Biology subjects

Toyoda, A.

Publications and source records attributed to Toyoda, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

Sequencing and chromosome-scale assembly of the giant Pleurodeles waltl genome

The Iberian ribbed newt (Pleurodeles waltl) constitutes a central model for probing the basis of vertebrate regeneration. Here, we present the sequencing and chromosome-scale assembly of the 20.3Gb P. waltl genome, which exhibits the highest level of contiguity and completeness among giant genome assemblies. We uncover that DNA transposable elements are the major contributors to its expansion, with hAT transposons comprising a large portion of repeats. Several hATs are actively transcribed and differentially expressed during adult P. waltl limb regeneration, along with domesticated hAT transposons of the ZBED transcription factor family. Despite its size, syntenic relationships are conserved. As an example, we show the high degree of conservation of the regeneration-associated Tig1 locus with several neighbouring genes. Together, the P. waltl genome provides a fundamental resource for the study of regenerative, developmental and evolutionary principles.

developmental biology↗

bioRxiv2022

Chromosome-scale genome assembly of a Japanese chili pepper landrace, Capsicum annuum 'Takanotsume'

Here, we report the genome sequence of a popular Japanese chili pepper landrace, Capsicum annuum Takanotsume. We used long-read sequencing and optical mapping, together with the genetic mapping technique, to obtain the chromosome-scale genome assembly of Takanotsume. The assembly consists of 12 pseudomolecules, which corresponds to the basic chromosome number of C. annuum, and is 3,058.5 Mb in size, spanning 97.0% of the estimated genome size. A total of 34,324 high-confidence genes were predicted in the genome, and 83.4% of the genome assembly was occupied by repetitive sequences. Comparative genomics of linked-read sequencing-derived de novo genome assemblies of two Capsicum chinense lines and whole-genome resequencing-derived genome assemblies of Capsicum species revealed not only nucleotide sequence variations but also genome structure variations (i.e., chromosomal rearrangements) between Takanotsume and its relatives. Overall, the genome sequence data generated in this study will accelerate the pan-genomics and breeding of Capsicum, and facilitate the dissection of genetic mechanisms underlying the agronomically important traits of Takanotsume.

genomics↗

bioRxiv2022

ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid cells

Recent advances in CRISPR technology have enabled us to perform gene knock-in in various species and cell lines. CRISPR-mediated knock-in requires donor DNA which serves as a template for homology-directed repair (HDR). For knock-in of short sequences or base substitutions, ssDNA donors are frequently used among various other forms of HDR donors, such as linear dsDNA. However, for insertion of long transgenes such as fluorescent reporters in human cells, the optimal type of HDR donors remains unclear. In this study, we established a simple and efficient CRISPR-mediated knock-in method for long transgenes using linear dsDNA and ssDNA donors, and systematically compared the performance of these two donors for endogenous gene tagging in human non-transformed diploid cells. Quantification using flow cytometry revealed higher efficiency of fluorescent tagging with dsDNA donors than with ssDNA. By analyzing knock-in outcomes using long-read amplicon sequencing and a classification framework, a variety of mis-integration events were detected regardless of the donor type. Importantly, the ratio of precise insertion was higher with dsDNA donors than with ssDNA. Moreover, in off-target integration analyses, dsDNA and ssDNA were comparably prone to non-homologous integration. These results indicate that ssDNA is not superior to dsDNA as long HDR donors for gene knock-in in human cells.

cell biology↗

bioRxiv2022

Long-read-resolved, ecosystem-wide exploration of nucleotide and structural microdiversity of lake bacterioplankton genomes

Reconstruction of metagenome-assembled genomes (MAGs) has become a fundamental approach in microbial ecology. However, an MAG is hardly complete and overlooks genomic microdiversity because metagenomic assembly fails to resolve microvariants among closely related genotypes. Aiming at understanding the universal factors that drive or constrain prokaryotic genome diversification, we performed an ecosystem-wide high-resolution metagenomic exploration of microdiversity by combining spatiotemporal (2 depths x 12 samples) sampling from a pelagic freshwater system, MAG reconstruction using long- and short-read metagenomic sequences, and profiling of single nucleotide variants (SNVs) and structural variants (SVs) through mapping of short and long reads to the MAGs, respectively. We reconstructed 575 MAGs, including 29 circular assemblies, providing high-quality reference genomes of freshwater bacterioplankton. Read mapping against these MAGs identified 100-101,781 SNVs/Mb, 0-305 insertions, 0-467 deletions, 0-41 duplications, and 0-6 inversions for each MAG. Nonsynonymous SNVs were accumulated in genes potentially involved in cell surface structural modification to evade phage recognition. Most (80.2%) deletions overlapped with a gene-coding region, and genes of prokaryotic defense systems were most frequently (>8% of the genes) involved in a deletion. Some such deletions exhibited a monthly shift in their allele frequency, suggesting a rapid turnover of genotypes in response to phage predation. MAGs with extremely low microdiversity were either rare or opportunistic bloomers, suggesting that population persistency is key to their genomic diversification. The results lead to the conclusion that prokaryotic genomic diversification is primarily driven by viral load and constrained by a population bottleneck.

microbiology↗

bioRxiv2021

Evolutionary history of sexual differentiation mechanism in insects

Gain of alternative splicing gives rise to functional diversity in proteins and underlies the complexity and diversity of biological aspects. However, it is still not fully understood how alternatively spliced genes develop the functional novelty. To this end, we infer the evolutionary history of the doublesex gene, the key transcriptional factor in the sexual differentiation of arthropods. doublesex is controlled by sex-specific splicing and promotes both male and female differentiation in some holometabolan insects. In contrast, doublesex promotes only male differentiation in some hemimetabolan insects. Here, we investigate ancestral states of doublesex using Thermobia domestica belonging to Zygentoma, the sister group of winged insects. We find that doublesex of T. domestica expresses sex-specific isoforms but is only necessary for male differentiation of sexual morphology. This result ensures the hypothesis that doublesex was initially only used to promote male differentiation during insect evolution. However, T. domestica doublesex has a short female-specific region and upregulates the expression of vitellogenin homologs in females, suggesting that doublesex may have already controlled some aspects of feminization in the common ancestor of winged insects. Reconstruction of the ancestral sequence and prediction of the protein structure show that the female-specific isoform of doublesex has a long C-terminal disordered region in holometabolan insects, but not in non-holometabolan species. We propose that doublesex acquired a female-specific isoform and then underwent a change in the protein motif structure, which became essential for female differentiation in sexual dimorphisms.

evolutionary biology↗

bioRxiv2021

The RNA helicase DDX6 controls early mouse embryogenesis by repressing aberrant inhibition of BMP signaling through miRNA-mediated gene silencing

The evolutionarily conserved RNA helicase DDX6 is a central player of post-transcriptional regulation, but its role during embryogenesis remains elusive. We here demonstrated that DDX6 enables proper cell lineage specification from pluripotent cells by analyzing Ddx6 KO mouse embryos and in vitro epiblast-like cell (EpiLC) induction system. Our study unveiled a great impact of DDX6-mediated RNA regulation on signaling pathways. Deletion of Ddx6 caused the aberrant transcriptional upregulation of the negative regulators of BMP signaling, which accompanied with enhanced Nodal signaling. Ddx6{bigtriangleup}/{bigtriangleup} pluripotent cells acquired higher pluripotency with a strong inclination toward neural lineage commitment. During gastrulation, abnormally expanded Nodal expression in the primitive streak likely promoted endoderm cell fate specification while inhibiting mesoderm development. We further clarified the mechanism how DDX6 regulates cell fate determination of pluripotent cells by genetically dissecting major DDX6 pathways: processing body (P-body) formation, translational repression, mRNA decay, and miRNA-mediated silencing. P-body-related functions were dispensable, but the miRNA pathway was essential for the DDX6 function. DDX6 may prevent aberrant transcriptional upregulation of the negative regulators of BMP signaling by repressing translation of certain transcription factors through the interaction with miRNA-induced silencing complexes (miRISCs). Overall, this delineates how DDX6 affects development of the three primary germ layers during early mouse embryogenesis and the underlying mechanism of DDX6 function. Author summaryGene expression occurs through the two steps: transcription (DNA to RNA) and translation (RNA to protein). Cells have very sophisticated regulatory processes working on various levels for the accurate gene expression. Post-transcriptional regulation, which includes all RNA-related controls, is crucial because it enables fine-tuning and rapid alteration of gene expression. RNA- binding proteins and non-coding RNAs are the two main players of post-transcriptional regulation. DDX6, the subject of our study, is an RNA-binding protein, more specifically an RNA helicase, which can unwind or rearrange RNA secondary structures. Its diverse molecular and cellular functions have been reported, but its embryogenic role is unknown. Here, we describe DDX6 function during early mouse embryogenesis and the underlying mechanism using genetic methodology. DDX6 enables proper cell lineage specification of pluripotent stem cells by mainly regulating BMP signaling through miRNA-mediated gene silencing. As DDX6- mediated RNA regulation affected signaling pathways, the loss of Ddx6 had a wide impact on developmental processes from pluripotency to embryo patterning. In addition, we identified which DDX6 molecular function is essential during early embryogenesis by genetically dissecting its main pathways.

genetics↗

bioRxiv2021

Repeated translocation of a supergene underlying rapid sex chromosome turnover in Takifugu fish

Recent studies have revealed a surprising diversity of sex chromosomes in vertebrates. However, the detailed mechanism of their turnover is still elusive. To understand this process, it is necessary to compare closely related species in terms of sex-determining genes and the chromosomes harboring them. Here, we explored the genus Takifugu, in which one strong candidate sex-determining gene, Amhr2, has been identified. To trace the processes involved in transitions in the sex determination system in this genus, we studied 12 species and found that while the Amhr2 locus likely determines sex in the majority of Takifugu species, three species have acquired sex-determining loci at different chromosomal locations. Nevertheless, the generation of genome assemblies for the three species revealed that they share a portion of the male-specific supergene that contains a candidate sex-determining gene, GsdfY, along with genes that potentially play a role in male fitness. The shared supergene span approximately 100 kb and are flanked by two duplicated regions characterized by CACTA transposable elements. These results suggest that the shared supergene has taken over the role of sex-determining locus from Amhr2 in lineages leading to the three species, and repeated translocations of the supergene underlie the turnover of sex chromosomes in these lineages. These findings highlight the underestimated role of a mobile supergene in the turnover of sex chromosomes in vertebrates. SignificanceAlthough turnover of sex chromosomes is very common in many vertebrate lineages, the transition process is still elusive. We studied the sex-determining region (SDR) of 12 congeneric fish species. We found that while nine species retained their ancestral SDR, three species had acquired derived SDRs. Although the derived SDRs resided in three different chromosomes, they harbored a shared supergene flanked by two putative transposable elements. The results highlight the underestimated role of a mobile supergene in turnover of sex chromosomes in vertebrates.

genomics↗

bioRxiv2021

Gene recruitments and dismissals in argonaut octopus genome provide insights to pelagic lifestyle adaptation and shell-like eggcase reacquisition

The paper nautilus, Argonauta argo, also known as the greater argonaut, is a species of octopods distinctly characterized by its pelagic lifestyle and by the presence of a spiral-shaped shell-like eggcase in females. The eggcase functions by protecting the eggs laid inside it, and by building and keeping air intakes for buoyancy. To reveal the genomic background of the species adaptation to pelagic lifestyle and the acquisition of its shell-like eggcase, we sequenced the draft genome sequence of the species. The genome size was 1.1 Gb, which is the smallest among the cephalopods known to date, with the top 215 scaffolds (average length 5,064,479 bp) covering 81% (1.09 Gb) of the total assembly. A total of 26,433 protein-coding genes were predicted from 16,802 assembled scaffolds. From these, we identified nearly intact HOX, Parahox, Wnt clusters and some gene clusters probably related to the pelagic lifestyle, such as reflectin, tyrosinase, and opsin. For example, opsin might have undergone an extensive duplication in order to adapt to the pelagic lifestyle, as opposed to other octopuses, which are mostly the benthic. Our gene models also discovered several genes homologous to those related to calcified shell formation in Conchiferan Mollusks, such as Pif-like, SOD, and TRX. Interestingly, comparative genomics analysis revealed that the homologous genes for such genes were also found in the genome of the octopus, which does not have a shell, as well as the basal cephalopods Nautilus. Therefore, the draft genome sequence of A. argo we presented here had not only helped us to gain further insights into the genetic background of the dynamic recruitment and dismissal of genes for the formation of an important, converging extended phenotypic structure such as the shell and the shell-like eggcase, but also the evolution of lifestyles in Cephalopods and the octopods, from benthic to pelagic.

genomics↗

bioRxiv2021

Development of a metatranscriptomic analysis method for multiple intestinal sites and its application to the common marmoset intestine

BackgroundThe intestinal microbiome is closely related to host health, and metatranscriptomic analysis can assess the functional activity of microbiomes by quantifying the bacterial gene expression level, which helps to elucidate the interaction between the microbiome and the environment. However, functional changes in the microbiome along the host intestinal tract remain unknown, and previous analytical methods have limitations, such as potentially overlooking unknown genes due to dependence on existing databases and being unable to take full advantage of metatranscriptome to reveal the functional change among multiple environments. ResultTo close these gaps, we develop a novel method that integrates metagenome and metatranscriptome to analyze the functional activity of microbiomes between intestinal sites. This method reconstructs a reference metagenomic sequence across multiple intestinal sites, allowing the gene expression levels of microbiome including unknown bacterial genes to be compared between multiple sites. As a result of applying this method to metatranscriptomic analysis in the intestinal tract of common marmoset, the reconstructed metagenome covered most of the expressed genes and it revealed that the changes in bacterial gene expressions among the caecum, transverse colon, and faeces were more dynamic and sensitive to environmental shifts than its abundance. In typical, the coenzyme synthesis gene and antibacterial resistance gene were more highly expressed in the caecum and transverse colon than in faeces, while there was no significant change in abundance of these genes. ConclusionOur findings demonstrate that an analytical method that integrates metagenome and metatranscriptome in multiple intestinal sites captures functional changes in the microbiomes at the gene resolution level.

microbiology↗

bioRxiv2021

Evolutionary origin of sex differentiation system in insects

The evolution of the functionality of genes and genetic systems is a major source of animal diversity. Its best example is insect sex differentiation systems: promoting male and female differentiation (dual-functionality) or only male differentiation (single-functionality). However, the evolutionary origin of such functional diversity is largely unknown. Here, we investigate the ancestral functions of doublesex, a key factor of insect sex differentiation system, using the apterygote insect, Thermobia domestica, and reveal that its doublesex is essential for only males at the phenotypic level, but contributes to promoting female-specific vitellogenin expression in females. This functional discordance between the phenotypic and transcription-regulatory levels in T. domestica shows a new type of functionality of animal sex differentiation systems. Then, we examine how the sex differentiation system transited from the single-functionality to the dual-functionality in phenotypes and uncover that a conserved female-specific motif of doublesex is detected in taxa with the dual-functional doublesex. It is estimated that the role of the sex differentiation system for female phenotypes may have evolved through accumulating mutations in the protein motif structures that led to the enhancement of its transcription-regulatory function.

evolutionary biology↗

bioRxiv2021

Genomic and transcriptomic analyses of the subterranean termite Reticulitermes speratus: gene duplication facilitates social evolution

Termites are model social organisms characterized by a polyphenic caste system. Subterranean termites (Rhinotermitidae) are ecologically and economically important species, including acting as destructive pests. Rhinotermitidae occupies an important evolutionary position within the clade representing an intermediate taxon between the higher (Termitidae) and lower (other families) termites. Here, we report the genome, transcriptome and methylome of the Japanese subterranean termite Reticulitermes speratus. The analyses highlight the significance of gene duplication in social evolution in this termite. Gene duplication associated with caste-biased gene expression is prevalent in the R. speratus genome. Such duplicated genes encompass diverse categories related to social functions, including lipocalins (chemical communication), cellulases (wood digestion and social interaction), lysozymes (social immunity), geranylgeranyl diphosphate synthase (social defense) and a novel class of termite lineage-specific genes with unknown functions. Paralogous genes were often observed in tandem in the genome, but the expression patterns were highly variable, exhibiting caste biases. Some duplicated genes assayed were expressed in caste-specific organs, such as the accessory glands of the queen ovary and frontal glands in soldier heads. We propose that gene duplication facilitates social evolution through regulatory diversification leading to caste-biased expression and subfunctionalization and/or neofunctionalization that confers caste-specialized functions. Significance StatementTermites are model social organisms characterized by a sophisticated caste system, where distinct castes arise from the same genome. Our genomics data of Japanese subterranean termite provides insights into the evolution of the social system, highlighting the significance of gene duplication. Gene duplication associated with caste-biased gene expression is prevalent in the termite genome. Many of the duplicated genes were related to social functions, such as chemical communication, social immunity and defense, and they often expressed in caste-specific organs. We propose that gene duplication facilitates social evolution through regulatory diversification leading to caste-biased expression and functional specialization. In addition, since subterranean termites are ecologically and economically important species including destructive pests in the world, our genomics data serves as a foundation for these studies.

genomics↗

bioRxiv2021

A chromosome-level genome sequence of a model chrysanthemum: evolution and 1 reference for hexaploid cultivated chrysanthemum

Chrysanthemums are one of the most industrially important cut flowers worldwide. However, their segmental allopolyploidy and self-incompatibility have prevented the application of genetic analysis and modern breeding strategies. We thus developed a model strain, Gojo-0 (Chrysanthemum seticuspe), which is a diploid and self-compatible pure line. Here, we present the 3.05 Gb chromosome-level reference genome sequence, which covered 97% of the C. seticuspe genome. The genome contained more than 80% interspread repeats, of which retrotransposons accounted for 72%. We identified recent segmental duplication and retrotransposon expansion in C. seticuspe, contributing to a relatively large genome size. Furthermore, we identified aretrotransposon, SbdRT, which was enriched in gene-dense genome regions and had experienced a very recent transposition burst. We also demonstrated that the chromosome-level genome sequence facilitates positional cloning in C. seticuspe. The genome sequence obtained here can greatly contribute as a reference for chrysanthemum in front-line breeding including genome editing.

plant biology↗

bioRxiv2021

Diverse DNA modification in marine prokaryotic and viral communities

Chemical modifications of DNA, including methylation, play an important role in prokaryotes and viruses. However, our knowledge of the modification systems in environmental microbial communities, typically dominated by members not yet cultured, is limited. Here, we conducted metaepigenomic analyses by single-molecule real-time sequencing of marine microbial communities. In total, 233 and 163 metagenomic assembly genomes (MAGs) were constructed from diverse prokaryotes and viruses, respectively, and 220 modified motifs and 276 DNA methyltransferases (MTases) were identified. Most of the MTases were not associated with the defense mechanism. The MTase-motif correspondence found in the MAGs revealed 10 novel pairs, and experimentally confirmed the catalytic specificities of the MTases. We revealed novel alternative motifs in the methylation system that are highly conserved in Alphaproteobacteria, illuminating the co-evolutionary history of the methylation system and host genome. Our findings highlight diverse unexplored DNA modifications that potentially affect the ecology and evolution of prokaryotes and viruses.

microbiology↗

bioRxiv2021

Unique metabolic strategies in Hadean analogues reveal hints for primordial physiology

Primordial microorganisms are postulated to have emerged in H2-rich alkaline Hadean serpentinite-hosted environments with homoacetogenesis as a core metabolism. However, investigation of two modern serpentinization-active analogues of early Earth reveals that conventional H2-/CO2-dependent homoacetogenesis is thermodynamically unfavorable in situ due to picomolar CO2 levels. Through metagenomics and thermodynamics, we discover unique taxa capable of metabolism adapted to the habitat. This included a novel deep-branching phylum, "Ca. Lithoacetigenota", that exclusively inhabits Hadean analogues and harbors genes encoding alternative modes of H2-utilizing lithotrophy. Rather than CO2, these metabolisms utilize reduced carbon compounds detected in situ presumably serpentinization-derived: formate and glycine. The former employs a partial homoacetogenesis pathway and the latter a distinct pathway mediated by a rare selenoprotein - the glycine reductase. A survey of serpentinite-hosted system microbiomes shows that glycine reductases are diverse and nearly ubiquitous in Hadean analogues. "Ca. Lithoacetigenota" glycine reductases represent a basal lineage, suggesting that catabolic glycine reduction is an ancient bacterial innovation for gaining energy from geogenic H2 even under serpentinization-associated hyperalkaline, CO2-poor conditions. This draws remarkable parallels with ancestral archaeal H2-driven methyl-reducing methanogenesis recently proposed. Unique non-CO2-reducing metabolic strategies presented here may provide a new view into metabolisms that supported primordial life and the diversification of LUCA towards Archaea and Bacteria.

microbiology↗

bioRxiv2021

Effects of short-term isolation on social animals' behavior: an experimental case study of Japanese macaque

One of the goals in animal socioecology is to understand the functions and dynamics of group living. While observations of free-ranging animals are a crucial source of information, an experimental investigation that manipulates the size or composition, or both, of animal groups in captivity can also bring complementary contributions to the research inquiry. When paired with an automatic data collection by biologging technology, experimental studies on captive animals also allow for big data analyses based on recent machine learning techniques. As an initial exploration of this research paradigm, the present study inquired to what extent isolation of captive Japanese macaques (Macaca fuscata) changed their movement patterns. Using three-dimensional location trajectories of the macaques that were systematically collected via Bluetooth Low Energy beacons and a deep neural network, we estimated the identifiability of whether a macaque was behaving in isolation or in group. We found that the neural network identified the isolation vs. in-group conditions with more than 90% accuracy from a five-minute location trajectory, suggesting that the isolation caused notable changes from the canonical group-living behaviors. In addition, the isolation made each individual more identifiable from one another based on their location trajectories.

animal behavior and cognition↗

bioRxiv2021

Integrated population genomic analysis and numerical simulation to estimate larval dispersal of Acanthaster cf. solaris between Ogasawara and other Japanese regions

The estimation of larval dispersal of marine species occurring on an ecological timescale is significant for conservation. In 2018, a semi-population outbreak of crown of thorns starfish, Acanthaster cf. solaris was observed on a relatively isolated oceanic island, Ogasawara. The aim of this study was to assess whether this population outbreak was caused by large-scale larval recruitment (termed secondary outbreak) from the Kuroshio region. We estimated larval dispersal of the coral predator A. cf. solaris between the Kuroshio and Ogasawara regions using both population genomic analysis and oceanographic dispersal simulation. Population genomic analysis revealed overall genetically homogenized patterns among Ogasawara and other Japanese populations, suggesting that the origin of the populations in the two regions is the same. In contrast, a simulation of 26-year oceanographic dispersal indicated that larvae are mostly self-seeded in Ogasawara populations and have difficulty reaching Ogasawara from the Kuroshio region within one generation. However, a connectivity matrix produced by the larval dispersal simulation assuming a Markov chain indicated gradual larval dispersal migration from the Kuroshio region to Ogasawara in a stepping-stone manner over multiple years. These results suggest that, while large-scale larval dispersal from an outbreak of the Kuroshio population spreading to the Ogasawara population within one generation is unlikely. This study also highlighted the importance of using both genomic and oceanographic methods to estimate larval dispersal, which provides significant insight into larval dispersal that occurs on ecological and evolutionary timescales.

ecology↗

bioRxiv2021

Genome-wide association study of gastric cancer- and duodenal ulcer-derived Helicobacter pylori strains reveals discriminatory amino acid differences and novel oncoprotein candidates

Genome-wide association studies (GWASs) can reveal genetic variations associated with a phenotype in the absence of any hypothesis of candidate genes. The problem of false-positive sites linked with the responsible site might be bypassed in bacteria with a high homologous recombination rate, such as Helicobacter pylori, which causes gastric cancer (GC). We conducted a GWAS followed by regression-based prediction of GC and duodenal ulcer H. pylori strains. We identified 14 single nucleotide polymorphisms (11 amino acid changes) that, combined, allowed effective disease discrimination. They were often informative of the underlying molecular mechanisms, such as electric charge alteration at the ligand-binding pocket, alteration in subunit interaction, and mode-switching of DNA methylation. We also identified three novel virulence factors/oncoprotein candidates. These results provide both defined targets for further informatic and experimental analyses to gain insights into GC pathogenesis and a basis for identifying a set of biomarkers for application in clinical settings.

physiology↗

bioRxiv2021

Evolutionary trajectories of three independent neo-sex chromosomes in Drosophila

Dosage compensation (DC) on the X chromosome is a mechanism to counteract the deleterious effects by gene loss from the Y chromosome. However, DC cannot work efficiently if the X chromosome also degenerates. This indeed occurs in the neo-sex chromosomes in Drosophila miranda, where neo-X as well as neo-Y chromosomes are under accelerated pseudogenization. To examine the generality of this pattern, we investigated the evolution of two additional neo-sex chromosomes that independently emerged in D. albomicans and D. americana and compared their evolutionary processes with that in D. miranda. Comparative genomic and transcriptomic analyses revealed that the pseudogenization rate on neo-X is also accelerated in the two species (though lesser extent in D. americana). We also found that neo-X-linked genes whose neo-Y homologs are pseudogenized tend to be upregulated more stringently than those whose neo-Y homologs remain functional. Moreover, the genes under strong functional constraints and highly expressed in the testis tended to remain functional on neo-X and neo-Y, respectively. Focusing on the D. miranda and D. albomicans neo-sex chromosomes that independently emerged from the same autosome, we further found that the same genes tend to have been pseudogenized in parallel on neo-Y. Those genes include Idgf6 and JhI-26 whose functions seem to be unnecessary or could be even harmful for males. These results indicate that neo-sex chromosomes in Drosophila share a common evolutionary trajectory after their emergence, which may be applicable to other sex chromosomes in a variety of organisms to avoid being an evolutionary dead-end.

evolutionary biology↗