Masafumi Nozawa

The evolution of animal chemosensory receptor gene repertoires: roles of chance and necessity.

Chemosensory receptors are essential for the survival of organisms that range from bacteria to mammals. Recent studies have shown that the numbers of functional chemosensory receptor genes and pseudogenes vary enormously among the genomes of different animal species. Although much of the variation can be explained by the adaptation of organisms to different environments, it has become clear that a substantial portion is generated by genomic drift, a random process of gene duplication and deletion. Genomic drift also generates a substantial amount of copy-number variation in chemosensory receptor genes within species. It seems that mutation by gene duplication and inactivation has important roles in both the adaptive and non-adaptive evolution of chemosensation.

The draft genomes of soft-shell turtle and green sea turtle yield insights into the development and evolution of the turtle-specific body plan

The unique anatomical features of turtles have raised unanswered questions about the origin of their unique body plan. We generated and analyzed draft genomes of the soft-shell turtle (Pelodiscus sinensis) and the green sea turtle (Chelonia mydas); our results indicated the close relationship of the turtles to the bird-crocodilian lineage, from which they split ∼267.9–248.3 million years ago (Upper Permian to Triassic). We also found extensive expansion of olfactory receptor genes in these turtles. Embryonic gene expression analysis identified an hourglass-like divergence of turtle and chicken embryogenesis, with maximal conservation around the vertebrate phylotypic period, rather than at later stages that show the amniote-common pattern. Wnt5a expression was found in the growth zone of the dorsal shell, supporting the possible co-option of limb-associated Wnt signaling in the acquisition of this turtle-specific novelty. Our results suggest that turtle evolution was accompanied by an unexpectedly conservative vertebrate phylotypic period, followed by turtle-specific repatterning of development to yield the novel structure of the shell.The unique anatomical features of turtles have raised unanswered questions about the origin of their unique body plan. We generated and analyzed draft genomes of the soft-shell turtle (Pelodiscus sinensis) and the green sea turtle (Chelonia mydas); our results indicated the close relationship of the turtles to the bird-crocodilian lineage, from which they split ∼267.9-248.3 million years ago (Upper Permian to Triassic). We also found extensive expansion of olfactory receptor genes in these turtles. Embryonic gene expression analysis identified an hourglass-like divergence of turtle and chicken embryogenesis, with maximal conservation around the vertebrate phylotypic period, rather than at later stages that show the amniote-common pattern. Wnt5a expression was found in the growth zone of the dorsal shell, supporting the possible co-option of limb-associated Wnt signaling in the acquisition of this turtle-specific novelty. Our results suggest that turtle evolution was accompanied by an unexpectedly conservative vertebrate phylotypic period, followed by turtle-specific repatterning of development to yield the novel structure of the shell.

The Neutral Theory of Molecular Evolution in the Genomic Era

The neutral theory of molecular evolution has been widely accepted and is the guiding principle for studying evolutionary genomics and the molecular basis of phenotypic evolution. Recent data on genomic evolution are generally consistent with the neutral theory. However, many recently published papers claim the detection of positive Darwinian selection via the use of new statistical methods. Examination of these methods has shown that their theoretical bases are not well established and often result in high rates of false-positive and false-negative results. When the deficiencies of these statistical methods are rectified, the results become largely consistent with the neutral theory. At present, genome-wide analyses of natural selection consist of collections of single-locus analyses. However, because phenotypic evolution is controlled by the interaction of many genes, the study of natural selection ought to take such interactions into account. Experimental studies of evolution will also be crucial.

Reliabilities of identifying positive selection by the branch-site and the site-prediction methods

Natural selection operating in protein-coding genes is often studied by examining the ratio (ω) of the rates of nonsynonymous to synonymous nucleotide substitution. The branch-site method (BSM) based on a likelihood ratio test is one of such tests to detect positive selection for a predetermined branch of a phylogenetic tree. However, because the number of nucleotide substitutions involved is often very small, we conducted a computer simulation to examine the reliability of BSM in comparison with the small-sample method (SSM) based on Fishers exact test. The results indicate that BSM often generates false positives compared with SSM when the number of nucleotide substitutions is ≈80 or smaller. Because the ω value is also used for predicting positively selected sites, we examined the reliabilities of the site-prediction methods, using nucleotide sequence data for the dim-light and color vision genes in vertebrates. The results showed that the site-prediction methods have a low probability of identifying functional changes of amino acids experimentally determined and often falsely identify other sites where amino acid substitutions are unlikely to be important. This low rate of predictability occurs because most of the current statistical methods are designed to identify codon sites with high ω values, which may not have anything to do with functional changes. The codon sites showing functional changes generally do not show a high ω value. To understand adaptive evolution, some form of experimental confirmation is necessary.

Origins and evolution of microRNA genes in plant species

MicroRNAs (miRNAs) are among the most important regulatory elements of gene expression in animals and plants. However, their origin and evolutionary dynamics have not been studied systematically. In this paper, we identified putative miRNA genes in 11 plant species using the bioinformatic technique and examined their evolutionary changes. Our homology search indicated that no miRNA gene is currently shared between green algae and land plants. The number of miRNA genes has increased substantially in the land plant lineage, but after the divergence of eudicots and monocots, the number has changed in a lineage-specific manner. We found that miRNA genes have originated mainly by duplication of preexisting miRNA genes or protein-coding genes. Transposable elements also seem to have contributed to the generation of species-specific miRNA genes. The relative importance of these mechanisms in plants is quite different from that in Drosophila species, where the formation of hairpin structures in the genomes seems to be a major source of miRNA genes. This difference in the origin of miRNA genes between plants and Drosophila may be explained by the difference in the binding to target mRNAs between plants and animals. We also found that young miRNA genes are less conserved than old genes in plants as well as in Drosophila species. Yet, nearly half of the gene families in the ancestor of flowering plants have been lost in at least one species examined. This indicates that the repertoires of miRNA genes have changed more dynamically than previously thought during plant evolution.

Evolutionary dynamics of olfactory receptor genes in Drosophila species

Olfactory receptor (OR) genes are of vital importance for animals to find food, identify mates, and avoid dangers. In mammals, the number of OR genes is large and varies extensively among different orders, whereas, in insects, the extent of interspecific variation appears to be small, although only a few species have been studied. To understand the evolutionary changes of OR genes, we identified all OR genes from 12 Drosophila species, of which the evolutionary time is roughly equivalent to that of eutherian mammals. The results showed that all species examined have similar numbers (≈60) of functional OR genes. Phylogenetic analysis indicated that the ancestral species also had similar numbers of genes, but there were frequent gains and losses of genes that occurred in each evolutionary lineage. It appears that tandem duplication and random inactivation of duplicate genes are the major factors of gene number change. However, chromosomal rearrangements have contributed to the establishment of genome-wide distribution of OR genes. These results suggest that the repertoire of OR genes in Drosophila has been quite stable compared with the mammalian genes. The difference in evolutionary pattern between Drosophila and mammals can be explained partly by the differences of gene expression mechanisms and partly by the environmental and behavioral differences.

Origins and Evolution of MicroRNA Genes in Drosophila Species

MicroRNAs (miRs) regulate gene expression at the posttranscriptional level. To obtain some insights into the origins and evolutionary patterns of miR genes, we have identified miR genes in the genomes of 12 Drosophila species by bioinformatics approaches and examined their evolutionary changes. The results showed that the extant and ancestral Drosophila species had more than 100 miR genes and frequent gains and losses of miR genes have occurred during evolution. Although many miR genes appear to have originated from random hairpin structures in intronic or intergenic regions, duplication of miR genes has also contributed to the generation of new miR genes. Estimating the rate of nucleotide substitution of miR genes, we have found that newly arisen miR genes have a substitution rate similar to that of synonymous nucleotide sites in protein-coding genes and evolve almost neutrally. This suggests that most new miR genes have not acquired any important function and would become inactive. By contrast, old miR genes show a substitution rate much lower than the synonymous rate. Moreover, paired and unpaired nucleotide sites of miR genes tend to remain unchanged during evolution. Therefore, once miR genes acquired their functions, they appear to have evolved very slowly, maintaining essentially the same structures for a long time.

Evolutionary dynamics of the immunoglobulin heavy chain variable region genes in vertebrates

Immunoglobulin heavy chains are polypeptides encoded by four genes: variable (IGHV), joining (IGHJ), diversity (IGHD), and constant (IGHC) region genes. The number of IGHV genes varies from species to species. To understand the evolution of the IGHV multigene family, we identified and analyzed the IGHV sequences from 16 vertebrate species. The results show that the numbers of functional and nonfunctional IGHV genes among different species are positively correlated. The number of IGHV genes is relatively stable in teleosts, but the intragenomic sequence variation is generally higher in teleosts than in tetrapods. The IGHV genes in tetrapods can be classified into three phylogenetic clans (I, II, and III). The clan III and/or II genes are relatively abundant, whereas clan I genes exist in small numbers or are absent in most species. The genomic organization of clan I, II, and III IGHV genes varies considerably among species, but the entire IGHV locus seems to be conserved in the subtelomeric or near-centromeric region of chromosome. The presence or absence of specific IGHV clan members and the lineage-specific expansion and contraction of IGHV genes indicate that the IGHV locus continues to evolve in a species-specific manner. Our results suggest that the evolution of IGHV multigene family is more complex than previously thought and that several factors may act synergistically for the development of antibody repertoire.

Roles of mutation and selection in speciation: from Hugo de Vries to the modern genomic era.

One of the most important problems in evolutionary biology is to understand how new species are generated in nature. In the past, it was difficult to study this problem because our lifetime is too short to observe the entire process of speciation. In recent years, however, molecular and genomic techniques have been developed for identifying and studying the genes involved in speciation. Using these techniques, many investigators have already obtained new findings. At present, however, the results obtained are complex and quite confusing. We have therefore attempted to understand these findings coherently with a historical perspective and clarify the roles of mutation and natural selection in speciation. We have first indicated that the root of the currently burgeoning field of plant genomics goes back to Hugo de Vries, who proposed the mutation theory of evolution more than a century ago and that he unknowingly found the importance of polyploidy and chromosomal rearrangements in plant speciation. We have then shown that the currently popular Dobzhansky–Muller model of evolution of reproductive isolation is only one of many possible mechanisms. Some of them are Oka’s model of duplicate gene mutations, multiallelic speciation, mutation-rescue model, segregation-distorter gene model, heterochromatin-associated speciation, single-locus model, etc. The occurrence of speciation also depends on the reproductive system, population size, bottleneck effects, and environmental factors, such as temperature and day length. Some authors emphasized the importance of natural selection to speed up speciation, but mutation is crucial in speciation because reproductive barriers cannot be generated without mutations.

A novel chimeric gene, siren, with retroposed promoter sequence in the Drosophila bipectinata complex.

Retrotransposons often produce a copy of host genes by their reverse transcriptase activity operating on host gene transcripts. Since transcripts normally do not contain promoter, a retroposed gene copy usually becomes a retropseudogene. However, in Drosophila bipectinata and a closely related species we found a new chimeric gene, whose promoter was likely produced by retroposition. This chimeric gene, named siren, consists of a tandem duplicate of Adh and a retroposed fragment of CG11779 containing the promoter and a partial intron in addition to the first exon. We found that this unusual structure of a retroposed fragment was obtained by retroposition of nanos, which overlaps with CG11779 on the complementary strand. The potential of retroposition to produce a copy of promoter and intron sequences in the context of gene overlapping was demonstrated.