genome encodes 37 genes usually found in insect mitogenomes. The mitogenome information for B. minax was compared to the homologous sequences of Bactrocera oleae, Bactrocera tryoni, Bactrocera philippinensis, Bactrocera carambolae, Bactrocera papayae, Bactrocera dorsalis, Bactrocera correcta, Bactrocera cucurbitae and Ceratitis capitata. The analysis indicated the structure and organization are typical of, and similar to, the nine closely related species mentioned above, although it contains the lowest genome-wide A+T content (67.3%). Four short intergenic spacers with a high degree of conservation among the nine tephritid species mentioned above and B. minax were observed, which also have clear counterparts in the control regions (CRs). Correlation analysis among these ten tephritid species revealed close positive correlation between the A+T content of zero-fold degenerate sites (P0FD), the ratio of nucleotide substitution frequency at P0FD sites to all degenerate sites (zero-fold degenerate sites, two-fold degenerate sites and four-fold degenerate sites) and amino acid sequence distance (ASD) were found. Further, significant positive correlation was observed between the A+T content of four-fold degenerate sites (P4FD) and the ratio of nucleotide substitution frequency at P4FD sites to all degenerate sites; however, we found significant negative correlation between ASD and the A+T content of P4FD, and the ratio of nucleotide substitution frequency at P4FD sites to all degenerate sites. A higher nucleotide substitution frequency at non-synonymous sites compared to synonymous sites was observed in nad4, the first time that has been observed in an insect mitogenome. A poly(T) stretch at the 5′ end of the CR followed by a [TA(A)]n-like stretch was also found. In addition, a highly conserved G+A-rich sequence block was observed in front of the poly(T) stretch among the ten tephritid species and two tandem repeats were present in the CR.

Phylogenomics of strongylocentrotid sea urchins

BackgroundStrongylocentrotid sea urchins have a long tradition as model organisms for studying many fundamental processes in biology including fertilization, embryology, development and genome regulation but the phylogenetic relationships of the group remain largely unresolved. Although the differing isolating mechanisms of vicariance and rapidly evolving gamete recognition proteins have been proposed, a stable and robust phylogeny is unavailable.ResultsWe used a phylogenomic approach with mitochondrial and nuclear genes taking advantage of the whole-genome sequencing of nine species in the group to establish a stable (i.e. concordance in tree topology among multiple lies of evidence) and robust (i.e. high nodal support) phylogenetic hypothesis for the family Strongylocentrotidae. We generated eight draft mitochondrial genome assemblies and obtained 13 complete mitochondrial genes for each species. Consistent with previous studies, mitochondrial sequences failed to provide a reliable phylogeny. In contrast, we obtained a very well-supported phylogeny from 2301 nuclear genes without evidence of positive Darwinian selection both from the majority of most-likely gene trees and the concatenated fourfold degenerate sites: ((P. depressus, (M. nudus, M. franciscanus), (H. pulcherrimus, (S. purpuratus, (S. fragilis, (S. pallidus, (S. droebachiensis, S. intermedius)). This phylogeny was consistent with a single invasion of deep-water environments followed by a holarctic expansion by Strongylocentrotus. Divergence times for each species estimated with reference to the divergence times between the two major clades of the group suggest a correspondence in the timing with the opening of the Bering Strait and the invasion of the holarctic regions.ConclusionsNuclear genome data contains phylogenetic signal informative for understanding the evolutionary history of this group. However, mitochondrial genome data does not. Vicariance can explain major patterns observed in the phylogeny. Other isolating mechanisms are appropriate to explore in this system to help explain divergence patterns not well supported by vicariance, such as the effects of rapidly evolving gamete recognition proteins on isolating populations. Our findings of a stable and robust phylogeny, with the increase in mitochondrial and nuclear comparative genomic data, provide a system in which we can enhance our understanding of molecular evolution and adaptation in this group of sea urchins.