Flight loss linked to faster molecular evolution in insects

The loss of flight ability has occurred thousands of times independently during insect evolution. Flight loss may be linked to higher molecular evolutionary rates because of reductions in effective population sizes (N_e) and relaxed selective constraints. Reduced dispersal ability increases population subdivision, may decrease geographical range size and increases (sub)population extinction risk, thus leading to an expected reduction in N_e. Additionally, flight loss in birds has been linked to higher molecular rates of energy-related genes, probably owing to relaxed selective constraints on energy metabolism. We tested for an association between insect flight loss and molecular rates through comparative analysis in 49 phylogenetically independent transitions spanning multiple taxa, including moths, flies, beetles, mayflies, stick insects, stoneflies, scorpionflies and caddisflies, using available nuclear and mitochondrial protein-coding DNA sequences. We estimated the rate of molecular evolution of flightless (FL) and related flight-capable lineages by ratios of non-synonymous-to-synonymous substitutions (dN/dS) and overall substitution rates (OSRs). Across multiple instances of flight loss, we show a significant pattern of higher dN/dS ratios and OSRs in FL lineages in mitochondrial but not nuclear genes. These patterns may be explained by relaxed selective constraints in FL ectotherms relating to energy metabolism, possibly in combination with reduced N_e.     

Host associations and evolutionary relationships of avian blood parasites from West Africa

The host specificity of blood parasites recovered from a survey of 527 birds in Cameroon and Gabon was examined at several levels within an evolutionary framework. Unique mitochondrial lineages of Haemoproteus were recovered from an average of 1.3 host species (maximum = 3) and 1.2 host families (maximum = 3) while lineages of Plasmodium were recovered from an average of 2.5 species (maximum = 27) and 1.6 families (maximum = 9). Averaged within genera, lineages of both Plasmodium and Haemoproteus were constrained in their host distribution relative to random expectations. However, while several individual lineages within both genera exhibited significant host constraint, host breadth varied widely among related lineages, particularly within the genus Plasmodium. Several lineages of Plasmodium exhibited extreme generalist host-parasitism strategies while other lineages appeared to have been constrained to certain host families over recent evolutionary history. Sequence data from two nuclear genes recovered from a limited sample of Plasmodium parasites indicated that, at the resolution of this study, inferences regarding host breadth were unlikely to be grossly affected by the use of parasite mitochondrial lineages as a proxy for biological species. The use of divergent host-parasitism strategies among closely related parasite lineages suggests that host range is a relatively labile character. Since host specificity may also influence parasite virulence, these results argue for considering the impact of haematozoa on avian hosts on a lineage-specific basis.     

Effective population size and the rate and pattern of nucleotide substitutions

Both the overall rate of nucleotide substitution and the relative proportions of synonymous and non-synonymous substitutions are predicted to vary between species that differ in effective population size (N_e). Our understanding of the genetic processes underlying these lineage-specific differences in molecular evolution is still developing. Empirical analyses indicate that variation in substitution rates and patterns caused by differences in N_e is often substantial, however, and must be accounted for in analyses of molecular evolution.     

GENOME SIZE IS NOT CORRELATED WITH EFFECTIVE POPULATION SIZE IN THE ORYZA SPECIES

Genome sizes vary widely across the tree of life and the evolutionary mechanism underlined remains largely unknown. Lynch and Conery (2003) proposed that evolution of genome complexity was driven mainly by nonadaptive stochastic forces and presented the observation that genome size was negatively correlated with effective population size (N_e) as a strong support for their hypothesis. Here, we analyzed the relation between N_e and genome size for 10 diploid Oryza species that showed about fourfold genome size variation. Using sequences of more than 20 nuclear genes, we estimated N_e for each species after correction for the effects of demography and heterogeneity of mutation rates among loci and species. Pairwise comparisons and correlation analyses did not detect a negative relationship between N_e and genome size despite about 6.5‐fold interspecies N_e variation. By calculating phylogenetically independent contrasts (PICs) for N_e, we repeated correlation analysis and did not find any correlation between N_e and genome size. These observations suggest that the genome size variation in the Oryza species cannot be explained simply by the effect of effective population size.     

Significance of Population Size on the Fixation of Nonsynonymous Mutations in Genes Under Varying Levels of Selection Pressure

Previous studies observed a higher ratio of divergences at nonsynonymous and synonymous sites (ω = d_N/d_S) in species with a small population size compared to that estimated for those with a large population size. Here we examined the theoretical relationship between ω, effective population size (N_e), and selection coefficient (s). Our analysis revealed that when purifying selection is high, ω of species with small N_e is much higher than that of species with large N_e. However the difference between the two ω reduces with the decline in selection pressure (s → 0). We examined this relationship using primate and rodent genes and found that the ω estimated for highly constrained genes of primates was up to 2.9 times higher than that obtained for their orthologous rodent genes. Conversely, for genes under weak purifying selection the ω of primates was only 17% higher than that of rodents. When tissue specificity was used as a proxy for selection pressure we found that the ω of broadly expressed genes of primates was up to 2.1-fold higher than that of their rodent counterparts and this difference was only 27% for tissue specific genes. Since most of the nonsynonymous mutations in constrained or broadly expressed genes are deleterious, fixation of these mutations is influenced by N_e. This results in a higher ω of these genes in primates compared to those from rodents. Conversely, the majority of nonsynonymous mutations in less-constrained or tissue-specific genes are neutral or nearly neutral and therefore fixation of them is largely independent of N_e, which leads to the similarity of ω in primates and rodents.     

The Genomic Selfing Syndrome Accompanies the Evolutionary Breakdown of Heterostyly

The evolutionary transition from outcrossing to selfing can have important genomic consequences. Decreased effective population size and the reduced efficacy of selection are predicted to play an important role in the molecular evolution of the genomes of selfing species. We investigated evidence for molecular signatures of the genomic selfing syndrome using 66 species of Primula including distylous (outcrossing) and derived homostylous (selfing) taxa. We complemented our comparative analysis with a microevolutionary study of P. chungensis, which is polymorphic for mating system and consists of both distylous and homostylous populations. We generated chloroplast and nuclear genomic data sets for distylous, homostylous, and distylous–homostylous species and identified patterns of nonsynonymous to synonymous divergence (d_N/d_S) and polymorphism (π_N/π_S) in species or lineages with contrasting mating systems. Our analysis of coding sequence divergence and polymorphism detected strongly reduced genetic diversity and heterozygosity, decreased efficacy of purifying selection, purging of large-effect deleterious mutations, and lower rates of adaptive evolution in samples from homostylous compared with distylous populations, consistent with theoretical expectations of the genomic selfing syndrome. Our results demonstrate that self-fertilization is a major driver of molecular evolutionary processes with genomic signatures of selfing evident in both old and relatively young homostylous populations.     

ASPM and the Evolution of Cerebral Cortical Size in a Community of New World Monkeys

The ASPM (abnormal spindle-like microcephaly associated) gene has been proposed as a major determinant of cerebral cortical size among primates, including humans. Yet the specific functions of ASPM and its connection to human intelligence remain controversial. This debate is limited in part by a taxonomic focus on Old World monkeys and apes. Here we expand the comparative context of ASPM sequence analyses with a study of New World monkeys, a radiation of primates in which enlarged brain size has evolved in parallel in spider monkeys (genus Ateles) and capuchins (genus Cebus). The primate community of Costa Rica is perhaps a model system because it allows for independent pairwise comparisons of smaller- and larger-brained species within two taxonomic families. Accordingly, we analyzed the complete sequence of exon 18 of ASPM in Ateles geoffroyi, Alouatta palliata, Cebus capucinus, and Saimiri oerstedii. As the analysis of multiple species in a genus improves phylogenetic reconstruction, we also analyzed eleven published sequences from other New World monkeys. Our exon-wide, lineage-specific analysis of eleven genera and the ratio of rates of nonsynonymous to synonymous substitutions (d_N/d_S) on ASPM revealed no detectable evidence for positive selection in the lineages leading to Ateles or Cebus, as indicated by d_N/d_S ratios of <1.0 (0.6502 and 0.4268, respectively). Our results suggest that a multitude of interacting genes have driven the evolution of larger brains among primates, with different genes involved in this process in different encephalized lineages, or at least with evidence for positive selection not readily apparent for the same genes in all lineages. The primate community of Costa Rica may serve as a model system for future studies that aim to elucidate the molecular mechanisms underlying cognitive capacity and cortical size.     

Simple life-history traits explain key effective population size ratios across diverse taxa

Effective population size (N_e) controls both the rate of random genetic drift and the effectiveness of selection and migration, but it is difficult to estimate in nature. In particular, for species with overlapping generations, it is easier to estimate the effective number of breeders in one reproductive cycle (N_b) than N_e per generation. We empirically evaluated the relationship between life history and ratios of N_e, N_b and adult census size (N) using a recently developed model (agene) and published vital rates for 63 iteroparous animals and plants. N_b/N_e varied a surprising sixfold across species and, contrary to expectations, N_b was larger than N_e in over half the species. Up to two-thirds of the variance in N_b/N_e and up to half the variance in N_e/N was explained by just two life-history traits (age at maturity and adult lifespan) that have long interested both ecologists and evolutionary biologists. These results provide novel insights into, and demonstrate a close general linkage between, demographic and evolutionary processes across diverse taxa. For the first time, our results also make it possible to interpret rapidly accumulating estimates of N_b in the context of the rich body of evolutionary theory based on N_e per generation.     

Evolutionary rates of mitochondrial genomes correspond to diversification rates and to contemporary species richness in birds and reptiles

Rates of biological diversification should ultimately correspond to rates of genome evolution. Recent studies have compared diversification rates with phylogenetic branch lengths, but incomplete phylogenies hamper such analyses for many taxa. Herein, we use pairwise comparisons of confamilial sauropsid (bird and reptile) mitochondrial DNA (mtDNA) genome sequences to estimate substitution rates. These molecular evolutionary rates are considered in light of the age and species richness of each taxonomic family, using a random-walk speciation–extinction process to estimate rates of diversification. We find the molecular clock ticks at disparate rates in different families and at different genes. For example, evolutionary rates are relatively fast in snakes and lizards, intermediate in crocodilians and slow in turtles and birds. There was also rate variation across genes, where non-synonymous substitution rates were fastest at ATP8 and slowest at CO3. Family-by-gene interactions were significant, indicating that local clocks vary substantially among sauropsids. Most importantly, we find evidence that mitochondrial genome evolutionary rates are positively correlated with speciation rates and with contemporary species richness. Nuclear sequences are poorly represented among reptiles, but the correlation between rates of molecular evolution and species diversification also extends to 18 avian nuclear genes we tested. Thus, the nuclear data buttress our mtDNA findings.     

Detecting positive selection within genomes: the problem of biased gene conversion

The identification of loci influenced by positive selection is a major goal of evolutionary genetics. A popular approach is to perform scans of alignments on a genome-wide scale in order to find regions evolving at accelerated rates on a particular branch of a phylogenetic tree. However, positive selection is not the only process that can lead to accelerated evolution. Notably, GC-biased gene conversion (gBGC) is a recombination-associated process that results in the biased fixation of G and C nucleotides. This process can potentially generate bursts of nucleotide substitutions within hotspots of meiotic recombination. Here, we analyse the results of a scan for positive selection on genes on branches across the primate phylogeny. We show that genes identified as targets of positive selection have a significant tendency to exhibit the genomic signature of gBGC. Using a maximum-likelihood framework, we estimate that more than 20 per cent of cases of significantly elevated non-synonymous to synonymous substitution rates ratio (d_N/d_S), particularly in shorter branches, could be due to gBGC. We demonstrate that in some cases, gBGC can lead to very high d_N/d_S (more than 2). Our results indicate that gBGC significantly affects the evolution of coding sequences in primates, often leading to patterns of evolution that can be mistaken for positive selection.     

Hotspots of Biased Nucleotide Substitutions in Human Genes

Genes that have experienced accelerated evolutionary rates on the human lineage during recent evolution are candidates for involvement in human-specific adaptations. To determine the forces that cause increased evolutionary rates in certain genes, we analyzed alignments of 10,238 human genes to their orthologues in chimpanzee and macaque. Using a likelihood ratio test, we identified protein-coding sequences with an accelerated rate of base substitutions along the human lineage. Exons evolving at a fast rate in humans have a significant tendency to contain clusters of AT-to-GC (weak-to-strong) biased substitutions. This pattern is also observed in noncoding sequence flanking rapidly evolving exons. Accelerated exons occur in regions with elevated male recombination rates and exhibit an excess of nonsynonymous substitutions relative to the genomic average. We next analyzed genes with significantly elevated ratios of nonsynonymous to synonymous rates of base substitution (d_N/d_S) along the human lineage, and those with an excess of amino acid replacement substitutions relative to human polymorphism. These genes also show evidence of clusters of weak-to-strong biased substitutions. These findings indicate that a recombination-associated process, such as biased gene conversion (BGC), is driving fixation of GC alleles in the human genome. This process can lead to accelerated evolution in coding sequences and excess amino acid replacement substitutions, thereby generating significant results for tests of positive selection.     

Rate heterogeneity in six protein-coding genes from the holoparasite Balanophora (Balanophoraceae) and other taxa of Santalales

Background and Aims

The holoparasitic flowering plant Balanophora displays extreme floral reduction and was previously found to have enormous rate acceleration in the nuclear 18S rDNA region. So far, it remains unclear whether non-ribosomal, protein-coding genes of Balanophora also evolve in an accelerated fashion and whether the genes with high substitution rates retain their functionality. To tackle these issues, six different genes were sequenced from two Balanophora species and their rate variation and expression patterns were examined.

Methods

Sequences including nuclear PI, euAP3, TM6, LFY and RPB2 and mitochondrial matR were determined from two Balanophora spp. and compared with selected hemiparasitic species of Santalales and autotrophic core eudicots. Gene expression was detected for the six protein-coding genes and the expression patterns of the three B-class genes (PI, AP3 and TM6) were further examined across different organs of B. laxiflora using RT-PCR.

Key Results

Balanophora mitochondrial matR is highly accelerated in both nonsynonymous (d_N) and synonymous (d_S) substitution rates, whereas the rate variation of nuclear genes LFY, PI, euAP3, TM6 and RPB2 are less dramatic. Significant d_S increases were detected in Balanophora PI, TM6, RPB2 and d_N accelerations in euAP3. All of the protein-coding genes are expressed in inflorescences, indicative of their functionality. PI is restrictively expressed in tepals, synandria and floral bracts, whereas AP3 and TM6 are widely expressed in both male and female inflorescences.

Conclusions

Despite the observation that rates of sequence evolution are generally higher in Balanophora than in hemiparasitic species of Santalales and autotrophic core eudicots, the five nuclear protein-coding genes are functional and are evolving at a much slower rate than 18S rDNA. The mechanism or mechanisms responsible for rapid sequence evolution and concomitant rate acceleration for 18S rDNA and matR are currently not well understood and require further study in Balanophora and other holoparasites.     

Molecular Phylogenies and Evolution of crt Genes in Algae

ABSTRACT

The carotenoids constitute the most widespread class of pigments in nature. Most previous work has concentrated on the identification and characterization of their chemical physical properties and bioavailability. In recent years, significant amounts of research have been conducted in an attempt to analyze the genes and the molecular regulation of the genes involved in the biosynthesis of carotenoids. However, it is important not to lose sight of the early evolution of carotenoid biosynthesis. One of the major obstacles in understanding the evolution of the respective enzymes and their patterns of selection is a lack of a well-supported phylogenic analysis. In the present research, a major long-term objective was to provide a clearer picture of the evolutionary history of genes, together with an evaluation of the patterns of selection in algae. These phylogenies will be important in studies characterizing the evolution of algae. The gene sequences of the enzymes involved in the major steps of the carotenoid biosynthetic pathway in algae (cyanobacteria, rhofophyta, chlorophyta) have been analyzed. Phylogenetic relationships among protein-coding DNA sequences were reconstructed by neighbor-joining (NJ) analysis for the respective carotenoid biosynthetic pathway genes (crt) in algae. The analysis also contains an estimation of the rate of nonsynonymous nucleotide substitutions per nonsynonymous site (d_N), synonymous nucleotide substitution per synonymous site (d_S), and the ratio of nonsynonmous (d_N/d_S) for the test of selection patterns. The phylogenetic trees show that the taxa of some genera have a closer evolutionary relationship with other genera in some gene sequences, which suggests a common ancient origin and that lateral gene transfer has occurred among unrelated genera. The d_N values of crt genes in the early pathway are relatively low, while those of the following steps are slightly higher, while the d_N values of crt genes in chlorophyta are higher than those in cyanobacteria. Most of the d_N/d_S values exceed 1. The phylogenetic analysis revealed that lateral gene transfer may have taken place across algal genomes and the d_N values suggest that most of the early crt genes are well conserved compared to the later crt genes. Furthermore, d_N values also revealed that the crt genes of chlorophyta are more evolutionary than cyanobacteria. The amino acids' changes are mostly adaptive evolution under the influence of positive diversity selection.     

Cospeciation in the triplex symbiosis of termite gut protists (Pseudotrichonympha spp.), their hosts, and their bacterial endosymbionts

A number of cophylogenetic relationships between two organisms namely a host and a symbiont or parasite have been studied to date; however, organismal interactions in nature usually involve multiple members. Here, we investigated the cospeciation of a triplex symbiotic system comprising a hierarchy of three organisms -- termites of the family Rhinotermitidae, cellulolytic protists of the genus Pseudotrichonympha in the guts of these termites, and intracellular bacterial symbionts of the protists. The molecular phylogeny was inferred based on two mitochondrial genes for the termites and nuclear small-subunit rRNA genes for the protists and their endosymbionts, and these were compared. Although intestinal microorganisms are generally considered to have looser associations with the host than intracellular symbionts, the Pseudotrichonympha protists showed almost complete codivergence with the host termites, probably due to strict transmissions by proctodeal trophallaxis or coprophagy based on the social behaviour of the termites. Except for one case, the endosymbiotic bacteria of the protists formed a monophyletic lineage in the order Bacteroidales, and the branching pattern was almost identical to those of the protists and the termites. However, some non-codivergent evolutionary events were evident. The members of this triplex symbiotic system appear to have cospeciated during their evolution with minor exceptions; the evolutionary relationships were probably established by termite sociality and the complex microbial community in the gut.     

Peeling Back the Evolutionary Layers of Molecular Mechanisms Responsive to Exercise-Stress in the Skeletal Muscle of the Racing Horse

The modern horse (Equus caballus) is the product of over 50 million yrs of evolution. The athletic abilities of the horse have been enhanced during the past 6000 yrs under domestication. Therefore, the horse serves as a valuable model to understand the physiology and molecular mechanisms of adaptive responses to exercise. The structure and function of skeletal muscle show remarkable plasticity to the physical and metabolic challenges following exercise. Here, we reveal an evolutionary layer of responsiveness to exercise-stress in the skeletal muscle of the racing horse. We analysed differentially expressed genes and their co-expression networks in a large-scale RNA-sequence dataset comparing expression before and after exercise. By estimating genome-wide d_N/d_S ratios using six mammalian genomes, and F_ST and iHS using re-sequencing data derived from 20 horses, we were able to peel back the evolutionary layers of adaptations to exercise-stress in the horse. We found that the oldest and thickest layer (d_N/d_S) consists of system-wide tissue and organ adaptations. We further find that, during the period of horse domestication, the older layer (F_ST) is mainly responsible for adaptations to inflammation and energy metabolism, and the most recent layer (iHS) for neurological system process, cell adhesion, and proteolysis.     

Molecular clock in neutral protein evolution

Background

A frequent observation in molecular evolution is that amino-acid substitution rates show an index of dispersion (that is, ratio of variance to mean) substantially larger than one. This observation has been termed the overdispersed molecular clock. On the basis of in silico protein-evolution experiments, Bastolla and coworkers recently proposed an explanation for this observation: Proteins drift in neutral space, and can temporarily get trapped in regions of substantially reduced neutrality. In these regions, substitution rates are suppressed, which results in an overall substitution process that is not Poissonian. However, the simulation method of Bastolla et al. is representative only for cases in which the product of mutation rate μ and population size N_e is small. How the substitution process behaves when μN_e is large is not known.

Results

Here, I study the behavior of the molecular clock in in silico protein evolution as a function of mutation rate and population size. I find that the index of dispersion decays with increasing μN_e, and approaches 1 for large μN_e . This observation can be explained with the selective pressure for mutational robustness, which is effective when μN_e is large. This pressure keeps the population out of low-neutrality traps, and thus steadies the ticking of the molecular clock.

Conclusions

The molecular clock in neutral protein evolution can fall into two distinct regimes, a strongly overdispersed one for small μN_e, and a mostly Poissonian one for large μN_e. The former is relevant for the majority of organisms in the plant and animal kingdom, and the latter may be relevant for RNA viruses.

     

Isolation of NBS-LRR RGAs from invasive Wedelia trilobata and the calculation of evolutionary rates to understand bioinvasion from a molecular evolution perspective

Supports for the molecular evolution of host–pathogen interactions on enemy release hypothesis are rare. According to the theory of plant immunity and the coevolution of hosts and pathogens, we hypothesized that the evolutionary rate (d_N/d_S) of resistance genes (R-genes) in invasive plants would be greater than in non-invasive plants, assuming that based on the enemy release hypothesis, the former would suffer less selection stress from co-evolutionary specialist pathogens. To test our hypothesis, we isolated and analyzed the conserved nucleotide-binding sites (NBS) of resistance gene analogues (RGAs) of an invasive weed, Wedelia trilobata (WTRGA). We then used the information in GenBank to compare the d_N/d_S of the NBS R-gene/RGAs in invasive and homologous non-invasive plants. Three W. trilobata NBS RGA sequences were obtained, belonging to the Toll/Interleukin-1 receptor (TIR) (WTRGA1 and WTRGA2) and non-TIR subclasses (WTRGA3). Compared with the homologous non-invasive plants, the invasive plants showed a significantly greater d_N/d_S for TIR NBS R-gene/RGAs (p < 0.0001), supporting our hypothesis. Future research should include an examination of R-genes/RGAs from more invasive plants on a population level to understand diversity and R-gene functions in invasive plant species, as well as to explore how disease resistance allows plants to adapt to changing pathogen stresses.     

Genetic and morphological diversity of Trisetacus species (Eriophyoidea: Phytoptidae) associated with coniferous trees in Poland: phylogeny,barcoding, host and habitat specialization

Eriophyoid species belonging to the genus Trisetacus are economically important as pests of conifers. A narrow host specialization to conifers and some unique morphological characteristics have made these mites interesting subjects for scientific inquiry. In this study, we assessed morphological and genetic variation of seven Trisetacus species originating from six coniferous hosts in Poland by morphometric analysis and molecular sequencing of the mitochondrial cytochrome oxidase subunit I gene and the nuclear D2 region of 28S rDNA. The results confirmed the monophyly of the genus Trisetacus as well as the monophyly of five of the seven species studied. Both DNA sequences were effective in discriminating between six of the seven species tested. Host-dependent genetic and morphological variation in T. silvestris and T. relocatus, and habitat-dependent genetic and morphological variation in T. juniperinus were detected, suggesting the existence of races or even distinct species within these Trisetacus taxa. This is the first molecular phylogenetic analysis of the Trisetacus species. The findings presented here will stimulate further investigations on the evolutionary relationships of Trisetacus as well as the entire Phytoptidae family.