Dosage compensation of the mammalian X chromosome (X) was proposed by Susumu Ohno as a mechanism wherein the inactivation of one X in females would lead to doubling the expression of the other. This would resolve the dosage imbalance between eutherian females (XX) versus male (XY) and between a single active X versus autosome pairs (A). Expression ratio of X- and A-linked genes has been relatively well studied in humans and mice, despite controversial results over the existence of upregulation of X-linked genes. Here we report the first comprehensive test of Ohno's hypothesis in bovine preattachment embryos, germline, and somatic tissues. Overall an incomplete dosage compensation (0.5 < X:A < 1) of expressed genes and an excess X dosage compensation (X:A > 1) of ubiquitously expressed "dosage-sensitive" genes were seen. No significant differences in X:A ratios were observed between bovine female and male somatic tissues, further supporting Ohno's hypothesis. Interestingly, preimplantation embryos manifested a unique pattern of X dosage compensation dynamics. Specifically, X dosage decreased after fertilization, indicating that the sperm brings in an inactive X to the matured oocyte. Subsequently, the activation of the bovine embryonic genome enhanced expression of X-linked genes and increased the X dosage. As a result, an excess compensation was exhibited from the 8-cell stage to the compact morula stage. The X dosage peaked at the 16-cell stage and stabilized after the blastocyst stage. Together, our findings confirm Ohno's hypothesis of X dosage compensation in the bovine and extend it by showing incomplete and over-compensation for expressed and "dosage-sensitive" genes, respectively.

The water skinks Eulamprus tympanum and Eulamprus heatwolei show thermally induced sex determination where elevated temperatures give rise to male offspring. Paradoxically, Eulamprus species reproduce in temperatures of 12-15 °C making them outliers when compared with reptiles that use temperature as a cue for sex determination. Moreover, these two species are among the very few viviparous reptiles reported to have thermally induced sex determination. Thus, we tested whether these skinks possess undetected sex chromosomes with thermal override. We produced transcriptome and genome data for E. heatwolei. We found that E. heatwolei presents XY chromosomes that include 14 gametologs with regulatory functions. The Y chromosomal region is 79-116 Myr old and shared between water and spotted skinks. Our work provides clear evidence that climate could be useful to predict the type of sex determination systems in reptiles and it also indicates that viviparity is strictly associated with sex chromosomes.

Although it is generally accepted that major changes in the earth's history are significant drivers of phylogenetic diversification and extinction, such episodes may also have long-lasting effects on genomic architecture. Here we show that widespread reductions in genome size have occurred in multiple lineages of mammals subsequent to the Cretaceous-Tertiary (KT) boundary, whereas there is no evidence for such changes in other vertebrate, invertebrate, or land plant lineages. Although the mechanisms remain unclear, such shifts in mammalian genome evolution may be a consequence of an increase in the efficiency of selection against excess DNA resulting from post-KT population size expansions. Independent historical changes in genome architecture in diverse lineages raise a significant challenge to the idea that genome size is finely tuned to achieve adaptive phenotypic modifications and suggest that attempts to use phylogenetic analysis to infer ancestral genome sizes may be problematical.

Inverted duplicates are a type of repetitive DNA motifs consist of two copies of reverse complementary sequences separated by a spacer sequence. They can lead to genome instability and many may have no function, but some functional small RNAs are processed from hairpins transcribed from these elements. It is not clear whether the pervasive numbers of such elements in genomes, especially those of mammals, is the result of high generation rates of neutral or slightly deleterious duplication events or positive selection for functionality. To test the functionality of intergenic inverted duplicates without known functions, we used mirror duplicates, a type of repetitive DNA motifs with few reported functions and little potential to form hairpins when transcribed, as a nonfunctional control. We identified large numbers of inverted duplicates within intergenic regions of human and mouse genomes, as well as 19 other vertebrate genomes. Structure characterization of these inverted duplicates revealed higher proportion to form stable hairpins compared with converted mirror duplicates, suggesting that inverted duplicates may produce hairpin RNAs. Expression profiling across tissues demonstrated that 7.8% of human and 5.7% of mouse inverted duplicates were expressed even under strict criteria. We found that expressed inverted duplicates were more likely to be structurally stable than both unexpressed inverted duplicates and expressed converted mirror duplicates. By dating inverted duplicates in the vertebrate phylogenetic tree, we observed higher conservation of inverted duplicates than mirror duplicates. These observations support the notion that expressed inverted duplicates may be functional through forming hairpin RNAs.

Understanding how mammalian genomes have been reshuffled through structural changes is fundamental to the dynamics of its composition, evolutionary relationships between species and, in the long run, speciation. In this work, we reveal the evolutionary genomic landscape in Rodentia, the most diverse and speciose mammalian order, by whole-genome comparisons of six rodent species and six representative outgroup mammalian species. The reconstruction of the evolutionary breakpoint regions across rodent phylogeny shows an increased rate of genome reshuffling that is approximately two orders of magnitude greater than in other mammalian species here considered. We identified novel lineage and clade-specific breakpoint regions within Rodentia and analyzed their gene content, recombination rates and their relationship with constitutive lamina genomic associated domains, DNase I hypersensitivity sites and chromatin modifications. We detected an accumulation of protein-coding genes in evolutionary breakpoint regions, especially genes implicated in reproduction and pheromone detection and mating. Moreover, we found an association of the evolutionary breakpoint regions with active chromatin state landscapes, most probably related to gene enrichment. Our results have two important implications for understanding the mechanisms that govern and constrain mammalian genome evolution. The first is that the presence of genes related to species-specific phenotypes in evolutionary breakpoint regions reinforces the adaptive value of genome reshuffling. Second, that chromatin conformation, an aspect that has been often overlooked in comparative genomic studies, might play a role in modeling the genomic distribution of evolutionary breakpoints.

Two articles published 5 years ago concluded that the genome of the lizard Anolis carolinensis is an amniote genome without isochores. This claim was apparently contradicting previous results on the general presence of an isochore organization in all vertebrate genomes tested (including Anolis). In this investigation, we demonstrate that the Anolis genome is indeed heterogeneous in base composition, since its macrochromosomes comprise isochores mainly from the L2 and H1 families (a moderately GC-poor and a moderately GC-rich family, respectively), and since the majority of the sequenced microchromosomes consists of H1 isochores. These families are associated with different features of genome structure, including gene density and compositional correlations (e.g., GC3 vs flanking sequence GC and intron GC), as in the case of mammalian and avian genomes. Moreover, the assembled Anolis chromosomes have an enormous number of gaps, which could be due to sequencing problems in GC-rich regions of the genome. In conclusion, the Anolis genome is no exception to the general rule of an isochore organization in the genomes of vertebrates (and other eukaryotes).

Mammalian male meiosis requires homologous recombination between the X and Y chromosomes. In humans, such recombination occurs exclusively in the short arm pseudoautosomal region (PAR1) of 2.699 Mb in size. Although it is known that complete deletion of PAR1 causes spermatogenic arrest, no studies have addressed to what extent male meiosis tolerates PAR1 size reduction. Here, we report two families in which PAR1 partial deletions were transmitted from fathers to their offspring. Cytogenetic analyses revealed that a ∼400-kb segment at the centromeric end of PAR1, which accounts for only 14.8% of normal PAR1 and 0.26% and 0.68% of the X and Y chromosomes, respectively, is sufficient to mediate sex chromosomal recombination during spermatogenesis. These results highlight the extreme recombinogenic activity of human PAR1. Our data, in conjunction with previous findings from animal studies, indicate that the minimal size requirement of mammalian PARs to maintain male fertility is fairly small.

Homologous synteny blocks (HSBs) and evolutionary breakpoint regions (EBRs) in mammalian chromosomes are enriched for distinct DNA features, contributing to distinct phenotypes. To reveal HSB and EBR roles in avian evolution, we performed a sequence-based comparison of 21 avian and 5 outgroup species using recently sequenced genomes across the avian family tree and a newly-developed algorithm. We identified EBRs and HSBs in ancestral bird, archosaurian (bird, crocodile, and dinosaur), and reptile chromosomes. Genes involved in the regulation of gene expression and biosynthetic processes were preferably located in HSBs, including for example, avian-specific HSBs enriched for genes involved in limb development. Within birds, some lineage-specific EBRs rearranged genes were related to distinct phenotypes, such as forebrain development in parrots. Our findings provide novel evolutionary insights into genome evolution in birds, particularly on how chromosome rearrangements likely contributed to the formation of novel phenotypes.

Mammalian sex chromosomes evolved from the degeneration of one homolog of a pair of ancestral autosomes, the proto-Y. This resulted in a gene dose imbalance that is believed to be restored (partially or fully) through upregulation of gene expression from the single active X-chromosome in both sexes by a dosage compensatory mechanism. We analyzed multiple genome-wide RNA stability data sets and found significantly longer average half-lives for X-chromosome transcripts than for autosomal transcripts in various human cell lines, both male and female, and in mice. Analysis of ribosome profiling data shows that ribosome density is higher on X-chromosome transcripts than on autosomal transcripts in both humans and mice, suggesting that the higher stability is causally linked to a higher translation rate. Our results and observations are in accordance with a dosage compensatory upregulation of expressed X-linked genes. We therefore propose that differential mRNA stability and translation rates of the autosomes and sex chromosomes contribute to an evolutionarily conserved dosage compensation mechanism in mammals.

The age of sex chromosomes is commonly obtained by comparing the substitution rates of XY gametologs. Coupled with phylogenetic reconstructions, one can refine the origin of a sex chromosome system relative to specific speciation events. However, these approaches are insufficient to determine the presence and duration of ancestral sex chromosome systems that were lost in some species. In this study, we worked with genomic and transcriptomic data from mammals and squamates and analyzed the effect of male mutation bias on X-linked sequences in these groups. We searched for signatures indicating whether monotremes shared the same sex chromosomes with placental mammals or whether pleurodonts and acrodonts had a common ancestral sex chromosome system. Our analyses indicate that platypus did not share the XY chromosomes with placental mammals, in agreement with previous work. In contrast, analyses of agamids showed that this lineage maintained the pleurodont XY chromosomes for several million years. We performed multiple simulations using different strengths of male mutation bias to confirm the results. Overall, our work shows that variations in substitution rates due to male mutation bias could be applied to uncover signatures of ancestral sex chromosome systems.

Many species have morphologically and genetically differentiated sex chromosomes, such as the XY pair of mammals. Y chromosomes are often highly degenerated and carry few functional genes, so that XY males have only one copy of most X-linked genes (whereas females have two). As a result, chromosome-wide mechanisms of dosage compensation, such as the mammalian X-inactivation, often evolve to reestablish expression balance. A similar phenomenon is expected in female-heterogametic species, where ZW females should suffer from imbalances due to W-chromosome degeneration. However, no global dosage compensation mechanisms have been detected in the two independent ZW systems that have been studied systematically (birds and silkworm), leading to the suggestion that lack of global dosage compensation may be a general feature of female-heterogametic species. However, analyses of other independently evolved ZW systems are required to test if this is the case. In this study, we use published genomic and expression data to test for the presence of global dosage compensation in Schistosoma mansoni, a trematode parasite that causes schistosomiasis in humans. We find that Z-linked expression is reduced relative to autosomal expression in females but not males, consistent with incomplete or localized dosage compensation. This gives further support to the theory that female-heterogametic species may not require global mechanisms of dosage compensation.

Plasmids are mobile genetic elements that play a key role in the evolution of bacteria by mediating genome plasticity and lateral transfer of useful genetic information. Although originally considered to be exclusively circular, linear plasmids have also been identified in certain bacterial phyla, notably the actinomycetes. In some cases, linear plasmids engage with chromosomes in an intricate evolutionary interplay, facilitating the emergence of new genome configurations by transfer and recombination or plasmid integration. Genome sequencing of Streptomyces clavuligerus ATCC 27064, a Gram-positive soil bacterium known for its production of a diverse array of biotechnologically important secondary metabolites, revealed a giant linear plasmid of 1.8 Mb in length. This megaplasmid (pSCL4) is one of the largest plasmids ever identified and the largest linear plasmid to be sequenced. It contains more than 20% of the putative protein-coding genes of the species, but none of these is predicted to be essential for primary metabolism. Instead, the plasmid is densely packed with an exceptionally large number of gene clusters for the potential production of secondary metabolites, including a large number of putative antibiotics, such as staurosporine, moenomycin, beta-lactams, and enediynes. Interestingly, cross-regulation occurs between chromosomal and plasmid-encoded genes. Several factors suggest that the megaplasmid came into existence through recombination of a smaller plasmid with the arms of the main chromosome. Phylogenetic analysis indicates that heavy traffic of genetic information between Streptomyces plasmids and chromosomes may facilitate the rapid evolution of secondary metabolite repertoires in these bacteria.

Since Haldane first noticed an excess of paternally derived mutations, it has been considered that most mutations derive from errors during germ line replication. Miyata et al. (1987) proposed that differences in the rate of neutral evolution on X, Y, and autosome can be employed to measure the extent of this male bias. This commonly applied method assumes replication to be the sole source of between-chromosome variation in substitution rates. We propose a simple test of this assumption: If true, estimates of the male bias should be independent of which two chromosomal classes are compared. Prior evidence from rodents suggested that this might not be true, but conclusions were limited by a lack of rat Y-linked sequence. We therefore sequenced two rat Y-linked bacterial artificial chromosomes and determined evolutionary rate by comparison with mouse. For estimation of rates we consider both introns and synonymous rates. Surprisingly, for both data sets the prediction of congruent estimates of alpha is strongly rejected. Indeed, some comparisons suggest a female bias with autosomes evolving faster than Y-linked sequence. We conclude that the method of Miyata et al. (1987) has the potential to provide incorrect estimates. Correcting the method requires understanding of the other causes of substitution that might differ between chromosomal classes. One possible cause is recombination-associated substitution bias for which we find some evidence. We note that if, as some suggest, this association is dominantly owing to male recombination, the high estimates of alpha seen in birds is to be expected as Z chromosomes recombine in males.

Mitochondria are essential organelles whose replication, development, and physiology are dependent upon coordinated gene interactions with both the mitochondrial and the nuclear genomes. The evolution of coadapted (CA) nuclear-mitochondrial gene combinations would be facilitated if such nuclear genes were located on the X-chromosome instead of on the autosomes because of the increased probability of cotransmission. Here, we test the prediction of the CA hypothesis by investigating the chromosomal distribution of nuclear genes that interact with mitochondria. Using the online genome database BIOMART, we compared the density of genes that have a mitochondrion cellular component annotation across chromosomes in 16 vertebrates. We find a strong and highly significant genomic pattern against the CA hypothesis: nuclear genes interacting with the mitochondrion are significantly underrepresented on the X-chromosome in mammals but not in birds. We interpret our findings in terms of sexual conflict as a mechanism that may generate the observed pattern. Our finding extends single-gene theory for the evolution of sexually antagonistic genes to nuclear-mitochondrial gene combinations.

The compact genome of fugu (Takifugu rubripes) has been used widely as a reference genome for understanding the evolution of vertebrate genomes. However, the fragmented nature of the fugu genome assembly has restricted its use for comparisons of genome architecture in vertebrates. To extend the contiguity of the assembly to the chromosomal level, we have generated a comprehensive genetic map of fugu and anchored the scaffolds of the assembly to the 22 chromosomes of fugu. The map consists of 1,220 microsatellite markers that provide anchor points to 697 scaffolds covering 86% of the genome assembly (http://www.fugu-sg.org/). The integrated genome map revealed a higher recombination rate in fugu compared with other vertebrates and a wide variation in the recombination rate between sexes and across chromosomes of fugu. We used the extended assembly to explore recent rearrangement events in the lineages of fugu, Tetraodon, and medaka and compared them with rearrangements in three mammalian (human, mouse, and opossum) lineages. Between the two pufferfishes, fugu has experienced fewer chromosomal rearrangements than Tetraodon. The gene order is more highly conserved in the three teleosts than in mammals largely due to a lower rate of interchromosomal rearrangements in the teleosts. These results provide new insights into the distinct patterns of genome evolution between teleosts and mammals. The consolidated genome map and the genetic map of fugu are valuable resources for comparative genomics of vertebrates and for elucidating the genetic basis of the phenotypic diversity of ~25 species of Takifugu that evolved within the last 5 My.

Placental mammals present 180 million-year-old Y chromosomes that have retained a handful of dosage-sensitive genes. However, the expression evolution of Y-linked genes across placental groups has remained largely unexplored. Here, we expanded the number of Y gametolog sequences by analyzing ten additional species from previously unexplored groups. We detected seven remarkably conserved genes across 25 placental species with known Y repertoires. We then used RNA-seq data from 17 placental mammals to unveil the expression evolution of XY gametologs. We found that Y gametologs followed, on average, a 3-fold expression loss and that X gametologs also experienced some expression reduction, particularly in primates. Y gametologs gained testis specificity through an accelerated expression decay in somatic tissues. Moreover, despite the substantial expression decay of Y genes, the combined expression of XY gametologs in males is higher than that of both X gametologs in females. Finally, our work describes several features of the Y chromosome in the last common mammalian ancestor.

The sequencing and comparison of vertebrate genomes have enabled the identification of widely conserved genomic elements. Chief among these are genes and cis-regulatory regions, which are often under selective constraints that promote their retention in related organisms. The conservation of elements that either lack function or whose functions are yet to be ascribed has been relatively little investigated. In particular, microsatellites, a class of highly polymorphic repetitive sequences considered by most to be neutrally evolving junk DNA that is too labile to be maintained in distant species, have not been comprehensively studied in a comparative genomic framework. Here, we used the UCSC alignment of the human genome against those of 11 mammalian and five nonmammalian vertebrates to identify and examine the extent of conservation of human microsatellites in vertebrate genomes. Out of 696,016 microsatellites found in human sequences, 85.39% were conserved in at least one other species, whereas 28.65% and 5.98% were found in at least one and three nonprimate species, respectively. An exponential decline of microsatellite conservation with increasing evolutionary time, a comparable distribution of conserved versus nonconserved microsatellites in the human genome, and a positive correlation between microsatellite conservation and overall sequence conservation, all suggest that most microsatellites are only maintained in genomes by chance, although exceptionally conserved human microsatellites were also found in distant mammals and other vertebrates. Our findings provide the first comprehensive survey of microsatellite conservation across deep evolutionary timescales, in this case 450 Myr of vertebrate evolution, and provide new tools for the identification of functional conserved microsatellites, the development of cross-species microsatellite markers and the study of microsatellite evolution above the species level.

Sensory gene families are of special interest for both what they can tell us about molecular evolution and what they imply as mediators of social communication. The vomeronasal type-1 receptors (V1Rs) have often been hypothesized as playing a fundamental role in driving or maintaining species boundaries given their likely function as mediators of intraspecific mate choice, particularly in nocturnal mammals. Here, we employ a comparative genomic approach for revealing patterns of V1R evolution within primates, with a special focus on the small-bodied nocturnal mouse and dwarf lemurs of Madagascar (genera Microcebus and Cheirogaleus, respectively). By doubling the existing genomic resources for strepsirrhine primates (i.e. the lemurs and lorises), we find that the highly speciose and morphologically cryptic mouse lemurs have experienced an elaborate proliferation of V1Rs that we argue is functionally related to their capacity for rapid lineage diversification. Contrary to a previous study that found equivalent degrees of V1R diversity in diurnal and nocturnal lemurs, our study finds a strong correlation between nocturnality and V1R elaboration, with nocturnal lemurs showing elaborate V1R repertoires and diurnal lemurs showing less diverse repertoires. Recognized subfamilies among V1Rs show unique signatures of diversifying positive selection, as might be expected if they have each evolved to respond to specific stimuli. Furthermore, a detailed syntenic comparison of mouse lemurs with mouse (genus Mus) and other mammalian outgroups shows that orthologous mammalian subfamilies, predicted to be of ancient origin, tend to cluster in a densely populated region across syntenic chromosomes that we refer to as a V1R "hotspot."

The loss of Y-linked genes during sex chromosome evolution creates a potentially deleterious low gene dosage in males. Recent studies have reported different strategies of dosage compensation. Unfortunately, most of these studies investigated taxa with comparatively old sex chromosome systems, which may limit insights into the evolution of dosage compensation and thus into the causes of different compensation strategies. Using deep RNA sequencing, we investigate differential expression patterns along the young XY chromosomes of threespine sticklebacks. Our strata-specific analyses provide new insights into the spatial patterns during the early stages of the evolution of dosage compensation. In particular, our results indicate systematic upregulation of male gene expression in stratum II, which in turn causes female hypertranscription in the same stratum. These findings are consistent with theoretical predictions that selection during early stages of sex chromosome evolution is stronger for a compensating upregulation in males than for the countercompensation of female hyperexpression. In contrast, no elevated gene expression is detectable in stratum I. We argue that strata-specific differences in compensating male gene expression may evolve in response to differences in the prevailing mechanism of Y chromosome degeneration.

There are large-scale variations of the GC-content along mammalian chromosomes that have been called isochore structures. Primates and rodents have different isochore structures, which suggests that these lineages exhibit different modes of GC-content evolution. It has been shown that, in the human lineage, GC-biased gene conversion (gBGC), a neutral process associated with meiotic recombination, acts on GC-content evolution by influencing A or T to G or C substitution rates. We computed genome-wide substitution patterns in the mouse lineage from multiple alignments and compared them with substitution patterns in the human lineage. We found that in the mouse lineage, gBGC is active but weaker than in the human lineage and that male-specific recombination better predicts GC-content evolution than female-specific recombination. Furthermore, we were able to show that G or C to A or T substitution rates are predicted by a combination of different factors in both lineages. A or T to G or C substitution rates are most strongly predicted by meiotic recombination in the human lineage but by CpG odds ratio (the observed CpG frequency normalized by the expected CpG frequency) in the mouse lineage, suggesting that substitution patterns are under different influences in primates and rodents.