Odd-Paired is Involved in Morphological Divergence of Snail-Feeding Beetles.

Body shape and size diversity and their evolutionary rates correlate with species richness at the macroevolutionary scale. However, the molecular genetic mechanisms underlying the morphological diversification across related species are poorly understood. In beetles, which account for one-fourth of the known species, adaptation to different trophic niches through morphological diversification appears to have contributed to species radiation. Here, we explored the key genes for the morphological divergence of the slender to stout body shape related to divergent feeding methods on large to small snails within the genus Carabus. We show that the zinc-finger transcription factor encoded by odd-paired (opa) controls morphological variation in the snail-feeding ground beetle Carabus blaptoides. Specifically, opa was identified as the gene underlying the slender to stout morphological difference between subspecies through genetic mapping and functional analysis via gene knockdown. Further analyses revealed that changes in opa cis-regulatory sequences likely contributed to the differences in body shape and size between C. blaptoides subspecies. Among opa cis-regulatory sequences, single nucleotide polymorphisms on the transcription factor binding sites may be associated with the morphological differences between C. blaptoides subspecies. opa was highly conserved in a wide range of taxa, especially in beetles. Therefore, opa may play an important role in adaptive morphological divergence in beetles.

Shared evolutionary trajectories of three independent neo-sex chromosomes in Drosophila.

Dosage compensation (DC) on the X Chromosome counteracts the deleterious effects of gene loss on the Y Chromosome. However, DC is not efficient if the X Chromosome also degenerates. This indeed occurs in Drosophila miranda, in which both the neo-Y and the neo-X are under accelerated pseudogenization. To examine the generality of this pattern, we investigated the evolution of two additional neo-sex chromosomes that emerged independently in D. albomicans and D. americana and reanalyzed neo-sex chromosome evolution in D. miranda Comparative genomic and transcriptomic analyses revealed that the pseudogenization rate on the neo-X is also accelerated in D. albomicans and D. americana although to a lesser extent than in D. miranda In males, neo-X-linked genes whose neo-Y-linked homologs are pseudogenized tended to be up-regulated more than those whose neo-Y-linked homologs remain functional. Moreover, genes under strong functional constraint and genes highly expressed in the testis tended to remain functional on the neo-X and neo-Y, respectively. Focusing on the D. miranda and D. albomicans neo-sex chromosomes that emerged independently from the same autosome, we further found that the same genes tend to become pseudogenized in parallel on the neo-Y. These genes include Idgf6 and JhI-26, which may be unnecessary or even harmful in males. Our results indicate that neo-sex chromosomes in Drosophila share a common evolutionary trajectory after their emergence, which may prevent sex chromosomes from being an evolutionary dead end.

Elucidating the functional evolution of heat sensors among Xenopus species adapted to different thermal niches by ancestral sequence reconstruction.

Ambient temperature fluctuations are detected via the thermosensory system which allows animals to seek preferable thermal conditions or escape from harmful temperatures. Evolutionary changes in thermal perception have thus potentially played crucial roles in niche selection. The genus Xenopus (clawed frog) is suitable for investigating the relationship between thermal perception and niche selection due to their diverse latitudinal and altitudinal distributions. Here we performed comparative analyses of the neuronal heat sensors TRPV1 and TRPA1 among closely related Xenopus species (X. borealis, X. muelleri, X. laevis, and X. tropicalis) to elucidate their functional evolution and to assess whether their functional differences correlate with thermal niche selection among the species. Comparison of TRPV1 among four extant Xenopus species and reconstruction of the ancestral TRPV1 revealed that TRPV1 responses to repeated heat stimulation were specifically altered in the lineage leading to X. tropicalis which inhabits warmer niches. Moreover, the thermal sensitivity of TRPA1 was lower in X. tropicalis than the other species, although the thermal sensitivity of TRPV1 and TRPA1 was not always lower in species that inhabit warmer niches than the species inhabit cooler niches. However, a clear correlation was found in species differences in TRPA1 activity. Heat-evoked activity of TRPA1 in X. borealis and X. laevis, which are adapted to cooler niches, was significantly higher than in X. tropicalis and X. muelleri which are adapted to warmer niches. These findings suggest that the functional properties of heat sensors changed during Xenopus evolution, potentially altering the preferred temperature ranges among species.

Transcriptional Activation of Glycogen Catabolism and the Oxidative Pentose Phosphate Pathway by NrrA Facilitates Cell Survival Under Nitrogen Starvation in the Cyanobacterium Synechococcus sp. Strain PCC 7002.

Cyanobacteria respond to nitrogen deprivation by changing cellular metabolism. Glycogen is accumulated within cells to assimilate excess carbon and energy during nitrogen starvation, and inhibition of glycogen synthesis results in impaired nitrogen response and decreased ability to survive. In spite of glycogen accumulation, genes related to glycogen catabolism are up-regulated by nitrogen deprivation. In this study, we found that glycogen catabolism was also involved in acclimation to nitrogen deprivation in the cyanobacterium Synechococcus sp. PCC 7002. The glgP2 gene, encoding glycogen phosphorylase, was induced by nitrogen deprivation, and its expression was regulated by the nitrogen-regulated response regulator A (NrrA), which is a highly conserved transcriptional regulator in cyanobacteria. Activation of glycogen phosphorylase under nitrogen-deprived conditions was abolished by disruption of the nrrA gene, and survival of the nrrA mutant declined. In addition, a glgP2 mutant was highly susceptible to nitrogen starvation. NrrA also regulated expression of the tal-zwf-opcA operon, encoding enzymes of the oxidative pentose phosphate (OPP) pathway, and inactivation of glucose-6-phosphate dehydrogenase, the first enzyme of the OPP pathway, decreased the ability to survive under nitrogen starvation. It was concluded that NrrA facilitates cell survival by activating glycogen degradation and the OPP pathway under nitrogen-deprived conditions.

Molecular phylogeny of ten intertidal hermit crabs of the genus Pagurus inferred from multiple mitochondrial genes, with special emphasis on the evolutionary relationship of Pagurus lanuginosus and Pagurus maculosus.

The hermit crab genus Pagurus exhibits high species diversity and a wide geological distribution. Despite the high species diversity of hermit crabs in the western Pacific coast of Japan, molecular phylogenetic studies of these species have yet to be conducted. To investigate their molecular phylogeny and genetic diversity, we obtained nearly complete mitochondrial genome sequences for ten Pagurus species found along the Pacific coast of Japan by next-generation sequencing, which were compared to other congeners deposited in the GenBank database. The genomes ranged from 13,458 to 16,401 base pairs in length, possessing 13 protein-coding genes, 2 rRNAs, and 22 tRNA genes. Based on the reconstructed phylogeny, we found that (1) Japanese Pagurus species separated into three groups, nested within the Northern Pacific species. (2) Pagurus lanuginosus and Pagurus maculosus, showed high morphological similarities, implying close kinship. Indeed, these two species were genetically closest to each other, compared to the remaining species studied. (3) An unspecified specimen sampled from the deep sea, which morphologically resembled Pagurus, might be a member of the Pagurus genus, but is genetically distant from the other Japanese Pagurus species. The novel data reported here may provide new perspectives for systematic studies of hermit crabs; these results provide important information that will facilitate population-level research and identifying intraspecific variation of these non-model, but ecologically important, decapod species.

Two-step evolution of endosymbiosis between hydra and algae.

In the Hydra vulgaris group, only 2 of the 25 strains in the collection of the National Institute of Genetics in Japan currently show endosymbiosis with green algae. However, whether the other non-symbiotic strains also have the potential to harbor algae remains unknown. The endosymbiotic potential of non-symbiotic strains that can harbor algae may have been acquired before or during divergence of the strains. With the aim of understanding the evolutionary process of endosymbiosis in the H. vulgaris group, we examined the endosymbiotic potential of non-symbiotic strains of the H. vulgaris group by artificially introducing endosymbiotic algae. We found that 12 of the 23 non-symbiotic strains were able to harbor the algae until reaching the grand-offspring through the asexual reproduction by budding. Moreover, a phylogenetic analysis of mitochondrial genome sequences showed that all the strains with endosymbiotic potential grouped into a single cluster (cluster γ). This cluster contained two strains (J7 and J10) that currently harbor algae; however, these strains were not the closest relatives. These results suggest that evolution of endosymbiosis occurred in two steps; first, endosymbiotic potential was gained once in the ancestor of the cluster γ lineage; second, strains J7 and J10 obtained algae independently after the divergence of the strains. By demonstrating the evolution of the endosymbiotic potential in non-symbiotic H. vulgaris group strains, we have clearly distinguished two evolutionary steps. The step-by-step evolutionary process provides significant insight into the evolution of endosymbiosis in cnidarians.

Tissue- and stage-dependent dosage compensation on the neo-X chromosome in Drosophila pseudoobscura.

Sex chromosome dosage compensation (DC) is widely accepted in various organisms. This concept is mostly supported by comparisons of gene expression between chromosomes and between sexes. However, genes on the X chromosome and autosomes are mostly not homologous, and the average gene expression level on these chromosomes may not be the same even under DC, which complicates comparisons between chromosomes. Many genes with sex-biased expression also make comparisons between sexes difficult. To overcome these issues, we investigated DC by comparing the expression of neo-X-linked genes in Drosophila pseudoobscura with those of their autosomal orthologs in other Drosophila species. The ratio of the former to the latter in males would be 1 under DC, whereas it becomes 0.5 without DC. We found that the ratio was ∼0.85 for adult whole bodies, indicating that the DC is incomplete on the neo-X chromosome in adults as a whole. The ratio (∼0.90) was also significantly less than 1 for adult bodies without gonads, whereas it was ∼1.0 for adult heads. These results indicate that DC varies among tissues. Our sliding-window analysis of the ratio also revealed that the upregulation of neo-X-linked genes in males occurred chromosome wide in all tissues analyzed, indicating global upregulation mechanisms. However, we found that gene functions also affected the levels of DC. Furthermore, most of the genes recently moved to the X were already under DC at the larval stage but not at the adult stage. These results suggest that DC in Drosophila species operates in a tissue/stage-dependent manner.

Microsatellite marker development by multiplex ion torrent PGM sequencing: a case study of the endangered Odorrana narina complex of frogs.

The endangered Ryukyu tip-nosed frog Odorrana narina and its related species, Odorrana amamiensis, Odorrana supranarina, and Odorrana utsunomiyaorum, belong to the family Ranidae and are endemically distributed in Okinawa (O. narina), Amami and Tokunoshima (O. amamiensis), and Ishigaki and Iriomote (O. supranarina and O. utsunomiyaorum) Islands. Because of varying distribution patterns, this species complex is an intrinsic model for speciation and adaptation. For effective conservation and molecular ecological studies, further genetic information is needed. For rapid, cost-effective development of several microsatellite markers for these and 2 other species, we used next-generation sequencing technology of Ion Torrent PGM™. Distribution patterns of repeat motifs of microsatellite loci in these modern frog species (Neobatrachia) were similarly skewed. We isolated and characterized 20 new microsatellite loci of O. narina and validated cross-amplification in the three-related species. Seventeen, 16, and 13 loci were cross-amplified in O. amamiensis, O. supranarina, and O. utsunomiyaorum, respectively, reflecting close genetic relationships between them. Mean number of alleles and expected heterozygosity of newly isolated loci varied depending on the size of each inhabited island. Our findings suggested the suitability of Ion Torrent PGM™ for microsatellite marker development. The new markers developed for the O. narina complex will be applicable in conservation genetics and molecular ecological studies.

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 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.

Testing immediate dosage compensation in Drosophila miranda via irradiation with heavy-ion beams.

Many organisms with heteromorphic sex chromosomes possess a mechanism of dosage compensation (DC) in which X-linked genes are upregulated in males to mitigate the dosage imbalance between sexes and between chromosomes. However, how quickly the DC is established during evolution remains unknown. In this study, by irradiating Drosophila miranda male flies, which carry young sex chromosomes (the so-called neo-sex chromosomes), with heavy-ion beams, we induced deletions in the neo-Y chromosome to mimic the condition of Y-chromosome degeneration, in which functional neo-Y-linked genes are nonfunctionalized; furthermore, we tested whether their neo-X-linked gametologs were immediately upregulated. Because the males that received 2-Gy iron-ion beam irradiation exhibited lower fertility, we sequenced the genomes and transcriptomes of six F1 males derived from these males. Our pipeline identified 82 neo-Y-linked genes in which deletions were predicted in the F1 males. Only three of them showed a one-to-one gametologous relationship with the neo-X-linked genes. The candidate deletions in these three genes occurred in UTRs and did not seriously affect their expression levels. These observations indirectly suggest that DC was unlikely to have operated on the neo-X-linked genes immediately after the pseudogenization of their neo-Y-linked gametologs in D. miranda. Therefore, the dosage imbalance caused by deletions in the neo-Y-linked genes without paralogs may not have effectively been compensated, and individuals with such deletions could have exhibited lethality. Future studies on sex chromosomes at different ages will further reveal the relationship between the age of sex chromosomes and the stringency of DC.

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 (omega) 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 Fisher's exact test. The results indicate that BSM often generates false positives compared with SSM when the number of nucleotide substitutions is approximately 80 or smaller. Because the omega 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 omega values, which may not have anything to do with functional changes. The codon sites showing functional changes generally do not show a high omega value. To understand adaptive evolution, some form of experimental confirmation is necessary.

Genomic drift and copy number variation of sensory receptor genes in humans.

The number of sensory receptor genes varies extensively among different mammalian species. This variation is believed to be caused partly by physiological requirements of animals and partly by genomic drift due to random duplication and deletion of genes. If the contribution of genomic drift is substantial, each species should contain a significant amount of copy number variation (CNV). We therefore investigated CNVs in sensory receptor genes among 270 healthy humans by using published CNV data. The results indicated that olfactory receptor (OR), taste receptor type 2, and vomeronasal receptor type 1 genes show a high level of intraspecific CNVs. In particular, >30% of the approximately 800 OR gene loci in humans were polymorphic with respect to copy number, and two randomly chosen individuals showed a copy number difference of approximately 11 in functional OR genes on average. There was no significant difference in the amount of CNVs between functional and nonfunctional OR genes. Because pseudogenes are expected to evolve in a neutral fashion, this observation suggests that functional OR genes also have evolved in a similar manner with respect to copy number change. In addition, we found that the evolutionary change of copy number of OR genes approximately follows the Gaussian process in probability theory, and the copy number divergence between populations has increased with evolutionary time. We therefore conclude that genomic drift plays an important role for generating intra- and interspecific CNVs of sensory receptor genes. Similar results were obtained when all annotated genes were analyzed.

Gene-by-Gene or Localized Dosage Compensation on the Neo-X Chromosome in Drosophila miranda.

Many organisms have a global mechanism for dosage compensation (DC) operating along the entire male X chromosome, which equalizes gene expression on the male X with that on the two Xs in females and/or on autosomes. At the initial stage of sex chromosome evolution, however, gene-by-gene (or localized) DC may also be necessary because the degeneration of Y-linked genes occurs independently at different times. We therefore tested whether the up-regulation of X-linked genes depends on the status of their Y-linked homologs, using the young sex chromosomes, neo-X and neo-Y, in Drosophila miranda. In support of the presence of gene-by-gene DC, the extent of up-regulation in males was indeed higher for neo-X-linked genes with pseudogenized neo-Y-linked homologs than for neo-X-linked genes with functional neo-Y-linked homologs. Further molecular evolutionary analysis also supports the idea that many individual neo-X-linked genes first acquired the potential for up-regulation, which then enabled the pseudogenization of neo-Y-linked homologs, without serious deleterious effects on male fitness.

Transcriptomic changes with increasing algal symbiont reveal the detailed process underlying establishment of coral-algal symbiosis.

To clarify the establishment process of coral-algal symbiotic relationships, coral transcriptome changes during increasing algal symbiont densities were examined in juvenile corals following inoculation with the algae Symbiodinium goreaui (clade C) and S. trenchii (clade D), and comparison of their transcriptomes with aposymbiotic corals by RNA-sequencing. Since Symbiodinium clades C and D showed very different rates of density increase, comparisons were made of early onsets of both symbionts, revealing that the host behaved differently for each. RNA-sequencing showed that the number of differentially-expressed genes in corals colonized by clade D increased ca. two-fold from 10 to 20 days, whereas corals with clade C showed unremarkable changes consistent with a slow rate of density increase. The data revealed dynamic metabolic changes in symbiotic corals. In addition, the endocytosis pathway was also upregulated, while lysosomal digestive enzymes and the immune system tended to be downregulated as the density of clade D algae increased. The present dataset provides an enormous number of candidate symbiosis-related molecules that exhibit the detailed process by which coral-algal endosymbiosis is established.

Functional divergence of duplicate genes several million years after gene duplication in Arabidopsis.

Lineage-specific duplicated genes likely contribute to the phenotypic divergence in closely related species. However, neither the frequency of duplication events nor the degree of selection pressures immediately after gene duplication is clear in the speciation process. Here, using Illumina DNA-sequencing reads from Arabidopsis halleri, which has multiple closely related species with high-quality genome assemblies (A. thaliana and A. lyrata), we succeeded in generating orthologous gene groups in Brassicaceae. The duplication frequency of retained genes in the Arabidopsis lineage was ∼10 times higher than the duplication frequency inferred by comparative genomics of Arabidopsis, poplar, rice and moss (Physcomitrella patens). The difference of duplication frequencies can be explained by a rapid decay of anciently duplicated genes. To examine the degree of selection pressure on genes duplicated in either the A. halleri-lyrata or the A. halleri lineage, we examined positive and purifying selection in the A. halleri-lyrata and A. halleri lineages throughout the ratios of nonsynonymous to synonymous substitution rates (KA/KS). Duplicate genes tended to have a higher proportion of positive selection compared with non-duplicated genes. Interestingly, we found that functional divergence of duplicated genes was accelerated several million years after gene duplication compared with immediately after gene duplication.

Comparative analysis of the brain transcriptome in a hyper-aggressive fruit fly, Drosophila prolongata.

Aggressive behavior is observed in many animals, but its intensity differs between species. In a model animal of genetics, Drosophila melanogaster, genetic basis of aggressive behavior has been studied intensively, including transcriptome analyses to identify genes whose expression level was associated with intra-species variation in aggressiveness. However, whether these genes are also involved in the evolution of aggressiveness among different species has not been examined. In this study, we performed de novo transcriptome analysis in the brain of Drosophila prolongata to identify genes associated with the evolution of aggressiveness. Males of D. prolongata were hyper-aggressive compared with closely related species. Comparison of the brain transcriptomes identified 21 differentially expressed genes in males of D. prolongata. They did not overlap with the list of aggression-related genes identified in D. melanogaster, suggesting that genes involved in the evolution of aggressiveness were independent of those associated with the intra-species variation in aggressiveness in Drosophila. Although females of D. prolongata were not aggressive as the males, expression levels of the 21 genes identified in this study were more similar between sexes than between species.

Evolutionary history of the extant amphioxus lineage with shallow-branching diversification.

Amphioxus or lancelets have been regarded as a key animal in understanding the origin of vertebrates. However, the evolutionary history within this lineage remains unexplored. As the amphioxus lineage has likely been separated from other chordates for a very long time and displays a marked left-right asymmetry, its evolutionary history is potentially helpful in better understanding chordate and vertebrate origins. We studied the phylogenetic relationships within the extant amphioxus lineage based on mitochondrial genomes incorporating new Asymmetron and Epigonichthys populations, and based on previously reported nuclear transcriptomes. The resulting tree patterns are consistent, showing the Asymmetron clade diverging first, followed by the Epigonichthys and Branchiostoma clades splitting. Divergence time estimates based on nuclear transcriptomes with vertebrate calibrations support a shallow diversification of the extant amphioxus lineage in the Tertiary. These estimates fit well with the closure of seaways between oceans by continental drift, ocean currents, and present geographical distributions, and suggest a long cryptic history from the origin of amphioxus to its most recent diversification. Deduced character polarities based on phylogenetic analyses suggest that the common ancestor of the extant amphioxus existed in a tiny epibenthic state with larva-like appearance of extant amphioxus, likely with ciliate epidermis.

Genome of the pitcher plant Cephalotus reveals genetic changes associated with carnivory.

Carnivorous plants exploit animals as a nutritional source and have inspired long-standing questions about the origin and evolution of carnivory-related traits. To investigate the molecular bases of carnivory, we sequenced the genome of the heterophyllous pitcher plant Cephalotus follicularis, in which we succeeded in regulating the developmental switch between carnivorous and non-carnivorous leaves. Transcriptome comparison of the two leaf types and gene repertoire analysis identified genetic changes associated with prey attraction, capture, digestion and nutrient absorption. Analysis of digestive fluid proteins from C. follicularis and three other carnivorous plants with independent carnivorous origins revealed repeated co-options of stress-responsive protein lineages coupled with convergent amino acid substitutions to acquire digestive physiology. These results imply constraints on the available routes to evolve plant carnivory.