Molecular Biology and Evolution

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January 19, 2015

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A fundamental question in evolutionary genetics concerns the roles of mutational pleiotropy and epistasis in shaping trajectories of protein evolution. This question can be addressed most directly by using site-directed mutagenesis to explore the mutational landscape of protein function in experimentally defined regions of sequence space. Here, we evaluate how pleiotropic trade-offs and epistatic interactions influence the accessibility of alternative mutational pathways during the adaptive evolution of hemoglobin (Hb) function in high-altitude pikas (Mammalia: Lagomorpha). By combining ancestral protein resurrection with a combinatorial protein-engineering approach, we examined the functional effects of sequential mutational steps in all possible pathways that produced an increased Hb–O2 affinity. These experiments revealed that the effects of mutations on Hb–O2 affinity are highly dependent on the temporal order in which they occur: Each of three β-chain substitutions produced a significant increase in Hb–O2 affinity on the ancestral genetic background, but two of these substitutions produced opposite effects when they occurred as later steps in the pathway. The experiments revealed pervasive epistasis for Hb–O2 affinity, but affinity-altering mutations produced no significant pleiotropic trade-offs. These results provide insights into the properties of adaptive substitutions in naturally evolved proteins and suggest that the accessibility of alternative mutational pathways may be more strongly constrained by sign epistasis for positively selected biochemical phenotypes than by antagonistic pleiotropy.

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An elaborated tripartite brain is considered one of the important innovations of vertebrates. Other extant chordate groups have a more basic brain organization. For instance, cephalochordates possess a relatively simple brain possibly homologous to the vertebrate forebrain and hindbrain, whereas tunicates display the tripartite organization, but without the specialized brain centers. The difference in anatomical complexity is even more pronounced if one compares chordates with other deuterostomes that have only a diffuse nerve net or alternatively a rather simple central nervous system. To gain a new perspective on the evolutionary roots of the complex vertebrate brain, we made here a phylostratigraphic analysis of gene expression patterns in the developing zebrafish (Danio rerio). The recovered adaptive landscape revealed three important periods in the evolutionary history of the zebrafish brain. The oldest period corresponds to preadaptive events in the first metazoans and the emergence of the nervous system at the metazoan–eumetazoan transition. The origin of chordates marks the next phase, where we found the overall strongest adaptive imprint in almost all analyzed brain regions. This finding supports the idea that the vertebrate brain evolved independently of the brains within the protostome lineage. Finally, at the origin of vertebrates we detected a pronounced signal coming from the dorsal telencephalon, in agreement with classical theories that consider this part of the cerebrum a genuine vertebrate innovation. Taken together, these results reveal a stepwise adaptive history of the vertebrate brain where most of its extant organization was already present in the chordate ancestor.

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The phylogenetic and taxonomic relationships among the Old World leaf-nosed bats (Hipposideridae) and the closely related horseshoe bats (Rhinolophidae) remain unresolved. In this study, we generated a novel approximately 10-kb molecular data set of 19 nuclear exon and intron gene fragments for 40 bat species to elucidate the phylogenetic relationships within the families Rhinolophidae and Hipposideridae. We estimated divergence times and explored potential reasons for any incongruent phylogenetic signal. We demonstrated the effects of outlier taxa and genes on phylogenetic reconstructions and compared the relative performance of intron and exon data to resolve phylogenetic relationships. Phylogenetic analyses produced a well-resolved phylogeny, supporting the familial status of Hipposideridae and demonstrated the paraphyly of the largest genus, Hipposideros. A fossil-calibrated timetree and biogeographical analyses estimated that Rhinolophidae and Hipposideridae diverged in Africa during the Eocene approximately 42 Ma. The phylogram, the timetree, and a unique retrotransposon insertion supported the elevation of the subtribe Rhinonycterina to family level and which is diagnosed herein. Comparative analysis of diversification rates showed that the speciose genera Rhinolophus and Hipposideros underwent diversification during the Mid-Miocene Climatic Optimum. The intron versus exon analyses demonstrated the improved nodal support provided by introns for our optimal tree, an important finding for large-scale phylogenomic studies, which typically rely on exon data alone. With the recent outbreak of Middle East respiratory syndrome, caused by a novel coronavirus, the study of these species is urgent as they are considered the natural reservoir for emergent severe acute respiratory syndrome (SARS)-like coronaviruses. It has been shown that host phylogeny is the primary factor that determines a virus’s persistence, replicative ability, and can act as a predictor of new emerging disease. Therefore, this newly resolved phylogeny can be used to direct future assessments of viral diversity and to elucidate the origin and development of SARS-like coronaviruses in mammals.

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Whether coding or regulatory sequence change is more important to the evolution of phenotypic novelty is one of biology’s major unresolved questions. The field of evo–devo has shown that in early development changes to regulatory regions are the dominant mode of genetic change, but whether this extends to the evolution of novel phenotypes in the adult organism is unclear. Here, we conduct ten RNA-Seq experiments across both novel and conserved tissues in the honey bee to determine to what extent postdevelopmental novelty is based on changes to the coding regions of genes. We make several discoveries. First, we show that with respect to novel physiological functions in the adult animal, positively selected tissue-specific genes of high expression underlie novelty by conferring specialized cellular functions. Such genes are often, but not always taxonomically restricted genes (TRGs). We further show that positively selected genes, whether TRGs or conserved genes, are the least connected genes within gene expression networks. Overall, this work suggests that the evo–devo paradigm is limited, and that the evolution of novelty, postdevelopment, follows additional rules. Specifically, evo–devo stresses that high network connectedness (repeated use of the same gene in many contexts) constrains coding sequence change as it would lead to negative pleiotropic effects. Here, we show that in the adult animal, the converse is true: Genes with low network connectedness (TRGs and tissue-specific conserved genes) underlie novel phenotypes by rapidly changing coding sequence to perform new-specialized functions.

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Gene degeneration or loss can significantly contribute to phenotypic diversification, but may generate genetic constraints on future evolutionary trajectories, potentially restricting phenotypic reversal. Such constraints may manifest as directional evolutionary trends when parallel phenotypic shifts consistently involve gene degeneration or loss. Here, we demonstrate that widespread parallel evolution in Penstemon from blue to red flowers predictably involves the functional inactivation and degeneration of the enzyme flavonoid 3',5'-hydroxylase (F3'5'H), an anthocyanin pathway enzyme required for the production of blue floral pigments. Other types of genetic mutations do not consistently accompany this phenotypic shift. This pattern may be driven by the relatively large mutational target size of degenerative mutations to this locus and the apparent lack of associated pleiotropic effects. The consistent degeneration of F3'5'H may provide a mechanistic explanation for the observed asymmetry in the direction of flower color evolution in Penstemon: Blue to red transitions are common, but reverse transitions have not been observed. Although phenotypic shifts in this system are likely driven by natural selection, internal constraints may generate predictable genetic outcomes and may restrict future evolutionary trajectories.

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Alternation of generations, in which the haploid and diploid stages of the life cycle are each represented by multicellular forms that differ in their morphology, is a defining feature of the land plants (embryophytes). Anciently derived lineages of embryophytes grow predominately in the haploid gametophytic generation from apical cells that give rise to the photosynthetic body of the plant. More recently evolved plant lineages have multicellular shoot apical meristems (SAMs), and photosynthetic shoot development is restricted to the sporophyte generation. The molecular genetic basis for this evolutionary shift from gametophyte-dominant to sporophyte-dominant life cycles remains a major question in the study of land plant evolution. We used laser microdissection and next generation RNA sequencing to address whether angiosperm meristem patterning genes expressed in the sporophytic SAM of Zea mays are expressed in the gametophytic apical cells, or in the determinate sporophytes, of the model bryophytes Marchantia polymorpha and Physcomitrella patens. A wealth of upregulated genes involved in stem cell maintenance and organogenesis are identified in the maize SAM and in both the gametophytic apical cell and sporophyte of moss, but not in Marchantia. Significantly, meiosis-specific genetic programs are expressed in bryophyte sporophytes, long before the onset of sporogenesis. Our data suggest that this upregulated accumulation of meiotic gene transcripts suppresses indeterminate cell fate in the Physcomitrella sporophyte, and overrides the observed accumulation of meristem patterning genes. A model for the evolution of indeterminate growth in the sporophytic generation through the concerted selection of ancestral meristem gene programs from gametophyte-dominant lineages is proposed.

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Opsins are ancient molecules that enable animal vision by coupling to a vitamin-derived chromophore to form light-sensitive photopigments. The primary drivers of evolutionary diversification in opsins are thought to be visual tasks related to spectral sensitivity and color vision. Typically, only a few opsin amino acid sites affect photopigment spectral sensitivity. We show that opsin genes of the North American butterfly Limenitis arthemis have diversified along a latitudinal cline, consistent with natural selection due to environmental factors. We sequenced single nucleotide (SNP) polymorphisms in the coding regions of the ultraviolet (UVRh), blue (BRh), and long-wavelength (LWRh) opsin genes from ten butterfly populations along the eastern United States and found that a majority of opsin SNPs showed significant clinal variation. Outlier detection and analysis of molecular variance indicated that many SNPs are under balancing selection and show significant population structure. This contrasts with what we found by analysing SNPs in the wingless and EF-1 alpha loci, and from neutral amplified fragment length polymorphisms, which show no evidence of significant locus-specific or genome-wide structure among populations. Using a combination of functional genetic and physiological approaches, including expression in cell culture, transgenic Drosophila, UV-visible spectroscopy, and optophysiology, we show that key BRh opsin SNPs that vary clinally have almost no effect on spectral sensitivity. Our results suggest that opsin diversification in this butterfly is more consistent with natural selection unrelated to spectral tuning. Some of the clinally varying SNPs may instead play a role in regulating opsin gene expression levels or the thermostability of the opsin protein. Lastly, we discuss the possibility that insect opsins might have important, yet-to-be elucidated, adaptive functions in mediating animal responses to abiotic factors, such as temperature or photoperiod.

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Evolutionary innovations are dependent on mutations. Mutation rates are increased by adverse conditions in the laboratory, but there is no evidence that stressful environments that do not directly impact on DNA leave a mutational imprint on extant genomes. Mutational spectra in the laboratory are normally determined with unstressed cells but are unavailable with stressed bacteria. To by-pass problems with viability, selection effects, and growth rate differences due to stressful environments, in this study we used a set of genetically engineered strains to identify the mutational spectrum associated with nutritional stress. The strain set members each had a fixed level of the master regulator protein, RpoS, which controls the general stress response of Escherichia coli. By assessing mutations in cycA gene from 485 cycloserine resistant mutants collected from as many independent cultures with three distinct perceived stress (RpoS) levels, we were able establish a dose-dependent relationship between stress and mutational spectra. The altered mutational patterns included base pair substitutions, single base pair indels, longer indels, and transpositions of different insertion sequences. The mutational spectrum of low-RpoS cells closely matches the genome-wide spectrum previously generated in laboratory environments, while the spectra of high RpoS, high perceived stress cells more closely matches spectra found in comparisons of extant genomes. Our results offer an explanation of the uneven mutational profiles such as the transition–transversion biases observed in extant genomes and provide a framework for assessing the contribution of stress-induced mutagenesis to evolutionary transitions and the mutational emergence of antibiotic resistance and disease states.

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Cyclic proteins have evolved for millions of years across all kingdoms of life to confer structural stability over their acyclic counterparts while maintaining intrinsic functional properties. Here, we show that cyclic miniproteins (or peptides) from Momordica (Cucurbitaceae) seeds evolved in species that diverged from an African ancestor around 19 Ma. The ability to achieve head-to-tail cyclization of Momordica cyclic peptides appears to have been acquired through a series of mutations in their acyclic precursor coding sequences following recent and independent gene expansion event(s). Evolutionary analysis of Momordica cyclic peptides reveals sites that are under selection, highlighting residues that are presumably constrained for maintaining their function as potent trypsin inhibitors. Molecular dynamics of Momordica cyclic peptides in complex with trypsin reveals site-specific residues involved in target binding. In a broader context, this study provides a basis for selecting Momordica species to further investigate the biosynthesis of the cyclic peptides and for constructing libraries that may be screened against evolutionarily related serine proteases implicated in human diseases.

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Termites have colonized many habitats and are among the most abundant animals in tropical ecosystems, which they modify considerably through their actions. The timing of their rise in abundance and of the dispersal events that gave rise to modern termite lineages is not well understood. To shed light on termite origins and diversification, we sequenced the mitochondrial genome of 48 termite species and combined them with 18 previously sequenced termite mitochondrial genomes for phylogenetic and molecular clock analyses using multiple fossil calibrations. The 66 genomes represent most major clades of termites. Unlike previous phylogenetic studies based on fewer molecular data, our phylogenetic tree is fully resolved for the lower termites. The phylogenetic positions of Macrotermitinae and Apicotermitinae are also resolved as the basal groups in the higher termites, but in the crown termitid groups, including Termitinae + Syntermitinae + Nasutitermitinae + Cubitermitinae, the position of some nodes remains uncertain. Our molecular clock tree indicates that the lineages leading to termites and Cryptocercus roaches diverged 170 Ma (153–196 Ma 95% confidence interval [CI]), that modern Termitidae arose 54 Ma (46–66 Ma 95% CI), and that the crown termitid group arose 40 Ma (35–49 Ma 95% CI). This indicates that the distribution of basal termite clades was influenced by the final stages of the breakup of Pangaea. Our inference of ancestral geographic ranges shows that the Termitidae, which includes more than 75% of extant termite species, most likely originated in Africa or Asia, and acquired their pantropical distribution after a series of dispersal and subsequent diversification events.

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Although parasitic organisms are found worldwide, the relative importance of host specificity and geographic isolation for parasite speciation has been explored in only a few systems. Here, we study Plasmodium parasites known to infect Asian nonhuman primates, a monophyletic group that includes the lineage leading to the human parasite Plasmodium vivax and several species used as laboratory models in malaria research. We analyze the available data together with new samples from three sympatric primate species from Borneo: The Bornean orangutan and the long-tailed and the pig-tailed macaques. We find several species of malaria parasites, including three putatively new species in this biodiversity hotspot. Among those newly discovered lineages, we report two sympatric parasites in orangutans. We find no differences in the sets of malaria species infecting each macaque species indicating that these species show no host specificity. Finally, phylogenetic analysis of these data suggests that the malaria parasites infecting Southeast Asian macaques and their relatives are speciating three to four times more rapidly than those with other mammalian hosts such as lemurs and African apes. We estimate that these events took place in approximately a 3–4-Ma period. Based on the genetic and phenotypic diversity of the macaque malarias, we hypothesize that the diversification of this group of parasites has been facilitated by the diversity, geographic distributions, and demographic histories of their primate hosts.

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Local protein interactions ("molecular context" effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and β-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations.

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Desaturase genes are essential for biological processes, including lipid metabolism, cell signaling, and membrane fluidity regulation. Insect desaturases are particularly interesting for their role in chemical communication, and potential contribution to speciation, symbioses, and sociality. Here, we describe the acyl-CoA desaturase gene families of 15 insects, with a focus on social Hymenoptera. Phylogenetic reconstruction revealed that the insect desaturases represent an ancient gene family characterized by eight subfamilies that differ strongly in their degree of conservation and frequency of gene gain and loss. Analyses of genomic organization showed that five of these subfamilies are represented in a highly microsyntenic region conserved across holometabolous insect taxa, indicating an ancestral expansion during early insect evolution. In three subfamilies, ants exhibit particularly large expansions of genes. Despite these expansions, however, selection analyses showed that desaturase genes in all insect lineages are predominantly undergoing strong purifying selection. Finally, for three expanded subfamilies, we show that ants exhibit variation in gene expression between species, and more importantly, between sexes and castes within species. This suggests functional differentiation of these genes and a role in the regulation of reproductive division of labor in ants. The dynamic pattern of gene gain and loss of acyl-CoA desaturases in ants may reflect changes in response to ecological diversification and an increased demand for chemical signal variability. This may provide an example of how gene family expansions can contribute to lineage-specific adaptations through structural and regulatory changes acting in concert to produce new adaptive phenotypes.

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Sex pheromones form an important facet of reproductive strategies in many organisms throughout the Animal Kingdom. One of the oldest known sex pheromones in vertebrates are proteins of the Sodefrin Precursor-like Factor (SPF) system, which already had a courtship function in early salamanders. The subsequent evolution of salamanders is characterized by a diversification in courtship and reproduction, but little is known on how the SPF pheromone system diversified in relation to changing courtship strategies. Here, we combined transcriptomic, genomic, and phylogenetic analyses to investigate the evolution of the SPF pheromone system in nine salamandrid species with distinct courtship displays. First, we show that SPF originated from vertebrate three-finger proteins and diversified through multiple gene duplications in salamanders, while remaining a single copy in frogs. Next, we demonstrate that tail-fanning newts have retained a high phylogenetic diversity of SPFs, whereas loss of tail-fanning has been associated with a reduced importance or loss of SPF expression in the cloacal region. Finally, we show that the attractant decapeptide sodefrin is cleaved from larger SPF precursors that originated by a 62 bp insertion and consequent frameshift in an ancestral Cynops lineage. This led to the birth of a new decapeptide that rapidly evolved a pheromone function independently from uncleaved proteins.

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Changes in exon–intron structures and splicing patterns represent an important mechanism for the evolution of gene functions and species-specific regulatory networks. Although exon creation is widespread during primate and human evolution and has been studied extensively, much less is known about the scope and potential impact of human-specific exon loss events. Historically, transcriptome data and exon annotations are significantly biased toward humans over nonhuman primates. This ascertainment bias makes it challenging to discover human-specific exon loss events. We carried out a transcriptome-wide search of human-specific exon loss events, by taking advantage of RNA sequencing (RNA-seq) as a powerful and unbiased tool for exon discovery and annotation. Using RNA-seq data of humans, chimpanzees, and other primates, we reconstructed and compared transcript structures across the primate phylogeny. We discovered 33 candidate human-specific exon loss events, among which six exons passed stringent experimental filters for the complete loss of splicing activities in diverse human tissues. These events may result from human-specific deletion of genomic DNA, or small-scale sequence changes that inactivated splicing signals. The impact of human-specific exon loss events is predominantly regulatory. Three of the six events occurred in the 5' untranslated region (5'-UTR) and affected cis-regulatory elements of mRNA translation. In SLC7A6, a gene encoding an amino acid transporter, luciferase reporter assays suggested that both a human-specific exon loss event and an independent human-specific single nucleotide substitution in the 5'-UTR increased mRNA translational efficiency. Our study provides novel insights into the molecular mechanisms and evolutionary consequences of exon loss during human evolution.

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Whole-genome resequencing of experimental populations evolving under a specific selection regime has become a popular approach to determine genotype–phenotype maps and understand adaptation to new environments. Despite its conceptual appeal and success in identifying some causative genes, it has become apparent that many studies suffer from an excess of candidate loci. Several explanations have been proposed for this phenomenon, but it is clear that information about the linkage structure during such experiments is needed. Until now only Pool-Seq (whole-genome sequencing of pools of individuals) data were available, which do not provide sufficient information about the correlation between linked sites. We address this problem in two complementary analyses of three replicate Drosophila melanogaster populations evolving to a new hot temperature environment for almost 70 generations. In the first analysis, we sequenced 58 haploid genomes from the founder population and evolved flies at generation 67. We show that during the experiment linkage disequilibrium (LD) increased almost uniformly over much greater distances than typically seen in Drosophila. In the second analysis, Pool-Seq time series data of the three replicates were combined with haplotype information from the founder population to follow blocks of initial haplotypes over time. We identified 17 selected haplotype-blocks that started at low frequencies in the base population and increased in frequency during the experiment. The size of these haplotype-blocks ranged from 0.082 to 4.01 Mb. Moreover, between 42% and 46% of the top candidate single nucleotide polymorphisms from the comparison of founder and evolved populations fell into the genomic region covered by the haplotype-blocks. We conclude that LD in such rising haplotype-blocks results in long range hitchhiking over multiple kilobase-sized regions. LD in such haplotype-blocks is therefore a major factor contributing to an excess of candidate loci. Although modifications of the experimental design may help to reduce the hitchhiking effect and allow for more precise mapping of causative variants, we also note that such haplotype-blocks might be well suited to study the dynamics of selected genomic regions during experimental evolution studies.